Human recombinant interleukin-1 alpha-mediated stimulation of procollagenase production and suppression of biosynthesis of tissue inhibitor of metalloproteinases in rabbit uterine cervical fibroblasts

Influence of human recombinant interleukin-1 (hrIL-1) on collagen metabolism was investigated with rabbit uterine cervical fibroblasts. Enzyme-linked immunosorbent assays for collagenase and tissue inhibitor of metalloproteinases (TIMP) indicated that hrIL-1 participates in both stimulation of procollagenase production and suppression of TIMP synthesis by uterine cervical cells. IL-1 did not modulate collagen synthesis. In addition, the sensitivity to IL-1 of uterine cervix from ovariectomized rabbits was augmented by estradiol-17 beta treatment. Thus it is proposed that IL-1 accelerates collagenolysis in the cervical tissue and its effect on uterine cervix is hormonally regulated.     

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)     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of metalloproteinase production, proliferation and chemotaxis of human lung fibroblasts by PDGF, interleukin-1beta and TNF-alpha

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.     

Interleukin-1 alpha and beta induce interleukin-1 beta gene expression in human dermal fibroblasts

The ability of the two forms of interleukin-1, IL-1 alpha and IL-1 beta, to induce IL-1 beta gene expression in human skin fibroblasts was studied in vitro, using Northern blot hybridization. Both recombinant IL-1 alpha and IL-1 beta caused a dramatic increase in IL-1 beta mRNA levels, IL-1 alpha being more efficient than IL-1 beta. Blockage of the prostaglandin synthesis by indomethacin reduced the basal level of IL-1 beta mRNA in control cultures and decreased also the stimulatory effect exerted by both IL-1s on IL-1 beta gene expression. These data suggest that IL-1 and prostaglandin (mainly PGE2) may act synergistically to stimulate IL-1 gene expression in dermal fibroblasts, contributing as a local amplifier system to the alterations of connective tissue in inflammatory processes.     

Alveolar type II cells inhibit fibroblast proliferation: role of IL-1alpha

Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway.     

Natural and recombinant interleukin 1 receptor antagonist does not inhibit human T-cell proliferation induced by mitogens, soluble antigens or allogeneic determinants.

Interleukin 1 receptor antagonist (IL-1ra) has been found in glycosylated form in the urine of febrile patients or of children with rheumatoid arthritis, and in the supernatant of monocytes cultured in the presence of immune complexes. It blocks competitively the binding of IL-1 alpha and beta to their receptors. Produced amongst others by mononuclear cell lines and matured monocytes and alveolar macrophages, it prevents prostaglandin E2 and collagenase production by fibroblasts and synovial cells. In mice, IL-1ra improves survival after lethal endotoxemia. In this study, both natural and recombinant human IL-1ra (rhIL-1ra) were tested in an allogeneic T-cell reaction, and in mitogen- or antigen-induced lymphocyte proliferation. Neither the natural nor the rhIL-1ra blocked T-cell proliferation, but rhIL-1ra abolished the effect of exogenous IL-1 beta. This was not due to a loss of bioactivity of IL-1ra in culture, as the IL-1ra of the supernatant still completely inhibited 125I-IL-1 alpha binding to EL 4-6.1 cells and markedly reduced PGE2 production during antigen presentation. We conclude that IL-1ra alone, even at high concentrations, is not sufficient to block human T-cell proliferation in vitro.     

Synergistic stimulation of fibroblast prostaglandin production by recombinant interleukin 1 and tumor necrosis factor

Mononuclear cell production of cytokines that stimulate fibroblast prostaglandin (PG) elaboration is an important mechanism by which mononuclear cells regulate fibroblast function. However, the soluble factors mediating these PG-stimulatory effects are incompletely understood. We characterized the effects on PG production by confluent normal lung fibroblasts of recombinant interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta) and tumor necrosis factor (TNF), alone and in combination. All three cytokines stimulated fibroblast PG production with both IL 1 peptides being significantly more potent than TNF. In addition, TNF interacted in a synergistic fashion with both IL 1 peptides to augment fibroblast PGE elaboration further. The stimulatory effects of the cytokines were almost entirely caused by an increase in PGE2 production and were reversed when the cytokine(s) were removed. These changes in PG production could not be explained by alterations in cell number and were completely negated by specific anticytokine antibodies. Recombinant gamma interferon, although synergizing with TNF in regulating other cellular functions, did not interact with TNF to augment fibroblast PGE elaboration. In addition, the synergistic interaction of IL 1 and TNF did not extend to all biologic effects of IL 1 since TNF did not augment the ability of IL 1 to stimulate thymocyte proliferation.     

