IFN-gamma Induction Research Articles

Induction of IL-4-dependent, anaphylactic-type and IL-4-Independent, non-anaphylactic-type IgG1 antibodies is modulated by adjuvants

Adjuvants can modulate the levels of anaphylactic- and non-anaphylactic-type IgG1 antibodies produced in response to a particular antigen. Mice immunized with ovalbumin (OVA) in Al(OH)(3) gel (alum) produced mostly the anaphylactic type, irrespective of the s.c. or i.p. route used, and this antibody was not detectable in IL-4(-/-) mice. In contrast, when OVA was injected in complete Freund's adjuvant (CFA), it induced substantial amounts of non-anaphylactic-type IgG1 in both IL-4(+/+) and IL-4(-/-) mice, and some anaphylactic IgG1 antibody in IL-4(+/+) mice only. When IFN-gamma was neutralized by specific mAb in wild-type mice immunized with OVA in CFA, the anaphylactic-type IgG1 antibody increased reaching the same levels as in alum-injected mice. This result indicates that the induction of IFN-gamma by the immunization with CFA down-regulates the production of IL-4-dependent, anaphylactic-type IgG1. Despite their different effects on IgG1 antibody production, both adjuvants dramatically increased the production of IgG2a in IL-4-deprived mice and did not induce any detectable IgE in these mice.

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection.

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

Individual diversities in interferon gamma production by human peripheral blood mononuclear cells stimulated with periodontopathic bacteria

Polarization of type 1 (Th1) or type 2 (Th2) immune responses determines the prognosis of many infectious diseases. Interferon gamma (IFN-gamma) and IL-4 are key cytokines for the development of type 1 and type 2 immune responses, respectively. The aim of this study was to examine individual diversities in the polarization of type 1 and type 2 responses against periodontopathic bacteria. Peripheral blood mononuclear cells (PBMCs) from adult periodontitis (AP) patients and healthy (H) subjects were stimulated with Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Bacteroides forsythus with or without polymyxin-B, CTLA-4 Ig and anti-IL-12 antibody. IFN-gamma, IL-4 and IL-12 in the culture supernatant were measured. IFN-gamma and IL-4 producing cells were also examined using a multiparameter flow cytometric assay. Bone resorption rate in AP patients was calculated using Schei's method, and the probing pocket depth was also measured. PBMCs from AP patients and H subjects produced IFN-gamma and IL-12, whereas the production of IL-4 was rarely observed. Among the bacteria tested, A. actinomycetemcomitans was the most potent inducer of IFN-gamma and IL-12, and the reaction was inhibited by polymyxin-B. IFN-gamma was found to be produced by T cells in the PBMCs, and the production was significantly reduced by CTLA-4 Ig and anti-IL-12 neutralizing antibody. The amount of IFN-gamma produced by the PBMCs of AP patients and H subjects varied among individuals, and was significantly correlated with the amount of IL-12 produced in a particular individual. The production of IFN-gamma was not related with periodontal condition which was evaluated using bone resorption and pocket depth. These results suggest that polarization of type 1 response against periodontopathic bacteria is dependent on the production of IL-12 by monocytes, and that IL-12 stimulates IFN-gamma production. However, individual diversities of IFN-gamma production might not be directly related to the severity of periodontitis.

The expression of MHC class II genes in macrophages is cell cycle dependent.

Using different drugs, we stopped the cell cycle of bone marrow-derived macrophages at different points. After IFN-gamma stimulation, macrophages arrested at the G(1) phase of the cell cycle did not increase cell surface expression of the MHC class II IA. This inhibition is specific, because, under the same conditions, IFN-gamma induces the expression of Fcgamma receptors and the inducible NO synthase mRNA. Treatments that inhibit macrophage proliferation by blocking the cell cycle at the G(1) phase, such as adenosine, forskolin, or LPS, blocked the IFN-gamma induction of IA. Under IFN-gamma treatment, the steady-state levels of IAalpha and IAss mRNA did not increase in cells arrested at the G(1) phase and the half-life of the MHC mRNA was not modified. These data suggest that the cell cycle modulation of IFN-gamma-induced MHC II gene expression occurs at the transcriptional level. The expression of the class II transactivator mRNA induced by IFN-gamma was also blocked when macrophages were arrested at the G(1) phase of the cell cycle, suggesting that the lack of IFN-gamma response occurs at the early steps of MHC class II expression. Finally, macrophages arrested at the G(1) phase showed increased basal levels of cell surface IA due to an increase of the translational efficiency. These data show that the expression of MHC class II genes is regulated by the cell cycle.

