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IMMUNOPATHOGENESIS OF SYSTEMIC SCLEROSIS
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IMMUNOPATHOGENESIS OF SYSTEMIC SCLEROSIS Barbara White, MD
INTRODUCTION
Over the last decade, it has become clear that an activated immune system plays a central role in causing tissue damage in patients with systemic sclerosis (SSc). What remains unknown is whether activation of the immune system is an initial or secondary event in the disease process. This article will review abnormalities of T cells, B cells, and nonspecific inflammatory cells in patients with SSc. The possible consequences of these abnormalities on blood vessel and fibroblast functions will be discussed. ABNORMAL CELLULAR IMMUNITY IN SSc
T cells are important in the development of tissue damage in SSc patients. Activated T cells dominate the inflammatory infiltrates in tissues of SSc patients. They are present very early in the course of the disease. They provide specificity to the immune response and show evidence of selection by antigen in SSc patients. Most importantly, when activated, they regulate functions of many hematopoietic and nonhematopoietic cells, including vascular cells and fibroblasts. Pathologic Evidence of T-cell Involvement in SSc
T cells infiltrate perivascular and interstitial spaces of the skin of patients h5 This infiltration precedes the characteristic findings of small with SSc (Fig. l).57, This work was supported in part by a Career Investigator Award from the Veterans Administration.
From the University of Maryland School of Medicine; and the Veterans Administration Medical Center, Baltimore, Maryland
RHEUMATIC DISEASE CLINICS OF NORTH AMERICA
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VOLUME 22 * NUMBER 4 NOVEMBER 1996
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Figure 1. Mononuclear cell infiltration
in the skin in SSc. Mononuclear cells are seen in the perivascular areas and scattered through the dermis of this patient with early diffuse cutaneous SSc. (Courtesy of Dr. C. L. Kauffman, University of Maryland.)
vessel vasculopathy and fibrosis. Both CD4+ and CD8+ T cells are increased in the skin, but CD4 + T cells are more frequent. Expression of human leukocyte antigen (HLA) class I1 molecules shows these infiltrating T cells have been recently activated. Infiltration with increased numbers of activated T cells also characterizes alveolitis in SSc patients. Patients with active lung disease have increased numbers and percentages of T cells in the interstitiumS2and in bronchoalveolar (BAL) fluids (Fig. 2).22,n*80 CD8 + T cells are increased more than CD4 + T cells in BAL fluids from patients with alveolitis.80The predominance of distinct T-cell subpopulations in the skin (CD4+) and lungs (CD8+) of SSc patients suggests that different T cells may contribute to the disease process in different organs. T-cell subsets in the blood have been studied extensively in SSc patients, with conflicting Absolute lymphocyte counts, absolute numbers and percentages of T cells, and absolute numbers and percentages of CD8+ T cells are reported to be normal or decreased in peripheral blood mononuclear cells (PBMC). Percentages of CD4 + T cells are increased, normal, or decreased. Total numbers of y/6 T cells are decreased in the blood of SSc patients, especially in early disease. Memory CD3 + or CD4 + T cells that are CD29 + or CD45RA are increased or normal in SSc patients, whereas CD29 + CD8 + T cells may be decreased. Evidence of activation of circulating T cells in SSc patients is in-
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Figure 2. Mononuclear cell infiltration in the lungs in SSc. Extensive mononuclear cell infiltration of the interstitium and alveolar spaces, with fibrosis, is seen.
creased expression of HLA-DR molecules, interleukin (1L)-2 receptors (R), and an increased frequency of in vivo hprt gene mutation. Other evidence of T cell activation in SSc patients includes increased serum levels of soluble IL-2R,8 14, 35, 56 CD4, CD8,'4 neopterin; and adenosine deaminase. Increased IL-2R levels may be a marker of internal organ i n v ~ l v e m e n tand ~ ~ may or may not* correlate with disease activity.
The T-cell Repertoire in SSc
Studies to date have found no difference in levels of expression of variable (V)a and Vp gene families in the blood;" skin, or lungsXoof SSc patients, compared to controls. Patients, however, have an increase in numbers and percentage of CD3+ T cells and total y/S T cells that express the V y l gene, in both peripheral blood and BAL fluids.x1CD4 - CD8 - a/P T cells preferentially use Vp5, 7, and 17 gene families in SSc patients.69 Analyses of the T-cell repertoire have been done to look for evidence of antigen-driven expansion of T cells bearing certain V gene families in SSc patients. Oligoclonal T-cell responses to conventional peptide antigens may be characterized by use of a few V gene families, with conservation of T-cell antigen receptor (TCR) V-diversity-joining region nucleotide lengths and DNA sequences. Analyses of TCR junctional region lengths and DNA sequences show two types of T cells are oligoclonal in SSc patients, suggesting evidence of selection by antigen. V y l + y/S T cells in the peripheral blood and tissuesR1and CD8+ T cells in the lungsBoof SSc patients have undergone oligoclonal expansion. Of note, both VS1+ y / S T cells and CD8+ T cells preferentially adhere to fibroblasts.1U6
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In Vivo and In Vitro T-cell Responses to Antigens
In vivo T-cell responses to exogenous antigens appear normal in SSc patients. Patients have a normal response to primary immunization and recall antigens with T-dependent antigens, with normal cutaneous delayed type hypersensitivity and antibody produ~tion.4~ In vitro testing of T cells in SSc patients has yielded contradictory results.R Reports suggest increased, normal, and decreased T-cell proliferative, helper, or suppressor responses to activation with a variety of stimuli. Unfractionated PBMC from SSc patients respond to potential target antigens in SSc, including type I and type IV collagen. NONSPECIFIC INFLAMMATORY CELLS IN TISSUES IN SSc
Cells such as natural killer (NK) cells, macrophages, mast cells, eosinophils, basophils, and neutrophils can contribute to tissue damage through nonspecific inflammation. All of these cell types are present in increased amounts or an activated state in SSc patients, although contradictory reports have also been published." Numbers and activity of NK cells in SSc patients are increased, normal, or decreased. Lymphokine-activated killer cell activity is increased or decreased. Of interest, one patient suffered a flare of her SSc when she received IL-2 and lymphokine-activatedkiller cells to treat a rnalignan~y.~~ Macrophages and Langerhans cells are increased in the skin of SSc patients, especially early in the disease. Patients with interstitial lung disease have increased numbers of macrophages in the interstitium, alveolar epithelial surfaces, and BAL fluids.11, 73 These pulmonary macrophages are activated. BAL fluids from patients with alveolitis contain more fibronectin and alveolar-macrophagederived growth factor than BAL fluids from patients without alveolitis or from 73 Monocytes from SSc patients behave abnormally, with decreased HLA-DR expression in response to interferon y (IFN-y) and reduced ability to stimulate angi~genesis.~~ Mast cells may be increased in the skin or BAL fluids of patients with SSC.~], 70 Degranulation of mast cells provides direct evidence of their activation within the skin of SSc patient^.^" Levels of histamine, tryptase, and hyaluronic acid are elevated in BAL fluids, which are compatible with mast cell activation within the lungs of SSc patients. Myocardial mast cell infiltration or degranulation has been associated with severe cardiac involvement in SSc. The circulating counterpart of mast cells, basophils, appear activated in SSc patients, with increased spontaneous histamine release and increased reactivity or sensitivity to activation signals. Basophil numbers are normal in BAL fluids." Numbers of eosinophils can be increased in the blood, clinically unaffected skin, and BAL fluids from SSc patients.z2* 64, 73 Evidence of eosinophil activation comes from increased major basic protein in the blood and lungs and increased eosinophil cationic protein in BAL fluids.Z2Eosinophilia in BAL fluids is associated with increased epithelial cell permeability in fibrosing alveolitis. Eosinophil activation is infrequent in skin biopsies. Neutrophils are not part of the inflammatory infiltrate in the skin of SSc patients. Neutrophilic infiltration, however, is common in alveolitis in SSc patients, with increased neutrophils in lung biopsies and BAL fluids.", 73 Neutrophils from SSc patients have an increased spontaneous release of hydrogen peroxide, suggesting in vivo activation. Neutrophilic alveolitis may correlate with the development of greater restrictive lung disease over time."
