TIMP-1 Production Research Articles

A critical balance between endothelins and nitric oxide regulates portal resistance after endotoxin pretreatment: [formula omitted]. Johns Hopkins Medical Institutions, Baltimore, Maryland

Production of tissue inhibitors of matrix metalloproteinases (TIMPs), a family of secreted proteins with inhibitory actions on matrix metalloproteinases (MMPs), is up-regulated following nerve injuries and is suggested to have protective effects against MMP-mediated tissue damages. To clarify the extracellular signals involved in TIMP production in the brain, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. I.c.v. administration of 500 pmol/day Ala1,3,11,15-ET-1, an ETB receptor agonist, increased the level of TIMP-1 mRNA in rat hippocampus, caudate-putamen and cerebrum. Ala1,3,11,15-ET-1 increased the level of TIMP-3 mRNA in the cerebrum, but not in the hippocampus or caudate-putamen. TIMP-2 mRNA was not affected in these brain regions. Ala1,3,11,15-ET-1 also stimulated the production of TIMP-1 and TIMP-3 proteins in the cerebrum. Immunohistochemical observations in the hippocampi of Ala1,3,11,15-ET-1-infused rats showed that NeuN-positive neurons and glial fibrillary acidic protein–positive astrocytes were immunoreactive for TIMP-1. In the cerebrum, astrocytes had TIMP-1 and TIMP3 reactivity, but neurons did not. In rat cultured astrocytes, both 100 nM Ala1,3,11,15-ET-1 and ET-1 increased the mRNA levels and protein release of TIMP-1 and TIMP-3 mRNAs. The effects of ET-1 on astrocytic TIMP-1 and TIMP-3 mRNAs were inhibited by BQ788, an ETB antagonist. These findings indicate that activation of brain ETB receptors causes production of TIMP-1 and TIMP-3, and suggest the involvement of astrocytes in ET-induced TIMP production.

Human Dermal Microvascular Endothelial Cells Produce Matrix Metalloproteinases in Response to Angiogenic Factors and Migration

Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.

Gelatinase A activity directly modulates melanoma cell adhesion and spreading.

Interaction of cells with the extracellular matrix (ECM) plays an important role in the regulation of cell behavior. Formation of adhesive contacts leads to transduction of signals into the cell and results in altered gene expression and modulation of the cellular phenotype. Specific adhesive interactions of the fibronectin and vitronectin receptors with their ligands in the matrix modulates expression of ECM-degrading metalloproteases. These proteases are involved in the acquisition of the invasive phenotype by a number of cell types. The activity of matrix metalloproteases (MMPs) is reduced by endogenous inhibitors referred to as tissue inhibitors of metalloproteases (TIMPs). Alterations in the balance between the activity of MMPs and TIMPs alters cellular invasion through effects on matrix degradation. In this study we demonstrate that inhibition of endogenous gelatinase A activity in A2058 human melanoma cells results in enhanced cellular adhesion. To further explore this phenomenon, we have used retroviral infection vectors to control the amount of the MMP inhibitor TIMP-2 in human melanoma A2058 cells. Altering the production of TIMP-2 modulates not only proteolysis of the extracellular matrix, but also the adhesive and spreading properties of the cells and results in altered cell morphology. These effects of TIMP-2 appear to be mediated by inhibition of gelatinase A activity. We conclude that gelatinase A, in addition to contributing to proteolysis of ECM components, also functions to proteolyse cell surface components that mediate attachment of A2058 cells to the ECM. Thus, gelatinase A may function to modulate cell attachment and facilitate cell migration and invasion.

Establishment of a new human urinary bladder mixed carcinoma cell line UMK-1 and its cell-density-dependent secretion of gelatinases and tissue inhibitor of metalloproteinase-1.

A new human carcinoma cell line, UMK-1, was established from primary urinary bladder carcinoma with histological diagnosis of transitional cell carcinoma grade 2, with squamous and adenocarcinomatous differentiation. These histologic features were maintained in the cultured cells and in xenografted tumors in nude mice. Ultrastructurally, the cultured cells were characterized by formation of glandular structure with well-formed junctional complexes and microvilli, intracytoplasmic lumina and tonofilament bundles. Upon continuous propagation in serum-free medium, UMK-1 cells secreted gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 into the conditioned medium. The gelatinolytic activities which were inhibited by EDTA, were composed of a major band at 95 kD and a minor band at 68 kD on gelatin zymography. The measurement of TIMP-1 in the conditioned medium was performed by the one-step sandwich enzyme immunoassay system. The production of gelatinases and TIMP-1 was observed during the late log phase of the cell growth and those productions showed similar kinetics. UMK-1 cells can be used to study the interaction of tumor cell-derived gelatinolytic proteinases and TIMP-1 in cancer invasion and metastasis.

