Constitutive STAT3 activation in peripheral CD3(+) cells from patients with primary Sjögren's syndrome.

OBJECTIVE: Signal transducers and activators of transcription (STATs) are crucial mediators of cytokine signalling. Constitutive activation of STATs, especially STAT3, has been reported in several diseases. Primary Sjögren's syndrome (pSS) is associated with overproduction of cytokines such as interleukin-10 (IL-10), although the mechanism by which this occurs is unknown. As STAT3 is a potent inducer of IL-10, this study focused on determining the pattern of STAT3 activation in peripheral lymphocytes from patients with pSS. METHODS: Twelve pSS patients and 12 healthy age-matched control subjects were studied. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation. Phosphorylated STAT3 (pSTAT3) and also STAT3 expression were determined by flow cytometry in gated CD3(+ )and CD19(+) lymphocytes. Similarly, pJak1 and pTyk2 were also determined in gated CD3(+) lymphocytes. RESULTS: Although the protein expression of STAT3 was similar among controls and pSS patients, we found that STAT3 was constitutively activated in CD3(+) lymphocytes from pSS patients. Neither Jak1 nor Tyk2 (the upstream activators of STAT3) was activated in pSS CD3(+) lymphocytes, suggesting that the constitutive activation of STAT3 observed in pSS patients might not depend on cytokine stimulation but instead might be the result of an abnormal inactivation of pSTAT3. CONCLUSIONS: These data provide evidence of abnormal STAT3 signalling in T cells from pSS patients.

Expression and function of IL-10R in mononuclear cells from patients with systemic lupus erythematosus.

OBJECTIVE: To assess the expression and function of the receptor for interleukin-10 (IL-10R) in immune cells from patients with systemic lupus erythematosus (SLE). METHODS: We assessed the expression and function of IL-10R in peripheral blood mononuclear cells (PBMCs) from 19 SLE patients and 15 healthy controls. The expression of IL-10R was assessed by flow cytometry, and the function of this receptor was determined by analysing both the activation of Jak-1, Tyk-2, Stat-1, and Stat-3 (Western blot) and the induction of gene expression (cDNA array test of 242 genes of cytokines, apoptosis and intracellular signalling) upon stimulation with IL-10. RESULTS: We found similar levels of IL-10R expression in SLE patients and controls. In addition, variable levels of Jak-1, Tyk-2, Stat-1, and Stat-3 activation were induced by IL-10 in PBMCs from SLE patients and controls, with no significant differences in protein phosphorylation or kinetics of activation. However, clear-cut differences in the gene expression induced through IL-10R were observed in SLE patients and controls, mainly in the genes involved in apoptosis and those encoding for cytokines and their receptors. CONCLUSIONS: Our data suggest that despite normal levels of IL-10R expression, and an apparent lack of abnormalities in the intracellular signals induced through this receptor, immune cells from SLE patients exhibit an aberrant pattern of gene expression induced through the IL-10R.

IL-15 and IL-15R in leucocytes from patients with systemic lupus erythematosus.

OBJECTIVE: To assess the functional status of the IL-15/IL-15Ralpha cytokine system in different leucocyte subsets from patients with systemic lupus erythematosus (SLE). METHODS: Eighteen patients with SLE (10 with inactive and eight with active disease) and 14 healthy individuals were studied. Serum levels and in vitro production of IL-15 were determined. In addition, the expression of IL-15 receptor alpha (IL-15Ralpha) and membrane-bound IL-15 was assessed and the in vitro effects of IL-15 on CD69 and CD64 expression, interferon-gamma and TNF-alpha synthesis, respiratory burst induction and apoptosis were studied. RESULTS: Serum levels of IL-15 were significantly increased in inactive and active patients with SLE. Accordingly, the in vitro synthesis and release of IL-15 by monocytes in response to IFN-gamma+lipopolysaccharide was significantly enhanced in SLE patients with active disease, as was the percentage of membrane-bound IL-15+ monocytes. On the other hand, enhanced basal expression of IL-15Ralpha was detected in leucocytes from SLE patients, with defective induction upon stimulation with phytohaemagglutinin or phorbol myristate acetate/ionomycin. Furthermore, diminished induction of CD69 expression and interferon-gamma and TNF-alpha synthesis by recombinant human IL-15 was detected in peripheral blood mononuclear cells from SLE, and there was defective induction of CD64 and priming for respiratory burst in neutrophils. The anti-apoptotic effect of IL-15 was diminished in leucocytes from SLE patients. CONCLUSION: Our data indicate that there is enhanced synthesis of IL-15 by immune cells from SLE patients, with a poor response to this cytokine by different leucocyte subsets. This abnormal function of IL-15/IL-15Ralpha may contribute significantly to the pathogenesis of SLE.

