Lymphocyte Interactions with the Vascular Endothelium in Systemic Sclerosis BASHAR KAHALEH, MD

L ymphocytes interact with the vascular endothe- lium in health and in disease. This interaction is documented substantially by in vivo and in vitro studies.* Recent studies have focused on the def-

inition of pathways for this interaction in the process of T-cell maturation when the primitive T-cell precursors reach the thymus, in lymphocytes homing to lymph nodes, and in memory T-cell recruitment to sites of tissue inflammation. The vascular endothelium facilitates this interaction by expressing surface molecules that interact congenially with corresponding ligands on the enticed lymphocytes. This process should, and usually does, leave the endothelium intact and ready to perform the next expected endothelial functional assignment. None- theless, only in scleroderma (SSc) does this cellular inter- active process leave the vascular endothelium in a state of functional and structural disarray. The chronology of en- dothelial dysregulation in SSc is beyond the scope of this review; still, it is important to remember that studies have described evidence of vascular damage and dysfunction in SSc, including ultrastructural and functional endothe- lial changes in association with alteration in endothelial cell (EC) products, “f-VIII-ag and ACE” 2-4 and evidence for platelet activation and release of platelet-specific pro- teins.5

Recent studies emphasized the fact that mononuclear cells are seen early in SSc around the small blood vessels, in association with signs of endothelial injury and en- hanced mRNA expression of Type I and III collagen by the surrounding fibroblasts.6 Mononuclear cells may in- fluence vascular and fibrotic events in SSc by direct cell -

From the Division of Rheumatology, Medical College of Ohio, Toledo, Ohio.

Address correspondence to Bashar Kahaleh, MD, Professor of Medicine and Chief Division of Rheumatology, Medical College of Ohio, P.O. Box 10008, Toledo, Ohio 43699-0008.

Supported by a grant from the Scleroderma Federation/United Sclero- derma Foundation Collaborative Research Fwding Program.

0 1994 by Elsevier Science Inc. l 0738-081x/94/$7.00

cell interaction and by the release of soluble mediators. The trigger mechanism for the cellular immune process is unknown; nonetheless, an antigen-driven mechanism is likely. Moreover, the pronounced EC damage sustained in this disease suggests a vascular nature for the autoanti- gen. Why, in scleroderma, lymphocyte - endothelial in- teraction leads to a devastated endothelial state is the subject of this review. The basic considerations in ap- proaching this question include the role of adhesion mol- ecules in lymphocyte - endothelial interaction, the role of cytokines, the role of endothelial cells as immune costi- mulators, the possibility that endothelial cells are the tar- get for lymphocyte action, and finally, the impact of this interaction on disease expression.

The Role of Adhesion Molecules Recent studies demonstrated an enhanced expression of adhesion molecules in SSc tissues. Thus, an increased endothelial expression of pi integrins has been reported’ and confirmed by other studies.8-10 Moreover, endothe- lial expression of the adhesion molecules, ICAM- and ELAM- 1, has also been described. ELAM-1 and ICAM- 1 are demonstrated by immunostains on EC of microves- sels in SSc skin biopsies taken from patients with rapidly progressive skin disease, while control biopsies were negative. Furthermore, the integrin /31 and 2 were seen expressed on infiltrating lymphocytes, indicating the ac- tivation of the integrin-ICAM-l/ELAM-1 pathway in lesional scleroderma.7

A preferential enhanced expression in the acute stages of SSc and a less intense expression in the chronic and atrophic phase of the disease are described,9 suggesting a causal relationship in disease pathogenesis. The dynamic nature of lymphocyte interaction with EC supports this observation. A sequential study of the pathological changes in the perivascular spaces in 60 SSc and 25 con- trol subjects recognized functional and structural endo-

361

Clinics in Dermatology 1994;12:361-367

362 KAHALEH

Table 1. Lymphocyte Binding to EC

Subject Scleroderma*

1 38 2 29 3 24 4 28 5 41 6 46

Control*

8.5 7 9.5

13 10

Mean 34 ?I 8.5 9.6 -c 2.5

Data expressed as number of lymphocytes bound per cell (EC). ‘Source of lymphocytes: EC is human umbilical vein (HWEQ.

thelial aberration with subendothelial edema as the first anomaly, followed by platelet aggregation and lympho- cyte migration of both the CD4+ and CD8+ subsets in association with additional signs of endothelial injury. Tissue fibrosis and decreased intensity of inflammation were recognized at later steps in the pathologic progres- sion.‘O

The expression of adhesion molecules in SSc signifies the utilization of the classic molecular adhesive cascade in the inflammatory process in SSc.

