Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.     

Cartilage and joint inflammation. Regulation of IL-8 expression by human articular chondrocytes.

Injury to cartilage is a recognized sequela of neutrophil activation in arthritic joints. This study examined the possibility that chondrocytes may play a direct role in intraarticular neutrophil activation. We demonstrate that IL-1 beta-stimulated primary and subcultured human articular chondrocytes, express the gene for the potent neutrophil chemotactic and activating cytokine, IL-8. Expression of IL-8 mRNA is also inducible by TNF-alpha and LPS and, to a lesser degree, by the chondrocyte growth factor, transforming growth factor-beta, but not by platelet-derived growth factor, acidic and basic fibroblast growth factor, or epidermal growth factor. Analysis of IL-1 beta-stimulated cartilage organ cultures by in situ hybridization demonstrates that chondrocytes in all zones of cartilage are rapidly induced to express the IL-8 gene in high copy number. Metabolically labeled IL-1 beta-stimulated chondrocytes synthesize IL-8 de novo, which comigrates on SDS-PAGE with IL-8 produced by synovial fibroblasts. Furthermore, the conditioned media of IL-1 beta-stimulated chondrocytes and cartilage organ cultures contain neutrophil chemotactic activity which is completely neutralized by a specific antibody to IL-8, establishing that a bioactive form of IL-8 is the major secreted neutrophil chemotactic factor. By using a specific RIA, we demonstrate that not only IL-1 beta, but also TNF-alpha and LPS can induce abundant IL-8 secretion from chondrocytes. In conclusion, articular chondrocytes are readily inducible to express the IL-8 gene and secrete biologically active IL-8 which can promote neutrophil-mediated inflammation and cartilage destruction.     

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.     

Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin 1 or tumor necrosis factor

Normal human dermal fibroblasts rapidly expressed (less than 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type.     

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed.CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA.These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.     

Soluble interleukin-6 receptor alpha inhibits the cytokine-Induced fractalkine/CX3CL1 expression in human vascular endothelial cells in culture

Soluble form of IL-6 receptor alpha (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor beta chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma. IL-1alpha-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the adhesion to monolayers stimulated with IL-1alpha. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1alpha or IFN-gamma was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

The effect of IL-1beta on the expression of inflammatory cytokines and their receptors in human chondrocytes

Cytokines released at sites of inflammation and infection can alter the normal processes of cartilage turnover, resulting in pathologic destruction or formation. Interleukin (IL)-1beta plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In the present study, we examined the effect of IL-1beta on the expression of IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor-alpha (TNF-alpha), and their receptors in human chondrocytes. The cells were cultured either with or without 100 U/ml of IL-1beta for up to 28 days. The level of expression of the cytokines and their receptors was estimated by determining mRNA levels using real-time PCR or by determining protein levels using ELISA. The expression of IL-1beta, IL-8, and TNF-alpha markedly increased in the presence of IL-1beta after day 14 of culture. The expression of IL-6 and IL-11 increased greatly in the presence of IL-1beta on day 1 and after day 14 of culture. The expression of IL-1beta, IL-8, IL-11, and TNF-alpha receptors significantly decreased in the presence of IL-1beta after day 14 of culture, whereas the expression of IL-6 receptor significantly increased. The expression of these cytokines, except for IL-6, decreased with the addition of human IL-1 receptor antagonist. These results suggest that IL-1beta promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6 receptor expression in the cells.     

Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules

Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1-10 micrograms/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1alpha and IL-1beta, and IL-6, IL-8 and TNF-alpha as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria.     

Interleukin-6 expression and regulation in rat enteric glial cells

As yet, little is known about the function of the glia of the enteric nervous system (ENS), particularly in an immune-stimulated environment. This prompted us to study the potential of cultured enteroglial cells for cytokine synthesis and secretion. Jejunal myenteric plexus preparations from adult rats were enzymatically dissociated, and enteroglial cells were purified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with recombinant rat interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, and IL-6 mRNA expression and secretion were assessed using RT-PCR and a bioassay, respectively. Stimulation with TNF-alpha did not affect IL-6 mRNA expression, whereas IL-1beta stimulated IL-6 mRNA and protein synthesis in a time- and concentration-dependent fashion. In contrast, IL-6 significantly and dose-dependently suppressed IL-6 mRNA expression. In summary, we have presented evidence that enteric glial cells are a potential source of IL-6 in the myenteric plexus and that cytokine production by enteric glial cells can be regulated by cytokines. These findings strongly support the contention that enteric glial cells act as immunomodulatory cells in the enteric nervous system.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

