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IMPACT OF SUBSTANCE P ON CELLULAR IMMUNITY
G.S. KATSANOS, A. ANOGEIANAKI, C. ORSO1, S. TETÈ2, V. SALINI1, P.L. ANTINOLFI3
and G. SABATINO4
Immunology Division, University of Chieti-Pescara, Chieti; 1Orthopaedics Division, University of Chieti-Pescara, Chieti; 2Dental School, University of Chieti-Pescara, Chieti; 3Orthopaedics Division, University of Perugia, Perugia; 4Paediatric Division, University of Chieti-Pescara,
Chieti, Italy
Received February 12, 2008 – Accepted April 24, 2008
The migration of activated T lymphocytes to sites of inflammation is part of a general process of leukocyte recruitment (1-5). There are numerous cellular contacts between nerve fibers and immune cells which can synthesize neuromediators and express receptors to these molecules (6). Neuromediators are able to modulate functions of immune cells and other cells. Communication between nerves and immune and inflammatory cells plays a major role in the modulation of several disfunctions including ion transport, mucosal permeability and cytokine production (7). Substance P (SP) peptide is involved in the pathogenesis of several inflammatory diseases, demonstrating that there is a narrow interrelation between the nervous system and immunity (8). SP released from sensory neurons and inflammatory cells, participates in
inflammation by interacting, directly or indirectly, with NK-1 receptor (NK-1R) expressed on nerves, epithelial cells, and immune and inflammatory cells, such as mast cells, macrophages and T cells (9).
SP has various immunomodulatory effects, including in vitro modification of lymphocyte proliferation and cytokine release (10). The principal effect of substance P on lymphocytes appears to be a stimulating one, both in vitro and in vivo. SP induces long-term potentiation of the immune response and raises the question of an involvement of SP as a major mediator of immunological memory. SP-induced enhancement of thymus cellularity parallels its ability of inhibiting the thymocyte apoptotic program. Indeed, exogenously administered SP completely nullified capsicin-induced apoptosis, suggesting a role for cellular protection.
Mailing address:Prof. G. Sabatino, Paediatric Division,University of Chieti-Pescara,via dei Vestini,Chieti, ItalyTel: ++39 0871 358339e-mail: [email protected]
Much evidence suggests a cross-talking between nerve fibers and the immunity system. The immunomodulation by substance P includes cell activation and proliferation of human cells, with cytokine and chemokine generation and release. Substance P was first isolated by Leeman et al. as an undecapeptide with important neurotransmitter-neuromodulator effects. In addition, substance P was shown to induce and mediate inflammation, angiogenesis, infections, intestinal mucosal immunity and stress. Substance P is able to activate several immune cells, such as CD4+ and CD8+ T lymphocytes, mast cells, NK cells and macrophages. In recent studies we have shown that substance P can activate interleukin-8, a CXC chemokine, demonstrating its involvement in immune cell chemoattraction. We believe that substance P is important in understanding the pathophysiology of inflammation.
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SP stimulates concanavalin A (ConA)-induced thymocyte proliferation, CD25 expression and IL-2 production, whereas SP antagonist exerts inhibitory effects on these functions. NK-1R is expressed on the majority of CD5+ thymocytes. SP administration also stimulated concanavalin A (Con A)-induced thymocyte proliferation of capsicin-treated rats, completely reversing the capsicin-induced inhibition. The SP-mediated stimulation of Con A-induced thymocyte proliferation is NK-1R-dependent. In conclusion, thymocytes spontaneously release SP, which is increased upon ConA or CD3 stimulation.
Expression of substance P (SP) receptors is markedly increased by infection, especially for T lymphocytes. CD4+ T lymphocytes predominantly express SP receptors in the secondary immune response (9). Several authors suggest that SP receptor expression may be important in the development of primary and secondary immune responses to infections (11). Moreover, SP receptor mRNA is expressed in intraepithelial and lamina propria T lymphocytes, as well as CD4+, CD8+ and CD4-CD8-CD3+ subsets. Studies suggest that SP production and SP receptor expression are distinctive for intestinal mucosal immunity and that SP may act as a modulator of an ongoing controlled inflammation in normal gut, by acting through its specific receptor on T lymphocytes in an autocrine and/or paracrine pattern (13).
Understanding the pathophysiology of inflammation and the role of chemical mediators such as SP, may improve inflammation management (14-16). Stress contributes to inflammation, an effect mediated by SP through the stimulation of macrophages and releasing cytokines (Fig. 1). It has been reported that macrophage functions are altered in stress (10). Macrophages involved in infection and activated by LPS are also involved in the stress response due to SP. SP released by sensory nerves plays a protective role against lipopolysaccharide (LPS)-induced hypotension. SP, which activates NK1 and stimulates the sympathetic axis, plays a protective role against endotoxin-induced hypotension and mortality (17). Moreover, Leeman et al. described that SP enhances LPS-induced macrophage TNF-alpha production from stressed animals, and recently we found that SP stimulates
the production of IL-8, a CXC chemokine in response to stress (in press). In addition, it has been found that SP induces cycloxygenase-2 expression in polymorphonuclear cells, demonstrating its effect in pain, fever and inflammation. Recently, chemokines such as CCL2 were identified as possible key integrators of neuropathic pain and inflammation after peripheral nerve lesion.
Increased histamine, SP, and monocyte chemoattractant protein (MCP)-1 levels caused inflammation and pain following severe injury. Evidence that neuropeptides contribute to inflammatory responses to injury include inflammatory cell chemotaxis, cytokine and growth factor production. The neuropeptide SP, which is released from nerve endings after injury, induces inflammation and mediates angiogenesis, keratinocyte proliferation, and fibrogenesis (18-19). In recent years, investigations have shown that, in addition to the classic factors, numerous endogenous peptides play a relevant regulatory role in angiogenesis. Such regulatory peptides, each of which exerts well-known specific biological activities, are present, along with their receptors, in the blood vessels and may take part in the control of the “angiogenic switch” (20-22). An in vivo and in vitro proangiogenic effect has been demonstrated for many regulatory peptides, such as SP. There is evidence that the angiogenic action of some of these peptides is at least partly mediated by their stimulating effect on VEGF. Specifically, we found for the first time that SP raises the expression of VEGF in a mast cell line, but VEGF blockade does not affect the proangiogenic action of SP. Investigations on “uncommon” regulators of angiogenesis could open new perspectives in the therapy of diseases coupled to dysregulation of angiogenesis.
The function of substance P in the communication of peripheral inflammation to the brain is very important. We believe that in the bidirectional loop of brain-immune interactions, substance P expression is a determinating factor in anxiety and depression, as well as having a potential role in the neural regulation of peripheral inflammation. Several components of the inflammatory system, including cytokines, chemokines, cell adhesion molecules and SP, have been reported to participate in adhesion formation (13, 23-24). Adhesion
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development depended upon the interferon-gamma (IFN-gamma) and signal transducer and activator of transcription-1 (STAT1) system (24). Natural killer T (NKT) cell-deficient mice developed adhesion poorly, whereas they developed severe adhesion after reconstitution with NKT cells from wild-type mice, suggesting that NKT cell IFN-gamma production is indispensable for adhesion formation. This response does not depend on STAT4, STAT6, interleukin-12 (IL-12), IL-18, tumor necrosis factor-alpha, Toll-like receptor 4 or myeloid differentiation factor-88-mediated signals. In addition, SP slightly induces LFA-1 mRNA translation and activation signals for integrins. The early-phase alteration of LFA-1 mRNA translation may be due to the changes of CD8+ T cells rather than changes of CD4+ cells (25-27). However, interaction of the integrin leukocyte function-associated antigen (LFA)-1 (CD11a/CD18) with its ligands, the intercellular adhesion molecules (ICAM)-1, -2, and -3 (CD54, CD102, and CD50), is pivotal to many leukocyte adhesion events. SP might be a physiological antagonist of the potent thymocyte apoptosis induced by glucocorticoids (28-29).
Hematopoiesis occurs in the bone marrow through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34 and methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1I and the neuropeptide, substance P (SP) (30-33).
The indirect method occurs by SP production, which stimulates CD34+ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators. It is particularly important that pro-inflammatory factors, such as inflammatory cytokines, leukocytes and oxidative stress, have been demonstrated to be involved in the development of gastroesophageal reflux disease (GERD) including non-erosive reflux disease (NERD) (29). Studies reveal that expression of IL-8 mRNA, a potent neutrophil chemotactic and activating peptide, is correlated with the endoscopic grade of esophagitis or with inflammatory cell infiltration (29, 34-37).
The neurokinin (NK) receptor family has been
proposed as targets for neural-related diseases. The experimental studies indicate that this family of receptors might also be targets for malignancies, both solid and hematological tumors (38).
Substance P is the most important member of the tachykinin family that constitutes the major endogenous ligand for the NK1 receptor type (39-40). The presence of functional NK1 receptors has been documented in malignant brain tumours of glial origin (40-41). It has been found that SP-NK1 receptor communication is involved in glioma development and progression, which is possible because tumor cells display SP-mediated autocrine activity, the ability of cytokine stimulation and MAP kinase activation. It has been suggested that SP receptor antagonist application might be useful in attempts directed at anti-cancer and anti-stress therapy.
Fig. 1. Correlation between stress, substance P and inflammation.
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ACKNOWLEDGEMENTS
We wish to thank Susan Leeman for her lecture given at our University of Chieti-Pescara, Italy, from which we amplified our knowledge of substance P.
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34. Karagiannides I, Kokkotou E, Tansky M, Tchkonia T, Giorgadze N, O’Brien M, Leeman SE, Kirkland JL, Pothoulakis C. Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes. Proc Natl Acad Sci USA 2006; 103(13):5207-12.
35. Papoff P, Mancuso M, Barbàra CS, Moretti C. The role of terlipressin in pediatric septic shock: a review of the literature and personal experience. Int J Immunopathol Pharmacol 2007; 20:213-222.
36. Hauswirth AW, Sonneck K, Florian ST, Krauth MT, Böhm A, Sperr WR, Valenta R, Schernthaner G-H, Printz D, Fritsch G, Bühring H-J, Valent P. Interleukin-3 promotes the expression of e-npp3/cd203c on human blood basophils in healthy subjects and in patients with birch pollen allergy. Int J Immunopathol Pharmacol 2007; 20:267-278.
37. Madonna R, Massaro M, Pandolfi A, Consoli A, Caterina RD. The prominent role of p38 mitogen-activated protein kinase in insulin-mediated enhancement of VCAM-1 expression in endothelial cells. Int J Immunopathol Pharmacol 2007; 20:539-555.
38. Rameshwar P. Implication of possible therapies targeted for the tachykinergic system with the
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ABNORMAL SYNTHESIS OF IgA IN COELIAC DISEASE AND RELATED DISORDERS
R. CIANCI, G. CAMMAROTA, S. LOLLI, G.B. GASBARRINI and F. PANDOLFI
Institute of Internal Medicine, Catholic University, Rome, Italy
Received March 5, 2008 – Accepted April 23, 2008
The pathogenesis of coeliac disease (CD) is complex. One controversial aspect is the role of IgA anti-endomysial (EMA) antibodies. Despite being the most reliable marker for CD diagnosis, its role in the pathogenesis (if any) remains obscure. The paradox is reinforced by the observation that CD is more common in IgA-deficient individuals. In this review, we discuss recent data suggesting that IgA autoantibodies may be related to aspecific dysregulation of IgA. In addition, new insights have elucidated new genes involved in IgA production and linked to CD. Allelic frequency of HS1,2 enhancer which regulates Ig synthesis is altered in CD and other IgA mediated disorders. We suggest that in CD, a T-cell mediated disease, the role of IgA anti-EMA autoantibodies remains elusive and could well be merely an epiphenomenon not directly related to pathogenic mechanisms, but rather to a state of heightened immunological responsiveness in genetically predisposed individuals.
Coeliac disease (CD) is a gluten-dependent entheropathy occurring in genetically predisposed individuals and resulting from inflammatory injury to the mucosa of the small intestine associated to a vast array of clinical manifestations. It has a major clinical impact since, with the recent increased awareness of its presentation, the frequency has been reported to be as high as 1/200 in Caucasians. CD is associated with a variety of other diseases.
Many of the events that generate the altered immune response to gluten have been well characterized. However, despite intensive investigation, there are several features relevant to CD pathogenesis that are still unclear. This is due in part to its complex pathogenesis: on one hand, T-cells are responsible for the ultimate tissue injury; on the other hand, B cells produce IgA and/or IgG anti-endomysial antibodies (EMA) whose role in pathogenesis is controversial and is herein reviewed in the context of a wider spectrum of diseases with
abnormal IgA regulation.The central role of CD4 T-lymphocytes
from genetically susceptible individuals in the pathogenesis of CD is now well established. The occurrence of the disease is strongly associated with HLA-DQ2, while most DQ2-negative patients express the DQ8 molecule. Gliadin-specific DQ2- and DQ8-restricted CD4 T-cells have been found at the site of the lesion in the gut (1). Taken together, these findings suggest that the intestinal damage in CD is caused by aberrant mucosal T-cell recognition of gliadin- derived peptides in the context of disease-associated Class-II molecules.
T-cell mediated injury A large body of evidence indicates that Th-
1-mediated cellular immunity is responsible for intestinal damage (1). As far as the cytokine pattern involved is concerned, this is a typical Th-1 pattern, accordingly, we have shown that treatment with
Mailing address: Prof. Franco Pandolfi, MDInstitute of Internal Medicine,Catholic University, Largo A. Gemelli, 800168 Rome, ItalyTel: ++39 06 30155671 Fax: ++39 06 35502775e-mail: [email protected]
EDITORIAL
Vol. 22, no. 2, 99-104 (2008)
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interferon may precipitate the onset of CD (2).The effector phases of tissue destruction appear
to be mediated by cytotoxic cells through both apoptosis-mediated tissue damage and perforins. The role of cellular cytotoxicity is supported by the finding that intra-epithelial lymphocytes (IEL) of the CD4- CD8+ phenotype are expanded in CD (1). Hue et al. showed that the toxic gliadin A p31–49 peptide in fact induced the expression of the non-classical MHC molecule MICA on the cell surface of villous epithelium in treated CD, but not in controls, thus T-cells are driven by the presence of tissue stress resulting in the production of MHC-like molecules such as MICA (3).
Serological diagnosis of CDCD diagnosis has been traditionally based on
gastroscopy. Thus, scientists have been involved for some time in the quest for a serological marker for its evident practical and economical benefits. During the last decades many efforts have been made to find serological markers for CD. Since the 1970s, anti-gliadin antibodies (AGA) of IgG and IgA types have been used for CD screening, but these antibodies are also present in a significant proportion of normal donors. Later, anti-endomysium antibodies (EMA) emerged as the most specific serological marker for CD. Tissue transglutaminase (tTG) type 2 (TG2) was eventually recognized as the target of anti-EMA antibodies. TG2 is a Ca-dependent enzyme responsible for the post-translational modification of proteins by transamidation or deamidation of specific polypeptide-bound glutamines and has been identified as the autoantigen recognized by anti-EMA antibodies. The phenomenon of endomysial antibodies has been explained by molecular mimicry between gliadin and the endomysial antigen, or by the unmasking of cryptic epitopes in the endomysial antigen upon exposure to gliadin (4).
THE PUZZLE OF IgA AUTOANTIBODIES
Anti-TG2 autoantibodies are the most common test for CD diagnosis, and are also the more specific marker for CD with a high sensitivity. Among anti-TG2 autoantibodies, those of the IgA class are considered to be the more specific, with 96% sensitivity in children and 97% in adults (5).
Available data suggest that these antibodies are produced solely in the intestinal mucosa, yet their role in CD has been defined by Woof and Kerr as a “contradiction” (6), since a T-cell mediated disease is commonly diagnosed by specific IgA autoantibodies. Sollid defines the presence of IgA antibodies as enigmatic, suggesting that their role is just an epiphenomenon (7).
The paradox of IgA anti-TG2 is reinforced by the intriguing observation that a disease diagnosed by the presence of specific IgA antibodies is more common in IgA-deficient individuals where only IgG anti-TG2 are present. Moreover, patients with CD may present anti-TG2 antibodies in the absence of intestinal villous atrophy and it is generally known that for every symptomatic patient with CD there are several asymptomatic cases. Moreover, anti-TG2 antibody synthesis is not tissue specific, but may be synthesized in different tissues including the cerebro spinal fluid (CSF) of patients with neurodegenerative diseases (8). Of peculiar interest is the fact that such autoantibodies are not found only in gluten-induced diseases, such as gluten ataxia (9) and dermatitis herpetiformis (DH). In addition, anti-tTG antibodies are higher in hepatic patients compared to controls, but their specificity for CD diagnosis in this group of patients is low (10), as also in patients with gluten-unrelated neurodegenerative diseases (9).
In addition, several studies report that anti-TG2 antibodies are deposited in what appears to be histologically normal small bowel mucosa even before they can be detected in the circulation and prior to the formation of the gluten-induced small intestinal lesion, thus strengthening the contention that gluten sensitivity is an immune-mediated systemic disease that can present with diverse manifestations and diverse organ involvement. Enteropathy is not a prerequisite for its existence (9) and autoantibodies to TG2 can be found in the absence of intestinal alterations (11).