The role of IL-4 and IL-6 in IL-1-dependent cartilage matrix degradation.

The objective of this paper was to study the effects of interleukin 4 (IL-4) and interleukin 6 (IL-6) on cartilage matrix degradation, the production of chondroitin-4-sulphate (C4S) and chondroitin-6-sulphate (C6S), metalloproteinase (stromelysin 1 = MMP-3) and metalloproteinase inhibitor (TIMP-1) production. Cartilage matrix degradation was assayed the release of 35SO4 from chondrocyte cultures. TIMP-1 and MMP-3 were measured by ELISA. C6S and C4S were measured by HPLC analysis. IL-1 beta significantly enhanced C4S production and significantly suppressed C6S production. Thus, the C4S/C6S ratio was significantly enhanced by IL-1 beta, and significantly suppressed by IL-4. IL-4 removed the suppressing effects of IL-1 beta for C6S and the enhancing effects of IL-1 beta for the C4S/C6S ratio. Whereas IL-1 beta stimulated the production of MMP-3, IL-4 and IL-6 had no effect on enzyme activity. IL-4, but not IL-6, removed the enhancing effects of IL-1 beta for MMP-3. In contrast, IL-4 and IL-6 significantly enhanced TIMP-1 production in chondrocytes, IL-4, but not IL-6, also significantly suppressed IL-1 beta-mediated cartilage matrix degradation. On the other hand, IL-6 significantly suppressed spontaneous cartilage matrix degradation which is supposed to be mediated by the autocrine IL-1 mechanisms. In conclusion our results suggest that IL-4 and IL-6 both protect the cartilage matrix degradation induced by IL-1.

Progesterone increases the production of tissue inhibitor of metalloproteinases-2 in rabbit uterine cervical fibroblasts

Rabbit uterine cervical fibroblasts in culture produces tissue inhibitor of metalloproteinases (TIMP)-l and TIMP-2. When cells were treated with physiological concentrations of progesterone, the production of two TIMPs increased, and essentially all TIMP-2 was found to be complexed with promatrix metalloproteinase 2 (proMMP-2)/progelatinase A. Progesterone did not modulate the production of proMMP-2 and resulted in the increased total amount of proMMP-2-TIMP-2 complex. These observations provide the first evidence that progesterone participates in maintaining the homeostasis of connective tissue matrix in uterine cervix by augmenting both TIMP-1 and TIMP-2 production along with the known suppressive effects on the proMMP-1 and proMMP-3 production.

Expression of metalloproteinases and tissue inhibitors of metalloproteinases in human osteoblast-like cells: differentiation is associated with repression of metalloproteinase biosynthesis.

To more clearly define the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) within the human osteoblast (hOB) lineage, normal hOB and human osteogenic sarcoma cells possessing various levels of alkaline phosphatase (a marker of commitment to the osteoblast lineage) were treated with bone-resorbing agents to determine their effect on the production of interstitial collagenase, stromelysin, 72-kilodalton (kDa) gelatinase, 92-kDa gelatinase, TIMP-1, and TIMP-2. The results revealed that 1) normal hOB release copious amounts of 72-kDa gelatinase, TIMP-1, and TIMP-2; 2) hOB production of 72-kDa gelatinase and TIMP-2 is not regulated by agents that promote bone resorption (e.g. phorbol-12-myristate 13-acetate, recombinant human interleukin-1 beta, tumor necrosis factor-alpha, PTH, and vitamin D3); 3) normal hOB fail to secrete collagenase, stromelysin, or 92-kDa gelatinase when cultured on plastic or a type I collagen substratum, even in response to bone-resorptive agents or mononuclear cell-conditioned medium; 4) in contrast, certain of the osteogenic sarcoma cell populations produce collagenase, stromelysin, and 92-kDa gelatinase, especially when exposed to bone-resorbing stimuli; 5) in general, the capacity for metalloenzyme production by osteogenic sarcoma cell lines varies inversely with their alkaline phosphatase expression; and 6) the most committed (highest alkaline phosphatase) osteogenic sarcoma cell line, SAOS-2, precisely mimics the metalloproteinase profile of normal hOB. The results suggest that the expression of most metalloproteinases is under strict repression within the differentiated normal hOB, and cellular development is associated with diminished capacity to elaborate such enzymes.

