Constitutive expression of a 92-kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines.

Synovial fibroblasts freshly isolated from the rheumatoid joint are characterized by their marked connective tissue degradative ability. This phenotype includes the ability to secrete large amounts of the matrix-degrading metalloproteinases, collagenase, and stromelysin. We have found that another aspect of this phenotype is the constitutive expression at both protein and mRNA levels of a 92-kD gelatinolytic metalloproteinase, which is not secreted by normal dermal or lung fibroblasts and is immunologically cross-reactive with a type V collagenase expressed by activated macrophages and neutrophils. Expression of this 92-kD metalloproteinase confers upon the fibroblasts the capacity to degrade collagenase- and stromelysin-resistant interstitial elements, such as collagen types IV, V and XI. In contrast to the 92-kD metalloproteinase, a 68-kD gelatinase (type IV collagenase) was expressed by all fibroblast types studied, indicating that its regulation is distinct from that of the 92-kD gelatinase. To identify what cytokines may be important in the induction of the rheumatoid synovial phenotype, including expression of the 92-kD gelatinase, we exposed normal dermal fibroblasts to a number of cytokines including many known or considered likely to be present in rheumatoid synovial fluid and tissue. Although IL-1 beta, tumor necrosis factor-alpha, lymphotoxin, platelet-derived growth factor, and basic fibroblast growth factor were capable of stimulating fibroblasts to secrete collagenase, only tumor necrosis factor-alpha, lymphotoxin, and IL-1 beta were able to induce expression of the 92-kD gelatinase, demonstrating discordant regulation of the two metalloproteinases. Expression of the 68-kD gelatinase was independent of that of the 92-kD gelatinase, as demonstrated at the protein and mRNA levels. Late passage rheumatoid synovial fibroblasts, which no longer constitutively expressed the 92-kD gelatinase, displayed an accentuated response to IL-1 beta when compared to normal dermal fibroblasts. Thus, in addition to IL-1 beta, tumor necrosis factor-alpha or lymphotoxin may contribute to the expression of a specific rheumatoid synovial phenotype in vivo that is associated with progressive matrix destruction.

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.

Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis.

The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.

One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing.

The role of intravenously administered recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) on the healing of incisional wounds in rats with impaired healing due to age or glucocorticoid administration was investigated. The administration of methylprednisolone to young adult rats decreased wound breaking strength to 50% of normal control. Breaking strength of incisional wounds from 19-mo-old rats was decreased approximately 27% compared with wounds from normal healing young adult rats. A single intravenous administration of rhTGF-beta 1 (100 or 500 micrograms/kg) increased wound breaking strength from old rats or young adult rats with glucocorticoid-induced impaired healing to levels similar to normal healing control animals when determined 7 d after injury. Even though the circulating half-life of systemically administered rhTGF-beta 1 is < 5 min, a sustained stimulatory effect on extracellular matrix secretion was evident in glucocorticoid-impaired rats when rhTGF-beta 1 was administered at the time of wounding, 4 h after wounding, or even 24 h before wounding. These observations indicate a previously unrecognized potential for the active form of TGF-beta 1 to profoundly influence the wound healing cascade after brief systemic exposure.

Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice.

Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.

Gamma-interferon inhibits collagen synthesis in vivo in the mouse.

Subcutaneous implantation of osmotic pumps into CAF1 mice resulted in the formation of thick fibrous capsules around the pumps. When pumps were loaded with recombinant murine gamma-interferon (rMuIFN-gamma) to deliver 2 X 10(3) U/h for 14 d, there was a marked decrease in thickness and collagen content of the capsules from rMuIFN-gamma-treated animals compared with capsules from animals receiving diluent alone. The collagen content of the capsules was estimated by hydroxyproline analysis of the tissue and by quantitative electron microscopy of collagen bundles. Heat-inactivated rMuIFN-gamma failed to reduce the fibrotic response in this assay. These results provide compelling evidence that gamma-interferon can down-regulate collagen synthesis in vivo and suggest the possibility that this lymphokine may be useful in the treatment of disease states characterized by excessive fibrosis.

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

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.

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.

Rapid publication. TGF-beta 1 induces bone closure of skull defects.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by increasing collagen synthesis and deposition as well as remodeling at sites of soft tissue repair. Large bony defects in the skull heal by fibrous connective tissue and never form bone unless osteoinductive bony fragments or powders are placed in the defect. We have found, however, that the single application of human recombinant TGF-beta 1 in a simple 3% methylcellulose gel to skull defects induced a dose-dependent increase in intramembranous bone formation. Complete bony bridging of defects occurred within 28 days after treatment with 2 micrograms TGF-beta 1. Sites treated with vehicle alone did not heal with bone formation but rather contained dense fibrous connective tissue between the defect margins.

Characterization of radioiodinated recombinant human TGF-beta 1 binding to bone matrix within rabbit skull defects.

Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-beta 1 induces bone closure of skull defects: temporal dynamics of bone formation in defects exposed to rhTGF-beta 1.

