The increased potential for the production of inflammatory cytokines by Kupffer cells and splenic macrophages eight days after thermal injury

Burn patients often experience a devastating inflammatory response to infection within the first two weeks after thermal injury. The inflammatory cytokines IL-6, TNF and IL-1 have been implicated in this condition but most studies have focused on the abnormal levels of cytokines in the plasma. In this study the production of cytokines was compared for Kupffer cells versus splenic macrophages; endotoxin (LPS) stimulation versus no stimulation; and burn (post burn days 1, 3 and 8) versus no burn (control). Corresponding serum levels of IL-6 were also determined. Kupffer cells from normal or burned animals were shown to produce much higher amounts of the inflammatory cytokines than that produced by splenic macrophages. An exception to this was the equal production of TNF by LPS-stimulated hepatic and splenic cells. Both LPS-stimulated Kupffer cells and splenic macrophages produced larger amounts of the cytokines than that produced by the unstimulated cells. There was a significant effect of thermal injury on cytokine production by LPS-stimulated Kupffer cells at post burn day 8 and on TNF production by stimulated splenic macrophages also at post burn day eight. Although there was a statistically significant effect of thermal injury at post burn day 8 on IL-1 production by unstimulated splenic macrophages, the absolute amount of cytokine produced was very small. The results suggest that by post burn day 8 the cells may have become primed to respond to a stimulus such as endotoxin (LPS), a condition that could arise in a burn patient from sepsis. Strangely, the large spike in serum IL-6 level occurred at post burn day one and the level of the cytokine returned nearly to the control value on post burn days 3 and 8.     

Nocardia brasiliensis induces an immunosuppressive microenvironment that favors chronic infection in BALB/c mice

Nocardia brasiliensis is an intracellular microorganism and the most common etiologic agent of actinomycetoma in the Americas. Several intracellular pathogens induce an immunosuppressive microenvironment through increases in CD4+ Foxp3+ regulatory T cells (Treg), thus downregulating other T-cell subpopulations and assuring survival in the host. In this study, we determined whether N. brasiliensis modulates T-lymphocyte responses and their related cytokine profiles in a murine experimental model. We also examined the relationship between N. brasiliensis immunomodulation and pathogenesis and bacterial survival. In early infection, Th17/Tc17 cells were increased at day 3 (P < 0.05) in footpad tissue and spleen. Treg subpopulations peaked at days 7 and 15 (P < 0.01) in the footpad and spleen, respectively. Transforming growth factor β1 (TGF-β1) and interleuki-10 (IL-10) are cytokines known for their immunosuppressive effects. During early and chronic infections, these cytokines were elevated with increased TGF-β1 levels from days 3 to 30 (P < 0.01) and sustained IL-10 expression throughout infection compared to uninfected mice. IL-6 production was increased at day 3 (P < 0.01), whereas gamma interferon (IFN-γ), IL-17A, and IL-23 levels were highest at day 15 postinfection (P < 0.01) when a decrease in the bacterial load (>1 log) was also observed (P < 0.05). After these changes, at 30 to 60 days postinfection, IFN-γ production was decreased, whereas the expression of anti-inflammatory cytokines and the bacterial load again increased (P < 0.05). The increment in Treg cells and the related cytokine profile correlated with reduced inflammation at day 15 (P < 0.05) in the footpad. We conclude that N. brasiliensis modulates the immune system to induce an immunosuppressive microenvironment that benefits its survival during the chronic stage of infection.     

Dendritic cells,but not macrophages,produce IL-12 immediately following Leishmania donovani infection

Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-γ from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-α production is induced by day 3 post infection. Not only do these data satisfactorily explain the dfferences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.     

Endogenous IL-10 regulates IFN-gamma and IL-5 cytokine production and the granulomatous response in Schistosomiasis mansoni-infected mice.

