Depressed CD40 ligand expression contributes to reduced gamma interferon production in human tuberculosis

Expression of CD40 ligand (CD40L) correlated directly with Mycobacterium tuberculosis-stimulated gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy tuberculin reactors. The CD40L agonist increased M. tuberculosis-induced IFN-gamma production by PBMC, and anti-CD40 or anti-CD40L antibodies reduced IFN-gamma production. CD40L expression on PBMC was reduced by exposure to B cells and to soluble factors from M. tuberculosis-infected monocytes. These findings suggest that CD40L dysregulation contributes to reduced IFN-gamma production in human tuberculosis.     

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.     

Ginseng modulates the immune response by induction of interleukin-12 production

In infections with intracellular microorganisms such as mycobacteria and Leishmania parasites as well as certain extracellular chronic infections such as Pseudomonas aeruginosa a Th1 response with activation of macrophages is desirable. Several studies indicate that such a response is associated with better recovery from infection, improved course of the chronic infection, and higher survival rate. In Th1 responses there is increased interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) production, whereas that of interleukin-10 (IL-10) is decreased. The present study indicated that Ginseng modulation of stimulated peripheral blood mononuclear cells (PBMC) results in a higher IL-12 production. The enhanced IL-12 production could induce a stronger Th1 response, resulting in better protection against infection with a variety of pathogens.     

IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-gamma expression.

Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-gamma (IFN-gamma) (Th2- and Th1-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P = 0.0075 and P = 0.0004, respectively) compared with non-atopic controls, whereas IFN-gamma production was not significantly different. In contrast to allergen, the prototypic Th1-type antigen M. tuberculosis PPD induced an excess of IFN-gamma over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-gamma or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.     

T-cell cytokine responses in human infection with Mycobacterium tuberculosis.

Compared with healthy tuberculin reactors, Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells from tuberculosis patients had diminished production and mRNA expression of the Th1 cytokines gamma interferon and interleukin 2 (IL-2), with no change in production and mRNA expression for the Th2 cytokines IL-4, IL-10, and IL-13. These results were confirmed by evaluation of T cells and CD4+ cells. At the level of systemic T cells, development of tuberculosis is associated with diminished Th1 but not enhanced Th2 responses.     

Depressed interleukin-12 (IL-12), but not IL-18, production in response to a 30- or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis

The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-gamma), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-gamma and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.     

Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.     

Interleukin-12 in human boutonneuse fever caused by Rickettsia conorii

Interleukin (IL)-12 contributes to the resistance against a number of intracellular pathogens. We examined the potential biological role of IL-12 by studying peripheral blood mononuclear cells (PBMC), its production and its effect on cytokine synthesis in 20 Sicilian patients with boutonneuse fever (BF) caused by Rickettsia conorii. Data indicate that PBMC from acute BF patients were able to produce IL-12 in response to in vitro stimulation with rickettsial antigen (Ag): this production was higher than that detected in healed patients. Monocytes were the main source of IL-12 by PBMC from BF patients. IL-12 secretion by in vitro Ag-stimulated PBMC from BF patients was potentiated by recombinant interferon gamma (IFN-gamma) or anti-IL-10 monoclonal antibodies (MoAbs). Furthermore, the treatment with anti-IL-12 MoAbs reduced the IFN-gamma synthesis. These results indicate that treatment of PBMC from acute BF patients with IL-12 shifted the response toward a Th1-type cytokine response. Furthermore, IL-12 and IFN-gamma are interdependent and they may be associated with the immunity against rickettsias.     

Absence of a prominent Th2 cytokine response in human tuberculosis.

Depressed Th1 responses are a prominent feature of human tuberculosis, but an enhanced Th2 response has not been detected in peripheral blood T cells stimulated in vitro with Mycobacterium tuberculosis. In disease due to Mycobacterium leprae, Th2 cells predominate in tissue lesions of patients with extensive disease but are absent from peripheral blood. To determine if Th2 cells are present in tissue lesions of tuberculosis patients, we evaluated patterns of cytokine expression in lymph nodes from tuberculosis patients with or without human immunodeficiency virus infection and in controls without tuberculosis. Gamma interferon and interleukin-10 (IL-10) mRNA expression in tuberculosis patients with or without human immunodeficiency virus infection was high, whereas IL-4 expression in the same patients was low. Immunolabeling studies showed that macrophage production of IL-12 was increased in lymph nodes from tuberculosis patients, that gamma interferon was produced by T cells, and that IL-10 was produced by macrophages rather than Th2 cells. These results indicate that Th2 responses are not enhanced either systemically or at the site of disease in human tuberculosis.     

