5-Azacytidine treatment of a murine cytotoxic T cell line alters interferon-gamma gene induction by interleukin 2

Genetic susceptibility to multiple sclerosis (MS) in Caucasians was previously shown to be correlated to the presence of given alleles at the HLA-DR and Gm loci. We now demonstrate that the humoral immune response in MS central nervous system (CNS) is modulated by both loci: the levels of IgG1 subclass and IgG1 allotypes in cerebrospinal fluid of MS patients depend on both their Gm genotype and their HLA-DR2 or HLA-DR7 phenotype. That HLA-DR molecules may either participate in a preferential recruitment of IgG1 allotype-producing B cells in MS CNS or act after such a selective homing is discussed. These results demonstrate that both HLA and Gm loci are synergistically involved in the modulation of the humoral immune response.     

Overlapping polypeptide induction in human fibroblasts in response to treatment with interferon-alpha, interferon-gamma, interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor

Cytokine-induced polypeptides were identified in whole cell lysates of human fibroblasts by computer-based analysis of two-dimensional gels with the use of the PDQuest System. Treatment with interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) enhanced the synthesis of 12 and 28 polypeptides, respectively. Exposure to interleukin 1 alpha (IL-1 alpha) or interleukin 1 beta (IL-1 beta) resulted in the increased synthesis of seven identical polypeptides. Treatment with tumor necrosis factor (TNF) at 100 U/ml led to enhanced expression of seven polypeptides, whereas exposure to TNF at 1000 U/ml increased the levels of these seven plus two additional polypeptides. The antiviral and antiproliferative effects of these cytokines in strain 153 fibroblasts were also assessed. Both IFN-alpha and IFN-gamma exhibited antiviral activity, whereas both IL-1 and TNF stimulated fibroblast growth. IFN-gamma was alone in inhibiting proliferation. Thus, although these cytokines exhibit low degrees of structural homology, they share some common functions, and a number of polypeptides were induced in common by two or more of these agents. The greatest similarities in polypeptide induction occur between IFN-alpha and IFN-gamma and between the IL-1s and TNF. However, polypeptides were also induced in common by IFN-alpha and TNF, IFN-gamma and IL-1, and IFN-gamma and TNF. These similarities in polypeptide induction may reflect the overlapping functions of these cytokines and may be indicative of common biochemical pathways in their mechanisms of action.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by interleukin 1 or tumor necrosis factor alpha in combination with cAMP inducing agents

This study demonstrates synergistic effects on Tac expression by interleukin 1 (IL-1) or tumor necrosis factor alpha (TNF alpha) in combination with the adenylate cyclase stimulator, forskolin (FK), as well as by IL-1 with TNF alpha in the human NK-like leukemic cell line YT. The maximal expression level (greater than 80% positive cells) obtained with FK plus IL-1 or FK plus TNF alpha could not be obtained by increasing the concentration of either agent alone. Furthermore, we demonstrate that Tac protein expression is correlated with increased steady-state Tac mRNA levels. Other agents that increase intracellular cAMP, such as prostaglandin E (PGE) or isobutyl-methylxanthine (IBMX), also synergized with IL-1 or TNF alpha (but not with FK). The findings suggest that cAMP plays a role in regulating Tac expression in YT cells, and that IL-1, TNF, and FK use distinct signal transduction mechanisms, all resulting in the same end point effect, namely, induction of Tac mRNA and cell surface protein expression.     

Interferons as macrophage-activating factors. III. Preferential effects of interferon-gamma on the interleukin 1 secretory potential of fresh or aged human monocytes

Human peripheral blood adherent leukocytes incubated with interferon (IFN) of three different species (alpha, beta, or gamma) show an enhanced potential of IL 1 synthesis and secretion that can be revealed by a second signal provided by endotoxins or Poly IC. We have shown that recombinant IFN-gamma, compared with recombinant IFN-alpha or purified IFN-beta, has preferential effects on IL 1 secretion in fresh monocyte cultures. We have observed a progressive and profound loss of the ability of adherent cell cultures to secrete IL 1 upon aging for 4 to 12 days in vitro. IFN-gamma was found to be more efficient than IFN-alpha or -beta at maintaining (when added at the onset of the cultures) or reversing the loss (when added on the fourth day of culture) of the IL 1 secretory function. These observations suggest that the secretion of IFN-gamma during the course of immune responses may have a critical role in feeding back the cascade of interleukins in a loop of amplification, and may thereby regulate macrophage-T lymphocyte interactions.     