IL-18 directs autoreactive T cells and promotes autodestruction in the central nervous system via induction of IFN-gamma by NK cells.

IL-18 promotes NK cell and Th1 cell activity and may bridge innate and adaptive immune responses. Myelin oligodendrocyte glycoprotein (MOG) is a myelin component of the CNS and is a candidate autoantigen in multiple sclerosis. In the present study we show that IL-18-deficient (IL-18-/-) mice are defective in mounting autoreactive Th1 and autoantibody responses and are resistant to MOG35-55 peptide-induced autoimmune encephalomyelitis. IL-18 administration enhances the disease severity in wild-type mice and restores the ability to generate Th1 response in the IL-18-/- mice. This restoration was abrogated in NK cell-depleted mice, indicating that the action of IL-18 in promoting the generation of MOG-specific Th cells was dependent on NK cells. Furthermore, transfer of NK cells from recombinase-activating gene 1-/- mice, but not from recombinase-activating gene 1/IFN-gamma-/- mice, rescued the defective Th1 responses in IL-18-/- mice and rendered IL-18-/- mice susceptible to the induction of autoimmune encephalomyelitis. Thus, IL-18 can direct autoreactive T cells and promote autodestruction in the CNS at least in part via induction of IFN-gamma by NK cells.

IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages.

Constitutive IL-18 expression is detected from many different cells, including macrophages, keratinocytes, and osteoblasts. It has been known that IL-18 gene expression is regulated by two different promoters (p1 promoter and p2 promoter). When RAW 264.7 macrophages were treated with IFN-gamma, IL-18 gene expression was increased in a dose- and time-dependent manner. IFN-gamma activated the inducible promoter 1, but not the constitutive promoter 2. Mutagenesis studies indicated that an IFN consensus sequence-binding protein (ICSBP) binding site between -39 and -22 was critical for the IFN-gamma inducibility. EMSA using an ICSBP oligonucleotide probe showed that IFN-gamma treatment increased the formation of DNA-binding complex, which was supershifted with anti-IFN regulatory factor-1 Ab and anti-ICSBP Ab. Another element, an AP-1 site between -1120 and -1083, was important. EMSA using an AP-1-specific oligonucleotide demonstrated that IFN-gamma or LPS treatment increased the AP-1-binding activity. The addition of anti-c-Jun Ab or anti-c-Fos Ab to IFN-gamma- or LPS-treated nuclear extracts resulted in the reduction of AP-1 complex or the formation of a supershifted complex. Taken together, these results indicate that IFN-gamma increased IL-18 gene expression via ICSBP and AP-1 elements.

Involvement of STAT-1 and Ets Family Members in Interferon-γ Induction of CD40 Transcription in Microglia/Macrophages

Cluster of differentiation (CD)-40 is a cell surface receptor belonging to the tumor necrosis factor receptor family that plays a critical role in the regulation of immune responses. We have previously shown that the cytokine interferon (IFN)-gamma induces CD40 expression in microglia. Herein, we have elucidated the molecular mechanisms underlying IFN-gamma induction of CD40 gene expression in microglia/macrophages. IFN-gamma up-regulates CD40 expression at the transcriptional level, and this regulation involves the STAT-1alpha transcription factor. Microglia from STAT-1alpha-deficient mice were refractive to IFN-gamma induction of CD40 expression, illustrating the importance of STAT-1alpha in this response. Functional analysis of the CD40 promoter indicates that two gamma activated sequence elements as well as two Ets elements are involved in IFN-gamma induction of CD40 promoter activity. STAT-1alpha binds to the gamma activated sequence elements, whereas PU.1 and/or Spi-B bind to the Ets elements. The expression of PU.1 and Spi-B, in conjuction with STAT-1alpha activation, correlates with IFN-gamma inducibility of CD40 expression. Collectively, our data demonstrate the involvement of STAT-1alpha, PU.1, and Spi-B in IFN-gamma induction of CD40 gene expression in cells of the macrophage lineage.