Furthermore, anti-TG2 antibodies are aspecifically expressed in other diseases. For instance, patients with Ullrich-Turner syndrome had positive tTG autoantibodies (12). Serum anti-tTG positivity can be found in subjects with non-Hodgkin’s lymphoma without CD (13). IgA and IgG anti-tTG occur in a large number of patients with end-stage heart failure without concomitant
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CD (14). Studying gastrointestinal diseases, Farrace et al. also concluded that the presence of anti-tTG antibodies is not a specific event occurring in CD patients, but is a general phenomenon related to mucosal lesions rather than to the autoimmune nature of CD (15). In keeping with this hypothesis they detected both circulating anti-tTG and induction of apoptosis in other pathologies leading to intestinal lesions such as Crohn’s disease and ulcerative colitis (15). In addition, Kazemi et al. (16) reported non-TG2 IgA autoantibodies shared by CD and inflammatory bowel diseases. All these data suggest that the presence of anti-TG2 autoantibodies is a wider hallmark of an autoimmune status leading to CD as well as other diseases. In keeping with this hypothesis, Metzger et al. found that elevated IgA anti-TG2 antibodies were associated with higher all-cause mortality risk in a cohort of over 4,500 subjects (KORA/MONICA Augsburg project) (17). They conclude that individuals with elevated IgA anti-tTG antibodies had a highly increased mortality risk, particularly due to cancer, and suggest that IgA anti-tTG antibody level is a marker of an aspecific autoreactivity which may be associated to serious diseases such as cancer, chronic liver disease or end-stage heart failure.
Autoantibodies are unlikely to have a direct pathogenetic mechanism also because they are insufficient to block tTG activity (18). However, the mechanism of tissue injury is complex and a role for autoantibodies can not be definitely ruled out. It has been suggested that anti-TG2 may mediate tissue injury by indirect mechanisms, therefore it can not be excluded that anti-TG2 antibodies may contribute to tissue damage.
Taken together, these data question a definitive role of anti-TG2 autoantibodies in the pathogenis of CD, and suggest that IgA autoantibodies may be related to an aspecific dysregulation of IgA synthesis rather than to discrete pathogenetic mechanisms occurring in various diseases. Further confirmation of dysregulation of IgA production in CD is supported by the presence of anti-Saccharomyces cerevisiae IgA antibodies and anti-casein antibodies (19).
Abnormal IgA synthesis as a result of systemic conditions has not received sufficient attention despite the fact that IgA synthesis is crucial to immune protection, being both the most abundant
immunoglobulin and the only one present in high concentration in secretions.
IgA nephropathy (IgAN)IgAN is the most common form of
glomerulonephritis, characterized by deposition of IgA in the glomerulus. The pathogenesis of IgAN remains elusive, but the basic abnormality of the disease has to be found in the IgA system and not in the kidney. IgAN is a disease characterized by hyper IgA production, with increased levels of serum IgA and IgA immuno complexes. Since IgA production alone is not sufficient to develop IgAN, physiochemical properties of circulating IgA, such as altered glycosylation, are likely to play a role (20).
IgA nephropathy occurs as a primary disease with no apparent trigger although it can be associated with liver cirrhosis. In both cases, the IgA deposited in the kidney is primarily polymeric IgA1. IgA in IgAN is abnormal in terms of its molecular size and specificity, further confirming the notion that abnormal regulatory relations exist in IgA synthesis in IgA related diseases.
Although clinical association between IgAN and CD are anecdotal (21), some relations among these two diseases have been described since anti-gliadin IgA antibodies with aberrant duodenal histopathological findings have been recently found in IgAN (22).
Other IgA autoantibodiesAlthough most autoimmune diseases
are commonly thought to be related to IgG autoantibodies, IgA autoantibodies are present in most of the major autoimmune diseases such as systemic lupus erythematosus, Sjiogren’s syndrome (23) and rheumathoid arthritis, where their presence can be related to clinical parameters (24). IgA autoantibodies are present in a variety of skin diseases. The precise role of IgA autoantibodies in human disease is largely unknown, thus confirming the need for additional investigation in the field of IgA dysregulation.
Anti-tTG in Dermatitis HerpetiformisDifferent types of tTG may also hold the key to
the diverse manifestations seen within the spectrum
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of gluten sensitivity. A recent study reported that patients with dermatitis herpetiformis (DH), a disease strongly associated to CD, have antibodies with low affinity for tissue transglutaminase (TG2) but very high affinity for epidermal transglutaminase (TG3) (25). Interestingly, at variance with CD, in DH, IgA autoantibodies appear to be directly involved in the pathogenesis through the action of granular IgA skin deposits (26). Other skin diseases, such as psoriasis, present IgA abnormalities and anti-tTG autoantibodies (27). They are also present in psoriatic arthritis and ankylosing spondylitis (28).
HS1,2 ENHANCER
The IgH cluster is regulated by 3’ Regulatory Region (3’RR) harbouring three enhancers which regulate Ig production and B cell maturation (29). Regulation of transcription is mediated partially by the interaction of trans-acting DNA-binding proteins with specific motifs in promoter and enhancer elements.
The human IgH 3’RR, located downstream of each of the two C alpha genes, modulates germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The immunoglobulin (Ig) heavy chain 3’RR-I in humans includes three enhancers, the central of which, HS1,2-A, is polymorphic (29). The structure, and possibly the function, of the enhancers situated in the immunoglobulin regulative region, at the 3’ end of the constant heavy chain cluster, is largely conserved, but with variable allele frequency in different human populations (30).
Mouse 3’ RR has been shown to play an important role in regulation of Ig isotype switching through their influence on the activity of the GL region promoters. Denizot et al. conclude that these enhancers play a role in the occurrence and evolution of various diseases involving IgA secretion (31).
Recently, considerable interest in HS1,2 gene in relation to diseases with abnormal IgA synthesis, has been generated by the pioneering observation by Aupetit et al. that HS1,2 Allele *2 frequency and homozygosity are increased in IgAN (32) and by our report of an increase in CD (33), dermatitis herpetiformis, psoriasis and psoriatic arthritis (34).
As far as the role of HS1,2-A is concerned, in vitro studies have demonstrated an increased enhancer activity on the luciferase gene by the HS1,2-A allele *2 compared with *1 as well as for all the alleles harboring a higher number of repeats in respect to the shorter ones. The role of HS1,2-A allele *2 may be then important in modifying immune or inflammatory responses in our patients. The most evident difference of allele *2 in regards to allele *1 is the presence of an extra consensus site for NF-κB and SP1 (34). Because of the possible role of HS1,2 in IgA production, we investigated HS1,2 allele frequency in patients with Selective IgA deficiency (IGAD). Our preliminary data suggest a statistically significant increase of allele *1 in these patients as well as a decrease of allele *2 frequency (manuscript in preparation).
In conclusion, in this review we suggest that in CD, a T-cell mediated disease, the role of IgA anti-TG2 autoantibodies remains elusive and could well be merely an epiphenomenon not directly related to pathogenic mechanisms, as supported by the presence of such antibodies in different diseases and could be rather related to a state of heightened immunological responsiveness in genetically predisposed individuals. Recent studies in IgA deficiency and diseases with IgA dysregulation have elucidated a complex set of genes related to IgA switch.
Clearly, further studies are needed to fully appreciate the role of these genes in the pathogenesis of CD and related disorders. In this line of research, we recently reported that HS1, 2-A allele *2, a gene regulating isotype switch, may contribute to the susceptibility to CD and related disorders.
The conclusion of this review is twofold: 1) clinically, available data suggest that IgA
anti-tTG autoantibodies are not linked to the pathogenesis of CD, but rather an associated marker of abnormal immune responses in CD and related disorders. Therefore, data of anti-tTG alone should be considered with caution; for instance, in a prospective cohort, 1% of children tested positive for anti-tTG were all asymptomatic to gluten sensitivity. The incidence in adults is also 1%, supporting that this test may be clinically unreliable (35). Although largely used as a first line screening for CD, the results of anti-tTG alone are limited and should be associated to intestinal endoscopy.
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2) genetically, as well as the well-known HLA genes which induce disease predisposition, very recent results suggest that in addition to HS1,2, several other genetic risk variants are being detected (36). Only a larger genetic analysis combining classical HLA typing with HS1,2 and the newly detected risk variants, will eventually be able to completely define a genetic risk for CD.
2. Cammarota G, Cuoco L, Cianci R, Pandolfi F, Gasbarrini G. Onset of coeliac disease during treatment with interferon for chronic hepatitis C. Lancet 2000; 356:1494-5.
3. Hue S, Mention JJ, Monteiro RC et al. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 2004; 21:367-77.
4. Natter S, Granditsch G, Reichel GL et al. IgA cross-reactivity between a nuclear autoantigen and wheat proteins suggests molecular mimicry as a possible pathomechanism in celiac disease. Eur J Immunol 2001; 31:918-28.
5. Rostom A, Dube C, Cranney A et al. The diagnostic accuracy of serologic tests for celiac disease: a systematic review. Gastroenterology 2005; 128:S38-46.
6. Woof JM, Kerr MA. The function of immunoglobulin A in immunity. J Pathology 2006; 208:270-82.
7. Sollid LM. Molecular basis of celiac disease. Ann Rev Immunol 2000; 18:53-81.
8. Schrodl D, Kahlenberg F, Peter-Zimmer K, Hermann W, Kuhn HJ, Mothes T. Intrathecal synthesis of autoantibodies against tissue transglutaminase. J Autoimmunity 2004; 22:335-40.
9. Hadjivassiliou M, Williamson CA, Woodroofe N. The immunology of gluten sensitivity: beyond the gut. Trends in Immunol 2004; 25:578-82.
10. Germenis AE, Yiannaki EE, Zachou K et al. Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases. Clin Diagn Lab Immunol 2005; 12:941-8.
11. Sollid LM, Jabri B. Is celiac disease an autoimmune
disorder? Curr Opin Immunol 2005; 17:595-600.12. Bettendorf M, Doerr HG, Hauffa BP et al. Prevalence
of autoantibodies associated with thyroid and celiac disease in Ullrich-Turner syndrome in relation to adult height after growth hormone treatment. J Pediatr Endocrinol Metabol 2006; 19:149-54.
13. Carroccio A, Iannitto E, Di Prima L et al. Screening for celiac disease in non-Hodgkin’s lymphoma patients: a serum anti-transglutaminase-based approach. Dig Dis Sci 2003; 48:1530-6.
14. Peracchi M, Trovato C, Longhi M et al. Tissue transglutaminase antibodies in patients with end-stage heart failure. Am J Gastroenterol 2002; 97:2850-4.
15. Farrace MG, Picarelli A, Di Tola M et al. Presence of anti-”tissue” transglutaminase antibodies in inflammatory intestinal diseases: an apoptosis-associated event? Cell Death Differ 2001; 8:767-70.
16. Kazemi-Shirazi L, Gasche CH, Natter S et al. IgA autoreactivity: a feature common to inflammatory bowel and connective tissue diseases. Clin Exp Allergy 2002; 128:102-9.
17. Metzger MH, Heier M, Maki M et al. Mortality excess in individuals with elevated IgA anti-transglutaminase antibodies: the KORA/MONICA Augsburg cohort study 1989-1998. Eur J Epidemiol 2006; 21:359-65.
18. Dieterich W, Trapp D, Esslinger B et al. Autoantibodies of patients with coeliac disease are insufficient to block tissue transglutaminase activity. Gut 2003; 52:1562-6.
19. Kristjansson G, Venge P, Hallgren R. Mucosal reactivity to cow’s milk protein in coeliac disease. Clin Exp Immunol 2007; 147:449-55.
20. Van der Boog PJ, Van Kooten C, De Fijter JW, Daha MR. Role of macromolecular IgA in IgA nephropathy. Kidney Int 2005; 67:813-21.
21. Helin H, Mustonen J, Reunala T, Pasternack A. IgA nephropathy associated with celiac disease and dermatitis herpetiformis. Arch Pathol Lab Med 1983; 107:324-7.
22. Almroth G, Axelsson T, Mussener E, Grodzinsky E, Midhagen G, Olcen P. Increased prevalence of anti-gliadin IgA-antibodies with aberrant duodenal histopathological findings in patients with IgA-nephropathy and related disorders. Ups J Med Sci
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2006; 111:339-52.23. Witte T, Matthias T, Arnett FC et al. IgA and IgG
autoantibodies against alpha-fodrin as markers for Sjogren’s syndrome. Systemic lupus erythematosus. J Rheumatol 2000; 27:2617-20.
24. Berglin E, Johansson T, Sundin U et al. Radiological outcome in rheumatoid arthritis is predicted by presence of antibodies against cyclic citrullinated peptide before and at disease onset, and by IgA-RF at disease onset. Ann Rheum Dis 2006; 65:453-8.
25. Sardy M, Karpati S, Merkl B, Paulsson M, Smyth N. Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis. J Exp Med 2002; 195:747-57.
26. Dieterich W, Laag E, Bruckner-Tuderman L et al. Antibodies to tissue transglutaminase as serologic markers in patients with dermatitis herpetiformis. J Invest Dermatol 1999; 113:133-6.
28. Riente L, Chimenti D, Pratesi F et al. Antibodies to tissue transglutaminase and Saccharomyces cerevisiae in ankylosing spondylitis and psoriatic arthritis. J Rheumatol 2004; 31:920-4.
29. Hu Y, Pan Q, Pardali E et al. Regulation of germline promoters by the two human Ig heavy chain 3’ alpha
enhancers. J Immunol 2000; 164:6380-6.30. Giambra V, Martinez-Labarga C, Giufre M et al.
Immunoglobulin enhancer HS1,2 polymorphism: a new powerful anthropogenetic marker. Ann Human Genet 2006; 70:946-50.
31. Denizot Y, Pinaud E, Aupetit C et al. Polymorphism of the human alpha1 immunoglobulin gene 3’ enhancer hs1,2 and its relation to gene expression. Immunology 2001; 103:35-40.
32. Aupetit C, Drouet M, Pinaud E et al. Alleles of the alpha1 immunoglobulin gene 3’ enhancer control evolution of IgA nephropathy toward renal failure. Kidney Int 2000; 58:966-71.
33. Frezza D, Giambra V, Cianci R et al. Increased frequency of the immunoglobulin enhancer HS1,2 allele 2 in coeliac disease. Scand J Gastroenterol 2004; 39:1083-7.
34. Cianci R, Giambra V, Mattioli C et al. Increased Frequency of Ig Heavy-Chain HS1,2-A Enhancer (*)2 Allele in Dermatitis Herpetiformis, Plaque Psoriasis, and Psoriatic Arthritis. J Invest Dermatol 2008; doi:10.1038/jid.2008.40.
35. Bentley DR. Genomes for medicine. Nature 2004; 429:440-5.
36. Hunt KA, Zhernakova A, Turner G et al. Newly identified genetic risk variants for celiac disease related to the immune response. Nat Gen 2008; 40:395-402.
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ETHANOL-INDUCED CHANGES IN PROFILE OF FATTY ACIDS IN ISOLATED RAT HEPATOCYTES AND THE INFLUENCE OF MAGNESIUM IONS
E. NIEDWOROK, M. MUC-WIERZGOŃ1, E. NOWAKOWSKA-ZAJDEL1, A. NOWOK1
and T. KOKOT1
Department of Human Nutrition, Faculty of Public Health, Medical University of Silesia, Bytom; 1Department of Internal Diseases, Faculty of Public Health, Medical University of Silesia, Bytom, Poland
Received April 8, 2007 – Accepted May 3, 2007
The influence of magnesium and ethanol on the fatty acid content in isolated rat hepatocytes was examined in this study. The isolated liver cells were obtained according to the Selgen method and then subjected to ethanol alone or both ethanol and magnesium activity. MgCl2 was used in two concentrations: 2 and 4 mM. The profile of fatty acids in the hepatocytes was evaluated after 5 hours of incubation. Our results revealed that magnesium ions presented together with ethanol in hepatocyte medium changed the hepatocyte fatty acid profile. The total amounts depended on the concentration of magnesium ions.
The fatty acids of the cell are the sources of energy and the components of its structure. The processes of their esterification and oxidation are pivotal in the metabolic conversions. Ethanol is the most important factor leading to peroxidation of fatty acids and creation of free radicals, and seems to be the main reason for the fatty acids profile changes (1-3). It is obvious that several free radicals are produced during the exposition to ethanol and its metabolism.
One of the major consequences of oxidative stress is starting and maintaining lipid peroxidation. Some authors have suggested that radical reaction was started by the reaction of polyunsaturated fatty acids with some kinds of oxygen free radicals (4). An alternative hypothesis stated that the free radicals involve an anion superoxide /O2/ in the onset of lipid peroxidation (5). Human and animal isolated hepatocytes have become an often used model to
investigate the metabolism and toxicity of many substances (6). The purpose of the present study is to find out if ethanol induces any changes in the composition of fatty acids and if the magnesium ions could prevent such changes. We used isolated rat hepatocytes as a model.
