Journal of Cystic Fibrosis 1(2002) 76–89

1569-1993/02/$ - see front matter� 2002 European Cystic Fibrosis Society. Published by Elsevier Science B.V. All rights reserved.PII: S1569-1993Ž02.00033-4

Immunopathogenesis of allergic bronchopulmonary aspergillosis in cysticfibrosis

Alan P. Knutsen *, Clifford Bellone , Henk Kauffmana, a b

Pediatric Research Institute, St. Louis University Health Sciences Center, 3662 Park Avenue, St. Louis, MO 63110, USAa

University Hospital Groningen, Groningen, The Netherlandsb

Abstract

Allergic bronchopulmonary aspergillosis(ABPA) is a hypersensitivity lung disease mediated by an allergic late-phaseinflammatory response toAspergillus fumigatus antigens. ABPA is characterized by markedly elevatedAspergillus-specific andtotal IgE levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates, and bronchiectasis and fibrosis, which afflictasthmatic and cystic fibrosis(CF) patients. We propose that ABPA develops in genetically susceptible CF patients due to HLA-DR2 and DR5 restriction, increased sensitivity to IL-4 stimulation, and increasedA. fumigatus allergen-specific Th2 CD4q T-cell-mediated responses. In addition,A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial celllayer by damaging the epithelial integrity and by a direct interaction with epithelial cell surface receptors, resulting in pro-inflammatory cytokine production and corresponding inflammatory responses.� 2002 European Cystic Fibrosis Society. Publishedby Elsevier Science B.V. All rights reserved.

Keywords: Allergic bronchopulmonary aspergillosis(ABPA); Cystic fibrosis(CF); Aspergillus fumigatus

1. Introduction

Allergic bronchopulmonary aspergillosis(ABPA) is ahypersensitivity lung disease mediated by an allergiclate-phase inflammatory response to certainAspergillusfumigatus antigens that occurs in approximately 1–2%of asthmatic and 7–10% of cystic fibrosis patientsw1–16x. ABPA is characterized by markedly elevatedAsper-gillus-specific IgE and total IgE levels and eosinophilia,and manifested by wheezing, pulmonary infiltrates,bronchiectasis and fibrosis. The immune response toAspergillus antigens in ABPA, as well as in allergicasthmatic and cystic fibrosis patients, is characterizedby a Th2 CD4q T-cell responsew1x. A central questionthen is how ABPA differs fromAspergillus-sensitiveatopic asthmatic and CF patients. It is proposed thatABPA develops in genetically susceptible cystic fibrosispatients due to increased frequency andyor activity ofAspergillus-specific Th2 CD4q T cells and to HLA-DR restriction. In addition,Aspergillus proteases damagethe respiratory epithelial barrier, exposing the bronchoal-

*Corresponding author. Tel.:q1-314-268-4014; fax:q1-314-577-5398.

E-mail address: knutsenm@slu.edu(A.P. Knutsen).

veolar lymphoid tissue(BALT) to high concentrationsof Aspergillus antigens that alter the immune response.The allergic inflammatory response in ABPA appears

to be quantitatively greater than inAspergillus-sensitivecystic fibrosis patients. In the model proposed of theimmunopathogenesis of ABPA, as illustrated in Fig. 1,A. fumigatus spores are inhaled into the bronchial airway,where they are trapped by the luminal mucus, germinateand form mycelia.A. fumigatus mycelia release allergensthat are processed by antigen-presenting cells(APC)bearing HLA-DR2 or DR5 and presented to T-cellswithin the bronchoalveolar lymphoid tissue(BALT).The T-cell response toAspergillus allergens becomesskewed toward a Th2 CD4q T-cell response with IL-4,IL-5 and IL-13 cytokine synthesis and secretion.

2. Interaction of respiratory epithelium andAspergillus

One of the characteristics features in patients withABPA is the observations thatA. fumigatus is foundbound to the surface epithelium growing on and betweenthe epithelial cells without being efficiently killed bymononuclear cells and eosinophilsw17x. It has also beenshown that spores ofA. fumigatus are attached to

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Fig. 1. Model of pathogenesis of allergic bronchopulmonary aspergillosis(from Knutsen et al.w1x).

epithelial surfaces cultured in vitrow18x. The physicalpresence ofA. fumigatus is possibly of importance forthe modulation of the immunological response towardsa Th2-type responsew19x. In order to evade the veryeffective natural defense system of the human airwaysagainst fungi, various virulence factors ofA. fumigatushave been detected that have been shown to interferewith, or even block, normal functions of the humoraland cellular defense of the airwaysw20,21x. Some ofthese virulence factors are the proteolytic enzymes ofA.fumigatus, which facilitate the immune response to thefungal antigens and play a role in the inflammatoryresponse.