In murine Schistosomiasis mansoni circumovum, granuloma formation is regulated by pro- and anti-inflammatory cytokines. Among the latter, interleukin-10 (IL-10) has been shown to regulate the inflammatory response. In this study we examined the role of endogenously produced IL-10 in T-helper 1 (Th1)- and Th2-type cytokine production and granuloma formation. The dynamics of IL-10 production through the course of the infection were different in granuloma versus splenic cells. In the former, production peaked during the early developmental stage (6 weeks of infection) of the granuloma and then declined. In splenocytes production peaked at 12 weeks, before down-modulation of the granuloma response. In the developing granuloma both macrophages and T cells secreted IL-10. In anti-IL-10 monoclonal antibody (mAb)-supplemented granuloma cell cultures endogenous IL-10-mediated regulation of interferon-gamma (IFN-gamma) was manifest only at 6 weeks; that of IL-2 continued throughout the infection (6-20 weeks). IL-4 production was unaffected, but IL-5 production was regulated at the 6 and 8 weeks time point. Splenocytes showed regulation of IFN-gamma and IL-2 production at the peak of the granulomatous response (8 weeks). IL-4 production was not regulated, whereas IL-5 production was regulated only at 6 weeks. Repeated injections of anti-IL-10 mAb given to mice at 6, 12 or 20 weeks of the infection significantly enhanced liver and lung granuloma growth, tissue eosinophilia, and IFN-gamma, IL-5 production at the early developmental phase (6 weeks) of the lesions. Thus, in schistosome-infected mice endogenous IL-10 is shown to regulate Th1- and Th2-type cytokine production and granuloma formation during the early Th0/Th1 phase of the immune response.     

Production of pro-inflammatory cytokines during the early stages of experimental Paracoccidioides brasiliensis infection.

Pro-inflammatory cytokines play an important role in both recruitment and activation of leukocytes migrating into tissues in response to invading pathogens. In this study the production of pro-inflammatory cytokines, determined by ELISA assays, and the recruitment of leukocytes into the lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia were evaluated during the early stages of infection. The results showed that infected mice had a significant increase in leukocytes in the lung during the first 4 days with a peak at day 2 post-challenge; infiltrates were composed mainly of polymorphonuclear neutrophils (PMN). Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha), interleukin (IL) 6, IL-1beta and macrophage inflammatory protein (MIP) 2 were produced at elevated levels during the first 4 days post-challenge, but only in pulmonary samples and not in sera. Additionally, during the early stages of infection, overall weight loss was recorded in infected mice. These results suggest that pro-inflammatory cytokines could be responsible for the recruitment of leukocytes into the lung during the early stages of P. brasiliensis infection. In addition, both pro-inflammatory cytokine production and leukocyte recruitment may participate in the control of infection by influencing the organization of the immune response in the host exposed to P. brasiliensis conidia.     

Augmentation of naive,Th1 and Th2 effector CD4 Responses by IL-6, IL-1 and TNF

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-γ, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-γ and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.     

Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.     

Superantigen activation and kinetics of cytokines in the Long-Evans rat.

This study was conducted to identify and quantify, over time, selected cytokine responses in Long-Evans rats that were exposed to staphylococcus enterotoxin B (SEB). The kinetics of selected cytokines [interleukin-2 (IL-2), IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)] and phenotype and cell cycle analysis of T lymphocytes were determined in Long-Evans rats administered a single intraperitoneal (i.p.) dose of either 50 microg or 500 microg of SEB. Rats injected with 50 microg SEB had significantly elevated levels of IL-2, IL-6 and IFN-gamma in their serum 2 hr post-injection. IL-2 serum levels were significantly elevated at 2 hr and returned to near control values by 12 hr while both IL-6 and IFN-gamma peaked at 6 hr but remained significantly increased at 24 hr post SEB exposure. A 500 microg dose of SEB did not further enhance these cytokine responses. When spleen cells were collected for culture 2 hr after rats were injected i.p. with 50 microg SEB and cocultured with SEB, TNF and IL-6 levels were significantly increased after 2 hr incubation, while IL-2 and IL-6 were significantly elevated at 6 hr. Production of all these cytokines in spleen cell cultures continued to increase over the 24 hr sampled. Peritoneal cells were collected for culture either at 1 hr or 2 hr after injection of either 50 microg or 500 microg of SEB. IL-6 was significantly increased after 1 hr in culture while TNF was significantly increased by 2 hr regardless of whether the cells were harvested 1 or 2 hr after SEB injection. The greatest response for both IL-6 and TNF occurred when cells from animals injected with 50 microg SEB were restimulated in vitro with SEB. The peak levels for IL-6 were at 12 hr post SEB exposure while TNF peaked at 6 hr. The percentage of CD4+ cells was significantly increased at 48 hr and 72 hr post SEB (50 microg) administration while the percentage of CD8+ cells remained similar to control values for the 168-hr test period. A similar pattern was observed in cell cycling where the CD4+ cells proliferated up to 2 days post SEB injection and then were significantly suppressed at day 3. The CD8+ cells were comparable to control values. These studies demonstrate that the cytokine responses in Long-Evans rats exposed to a superantigen are somewhat similar to those that occur in mice and humans, e.g. a rapid short increase in the production of IFN-gamma and TNF that was accompanied by an increase in the production of IL-2. Additional responses noted in this species, however, were a marked increase in IL-6 production, as well as an early increase in the number and cycling of CD4+ cells followed by a down-regulation of these events. These activities occurred in the absence of notable histopathological alteration of lymphoid organs. The results indicate that the Long-Evans rat is an acceptable animal model to investigate the pathogenesis of superantigen-induced disease and that IL-6 may be an active mediator of this process.     