Involvement of protein kinase C in the interleukin 1α-induced gene expression of matrix metalloproteinases and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibroblasts

The role of protein kinase C in the interleukin 1 (IL-1)-mediated production of pro-matrix metalloproteinases (proMMPs) and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibro-blasts has been investigated. IL-1 and a protein kinase C activator, 12- O-tetradecanoylphorbol 13-acetate (TPA) augmented the production of proMMP-1 (interstitial procollagenase), proMMP-3 (prostromelysin-1) and TIMP-1, but their effects were inhibited by the protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine in a dose-dependent manner. The suppressive effect of H-7 and staurosporine on the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 resulted from the decrease in the steady-state levels of their mRNAs. When protein kinase C was down-regulated by treating the cells with a high level of TPA, the inductive effect of IL-1 upon proMMP-3 production was reduced considerably. These results indicate that protein kinase C mediates the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 at the pretranslational level in human uterine cervical fibroblasts. On the other hand, neither IL-1 nor TPA modulated the production of proMMP-2 (progelatinase A). Both IL-1 and TPA also accelerated the productoin of prostaglandin E 2 (PGE 2) by cervical fibroblasts. However, the treatment of the cells with staurosporine in the presence of IL-1 or TPA further augmented PGE 2 synthesis, suggesting that the increased synthesis of PGE 2 by IL-1 treatment is mediated via signalling pathways distinct from those of proMMPs-1 and -3 and TIMP-1.

Metalloproteinase and TIMP expression by the human breast carcinoma cell line 8701-BC.

It is widely accepted that collagenolytic enzymes are required to facilitate the invasion and spread of tumour cells into host tissues. Immunohistochemical, zymographic and PCR analyses have produced evidence that the recently established human mammary carcinoma cell line, 8701-BC, expresses several metalloproteinases (MMP-1, -2, -9 and -10) and their tissue inhibitors (TIMP-1 and -2). Application of these different techniques has led to several observations, both complementary and dissimilar. Whereas PCR analysis showed that mRNA was detected for each of the proteins, the immunolocalization study demonstrated that MMP-1, MMP-2, MMP-9 and TIMP-1 production was restricted to only a proportion of the tumour cells, with no evidence of MMP-3 or TIMP-2 synthesis. Such observations suggested phenotypic heterogeneity within the cell line, which was further examined by use of the tumour cell clones BC-3A and BC-61 derived from the parental 8701-BC line. Comparative studies using zymography and PCR analysis demonstrated differences in MMP-2 and MMP-10 expression between the 3 cultures. The data indicate that the 8701-BC cell line retains an inherent capacity for metalloproteinase and TIMP expression, with the production of both interstitial collagenase (MMP-1) and the 2 basement-membrane-degrading enzymes (MMP-2 and MMP-9) representing an aggressive collagenolytic phenotype. The concomitant production of TIMP-1 by these cell cultures, and the apparent phenotypic heterogeneity displayed by these lines, suggest that metalloproteinase dysregulation may represent an important feature of clonal heterogeneity. Although the 8701-BC and BC-61 cells were much more invasive than those of the BC-3A clone, as judged by the penetration of "Matrigel", it has not yet been possible to relate this invasive potential to the metalloproteinase and TIMP profiles reported here for each cell line.

Selective regulation of metalloproteinase inhibitor (TIMP-1) by oncostatin M in fibroblasts in culture.

Tissue inhibitor of metalloproteinases (TIMP-1) is a potent inhibitor of activated matrix metalloproteinases (MMP) such as collagenase, stromelysin, and gelatinase, and thus helps to control extracellular matrix metabolism and deposition by connective tissue cells. Since various cytokines and growth factors can modify the production of MMP and TIMP-1, we explored the action of oncostatin M (OM), a unique lymphocyte- and monocyte-derived cytokine, on expression of these proteins. We examined the regulation of TIMP-1 expression in cultured human fibroblasts by cytokines including OM, IL-6, leukemia inhibitory factor (LIF), and IL-1 alpha. When used at levels of 5 to 50 ng/ml, OM, IL-6, LIF, and IL-1 alpha elevated the TIMP-1 expression at the RNA level in fibroblasts of lung or synovial origin. Interestingly, OM stimulation resulted in highest levels of TIMP-1 RNA and protein synthesis. However, unlike IL-1 alpha, the cytokines OM, IL-6, and LIF did not induce MMP or PGE2 release. OM also enhanced TIMP-1 mRNA levels in the H2981 lung carcinoma and HepG2 hepatoma cell lines. The results suggest that OM as well as IL-6 and LIF, other cytokines acting through similar receptor pathways, may act to inhibit net MMP activity by specifically up-regulating TIMP-1 expression. The selective induction of TIMP-1 by OM may be influential in altering matrix destruction in chronic inflammation and tumor metastasis.

