Drugs of the future: the hormone relaxin.

The peptide hormone relaxin is emerging as a multi-functional factor in a broad range of target tissues including several non-reproductive organs, in addition to its historical role as a hormone of pregnancy. This review discusses the evidence that collectively demonstrates the many diverse and vital roles of relaxin: the homeostatic role of endogenous relaxin in mammalian pregnancy and ageing; its gender-related effects; the therapeutic effects of relaxin in the treatment of fibrosis, inflammation, cardioprotection, vasodilation and wound healing (angiogenesis) amongst other pathophysiological conditions, and its potential mechanism of action. Furthermore, translational issues using experimental models (to humans) and its use in various clinical trials, are described, each with important lessons for the design of future trials involving relaxin. The diverse physiological and pathological roles for relaxin have led to the search for its significance in humans and highlight its potential as a drug of the future.

Implantation and pregnancy following in vitro fertilization and the effect of recombinant human relaxin administration in Macaca fascicularis.

Implantation and early pregnancy, and the potential effects of the reproductive-hormone relaxin, were examined in the cynomolgus macaque (Macaca fascicularis) following in vitro fertilization and embryo transfer. Mature oocytes were collected from regularly cycling, female cynomolgus monkeys subjected to ovarian superovulation using recombinant human FSH and hCG. Oocytes fertilized in vitro were cultured to the 4- to 8-cell stage, slow-cooled, and stored in liquid nitrogen before thawing and embryo transfer. Regularly cycling recipients were administered recombinant human relaxin or vehicle for 21 days through the peri-implantation period (Day 0 = pump implantation), during which time the thawed embryos were transferred (Day 7). Endometrial thickness and the number of gestational sacs were monitored by ultrasound at three time points (Days 7, 21, and 28). The number of days of placental sign (implantation bleeding) in pregnant females and menses in nonpregnant females were also recorded. Implantation (gestational sacs/embryo transferred) and multiple pregnancy (multiple gestations/ pregnant recipient) rates were slightly higher in relaxin-treated recipients compared to vehicle-treated recipients. Administration of relaxin was associated with increased implantation bleeding in pregnant females. Endometrial thickness was increased in relaxin-treated recipients at Days 7 and 28 compared to Day 0, but these differences were not observed at the same time points in vehicle-treated females. Systemic administration of recombinant human relaxin in an in vitro fertilization/embryo transfer setting was associated with effects consistent with a role for this hormone in endometrial physiology in primates.

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo.

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.

Relaxin decreases renal interstitial fibrosis and slows progression of renal disease.

BACKGROUND: Relaxin, a hormone of the insulin-growth factor family, promotes collagen remodeling. In rodent models of pulmonary and dermal fibrosis, relaxin reduced interstitial fibrosis. To study relaxin's effect in renal disease, we used the experimental bromoethylamine (BEA) model that leads to severe renal interstitial fibrosis, a decrease in glomerular filtration rate, and albuminuria at one month. METHODS: Rats were injected with BEA one week prior to implantation of an osmotic pump delivering relaxin (2 microg/hour) or vehicle continuously for 28 days. RESULTS: BEA caused a significant decrease in creatinine clearance, which was partially prevented by relaxin. In the relaxin-treated BEA rats, serum creatinine was normal, and albumin excretion was slightly decreased. By morphometric measurement, relaxin administration was associated with a significant decrease in interstitial fibrosis at the corticomedullary junction. This was accompanied by a decrease in the number of ED-1 positive cells (an index of macrophage infiltration) and in the intensity of immunohistochemical staining for transforming growth factor-beta. This antifibrotic effect of relaxin did not appear to be mediated by systemic hemodynamic changes since the mean arterial pressure was not significantly different among the groups. CONCLUSIONS: Relaxin may have a useful application in decreasing interstitial fibrosis and thereby slowing the progression of renal disease.

Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites.