Our preliminary studies demonstrate an enhanced in vitro binding of scleroderma lymphocytes to EC. The mean number of lymphocytes bound to EC was 9.6 + 2.5 (mean + SD) lymphocyte/EC in five controls, and 34 Z!Z 8.5 lymphocytes/EC in six scleroderma patients (Table 1). This binding was not dependent on EC synthesis of new proteins, since both irradiated and cycloheximide- treated EC supported lymphocyte binding to a similar degree. Furthermore, binding was not due to EC expres- sion of the intercellular adhesion molecule (ICAM-l), since addition of antibody to ICAM- did not abolish binding nor did an antibody added to Class II MHC anti- gen. The exact molecular adhesive system governing this observation is not yet determined. It is important to ap- preciate the complex nature of a cell adhesion system in order to understand this aspect of SSc pathogenesis.

A rapidly expanding body of data disclosed the struc- ture, function, and expression of endothelial surface mol- ecules which support cellular adhesion.” The data indi- cate that the process of cell adhesion is extremely complex, as illustrated by the large number of molecules involved in this process (Table 2). Most of the information is derived from the study of EC in culture. Data obtained in vitro may not be a true reflection of cellular behavior in vivo. Accordingly, cultured EC in the resting state pro- motes lymphocyte adhesions, while unstimulated EC in vivo does not support cell adhesion.12 Thus, it is impor- tant to recognize that there are qualitative and quantita- tive differences between the in vivo and in vitro cellular performance.

It is now clear that the simple expression of an adhe- sion molecule is only a modest step in the process of cell adhesion. Cell adhesion is a complex multistep process that requires a host of regulatory factors determining the specificity of cellular attraction to a given local.13

Different cell types and subsets use different pathways in their interaction with EC; thus, CD4+ cells utilize a different adhesion molecule system than do CD8+ cells.14 The most dramatic difference is between naive T-cells (CD45RA) and memory cells (CD45RO). Memory cells traffic in inflamed tissue and lymph nodes, while naive cells traffic in lymph nodes. This difference in cel- lular placement is attributed to a higher memory cell sur- face expression of LFA-1 and VLA-4 antigens, leading to a higher binding capacity to ICAM-1, VCAM-1, laminin, and fibronectin,15 markers expressed on inflamed EC. In inflammation, dramatic events modify EC biologic func- tion, including an up-regulation of ICAM- and fibro- nectin expressions and induction of VCAM-1 and E- selectin expression. This process is mediated by a variety of cytokines, including interleukin-1 (IL-l), IL-4, tumor necrosis factor (TNF), and interferon y (IF-y). It is interest- ing to note that even ligands used in normal homing of lymphocytes to lymph nodes are used in inflammation because of a structural and functional change in EC that leads to the appearance of “a high lymph node venular EC” -like appearance. This change is partially achieved by EC expression of L-selectin and VLA-4.

