Inhibition by IL-1 of endothelial cell activation induced by tumor necrosis factor or lymphotoxin

Previous studies in this and other laboratories have demonstrated that IL-1, lymphotoxin (LT), and TNF rapidly stimulate a number of proinflammatory properties in cultured endothelial cells (EC) including cell-surface procoagulant activity and increased adhesivity for lymphocytes, monocytes, and polymorphonuclear leukocytes. In addition, we have demonstrated that LT and TNF, but not IL-1, stimulate increases in EC RNA synthesis, protein synthesis, and cellular volumes, changes which may correspond to the hypertrophy of EC seen at sites of inflammation in vivo. It is reported here that both human rIL-1 alpha and rIL-1 beta totally inhibit the increases in EC RNA synthesis, protein synthesis, and cell volumes induced by either TNF or LT. As little as 0.1 ng/ml of either IL-1 was sufficient to totally block the activation of EC induced by 100-fold higher concentrations (10 ng/ml) of either LT or TNF. The relevance of these findings to the regulation of inflammatory responses is discussed.     

Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1

We have previously characterized more than 20 proteins induced by the immunoregulatory lymphokine IFN-gamma in human fibroblasts by their m.w. and isoelectric points determined in two-dimensional gels. Some of these proteins are induced uniquely by IFN-gamma, whereas others are also induced by IFN-alpha, TNF, or IL-1. Recent technologic advances have allowed us to begin to rapidly identify proteins induced by IFN-gamma and other cytokines by sequencing the induced proteins from blots of preparative two-dimensional gels of total cell lysates. In this study, we show that the approximately 21 kDa, isoelectric point greater than 7 protein induced by IFN-gamma is manganese superoxide dismutase (Mn-SOD), a mitochondrial protective enzyme encoded by a nuclear gene. Mn-SOD is induced by IFN-gamma and also by TNF in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of Mn-SOD mRNA is a primary, rapid, and dose-dependent response to IFN-gamma. In ACHN renal carcinoma cells, Mn-SOD mRNA and protein are induced synergistically by IFN-gamma in combination with either TNF or IL-1, and the induced protein is enzymatically active. IFN-gamma and TNF together induce Mn-SOD mRNA by more than 100-fold relative to its level in untreated ACHN cells. The induction of Mn-SOD by IFN-gamma and its synergistic induction by IFN-gamma in combination with TNF and IL-1 should protect healthy cells from the toxicity of O2- during an immune response, and may provide a mechanism for selective killing of infected cells.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

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.     

Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.

The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation.     

A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide

The alteration in the surface of endothelial cells (EC) in response to cytokines is likely to be of great importance to the regulation of cell migration and thereby to the evolution of inflammatory processes. We have generated three mAb against cytokine inducible Ag on EC. Whereas mAb 1.2B6 and 6.5B5 were found to react with ELAM-1 and ICAM-1, respectively, mAb 1.4C3 reacted with a novel molecule that showed a different pattern of expression from ELAM-1 or ICAM-1 after stimulation of EC by TNF, IL-1, or LPS. Like ELAM-1, the 1.4C3 Ag was minimally expressed on resting EC, whereas ICAM-1 was moderately expressed. After stimulation with IL-1, TNF, or LPS, ELAM-1 expression was maximal after 4 to 6 h, 1.4C3 Ag after 6 to 10 h, and ICAM-1 after 10 to 24 h. The duration of 1.4C3 expression was intermediate between ELAM-1 and ICAM-1, and was more prolonged in response to TNF than IL-1 or LPS. Whereas the expression of the three Ag showed a similar dose response to varying concentrations of IL-1 or LPS, EC required a 10-fold higher concentration of TNF for half maximal expression of ELAM-1 than for half maximal expression of 1.4C3 Ag or ICAM-1 (5 ng/ml compared to 0.5 ng/ml). Of the three Ag, only ICAM-1 was enhanced by IFN-gamma. SDS-PAGE under reducing conditions showed the 1.4C3 Ag to migrate as a single band with a relative molecular mass of approximately 95 kDa. mAb 1.4C3 adds to our understanding of the kinetics of the EC response to different cytokines and will be useful in studying the regulation of EC activation. Furthermore, the 1.4C3 molecule may have an important role in leukocyte-EC interactions.     

Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon

We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.     

Stimulation of endothelial cell binding of lymphocytes by tumor necrosis factor

Lymphokines and monokines have been reported to affect endothelial cell (EC) morphology and function. In experiments here described, we have demonstrated that recombinant tumor necrosis factor (TNF) stimulates the adhesion of T lymphocytes to confluent monolayers of human umbilical vein EC. The increase in adhesion induced by TNF was EC-specific inasmuch as preincubation of the lymphocytes with TNF did not alter binding, and preincubation of human dermal fibroblasts with TNF did not increase their inherently low adhesiveness for lymphocytes. Stimulation of T-EC binding occurred after treatment of the EC with as little as 0.01 U/ml (1 pg/ml) of TNF. In kinetic experiments, preincubation of EC with TNF for 4 hr resulted in optimal adhesion. TNF-treated EC retained their increased adhesiveness after fixation with paraformaldehyde, suggesting that TNF stimulated binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to the lymphocyte function-associated antigen 1 alpha- or beta-chains on the T cell markedly inhibited unstimulated T-EC binding, such antibodies had no effect on the increase in EC adhesiveness induced by TNF, indicating that the increased binding resulted from the generation of an alternate binding receptor on the EC membrane. These findings provide additional evidence that cytokines participate in the mobilization of mononuclear cells in the chronic inflammatory reaction by stimulation of the adhesiveness of endothelium for circulating lymphocytes.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

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.     

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.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.     

IL-7 is requisite for IL-1-induced thymocyte proliferation. Involvement of IL-7 in the synergistic effects of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor with IL-1.

In the absence of artificial comitogens murine thymocytes proliferate significantly in response to IL-1 at high but not at low cell densities. This observation has led us to examine a possible indirect mechanism requiring other thymocyte-growth factors, such as IL-2, IL-4, IL-6, and IL-7, in this phenomenon. Our data provide evidence that IL-7 is requisite for the IL-1-induced proliferative response because on the one hand the growth-promoting activity of IL-1 is completely inhibited by an anti-IL-7 mAb, and on the other hand IL-7 synergizes with IL-1 on thymocyte growth. This synergy is observed even at concentrations at which IL-7 is not detected in the pre-B cell proliferation assay, and results, at optimal doses, in TdR incorporation levels similar to those attained in response to IL-1 + IL-2. The anti-IL-7 mAb acts in a dose-dependent manner and does not affect other activities of IL-1, such as its capacity to sustain the growth of the U373 astrocytoma cell line. It is also noteworthy that this mAb does not significantly impair thymocyte growth in response to IL-2 and that the growth-promoting activity of IL-1 is not affected by neutralizing mAb against IL-2, IL-4, and IL-6. In addition, we show that the potentiating effect of granulocyte-macrophage (GM)-CSF and TNF-alpha on IL-1-induced thymocyte growth is dependent on IL-7 because i) the anti-IL-7 mAb abrogates the respective synergistic interactions and ii) both factors potentiate the proliferative response to IL-7. Finally, depletion of thymocyte suspensions for Ia+ Mac-1+ accessory cells results in a considerable decrease in IL-1- and IL-1 + GM-CSF-induced TdR uptake, whereas IL-7-induced growth remains unchanged. Taken together, these results support the notion that, in the absence of artificial comitogens, thymocyte proliferation in response to IL-1 alone or in combination with GM-CSF is dependent on accessory cell-derived IL-7.