Identification of IFN-gamma-producing cells in IL-12/IL-18-treated mice

Both IL-12 and IL-18 have been characterized as effective IFN-gamma-inducing cytokines. Concomitant treatment with IL-12 and IL-18 has been shown to synergistically induce IFN-gamma and may be an effective therapy for treating cancer, allergy, and infectious diseases. To understand the mechanisms underlying the strong induction of IFN-gamma by IL-12/IL-18 in mice, we focused our studies on the IFN-gamma-producing cells in various lymphoid organs and tissues and utilized the intracellular cytokine staining method to detect such cells in situ. After combined treatment with IL-12 and IL-18, IFN-gamma-positive cells in C57BL/6 mice were detected in the liver (12.18%), spleen (0.68%), bone marrow (1.80%), and peritoneum (2.12%), but not in the thymus or lymph nodes (<0.05 and <0.08%, respectively). A two-color staining method revealed that the majority of IFN-gamma-producing cells in the liver were NK1.1(+) cells, while those in the spleen were mostly CD3(+) cells, and to a lesser degree NK1.1(+) cells. Both CD4(+) and CD8(+) cells in the liver and in the spleen produced IFN-gamma. The CD19(+) B cell population was not definitely shown to produce IFN-gamma in our induction experiments. NKT cells, which are a subpopulation of NK1. 1(+) CD3(+) cells, were diminished in the liver and did not seem to contribute to IFN-gamma production arising from IL-12/IL-18 treatment. Further in vitro experiments confirmed the responsiveness of hepatic mononuclear cells to IL-12/IL-18 stimulation. This study is the first to show the IFN-gamma-producing mechanisms of IL-12/IL-18 treatment at the phenotypic level.     

Differential effects of LPS and TGF-beta on the production of IL-6 and IL-12 by Langerhans cells, splenic dendritic cells, and macrophages

We examined modulatory effects of lipopolysaccharide (LPS) on IL-6 and IL-12 production by mouse Langerhans cells (LC), spleen-derived CD11c+ dendritic cells (DC), and macrophages (Mphi). Low dose LPS (1 ng/ml) increased IL-6 and IL-12 p40 production by Mphi. LPS slightly augmented IL-6 production but showed no effect on IL-12 p40 production by DC. In contrast, only high dose LPS (1 microg/ml) induced IL-6 but not IL-12 p40 production by LC. CD14 expression was the highest on Mphi and then on DC, but not on LC, which may explain the difference in responsiveness to LPS. We also found that TGF-beta inhibited IL-6 and IL-12 p40 production by LPS-stimulated Mphi. However, TGF-beta did not inhibit IL-6 production and even enhanced IL-12 p40 production by anti-CD40/IFN-gamma-stimulated Mphi. Concerning LC, TGF-beta enhanced IL-6 and IL-12 p40 production when stimulated with anti-CD40/IFN-gamma alone or with anti-CD40/IFN-gamma and LPS. Taken together, these findings indicate diverse effects of LPS and TGF-beta on these antigen presenting cells, which probably represents their differential roles in the innate immunity.     

Plasma levels of interleukin-12 (IL-12), interleukin-18 (IL-18) and transforming growth factor beta (TGF-beta) in Plasmodium falciparum malaria

The interaction between pro- and anti-inflammatory cytokines such as interleukin 12 (IL-12), interleukin 18 (IL-18) and transforming growth factor beta (TGF-beta) may play an important role in malaria pathogenesis and outcome. IL-18 cooperates with IL-12 in the IFN-gamma production by T, B, and NK cells, and synergizes with IL-12 for IFN-gamma production by Th1 cells. Recently it has been demonstrated that these cytokines modulate the immunoresponse in Plasmodium falciparum malaria. The aim of this study was to measure the plasma levels of IL-12, IL-18 and TGF-beta in 105 African children with various degrees of malaria, and correlate the production of these cytokines with the severity of the disease. IL-12, IL-18 and TGF-beta levels were determined using enzyme-linked immunosorbent assay. The severity of malaria was established by parasitemia, clinical symptoms and haematological parameters. The levels of IL-12, IL-18 and TGF-beta were found to be significantly elevated (15.6 + / - 12.3, 22.7 + / - 13.8 pg/ml and 25.14 + / - 13.22 pg/ml respectively) in all of the children. IL-12 and IL-18 levels were significantly lower (13.2 + / - 5.53 and 21.5 + / - 10 pg/ml pg/ml) in children with severe disease, whereas the level of TGF-beta was higher (28.09 + / - 12.39 pg/ml). In contrast, IL-12 and IL-18 levels were found to be higher (17.32 + / - 7.8 pg/ml and 25.7 + / - 7.6 pg/ml) in patients with mild disease, whereas the level of TGF-beta was lower (20.92 + / - 12.76 pg/ml) compared to the severe malaria group. The correlation between IL-12 and IL-18 demonstrated a progressive relationship up to a value of IL-12 < 25 pg/ml, while IL-18 remained stable at higher levels of IL-12. An inverse correlation was found between IL-12 and TGF-beta up to a value of IL-12 < 30, after which the level of TGF-beta remained stable. This finding suggests that fine mechanisms regulate the interaction between IL-12, IL-18 and TGF-beta in the immune response to Plasmodium falciparum.     