2.1. Aspergillus proteolytic enzymes

It has been found that certain strains ofA. fumigatusrelease proteolytic enzymes with elastolytic and colla-genolytic activity. The possible role of these proteolyticenzymes as a pathogenic factor in fatal invasive asper-gillosis is still uncertain. However, findings in patientswith ABPA or aspergilloma indicate that these proteasesmay be involved in the pathogenesis of these diseases.In studies of ABPA, culture filtrate extracts with markedelastase and collagenase activity were examined. InWestern blot experiments, binding of IgG antibodies toa 32-kDa elastase protein was found with sera frompatients with ABPA or aspergillomaw22x. The pro-

nounced binding of IgG antibodies to the 32-kDa fungalelastase suggests that these proteases are produced invivo in patients with ABPA and aspergilloma. Further-more, during exacerbation of aspergilloma, antibodyconcentrations against different antigens, including the32- and 40-kDa proteases, were markedly increasedw23x. The observation that the antibody concentrationsagainst the 32- and 40-kDa fungal proteases areincreased during the exacerbation phase indicates thatthese proteases may play a role in the pathogenicity ofthe disease.

2.2. Aspergillus proteases and host cytokines

An important feature of pathogenic microorganismsis their capacity to interact with epithelial cells of themucosal surface. Previously, it has been shown thatproducts released in vitro byA. fumigatus are able tocause epithelial cell detachmentw24,25x. This capacityto induce epithelial cell detachment is also characteristicof other proteases released by different fungi, e.g.Alternaria andCladosporium, but Aspergillus proteasesare more active at much lower concentrationsw26x.Recent studies performed with proteases from varioussources, e.g. Der p1 from house dust mitew27x, haveshown that degradation of epithelial cell structuresresults in facilitated transport of antigens and allergensacross the epithelium, leading to enhanced exposure to

78 A.P. Knutsen et al. / Journal of Cystic Fibrosis 1 (2002) 76–89

antigen-presenting cells with concurrent immuneresponses.In addition to damaging the epithelial cell-layer integ-

rity, recent studies have shown that human bronchialand alveolar epithelial cell lines produce pro-inflamma-tory cytokines, such as IL-8, IL-6 and MCP-1, afterincubation with protease-containing culture filtrates ofA. fumigatus. This cytokine-releasing activity could beascribed to the proteolytic activity of these extractsw25x.These observations suggest that proteolytic enzymesreleased byAspergillus growing on and between epithe-lial cells may be responsible for the induction ofchemoattractive cytokines by epithelial cells and corre-sponding inflammatory responses. It has been proposedthat induction of a severe inflammatory response by thedirect activation of epithelial cells may induce additionalepithelial injury w20x. Destruction of the epithelial cellbarrier by proteases from either the fungus, or from theeosinophilic and neutrophilic inflammation, is followedby repair mechanisms, resulting in the influx of serumproteins and extracellular matrix proteins to the luminalsite of the epitheliumw28x. Since spores and mycelia ofA. fumigatus have surface structures that are able tointeract with extracellular matrix molecules, damage andconcurrent repair of the airway mucosa, with simulta-neous release of repair molecules, may facilitate thebinding of Aspergillus to the damaged sites of theairways. The interaction of enhanced release of proteo-lytic enzymes and allergens on the epithelial surfacewill induce a continuous inflammatory response andmast cell degranulation, resulting in severe and long-lasting periods of exacerbation of ABPA.In addition to the induction of cytokine responses of

epithelial cells, it was shown that proteases fromA.fumigatus at higher concentrations also inhibit epithelialcell cytokine productionw26x. This inhibition was spe-cific for the elastaseycollagenase-containing extracts ofA. fumigatus, which may represent an additional viru-lence factor by preventing effective targeting by infiltra-tive phagocytic cells. This would lead to continuousrelease of antigens and allergens, and strong activationof the Th2-type immunological response, with very highproduction of total and specific IgE antibody, as well asan additional Th1 response, with the formation of IgGand IgA antibodies to antigens ofA. fumigatus, as isobserved in patients with ABPA.

3. Host immune responses to Aspergillus

The allergic inflammatory response in ABPA appearsto be quantitatively greater than inAspergillus-sensitivecystic fibrosis patients. In the model proposed of theimmunopathogenesis of ABPA(Fig. 1), A. fumigatusspores are inhaled into the bronchial airway, where theyare trapped by the luminal mucus, germinate and formmycelia.A. fumigatus mycelia release allergens that are

processed by antigen-presenting cells(APC) bearingHLA-DR2 or DR5 and presented to T-cells within thebronchoalveolar lymphoid tissue(BALT). The T-cellresponse toAspergillus allergens becomes skewedtoward a Th2 CD4q T-cell response, with IL-4, IL-5and IL-13 cytokine synthesis and secretion.

3.1. Th2 CD4q T-cells in ABPA

Several groups have observed T-cell lymphoprolifer-ative responses to crudeAspergillus extractsw29–32x.Subsequently,Aspergillus-specific T-cell responses wereexamined. In these studies, T-cells were stimulated witha crudeAspergillus extract for 48 h, and theAspergillus-stimulated T-cell supernatant was then co-cultured withatopic control B-cells for 10 days. These supernatantsobtained from Aspergillus-stimulated T-cells from ABPApatients enhanced B-cell IgE synthesisw33x. Subsequent-ly, T-cell lines were generated from ABPA patients withanAspergillus allergen, Asp f1w34x. In these studies, T-cell phenotype was exclusively CD4qCD25qHLA-DRq T-cells (Table 1). The cytokine profile of theseT-cells was IL-4q, IFN-gy, i.e. Th2 CD4q T-cells.Indeed, the lymphoproliferative stimulus for Asp f1 T-cell lines was predominantly IL-4-mediated, i.e. anautocrine pattern(Fig. 2). However, atopicAspergillus-sensitive CF patients also developed Th2 CD4q T-cells.Subsequent studies performed by Chauhan and co-workersw35,36x demonstrated that T-cell clones obtainedfrom asthmatic ABPA patients were either Th2(IL-4q, IFN-gy) or Th0 (IL-4q, IFN-gq) T-cells. Further-more, tetanus toxoid-generated T-cell clones were theexpected Th1 phenotype, namely IFN-gq. Thus, theTh2 CD4q T-cell response in ABPA is specific toAspergillus antigens, and not a generalized Th2 cellresponse to all antigens.

3.2. IL-4 cytokine in ABPA

IL-4 plays a critical role in the allergic inflammatoryresponse in ABPA(Fig. 1). IL-4 up-regulates cellularactivity via binding to IL-4 receptor(IL-4R) found ona variety of cells, including B-cells, NK-cells, mastcells, endothelial cells and a subpopulation of T-cellsw37–42x. IL-4 and IL-13 induce IgE isotype switchingof B-cells w43–48x. Although IL-4 is necessary for IgEisotype switching, it is not sufficient. In order for IgEsecretion to occur, a second signal mediated by cell–cell T- and B-cell interactions via CD40L::CD40 andCD28::CD86 ligand–receptor interactions occursw46–58x. IL-4 also induces the low-affinity IgE receptorCD23 and soluble CD23, which augments B-cell IgEsynthesisw46,47,59–64x. In addition, T-cell CD23 andB-cell CD21 cognate ligation also augments B-cell IgEsynthesis. CD86 expression on B-cells is also up-regu-lated by IL-4 in atopic patientsw49–57x. CD86 on B-

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Table 1Asp f1 T-cell lines from patients with ABPA

Study Cystic fibrosis patients Asthmatic patients

1 2 3 4 1 2

Diagnosis of ABPA q q q Atopic q qSerum IgE(IUyml) 3800 1950 2091 634 7000 450

Phenotype (%)a

CD3q 97 99 100 100 99 97CD4q 100 99 98 100 100 100CD8q 0 0 0 0 0 0CD25q 96 99 97 97DRq 91 100 97 95TCRabq 99 99 100 99CD45ROq 100 100CD23qCD3q 1.3 0.7 1.3 1.6

Lymphoproliferationb

Asp f1 (cpm) 37481 16219 24481 57168 9644(SI) (21.0) (3.3) (20.8) (4.3) (11.4)Tetanus toxoid(cpm) y1137 1009 27 10712 278(SI) (0.4) (0.4) (1.0) (1.6) (1.3)

Cytokine synthesis supernatantc

IL-4 (pgym)l 300 70000 6600 13800 16600IFN-g (pgyml) -100 1200 -100 -100 -100IL-2 (Uyml) ND ND -300 ND -300

From Knutsen et al.w34x.Analyzed by flow cytometry using FITC or PE labeled monoclonal antibodies.a