Bacterial lipopolysaccharide-induced interferon-gamma production: roles of interleukin 1 and interleukin 2

Bacterial lipopolysaccharide (LPS) induced human peripheral blood mononuclear cells (PBMC) to produce interferon-gamma (IFN-gamma). Monocytes play a mandatory accessory role in this process, because purified T lymphocytes failed to produce IFN-gamma in response to LPS and the addition of 2% monocytes to T cell cultures resulted in an optimal LPS-induced IFN-gamma production. IFN-gamma production was abolished in the presence of monoclonal antibodies specific for HLA-DR antigen. Addition of exogenous interleukin 2 (IL 2) markedly enhanced IFN-gamma secretion by PBMC induced with LPS. The addition of anti-Tac antibody specific for IL 2 receptors abrogated IFN-gamma production, suggesting that an interaction of IL 2 with IL 2 receptors was involved. By using a specific antibody binding assay, LPS was shown to amplify IL 2 receptor expression on PBMC, whereas exogenous IL 2 showed only a negligible enhancing effect on the expression of its own receptors. Interleukin 1 (IL 1), a product of LPS-stimulated monocytes, potentiated IL 2-induced IFN-gamma production in the absence of LPS. Neither IL 1 nor IL 2 alone induced IFN-gamma production in purified T lymphocyte cultures. When added together, however, substantial levels of IFN-gamma were induced. An enhanced IL 2 receptor expression on T cells was also demonstrated as a result of the combined action of IL 1 and IL 2. These results suggest that induction of IFN-gamma by LPS is due mainly to the generation of IL 1 and an enhanced expression of IL 2 receptors.     

Synergistic effect of recombinant IL 2 and interferon-gamma on the proliferation of human monoclonal lymphocytes

We studied the effect of interferon-gamma (IFN-gamma) on the proliferation of lymphocytes from 10 B-type chronic lymphocytic leukemia (B-CLL) patients. In no instance did IFN-gamma induce a proliferative response whether used alone or in combination with anti-mu antibody (Ab). This was observed regardless of the responsiveness of a given patient's cells to interleukin 2 (IL 2) and to B cell growth factor (BCGF). In contrast IFN-gamma strongly and reproducibly synergized with IL 2 (but not with BCGF) to support B-CLL proliferation in five of the 10 patients. The effect of IFN-gamma was dose related and could be inhibited by an anti-IFN-gamma monoclonal Ab. A monoclonal Ab toward the IL 2 receptor molecule was also inhibitory. Preincubation with IFN-gamma potentiated the responsiveness of B-CLL to IL 2 in secondary cultures, showing that IFN-gamma exerts its effect before that of IL 2.     

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Antigen-induced human interferon-gamma production. Differential dependence on interleukin 2 and its receptor

Once stimulated with Toxoplasma gondii or cytomegalovirus (CMV) antigens, peripheral blood mononuclear cells from healthy seropositive donors secrete comparable levels of interferon-gamma (IFN-gamma). Both antigens also stimulated specific production of interleukin 2 (IL-2), a lymphokine believed to be important in IFN-gamma generation. T. gondii antigen, however, induced ninefold more IL-2 than did CMV antigen suggesting different mechanisms for antigen-stimulated IFN-gamma production. Therefore, we examined for both antigens 1) the cellular sources of IL-2 and IFN-gamma, 2) the kinetics of IL-2 production and IL-2 receptor (IL-2R) expression, and 3) the effect of antibodies to IL-2 and IL-2R on IFN-gamma secretion. For both antigens, IL-2 and IFN-gamma secretion was T4+ cell-dependent. T. gondii antigen induced high levels of IL-2 at 24 hr which increased further at 48 hr, and IFN-gamma production was strongly inhibited by antibodies to both IL-2 (90 +/- 2%) and IL-2R (80 +/- 5%). In contrast, CMV antigen stimulated low levels of IL-2 at 24 hr which declined still further by 48 hr, and CMV-stimulated IFN-gamma generation was appreciably less well inhibited by antibodies to IL-2 (47 +/- 2%) and IL-2R (31 +/- 8%). These results suggest the possibility of two mechanisms for antigen-induced IFN-gamma production--one primarily dependent on and the other largely independent of IL-2 and its receptor. Both mechanisms, however, require the activity of sensitized T4+ cells.     

Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts

Human endothelial cells and dermal fibroblasts both expressed a membrane-associated interleukin 1 (IL-1) activity when stimulated with either recombinant tumor necrosis factor (TNF) or recombinant lymphotoxin but stimulated endothelial cells expressed significantly more membrane IL-1 per cell than did fibroblasts. Lipopolysaccharide induced membrane IL-1 activity on endothelial cells but not fibroblasts. Interferon-gamma treatment of endothelial cells and fibroblasts had no direct effect on membrane IL-1 expression and little effect when used as a pretreatment for TNF or lipopolysaccharide stimulation. Endothelial cell membrane IL-1 activity was induced within 24 hr of culture with TNF or lipopolysaccharide, and increased up to 72 hr of incubation. Antibodies raised against human monocyte-derived IL-1 species neutralized the membrane IL-1 activity of TNF-stimulated endothelial cells. Both absorption studies and neutralization with specific sera indicated that endothelial cell membrane IL-1 is structurally related to IL-1 alpha. Endothelial cells expressed both IL-1 beta mRNA in response to TNF, lymphotoxin, and recombinant IL-1 species, as detected by Northern blot analysis. These studies demonstrate that endothelial cells can be activated to express a cell-surface IL-1 activity which is structurally, as well as functionally, related to the secreted form of IL-1.     

