Lack of effect of different cytokines on expression of membrane-bound regulators of complement activity on human uveal melanoma cells

Tumor cells are protected from antibody-dependent complement-mediated lysis by membrane-bound regulators of complement activation (m-RCA). m-RCA are expressed on uveal melanoma cells. We determined whether cytokine treatment affects expression of m-RCA on these cells in vitro. m-RCA expression on uveal melanoma cell lines was studied by flow cytometry, using monoclonal antibodies directed against CD46, CD55, and CD59. Cytokines studied were interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1B (IL-1B), IL-12, and tumor necrosis factor-alpha (TNF-alpha). All three m-RCA were expressed on the uveal melanoma cell lines (CD59>CD46>CD55), although in variable amounts. With a few exceptions, the cytokines had no effect on m-RCA expression. CD55 expression was not influenced by any of the cytokines. IFN-gamma downregulated expression of CD46 on one cell line (p < 0.01). TNF-alpha upregulated CD59 expression on two of the five cell lines (p < 0.012 and p < 0.001, respectively), which effect was dose dependent. IFN-alpha, IFN-gamma, IL1-beta, IL12, and TNF-alpha had limited effects on m-RCA expression on uveal melanoma cells in vitro.     

Differential modulation of B7-1 and B7-2 isoform expression on human monocytes by cytokines which influence the development of T helper cell phenotype

The co-stimulatory molecules B7-1/B7-2 expressed on the surface of antigen-presenting cells have been suggested to influence the development of T helper 1 (Th1)-versus Th2-immune responses. These studies were conducted to elucidate the effect of immunoregulatory cytokines which influence the development of Th1/Th2 immune responses on the expression of the B7 isoforms B7-1 and B7-2 on resting and activated human monocytes and B cells. Interleukin (IL)-4 and IL-10, which induce the development of Th2 immune responses, down-regulated B7-2 and moderately up-regulated B7-1 expression on resting CD14+ monocytes in peripheral blood mononuclear cells. Interferon-gamma (IFN-gamma), which induces the development of Th1 immune responses, enhanced the expression of both B7-1 and B7-2 isoforms. Tumor necrosis factor (TNF)-alpha, which elicits both Th1- and Th2 characteristics depending on experimental conditions, down-regulated B7-2 but did not alter B7-1 expression. The effect of TNF-alpha and B7-2 expression is not mediated through endogenously produced IL-10, as addition of anti-IL-10 antibodies did not restore B7-2 expression. None of the other cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-5, IL-6, IL-12, granulocyte/macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-alpha, modulated the expression of B7 isoforms on resting monocytes. Lipoolysaccharide stimulation of monocytes down-regulated B7-2 and up-regulated B7-1 expression in a manner similar to IL-10. The expression of B7-1 and B7-2 on purified B cells were not altered by any of the cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-gamma, TNF-alpha, TGF-alpha and GM-CSF. Taken together, our results suggest that the cytokines which induce Th1/Th2 immune responses exert differential effects on B7 isoform expression on resting monocytes but have no effect on resting or activated B cells.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Cytokines modulate expression of cell-membrane complement inhibitory proteins in human lung cancer cell lines

Human lung cancers overexpress several cell-membrane complement inhibitory proteins (CIP). These complement inhibitory proteins are membrane cofactor protein (CD46), decay-accelerating factor (DAF; CD55), and CD59 (protectin). These cell-membrane proteins have a wide normal tissue distribution, are known to protect normal host cells from homologous complement-mediated lysis, and are thought to facilitate tumor escape from immunosurveillance. To study whether proinflammatory cytokines that are involved in cancer growth can modulate cell-membrane CIP expression in lung cancer cells, we studied the effect of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma on two human lung cancer cell lines. ChaGo K-1 and NCI-H596 cell lines, undifferentiated carcinoma and lung adenosquamous carcinoma, respectively, were stimulated with different cytokines, and the effects of incubation time and cytokine concentration on cell-membrane CIP expression were studied. Cell-membrane CIP expression was evaluated using flow cytometry and cytokine effect was calculated as percent change in mean fluorescence intensity of each CIP molecule from its untreated control. We found that DAF was the lung cancer cell-membrane CIP molecule that was the most responsive to cytokine stimulation. Maximal stimulatory effect was usually noted 72 h after a cytokine was introduced. In ChaGo K-1 and NCI-H596 lung cancer cell lines, IL-1alpha and TNF-alpha increased DAF expression. IL-1alpha (100 U/ml/72 h) increased DAF expression up to a maximal mean of 45 and 48%, respectively, in comparison with untreated cells. TNF-alpha (1, 000 U/ml/72 h) increased DAF expression up to a mean of 131 and 46%, respectively. IFN-gamma (1 U/ml/72 h) increased DAF expression in NCI-H596 cells up to a mean of 100%, but had a slight inhibitory effect on DAF expression in ChaGo K-1 cells, decreasing expression by a mean of 17% in comparison with untreated cells. We conclude that cell-membrane DAF expression in the studied human lung cancer cell lines is modulated by IL-1alpha, TNF-alpha, and IFN-gamma, and speculate that cytokine-mediated modulation of cell-membrane DAF in human lung cancer cells might affect lung cancer cell biology.     

Complement regulatory proteins in glomerular diseases

Complement activation plays a critical role in the pathogenesis of many forms of glomerulonephritis. Complement activation leads to tissue injury through various mechanisms including the generation of chemotactic factors and activation of the resident glomerular cells following C5b-9 insertion. Recent advances have disclosed the mechanisms of regulation of complement activation by discovery of a number of complement regulatory proteins. Decay accelerating factor (DAF), membrane cofactor protein (MCP), and complement receptor type 1 (CR1) act by inactivating C3/C5 convertase. They belong to the gene superfamily known as the regulators of complement activation (RCA), and share a common structural motif called a short consensus repeat (SCR). In contrast, CD59 works by inhibiting formation of C5b-9. The glomerulus is particularly well endowed with these membrane-bound complement regulatory proteins. DAF, MCP, and CD59 are ubiquitously expressed by all three resident glomerular cells, while CR1 is localized exclusively in podocytes. Expression of complement regulatory proteins can be changed by many factors including complement attack itself, and their expression levels are affected in various glomerular disorders. Studies utilizing cultured glomerular cells and animal models of glomerular diseases suggest important protective roles of complement regulatory proteins against immune-mediated renal injury. Recent progress in molecular biological techniques has made new therapeutic strategy feasible. Systemic administration of soluble recombinant complement regulatory proteins and local overexpression of complement regulatory proteins are promising therapeutic approaches.     

Role of liver NK cells and peritoneal macrophages in gamma interferon and interleukin-10 production in experimental bacterial peritonitis in mice

Gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-gamma, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-gamma and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-alpha levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-gamma levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-gamma producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-gamma and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.     

Type 1- and type 2-like lesional skin-derived Mycobacterium leprae-responsive T cell clones are characterized by coexpression of IFN-gamma/TNF-alpha and IL-4/IL-5/IL-13, respectively

In an earlier study, we generated a large number of Mycobacterium leprae-responsive and M. leprae-nonresponsive T cell clones (TCC) from the lesional skin of immunologic unstable borderline leprosy patients. In that study, we divided TCC into type 1- and type 2-like on the basis of their IFN-gamma and IL-4 expression. To explore whether other cytokines are coproduced along with IFN-gamma and IL-4, we investigated the secretion of a panel of other cytokines (TNF-alpha, IL-5, IL-6, IL-10, and IL-13) by a large number of these TCC. Upon analysis of 139 M. leprae-responsive TCC, we observed a positive correlation in the coproduction of IFN-gamma/TNF-alpha (r = 0.81), and in that of IL-4/IL-5 (r = 0.83), IL-4/IL-13 (r = 0.80), and IL-5/IL-13 (r = 0.82). Polarized type 1-like TCC produced dominantly IFN-gamma/TNF-alpha, and polarized type 2-like TCC predominantly IL-4/IL-5/IL-13. Most type 0-like TCC produced both sets of cytokines. In contrast, type 1- and type 2-like subsets of M. leprae-nonresponsive TCC (n = 58) did not show the same coexpression of these cytokines. Furthermore, when the differential expression of a broad panel of cytokines by individual M. leprae-responsive TCC is considered, it appeared that additional phenotypes could be recognized. These results suggested that distinct isotypes of type 1- and type 2-like T cells, based on the secretion of a panel of cytokines, may reflect M. leprae-specific characteristics.     

Differential regulation of tissue-specific lymph node high endothelial venule cell adhesion molecules by tumour necrosis factor and transforming growth factor-beta 1

Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). We have previously shown that pretreatment of LN HEV cells with pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), augments their adhesiveness for thoracic duct lymphocytes (TDL). Here we report that a mouse monoclonal antibody, 3C10, recognized tissue-specific endothelial determinants on rat LN HEV cells and blocked their adhesiveness for TDL and EL-4J cells transfected with rat L-selectin. In contrast, 3C10 antibody did not inhibit lymphocyte attachment to Peyer's patch (PP) frozen sections or cultured PP HEV cells. The antibody immunoprecipitated from LN HEV cells two proteins with apparent molecular weights of 90,000 and 50,000. The expression of 3C10 antigen on LN HEV cells was increased by incubation with TNF-alpha or IFN-gamma. Furthermore, pretreatment of cytokine-stimulated LN HEV cells with 3C10 antibody blocked TDL binding in a dose-dependent manner. In contrast, 3C10 antigen expression on LN HEV cells was significantly decreased following incubation of cells with transforming growth factor-beta 1 (TGF-beta 1). In addition, TGF-beta 1 also abrogated the adhesiveness of LN HEV cells stimulated with TNF-alpha, IFN-gamma or both cytokines. Together, these data suggest that endothelial determinants recognized by the 3C10 antibody are tissue-specific ligands for lymphocyte adhesion and cytokines such as TNF-alpha and TGF-beta differentially regulate their expression and function.     

Human eosinophils produce biologically active IL-12: implications for control of T cell responses

The present study assessed the capacity of eosinophils (EOS) to synthesize the cytokine IL-12. Blood-derived, highly purified human EOS from six atopic patients and two nonatopic individuals were treated in culture with IL-4, IL-5, granulocyte-macrophage CSF, IFN-gamma, TNF-alpha, IL-1alpha, RANTES, and complement 5a, respectively. The expression of both IL-12 protein and mRNAs for the p35 and p40 IL-12 subunits was strongly induced in all donors by the Th2-like cytokines IL-4 and granulocyte-macrophage CSF and was also moderately induced by TNF-alpha and IL-1alpha. IL-5 treatment resulted in IL-12 synthesis in four atopic donors and one nonatopic donor, whereas IFN-gamma induced IL-12 synthesis in only two atopic donors. In contrast, RANTES exclusively induced mRNA for the p40 subunit without detectable protein release, and complement 5a had no effect on IL-12 mRNA or protein expression. EOS-derived IL-12 was biologically active, because supernatants derived from IL-4-treated EOS superinduced the Con A-induced expression of IFN-gamma by a human Th1-like T cell line. This activity was neutralized by anti-IL-12 Abs. In conclusion, EOS secrete biologically active IL-12 after treatment with selected cytokines, which mainly represent the Th2-like type. Consequently, EOS may promote a switch from Th2-like to Th1-like immune responses in atopic and parasitic diseases.     

Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan

Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.