IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

Regulation of lymphocyte tumor necrosis factor receptors by IL-2

Activated lymphocytes are known to express TNF receptors. The precise stimuli involved in induction and regulation of these receptors have not been elucidated. Our findings demonstrate that IL-2, alone and in serum-free conditions, can trigger and regulate TNF receptor expression on normal lymphocytes. Flow cytometric analyses demonstrated that the receptor was rapidly induced on CD4, CD8, and CD16+ cells after IL-2 stimulation. Receptors increased with culture duration, became maximal between days 5 and 9, and were maintained for 18 to 20 days in the presence of IL-2. By using 125I-TNF and FITC-TNF binding, we present evidence that IL-2 concentration determines the magnitude of lymphoid TNF receptor expression--influencing both the percentage of TNF-positive cells within the population and the number of receptors/cell. Collectively, our results are persuasive for consideration of IL-2 as a central mediator in the regulation of lymphocyte TNF receptors.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages.

Human alveolar macrophages (AM) are susceptible to infection with respiratory syncytial virus (RSV), but the infection is abortive after the initial cycles of virus replication. We have investigated if RSV infection of AM results in the production of cytokines TNF, IL-6, and IL-8, all of which may modulate inflammatory and immune responses to the virus, as well as may directly protect respiratory epithelial cells against spread of infection. Within 1 h after interaction with RSV, increased mRNA levels were found for all three cytokines. Peak expression of the mRNAs occurred at 3 to 6 h. The virus most effectively induced TNF mRNA expression greater than IL-6 mRNA greater than IL-8 mRNA, as compared to cytokine mRNA expression induced by bacterial endotoxin. Inactivated virus was almost as effective as live virus in inducing and maintaining increased IL-6 and IL-8 mRNA over 16 h, whereas live infectious RSV was necessary for maintaining TNF mRNA expression over the same time. Protein concentrations of the different cytokines in the supernatants of infected AM reflected the increased levels of mRNA in the cells. Despite the high levels of cytokines with possible antiviral activity (TNF and IL-6) in the AM supernatants, neither supernatants nor rTNF when added to bronchial epithelial cells protected them from infection with RSV. However, TNF, IL-1, and RSV, but not IL-6, induced IL-8 and IL-6 mRNA expression by the bronchial epithelial cells suggesting that cytokines produced by RSV-infected AM may be more important in modulating the inflammatory response in infection than directly interfering with virus infection/replication of airway epithelium.     

Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors, and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells

The effect of human TNF on cultured human microvascular endothelial (HME) cells was examined. Incubation with TNF alone transformed the morphology of HME cells from a cobblestone-like appearance into a disordered array of criss-crossed, elongated, spindle-shaped cells. Coadministration of epidermal growth factor (EGF) and TNF caused even more dramatic morphologic changes than TNF alone. Addition of basic fibroblast growth factor or insulin-like growth factor-I showed rather weak effects on cell morphology than EGF. Cell growth of HME cells was stimulated up to two-fold by TNF whereas addition of EGF additively enhanced the growth rate. Treatment of HME cells with 10 ng/ml EGF increased the binding of 125I-TNF, and Scatchard analysis showed increased TNF-R number by EGF treatment. Cellular response to TNF in the absence or presence of EGF was assessed by analyzing SDS-PAGE patterns of secreted proteins from HME cells. TNF enhanced the secretion of a protein of molecular weight 25,000 Da (25 kDa) which was found to be IL-6. In contrast, secretion of a polypeptide of 29 kDa was significantly increased when HME cells were treated with EGF, but not with TNF. Coadministration of TNF and EGF synergistically increased the secretion of the 29-kDa protein. This 29-kDa protein was found to be tissue inhibitor of metalloproteinases when assayed with antitissue inhibitor of metalloproteinases antibody. TNF and EGF also enhanced secretion of collagenase with Mr of approximately 55 kDa. Increased steady state levels of the inhibitor mRNA were observed when HME cells were treated with EGF, and coadministration of TNF further increased the levels. The morphologic transformation of HME cells by TNF and/or EGF is discussed in relation to their expression of the secreted proteins.     

Modulation of IL-2- and IL-4-induced cytotoxicities in human T helper lymphocyte clones by tumor necrosis factor-alpha.

A set of alloreactive IL-2-dependent human CD4+ 45RA-w29+56- Th cell clones was divided into two groups according to their ability to respond to IL-4 by proliferation and their susceptibility to inhibition by TNF-alpha. The latter cytokine blocked proliferative responses to IL-2 of IL-4-nonresponsive clones, but did not affect proliferation of IL-4-responsive clones. In the present communication, it is demonstrated that exposure of apparently non-cytotoxic Th cells to IL-4 resulted in the dose-dependent induction of allospecific CTX in clones previously shown to be capable of responding to IL-4 by proliferation. In contrast, IL-2 induced both allospecific and MHC-unrestricted "NK-like" CTX in both IL-4 responder and nonresponder TCC. However, coculture with IL-4 in addition to IL-2 down-regulated this induction of NK-like CTX by the IL-2 (in those clones capable of responding to IL-4). Acquisition of these two types of CTX by the same TCC was additionally modulated by TNF-alpha, which also blocked the induction of NK-like CTX but had no effect on the induction of allospecific CTX by either IL-2 or IL-4. In contrast, IFN-gamma was unable to block induction of either type of CTX in this model system. These data suggest that even at the clonal level, the relative availability of a number of different up- and down-regulatory cytokines influences the outcome of an immune response. In the present model, IL-2 up-regulates specific and NK-like CTX, the latter component of which is down-regulated by TNF-alpha or IL-4, whereas IL-4 itself can up-regulate specific but not NK-like CTX.     

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.     

Effects of eleven cytokines and of IL-1 and tumor necrosis factor inhibitors in a human B cell assay.

The effects of different recombinant human cytokines and cytokine inhibitors were compared in a culture system in which cell contact with mutant EL-4 thymoma cells of murine origin efficiently stimulates human B cell proliferation and Ig secretion in conjunction with human T cell supernatant. IL-1 alpha, IL-1 beta, TNF-alpha, and IL-2 co-stimulated B cell proliferation and IgM, IgG, and IgA secretion, whereas IL-3, IL-4, IL-5, IL-6, IFN-gamma, or GM-CSF had weak or no activity in this regard. In contrast, TGF-beta 1 was strongly inhibitory. A very strict hierarchy of cytokine interactions was found in that IL-1 was necessary to induce TNF-alpha responsiveness, and TNF-alpha the IL-2 responsiveness, of the B cells. Most likely the small number of starting B cells in the present assay (300 FACS-separated B cells/200 microliters) minimized the effects of autocrine B cell factors. IL-4 together with IL-1 induced IgE secretion, and the IgE secretion was further increased by TNF-alpha. IFN-gamma had no modulatory effect on the IL-4 dependent IgE response in this system. Pretreatment of B cells with IL-1R antagonist (IL-1ra, which binds to IL-1R) or addition of soluble TNF receptor type 1 (sTNF-R55, which binds to TNF) completely inhibited the IL-1 or TNF-alpha effects, respectively. This occurred in a specific manner; the inhibition was reversed by a large excess of cytokine. IL-1ra also inhibited a B cell response induced by PMA-preactivated EL-4 cells alone. Because B cells responding to such preactivated EL-4 cells did not acquire TNF-alpha responsiveness, no IL-1 was apparently involved under this assay condition. It appears, therefore, 1) that IL-1ra can act on B cells and 2) that this antagonist may not only block IL-1R, but may provide a direct or indirect inhibitory signal interfering even with IL-1-independent B cell activation.     

The effects of IL-1, IL-2, and tumor necrosis factor on polymorphonuclear leukocyte Fc gamma receptor-mediated phagocytosis. IL-2 down-regulates the effect of tumor necrosis factor

It has been reported that the Fc gamma R-mediated phagocytic activity of polymorphonuclear leukocytes (PMN) from patients with acute bacterial infections is markedly enhanced when compared with healthy controls. Inasmuch as several potent cytokines are known to be involved in inflammatory and infectious processes, we studied the effects of three such cytokines (IL-1 beta, IL-2, and TNF-alpha) on normal PMN Fc gamma R-mediated phagocytosis. IL-1 beta and TNF alpha both caused a significant increase in the ingestion of EIgG by adherent PMN. In combination, IL-1 beta and TNF-alpha had an additive effect, even when each was used at its optimal concentration. In contrast to the enhancing effects mediated by IL-1 beta and TNF-alpha, IL-2 alone had no significant effect on PMN phagocytosis. Notably, however, IL-2 at a concentration of 10(4) U/ml partially inhibited TNF-alpha-mediated enhancement of phagocytosis by decreasing TNF binding to the PMN cell surface. This inhibitory effect of IL-2 on TNF was reversed by anti-IL-2 antibody and mAb directed against the low affinity IL-2R (anti-Tac), whereas mAb directed against the intermediate affinity receptor (mik-beta 1) had no such effect. These findings may have important physiologic implications, because patients receiving IL-2 therapy have been shown to have increased susceptibility to infection.     