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ADHESION MOLECULE EXPRESSION IN SSc
SSc is characterized by increased expression of adhesion molecules from the selectin, integrin, and immunoglobulin (Ig) gene superfamilies. Increased expression of adhesion molecules indicates activation of cells bearing those molecules and may facilitate homing and retention of lymphocytes and nonspecific inflammatory cells in the tissues. Of note, SSc patients have reduced numbers of circulating T cells capable of adhering to endothelial cells in vitro, which may be the result of depletion of T cells by increased adherence to vascular endothelium in v i v ~ . ~ ~ Expression of E-selectin and P-selectin is increased in SSc patients. E-selectin expression is increased on endothelial cells in the skin and minor salivary glands.20,21, 21 Expression of E-selectin by endothelial cells indicates those cells have been activated to cytokines. E-selectin mediates endothelial cell interactions with neutrophils, granulocytes, monocytes, and CD4 + T cells. Therefore, it is not surprising that endothelial cell expression of E-selectin in the skin from patients with early SSc correlates with the amount of mononuclear cell infiltration.21Soluble E-selectin is increased in the blood of SSc 21 P-selectin is expressed by endothelial cells in skin of SSc patients with early disease,"" and soluble P-selectin is elevated.2o Integrins are transmembrane heterodimeric molecules consisting of an a and p chain. They bind extracellular matrix proteins, which transmit information about the environment to the cell. Integrins also bind receptors of the Ig gene superfamily. Expression of integrins is increased on endothelial cells, perivascular lymphocytes, fibroblasts, and dendritic cells in SSc skin. Endothelial cells have increased expression of p l integrins.z' Dermal mononuclear cells express increased amounts of pl, p2, including lymphocyte function-associated antigen 1 (LFA-l), and p3 integrins,2I with expression greatest in early skin disease. Dermal fibroblasts and dendritic cells from SSc patients have increased expression of p l and p3 integrins.*l Intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-l (VCAM-l) are members of the Ig gene superfamily. Receptor-ligand interactions between ICAM-1 on endothelial cells and LFA-1 on T cells are important in the homing of T cells to peripheral tissues. Similarly, adhesion of T cells to fibroblasts is mediated in part by interactions between ICAM-1 on the fibroblasts and LFA-1 on the T cells. ICAM-1 expression is increased on both endothelial cells and fibroblasts in SSC.~", 2 1 , 52, 71 Greater than normal responsiveness to cytokine stimulation may cause some of the increased expression of ICAM-1 on SSc fibroblasts. Given the increases in both ICAM-1 and LFA-1 expression in SSc, it is not unexpected that SSc skin biopsies with more ICAM1 on the endothelial cells have more pronounced mononuclear cell infiltrates.2n Nor should it be unexpected that SSc fibroblasts bind more T cells than do control fibroblasts.' Of note, circulating ICAM.1 levels are increased in about one third of SSc patients.2nIncreased shedding of soluble ICAM-1 from fibroblasts in SSc may be a contributing f a ~ t o r .Similar ~' to the findings with ICAM-1, VCAM1 is expressed in increased amounts on endothelial cells in the skin,'" and soluble VCAM-1 is elevated in SSc patients with early disease.2" POTENTIAL CONTRIBUTIONS OF T CELLS AND NONSPECIFIC INFLAMMATORY CELLS TO TISSUE DAMAGE IN SSc
Temporal and physical associations suggest that activated T cells and inflammatory cells cause tissue damage in SSc patients. Infiltration of the skin
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with activated mononuclear cells, which are largely T cells, precedes the development of characteristic vasculopathy and fibrosis. Fibroblasts that are producing procollagen mRNA are located next to these mononuclear cell infiltratesM Alveolitis of the lungs, with infiltration with lymphocytes, macrophages, neutrophils, and eosinophils, precedes and predicts the development of pulmonary fibrosis.73Soluble mediators and cellular cytotoxity are the major mechanisms by which cells of the immune system cause tissue damage. Soluble Mediators of the Immune System in SSc
Cells of the immune system secrete many different mediators that can alter functions of vascular cells or fibroblasts. Soluble mediators reported to be present in abnormally high amounts in SSc patients include IL-1, IL-2, IL-4, IL-6, IL8, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-P, platelet derived growth factor (PDGF), granzyme A, and leukotriene B4.n All of these can be made by T cells or other inflammatory cells. Target cells in SSc, including endothelial cells, vascular smooth muscle cells, and fibroblasts, may themselves contribute to the production of some of these mediators. To date, the cellular sources of these soluble mediators in SSc patients have not been fully elucidated. Existing data suggest a T-helper 2 pattern of cytokine production by T cells in blood and BAL fluids from SSc patients, with increased IL-4I3,54 and reduced production of IFN-Y.~~ This is of interest because interleukin-4 stimulates proliferation, chemotaxis, and extracellular matrix production by fibroblasts and increases T-cell adhesion to endothelial cells. It also induces a collapse of vimentin intermediate filaments in endothelial cells, which is the first morphologic abnormality seen in the endothelium in SSc skin.57IL-2 levels are increased in the sera of some SSc patientsM,72 and may correlate with more aggressive skin disease.44 Several proinflammatory cytokines, IL-1, TNF-a, and IL-6, are increased in SSc patients. Among the many properties of IL-1 is the ability to increase proliferation of T and B cells, stimulate prostaglandin E2 (PGE,) and extracellular matrix production by fibroblasts, increase endothelial cell expression of ICAM1, and promote vascular smooth muscle proliferation. Some, but not all, reports suggest serum levels, monocyte produ~tion,3~ and fibroblast production of IL-a or IL-p are increased in SSc. SSc fibroblasts express more IL-1R.38 This may make them more sensitive to IL-1 stimulation, with greater than normal increases in ICAM-1, IL-6, PGE2, and IL-lp.38 TNF-a has pleotrophic effects similar to IL-1. For example, it increases fibroblast proliferation and production of PGE, and IL-p. It also increases endothelial cell expression of E-selectin, ICAM-1, VCAM, and release of endothelin1. TNF-a decreases fibroblast production of type I and I1 collagens, however, although increasing production of collagenase. Some, but not all, reports indicate that serum levels35and production of TNF-a by PBMC35and mon~cytes"'~ are increased in SSc. Expression of TNF-a is increased in the skin of SSc patients?" SSc fibroblasts have normal decreases in collagen production in response to TNF-a, but an abnormal increase in ICAM-1 expression. TNF-a receptors are quantitatively and qualitatively normal on SSc fibroblasts4 IL-6 has a broad range of activities that overlap with IL-1 and TNF-a. Of note, IL-6 may increase function of cytolytic cells because of its ability to induce perforin and serine esterases. Serum levels72and production of IL-6 by PBMC,'O fibroblasts,16and endothelial cells40 may be increased in SSc. Alveolar macrophages from SSc patients produce normal amounts of IL-6 and have normal levels of IL-6R.I" Expression of IL-6 is increased in the epidermis63of SSc patients.