Interferon gamma induction during oral tolerance reduces T-cell migration to sites of inflammation.

Previous data suggest that oral antigen induces interferon (IFN)-gamma production in intestinal T cells. However, oral tolerance is associated with decreased production of IFN-gamma by T cells after antigen sensitization. The aim of this study was to examine the role of IFN-gamma in oral tolerance. Oral tolerance was examined in BALB/c mice after the adoptive transfer of T cells from chicken ovalbumin (OVA(323-339))-specific, DO11.10 x RAG-1(-/-) T-cell receptor transgenic mice. OVA feeding induced systemic tolerance of delayed-type hypersensitivity (DTH) and antibody responses. OVA feeding up-regulated IFN-gamma production by transgenic T cells in Peyer's patch and mesenteric lymph node but not splenic tissues. Treatment of OVA-fed mice with neutralizing monoclonal antibody to IFN-gamma prevented tolerance of DTH responses. Analysis of transgenic T-cell numbers in DTH sites by immunohistochemical staining suggested that induction of IFN-gamma by oral antigen decreased accumulation of transgenic T cells in cutaneous sites of antigen injection. IFN-gamma-deficient or wild-type DO11.10 and BALB/c mice were used to show that IFN-gamma production by donor transgenic T cells was critical for oral tolerance. These data suggest that the induction of IFN-gamma by oral antigen contributes to systemic tolerance by decreasing migration of T cells to peripheral sites of inflammation.

Influence of beta(2)-integrin adhesion molecule expression and pulmonary infection with Pasteurella haemolytica on cytokine gene expression in cattle.

beta(2)-Integrins are leukocyte adhesion molecules composed of alpha (CD11a, -b, -c, or -d) and beta (CD18) subunit heterodimers. Genetic CD18 deficiency results in impaired neutrophil egress into tissues that varies between conducting airways and alveoli of the lung. In this study, we investigated whether CD18 deficiency in cattle affects proinflammatory cytokine (PIC) expression in pulmonary tissue after respiratory infection with Pasteurella haemolytica. Cattle were infected with P. haemolytica via fiberoptic deposition of organisms into the posterior part of the right cranial lung lobe. Animals were euthanized at 2 or 4 h postinoculation (p.i.), and tissues were collected to assess PIC gene expression using antisense RNA probes specific for bovine interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) along with the beta-actin (beta-Act) housekeeping gene. Expression of PIC was induced at 2 h p.i. in P. haemolytica-infected cattle and continued to 4 h p.i. At 2 h p.i., induction of gene expression and increase of cells that expressed PIC were observed both in CD18(+) and CD18(-) cattle after inoculation of P. haemolytica. The induction of gene expression with P. haemolytica inoculation was more prominent in CD18(-) cattle than in CD18(+) cattle by comparison to pyrogen-free saline (PFS)-inoculated control animals. At 4 h p.i., however, the induction of PIC, especially IL-1alpha, IL-6, and IFN-gamma, in the lungs of CD18(+) cattle inoculated with P. haemolytica was greater than that in lungs of the CD18(-) cattle. IFN-gamma and TNF-alpha genes were not increased in P. haemolytica-inoculated CD18(-) cattle lungs compared to the PFS-inoculated control lungs at 4 h p.i. In PFS-inoculated lungs, we generally observed a higher percentage of cells and higher level of gene expression in the lungs of CD18(-) cattle than in the lungs of CD18(+) cattle, especially at 4 h p.i. The rate of neutrophil infiltration into the lungs of CD18(-) cattle at 2 h p.i. was significantly higher than that of CD18(+) cattle; at 4 h p.i., there was no difference between the two groups. These data suggest that beta(2)-integrins may contribute to the induction of expression of some PIC genes, as a consequence of P. haemolytica infection.

