Interleukin-1 beta- and tumor necrosis factor-alpha-independent monocyte stimulation of fibroblast collagenase activity

To investigate the mechanism of cyclosporine (Cs)-induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM-LPS) that contained 665 pg/ml IL-1 beta and 16 pg/ml TNF alpha and significantly (P less than 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM-LPS-Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose-dependent manner, with MCM-LPS-Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL-1 beta and TNF alpha production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM-LPS-Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM-LPS (no Cs). This suggested that factor(s) other than or in addition to IL-1 beta and TNF alpha might be responsible for the stimulation of GN 23 collagenase activity. MCM-LPS depleted of IL-1 beta by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL-1 beta and TNF alpha, when tested alone or together at levels found in the stimulatory MCM-LPS and MCM-LPS-Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL-1 beta and TNF alpha were not necessarily involved. Cs may alter the synthesis of other collagenase-stimulating cytokines, accounting for the diminished ability of Cs-treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts

Relaxin is believed to play a role in connective tissue remodeling during pregnancy (Bell, R.J., Eddie, L. W., Lester, A. R., Wood, E. C., Johnston, P.D., and Niall, H. D. (1987) Obstet. Gynecol. 69, 585-589; MacLennan, A. H. (1983) Clin. Reprod. Fertil. 2, 77-95). In the present study, normal human fibroblasts exposed to concentrations of a synthetic bioactive relaxin peptide from 0.1 to 10 ng/ml synthesized and secreted the metalloproteinase procollagenase, which was immunoprecipitable as a doublet of 52 and 57 kDa by a monoclonal antibody to human collagenase. The stimulation in procollagenase protein expression was reflected in an elevation in procollagenase mRNA levels. Media conditioned for 48 h by relaxin-treated fibroblasts (0.1 ng/ml) contained 1.7 units/ml activatable collagenase compared with 0.2 units/ml by untreated fibroblasts. In addition, relaxin caused a modest decrease in the levels of tissue inhibitor of metalloproteinases, as detected by reverse zymography and Northern analysis. Relaxin was also a potent modulator of the collagen secretory phenotype of these fibroblasts. Relaxin at 100 ng/ml down-regulated collagen secretion by 40%. When fibroblasts were treated simultaneously with cytokines such as transforming growth factor beta or interleukin 1 beta, which stimulated collagen synthesis to at least 9-fold of basal levels, relaxin at 100 ng/ml was able to down-regulate collagen expression by up to 88%. This decrease was reflected by changes at the mRNA level. These results indicate that relaxin can cause significant collagen turnover both by stimulating collagenase expression and by down-modulating collagen synthesis and secretion.     

Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.     

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.     

Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells.

In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.     

Interferon-gamma inhibits steroidogenesis and accumulation of mRNA of the steroidogenic enzymes P450scc and P450c17 in cultured porcine Leydig cells

The inhibitory effects of recombinant porcine interferon-gamma (IFN gamma) on human CG (hCG)-stimulated testosterone production, and on mRNA concentrations of cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20lyase (P450c 17) were investigated using porcine primary Leydig cell culture as a model. After preincubation of Leydig cells for 24 h with 1000 pM IFN gamma, hCG-stimulated (10 ng/ml, 2 h) testosterone production was inhibited by 50%, whereas no significant changes were seen in hCG-stimulated cAMP production. Incubation with 10 microM 5-cholestene-3 beta,22(R)-diol or 10 microM 5-cholestene-3 beta,20 alpha-diol together with hCG (10 ng/ml, 2 h) reversed most of the inhibitory effect of IFN gamma, suggesting that IFN gamma inhibits P450scc activity, possibly by inhibiting the substrate (cholesterol) availability for P450scc. Incubation with IFN gamma also decreased basal concentrations of P450scc (45%) and P450c 17 (35%) mRNA, although these changes probably did not contribute to the decreased testosterone production. Long-term treatment with hCG (100 ng/ml, 24 h) increased P450scc mRNA (3- to 4-fold) and P450c 17 mRNA (4- to 5-fold) concentrations. Simultaneous treatment with IFN gamma attenuated these hCG-induced increases in P450scc mRNA (50%) and P450c 17 mRNA (40-100%) concentrations, as well as in testosterone production (77%). This inhibition of testosterone production could only be partly reversed by the hydroxylated cholesterol derivatives. This suggests that in addition to possible suppression of cholesterol availability, decreased P450scc and/or P450c 17 activities (through decreased mRNA concentrations) were also involved in the IFN gamma suppressed steroidogenic capacity of porcine Leydig cells during long-term hCG stimulation.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)