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IL-6 may be responsible for the increased IL-2R expression on PBMC from SSc patients. Transforming growth factor+ is a logical candidate for a cytokine that contributes to tissue damage in SSc. It stimulates collagen production by fibroblasts and vascular smooth muscle cells and induces endothelial cell production of endothelin-1. Endothelin-1 is a potent vasoconstrictor that is increased in sera3’ and BAL fluids7 from SSc patients. Endothelin-1 stimulates collagen production by fibroblasts7,31 Some, but not all, reports find increased TGF-P proteins or mRNAs in the blood,” skin24,42, h7 and BAL fluids’ from SSc patients. TGF-P mRNA colocalizes with proal(1) and proa(II1) collagen mRNAsHand Type VII collagenh7in skin from some SSc patients. The cellular sources of TGF-P remain unknown. The TGF-P in the lungs of patients with alveolitis may come from regenerating alveolar epithelium. SSc fibroblasts make normal amounts of TGF-P in ~ i t r o . ~ ~ When considering the potential role of TGF-P in SSc, it is important to note that Higley et aIz4did not find TGF-P in skin from patients with active diffuse cutaneous SSc, although it was found in skin in later stages of disease and in skin from patients with limited cutaneous SSc. This observation, plus the knowledge that TGF-P binds extracellular matrix, raises the possibility that the increase in TGF-P is secondary to an increase in extracellular matrix in the tissues of SSc patients. Moreover, it is not possible to induce or sustain a SSc-like phenotype in normal fibroblasts with in vitro exposure to TGF-P. SSc fibroblasts may be unusually sensitive to TGF-P, with greater than normal proliferation, synthesis of type-a PDGF receptor, binding of PDGF-BB, and production of connective 79 tissue growth factor.3y, Interleukin-8 is chemotactic for neutrophils, lymphocytes, and endothelial cells. Levels of IL-8 are elevated in the sera”’ and BAL fluids” of SSc patients, as well as on dermal endothelial cells?” IL-8 levels in BAL fluids correlate with the degree of neutrophilia,” suggesting IL-8 contributes to cellular inflammation in SSc patients with alveolitis. Cellular Cytotoxicity
Relatively few studies have been done of cellular cytotoxicity in SSc. Cytotoxic responses to fibroblasts, epithelial cells, and muscle cells have been reported.Iz Granzyme A levels are elevated in the sera of SSc patients.’’ This serine protease, which is released by cytolytic CD8+ T cells as part of the perforin pathway of cytotoxicity, may contribute to endothelial cell death in SSc.” ABNORMAL HUMORAL IMMUNITY IN SSc
T cell-dependent and -independent antigen-specific antibody responses are normal in vivo in SSc patients.45In contrast, hypergammaglobulinemia is very common and autoantibody production is an early, nearly universal finding. There are multiple autoantigen targets in SSc, some of which are disease-specific. Evidence that autoantibodies cause tissue damage in SSc patients is limited. Pathologic Evidence of B-Cell Involvement in SSc
Hypergammaglobulinemia and autoantibodies are found in the sera of SSc patients and increased numbers of plasma cells are seen within their skin17,s7
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and Polyclonal B-cell activation causes increases in IgG, IgA, IgM, and IgE.l7IgG is also increased in BAL fluids of SSc patients, with no correlation to disease activity.” Antinuclear antibodies occur in 90% to 98% of patients with SSc. The isotype is usually IgG, especially the IgG3 subclass, although IgM, IgE, and IgA antinuclear antibodies are also seen.25 Autoantibody production may be an epiphenonemon in SSc. In silica-associated SSc, autoantibodies occur only in patients with SSc and not in individuals with just silicosis.a Thus, the production of the autoantibodies appears to result from events that cause SSc and is not the forerunner of the disease. In spontaneous SSc, titers of antitopoisomerase 1 and anticentromere antibodies remain relatively stable for years, which suggests that these SSc-specific autoantibodies are not involved in disease activity. As another example, an illness with some characteristics of SSc occurs in mice with chronic graft versus host disease. The initiating event is activation of T cells by alloantigens. This leads to secondary production of autoantibodies directed against proteins involved with rRNA transcription (Nor:90 and fibrillarh~),’~ similar to those seen in SSc. Autoantibody Production in SSc
Autoantibody targets that are quite specific for SSc include the self-antigens DNA topoisomerase 1 centromeric proteins,5O RNA polymerases I and 111,26,43, 68 PM-Scl antigenz, and myenteric neurons.28The reasons for specific targeting of these particular proteins are unknown and may have to do with subcellular localization or function of the target antigen. An enormous range of other autoantigens can be targets of autoantibody production in SScn These include endothelial cells, fibroblasts, smooth muscle, granulocytes, red blood cells, platelets, thyroid tissue, salivary gland tissue, neutrophil cytoplasmic antigens, heat shock protein, types I, 111, and IV collagen, IL-6, IL-8, IgG, cardiolipin, FcyR, histones, mitochondria, laminin, single stranded RNA, U3, and U11 nuclear ribonucleoproteins, Th ribonucleoproteins, upstream binding factor (NOR-90), high motility group (HMG)17 nucleosome protein, the Ku antigen, and RNA polymerase 11. Targets of autoantibody production in SSc appear to be presented to the immune system in a native state. Evidence for this is antibody recognition of 49, 69 and active sites, includconformational multiple epit0pes,2~, ing phosphorylation sites, on target molecule^.^, 15, 26, 29, 43, In addition, different components of multiunit complexes may be recognized,’8,43 which suggests the entire complex is processed and presented. The HLA alleles of the patient influence which SSc-specific autoantibodies are produced, perhaps because certain HLA alleles are required to activate those T cells that provide the necessary help for antibody production by B cells. The anticentromere antibody response is tightly associated with HLA-DQ alleles with a polar glycine or tyrosine at position 26 of the HLA-DQP chain.80 In whites, black and Japanese,& the antitopoisomerase 1 antibody response is associated with HLA-DQ alleles with a tyrosine residue at position 30. The HLA-DR60and HLA-DP6f3 chains influence the topoisomerase I autoantibody response. Autoantibodies to PM-Scl correlate with the presence of the HLA-DRB1*0301, DQA1-*0501, DQB1*0201 h a p l ~ t y p e . ~ ~ Homology Between Autoantigen Targets and Viruses
Evidence that molecular mimicry plays a role in the development of autoantibodies in some SSc patients is the homology between some SSc autoantibody
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Table 1. AUTOANTIBODY TARGETS IN SYSTEMIC SCLEROSIS AND
HOMOLOGOUS VIRUSES ~~~~
Autoantibody Target
DNA Topoisomerase 1
PM-Scl antigen U1 snRNP
Fibrillarin (U3 snRNP)
Homologous Viruses
p30QaQ protein from feline sarcoma virus UL70 protein of human cytomegalovirus SV-40 large T antigen Human immunodefiency virus tat protein Herpes simplex virus type I ICP4 protein Herpes simplex virus type I P40 protein Epstein-Barr virus nuclear antigen 1
targets and viruses (Table 1).B-cell epitopes on DNA topoisomerase I include amino acids 121-126 and 741-746, which have homology to the UL70 protein of human cytomegalovirussl and the p30gag protein from feline sarcoma virus,4i respectively. B-cell epitopes on PM-Scl antigen include a 138-amino acid fragment with homology to the nuclear localization signal of SV-40 large T antigen and a molecular signal found in the HIV tat protein.2 A universal B-cell epitope on U1 RNP is similar to herpes simplex virus type 1 ICP4 protein.4yThe carboxy terminus of fibrillarin shares sequence homology with P40, a capsid protein A hexapeptide sequence, GRGRGG, encoded by herpes simplex virus type l.36 in the amino terminus of fibrillarin is shared with Epstein-Barr virus (EBV) nuclear antigen.3hA quarter of SSc patients have weak antibody reactivity to HIV retroviral proteins by Western blots, without signs of direct infection.13 POTENTIAL CONTRIBUTIONS OF HUMORAL IMMUNITY TO TISSUE DAMAGE IN SSc
There is little evidence to date that autoantibodies damage cells or tissues in SSc patients through complement activation or immune complex formation. Many of the autoantibodies do not activate the complement cascade, and serum complement levels are normal. Although immune complexes can be found in the sera of approximately 25% of SSc patients, immune complexes and complement are not typically deposited in tissues. Immune complexes can be identified in BAL fluids from SSc patients, but there is no correlation with disease a ~ t i v i t y . ~ ~ The anti-red cell, antigranulocyte, and antiplatelet antibodies that occur in SSc patients are not usually associated with c y t ~ p e n i a s . ~ ~ Autoantibodies in SSc patients have the potential to activate cells that bear the target autoantigen, although there is no evidence to date of this happening in vivo. An anti-FcyRIII antibody secreted by an EBV-transformed B cell from a SSc patient triggered in vitro release of P-glucuronidase, arylsulfatase, and alkaline phosphatase from ne~trophils.’~ A potential pathologic role for these antibodies might be through triggering neutrophils, NK cells, or macrophages to release cytokines. The ability of SSc antibodies to modify intracellular events in vivo is unexplored. Antibodies against centromeric proteins, topoisomerase 1, and RNA polymerases all inhibit the function of their respective targets in vitro.5,15, 2h, 2y,43,6R This inhibition could have clinical consequences. Jabs et al”] found a correlation between anticentromeric antibodies and aneuploidy in SSc patients. Anticentromeric antibodies could disrupt the functional centromere and allow for malsegregation of chromosomes during mitosis.
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The final mechanism through which antibodies might contribute to tissue damage in SSc is antibody-dependent cellular cytotoxicity. Antibodies from SSc patients are capable of mediating antibody-dependent cellular cytotoxicity of endothelial cells in v i t r ~ .Similar ~ ~ , ~damage ~ in vivo might contribute to the vasculopathy of SSc.
SUMMARY The information outlined above can be used to generate a model of the immunopathogenesis of SSc (Fig. 3). This model includes a susceptible host, with age greater than 25 and female gender being risk factors. The model also includes exposure to exogenous agents, which could be different in different individuals and may include inhaled or ingested chemicals or infectious agents. An early event is T-cell activation, with infiltration in the skin and internal organs. Activation of the T cells is a selective process that appears to be influenced by antigen in SSc patients. The importance of a particular T-cell subpopulation may depend upon the organ involved and the stage of the disease. CD4 T cells predominate in the skin. In contrast, CD8+ T cells are increased in the lungs of patients with alveolitis, where they are oligoclonal, showing evidence of antigen-driven selection. V61 +y/6 T cells are increased in both the blood and lungs of SSc patients and also show evidence of selection by antigen. B cells are activated early, with polyclonal activation leading to hypergammaglobulinemia. SSc-specific autoantibodies target DNA topoisomerase I, centromeric proteins, and RNA polymerases I and 111. Characteristics of autoantibodies in SSc suggest that the target antigens are presented to the immune system as native molecules or even part of a multiunit complex. There is some homology between viruses and autoantibody targets in SSc, which suggests that molecular mimicry may play a role in initiating the antibody response. Many nonspecific inflammatory cells infiltrate the tissues and show evidence of activation. These
+
SUSCEPTIBLE HOST (usually woman > 25 years of age)
IExogenous triggers
B-cell activation
T-CELL ACTIVATION
I \
Autoantidodies, t Ig
Activation of nonsuecific inflammatory cells Inflammatory infiltrates
I
Soluble mediators, cytotoxicity
FIBROBLASTS
ENDOTHELIAL CELLS
FIBROSIS
I
Activation, injury
ISCHEMIC VASCULOPATHY
Figure 3. Model of the immunopathogenesis of SSc.
