Increased secretion of IL-18 in vitro by peripheral blood mononuclear cells of patients with bronchial asthma and atopic dermatitis.

This study was performed to determine whether or not IL-18, formerly called IFN-gamma-inducing factor, is involved in the pathogeneses of allergic disorders. Peripheral blood mononuclear cells (PBMC) were obtained from patients with allergic bronchial asthma (BA), patients with atopic dermatitis (AD) and controls who did not have any allergic disease, and then cultured with lipopolysaccharide (LPS) or phytohaemagglutinin (PHA). The concentrations of IL-18, IFN-gamma and IL-13 in supernatant fluids were determined by enzymatic immunoassaying, and the expression of IFN-gamma messenger (m) RNA in the cells was measured by colorimetric microplate assaying. IL-18 secretion in the BA patients (geometric mean (gm) = 189 pg/ml) and AD patients (gm = 172 pg/ml) was significantly higher than that in non-allergic controls (gm = 118 pg/ml). In contrast, IFN-gamma secretion in the BA patients (gm = 7.3 IU/ml) and AD patients (gm = 6.8 IU/ml) was significantly lower than that in non-allergic controls (gm = 20.7 IU/ml). The amounts of IL-13 in supernatant fluids and IFN-gamma mRNA in cells were not statistically different among the BA patients, AD patients and non-allergic controls. The possible involvement of IL-18 in allergic disorders is discussed.     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Effect of IL-4 and interferon-gamma (IFN-gamma) on IL-3- and IL-5-induced eosinophil differentiation from human cord blood mononuclear cells.

IL-4 and IFN-gamma positively and negatively regulate allergic inflammation. To determine the regulatory mechanisms of eosinopoiesis by cytokines, we examined the effect of recombinant IL-4 and IFN-gamma and of anti-IL-4 and anti-IFN-gamma antibodies on IL-3- and IL-5-induced eosinophil differentiation from human umbilical cord blood mononuclear cells. rhIL-4 (10-300 U/ml) inhibited IL-3- and IL-5-induced eosinophil differentiation from cord blood mononuclear cells on day 28 of culture by 62-81% in a concentration-dependent manner. rhIFN-gamma (5-500 U/ml) also inhibited IL-3- and IL-5-induced eosinophil differentiation by 80-99% in a concentration-dependent manner. The inhibitory effect of rhIL-4 and rhIFN-gamma was observed only when rhIL-4 or rhIFN-gamma were present in the culture from day 0 to day 14, but not from day 15 to day 28. Addition of anti-IL-4 antibody to the culture enhanced IL-3- and IL-5-induced eosinophil differentiation on day 28 of culture by 30%, whereas anti-IL-2 MoAb and anti-IFN-gamma MoAb had no significant effect. These results indicate that IL-4 and IFN-gamma have inhibitory effects on IL-3- and IL-5-induced eosinophil differentiation from its progenitor cells.     

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.     

Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes.

Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-gamma secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-gamma was markedly reduced. IFN-gamma production was completely blocked by an anti-IL-12 antibody, indicating that IFN-gamma secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40-CD40L or CD28-B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.     

Elevated serum interleukin (IL)-12p40 levels in common variable immunodeficiency disease and decreased peripheral blood dendritic cells: analysis of IL-12p40 and interferon-gamma gene

Common variable immunodeficiency disease (CVID) is a heterogeneous syndrome characterized by low immunoglobulin serum levels and recurrent bacterial infections. Several studies suggest that CVID patients have a polarized immune response towards a T helper type 1 phenotype (TH1). However, the factors causing the TH1 polarization remain to be determined in this disease. In the present study, serum interleukin (IL)-12, interferon (IFN)-gamma levels and the IL-12p40 and IFN-gamma gene were studied in CVID patients. Furthermore, we evaluate dendritic cells (DCs) compartment, myeloid dendritic cells (mDCs) and plasmocytoid dendritic cells (pDCs), which help to differentiate naive T cells preferentially into TH1 and TH2, respectively. The serum IL-12p40 subunit levels were increased significantly in CVID patients compared to healthy controls. We examined whether these elevated serum IL-12p40 levels are associated with IFN-gamma or IL-12p40 gene polymorphisms, or with new mutations in the IL-12p40 promoter gene. In our hands, no new mutations were found and gene polymorphisms frequencies in CVID patients were similar to the control population. In conclusion, the elevated serum levels of IL-12p40 found in our CVID patients were not related to these genetic variations. The DC compartment analysis did not show an imbalance between pDCs and mDCs, but revealed the presence of low numbers and percentage of both DC populations in CVID.     

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.     

Ribavirin inhibits DNA,RNA, and protein synthesis in PHA-stimulated human peripheral blood mononuclear cells: possible explanation for therapeutic efficacy in patients with chronic HCV infection

The treatment of choice for patients infected chronically with HCV is the combination of IFN-alpha and ribavirin. Monotherapy with ribavirin leads to a clinical and histological improvement, but its exact mechanism of action is unknown. Therefore, the effect of ribavirin on synthesis of inflammatory cytokines and on apoptosis in stimulated peripheral blood mononuclear cells (PBMCs) was investigated. PBMCs were isolated from the blood of HCV infected patients and from healthy volunteers. The effect of ribavirin on IFN-gamma and IL-1beta release in the supernatant of unstimulated and phytohemagglutinin (PHA) stimulated PBMCs was investigated by enzyme linked immunosorbent assay (ELISA). The effect on total DNA, RNA, and protein synthesis was analyzed by measurement of 3H-thymidine, 3H-uridine and 3H-leucine incorporation into cellular macromolecules. Ribavirin led to a dose-dependent decrease of the IFN-gamma but an increase of IL-1beta release into the supernatant of PHA-stimulated PBMCs. At the same time, a dose-dependent decrease of total DNA, RNA, and protein synthesis in cultures of PHA-stimulated PBMCs was demonstrated. These effects could be compensated by the addition of equimolar amounts of guanosine. The rate of apoptotic CD45+ and CD14+ cells in PBMCs cultures increased in a dose-dependent manner. Our data suggest that ribavirin administration to chronically HCV-infected patients could lead to a decrease of the synthesis of proinflammatory cytokines (e.g., IFN-gamma) by an inhibition of total DNA-, RNA-, and protein-synthesis and by induction of apoptosis in the cells of the inflammatory infiltrate. Furthermore, ribavirin could influence the synthesis of viral particles in the hepatocytes.     

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.     

Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.     

Low numbers of interleukin-12-producing cord blood mononuclear cells and immunoglobulin E sensitization in early childhood

BACKGROUND: Successful pregnancies are associated with skewing towards a Th2 cytokine profile. Cytokine responses to allergens can be detected in cord blood mononuclear cells (CBMC), suggesting allergen priming already in utero. OBJECTIVE: To investigate the cytokine profile in CBMC after in vitro stimulation with allergens and to relate the responses to the outcome in terms of allergic disease at 2 years of age. METHODS: CBMC were isolated from 82 children. The responses to ovalbumin (OVA), birch, cat and phytohaemagglutinin (PHA) were investigated by the ELISpot technique. The numbers of IFN-gamma-, IL-4- and IL-12-producing CBMC were counted for each stimulation. The children were followed prospectively; skin prick test (SPT) and RAST towards food and inhalant allergens were assessed at 24 months of age. RESULTS: Sixteen (19.5%) children were classified as IgE sensitized (positive SPT; > or =3 mm and/or RAST; > or =0.35 kUA/L). The numbers of IL-12-producing CBMC after stimulation with birch, OVA and cat were lower among IgE-sensitized children, statistically significant for cat. IFN-gamma-producing cells, did not differ in numbers between the sensitized and non-sensitized children. Children who had atopic eczema/dermatitis syndrome (AEDS) during the observation (n=53) had significantly lower numbers of IFN-gamma-producing CBMC after stimulation with OVA and cat than their non-AEDS counterparts. CONCLUSIONS: Although the numbers of infants in our study are limited our data suggest that a low number of IL-12-producing CBMC is associated with IgE sensitization during early childhood and that a reduced number of IFN-gamma-producing CBMC promotes the development of AEDS during the first 2 years of life.     

Human T helper (Th) cell lineage commitment is not directly linked to the secretion of IFN-gamma or IL-4: characterization of Th cells isolated by FACS based on IFN-gamma and IL-4 secretion

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.     

