- Email: [email protected]
The Second International Workshop on Scleroderma Research*
Matrix Vol. 13/1993, pp. 427 -429
Meetin;J Report
© 1993 by GustavFischer Verlag, Stuttgart· Jena . New York
The Second International Workshop on Scleroderma Research*
The etiology and pathogenesis of scleroderma is much less well understood than that of other rheumatic and autoimmune diseases. Prominent disease characteristics include abnormal vascular reactivity and vascular damage, diffuse cutaneous and visceral fibrotic disease, a stereotypic pattern of autoantibody formation and evidence of T cell activation. These features, along with results of previous in vivo and in vitro studies, do not readily allow a coherent and simple theory of disease pathogenesis. The coexistence of scleroderma with systemic lupus erythematosus in individual patients, as well as family studies with multiple connective tissue disease patients in a kindred, suggests an immune etiology; however, clinically, fibrotic and vascular features of the disease predominate. The Second International Workshop on Scleroderma Research explored genetic, immunologic, vascular, and metabolic factors involved in the pathogenesis of the disease in an attempt to help unravel pathogenetic mechanisms in the disorder. Presentation and discussion of largely unpublished data and promotion of collaborative ventures was a major goal.
Immunogenetics Previous studies have demonstrated assoCIatlOns of some HLAantigens with scleroderma, including Class I (HLA-A9, B8, and B35), Class II (DRl, DR3, DR5, and DR52) and Class III (C4 null) antigens. Associations of particular autoantibodies with HLA antigens have also been found, for example, DR5 with antitopoisomerase I and DR3 with anti-PM-Scl. Frank Arnett, of the University of Texas at Houston, reported on association of DQ ~ chain antigens with scleroderma. DQw5 and DQw7 were found more frequently only in scleroderma patients with anti-centromere antibodies (ACA) compared to controls. DQw5 and DQw7 share a key amino acid in the "cleft" of the DQ~ antigen binding region. Petros Tsipouras (University of Connecticut) discussed the possible use of anonymous probes in uncovering " The workshop was sponsored by and supported by the Scleroderma Foundation of Greater Chicago.
genes that may be linked to scleroderma. If kindreds of sufficient size with several affected individuals can be studied, probes for VNDRs and VNTRs (variable number dinucleotide repeats and variable number tandem repeats) can establish chromosomal linkages. Ken Welsh (Oxford Transplant Center, England) and Ethylin Wan Jabs Oohns Hopkins) discussed chromosomal alterations that may playa role in the development of autoantibodies and scleroderma. Earlier reports had demonstrated increased chromosomal breaks and aneuploidy in scleroderma patients. Jabs found a correlation of aneuploidy and increased chromosomal loss with anticentromere antibodies (ACA). Patients with antibodies to CENP-C exhibited more aneuploidy than patients those with antibodies to CENP-A or CENP-B. CENP-C, but not CENP-A and CENP-B is present at the kinetochore of functional centromeres; aneuploidy might thus result from centromeric dysfunction in the presence of anticentromere antibodies. Welsh found that lengths of telomere repeat arrays, which are protective of chromosomes, were shorter in scleroderma fibroblasts than in normal fibroblasts. Chromosomal breaks were found at sites of telomere repeat arrays.
Animal models Several animal models for scleroderma have been studied although none recapitulates the mixed vascularimmunologic-fibrotic nature of the human disease. The tight skin (Tsk) mouse is one such model, its phenotype of tight skin inherited as an autosomal dominant trait. A role for the immune system in this model is suggested by recent reports that bone marrow or spleen cell transfer from Tsk into syngeneic normal mice leads to expression of the tight skin phenotype. Steven Clark (University of Connecticut) reported on use of a' collagen promoterchloramphenicol acetyl transferase (COLCAT) transgene, bred into Tsk mice, in studying the regulation of collagen synthesis in this model. Tsk mice carrying the transgene, expressed increased CAT activity in skin, in vivo, and in cultured dermal fibroblasts in vitro. Graft versus host disease (GVHD) has been another
428
J. H. Korn and C. Black
model for scleroderma featuring both vascular and cutaneous changes. When animals are treated with cyclosporine A, a syndrome of GVHD can develop even with autologous bone marrow transplantation. Cyclosporin A treatment prevents clonal deletion of T cells reactive to self MHC Class II antigenes. Alan Hess (University of Maryland) reported on the role of T cells in this rat model of cyclosporine augmented GVHD. GVHD in cyclosporine-treated bone marrow recipients can be adoptively transferred; both CD8+ and CD4+ cells are required for full expression of the disease. Antibodies to Class II antigens, especially the IA subregion, block GVHD. Thalidomide prevents acute GVHD and allograft rejection; thalidomide both blocks TNFa release from macrophages and inhibits expression of adhesion molecules. Thus, although both cyclosporine and thalidomide are immunnosuppressive, one promotes and the second inhibits GVHD. Keith Elkon (Cornell) reported on the transfer of scleroderma lymphocytes to SCID (severe combined immunodeficiency) mice. SCID mice recipients of both normal and scleroderma lymphocytes had increased skin thickness but changes were modestly greater in recipients of scleroderma lymphocytes.
Growth Factors, Cytokines and Immune Cells A role has long been postulated for immune cell derived and other cytokines and growth factors in the pathogenesis of scleroderma. Evidence for activation of the immune system in scleroderma has grown over the last several years with reported data showing increased levels of cytokines and T cell surface molecules (IL-2 receptor and CD4 and CD8 antigens). The potential exists for these immune cell products to both "activate" connective tissue cells leading to increased matrix synthesis and to lead to endothelial cell damage. Platelet-derived growth factor (PDGF) is one growth factor that may be important in regulation of fibroblast metabolism in the skin. In particular, PDGF regulates production of type V collagen. The distribution of PDGF in scleroderma skin was discussed by Steffen Gay (Birmingham, AL). By immunocytochemistry, PDGF is distributed widely including endothelial cells, monocytes, and stromal fibroblasts. It is found most extensively in the skin of patients with short disease duration. Another growth factor, basic fibroblast growth factor (bFGF), largely co-localizes with PDGF; indeed PDGF is known to upregulate bFGF expression. The physical interaction of bFGF, heparin sulfate proteoglycan, and type V collagen may serve to protect bFGF from degradation. TGF~ was shown to increase expression of the PDGFa receptor in scleroderma, but not in normal, fibroblasts (Trojanowska, Medical University of South Carolina). Associated with this, is an enhanced proliferate response
to PDGF after TGF~ pretreatment. Conversely, TGF~ increases expression of the bFGF receptor to a much greater extent in normal compared to scleroderma fibroblasts. However the induction is of the low affinity bFGF receptor. . Another inducer of BFGF is the ras gene product. Ras transformed cell lines make both the common 18 kD and the 22kD bFGF. Ras also stimulates production of cystatins which inhibit activation of collagenase by other metalloproteinases. Thus the ras gene product indirectly inhibits collagenase activation. Both ras and cystatin C were shown by Gay to be present in scleroderma skin. In the discussion it was noted that regulation by ras is complex. In some cases, ras-transformed cell lines make increased levels of metalloproteinases. Several immune cell derived cytokines have been shown to modulate connective tissue cell metabolism. Among the best studied of these is IL-1 which affects a range of fibroblast functions including proliferation and synthesis of collagen, proteoglycans, collagenase, and prostaglandins. The multiple activities can be effected not only by the intact IL-1 molecule but also by distinct peptides (Postlethwaite, Univ. Tennessee, Memphis). Furthermore, one of these peptides blocks binding of the intact molecule, another mediates fibroblast proliferation and yet a different peptide stimulates collagenase and glycosaminoglycans. The IL-1 receptor antagonist (IRAP) blocks the action of the hyaluronic acid stimulating peptide although the latter does not itself bind to fibroblasts. These data suggest that multiple epitopes on the fibroblast cell surface receptor for IL-1 are involved in binding and signal transduction. The role of the immune system in the vascular disease of scleroderma has been less well understood than immune cell activities in relation to fibroblast activation. Kahaleh (Medical College of Ohio, Toledo) presented data on granzyme A, a T cell product which may be involved in endothelial cell injury. Granzyme A binds to endothelial cells and extracellular matrix. It is also cytotoxic to fibroblasts. Circulating granzyme A was found in the serum of scleroderma patients. Using RTPCR, mRNA for granzyme was found in the tissue of the majority of scleroderma patients but not in control skin. Granzyme A is not found in circulating T cells suggesting that activation of T cells and granzyme A transcription occurs in the skin. Preliminary data were also presented suggesting that an antigen isolated from endothelial cells may stimulate scleroderma but not normal T cells in vitro. Presumably, such stimulation could lead to granzyme production and subsequent endothelial damage. One measure of such damage might be circulating adhesion molecules. Haskard (Hammersmith Hospital, London) in fact found that levels of circulating E-selectin, ICAM-1, and VCAM-1 were raised in some patients with diffuse scleroderma, limited scleroderma, or Raynaud's.
