Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma

Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We also studied the regulation of factor H synthesis in keratinocytes by several cytokines, namely IL-1alpha, IL-2, IL-6, TGF-beta1, TNF-alpha and IFN-gamma. Human keratinocytes expressed factor H mRNA and constitutively released small amounts of factor H protein into the culture medium. This release was strongly upregulated by IFN-gamma but not by other cytokines tested. Western blot analysis revealed that IFN-gamma augments the synthesis of both molecular species, factor H (FH; 155kDa) and factor H-like protein-1 (FHL-1; 45kDa), of factor H. Factor H released in response to IFN-gamma was functionally active. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor H and that this synthesis is regulated by IFN-gamma.     

Constitutive expression and regulation of rat complement factor H in primary cultures of hepatocytes, Kupffer cells, and two hepatoma cell lines

The 155-kd soluble complement regulator factor H (FH), which consists of 20 short consensus repeats, increases the affinity of complement factor I (FI) for C3b by about 15 times. In addition to its cofactor activity, it prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. The primary site of synthesis of FH, as well as of FI, is the liver, but the cell types responsible for the hepatic synthesis of both factors have not yet been clearly identified. In contrast to FI-mRNA, which was detectable only in hepatocytes (HC), FH-specific mRNA was identified in both HC and Kupffer cells (KC). As calculated for equal amounts of mRNA isolated from both cell types, FH-specific mRNA was found to be nearly 10-fold higher in KC than in HC, leading to the conclusion that KC are an abundant source of FH. Of the investigated proinflammatory cytokines IL-6, TNF-alpha, IL-1beta, and IFN-gamma, only IFN-gamma up-regulated FH-specific mRNA up to 6-fold in both primary HC and KC. This was also demonstrable on the protein level. However, FH-specific mRNA was not inducible in the rat hepatoma cell line H4IIE, which did not express FH-specific mRNA and could not be up-regulated in FAO cells that constitutively expressed FH-specific mRNA. This demonstrates that transformed cell lines do not reflect FH regulation in isolated primary HC. In addition to IFN-gamma, the endotoxin lipopolysaccharide (LPS) up-regulated FH-specific mRNA nearly 10-fold in KC after stimulation at concentrations of 10 or 1 ng/ml. In contrast, concentrations of up to 2 microg LPS/ml did not show any effect on HC. Our data suggest that LPS does not regulate the expression of FH in HC.     

Expression of terminal complement components by human keratinocytes

Human keratinocytes are important constituents of the skin immune system. They produce several cytokines, chemokines as well as some complement proteins. As regards soluble complement proteins, so far keratinocytes have been shown to synthesize only C3, factor B, factor H and factor I. Synthesis and regulation of synthesis of other complement proteins has not yet been studied. Here we studied the synthesis of terminal complement components, C5-C9 by human keratinocytes. We also studied the regulation of terminal complement synthesis in keratinocytes by several cytokines, namely, IL-1alpha, IL-2, IL-6, TGF-beta1, TNF-alpha, and IFN-gamma. Human keratinocytes constitutively expressed C5, C7, C8gamma and C9 mRNA but not C6, C8alpha and C8beta mRNA. They released C7 and C9, but not C5, C6 and C8. None of the cytokines tested had any influence on the synthesis of terminal components except TNF-alpha, which strongly upregulated C9 production. In conclusion, we demonstrate that keratinocytes are capable of synthesizing some of the terminal complement components and that the synthesis of C9 is regulated by TNF-alpha.     

Synthesis of complement proteins in the human chorion is differentially regulated by cytokines

The aim of the current paper was to determine the chorion's contribution to complement synthesis in the placenta and its regulation by cytokines. Biosynthetic labeling followed by immunoprecipitation with polyclonal antibodies was performed in chorionic tissue and chorion-derived cells. Eight complement proteins, factor B, C3, C1r, C1s, C1 inhibitor, factor H, C4 and C2 were detected in chorionic tissue and were secreted extracellularly. In chorion-derived cells, IL-1beta stimulated factor B synthesis but had no effect on C1r, C1 inhibitor, C1s, factor H and C4. TNFalpha had no stimulative effect on any of the complement proteins tested. In contrast, both IL-1beta and TNFalpha highly induced IL-6 secretion in chorion-derived cells, demonstrating the overall responsiveness of these cells to these stimuli. Interestingly, IFN-gamma increased the synthesis of C1s, C1r, C1 inhibitor, C4 and factor H in chorion-derived cells. The fact that the latter two complement proteins have opposing effects on immune activation of the complement cascade demonstrates the complex balance required to both maintain an ability to ward off infections but simultaneously suppress the immune response to enable tolerance of the allograft fetus.     