The multiplicity of pathways of T-cell interaction may facilitate efficient capturing of cells at once and influence the selection of a specific cell subset. Lymphocytes, unlike polymorphonuclear cells, do not roll on EC, but rapidly stop after several collisions. Selectins L and E and CD44 are the likely candidates for the initial interaction.16 How T-cell activation follows is not known; however, engage- ment of CD3, TCR, CD2, CD7, CD28, and CD31 may lead to the induction and amplification of integrin ex- pression on T-cells. l’ Once expressed, integrins form the glue that mediates the strongest adhesion by interaction of VAL-4-ICAM-1, LFA-1 -ICAM-1, and ICAM-2. Transendothelial migration is poorly understood and needs further studies; integrins may be important in this process. Factors such as platelet-activating factor (PAF) that influence local circulation, T-cell motility, ligand- binding avidity, other cell-cell interaction, exchange of cytoplasmic proteins, and EC release of cytokines can stimulate T-cell transmigration through the endothe- lium.‘*

The expression of adhesion molecules in SSc and in other disease states is not sufficient to induce the disease, as illustrated by the reported expression of the molecules in both involved and clinically uninvolved ~kirt.~~ It is likely that circulating cytokines induce this expression

Clinics in Dermatology KAHALEH 363 1994;12:361-367 LYMPHOCYTE INTERACTIONS WITH THE VASCULAR ENDOTHELIUM

Table 2. Molecules Involved in Lymphocyte Adhesion to EC

1. Selectin L-selectin

E-selectin “ELAM-1” P-selectin “GMP-140”

2. B2 integins CDlla “LFA-1” CD18 A4Bl VLA-4

Expression

PMNs, monocytes

EC

EC, platelets

Lymphocytes

Lymphocytes Lymphocytes Lymphocytes

Target Cell

high EC Lymphocytes PMNs, monocytes Lymphocytes PMNs, monocytes Lymphocytes

EC

EC EC EC

Ligand

$3350

SLeX

~150 SLeX

ICAM- 1

ICAM- VCAM-1 VCAM-1

which, by itself, is not enough to mediate SSc progres- sion. Nonetheless, understanding this system may pro- vide us with a therapeutic opportunity to modify this aspect of the disease and to test the validity of the im- mune hypothesis in SSc pathogenesis.

Effect of Cytokines

Cytokines mediate a large spectrum of biologic effects. Their relevant impact on lymphocyte-EC interaction in- cludes their effect on adhesion molecule induction, MHC antigen expression, and a unique direct effect on EC function.

induction of Adhesion Molecules In vitro studies documented the effect of a variety of cytokines on the induction of adhesion molecule expres- sion. Accordingly, lymphocyte adhesion to EC increases two- to threefold following EC treatment with IL-l, TNF- cy and IF-Y.~O Careful studies demonstrated that different cytokines induce different adhesion molecules on EC and lymphocytes. Thus, VCAM-1, present in small quantity on resting EC, is induced by IL-l, TNF-a, and IL-4. ICAM-1, also present on resting EC, is up-regulated by IL-l, TNF-cu, and IF-), whereas ICAM- is not regulated by cytokines. *l Moreover, IL-8 induces /32-integrin on leukocytes, macrophage inflammatory protein-l (MIP-1) induces pl-integrin expression on T-cells, and PAF in- duces E-selectin expression. ** IL-l and TNF-a! activated EC bind memory T-cells of both CD4 and CDS pheno- type through expression of VAL-4 on T-cells and VCAM- 1 on EC, with a minor contribution by ELAM-1 and ICAM- .23

Investigations demonstrated the presence of all the influential cytokines in SSc sera.24,25 However, since re- leased cytokines have a very short half-life and can drift

away in the bloodstream after local release, they have to be immobilized on the vascular EC to be effective in me- diating the interaction with circulating lymphocytes. This immobilization is achieved by cytokine attachment to en- dothelial proteoglycans, including CD44.26 The differen- tial expression of various proteoglycans on EC in differ- ent pathologic conditions may be essential for the specificity of a certain lymphocyte subset attraction to tissue through specific immobilized cytokine on EC. This important aspect of EC-lymphocyte interaction has not been explored in SSc. A better understanding of EC regu- lation of lymphocyte trafficking may explain the peculiar differential vascular bed involvement in SSc and, ulti- mately, the various patterns of disease presentation.