Chloroquine-induced inhibition of the production of TNF, but not of IL-6, is affected by disruption of iron metabolism.

There is now considerable evidence that cerebral malaria may be related to the over-production of tumour necrosis factor (TNF). Nevertheless, our knowledge is very poor concerning the biological events which lead up to this TNF over-production. Furthermore, interleukin-6 (IL-6) is produced in large amounts during malaria infection and seems to have inhibitory action on TNF production. Anti-malarial drugs were investigated for their ability to interfere with TNF and IL-6 secretion by human non-immune macrophages stimulated by lipopolysaccharides (LPS) or Plasmodium falciparum culture supernatant. Macrophages were pretreated with chloroquine, quinine, proguanil, mefloquine or halofantrine before stimulation. TNF and IL-6 production were suppressed in a dose-dependent manner when macrophages were treated with chloroquine, but not with other anti-malarial drugs. Considering that chloroquine probably acts via lysosomotropic mechanisms, and that iron metabolism may interfere with the non-specific immune response, we focused our attention on these biochemical events in order to investigate the mechanisms by which chloroquine inhibits cytokine production. Our results demonstrated that chloroquine-induced inhibition of TNF and IL-6 production is not mediated through a lysosomotropic mechanism, and that chloroquine probably acts on TNF secretion by disrupting iron homeostasis. Inhibition of IL-6 production seems not to be mediated through these pathways. These observations suggest that chloroquine may help to prevent cerebral malaria whatever the drug sensitivity of the parasite strain, and may provide new tools for an anti-disease therapy regardless of the emergence of parasite multi-drug resistance.     

Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins

The capacity of endothelial cells to produce and release cytokines (IL-6, IL-8 and G-CSF) in response to exposure to Staphylococcus aureus strains or staphylococcal exotoxins (alpha-toxin, enterotoxin A and TSST-1) was investigated. An endothelial cell culture model of human umbilical vein endothelial cells (HUVEC) was used. Five out of ten clinical isolates of S. aureus were found to induce cytokine production and release from endothelial cells. Four of the five isolates that induce cytokine release produced enterotoxin A, B, C, D and/or TSST-1, compared with two of those that did not induce release. Purified staphylococcal exotoxins (1 pg/ml-1 microg/ml) did not act as primary stimuli and induced no detectable cytokine secretion. When endothelial cells were prestimulated with IL-1beta or TNF alpha at a concentration of 1 ng/ml for 2 h, IL-1beta served as a potent primary stimulus for IL-6, IL-8 and G-CSF production, whereas TNF alpha did not induce any significant cytokine release during the subsequent 24 h. A further increase in IL-6 and G-CSF release, but not of IL-8, was observed when IL-1beta prestimulated cells were exposed to alpha-toxin or TSST-1. However, to potentiate cytokine production (IL-6 and IL-8) by SEA, both IL-1beta and the toxin had to be present simultaneously. Our data show that S. aureus, but not staphylococcal exotoxins, have the capacity to act as primary stimuli of endothelial cells and induce production and release of cytokines. IL-1beta may prime HUVEC to release IL-6, IL-8 and G-CSF prior to subsequent stimulation with staphylococcal exotoxins.     