Human keratinocytes produce the complement inhibitor factor I: Synthesis is regulated by interferon-gamma

Extrahepatic complement synthesis is believed to play an important role in host defense and inflammation at tissue and organ level. In the epidermis the most abundant cell type, keratinocytes have been shown to produce C3, factor B and factor H. In the present study, we investigated the synthesis of factor I by human keratinocytes. We also studied whether proinflammatory cytokines IL-1alpha, IL-6, TGF-beta1, TNF-alpha and IFN-gamma regulate factor I synthesis in keratinocytes. Human keratinocytes constitutively expressed factor I mRNA and produced factor I protein. Amongst the above-mentioned cytokines, only IFN-gamma regulated the synthesis of factor I, and this effect occurred predominantly at pre-translational level. Factor I produced by keratinocytes was functionally active in cleaving C3b. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor I, and that this synthesis is regulated by IFN-gamma.     

IL-13 results in differential regulation of the complement proteins C3 and factor B in tumour necrosis factor (TNF)-stimulated fibroblasts.

IL-13, like IL-4, a product of activated T cells, has multiple biological actions, primarily on B cells and monocytes. The purpose of the present study was to compare the effects of IL-13 with those of IL-4 on the synthesis of complement proteins in fibroblasts. Dermal fibroblasts were developed from skin biopsies. Confluent monolayers were stimulated with the relevant cytokine or combinations of cytokines and biosynthetically labelled with 35S-methionine. The specific proteins were analysed using immunoprecipitation and SDS-PAGE. Addition of IL-13 to fibroblast cultures treated with TNF-alpha resulted in a dose-dependent increase in C3 protein biosynthesis and a concomitant down-regulation of factor B protein biosynthesis. In TNF-stimulated fibroblasts, the addition of IL-13, 100 ng/ml, induced a 2.45-fold increase in the synthesis of C3, while in the same cells under identical conditions the synthesis of factor B was only 42% of the level without IL-13. Similar effects of IL-13 were noted on IL-1-treated fibroblasts. These effects were specific for C3 and factor B, and no alteration of the constitutive or TNF-induced synthesis of C1s or C1 inhibitor proteins was observed. IL-13 altered the synthesis of C3 and factor B proteins also in fibroblasts stimulated with interferon-gamma (IFN-gamma) in addition to TNF, in the same direction as it did in cells stimulated with TNF alone. IL-13 has similar effects to those of IL-4 on the synthesis of C and factor B in TNF- and IL-1-stimulated fibroblasts. The observed effects of IL-13 are IL-4-independent, as anti-IL-4 antibody abrogates IL-4-induced effects, but has no effect on IL-13-induced responses. This interaction between different cytokines on the synthesis of proinflammatory and immunoregulatory proteins may have significance, particularly at local sites of inflammation, and may affect the synthesis of complement proteins in inflamed joint as in rheumatoid arthritis.     

Anti-dsDNA antibody up-regulates interleukin 6, but not cyclooxygenase, gene expression in glomerular mesangial cells: a marker of immune-mediated renal damage?

OBJECTIVE AND DESIGN: To determine whether anti-double stranded DNA antibody (anti-dsDNA) can affect the synthesis of eicosanoids and cytokines in rat glomerular mesangial cells (RMC). MATERIALS OR SUBJECTS: Glomerular mesangial cells were isolated and subcultured from Sprague-Dawley rats. Monoclonal anti-dsDNA (12B3 clone) was derived from autoimmune MRL-lpr/lpr mouse by hybridoma technology. METHODS: The mRNA expression of cyclo-oxygenase type 1 (COX-1), type 2 (COX-2), Th1 (IL-2 and IFN-gamma)/Th2 (IL-4 and IL-10) and proinflammatory (IL-6 and TNF-alpha) and anti-inflammatory (TGF-beta) cytokines of RMC +/- anti-dsDNA was detected by RT-PCR. The PGE2 production by RMC +/- anti-dsDNA was measured by ELISA. The statistical significance was assessed by non-parametric Wilcoxon signed rank test. RESULTS: We found RMC spontaneously expressed COX-1, but not COX-2. The incubation of RMC with anti-dsDNA (50 ng/ml) did not affect COX expression and PGE2 production by RMC. RMC also spontaneously expressed IL-6, TNF-alpha and TGF-beta mRNA. However, only IL-6 was up-regulated by anti-dsDNA. CONCLUSIONS: Increased IL-6 expression in RMC may become a marker of anti-dsDNA-mediated immune damage of mesangial cells.     

MHC class II-dependent activation of CD4+ T cell hybridomas by human mast cells through superantigen presentation.

