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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.

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.