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include macrophages and monocytes, mast cells, eosinophils, basophils, and natural killer cells. Soluble mediators made by these T cells, B cells, and nonspecific inflammatory cells can activate and damage fibroblasts, endothelial cells, and other vascular cells. The relative importance of the various candidate cytokines, the temporal sequence of their production, and their cellular sources remain largely to be defined. There may be some contribution of direct Tcell cytotoxicity or antibody-dependent cellular cytoxicity to the tissue damage that occurs.
References 1. Abraham D, Lupoli S, McWhirter A, et al: Expression and function of surface antigens on scleroderma fibroblasts. Arthritis Rheum 34:1164, 1991 2. Alderuccio F, Chan EK, Tan EM: Molecular characterization of an autoantigen of PMScl in the polymyositis/scleroderma overlap syndrome: A unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex. J Exp Med 173:841, 1991 3. Alms WJ, James SP, Wigley FM, et al: Cytokine mRNAs produced spontaneously by peripheral blood mononuclear cells and bronchoalveolar lavage cells from patients with scleroderma. Clin Res 40:396A, 1992 4. Berman B, Wietzerbin J: Tumor necrosis factor-a (TNF-a), interferon-a (IFN-a) and interferon-y (IFN-y) receptors on human normal and scleroderma dermal fibroblasts in vitro. J Dermatol Sci 3:82, 1992 5. Bernat RL, Borisy GG, Rothfield NF, et al: Injection of anticentromeric antibodies in interphase disrupts events required for chromosome movement in mitosis. J Cell Biol 111:1519, 1990 6. Briggs D, Stephens C, Vaughan R, et al: A molecular and serologic analysis of the major histocompatibility complex and complement C4 in systemic sclerosis. Arthritis Rheum 36:943, 1993 7. Cambrey AD, Harrison NK, Dawes KE, et al: Increased levels of endothelin-l in bronchoalveolar lavage fluid from patients with systemic sclerosis contribute to fibroblast mitogenic activity in vitro. Am J Respir Cell Mol Biol 11:439, 1994 8. Clements P, Peter J, Agopian M, et al: Elevated serum levels of soluble interleukin 2 receptor, interleukin 2 and neopterin in diffuse and limited scleroderma: Effect of chlorambucil. J Rheumatol 17:908, 1990 9. Corrin B, Butcher D, McAnulty BJ, et al: Immunohistochemical localization of transforming growth factor-pl in the lungs of patients with systemic sclerosis, cryptogenic fibrosing alveolitis, and other lung disorders. Histopathology 24:145, 1994 10. Crestani B, Seta N, De Bandt M, et al: Interleukin-6 secretion by monocytes and alveolar macrophages in systemic sclerosis with lung involvement. Am J Respir Crit Care Med 149:1260, 1994 11. Crestani B, Seta N, Palazzo E, et al: Interleukin-8 and neutrophils in systemic sclerosis with lung involvement. Am J Resp Crit Care Med 150:1363, 1994 12. Currie S, Saunders M, Knowles M: Immunologic aspects of systemic sclerosis: In vitro activity of lymphocytes from patients with the disorder. Br J Dermatol 84:400, 1970 13. Dang H, Dauphinee MJ, Tala1 N, et al: Serum antibody to retroviral gag proteins in systemic sclerosis. Arthritis Rheum 341337, 1991 14. Degiannis D, Seibold JR, Czarnecki M, et al: Soluble interleukin-2 receptors in patients with systemic sclerosis. Clinical and laboratory correlations. Arthritis Rheum 33:375, 1990 15. Eng WK, Pandit SD, Sternglanz R: Mapping of the active site tyrosine of eukaryotic DNA topoisomerase I. J Biol Chem 264:13373, 1989 16. Feghali CA, Bost KL, Boulware DW, et al: Mechanisms of pathogenesis in scleroderma: I. Overproduction of interleukin-6 by fibroblasts cultured from affected skin sites of patients with scleroderma. J Rheumatol 19:1207, 1992
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17. Fleishmajer R, Perlish JS, Reeves JRT: Cellular infiltrates in scleroderma skin. Arthritis Rheum 20975,1977 18. Ge Q, Wu Y, Trieu EP, et al: Analysis of the specificity of anti-PM-Scl autoantibodies. Arthritis Rheum 371445, 1994 19. Gelpi C, Martinez MA, Vidal S, et al: Autoantibodies to a transfer RNA-associated protein in a murine model of chronic graft vs. host disease. J Immunol 152:1989, 1994 20. Gruschwitz MS, Hornstein OP, Von Den Driesch P: Correlation of soluble adhesion molecules in the peripheral blood of scleroderma patients with their in situ expression and with disease activity. Arthritis Rheum 38:184, 1995 21. Gruschwitz M, Von Den Driesch P, Kellner P, et al: Expression of adhesion proteins involved in cell-cell and cell-matrix interactions in the skin of patients with progressive systemic sclerosis. J Am Acad Dermatol 27169, 1992 22. Gustafsson R, Fredens K, Nettelbladt 0, et a1 Eosinophil activation in systemic sclerosis. Arthritis Rheum 34:414, 1991 23. Hebbar M, Lassalle P, Janin A, et al: E-Selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis. Role of resident mast cell-derived tumor necrosis factor a.Arthritis Rheum 38:406, 1995 24. Higley H, Persichitte K, Chu S, et al: Immunocytochemical localization and serologic detection of transforming growth factor p 1. Association with type I procollagen and inflammatory cell markers in diffuse and limited systemic sclerosis, morphea, and Raynaud’s phenomenon. Arthritis Rheum 37278,1994 25. Hildebrandt S, Weiner E, Seneca1 JL, et al: The IgG, IgM, and IgA isotypes of antitopoisomerase I and anticentromere antibodies. Arthritis Rheum 33:724, 1990 26. Hirakata M, Okano Y, Pati U,et al: Identification of autoantibodies to RNA polymerase 11. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerase I and 111. J Clin Invest 912665, 1993 27. Holt CM, Lindsey N, Moult J, et al: Antibody-dependent cellular cytotoxicity of vascular endotheliurn: Characterization and pathogenic associations in systemic sclerosis. Clin Exp Med 78:359, 1989 28. Howe S, Eaker EY, Sallustio JE, et al: Antimyenteric neuronal antibodies in scleroderma. J Clin Invest 94:76, 1994 29. Imai H, Fritzler MJ, Neri R, et al: Immunocytochemical characterization of human NOR-90 (upstream binding factor) and associated antigens reactive with autoimmune sera. Two MR forms of NOR-90/hUBF autoantigens. Mol Biol Rep 19:115, 1994 30. Jabs EW, Tuck-Muller CM, Anhalt GJ, et al: Cytogenetic survey in systemic sclerosis: Correlation of aneuploidy with the presence of anti-centromere antibodies. Cytogenet Cell Genet 63:169, 1993 31. Kahaleh MB: Endothelin, an endothelial-dependent vasoconstrictor in scleroderma: Enhanced production and profibrotic action. Arthritis Rheum 34978, 1991 32. Kahaleh MB, LeRoy EC: Interleukin-2 in scleroderma: Correlation of serum level with extent of skin involvement and disease duration. Ann Intern Med 110:446, 1989 33. Kahaleh MB, Yin T: The molecular mechanism of endothelial cell (EC) injury in scleroderma (SSc): identification of granzyrne 1 (a product of cytolytic T-cell) in SSc. Arthritis Rheum 33:S67, 1990 34. Kahari V-M, Sandberg M, Kalimo H, et al: Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J Invest Dermatol 90:664, 1988 35. Kantor TV, Friberg D, Medsger TA Jr, et al: Cytokine production and serum levels in systemic sclerosis. Clin Immunol Immunopathol 65:278, 1992 36. Kasturi KN, Hatakeyama A, Spiera H, et al: Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes. J Exp Med 181:1027, 1995 37. Kawaguchi Y, Harigai M, Hara M, et al: Increased interleukin-1 receptor, type I, at messenger RNA and protein level in skin fibroblasts from patients with systemic sclerosis. Biochem Biophys Res Cornmun 184:1504, 1992 38. Kawaguchi Y, Harigai M, Suzuki K: Interleukin-1 receptor on fibroblasts from systemic sclerosis patients induces excessive functional responses to interleukin lp. Biochem Biophys Res Commun 190:154, 1993