Paradoxical priming effects of IL-10 on cytokine production.

IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37 degrees C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1beta, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NFkappaB translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68(+) cells among the CD14(+) cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers.     

Serum levels of IL-1, IL-6 and tumour necrosis factors in patients undergoing coronary artery bypass grafts or cholecystectomy.

Plasma levels of biologically active IL-1, tumour necrosis factor (TNF) and IL-6 were measured before, during and after coronary artery bypass graftings (CABG) (n = 9) and cholecystectomy (CHO, n = 9), and in normal controls (nine healthy volunteers). Mean pre-operative IL-1 concentration in four of the nine CABG patients was 0.452 + 0.03 ng/ml, significantly (P less than 0.001) higher than that of the other five (0.045 +/- 0.009 ng/ml), CHO patients (0.035 +/- 0.005 ng/ml) and controls (0.029 +/- 0.008 ng/ml). Three of the four patients with high pre-operative IL-1 had functional capacity IV, while the other five had functional capacity IIa or IIb. Slight IL-1 elevation after anaesthesia, followed by reduction after initiation of bypass, elevation on completion of surgery and reduction to basal levels after 7 days was found in patients undergoing CABG. Mean basal TNF levels of CABG and CHO patients did not differ, but were higher than those of controls (2.85 +/- 0.5 ng/ml for CABG, 2.05 +/- 0.06 ng/ml for CHO, 0.72 +/- 0.07 ng/ml for normals, P less than 0.001). A unique kinetics of release during CABG was observed also for TNF. Mean pre-operative IL-6 levels were normal (50 +/- 3 ng/ml for CABG, 50 +/- 0.5 ng/ml for CHO and 65 +/- 10 ng/ml for controls). Gradual elevation to a mean peak of 725 +/- 100 ng/ml on completion of CABG was observed as compared with 275 +/- 50 ng/ml in CHO (P less than 0.01). On the seventh post-operative day mean IL-6 levels returned to normal. Two patients with post-operative low-grade fever (38 degrees C) had high, late cytokine levels. One of these two patients had leucocytosis, sterile discharge from the operative wound and was diagnosed as suffering from the Dressler syndrome. In this study elevated cytokine values and unique kinetics of release into the serum were found in patients undergoing CABG.     

Constitutive Secretion of Interleukin-6 by Human Decidual Stromal Cells in Culture. Regulatory Effect of Progesterone

PROBLEM : Although several studies have demonstrated that decidual stromal cells (DSC) can secrete cytokines in culture, none of these studies documented the purity of the cultures. Since other cells of the decidua, such as macrophages and epithelial cells, also produce cytokines, it is important to ensure purity of the culture so that cytokine production can be ascribed with confidence to DSC. METHOD : DSC from early human pregnancies were highly purified and maintained in culture. Basal secretion by these cells of IL-6, together with other cytokines considered critical for pregnancy (IL-1β, TNFα and IFNγ), was measured with immunological techniques. RESULTS : We found that DSC in culture produce insignificant quantities of IL-1β, TNFá and IFNΓ, but appreciable amounts of IL-6. The production of this later cytokine was, however, inhibited by the effect of progesterone. CONCLUSIONS : Basal production of IL-6 by DSC may be involved in physiological functions at the maternal-fetal interface. Nevertheless, the secretion of this cytokine is regulated by progesterone, probably to prevent excessive production of this cytokine from triggering an inflammatory response that might compromise pregnancy.     

Developmental regulation of the cytokine repertoire in human macrophages: IL-1, IL-6, TNF-alpha, and M-CSF

We were interested in the dependence of constitutive and stimulated cytokine secretion on the stage of macrophage (MAC) differentiation in vitro. Elutriation-purified blood MO were cultured up to 28 days and their secretory repertoire was analyzed under adherence conditions at various culture stages. For each of the cytokines tested, interleukin (IL)-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage colony-stimulating factor (M-CSF), a different pattern of regulation was observed. During the initial phase of maturation (up to day 7 in culture) within which the characteristics of normal MO to MAC transformation are achieved, M-CSF was the only cytokine to be secreted constitutively. From the LPS-dependent cytokines, IL-1 beta and IL-6 were downregulated whereas TNF-alpha levels increased severalfold. For the release of IL-1 beta, IL-6, and TNF-alpha a synergistic effect of interferon-gamma (IFN-g) and lipopolysaccharide (LPS) was observed. M-CSF release increased until day 7 in culture with LPS being stimulatory for this particular cytokine only during the first days of differentiation. Upon further cultivation of MAC up to 28 days, LPS-induced IL-1 beta levels remained very low, but IL-6 levels increased again reaching that of blood MO, and TNF-alpha continued to rise reaching levels up to 30-fold higher than in blood MO. M-CSF secretion stayed high with LPS even suppressing constitutive secretion. Long-term cultured MAC started to release IL-6 and TNF-alpha also in the absence of a stimulus and, furthermore, became responsive to IFN-g alone. Our data show that the release of each cytokine investigated is differently regulated during maturation. These results document the functional plasticity of human MAC and emphasize the impact that MO to MAC differentiation may have in vivo.     

Dual role of interleukin-4 (IL-4) in pulmonary paracoccidioidomycosis: endogenous IL-4 can induce protection or exacerbation of disease depending on the host genetic pattern

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.     

Chronic mucocutaneous candidiasis. I. Altered antigen-stimulated IL-2, IL-4, IL-6 and interferon-gamma (IFN-γ) production

Patients with chronic mucocutaneous candidiasis (CMC) present with persistent infections with the opportunistic yeast Candida. Impaired cell-mediated responses to Candida have been documented in CMC patients, but the defect remains poorly understood. The importance of Th1 cytokines in resistance and Th2 in susceptibility to Candida infections has recently been demonstrated in murine models. In our studies we evaluated production of IL-2 and IFN-γ (markers of Th1 type responses) as well as IL-4 and IL-6 (Th2 type markers) following stimulation with two kinds of Candida antigens (CAgs), polysaccharide antigens, tetanus toxoid and pokeweed mitogen. Our results demonstrate that CMC patients have impaired cytokine production upon in vitro stimulation with CAgs resulting in low or absent IL-2, increased IL-6 and either absent or increased IFN-γ production. Cytokine production following stimulation by other antigens was unaltered. The overall cytokine-producing capacity assessed through mitogen stimulation was also intact. Addition of IFN-α or IFN-γ to culture in an attempt to modify cytokine production did not have significant effects. Levels of soluble IL-6 receptors were not increased and could not account for increased IL-6 production. Our studies support the hypothesis that Candida antigens trigger a predominantly Th2 instead of a Th1 cytokine response in patients with CMC.     

IL-35 promoted STAT3 phosphorylation and IL-10 production in B cells,but its production was reduced in patients with coronary artery diseases

Interleukin (IL)-35 is a heterodimeric cytokine composed of the IL-12A subunit and the Epstein-Barr virus induced gene 3 (EBI3) subunit. Binding of IL-35 with IL-12 receptor subunit beta 2 (IL-12RB2) and IL-6 signal transducer (IL-6ST) occupies the binding sites of IL-6, IL-12, and IL-27 and prevents their signal transduction. IL-35 is also shown to promote the development of regulatory T cells (Tregs) and regulatory B cells (Bregs). In this study, we investigated B cell-mediated IL-35 production in patients with coronary artery disease (CAD). The expression levels of IL-35 subunits and IL-10 were significantly lower in B cells from CAD patients than in B cells from healthy control individuals. Exogenous IL-35 could effectively increase the IL-10 production by B cells in a concentration-dependent manner. IL-35 promoted the phosphorylation of STAT1 and STAT3 in B cells, and the inhibition of STAT3 phosphorylation suppressed IL-10 production. Raising the IL-35 concentration in cell culture eliminated the difference in IL-10 expression between CAD B cells and healthy B cells. We also demonstrated that B cells from CAD patients presented lower capacity to suppress interferon gamma (IFNG) and tumor necrosis factor (TNF) expression by T cells than B cells from healthy controls. Exogenous IL-35 could significantly improve the suppressive capacity of B cells in both healthy controls and CAD patients. Together, these results demonstrated that a reduction in IL-35 production was associated with Breg defects in CAD patients. IL-35 and IL-35 targets may serve as therapeutic candidates in the treatment of CAD and related diseases.