Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

IL-10 enhances NK cell proliferation, cytotoxicity and production of IFN-gamma when combined with IL-18.

Previous studies have shown that IL-10 inhibits the accessory cell functions required for production of IFN-gamma by T cells and NK cells. Our results show that although IL-10 did not induce the production of IFN-gamma by NK cells, it did enhance the ability of IL-18 to stimulate NK cell production of IFN-gamma. In addition, IL-10 augmented NK cell proliferation and cytotoxic activity when combined with IL-18. However, IL-10 did not affect the ability of IL-12 to stimulate NK cells to produce IFN-gamma or proliferate, but there was an additive effect with IL-12 to increase NK cell cytotoxic activity. Interestingly, the type I IFN, whose receptors (R) are related to the IL-10R, also enhanced the effects of IL-18 on NK cell production of IFN-gamma and NK cell cytotoxicity. The ability of IL-10 to elevate the production of IFN-gamma appeared to be specific for NK cells since IL-10 had no effect on the production of IFN-gamma by Th1 clones stimulated with IL-18 or IL-12 in the presence of a monoclonal antibody specific for CD3. These latter results correlated with lower mRNA levels for the alpha and beta chains of the IL-10R in Th1 cells than observed in NK cells. Thus, the ability of IL-10 and IL-18 to up-regulate NK cell function, but not Th1 cell activity, appears to be based on expression of the IL-10R.     

Bone marrow-derived macrophages grown in GM-CSF or M-CSF differ in their ability to produce IL-12 and to induce IFN-gamma production after stimulation with Trypanosoma cruzi antigens

Trypanosoma cruzi is the etiological agent of Chagas' disease in man. Control of parasitism at the beginning of experimental infection depends on cytokine-activated macrophages that synthesize nitric oxide (NO). We investigated macrophage populations derived in the presence of M-CSF (M-M?) or GM-CSF (GM-M?) regarding their ability to control intracellular parasitism by T. cruzi and to synthesize IL-12 and NO. Both macrophage populations supported intracellular multiplication of the parasite; when activated by IFN-gamma, GM-M? exerted better control of parasitism. Stimulation of GM-M? with T. cruzi or Staphylococcus aureus resulted in IL-12 production and higher levels of NO synthesis in comparison with stimulated M-M?. Mice immunized with parasite-Ag-pulsed GM-M? but not with pulsed M-M? had increased IFN-gamma and IL-2 production in lymph nodes. However, when immunization was followed by infection with live parasites, transient elevation of IFN-gamma production was observed in both GM-M?- and M-M?-immunized mice, without reduction of blood parasite levels.     

Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection.

Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcR alpha beta and TcR gamma delta cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4lowCD8- and CD4-CD8low cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection.     

IL-18-independent cytotoxic T lymphocyte activation and IFN-gamma production during experimental acute graft-versus-host disease

Acute graft-versus-host disease (GvHD) is a serious complication after allogeneic bone marrow transplantation. Donor-derived T cells infiltrate recipient target organs and cause severe tissue damage, often leading to death of the affected patient. Tissue destruction is a direct result of donor CD8+ T cell activation and cell-mediated cytotoxicity. IL-18 is a novel pro-inflammatory cytokine with potent T(h)1 immune response-promoting and cytotoxic T lymphocyte (CTL)-inducing activity. IL-18 is strongly induced in experimental mouse models and human patients with acute GvHD. However, the precise role of IL-18 in the development of acute GvHD is still unknown. In this study, we have used IL-18-binding protein, a soluble IL-18 decoy receptor, to specifically neutralize IL-18 in vivo and in vitro. Our results demonstrate that IL-18 is induced during GvHD. However, its effect in the induction of GvHD appears to be redundant, since neutralization of IL-18 does not alter any disease parameter analyzed. Our study further shows that IFN-gamma production and CTL induction upon activation by T cell mitogens or by alloantigen does not involve IL-18-mediated amplification, in contrast to lipopolysaccharide-induced IFN-gamma production. We conclude that IL-18 expression correlates with the course of GvHD; however, its effect is dispensable for IFN-gamma and CTL induction for the initiation phase of this disease, most likely due to direct, IL-18-independent, CTL activation.     