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39. Kikuchi K, Kadono T, Ihn H, et al: Growth regulation in scleroderma fibroblasts: Increased response to transforming growth factor-p 1. J Invest Derm 105:128, 1995 40. Koch AE, Kronfeld-Harrington LB, Szekanecz Z , et al: In situ expression of cytokines and cellular adhesion molecules in the skin of patients with systemic sclerosis. Their role in early and late disease. Pathobiology 61:239, 1993 41. Koch AE, Litvak MA, Burrows JC, et al: Decreased monocyte-mediated angiogenesis in scleroderma. Clinical Immunol Immunopathol 64:153, 1992 42. Kulozik M, Hogg A, Lankat-Buttgereit B, et al: Colocalization of transforming growth factor p2 with al(1) procollagen mRNA in tissue sections of patients with systemic sclerosis. J Clin Invest 86:917, 1990 43. Kuwana M, Kaburaki J, Mimori T, et al: Autoantibody reactive with three classes of RNA polymerases in sera from patients with systemic sclerosis. J Clin Invest 91:1399, 1993 44. Kuwana M, Kaburaki J, Okano Y, et al: The HLA-DR and DQ genes control the autoimmune response to DNA topoisomerase I in systemic sclerosis (scleroderma). J Clin Invest 92:1296, 1993 45. Lupoli S, Amlot P, Black C: Normal immune responses in systemic sclerosis. J Rheumato1 17323, 1990 46. Marks RM, Czerniecki M, Andrews BS, et al: The effects of scleroderma serum on human microvascular endothelial cells. Induction of antibody-dependent cellular cytotoxicity. Arthritis Rheum 31:1524, 1988 47. Maul GG, Jimenez SA, Riggs E, et al: Determination of an epitope of the diffuse systemic sclerosis marker antigen DNA topoisomerase 1: Sequence similarity with retroviral p30gag protein suggests a possible cause for autoimmunity in systemic sclerosis. Proc Natl Acad Sci USA 86:8492, 1989 48. McHugh NJ, Whyte J, Harvey G, et al: Anti-topoisomerase I antibodies in silicaassociated systemic sclerosis. Arthritis Rheum 37:1198, 1994 49. Misaki Y, Yamamoto K, Yanagi K, et al: B-cell epitope on the U1 snRNP-C autoantigen contains a sequence similar to that of the herpes simplex virus protein. Eur J Immunol 23:1064, 1993 50. Moroi Y, Peebles C, Fritzler MJ, et al: Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 771627, 1980 51. Muryoi T, Kasturi KN, Kafina MJ, et al: Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes. J Exp Med 175:1103,1992 52. Needleman BW: Increased expression of intercellular adhesion molecule 1 on the fibroblasts of scleroderma patients. Arthritis Rheum 33:1847, 1990 53. Needleman BW, Choi J, Burrows-Mezu A, et al: Secretion and binding of transforming growth factor-p by scleroderma and normal dermal fibroblasts. Arthritis Rheum 33:650, 1990 54. Needleman BW, Wigley FM, Stair RW: Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor a, and interferon-? levels in sera from patients with scleroderma. Arthritis Rheum 35:67, 1992 55. Oddis CV, Okano Y, Rudert WA, et al: Serum autoantibody to the nucleolar antigen PM-Scl. Clinical and immunogenetic associations. Arthritis Rheum 35:1211, 1992 56. Patrick MR, Kirkham BW, Graham M, et al: Circulating interleukin 1 p and soluble interleukin-2 receptor: Evaluation as markers of disease activity in scleroderma. J Rheumatol 22:654, 1995 57. Prescott RJ, Freemont AJ, Jones CJP, et al: Sequential dermal microvascular and perivascular changes in the development of scleroderma. J Pathol 166:255, 1992 58. Puett DW, Fuchs HA: Rapid exacerbation of scleroderma in a patient treated with interleukin-2 and lymphokine-activated killer cells for renal cell carcinoma. J Rheumato1 21:752, 1994 59. Query CC, Keene J D A human autoimmune protein associated with U1 RNA contains a region of homology that is crossreactive with retroviral p30gag antigen. Cell 51:211, 1988 60. Reitamo S, Remitz A, Varga J, et al: Demonstration of interleukin-8 and auto-antibodies to interleukin-8 in the serum of patients with systemic sclerosis and related disorders. Arch Dermatol Res 129:189, 1993
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61. Reveille JR, Durban E, MacLeod-St. Clair MJ, et al: Association of amino acid sequences in the HLA-DQB1 first domain with the antitopoisomerase I autoantibody response in scleroderma (progressive systemic sclerosis). J Clin Invest 90:973, 1992 62. Reveille JR, Owerbach D, Goldstein R, et al: Association of polar amino acids at position 26 of the HLA-DQBI first domain with the anticentromere autoantibody response in systemic sclerosis (scleroderma). J Clin Invest 89:1209, 1992 63. Romero LI, Pincus SH In situ localization of interleukin-6 in normal skin and atrophic cutaneous disease. Int Arch Allergy Immunol 9944, 1992 64. Rossi GA, Bitterman PB, Rennard SI, et al: Evidence for chronic inflammation as a component of the interstitial lung disease associated with progressive systemic sclerosis. Am Rev Respir Dis 131:612, 1985 65. Roumm AD, Whiteside TL, Medsger TA, Jr, et al: Lymphocytes in the skin of patients with progressive systemic sclerosis:Quantification, subtyping, and clinical correlations. Arthritis Rheum 27645,1984 66. Rudnicka L, Majewski S, Blasczczyk M, et al: Adhesion of peripheral blood mononuclear cells to vascular endothelium in patients with systemic sclerosis (scleroderma). Arthritis Rheum 35:771, 1992 67. Rudnicka L, Varga J, Christian0 AM, et al: Elevated expression of type VII collagen in the skin of patients with systemic sclerosis. Regulation by transforming growth factorp. J Clin Invest 93:1709, 1994 68. Satoh M, Kuwana M, Ogasawara T, et al: Association of autoantibodies to topoisomerase 1 and the phosphorylated (110) for RNA polymerase I1 in Japanese scleroderma patients. J Immunol 153:5838, 1994 69. Sakamoto A, Sumida T, Maeda T, et a1 T cell receptor V@ repertoire of doublenegative a/@ T cells in patients with systemic sclerosis. Arthritis Rheum 35:944, 1992 70. Seibold JR, Giomo RC, Clamon HN: Dermal mast cell degranulation in systemic sclerosis. Arthritis Rheum 33:1702, 1990 71. Shi-Wen X, Panesar M, Vancheeswaran R, et al: Expression and shedding of intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 by normal and scleroderma fibroblasts. Effects of interferon-y, tumor necrosis factor-a, and estrogen. Arthritis Rheum 371689, 1994 72. Sfikakis PP, McCune BK, Tsokas G, et al: Immunohistologic demonstration of transforming growth factor-@isoforms in the skin of patients with systemic sclerosis. Clin Immunol Immunopathol 69:199, 1993 73. Silver RM, Miller KS, Kinsella MB, et al: Evaluation and management of scleroderma lung disease using bronchoalveolar lavage. Am J Med 88:470, 1990 74. Sipos A, Czijak L, Lorincz G, et al: Studies on anti-granulocyte and anti-platelet antibodies in patients with systemic sclerosis. Scand J Rheumatol 1743, 1988 75. Szegedi A, Boros P, Chen J, et al: An Fca RIII[CDl6]-specific autoantibody from a patient with progressive systemic sclerosis. Immunol Lett 35:69, 1993 76. Tan EM, Rodnan GP, Garcia I, et a1 Diversity of antinuclear antibodies in progressive systemic sclerosis. Arthritis Rheum 23:617, 1980 77. White 8: Immune Pathogenesis. In Clements P, Furst D (eds): Systerpic Sclerosis. Malvern, PA, Lea & Febiger, 1996, pp 229-250 78. White B, Kom JH, Piela-Smith T H Preferential adherence of human y/6, CD8 + and memory T cells to fibroblasts. J Immunol 152:4912, 1994 79. Yamakage A, Kikuchi K, Smith EA, et al: Selective upregulation of platelet-derived growth factor (Y receptors by transforming growth factor @ in scleroderma fibroblasts. J Exp Med 175:1227, 1992 80. Yurovsky VV, Wigley FM, Wise RA, et al: Skewing of the CD8 + T cell repertoire in the lungs of systemic sclerosis patients. Hum Immunol 48:84, 1996 81. Yurovsky W, Sutton PA, Schulze DH, et al: Expansion of selected V61+ y/S T cells in systemic sclerosis patients. J Immunol 153881, 1994
Address reprint requests to Barbara White, MD University of Maryland MSTF Room 8-34 10 South Pine Street Baltimore, MD 21201
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IMMUNOPATHOGENESIS OF SYSTEMIC SCLEROSIS Barbara White, MD
INTRODUCTION
Over the last decade, it has become clear that an activated immune system plays a central role in causing tissue damage in patients with systemic sclerosis (SSc). What remains unknown is whether activation of the immune system is an initial or secondary event in the disease process. This article will review abnormalities of T cells, B cells, and nonspecific inflammatory cells in patients with SSc. The possible consequences of these abnormalities on blood vessel and fibroblast functions will be discussed. ABNORMAL CELLULAR IMMUNITY IN SSc
T cells are important in the development of tissue damage in SSc patients. Activated T cells dominate the inflammatory infiltrates in tissues of SSc patients. They are present very early in the course of the disease. They provide specificity to the immune response and show evidence of selection by antigen in SSc patients. Most importantly, when activated, they regulate functions of many hematopoietic and nonhematopoietic cells, including vascular cells and fibroblasts. Pathologic Evidence of T-cell Involvement in SSc
T cells infiltrate perivascular and interstitial spaces of the skin of patients h5 This infiltration precedes the characteristic findings of small with SSc (Fig. l).57, This work was supported in part by a Career Investigator Award from the Veterans Administration.