Induction of MIX Antigens T-cells recognize antigens after they are presented by an- tigen-presenting cells in association with self-MHC mol- ecules. Thus, CD8+ cells recognize endogenous antigens that are bound to Class I MHC, while CD4+ cells recog- nize proteins in association with Class II MHC. A variety of cytokines up-regulate Class I expression, including IL- 1, TNF-a, LT, and the interferons (IFN) (Y, j3, and y. Still, IFN-y is the only well-characterized cytokine that stimu- lates Class II MHC antigen expression.27 IFN-y induces the expression of Class II antigen in a coordinated fashion of DR, DQ, and DP. Impairment in INF-), production in SSc has been reported. *Key Decreased IF-y production may enhance tissue fibrosis and support the rationale for its use in therapy; still, impaired IFN-y production may contribute to the perpetuation of immune reactions, be- cause it can terminate immune processes in certain sys- tems. Impaired IFN-), production is reported by all inves- tigators who studied this cytokine in SSc; yet several studies in the past, as well as a recent study,3o docu- mented expression of Class II MHC antigens in SSc. Since

364 KAHALEH Clinics in Dermatology 1994;12:361-367

IFN-1/ is the most potent mediator of HLA Class II induc- tion, it follows that either there is a difference between the in vitro and in vivo environments for INF-y produc- tion or another cytokine may be responsible for the in vivo expression. This question is relevant not only to the immune pathogenesis of SSc, but also to other autoim- mune rheumatic disorders in which a similar impairment in IFN-7 synthesis is noted.

Cytokine Modulation of EC Function The past two decades have witnessed dramatic advances in our understanding of the dynamic nature of the vascu- lar endothelium and the multiplicity of the biological functions performed by endothelial cells. It is now clear that EC not only interact, but also participate in the func- tion of the immune system, in the coagulation and fibri- nolytic system, in regulation of vascular permeability, and in the control of vascular tone and substantially in- fluences surrounding cell function and growth behavior.

EC undergo dramatic changes at the sites of inflam- matory infiltration characteristic of T-cell-mediated re- sponses. This alteration in EC appearance has long been recognized and considered a passive phenomenon. These changes consist of changes in EC morphology, “swollen EC,” and of evidence for increased metabolic activity. Now we know that EC are essential participants in the inflammatory process, as represented by the diverse spectrum of EC biologic function. Nonetheless, EC un- dergo substantial modifications in this interactive affair. These changes are mediated principally by the released cytokines. The outcome of this effect is designated “EC activation,” in which EC undergo a major shift in meta- bolic and biologic function, leading to a switch in EC functional profile from an anticoagulant to a procoagu- lant, from a promotor of vasorelaxation to booster of va- sospasm, and from anti-inflammatory to a proinflamma- tory organ. 31 Moreover, changes in EC shape with opening of the endothelial gap junctions result in in- creased vascular permeability. These pathologic changes can be attained by an increase or decrease in normal con- stitutive functions of the endothelium or by induction of new functions and molecules. The end result is a shift in the profile of thebiologic function of endothelium toward a pathologic profile. This shift can be subtle, as best illus- trated in the chronic connective tissue disorders, or frank and acute, as seen in adult respiratory distress syndrome. An example of this effect is depicted by the effect of IL- 1 o and /?, TNF-a, LT and IFN-), on EC morphology, where the cells change from a polygonal contact-inhibited mor- phology to elongated fibroblasticliie cells overlapping and spindle-shaped. 32 This is accomplished by rear-

rangement of actin filaments and disappearance of fibro- nectin from the EC matrix.

EC activation benefits the host by facilitating the de- velopment of cell-mediated immunity; however, when the process is intense or chronic, or when EC become the target, these changes may become detrimental to the host, as may be the case in SSc. An example of a detrimental effect of EC activation on the host is seen in Kawasaki syndrome, in which IFN-y induces EC antigen expression which is recognized by an IgM antibody; yet another EC antigen is induced by IL-l and recognized by IgM and IgG antibodies. Both antibodies activate the complement cascade, leading to EC lysis.33

Our understanding of the harmful effect of prolonged uncontrolled EC participation in the immune process is critical. In SSc, prolonged EC activation may perpetuate the disease, encourage intravascular coagulation, and lead to an extensive interstitial changes.

EC as an Immune Costimulator

Endothehal cell immunologic properties are enormous; however, their impact on lymphocyte function includes their ability to present antigens and to stimulate T-cell activation.

EC as an Antigen-Presenting Cell

All cell types are capabIe of presenting antigens in associ- ation with Class I MHC, whereas only a limited numbers of specialized cells present antigens in association with Class II MHC. EC spontaneously express Class I antigens in vitro, but Class II antigens are expressed only after stimulation with cytokines.

In vivo, human arterial EC express Class II antigens during the process of inflammation, as seen in Kawasaki disease and in allograft rejection.34

Moreover, EC can stimulate proliferation of resting allogenic T-cells from a sensitized host after addition of antigen, while dermal fibroblasts and other cell types ca- pable of expressing Class II are unable to perform this task, indicating that EC are capable of processing the antigen.35

The fate of EC in the process of antigen presentation in vivo is not known. Can there be vast EC damage follow- ing EC presentation of an antigen to cytotoxic T-cells? This aspect of EC biologic function may be quite relevant to SSc pathogenesis and should be carefully evaluated.

Stimulation of T-cell Activation Endothelial cells amplify T-cell responses to antigens and to polyclonal cell activators. Thus, EC augment IL-2 syn-

Clinics in Dermatology KAHALEH 365 2994;22:361-367 LYMPHOCYTE INTERACTIONS WITH THE VASCULAR ENDOTHELIUM

Table 3. 3H-TdR, Uptake by Lymphocyte Cultured with irradiated EC

Control

Spontaneous Cultured with EC

n=4 Scleroderma

237+33 826f102

1 1185f131 31981k162 2 1408t406 8174It193 3 627f31 4479 2340 4 814-+182 3218zk420

Data presented as mean +SD of quadruplicate experiments.

thesis by PHA-stimulated CD4+ cells or by an anti-CD3 antibody-stimulated PBM. 36 This stimulation requires cell-cell interaction in which the stimulatory signal is shown to be an EC surface l&and, since paraformalde- hyde-fixed EC can provide the stimulatory activity. Our preliminary studies demonstrate an enhanced SSc lym- phocyte proliferation in culture with irradiated EC, indi- cating an amplification of EC stimulatory mechanisms (Table 3).

The exact nature of the unique EC stimulatory signal is not known; however, antibodies to LFA-3 (CD-58) or CD-2 partially inhibit the EC effect.37 Moreover EC in- crease c-fos mRNA levels in PHA-stimulated T-cells in- dependent of LFA-3, indicating the presence of a second stimulatory signal. 38 EC-derived cytokines IL-1 and 6, released upon activation of EC with IL-l, TNF-a, or LT, are T-cell stimulators in certain settings. Furthermore, EC play a major role in lymphocyte recruitment in a tissue- specific manner, convert T-cells to a motile form, and help in the extravasation of selected cell types in mecha- nisms that are poorly understood.

Appreciation of the vital position of vascular EC and its integral role in the cellular immune system is essential for understanding the pathogenetic steps of a disease as mysterious as SSc.

EC as a Target of Immune Activity Can EC be the target for the immune-inflammatory ac- tion in SSc? This is a fundamental question that has negli- gible supporting data. Nonetheless, it is important to note that autoimmune mechanisms have a specific target tis- sue and that autoimmunity, in general, seeks to obliterate the target tissue. In SSc the only cell type that sustains the major brunt of immune injury is the vascular endothe- lium. Can this result from recognition of a vascular au- toantigen by the immune system, or is it the result of uncontrolled and failed immune reaction? We propose that a product of activated cytotoxic T-cells (granzyme 1) mediates EC toxicity. 3g This finding indicates the pres-

ence of active recognition of an antigen via Class 1 MHC cytotoxic effector mechanism. The nature of the antigen is not clear; however, a vascular or perivascular makeup for the antigen is likely. Whether the vascular antigen is a true autoantigen or an invading organism is not clear. Lessons learned from AIDS should lead us to reexamine our concept of autoimmunity in connective tissue dis- eases. A casual look at early stage AIDS may find an active autoimmune process not dissimilar to SSc with circulating cytokines and a variety of immune phenom- ena, yet the disease has a known viral trigger.

On the other side is the argument that the disease is predominantly a fibrotic process in which the vascular disease results from a nonspecific and uncontrolled con- stant EC activation. Examples of nonspecific EC injury can be shown in vitro by the toxic effects of a large con- centration of IL- 1 or TNF-cr on cultured EC,32 and in vivo where overexpression of TNF-a and IFN-y in cerebral malaria is associated with the most severe disease. More- over, vascular injury in sepsis is closely related to the load of TNF in this condition. Can the vascular damage in SSc result from a similar nonspecific mechanism? Or is it due to an organized cellular immune process targeting EC? This is a crucial pathogenetic question yet to be resolved.

Impact of EC-Lymphocyte Interaction on Disease Manifestation Impact on Fibroblasts and Tissue Fibrosis

The impact of immunologically activated EC on the biol- ogy of fibroblasts is immense. Activated fibroblasts dis- playing elevated levels of type I and III collagen mRNA in close proximity to EC, which are surrounded by infiltrat- ing lymphocytes, are seen in the early stages of SSC.~ Moreover, capillary EC are shown to express PDGF in SSc biopsies.41 EC expression of other cytokines and growth factors has not been evaluated systematicly; however, it would not be surprising to find enhanced expression of a multitude of mediators, such as IL-l, TNF-a: and most significantly TGF-/I. It can be argued that EC-derived mediators create a chemotactic gradient that attracts fi- broblasts toward the blood vessel, initiates their prolifer- ation, and activates their cellular mechanisms, including collagen synthetic capacity. Another likely source of fi- broblast-activating mediators may result from the en- hanced vascular permeability. Extravasation of plasma proteins to the interstitium, including platelet release products and other plasma cytokines, may generate the optimal milieu for the induction of fibroblast activation. Furthermore, EC expression of surface adhesion mole- cules can entice the trafficking of virtually any and all

366 KAHALEH Clinics in Dermatology 1994;12:362-367

circulating cell types to the interstitium where they may activate fibroblasts. Appreciation of the vital position for the vascular endothelium and its key role in developing fibroblast activation is essential for the understanding of tissue fibrosis.

impact on the lnfimal Arterial Lesion

In the response to injury hypothesis of Ross and asso- ciates,“* endothelial denudation and exposure of the su- bendothelium, with subsequent platelet adhesion and aggregation, is the primary trigger for smooth muscle cell migration, proliferation, and the evolution of intimal hy- perplasia. After years of progressive intimal proliferation, the blood vessel may eventually occlude with a final thrombus formation. A similar sequence of events is likely in SSc.

Immune factors involved in the developing of intimal lesion are multiple, including immune complexes, mast cells products, and cellular immune mechanisms, as best illustrated by the profound arteriolar intimal proliferative lesions in allograft rejection. EC contributions to this pro- cess include increased vascular permeability, enhanced growth factor synthesis (particularly platelet-derived growth factor), and the procoagulant role of an injured endothelium.

Conclusion This review advances many issues in SSc pathogenesis. Data suggest the presence of an antigen-driven process in the disease that leads to lymphocytes activation and en- dothelial damage. The fact that the vascular tree, particu- larly in the microcirculation, is the target tissue in this disease is well established now. It is likely that the im- mune process is relentlessly aimed at the destruction of microvessels, leading to the clinically recognized state of chronic organ ischemia and tissue underperfusion in SSc. Identification of the initial vascular trigger of immune stimulation is fundamental to our understanding of the disease. Nonetheless, understanding the multifaceted properties of immune activation and involvement in dis- ease pathogenesis offers us ample opportunity to develop a multiple-step strategy for therapeutic intervention in this most unfortunate disease. Blocking lymphocyte ad- hesion, EC activation, or therapy for the consequences of the EC biological dysfunction and immunosuppressive therapy may, in time, prove to be effective in sclero- derma.

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