Human mast cells (MC) were examined for expression of MHC class II antigens and for their ability to activate CD4+ T cell hybridomas through presentation of superantigen (SAg). HMC-1, a leukemic immature MC line expressing class II Ags, was shown to efficiently present the staphylococcal enterotoxin B (SEB) SAg to responding T cell hybridoma on treatment with interferon-gamma (IFN-gamma), which up-regulated class II molecules. The study was then extended to human normal MC. Almost pure (>99%) cord blood-derived MC (CBMC) were shown to express class II Ags (HLA-DR and HLA-DQ) and CD80, which were up-regulated by IFN-gamma treatment and, to a lesser extent, by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). CBMC directly activated CD4+ T cell hybridomas through presentation of SEB and TSST1 SAgs. The production of IL-2 required a cell-to-cell contact between T cells and CBMC and it was inhibited by anti-class II antibodies. Furthermore, an additional pretreatment of CBMC by IFN-gamma or GM-CSF or IL-4 had no effect on their presenting efficiency. This previously unknown function of human MC, i.e., MHC class II-dependent activation of CD4+ T cells, may be critical in subsequent cellular activation events because colocalization of mast and T cells is frequently observed at sites of antigen entry.     

Stimulation of Histamine Receptors of Human Monocytoid and Hepatoma-Derived Cell Lines and Mouse Hepatocytes Modulates the Production of the Complement Components C3, C4, Factor B, and C2

The influence of histamine (and the related agonists and antagonists) alone or in the presence of recombinant human interleukin 1 alpha (IL-1 alpha) and gamma interferon (IFN-gamma) was studied on the production of complement components C3, C2, factor B, and C4 in vitro with human monocytoid cell line U937, hepatoma-derived cell line HepG2, and mouse hepatocytes. Both U937 and HepG2 cells responded to histamine through H1 and H2 histamine receptors. The effect of histamine on the biosynthesis and gene expression of complement proteins was predominantly enhancing via the H1 histamine receptors and inhibitory through the H2 receptors. The actual predominance of the histamine receptor involved (and the outcome of the ligand interaction) seemed to be greatly affected by the simultaneous activation of the cells by IL-1 or IFN-gamma.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.     

Effects of associated cytokines (IL-1, TNF-alpha, IFN-gamma and TGF-beta) on collagen and glycosaminoglycan production by cultured human synovial cells

The production of collagen and glycosaminoglycans (GAG) was studied in cultured human synovial cells exposed to four cytokines, alone or in dual combination, namely interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Among these cytokines, only TGF-beta (0.1-10 ng/ml) induced a significant and dose-dependent increase of collagen synthesis in a 24-h incubation. This effect was reversed when the factor was associated with either IL-1 beta (100-500 pg/ml), TNF-alpha (1-100 ng/ml) or IFN-gamma (100 U/ml). Except IFN-gamma which clearly inhibits the collagen production, the other cytokines IL-1 and TNF-alpha were not very effective when tested separately, although they generally induced a small reduction in collagen amount. IL-1 beta and TNF-alpha were found to be more efficient than TGF-beta in stimulating the production of GAG by the synovial cells. IFN-gamma exerted an antagonistic effect on the TGF-beta-induced stimulation of GAG synthesis. TNF-alpha and IL-1 beta were shown to have an additive effect on that production. The results indicate that interactions between cytokines present in the inflamed synovial tissue may modulate their respective actions and thus introduce differentials in their effect on collagen and GAG metabolism which are responsible for the alterations of synovial extracellular matrix in rheumatoid arthritis.     

Erythropoietin enhances immunoglobulin production and proliferation by human plasma cells in a serum-free medium

The effect of recombinant human erythropoietin (Epo) on plasma cells was studied in a serum-free medium, COSMEDIUM-001 (Cosmedium). Epo enhanced both Ig production and thymidine uptake by human plasma cell lines, AF-10 and IM-9. Interleukin-6 (IL-6) enhanced both Ig production and thymidine uptake by AF-10 and IM-9, while other cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha (IFN-alpha) or IFN-gamma, failed to do so. However, the Epo effect was specific since Epo-induced enhancement of Ig production and thymidine uptake was blocked by the anti-Epo antibody but not by the anti-IL-6 antibody or the control antibody. Conversely, IL-6-induced enhancement was blocked by the anti-IL-6 antibody but not by the anti-Epo antibody. Epo also enhanced Ig production (IgG, IgM, and IgA) and thymidine uptake by PCA-1+ plasma cells generated in vitro. This enhancement was also blocked by the anti-Epo antibody but not by the anti-IL-6 antibody. Taken together, these results suggest that Epo enhances plasma cell responses by a different mechanism than does IL-6.     

Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

Activated T-lymphocytes induce growth inhibition and prostaglandin E2 release from syngeneic glomerular mesangial cells.