From the University of Maryland School of Medicine; and the Veterans Administration Medical Center, Baltimore, Maryland
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Figure 1. Mononuclear cell infiltration
in the skin in SSc. Mononuclear cells are seen in the perivascular areas and scattered through the dermis of this patient with early diffuse cutaneous SSc. (Courtesy of Dr. C. L. Kauffman, University of Maryland.)
vessel vasculopathy and fibrosis. Both CD4+ and CD8+ T cells are increased in the skin, but CD4 + T cells are more frequent. Expression of human leukocyte antigen (HLA) class I1 molecules shows these infiltrating T cells have been recently activated. Infiltration with increased numbers of activated T cells also characterizes alveolitis in SSc patients. Patients with active lung disease have increased numbers and percentages of T cells in the interstitiumS2and in bronchoalveolar (BAL) fluids (Fig. 2).22,n*80 CD8 + T cells are increased more than CD4 + T cells in BAL fluids from patients with alveolitis.80The predominance of distinct T-cell subpopulations in the skin (CD4+) and lungs (CD8+) of SSc patients suggests that different T cells may contribute to the disease process in different organs. T-cell subsets in the blood have been studied extensively in SSc patients, with conflicting Absolute lymphocyte counts, absolute numbers and percentages of T cells, and absolute numbers and percentages of CD8+ T cells are reported to be normal or decreased in peripheral blood mononuclear cells (PBMC). Percentages of CD4 + T cells are increased, normal, or decreased. Total numbers of y/6 T cells are decreased in the blood of SSc patients, especially in early disease. Memory CD3 + or CD4 + T cells that are CD29 + or CD45RA are increased or normal in SSc patients, whereas CD29 + CD8 + T cells may be decreased. Evidence of activation of circulating T cells in SSc patients is in-
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Figure 2. Mononuclear cell infiltration in the lungs in SSc. Extensive mononuclear cell infiltration of the interstitium and alveolar spaces, with fibrosis, is seen.
creased expression of HLA-DR molecules, interleukin (1L)-2 receptors (R), and an increased frequency of in vivo hprt gene mutation. Other evidence of T cell activation in SSc patients includes increased serum levels of soluble IL-2R,8 14, 35, 56 CD4, CD8,'4 neopterin; and adenosine deaminase. Increased IL-2R levels may be a marker of internal organ i n v ~ l v e m e n tand ~ ~ may or may not* correlate with disease activity.
The T-cell Repertoire in SSc
Studies to date have found no difference in levels of expression of variable (V)a and Vp gene families in the blood;" skin, or lungsXoof SSc patients, compared to controls. Patients, however, have an increase in numbers and percentage of CD3+ T cells and total y/S T cells that express the V y l gene, in both peripheral blood and BAL fluids.x1CD4 - CD8 - a/P T cells preferentially use Vp5, 7, and 17 gene families in SSc patients.69 Analyses of the T-cell repertoire have been done to look for evidence of antigen-driven expansion of T cells bearing certain V gene families in SSc patients. Oligoclonal T-cell responses to conventional peptide antigens may be characterized by use of a few V gene families, with conservation of T-cell antigen receptor (TCR) V-diversity-joining region nucleotide lengths and DNA sequences. Analyses of TCR junctional region lengths and DNA sequences show two types of T cells are oligoclonal in SSc patients, suggesting evidence of selection by antigen. V y l + y/S T cells in the peripheral blood and tissuesR1and CD8+ T cells in the lungsBoof SSc patients have undergone oligoclonal expansion. Of note, both VS1+ y / S T cells and CD8+ T cells preferentially adhere to fibroblasts.1U6
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In Vivo and In Vitro T-cell Responses to Antigens
In vivo T-cell responses to exogenous antigens appear normal in SSc patients. Patients have a normal response to primary immunization and recall antigens with T-dependent antigens, with normal cutaneous delayed type hypersensitivity and antibody produ~tion.4~ In vitro testing of T cells in SSc patients has yielded contradictory results.R Reports suggest increased, normal, and decreased T-cell proliferative, helper, or suppressor responses to activation with a variety of stimuli. Unfractionated PBMC from SSc patients respond to potential target antigens in SSc, including type I and type IV collagen. NONSPECIFIC INFLAMMATORY CELLS IN TISSUES IN SSc
Cells such as natural killer (NK) cells, macrophages, mast cells, eosinophils, basophils, and neutrophils can contribute to tissue damage through nonspecific inflammation. All of these cell types are present in increased amounts or an activated state in SSc patients, although contradictory reports have also been published." Numbers and activity of NK cells in SSc patients are increased, normal, or decreased. Lymphokine-activated killer cell activity is increased or decreased. Of interest, one patient suffered a flare of her SSc when she received IL-2 and lymphokine-activatedkiller cells to treat a rnalignan~y.~~ Macrophages and Langerhans cells are increased in the skin of SSc patients, especially early in the disease. Patients with interstitial lung disease have increased numbers of macrophages in the interstitium, alveolar epithelial surfaces, and BAL fluids.11, 73 These pulmonary macrophages are activated. BAL fluids from patients with alveolitis contain more fibronectin and alveolar-macrophagederived growth factor than BAL fluids from patients without alveolitis or from 73 Monocytes from SSc patients behave abnormally, with decreased HLA-DR expression in response to interferon y (IFN-y) and reduced ability to stimulate angi~genesis.~~ Mast cells may be increased in the skin or BAL fluids of patients with SSC.~], 70 Degranulation of mast cells provides direct evidence of their activation within the skin of SSc patient^.^" Levels of histamine, tryptase, and hyaluronic acid are elevated in BAL fluids, which are compatible with mast cell activation within the lungs of SSc patients. Myocardial mast cell infiltration or degranulation has been associated with severe cardiac involvement in SSc. The circulating counterpart of mast cells, basophils, appear activated in SSc patients, with increased spontaneous histamine release and increased reactivity or sensitivity to activation signals. Basophil numbers are normal in BAL fluids." Numbers of eosinophils can be increased in the blood, clinically unaffected skin, and BAL fluids from SSc patients.z2* 64, 73 Evidence of eosinophil activation comes from increased major basic protein in the blood and lungs and increased eosinophil cationic protein in BAL fluids.Z2Eosinophilia in BAL fluids is associated with increased epithelial cell permeability in fibrosing alveolitis. Eosinophil activation is infrequent in skin biopsies. Neutrophils are not part of the inflammatory infiltrate in the skin of SSc patients. Neutrophilic infiltration, however, is common in alveolitis in SSc patients, with increased neutrophils in lung biopsies and BAL fluids.", 73 Neutrophils from SSc patients have an increased spontaneous release of hydrogen peroxide, suggesting in vivo activation. Neutrophilic alveolitis may correlate with the development of greater restrictive lung disease over time."