For lymphoproliferation assay of Asp f1 T-cell lines, the cell lines were rested, then incubated with media, Asp f1 or tetanus toxoid for 72b

h. Counts per minute(cpm) are net cpm calculated as stimulated minus media.For cytokine synthesis of Asp f1-stimulated T-cell lines, the cell lines were rested, then incubated with Asp f1 for 48 h.c

cells is an important co-stimulatory molecule foraugmentation of IgE synthesis. The ligand for CD86 isCD28 on T-cells. In addition, several studies haveobserved CD86 to be critical in promoting Th2 CD4qT-cell responses and cytokine synthesis, eosinophil air-way inflammation and airway hyperresponsiveness afterallergen challenge. Since ABPA is characterized by aheightened Th2 CD4q T-cell response toA. fumigatusallergens and a hyper-IgE state, it is hypothesized thatone reason for this response is increased sensitivity toIL-4 stimulation in ABPA, resulting in increased expres-sion of CD23 and CD86, leading to a positive feedbackamplification mechanism, which also increases Th2CD4q T-cell responses. In recent studies, it wasobserved that the B-cells from ABPA patients weresignificantly more sensitive to IL-4 stimulation com-pared to atopic and non-atopic patients, with up-regula-tion of CD23 and CD86 expressionw65x. Recently, itwas demonstrated that ABPA CF patients had signifi-cantly increased rates of CD23 expression per B-cell toIL-4, but not to IL-13 stimulation compared to non-ABPA CF patients and non-atopic controls(Figs. 3 and4) w66x. Furthermore, ABPA CF patients also hadsignificantly increased CD23 expression on CD86q B-cells compared to non-ABPA CF and control patients(Fig. 3). At day 0 prior to culture, the number of CD23molecules per B-cell and CD86q B-cell were signifi-

cantly elevated in vivo in ABPA CF patients comparedto non-ABPA CF and to non-atopic patients(Fig. 3).Thus, ABPA patients had increased sensitivity to IL-4,but not IL-13 stimulation, with up-regulation of CD23and CD86 expression compared to non-ABPA CFpatients and non-atopic controls, such that ABPA CF)non-ABPA CF4non-atopic patients. We propose thatin ABPA there is a positive amplification feedback loopmechanism of B-cell CD86 and Th2 CD4q T-cell IL-4stimulation.The model proposes thatA. fumigatus growing within

the airways releases high levels of allergens in theairway and lung parenchyma, which in turn produces aheightened and prolonged late-phase allergic inflamma-tory response. Furthermore, ABPA patients develop IgEantibodies to specificAspergillus proteins, Asp f2, Aspf4 andyor Asp f6, whereas atopic patients develop IgEantibodies to Asp f1 andyor Asp f3 w67–72x. It ishypothesized that mycelial formation and secretion ofproteins in ABPA is necessary, suggesting that thecolonization in ABPA is greater than inAspergillus-sensitive atopic patients. This increased exposure toAspergillus allergens occurring in a genetically suscep-tible host then drives the skewed Th2 CD4q T-cell andhyper-IgE responses observed in ABPA. Recently, stud-ies to determine whether there is increased frequency ofTh2 CD4q T-cells in patients with ABPA have been

80 A.P. Knutsen et al. / Journal of Cystic Fibrosis 1 (2002) 76–89

Fig. 2. Asp f1-specific T-cell line was rested for 2–17 days after IL-2 and Asp f1 stimulation(top panel). Supernatant IL-4 concentrationwas measured serially. Significant amounts of IL-4 were synthesizedfor up to 10–14 days of rest. Monoclonal anti-IL-4 antibody inhibitedAsp f1-stimulated lymphoproliferation of antigen-specific T-cell linesin a dose–response manner(bottom panel). Maximum inhibition was84% at 5% anti-IL-4 antibody concentration(vyv) in this experimentand 78% in a second experiment. Anti-IL-2 had little inhibition ofproliferation, indicating an IL-4-dependent autocrine lymphoprolifer-ative response(from Knutsen et al.w34x).

initiated w66x. As observed in Fig. 5, the frequency ofcytoplasmic IFN-gq and IL-4q CD3q T cells inPMA- and ionomycin-stimulated cultures was compa-rable in CF patients with ABPA compared to non-ABPACF patients, indicating that there was not a skewing ofTh2 CD4q T-cell responses(Fig. 5a). Interestingly,

IFN-gq CD3q T-cells were significantly decreased inboth ABPA and non-ABPA CF patients compared tonon-atopic controls. When antigen-specific frequency ofTh1 and Th2 responses were evaluated, a differentpicture emerged. The frequency of IFN-gq CD3q T-cells in tetanus toxoid-stimulated cultures was similar inABPA and non-ABPA CF patients and was decreasedcompared to non-atopic controls. However, in Asp f2yf3yf4-stimulated cultures, the frequency of IL-4qCD3q T-cells was significantly increased in ABPA CFpatients compared to non-ABPA CF patients(Fig. 5b).This suggests that there is an increased frequency ofAspergillus-specific Th2 CD4q T-cells in ABPA com-pared to non-ABPA CF patients.

