Control by IFN-gamma and PGE2 of TNF alpha and IL-1 production by human monocytes.

There have been suggestions that the production of pro-inflammatory mediators by human monocytes in response to interferon-gamma (IFN-gamma) may be controlled by changes in prostaglandins. Therefore we investigated tumour necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) activities and prostaglandin E2 (PGE2) levels in the supernatants of highly purified human monocytes cultured for 18 hr with recombinant human IFN-gamma. IFN-gamma (100 U/ml) did not stimulate monocytes isolated by counter-current centrifugal elutriation for detectable TNF alpha or IL-1 activities, or PGE2 production. However, IFN-gamma synergistically enhanced lipopolysaccharide (LPS)-induced TNF alpha and IL-1 activities. In contrast, there was no consistent change in PGE2 levels upon addition of IFN-gamma to LPS-treated monocyte cultures. The TNF alpha and IL-1 activities induced by LPS and by LPS with IFN-gamma were reduced by PGE2, and stimulated by indomethacin. As reported previously for IL-1 activities, the regulation by cyclo-oxygenase products of TNF alpha activities reflected predominantly a control of the production of immunoreactive TNF alpha, rather than the measurement of TNF alpha bio-activity. However, the addition of indomethacin or PGE2 to monocyte cultures did not change the extent of IFN-gamma synergy with LPS for increased TNF alpha and IL-1 activities. The results of this study suggest that, despite control by cyclo-oxygenase products of TNF alpha and IL-1 production in human monocytes, IFN-gamma may enhance TNF alpha and IL-1 activities independently of this regulatory mechanism. These findings are contrary to those suggested for the regulation by prostanoids of IL-1 production by murine macrophages.     

Interferon-beta induces selective enhancement of antigen-specific T cell responses.

Interferon-beta (IFN-beta) inhibits mitogen-induced T cell responses, in part through downregulation of interleukin-12 (IL-12) or upregulation of IL-10. We have reexamined these findings using ragweed (RW) stimulated or tetanus toxoid (TT)-stimulated human peripheral blood mononuclear cells (PBMC) and nontransformed, antigen-specific, human Th0, Th1, and Th2 clones. IFN-beta induced concentration-dependent inhibition of phytohemagglutinin (PHA)-stimulated PBMC proliferation and enhancement of RW-stimulated or TTstimulated PBMC proliferation. Monocyte depletion of PBMC isolates resulted in concentration-dependent inhibition of RW-driven or TT-driven proliferation by IFN-beta. This response was unaltered by the addition of either exogenous recombinant human IL-12 (rHuIL-12) or saturating concentrations of anti-IL-10. Moreover, addition of exogenous rHuIL-10 to nondepleted RW-driven or TT-driven PBMC cultures did not alter the concentration-dependent enhancement of antigen-driven proliferation induced by IFN-beta. Th0, Th1, and Th2 clones stimulated in the presence of antigen and autologous, irradiated PBMC displayed concentration-dependent inhibition of proliferation in the presence of IFN-beta that was unaltered by the addition of either exogenous rHuIL-12 or a saturating concentration of anti-IL-10. Finally, whereas IFN-beta inhibited antigen-driven generation of IL-5, IL-12, IL-13, and IFN-gamma, IFN-beta enhanced generation of both IL-4 and IL-10. Thus, IFN-beta, induces a selective, IL-10-independent and IL-12-independent upregulation of antigen-specific T cell responses, supporting the role of IFN-beta as an immunomodulatory rather than an antiproliferative/immunosuppressive cytokine.     

Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells. Introduction of an amplification step using ionomycin and phorbol myristate acetate.