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ADHESION MOLECULE EXPRESSION IN SSc
SSc is characterized by increased expression of adhesion molecules from the selectin, integrin, and immunoglobulin (Ig) gene superfamilies. Increased expression of adhesion molecules indicates activation of cells bearing those molecules and may facilitate homing and retention of lymphocytes and nonspecific inflammatory cells in the tissues. Of note, SSc patients have reduced numbers of circulating T cells capable of adhering to endothelial cells in vitro, which may be the result of depletion of T cells by increased adherence to vascular endothelium in v i v ~ . ~ ~ Expression of E-selectin and P-selectin is increased in SSc patients. E-selectin expression is increased on endothelial cells in the skin and minor salivary glands.20,21, 21 Expression of E-selectin by endothelial cells indicates those cells have been activated to cytokines. E-selectin mediates endothelial cell interactions with neutrophils, granulocytes, monocytes, and CD4 + T cells. Therefore, it is not surprising that endothelial cell expression of E-selectin in the skin from patients with early SSc correlates with the amount of mononuclear cell infiltration.21Soluble E-selectin is increased in the blood of SSc 21 P-selectin is expressed by endothelial cells in skin of SSc patients with early disease,"" and soluble P-selectin is elevated.2o Integrins are transmembrane heterodimeric molecules consisting of an a and p chain. They bind extracellular matrix proteins, which transmit information about the environment to the cell. Integrins also bind receptors of the Ig gene superfamily. Expression of integrins is increased on endothelial cells, perivascular lymphocytes, fibroblasts, and dendritic cells in SSc skin. Endothelial cells have increased expression of p l integrins.z' Dermal mononuclear cells express increased amounts of pl, p2, including lymphocyte function-associated antigen 1 (LFA-l), and p3 integrins,2I with expression greatest in early skin disease. Dermal fibroblasts and dendritic cells from SSc patients have increased expression of p l and p3 integrins.*l Intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-l (VCAM-l) are members of the Ig gene superfamily. Receptor-ligand interactions between ICAM-1 on endothelial cells and LFA-1 on T cells are important in the homing of T cells to peripheral tissues. Similarly, adhesion of T cells to fibroblasts is mediated in part by interactions between ICAM-1 on the fibroblasts and LFA-1 on the T cells. ICAM-1 expression is increased on both endothelial cells and fibroblasts in SSC.~", 2 1 , 52, 71 Greater than normal responsiveness to cytokine stimulation may cause some of the increased expression of ICAM-1 on SSc fibroblasts. Given the increases in both ICAM-1 and LFA-1 expression in SSc, it is not unexpected that SSc skin biopsies with more ICAM1 on the endothelial cells have more pronounced mononuclear cell infiltrates.2n Nor should it be unexpected that SSc fibroblasts bind more T cells than do control fibroblasts.' Of note, circulating ICAM.1 levels are increased in about one third of SSc patients.2nIncreased shedding of soluble ICAM-1 from fibroblasts in SSc may be a contributing f a ~ t o r .Similar ~' to the findings with ICAM-1, VCAM1 is expressed in increased amounts on endothelial cells in the skin,'" and soluble VCAM-1 is elevated in SSc patients with early disease.2" POTENTIAL CONTRIBUTIONS OF T CELLS AND NONSPECIFIC INFLAMMATORY CELLS TO TISSUE DAMAGE IN SSc
Temporal and physical associations suggest that activated T cells and inflammatory cells cause tissue damage in SSc patients. Infiltration of the skin
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with activated mononuclear cells, which are largely T cells, precedes the development of characteristic vasculopathy and fibrosis. Fibroblasts that are producing procollagen mRNA are located next to these mononuclear cell infiltratesM Alveolitis of the lungs, with infiltration with lymphocytes, macrophages, neutrophils, and eosinophils, precedes and predicts the development of pulmonary fibrosis.73Soluble mediators and cellular cytotoxity are the major mechanisms by which cells of the immune system cause tissue damage. Soluble Mediators of the Immune System in SSc
Cells of the immune system secrete many different mediators that can alter functions of vascular cells or fibroblasts. Soluble mediators reported to be present in abnormally high amounts in SSc patients include IL-1, IL-2, IL-4, IL-6, IL8, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-P, platelet derived growth factor (PDGF), granzyme A, and leukotriene B4.n All of these can be made by T cells or other inflammatory cells. Target cells in SSc, including endothelial cells, vascular smooth muscle cells, and fibroblasts, may themselves contribute to the production of some of these mediators. To date, the cellular sources of these soluble mediators in SSc patients have not been fully elucidated. Existing data suggest a T-helper 2 pattern of cytokine production by T cells in blood and BAL fluids from SSc patients, with increased IL-4I3,54 and reduced production of IFN-Y.~~ This is of interest because interleukin-4 stimulates proliferation, chemotaxis, and extracellular matrix production by fibroblasts and increases T-cell adhesion to endothelial cells. It also induces a collapse of vimentin intermediate filaments in endothelial cells, which is the first morphologic abnormality seen in the endothelium in SSc skin.57IL-2 levels are increased in the sera of some SSc patientsM,72 and may correlate with more aggressive skin disease.44 Several proinflammatory cytokines, IL-1, TNF-a, and IL-6, are increased in SSc patients. Among the many properties of IL-1 is the ability to increase proliferation of T and B cells, stimulate prostaglandin E2 (PGE,) and extracellular matrix production by fibroblasts, increase endothelial cell expression of ICAM1, and promote vascular smooth muscle proliferation. Some, but not all, reports suggest serum levels, monocyte produ~tion,3~ and fibroblast production of IL-a or IL-p are increased in SSc. SSc fibroblasts express more IL-1R.38 This may make them more sensitive to IL-1 stimulation, with greater than normal increases in ICAM-1, IL-6, PGE2, and IL-lp.38 TNF-a has pleotrophic effects similar to IL-1. For example, it increases fibroblast proliferation and production of PGE, and IL-p. It also increases endothelial cell expression of E-selectin, ICAM-1, VCAM, and release of endothelin1. TNF-a decreases fibroblast production of type I and I1 collagens, however, although increasing production of collagenase. Some, but not all, reports indicate that serum levels35and production of TNF-a by PBMC35and mon~cytes"'~ are increased in SSc. Expression of TNF-a is increased in the skin of SSc patients?" SSc fibroblasts have normal decreases in collagen production in response to TNF-a, but an abnormal increase in ICAM-1 expression. TNF-a receptors are quantitatively and qualitatively normal on SSc fibroblasts4 IL-6 has a broad range of activities that overlap with IL-1 and TNF-a. Of note, IL-6 may increase function of cytolytic cells because of its ability to induce perforin and serine esterases. Serum levels72and production of IL-6 by PBMC,'O fibroblasts,16and endothelial cells40 may be increased in SSc. Alveolar macrophages from SSc patients produce normal amounts of IL-6 and have normal levels of IL-6R.I" Expression of IL-6 is increased in the epidermis63of SSc patients.
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IL-6 may be responsible for the increased IL-2R expression on PBMC from SSc patients. Transforming growth factor+ is a logical candidate for a cytokine that contributes to tissue damage in SSc. It stimulates collagen production by fibroblasts and vascular smooth muscle cells and induces endothelial cell production of endothelin-1. Endothelin-1 is a potent vasoconstrictor that is increased in sera3’ and BAL fluids7 from SSc patients. Endothelin-1 stimulates collagen production by fibroblasts7,31 Some, but not all, reports find increased TGF-P proteins or mRNAs in the blood,” skin24,42, h7 and BAL fluids’ from SSc patients. TGF-P mRNA colocalizes with proal(1) and proa(II1) collagen mRNAsHand Type VII collagenh7in skin from some SSc patients. The cellular sources of TGF-P remain unknown. The TGF-P in the lungs of patients with alveolitis may come from regenerating alveolar epithelium. SSc fibroblasts make normal amounts of TGF-P in ~ i t r o . ~ ~ When considering the potential role of TGF-P in SSc, it is important to note that Higley et aIz4did not find TGF-P in skin from patients with active diffuse cutaneous SSc, although it was found in skin in later stages of disease and in skin from patients with limited cutaneous SSc. This observation, plus the knowledge that TGF-P binds extracellular matrix, raises the possibility that the increase in TGF-P is secondary to an increase in extracellular matrix in the tissues of SSc patients. Moreover, it is not possible to induce or sustain a SSc-like phenotype in normal fibroblasts with in vitro exposure to TGF-P. SSc fibroblasts may be unusually sensitive to TGF-P, with greater than normal proliferation, synthesis of type-a PDGF receptor, binding of PDGF-BB, and production of connective 79 tissue growth factor.3y, Interleukin-8 is chemotactic for neutrophils, lymphocytes, and endothelial cells. Levels of IL-8 are elevated in the sera”’ and BAL fluids” of SSc patients, as well as on dermal endothelial cells?” IL-8 levels in BAL fluids correlate with the degree of neutrophilia,” suggesting IL-8 contributes to cellular inflammation in SSc patients with alveolitis. Cellular Cytotoxicity
Relatively few studies have been done of cellular cytotoxicity in SSc. Cytotoxic responses to fibroblasts, epithelial cells, and muscle cells have been reported.Iz Granzyme A levels are elevated in the sera of SSc patients.’’ This serine protease, which is released by cytolytic CD8+ T cells as part of the perforin pathway of cytotoxicity, may contribute to endothelial cell death in SSc.” ABNORMAL HUMORAL IMMUNITY IN SSc
T cell-dependent and -independent antigen-specific antibody responses are normal in vivo in SSc patients.45In contrast, hypergammaglobulinemia is very common and autoantibody production is an early, nearly universal finding. There are multiple autoantigen targets in SSc, some of which are disease-specific. Evidence that autoantibodies cause tissue damage in SSc patients is limited. Pathologic Evidence of B-Cell Involvement in SSc
Hypergammaglobulinemia and autoantibodies are found in the sera of SSc patients and increased numbers of plasma cells are seen within their skin17,s7
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and Polyclonal B-cell activation causes increases in IgG, IgA, IgM, and IgE.l7IgG is also increased in BAL fluids of SSc patients, with no correlation to disease activity.” Antinuclear antibodies occur in 90% to 98% of patients with SSc. The isotype is usually IgG, especially the IgG3 subclass, although IgM, IgE, and IgA antinuclear antibodies are also seen.25 Autoantibody production may be an epiphenonemon in SSc. In silica-associated SSc, autoantibodies occur only in patients with SSc and not in individuals with just silicosis.a Thus, the production of the autoantibodies appears to result from events that cause SSc and is not the forerunner of the disease. In spontaneous SSc, titers of antitopoisomerase 1 and anticentromere antibodies remain relatively stable for years, which suggests that these SSc-specific autoantibodies are not involved in disease activity. As another example, an illness with some characteristics of SSc occurs in mice with chronic graft versus host disease. The initiating event is activation of T cells by alloantigens. This leads to secondary production of autoantibodies directed against proteins involved with rRNA transcription (Nor:90 and fibrillarh~),’~ similar to those seen in SSc. Autoantibody Production in SSc
Autoantibody targets that are quite specific for SSc include the self-antigens DNA topoisomerase 1 centromeric proteins,5O RNA polymerases I and 111,26,43, 68 PM-Scl antigenz, and myenteric neurons.28The reasons for specific targeting of these particular proteins are unknown and may have to do with subcellular localization or function of the target antigen. An enormous range of other autoantigens can be targets of autoantibody production in SScn These include endothelial cells, fibroblasts, smooth muscle, granulocytes, red blood cells, platelets, thyroid tissue, salivary gland tissue, neutrophil cytoplasmic antigens, heat shock protein, types I, 111, and IV collagen, IL-6, IL-8, IgG, cardiolipin, FcyR, histones, mitochondria, laminin, single stranded RNA, U3, and U11 nuclear ribonucleoproteins, Th ribonucleoproteins, upstream binding factor (NOR-90), high motility group (HMG)17 nucleosome protein, the Ku antigen, and RNA polymerase 11. Targets of autoantibody production in SSc appear to be presented to the immune system in a native state. Evidence for this is antibody recognition of 49, 69 and active sites, includconformational multiple epit0pes,2~, ing phosphorylation sites, on target molecule^.^, 15, 26, 29, 43, In addition, different components of multiunit complexes may be recognized,’8,43 which suggests the entire complex is processed and presented. The HLA alleles of the patient influence which SSc-specific autoantibodies are produced, perhaps because certain HLA alleles are required to activate those T cells that provide the necessary help for antibody production by B cells. The anticentromere antibody response is tightly associated with HLA-DQ alleles with a polar glycine or tyrosine at position 26 of the HLA-DQP chain.80 In whites, black and Japanese,& the antitopoisomerase 1 antibody response is associated with HLA-DQ alleles with a tyrosine residue at position 30. The HLA-DR60and HLA-DP6f3 chains influence the topoisomerase I autoantibody response. Autoantibodies to PM-Scl correlate with the presence of the HLA-DRB1*0301, DQA1-*0501, DQB1*0201 h a p l ~ t y p e . ~ ~ Homology Between Autoantigen Targets and Viruses
Evidence that molecular mimicry plays a role in the development of autoantibodies in some SSc patients is the homology between some SSc autoantibody
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Table 1. AUTOANTIBODY TARGETS IN SYSTEMIC SCLEROSIS AND
HOMOLOGOUS VIRUSES ~~~~
Autoantibody Target
DNA Topoisomerase 1
PM-Scl antigen U1 snRNP
Fibrillarin (U3 snRNP)
Homologous Viruses
p30QaQ protein from feline sarcoma virus UL70 protein of human cytomegalovirus SV-40 large T antigen Human immunodefiency virus tat protein Herpes simplex virus type I ICP4 protein Herpes simplex virus type I P40 protein Epstein-Barr virus nuclear antigen 1
targets and viruses (Table 1).B-cell epitopes on DNA topoisomerase I include amino acids 121-126 and 741-746, which have homology to the UL70 protein of human cytomegalovirussl and the p30gag protein from feline sarcoma virus,4i respectively. B-cell epitopes on PM-Scl antigen include a 138-amino acid fragment with homology to the nuclear localization signal of SV-40 large T antigen and a molecular signal found in the HIV tat protein.2 A universal B-cell epitope on U1 RNP is similar to herpes simplex virus type 1 ICP4 protein.4yThe carboxy terminus of fibrillarin shares sequence homology with P40, a capsid protein A hexapeptide sequence, GRGRGG, encoded by herpes simplex virus type l.36 in the amino terminus of fibrillarin is shared with Epstein-Barr virus (EBV) nuclear antigen.3hA quarter of SSc patients have weak antibody reactivity to HIV retroviral proteins by Western blots, without signs of direct infection.13 POTENTIAL CONTRIBUTIONS OF HUMORAL IMMUNITY TO TISSUE DAMAGE IN SSc
There is little evidence to date that autoantibodies damage cells or tissues in SSc patients through complement activation or immune complex formation. Many of the autoantibodies do not activate the complement cascade, and serum complement levels are normal. Although immune complexes can be found in the sera of approximately 25% of SSc patients, immune complexes and complement are not typically deposited in tissues. Immune complexes can be identified in BAL fluids from SSc patients, but there is no correlation with disease a ~ t i v i t y . ~ ~ The anti-red cell, antigranulocyte, and antiplatelet antibodies that occur in SSc patients are not usually associated with c y t ~ p e n i a s . ~ ~ Autoantibodies in SSc patients have the potential to activate cells that bear the target autoantigen, although there is no evidence to date of this happening in vivo. An anti-FcyRIII antibody secreted by an EBV-transformed B cell from a SSc patient triggered in vitro release of P-glucuronidase, arylsulfatase, and alkaline phosphatase from ne~trophils.’~ A potential pathologic role for these antibodies might be through triggering neutrophils, NK cells, or macrophages to release cytokines. The ability of SSc antibodies to modify intracellular events in vivo is unexplored. Antibodies against centromeric proteins, topoisomerase 1, and RNA polymerases all inhibit the function of their respective targets in vitro.5,15, 2h, 2y,43,6R This inhibition could have clinical consequences. Jabs et al”] found a correlation between anticentromeric antibodies and aneuploidy in SSc patients. Anticentromeric antibodies could disrupt the functional centromere and allow for malsegregation of chromosomes during mitosis.
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The final mechanism through which antibodies might contribute to tissue damage in SSc is antibody-dependent cellular cytotoxicity. Antibodies from SSc patients are capable of mediating antibody-dependent cellular cytotoxicity of endothelial cells in v i t r ~ .Similar ~ ~ , ~damage ~ in vivo might contribute to the vasculopathy of SSc.
SUMMARY The information outlined above can be used to generate a model of the immunopathogenesis of SSc (Fig. 3). This model includes a susceptible host, with age greater than 25 and female gender being risk factors. The model also includes exposure to exogenous agents, which could be different in different individuals and may include inhaled or ingested chemicals or infectious agents. An early event is T-cell activation, with infiltration in the skin and internal organs. Activation of the T cells is a selective process that appears to be influenced by antigen in SSc patients. The importance of a particular T-cell subpopulation may depend upon the organ involved and the stage of the disease. CD4 T cells predominate in the skin. In contrast, CD8+ T cells are increased in the lungs of patients with alveolitis, where they are oligoclonal, showing evidence of antigen-driven selection. V61 +y/6 T cells are increased in both the blood and lungs of SSc patients and also show evidence of selection by antigen. B cells are activated early, with polyclonal activation leading to hypergammaglobulinemia. SSc-specific autoantibodies target DNA topoisomerase I, centromeric proteins, and RNA polymerases I and 111. Characteristics of autoantibodies in SSc suggest that the target antigens are presented to the immune system as native molecules or even part of a multiunit complex. There is some homology between viruses and autoantibody targets in SSc, which suggests that molecular mimicry may play a role in initiating the antibody response. Many nonspecific inflammatory cells infiltrate the tissues and show evidence of activation. These
+
SUSCEPTIBLE HOST (usually woman > 25 years of age)
IExogenous triggers
B-cell activation
T-CELL ACTIVATION
I \
Autoantidodies, t Ig
Activation of nonsuecific inflammatory cells Inflammatory infiltrates
I
Soluble mediators, cytotoxicity
FIBROBLASTS
ENDOTHELIAL CELLS
FIBROSIS
I
Activation, injury
ISCHEMIC VASCULOPATHY
Figure 3. Model of the immunopathogenesis of SSc.
IMMUNOPATHOGENESIS OF SYSTEMIC SCLEROSIS
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include macrophages and monocytes, mast cells, eosinophils, basophils, and natural killer cells. Soluble mediators made by these T cells, B cells, and nonspecific inflammatory cells can activate and damage fibroblasts, endothelial cells, and other vascular cells. The relative importance of the various candidate cytokines, the temporal sequence of their production, and their cellular sources remain largely to be defined. There may be some contribution of direct Tcell cytotoxicity or antibody-dependent cellular cytoxicity to the tissue damage that occurs.
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