Expression of Interstitial Collagenase, 92-kDa Gelatinase, and Tissue Inhibitor of Metalloproteinases-1 in Granuloma Annulare and Necrobiosis Lipoidica Diabeticorum

Granuloma annular (GA) and necrobiosis lipoidica diabeticorum (NLD) are disorders characterized by granulomatous inflammation and degenerative changes in collagen and elastic fibers. Because these disorders have often been described as being associated with altered extracellular matrix deposition, we studied the in situ expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases (TIMP)-1. Twelve lesions each of GA and NLD of different histopathologic types and durations were examined. Interstitial collagenase mRNA was seen in histiocyte-like cells in one-third of the cases of both diseases, typically in younger lesions. In GA, collagenase mRNA was only detected in lesions of the palisading type. Signal for 92-kDa gelatinase mRNA was observed in eosinophils, which were present in low numbers in five of 12 GA and three of 12 NLD samples. The signal for this enzyme and the presence of eosinophils did not correlate with the age of lesion. TIMP-1 mRNA was consistently expressed by histiocyte-like cells in both disorders. In GA, TIMP-1 mRNA was detected at the outer edge of the palisading granulomas, but in NLD, inhibitor expression was seen in the perivascular and periadnexal accumulation of inflammatory cells. Our data indicate that collagenase and TIMP are expressed early in these disorders and that these proteins may contribute to stromal remodeling associated with necrobiotic lesions. Our results further indicate that the localization of TIMP-1 production may provide a distinction between the two disorders, whereas metalloproteinase expression is not sufficiently specific to aid in the differential diagnosis of GA and NLD.

Trophoblastic localization and production of immunoreactive endothelin-1 in human placenta

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO2 values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology.Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll® gradient method and cultured on Matrigel® to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h.Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen.These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.

血管内皮細胞のＴＩＭＰ

Vascular endothelial cells (EC) have been shown to produce various biologically active substances and play an important role in both physiology and pathology of the blood vessels. EC have been shown to produce metalloproteinases (MMP) and tissue inhibitor of MMP TIMP. Vascular tissue may be composed of type N collagen which is a main conponent of vascular basement membrane and type I or II collagen, a main stromal matrix component. These different matrix conponents are digested by different MMP. Imbalance of MMP and TIMP produced by EC may cause vascular events: for example, increased MMP and decreased TIMP are a predisposing factor leading to vascular damage, inflammatory cell infiltration and metastasis of malignant cells, while decreased MMP and increased TIMP may lead to fibrosis, atherosclerosis or wound healing.It has been shown that cytokines modulate production of TIMP by EC. Our data imply that IL-1 or tumor necrosis factor inhibit TIMP production by EC, while IL-6 promote it. Therefore, regulation of matrix conponent, MMP and TIMP may be important for tissue repair, wound healing, atherosclerosis and inflammation.

Modulation by interleukin 1 and tumor necrosis factor α of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes

The actions of interleukin 1 (IL1) and tumor necrosis factor α (TNFα) on several parameters of the collagen metabolism of rabbit articular chondrocytes were studied by comparing the responses of either differentiated chondrocytes in primoculture or dedifferentiated cells in late passage culture to human recombinant (hr) IL1α, hr-TNFα and cytokine-enriched fractions of rabbit macrophage-conditioned media. In response to IL1 or TNFα, differentiated chondrocytes (i.e., producing the cartilage-specific collagens, types II and XI, but no type I), sharply reduced their synthesis of collagen, a reduction which involved both types II and XI collagens, without consistently changing their production of non-collagenous proteins; they also incorporated a smaller proportion of collagen into the matrix. Similar levels of response were obtained for hr-IL1α at picomolar and for hr-TNFα at nanomolar concentrations. However, the action of TNFα, but not of IL1, was manifested only in the presence of serum. Simultaneously, IL1, but not TNFα, induced the chondrocyte production of procollagenase (a difference which contrasted with the similar levels of procollagenase induced by both cytokines in synovial and skin fibroblasts) but neither cytokine influenced the accumulation of the collagenase inhibitor TIMP. These effects were not affected by indomethacin and are thus unlikely to be prostaglandin-mediated. During their dedifferentiation in monolayer subcultures, chondrocytes became more sensitive to the procollagenase-inducing ability of IL1 and TNFα, but their response to TNFα was lower than to IL1. They also increased their production of TIMP, which remained unaffected by the cytokines. Simultaneously, they decreased their production of collagen and substituted progressively the synthesis of fibroblast-specific collagens, types I, III and V, for types II and XI. Acting on dedifferentiated cells, even in the presence of indomethacin, IL1 and TNFα further decreased the synthesis of collagen, reducing the production of both typical type I (i.e. [ α1(I)] 2 · α2(I) molecules) and type V collagens as well as their incorporation into the matrix, but increasing the synthesis of type III collagen. Therefore not only IL1, but also TNFα can exert profound influences on the collagen degradation and repair processes occurring in the pathology of articular cartilage.

Messenger function of prostaglandins in cell to cell interactions and control of proteinase activity in the rheumatoid joint

Destruction of joint structures in arthritis may result from failure of normal mechanisms controlling interactions among cells of the various tissues of the joint. Normal synovium in culture produces less prostaglandin E (PGE) and collagenase than rheumatoid. When rheumatoid synovium is dissociated into cells, the adherent cell cultures rapidly lose the ability to synthesize large amounts of PGE and collagenase and become indistinguishable from normal synovial cells. A mononuclear cell factor (MCF) derived from supernatant media of cultured human blood mononuclear cells and a ‘synovial factor(s)’ (SF) from cultures of either normal or rheumatoid synovial fragments both stimulate production of PGE and proteinase by cells derived from human synovium, cartilage and bone. The activities of factors which may be present in these stimulatory supernatants may be unmasked in vitro when they are removed from the normal control present in vivo. Normal synovium probably contains cells which, with the appropriate stimulus, may be recruited to participate in joint tissue degredation. Normal connective tissue turnover may also be controlled by a neutral metallo-proteinase inhibitor (TIMP), which is produced in considerable amounts by normal synovium, but which cannot be detected in cultures of rheumatoid synovium. While corticosteroids inhibit the production and action of MCF and SF, they stimulate production of TIMP by normal or rheumatoid synovial tissue in vitro and may contribute to the endogenous control mechanisms. PGE may also have a modulatory role in these cellular interactions.

Production of collagenase and inhibitor (TIMP) by normal, rheumatoid and osteoarthritic synovium in vitro: effects of hydrocortisone and indomethacin.

1. The amounts of latent and active collagenase and of collagenase inhibitor (TIMP) produced by two normal, three rheumatoid and two osteoarthritic synovial specimens in culture were compared. Normal synovia produced TIMP, but little latent enzyme. Rheumatoid synovia produced higher levels of total collagenase activity than normal, of which up to 50% in one sample was present in the medium in an active form, whereas no specific inhibitory activity due to TIMP was detectable. The amounts of collagenase and TIMP produced by osteoarthritic synovia were more variable and appeared to reflect the degree of inflammation in the tissue at the time of initiating the cultures. 2. Concentrations of TIMP were usually higher in the culture media of normal, rheumatoid and osteoarthritic synovia when hydrocortisone was present. Correspondingly, amounts of total collagenase were reduced. Production of prostaglandin E (PGE) were inhibited in a dose-dependent manner by hydrocortisone. 3. Indomethacin had no consistent effect on the production of TIMP by rheumatoid and osteoarthritic synovia, although it tended to depress production of collagenase. The production of TIMP by normal synovia was depressed by indomethacin. No PGE was detectable in the media when indomethacin was present. 4. These results are consistent with those from previous animal studies, and we conclude that the balance between production of collagenase and TIMP may be critical in determining the extent of the destructive processes in arthritis. The ability of hydrocortisone to suppress production of collagenase and to increase free TIMP concentration, as well as to inhibit synthesis of prostaglandin, may explain in part how the drug exerts its therapeutic effects in patients with rheumatoid arthritis.