Different staphylococcal strains elicit different levels of production of T-helper 1-inducing cytokines

Cytokine production has been implicated in the pathogenic mechanisms of infections caused by the staphylococci, since these bacteria may act as strong cytokine inducers. To gain deeper insight into the Th1 immune response activated by these bacteria, we have analyzed the interferon (IFN), interleukin-12 (IL-12) and IL-18-inducing activities of different Staphylococcus aureus (S. aureus), S. epidermidis and S. saprophyticus strains in human monocytes and murine bone marrow macrophages. A large majority of the S. aureus strains elicited the simultaneous production of IL-12 p70 and IFN-alpha in the human monocytes, while the S. epidermidis and S. saprophyticus strains induced only a low level of production, if any, of these cytokines. Furthermore, a majority of the S. aureus strains induced significantly higher IL-12 p70 and IL-18 titers in the murine bone marrow macrophages than did the S. epidermidis and S. saprophyticus strains. As IL-12, IL-18 and IFN-alpha stimulate Th1 differentiation synergistically, we suggest that S. aureus strains bias the immune response toward a Th1 phenotype, whereas S. epidermidis and S. saprophyticus strains provide a weaker stimulus for the production of Th1-inducing cytokines, and accordingly possibly elicit a less extensive Th1-associated adaptive immunity.     

Interleukin-1-inducing activity of the polysaccharide-containing antigens of the cell wall in Yersinia pestis

The induction of the synthesis of interleukin-1 (IL-1) in human monocytes under the influence of the endotoxic preparations (LPS) and Y. pestis basis somatic antigen has been experimentally studied. The results obtained in this study make it possible to come to the conclusion that the capacity of the endotoxin of Y. pestis cell wall, consisting of LPS of type R, for inducing the synthesis of IL-1 in human monocytes is not different from the corresponding capacity of Salmonella and Shigella LPS, type S. Y. pestis O-specific polysaccharide in a discrete state has considerably greater IL-1-inducing activity in comparison with other preparations used in this experimental study. Such typical changes, characteristic of the initial stage of Y. pestis infection, as a sharp rise in temperature, transitory neutropenia, significant primary suppression of the oxygen-dependent metabolism of polymorphonuclear neutrophils are probably due to the induction of the synthesis of IL-1 by the polysaccharide-containing antigen of Y. pestis cell wall (LPS, basic somatic antigen) in cells of the mononuclear phagocytizing system.     

Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction

Dendritic cells (DC) are key initiators of primary immune responses. Myeloid DC can secrete IL-12, a potent Th1-driving factor, and are often viewed as Th1-promoting APC. Here we show that neither a Th1- nor a Th2-inducing function is an intrinsic attribute of human myeloid DC, but both depend on environmental instruction. Uncommitted immature DC require exposure to IFN-gamma, at the moment of induction of their maturation or shortly thereafter, to develop the capacity to produce high levels of IL-12p70 upon subsequent contact with naive Th cells. This effect is specific for IFN-gamma and is not shared by other IL-12-inducing factors. Type 1-polarized effector DC, matured in the presence of IFN-gamma, induce Th1 responses, in contrast to type 2-polarized DC matured in the presence of PGE2 that induce Th2 responses. Type 1-polarized effector DC are resistant to further modulation, which may facilitate their potential use in immunotherapy.     

Production of interleukin 1 by adult T cell leukemia (ATL) cell lines

The accessory function for T cell activation and the production of interleukin 1 (IL 1) of adult T cell leukemia (ATL) cell lines were studied in vitro. ATL cell lines such as Hut-102, MT-1, and MT-2 functioned as accessory cells for the stimulation of human T cell proliferative response induced with concanavalin A (Con A) and induced allogeneic mixed lymphocyte reaction. Cell lysates of three ATL cell lines and the culture supernatant of MT-2 cells had activities to stimulate murine thymocyte proliferative response. Then we studied physicochemical properties of the factors produced by MT-2 cells. The m.w. of the factors were approximately 15,000 by Sephacryl S-200 column chromatography, and their isoelectric point values were 5.4 and 4.8 by chromatofocussing technique. No fraction contained interleukin 2 (IL 2) activities to stimulate IL 2-dependent murine cytotoxic T cell line. The thymocyte-stimulating activities of the factors were absorbed with rabbit anti-IL 1 alpha antiserum, but not with anti-IL 1 beta antiserum. Furthermore, messenger RNA extracted from MT-2 cells hybridized to complementary DNA of IL 1 alpha, but not of IL 1 beta, by Northern blot hybridization analysis. The factors from MT-2 cells could stimulate the production of IL 2 and the expression of IL 2 receptors of human T cells in the presence of Con A as well as recombinant IL 1 alpha and IL 1 beta did, and these activities were also blocked by rabbit anti-IL 1 alpha antiserum, but not by anti-IL 1 beta antiserum. These results suggest that the factors produced by MT-2 cells correspond to IL 1 alpha. However, the accessory function of MT-2 cells for T cell activation was not blocked by rabbit anti-IL 1 antiserum. These results suggest that ATL cell lines produce IL 1-like factors, but the accessory function of ATL cell lines for T cell activation is mediated by some other mechanisms rather than by secreted IL 1-like factors.     