The temporal dynamics of bone repair in a skull defect in rabbits was examined to characterize the in vivo cellular events occurring following a single local application of recombinant human TGF-beta 1 (rhTGF-beta 1). Rabbits received vehicle or 0.4, 1, 2, or 5 micrograms rhTGF-beta 1 applied to 12 mm defects at the time of surgery. The defect sites were subsequently evaluated by radiography and qualitative and quantitative histology at time points ranging from 1 to 180 days. Based on radiographic assessment, the defect area decreased rapidly in a dose-dependent manner through 35 days after surgery in the rhTGF-beta 1-treated groups. Minimal closure occurred in sites administered vehicle control at all time points examined. Sites treated with rhTGF-beta 1 were characterized histologically by an increase in parameters of active bone formation through 49 days, including percentage osteoid surface, percentage osteoblast/total surface, and an increase in the trabecular bone volume. Bone resorption parameters were increased at 16 and 49 days with histologic evidence of remodeling from woven to lamellar bone. By 70 days, no differences were observed among the groups for parameters of either bone formation or resorption. Bone formation rate was not altered with rhTGF-beta 1 treatment at any time point. These results indicate that exogenously applied rhTGF-beta 1 stimulated the recruitment and proliferation of osteoblasts at the defect site, resulting in a rapid deposition of bony matrix, with normal remodeling processes occurring thereafter. This study supports the hypothesis that TGF-beta 1 is a potent osteoinductive growth factor in vivo and may have potential application as a therapeutic aid to nonhealing bony defects.

Relaxin alone and in conjunction with interferon-gamma decreases collagen synthesis by cultured human scleroderma fibroblasts.

Fibroblasts derived from the involved skin of scleroderma patients frequently display a phenotype of supernormal collagen expression when cultured. Fibroblasts displaying this phenotype derived from seven patients were treated with relaxin (1-100 ng/ml) and interferon-gamma (1-100 U/ml), individually and in combination, to assess the relative abilities of these cytokines to down-modulate collagen synthesis and secretion. Scleroderma fibroblasts displayed varying sensitivities to both relaxin and interferon-gamma. Relaxin (100 ng/ml) decreased expression of collagen by six of seven lines tested from 8 to 59% compared to untreated cultures. Interferon-gamma (100 U/ml) depressed collagen secretion by all seven lines in a range from 7 to 89%. When relaxin and interferon-gamma were used in combination, relaxin augmented IFN-gamma-induced decreases in collagen secretion in four of seven lines. In three of these lines, the use of relaxin in conjunction with suboptimal doses of interferon-gamma resulted in decreases equivalent to or greater than that seen with a tenfold higher concentration of interferon-gamma. This study demonstrates the ability of relaxin to directly alter the excessive collagen-producing phenotype of scleroderma fibroblasts. In addition, in some cases, combining relaxin and interferon-gamma resulted in a cooperative effect in decreasing collagen expression by scleroderma cells in vitro.

Interferons and collagen production.

The immunoregulatory, antiviral, and antiproliferative agents known as the interferons have profound effects on collagen synthesis. Interferons alpha, beta, and gamma suppress collagen synthesis by dermal fibroblasts. In addition, interferon gamma (IFN-gamma) inhibits the constitutively increased collagen synthesis characteristic of fibroblasts derived from lesions of patients with scleroderma. IFN-gamma also inhibits collagen synthesis by myofibroblasts and synovial fibroblast-like cells. Inhibition of collagen synthesis by IFN-gamma is associated with a coordinate inhibition of transcription for types I and III collagen. In addition, IFN-gamma suppresses levels of procollagen mRNA and type II collagen synthesis in human articular chondrocytes. In vivo studies in mice have demonstrated that IFN-gamma inhibits the collagen synthesis associated with the fibrotic response to an implanted foreign body, bleomycin-induced pulmonary fibrosis, and the healing response to cutaneous thermal burns. In the latter case, while collagen content of the wound scar was decreased, hyaluronic acid was increased in mice receiving IFN-gamma compared to controls. This is in accord with in vitro studies showing that, while interferons alpha and beta decrease production of glycosaminoglycans, IFN-gamma increases production of glycosaminoglycans. Of interest, acute inflammation at sites of thermal injury, or when elicited by proinflammatory agents in separate experiments, also was suppressed in mice treated with IFN-gamma. The means by which IFN-gamma inhibits collagen synthesis involves transcriptional regulation. There is a single report that interferon alpha can decrease the size of a keloid of recent onset in a human patient. Because the interferons can inhibit collagen synthesis in vivo, further studies may be warranted to evaluate the usefulness of these agents in the treatment of disease states characterized by abnormal fibrotic responses as well as their potential for altering the healing response associated with particular therapeutic interventions.

Induction of dermal and subcutaneous inflammation by recombinant cachectin/tumor necrosis factor (TNF alpha) in the mouse.