Second International Workshop on Scleroderma Research
Integrins and cell adhesion The growth of fibroblasts in monolayer cultures, while traditional, has limited resemblance to the in vivo situation where fibroblasts grow in and interact with a 3-dimensional matrix. The study of fibroblasts grown in collagen gels has been an attempt to more closely simulate the in vivo environment. Fibroblasts grown in this way contract the surrounding gels. This contraction is stimulated by PDGF treatment and blocked by antibodies to ~1 integrins (K. Rubin, Uppsala, Sweden). Scleroderma fibroblasts have decreased levels of a1~1 integrins on their surface. Normal fibroblasts grown in collagen gels downregulate collagen synthesis in concert with gel contraction. There is a striking linear relationship between the ability of normal fibroblasts to contract a collagen gel and the extent to which pro collagen mRNA is down-regulated. This correlation is decoupled in scleroderma fibroblasts which contract the gel normally but do not coordinately down-regulate collagen synthesis. This regulatory abnormality was reported by several attendees (T. Krieg, Univ. of Kaln, Germany; McWhirter and Rubin). Furthermore, when scleroderma fibroblasts are grown in the presence ofTGF~, there is upregulation (rather than downregulation) of collagen synthesis in collagen gels, an effect that persists after removal of TGF~ (A. McWhirter, Royal Free Hospital, London). In normal fibroblasts collagen synthesis in gels as compared to monolayer culture is decreased by greater than 90%. This downregulation is due, in part, to decreased transcription and in part to decreased mRNA stability (T. Krieg). Some of the decrease in collagen was due to decreased mRNA stability. In 4/10 scleroderma strains, there was defective downregulation of collagen synthesis in gels suggesting a defect in interaction with extracellular matrix. This might be due to the decreased a2 integrin, compared to normal cells, on the surface of scleroderma fibroblasts. When scleroderma fibroblasts are grown in the presence of TGF~, there is up regulation (rather than downregulation) of collagen synthesis in collagen gels, an effect that persists after removal of TGF~ (A. McWhirter, Royal Free Hospital, London).