A 63 kDa tumor necrosis factor inhibitor released from a human monocytic leukemia cell line, THP-1

A human monocytic cell line, THP-1-S, was cultured in a serum-free medium. The effect of the culture supernatant of THP-1-S on the cytotoxicity of rTNF-alpha to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF-alpha when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of 125I-rTNF-alpha to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF-alpha and the inhibitory factor which formed during incubation with the culture supernatant from THP-1-S cells. However, the supernatant did not bind to or down-regulate the receptor for TNF-alpha on the cell surface of L-M-2d6 cells. This factor eluted with an apparent molecular mass of 63,000 Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF-alpha in serum-free culture conditions.     

P48 induces tumor necrosis factor and IL-1 secretion by human monocytes

Bacterial products are potent stimulators of TNF and IL-1 release, however, the factors that regulate cytokine secretion in the absence of bacterial products are not well defined. P48 is a cytokine recently identified in the supernatant of the human null cell leukemia cell line Reh, which induces differentiation and cytolytic activity in HL-60 cells. P48 has been purified to homogeneity and is distinct from TNF-alpha TNF-beta, IFN-gamma, IL-6, and macrophage CSF. In the present study we examined the ability of P48 to stimulate cytokine release by human peripheral blood monocytes. P48 stimulated the secretion of TNF and IL-1 in a dose-dependent manner. Priming the monocytes with IFN-gamma enhanced P48-induced cytokine release but was not a requirement for secretion. Cytokine secretion was in response to P48 and was not caused by endotoxin contamination. The cytokine-inducing activity of P48 was extremely sensitive to heat treatment but could not be eliminated by using polymyxin B. Polyclonal antisera to P48 completely blocked the cytokine-inducing activity. P48 may be an important new member of the cytokine network involved in the regulation of cytokine secretion by monocytes.     

Contribution of IL-6 to the antiproliferative effect of IL-1 and tumor necrosis factor on tumor cell lines

The role of IL-6 in the antiproliferative effect of IL-1 for tumor cell lines was investigated using IL-1-sensitive cell lines. Human recombinant IL-1 alpha and IL-6 both inhibited the growth of an IL-1-sensitive cloned human melanoma cell line (A375-C6). However, IL-1 has greater maximum growth inhibitory activity than IL-6. Conditioned medium of the tumor cells that were treated with IL-1 contained IL-6 as determined by ELISA. Northern blot analysis revealed that IL-6 mRNA expression increased in IL-1-treated cells. In addition, antibody against human IL-6 neutralized about 50% of the antiproliferative effect of IL-1. The growth of an IL-1-resistant clone of A375 cells (A375-C5), which cannot be shown to express any detectable IL-1R, was inhibited by IL-6 to the same degree as A375-C6 cells. The A375-C5 cell line did not produce IL-6 or increase IL-6 mRNA after stimulation with IL-1. These results indicate that IL-6 mediates in part the antiproliferative effect of IL-1 on A375-C6 cells by acting as an autocrine antiproliferative factor. IL-1 also inhibited the growth of a malignant human mammary cell line (MDA-MB-415). IL-6 exhibited only slight growth inhibition in this cell line. Neither IL-6 production nor IL-6 mRNA expression was induced in this cell line by IL-1. Antibody against IL-6 did not neutralize the antiproliferative effect of IL-1. Therefore, for MDA-MB-415 cells IL-6 appeared not to be involved in the antiproliferative effect of IL-1. These results indicate that the antiproliferative effect of IL-1 involves at least two pathways, one IL-6 dependent and another IL-6 independent. The contribution of IL-6 to the antiproliferative effect of TNF was also examined. IL-6 appeared not to play a role in the antiproliferative effect of TNF in these cell lines.     

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1beta and tumor necrosis factor alpha

The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1beta and TNFalpha on the production of IL-8. The stimulatory effect of BK on the IL-1beta- or TNFalpha-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1beta and TNFalpha on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1beta and TNFalpha increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1beta as well as the IL-1beta-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1beta- and TNFalpha-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.