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.     

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.     

Cytokine production at the site of disease in human tuberculosis.

Clinical and immunologic evidence suggests that tuberculous pleuritis provides a model to understand protective immune mechanisms against Mycobacterium tuberculosis. We therefore evaluated the pattern of cytokine mRNA expression and cytokine production in pleural fluid and blood of patients with tuberculous pleuritis. RNA was extracted from mononuclear cells, reverse transcribed to cDNA, and amplified by polymerase chain reaction (PCR). After normalization for T-cell cDNA, cDNA from pleural fluid cells and peripheral blood mononuclear cells (PBMC) was amplified with cytokine-specific primers. PCR product was quantified by Southern blot. For the Th1 cytokines gamma interferon (IFN-gamma) and interleukin-2 (IL-2), PCR product was greater in pleural fluid than in blood, whereas PCR product for the Th2 cytokine IL-4 was decreased in pleural fluid compared with blood. Concentrations of IFN-gamma were elevated in pleural fluid compared with serum, but IL-2, IL-4, and IL-5 were not detectable. Mean concentrations of IFN-gamma and IL-2 in supernatants of M. tuberculosis-stimulated pleural fluid cells were significantly greater than corresponding concentrations in supernatants of stimulated PBMC. In situ hybridization showed that increased IFN-gamma production by pleural fluid cells was associated with a 20- to 60-fold increase in the frequency of antigen-reactive IFN-gamma-mRNA-expressing cells. Because IL-10 can be produced by T cells and macrophages, pleural fluid cells and PBMC were normalized for beta-actin cDNA content and then amplified by PCR with IL-10-specific primers. IL-10 mRNA was greater in pleural fluid cells than in PBMC and was expressed predominantly by macrophages. IL-10 concentrations were elevated in pleural fluid versus serum. These data provide strong evidence for compartmentalization of Th1 cytokines and IL-10 at the site of disease in humans with a resistant immune response to mycobacterial infection.     

Dynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment

Pro- and anti-inflammatory cytokines and their signaling pathways play key roles in protection from and pathogenesis of mycobacterial infection, and their balance and dynamic changes may control or predict clinical outcome. Peripheral blood cells' capacity to produce proinflammatory (tumor necrosis factor alpha [TNF-alpha], interleukin-12/23p40 [IL-12/23p40], and gamma interferon [IFN-gamma]) and anti-inflammatory (IL-10) cytokines in response to Mycobacterium tuberculosis or unrelated stimuli (lipopolysaccharide, phytohemagglutinin) was studied in 93 pulmonary tuberculosis (TB) patients and 127 healthy controls from Indonesia. Their cells' ability to respond to IFN-gamma was examined to investigate whether M. tuberculosis infection can also inhibit IFN-gamma receptor (IFN-gammaR) signaling. Although there was interindividual variability in the observed responses, the overall results revealed that M. tuberculosis-induced TNF-alpha and IFN-gamma levels showed opposite trends. Whereas TNF-alpha production was higher in active-TB patients than in controls, IFN-gamma production was strongly depressed during active TB, correlated inversely with TB disease severity, and increased during therapy. By contrast, mitogen-induced IFN-gamma production, although lower in patients than in controls, did not change during treatment, suggesting an M. tuberculosis-specific and reversible component in the depression of IFN-gamma. Depressed IFN-gamma production was not due to decreased IL-12/IL-23 production. Importantly, IFN-gamma-inducible responses were also significantly depressed during active TB and normalized during treatment, revealing disease activity-related and reversible impairment in IFN-gammaR signaling in TB. Finally, IFN-gamma/IL-10 ratios significantly correlated with TB cure. Taken together, these results show that M. tuberculosis-specific stimulation of IFN-gamma (but not TNF-alpha) production and IFN-gammaR signaling are significantly depressed in active TB, correlate with TB disease severity and activity, and normalize during microbiological TB cure. The depression of both IFN-gamma production and IFN-gammaR signaling may synergize in contributing to defective host control in active TB.     