3.3. BALT immune response in ABPA

Since the immune response toAspergillus antigensoriginates in the bronchoalveolar lymphoid-associatedtissue(BALT), investigators have examined bronchoal-veolar immunity. Analysis of the cells obtained frombronchoalveolar lavage fluid(BALF) in ABPA revealsan admixture of alveolar macrophages, eosinophils andlymphocytes similar to that found in asthmaw44,45,73,74x. Eosinophil infiltration predominates, bothin BALF and lung tissue, as is evident on lung biopsyw75x. In addition, eosinophils are activated and havereleased their mediators, such as major basic protein.Thus, eosinophils are a major effector cell causinginflammation. Lymphocytes found in BALF are com-posed of T-, B- and NK cells. The T-cells are anadmixture of CD4q and CD8q T-cells, approximatelyin a 2:1 ratio. Interestingly, we have observed increasedCD23q NK cells and CD23qCD4q T-cells obtainedfrom BALF of ABPA patients, indicating in vivo IL-4stimulation w1x. Recently, in preliminary studies,increased in vivo CD23q expression (;10%) onCD4q T-cells in CF ABPA patients has been observed.The significance of CD23q T-cells is probably T-cellCD23 and B-cell CD21 T::B ligand–counterligand inter-action and augmentation of IgE synthesis. Similarly,CD23q NK cells are an important source of sCD23andyor NK CD23::B cell CD21 interaction, increasingimmunoblast IgE secretion. Recently, we have observedinduction of CD23qCD69qCD4q T-cells inAspergil-lus-stimulated cultures of CF ABPA patients(unpubli-shed results). In addition, the expression of CD23 isextremely elevated on addition of IL-4. The role of thisCD4q T-cell subpopulation in ABPA is unknown, butis under study.

3.4. B-cell responses in ABPA

In ABPA, extremely elevated total serum IgE concen-trations and elevated IgE anti-Aspergillus antibodies arecharacteristic of ABPA. There appear to be quantitative,

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Fig. 3. IL-4 induction of CD23q molecules on CD20q and on CD86q CD20q B-cells. ABPA CF patients had significantly increased numberof CD23 molecules(a) per B-cell and(b) per CD86q B-cell on day 0 compared to non-ABPA CF patients(P-0.01) and non-atopic controlpatients(P-0.01). Following IL-4 stimulation, ABPA CF patients had a significantly increased rate of CD23q expression(a) per B-cell and(b) per CD86q B-cell compared to non-ABPA CF(P-0.01) and non-atopic control patients(P-0.01). Data presented as mean"S.E. (fromKnutsen et al.w66x).

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Fig. 4. IL-13 induction of CD23q molecules on CD20q and on CD86q CD20q B-cells. Following IL-13 stimulation, ABPA CF patients hadno significantly increased rate of CD23q expression(a) per B-cell and(b0 per CD86q B-cell compared to non-ABPA CF and non-atopic controlpatients(P-0.01). Data presented as mean"S.E.(from Knutsen et al.w66x).

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Fig. 5. Th1 and Th2 CD3q T cells. (a) Following PMA and ionomycin stimulation, ABPA CF patients had comparable percentages of IFN-g

and IL-4 CD3q T-cells compared to non-ABPA CF patients. However, both ABPA and non-ABPA CF patients had significantly decreasedpercentages of IFN-g CD3q T-cells compared to normal control subjects(P-0.001,P-0.001 respectively). (b) Following tetanus toxoid andAsp f2y3y4 stimulation, both ABPA and non-ABPA CF patients had significantly decreased IFN-g CD3q T-cells compared to normal controlsubjects(P-0.05,P-0.05, respectively) but not different from each other. However, ABPA CF patients had significantly increased Asp f2y3y4-stimulated IL-4 CD3q T-cells compared to non-ABPA CF patients and normal control subjects(P-0.01,P-0.01, respectively). Data presentedas mean"S.E.(from Knutsen et al.w66x).

