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We have studied cells dispersed with proteolytic enzymes from rheumatoid arthritic synovectomy specimens to determine the cell type(s) responsible for joint destruction. Initially 10-50% of these cells adhered to culture dishes within 24 hr and were of two main types: small, round cells and larger, stellate cells. During 1-4 days of culture, 5-25% had Fc receptors and 25-50% showed brisk phagocytosis. Daily producition, per 10(6) cells of collagenase (EC 3.4.24.3) (after trypsin pretreatment) was up to 70 mug of collagen fibrils lysed per min at 37 degrees (70 units), of prostaglandin (PGE2), up to about 1200 ng, and of lysozyme, up to about 100 mug. Under identical conditions of assay, fibroblasts grown from explants of synovium produced no detectable collagenase or lysozyme, and PGE2 was only 2-4 ng. With the dispersed cell preparations, macrophage markers (Fc receptors and lysozyme) were undetectable after 4 days and PGE2 decreased rapidly after about 7 more days. However, collagenase production continued for 3-25 weeks, and in some cultures, after cell passage. At these later stages, large, slow-growing stellate cells were predominant and could phagocytose carbon particles if incubated for greater than 6-8 hr. Indomethacin (14 muM) inhibited PGE2 but stimulated collagenase production whereas dexamethasone (10 nM) inhibited both. Production of PGE2 and collagenase in large amounts in vitro by these cells suggests that they may be involved in joint destruction in vivo. The precise origin of these synovial cells and the mechanisms responsible for the sustained production of collagenase at a high rate remain unidentified.

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor α inhibitor (TNFα INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNFα INH purified by ion-exchange chromatographies, gel filtration, TNFα affinity column, and reverse-phase chromatography. The TNFα INH migrates with an apparent Mrof ∼33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNFα INH activity from the gel yields also a ∼33,000-Da inhibitory fraction. Besides inhibiting TNFα-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNFα INH impeded in a dose-dependent manner prostaglandin E2production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNFα INH is active on both actinomycin D-treated and untreated cells. In contrast to TNFα, TNFβ-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1α nor interleukin-1β biologic activities. The mechanism of action of TNFα INH involves blocking of 125I-TNFα binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNFα with TNFα INH increased binding inhibition, suggesting an interaction between TNFα and the inhibitor.

In many inflammatory diseases where tissue remodeling occurs, T cells are in close contact with mesenchymal cells. We investigated the effect of direct cell-cell contact between peripheral blood T lymphocytes or HUT-78 lymphoma cells and dermal fibroblasts or synoviocytes on the deposition of the major extracellular matrix components: types I and III collagen. Incubation of dermal fibroblasts and synoviocytes with plasma membrane preparations from resting T cells slightly increased the production of collagen I but did not significantly affect that of collagen III. Conversely, direct contact with either plasma membranes or fixed phytohemagglutinin/phorbol myristate acetate-activated T cells markedly inhibited the synthesis of types I and III collagen by 60-70% in untreated dermal fibroblasts and synoviocytes and by 85% in transforming growth factor beta-stimulated fibroblasts. This decrease of collagen synthesis was abrogated when fixed T cells were separated physically from fibroblasts, demonstrating that direct contact between the two cell types was necessary. This inhibition was associated with a marked decrease in steady-state levels of pro-alpha1(I) and pro-alpha1(III) collagen mRNAs. T cell contact decreased the transcription rate but did not significantly alter the stability of the alpha1(I) and alpha1(III) transcripts. Finally, using neutralizing antibodies or cytokine inhibitors we provide evidence that this inhibition of extracellular matrix production mediated by T cell contact was partially due to additive effects of T cell membrane-associated interferon gamma, tumor necrosis factor alpha, and interleukin-1alpha.