In Vitro Hydroxyurea Decreases Th1 Cell-Mediated Immunity

Hydroxyurea (HU) is used in the treatment of hematologic disorders and is sometimes added to antiretroviral combination therapy to potentiate human immunodeficiency virus (HIV) suppression. However, HU has toxic effects on rapidly dividing cells, including the effectors of the immune response. To determine whether HU affects specific T-cell responses, we measured lymphocyte proliferation and cytokine production in response to microbial antigen and mitogen stimulation in the presence of added HU (10 to 1,000 μM). HU treatment of peripheral blood mononuclear cells obtained from HIV-infected patients and uninfected controls decreased lymphocyte proliferation and gamma interferon production compared with untreated cells. Interleukin-2 (IL-2) and IL-10 production was not affected by HU. The HU-mediated decrease of lymphocyte proliferation was similar in peripheral blood mononuclear cells from HIV-infected patients and from uninfected controls. The inhibitory effect of HU required continuous exposure to the drug and could be reverted by washing the drug out of the culture environment. These findings suggest that HU-containing therapeutic regimens might decrease Th1-cell-mediated immune responses in vivo.     

An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis

Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.     

Endogenous Inhibition of Antimycobacterial Immunity by IL-10 Varies between Mycobacterial Species

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10-/- mice. M. avium infection in IL-10-/- mice resulted in sustained increases in interferon (IFN)-gamma-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10-/- mice led to a transient increase in IFN-gamma T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10-/- macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-gamma T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.     

The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection.

The early phase of acquired cellular immunity to Mycobacterium tuberculosis infection is mediated by the emergence of protective CD4 T lymphocytes that secrete cytokines including interferon-gamma (IFN-gamma), a molecule which is pivotal in the expression of resistance to tuberculosis. Recent evidence demonstrates that infection with M. tuberculosis induces peripheral blood mononuclear cells to release the cytokine interleukin-12 (IL-12), a molecule that promotes the emergence of T-helper type-1 (Th1), IFN-gamma-producing T cells. We demonstrate here that IL-12 mRNA expression was induced by M. tuberculosis infection both in vivo and in vitro and that exogenous administration of IL-12 to mice transiently resulted in increased resistance to the infection. IL-12 also increased the production of IFN-gamma by both splenocytes derived from infected animals treated in vivo and by antigen-stimulated CD4 cells from untreated infected animals, with maximal effects at times associated with the expansion of antigen-specific CD4 T cells in vivo. In the absence of a T-cell response, as seen in SCID mice or nude mice, IL-12 only slightly augmented the moderate bacteriostatic capacity of these immunocompromised mice. Neutralization of IL-12 by specific monoclonal antibodies resulted in a reduction in granuloma integrity and slowing of the capacity of the animal to control bacterial growth.     

Evaluation of T cell immune responses in multi-drug-resistant tuberculosis (MDR-TB) patients to Mycobacterium tuberculosis total lipid antigens

Mycobacterium tuberculosis lipid antigens produce significant T cell responses in healthy tuberculin reactor [purified protein derivative (PPD-positive] individuals. In the present study, proliferation and interferon (IFN)-gamma/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipid antigens in T lymphocytes from 25 patients with multi-drug-resistant tuberculosis (MDR-TB). The obtained results were compared with those of 30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin test negative (PPD-negative) subjects. Peripheral blood mononuclear cells (PBMCs) and T cells (CD4(+) and CD8(+)) were stimulated using autologous immature dendritic cells. Proliferation responses were assessed using 3-{4,5-dimethylthiazol-2-yl}-2,5 diphenyl tetrazolium bromide (MTT). IFN-gamma/IL-4 concentrations in the supernatant of the CD4(+) and CD8(+)T cells were measured by enzyme-linked immunosorbent assay. Proliferation assay showed that the peripheral blood mononuclear cells and CD4(+) T cells from the MDR-TB patients responded significantly less to the M. tuberculosis total lipid antigens than to the CD4(+) T cells in the PPD-positive subjects. Total lipid antigen-specific proliferative responses in the CD8(+) T cells from the MDR-TB patients were minimally detected and the responses were similar to those of the PPD-positive subjects. IFN-gamma production by the CD4(+) T cells stimulated by total lipid antigens from the MDR-TB patients was decreased significantly compared with the PPD-positive individuals, whereas IL-4 production in the patients was elevated. IFN-gamma and IL-4 production in the CD8(+) T cells of the MDR-TB patients was similar to those of the PPD-positive subjects. In conclusion, it is suggested that stimulated CD4(+) T cells by M. tuberculosis total lipid antigens may be shifted to T helper 2 responses in MDR-TB patients.     

Regulatory effects of Th1-type (IFN-γ, IL-12) and Th2-type cytokines (IL-10, IL-13) on parasite-specific cellular responsiveness in Onchocerca volvulus-infected humans and exposed endemic controls

The present study investigated in vitro the regulatory effects of T helper 1 (Th1)-type (interferon-gamma, IFN-gamma; interleukin-12, IL-12) and Th2-type cytokines (IL-10, IL-13) on Onchocerca volvulus-specific cellular reactivity in onchocerciasis patients, and in exposed endemic control individuals presenting no clinical and parasitological signs of disease. In both patients and controls, addition of IL-10 dose-dependently depressed O. volvulus antigen (OvAg)-specific cellular proliferation, and peripheral blood mononuclear cells (PBMC) from patients who were more sensitive to the suppressive effect of IL-10 than those from endemic controls. However, neutralization of IL-10 by specific antibody did not reverse cellular hyporesponsiveness. In contrast to the inhibitory effects of IL-10, exogenous IL-12 and IL-13 augmented PBMC proliferative responses to OvAg both in patients and controls (P<0. 01) and neutralizing of IL-12 or IL-13 significantly decreased OvAg-specific proliferation in both groups. Exogenous IFN-gamma did not activate OvAg-specific proliferative responses in patients, but anti-IFN-gamma antibodies abolished cellular reactivity to OvAg. Antibody to IL-10 increased (P<0.05) OvAg-specific production of IL-5, IL-12 and IFN-gamma, and inversely, anti-IFN-gamma enhanced IL-10 (in patients only) and IL-5 and IL-13 in both patients and controls. Neutralization of IL-12 activated OvAg-specific production of IL-10, IL-2 and IFN-gamma. In conclusion, despite of an overproduction of IL-10, which suppressed cellular reactivity in patients and control individuals, OvAg-specific cellular responses were activated in vitro by exogenous supplementation with IL-12 and IL-13, and cytokine neutralization experiments confirmed that distinct type 1 and type 2 T helper cytokines cross-regulate expression and magnitude of O. volvulus-specific cellular responsiveness in humans.     

IL-15 in human visceral leishmaniasis caused by Leishmania infantum

Interleukin (IL)-15 is a recently discovered cytokine with the ability to stimulate the proliferation activity of Th1 and/or Th2 lymphocytes. Here, we investigated the involvement of IL-15 in the immune response to Leishmania infantum infection by studying patients with visceral leishmaniasis (VL). We found that IL-15 is produced by leishmanial antigen (LAg)-stimulated peripheral blood mononuclear cells (PBMC) from active VL patients at a significantly higher level than those produced by cells from healed VL subjects or healthy controls. A significant increase in IL-15 serum blood levels was also observed in acute VL patients compared with healed ones. Furthermore, recombinant IL-15 had an appreciable effect in vitro in reducing IL-4 and increasing the production of IL-12 in response to LAg, but it was ineffective in altering the production of interferon-gamma (IFN-gamma). The production of endogenous IL-15 in acute VL patients appeared to be insufficient to activate both IFN-gamma and IL-12, as attested by the absence of modification of these two cytokines by neutralization experiments in the presence of anti-IL-15 monoclonal antibodies (MoAB). On the contrary, the neutralization of IL-15 increased IL-4 production. Together, these results indicate that endogenous IL-15 plays a role in the suppression of Th2-type cytokines, even though it does not enhance the production of Th1 cytokines in acute VL patients. Since IL-15, in the presence of anti-IL-4 MoAb, caused a further increase in IL-12 production and led to a significant production of IFN-gamma, one of its indirect effects on Th1 cell activation could be due to the latter's effect on Th2 cytokines such as IL-4. Therefore, our observations indicate that there is a potential for IL-15 to augment the T-cell response to human intracellular pathogens.