Requirements for in vivo IFN-gamma induction by live microfilariae of the parasitic nematode, Brugia malayi.

Lymphatic filariasis caused by the parasitic nematode, Brugia malayi, is a chronic human disease immunologically characterized by stimulation of Th2 cells and reduced antigen-specific T cell responses. Single stage intra-peritoneal infections with infective larvae (L3) or adult nematodes induce Th2 cells, while the microfilarial stage (Mf) stimulates IFN-gamma and Mf-specific IgG1, IgG2a, IgG2b, IgG3 and IgM, but not IgE. To investigate whether IFN-gamma is elicited by live Mf in their natural site of infected, mice were infected intravenously. Intravenous infection had a striking effect on the response to Mf and high levels of IgE were induced even in the presence of IFN-gamma. Indeed IgE levels to Mf increased markedly with the number of immunizations, higher doses of Mf and prolonged exposure to Mf suggesting that under conditions of chronic antigen exposure, typical of human disease, Mf will stimulate high levels of IgE. The ability of Mf-induced IFN-gamma to modulate or regulate a pre-existing Th2 response, was investigated by infecting mice initially with adult male worms to induce a Th2 response, followed 14 days later by infection with Mf. Although Mf stimulated IFN-gamma in the presence of male adults, the antibody isotypes elicited did not reflect IFN-gamma induction and IgG1 and IgE dominated the response. Although it cannot be discounted that IFN-gamma induction by Mf may act locally as an inflammatory mediator or modulator of Th2 cells, these data suggest that Mf-stimulated IFN-gamma does not have a profound effect overall on progression of the Th2-dominated immune response to filarial infection.

α-MSH inhibits induction of C/EBPβ-DNA binding activity and NOS2 gene transcription in macrophages

alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous tridecapeptide that exerts anti-inflammatory actions and abrogates postischemic renal injury in rodents. alpha-MSH inhibits lipopolysaccharide (LPS)-induced gene expression of several cytokines, chemokines, and nitric oxide synthase-2 (NOS2), but the molecular mechanisms underlying these effects have not been clearly defined. To test the hypothesis that alpha-MSH inhibits the expression of inducible trans-activating factors involved in NOS2 regulation, we used RAW 264.7 macrophage cells to examine the effects of alpha-MSH on the activation of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer binding protein-beta (C/EBPbeta), trans-acting factors known to be involved in LPS + interferon (IFN)-gamma induction of the NOS2 gene. Gel shift assays were performed to identify NF-kappaB and C/EBP DNA binding activities in LPS + IFN-gamma-treated RAW 264.7 cells in the presence and absence of alpha-MSH. NOS2 promoter assays were conducted to identify the effects of alpha-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription. Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappaB and C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the C/EBP complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBPbeta, but not that of NF-kappaB (in contrast to reports in other cell types), as well as the activity of a murine NOS2 promoter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect on the induction of NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box. These data indicate that alpha-MSH inhibits the induction of C/EBPbeta DNA binding activity and that this effect is a major mechanism by which alpha-MSH inhibits the transcription of the NOS2 gene. The inability of alpha-MSH to inhibit LPS + IFN-gamma induction of NF-kappaB in murine macrophage cells, which contrasts with inhibitory effects of the neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.

Identification of activated tumor antigen-reactive CD8+ cells in healthy individuals.