MATERIALS AND METHODS
The researchers obtained permission from the Silesian Medical University Committee of Bioethics to carry out this study. Isolated hepatocytes of male rats were used in our experiment. Hepatocytes of adult rats were isolated according to the Selgen method (1). The cell viability ranged from 85 to 98% and was assessed by trypan blue exclusion. The hepatocytes (n=1.3x107 ) were then placed in 10 cm3 Nuncon flasks in a Hepatocyte Medium. Culture media were supplemented with Penicillin 200 U/cm3
Mailing address: Dr. Elisabeth Niedworok,Dept. Human Nutrition,Faculty of Public Heath, Silesian Medical University,41-902 Bytom Piekarska str 18, Polande-mail: [email protected]
Vol. 22, no. 2, 105-108 (2008)
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The following substances were added to the medium:A) 80 µL of ethanol,B) MgCl2 solution (to concentration of 2mM) + 80 µL
of ethanol, C) MgCl2 solution (to concentration of 4mM) + 80
µL of ethanol.The control samples consisted of hepatocytes
incubated in the same conditions without any substances added to the medium. All operations connected with the isolation of the cells and preparation of the medium were realised aseptically in a laminar chamber. The samples were incubated in a temperature of 37 0C for 5 hours in the chamber (Heraus instruments BB16) in an atmosphere of 5% CO2. After incubation the hepatocytes were centrifuged at 50x g for 10 min, then the supernatant was removed and the cell sediment was washed three times with physiological saline. Disintegration of the cells was achieved through repeated freezing in liquid nitrogen
and defrosting of the examined samples. They were then homogenised in an Ultra –Turrex T8 mechanical homogenisator. The effect of the process was observed under the microscope after the cells had been liophilized (Heto FD 25 liophilizator ). Qualitative and quantitative analyses of fatty acids in the obtained samples were then carried out. The procedures described by Wollenweber and Rietschel (2) to determine the fatty acids and ester bound fatty acids in the tested samples were used.
Each experiment was repeated 3 times. Chi- square test version 6 [StatSoft, Inc. (2001) and STATISTICA (data analysis software system)] were used for statistical analysis. A value of p< 0.05 was considered statistically significant.
RESULTS
Table I shows that ethanol added to the medium increased the amount of the analysed fatty acids in comparison with the control sample. Simultaneously, it appeared that it significantly decreased in the hepatocytes incubated in the mixture of ethanol and MgCl2 (samples B and C). Table II illustrates the Mg2+ effected the profile of hepatocyte fatty acid changes induced by ethanol. The values of FA, SFA, MUFAs and PUFAs in the cells incubated with ethanol were significantly higher than those obtained in the control. It can be observed that the presence of MgCl2 in the medium in higher concentration significantly reduced the amount of fatty acid in comparison with sample B.
DISCUSSION
The most distinct changes of the content of the amounts of fatty acids were observed in the hepatocytes incubated with ethanol (p<1x 10-4) (Table I). After using ethanol together with 2 mM of MgCl2 some changes were observed compared to the control (p=0.996). In hepatocytes incubated in the presence of a mixture of ethanol and 4 mM of MgCl2 the total amount of the fatty acids was significantly lower than in the control samples (p<10-3). This suggests that Mg2+ has a stabilising influence on the proper metabolism of fatty acids and their essential quantitative changes caused by ethanol.
For estimation of ethanol and Mg2+ effects on the amount and degree of saturation of hepatocytes fatty acid quantitative changes of their particular fractions saturated (SFA), monounsaturated (MUFA),
Table I. Influence of ethanol and ions of magnesium on amounts in pool of total fatty acids in hepatocytes/area under the peaks x 106/ 1.3x107/, the mean values from 3 experiments.
Fatty acid 4 mM MgCl2+ethanol
2 mM MgCl2+ethanol
Ethanol Control
C14:0 7.7 8.6 48.8 6.0
C15:0 2.7 4.0 25.8 2.6
C16:0 161.0 186.0 2226.1 194.0
C17:0 4.1 6.0 59.0 4.8
Ci18:0 1.3 2.0 16.0 2.1
C18:2 64.4 80.0 1217.4 86.4
C18:1b 25.0 32.0 500.0 33.3
C18:0 162.0 193.4 1799.0 187.0
C20:4a 2.6 168.0 2183.0 179.0
C20:3a 6.1 8.0 43.0 6.0
C20:0 1.7 2.3 - 1.2
C22:6b 29.0 39.0 461.0 39.0
C22:6c 5.0 6.4 55.2 6.1
C22:0 2.6 3.5 - 3.4
C23:0 2.7 3.8 - 3.8
C24:1 3.4 5.3 - 5.0
C24:0 8.7 11.8 - 11.4
Table I. Influence of ethanol and ions of magnesium on amounts in pool of total fatty acids in hepatocytes/area under the peaks x 106/ 1.3x107/, the mean values from 3 experiments.
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polyunsaturated (PUFF)] and their total amount (FA) were qualified in comparison with control samples. The obtained outcomes (summed AUPs) revealed that better stabilising fatty acid metabolism results were achieved using lower MgCl2 concentration (2 mM). Coefficients of saturation degree of FA of hepatocytes incubated in the presence of ethanol and Mg2+, and in the control samples were as given: 1.93 and 1.17 (Table II).
The coefficient of saturation decreased to 0.94 in the liver cells incubated in the presence of ethanol. It is an effect of the changes of saturated to unsaturated fatty acids ratio, to the advantage of the latter. Many Authors indicate that excessive ethanol consumption upsets liver lipid metabolism (7-8). 75 to 90 % of ethanol is metabolised in hepatocytes. Numerous researches have revealed the enlargement of fatty acid content in liver cells incubated in the presence of ethanol. Our investigation also confirmed this. We noticed over 11 times enlargement of fatty acid content in the hepatocytes incubated in the medium
containing ethanol in comparison with the control sample.
We hypothesize that the observed effect was a consequence of stimulation of synthesis of fatty acids and triglicerides (TG) in response to enlarging relation of NADH+H+/NAD+, the products of ethanol metabolism, proceeding on the way catalysed by cytosolic and mitochondrial alcohol and mitochondrial aldehyde dehydrogenase. The reason of enlargement of FA content in liver cell response to ethanol may be lower β-oxidation of fatty acids as an effect of competing with the preferred route of ethanol oxygenation.
Many Authors indicate the positive correlation between ethanol concentration and the amount of oxygen reactive forms in tissues (7-10). The influence of ethanol and lipid peroxidation in liver cells causes controversy. Other data have suggested that ethanol stimulates peroxidation of lipids in hepatocytes. Our research proves that magnesium ions influence the stabilisation of the metabolism of FA in liver cells.
It was affirmed that in the examined specimens incubated in ethanol and 2 mM solution of MgCl2, the quantities of FA present did not differ significantly from their content in the control group. When only ethanol was present in the medium this value was about 11 times higher. We observed similar results when SFA, MUFA and PUFA were taken into consideration. Günter et al. (11) turned attention to the profitable activity of Mg ions also in a field of liver lipids. In conclusion, our examinations confirm that Mg ions exert an influence on hepatocyte lipid metabolism changed by ethanol.
REFERENCES
1. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976; 13:29-83.
4. Retterstol K, Lund AM, Tyerdal S, Cristophersen BO. Metabolism of some radiolabeled essential fatty acids in isolated rat hepatocytes is affected by dietary ethanol. Alcohol 2000; 21:19-26.
5. Mikkelsen L, Hansen HS, Grunnet N, Dich J.
Table II. The amount of fatty acids in hepatocytes/area under the peaks x 106/ 1.3x107/: the mean values from 3 experiments.
Fatty acids 4 mM MgCl2
+ethanol
2 mM MgCl2
+ethanol
Ethanol Control
Total FA 490.0 760.1 8631.3 771.1
SFA 354.5 501.4 4174.7 416.3
MUFA 28.4 37.3 500.0 38.3
PUFA 107.1 221.4 3956.6 316.5
*SFA/UFA 2.6 1.93 0.94 1.17
*SFA/UFA coefficient of degree of saturation of fatty acids as ratio summarizied AUPs of saturated fatty acids to summarizied AUPs of unsaturated fatty acids
Table II. The amount of fatty acids in hepatocytes/area under the peaks x 106/ 1.3x107/: the mean values from 3 experiments.
*SFA/UFA coefficient of degree of saturation of fatty acids as ratio summarizied AUPs of saturated fatty acids to summarizied AUPs of unsaturated fatty acids
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Inhibition of fatty acid synthesis in rat hepatocytes by exogenous polyunsaturated fatty acids is caused by lipid peroxidation. Biochim Biophys Acta 1993; 1166:99-104.
6. Mitaka T. The current status of primary hepatocyte cultures. Int J Exp Path 1998; 79:393-409.
7. Sergent O, Morel I, Chevanne M, Cillard P, Cillard J. Oxidative stress induced by ethanol in rat hepatocyte cultures. Biochem Mol Biol Int 1995; 35:575-583.
8. Sergent O et al. Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures. Relationship with lipid peroxidation. Biol Trace Elem Res 1995; 47:185-192.
9. Niedworok E. The influence of magnesium and ethanol on the peroxidation of lipids in isolated rat hepatocytes. Pol J Environm Stud 2004; 13(SII):348-349.
10. Niedworok E, Wardas M, Maciejewska-Paszek I. Bioavailability of magnesium ions in aspect suplementation. Vitamine und Zuzatzstoffe in der Ehnarhrung von Mensch und Tier. Friedrich-Schiller Universitat Jena 2005; 413-416.
11. G nther T, Vormann J, H�lriegl V. Effects of magnesium and iron on lipid peroxidation in cultured hepatocytes. Moll Cell Biochem 1995; 144:141-145.
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ACCELERATED WOUND HEALING OF ORAL SOFT TISSUES AND ANGIOGENIC EFFECT INDUCED BY A POOL OF AMINOACIDS COMBINED TO SODIUM
HYALURONATE (AMINOGAM)
G. FAVIA, M.A. MARIGGIÒ1, E. MAIORANO2, A. CASSANO1, S. CAPODIFERROand D. RIBATTI3
Department of Dental Sciences and Surgery, 1Department of Biomedical Sciences and Human Oncology, 2Department of Pathological Anatomy, 3Department of Human Anatomy and Histology,
University of Bari, Bari, Italy
Received February 1, 2008 – Accepted June 16, 2008
In this study we investigated the property of a new medical substance, in the form of a gel compound containing four aminoacids (glycine, leucine, proline, lysine) and sodium hyaluronate (AMINOGAM), to accelerate the wound healing process of the soft oral tissues and to promote angiogenesis in vivo in the vascular proliferation in chick embryo chorioallantoic membrane (CAM) assay. Furthermore, we investigated the capacity of AMINOGAM to induce the expression of an angiogenic cytokine, namely vascular endothelial growth factor (VEGF) in human fibroblasts in vitro. Results showed that AMINOGAM promoted wound healing in post-surgical wounds (after teeth extraction, oral laser surgery with secondary healing without direct suture of the surgical wound, and after dental implant insertion). Stimulated angiogenesis in vivo in the CAM assay and the response was similar to that obtained with vascular endothelial growth factor, a well-known angiogenic cytokine, tested in the same assay, and confirmed by clinical outcomes, which showed reduction of the healing time of oral soft tissues after three different kinds of surgery and also the absence of post-operative infections.
Wound healing is a specific biological process related to the general phenomenon of growth and tissue regeneration. It is characterized by the formation of a granulation tissue consisting of inflammatory cells, newly formed blood vessels and fibroblasts embedded in a loose collageneous extracellular matrix. Re-epithelization, angiogenesis and matrix deposition are critical events controlling this process (1).
Angiogenesis is confined to the wound site and plays a pivotal role for successful wound healing (2). Indeed, re-vascularization is required to furnish
the new tissue and to dispose of the waste products of metabolism. Angiogenesis occurs as a highly regulated process which is rapidly stimulated after injury and ceases when wound healing is complete. Wound angiogenesis is believed to be initiated by the early release of preformed growth factors, such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) (3-4).
Wound management after surgery implies obtaining healing in the shortest time, with minimal pain, discomfort, and scarring for the patient, leading to the wound closure with a flexible and
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fine scar with high tensile strength. Several factors could impede tissue repair and regeneration such as hypoxia, infection, tumors, metabolic disorders such as diabetes mellitus, the presence of debris and necrotic tissue, whereas certain types of medications and a diet deficient in protein, vitamins, or minerals could delay this process. It is generally accepted that a high availability of aminoacids is necessary in wound repair due to an increased metabolic activity.
Hyaluronic acid is also involved in wound healing. It is a glycosaminoglycan composed of repeating disaccharide units on D-glucoronate and N-acethylglucosamine and is one of the most abundant constituents of the extracellular matrix. Hyaluronic acid is involved in a number of developmental processes and has been shown to promote cell proliferation, differentiation and motility.
Hyaluronic acid is naturally biocompatible, biodegradable and lacks immunogenicity (5). Hyaluronic acid-modified liposomes as bioadhesive carriers for delivering growth factors to wound sites have been studied and reported (6) and more recently, hyaluronic acid demonstrated effective for managing acute wounds, particularly in terms of its safety and efficacy (7).
Traditional wound healing agents include topical liquid and semi-liquid formulations as well as dry traditional dressings. In this study we investigated the property of a new medical substance, in form of a gel compound containing four aminoacids (glycine, leucine, proline, lysine) and sodium hyaluronate (AMINOGAM), to accelerate the wound healing process of the soft oral tissues and to promote angiogenesis in the vascular proliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay for angiogenesis and anti-angiogenesis (8). Furthermore, we investigated the capacity of AMINOGAM to induce the expression of VEGF in human fibroblasts in vitro.
MATERIALS AND METHODS
Clinical study120 patients (60 for the experimental group and 60
for the control group) were selected. The 60 patients of the experimental group were divided into three subgroups of 20 patients who had undergone three different surgical procedures: subgroup A, underwent dental extraction of molars on both side of the jaw and were treated
with AMINOGAM (three applications/die) upon the extraction sites exclusively on one side; subgroup B, received endosseous dental implants with flap elevations and were treated with AMINOGAM (three applications/die) until complete healing of the surgical incision; subgroup C received diode laser treatment of benign neoplasms of the oral mucosa without direct suture of the surgical margins (diameter of the soft tissue exposed approximately was 20 mm) and were treated with AMINOGAM (three applications/die) until secondary healing. For experimental subgroups B and C, excluding subgroup A who had internal control of extraction sites with the opposite sites, an equivalent number of patients were selected as control subgroup. To compare the healing times between the control subgroups and the experimental subgroups, in the former the normal healing times of soft tissues for each surgical procedure were established as follows: for subgroup A, 15 days with complete closure of the socket and 30 days for keratinization of the gingiva; for subgroup B, 12 days with full thickness closure of the edges of the elevated flaps; for subgroup C, 21 days with complete reconstruction of the covering mucosa and keratinization.
In vivo study
Fifty fertilized White Leghorn chicken eggs were incubated at 37°C at constant humidity. On day 3 of incubation a square window was opened in the egg shell after removal of 2-3 ml of albumin so as to detach the developing CAM from the shell. The window was sealed with glass and the eggs were returned to the incubator. At day 8 of incubation, gelatin sponges (Gelfoam, Upjohn Company, Kalamazoo, MI) were cut to 1 mm3 pieces and placed on top of a growing CAM under sterile conditions. AMINOGAM in phosphate buffered saline (PBS) was then adsorbed on to the sponges. Sponges containing PBS alone or 200 ng of VEGF (R & D Systems, Abington, UK) were used, respectively, as negative and positive controls. On day 12, the CAM were photographed using a stereomicroscope (SZ-CTV Olympus Optical Co, Rozzano, Italy) connected to a digital camera. Blood vessels entering the sponges within the focal plane of the CAM were counted by two observers in a double-blind fashion at a magnification of x 50, and means values ± 1 standard deviation (SD) were determined. The significance of differences between experimental and control CAM counts was determined by Student’s t test for unpaired data.
ImmunohistochemistryTwo murine monoclonal antibodies (MAb) against
the endothelial cell marker CD31 (MAb 1A10, Dako Cytomation, Glostrup, Denmark) and against the
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4-6 fields covering the whole of each of three sections adjacent to those stained for microvessels and mean ± 1 SD and were determined for each section. The statistical significance of differences between experimental and control counts was determined by Student’s t test for unpaired data.
VEGF mRNA expression in human fibroblastsHuman foetal lung fibroblasts (MRC-5) were cultured
in MEM (Minimum Essential Medium with Earle’s salts; Euroclone, Ltd.) supplemented with 10% foetal calf serum, 100 μg /ml penicillin, 250 μg /ml streptomycin and 2 mmol L-glutamine, at 37°C in a humidified atmosphere of 5% CO2. MRC-5 were utilized between the 26th and 28th passages. Cells (5x105) were plated into 25 cm2 flasks for three days. Then medium was replaced with a fresh one. A solution containing 0.125 % aminoacids (glycine, leucine, proline, lysine) and 0.09 % sodium hyaluronate, at the same ratio of AMINOGAM, in H2Od was freshly made. This solution was added into the medium at the ratio 1:10 (v/v; final volume 6 ml). Three and four days after starting the assay, cells were harvested, washed twice in PBS and counted. Total RNA was isolated from 5x105 cells by TRIzol reagent (Invitrogen), and spectrophotometrically measured. 2 ng of total RNA primed with oligo (dT), incubated at 42°C for 45 min, were used to synthesize c-DNA by SuperScript III First-strand Synthesis System for RT-PCR (Invitrogen, USA). Reverse transcription was terminated by heating at 70°C for 10 min and 1 μl of c-DNA was used as the template for PCR. Primers to evaluate VEGF expression were: For: 5’- TGTCTTGCTCTATCTTTCTT-3’ Rev 5’- CTTGCCTTGCTGCTCTACCT-3’, (amplification product of 383 bp). GAPDH was used as housekeeping gene: For: 5’-TTGGTATCGTGGAAGGACTCA-3’; Rev: 5’-TGTCATCATATTTGGCAGGTTT-3’ (amplification product of 269 bp). The PCR reaction was done at 94°C for 15 sec, 60°C for 1 min, and 72°C for 1 min for 40 cycles for VEGF amplification, 94°C for 15 sec, 55°C for 30 sec, and 68°C for 1 min for 30 cycles for GAPDH amplification. The PCR products were analyzed on 1.5 % agarose gel and stained by ethidium bromide.
RESULTS
Clinical studyAll clinical results are summarized in Table 1.In the patients of subgroup A, who underwent
dental extraction of molars on both side of the jaw and were treated with AMINOGAM (three applications/die) upon the extraction sites exclusively on one side, a reduction of the elapsed
macrophage marker CD68 (NCL-CD68-KP1, Novocastra Laboratories Ltd, Newcastle, UK) were used. Briefly, some bioptic specimens obtained from experimental and control subgroup A were collected on 3-amino-propyl-triethoxysilane coated slides, deparaffinized by the xylene-ethanol sequence, rehydrated in a graded ethanol scale and in Tris-buffered saline (TBS, pH 7.6), and incubated overnight at 4°C with the MAb 1A10 (1:25 in TBS) and with the Mab KP1 (1:200 in TBS), after prior antigen retrieval by heating the sections in a pressure cooker in 1mmol/l EDTA buffer, pH 8.0 for 1.3 min. For CD31 immunostaining, the sections were incubated with biotinylated IgG and then with peroxidase-conjugated streptavidin (LSAB2, DakoCytomation). The colour was developed by diaminobenzidine. The immunodetection of CD68 was performed with alkaline phosphatase anti-alkaline phosphatase (APAAP, DakoCytomation) and fast red as chromogen, followed by hematoxylin counterstaining. Negative controls included an unrelated monoclonal IgG1 produced by the P3X63/Ag8 mouse secretory myeloma replacing the antibody, for the MAb against CD31 (9) and preincubation with a 10-fold excess of specific blocking peptide (Santa Cruz) for the antibody against CD68.
Microvessel density and CD68 positive cells countingThese were simultaneously assessed without
knowledge of the final pathological diagnosis by two investigators with a double-headed light microscope (Axioplan II, Zeiss, Oberkochen, Germany). Four to six 200X fields covering almost the whole of each of three sections per sample were examined with a 144-intersection point square reticulum (0.78 mm2) inserted in the eyepiece. Care was taken to select microvessels, i.e. capillaries and small venules, from all the CD31-stained vessels. They were identified as transversally sectioned tubes with a single layer of endothelial cells, with or without a thin basement membrane. Each assessment was agreed upon in turn. Microvessels were counted with a planimetric point-count method with slight modifications to restrict counting to transversally cut microvessels occupying the reticulum intersection points (10). As the microvessel diameter was smaller than the distance between adjacent points, only one transversally sectioned microvessel could occupy a given point. Microvessels transversally sectioned outside the points and those longitudinally or tangentially sectioned were omitted. Therefore, it was sufficiently certain that a given microvessel was counted only once, even in the presence of several of its section planes. As almost the entire section was analysed per sample, and as transversally sectioned microvessels hit the intersection points randomly, the method allowed objective counts. Cells stained with anti-CD68 antibody was counted on
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B GROUP: 20 patients who received endosseous implants of 4.1 mm in diameter by full thickness flap elevation with suture, using AMINOGAM and 20 controls (not users) with Standard Deviation (SD)
C GROUP: 20 patients who underwent to diode laser surgery without suture and used AMINOGAM and 20 controls (not users) with Standard Deviation (SD)
Data are expressed as median value. Comparison between control and case groups have been run by T-Test Student for impaired data or, when normality test failed, by Mann-Whitney Rank Sum Test. Comparison of proportion (for complications) has been evaluated by z-test, with Yates correction for small numbers. SigmaStat software has been used for calculations.
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time between surgical procedure and suture removal (median time 6.9 days vs 7.6 days in the contralateral control sites) (p 0.007) was obtained. Moreover, in the experimental group faster healing (median time 11.5 days vs 14.4 days in the control group)
(p < 0.001) of the extraction sockets with gingival keatinization was recognizable without post-surgical complications, whereas in 25% of the control subgroup delayed healing, post-extractive sicca alveolitis and infection were observed.
In the patients of subgroup B, who received endosseous dental implants with flap elevations and were treated with AMINOGAM (three applications/die) until complete healing of the surgical incision, the removal of the suture was performed earlier (median time 6.9 days vs 10.5 days in control subgroup) (p < 0.001), gingival healing was faster (median time 8.4 days vs 14.5 days in the control subgroup) (p < 0.001), and no complications were found in either experimental or control subgroup.
In the patients of subgroup C, who received diode laser treatment of benign neoplasms of the oral mucosa without direct suture of the surgical margins and were treated with AMINOGAM (three applications/die) until secondary healing, the healing time was shorter (median time 17.5 days vs 21.1 days in the control subgroup) (p < 0.001), without complications, whereas in 15% of the control group mycotic infections were observed.
In bioptic specimens obtained from patients of the experimental subgroup A, a significant increase of the microvascular density (mean value 12.5% vs 8.2%, p < 0.001 vs control) and of the number CD68-positive macrophage (mean value 12±4 vs 6±2, p < 0.001 vs control) as compared to the control subgroup was demonstrated (Fig. 1). In bioptic specimen obtained from patients of experimental subgroup B (gingiva removed to expose endosseous implants after osteointegration), a good collagen neo-synthesis, especially around new formed vassels, was observable in haematoxylin and eosin stains, confirmed by immunohistochemical findings on collagen IV stain and confocal laser scanning analysis (Fig. 2).
In vivo CAM assayOn day 12, macroscopic examination showed
that sponges treated with AMINOGAM were surrounded by numerous allantoic vessels, which developed radially towards the implant in a ‘spoked-wheel’ pattern (mean number of vessels around the implants 25±3) (Fig. 3). A similar picture was produced by sponges loaded with VEGF (mean
Fig. 1. Immunohistochemical reactivity for CD31 (A, B) and CD68 (C, D) in bioptic specimens obtained from experimental (B, D) and control subgroup A (A, C). Note in the experimental subgroup a higher microvascular density and higher number of CD68-positive cells as compared to control one. Original magnification, 250 X.
Fig. 2. Collagen neo-synthesis in human gingiva treated with AMINOGAM; tissue was obtained from patients of the experimental subgroup B (gingiva removed to expose endosseous implants after osteointegration); collagen deposition was observed above all around new formed vassels (A) as confirmed by immunohistochemical reactivity for collagen IV (B) and confocal laser scanning analysis (C, D, E).
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number of vessels around the implants 28±4) (Fig. 3). There was a statistically significant difference between AMINOGAM and PBS loaded sponges (mean number of vessels around the implant 8±2, p < 0.001 vs AMINOGAM treated sponges), whereas the angiogenic response induced by AMINOGAM
implants was comparable to that obtained with VEGF.
VEGF mRNA expression in human fibroblastsThe expression of VEGF gene in MRC-5 treated
with AMINOGAM solution was analyzed by RT-PCR. VEGF amplification product was slightly augmented on the third day, while a noteworthy rise was seen on the fourth day of incubation. All experiments were controlled by the presence of GAPDH expression (Fig. 4).
DISCUSSION
In this study for the first time we have demonstrated that a new medical substance, in form of a gel compound containing four aminoacids (glycine, leucine, proline, lysine) and sodium hyaluronate (AMINOGAM), is able to accelerate the wound healing process of the soft oral tissues. To this purpose we studied three experimental conditions, dental extraction of molars on both side of the jaw, endosseous dental implants with flap elevations and diode laser treatment of benign neoplasms of the oral mucosa without direct suture of the surgical margins. In all the experimental conditions, AMINOGAM
treatment reduced the time of wound healing and was not associated to post-surgical complications, as compared to the same cases not treated with the compound. Moreover, in healing of gingival tissues covering endosseous implants after their insertion, an optimal gingival integrity obtained in a short time is essential to reduce infective complications which can lead to the damage and/or failure of implants. Moreover, it also promotes bone regeneration around the implants in the coronal portion, a critical zone for osteointegration and subsequently restoration. In the meantime, after diode laser surgery of soft oral tissues, the use of AMINOGAM provides a double effect through mechanical protection of the wound and the acceleration of the healing process.
We also demonstrated by immunohistochemsitry
a significant increase of the microvascular density and of the number CD68-positive macrophage in bioptic specimens obtained from patients of the first experimental subgroup as compared to those of the control subgroup, to confirm an increased vascularization and macrophage infiltration in the experimental specimens. Macrophages, in turn, may be involved in the angiogenic response taking place in the wound healing process, through the release of angiogenic cytokines, such as VEGF, contained in their cytoplasm.
To further investigate the possible direct angiogenic effect exerted by AMINOGAM, we also tested this compound in vivo in the CAM assay and we demonstrated that AMINOGAM stimulates angiogenesis, and that the angiogenic response is similar to that obtained with VEGF, a well-known angiogenic cytokine. Furthermore, AMINOGAM
solution is able to stimulate the expression of VEGF in human fibroblasts.
Among the components of AMINOGAM, sodium hyaluronate is involved in angiogenesis. In fact, it enhances keratinocyte proliferation and migration, as well as the angiogenic response from the wound bed. In 1991, for the first time, West et al. (11) demonstrated that the degradation products of hyaluronic acid were pro-angiogenic. The angiogenic response was further confirmed (12-14) and then attributed to an intracellular effect upon signaling pathways (15-16) enhanced by co-application of VEGF (17). The involvement of hyaluronic acid in the regulation of angiogenesis is also supported by the observation that blocking the interaction of endogenous hyaluronic acid with cell surface hyaluronic acid-binding proteins inhibits both endothelial cell migration form the edge of a wounded monolayer and formation of tubular structures by endothelial cell clumps suspended in collagen gels (18).
Hyaluronic acid has been used extensively in a number of fields, including ophthalmic and joint surgery and, particularly, wound healing. Among the other components of AMINOGAM there are four amino acids, namely glycine, leucine, proline and lysine, which seem to be involved in favoring wound healing by collagenogenosis and extracellular matrix formation (which is particularly promoted by the so-called Small Leucin Rich Proteoglicans
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recently identified). In fact, the relationship between nutrition and wound healing, after injury or surgical intervention, has been recognized for centuries (19).
Finally, we have demonstrated that AMINOGAM
solution is able to stimulate the expression of VEGF in culture of human fibroblasts, also involved in the wound healing process. We hypothesize that fibroblasts and macrophages, which enter the composition of the cellular infiltrate in wound healing, may be involved in the stimulation of angiogenesis through the release of a pro-angiogenic cytokine, such as VEGF.
Overall, these clinical and experimental data, indicating that AMINOGAM accelerates wound healing, promote angiogenesis and the release of VEGF by fibroblasts, suggest that this new compound may be successfully used in the treatment of several oral pathological conditions in which an accelerated repair of the surgical solution is of benefit for the patient.
ACKNOWLEDGEMENTS
This study was supported by ERREKAPPA Euroterapici, Milan, Italy.
2. Banda WJ, Knighton DR, Hunt TK, Werb Z. Isolation of a non mitogenic angiogenic factor from wound fluid. Proc Natl Acad Sci USA 1992; 79:7773-7777.
3. Brown LF, Kiang-Teck Y, Berse B et al. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 1992; 176:1375-1379.
4. Niessen NN, Gamelli RL, Polverini PJ, et al. Basic fibroblast growth factor mediates angiogenic activity in early surgical wounds. Surgery 1996; 119:547-465.
5. Vercruysse KP, Prestwich GD. Hyaluronate derivatives in drug delivery. Crit Rev Ther Carrier Syst 1998; 15:513-555.
6. Yerushalmi N, Arad A, Margalit R. Molecular and cellular studies of hyaluronic acid-modified liposomes as bioadhesive carriers for topical drug
delivery in wound healing. Arch Biochem Biophys 1994; 313:267-273.
7. Voinchet V, vasseur P, Kern J. Efficacy and safety of hyaluronic acid in the management of acute wounds. Am J Dermatol 2006 ; 7:353-357.
8. Ribatti D, Nico B, Vacca A, Presta M. The gelatin sponge-chorioallantoic membrane assay. Nat Protocols 2006; 1:85-91.
9. Vacca A, Ribatti D, Roncali L, et al. Melanocyte tumor progression is associated with changes in angiogenesis and expression of the 67-kilodalton laminin receptor. Cancer 1993; 72:455-461.
10. Ribatti D, Nico B, Vacca A, et al. Do mast cells help to induce angiogenesis in B-cell non Hodgkin’s lymphoma? Br J Cancer 1998; 77:900-1906.
11. West DC, Hampson IN, Arnold F, et al. Angiogenesis induced by degradation products of hyaluronic acid. Science 1991; 228:1324-1326.
12. Sattar A, Rooney P, Kumar S, et al. Application of angiogenic oligosaccharides of hyaluronic acid increases blood vessel number in rat skin. J Invest Dermatol 1994; 103:576-579.
13. Trochon V, Mabilat-Pragnon C, Bertrand P, et al. Hyaluronectin blocks the stimulatory effect of hyaluronan-derived fragments on endothelial cells during angiogenesis in vitro. FEBS Lett 1997; 418:6-10.
14. Deed R, Rooney P, Kumar P, Norton JD, Smith J, Freemont AJ, Kumar S. Early-response gene signaling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by on-angiogenic, high-molecular-weight hyaluronan. Int J Cancer 1997; 71:251-256.
15. Slevin M, Krupinski J, Kumar S, Gaffney J. Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplsmic signal transduction pathway resulting in proliferation. Lab Invest 1998; 78:987-1003.
16. Slevin M, Kumar S, Gaffney J. Angiogenic oligosaccharides of hyalurona induce multiple signaling oathways affecting vascular endothelial cell mitogenic and woun healing responses. J Biol Chem 2002; 277:41046-41059.
17. Montesano R, Kumar S, Orci L, Pepper MS. Synergistic effect of hyaluronan oligosaccharides and
116 JOURNAL OF BIOLOGICAL REGULATORS & HOMEOSTATIC AGENTS
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SERUM IL-4 AS A MARKER OF IMMUNOLOGICAL RESPONSE TO SUBLINGUAL IMMUNOTHERAPY
G. CIPRANDI, M. DE AMICI1, G. MURDACA, B.M. COLOMBO, S. QUAGLINI2, G. MARSEGLIA1 and M. DI GIOACCHINO3
Department of Internal Medicine, Azienda Ospedaliera Universitaria San Martino, Genoa University, Genoa; 1Department of Pediatric Science, Pediatric Clinic, Pavia University, IRCCS
San Matteo Foundation; 2Department of Computer Science and Systems, Pavia University, Pavia; 3Allergy Related Disease Unit, “G. d’Annunzio University” Foundation, Chieti, Italy
Received January 19, 2008 – Accepted April 14, 2008
Allergic rhinitis (AR) is characterized by a Th2 polarized immune response. Specific Immunotherapy modifies this bias restoring a physiologic Th1 profile. Sublingual immunotherapy (SLIT) is widely prescribed, but there is no early, simple marker of response. This study was undertaken in order to determine whether serum IL-4 might be a possible marker of SLIT immunological response in order to quickly and easily detect responder patients. Thirty-nine AR patients with a pollen allergy assumed pre-seasonal SLIT for 3 months. VAS for symptoms and medication efficacy were evaluated. Serum IL-4 was assessed before and 3 and 6 months after SLIT initiation. Eighty-two percent of patients (32/39) showed a clinical response to SLIT. Serum IL-4 significantly decreased at 6 months post-therapy in responders, whereas it increased in non-responders. In conclusion, these results may be considered clinically relevant proof that SLIT treatment induces a quick reduction in Th2 polarization. Serum IL-4 appears to be an early marker of immunological response to SLIT.