Chondrocyte activation by interleukin-1: analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate

Following activation, monolayers of lapine articular chondrocytes secreted into their culture media large amounts of prostaglandin E2 (PGE2) and the neutral metalloproteinases collagenase and gelatinase. Partially purified preparations of synovial "chondrocyte activating factors" (CAF), which contain interleukin-1 (IL-1), generally proved stronger activators of chondrocytes than recombinant, human, IL-1 alpha (rHIL-1 alpha) or IL-1 beta (rHIL-1 beta). The presence of synergistic cytokines within the synovial material provides one possible explanation of this discrepancy. As first reported by K. Phadke (1987, Biochem. Biophys. Res. Commun. 142, 448-453) fibroblast growth factor (FGF) synergized with rHIL-1 in promoting the synthesis of neutral metalloproteinases. In our hands FGF alone did not induce neutral metalloproteinases and increased PGE2 synthesis only modestly. However, at doses from 1 ng/ml to 1 microgram/ml, FGF progressively enhanced the synthesis of PGE2, collagenase, and gelatinase by chondrocytes responding to rHIL-1. Acidic and basic FGF synergized equally well with both rHIL-1 alpha and rHIL-1 beta. Phorbol myristate acetate (PMA), but not the Ca2+-ionophore A23187, could substitute for FGF as a synergist. PMA alone was a poor inducer of collagenase or gelatinase but, unlike FGF, it greatly enhanced the synthesis of PGE2 by chondrocytes. Dot-blot analyses with a cDNA probe to collagenase mRNA confirmed that partially purified synovial CAF induced collagenase mRNA more effectively than rHIL-1, with rHIL-1 alpha being superior to rHIL-1 beta in this regard. The synergistic effects of both FGF and PMA upon IL-1-mediated collagenase induction were associated with increased abundance of collagenase mRNA.     

Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA).

This study examines the role of interleukin-6 (IL-6) in connective tissue metabolism. Effects of different preparations of IL-6 on production of collagenase and tissue inhibitor of metalloproteinases-1/erythroid potentiating activity production are studied in human fibroblasts, synoviocytes, and articular chondrocytes. In contrast to interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha), IL-6 does not stimulate the production of collagenase, nor does it modulate the stimulatory effects of IL-1 beta and TNF alpha on the production of this proteinase. Furthermore, IL-6 has no detectable effect on prostaglandin E2 production, an additional proinflammatory response induced by IL-1 beta and TNF alpha. IL-6, however, is identified as a potent inducer of de novo synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity in all types of connective tissue cells examined. These results define new biological activities of IL-6 and provide further insight into the regulation of connective tissues by cytokines.     

Modulation by interleukin 1 and tumor necrosis factor alpha 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 alpha (TNF alpha) 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) IL1 alpha, hr-TNF alpha and cytokine-enriched fractions of rabbit macrophage-conditioned media. In response to IL1 or TNF alpha, 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-IL1 alpha at picomolar and for hr-TNF alpha at nanomolar concentrations. However, the action of TNF alpha, but not of IL1, was manifested only in the presence of serum. Simultaneously, IL1, but not TNF alpha, 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 TNF alpha, but their response to TNF alpha 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 TNF alpha further decreased the synthesis of collagen, reducing the production of both typical type I (i.e. [alpha 1(I)]2 x alpha 2(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 TNF alpha can exert profound influences on the collagen degradation and repair processes occurring in the pathology of articular cartilage.     

A synthetic peptide corresponding to 86-93 of the human type I IL-1 receptor binds human recombinant IL-1 (alpha and beta) and inhibits IL-1 actions in vitro and in vivo.

A synthetic peptide corresponding to 86-93 of the human type I IL-1 receptor and its analogues bound human recombinant (hr) IL-1 (alpha and beta) and inhibited dose-dependently both Con A-stimulated proliferation of mouse spleen cells and hrIL-1 beta-stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in rat bone marrow cell cultures. Furthermore, hrIL-1 beta-induced mouse paw edema was dose-dependently inhibited by systemic administration (ip) of the synthetic peptide. These results suggest that one of the IL-1 binding sites of the human type I IL-1 receptor comes to the region of 86-93 and the synthetic peptide having the ability to bind hrIL-1 (alpha and beta) blocks the biological activities of exogenous hrIL-1 beta and endogenous mouse IL-1.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

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.     

Induction of interleukin-1 beta production in human dermal fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha. Involvement of protein kinase-dependent and adenylate cyclase-dependent regulatory pathways.

It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.