We investigated the ability of HER-2 peptide E75, which maps an immunodominant CTL epitope for ovarian and breast tumor-associated lymphocytes (TAL), to activate effector functions in freshly isolated CD8+ cells from healthy individuals. IFN-gamma was rapidly induced by E75 within 20-24 h, in five of six healthy donors, in the presence of IL-12 and was detectable as early as 6 h. The IFN-gamma levels were Ag-concentration dependent. Similar results were obtained with peptides mapping CTL epitopes from two other tumor Ag: folate binding protein (FBP) and amino-enhancer of split of Notch (AES). IFN-gamma was also detected, from freshly isolated, unstimulated PBMC in response to HLA-A2 matched tumors + IL-12 but not of IL-12 alone. The major source of IFN-gamma were CD45RO+ CD8+ cells. Induction of IFN-gamma and IL-2 from CD8+ cells and of IL-12 from dendritic cells (DC) by CD8+ cells reactive with E75 mirrored their induction by the influenza matrix peptide (M1: 58-66) in the same individual. Responses to M1 are used to define the presence of activated memory cells in healthy individuals. Compared to M1 responses E75 recognition induced 2-4-fold lower levels of IL-12 from the same APC and IFN-gamma and IL-2 from the same CD8+ cells. At lower Ag concentrations the endogenous IL-12 induced by E75-reactive CD8+ cells did not reach the threshold required to co-stimulate for IFN-gamma. alphaB7.1 synergized with E75 in increasing the overall levels of IL-2 induced within 24 h. The presence of tumor Ag-reactive activated CD8+ cells in healthy individuals may improve our understanding of the mechanisms of immunosurveillance and regulation of immune responses by tumors.

Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control.

Inhibition of class II trans-activator (CIITA) expression prevents embryonic trophoblast cells from up-regulating MHC class II genes in response to IFN-gamma. This is thought to be one mechanism of maternal tolerance to the fetal allograft. The CIITA gene is regulated by four distinct promoters; promoter III directs constitutive (B cell) expression, and promoter IV regulates IFN-gamma-inducible expression. Using in vivo genomic footprinting, promoter-reporter analysis, Southern blot analysis, and RT-PCR, we have examined the cause of CIITA silencing in a trophoblast-derived cell line. We report here that methylation of promoter IV DNA at CpG sites in Jar cells prevents promoter occupancy and IFN-gamma-inducible transcription. The inhibition of CpG methylation in Jar cells by treatment with 5-aza-2'-deoxycytidine restores IFN-gamma inducibility to CIITA. This is the first description of an epigenetic mechanism involved in regulation of CIITA and MHC class II gene expression.

The antiangiogenesis effect of interleukin 12 during early growth of human pancreatic cancer in SCID mice.

Interleukin 12 (IL-12) is a heterodimeric cytokine that exerts a potent antitumor effect through its pleiotropic actions. It was recently reported that IL-12 has also a potent antiangiogenic effect through the induction of IFN-gamma, which triggers the production of chemokines such as IP-10 that has been shown to have antiangiogenesis properties. In this study we transfected the IL-12 gene into a human pancreatic adenocarcinoma cell line (PK-1). PK-1 cells transfected with the green fluorescence protein (gfp) gene were used as positive controls. The in vitro growth curve and in vivo tumor growth of transfectants (IL-12/PK-1 and gfp/PK-1) were compared with those of parental cells. The SCID mice used in this study were administered antiasialo GM-1 Ab (100 microg, i.p., twice weekly) to deplete the remaining immunoeffector cells, NK cells. Using a skinfold chamber model, we observed and recorded tumor angiogenesis by intravital microscopy. In vitro growth of IL-12/PK-1 and gfp/PK-1 cells was not different from that of wild-type PK-1 cells (wt/PK-1). However, IL-12 transfected PK-1 cells did not develop into tumors as did the wt/PK-1 cells after subcutaneous inoculation in antiasialo GM-1 Ab administered SCID mice. The growth of IL-12/PK-1 tumors was restored in mice treated with anti-IL-12 antibody. We found that IL-12/PK-1, in contrast to gfp/PK-1 and wt/PK-1, failed to initiate an angiogenic response, as observed in the skinfold chamber model. These results indicate that the antiangiogenesis effect of IL-12 alone, without immune system involvement, is sufficient to block the growth of human pancreatic cancer.