Allergic rhinitis (AR) is characterized by mucosal inflammation, sustained by mast cell and eosinophil activation. This inflammatory reaction displays a predominant T-helper (Th) 2 cell profile with greater interleukin (IL)-4 production than interferon (IFN)-γ expression by Th1 cells: the so called Th2 polarization (1). AR management includes patient education, allergen avoidance, drug therapy and, when appropriate, allergen-specific immunotherapy (SIT) (2). SIT is the practice of administering gradually increasing doses of the causal allergen in order to reduce allergic symptoms and the need for medication (3). Treatment efficacy depends on
achieving clinical tolerance of the causal allergen (4). There is evidence that SIT is effective in patients with allergic rhinitis and mild forms of asthma as stated in a World Health Organization (WHO) position paper (5).
SIT is classically based on the subcutaneous administration of allergen extracts (SCIT), but as this route of administration has exhibited a risk for serious adverse reactions, alternative routes of administration, including oral, sublingual (SLIT), and nasal, have been investigated and developed (6). The rationale of SLIT is that oral administration of high-dose allergens may reduce and prevent
Mailing address: Giorgio Ciprandi, M.D.Ospedale San Martino,Largo R. Benzi 10, 16132 Genova, ItalyTel: ++39 10 35338120Fax: ++39 10 3537573e-mail: [email protected]
Vol. 22, no. 2, 117-123 (2008)
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IgE responses. Methodologically, the extract is kept under the tongue for a few minutes and then swallowed: the “sublingual-swallow”. SLIT efficacy has been evaluated in one WHO position paper (5), in the EAACI/ESPACI document (6), in the ARIA document (7), and investigated in two meta-analyses on patients with AR and asthma (8-9).
The exact SIT mechanism of action is not completely understood (10). SCIT has been in practice for many years, and several studies have shown that subcutaneous administration of the allergen induces hyposensitization by redirecting peripheral and mucosal Th2-responses to a prevalent Th1-polarization (11-13). However, regarding SLIT, only two studies have demonstrated that it was able to increase allergen-driven IFN-γ production (14-15); other studies have not reported cellular changes (16-17). Moreover, a number of recent studies indicate that regulatory T cells (Tregs) play an important role in controlling the Th2-biased response (18). Indeed, a defect in T regulatory cells has been proposed by one study in allergic subjects (19). Very recently, also regarding SLIT, it was shown that the appearance of allergen-specific tolerance induction is highlighted by restored allergen-specific IL-10 and IFN-γ production (15, 19-20). On the other hand, only two studies have reported decreased allergen-induced Th2-dependent cytokine (IL-4 and IL-13) production after SLIT (21-22), while other studies did not confirm this result (15-17, 20, 23).
Both forms of SIT: SCIT and SLIT are long-term and expensive treatments as their administration is projected for 3-5 years. Moreover, a discrete percentage of allergic patients (about 20-30%) do not respond to this therapy (24). Considering that SIT prescriptions are numerically relevant in Europe and SLIT is more prescribed than SCIT, the aim of this study is to investigate whether serum IL-4 may be an easily detectable early marker of immunological response to SLIT. In addition, this information would be useful in identifying candidate responders.
MATERIALS AND METHODS
PatientsThirty-nine patients with AR due to pollen sensitisation
were enrolled; each subject underwent a complete evaluation and was then included in the study. The study was conducted with the approval of the local Ethics
Committee and performed with the written informed consent of all participants. AR was diagnosed according to validated criteria (7). The patients’ blood samples for assessing serum IL-4 were collected before initiating SLIT (baseline, T0), at three months (corresponding to the end of the SLIT schedule, T1) and six months (corresponding to the pollen season peak, T2).
Specific Immunotherapy Subjects undergoing SLIT received either a Betula
alba pollen allergen extract (Anallergo, Florence, Italy; 17 cases, cumulative dose was 30 mcg), Parietaria judaica (15 cases, cumulative dose was 12 mcg), or a mix of three Graminacee (7 cases, cumulative dose was 12 mcg) for at least three months before the pollen season, as pre-seasonal immunotherapy. The dose during the maintenance phase was 1,000 U.RAST/ml (internal reference): five drops 3 times per week.
Clinical evaluationAllergy symptoms and the use of medication were
scored at the end of the pollen season. The patient questionnaire included a visual analogue scale (VAS) of nasal symptoms (25). During the pollen season patients could assume lyophilised ebastine tablets (10 mg, 1 or 2) on demand as rescue medication. The medication scoring (MS) was: not at all (0), occasionally/seldom (1), occasionally/often (2), almost daily (3), continuously with the lowest dose (4), continuously with the maximal dose (5). Patients were arbitrarily considered SLIT responders when VAS was ≤ 5 and MS ≤ 2.
Serum IL-4 assayThe human IL-4 Immunoassay (R&D Systems)
employs the quantitative sandwich enzyme immunoassay technique and was performed according to the manufacturer’s instructions and expressed as pg/mL. The minimum detectable dose, corresponding to the zero standard concentration, was arbitrarily assigned as 0.09 pg/ml. Reference values for IL-4 were 0-31.2 pg/ml.
Statistical analysisDescriptive statistics were calculated on data at
baseline (T0), at three months (T1) and six months (T2) following SLIT. Moreover, the differences in IL-4 serum levels were calculated at the different times: ∆T1-
T0, ∆T2-T0, and ∆T2-T1 for each patient. The non-parametric Wilcoxon test was performed to evaluate the difference between responder and non-responder patients for both absolute values of IL-4 and ∆s. The same test was used to investigate the relationship between IL-4 and VAS. In addition, logistic regression was used to build a predictive model for clinical response to SLIT starting
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from a temporal trend in serum IL-4. The logistic model result was considered correct if a patient’s response/non-response was predicted with a probability higher than 0.5. All statistical analyses were performed using S-Plus (26).
RESULTS
The mean age of the analyzed AR patients (17 females and 22 males) was 37.5 years (range 14-75; median age 39).
Serum IL-4Table I shows the descriptive statistics of IL-4
serum levels at T0, T1 and T2. ∆T1-T0 and ∆T2-T1 were significantly different between responder and non-responder (p=0.042 and 0.018 respectively), while ∆T2-T0 was not significantly different in the two groups. The model estimates the probability of clinical response by the following formula:
p(response)=exp(2.285+0.028*∆T1-T0-0,032*∆T2-
T1)/[1+exp(2.29+0.028*∆T1-T0-0.032*∆T2-T1)] . Logistic regression shows that an IL-4 temporal trend, in terms of ∆T1-T0 and ∆T2-T1, significantly correlated with clinical response (Table II). The model prediction was considered correct when p(response) was less than 0.5 for a non-responder patient or ≥ 0.5 for a responder patient. Fig. 1 shows the model performance: its specificity (true negative rate) was 97% (31/32) and sensibility (true positive rate) 57% (4/7), with an overall efficiency of 90%.
IL-4 serum levels at base line, three months and six months following SLIT treatment, for responders and non-responders are reported in Fig. 2, together with linear regression lines. The two trends are clearly different as there is a significant decrease in serum IL-4 present in responder patients, whereas a significant increase is present in non-responders.
The relationship between VAS and IL-4 values at six months was also investigated, assuming an IL-4 cut-off value of 31.2 pg/ml, patients with normal IL-4 values showed a lower VAS (better clinical response), approaching statistical significance (Wilcoxon test p=0.08). Median and inter-quartile ranges for VAS in the groups were (5, 4-5) and (6, 5-7.75), respectively.
DISCUSSION
Allergic rhinitis is characterized by Th2-polarized inflammation. Th2-derived cytokines, such as IL-4 and IL-13, are the primary pathogenic factors which induce, maintain and amplify allergic inflammation. IL-4 and IL-13 orchestrate this inflammation by promoting IgE synthesis, up-regulating adhesion molecules selective for eosinophil recruitment, and causing increased mucus production and airway hyperreactivity (27). On the other hand, there is accumulating evidence that Th1-related cytokines, such as IFN-γ and IL-12, may suppress and counteract this Th2 response and vice-versa, as there is functional dichotomy between Th1 and Th2 cells (28).
SIT is highly effective in the treatment of AR, and is the only treatment which promotes life-long tolerance against previously disease-causing allergens, through the restoration of normal immunity (29). SCIT was found to be associated with a decrease in IL-4 and IL-5 production by CD4+ Th2 cells, and a shift towards increased IFN-γ production by Th1 cells (11-13). SLIT is the preferred version of SIT, with a safer and more effective route of administration, however, its mechanism of action is still poorly understood, as there are few data
Table I. Temporal trend in IL-4 serum levels at T0 (base line), T1 (3 months) and T2 (6 months).
Table I. Temporal trend in IL-4 serum levels at T0 (base line), T1 (3 months) and T2 (6 months).
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Fig. 1. The performance of the logistic model for clinical response prediction (0=no, 1=yes). Table inside the figure shows the number of cases correctly and incorrectly classified, using the response probability threshold of 0.5.
Table I. Temporal trend in IL-4 serum levels at T0 (base line), T1 (3 months) and T2 (6 months).
Table II. Logistic regression model of clinical response as a function of ∆T1-T0 and ∆T2-T1.
Fig. 1. The performance of the logistic model for clinical response prediction (0=no, 1=yes). Table inside the figure shows the number of cases correctly and incorrectly classified, using the response probability threshold of 0.5.
Fig. 2. Regression lines showing the decreasing and increasing trend of IL-4 in responders (�black diamonds) and non-responders (� white diamonds), respectively. Logarithmic transformation of IL-4 has been used to improve distribution symmetry.
��
Fig. 2. Regression lines showing the decreasing and increasing trend of IL-4 in responders (♦black diamonds) and non-responders (◊ white diamonds), respectively. Logarithmic transformation of IL-4 has been used to improve distribution symmetry.
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concerning the immunologic changes induced by SLIT (2). A decrease in serum levels of IL-13, as well as IL-4 has been demonstrated after SLIT therapy (21-22).
Regarding the balance between Th1-Th2 cells, some studies have shown a shift from Th2 towards a Th1 response (14-15, 20, 30), whereas others have failed to confirm this result (16-17). The discrepancies among these studies may depend on several methodological factors, including the type of allergen, duration of SLIT, age of patients and AR duration, dose of allergen extracts used in in vitro experiments, incubation kinetics, and the methods used to assess cytokines.
A very recent study indirectly supports the relevance of the Th2-Th1 shift through the increased expression of both IL-18 and the signalling lymphocytic activation molecule (SLAM) (30). IL-18 is a macrophage-derived cytokine which induces IFN-γ production from Th1 cells (31) and SLAM is a transmembrane, cell surface glycoprotein, whose activation promotes T-cell proliferation, and IFN-γ production, the latter is preferentially expressed in Th1 cells (32). Therefore, Th1 polarization represents an important change in the immune response after SLIT. However, all these studies were performed in the allergen-induced cytokine production model that is complicated and reserved only for in vitro studies as it can not be applied in vivo.
Importantly, there is still no prognostic parameter capable of identifying responders, which is an important draw back to this line of therapy, as a discrete percentage of subjects do not show any clinical benefit. Moreover, there is no agreement over the length of time necessary to detect responders. Thus, the definition of an easily detectable and early marker of immune response to SLIT would be clinically relevant. Therefore, this study was also designed to assess this parameter.
The first finding is that 82% of patients assuming SLIT showed a clinical response. This high percentage might depend on different factors, such as adequate screening of patients, their education and motivation. Overall, the most important outcome is that within six months of SLIT initiation, serum IL-4 levels significantly decreased in responder subjects. The trend of serum IL-4 values had a predictive correlation with response established as 90% efficient.
Moreover, it is noteworthy that serum IL-4 levels significantly increased during the peak pollen season in non- responder patients, whereas it significantly decreased in responders. Therefore, the present study highlighted that effective SLIT is capable of inducing an immunologic response directed at reducing Th2 polarization in allergic patients presenting with AR due to pollen sensitisation. Therefore, serum IL-4 reduction may be considered as an early marker of SLIT effectiveness.
In conclusion, these results may be considered clinically relevant proof that SLIT treatment induces a quick reduction in the Th2 response. This Th2 response may be useful to precociously identify responder patients. Therefore, the early evaluation of serum IL-4 during SLIT, i.e. at baseline and after 3/6 months, may help guide decisions in SLIT treatment. These preliminary findings should however be confirmed in further randomized studies.
ACKNOWLEDGEMENTS
The authors wish to thank Cristina Torre (Clinica Pediatrica, Fondazione IRCCS Policlinico S. Matteo) for outstanding technical support, Vania Giunta (Dipartimento di Informatica e Sistemistica, Università di Pavia) for data analysis and Laurene Kelly for correction of the English.
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13. Wilson DR, Nouri-Aria KT, Walker SM, Pajno GB, O’Brien F, Jacobson MR et al. Grass pollen immunotherapy: symptomatic improvement correlates with reduction in eosinophils and IL-5 mRNA expression in the nasal mucosa during the pollen season. J Allergy Clin Immunol 2001; 107:971-976.
14. Arikan C, Bahceciler NN, Deniz G, Akdis M, Akkoc T, Akdis CA et al. Bacillus Calmette-guerin-induced interleukin-12 did not additionally improve clinical and immunologic parameters in asthmatic children treated with sublingual immunotherapy. Clin Exp Allergy 2004; 34:398-405.
15. Cosmi L, Santarlasci V, Angeli R, Liotta F, Maggi L, Frosali F et al. Sublingual immunotherapy with
Dermatophagoides monomeric allergoid down-regulates allergen-specific immunoglobulin E and increases both interferon-γ- and interleukin-10-production. Clin Exp Allergy 2006; 36:261-72.
16. Rolinck-Weminghaus C, Kopp M, Liebke C, Lange J, Wahn U, Niggemann B. Lack of detectable alterations in immune response during sublingual immunotherapy in children with seasonal allergic rhinoconjunctivitis to grass pollen. Int Arch Allergy Immunol 2005; 136:134-141.
17. Dehlink E, Eiwegger T, Gerstmayr M, Kampi E, Bohle B, Chen KW et al. Absence of systemic immunologic changes during dose build-up phase and early maintenance period in effective specific sublingual immunotherapy in children. Clin Exp Allergy 2006; 36:32-9.
18. Umetsu DT, DeKruyff RH. The regulation of allergy and asthma. Immunological reviews 2006; 212:238-55.
19. Ciprandi G, Fenoglio D, Cirillo I, Vizzaccaro A, Ferrera A, Tosca MA et al. Induction of interleukin 10 by sublingual immunotherapy for house dust mites: a preliminary report. Ann Allergy Asthma Immunol 2005; 95:38-44.
20. Savolainen J, Jacobsen L, Valovirta E. Sublingual immunotherapy in children modulates allergen-induced in vitro expression of cytokine mRNA in PBMC. Allergy 2006; 61:1184-1190.
21. Ippoliti F, De Santis W, Volterrani A et al. Immunomodulation during sublingual therapy in allergic children. Pediatr Allergy Immunol 2003; 14:216-221.
22. Bohle B, Kinaciyan T, Gerstmayr M, Radakovics A, Jahn-Schmid B, Ebner C. Sublingual immunotherapy induces IL-10-producing T regulatory cells, allergen-specific T-cell tolerance, and immune deviation. J Allergy Clin Immunol 2007; 120:707-713.
23. Fanta C, Bohle B, Hirt W, et al. Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy. Int Arch Allergy Immunol 1999; 120:218-224.
24. Malling HJ. Sublingual immunotherapy: efficacy-methodology and outcome of clinical trials. Allergy 2006; 61:24-28.
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Mechin H, Daures JP, Bousquet J. Visual analog scales can assess the severity of rhinitis graded according to ARIA guidelines. Allergy 2007; 62:367-372.
26. Venables WN, Ripley BD. Modern Applied Statistics with S. Fourth Edition. Springer. ISBN 0-387-95457-0, 2002.
27. Bachert C, Van kempen M, Van Cauwenberge P. Regulation of proinflammatory cytokines in seasonal allergic rhinitis. Int Arch Allergy Immunol 1999; 118:375-379.
28. Tang C, Inman MD, van Rooijen N et al. Th type-1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism. J Immunol 2001; 166:1471-1481.
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30. Savolainen J, Nieminem K, Laaksonen K, Laiho T, Jacobsen L, Lahesmaa R et al. Allergen-induced in vitro expression of IL-18, SLAM and GATA-3 mRNA in PBMC during sublingual immunotherapy. Allergy 2007; 62:949-953.
31. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 1995; 6552:88-91.
32. Cocks BG, Chang C-Cj, Carballido JM, Yssel H, de Vries JE, Aversa G. A novel receptor involved in T-cell activation. Nature 1995; 376:260-263.
JOURNAL OF BIOLOGICAL REGULATORS & HOMEOSTATIC AGENTS
S. TORTORICI, A. MAURO1, F. BURRUANO, P. DIFALCO, A. LEONE1, A. GERBINO1,M. BUSCEMI1, P. CONTI2, F. MASTRANGELO3, and S.TETÈ3
Department of Oral Surgery, 1Department of Experimental Medicine, Histology and Embryology Section, Medical and Surgery Faculty, University of Palermo, Palermo; 2Immunology Division,
3Department of Oral Science, University of Chieti, Chieti, Italy
Received February 13, 2008 – Accepted April 24, 2008
Oral leukoplakia is the most common and potentially malignant disorder of the oral mucosa. The definition of leukoplakia given by the World Health Organization is “a white plaque that cannot be characterized either from a clinical or from a histopathological point of view”, thus the diagnosis of leukoplakia is based on the exclusion of other lesions of the oral mucosa. We believe it is necessary to identify molecular and immunohistochemical parameters that can contribute to discriminating between the different leukoplakia clinical subtypes coded by the epidemiology. In the present work we show the preliminary results of this research project. We investigated the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and inducible nitric oxide synthase (iNOS) in a verrucous proliferative leukoplakia sample. By immunohistochemistry we detected the presence of all the three proteins both in the leukoplakia samples and in healthy oral mucosa, while the reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed in both samples only the expression of MMP-2 and MMP-9 but not iNOS.
Oral leukoplakia is the most common and potentially malignant disorder of the oral mucosa (1-2). Leukoplakia is a clinical entity defined by the World Health Organization as “a white plaque that does not wipe off and cannot be characterized clinically or pathologically as any other disease and is not associated with any physical or chemical agent except tobacco” (3-5).
Although the etiology of leukoplakia remains unknown, many physical agents have been proposed to be risk factors: tobacco, alcohol, chronic friction, electrogalvanic reaction between unlike restorative
metals, diets low in antioxidants, ultraviolet radiation and human papillomavirus infections (6-9).
Clinically leukoplakias are divided into homogeneous and non-homogeneous (fissured leukoplakia, nodular leukoplakia, erythroleukoplakia, speckled leukoplakia and verrucous proliferative leukoplakia) types. Among the clinical sub-types of non-homogeneous leukoplakias, verrucous proliferative leukoplakia is important because it is a very aggressive lesion (2). The clinical type of leukoplakia has a bearing on the prognosis, because non-homogenous leukoplakia has a higher incidence
Mailing address: Prof. Silvia Tortorici, Dipartimento di Scienze StomatologicheSezione di Chirurgia Orale,Facoltà di Medicina e Chirurgia, Università degli Studi di Palermo,Largo degli Abeti, 10 90141 Palermo, ItalyTel: ++39 091/6552213 Fax: ++39 091/6552213e mail: [email protected]
Vol. 22, no. 2, 125-130 (2008)
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of dysplasia (2, 10-11). Histologically, oral leukoplakia is a
hyperorthokeratosis or parakeratosis with or without acanthosis, with or without inflammation, and with or without dysplasia (11). According to the degree of dysplasia, the oral leukoplakia histologically is classified as mild, moderate or severe, based on histopathological findings (3-4). At the present time, conservative surgical excision remains the treatment of choice for small leukoplakias, but some authors have proposed pharmacologic systemic therapies with Vitamin A or folate. To better understand this serious pathology of oral mucosa we are beginning to study some histochemical and molecular biological features of leukoplakias. For this reason it could be interesting to investigate through immunohistochemistry and RT-PCR analysis the expression of iNOS, an enzyme involved in inflammatory processes and pre-carcinogenic events (12-13) and some matrix metalloproteinases (MMP-2, MMP-9), that through their remodelling function could be involved in such conditions (14-16).
iNOS is the inducible form of nitric oxide synthase which is expressed by macrophages and activated by inflammatory signals such as cytokines and interferon-γ or oxidative stresses such as exposure to H2O2. MMP-2 and MMP-9 are matrix metalloproteinases involved in the breakdown of extracellular matrix both in physiological and pathological processes. MMP-2 degrades type IV and V collagen, denatured collagen and elastin; MMP-9 degrades type IV and V collagen.
MATERIALS AND METHODS
Epidemiology This is a retrospective study on 170 patients with oral
leukoplakia observed in the Department of Stomatology and Oral Science, Division of Oral Surgery “G. Messina”, University of Palermo, during a 5-year period. According to the criteria of the World Health Organization 2005 (4), we selected 170 cases of white oral lesions (excluding other benign disorders confirmed by biopsies). The mean age at the first visit was 45 years, 120 male, and 50 female, white European. There was a male:female ratio of 2:1. The majority of patients (90%) had used tobacco and were current smokers. Seven were alcohol misusers. Patients were divided into 5 groups on the basis of biopsies: 1st) leukoplakia with no dysplasia (n = 62); 2nd) leukoplakia with mild dysplasia (n = 47); 3rd) leukoplakia with
moderate dysplasia (n = 29); 4th) leukoplakia with severe dysplasia (n = 22); and 5th) oral squamous cell carcinomas (n = 10). The lesions of the 2nd, 3rd, 4th and 5th groups were surgically removed and the patient were monitored every 30 days for a follow-up of at least 12 month. For 5 patients of the 5th group the histological diagnosis was micro-invasive carcinoma.
ImmunohistochemistryThe analysis was performed on a single sample of
human oral mucosa affected by leukoplakia and one sample of healthy oral mucosa. Immediately after surgical dissection the sample was fixed in Bouin’s solution. After fixation the tissue was dehydrated in a graded series of alcohols, cleared in xylene, and paraffin embedded. Sections of 7 μm were cut on to a Leica microtome RM2145, dried overnight at 37°C and then stored at room temperature until use. On the day of the experiment, slides were de-waxed in xylene and re-hydrated in a graded series of alcohols. Slides were then transferred into distilled water for 5 min. The immunohistochemistry was performed using the “DakoCytomation EnVision + System-HRP (AEC)” kit from Dako, following the manufacturer’s instructions.
Mouse anti-human MMP-2 monoclonal antibody (Chemicon, cat n. MAB3308) (1:800 dilution), rabbit anti-mouse MMP-9 full length polyclonal antibody (Chemicon, cat n. AB19047) (1:100 dilution), rabbit anti iNOS/ NOS type II polyclonal antibody (BD biosciences, cat n. 610332) (1:25 dilution) were used. The antibodies were diluted in a 0.1% BSA (Bovine Serum Albumine) solution. Negative control sample was treated in the same way, omitting primary antibody. Slides were coverslipped using the “Dako Cytomation Faramount Aqueous Mounting Medium”. The specimens were observed under a Leica DM1000 light microscope.
Total RNA extractionThe two samples were frozen in liquid nytrogen
immediately after surgical dissection and stored at -80°C until use. Total RNA extraction was accomplished using the “illustra RNAspin Mini Kit” (Amersham Biosciences, Milan) following the manufacturer’s instructions. RNA yield was evaluated spectrophotometrically (A260/A280) and RNA aliquots were stored at -80°C until use.
For RT reaction 2 μg RNA was used. To avoid DNA contamination in the RNA samples DNAse digestion was performed using “AMPD1 kit” (SIGMA). RT reaction was performed using the “Enhanced avian HS RT-PCR kit” (SIGMA) following the manufacturer’s instructions.
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Briefly: 1 μl random nonamers and 1 μl anchored oligo (dT)23 were added to the DNAse digestion product and incubated at 70°C for 10 min. to denature the sample. Then 2 μl 10x Buffer, 1 μl deoxynucleotide mix, 1 μl RNAse inhibitor, 1 μl Enhanced AMV Reverse Transcriptase enzyme and DEPC water were added to the sample. The RT reaction was performed in 20 μl total volume at 42°C for 50 min, followed by 95°C for 5 min to inactivate the enzyme. The PCR was performed using the “PCR enzyme Selection Kit-High specificity” (Invitrogen) following the manufacturer’s instructions. Briefly: 2 μl of template DNA, 0.5 μl of the Primers mix (200 nM final concentration) and 22.5 μl of the “Platinum Super Mix” were mixed together. The reaction was cycled for 94°C for 3 min, then 40 cycles of 94°C for 60 sec., 56°C for 60 sec., 72°C for 60 sec., with a final extension at 72°C for 10 minutes. GAPDH gene was used as internal positive control.
The PCR products were visualized on 2% agarose gels, stained with ethidium bromide.
Primers sequences are shown in Table I.
RESULTS
iNOS expression is uniformly localized as brownish granules in the stratified epithelium, particularly in the cells of the stratum spinosum (Fig.1 A). The dermal region shows a immunoreactivity localized mainly in the endothelial cells of the vessels and in some isolated connective cells (Fig. 1B). We could not detect any difference in the spatial distribution and in the amount of iNOS expression between leukoplakia and control.
MMP immunoreactivity usually has the appearance of isolated microgranules inside the cytoplasm. The immunohistochemical reactivity for MMP-2 is localized in the cytoplasm of the epithelial cells. Moreover, the expression of MMP-2 is also evident in the endothelium of the vessels and
in many isolated cells of the connective tissue. We detected a strong increase of MMP-2 expression in the leukoplakia sample (Fig. 1C) where the number of positive connective cells is much bigger compared to the control (Fig. 1D) and even the intensity of the staining seems to be stronger.
MMP-9 expression appears to be localized, as MMP-2, in the epithelial cells, in the endothelium of the vessels and in many connective cells of the dermal region. The distribution of MMP-9 has the same features both in the healthy oral mucosa and in the leukoplakia sample, but the immunoexpression is stronger in the leukoplasic tissue (Fig. 1E, 1F). In the superficial epithelial cells of the leukoplasic tissue the nucleus is negative and begins to be pycnotic while a diffuse reactivity is distributed into the cytoplasm, but in some case the reactivity is present as well-defined cytoplasm granules. On the border of the leukoplasic tissue the epithelial cells show a weak and much less homogeneous reactivity.
RT-PCR analysis revealed a strong expression of the two matrix metalloproteinases investigated (MMP-2 and MMP-9) in both samples; healthy oral mucosa and oral mucosa with leukoplakia, while iNOS enzyme was not detected in either of the two samples (Fig. 1G, 1H).
DISCUSSION
The expression of iNOS we report in leukoplakia through immunohistochemistry is in agreement with previous investigations from other authors (12), while iNOS expression in leukoplakia has never been reported through RT-PCR. Chaiyarit et al. (13) suggest that iNOS over-expression could contribute to the development of oral carcinogenesis from leukoplakia
(Amersham Biosciences, Milan) following the manufacturer's instructions. RNA yield was evaluated
spectrophotometrically (A260/A280) and RNA aliquots were stored at -80°C until use.
For RT reaction 2 µg RNA was used. To avoid DNA contamination in the RNA samples DNAse
digestion was performed using �AMPD1 kit� (SIGMA). RT reaction was performed using the
�Enhanced avian HS RT-PCR kit� (SIGMA) following the manufacturer's instructions. Briefly: 1 µl
random nonamers and 1 µl anchored oligo (dT)23 were added to the DNAse digestion product and
incubated at 70°C for 10 min. to denature the sample. Then 2 µl 10x Buffer, 1 µl deoxynucleotide mix,
1 µl RNAse inhibitor, 1 µl Enhanced AMV Reverse Transcriptase enzyme and DEPC water were
added to the sample. The RT reaction was performed in 20 µl total volume at 42°C for 50 min,
followed by 95°C for 5 min to inactivate the enzyme. The PCR was performed using the �PCR enzyme
Selection Kit-High specificity� (Invitrogen) following the manufacturer�s instructions. Briefly: 2 µl of
template DNA, 0.5 µl of the Primers mix (200 nM final concentration) and 22.5 µl of the �Platinum
Super Mix� were mixed together. The reaction was cycled for 94°C for 3 min, then 40 cycles of 94°C
for 60 sec., 56°C for 60 sec., 72°C for 60 sec., with a final extension at 72°C for 10 minutes. GAPDH
gene was used as internal positive control.
The PCR products were visualized on 2% agarose gels, stained with ethidium bromide.
Primers sequences are shown in Table I.
Table I. Names, sequences and amplification product size of the PCR primers used in this work.
Primer name Primer sequence Product size MMP-2 human forward TGA TGG TGT CTG CTG GAA AG 280 bp MMP-2 human reverse GAC ACG TGA AAA GTG CCT TG MMP-9 human forward CAT TTC GAC GAT GAC GAG TTG 554 bp MMP-9 human reverse AAG CCC CAC TTC TTG TCG CT iNOS human forward TGT AGC GAG TCG AAA ACT GAG G 532 bp iNOS human reverse CTG CCC CAG TTT TTG ATC CT GAPDH human forward GAG TCA ACG GAT TTG GTC GT 238 bp GAPDH human reverse TTG ATT TTG GAG GGA TCT CG
Table I. Names, sequences and amplification product size of the PCR primers used in this work.
128 129Journal of Biological Regulators & Homeostatic Agents
both through oxidative stress and through iNOS-mediated nitrative stress and DNA damage.
The strong expression of the two matrix metalloproteinases (MMP-2 and MMP-9) detected through immunohistochemistry and RT-PCR in both leukoplasic and healthy oral
mucosa could be related to the remodelling of the extracellular matrix that takes place both in physiological and pathological conditions (14-16). Moreover, the immunohistochemical data seem to indicate an increase in the expression of the two metalloproteinases in leukoplakia, thus suggesting a
Fig. 1. Immunohistochemistry: A) leukoplakia sample 40X, iNOS in the stratified epithelium; B) healthy oral mucosa 40X, iNOS in the vessel endothelium; C) leukoplakia sample 40X, MMP-2 in the vessel endothelium and in some isolated connective cells; D) healthy oral mucosa 40X, MMP-2 in the vessel endothelium and in some connective cells; E) leukoplakia sample 40X, MMP-9 in the vessel endothelium and in some connective cells; F) healthy oral mucosa 40X, MMP-9 in the vessel endothelium and in some connective cells; G) RT-PCR analysis in leukoplakia sample. Lane 1: MMP-2 amplification product (280 bp); lane 2: MMP-9 amplification product (554 bp); lane 3: 100 bp DNA ladder; lane 4: no amplification product for iNOS; lane 5: GAPDH amplification product (238 bp); H) RT-PCR analysis in healthy oral mucosa. Lane 1: MMP-2 amplification product (280 bp); lane 2: 100 bp DNA ladder; lane : MMP-9 amplification product (554 bp); lane 4: no amplification product for iNOS; lane 5: GAPDH amplification product (238 bp).
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more dynamic condition in which the tissue is more involved in degradation and remodelling processes.
The result of the histo-pathological analysis, from the Laboratory of Pathological Histology of “Paolo Giaccone” hospital, reported a condition of verrucous proliferative leukoplakia with hyperkeratosis, but without any sign of dyplasia which could explain the fact that in our two different samples (leukoplakia and control tissue) we detected the same gene expression with presence of MMP-2, MMP-9 and iNOS at the immunohistochemical assay and presence of MMP-2 and MMP-9 at the PCR analysis. It was surprising to find iNOS immuno-expression even in the healthy sample of oral mucosa since iNOS is the inducible isoform of nitric oxide synthase usually induced and synthesized in inflammatory conditions. Our data is reported considering that the commercially available iNOS antibody used is a polyclonal antibody and that the expression of iNOS detected through immunohistochemistry was not paralleled by the molecular data (PCR analysis).
The definition of leukoplakia given by the World Health Organization, as mentioned in the introduction, is “a white plaque that cannot be characterized either from a clinical or from a histopathological point of view”, thus the diagnosis of leukoplakia is based on the exclusion of other lesions of the oral mucosa. Our preliminary data concerning a single case represent the beginning of a broader study in which we plan to investigate these three genes and other molecular markers in a wide range of samples. In fact, we believe it can be useful to identify molecular and immunohistochemical parameters that can contribute to further discrimination between the different leukoplakia clinical subtypes coded by the epidemiology.
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3. WHO Collaborating Centre for Oral Precancerous Lesions. Definitions of leukoplakia and related
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5. Axéll T, Pindborg JJ, Smith CJ, van der Waal I. An International Collaborative Group on oral white lesions. Oral white lesions with special reference to precancerous and tabacco-related lesions: conclusions of an International Symposium held in Uppsala (Sweden, May 18–21 1994). J Oral Pathol Med 1996; 25:25-49.
6. Kingsley K, Johnson D, O’Malley S. Transfection of oral squamous cell carcinoma with human papillomavirus-16 induces proliferative and morphological changes in vitro. Cancer Cell Int 2006; 6:14-18.
7. Pimenta FJ, Gomes DA, Perdigão PF, Barbosa AA, Romano-Silva MA, Gomez MV, Aldaz CH, De Marco L, Gomez RS. Characterization of the tumor suppressor gene WWOX in primary human oral squamous cell carcinomas. Int J Cancer 2006; 118:1154-8.
8. Poate TWJ, Warnakulasuriya S. Effective management of smoking in an oral dysplasia clinic in London. Oral Diseases 2006; 12:22-26.
9. Al Madori G, Bussu F, Navarra P. Pilot phase IIA study for evaluation of the efficacy of folic acid in the treatment of laryngeal leucoplakia. Cancer 2006; 107:328-36.
10. Ghazali N, Bakri MM, Zain RB. Aggressive, multifocal oral verrucous leukoplakia: proliferative verrucous leukoplakia or not? J Oral Pathol Med 2003; 32:383-92.
11. Bagan JV, Jimenez Y, Sanchis JM, Poveda R, Milian MA, Murillo J, Scully C. Proliferative verrucous leukoplakia: high incidence of gingival squamous cell carcinoma. J Oral Pathol Med 2003; 32:379-82.
12. Ma N, Tagawa T, Hiraku Y, Murata M, Ding X, Kawanishi S. 8-Nitroguanine formation in oral leukoplakia, a premalignant lesion. Nitric Oxide 2006; 14:137-43.
13. Chaiyarit P, Ma N, Hiraku Y, Pinlaor S, Yongvanit P, Jintakanon D, Murata M, Oikawa S, Kawanishi S.
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Unauthorized reproduction may result in financial and other penalties131
Nitrative and oxidative DNA damage in oral lichen planus in relation to human oral carcinogenesis. Cancer Sci 2005; 96:553-9.
14. Cataldo DD, Gueders MM, Rocks N, Sounni NE, Evrard B, Bartsch P, Louis R, Noel A, Foidart JM. Pathogenic role of matrix metalloproteases and their inhibitors in asthma and chronic obstructive pulmonary disease and therapeutic relevance of matrix metalloprotease inhibitors. Cell Mol Biol
2003; 49:875-84. 15. Dahan M, Nawrocki B, Elkaïm R, Soell M,
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16. Xue M, Le NT, Jackson CJ. Targetting matrix metalloproteases to improve cutaneous wound healing. Expert Opin Ther Targets 2006; 10:143-55.
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EDITORIAL
IMMUNOLOGY OF HUMAN RICKETTSIAL DISEASES
P. MANSUETO, G. VITALE, G. DI LORENZO, F. ARCOLEO1, S. MANSUETOand E. CILLARI1
Dipartimento di Medicina Clinica e delle Patologie Emergenti, University of Palermo;1U.O. di Patologia Clinica, Azienda Ospedaliera “V. Cervello”, Palermo, Italy
Received November 3, 2007 - Accepted April 24, 2008
Among human rickettsial diseases caused by micro-organisms of the genus Rickettsia (Order Rickettsiales; Family Rickettsiaceae), transmitted to human hosts through arthropod vectors, Mediterranean Spotted Fever, or Boutonneuse Fever, and Rocky Mountain Spotted Fever are considered to be important infectious diseases due to continued prevalence in the developed world, and potentially fatal outcome in severe cases. Proliferation of rickettsiae, at the site of the tick bite, results in focal epidermal and dermal necrosis (tache noire). Rickettsiae then spread via lymphatic vessels to the regional lymph nodes, and, via the bloodstream, to skin, brain, lungs, heart, liver, spleen and kidneys. The pathogen invades and proliferates in the endothelial cells of small vessels, target cells of rickettsial infection, destroying them, and spreading the infection to the endothelia of the vascular tree. The damage of the endothelium, and the subsequent endothelia dysfunction, is followed by the activation of acute phase responses, with alteration in the coagulation and in the cytokine network, together with a transient immune dysregulation, characterized by the reduction in peripheral CD4+ T lymphocytes.
The human rickettsial diseases are a variety of clinical entities caused by micro-organisms of the genus Rickettsia (Order Rickettsiales; Family Rickettsiaceae). The pathogen invades and proliferates in the endothelial cells (ECs) of small vessels, target cells of rickettsial infection, destroying them, and spreading the infection to the endothelia of the vascular tree. The damage of the endothelium, and the subsequent endothelia dysfunction, is followed by the activation of acute phase responses, with alteration in the coagulation and in the cytokine network, together with a transient immune dysregulation, characterized by the reduction in peripheral CD4+ T lymphocytes. The mechanisms
of host defence are not yet completely understood, although cell-mediated immunity is believed to play a crucial role (1).
ENDOTHELIAL CELL INVASION AND INJURY
Vasculitis of small vessels of numerous organs (including central nervous system, lung, myocardium, liver, and kidney) is a typical event during Boutonneuse Fever (BF), as seen by histopathologic examination, and the complications are attributed to changes in vascular permeability and oedema due to endothelial injury (2).
Mailing address: Dr Pasquale Mansueto, M.D.Dipartimento di Medicina Clinica e delle Patologie Emergenti,Via del Vespro n° 141, 90127 Palermo, ItalyTel: ++39 91 6552970 Fax: ++39 91 6555995e-mail: [email protected]
Vol. 22, no. 2, 131-139 (2008)
132 133Journal of Biological Regulators & Homeostatic Agents
EC invasionAlthough some signalling events involved in
rickettsial entry into the ECs have been documented (i.e. activation of Src-family tyrosine kinases, tyrosine phosphorylation of focal adhesion kinase, and β1-integrin activation), host proteins mediating entry are not known (2).
In some studies it has been reported that Ku70 subunit of DNA-dependent protein kinase is a receptor involved in R. conorii internalization (Fig. 1). Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. R. conorii infection also stimulates the ubiquitination of Ku70. In addition, an ubiquitin ligase, called c-Cbl, is recruited to R. conorii entry foci, and downregulation of c-Cbl blocks rickettsial invasion and Ku70 ubiquitination (3).
Experimental approaches have identified the rickettsial outer membrane protein B (OmpB) as a ligand for Ku70. Antibodies against OmpA and OmpB are able to protect against reinfection, but they do not play a key role in immunity against primary infection (4).
The rickettsiae induce internalization by phagocytosis, associated with phospholipase A2 activity, apparently of rickettsial origin, and escaping from the phagosome into the cytosol by an unidentified mechanism, even if some experimental data suggest a role for the tlyC and pld genes, with potential membranolytic activities, in the process of phagosomal escape by R. prowazekii (5). In the cytosol, R. conorii stimulates the polymerization of host cell F-actin, controlled by the Arp2/3 complex, using the bacterial surface RickA protein, which propels the bacterium through the cytoplasm and across the cell membrane into the adjacent endothelial cell or extracellular space (Fig. 1) (6).
EC injuryThe infection of cultured ECs causes the
induction of oxidative stress mechanisms, as evidenced by accumulation of reactive oxygen species (ROS), induced by the combination of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) production (7), altered levels of antioxidant enzymes, and depleted levels of intracellular reduced thiols, together with alterations in the levels of mRNA expression of selected antioxidant enzymes
(8) (Fig. 2).
Production of cytokines and chemokines by ECsHuman umbilical vein endothelial cells
(HUVECs), infected by R. conorii, actively secrete large amounts of IL-8 and IL-6, via an IL-1α dependent pathway (Fig. 3) (9).
IL-1α, produced by HUVECs, is mainly in a precursor form. Therefore, IL-1α precursor can be secreted and immediately bound to the type I IL-1 receptor (10). Alternatively, IL-1α can be active without being secreted and fixed on endothelial surface membranes in cell-cell membrane interaction. Thus, IL-1α can act as an autocrine factor on HUVECs. IL-1α activates ECs, and increases the expression of adhesion molecules on both ECs and leukocytes by inducing the production of other cytokines, such as IL-8 and IL-6 (10).
IL-8 and IL-6 might play a role in the development of vasculitis induced by rickettsial infection. IL-8 is a potent chemotactic agent for polymorphonuclear leukocytes, stimulates polymorphonuclear leukocyte transendothelial migration, and activates their functions, but it seems to have no effect on endothelial cells (11). IL-6, also secreted by R. conorii-infected HUVECs, may mediate the acute phase protein production associated with BF. IL-6 might also be implicated in the local differentiation and proliferation of T lymphocytes, through its stimulatory effects on IL-2 production and IL-2 receptor (IL-2R) expression in T cells, and in B lymphocyte stimulation (12).
In addition, the expression of the chemokines CXCL9 and CXCL10, known to target CD8+ and CD4+ T lymphocytes, is significantly high in ECs of C3H/HeN mice infected with R. conorii. The peak of expression of these chemokines occurred 4 days before CD8+ T cells infiltrated the infected tissues. Therefore, CXCL9 and CXCL10 may play a role early during the immune response against rickettsial infections (13). Similarly, the peak of expression of fractalkine (CX3CL1), another chemokine expressed mainly by endothelial cells, on day 3 of infection, coincided with the time of infiltration of macrophages into infected tissues and preceded the peak of rickettsial content in tissues (14).
Induction of cellular adhesion moleculesIn vitro R. conorii infection of ECs, through the
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release of the early response cytokines (IL-1α, IL-6, and TNF-α) and other components of the acute phase reaction, enhances the expression on the surface of ECs and polymorphonuclear leukocytes of cellular adhesion molecules (CAM), i.e. E-selectin, VCAM-1, and ICAM-1, which are responsible for enhanced rolling and the adherence of mononuclear cells to infected ECs (Fig. 3) (15).
In addition, during the acute phase of BF, soluble forms of these adhesion molecules (sL-selectin, sE-selectin, s-VCAM-1, and sICAM-1) are shed into the plasma by the proteolytic cleavage of their membrane-bound counterparts from activated leukocytes and ECs, and/or by the differential splicing of their mRNA (Fig. 3). The shed forms are functionally active and could be involved in the control of adhesive interactions between the cells. The persistence of high levels of these CAM has negative prognostic value, and could be useful to monitor disease evolution (15).
Platelet activation In the acute phase of BF, through measurements of
a major metabolite of thromboxane (TX) in the urine (11-dehydro-TXB2), a marker of platelet activation, and of plasma prothrombin fragment 1+2, whose levels reflect activation of prothrombin to thrombin, biochemical evidence has been demonstrated for the occurrence of a TXA2-dependent platelet activation and thrombin generation in vivo. These phenomena could, at least in part, account for clinical manifestations of BF, such as vasculitis and focal microthrombus formation (16).
Mechanism of intracellular killing of R. conoriiPerivascular infiltrations of CD4+ and CD8+
T lymphocytes, macrophages, natural killer (NK) cells, infected ECs themselves, and marginated elements of blood secrete cytokines and chemokines that activate infected ECs, by paracrine and autocrine stimulation, to kill intracellular rickettsiae. Human cells might be capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by a cytokine- and chemokine-dependent, nitric oxide-dependent mechanism, and, in particular, by one or a combination of these three
Fig. 1. Endothelial cell invasion.
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mechanisms, involving: 1) nitric oxide synthesis; 2) hydrogen peroxide production; 3) tryptophan degradation, resulting in its intracellular depletion and starvation for the parasite (17).
ACUTE PHASE RESPONSE
In BF, the invasion and proliferation of rickettsiae in the ECs is the early event responsible for acute phase response activation. Some acute phase responses appear to be involved in the promotion of inflammatory events (IL-1α, IL-8, IFN-γ, complement proteins, and fibrinogen), while others appear to moderate them (ceruloplasmin, and α1-antitrypsin). After the resolution of the infection, all mediators reach the normal range, acute phase response subsides, and the homeostatic balance is restored (Fig. 4) (18).
The analysis of cytokine serum levels indicates that IL-1α is not detectable in the blood of acute BF patients, which probably depends on the fact that stimulation in ECs causes the production of IL-1α predominantly in cell-associated form, thus
contributing to the localized procoagulant and inflammatory responses which occur in the course of rickettsial disease. The undetectable serum levels of IL-8 may be due to its prevalent concentrations on endothelial protrusions, either in vesicles or on the plasma membrane (18).
As for as IFN-γ, its increase has been detected in the firsts week, followed by its normalization. Furthermore, IFN-γ appears to be associated with complement protein modifications. In fact, IFN-γ is the main inducer of C4 gene expression (18-19). Complement could prove useful for the elimination of infected, damaged or compromised cells, exerting anti-inflammatory activities (18).
The detection of high levels of serum fibrinogen, in the first week of BF infection, constitutes the primary event inducing a local activation of haemostasis, through platelet activation, and it is a cursory indicator of inflammation in most human infections (18).
Also anti-inflammatory mediators (i.e. ceruloplasmin and α1-antitrypsin) are detected in patients affected by BF. The strong release
Fig. 2. Endothelial cell injury.
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of ceruloplasmin might minimize the level of inflammatory response, acting as scavenger of oxygen free radicals, or ROS, produced by leukocytes (18). α1-antitrypsin persists at high levels in BF patients until the third week and could indicate that this proteinase inhibitor is required for a long period, both to neutralize the lysosomal hydrolases released following the infiltration of macrophages and, more importantly, to induce the release of anti-inflammatory cytokines (18).
TOLL-LIKE RECEPTORS AND THE INNATE IMMUNE SYSTEM
One of the mechanisms by which the innate immune system senses the invasion of pathogenic microrganisms is through the pattern recognition receptors (PRRs), which are germ line-encoded receptors, including transmembrane Toll-like receptors (TLRs), that can sense pathogen-associated molecular patterns, and initiate signalling cascades that lead to an innate immune response. The Toll-like receptor 4 (TLR4) plays an important
role in inflammation and immunity. It has been demonstrated that the +869G single-nucleotide polymorphism of the TLR4 gene, known to attenuate receptor signalling, decrease endotoxin responsiveness and determine poor outcome from sepsis, is overexpressed in BF patients, with significant differences, in the frequency of TLR genotypes and alleles, compared with age-matched controls. These data might be interpreted as one of the hypothetical bases for a genetic predisposition to BF (20).
IMMUNOLOGIC ALTERATIONS
Rickettsial-dendritic cell interactionThe role played by dendritic cells (DCs), known
as specialized antigen presenting cells, initiators and orchestrators of the immune response, remains unclear in rickettsial infections. It seems that a vigorous proinflammatory response of DCs, associated with up-regulation of CD40, CD80, CD86, and major histocompatibility complex class II, production of IL-2, IL-12, and IL-23, and
Fig. 3. Production of cytokines by ECs and induction of cellular adhesion molecules.
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mediated by the increase in TH1-type (TNF-α and IFN-γ), and in TH2-type cytokines (IL-10, and IL-6), appear to be important for the healing process (Fig. 5). In detail, TNF-α, IFN-γ, IL-10, and IL-6 are significantly increased in the blood of patients with acute BF compared with those detected from control healthy subjects (22).
In addition to the above, also in mild/moderate Mountain Spotted Fever (MSF) a strong and balanced intralesional proinflammatory and anti-inflammatory response is demonstrable, with a dominant type 1 immunity, whereas severe MSF is associated with increased expression of chemokine, i.e. RANTES, mRNA (23).
The dramatic increase in TNF-α, detected in the first week after the onset of symptoms, together with IFN-γ, is one of the early events observed in acute BF patients, followed by the increase of IL-10 and IL-6 (Fig. 5) (22). Activated antigen presenting cells (i.e. activated macrophages) produce high levels of TNF-α. TNF-α induces IFN-γ production from activated CD4+ T cells and NK cells. One important
production of antigen-specific IFN-γ in CD4+ T cells, is involved in the appearance of a protective immunity to rickettsiae (21).
Analysis of lymphocyte subpopulationsIn acute BF patients there is a reduction of
circulating T cells and, in particular, of CD4+ (helper/inducer T cells), CD4+/CD45RO+ (memory T cells), and CD4+/CD45+ (naive T cells) subsets. These modifications may be due to the cell adhesion to vascular endothelium, followed by their entrance into the sites of inflammation. Also apoptosis, spontaneous and activation-induced, could have a role in this decrease. The other lymphocyte subsets (CD8+ [suppressor/cytotoxic T cells], CD16+ [NK cells], and CD20+ [B cells]) tend to decrease in a non-significant way, whereas monocytes/macrophages (CD14+/HLA-DR+) are significantly increased. All cell subsets return to the normal levels after successful treatment, except for monocytes, that are persistently high after recovery (22).
The inflammatory and immunologic responses,
Fig. 4. Acute phase response.
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effect of IFN-γ on macrophages is the induction of receptors for TNF-α, so that the binding of TNF-α on macrophages might further activate these cells by an autocrine TNF-α loop, thus increasing their anti-rickettsial activity, via the induction of NO synthesis and incretion (24). Moreover, the persistence of high serum levels of TNF-α, in recovered patients, could be a sign of residual local lesions. A relationship has also been observed between serum TNF-α and a high production of one the two soluble TNF receptors, sTNF-RI; persisting high values of sTNF-RI, in BF, could be indicator of disease activity (25).
Because IL-10 (a TH2-type cytokine) suppresses the ability of IFN-γ-activated macrophages to produce inflammatory mediators, the persistence of high levels of IL-10 could be due to its ability to down-regulate the potential tissue-damaging effects, mediated by the increase in TH1-type cytokines (26).
The profile of serum IL-6 (another TH2-type cytokine) production, in patients with acute BF and after recovery, is similar to that of IL-10. In acute BF, IL-6 could acts as an anti-inflammatory cytokine, rather than as a growth and differentiation factor for
B cell and immunoglobulin production (27). Therefore, in BF patients, TH1- and TH2-type
responses are not characteristically polarised, as both activating (i.e. TNF-α, IFN-γ) and suppressive (i.e. IL-10 and IL-6) cytokines are increased. In BF, as in any other inflammatory environment, counterbalancing mechanisms are normally produced to curtail the process, thus IL-10 and IL-6 could be capable of derailing TH1-type responses and deactivating macrophages, thereby moderating tissue injury (28).
Finally, in BF patients, IL-12, predominantly produced by monocytes, appears to act very early, favouring TH1 differentiation and TH1-type cytokine production, primarily of IFN-γ from T cells and NK, acting as a growth factor for activated T cells and NK, enhancing cytotoxic activity of NK, and contributing to the phagocyte cell activation and the innate resistance to intracellular pathogens (29).