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Activation of virus-specific CD8+ T cells by lipopolysaccharide-induced IL-12 and IL-18

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-gamma production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8(+) T cells directly ex vivo. Following acute viral infection, 70-80% of virus-specific T cells will produce IFN-gamma after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-gamma production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

Induction of CD4+ T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-beta.

Induction and maintenance of Ag-specific tolerance are pivotal for immune homeostasis, prevention of autoimmune disorders, and the goal of transplantation. Recent studies suggest that certain cytokines, notably IL-10 and TGF-beta, may play a role in down-regulating immune functions. To further examine the role of cytokines in Ag-specific hyporesponsiveness, murine CD4+ T cells were exposed ex vivo to alloantigen-bearing stimulators in the presence of exogenous IL-10 and/or TGF-beta. Primary but not secondary alloantigen proliferative responses were inhibited by IL-10 alone. However, the combined addition of IL-10 + TGF-beta markedly induced alloantigen hyporesponsiveness in both primary and secondary MLR cultures. Alloantigen-specific hyporesponsiveness was observed also under conditions in which nominal Ag responses were intact. In adoptive transfer experiments, IL-10 + TGF-beta-treated CD4+ T cells, but not T cells treated with either cytokine alone, were markedly impaired in inducing graft-vs-host disease alloresponses to MHC class II disparate recipients. These data provide the first formal evidence that IL-10 and TGF-beta have at least an additive effect in inducing alloantigen-specific tolerance, and that in vitro cytokines can be exploited to suppress CD4+ T cell-mediated Ag-specific responses in vivo.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

Mononuclear phagocyte-derived IL-10 suppresses the innate IL-12/IFN-gamma axis in lung-challenged aged mice

Previously, we reported that IL-10-producing mononuclear phagocytes increase in lungs of aged mice, causing impaired innate cytokine expression. Since dendritic cells (DCs) contribute to innate NK cell and adaptive T cell immunity, we tested the hypothesis that age-related IL-10 might influence DC function with effects on NK and T cell activation. The results showed that DC recruitment to sites of lung inflammation was normal in aged mice (>20 mo). However, IFN-gamma-producing NK cells in LPS-challenged lungs were decreased in aged as compared with young mice, which was associated with increased IL-10(+)CD11b(+)Gr-1(low)CD11c(-) cells consistent with mononuclear phagocytes. In vivo or in vitro blockade of IL-10 signaling restored IFN-gamma-producing NK cells. This restoration was reversed by IL-12 neutralization, indicating that IL-10 suppressed sources of IL-12 in aged mice. To probe DC function in adaptive immunity, we transferred young naive OVA-specific TCR transgenic T cells to old mice. Following challenge with OVA plus LPS, Ag presentation in the context of MHC-I and MHC-II occurred with similar kinetics and intensity in draining lymph nodes of young and old recipients as measured by proliferation. Despite this, aged hosts displayed impaired induction of IFN-gamma(+)CD4(+), but not IFN-gamma(+)CD8(+), effector T cells. Blockade of IL-10 signaling reversed age-associated defects. These studies indicate that the innate IL-12/IFN-gamma axis is not intrinsically defective in lungs of aged mice, but is rather suppressed by enhanced production of mononuclear phagocyte-derived IL-10. Our data identify a novel mechanism of age-associated immune deficiency.     

IL-10- and TGF-beta-mediated susceptibility in kala-azar and post-kala-azar dermal leishmaniasis: the significance of amphotericin B in the control of Leishmania donovani infection in India

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.     

TGF-beta 1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes

TGF-beta1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-beta1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-beta1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1(-/-) mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-gamma and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1(-/-) T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1(-/-) T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4(+) T cells have elevated intracellular Ca(2+) levels. However, upon subsequent stimulation in vitro, increases in Ca(2+) levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1(-/-) splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca(2+) levels.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.     

IL-12 and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs

Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN-gamma production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-gamma, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN-gamma induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN-gamma and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN-gamma production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN-gamma production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN-gamma production, indicating important cytokines in the Th1 response of sarcoidosis.     

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.