Intracellular IFN-gamma production and IL-12 serum levels in latent autoimmune diabetes of adults (LADA) and in type 2 diabetes.

Th1 cytokines, such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and Th1-inducing cytokines, such as IL-12, are involved in the pathogenesis of various organ-specific autoimmune diseases, including autoimmune diabetes. In this study, we investigated intracellular IFN-gamma release by T lymphocytes and IL-12 serum levels in 48 type 2 and 36 latent autoimmune diabetes of adults (LADA) diabetics and 25 control subjects in an attempt to evaluate their role in the pathogenesis of these clinical entities. Ionomycin (ION) and phorbol-12-myristate-13-acetate (PMA)-activated peripheral blood mononuclear cells (PBMCs) were stained with anti-CD4-FITC or anti-CD8-FITC and anti-IFN-gamma phycoerythrin (PE) monoclonal antibodies (mAbs) and analyzed by flow cytometry. IL-12 serum levels were determined by enzyme-linked immunosorbent assay (ELISA). In all study groups, IFN-gamma content of CD4(+) and CD8(+) lymphocytes was significantly upregulated by stimulation. Furthermore, it was observed that CD4(+) and CD8(+) lymphocytes from type 2 diabetics produced significantly lower levels of IFN-gamma compared with LADA patients and controls. However, the percentages of CD4(+)/IFN-gamma(+) and CD8(+)/IFN-gamma(+) cells from type 2 diabetics were significantly higher compared with controls. The flow cytometric picture of intracellular IFN-gamma release in LADA patients did not differ from that observed in controls. However, IL-12 serum levels in type 2 and LADA diabetics were lower than in controls. Because Th1 cytokines have been associated with the pathogenesis of autoimmune diabetes, these results preclude Th1 involvement in the autoimmune phenomena observed in LADA patients. In contrast, the low IFN-gamma levels observed in type 2 diabetics in combination with the low IL-12 serum levels might be a contributing factor in the frequently observed chronic complications in these patients.

Flow cytometric detection of intracellular IL-12 release: in vitro effect of widely used immunosuppressants.

Interleukin 12 (IL-12) is a potent regulator of the Th1/Th2 pathway, enhancing alloantigen-specific immune functions. In the present study, we developed a flow cytometric assay detecting intracellular IL-12 production by human CD14+ monocytes in order to assess the in vitro effects of widely used immunosuppressants, such as cyclosporine (CsA), sirolimus (SRL) and dexamethasone (DXM). For the purpose of the study, a two-step activation procedure was developed involving the preactivation of peripheral blood mononuclear cells (PBMC) with interferon-gamma (IFN-gamma) and reactivation with IFN-gamma and lipopolysaccharide (LPS). All immunosuppressive agents were added at the initiation of the preactivation or the reactivation step. Following this activation protocol, a fourfold to fivefold up-regulation of the percentage of CD14+/IL-12+ cells and of the mean fluorescence intensity was observed. CsA did not significantly affect the intracellular IL-12 release by CD14+ cells, independent of the time point of the addition. SRL exerted an up-regulatory effect when added at the initiation of the IFN-gamma pre-incubation, and this was manifested as a significant increase in the percentage of CD14+/IL-12+ cells. In contrast, DXM effectively repressed both the percentage and the fluorescence intensity of IL-12-producing CD14+ cells when added at the initiation of the reactivation step. Since only the steroid preparation was shown to down-regulate the intracellular release of IL-12, it is tempting to assume that steroid addition in immunosuppressive schemes is beneficial for the suppression of Th1-inducing cytokine production, as well as for the compensation of possible up-regulation induced by other immunosuppressive agents administered concurrently.

Correlation between intracellular interferon-gamma (IFN-gamma) production by CD4+ and CD8+ lymphocytes and IFN-gamma gene polymorphism in patients with type 2 diabetes mellitus and latent autoimmune diabetes of adults (LADA).

IFN-gamma is considered to be involved in the pathogenesis of diabetes mellitus. In this study, the presence of T/A mutation at position -874 in IFN-gamma gene was assessed in patients with latent autoimmune diabetes of adults (LADA), in patients with type 2 diabetes and in healthy individuals. Subsequently, an attempt was made to correlate the presence of this mutation with the ability of CD4+ or CD8+ lymphocytes from these individuals to release IFN-gamma following mitogenic stimulation. There were no significant differences in the distribution of genotypes and haplotypes in the three study groups. However, the frequency of the low IFN-gamma production allele (IFN-gamma 874( *)A) was significantly higher in type 2 diabetics compared to controls. CD4+ and CD8+ cells obtained from type 2 diabetics released significantly lower amounts of IFN-gamma in the intracellular space, compared to those released by cells obtained from LADA patients and healthy volunteers. Furthermore, even CD4+ and CD8+ from type 2 diabetics bearing the TT genotype (high producers) released significantly lower amounts of IFN-gamma than LADA patients carrying the same genotype, probably due to the activity of molecules directly or indirectly inhibiting IFN-gamma production. The results of this study indicate that IFN-gamma may contribute to the development of type 2 diabetes, based on a combination of molecular and immunological observations.

TNF-alpha, TGF-beta1, IL-10, IL-6, gene polymorphisms in latent autoimmune diabetes of adults (LADA) and type 2 diabetes mellitus.

Abundant evidence suggests that cytokines involve in the pathogenesis of latent autoimmune diabetes of adults (LADA). This is a slowly progressive form of type 1 diabetes, which is initially diagnosed as type 2 diabetes. In this study, healthy individuals LADA and type 2 diabetic patients were genotyped for IL-6-174G/C, TNF-alpha-308A/G, TGF-beta1-codon10T/C, TGF-beta1-codon25G/C, IL-10-1082A/G, IL-10-819T/C, IL-10-592A/C gene polymorphisms, by sequence-specific-primer polymerase chain reaction methodology. A significant difference in the frequencies of -1082A/G IL-10 alleles was observed, with the -1082*A allele (known to be associated with low IL-10 production), predominating in LADA diabetics than type 2 diabetics (p=0.036). No significant differences of genotypes, phenotypes, or haplotype frequencies in the remaining cytokine polymorphisms were observed. Analysis of allele combinations revealed a significant involvement of the low and high in vitro production IL-10 alleles in the development of LADA and type 2 diabetes, respectively. These results suggest that the G/A mutation at position -1082 of IL-10 promoter gene region might be one of the factors participating to the pathogenesis of LADA diabetes and that identification of cytokine gene polymorphisms might contribute to the characterization of the different types of diabetes mellitus.