Receptor down-regulation is the result of various cellular processes including receptor internalization, new synthesis, and recycling. Monensin, a monocarboxylic acid ionophore, has been used to characterize the role of recycling in the metabolism of insulin receptors on two cultured human cell lines, U-937 and IM-9, which have different rates of internalization. The U-937 monocyte-like cell internalizes insulin receptors readily. Incubation with monensin at low doses (10(-6) to 10(-7) M) for 2 h did not affect subsequent surface insulin binding. However, the drug markedly enhanced insulin-induced down-regulation. Monensin had little effect on ligand internalization in this cell line as demonstrated by quantitative morphometric analysis. The IM-9 lymphocyte, a slow internalizer, was less sensitive to monensin exposure. Prolonged exposure (12 h) to this compound of either cell line resulted in apparent inhibition of insertion into the surface membrane of both newly synthesized and recycled receptors. When solubilization was used to quantitate total cell receptors, there was essentially no difference in intact cell binding (i.e. surface receptors) and total cell binding in IM-9 cells when insulin-induced down regulation alone was compared to insulin and monensin. By contrast for the U-937 cells there was only a small further decrease in binding when monensin was added to insulin in the solubilized cells compared to the marked augmentation of down-regulation when monensin was added to insulin in intact cells. These data demonstrate that cells with a rapid internalization rate have an associated active recycling process. By contrast cells with a slow internalization rate have a similarly slow recycling rate. This is consistent with relatively equal rates of receptor biosynthesis and plasma membrane insertion in both cell types.

To define the potential role of interleukin-6 (IL-6) and its soluble receptor alpha in cartilage metabolism, we analyzed their effects on tissue inhibitor of metalloproteases (TIMP) synthesis by synoviocytes and chondrocytes. TIMP-1 production by isolated human articular synovial fibroblasts and chondrocytes, stimulated by IL-6 and/or its soluble receptor, was first assayed by specific enzyme-linked immunosorbent assay; the slight stimulatory effect of IL-6 on TIMP-1 production by both types of cells was markedly amplified by the addition of soluble receptor, the maximal secretion being observed only at 96 h. TIMP-1 mRNA expression, determined by ribonuclease protection assay, was induced by IL-6 together with its soluble receptor, but TIMP-2 and -3 mRNAs were not affected by these factors. A specific neutralizing antibody abolished the effects of the soluble receptor. Finally, supernatant from synoviocytes stimulated by IL-6 plus its soluble receptor blocked almost completely the collagenolytic activity of supernatant from IL-1-induced synoviocytes. These observations indicate that IL-6 and its soluble receptor have a protective role in the metabolism of cartilage. Given the high levels of soluble receptor in synovial fluid and the marked induction of IL-6 by IL-1 or TNF-alpha, it is likely that IL-6 and its soluble receptor are critical in controlling the catabolic effects of pro-inflammatory cytokines.

Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form monolayers and maintain morphological features characteristic of epithelia. Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by incubation in the presence of 45 mM K+. Freeze-dry radioautographic localization of [3H]ouabain-binding sites revealed grains distributed only along that fraction of the plasmalemma directly facing the culture-dish surface. Binding and localization of [3H]ouabain were correlated with an inhibition of the Na+ pump in these cells because analysis of cellular electrolytes in control cultures versus those exposed to 10(-3) M ouabain revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a reciprocal increase in Na+. There was no change in cell H2O. Similarly, oxygen consumption was reduced by 32% after exposure to ouabain. These results provide direct evidence that in epithelial cells in culture the membrane facing the culture dish corresponds to the basolateral membrane of epithelial cells in vivo.

Marked connective tissue remodelling involves both destruction and repair in inflammatory lung diseases. Throughout the remodelling event, it was reasoned that alveolar macrophages may release substances similar to those produced by blood monocyte‐macrophages that affect fibroblast functions, ie, the interleukin 1 family of monokines (or cytokines). We have examined human alveolar macrophage cultures obtained after bronchoalveolar lavage of freshly excised lungs from heavy smokers with bronchial carcinoma. Crude culture media contained fibroblast proliferative activity and collagenase‐ and PGE2‐ production‐stimulating activity. The main peak of these biological activities was located around ‐ I8 kilodaltons (kD) on gel filtration chromatography. Resolution of this peak by high performance liquid chromatography showed the presence of three distinct peaks, with quantitative and qualitative differences in biological activities. This suggests the presence of heterogeneous factors.