Collagen metabolism Gerard Karsenty (M. D. Anderson Hospital, Houston) presented data on the cloning of a cDNA for a transcription factor binding to the IF-1 site. The factor is a new zinc finger protein with homology to the Drosophila Kruppel gene, and binds to the a1(I) promoter, in a region between 130 and 200 bp 5' to the transcription start site. Using a1(I) promoter-CAT (chloramphenicol acetyl transferase) constructs, the binding site was shown to be involved in regulation of collagen gene transcription. The role of the a2(I) collagen promoter was addressed in a different way by Benoit DeCrombrugghe (M. D. Ander-
429
son Hospital, Houston). Using the collagen promoter linked to lacZ or luciferase as a transgene, he explored the question of what were the minimal promoter sequen~es required for in vivo tissue specific expression. Even With very short promoter sequences, tissue specific expression of the transgene was achieved. Expression of the reporter gene in transgenic mice paralleled expression of TGF~. One factor that stimulates TGF~ transcription is hypoxia. In experiments with TGF~ promoter-CAT constructs, the response element for hypoxia appears to be between -453 and the transcription start site. Fibroblasts show a small increase in collagen synthesis when grown in low p02, possibly due to endogenous TGF~. Using low p02 concentrations, Falanga (University of Miami) was able to obtain clonal growth of scleroderma fibroblasts not achievable in ambient air. The mechanism by which hypoxia exerts its effect on the TGF~ promoter is unclear. However, lactate, which is increased in hypoxia, also increases collagen protein synthesis and, to a lesser extent, pro collagen mRNA (Amento, Genentech, South San Francisco). Lactate appears to increase TGF~ binding to cells. TGF~ could act in a self-regulatory autocrine positive feedback loop. When scleroderma cells are grown in vitro, the increase in collagen synthesis, compared to normal lines, may abate with serial passage. Vuorio (Turku, Finland) suggested that the activated state for transcription factors may, in fact, last longer than the activated state for collagen synthesis per se. In addition, some scleroderma lines with high collagen synthesis show an increase in collagen mRNA half-life as well as an increased rate of transcription. Hatamochi (Kawasaki Medical School, Japan) presented data showing that the increased synthesis of collagen in scleroderma lines he studied was not as prevalent as suggested by some centers. Only 2/8 patients had increased collagen synthesis. However, he found that all had decreased production of collagenase, albeit with normal collagenase steady state mRNA. Amento (Genentech, South San Francisco) noted that interferon-y suppresses collagenase protein without altering mRNA. Interferon-y also acts in synergy with relaxin to decrease collagen synthesis. Data presented at the conference certainly indicate that multiple mechanisms are at play in the pathogenesis of scleroderma and their interactions are complex. A possible scenario is immune activation, in the right genetic background, leading subsequently to vascular injury. Whether this is directly T cell mediated (e. g. granzyme) or indirect, is unclear. Subsequent hypoxia leads to increased synthesis of collagen and matrix proteins. In addition, immune factors contribute to abnormalities in fibroblast metabolism and may lead to permanent changes in the connective tissue phenotype. Joseph H. Korn, M.D. Workshop Co-Chairs
Carol Black, M. D.
Meetin;J Report
© 1993 by GustavFischer Verlag, Stuttgart· Jena . New York
The Second International Workshop on Scleroderma Research*
The etiology and pathogenesis of scleroderma is much less well understood than that of other rheumatic and autoimmune diseases. Prominent disease characteristics include abnormal vascular reactivity and vascular damage, diffuse cutaneous and visceral fibrotic disease, a stereotypic pattern of autoantibody formation and evidence of T cell activation. These features, along with results of previous in vivo and in vitro studies, do not readily allow a coherent and simple theory of disease pathogenesis. The coexistence of scleroderma with systemic lupus erythematosus in individual patients, as well as family studies with multiple connective tissue disease patients in a kindred, suggests an immune etiology; however, clinically, fibrotic and vascular features of the disease predominate. The Second International Workshop on Scleroderma Research explored genetic, immunologic, vascular, and metabolic factors involved in the pathogenesis of the disease in an attempt to help unravel pathogenetic mechanisms in the disorder. Presentation and discussion of largely unpublished data and promotion of collaborative ventures was a major goal.
Immunogenetics Previous studies have demonstrated assoCIatlOns of some HLAantigens with scleroderma, including Class I (HLA-A9, B8, and B35), Class II (DRl, DR3, DR5, and DR52) and Class III (C4 null) antigens. Associations of particular autoantibodies with HLA antigens have also been found, for example, DR5 with antitopoisomerase I and DR3 with anti-PM-Scl. Frank Arnett, of the University of Texas at Houston, reported on association of DQ ~ chain antigens with scleroderma. DQw5 and DQw7 were found more frequently only in scleroderma patients with anti-centromere antibodies (ACA) compared to controls. DQw5 and DQw7 share a key amino acid in the "cleft" of the DQ~ antigen binding region. Petros Tsipouras (University of Connecticut) discussed the possible use of anonymous probes in uncovering " The workshop was sponsored by and supported by the Scleroderma Foundation of Greater Chicago.
