Interleukin 1-driven secretion of interleukin 2 is highly temperature-dependent

To investigate the temperature dependence of T lymphocyte activation, we have examined the IL 1-induced secretion of IL 2 by the murine T lymphoma cell line LBRM-33-1A5. During 24-hr incubations, there are modest increases in IL 2 secretion as culture temperatures are increased from 33 degrees to 37 degrees C, but IL 2 secretion declines at higher temperatures. However, the kinetics of IL 2 release during the first 8 hr of culture are highly temperature-dependent. The rate of IL 2 release increases linearly with temperature over the range from 33 degrees to 41 degrees C, demonstrating temperature coefficients (Q10) greater than 70. In contrast, IL 2-promoted proliferation of a continuous T cell line is much less temperature-dependent with Q10 values of less than 4.0.     

Interleukin 2 receptor expression by T cells in human aging

Aged individuals have depressed cell-mediated immunity and diminished T cell proliferation to mitogenic and antigenic stimuli. Because T cell responses depend on the surface expression and normal function of interleukin 2 receptors, we measured the quantities and affinities of cell surface IL-2R and the amount of soluble IL-2R alpha chain (p55) release in vitro in PHA-stimulated mononuclear cells from healthy aged (greater than or equal to 65 years old) and young (less than or equal to 39 years old) donors. At the peak of the PHA response, the fraction of cells expressing IL-2R alpha chain (CD25+) was lower in the aged (43% vs 56%, P = 0.033). Relative to the lower proliferation and CD25 expression, old donor cells released unexpectedly high quantities of soluble alpha chain into culture supernatants. However, the average affinities and the mean numbers of high- and low-affinity surface receptors per CD25+ cell were equivalent in cells from eight pairs of aged and young donors (1850 vs 1586 high affinity, and 20,655 vs 23,466 low affinity, P greater than 0.2 for both). The soluble IL-2R released by stimulated cells had no effect on proliferative responses, because addition of saturating doses of exogenous recombinant IL-2 did not increase cellular proliferation, and addition of soluble anchor-minus recombinant IL-2R alpha chain did not suppress it. These results indicate that in healthy older individuals, diminished numbers of T cells can be induced to express cell surface IL-2R following mitogenic stimulation, although aged CD25+ can express a normal complement of IL-2R molecules. In the aged, either CD25+ cells release excessive quantities or a subset of cells synthesizes and releases soluble IL-2R alpha chain into the extracellular environment without expressing it on the cell surface.     

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

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.     

Interleukin 2-dependent phosphorylation of interleukin 2 receptors and other T cell membrane proteins

The addition of IL 2 to Con A-activated splenic T cells induced the rapid and time-dependent phosphorylation of membrane proteins with m.w. of 115,000 to 105,000, 90,000, and 66,000, and to a lesser extent 55,000 to 58,000, 40,000, and 34,000. Immunoprecipitations conducted with an anti-IL 2 receptor antibody indicated that the murine IL 2 receptor (55,000 to 58,000) was included in the set of IL 2-dependent phosphoproteins. Phosphorylation of these same proteins was also seen after IL 2 treatment of PHA-activated T cells and of the IL 2-dependent line CTLL-2. Membrane phosphorylation was dependent on physiologically relevant IL 2 concentrations (0.2 to 1 ng/ml), and was detected as early as 1 min after IL 2 addition, with maximal levels of phosphorylation achieved by 15 min. In contrast to these observations, the pattern of cytoplasmic protein phosphorylation remained unchanged after IL 2 addition, although IL 2 did augment the level of preexisting cytoplasmic phosphorylation induced by lectin. The pattern of membrane protein phosphorylation induced by IL 2 also overlapped in part with that induced after stimulation of Con A-activated T cells with the phorbol ester PMA. IL 2-stimulated phosphorylation was inhibited by the addition of agents that both stimulate cyclic AMP-dependent protein kinases and block lymphocyte mitogenesis. No effect was seen upon addition of agents that enhance cyclic GMP-dependent protein kinases. These observations support a role for specific membrane as opposed to cytoplasmic protein phosphorylation in the regulation of lymphocyte growth by IL 2, and also suggest that protein kinase A, and perhaps protein kinase C, participate as regulators of the IL 2 signaling mechanism.     

Interleukin 1 responsiveness and receptor expression by murine TH1 and TH2 clones

Murine Th1 and Th2 T cell lines differ in their responses to interleukin 1 (IL 1). Therefore, we examined two T-cell lines, D10.G4.1 (Th2) and MTg12B (Th1) in an attempt to correlate IL 1 receptor (IL 1R) expression with their IL 1 responsiveness. D10.G4.1 cells, which respond to IL 1, expressed two forms of the IL 1R, with molecular masses of approximately 80 kDa and approximately 60 kDa. In contrast, MTg12B cells failed to respond to IL 1 and only expressed the approximately 60 kDa receptor form. This suggests that the approximately 80 kDa receptor is essential for signaling. Expression of both IL 1R forms on D10.G4.1 cells could be inhibited by the anti-IL 4 antibody, 11B11. Antigen presentation reversibly upregulated both forms of the IL 1R, whereas stimulation with concanavalin A (ConA) and anti-CD3 only upregulated the approximately 60 kDa moiety. Upregulation of the approximately 80-kDa IL 1R by repeated antigenic stimulation resulted in a marked increase in sensitivity of D10.G4.1 cells to IL 1.     

Interleukin 2 promotes expression of an interleukin 2 receptor and proliferation of T cells stimulated with non-mitogenic dose of concanavalin A

Interleukin 2 (IL-2) initiated proliferation of the cells of T-enriched population stimulated with non-mitogenic dose of concanavalin A (Con A), whereas it could not induce proliferation of the cells treated with non-mitogenic lectin wheat germ agglutinin (WGA). Use of monoclonal antibody (MAb) directed against interleukin 2 receptor (IL-2R) showed that IL-2 significantly increased expression of IL-2R on the cells of T-enriched population stimulated with Con A, whereas it could not cause any significant enhancement of expression of IL-2R on the cells treated with WGA. We concluded that IL-2 activated T cells in combination with non-mitogenic does of Con A, and induced both expression of IL-2R and proliferation.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. I. Functional characterization by monoclonal antibodies

Three rat monoclonal antibodies (MAb) capable of stimulating interleukin 2 (IL 2) production by a variant subline of EL4 thymoma cells (EL4-6.1) have been produced. The stimulatory capacity of these MAb (designated RL73, RL119, and RL388) was originally found to be dependent on the presence of irradiated peritoneal exudate cells; however, this requirement could be replaced by the cellfree supernatant of the "macrophage-like" cell line P388D1 or by biochemically purified human interleukin 1 (IL 1). A number of other rat MAb directed against cell surface structures did not stimulate IL 1-dependent IL 2 production by EL4-6.1 cells; however, certain MAb directed against Thy-1 as well as the lectin phytohemagglutin did have this capacity. Furthermore, the stimulatory activity of MAb RL73, RL119, and RL388 appeared to be restricted to the EL4-6.1 variant line, because neither the parental EL4 line from which it was derived nor a series of ovalbumin-specific T-T hybrids responded to these MAb. The cell surface antigens recognized by MAb RL73, RL119, and RL388 were present on a wide variety of T cell lines and T-T hybrids, as well as on lines of B cell, macrophage, and fibroblast origin. Interestingly, the MAb reacted with the majority (approximately 85%) of thymocytes but not (or only to a very small extent) with resting T lymphocytes. After stimulation by concanavalin A, however, the three MAb reacted strongly with activated T lymphoblasts. The latter data suggest that MAb RL73, RL119, and RL388 may react with cell surface structures that are normally expressed as a consequence of lymphocyte activation.     

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.     

Interleukin 2 receptor blockade by anti-Tac antibody inhibits IFN-gamma induction

Anti-Tac antibody, which binds to the interleukin 2 (IL-2) receptor and thus blocks IL-2 binding to and activation of T lymphocytes, was used to investigate the role of IL-2 in interferon-gamma (IFN-gamma) production. Three T-cell mitogens (phytohemagglutinin, concanavalin A, and the pan-T monoclonal antibody OKT3) were used as IFN-gamma inducers. In each case, anti-Tac antibody clearly inhibited IFN-gamma production. This occurred even under conditions where cellular proliferation (as measured by incorporation of [3H]thymidine) was only slightly inhibited. The inhibitory effects of anti-Tac were reversed by the addition of purified IL-2. Therefore, endogenous production of IL-2 and its binding to the IL-2 receptor are needed for maximum IFN-gamma production.     

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.     

Interleukin 2 induction of antigen-nonspecific suppressor cells

Although Interleukin 2 (IL-2) is essential to the generation of immune responses it may also be important as a regulator of these same responses, as both primary and secondary anti-SRBC responses are greatly diminished when IL-2 is included in culture. IL-2 must be present within the first 24 hr of culture to affect maximum suppression. This inhibition is mediated by suppressor cells which are expanded by pulsing spleen cells with IL-2 for 48-72 hr. Their development is not antigen dependent and their action is antigen nonspecific. Suppressor cell activity can be generated from either naive or primed animals which are equally effective in inhibiting primary or secondary anti-SRBC responses. Suppressor cells can be propagated for long periods of time in T-cell growth factor-containing medium. These long-term cultured cells retain the ability to inhibit various immune responses such as mitogen- and alloantigen-induced proliferation, the generation of cytotoxic T lymphocytes and humoral responses. These cells suppress these responses by absorbing IL-2, as demonstrated by their ability to remove IL-2 upon incubation at 4 degrees C, and the reversal of suppression by the addition of supraoptimal amounts of IL-2.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. II. Structure, biosynthesis, and maturation

In the present study, we examined the biosynthesis and the maturation of two distinct membrane glycoproteins detected by two monoclonal antibodies (RL388 and RL119), which were selected on the basis of their ability to stimulate the production of interleukin 2 by a subline of the murine EL4 thymoma. RL388 detected a disulfide-linked heterodimer complex (Mr = 130,000) composed of a glycosylated heavy (Mr = 86,000) and a nonglycosylated light (Mr = 39,000) subunit. The unglycosylated precursor of the heavy chain was a polypeptide of Mr = 57,500, which was converted upon maturation into a Mr = 73,000 core-glycosylated intermediate, and then into the Mr = 86,000 surface-expressed molecule. Partial endo-H digestion of the core-glycosylated form suggested the presence of four N-linked glycan units. The antibody reacted with a protein determinant expressed on the mature form as well as the unglycosylated precursor of the heavy chain. Moreover, both subunits assembled rapidly during biosynthesis, and the glycosylation of the heavy chain was not required for this association. Taken together, these data suggest that the antigen detected by RL388 may be the murine homologue of the human 4F2 antigen. The antigen identified by RL119 was a surface glycoprotein of Mr = 55,000 with three to five N-linked glycan units. The unglycosylated precursor polypeptide was of Mr = 29,000. The fully core-glycosylated form of Mr = 41,000, which was detected after inhibition of glucosidase I with 1-deoxynojirimycin, was converted into a Mr = 39,000 intermediate, and upon further trimming, into a Mr = 36,000 endo-H-sensitive form. The latter could be detected for chase periods of over several hours, thus suggesting a low rate of intracellular processing. The wide cellular distribution of the molecules identified by RL388 and RL119 and their preferential expression on the surface of growing cells suggests that they may be associated with cell activation events.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

Prostaglandin E2-dependent induction of granulocyte-macrophage colony-stimulating factor secretion by cloned murine helper T cells

PG are known to inhibit T cell proliferation, at least in part by suppressing IL-2 production, but effects of PG on the production of other lymphokines have not been well studied. We have found that PGE2 and PGE1, but not PGF2 alpha, inhibit both proliferation and production of granulocyte-macrophage (GM)-CSF by murine TH clones stimulated with Ag or anti-CD3 antibody. Thus, signals generated via the Ag receptor:CD3 complex were inhibited by PGE. Most interesting, however, was the finding that PGE2 and PGE1 could act synergistically with IL-2 for the induction of GM-CSF in some TH1 clones. Dependence on PGE2 for this response was not found in all clones, as some TH1 cells could produce GM-CSF after IL-2 alone, and some cells did not produce GM-CSF even in the presence of PGE2 and IL-2. These observations indicate that there is a subset of TH1 cells receptive to a stimulating activity of PGE2 in the presence of IL-2. PGE2 is known to elevate cAMP levels in T cells. Therefore, we tested whether other agents known to increase cAMP, such as forskolin and cholera toxin, could act in conjunction with IL-2 to induce GM-CSF secretion. As was found with PGE2, these compounds also induced GM-CSF activity in the presence of IL-2, suggesting a critical role for cAMP in this process. Overall these data indicate that the requirements for activation of GM-CSF secretion vary among individual T cells. Most importantly they provide the first evidence that E-series PG are positive signals for lymphokine induction in certain T cells, whereas simultaneously acting as negative signals limiting proliferation. This result also suggests that treatment with anti-inflammatory drugs that decrease PGE2 concentrations may inhibit lymphokine secretion normally stimulated by this pathway.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by simultaneous activation of distinct signal transduction pathways

The human NK-like leukemic cell line YT was used to study interleukin 2 receptor (IL-2R; Tac) expression induced by activators of distinct signal transduction pathways. Tac expression was induced by active phorbol esters (12-O-tetradecanoylphorbol 13-acetate [TPA] and 4 beta-phorbol 12,13-didecanoate), which directly activate protein kinase C (PKC), as well as forskolin (FK), a stimulator of adenylate cyclase. A synergistic effect on Tac expression was obtained by simultaneous stimulation with optimal concentrations of phorbol esters and FK. Inactive phorbol esters (4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate) and the inactive analog of FK (1,9-dideoxyforskolin) had no effect on Tac expression. The active phorbol esters synergized also with interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in Tac expression. Staurosporine, a potent inhibitor of PKC in vitro, inhibited Tac expression marginally in YT cells stimulated with FK, and enhanced Tac expression in cultures treated with TPA, TNF alpha, or IL-1. Based on the assumption that synergistic effects are observed when two agonists use different signaling pathways, these findings provide evidence that IL-1, TNF, and TPA use different pathways/regulatory elements to regulate Tac expression on the cell surface. Synergistic upregulation of Tac expression by simultaneous activation of distinct pathways may be an important mechanism to modulate the immune response.     

Interleukin 1 and protein kinase C activator are dissimilar in their effects on Il-2 receptor expression and Il-2 secretion by T lymphocytes

T lymphocytes respond to mitogenic stimulation by expressing the receptor for interleukin 2 (Il-2) and secreting Il-2; once the receptor is expressed, Il-2 induces these cells to proliferation. In the present report using mouse T lymphocytes, thymocytes, and the lymphoma cell line EL4, we studied receptor expression and Il-2 secretion as early parameters for T-lymphocyte activation in response to ionomycin, concanavalin A (Con A), 12-O-tetradecanoyl-phorbol 13-acetate (TPA), and interleukin 1 (Il-1). Il-1 is required for mitogenic response of lymphocyte preparations that are rigorously depleted of macrophages. On its own, Il-1 had very little effect on Il-2 secretion and Il-2 receptor expression by T lymphocytes. TPA strongly synergized with ionomycin both for Il-2 secretion and for Il-2 receptor expression whereas Il-1 did not. Il-1 required the simultaneous presence of ionomycin and TPA to have any demonstrable effect on T lymphocytes from spleen and on thymocytes. However, on EL4 cells which were also partially responsive to TPA alone, Il-1 showed strong synergy with TPA to induce Il-2 secretion and Il-2 receptor expression. The effect of Il-1 on EL4 cells was dose dependent where increasingly higher concentrations of Il-1 in the presence of a fixed concentration of TPA caused higher percentage of EL4 cells to become Il-2 receptor positive. The present results suggest that Il-1 does not cause its effect on T lymphocytes via the same mechanism of protein kinase C activation that has been proposed for TPA.