Type 1 vs. type 2 cytokine secretion in vitro and its regulation by hydrocortisone in humans subjected to 120-day anti-orthostatic bed-rest regime

Head-Down Bed-Rest (HDBR) mimics some of the physiological stress effects of microgravity. Six healthy volunteers were subjected to bed-rest for 120 days. Blood samples were collected one month before (PRE), on day 110 of HDBR (DAY 110), and on the 7th day after bed-rest regime ends (POST). Distribution of T-cell subsets, NK-, B-cells and monocytes was assessed in the whole blood. Distribution of cytokine secreting T-cells was assessed in PMA/ionomycin cell culture. Peripheral Blood Mononuclear Cells (PBMC) and whole blood cells (WB) were activated with a combination of PHA and LPS to assess cytokine secretion. In addition, PHA/LPS activated cell cultures were treated with 10(-6) M of hydrocortisone (HCS) in order to study stress-induced alterations in the cortisol-sensitivity of immunocytes. Results from HCS culture were compared to non-treated control cultures. Stress factors of HDBR affect immune responsiveness and immune-endocrine homeostatic interrelations in vitro as follow: 1) alter expression of surface receptor to IL-2 (CD25) by CD4+ and CD8+ T-cell subsets in PHA/LPS activated PBMC culture; 2) alter distribution of IL-2 and/or IFN-gamma producing CD4+ and CD8+ T-cells in PMA/ionomycin activated culture; 3) significantly affect secretion of IL-2, IFN-gamma, and IL-4, but not IL-10 and soluble IL-2 receptor alpha in PHA/LPS activated PBMC culture; 4) shift Type 1 vs. Type 2 cytokine balance in PHA/LPS activated culture toward to Type 1 response; 5) in vitro treatment with hydrocortisone unequally modulate expression of CD25 on CD4+, and CD8+ T-cells, as well as secretion of Type 1 and Type 2 cytokines in PHA/LPS activated PBMC culture during bed-rest regime; 6) assessment of immune profile depends from the cellular and humoral milieu of cell culture.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

Dysregulation of interleukin-10 and interleukin-12 are involved in the reduced host resistance to Listeria monocytogenes infection in alymphoplastic aly mutant mice

The aly is a unique spontaneous autosomal recessive mutation in mice that causes a systemic defect of lymph nodes and Peyer's patches and disorganized splenic and thymic structures with immunodeficiency. Our previous study demonstrated that resistance to Listeria monocytogenes infection and interferon-gamma (IFN-gamma) production are attenuated in the mutant mice. In this study, we investigated the mechanism of decrease in antilisterial resistance and IFN-gamma production in aly mice. Interleukin (IL)-12 production in response to heat-killed L. monocytogenes (HK-LM) was decreased but IL-10 production was increased in aly/aly macrophage cultures, compared with those in aly/+ macrophages. Nonadherent cells and macrophages obtained from the spleens of naive aly/+ mice and aly/aly mice were reconstituted and stimulated with HK-LM. IFN-gamma production was markedly decreased when macrophages derived from aly/aly mice were used. IFN-gamma production in aly/aly spleen cell cultures was recovered in the presence of anti-IL-10 monoclonal antibody (mAb) or recombinant IL-12. When aly/+ mice and aly/aly mice were injected with mAb against IL-10 or IL-12 p40, antilisterial resistance was inhibited by injection of anti-IL-12 p40 mAb, while anti-IL-10 mAb treatment augmented the resistance. Administration of anti-IFN-gamma mAb attenuated antilisterial resistance in aly/+ mice but not in aly/aly mice. The present results suggest that downregulation of IL-12 and upregulation of IL-10 in macrophages might be involved in the decrease in antilisterial resistance and IFN-gamma production in aly/aly mice in addition to the structural defect in lymphoid organs. Moreover, the results predict that an IL-12-dependent and IFN-gamma-independent mechanism may be also involved in the decrease in antilisterial resistance in aly/aly mice.     

Antigen concentration determines helper T cell subset participation in IgE antibody responses.