genes that may be linked to scleroderma. If kindreds of sufficient size with several affected individuals can be studied, probes for VNDRs and VNTRs (variable number dinucleotide repeats and variable number tandem repeats) can establish chromosomal linkages. Ken Welsh (Oxford Transplant Center, England) and Ethylin Wan Jabs Oohns Hopkins) discussed chromosomal alterations that may playa role in the development of autoantibodies and scleroderma. Earlier reports had demonstrated increased chromosomal breaks and aneuploidy in scleroderma patients. Jabs found a correlation of aneuploidy and increased chromosomal loss with anticentromere antibodies (ACA). Patients with antibodies to CENP-C exhibited more aneuploidy than patients those with antibodies to CENP-A or CENP-B. CENP-C, but not CENP-A and CENP-B is present at the kinetochore of functional centromeres; aneuploidy might thus result from centromeric dysfunction in the presence of anticentromere antibodies. Welsh found that lengths of telomere repeat arrays, which are protective of chromosomes, were shorter in scleroderma fibroblasts than in normal fibroblasts. Chromosomal breaks were found at sites of telomere repeat arrays.
Animal models Several animal models for scleroderma have been studied although none recapitulates the mixed vascularimmunologic-fibrotic nature of the human disease. The tight skin (Tsk) mouse is one such model, its phenotype of tight skin inherited as an autosomal dominant trait. A role for the immune system in this model is suggested by recent reports that bone marrow or spleen cell transfer from Tsk into syngeneic normal mice leads to expression of the tight skin phenotype. Steven Clark (University of Connecticut) reported on use of a' collagen promoterchloramphenicol acetyl transferase (COLCAT) transgene, bred into Tsk mice, in studying the regulation of collagen synthesis in this model. Tsk mice carrying the transgene, expressed increased CAT activity in skin, in vivo, and in cultured dermal fibroblasts in vitro. Graft versus host disease (GVHD) has been another
428
J. H. Korn and C. Black
model for scleroderma featuring both vascular and cutaneous changes. When animals are treated with cyclosporine A, a syndrome of GVHD can develop even with autologous bone marrow transplantation. Cyclosporin A treatment prevents clonal deletion of T cells reactive to self MHC Class II antigenes. Alan Hess (University of Maryland) reported on the role of T cells in this rat model of cyclosporine augmented GVHD. GVHD in cyclosporine-treated bone marrow recipients can be adoptively transferred; both CD8+ and CD4+ cells are required for full expression of the disease. Antibodies to Class II antigens, especially the IA subregion, block GVHD. Thalidomide prevents acute GVHD and allograft rejection; thalidomide both blocks TNFa release from macrophages and inhibits expression of adhesion molecules. Thus, although both cyclosporine and thalidomide are immunnosuppressive, one promotes and the second inhibits GVHD. Keith Elkon (Cornell) reported on the transfer of scleroderma lymphocytes to SCID (severe combined immunodeficiency) mice. SCID mice recipients of both normal and scleroderma lymphocytes had increased skin thickness but changes were modestly greater in recipients of scleroderma lymphocytes.
Growth Factors, Cytokines and Immune Cells A role has long been postulated for immune cell derived and other cytokines and growth factors in the pathogenesis of scleroderma. Evidence for activation of the immune system in scleroderma has grown over the last several years with reported data showing increased levels of cytokines and T cell surface molecules (IL-2 receptor and CD4 and CD8 antigens). The potential exists for these immune cell products to both "activate" connective tissue cells leading to increased matrix synthesis and to lead to endothelial cell damage. Platelet-derived growth factor (PDGF) is one growth factor that may be important in regulation of fibroblast metabolism in the skin. In particular, PDGF regulates production of type V collagen. The distribution of PDGF in scleroderma skin was discussed by Steffen Gay (Birmingham, AL). By immunocytochemistry, PDGF is distributed widely including endothelial cells, monocytes, and stromal fibroblasts. It is found most extensively in the skin of patients with short disease duration. Another growth factor, basic fibroblast growth factor (bFGF), largely co-localizes with PDGF; indeed PDGF is known to upregulate bFGF expression. The physical interaction of bFGF, heparin sulfate proteoglycan, and type V collagen may serve to protect bFGF from degradation. TGF~ was shown to increase expression of the PDGFa receptor in scleroderma, but not in normal, fibroblasts (Trojanowska, Medical University of South Carolina). Associated with this, is an enhanced proliferate response
to PDGF after TGF~ pretreatment. Conversely, TGF~ increases expression of the bFGF receptor to a much greater extent in normal compared to scleroderma fibroblasts. However the induction is of the low affinity bFGF receptor. . Another inducer of BFGF is the ras gene product. Ras transformed cell lines make both the common 18 kD and the 22kD bFGF. Ras also stimulates production of cystatins which inhibit activation of collagenase by other metalloproteinases. Thus the ras gene product indirectly inhibits collagenase activation. Both ras and cystatin C were shown by Gay to be present in scleroderma skin. In the discussion it was noted that regulation by ras is complex. In some cases, ras-transformed cell lines make increased levels of metalloproteinases. Several immune cell derived cytokines have been shown to modulate connective tissue cell metabolism. Among the best studied of these is IL-1 which affects a range of fibroblast functions including proliferation and synthesis of collagen, proteoglycans, collagenase, and prostaglandins. The multiple activities can be effected not only by the intact IL-1 molecule but also by distinct peptides (Postlethwaite, Univ. Tennessee, Memphis). Furthermore, one of these peptides blocks binding of the intact molecule, another mediates fibroblast proliferation and yet a different peptide stimulates collagenase and glycosaminoglycans. The IL-1 receptor antagonist (IRAP) blocks the action of the hyaluronic acid stimulating peptide although the latter does not itself bind to fibroblasts. These data suggest that multiple epitopes on the fibroblast cell surface receptor for IL-1 are involved in binding and signal transduction. The role of the immune system in the vascular disease of scleroderma has been less well understood than immune cell activities in relation to fibroblast activation. Kahaleh (Medical College of Ohio, Toledo) presented data on granzyme A, a T cell product which may be involved in endothelial cell injury. Granzyme A binds to endothelial cells and extracellular matrix. It is also cytotoxic to fibroblasts. Circulating granzyme A was found in the serum of scleroderma patients. Using RTPCR, mRNA for granzyme was found in the tissue of the majority of scleroderma patients but not in control skin. Granzyme A is not found in circulating T cells suggesting that activation of T cells and granzyme A transcription occurs in the skin. Preliminary data were also presented suggesting that an antigen isolated from endothelial cells may stimulate scleroderma but not normal T cells in vitro. Presumably, such stimulation could lead to granzyme production and subsequent endothelial damage. One measure of such damage might be circulating adhesion molecules. Haskard (Hammersmith Hospital, London) in fact found that levels of circulating E-selectin, ICAM-1, and VCAM-1 were raised in some patients with diffuse scleroderma, limited scleroderma, or Raynaud's.
Second International Workshop on Scleroderma Research
Integrins and cell adhesion The growth of fibroblasts in monolayer cultures, while traditional, has limited resemblance to the in vivo situation where fibroblasts grow in and interact with a 3-dimensional matrix. The study of fibroblasts grown in collagen gels has been an attempt to more closely simulate the in vivo environment. Fibroblasts grown in this way contract the surrounding gels. This contraction is stimulated by PDGF treatment and blocked by antibodies to ~1 integrins (K. Rubin, Uppsala, Sweden). Scleroderma fibroblasts have decreased levels of a1~1 integrins on their surface. Normal fibroblasts grown in collagen gels downregulate collagen synthesis in concert with gel contraction. There is a striking linear relationship between the ability of normal fibroblasts to contract a collagen gel and the extent to which pro collagen mRNA is down-regulated. This correlation is decoupled in scleroderma fibroblasts which contract the gel normally but do not coordinately down-regulate collagen synthesis. This regulatory abnormality was reported by several attendees (T. Krieg, Univ. of Kaln, Germany; McWhirter and Rubin). Furthermore, when scleroderma fibroblasts are grown in the presence ofTGF~, there is upregulation (rather than downregulation) of collagen synthesis in collagen gels, an effect that persists after removal of TGF~ (A. McWhirter, Royal Free Hospital, London). In normal fibroblasts collagen synthesis in gels as compared to monolayer culture is decreased by greater than 90%. This downregulation is due, in part, to decreased transcription and in part to decreased mRNA stability (T. Krieg). Some of the decrease in collagen was due to decreased mRNA stability. In 4/10 scleroderma strains, there was defective downregulation of collagen synthesis in gels suggesting a defect in interaction with extracellular matrix. This might be due to the decreased a2 integrin, compared to normal cells, on the surface of scleroderma fibroblasts. When scleroderma fibroblasts are grown in the presence of TGF~, there is up regulation (rather than downregulation) of collagen synthesis in collagen gels, an effect that persists after removal of TGF~ (A. McWhirter, Royal Free Hospital, London).
Collagen metabolism Gerard Karsenty (M. D. Anderson Hospital, Houston) presented data on the cloning of a cDNA for a transcription factor binding to the IF-1 site. The factor is a new zinc finger protein with homology to the Drosophila Kruppel gene, and binds to the a1(I) promoter, in a region between 130 and 200 bp 5' to the transcription start site. Using a1(I) promoter-CAT (chloramphenicol acetyl transferase) constructs, the binding site was shown to be involved in regulation of collagen gene transcription. The role of the a2(I) collagen promoter was addressed in a different way by Benoit DeCrombrugghe (M. D. Ander-
429
son Hospital, Houston). Using the collagen promoter linked to lacZ or luciferase as a transgene, he explored the question of what were the minimal promoter sequen~es required for in vivo tissue specific expression. Even With very short promoter sequences, tissue specific expression of the transgene was achieved. Expression of the reporter gene in transgenic mice paralleled expression of TGF~. One factor that stimulates TGF~ transcription is hypoxia. In experiments with TGF~ promoter-CAT constructs, the response element for hypoxia appears to be between -453 and the transcription start site. Fibroblasts show a small increase in collagen synthesis when grown in low p02, possibly due to endogenous TGF~. Using low p02 concentrations, Falanga (University of Miami) was able to obtain clonal growth of scleroderma fibroblasts not achievable in ambient air. The mechanism by which hypoxia exerts its effect on the TGF~ promoter is unclear. However, lactate, which is increased in hypoxia, also increases collagen protein synthesis and, to a lesser extent, pro collagen mRNA (Amento, Genentech, South San Francisco). Lactate appears to increase TGF~ binding to cells. TGF~ could act in a self-regulatory autocrine positive feedback loop. When scleroderma cells are grown in vitro, the increase in collagen synthesis, compared to normal lines, may abate with serial passage. Vuorio (Turku, Finland) suggested that the activated state for transcription factors may, in fact, last longer than the activated state for collagen synthesis per se. In addition, some scleroderma lines with high collagen synthesis show an increase in collagen mRNA half-life as well as an increased rate of transcription. Hatamochi (Kawasaki Medical School, Japan) presented data showing that the increased synthesis of collagen in scleroderma lines he studied was not as prevalent as suggested by some centers. Only 2/8 patients had increased collagen synthesis. However, he found that all had decreased production of collagenase, albeit with normal collagenase steady state mRNA. Amento (Genentech, South San Francisco) noted that interferon-y suppresses collagenase protein without altering mRNA. Interferon-y also acts in synergy with relaxin to decrease collagen synthesis. Data presented at the conference certainly indicate that multiple mechanisms are at play in the pathogenesis of scleroderma and their interactions are complex. A possible scenario is immune activation, in the right genetic background, leading subsequently to vascular injury. Whether this is directly T cell mediated (e. g. granzyme) or indirect, is unclear. Subsequent hypoxia leads to increased synthesis of collagen and matrix proteins. In addition, immune factors contribute to abnormalities in fibroblast metabolism and may lead to permanent changes in the connective tissue phenotype. Joseph H. Korn, M.D. Workshop Co-Chairs
Carol Black, M. D.