Alterations in production of immunoglobulin classes and subclasses during experimental Trypanosoma cruzi infection

The spleens of mice infected with Trypanosoma cruzi were tested for their contents of cells producing IgM, IgG1, IgG2a, IgG2b, and IgG3 specific for the trinitrophenyl hapten after immunization with trinitrophenyl-Ficoll and trinitrophenyl-bovine serum albumin at various times during the acute and chronic phases of the disease. Reduced splenic contents of all of these cells was the characteristic of the acute period, and a return to normal levels occurred during the chronic stage. However, the density of IgG2a- and IgG2b-producing plaque-forming cells in the spleen was not restored to normality until 130 days postinfection, i.e., long after the chronic phase had been attained, reflecting a diluting effect of splenomegaly on cells that produce immunoglobulin isotypes known to be cytotoxic for the blood form of the parasite.     

Requirement for both IL-12 and IFN-gamma signaling pathways in optimal IFN-gamma production by human T cells

Phytohemagglutinin (PHA)-derived T lymphoblasts or T cell clones from patients genetically deficient in IL-12R beta 1 (IL-12R beta 1(-/-)) or IFN-gamma R1 (IFN-gamma R1(-/-)) produced two- to threefold reduced IFN-gamma levels compared to the corresponding cells from healthy individuals after anti-CD3 and PMA stimulation. Moderate IFN-gamma production was observed in PHA-derived T lymphoblasts or T cell clones derived from healthy subjects in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, whereas it was negligible in the presence of both mAb. However, when anti-IFN-gamma R1 and/or anti-IL-12 mAb were added during restimulation, the cells produced normal levels of IFN-gamma, indicating that both IFN-gamma and IL-12 had an effect on the priming phase. Moderate production of IFN-gamma was partially enhanced only in IFN-gamma R1(-/-) T cell clones generated in the presence of IL-12, but was almost completely abolished when IL-12R beta 1(-/-) and IFN-gamma R1(-/-) T cell clones were generated in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, respectively. IL-4 production was enhanced in T cell clones from IL-12R beta 1(-/-),but not from IFN-gamma R1(-/-) patients, whereas IL-10 and IL-2 production did not differ significantly in polyclonal T cells or clones from healthy and deficient individuals. These results indicate that IL-12R beta 1- and IFN-gamma R1-dependent signals co-ordinately regulate IFN-gamma, but not IL-2 and IL-10 production, whereas only IL-12 negatively controls IL-4 production by in vitro-generated T cell clones. Thus, although IL-12 and IFN-gamma signals are each sufficient for moderate production of IFN-gamma by human T cells, both are needed for optimal IFN-gamma production, and in the absence of both IFN-gamma production is completely abrogated.     

Gamma interferon suppresses acute and chronic Trypanosoma cruzi infection in cyclosporin-treated mice.

To determine if exogenous gamma interferon is effective in immunosuppressed mice infected with Trypanosoma cruzi, recombinant murine gamma interferon was administered to cyclosporin-treated mice with either acute or chronic T. cruzi infection. Gamma interferon significantly decreased parasitemia and prevented death in acutely infected mice. Parasitemias and mortality of mice treated with both gamma interferon and cyclosporin were similar to those of immunocompetent controls. In chronically infected mice, cyclosporin treatment produced significantly more organ explant cultures positive for T. cruzi. Fewer positive cultures, particularly for spleen and heart, were obtained from cyclosporin-treated mice when they also received gamma interferon. Ketoconazole treatment of mice resulted in no positive cultures. Cyclosporin treatment did not prevent activation of peritoneal macrophages by parenteral gamma interferon, nor did it have a consistent effect on serum titers of alpha/beta or gamma interferon in response to a second challenge inoculum of T. cruzi. These data indicate that exogenous gamma interferon suppresses acute and chronic T. cruzi infection in cyclosporin-treated mice but that gamma interferon is not as effective as the relatively specific antimicrobial ketoconazole. Gamma interferon activates macrophages despite cyclosporin treatment, and its effects appear to be tissue specific.     