ROLE OF CD8+ T CELLS
CD8+ T lymphocytes contribute to
Fig. 5. Immunologic alterations.
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protective immunity to rickettsiae by both Major Histocompatibility Complex (MHC)-I-restricted Cytotoxic T-lymphocyte (CTL) activity and by production of IFN-γ. However, CTL activity may be more critical for the recovery from rickettsial infection than the effects of IFN-γ production (Fig. 5) (30).
Infected cells are eliminated by apoptosis associated with CTL activity. Although apoptotic loss of endothelium could be considered a pathologic event, it favours the human host, more than the survival or the necrosis of an infected cell. Survival of infected ECs would permit further growth of the rickettsiae, while necrosis would release rickettsiae, allowing their spread to establish foci of infection in other cells. In contrast, rickettsiae-containing apoptotic bodies would be phagocytised rapidly and digested intracellularly (30).
REFERENCES
1. Feng H, Popov VL, Yuoh G, Walker DH. Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae. J Immunol 1997; 158:5314-20.
2. Martinez JJ, Cossart P. Early signaling events involved in the entry of Rickettsia conorii into mammalian cells. J Cell Sci 2004; 117:5097-5106.
3. Martinez JJ, Seveau S, Veiga E, Matsuyama S, Cossart P. Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii. Cell 2005; 123:1013-23.
4. Feng HM, Whitworth T, Popov V, Walker DH. Effect of antibody on the rickettsia-host cell interaction. Infect Immun 2004; 72:3524-30.
5. Whitworth T, Popov VL, Yu XJ, Walker DH, Bouyer DH. Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape. Infect Immun 2005; 73:6668-73.
6. Gouin E, Egile C, Dehoux P et al. The RickA protein of Rickettsia conorii activates the Arp2/3 complex. Nature 2004; 427:457-61.
7. Santucci LA, Gutierrez PL, Silverman DJ. Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells. Infect Immun 1992; 60:5113-8.
8. Rydkina E, Sahni SK, Santucci LA, Turpin LC,
Baggs RB, Silverman DJ. Selective modulation of antioxidant enzyme activities in host tissues during Rickettsia conorii infection. Microb Pathog 2004; 36:293-301.
9. Kaplanski G, Teysseire N, Farnarier C et al. IL-6 and IL-8 production from cultured human endothelial cells stimulated by infection with Rickettsia conorii via a cell-associated IL-1 alpha-dependent pathway. J Clin Invest 1995; 96:2839-44.
10. Kaplanski G, Farnarier C, Kaplanski S et al. Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism. Blood 1994; 84:4242-8.
11. Fibbe WE, Pruijt JF, Velders GA et al. Biology of IL-8-induced stem cell mobilization. Ann NY Acad Sci 1999; 872:71-82.
12. Kishimoto T. IL-6: from laboratory to bedside. Clin Rev Allergy Immunol 2005; 28:177-86.
13. Valbuena G, Bradford W, Walker DH. Expression analysis of the T-cell-targeting chemokines CXCL9 and CXCL10 in mice and humans with endothelial infections caused by rickettsiae of the spotted fever group. Am J Pathol 2003; 163:1357-69.
14. Valbuena G, Walker DH. Expression of CX3CL1 (fractalkine) in mice with endothelial-target rickettsial infection of the spotted-fever group. Virchows Arch 2005; 446:21-7.
15. Vitale G, Mansueto S, Gambino G et al. Differential up-regulation of circulating soluble selectins and endothelial adhesion molecules in Sicilian patients with Boutonneuse fever. Clin Exp Immunol 1999; 117:304-8.
16. Davi G, Giammarresi C, Vigneri S et al. Demonstration of Rickettsia Conorii-induced coagulative and platelet activation in vivo in patients with Mediterranean spotted fever. Thromb Haemost 1995; 74:631-4.
17. Feng HM, Walker DH. Mechanisms of intracellular killing of Rickettsia conorii in infected human endothelial cells, hepatocytes, and macrophages. Infect Immun 2000; 68:6729-36.
18. Vitale G, Mansueto S, Gambino G et al. The acute phase response in Sicilian patients with boutonneuse fever admitted to hospitals in Palermo, 1992-1997. J Infect 2001; 42:33-9.
19. Mansueto S, Vitale G, Cillari E et al. High levels of
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interferon-gamma in boutonneuse fever. J Infect Dis 1994; 170:1637-8.
20. Balistreri CR, Candore G, Lio D et al. Role of TLR4 receptor polymorphisms in Boutonneuse fever. Int J Immunopathol Pharmacol 2005; 18:655-60.
21. Jordan JM, Woods ME, Feng HM, Soong L, Walker DH. Rickettsiae-stimulated dendritic cells mediate protection against lethal rickettsial challenge in an animal model of spotted fever rickettsiosis. J Infect Dis 2007; 196:629-38.
22. Cillari E, Milano S, D’Agostino P et al. Depression of CD4 T cell subsets and alteration in cytokine profile in boutonneuse fever. J Infect Dis 1996; 174:1051-7.
23. de Sousa R, Ismail N, Nobrega SD, França A, Amaro M, Anes M, Poças J, Coelho R, Torgal J, Bacellar F, Walker DH. Intralesional expression of mRNA of interferon-gamma, tumor necrosis factor-alpha , interleukin-10, nitric oxide synthase, indoleamine-2,3-dioxygenase, and RANTES is a major immune effector in Mediterranean spotted fever rickettsiosis. J Infect Dis 2007; 196:770-81.
24. Oristrell J, Amengual MJ, Font-Creus B, Casanovas A, Segura-Porta F. Plasma levels of TNF alpha in patients with mediterranean spotted fever. Clin Infect
Release of soluble tumor necrosis factor receptors in Mediterranean spotted fever rickettsiosis. Clin Diagn Lab Immunol 1996; 3:233-5.
26. Capsoni F, Minonzio F, Ongari AM, Carbonelli V, Galli A, Zanussi C. IL-10 up-regulates human monocyte phagocytosis in the presence of IL-4 and IFN-gamma. J Leukoc Biol 1995; 58:351-8.
27. Oristrell J, Sampere M, Amengual MJ, Font-Creus B and Segura-Porta F. Plasma interleukin-6 levels in Mediterranean spotted fever. Europ J Clin Microbiol Infect Dis 2004; 23:417-8.
28. Kutlu A, Bozkurt B, Ciftci F, Bozkanat E. Th1-Th2 interaction: is more complex than a see-saw? Scand J Immunol 2007; 65:393-5.
29. Billings AN, Feng HM, Olano JP, Walker DH. Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon. Am J Trop Med Hyg 2001; 65:52-6.
30. Walker DH, Olano JP, Feng HM. Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection. Infect Immun 2001; 69:1841-6.
JOURNAL OF BIOLOGICAL REGULATORS & HOMEOSTATIC AGENTS
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IMPETIGO HERPETIFORMIS OCCURRING DURING N-BUTYL-SCOPOLAMMONIUM BROMIDE THERAPY IN PREGNANCY:
CASE REPORT
C. GUERRIERO, S. LANZA SILVERI, T. SISTO, D. ROSATI, C. DE SIMONE, B. FOSSATI, F. POMINI1, M. ROTOLI, P. AMERIO and R. CAPIZZI
Department of Dermatology,Catholic University of the Sacred Heart; 1Department of Gynecology and Obstetrics, Catholic University of the Sacred Heart, Rome, Italy
Received February 4, 2008 – Accepted May 9, 2008
LETTER TO THE EDITOR
Impetigo herpetiformis (IH) is a rare dermatosis arising during the third trimester of pregnancy which is generally considered as a form of pustular psoriasis of unknown aetiology. Clinically it is characterized by erythematous plaques surrounded by sterile pustules associated with fever, diarrhea, sweating and increasing risk of stillbirth for placental insufficiency. We describe a case of developed erythematous plaques surrounded by pustules localised initially to the trunk of a 35-year-old woman at the 34th week of gestation after 5 days of treatment with N-Butyl-Scopolammonium, and which later involved the upper and lower limbs. Skin histology confirmed the diagnosis of generalised pregnancy pustular psoriasis (impetigo herpetiformis). IH is reported to be associated with hypocalcemia, hypoparathyroidism, use of oral contraceptives and bacterial infections. This is the first report suggesting the potential role of drugs other than oral contraceptives in the pathogenetic mechanism of this disease. In this case an adverse cutaneous reaction to BB could be the cause of the development of Koebner isomorphism.
We report the case of a 35-year-old woman, febrile and asthenic, at the 34th week of gestation who presented with erythematous plaques surrounded by non-follicular pustules, localised initially to the trunk and subsequently spreading to the upper and lower limbs, which appeared 5 days after starting treatment with N-Butyl Scopolammonium-Bromide (BB) for early uterine contractions. Mucosal tissues were not involved. Her medical history was negative for skin disorder, and family history was negative for psoriasis. She has had two previous normal pregnancies, both with uncomplicated vaginal delivery at term. The current
pregnancy had also been uneventful with normal ultrasound scans, haematological values and vaginal cultures. Laboratory findings showed neutrophilic leukocytosis (13300/mm3 equivalent to 80.6% WBC), ESR of 52 mm/h, microcytic hypochromic anaemia (Hb: 9.5 g/dl). Serum calcium, liver and renal function tests, and autoimmune profile were normal. Bacteriological culture on the fluid from one of the pustules on the trunk was negative. Upon presentation at 34 weeks of gestation, an ultrasound scan revealed a normal growing fetus of an estimated weight of 2078 g corresponding to the 30th percentiles (1). The Amniotic Fluid Index was within
Mailing address: Cristina Guerriero M.D.,Department of Dermatology,Catholic University of the Sacred Heart, L.go A. Gemelli, 8 00168 Roma, ItalyTel: ++390630154227 Fax: ++39063016293e-mail: [email protected]
Vol. 22, no. 2, 141-144 (2008)
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normal values at 140 mm3. Color Doppler resistance indexes of both Umbilical arteries (mean Pulsatility Index (PI) 1.12), Mean Cerebral Artery (PI 1.82) and right and left uterine arteries (Resistance Index (RI) 0.45 and 0.32, respectively) were normal (2). A normal pattern of fetal movements and no structural fetal abnormalities were detected. Fetal Heart Rate monitor was reassuring and no uterine contractions were detected. Vaginal examination was also normal. Skin biopsy from one of the plaques on the trunk showed epidermic hyperplasia with parakeratosis and neutrophilic exocitosis. Intraepidermic pustules were present. (Fig. 1). The patient was treated with topical high potency steroid (clobetasole propionate) and systemic methylprednisolone (1 mg/kg/die i.v.) which were gradually reduced over two weeks. The cutaneous lesions disappeared two weeks later and the patient was discharged home without any therapy. No relapse occurred after the end of the therapy. At 39 weeks the patients was admitted to the maternity ward with painful and frequent uterine contractions, and after three hours delivered a healthy male baby of 3300 g with 9/9 apgar scores and an umbilical venous ph of 7.39.
DISCUSSION
Impetigo Herpetiformis (IH) was first described by Von Hebra (3) as a rare pustular dermatitis
occurring during pregnancy with a dramatic clinical presentation and serious systemic features including fetal distress, due to placental insufficiency. Approximately 350 cases of IH have been reported in the literature (4). Its aetiology is currently unknown (5). However, hypocalcemia, hypoparathyroidism, bacterial infections and oral contraceptives can trigger this disease (6). IH occurs more frequently in second pregnancy, commonly during the third trimester or post-partum, although earlier onset in successive pregnancies has been reported (7).
The classification of IH is controversial (5, 8). Some authors consider the disease as an entity distinct from pustular psoriasis. Others suggest that IH is not a separate entity but should be considered as an acute pustular flare of psoriasis precipitated by the endocrine changes in pregnancy (8), or a rare form of generalized pustular psoriasis similar in pathogenesis to some stress-induced exacerbations (9).
Clinically, the eruption is characterized by erythematous plaques with grouped sterile pustules on the margins, localised symmetrically at the flexural and intertriginous areas extending centrifugally. Plaques can evolve in erosions which can become infected. Mucous involvement can be present and is characterized by erosive or circinate lesions. Systemic symptoms such as fever, malaise, diarrhea, vomiting and sweating may also be present.
Fig. 1. Epidermic hyperplasia with parakeratosis (�), neutrophilic exocitosis and intraepidermic pustules ( ).
Fig. 1. Epidermic hyperplasia with parakeratosis (→), neutrophilic exocitosis and intraepidermic pustules (▬►).
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In severe cases cardiac and renal failure can occur. Typical laboratory findings can include leukocytosis, elevated sedimentation rate, hypocalcemia and hyperphosphatemia, hypoalbuminaemia and microcytic anemia. Low serum levels of vitamin D are rarely reported. Histological examination of the lesions reveals intraepidermal non-follicular and sterile pustules containing neutrophils and sometimes mononuclear cells. Lymphocyte infiltrates and neutrophils are found in the papillary dermis.
IH may be complicated by sepsis or placental insufficiency, with an increasing risk of fetal abnormalities, stillbirths and neonatal deaths (10). Hypocalcemia is associated with the risk of tetania and death. IH must be differentiated from a wide spectrum of cutaneous diseases with similar presentation such as acute generalised exanthematous pustulosis (AGEP), dermatitis herpetiformis, herpes gestationis, Sneddon-Wilkinson syndrome, pustular vasculitis and DRESS.
AGEP is an acute, spontaneously healing reaction to drugs, usually antibiotics, characterized by haematological hypereosinophilia. EuroSCAR study (11) proposed more sophisticated criteria for a diagnostic score based on lesion morphology, disease course and histological features. Skin histology typically shows an inflammatory infiltrate with neutrophils and eosinophils. AGEP is characterized by spontaneous remission within 2-3 weeks of onset. Sneddon-Wilkinson disease is a subcorneal pustular dermatosis with flaccid pustules arising on normal skin or a slightly erythematous base forming characteristic anular or serpiginous patterns with a scaly edge. While spongiosis is not a feature of Sneddon Wilkinson disease, spongiform pustules are a common finding in IH.
IH can be differentiated also by drug rash with eosinophilia and systemic symptoms (DRESS syndrome) because of the absence of haematolgical and histological hypereosinophilia and the typical clinical and anamnestic features of DRESS (12). Dermatitis herpetiformis and herpes gestationis are characterised by the presence of vesicles and bullous lesions that are absent in IH. Occasionally, pustular vasculitis can be confused with IH, but skin biopsy shows inflammatory infiltrates with eosinophils and presence of vasculitis. Infective follicular eruptions such as impetigo can be easily distinguished, because
in IH the pustules are sterile and not follicular. The use of drugs for the treatment of IH is limited
because of the potential for systemic complications. Therapy is based on the use of local and systemic steroids (prednisone at a dosage of 1 mg/kg daily), fluid integration and antibiotics in the presence of infection. Fetal monitoring is important for the increased risk of fetal mortality. Other treatments that can be used in the post-partum period are oral cyclosporine (13), PUVA (14) and systemic retinoids (5, 15) (isotretinoin, etretinate, acetretin), alone or in combination with oral corticosteroids. In the literature the treatment of IH with methotrexate is also described, especially in those cases non-responsive to conventional treatment with corticosteroids.
In this case, the early diagnosis and the prompt onset of therapy with systemic steroids led to a significant clinical improvement in approximately 2 weeks without fetal complications. Careful fetal monitoring by ultrasound and colour-doppler ultrasound showed regular intrauterine growth and no signs of placental insufficiency. In our patient no risk factor commonly associated with the ethiopathogenesis of IH (6) could be recognised, and the only anamnestic finding was the start of therapy with BB for uterine contractions and preterm labour.
According to the literature, the only drugs associated with the development of IH are oral contraceptives, although the pathological mechanism remains unknown. On the other hand, treatment with certain drugs is a recognised risk factor for new onset or exacerbation of psoriasis. These include angiotensin-converting enzyme (ACE) inhibitors, non-steroidal anti-inflammatory drugs, benzodiazepines, organic nitrates, beta-blockers, dipyrone, lithium and gold (16).
In the literature BB has not been previously reported as a trigger for IH or psoriasis. The temporal link between the administration of BB and development of the cutaneous lesions, suggests that BB could have been at least partially responsible for the development of IH in our patient. To the best of our knowledge this could be the first case in which therapy with BB induced IH.
In our case, the early diagnosis and adequate treatment led to a complete resolution without complications for both mother and child. However,
144
further studies on IH are needed to improve early diagnosis and minimise systemic complications.
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13. Finch TM, Tan CY. Pustular psoriasis exacerbated by pregnancy and controlled by cyclsporin A. Br J Dermatol 2000; 142:582-584.
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15. Bukhari IA. Impetigo herpetiformis in a primigravida: successful treatment with etretinate. J Drug Dermatol 2004; 3:449-51.
16. Cohen AD, Bonneh DY, Reuveni H, Vardy AD, Naggan L, Halevy S. Drug exposure and psoriasis vulgaris: case-control and case-crossover studies. Acta Derm Venereol 2005; 85:299-303.