Relaxin is a reproductive hormone that has historically been characterized as being responsible for pubic ligament loosening and cervical ripening. Recently, relaxin has been associated with neovascularization of the endometrial lining of the uterus, potentially via specific induction of vascular endothelial growth factor. Previously conducted clinical studies using partially purified porcine relaxin have described relaxin's ability to stimulate the healing of ischemic wounds, suggesting that relaxin may also have angiogenic effects at sites of ischemic wound healing. In the present study, relaxin's angiogenic effects in the context of wound repair were tested in rodent models of angiogenesis and wound healing. Relaxin showed an ability to stimulate new blood vessel formation, particularly at ischemic wound sites, and to induce both vascular endothelial growth factor and basic fibroblast growth factor specifically in cells, presumably including macrophages, collected from wound sites. Resident macrophages collected from nonwound sites, such as the lung, did not show altered expression of these cytokines following relaxin administration. Because angiogenic wound cells are frequently macrophages, THP-1 cells, a cell line of monocyte lineage that binds relaxin specifically, were tested for and shown to induce vascular endothelial growth factor and basic fibroblast growth factor in response to relaxin. In conclusion, relaxin may be useful in the treatment of ischemic wounds by stimulating angiogenesis via the induction of vascular endothelial growth factor and basic fibroblast growth factor in wound macrophages.

Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women.

Although the role of the reproductive hormone, relaxin, in rodents is well documented, its potential contribution to human reproduction is less well defined. In this study, we examine the effects of relaxin on human endometrial cells in vitro and describe the clinical effects of relaxin on menstrual flow in women. In cultured endometrial cells, relaxin specifically induces the expression of an angiogenic agent, vascular endothelial growth factor (VEGF). cAMP is implicated as a second messenger involved in VEGF stimulation. VEGF expression is temporally regulated in the endometrium, and our results suggest that relaxin, which is secreted by the corpus luteum and is present in the endometrium during the menstrual cycle and pregnancy, may be involved in regulating endometrial VEGF expression. Relaxin was recently tested in a clinical trial for efficacy in the treatment of progressive systemic sclerosis, and was administered at levels up to 10 times higher than that measured during pregnancy. The most frequent relaxin-related adverse event reported during the course of the study was the onset of menometrorrhagia, defined in this study as heavier-than-usual or irregular menstrual bleeding. The intensification of menstrual flow observed in these patients is consistent with the hypothesis that relaxin mediates neovascularization of the endometrial lining.

Transcriptional inhibition of stromelysin by interferon-gamma in normal human fibroblasts is mediated by the AP-1 domain.

The expression of the major matrix-degrading metalloproteinase, stromelysin (SL), is modulated by a variety of cytokines and growth factors. Interferon-gamma (IFN-gamma) is a potent modulator of SL expression, either inhibiting or activating expression in a cell-specific manner. We have investigated the mechanisms involved in the regulation of SL gene expression in cultured human fibroblasts by IFN-gamma. Reverse transcription-polymerase chain reaction (RT-PCR) assays confirmed the previously reported profound inhibitory response of SL mRNA expression to IFN-gamma [Amaldi et al., 1989]. For evaluation in transient gene expression assays, 1.2-kilobase (kb) pairs (-1214 to +14 relative to the transcription start site), and shorter, deletion mutant fragments of the SL promoter were cloned into appropriate chloramphenicol acetyltransferase transferase (CAT) expression vectors. The SL promoter along this region contains an active polyomavirus enhancer A-binding protein-3 (PEA-3) site at -216 and an activator protein-1 (AP-1) site at -70. Treatment of transfected neonatal foreskin fibroblasts with 300-500 U/ml IFN-gamma resulted in down-regulation of both basal and IL-1beta-induced CAT gene expression. IFN-gamma also decreased CAT expression when placed under the control of a synthetic multimeric AP-1 site construct. Gel-shift assay data indicate a decrease in specific binding to AP-1 oligonucleotide of nuclear extract from IFN-gamma and PMA/IFN-gamma-treated cells. The suppression of SL expression by IFN-gamma, in human fibroblasts therefore is mediated through the AP-1 element.

Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.

Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1.

Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (Kd = 102 pM) to a specific receptor on THP-1 cells. Receptor density was low ( approximately 275 receptors/cell), but binding of relaxin triggered intracellular signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.

Connective tissue metabolism in scleroderma, including cytokines.

The recent literature on scleroderma emphasizes the importance of cytokine dysregulation, cell-cell interactions, and extracellular matrix overproduction in the pathogenesis of this disease. Advances in our understanding of these areas are discussed here.

Monocyte chemotactic protein-1 (MCP-1) mRNA is down-regulated in human dermal fibroblasts by dexamethasone: differential regulation by TGF-beta.

Macrophages are a source of cytokines driving repair. Wound macrophages are derived from circulating monocytes. Monocyte chemotactic protein-1 (MCP-1) is a potent specific monocyte chemoattractant. Treatment of serum stimulated dermal fibroblasts with dexamethasone led to a dose dependent down-regulation of MCP-1 mRNA levels. Such an anti-inflammatory effect may partially explain the negative influence of glucocorticoid treatment on wound repair. Topical or parenteral of fibroblasts cultured in serum free media with TGF-beta increased MCP-1 mRNA levels. TGF-beta treatment of fibroblasts cultured in serum also partially overcame the dexamethasone mediated decrease in MCP-1 mRNA levels. In glucocorticoid treated animals TGF-beta may stimulate repair by an indirect pro-inflammatory action following transcriptional up-regulation of MCP-1.

Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion.

Vascular matrix remodeling occurs during development, growth, and several pathological conditions that affect blood vessels. We investigated the capacity of human smooth muscle cells (SMCs) to express matrix metalloproteinases (MMPs), enzymes that selectively digest components of the extracellular matrix (ECM), in the basal state or after stimulation with certain cytokines implicated in vascular homeostasis and pathology. Enzymatic activity associated with various proteins secreted in the culture media was detected by gelatin or casein sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Proteins were identified by immunoprecipitation and mRNA by Northern blotting. SMCs constitutively secreted a 72-kD gelatinase and the tissue inhibitors of MMPs (TIMPs) types 1 and 2. SMCs stimulated with interleukin-1 or tumor necrosis factor-alpha synthesized de novo 92-kD gelatinase, interstitial collagenase, and stromelysin. Several lines of evidence suggest that when stimulated by cytokines, SMCs produce activated forms of MMPs. Together, the constitutive and the cytokine-induced enzymes can digest all the major components of the vascular ECM. Moreover, since these mediators augment the production of MMPs without appreciably affecting the synthesis of TIMPs, locally secreted cytokines may tip the regional balance of MMP activity in favor of vascular matrix degradation.

Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis.

Angiogenesis is important in the proliferation of inflammatory synovial tissue. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that is also angiogenic in vivo. We examined the potential role of VEGF in mediating chemotaxis and proliferation of endothelial cells in rheumatoid arthritis (RA) using samples of synovial tissue and synovial fluid from 55 arthritic patients. Synovial fluid VEGF by ELISA was higher in RA synovial fluids (386 +/- 122 ng/ml) (SE) compared with osteoarthritis (OA) synovial fluids (< 0.8 ng/ml) (p < 0.05) or synovial fluids from patients with other arthritides (6.6 +/- 2 ng/ml). In addition to its known mitogenic properties, we found that human rVEGF was chemotactic for HUVECs at concentrations above 0.02 nM. Incubation of RA synovial fluids with neutralizing anti-VEGF resulted in 23 to 66% (mean 53 +/- 4%) inhibition of HUVEC chemotaxis. Conditioned medium from four of five RA synovial tissue explants was mitogenic for bovine adrenal capillary endothelial cells. Anti-VEGF neutralized from 19 to 42% (mean 28 +/- 4%) of this mitogenic activity. To determine the cellular source of VEGF in synovial tissue, we employed immunohistochemistry. VEGF+ cells were rarely (< 1%+) found in normal synovial tissues. In contrast, RA and OA synovial tissues exhibited VEGF+ lining cells (8% and 13%, respectively). A few synovial tissue macrophages were VEGF+ in both RA and OA (5% and 2%, respectively). These results elucidate a newly described function for VEGF as a potent chemotaxin for endothelial cells as well as a role for VEGF in RA-associated endothelial migration and proliferation.