Human macrophages produce in culture a factor termed mononuclear cell factor (MCF) that increases the production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. A factor with similar biologic activity is also produced by the murine macrophage cell line P388D1. By using a sequential purification scheme involving ammonium sulfate fractionation; chromatography on DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose; and discontinuous polyacrylamide gel electrophoresis, the P388D1 cell-derived, synovial cell-stimulating factor was copurified with the lymphocyte-activating factor [LAF; interleukin 1 (IL 1)]. The specific activity of the partially purified LAF (IL 1) was approximately 15,000-fold higher than that of the LAF (IL 1) in the original P388D1 cell culture supernatant. On the basis of (i) the copurification of the P388D1 cell-derived LAF (IL 1) and synovial cell-stimulating factors; (ii) the similarity in cell of origin, molecular weight, and phenylglyoxal sensitivity of human MCF and murine LAF (IL 1); and (iii) the presence of LAF (IL 1) activity in preparations of partially purified human MCF, we have postulated that LAF (IL 1) may have effects on cell targets that are nonlymphoid in nature and also that human MCF may be similar to, or identical with human LAF (IL 1). The results of these studies have raised the possibility that LAF (IL 1) may play a role in macrophage-mediated activation of synovial cells and lymphocytes which are involved in the inflammatory responses associated with rheumatoid arthritis.

The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

Urine of some febrile patients exhibits a TNF-alpha inhibitory activity (TNF-alpha INH), sensitive to heat and trypsin, with an apparent mol wt of 40-60 X 10(3) and a pI range of 5.5-6.1. As for the Il-1 INH, the TNF INH activity involves a competitive mechanism of action suggesting the existence of a family of negative feedback-regulating molecules interfering with cytokines actions.

Cachectin/TNF (tumor necrosis factor), an endotoxin-induced murine macrophage hormone implicated in the pathogenesis of cachexia and shock, has been found capable of stimulating collagenase and prostaglandin E2 (PGE2) production by isolated human synovial cells and dermal fibroblasts. This bioactivity associated with cachectin is comparable to that observed with the monokine interleukin 1 (IL-1), previously suggested as the major mediator of proteolysis. The ability of cachectin/TNF to stimulate collagenase and PGE2 production suggests that it may play a role in tissue destruction and remodelling, as these processes occur in inflammatory diseases.

Human peripheral blood mononuclear cells (lymphocyte-monocyte) in culture release a solube factor which can stimulate, up to 200-fold, production of prostaglandin E2 by isolated, adherent, rheumatoid synovial cells. Production of the factor by the mononuclear cells is enhanced by phytohemagglutinin. This factor is similar in apparent mol vt (10,000-20,000) to that which also stimulates collagenase production by the same cells.

Cultured cells depend on cytokine mediators for sustained growth and maintenance and are routinely employed in bioassays to detect and measure minute changes in biological mediators, e.g. the interferons and interleukins. We evaluated the effects of mycoplasma infection on the steady-state mRNA levels of two cytokines IL-1 alpha and beta. Noninfected human squamous carcinoma cell line A431 expressed constitutively IL-1 alpha and beta mRNA. In contrast freshly isolated peripheral blood mononuclear cells and the monocytic cell line U937 expressed abundant IL-1 mRNA only after the appropriate stimulation. Peripheral blood mononuclear cells and U937 steady-state IL-1 beta mRNA levels were considerably greater than IL-1 alpha mRNA levels, whereas nearly equivalent high levels of IL-1 alpha and beta mRNA were detected in A431 cells. Mycoplasma infection of cultured A431 cells reduced the steady-state levels of IL-1 alpha and beta mRNA. This effect was nonspecific for A431 cells as actin mRNA steady-state levels showed similar decreases to mycoplasma contamination. However, this response was cell specific since mycoplasma-free and contaminated U937 cells differed little in IL-1 mRNA expression. These results show that the response to mycoplasma infection is at least partly cell-type dependent.

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.