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Table 2In vitro IgE synthesis by B-cells of patients with cystic fibrosis and allergic bronchopulmonary aspergillosis

Patient group Serum IgE IgE (pgyml)(IUyml)

B cells alone Autologous BqT

Preformed NetNet

ABPA (5) 4870"836 1754"595 1980"617 675"393STyPPT positive(9) 291"56 566"205 220"112 90"35STyPPT negative(13) 58"22 167"66 13"10 140"98Control (23) – 355"58 76"34 139"39P (ANOVA) -0.01 -0.01 -0.01 -0.05

From Knutsen et al.w33x. Abbreviations: ST, Aspergillus prick skin test; PPT,Aspergillus precipitins. The number of individuals studied is inparentheses. Purified-B cells were cultured for 10 days. The net B-cell IgE synthesis was calculated from IgE concentrations of day 10 minuspreformed day 0 cultures. Preformed day 0 IgE concentration determined by lysis of day 0 B-cells. Data are presented as mean"S.E.M.P valueswere determined by ANOVA.P-0.05 comparing ABPA vs. STyPPT positive and STyPPT negative using the post hoc Sheffe test.

and perhaps qualitative, differences of the B-cell IgEantibody responses in ABPA compared toAspergillus-sensitized non-ABPA atopic patients. The heightenedtotal and specific anti-Aspergillus IgE antibodyresponses have been described by several groupsw4,10,76–80x. In ABPA, there are also increasedamounts of IgG and IgA anti-Aspergillus antibodies,reflective of the Th2 humoral vs. Th1 cellular responseto Aspergillus antigensw81–88x. Although otherAsper-gillus-exposed groups develop IgE, IgG and IgA anti-Aspergillus antibodies, there is a quantitative increase inIgE anti-Aspergillus antibodies in ABPA patients. Asobserved in Table 2, it was previously reported that B-cells obtained from patients with ABPA spontaneouslysynthesize increased amounts of IgE in vitro comparedto Aspergillus-sensitized non-ABPA patients groups,indicating in vivo activation of IgE immunoblastsw33x.In recent studiesw66x, we have observed increasedCD23q CD86q B-cells in ABPA that probablyaccounts for this previous observation and for this hyper-IgE statew65x. Furthermore, Greenberger and Pattersonw77x demonstrated that specific anti-Aspergillus fumiga-tus IgE and IgA antibodies are produced within BALT.In contrast, IgG anti-Aspergillus antibodies obtainedfrom BAL fluid were predominantly exudative, derivedfrom the peripheral systemic lymphoid system. Slavinet al. w89x demonstrated lymphoid follicles that stainedwith anti-IgE in a lung biopsy of a patient with ABPA,indicating in vivo IgE-bearing B-cells and immunob-lasts. Greenberger and Pattersonw77x further demonstrat-ed that anti-Aspergillus IgE antibodies comprised onlya small fraction of the total IgE in the peripherallymphoid system. This implies that the CD4q Th2 T-cells have traveled to the systemic immune system andhave activated other clones of B-cells, in addition tothose withAspergillus specificity.

3.5. IL-4 receptor (IL-4R)

As a potential mechanism for increased B-cell IgEsynthesis and secretion, mutations of IL-4 receptor alpha

chain(IL-4Ra) were evaluated in collaboration with DrTalal Chatila. Mutations or polymorphism of IL-4Rahave been identified in atopic individuals with elevatedIgE levels w40–42x. These polymorphisms increase IL-4 and IL-4R interactions, resulting in a gain-of-functionof IL-4Ra that promotes B-cell IgE isotope switching.Subsequently, seven mutations have been identified thatresult in increased IL-4R activityw40,90,91x. In addition,increased IL-4 activity would result in increased expres-sion of other receptors, including CD23 and CD86 onB-cells, eosinophils and NK cells, VLA-4 on eosinophilsand T-cells, VCAM on endothelial cells, CCR-3, CCR-4 and eotaxin secretion(Fig. 1). In preliminary studies,homozygous mutations of IL-4Ra chain in 2y2 ABPApatients and heterozygous mutations in 3y5 atopicpatients and 2y5 non-atopic control patients have beenreported. However, increased sensitivity to IL-4 stimu-lation was observed in ABPA and atopic patients withIL-4Ra chain mutations and wild type. IL-4R is aheterodimer, consisting of IL-4Ra chain and the com-mon gamma chain(Cg) w46–48x. The IL-13R is also aheterodimer, consisting of IL-4Ra and IL-13Ra chains.IL-4 stimulates both the IL-4R and the IL-13R receptors,whereas both IL-4 and IL-13 stimulate the IL-4ayIL-13Ra receptor. IL-13, like IL-4, increases CD23 expres-sion, IgE isotype switching and IgE synthesis. However,IL-13 does not activate and skew Th2 responses. Thereis evidence that IL-4Ra and IL-13Ra interact with theJanus kinase 1(Jak-1), whereas the Cg chain interactswith Jak-3. Upon IL-4 stimulation, IL-4a and Cg chainsundergo phosphorylation by Jak-1 and Jak-3. After IL-13 stimulation, the IL-13Ra chain is phosphorylated byJak-1. Phosphotyrosines in the IL-4Ra and IL-13Raserve as docking sites for the SH2 domains of the signaltransducer and activator of transcription(STAT6) mol-ecule. STAT6 is subsequently phosphorylated by the Jakkinases, when it is then released as a dimer andtranslocates to the nucleus activating IL-4 and IL-13responsive transcription. Our studies demonstratingincreased CD23 expression with IL-4 stimulation sug-

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gest that other mutations or polymorphisms of the IL-4Ra, IL-13a, Jak-1 or STAT6 pathway may exist.

3.6. Eosinophils and mast cells in ABPA

The effector cells responsible for the allergic inflam-matory responses in ABPA are predominantly mast cellsand eosinophils. In our model, whenAspergillus anti-gens cross-link IgE bound to mast cells, mast cellsrelease a variety of mediators, such as histamine, leu-kotrienes and PAF, which induce bronchial smooth-muscle contraction and vascular permeabilityw1x. Anumber of mast cell cytokines, such as leukotriene B4and PAF, are chemoattractant for eosinophils. In addi-tion, chemokines, such as eotaxin, RANTES and MCP-3, derived from a variety of cell types, such as epithelialand phagocytic cells, induce eosinophil chemotaxis andactivation w43–45,92x. Basophil hyperreactivity withincreased histamine release also has been reported inABPA, probably due to IL-4 stimulationw93x. Th2CD4q T-cells secrete the cytokines IL-3 and IL-5 thatpromote bone marrow maturation of eosinophils andactivation of eosinophilsw74,94–98x. Furthermore, IL-4induces expression of VCAM-1 on vascular endothelialcells and its ligand VLA-4 on T-cells and eosinophils.Recent studies have demonstrated selective expressionof the eotaxin receptor, chemokine receptor 3(CCR3),on eosinophils, basophils, and Th2 T-cells, and CCR3expression is up-regulated by IL-4 polarizing conditionsw99,100x. Thus, both chemotactic and cell-surface adhe-sion molecules promote recruitment of Th2 T-cells andeosinophils within the allergic inflammatory sitew44,45,101,102x. Eosinophils possess Fc receptors forIgE, IgG and IgA, and IL-4 also induces increasedexpression of the low-affinity IgE receptor CD23 oneosinophilsw74x. In addition, IL-4 and IL-5 induce Fcreceptors for IgA w74,103x. In ABPA, significantamounts ofAspergillus-specific IgA antibodies are pro-duced within BALT and are present within the bronchialmucus. Thus, both IgE and IgA anti-Aspergillus anti-bodies bind to their Fc receptors on eosinophils andtrigger mediator release when they engage allergenw74,104x. When eosinophil-bound IgE, IgA and IgG arecross-linked byAspergillus antigens, the eosinophils aretriggered to secrete inflammatory mediators, such asmajor basic protein and eosinophil-derived neurotoxinw74x.

3.7. HLA-DR restriction in ABPA

Chauhan et al.w35x investigated whether there isunique TCR recognition(T-cell epitopes), TCR-Vb

restriction or HLA-class II restriction that would pro-mote enhanced Th2 responses. Analysis of T-cell epitopemapping has revealed that there are three immunodom-inant regions of the Asp f1 protein in ABPA patients

that are recognized by TCRw35x. Their findings weresimilar to that found in other allergen models. O’Hehirand Lamb’s group has evaluated T-cell responses topurified house dust mite allergens. In their model, T-cellclones were generated from atopic and non-atopic indi-viduals w105x. Significantly, the T-cell clones from non-atopic individuals proliferated in response to allergenstimulation, but did not support IgE synthesis, whereasthe T-cell clone from atopic patients did. Furthermore,TCR epitope mapping studies revealed a limited numberof epitopes reacting with TCRw106,107x, TCR Vb

restriction or usagew108,109x, and HLA class II restric-tion w109x. Four major Vb chains, Vb 3, 6, 13 and 14,react to Asp f1. This will allow for evaluation of whethermutations of the epitope might alter the T-cell cytokineandyor lymphoproliferative responses for potentialimmunotherapy of ABPA. Recently, Chauhan and co-workersw35,36x identified that there is HLA-DR2(splitinto HLA-DR15 and DR16) and HLA-DR5 restrictionin patients with ABPA. Furthermore, within HLA-DR2and HLA-DR5, there are restricted genotypes. In partic-ular, HLA-DRB1*1501 and 1503 were reported to pro-vide high relative risk. On the other hand, 40–44% ofnon-ABPA atopicAspergillus-sensitive individuals havethe HLA-DR2 andyor DR5. Further studies indicatedthat the presence of HLA-DQ2(especially DQB1*0201)provided protection from the development of ABPA.These results are similar to those found with purifiedhouse dust mite allergensw110–112x. Thus, certaingenotypes of HLA-DR2 and DR5 may be necessary, butnot sufficient to cause ABPA. Furthermore, Chauhan etal. w36x demonstrated that Asp fl allergen has lowaffinity of binding to HLA-DR. This is consistent withTh2 T-cell response previously reported by others, inthat strong antigen HLA-DR-Ag-TCR affinity bindingfavors a Th1 cellular response and low-affinity bindingfavors a Th2 humoral responsew111–115x.

3.8. CFTR mutations in ABPA

Since ABPA is found in highest incidence in atopicCF patients, Miller et al.w116x examined CFTR muta-tions in asthmatic ABPA patients. Six of 11 patients hadmutations of the CFTR gene. Clearly, there wasincreased frequency of heterozygous mutations of theCFTR gene in these asthmatic patients. It has beenhypothesized that, in CF, the abnormal mucus promotesthe trapping ofAspergillus spores within the bronchialairway, permitting and perhaps promotingAspergillusmycelia growth. The significance of the heterozygousCFTR mutation in asthmatic patients is unclear as tothe mucus properties of these asthmatics. Thus, theabnormal mucus may allow increasedAspergillus colo-nization within the bronchial airway of CF and asth-matics, and, in a genetically susceptible individual,stimulate a Th2 T-cell response and subsequent ABPA.

86 A.P. Knutsen et al. / Journal of Cystic Fibrosis 1 (2002) 76–89

Table 3Summary of immunopathogenesis of ABPA

≠ Th2 CD4q T-cell response toAspergillus fumigatusBALT mucosal immune response≠ IL-4, IL-13, IL-5≠ IgE total and anti-Aspergillus antibodies≠ IgA and IgG anti-Aspergillus antibodies≠ Eosinophil BALT and peripheral≠ VLA-4 and VCAM integrinsMigration of eosinophils and lymphocytes into inflammatory lesions

≠ Th2 Af-specificHLA-DR2 (DR15, DR16) and HLA-DR5 restrictionGenotype restriction(DRB1*1501 and 1503)Low-affinity bindingPresence of HLA-DQ2 is protective(DQB1*0201)

TCR-Vb restriction(Vb3, b6, b13, b14)≠ IL-4 activity

≠ IL-4Ra homozygousb chain mutations(preliminary data)Mast cell degranulationBasophil hyperreactivity

≠ sCD25 during ABPA flares≠ T suppressor functionAnergy toA. fumigatus by DTHCFTR heterozygous mutations? effect on mucus properties

In summary, a quantitative increased Th2 CD4q T-cell response toAspergillus in both the BALT andsystemic immune systems characterize ABPA(Table 3).Perhaps key in the immunopathogenesis is that theBALT is exposed to high levels ofAspergillus allergens,perhaps because of abnormal mucus properties due toCFTR mutations. Antigen presentation to T-cells ischaracterized by HLA-DR 2 and DR5 restriction of low-affinity antigen binding. In addition, there is restrictedTCR-Vb usage. Thus, there is an immunogenetic sus-ceptibility to develop ABPA, which resides within theHLA-DR-Ag-TCR signaling of the T-cells toward a Th2CD4q T-cell response. In addition, there may increasesensitivity of T-cells, B-cells, NK cells and eosinophilsto IL-4 stimulation due to mutations of IL-4Ra andyorthe JakySTAT pathway, accounting for the allergicinflammatory response toAspergillus. This leads to apositive feedback amplification loop of Th2 CD4q T-cells™IL-4 synthesis™CD23qCD86q B-cells. If thehypothesis is confirmed, it would suggest therapeutictargeting at IL-4, IL-4R andyor CD86. The results ofthese studies have significance for the atopic state ingeneral: they suggest that the airway changes observedin ABPA are an example of allergen-induced allergicinflammation airway remodeling observed in asthmaticpatients.

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