A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

Inhibition of macrophage-induced antigen-specific T-cell proliferation by interferon-gamma

The effects of IFN-gamma on macrophage (M phi)-mediated antigen-specific T-cell proliferation was investigated. A well-defined assay system using purified resident populations of antigen-pulsed peritoneal M phi and immune T cells was used to measure M phi-induced antigen-specific T-cell proliferation. Antibody affinity purified or recombinant IFN-gamma inhibited M phi-induced T-cell proliferation when KLH-pulsed M phi from mice given IFN-gamma prior to KLH were cultured with KLH immune T cells from normal mice. Monoclonal rat anti-IFN-gamma antibody neutralized the inhibitory effect of IFN-gamma. This inhibition of T-cell proliferation occurred despite the fact that these M phi appeared to be activated by IFN-gamma treatment as measured by increased tumoricidal activity. The mechanism for the inhibition was unrelated to class II (Ia) expression, IL-1 secretion, and prostaglandin secretion. These results demonstrate the complex and sensitive role IFN-gamma has in regulating the immune response.     

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.     

Differential signal requirements in T-cell activation by mitogen and superantigen

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca²⁺ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca²⁺ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

Differential effects of concanavalin A, serum and cytokines on proteoglycan elaboration and DNA synthesis by mononuclear cells from human peripheral blood.

Human blood derived mononuclear cell (MC) cultures required concanavalin A (Con A) stimulation to synthesize and secrete into the medium high levels of a protease-resistant proteoglycan (PG) containing predominantly chondroitin sulfate (CS), which was elaborated largely by T-cells in culture. PG and DNA synthesis were studied in MC cultures in the absence and presence of Con A as well as serum and some biologically active polypeptide factors. In the presence of Con A, stimulation of PG synthesis was substantially greater in T-cell enriched cultures than in B-cell enriched cultures. DNA synthesis was also stimulated in the presence of Con A. This stimulation was concentration-dependent, but required the presence of serum for additional responses. DNA and cell proliferation were stimulated by interleukin-2 (IL-2), but PG production was not stimulated by conditioned media, IL-1, IL-2, IL-3, or transforming growth factor-beta (TGF-beta). Our results indicate that the elaboration of PG from T-cells of human MC is independent of the effects of regulatory peptides on cell proliferation and DNA synthesis.     

Age-related changes in the activation requirements of human CD4+ T-cell subsets.

In agreement with previous studies, we found that the proliferative response of unfractionated T-cells to phytohaemagglutinin (PHA) was severely impaired in healthy aged individuals (70-85 years). On the other hand, we did not observe significant differences between aged and young adults in T-cell responsiveness to mab OKT3 (anti-CD3). PHA responses in "old" T-cells could be substantially improved, however, by the addition of recombinant interleukin-2 (rIL-2) or KOLT2 (anti-CD28 mab). When individual CD4+ T-cell subpopulations were isolated from young and old donors and stimulated with PHA in the presence of autologous accessory cells, age-related deficiencies were seen in both CD4+CD45RA+ (naive) and CD4+CD45RO+ (memory) cell populations. Further analysis using a panel of coactivators in cultures depleted of accessory cells identified specific abnormalities in the CD2 or alternate pathway of T-cell activation. These were predominantly seen in CD4+ naive T-cells. The capacity of rIL-2, KOLT2, and PMA to restore, at least partially, T-cell responsiveness in the aged suggests a defect(s) in an early signal transduction mechanism.     

Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages

The ability of Shigella dysenteriae type 1 porin to induce the release of nitric oxide (NO) and interleukin-1 (IL-1) from peritoneal macrophages of mouse and to regulate lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) mediated release of the two proinflammatory mediators was investigated. Porin released nitrite when added to macrophage cultures. A maximum of 3.2-fold nitrite release by macrophages was observed with 100 ng ml(-1) of porin. The nitrite release of LPS was enhanced significantly by lower concentrations of porin, whereas the effect of IFN-gamma was enhanced by porin at higher concentrations. Polysaccharide (PS) moiety of LPS stimulated the nitrite release of elicited macrophages by 1.6-fold compared to untreated control. It also enhanced the stimulatory effect of 1 and 10 ng ml(-1) of porin by 1.3-fold. Lipid A (LPA) moiety of LPS did not release nitrite, nor did it increase the porin mediated nitrite production. Porin treated 24 h old macrophage culture supernatants were applied for ConA activated thymocyte proliferation as a measure for determination of IL-1 release. Sixty percent depletion of thymocyte proliferation was observed when the porin treated macrophage supernatants were absorbed with anti-IL-1 antibody. A maximum of 5.5-fold increase of thymocyte proliferation over control was found with 1 and 10 ng ml(-1) of porin. One or 10 ng ml(-1) of porin and LPS augmented the thymocyte growth, 1.5-fold beyond that obtained by porin and 1.8-/1. 7-fold more than that obtained by LPS, alone. Similarly, porin and IFN-gamma co-stimulated the cell growth also. PS enhanced the thymocyte proliferation by 5-fold. It also enhanced the thymocyte growth by co-stimulating 1.4-fold the effect observed by 1 or 10 ng ml(-1) of porin alone. LPA could not participate in the cell proliferating activity nor did it enhance the stimulatory effect of porin. Therefore, both nitrite release and thymocyte proliferation by LPS could be substituted by PS only. The tight association of the two bacterial outer membrane components, porin and LPS, could be a necessary co-signal for boosting the release of the two proinflammatory mediators, namely NO and IL-1, which may be associated with the inflammatory response of the colon during Shigella invasion.     

Different effects of IL-2 addition or antibody crosslinking on T-cell subset stimulation by CD3 antibodies

The effect of exogenous recombinant interleukin-2 (IL-2) or of antibody crosslinking on the activation of human T-cell subsets by IgG2a (OKT3/BMA030), IgG1 (Leu4 and UCHT1), or IgG2b (BMA031) anti-T3 antibodies (CD3) was investigated. In so-called nonresponder cultures as well as in monocyte-depleted cell cultures addition of IL-2 increased the CD3-induced activation and proliferation of T4 and T8 cell subsets. Relatively more T8 than T4 cells were stimulated by antibody binding and IL-2. Crosslinking the cell-bound CD3 antibodies by plastic bound goat anti-mouse antibodies activated both T-cell subsets optimally and increased the IL-2 production of the IgG1-CD3 stimulated cultures. The data show that T cells (T8 greater than T4) can be stimulated by CD3 antibody binding and IL-2, but that crosslinking the cell-bound CD3 antibodies is crucial for optimal T4 cell stimulation and IL-2 production.     

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.     

Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion.

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.     

In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall.

Mannoprotein components from Candida albicans were investigated for their ability to induce production of tumor necrosis factor (TNF) by cultured splenocytes from naive or Candida-infected mice. Two chromatographically separated mannoproteins preparations, designated F1 and F2, were as able as the heat-inactivated Candida cells to induce the production of TNF from splenocytes of naive animals. In addition, they caused a significant augmentation of basic TNF secretion by splenocytes of Candida-infected animals. Experiments using plastic and/or nylon wool adherence, as well as treatments with antibodies depleting T or NK cells, consistently indicated that most if not all TNF was produced by splenic macrophages. In cultures of splenocytes from Candida-infected mice, mannoprotein addition also stimulated interferon-gamma (IFN-gamma) production by Thy 1.2 positive cells. Depletion of these cells or addition of anti-IFN-gamma antibodies abolished IFN production and reduced TNF secretion by adherent cells to the levels found in the cultures of mannoprotein-stimulated spleen cells from naive mice. These data add further evidence to the immunomodulatory properties possessed by some cell wall constituents of the human commensal microorganism C. albicans and suggest that IFN-gamma is endowed with a regulatory role in TNF production by mouse macrophages in vitro.     

Enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice.

The role of gamma interferon (IFN-gamma) induced during a viral infection in the ability of the host to acquire antiviral immunity was studied in mice. They were injected subcutaneously daily with an ammonium sulfate-precipitated sheep anti-IFN-gamma antibody preparation able to neutralize 10(4) U of IFN-gamma. Specificity of the anti-IFN-gamma antiserum was demonstrated by absence of detectable activity against natural IFN-alpha and -beta. Controls were treated with a similarly prepared normal sheep serum. Treatment with the IFN-gamma-specific antibody preparation had no influence on the ability of mice to generate anti-vaccinia virus- or anti-vesicular stomatitis virus (VSV)-specific cytotoxic T-cell (CTL) responses or T helper-dependent immunoglobulin G responses to VSV. In contrast, treatment of mice with sheep anti-IFN-gamma impaired CTL responses against lymphocytic choriomeningitis (LCM) virus (LCMV, Aggressive isolate); in addition, under the experimental conditions used, it prevented lethal LCM. Cytotoxic T-cell activity measured in the spleens of anti-IFN-gamma-treated mice was comparable to that found in mice initially infected with a 100-fold-larger dose of LCMV. Evaluation of the effects of treatment on the kinetics of virus replication revealed that in both euthymic and athymic nude C57BL/6 mice, anti-IFN-gamma treatment led to an increase of virus titers up to 100-fold compared with control mice. Therefore, IFN-gamma may play a role in controlling viruses with tropism for lymphocytes and monocytes/macrophages, such as LCMV.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.