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.     

Membrane-associated IL 1-like activity on rat dendritic cells

The secretion of interleukin 1 (IL 1) by rat dendritic cells (DC) was studied in relation to their ability to induce the production of interleukin 2 (IL 2) and to induce IL 2 responsiveness. IL 1 (or IL 1-like activity) was measured by its capacity to enhance IL 2 production by EL4 cells. In contrast to peritoneal exudate cells (PEC) or splenic adherent cells, DC from thoracic duct lymph (TD-DC) or from spleen did not secrete detectable amounts of IL 1 on stimulation with LPS/Silica. However, TD-DC and splenic DC were able to enhance IL 2 production by EL4 cells directly, and were only two times less effective than PEC. By preventing cell-to-cell contact between stimulator cells and EL4 cells, it was demonstrated that most of the IL 2-inducing activity of TD-DC and PEC was associated with the cell membrane. Treatment with 1% paraformaldehyde (PFA) to abolish metabolic activity resulted in a 50% decrease (or inactivation) of IL 2-inducing activity of TD-DC in the EL4 assay. Moreover, UVB-irradiation (300 mJ/cm2) of TD-DC, which has been described to inhibit the release of IL 1 by macrophages, caused a 70% decrease in IL 2-inducing activity. In a primary allogeneic mixed leukocyte reaction, neither PFA-treated TD-DC nor UV-irradiated TD-DC were able to induce T cell proliferation or IL 2 production. The cells were, however, able to induce IL 2 responsiveness, i.e., T cell proliferation in the presence of excess human recombinant IL 2. The finding that IL 1 enhanced T cell responses to PFA-treated or UV-irradiated TD-DC in the absence and in the presence of excess IL 2 indicates that loss of stimulatory activity of TD-DC may be due in part to loss or inactivation of IL 1. These results suggest that membrane-associated structures, that are identical to or mimic IL 1, are involved in the activation of T cells by DC.     

Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release

The objective of this in vitro study was to examine the immunomodulatory impact of human periodontal ligament (PDL) cells on the nature and magnitude of the leukocyte infiltrate in periodontal inflammation, particularly with regard to Th17 cells. PDL cells were challenged with pro-inflammatory cytokines (IL-1ß, IL-17A, and IFN-γ) and analyzed for the expression of cytokines involved in periodontal immunoinflammatory processes (IL-6, MIP-3 alpha, IL-23A, TGFß1, IDO, and CD274). In order to further investigate a direct involvement of PDL cells in leukocyte function, co-culture experiments were conducted. The expression of the immunomodulatory cytokines studied was significantly increased under pro-inflammatory conditions in PDL cells. Although PDL cells did not stimulate leukocyte proliferation or Th17 differentiation, these cells induced the recruitment of leukocytes. The results of our study suggest that PDL cells might be involved in chronic inflammatory mechanisms in periodontal tissues and thus in the transition to an adaptive immune response in periodontitis.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8(+) DC preferentially induce Th1 responses, whereas CD8(-) DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8(-) than CD8(+) DC produced IL-12 after HKBA challenge, whereas only CD8(+) DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11c(low) cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8(+) and CD8(-) DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

Dissociation between the interleukin 1-inducing capacity and Limulus reactivity of lipopolysaccharides from gram-negative bacteria

In this study we compared the interleukin 1 (IL 1)-inducing capacity and the reactivity in the Limulus amoebocyte assay (LAL) of purified lipopolysaccharides (LPSs) from various bacterial strains. LPSs differed greatly in their capacities (on a weight basis) to induce IL 1 release from serum-free cultured human monocytes. LPS species that induced high levels of IL 1 release from human monocytes exhibited a high thiobarbiturate-reactive 2-keto-3-deoxy-octonic acid (KDO) content. No relationship was found between the IL 1-inducing activity and the LAL reactivity of purified LPSs. Filtration experiments in which membranes of decreasing size-exclusion limits were used demonstrated that molecular species of LPS with an apparent Mr below 3,000 may induce IL 1, whereas only species with an apparent Mr above 8,000 are recognized in the LAL assay. The latter observation suggests that the reaction with LAL requires an aggregated form of LPS. These results indicate that biologically active LPS species can cross dialysis membranes in vivo although no LAL reactive material is detected in the blood compartment. The Limulus assay is an insufficient criterion for the absence of LPS in biological fluids.     

The regulation of gamma-interferon production by interleukins 1 and 2

Purified interleukins 1 and 2 (IL-1 and IL-2) were used to investigate their role in the production of gamma-interferon (gamma-IFN). Macrophage depletion from human peripheral blood mononuclear leukocytes (PBML) inhibited gamma-IFN production. Addition of purified IL-1 partially restored IFN production of macrophage-depleted PBML induced by three T cell mitogens (phytohemagglutinin, PHA; concanavalin A, con A; and staphylococcal enterotoxin A, SEA), but had no effect on induction of IFN production by undepleted PBML. Therefore endogenous IL-1 production by macrophages is probably one of the mechanisms by which they act as accessory cells for IFN production by lymphocytes. A monoclonal antibody 9.6 which binds to the sheep erythrocyte (E) receptor found on human T cells inhibited IFN production. Addition of IL-2, but not IL-1, was found to reverse this inhibition. Prostaglandin E2, a macrophage product, inhibited gamma-IFN production induced by PHA, Con A, and OKT3 but usually not SEA. This inhibitory effect was reversible by the addition of IL-2 but not IL-1. In the absence of mitogen IL-1 alone rarely induced any IFN production, although some IFN was produced by PBML from a small minority of donors. Without mitogen IL-2 induced IFN production only at very high concentrations and the added presence of IL-1 did not enhance this induction.     

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by Semliki Forest virus

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.     

Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Difference in requirement of macrophage products for concanavalin A-induced production of eosinophil chemotactic factor and macrophage chemotactic factor from guinea pig lymphocytes in vitro

Differences between the conditions for an eosinophil chemotactic factor (ECF) and macrophage chemotactic factor (MCF) production by lymphoid cells of mesenteric lymph nodes and spleen were studied in guinea pigs. If lymphoid cells were washed less than 4 hr after concanavalin A (Con A) stimulation and were cultured for an additional 24 hr, they failed to produce ECF, whereas Con A stimulation for 1 hr before washing was sufficient to stimulate them to produce MCF. Subsequently, it was shown that heat-labile soluble factors (termed ECF-PF) with potentiating activity for ECF production are produced from macrophages by 5 micrograms/ml Con A activation. When ECF-PF were added to the cell culture with 5 micrograms/ml Con A, the lymphoid cells could produce ECF even when they were washed 2 hr after Con A stimulation and were cultured for an additional 24 hr, suggesting that ECF-PF plays a critical role in the early stage of ECF production. The lymphoid cells were also able to produce ECF even when they were cultured with ECF-PF and a suboptimal dose of Con A (1 microgram/ml) for ECF production. Protein synthesis seemed to be essential for ECF-PF production. The ECF-PF activity was associated with two separated molecular fractions with m.w. of about 50,000 to 70,000 and of 10,000 to 20,000. It is thus suggested that ECF is produced from T cells by Con A stimulation under conditions which differ, at least, from those for MCF in the requirement of ECF-PF.     

Con A-stimulated thymocytes produce interleukin 2 after removal of Lyt-1 positive cells

Mouse lymphocytes produce several lymphokines, including interleukin 2 (IL-2) and colony-stimulating factors (CSFs) following stimulation with T-cell mitogens. However, very little IL-2 is produced by thymocytes upon concanavalin A (Con A) stimulation. Strong selective inhibition of IL-2 production was observed when fresh spleen cells were mixed with Con A-activated thymocytes. Sorting of populations on the basis of antigenic phenotype showed that the cell mediating the blockage in IL-2 secretion is a large T cell expressing markers for both Lyt-1 and Lyt-2. This specific inhibition of IL-2 accumulation was not mediated by a soluble product, or by absorption on expressed IL-2 receptors on the activated thymocytes. Removal of the Lyt-1 positive cells from a thymocyte population renders it capable to produce IL-2 upon Con A stimulation, indicating a functional role of these cells.     

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.