The concept of an active role of T lymphocytes in the initiation and development of autoimmune glomerulonephritis has gradually evolved from recent investigations. In the present study we started in a murine coculture system to directly examine cellular interactions of intrinsic glomerular mesangial cells (MC) and syngeneic T lymphocytes. Lymph node lymphocytes and, moreover, cloned T helper cells specifically affected syngeneic proliferating MC, causing growth inhibition and prostaglandin E2 (PGE2) release. The T cell specificity of mesangial cell responses was confirmed by demonstrating (i) that MC cocultured with other cell types showed no reaction and (ii) that additional activation of T lymphocytes by IL-2 or concanavalin A significantly enhanced the MC responses. Subsequently, we confirmed the presence of T cell factors in the supernatants responsible for the observed effects: interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). Experiments with combinations of recombinant mouse IFN-gamma and human lymphotoxin or TNF-alpha showed that these lymphokines could substitute for the direct T lymphocyte effects causing a synergistic growth inhibition and PGE2 release from mouse MC. The observed lymphokine activities were not due to mesangiolysis as shown by neutral red uptake of MC. Pointing to the essential role of T helper cell-specific products, IFN-gamma antibodies abolished both the IFN-gamma and the combined IFN-gamma/TNF-alpha effect. Thus, our investigations with syngeneic MC-lymphocyte cocultures demonstrated that cultured MC specifically responded to T lymphocytes and their products.     

Expression and regulation of complement factors H and I in rat and human cells: some critical notes

The complement factors I (FI) and H (FH) are complement regulatory proteins. FI, a highly glycosylated serine protease of 88 kDa cleaves the alpha-chains of both complement components C3b and C4b, thereby inactivating them. Complement FH, a glycoprotein of 150 kDa which is composed of 20 short consensus repeats synergizes with FI by increasing the affinity of FI for C3b in the C3b/FH complex by about 15-fold as compared to free C3b. Furthermore, FH prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. Both, FI and FH are mainly synthesized in the liver. According to the quantification of specific mRNA of both factors, various amounts are produced by different liver cell types, i.e. hepatocytes (HC) and Kupffer cells (KC). Investigations of cultured primary HC and KC from rat liver showed that FI is exclusively synthesized and secreted by HC whereas FH is synthesized by both HC and KC. Using quantitative-competitive PCR for the quantification of FH-specific mRNA, its constitutive rate of synthesis was found to be nearly ten times higher in KC than in HC. An extrahepatic source of both proteins are human umbilical vein endothelial cells (HUVEC) in which the synthesis of FI is upregulated by IL-6 which is in accord with the upregulation observed in rat HC and two rat hepatoma cell lines (FAO and H4IIE). Three other proinflammatory cytokines, IL-1beta, IFN-gamma and TNF-alpha, were alone or in combination, without any effect on the regulation of FI. This demonstrates that the regulation of FI is similar in HUVEC and HC. These results are in contrast to a previously described IFN-gamma-mediated upregulation of FI in HUVEC and suggest, in accordance with other investigations on extrahepatic sources of FI (e.g. myoblasts), that IFN-gamma has probably no prominent role in the regulation of FI. Instead, IL-6 appears to be the main upregulating cytokine of FI mRNA and of FI protein synthesis in HC as well as in rat and human hepatoma cells and in HUVEC. Of note are experiments by others and us who could not identify FI-specific mRNA in peripheral blood-derived monocytes, granulocytes, or B- and T-cells of man or rat and in rat peritoneal macrophages. FI-specific mRNA could also not be detected in B- or T-cell lymphoma cells, whereas FH-specific mRNA was easily detectable in both human and rat monocytes, and in rat peritoneal macrophages. These data support the notion that FI in contrast to FH is not expressed by cells of the monocyte-macrophage lineage or by other leukocytes of peripheral blood, at least in the absence of additional stimulants.     

Panax ginseng induces production of proinflammatory cytokines via toll-like receptor.

Ginseng radix, the dried root of Panax ginseng C. A. Meyer, has been shown to enhance the ability to resist microbial infections. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and interferon-gamma (IFN-gamma) are produced by macrophages treated with ginseng radix extract (GRE) in vitro as well as in vivo. However, the molecular mechanisms of the production are still not clear. In the present study, we demonstrated that production of TNF-alpha and IFN-gamma was induced by GRE in spleen cells and peritoneal macrophages from C3H/HeN mice but was impaired in C3H/HeJ mice carrying a defective toll-like receptor-4 (TLR-4) gene. In addition to these cytokines, the expression of IFN-beta and inducible nitric oxide synthase (iNOS) mRNAs was also increased in GRE-treated C3H/HeN spleen cells. We investigated the possibility that GRE contains a lipopolysaccharide (LPS)-like component. However, GRE induced production of TNF-alpha and IFN-gamma in the presence of polymyxin B, an LPS inhibitor. Furthermore, a Limulus amebocyte lysate assay showed that the endotoxin content of GRE was below the threshold level of 1 ng/ml LPS. These results suggest that GRE contains a non-LPS agent that enhances innate immunity through production of proinflammatory cytokines via TLR-4.