The ability of cachectin/tumor necrosis factor (TNF alpha) to induce acute dermal and subcutaneous inflammation was examined in a murine model. A number of other proteins, and diluent alone were examined as controls. After subcutaneous injection into the mouse footpad, recombinant human TNF alpha (rHuTNF alpha) induced acute inflammation with an initial marked dermal and subcutaneous neutrophil infiltrate by approximately 3 h, with a peak between 4 and 24 h and resolution by 79 h. Recombinant interleukin-2, cytochrome c, and heat-inactivated rHuTNF alpha induced negligible inflammation. Recombinant human lymphotoxin (TNF beta), another control protein, also induced acute inflammation in our system. Because TNF alpha and TFN beta are partially homologous, they may be acting through a similar mechanism. This pro-inflammatory effect of TNF alpha may result from chemotactic activity as well as by induction of secondary mediators. Inflammation induced by TNF alpha was partially suppressed by indomethacin treatment, suggesting that products of the cyclo-oxyganase pathway may mediate a portion of the inflammation involved. Five daily injections of rHuTNF alpha into the mouse footpad resulted in a predominantly mononuclear infiltrate and focal fibrosis. These results suggest that TNF alpha may be an important mediator of acute inflammation in vivo and might provide a signal for the production of collagen.

The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model.

The ability of gamma interferon (IFN-gamma) to affect cutaneous collagen synthesis in vivo was examined in a murine wounding model. Reproducible areas of full-thickness skin necrosis were produced by argon laser radiation. Mice received recombinant murine IFN-gamma (rMuIFN-gamma) (8.7 X 10(3) units/hr) over 14 d via osmotic pumps implanted subcutaneously or intraperitoneally. At 14 and 21 d after wounding, there was less fibrous tissue in healing scars of treated animals as determined by light and transmission electron microscopy. Associated with the decrease in connective tissue was an increase in the acid mucopolysaccharide content of healing scars, which was largely hyaluronate. Quantitative image analysis of electron micrographs confirmed that less collagen was present in healing scars of animals receiving rMuIFN-gamma. The mean cross-sectional area of collagen fibers was smaller in specimens from treated mice, but no difference was seen in the size of collagen fibrils. The time required to obtain full skin closure was also delayed 23%-27% in treated animals. Using this injury model, we also found that rMuIFN-gamma significantly reduced the degree of perilesional erythema surrounding the laser injury sites and, in the first 6 d after wounding, the degree of polymorphonuclear infiltrate present histologically at lesional sites. Indeed, rMuIFN-gamma also decreased the cutaneous accumulation of neutrophils induced by known proinflammatory mediators, such as interleukin 1 and activated serum. Thus, systemically administered IFN-gamma not only down-regulates collagen synthesis in the skin but also modulates in a previously unrecognized manner: neutrophil accumulation at sites of tissue injury in vivo.

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.

Specific high-affinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation.

Human peripheral blood monocytes have high affinity binding sites for 1,25-(OH)2D3 (Kd 0.14 nM, sedimentation coefficient 3.7S). Resting human peripheral blood T lymphocytes, however, do not have a demonstrable 1,25-(OH)2D3 receptor. After activation with phytohemagglutinin the T cells exhibit the receptor within 24 h, and this expression is blocked by cycloheximide. The receptor in activated T lymphocytes has a sedimentation coefficient of 3.7S and a high affinity (Kd 0.10 nM) for the ligand.

The role of relaxin in glycodelin secretion.

Glycodelin is a glycoprotein named for its unique carbohydrate structure. Glycodelin is produced by the secretory endometrium during the late luteal phase and returns to baseline during menses of the ensuing cycle, whereas in conceptive cycles it rapidly increases. Although progesterone and possibly estradiol are required for glycodelin production, they are not directly involved in the synthesis and release of this protein. Their role may be development of the endometrial secretory glandular elements, whereas other factors are required to initiate and maintain glycodelin secretion. The pattern of relaxin secretion during the luteal phase and early pregnancy is similar to that of glycodelin, but their profiles have not been determined simultaneously. To investigate the relationship of relaxin and glycodelin, two studies were conducted. In the first study, relaxin, glycodelin, and ovarian steroids were measured in daily serum samples from nonconceptive and conceptive natural cycles. Profiles of relaxin and glycodelin were closely associated, with the onset of relaxin preceding glycodelin secretion by 1-2 days in nonconceptive cycles, and the pregnancy-associated increases in each hormone differing by about 2 days. The second study tested the hypothesis that relaxin stimulates glycodelin secretion. Samples were obtained from patients injected with human relaxin for 28 days. In subjects demonstrating ovarian cyclicity, glycodelin secretion was elevated, but it was not detected in subjects without ovarian cyclicity or in placebo-treated control subjects. This study reveals a close temporal and quantitative relationship between relaxin and glycodelin profiles in the late luteal phase and early pregnancy. It also demonstrates that relaxin administration can stimulate glycodelin production from a developed endometrium. This is the first report of a nonsteroidal ovarian factor that controls glycodelin secretion, and these results suggest a function for relaxin during early pregnancy. Glycodelin is a potent inhibitor of sperm zona pellucida binding by virtue of its extensive carbohydrate structure, but it is normally at a nadir in the periovulatory period. The data demonstrate that relaxin can stimulate glycodelin secretion throughout the menstrual cycle, including the periovulatory period, when relaxin-induced glycodelin secretion could have a contraceptive effect.

Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1.

In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis.