The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production

CD150 (signaling lymphocyte activation molecule [SLAM]) is a self-ligand cell surface glycoprotein expressed on T cells, B cells, macrophages, and dendritic cells. To further explore the role of CD150 signaling in costimulation and T(H)1 priming we have generated a panel of rat antimouse CD150 monoclonal antibodies. CD150 cell surface expression is up-regulated with rapid kinetics in activated T cells and lipopolysaccharide/interferon gamma (IFN-gamma)-activated macrophages. Anti-CD150 triggering induces strong costimulation of T cells triggered through CD3. DNA synthesis of murine T cells induced by anti-CD150 is not dependent on SLAM-associated protein (SAP, SH2D1A), because anti-CD150 induces similar levels of DNA synthesis in SAP(-/-) T cells. Antibodies to CD150 also enhance IFN-gamma production both in wild-type and SAP(-/-) T cells during primary stimulation. The level of IFN-gamma production is higher in SAP(-/-) T cells than in wild-type T cells. Anti-CD150 antibodies also synergize with interleukin 12 (IL-12) treatment in up-regulation of IL-12 receptor beta(2) mRNA during T(H)1 priming, and inhibit primary T(H)2 polarization in an IFN-gamma-dependent fashion. Cross-linking CD150 on CD4 T cells induces rapid serine phosphorylation of Akt/PKB. We speculate that this is an important pathway contributing to CD150-mediated T-cell proliferation.     

Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells.

The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)-alpha and PMA. The concentration of PMA was found to be critical and showed a sharp optimum. In most cases maximal proliferation was obtained with as little as 0.1 ng/mL PMA. In all cases tested, TNF-alpha-induced proliferation could be inhibited completely by the addition of low doses of interleukin-4 (IL-4). Maximal inhibition was already found with 400 pg/mL IL-4. Inhibition by IL-4 was not caused by a downmodulation of TNF receptors. Apart from TNF-alpha, IL-2 was also in synergy with PMA able to induce proliferation in B-CLL cells of some patients. This IL-2-induced proliferation could be inhibited both by IL-4 and by neutralizing anti-TNF-alpha antibodies. This shows that TNF-alpha also can act as an autocrine growth factor. These data indicate that TNF-alpha is an important growth factor for neoplastic B-CLL cells and that IL-4 provides a tool to interfere with this TNF-alpha response.     

T-cell-replacing factor (interleukin 5) induces expression of interleukin 2 receptors on murine splenic B cells.

Small, resting B lymphocytes express few, if any, interleukin 2 (IL-2) receptors, but activated B cells may express such receptors. This paper examines the requirements for receptor expression. Normal murine splenocyte populations were enriched for B cells and cultured at relatively low density. IL-2 receptor expression was studied by measuring the binding of the anti-IL-2 receptor monoclonal antibody PC61. Lymphoblasts arising through stimulation by Escherichia coli lipopolysaccharide failed to express IL-2 receptors. B cells cultured with conditioned medium from concanavalin A-stimulated EL4 thymoma cells, with or without LPS, displayed IL-2 receptors. This bioactivity of EL4 conditioned medium could not be replaced by any concentration of B-cell-stimulatory factor 1 (IL-4), IL-1, IL-2, or IL-3 tested. However, the recently cloned lymphokine T-cell-replacing factor (IL-5) was a potent inducer of IL-2 receptor expression, as was the probably identical material known as eosinophil differentiation factor. The receptors so induced appeared to be functional, as receptor-expressing (but not control) lymphoblasts, responded to IL-2 by proliferation, indicative of high-affinity-receptor expression.     

Interleukin 4 is a growth factor for activated thymocytes: possible role in T-cell ontogeny.

We have shown that recombinant or natural interleukin 4 (IL-4) (formerly called B-cell stimulatory factor 1) induces proliferation of activated adult or fetal thymocytes. In the case of adult thymocytes, IL-4 in combination with Con A or phorbol 12-myristate 13-acetate (PMA) stimulated the proliferation of peanut agglutinin (PNA)-negative (-) thymocytes, while PNA-positive (+) thymocytes showed only marginal responses. Further investigation revealed that day 14-17 fetal thymocytes, purified L3T4- LyT2- double-negative adult thymocytes, and single positive L3T4+ LyT2- or L3T4- LyT2+ thymocytes failed to respond to IL-4 or PMA alone but proliferated strongly with both IL-4 and PMA. In contrast, purified double-positive L3T4+ LyT2+ adult thymocytes showed only a marginal proliferative response to these stimuli. Responsiveness of thymic subpopulations to PMA and IL-4 could be inhibited with anti-IL-4 but not with anti-IL-2 monoclonal antibodies, indicating that they were IL-2 independent. Finally, we have observed that supernatants from calcium ionophore and PMA-stimulated adult double-negative L3T4- LyT2- thymocytes induce proliferation of double-negative adult thymocytes. This latter response is inhibited by anti-IL-4 monoclonal antibodies, suggesting that under appropriate stimulation conditions, these immature thymocytes are able to produce IL-4. These observations suggest a role for IL-4 in T-cell ontogeny.     

Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor.

CD28 is a costimulatory receptor found on the surface of most T lymphocytes. Engagement of CD28 induces interleukin 2 (IL-2) production and cell proliferation when combined with an additional signal such as treatment with phorbol ester, an activator of protein kinase C. Recent studies have established that after CD28 ligation, the cytoplasmic domain of CD28 can bind to the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3 kinase). There is a concomitant increase in PI3 lipid kinase activity that may be important in CD28 signaling. Despite the requirement of phorbol 12-myristate 13-acetate (PMA) for effector function, we have found, however, that treatment of Jurkat T cells with the phorbol ester PMA dramatically inhibits (i) the association of PI3 kinase with CD28, (ii) the ability of p85 PI3 kinase to be immunoprecipitated by anti-phosphotyrosine antibodies, and (iii) the induction of PI3 kinase activity after stimulation of the cells with the anti-CD28 monoclonal antibody 9.3. These changes occur within minutes of PMA treatment and are persistent. In addition, we have found that wortmannin, a potent inhibitor of PI3 kinase, does not interfere with the induction of IL-2 after stimulation of Jurkat T cells with anti-CD28 monoclonal antibody and PMA. We conclude that PI3 kinase activity may not be required for CD28-dependent IL-2 production from Jurkat T cells in the presence of PMA.     

Interleukin-6 and granulocyte-macrophage colony-stimulating factor are candidate growth factors for chronic myelomonocytic leukemia cells

Previous studies suggest that malignant cells from some patients with myeloid leukemias produce colony-stimulating factors (CSFs) that can function as autocrine growth factors in vitro. We have examined the roles of interleukin-6 (IL-6) and granulocyte-macrophage CSF (GM-CSF) in the proliferation of myeloid leukemia cells. IL-6 activity was assessed in conditioned medium (CM) from myeloid leukemia cell cultures or cell lysates using IL-6-dependent KD83 and 7TD1 murine cell lines. Media conditioned by cells from patients with chronic myelomonocytic leukemia (CMMoL), but not by normal monocytes, chronic myelogenous leukemia (CML), or acute myelogenous leukemia (AML) cells, contained substantial levels (50 to 1,000 U/10(6) cells) of IL-6. The IL-6 content of CM correlated directly with donor peripheral blood WBC count. CM from two of five CMMoL samples also contained greater than 350 pg/mL GM-CSF. Moreover, CMMoL cells spontaneously formed colonies in semisolid medium. CMMoL colony formation could be partially inhibited by antibodies to IL-6 or GM-CSF, whereas combination of these antibodies gave additive, and nearly complete (greater than 93%), inhibition of spontaneous colony formation. Cell lysates from uncultured CMMoL cells from one patient contained abundant GM-CSF protein but no detectable IL-6. These data suggest that IL-6 and GM-CSF act in vitro as autocrine growth factors for CMMoL cells, and that CMMoL cells in vivo may represent a GM-CSF-dependent autocrine growth system.     

The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.

Late-phase human T-cell lymphotropic virus I-associated adult T-cell leukemia cells express IL-2 receptors (IL-2R) but no longer produce IL-2. We have reported that the IL-2-independent adult T-cell leukemia line HuT-102 secretes a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and lymphokine-activated killer cell activity. Stimulation of proliferation of the cytokine-dependent human T-cell line Kit-225 mediated by HuT-102-conditioned medium or by 3200-fold-purified IL-T was not blocked by the addition of antibodies against IL-2 or IL-2R alpha subunit. However, IL-T-mediated stimulation of this human T-cell line was inhibited by addition of Mik-beta 1, an antibody that binds specifically to IL-2R beta subunit. In addition, the activation of large granular lymphocytes to lymphokine-activated killer cells mediated by IL-T-containing conditioned medium was not blocked by antibodies directed toward IL-2 or IL-2 alpha but was inhibited by an antibody to IL-2R beta, suggesting the requirement of this receptor subunit for IL-T action. This conclusion was confirmed using an IL-3-dependent murine myeloid precursor cell line, 32D, that expresses IL-2R alpha and IL-2R gamma, but not IL-2R beta. Neither IL-2 nor IL-T stimulated 32D cell proliferation. However, after transfection with the gene encoding human IL-2R beta, 32D beta cells proliferated on addition of either cytokine. The IL-T-mediated stimulation of 32D beta proliferation was inhibited by an anti-IL-2R beta antibody but not by an anti-IL-2 antibody. Thus, the IL-T-mediated stimulation of T-cell and lymphokine-activated killer cell activation requires the expression of the IL-2R beta subunit.     

Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleukin-10 producing CD5+ B cells

Recent studies indicate that normal B cells can be primed to differentiate into two distinct cytokine-secreting effector subsets, Be1 and Be2. The aim of this study was to analyse, for the first time, Be1 and Be2 cells at the single cell level in SLE patients using the recently developed technique of flow cytometry for intracellular cytokines. Peripheral blood mononuclear cells (PBMC) from SLE patients and age- and sex-matched normal controls were cultured for 24 h in the presence or absence of phorbal myristate acetate and ionomycin (PMA/I) or lipopolysaccharide (LPS). The production of type I (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-6, IL-10, IL-13) cytokines by B cells (and IL-10 production by fractionated CD5+ and CD5- B cells) was investigated using an intracellular cytokine staining technique and flow cytometry. In the absence of PMA/I stimulation, the percentage of B cells from SLE patients was significantly lower than those of normal subjects and significantly more SLE B cells spontaneously produced IL-10 than controls. Moreover, CD5+ B cells from SLE patients were enriched for cells with signs of previous in vivo activation and for high levels of IL-10 production. A significant positive correlation was observed between the percentage of IL-10- and IL-6-producing PMA/I-stimulated B cells in SLE patients, but not in controls. There were no significant differences in the production of other cytokines by B cells of SLE patients and normal subjects. In conclusion, a general alteration of type 1 and type 2 cytokine production by B cells is not observed in SLE patients. The role of B cell cytokines in the pathogenesis of SLE appears to be exerted by elevated secretion of in vivo IL-10, which may play an important role in the immune dysregulation observed in SLE patients. Moreover, the cross regulation of IL-10 and IL-6 is disrupted in SLE patients.     

Synergistic action of calcium ionophore A23187 and phorbol ester TPA on B-chronic lymphocytic leukemia cells

The peripheral blood mononuclear cells from 16 patients with B-chronic lymphocytic leukemia (B-CLL, n = 13), B-prolymphocytic leukemia (B-PLL, n = 2) or hairy cell leukemia (n = 1) were incubated in the presence of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore A23187. A synergy between these inducers was found with respect to morphological changes and B cell proliferation and differentiation. A23187 used alone did not activate the cells. B-CLL cells treated with the double stimulus acquired a plasmacytoid morphology, showed significantly higher incorporation of 3H-thymidine and 3H-uridine, and produced significantly higher amounts of monoclonal immunoglobulin compared with the same cells exposed to either of the inducers alone. These results indicate that phorbol ester and calcium ionophore act synergistically on B-CLL cells to induce proliferation and differentiation. B-PLL cells responded more vigorously to the signals provided by TPA and A23187. Previous studies showed that TPA and A23187 can mimic the two physiological second messengers diacylglycerol and inositol trisphosphate in the transduction of signals leading to cell activation, proliferation, and differentiation in normal B cells. The present findings suggest that the capacity of B- CLL and B-PLL cells to differentiate in response to signals of the second messenger pathway is intact.     

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.     

Inhibition of human granulocyte-macrophage colony formation by interleukin 2-treated lymphocytes is mediated by interferon gamma and tumor necrosis factor alpha

We previously demonstrated that human granulocyte-macrophage colony (granulocyte-macrophage colony-forming units, CFU-GM) formation was inhibited by interleukin 2 (IL-2)-treated lymphocytes and their conditioned medium (CM). In the present study, the mechanism of this suppression was investigated. When anti-interferon (IFN)-gamma antibody or anti-tumor necrosis factor (TNF)-alpha antibody was added to CFU-GM agar culture with IL-2-treated lymphocytes or their CM, the inhibition of CFU-GM colony formation was partially abrogated, whereas anti-TNF-beta antibody did not abolish the inhibitory effects. When anti-IFN-gamma and anti-TNF-alpha antibodies were added simultaneously, full recovery of colony formation was observed. In the CM of IL-2-treated lymphocytes, detectable levels of IFN-gamma (81 +/- 15 U/ml) and TNF (3.1 +/- 1.1 U/ml) were found. Addition of IFN-gamma and TNF-alpha at these concentrations to the agar culture inhibited CFU-GM colony formation. Taken together, these results indicate that inhibition of human CFU-GM colony formation by IL-2-treated lymphocytes and their CM is mediated by IFN-gamma and TNF-alpha generated from IL-2-treated lymphocytes.     

Hepatitis B virus-specific T-cell proliferation and cytokine secretion in chronic hepatitis B e antibody-positive patients treated with ribavirin and interferon alpha

Immune elimination of hepatitis B virus (HBV) during antiviral therapy depends on the activation of T-cell responses, which are generally impaired in chronic hepatitis B. HBV-specific T helper (Th)-cell reactivity has been assessed post-treatment in liver and peripheral blood of 18 anti-HBe-positive patients with chronic hepatitis B administered combined ribavirin/interferon alfa (IFN-alpha) therapy. The results showed that patients with undetectable HBV DNA by quantitative polymerase chain reaction under combination therapy were able to mount an HBV-specific CD4(+) Th-cell proliferative response and such T-cell reactivity is detectable 1 year after HBV DNA clearance. Hepatitis B virus core (HBcAg) and e (HBeAg) antigen-specific Th-cell proliferation was found more frequently in the liver and peripheral blood in those patients who sustained the alanine aminotransferase (ALT) normalization together with HBV DNA loss. However, HBV-specific IFN-gamma production in vitro in peripheral blood mononuclear cells augmented in 4 of 5 sustained responders and all 13 nonresponders, interleukin 10 (IL-10) production decreased in all 5 sustained responders but increased in 7 of 13 nonresponders. Furthermore, intrahepatic HBcAg plus HBeAg-specific Th-cell proliferation only occurred in sustained responders (2 of 3, 67%, vs. 0 of 9; P =.045) whose cells showed in vitro significantly increased productions in HBcAg/HBeAg-specific IFN-gamma and IL-12 compared with nonresponders in whom IFN-gamma and IL-12 productions decreased together with increased IL-10 secretion. In conclusion this study indicates that combined therapy with ribavirin and IFN-alpha for chronic hepatitis B not only significantly reduces viremia levels but also induces lasting CD4(+) T-cell proliferation and Th1 cytokine release at the site of infection, which may lead to sustained eradication of the HBV.     

Interferon-gamma suppresses PDGF production from THP-1 cells and blood monocyte-derived macrophages.