The study of T cell responses in parasitic disease and allergy in humans has been limited by difficulties in the measurement of interleukin-4 (IL-4) in supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC). To obtain measurable amounts of IL-4 in vitro, we have added an amplification step to the antigen-specific response. Human PBMC were stimulated by tetanus toxoid (TT) or tuberculin (PPD) for 6 days and then pulsed with ionomycin and phorbol myristate acetate (PMA) for 24 h. TT-stimulated cells from nine revaccinated donors but not from seven unvaccinated donors and four that had only received childhood vaccinations against tetanus produced high levels of IL-4 (median (range) 1500 (300-3800), 316 (0-1600), and 270 (100-410) pg/ml, respectively, as measured by an enzyme linked immunosorbent assay, P = 0.005). PPD did not increase IL-4 production above the background level, although the majority of PPD-stimulated PBMC proliferated and produced interferon-gamma (IFN-gamma) in cultures without ionomycin and PMA. TT-induced IL-4 production correlated positively with proliferation. Culture supernatants did not interfere with IL-4 immunoreactivity and failed to affect ionomycin and PMA induced IL-4 production. The findings suggest that proliferating antigen-specific T cells were the source of IL-4 in these experiments. The method should prove useful for comparing the IL-4 producing ability of antigen-specific T cells from different individuals.     

High dose allergen stimulation of T cells from house dust mite-allergic subjects induces expansion of IFN-gamma+ T Cells, apoptosis of CD4+IL-4+ T cells and T cell anergy

BACKGROUND: During clinically effective allergen-specific immunotherapy a shift in cytokine dominance from IL-4, IL-5 predominant to IFN-gamma predominant has been observed. As antigen concentration influences Th cell priming, this study aimed to determine the effect of different allergen concentrations on human house dust mite (HDM)-specific T cell production of IL-4 and IFN-gamma, proliferation and apoptosis. METHODS: HDM-allergic donor PBMC were cultured for 14 days with different concentrations of HDM extract (1, 10 and 100 microg/ml). T cell intracellular IL-4 and IFN-gamma, division (CFSE labelling) and apoptosis (active caspase-3 staining) were analysed by flow cytometry. Proliferation was assessed by (3)H-thymidine incorporation. RESULTS: Increased CD4+IFN-gamma+ and CD8+IFN-gamma+ T cell numbers were observed in high allergen concentration cultures compared with low concentration cultures whereas there were no differences in CD4+IL-4+ and CD8+IL-4+ T cell numbers. CFSE cell labelling revealed that high allergen concentration favours the expansion of IFN-gamma-producing CD4+ T cells. The proportion of apoptotic cells increased with allergen concentration and there was preferential apoptosis of CD4+IL-4+ T cells. HDM-induced proliferation was decreased in high allergen concentration cultures; this was reversible by IL-2 consistent with anergy. CONCLUSION: These results show that T cell division and apoptosis contribute to the cytokine skewing to predominant IFN-gamma production by T cells observed at high allergen concentration. Thus the use of hypoallergenic T cell reactive preparations which can be given safely at higher doses than natural extracts may enhance efficacy of allergen-specific immunotherapy.     

Interleukin-1 production by mononuclear cells from patients with scleroderma.

Interleukin-1 (IL-1) production by peripheral blood mononuclear cells (PBMC) from patients with scleroderma and healthy controls was studied. Supernatants from unstimulated PBMC cultures from 10 of 13 patients with progressive systemic sclerosis (PSS) had significantly less IL-1 activity as measured by thymocyte proliferation than controls. IL-1 activity per monocyte/macrophage in both patients and controls was 10 times greater when PBMC were cultured at 10(5) cells/ml compared to 10(6) cells/ml. Five-fold dilution of supernatants from PBMC cultured at 10(6) cells/ml revealed more IL-1 activity than undiluted supernatant and addition of indomethacin increased IL-1 activity primarily of the undiluted supernatant. The results show that IL-1 activity from crude PBMC supernatants from PSS patients is low and may be regulated by non-dialysable inhibitors produced by PBMC and/or cell interactions.     

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.     

Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma.

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.     

Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities.

The in vitro production of interferon-gamma (IFN-gamma) in 19 atopic dermatitis (AD) patients was compared with that of 12 controls. IFN-gamma production by phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) was profoundly diminished in AD patients, whereas the proliferative response was similar to that of control PBMC. The addition of 40 U/ml of interleukin-2 (IL-2) to the cultures failed to restore IFN-gamma production. Similarly, removal of adherent cells also had no effect. Reduced IFN-gamma secretion was observed after stimulation with the CD3 monoclonal antibody OKT3, ionomycin + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with high levels of IL-2 (200 U/ml). There were increased proportions of CD4+ T helper/inducer cells and decreased proportions of CD8+ T cytotoxic-/suppressor cells and CD16+ natural killer (NK) cells in AD patients. This resulted in an increased CD4/CD8 ratio as compared with controls, but no correlation was observed between numbers of T cell subpopulations and IFN-gamma generation. However, a significant correlation was found between IFN-gamma generation in vitro and IgE serum concentration in AD patients. The data suggest that the decreased production of IFN-gamma by AD patients is due to intrinsic differences in capacity to produce this cytokine and is not the result of differences in regulatory cell interactions. Moreover, the findings indicate that decreased production of IFN-gamma may be an important factor in the pathogenesis of this disease.     

The immunosuppressive drug thalidomide induces T helper cell type 2 (Th2) and concomitantly inhibits Th1 cytokine production in mitogen- and antigen-stimulated human peripheral blood mononuclear cell cultures.

Thalidomide is an effective immunomodulatory drug in man, but its mechanism of action remains unclear. We hypothesized that, in addition to its reported inhibitory effects on production of monocyte-derived tumour necrosis factor-alpha (TNF-alpha), thalidomide might be effective at the level of Th immunoregulation. In a comparative study with the immunosuppressant cyclosporin A, we have demonstrated a potent and specific effect of thalidomide on cytokine production relating to the distinct Th1 and Th2 subsets. It induced and enhanced the production of IL-4 and IL-5 and, at the same dose (1000 ng/ml), significantly inhibited interferon-gamma (IFN-gamma) production in phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell (PBMC) cultures. Stimulation of PBMC with recall antigen (streptokinase:streptodornase (SKSD)) at 144 h in the absence of thalidomide resulted in a predominantly Th1 response, with the production of IFN-gamma and IL-2. Thalidomide switched this response from a Th1 to a Th2 type. The effect was most pronounced at 1000 ng/ml thalidomide, where inhibition of IFN-gamma and enhancement of IL-4 production was maximal. In unstimulated cultures thalidomide alone induced IL-4 production. Cyclosporin A, in contrast, inhibited both Th1 and Th2 cytokine production by PHA-stimulated PBMC. Time course data from thalidomide-treated cultures revealed that the augmented IL-4 production diminished as the culture time increased, whereas IFN-gamma production was significantly increased. This response might be due to activation-induced apoptosis of Th2 cells or the induction of Th2 cell anergy, in the continued presence of stimulating agents, with the emergence of IFN-gamma-secreting Th1 cells when Th2 antagonism declines. The effects of thalidomide and related compounds may enhance our understanding of the mechanisms of T helper cell selection, offer the possibility of controlled therapeutic switching between Th1 and Th2 responses, and may lead to a rational approach for the treatment of some T cell-mediated immunological disorders.     

Hepatitis C virus non-structural protein 4 suppresses Th1 responses by stimulating IL-10 production from monocytes

The majority of hepatitis C virus (HCV) infections become chronic, despite the presence of HCV-specific cellular and humoral immune responses. We have previously suggested that IL-10-secreting antigen-specific regulatory T cells may contribute to viral persistence, and demonstrate here that peripheral blood mononuclear cells (PBMC) from chronically HCV-infected patients secrete IL-10, but not IFN-gamma, in response to HCV nonstructural protein 4 (NS4). A neutralizing anti-IL-10 antibody restored this defective antigen-specific IFN-gamma production in vitro. Furthermore, PBMC from normal individuals secreted IL-10 in response to NS4, suggesting that cells of the innate immune system, in addition to T cells, produced IL-10 in the HCV-infected patients. Cell separation experiments revealed that the innate IL-10 was produced by blood monocytes, but not dendritic cells (DC). In addition, NS4 inhibited IL-12 production by PBMC in response to LPS and IFN-gamma, and Th1 responses to recall antigens in normal individuals. Furthermore, supernatants from NS4-stimulated monocytes inhibited LPS-induced maturation of DC and suppressed their capacity to stimulate proliferation and IFN-gamma production by allospecific T cells. Our data suggest that HCV subverts cellular immunity by inducing IL-10 and inhibiting IL-12 production by monocytes, which in turn inhibits the activation of DC that drive the differentiation of Th1 cells.     

APC-dependent impairment of T cell proliferation in aging: role of CD28- and IL-12/IL-15-mediated signaling

The age-related impairment of phytohaemagglutinin (PHA)-triggered peripheral blood mononuclear cell (PBMC) proliferation was paralleled by an expansion of CD28 (-) T lymphocytes with a poor capacity to undergo lectin-induced blastogenesis. However, both CD28 (-) and CD28 (+) T cells isolated from aged individuals exhibited a significant reduction of proliferative response to PHA in comparison with young controls, this implies that the CD28-mediated signaling is not the only defective pathway in the elderly. Thus, PBMC or T cell subsets plus monocytes from aged donors were stimulated with PHA and assayed for the production of, or the response to cytokines known to regulate T cell functions. Results can be so summarized: (i). interleukin (IL)-2 as well as IL-10 release was unaffected by age; (ii). in both groups of subjects, IL-15 concentrations were similar to those spontaneously released by PBMC; (iii). surprisingly, IL-12 p70 and IL-12 p40 production by PBMC was markedly increased in the aged group; (iv) in spite of this finding and of the experimental outcome that IFN-gamma synthesis was almost completely dependent on IL-12. PBMC from old individuals did not release higher amounts of IFN-gamma in comparison with young controls; (v). moreover, only a slight increase in IFN-gamma production was observed in PBMC cultures from the aged group as a result of IL-12 and/or IL-15 costimulation; (vi) at the same time, even though IL-12 as well as IL-15 were necessary for an efficient T cell proliferation, the addition of exceeding doses of cytokines proved to be ineffective in enhancing the proliferative outcome of PBMC or of both CD28 (+) and CD28 (-) T cells in the aged group. Taken together, the data outline the role of CD28 and IL-12/IL-15 signaling impairment in T cell proliferative deficiency during senescence.     

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.     

Brucella abortus regulates bovine macrophage-T-cell interaction by major histocompatibility complex class II and interleukin-1 expression

T-cell activation is dependent on nominal antigen associated with major histocompatibility complex (MHC) class II molecules and interleukin-1 (IL-1), both provided by antigen-presenting cells. We have studied the effects of Brucella abortus and recombinant bovine gamma interferon (IFN-gamma) on bovine macrophage expression of MHC class II and IL-1 molecules and subsequent T-cell proliferation in response to B. abortus. When peripheral blood mononuclear cells were cocultured with B. abortus and IFN-gamma, increasing amounts of IFN-gamma, from 1 to 100 U/ml, down regulated T-cell proliferation. Expression of MHC class II molecules on macrophages was increased independently by IFN-gamma or B. abortus treatment. Thus, class II molecule expression was not the cause of down regulation of T-cell proliferation as observed in other systems. However, B. abortus-IFN-gamma-treated macrophages obtained from overnight cultures had minimal membrane IL-1 compared with macrophages treated with B. abortus alone. Loss of membrane IL-1 required IFN-gamma and the o-polysaccharide of the lipopolysaccharide. IFN-gamma at 1 U/ml in combination with B. abortus produced a 32% decrease in T-cell response, while IFN-gamma at 100 U/ml added to B. abortus-treated cultures produced an 82% reduction in T-cell response. Membrane IL-1 levels were not altered when recombinant bovine IFN-alpha or the rough strain 45/20 of B. abortus, which lacks the o-polysaccharide, was used. Secreted IL-1 levels were unaffected by IFN-gamma and B. abortus treatment. The addition of recombinant bovine IL-1 beta (0.001 to 0.1 ng/ml) to B. abortus- and IFN-gamma-treated cultures failed to provide a signal necessary for T-cell proliferation. These data suggest that membrane IL-1 has a key role in T-cell activation in response to B. abortus. When the o-polysaccharide of B. abortus lipopolysaccharide is combined with IFN-gamma at an inappropriate time during an immune response, T-cell proliferation is prevented and cannot be restored by the addition of exogenous IL-1.     

Interferon-gamma inhibits the proliferation but not the differentiation of murine B cells in response to IL-5

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.     

Autoregulatory role of interleukin-10 in hepatitis C patients treated with IFN-alpha.

Interferon-alpha2 (IFN-alpha2) is used as standard treatment of patients with chronic hepatitis C (cHCV), but little is known about the immunomodulatory effects of this cytokine in vivo. We have studied immunologic parameters in freshly isolated peripheral blood mononuclear cells (PBMC) of 26 patients with cHCV 12 h before and 12 h after the first s.c. injection of 5-6 MU IFN-alpha2. In PBMC obtained after IFN injection, a substantial increase in IL-10 production after antigen-specific and nonspecific stimulation was observed, whereas IFN-gamma production and proliferation were significantly diminished compared with PBMC obtained before IFN injection. Patients were stratified according to single nucleotide polymorphisms (SNPs) in the interleukin-10 (IL-10) promoter, which have been associated with the response to IFN therapy. Induction of IL-10 and suppression of IFN-gamma levels were more prominent in patients with genotype CC at position -592 (n = 15) compared with patients with genotype AA/AC (n = 11). In conclusion, our data indicate that IFN-alpha2 therapy can potently enhance IL-10 and suppress IFN-gamma production of PBMC, which is, at least partially, dependent on an SNP in the IL-10 promoter. This suggests an autoregulatory role of IL-10 in IFN therapy.     

Interaction of Mycobacterium tuberculosis-Induced Transforming Growth Factor β1 and Interleukin-10

Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-beta1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-beta1 independently suppressed the production of PPD-induced gamma interferon (IFN-gamma) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-gamma in cultures containing both rTGF-beta1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-beta1 but not with rIL-10. Suppression of PPD-induced IFN-gamma in PBMC containing both rTGF-beta1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P < 0.05) than cultures containing TGF-beta1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-beta1 and IL-10 together enhanced PPD-induced IFN-gamma in PBMC in a synergistic manner. Thus, TGF-beta1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-gamma, and TGF-beta1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.     

Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6. Effect of IL-1 receptor antagonist.

The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells. Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor. In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity. In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition. We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC). We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells. The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity. We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta. The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood. Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing. In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures. The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production.     

Interferon-gamma (IFN-gamma) and prostaglandin E2 (PGE2) regulate differently IL-12 production in human intestinal lamina propria mononuclear cells (LPMC).

IL-12 modulates Th1 immune response during chronic colitis. Mechanisms regulating IL-12 synthesis in human intestine are poorly understood. The aim of this study was to investigate the effect of IFN-gamma and PGE2 on lipopolysaccharide (LPS)-stimulated LPMC IL-12 production. Normal LPMC cultures were run in the presence or absence of IFN-gamma and/or PGE2 before LPS stimulation. To examine the role of endogenous PGE2 on LPS-stimulated IL-12 release, LPMC cultures were added of indomethacin before LPS stimulation. IL-12, IL-10 and IL-8 were measured by ELISA. No IL-12 was detected in either unstimulated or LPS-stimulated LPMC cultures. In contrast, LPMC released IL-8 (650 +/- 125 pg/ml) and IL-10 (75 +/- 25 pg/ml) in response to LPS. Treatment of LPMC with IFN-gamma facilitated LPS-stimulated IL-12, whereas it completely abrogated IL-10 production. IL-12 release by LPMC stimulated with IFN-gamma and LPS was significantly inhibited by exogenous IL-10. The addition of PGE2 to IFN-gamma-treated LPMC cultures inhibited in a dose-dependent manner LPS-induced IL-12 secretion. Furthermore, IL-12 was detectable (85 +/- 25 pg/ml) in the supernatants of LPMC cultures treated with indomethacin and LPS. In contrast to the effect on IL-12, PGE2 significantly augmented LPS-stimulated LPMC IL-10 production. However, the inhibition of IL-12 by PGE2 was only partially reversed by anti-IL-10. In a simplified model of LPS tolerance, we finally showed that monocyte-derived macrophages exhibited reduced IL-12 production after repeat LPS stimulation. In these cell cultures, indomethacin abrogated the induction of LPS desensitization. IFN-gamma and PGE2 modulate differently the LPMC responsiveness to LPS in terms of IL-12 synthesis.     

Eukaryotic heat shock proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells

Heat shock proteins (HSP) like Hsp60, Hsp70 and gp96 act directly on antigen-presenting cells (APC), e.g. by inducing the secretion of cytokines. Here we analyzed the impact of Hsp60 on the antigen-specific activation of CD8(+) T cells in a TCR transgenic system. Hsp60 induced low amounts of IFN-gamma in the absence of antigenic peptide; however, the release of IFN-gamma is increased by a factor of 3-10 following the addition of Hsp60 to purified populations of OT-1 [ovalbumin (OVA)257-264/H2-K(b)-restricted] T cells and antigen-pulsed peritoneal exudate cells (PEC) as APC. This effect is strictly correlated with the PEC ability to produce IL-12. In contrast, antigen-specific IL-2 secretion and T cell proliferation was not changed in the presence of Hsp60. Hsp60-containing OT-1 T cell cultures produced IFN-gamma even when the number of antigenic MHC class I complexes was too low to be stimulatory and could not be detected with specific mAb. Hsp60, thus, acts as a catalyzing molecule to initiate both innate and adaptive immune responses, and its presence (e.g. during an infection with cellular destruction) has direct consequences for the activation of otherwise 'ignorant' antigen-specific T cells.     

Increased IL-2, IL-4 and interferon-gamma (IFN-gamma) in steroid-sensitive nephrotic syndrome.

We investigated the production of cytokines by peripheral blood mononuclear cells (PBMC) and serum cytokine concentrations in children with steroid-sensitive idiopathic nephrotic syndrome (SSNS). PBMC from patients off treatment were collected during remission and relapse and cultured in medium alone or stimulated with calcium ionophore plus phorbol myristate acetate. Control PBMC were taken from healthy age-matched children. IL-2 was measured by bioassay, IL-4 by immunoradiometric assay, and IL-8 and IFN-gamma by ELISA. After 24 h culture without stimulation, IL-2, IL-4 and IFN-gamma were not detectable in the supernatant in any of the children. After stimulation, the supernatant concentrations of IL-2 (median 172 U/ml at 24 h) and IL-4 (160 pg/ml at 24 h; 210 pg/ml at 72 h) were significantly increased in relapse compared with remission (IL-2 37 U/ml; IL-4 65 pg/ml and 60 pg/ml) and controls (IL-2 69 U/ml; IL-4 40 pg/ml and 40 pg/ml) (P < 0.05). The concentration of IFN-gamma was not significantly increased in relapse compared with remission and controls (600, 325, and 145 U/ml, respectively, at 72 h). IL-8 concentrations were similar in relapse, remission and controls with stimulation (median 32, 40 and 40 ng/ml, respectively) and without (30, 17 and 10 ng/ml). IL-2 was not detectable in serum, but IL-4, IL-8 and IFN-gamma were measurable in about half the patients, both in relapse and remission, though were virtually undetectable in controls. We conclude that relapse of SSNS in children is associated with T lymphocyte activation with release of IL-2, IL-4 and IFN-gamma.