Up-regulation of IL-18 and predominance of a Th1 immune response is a hallmark of lupus nephritis

There is evidence that nephritis is dominated by a Th1 immune response in systemic lupus erythematosus. Since IL-18 promotes polarization of the immune response toward Th1, we investigated the role of this cytokine in lupus nephritis (LN). A total of 133 lupus patients and 44 healthy subjects were enrolled. Demographic and clinical characteristics with renal biopsy data were recorded. IL-18 along with IFN-gamma and IL-4, two prototypical of Th1 and Th2 cytokines, were measured in serum by ELISA. Peripheral blood lymphocytes were analysed by flow cytometry for IFN-gamma and IL-4. IL-18 expression was determined by immunohistochemistry in 13 renal biopsy specimens from patients with LN and 2 controls. Serum IL-18 was higher in lupus patients than in controls. Levels of IL-18 correlated with urinary microalbumin and were increased in patients with LN when compared to those without LN. IL-18 expression was also increased within the glomeruli of nephritic patients and was primarily detected within the mesangial matrix and in infiltrating mononuclear cells. Measurement of IFN-gamma and IL-4 in either sera or peripheral blood lymphocytes showed high IFN-gamma along with low IL-4 expression in LN patients compared to patients without nephritis. A positive correlation between serum IL-18 and IFN-gamma levels was found. IL-18 may play a prominent role in the pathogenesis of LN by promoting a cytokine imbalance towards a Th1 immune response. Measurement of IL-18 may be helpful for the early identification of lupus patients with LN and may help gauge the response to treatment in patients with active LN undergoing treatment.     

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.     

IL-12 receptor deficiency revisited: IL-23-mediated signaling is also impaired in human genetic IL-12 receptor beta1 deficiency

IFN-gamma and IL-12 are crucial cytokines for cell-mediated immunity against intracellular pathogens. We have previously shown that human IL-12Rbeta1-deficiency leads to impaired IL-12 responsiveness and unusual susceptibility to infections due to mycobacteria and salmonellae. IL-23 is a cytokine with functions that partially overlap with those of IL-12. IL-23 consists of IL-12p40 and a novel p19 protein, and binds to a receptor complex comprising IL-12Rbeta1 and IL-23R. Thus, IL-12Rbeta1-deficiency may impair both IL-12- and IL-23 signaling, and both may contribute to the immunological phenotypes. To examine whether IL-12Rbeta1 is essential for IL-23 signaling in human T cells, we have studied IL-23 responsiveness of four IL-12Rbeta1-deficient individuals. Whereas IL-23 promoted IFN-gamma production by CD4(+) and CD8(+) T cells in controls, IL-12Rbeta1-deficient T cells lacked IL-23-induced IFN-gamma secretion, but responded normally to IL-2, IL-4, IL-15 and IL-18. We also show that induction of IFN-gamma production by IL-23 depends upon TCR-ligation and is enhanced by CD28-costimulation. Furthermore, IL-23 cooperates with IL-12 and IL-18 in promoting IFN-gamma production in controls, but not in patients. We conclude that IL-12Rbeta1-deficiency impairs IL-12- and IL-23-dependent signaling in human T cells. The syndrome caused by IL-12Rbeta1-deficiency thus needs to be reinterpreted as resulting from defective IL-12-as well as IL-23-mediated immunity.     

Differential role of IL-18 and IL-12 in the host defense against disseminated Candida albicans infection

IFN-gamma plays a crucial role in the defense against infection with Candida albicans. Since IL-18 and IL-12 are strong stimuli of IFN-gamma production, we investigated whether endogenous IL-18 and IL-12 are involved in the host defense during disseminated candidiasis. IL-18 knockout (IL-18-/-) mice, but not IL-12-/- mice, displayed an increased mortality due to C. albicans infection, accompanied by a decreased clearance of the yeasts from the kidneys late during the course of infection. Histopathology of the organs, combined with phagocyte recruitment experiments, showed a decreased influx of monocytes at the sites of Candida infection, mainly in the IL-18-/- mice. Whereas production of the chemokine KC was decreased in both IL-18-/- and IL-12-/- mice, MIP-2 production was deficient only in IL-18-/- animals, which may explain the differences in phagocyte recruitment. In addition, although IFN-gamma production capacity, as a parameter of the Th1-protective immunity, was reduced by 65 to 80% in the IL-12-/- mice, this defect was even more pronounced in the IL-18-/- mice (85 to 95% down-modulation). In conclusion, the anticandidal effects of endogenous IL-18 are mediated late during the infection by assuring a proper IFN-gamma response and promoting the infiltration of the site of infection by monocytes.