Effects of immune complexes on production by human monocytes of interleukin 1 or an interleukin 1 inhibitor

The objective of these studies was to examine the ability of phorbol myristic acetate (PMA), Fc fragments, and various forms of immune complexes to induce the production by human monocytes of factors stimulatory to chondrocytes or thymocytes. All of these materials were prepared free of detectable contamination with bacterial lipopolysaccharides (LPS) at the level of less than 0.1 ng/ml. Supernatants and lysates from stimulated human monocytes were assayed for their ability to induce collagenase production in cultured rabbit articular chondrocytes or to augment mitogen-induced proliferation of murine thymocytes. The activity detected by these assays exhibited an m.w. of approximately 15,000, and electrophoretic heterogeneity in the pH ranges of 5 to 5.5 and 6.5 to 7.0, characteristics of human interleukin 1 (IL 1) or IL 1-like factors. Monocytes cultured with 2 ng/ml LPS produced chondrocyte and thymocyte stimulatory factors. PMA, Fc fragments, and soluble, precipitated, particulate, or adherent immune complexes were inactive in stimulating the monocytes. However, complement fixation by precipitated immune complexes did generate activity capable of inducing monocytes to synthesize and secrete chondrocyte and thymocyte stimulatory factors. Adherent immune complexes and PMA were biologically active, as evidenced by induction of superoxide generation in the human monocytes. Supernatants from monocytes cultured on adherent immune complexes contained a factor inhibitory to chondrocyte and thymocyte responsiveness. This factor had a m.w. approximately 22,000 and appeared to inhibit specifically IL 1 stimulation, not interleukin 2 stimulation or cell proliferation. It was concluded that PMA, Fc fragments, and various forms of immune complexes in the absence of complement do not induce IL 1 production in human monocytes. However, complement fixation by immune complexes does lead to activation of monocytes to produce IL 1. Monocytes cultured on adherent immune complexes produce an IL 1 inhibitor.     

Synergistic increases in IL-1 synthesis by the human monocytic cell line THP-1 treated with PAF and endotoxin

The capacity to stimulate cytokine release may be important to the long-term effects of platelet-activating factor (PAF), which has a very short half-life. Previous studies have shown that PAF stimulates interleukin 1 (IL-1) release by human monocytes. IL-1 and other cytokines produced in response to PAF may be important to the long-term effects of this short-lived lipid. The THP-1 human monocytic leukemia cell line, was used to study the mechanism by which PAF stimulates IL-1 release. PAF stimulates the release of IL-1 beta activity into THP-1 cell supernatants with a multiphasic dose-response curve very similar to that for monocytes. When THP-1 cells are treated with PAF and LPS in combination, these two stimuli interact synergistically to greatly increase the release of IL-1 activity. To assess the effect of PAF on IL-1 beta synthesis, THP-1 cell pellet proteins were separated by SDS-PAGE, blotted, and immunostained to detect IL-1 beta. Immunostaining revealed that PAF increases intracellular IL-1 beta precursor and that the combination of PAF and LPS increases IL-1 beta precursor synergistically. PAF increases IL-1 beta release mainly by increasing IL-1 beta synthesis.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.     

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.     

Interleukin 5 up-regulates high-affinity interleukin 2 receptor expression by human resting peripheral T cells: a comparison with the effect of interleukin 4 on B cells.

The separate regulation mechanisms of cytokines on two classes of interleukin 2 receptors (IL-2R) on human peripheral T and B cells were analyzed by a flow cytometer using a double stain with IL-2R alpha (55 kilodalton Tac) and IL-2R beta (75 kilodalton mik beta 1, mik beta 3). Although the expression of IL-2R alpha by T cells was slightly enhanced by IL-2 and IL-4, expression of the beta chain was diminished by both cytokines. IL-5 by itself did not alter the expression of either IL-2R alpha or beta, but preculturing with IL-2 for 24 h followed by IL-5 for another 24 h induced an increase in IL-2R alpha expression and in simultaneous alpha/beta chain expression. Increased numbers of high-affinity IL-2R were confirmed by 125I binding assays. On B cells, IL-4 increased alpha, beta, and simultaneous alpha/beta chain expression, but IL-4-treated B cells did not show an increased number of high-affinity IL-2R.     

Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro

Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption.