Regulation of interleukin-12 expression in human monocytes: selective priming by interferon-gamma of lipopolysaccharide-inducible p35 and p40 genes

Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that is critical for T lymphocyte and natural killer cell activities and functions. In this study, we examined the regulation of IL-12 expression by human monocytes in response to bacterial lipopolysaccharide (LPS). Several novel aspects of IL-12 induction from monocytes were shown. Optimal expression of IL-12 mRNA and bioactivity required specific priming of monocytes by interferon-gamma (IFN-gamma) before LPS stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) provided an equivalent priming stimulus for LPS-induced tumor necrosis factor (TNF) and IL-12 p40 mRNA, but primed poorly for LPS-inducible p35 message and secreted IL-12 activity. Macrophage colony-stimulating factor (M-CSF), although a potent survival factor for monocytes, showed no priming activity for IL-12 production. Time course experiments demonstrated independent regulation of p40 and p35 by IFN-gamma and LPS. LPS inducibility of p40 expression required only a brief exposure to IFN-gamma (2 hours), while prolonged exposure (+/- 24 hours) to IFN-gamma resulted in diminishing levels of p40 mRNA. p35 inducibility (by LPS) required a longer exposure to IFN-gamma (8 to 16 hours), and continued to be inducible up to 40 hours following IFN- gamma priming. Both mRNAs were rapidly induced (1 to 2 hours) in IFN- gamma-primed monocytes; p35 message reached a plateau by 2 hours, while p40 continued to accumulate. Finally, both p40 and p35 were directly induced by LPS in the presence of cycloheximide. These results indicated that both p40 and p35 are LPS-inducible in monocytes following IFN-gamma pretreatment, and that the regulated expression of p35 controls the level of active IL-12 protein in purified human monocytes. The selectivity of priming by IFN-gamma is in accord with a putative role for IL-12 in the initiation and amplification of TH1-type responses.     

IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2.

The effect of human recombinant interleukin 4 (IL-4) on antibody production by normal peripheral blood mononuclear cells enriched for B cells was investigated. IL-4 preferentially induced IgE synthesis in vitro. In addition, a low induction of IgG production was observed, whereas IL-4 had no effect on IgA and IgM synthesis. The IL-4-induced IgE production by B cells required T cells and monocytes but was specifically inhibited by an anti-IL-4 antiserum indicating that, although IL-4 acts indirectly, it is responsible for the induction of IgE synthesis. IL-4-induced IgE production was blocked in a dose-dependent way by interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), and prostaglandin E2. IFN-gamma also inhibited IL-4-induced IgG production. These inhibitory effects of IFN-gamma and IFN-alpha on IgE production cannot be attributed to toxic effects since IFN-alpha induced IgM production in the presence of IL-4, whereas IFN-gamma was ineffective in inhibiting IgG production induced by IL-2. IFN-gamma, IFN-alpha, and prostaglandin E2 also inhibited IL-4-induced expression of the low-affinity receptor for the Fc portion of IgE (CD23) on B cells, indicating that there is an association between CD23 expression and IL-4-induced IgE production. This theory was supported by the finding that IL-4-induced IgE production was inhibited by F(ab')2 fragments of an anti-CD23 monoclonal antibody.     

Interleukin-11 promotes accessory cell-dependent B-cell differentiation in humans.

Interleukin-11 (IL-11) is a recently described stromal-derived cytokine that supports the growth of an IL-6-dependent murine plasmacytoma line in the presence of antibody to IL-6 and appears to act in a manner similar to IL-6 on hematopoietic stem cells. Because IL-6 is known to promote differentiation of normal human B cells, the role of IL-11 on B-cell differentiation in vitro was characterized. IL-11 does not result in significantly increased DNA synthesis or Ig secretion by purified B cells alone or B cells cultured with Staphylococcus Cowan I, a T-cell-independent B-cell mitogen. In contrast, purified B cells cultured in the presence of pokeweed mitogen (PWM), irradiated T cells, and monocytes show increased DNA synthesis at day 3 and increased IgG and IgM secretion at day 7 of culture; addition of IL-11 further augments Ig secretion without change in DNA synthesis, an effect that can only be partially blocked by monoclonal antibody to IL-6. Similar experiments confirmed that increased IgG secretion was demonstrable when either IL-11 or IL-6 was added to B cells + CD4+/45RA- T cells + monocytes + PWM; in contrast, Ig secretion was low and equivalent when CD4+/45RA+ T cells were cultured with B cells+monocytes+PWM with or without IL-6 or IL-11. Neither IL-6 nor IL-11 could significantly increase phytohemagglutinin (PHA)-induced DNA synthesis by CD4+/45RA- or CD4+/45RA+ T cells. Although PWM or IL-11 induced IL-6 mRNA expression in both CD4+/45RA- T cells and monocytes, in neither cell did IL-11 increase IL-6 mRNA expression over that noted to PWM alone. These observations support the view that IL-11 promotes differentiation of human B lymphocytes only in the presence of accessory T cells and monocytes and that a minor component of this effect may be through stimulation of IL-6 production by CD4+/45RA- T cells and monocytes.     

Regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist

We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM- CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G- CSF production was only moderately inhibited. In contrast, IFN-alpha markedly stimulated the production of IL-1 receptor antagonist (IL-1RA) by stromal cells. The inhibition of cytokine expression resulted in a reduced hematopoietic activity of stromal cells, indicated by a reduced proliferation of the factor dependent cell line MO7e on IFN-alpha- treated stromal cells. In the presence of cycloheximide (CHX), IFN- alpha failed to inhibit IL-1 mRNA expression, whereas the regulation of GM-CSF and IL-1RA by IFN-alpha was not affected. Our results indicate that the myelosuppressive effects of IFN-alpha, as observed in therapeutic applications or associated with viral infections, are, in part, indirectly mediated by inhibition of the paracrine production of hematopoietic growth factors.     

Type I interferons in combination with bacterial stimuli induce apoptosis of monocyte-derived dendritic cells

Both type I interferons (IFNs) as well as lipopolysaccharide (LPS) individually compromise selected monocytic or dendritic cell (DC) functions. This study investigates the influence of these agents on the differentiation and the regulation of cell death of monocyte-derived DCs generated in the presence of granulocyte-macrophage colony-stimulating factor plus interleukin-4 (IL-4). It is reported that excessive apoptosis occurred rapidly in monocyte-derived DC cultures, if IFN-alpha or IFN-beta was added in combination with LPS or lipoteichoic acid (LTA). The small fraction of cells surviving in such cultures displayed a mature DC phenotype with expression of CD83, CD80, and CD86. IL-10 was found in the supernatants of monocyte-derived DC cultures, if supplemented with LPS or IFN-alpha plus LPS but not in control cultures. When monocyte-derived DCs were generated in the presence of IFN-alpha without LPS, these cells displayed an immature DC phenotype with a reduction of cell recovery but no overt apoptosis. However, the addition of LPS, LTA, LPS plus IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 to such cells again resulted in the rapid induction of apoptosis in the majority of cells, together with a reduced production of IL-12 p70 and TNF-alpha. Together, these data indicate an exquisite sensitivity of monocyte-derived DCs to activation-induced cell death if generated in the presence of IFN-alpha, indicating the existence of an important mechanism of immunosuppression caused by IFN-alpha-inducing agents, such as viral or bacterial stimuli. (Blood. 2001;98:736-742)     

Th1 and Th2 T-helper cells exert opposite regulatory effects on procoagulant activity and tissue factor production by human monocytes

The role of T-cell subsets in the induction of tissue factor (TF) production by human monocytes in vitro was investigated. Mitogen stimulation enabled both unfractionated T cells and their CD4+ or CD8+ subsets to promote procoagulant activity (PCA). After mitogen or antigen activation, all seven T-cell clones with Th1 cytokine profile, but none of seven Th2 clones, induced TF production and PCA. T-cell blasts from four Th1 activated clones were fixed with paraformaldehyde and added to monocytes in the presence of medium alone or their supernatants. Addition of either fixed Th1 cells or their supernatants induced low TF production (0.2 to 0.6 ng/mL), whereas addition of both resulted in much higher TF synthesis (1.8 to 3.4 ng/mL). Among Th1-type cytokines, only interferon-gamma (IFN-gamma) induced minimal TF production (0.1 to 0.4 ng/mL). No TF synthesis was induced by activated and fixed Th2 cells and/or their supernatants, whereas combined addition of fixed Th2 cells and Th1 supernatants or IFN-gamma induced noticeable TF production. The addition of either anti-IFN-gamma antibody or Th2 supernatants to monocytes stimulated with activated and fixed Th1 cells plus their supernatant resulted in a dose-dependent inhibition of TF synthesis, which was partially restored by neutralization of interleukin-4 (IL-4) or IL-10. Addition of recombinant IL-4, IL-13, or IL-10, but not IL-5, inhibited the Th1- induced TF production by monocytes. Data indicate that both CD8+ and CD4+ Th1, but not Th2, T cells can help TF production and PCA. Both cell-to-cell contact with activated T cells and Th1-type cytokines, in particular IFN-gamma, are required for optimal TF synthesis, whereas Th2-derived cytokines (IL-4, IL-13, and IL-10) are inhibitory. This may be of potential interest for future therapeutic strategies.     

IFN-alpha promotes the rapid differentiation of monocytes from patients with chronic myeloid leukemia into activated dendritic cells tuned to undergo full maturation after LPS treatment

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease arising from the clonal expansion of a stem cell expressing the bcr/abl oncogene. CML patients frequently respond to treatment with interferon-alpha (IFN-alpha), even though the mechanisms of the response remain unclear. In the present study, we evaluated the role of IFN-alpha in differentiation and activity of monocyte-derived dendritic cells (DCs) from CML patients as well as in modulation of the cell response to lipopolysaccharide (LPS). Treatment of CML monocytes with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in the rapid generation of activated DCs (CML-IFN-DCs) expressing interleukin-15 (IL-15) and the antiapoptotic bcl-2 gene. These cells were fully competent to induce IFN-gamma production by cocultured autologous T lymphocytes and expansion of CD8(+) T cells. LPS treatment of CML-IFN-DCs, but not of immature DCs generated in the presence of IL-4/GM-CSF, induced the generation of CD8(+) T cells reactive against autologous leukemic CD34(+) cells. Altogether, these results suggest that (1) the generation of highly active monocyte-derived DCs could be important for the induction of an antitumor response in IFN-treated CML patients and (2) IFN-alpha can represent a valuable cytokine for the rapid generation of active monocyte-derived DCs to be utilized for vaccination strategies of CML patients.     

Interleukin-10 expression: is there a neglected contribution of CD8+ T cells in rheumatoid arthritis joints?

OBJECTIVE: To search for RA specific processes among T cell accumulation, T cell activation, or cytokine expression in CD4+ and CD8+ synovial fluid (SF) T cells. METHODS: Flow cytometry of CD4+, CD8+, CD45RA+, CD45RO+, CD69 double or triple stained peripheral blood (PB) and SF T cells. IL-2, IL-10, and IFN-gamma expression was determined in PMA + ionomycin stimulated T cells on the single cell level. Concentrations of secreted IL-2, IL-4, IL-10, and IFN-gamma were quantified in the sera and synovial fluids by enzyme linked immunosorbent assay (ELISA). RESULTS: A preferential recruitment of CD45RO+ memory T cells was found for CD4+ helper T cells, and in similar also for CD8+ suppressor T cells. An elevated CD69 expression was detected in memory, but also in CD45RA+ naive CD4+ and CD8+ SF T cells, whilst IL-2 expression was only demonstrable in a minor proportion of T cells populations. Preferential recruitment of memory T cells, but incomplete activation of naive and memory, CD4+ and CD8+ T cells were in similar found in RA and control patients. In RA but not in the control patients, a relevant proportion of CD4+ and CD8+ PB and SF T cells expressed IL-10 and IFN-gamma. High concentrations of IL-10, that were correlated with the amounts of secreted TNF-alpha, were only detected in RA joints. CONCLUSION: Memory and naive T cell state of CD4+ and CD8+ T cell accumulates in the joints, and early T cell activation occur in similar patterns in RA and control patients. High IL-10 SF concentrations in contrast, and elevated percentages of IFN-gamma and IL-10 expressing CD4+ and CD8+ T cells in the PB and SF were characteristic for RA. Here, CD8+ T cells may contribute to high IL-10 concentrations in RA joints.     

Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo

Peripheral blood stem cell (PBSC) transplantation is successful in improving engraftment without increasing acute graft-versus-host disease (GVHD), despite much larger numbers of T cells in unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte colony-stimulating factor (G-CSF) therapy has been associated with less acute GVHD. We studied the effect of G-CSF on interferon (IFN)-gamma and IL-4 expression in CD4(+) lymphocytes. CD4(+) cells co-cultivated with G-CSF and stimulated with PHA or CD3 monoclonal antibodies showed significant decreases in IFN-gamma and increases in IL-4 expression (n = 13; P <. 01). G-CSF appeared to have a direct effect on CD4(+) cells independent of monocytes present in the culture because purified CD4(+) cells exposed to G-CSF, washed, and cocultivated with untreated monocytes demonstrated similar changes in IFN-gamma and IL-4 expression, whereas untreated CD4(+) cells cocultured with G-CSF-stimulated monocytes behaved as controls. We then studied peripheral blood mononuclear cells (PBMCs) from G-CSF-mobilized PBSC donors. When their PBMCs were cultured with PHA or CD3 monoclonal antibody, the percent of IFN-gamma-expressing cells decreased by a mean of 55% and 42%, respectively, whereas the percent of IL-4-containing cells increased by a mean of 39% and 58%, respectively, following G-CSF stimulation. Increased apoptosis of IFN-gamma-producing CD4(+) cells was not responsible for the shift in TH1/TH2 subsets. G-CSF-R mRNA was present in both CD4(+) and CD8(+) cells. These results suggest that G-CSF decreases IFN-gamma and increases IL-4 production in vitro and in vivo and likely modulates a balance between TH1 and TH2 cells, an effect that may be important in PBSC transplantation.     

Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes

We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN- gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.     

Suppression of interleukin-12 production by human monocytes after preincubation with lipopolysaccharide.

Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern. Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation. We show here for the first time that the production of IL-12 by IFN-gamma- or GM-CSF-primed human monocytes can be completely suppressed by preincubation with LPS (from Escherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure. A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion. mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits. The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), or nitric oxide (NO) production induced by LPS. Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor. LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-gamma-induced upregulation of CD54 did not decline. Downregulation of IL-12 was not due to changes in mRNA stability. These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.     

Cytokine-mediated regulation of iron transport in human monocytic cells

Under chronic inflammatory conditions cytokines induce a diversion of iron traffic, leading to hypoferremia and retention of the metal within the reticuloendothelial system. However, the regulatory pathways underlying these disturbances of iron homeostasis are poorly understood. We investigated transferrin receptor (TfR)-dependent and -independent iron transport mechanisms in cytokine-stimulated human monocytic cell lines THP-1 and U937. Combined treatment of cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) reduced TfR mRNA levels, surface expression, and iron uptake, and these effects were reversed by interleukin-10 (IL-10), thus stimulating TfR-mediated iron acquisition. IFN-gamma and LPS dose-dependently increased the cellular expression of divalent metal transporter-1, a transmembrane transporter of ferrous iron, and stimulated the uptake of nontransferrin bound iron (NTBI) into cells. At the same time, IFN-gamma and LPS down-regulated the expression of ferroportin mRNA, a putative iron exporter, and decreased iron release from monocytes. Preincubation with IL-10 partly counteracted these effects. Our results demonstrate that the proinflammatory stimuli IFN-gamma and LPS increase the uptake of NTBI via stimulation of divalent metal transporter-1 expression and cause retention of the metal within monocytes by down-regulating ferroportin synthesis. Opposite, the anti-inflammatory cytokine IL-10 stimulates TfR-mediated iron uptake into activated monocytes. The regulation of iron transport by cytokines is a key mechanism in the pathogenesis of anemia of chronic disease and a promising target for therapeutic intervention.     

成纤维滑膜细胞p53基因表达对类风湿关节炎患者CD4+T淋巴细胞的调节

目的 研究人成纤维滑膜细胞(FLS)p53基因表达对类风湿关节炎(RA)患者CD4+T淋巴细胞的调节作用.方法 采用小分子干扰RNA(siRNA)转染抑制FKS内p53基因表达,并与RA患者外周血CD4+T淋巴细胞共同培养.检测转染后FLS中骨保护素(OPG)表达及白细胞介素(IL)-6分泌.并对与之共培养的CD4+细胞膜胞质内蛋白干扰素(IFN)-γ、IL-17、IL-4和CD25以及IFN-γ孤儿核受体γt(RORγt)、IL-17、Foxp3 RNA水平进行测定.结果 p53基因被抑制后,FLS分泌IL-6减少,但OPG表达未受影响.p53基因被抑制的FLS使共培养的CD4+T淋巴细胞内IL-17及IFN-γ蛋白和RNA表达上调,但对CD4+T淋巴细胞RORγt RNA影响不大.虽可上调Foxp3表达,但CD4+CD25high细胞阳性率并无明显变化.结论 FKS内D53表达对RA外周血Th1、Th17有调节作用.     

Effect of 17β-oestradiol on expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar cells

Oestradiol (E(2)) alters lymphocyte functionin vitro including T cell DNA synthesis and B cell immunoglobulin production in human tonsillar, splenic and peripheral blood cells. We have investigated whether one mechanism for this effect is that E(2) modifies the expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar mononuclear cells. Without E(2), addition of PHA (1 μg ml(-1)) for 10 h increased the expression of IL-2 and IL-6 mRNA but had no significant effect on IFN-γ mRNA. In separated T cells after 24 h incubation, E(2) (7×10(-8) M: ) increased only the IFN-γ mRNA levels. However, when E(2) was present in PHA-stimulated T cell cultures, mRNA levels from all cytokines were suppressed. E(2) decreased IL-2 mRNA levels in the T cell preparation after 24 h culture. For IL-6, E(2) decreased mRNA both in mononuclear cells and T cells after 10 h incubation. For IFN-γ, E(2) decreased mRNA levels in the mononuclear cell preparation after 24 h culture. Stimulation of the T cell preparation with PHA after 24 h incubation with E(2) decreased the IFN-γ mRNA levels compared to the cultures incubated with E(2) only. One part of the action of E(2) may be through a block in the up-regulation of the mRNA of IL-2, IL-6 and IFN-γ in activated cells.     

Cytokine profile of liver-infiltrating CD4+ T cells separated from murine primary biliary cirrhosis-like hepatic lesions induced by graft-versus-host reaction

BACKGROUND AND METHODS: We have previously reported that CD4+ T cells induced primary biliary cirrhosis (PBC)-like hepatic lesions in mice with graft-versus-host reaction due to major histocompatibility complex class II disparity. To clarify the relationship between the cytokine profile produced by CD4+ T cells and the formation of hepatic lesions, we sorted CD4+ T cells from the liver by using flow cytometry and examined their cytokine mRNA expression at various times after GVHR induction. We also examined the associated changes in the serum levels of antimitochondrial antibodies (AMA). RESULTS: Histologically, the infiltration of CD4+ T cells around the bile ducts was observed from day 5, and the lesions deteriorated gradually until day 14. On day 14, CD8+, B220+ and Mac-1+ cells, as well as CD4+ T cells were seen around the bile ducts. In the liver-infiltrating CD4+ T cells, the expression level of interferon-gamma (IFN-gamma) mRNA was observed to increase at an early phase (day 3), whereas that of interleukin (IL)-10 mRNA was elevated at a later phase (day 14). The elevation of IFN-gamma mRNA expression at an early phase before the appearance of non-suppurative destructive cholangitis suggests that IFN-gamma may be related to the pathogenesis of PBC in this model. Serum levels of AMA on day 14 were significantly higher than those on day 5. Interleukin-10 was considered to stimulate antibody production, to show an inhibitory effect upon the function of T helper 1 cells, and to inhibit fibrosis. CONCLUSIONS: Interferon-gamma may play an important role in the pathogenesis of this model. Moreover, delayed expression of IL-10 mRNA may control PBC-like hepatic lesions.     

Serum cytokine levels and type 1 and type 2 intracellular T cell cytokine profiles in mixed connective tissue disease

OBJECTIVE: To determine serum cytokine concentrations and intracellular cytokine production of CD4+ and CD8+ T cells in 20 patients with mixed connective tissue disease (MCTD). METHODS: Detailed analysis of cytokine production; 8 patients were in the active stage, 12 in the inactive stage of the disease. Serum concentrations of interferon-gamma (IFN-gamma), interleukin 4 (IL-4), and IL-10 were assessed by ELISA. Intracellular content of IFN-gamma, IL-4, and IL-10 in CD4+ and CD8+ peripheral blood T cell and lymphocyte subsets was determined by flow cytometry. RESULTS: Serum concentrations of both type 1 and type 2 cytokines were significantly higher in patients with MCTD than in healthy controls. IFN-gamma expression of CD4+ and CD8+ T cells did not differ comparing all patients to controls. In patients with active MCTD, the percentage of CD8+/IFN-gamma+ Tc1 cells was significantly increased compared to inactive disease or to controls (p < 0.05). IL-4 expression of CD4+ T cells was scarcely detectable in MCTD, while a subpopulation of CD8+ Tc2 cells produced IL-4. A higher percentage of these CD8+/IL-4+ Tc2 cells were detected in patients with MCTD, especially in active disease, compared to controls (p < 0.05). The percentage of IL-10-expressing CD4+ and CD8+ T cells was higher in patients than in controls (p < 0.05). Again, CD4+ and CD8+ T cells from patients with active MCTD produced significantly more IL-10 than cells in patients with inactive disease or in controls (p < 0.05). CONCLUSION: Our results suggest that MCTD is associated with increased production of both type 1 (IFN-gamma) and type 2 cytokines (IL-4, IL-10). Cytokine production is usually higher in active MCTD than in inactive disease. CD8+ T cells may produce more IFN-gamma and IL-10 in comparison to CD4+ T cells. Patients with active disease have more IL-4-expressing Tc2 cells and IL-10-expressing Th2 and Tc2 cells than patients with inactive MCTD or controls. In MCTD, increased IL-10 production by Th2 and Tc2 cells may be an attempt by the immune system to downregulate the inflammatory reaction, although this effect may not be sufficient to restore the physiological Th1/Th2 balance in MCTD.     

Leishmania infantum released proteins specifically regulate cytokine expression and production patterns by CD4+ and CD8+ T cells

Specific immune responses by CD4+ and CD8+ T cells, from two infected mice strains (BALB/c and C57BL/6), induced by High, Inter and Low protein fractions released by Leishmania infantum, were assessed through the evaluation of IL-12, IFN-gamma and IL-10 mRNA by real-time PCR and respective protein production by ELISA. During infection establishment, High and Inter fractions directed both mice strains T cells subsets to increase the production of IFN-gamma, associated to IL-12 release. Later on, parasite replication augmented in BALB/c and stabilised in C57BL/6 mice. Inter fraction induced CD4+ T cells to maintain IFN-gamma production, with the simultaneous release of IL-12 by both cell subsets in BALB/c mice and by CD8+ T cells in C57BL/6 mice. These observations suggested a prophylactic potential for Inter fraction which was able to induce Th1 response with IL-12 involvement, required for the maintenance of memory cells, in mice strains with different parasitic evolution.