Comparative study of DNA vaccines encoding various antigens against Leishmania mexicana.

Leishmaniases represent an important public health problem in large parts of the world. In the south-east of Mexico, the major species isolated from patients is Leishmania mexicana mexicana, causing localised cutaneous leishmaniasis, and the development of a vaccine is a key objective for the control of this parasite. We thus performed a comparative study of DNA vaccines encoding L. m. mexicana gp63 and CPb, L. m. amazonensis gp46, and L. major LACK to define the best antigen(s) candidate(s). cDNAs encoding these antigens were subcloned into the VR1012 plasmid, and susceptible BALB/c mice were immunised with two i.m. injections of 100 microg of plasmid DNA. All mice immunised with VR1012-GP46, VR1012-CPb and VR1012-GP63 showed increased IgG levels against L. m. mexicana, but not those immunised with VR1012-LACK. Two to three weeks after the last immunisation, mice were challenged by the injection of 4 x 10(6) L. m. mexicana parasites in the foot pad to evaluate protection. Measurement of lesion size indicated that mice immunised with VR012-GP46, VR012-GP63 and VR1012-CPb were partially protected against infection, whereas the other plasmids had no effect. Thus, these plasmids represent good candidates for further development of DNA immunisation against L. m. mexicana.

In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis.

Th1-type cellular immune responses (interferon-gamma) play a critical role in protection against Leishmania spp. infection, whereas Th2-type cytokines (interleukin (IL)-4, IL-10) have a counter-protective effect. IL-12, a potent inducer of Th1-type cellular immune responses, may play a pivotal role in the development of a protective response. We found that IL-10 and IL-12 mRNAs were expressed in most lesions of individuals with active cutaneous leishmaniasis. The quantity of IL-12 mRNA was highly variable but correlated strongly with the level of interferon-gamma expression. IL-12 expression also paralleled the expression of IL-10, a potent in vitro suppressor of IL-12 and interferon-gamma production. The more chronic, non-healing lesions generally had higher levels of IL-12 mRNA indicating that the expression of this cytokine alone was not sufficient to induce healing. Although the in situ production of IL-10 did not appear to block IL-12 expression, IL-10 may still promote disease by direct suppression of macrophage activation.

Induction of macrophage killing of Leishmania donovani by human CD4+ T cell clones.

Immunity in leishmaniasis is mediated by T cells, but protective responses in humans have not been fully defined. In this study, the functional activity of CD4+ T cell clones derived from an immune individual was investigated to identify potentially protective responses. The T cells proliferated and produced interferon-gamma (IFN-gamma) in response to a soluble Leishmania donovani antigen extract and live amastigotes. There was considerable variation in the anti-leishmanial activity of the T cell clones when they were co-cultured with L. donovani infected monocytes isolated from an HLA-DR,DQ matched donor. All of the clones which demonstrated antigen specific reactivity by proliferation or cytokine production induced some degree of inhibition of intracellular parasite replication, but only a few of the clones induced pronounced leishmanicidal activity. There was strong correlation between the level of amastigote-induced IFN-gamma secretion and anti-leishmanial activity. This approach enables the identification of potentially protective immune responses in humans at the clonal level, and offers a means for the identification of the relevant antigen(s).

Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis.

The nature of the host cellular immune response largely determines the expression of disease following infection with the intracellular protozoans Leishmania spp. In experimental animals control and resolution of infection are mediated by gamma interferon and tumor necrosis factor alpha (TNF-alpha), whereas disease progression is associated with the production of interleukin 4 (IL-4), IL-5, IL-10, and transforming growth factor beta (TGF-beta). We have analyzed the profile of cytokine gene expression directly in the lesions of 13 patients with localized cutaneous leishmaniasis due to Leishmania mexicana. All but one patient had a single lesion, and the time of evolution ranged from 8 days to 18 months. Cytokine gene expression was quantitated by reverse transcriptase PCR and interpolation from a standard curve. Gamma interferon, TNF-alpha, IL-1 alpha, IL-6, IL-10, and TGF-beta gene expression was present in all samples. IL-3 and IL-4 gene expression was barely detectable in 1 and 3 of 13 samples, respectively. IL-2 and IL-5 mRNAs were not found. A significant increase in the expression of IL-1 alpha, TNF-alpha, IL-10, and TGF-beta was observed in late lesions (> or = 4 months) compared with that in early lesions (< or = 2 months). Because of their inhibitory effects on macrophage function, the expression of IL-10 and TGF-beta may play a role in the immunopathogenesis of chronic cutaneous leishmaniasis.