Characterization of fibroblast proliferation factors elaborated by antigen- and mitogen-stimulated guinea pig lymph node cells: Differentiation from lymphocyte-derived chemotactic factor for fibroblasts,lymphocyte mitogenic factor,and interleukin 1

Guinea pig lymph node cells stimulated in culture by T-cell mitogens or sensitizing antigens release ~60,000- and ~16,000-mol wt proteins that induce normal guinea pig fibroblasts to proliferate in vitro. These fibroblast proliferation factors can be separated from lymphocytederived chemotactic factor for fibroblasts and from lymphocyte mitogenic factor by gel filtration employing Sephadex G-100. The 16,000-mol wt fibroblast proliferation factor was found to coelute with interleukin 1 (IL 1) from gel filtration columns. When the 16,000 molecular weight factor was further analyzed by anion exchange-high-performance liquid chromatography five major peaks containing IL 1 activity were obtained, only one contained fibroblast proliferation activity, suggesting forms of IL 1 exist that are not mitogenic for fibroblast. Occasionally, a large-molecular-weight inhibitor of fibroblast proliferation was detectable in void volume fractions from gel filtration of supernatant from antigen-stimulated lymph node cell cultures. This inhibition was accompanied by gross aggregation of fibroblasts. These studies suggest that fibroblast accumulation at sites of certain cell-mediated immune reactions in vivo may in part be attributable to the release of mediators by lymphocytes and, or macrophages that induce fibroblast growth.     

Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.     

Lymphocyte chemotactic activity of human interleukin 1

In the cellular immune response, there is an accumulation of mainly nonantigen-specific mononuclear cells that presumably is dependent on the local secretion of chemotactic factors. In view of the presence of large numbers of macrophages early in the delayed hypersensitivity response, the possible role of these cells in the chemotaxis of lymphocytes was investigated by studying the chemotactic activity of purified human interleukin 1 (IL 1) on T and B cells. Chemotactic activity for T and B cells was observed, the effect on B cells being greater than on T cells. At low concentrations (less than 1 U/ml), IL 1 had predominantly chemotactic activity for B cells and chemokinetic activity for T cells. At high concentrations (10 to 20 U/ml), IL 1 had pure chemotactic activity for both cell types. A relationship was found between levels of migration of T and B cells and mouse thymocyte proliferation induced by purified IL 1 and by lipopolysaccharide-stimulated monocyte supernatants. The principal peaks of both activities were found in 16,000 to 18,000 m.w. fractions. In additional studies, the chemotactic response to IL 1 was inhibited by preincubation of T and B cells with IL 1 or stimulated monocyte supernatant, demonstrating the role of binding of IL 1 in the chemotactic response.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Chemotactic cytokines: the role of leukocytic pyrogen and epidermal cell thymocyte-activating factor in neutrophil chemotaxis

Macrophage-derived leukocytic pyrogen (LP) is thought to be similar if not identical to interleukin 1 (IL 1). In addition to macrophages, keratinocytes produce a factor that has similar biologic and biochemical characteristics to IL 1, called epidermal cell thymocyte-activating factor (ETAF). Because many diseases affecting the skin are characterized by infiltration of polymorphonuclear leukocytes (PMN) and some of these disorders are associated with fever, we investigated whether ETAF like LP had pyrogenic activity, and whether ETAF or LP were chemoattractants. ETAF or LP purified by column chromatography and isoelectric focusing were found to have chemotactic activity for PMN. The fractions containing maximal chemotactic activity corresponded to maximal fever-inducing activity and maximum thymocyte-activating activity. Furthermore, the chemotactic activity of ETAF and LP could be blocked by an antibody directed against LP. The results of this study indicate that these mediators, which arise from distinct cell populations, are closely related and may play a vital role in skin as well as distant inflammatory and immunologic events.     

Expression of interleukin 1 receptors on human peripheral T cells

The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes.