IL-4 acts as a homeostatic regulator of IL-2-induced TNF and IFN-gamma.

Interleukin-4 (IL-4) is a cytokine secreted by interleukin-2 (IL-2)-activated lymphocytes. IL-2-stimulated lymphocytes also secrete two cytokines, tumour necrosis factor (TNF) and gamma-interferon (IFN-gamma), which contribute to effector function and which may themselves recruit fresh, cytokine-secreting effector cells. We have now investigated whether the IL-4 induced is able to homeostatically regulate secretion of the TNF and IFN-gamma. Peripheral blood mononuclear cells or lymphocytes from normal donors and from patients with neoplastic disease were cultured in the presence of IL-2 alone, IL-4 alone or with both cytokines. IL-2 induced high levels of TNF and IFN-gamma secretion in both groups. The addition of recombinant IL-4 to these IL-2-stimulated cultures lead to significant inhibition of IFN-gamma and TNF production. IFN-gamma secretion was reduced by 50-99% in normal donors and by between 11% and 99% in patients (P less than 0.001). TNF levels induced by IL-2 were similarly reduced by IL-4 both in normal donors (P less than 0.003) and in patients (P less than 0.01). These inhibitory effects were produced by IL-4 at doses of IL-2 attainable in vivo. Inhibition appears to represent a homeostatic regulatory mechanism which may limit recruitment of fresh activated killer (AK) cells. When endogenous IL-4 activity in IL-2-activated lymphocytes was blocked by anti-IL-4 antibody, significantly higher levels of IFN-gamma and TNF were secreted (P less than 0.05). Since both TNF and IFN-gamma may contribute to the anti-neoplastic action of IL-2, manipulating the level of IL-4 activity in vivo could augment the benefits of IL-2 immunotherapy.     

Induction of interleukin-12 and gamma interferon requires tumor necrosis factor alpha for protective T1-cell-mediated immunity to pulmonary Cryptococcus neoformans infection

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-alpha) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-alpha expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-alpha-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-alpha prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-alpha monoclonal antibody (day 0) diminished the lung levels of TNF-alpha, interleukin-12 (IL-12), and gamma interferon (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-alpha (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-alpha-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-alpha-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-alpha production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-alpha results in a T2 shift of the T1/T2 balance of antifungal immunity.     

Interleukin 2 leads to dose-dependent expression of the alpha chain of the IL-2 receptor on CD25-negative T lymphocytes in the absence of exogenous antigenic stimulation

Expression of the alpha chain of the interleukin 2 receptor on T lymphocytes is restricted, increasing in the setting of activation, particularly after antigenic stimulation via the TCR. The effects of IL-2 in vitro on the expression of CD25 and proliferation as well as the cytokine induction in CD25-depleted T cells were studied. CD25-depleted and PBMC of healthy donors were cultured for 7 days with 0, 10, or 100 IU/ml of IL-2. Phenotypic analysis and measurement of cytokines in the culture supernatants were performed. IL-2 led to a dose-dependent induction of the IL-2R alpha chain on both CD4 and CD8 T lymphocytes. In the CD25-depleted cultures, IL-2 treatment (100 IU/ml) increased the percentage of CD4 T cells expressing CD25 by 30.6% (P = 0.05) and of CD8 T cells by 48.2% (P = 0.01) on day 7 compared to no treatment. In the PBMC cultures the increase on day 7 was 36.4% for CD4 (P = 0.01) and 50.8% (P = 0.025) for CD8 T lymphocytes. The patterns of cytokine induction in the CD25-depleted and control cultures were similar with increases of IFN-gamma, GM-CSF, IL-16, TNF alpha, and soluble IL-2 receptor in the IL-2-containing cultures. CFSE experiments demonstrated the proliferative capacity of both CD25-positive and -negative T cells. Interleukin 2 alone can lead to a dose-dependent induction of the alpha chain of its receptor on resting CD4 and CD8 T lymphocytes. IL-2 as a sole stimulant is also associated with generation of a cytokine milieu that includes IFN-gamma, GM-CSF, IL-16, and TNF alpha.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Effect of cytokines on antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis.

In our previous study, it was observed that human peripheral blood polymorphonuclear leucocytes (PMNs) exhibited a fungistatic effect on yeast cells of Paracoccidioides brasiliensis, and that interferon-gamma (IFN-gamma), but not tumor necrosis factor-alpha or interleukin-8 (IL-8), enhanced the antifungal activity of PMNs. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) also enhanced the PMN activity. GM-CSF-activated PMNs exhibited a killing effect on P. brasiliensis isolate Bt-4 and an enhanced fungistatic effect on isolate Aoki. IL-1beta activated PMNs to kill isolate Bt-4. Granulocyte colony-stimulating factor had no effect. Combinations of IFN-gamma with GM-CSF or IL-1beta, but not a combination of GM-CSF and IL-1beta, exhibited a synergistic effect in enhancing the antifungal activity of PMNs. These results strongly suggest that PMNs activated with IFN-gamma, GM-CSF and/or IL-1beta might play an important role in host defense in early infection with P. brasiliensis due to their enhanced antifungal activity.     

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells

We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.     

Staphylococcal enterotoxin A acts through nitric oxide synthase mechanisms in human peripheral blood mononuclear cells to stimulate synthesis of pyrogenic cytokines

The pyrogenic response to supernatant fluids obtained from human peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA) was characteristic of a response to an endogenous pyrogen in that it was brief and monophasic and was destroyed by heating supernatant fluids at 70 degrees C for 30 min. The febrile responses were in parallel with the levels of interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, and IL-6 in supernatant fluids obtained from PBMC treated with SEA. Both the pyrogenicity and the levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids started to rise at 6 to 18 h and reached their peak levels at 24 to 96 h after SEA incubation. Both the fever and the increased levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with anisomycin (a protein synthesis inhibitor), aminoguanidine (an inhibitor of inducible nitric oxide synthase [NOS]), or dexamethasone (an inhibitor of NOS). The febrile response to supernatant fluids obtained from the SEA-stimulated PBMC was attenuated by adding either anti-IL-1beta, anti-TNF-alpha, or anti-IFN-gamma monoclonal antibody (MAb) to supernatant fluids. The antipyretic effects exerted by anti-IL-1beta MAb were greater than those exerted by anti-TNF-alpha or anti-IFN-gamma MAb. The data suggest that SEA acts through the NOS mechanisms in PBMC to stimulate synthesis of pyrogenic cytokines (in particular, the IL-1beta).     

Cytokine induction of neopterin production.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.     

Dexamethasone promotes type 2 cytokine production primarily through inhibition of type 1 cytokines.

Glucocorticoids, at concentrations mimicking stress-physiologic plasma levels, cause an in vitro shift in the type 1/type 2 cytokine balance of human peripheral blood mononuclear cells (PBMC) toward a predominant type 2 response. The mechanisms of these immune alterations are currently unknown but may involve modulation of key cytokines known to regulate the type 1/type 2 cytokine balance. Therefore, we sought to determine the role of cytokines previously reported to regulate the type 1/type 2 cytokine balance, including interleukin-12 (IL-12), interferon-gamma (IFN-gamma, IL-10, IL-4, and IL-13, in the glucocorticoid-mediated human type 1/type 2 cytokine alterations. Human PBMC were stimulated in vitro with tetanus toxoid in the presence of 10(-8) M dexamethasone (DEX). Cultures were supplemented with recombinant human (rHuIL-12), rHuIFN-gamma, or neutralizing monoclonal antibodies (mAb) against IL-4, IL-10, or IL-13. DEX decreased IFN-gamma production and increased IL-4 and IL-10 production by tetanus-stimulated PBMC. The addition of either recombinant IL-12p70 or IFN-gamma abrogated the DEX-mediated decrease in IFN-gamma and increase in IL-4 production. Neutralization of IL-4 activity partially abrogated the DEX-induced alterations in IFN-gamma and IL-4, but not IL-10, production. Neutralization of IL-10 or IL-13 had no effect on the Dex-mediated type 1/type 2 cytokine alterations. Therefore, the DEX-mediated type 1/type 2 cytokine alterations in tetanus-stimulated PBMC are primarily the result of downregulation of type 1 cytokines, subsequently permitting the production of type 2 cytokines.     

Different cytokine profiles of peripheral blood mononuclear cells from patients with persistent and self-limited hepatitis C virus infection

An imbalance between T helper cell Th1 and Th2 like cytokines has been described in several chronic infectious diseases. In an attempt to characterise the mechanism responsible for viral persistence in hepatitis C virus (HCV)-related chronic infection, we analyzed Th1 cytokines (IL-2, IL-12, IFN-gamma) and Th2 cytokines (IL-4, IL-10) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) derived from ten patients with viremic chronic hepatitis C, five healthy HCV seropositive individuals and four HCV seronegative individuals. Cytokine production was determined by enzyme-linked immunosorbent assay (ELISA) after 72 h of stimulation. The results showed that the production of IFN-gamma by PHA-stimulated PBMC was decreased in patients with hepatitis C infection (P=0.05). IL-4 production was not detected in both patients and controls, while no difference was observed for IL-2, IL-10 and IL-12 production between patients and controls. Furthermore, IL-12 and IFN-gamma production was weaker in patients with viremic chronic hepatitis C than in subjects who were able to clear the virus (P=0.01; P=0.03, respectively). These results clearly indicate that a defect both in IL-12 and IFN-gamma production may contribute to the persistence of HCV infection.     

Regulation of cytokine production in human peripheral blood mononuclear cells and allergen-specific th cell clones by 1alpha,25-dihydroxyvitamin D3

BACKGROUND: The steroid hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol), in addition to its crucial role in calcium homeostasis, exerts several effects on the immune system by regulating cell proliferation, differentiation, and maturation. These effects may be exerted through the control of protooncogenes and the regulation of cytokine production. METHODS: The influence of calcitriol on cytokines secretion by human peripheral blood mononuclear cells (PBMC) isolated from healthy donors, and by allergen-specific T helper (Th) cell clones was studied. PBMC were cultured for 48 h with phorbol myristate acetate (PMA) and ionomycin in the presence or absence of calcitriol. Human Th cell clones were stimulated with either Bet v 1 allergen or anti-CD3 antibodies and PMA. Cytokines were measured in the supernatants by ELISA, and at single-cell level by FACS. RESULTS: Calcitriol significantly inhibited the production of IL-2, IFN-gamma and IL-12 by PBMC, as well as the percentage of CD4+ T cells containing intracytoplasmic IL-2 and IFN-gamma. Interestingly, calcitriol-treated PBMC induced the production of IL-10 and IL-5, but not of IL-4. The effect of calcitriol was maximal at 10(-7) to 10(-9) and noneffective at 10(-11) M. Calcitriol diminished the secretion of IL-1, TNF-alpha, and MG-CSF in PBMC. Furthermore, calcitriol also decreased the secretion of IL-2 and IFN-gamma by Th1 clones, and of IL-4 by Th2 clones. CONCLUSIONS: Our data strongly support the notion that calcitriol modulates the production of cytokines in a time- and concentration-dependent manner, and suggest that nonhypercalcemic derivatives of 1alpha,25-dihydroxyvitamin D(3) may be used for new immunosuppressive therapies.     

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.     

Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.     

The effects of the antifungal azoles itraconazole, fluconazole, ketoconazole and miconazole on cytokine gene expression in human lymphoid cells.

The antifungal azole drugs itraconazole (itra; R51,211), fluconazole (flu; UK-49,858), ketoconazole (keto) and micronazole (mico) have been investigated for their suppressive influence on the gene expression of the immunoregulatory cytokines IL2, IL4, IL9, GM-CSF, TNF-alpha, IFN-gamma, as well as both chains of the IL2 receptor in human PBMC and of the cytokines in the human keratinocyte cell line HaCat 17.5. The results obtained in Northern blot analysis were compared with the effects of the established immunosuppressant drug CSA and the new immunosuppressive drug FK 506, as well as the cytokine TGF-beta, which is also immunosuppressive. While 1 microgram/ml CSA and 0.1 microgram/ml FK 506 completely suppressed PHA-stimulated accumulation of mRNA for IL2, IL4, IL9, GM-CSF, TNF-alpha and IFN-gamma in PBMC, flu, keto and TGF-beta failed to inhibit any (except TNF-alpha blocked by TGF-beta). Itra and mico did suppress accumulation of mRNA, but unlike CSA and FK 506, only at high doses (10 micrograms/ml) and after extended incubation (24 h). None of the drugs nor TGF-beta suppressed the expression of the IL2R-alpha and IL2R-beta genes or TNF-alpha-stimulated cytokine gene expression in keratinocytes. Itra and mico, 1 mg/ml (achievable serum level), caused only slight inhibition of the cytokines in PBMC after 6 and 24 h of incubation. These results demonstrate that the mode of action of the azoles is different from CSA and FK 506.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同刺激剂诱导肺癌患者引流淋巴结细胞分泌细胞因子的研究

目的:探讨多种刺激剂对肿瘤引流淋巴结(TDLN)细胞分泌细胞因子的影响及其抗肿瘤效应和机制。方法:采用ELISA和Griess法测定不同刺激组(分别为IL2、IL2 自身肺癌细胞抗原、IL2 GMCSF IL4 自身肺癌细胞抗原和IL2 GMCSF IL4 LPS)刺激TDLN后第7、14、21天,分泌IL12p70、IFNγ、TNFα和NO的水平。结果:IL2 GMCSF IL4 自身肺癌细胞抗原和IL2 GMCSF IL4 LPS组刺激的TDLN细胞中,CD83 细胞的比率明显增多,分泌IL12p70、IFNγ及TNFα的水平也明显高于IL2和IL2 自身肺癌细胞抗原刺激组,IL2 GMCSF IL4 LPS组刺激的TDLN细胞分泌NO的水平明显高于其他3组。各种刺激剂刺激TDLN细胞后,分泌IL12p70、IFNγ和NO的水平在第14天时达到高峰。结论:不同刺激剂诱导TDLN细胞中的CD83 细胞数量不同,故具有不同的抗肿瘤活性。     

Interferon-γ-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-γ production

The novel cytokine interferon-γ-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-γ (IFN-γ) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-γ had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-γ; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-γ production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-γ, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-γ production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.     

Characterization of cytokine production in infectious mononucleosis studied at a single-cell level in tonsil and peripheral blood.

Cytokine profile and production was studied at a single-cell level in cells obtained from 14 patients with acute infectious mononucleosis (IM), with less than 7 days of symptomatic disease, by use of cytokine-specific MoAbs and indirect immunofluorescence technique. In producer cells, all the studied cytokines, except IL-1, accumulated in the Golgi system, which resulted in a characteristic morphology of the staining. Less than one in a thousand mononuclear cells obtained directly from IM blood and stained within 2 h of sampling produced IL-2, interferon-gamma (IFN-gamma), IL-4, IL-5, IL-6, IL-10, GM-CSF, tumour necrosis factor-alpha (TNF-alpha) or TNF-beta, spontaneously. However, these cells were induced to cytokine synthesis by T cell receptor ligation in vitro using immobilized anti-CD3 MoAbs for 2-3 h restimulation under conditions which did not activate normal cells. By this approach 168 +/- 120 cells/10,000 peripheral blood mononuclear cells produced IFN-gamma as compared with 10 +/- 8 cells/10,000 non-stimulated cultured cells obtained from IM patients (P < 0.001) and 1/10,000 cells obtained from healthy controls, respectively. No induced production of IL-2, IL-3, IL-4, IL-5, IL-10, GM-CSF or TNF-beta was detected in IM cells obtained from peripheral blood by this restimulation. In contrast, a spontaneous cytokine production was evident in tonsil material obtained from four IM patients tonsilectomized because of respiratory obstruction. From this site 160 +/- 40 cells/10,000 cells produced IL-2, 40 +/- 30 cells IL-6, 30 +/- 30 cells TNF-beta and 35 +/- 25 cells IFN-gamma, respectively. No such spontaneous IL-2, IL-6, TNF-beta or IFN-gamma production was evident in control cells obtained from patients tonsilectomized because of chronic tonsil hyperplasia.     

Mechanisms of cytokine synergy essential for vaccine protection against viral challenge.

The ability of cytokines to steer CD4(+) T(h) cell responses toward a T(h)1 or T(h)2 phenotype and enhance the magnitude of both CD8(+) cytotoxic T lymphocytes (CTL) and antibody responses has clearly been demonstrated by our lab and others, but the influence of cytokines on protective immune responses is much less clear. Here we show an essential role for CD4(+) T(h)1 helper cell induction and IFN-gamma production in protection from viral challenge with a recombinant vaccinia virus expressing HIV-1MN viral envelope glycoprotein gp160. Complete protection from viral challenge is achieved only when the triple combination of exogenous cytokines granulocyte macrophage colony stimulating factor (GM-CSF), IL-12 and tumor necrosis factor (TNF)-alpha are co-administered with the peptide vaccine. In vivo depletion of CD4(+) cells or immunization of IFN-gamma-deficient mice abrogates protection. GM-CSF, IL-12 and TNF-alpha also synergize for the enhanced induction of CTL; however, adoptive transfer of a CD8(+) CTL line afforded only partial protection in this viral challenge model. As a possible mechanism of in vivo protection we show that GM-CSF increases the percentage and activity of antigen-presenting dendritic cells in draining lymph nodes where the immune response is initiated. We further demonstrate synergy between IL-12 and the proinflammatory cytokine TNF-alpha in driving IFN-gamma production. Thus, a combination of IL-12 and TNF-alpha is essential for the optimal development of T(h)1 responses and help for CTL induction in BALB/c mice, and is complemented by a third cytokine, GM-CSF, which enhances antigen presentation.     

Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides.

Cytokines such as tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 8 (IL-8), IL-6, IL-1 alpha, and IL-1 beta produced during the immune and inflammatory responses to bacterial stimuli have been reported to interact with polymorphonuclear neutrophil (PMN) activities. However, contradictory findings on their direct and priming effects on the PMN oxidative burst, which is essential for bacterial killing, have been reported. We have used a flow cytometry method to study the effects of these cytokines on the oxidative burst of PMN in whole blood to avoid PMN activation related to isolation procedures. None of the cytokines tested directly activated the PMN oxidative burst, but they did have differential priming effects on the oxidative burst in response to bacterial N-formyl peptides. TNF, GM-CSF, and IL-8 strongly primed a subpopulation of PMN to produce H2O2 in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP), while IL-1 alpha, IL-1 beta, and IL-6 failed to do so. Furthermore, the addition of TNF, GM-CSF, or IL-8 to whole blood increased the capacity of a subpopulation of PMN to bind N-formyl peptides, a phenomenon that could account, at least in part, for the strong H2O2 production in response to FMLP after priming by the cytokines. The size of the primed hyperresponsive subpopulation was greater after priming with TNF or GM-CSF than after priming with IL-8. However, GM-CSF, TNF, and IL-8 at suboptimal concentrations cooperated in the induction of a subpopulation hyperresponsive to FMLP. These results show that, of the various proinflammatory cytokines tested, TNF, GM-CSF, and IL-8 strongly prime the PMN oxidative burst in response to bacterial peptides in whole blood and suggest that these cytokines may play a critical role in bacterial killing in vivo.