Constitutive activation of the collagenase promoter in recessive dystrophic epidermolysis bullosa fibroblasts: role of endogenously activated AP-1.

Recessive dystrophic epidermolysis bullosa (RDEB) is a mutilating disease of the skin characterized by recurrent blistering and erosions that result from compromised integrity of the basement membrane zone. In this study, fibroblasts derived from the skin of RDEB patients were characterized for expression of the major metalloproteinases, particularly interstitial collagenase. Consistent with previous reports on increased collagenase protein levels in fibroblasts from some RDEB patients, we found that steady-state levels of collagenase mRNA were significantly increased in fibroblast strains derived from three of five RDEB patients compared to fibroblasts obtained from normal donors. Stromelysin mRNA was elevated in the same three fibroblast strains, whereas expression of neither the 72- nor the 92-kDa type IV collagenases was different from that of controls. Tissue inhibitor of metalloproteinases was expressed in RDEB fibroblasts at levels similar to those observed in normal fibroblasts. To investigate the mechanism behind the steady-state elevation in collagenase and stromelysin expression, AP-1 expression and activation were studied. Although levels of Jun expression were not different from those seen in normal fibroblasts, AP-1 activity, as assessed by ability to bind to a TPA response element-containing oligonucleotide, was endogenously elevated in RDEB fibroblasts compared to normal fibroblasts. Transfection studies using a plasmid construct containing the collagenase promoter linked to a CAT reporter gene demonstrated that RDEB fibroblasts were able to support active transcription of the promoter compared to normal fibroblasts. These studies support the hypothesis that RDEB fibroblasts contain chronically activated AP-1, and perhaps other transactivating factors, that contribute to the cellular phenotype of collagenase and stromelysin overexpression.

Interleukin-1 and transforming growth factor-alpha: synergistic stimulation of metalloproteinases, PGE2, and proliferation in human fibroblasts.

Wound healing and other inflammatory processes are driven by a complex series of interactions among cells, the extracellular matrix, and secreted products of various cell types. Cytokines, such as interleukin-1 and transforming growth factor-alpha, are present at wound sites and contribute to the proinflammatory milieu of these sites. In the present study, we have investigated the effect of these cytokines, individually and in concert, on fibroblast expression of matrix metalloproteinases, which contribute to extracellular matrix remodeling, and of prostaglandin E2, which alters vascular tone and permeability. The metalloproteinases, procollagenase (matrix metalloproteinase-1) and prostromelysin (matrix metalloproteinase-3), are induced by exposure of dermal fibroblasts to interleukin-1, not stimulated by transforming growth factor-alpha, but are synergistically induced by the combination of cytokines. The 92-kDa type IV procollagenase (matrix metalloproteinase-9, progelatinase B), is also stimulated in synergistic fashion. Prostaglandin E2 is induced in rheumatoid synovial fibroblasts by interleukin-1 beta, not altered by transforming growth factor-alpha, and is synergistically released by the combination of the two cytokines. Fibroblast proliferation, which is also a component of normal wound healing, is also synergistically stimulated by the action of the two cytokines in concert. These results indicate that interleukin-1 beta and transforming growth factor-alpha synergize to elicit a number of phenotypic responses in fibroblasts which are relevant to normal wound healing and chronic inflammation.

Transforming growth factor beta (TGF-beta) and dexamethasone have direct opposing effects on collagen metabolism in low passage human dermal fibroblasts in vitro.

Collagen metabolism is a balance between synthesis and lysis. Both are under tight regulatory control. TGF-beta reverse the impairment of healing seen after glucocorticoid treatment in vivo. Both TGF-beta and glucocorticoids are known to regulate collagen metabolism directly. We have examined the effect of dexamethasone and of TGF-beta individually and in combination on the regulation of procollagen type 1, interstitial collagenase and tissue inhibitor of metallo-proteinase-1 (TIMP-1) synthesis at both the protein and mRNA levels in low passage human dermal fibroblasts. Dexamethasone treatment decrease synthesis of procollagen and caused a dose dependent down-regulation of TIMP-1 synthesis. Interstitial collagenase synthesis by fibroblasts was detectable but low. Thus, glucocorticoid treatment of fibroblasts tilts the balance of collagen metabolism away from accumulation. TGF-beta had opposing effects, stimulating both procollagen and TIMP-1 synthesis at the protein and mRNA levels. TGF-beta was able to cause a dose-dependent reversal of the glucocorticoid induced decrease in procollagen and TIMP-1 synthesis. Stimulation of healing in glucocorticoid treated animals by TGF-beta may be by the direct action of this agent upon fibroblast collagen metabolism.

Human relaxin decreases collagen accumulation in vivo in two rodent models of fibrosis.

The reproductive hormone, relaxin, inhibits collagen synthesis in vitro by normal human dermal fibroblast. In the present study, recombinant human relaxin is shown to modulate collagen accumulation and organization by mesenchymal cells in vivo in two rodent models of fibrosis: 1) fibrotic infiltration of polyvinyl alcohol sponge implants in rats, and 2) capsule formation around implanted osmotic pumps in mice. In the sponge, relaxin inhibits collagen accumulation, a measured by hydroxyproline content, in a dose-responsive manner by up to 25-29% in animals receiving 30 ng/ml relaxin, a finding supported by a decrease in collagen-specific trichrome staining in sections of sponges from relaxin-treated animals. In mice, the capsules surrounding relaxin-containing pumps are thinner and less dense than are capsules from control pumps. Ultrastructurally, control capsules are composed of densely packed parallel arrays of collagen fibrils, whereas fibrils more frequently are not packed in parallel arrays and are less abundant in treated capsules.

Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA). Evidence for a novel MGSA receptor.

Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.

Melanoma growth-stimulatory activity/GRO decreases collagen expression by human fibroblasts. Regulation by C-X-C but not C-C cytokines.

Melanoma growth-stimulatory activity (MGSA)/GRO is well characterized as a potent neutrophil chemoattractant. In the present study, we have demonstrated that MGSA induced a dose-dependent decrease in the expression of interstitial collagens by rheumatoid synovial fibroblasts. The decrease was observed over a dose range of 0.6-6.0 nM MGSA. This effect was specific, as MGSA had no demonstrable effect on the expression of collagen-degrading metalloproteinases, nor did it affect the collagenase inhibitor, tissue inhibitor of metalloproteinases. It also had no effect on the proliferation rate of these fibroblasts, unlike its mitogenic effect on melanoma cells. The ability to inhibit collagen expression was also demonstrated by another member of the C-X-C branch of the platelet factor 4 superfamily, interleukin-8 (IL-8), but not by RANTES, MIP-1 alpha, or MIP-1 beta, which belong to the C-C branch. Steady-state levels of expression of MGSA and IL-8 transcripts in normal adult tissues were dissimilar, suggesting that expression may be an important level at which the activity of these cytokines is regulated. Direct binding experiments with 125I-MGSA on synovial fibroblasts have allowed us to identify an MGSA receptor with a KD of 10.1 nM and approximately 75,000 binding sites/fibroblast. 125I-MGSA binding was specific and could not be displaced by unlabeled IL-8. These results suggest that MGSA, as well as IL-8, may play a role other than that of neutrophil chemo-attractant and more specifically, may be important in the regulation of collagen turnover.