Involvement of the immunological mechanisms in atherogenesis has recently been suggested by immunohistological detection of macrophages and T lymphocytes in atherosclerotic lesions. In the present study, we have investigated the regulatory effect of interferon-gamma (IFN-gamma), a cytokine secreted by activated T cells, on the production and secretion of platelet-derived growth factor (PDGF) from macrophages in culture. The human monocytic leukemia cell line, THP-1, was treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce macrophage differentiation and PDGF production, and then various doses of recombinant human IFN-gamma (0-1000 I.U./ml) were added to the culture. After 48 h, the conditioned medium and the cells were harvested and analyzed for PDGF production. PDGF-dependent mitogenic activity in the conditioned medium, estimated by neutralization of mitogenic activity with anti-PDGF antibody, was suppressed by IFN-gamma treatment. Radioimmunoassays for PDGF also revealed a decrease in both PDGF-AA and -BB in the conditioned medium with IFN-gamma treatment, whereas neither total cell DNA as an indication of cell number nor overall protein synthesis based on [3H]leucine incorporation were decreased. Northern analysis of total RNA extracted from the cells demonstrated that IFN-gamma suppressed the level of PDGF mRNA. Analysis of mRNA degradation in the presence of actinomycin D demonstrated that the decrease in PDGF mRNA was not due to enhanced degradation of mRNA. A similar inhibitory effect of IFN-gamma on PDGF mRNA levels was also found in monocyte-derived macrophages cultured in the presence of granulocyte-macrophage colony stimulating factor. These results suggest that IFN-gamma modulates production and secretion of PDGF from macrophages and that the functions of macrophages in atherogenesis may be regulated by the cellular interactions between T cells and macrophages through the action of cytokines such as IFN-gamma.     

Interferon-gamma-like molecule induces Ia antigens on cultured mast cell progenitors.

Persisting (P) cells (murine cells that resemble mast cells and grow continuously in vitro for prolonged periods in the presence of a specific growth factor) did not express detectable levels of Ia antigens (murine class II major histocompatibility antigens) when their growth was supported by partially purified P cell-stimulating factor. However, when these Ia-negative P cells were transferred to medium conditioned by concanavalin A-stimulated spleen cells, Ia antigens appeared within 24 hr. The increase in Ia antigens was due to induction of synthesis of Ia antigens by P cells and not to absorption of Ia antigens from the conditioned medium or selective growth of Ia-positive cells from a low number of Ia-positive cells in the original population. The Ia-inducing activity was also found in supernatants from antigen-stimulated cloned T-cell lines, but not from certain T-cell hybridomas or the T lymphoma EL-4. The presence of Ia-inducing activity correlated with the presence of interferon-gamma (IFN-gamma). The gel filtration profiles of IFN-gamma activity and Ia-inducing activity were coincident and corresponded to an apparent molecular weight of 40,000-45,000. Both the IFN-gamma and Ia-inducing activity were destroyed by treatment at pH 2. These results indicate that IFN-gamma or a closely related molecule induces Ia antigens on P cells and suggest that regulation of Ia antigen expression may be an important aspect of the effects of IFN-gamma on the immune and hemopoietic systems.     

Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation.

Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3.     

Epstein-Barr virus-containing B-cell line produces an interleukin 1 that it uses as a growth factor

We report the establishment of a spontaneous interleukin 1 (IL-1)-producing subclone derived from the human Epstein-Barr virus (EBV)-containing B-lymphoblastoid cell line (721 LCL) and show that the IL-1 produced by this B-cell subclone is distinct from other types of IL-1. The parental cell line 84.5, a deletion mutant of the 721 LCL cell line, can be induced to produce IL-1 activity when stimulated by certain inducers such as phorbol 12-myristate 13-acetate in the presence of fetal calf serum. From this parental 721/84.5 clone, a subclone, termed 3B6, has been developed. This 3B6 subclone has an immature B-cell phenotype, expresses only HLA class II DP subregion antigens, and spontaneously releases IL-1 in the culture supernatant with relatively few inhibitory molecules under serum-free culture conditions. The 3B6-derived IL-1 was purified from 3B6 conditioned medium with a three-step procedure. The molecular weight of this IL-1 is 13,500, and the isoelectric point values are pH 4.9 and 5.1 without any component focusing near pH 7. The N-terminal amino acid sequence differs markedly from those reported for the two IL-1 species produced by monocytes. The purified material shares several biological properties with monocyte IL-1, since it could induce the proliferation of murine thymocytes, the production of interleukin 2 by phytohemagglutinin-stimulated cloned HSB2 T cells, and the proliferation of human fibroblasts. However, this IL-1 activity could not be blocked by polyclonal anti-monocytic IL-1 antibodies, and, more importantly, it was not pyrogenic in rabbits. Finally, it promotes the growth of B-cell clones derived from the parental 721/84.5 lines in the absence of fetal calf serum, which suggests that it could act as an autocrine growth factor in this Epstein-Barr virus-transformed B-cell line.