The role of the thymus in modulating gammadelta T cell suppressor activity during experimental Trypanosoma cruzi infection

We have previously shown that splenic gammadelta T cells from young but not aged BALB/c mice possess suppressor activity in vivo and in vitro during the acute phase of Trypanosoma cruzi infection. The present work was undertaken to investigate the suppressor activity of gammadelta T cells from T. cruzi-infected euthymic or athymic mice and the role of the thymus in modulating non-adherent spleen cell suppressor activity during the acute phase of infection. Splenic gammadelta T cells from aged or athymic BALB/c mice reconstituted with total spleen cells or non-reconstituted did not exhibit suppressor activity when added to full allogeneic, mixed lymphocyte cultures. In contrast, splenic gammadelta T cells from young euthymic BALB/c mice showed suppressor activity in vitro. Thymectomy reduced the splenic gammadelta T cell suppressor activity of young BALB/c mice in a time-dependent manner, following a T. cruzi challenge. The continuous provision of thymocytes to aged mice, young thymectomized mice or total spleen cell- reconstituted athymic mice could re-establish the gammadelta T cell suppressor activity. Of particular significance was the observation that the depletion of gammadelta T cells during the acute phase of T. cruzi infection restored the capacity of these mice to mount a humoral immune response to a non-related antigen such as ovalbumin. These results indicate that gammadelta T cells of extrathymic origin cannot mediate suppression and that the thymus has a role in the regulation of suppression during the acute phase of T. cruzi infection.      

IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.     

Effect of acute pain on splenic NK cell activity, lymphocyte proliferation and cytokine production activities

Various types of physical and physiological stress in animals have been shown to affect their humoral and cell-mediated immune responses. The present study was designed to investigate the possible influence of acute pain on the immune system. BALB/c mice were exposed to an increasing number of heat shocks using a Tail Flick apparatus; an equal number of control mice received no shock treatments. After each of the regimens was completed, the spleen of each mouse was recovered and various cell populations isolated to assess: the proliferative response to phytohemagglutinin by lymphocytes; cytotoxic activities of natural killer (NK) cells; and, the production of select important cytokines by splenic lymphocytes. The results indicated that NK cell activity and proliferation of lymphocytes were significantly (p < 0.001) increased due to the shock regimens after only a single day's rounds of stimulation (i.e., 3 rounds of approximately 12 equally time-spaced shocks/hr with 30-45 min gap between rounds). After 2 and 3 days' rounds of stimulations, no significant changes were detected in the proliferative response of isolated lymphocytes; conversely, the activity of NK cells remained significantly elevated compared to the controls hosts' cells, even on the second day of stimulation but not on the third. Regarding effects on cytokines, no significant changes were detected in the amount of Interferon-gamma (IFNgamma) and Interlukin-10 (IL-10) produced by lymphocytes obtained from the spleens of any of the shocked mice. These results could suggest that certain acute stressors might actually strengthen a host's immunological reactivity and, possibly, result in an enhanced capacity to resist pathogens that might infect the body.     

Benzylpenicillin differentially conjugates to IFN-gamma,TNF-alpha,IL-1beta,IL-4 and IL-13 but selectively reduces IFN-gamma activity

It is known that beta-lactam antibiotics can conjugate to lysine and histidine residues on proteins via the carbonyl group of the opened beta-lactam ring. However, it is not known which proteins these drugs target and there is little work addressing whether conjugation is preferential for some proteins over others or if conjugation has functional consequences for the protein. We have previously shown that the beta-lactam antibiotic benzylpenicillin (BP) conjugates to IFN-gamma and reduces its activity. This interaction demonstrates selectivity, as BP does not bind to IL-4. Here, we extend our study to include other Th1 and Th2 cell-associated cytokines and two cytokines associated with inflammatory responses. We demonstrate by Western blotting that BP also conjugates to IL-1beta, IL-2, IL-5, IL-13 and TNF-alpha but not to IL-10. Densitometric analysis of leading cytokine bands on blots revealed that IFN-gamma always gave more intense BP-positive bands than any other cytokine analysed. Cytokines pre-incubated with BP at 37 degrees C in a protein-containing, serum-free medium were assayed for their biological activity. By in vitro bioassay, BP inhibited the ability of IFN-gamma but not IL-1beta or TNF-alpha to induce CD54 expression on epithelial cells. In addition, BP did not affect IL-4 or IL-13 inhibition of mast cell proliferation. When the pre-incubation temperature was reduced to 4 degrees C, BP did not conjugate to IFN-gamma or modulate its activity. BP retained its inhibitory effect on IFN-gamma activity when 20% FCS was added to the pre-incubation medium. In conclusion, BP conjugates to some cytokines but not others and this does not appear to be related to primary protein structure. Furthermore, of the cytokines studied, conjugation only to IFN-gamma is accompanied by inhibition of activity. This phenomenon is temperature dependent and occurs in the presence of serum. These findings provide further evidence for differential, direct drug-cytokine interactions. Such interactions may have therapeutic implications in terms of targeting cytokines to regulate their activity.     

Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-gamma production

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.     

Ovalbumin-induced IL-4, IL-5 and IFN-gamma production in infants with atopic dermatitis

BACKGROUND: Although hen's eggs are considered a cause of infantile atopic dermatitis (AD), little is known about cytokine production upon egg stimulation in infants with AD. Objective: This study aimed to characterize the production of IL-4, IL-5 and IFN-gamma upon stimulation with ovalbumin (OVA), a representative allergenic protein of egg, in infants with AD. METHODS: Peripheral blood mononuclear cells (PBMCs) from 68 children with AD, including 46 infants (<1 year), were stimulated with OVA and the production of IL-4, IL-5 and IFN-gamma was measured with ELISA kits. RESULTS: Upon stimulation with OVA, the production of IL-4 and IL-5, but not IFN-gamma, by PBMCs was significantly higher in infants with AD than in non-atopic controls. OVA-induced IL-5 production peaked in younger infants (2-5 months) and then decreased with age increase. In contrast, OVA-induced IL-4 production peaked at the age of 1-2. This coincided with the serum level of egg white-specific IgE (EW-IgE). There was a significant positive correlation between IL-5 production and the severity of symptoms in infants with AD, while IL-4 production significantly correlated with the serum level of EW-IgE. CONCLUSION: These results demonstrate that OVA-induced IL-5 production fluctuates with age in a different manner than IL-4 or EW-IgE. Our results suggest that egg contributes to the development of AD in younger infants by inducing the production of IL-5, but not IL-4.     

NK cell activation and protection occur independently of natural killer T cells during Trypanosoma cruzi infection

Natural killer T (NKT) cells regulate aspects of pro-inflammatory and anti-inflammatory responses and contribute to the control of infections and chronic inflammatory diseases. During Trypanosoma cruzi infection both NKT cells and NK cells are critical to the protective response. How NKT cells interact and possibly regulate NK cells during infections remains uncertain. In vivo studies have demonstrated that specific activation of NKT cells with alpha-galactosylceramide (alpha-GalCer) leads to NK cell activation. These results suggest that during some infections activated NKT cells might regulate NK cell activation and functions. Therefore, using gene-deficient mice that lack NKT cells and antibody-treated mice that lack NK cells, we investigated the interactions of NKT cells and NK cells during experimental T. cruzi infection. We report here that during acute T. cruzi infection spleen and liver NK cell activation and cytolytic activity occur independently of NKT cells. Moreover, NK cell protection occurs independently of NKT cells. In contrast to these results that fail to demonstrate an interdependence, at day 4 of infection the number of liver NK cells is controlled by NKT cells. Thus, during T. cruzi infection, regulation of the number of liver NK cells requires NKT cells, but the activation of NK cells and protection by NK cells does not. The data presented here argue that during infections NK cell activation and protection occur independently of NKT cells.