Regulation and antimetastatic functions of liver-associated natural killer cells

The liver is a complex organ composed of hepatic parenchymal cells and a variety of non-parenchymal cells that consist of endothelial cells, Kupffer cells, and several subsets of resident lymphocytes, including natural killer (NK), T, and NK1.1+/CD3+ (NK/T) cells. The regulation of these various lymphoid subpopulations and their relative contributions to antiviral, antitumor and pathogenic inflammatory responses in the liver remain topics of much interest. Studies from our laboratory have shown that various immune stimulants and cytokines can augment liver-associated NK activity at least partially through the mobilization of NK cells from the bone marrow to the liver. The mobilization process can be dependent on the induction of interferon (IFN)-gamma and/or tumor necrosis factor-alpha and on very late activation antigen-4/vascular cell adhesion molecule-1 interaction. The induction of IFN-gamma by cytokines such as interleukin (IL)-12 also rapidly triggers the induction of chemokine genes in parenchymal cells that may contribute to the localization of NK and T cells. Both IL-2 and IL-12 trigger changes in the number and functions of liver-associated leukocyte subsets, and induce antimetastatic effects that are likely mediated through several direct and indirect mechanisms. The overall goal of these studies is to understand the interactions and functions of liver-associated NK1.1+ cells in the context of innate and adaptive immune responses to neoplasia.

Secretion of CXC chemokine IP-10 by peripheral blood mononuclear cells from healthy donors and breast cancer patients stimulated with HER-2 peptides.

CXC chemokines play an important role in recruitment of T cells to the site of activation and regulation of angiogenesis. CXC chemokines are secreted by T cells stimulated with cytokines or by established cytotoxic T lymphocyte (CTL) lines at recognition of conventional antigen (Ag), but the activation requirements and the relationship of interferon-gamma (IFN-gamma) inducible protein (IP-10) secretion with IFN-gamma induction in lymphocytes are still unclear. We studied the induction of IP-10 from nonadherent peripheral blood mononuclear cells (PBMC) by IFN-gamma, interleukin-12 (IL-12), and the HER-2 peptide E75, which forms a CTL-defined antigen. We found that IFN-gamma alone was a weak inducer of IP-10 in these cells, whereas IL-12 was a significantly stronger inducer of IP-10. In the presence of IL-12, the tumor peptide E75 (HER-2, 369-377) was a stronger inducer of IP-10 than was IL-12 alone. E75 and its variants mutated at position 5 could also induce IP-10 in the absence of exogenous IL-12 or IFN-gamma. IP-10 induction by E75 required HLA-A2 presentation and B7-CD28 interactions and was partially inhibited by blocking of CD40-CD40L interactions. These results indicate that presentation of tumor peptides to peripheral T cells can induce a fast chemokine response, which in its early phase may be higher than the IFN-gamma response. This shows that the IP-10 response was independent of any early-phase IFN-gamma response in peripheral T cells. This may be important for understanding the regulation of the balance between chemoattractant chemokines (CC) and CXC chemokines by tumor Ag and may have implications for understanding the mechanisms of polarization of T cells and conditioning of antigen-presenting cells (APC) by tumor antigens.

Interferon consensus sequence binding protein confers resistance against Yersinia enterocolitica.

Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-gamma) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacterium Yersinia enterocolitica. ICSBP(-/-) mice failed to produce IL-12 and IFN-gamma, but also IL-4, after Yersinia challenge. In addition, granuloma formation was highly disturbed in infected ICSBP(-/-) mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP(-/-) mice rapidly succumbed to acute Yersinia infection. In vitro treatment of spleen cells from ICSBP(-/-) mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-gamma induction. In experimental therapy of infected ICSBP(-/-) mice, we observed that administration of rIL-12 induced IFN-gamma production which was associated with improved resistance to Yersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-gamma production but nevertheless reduced bacterial burden in ICSBP(-/-) mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course of Yersinia infection in ICSBP(-/-) mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense against Yersinia. These results are important for understanding the complex host immune responses in bacterial infections.

Modulation of major histocompatibility class II protein expression by varicella-zoster virus.

We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-gamma)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-gamma treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-gamma. In contrast, cells that were treated with IFN-gamma before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-gamma treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-alpha) RNA expression in response to IFN-gamma stimulation revealed that MHC class II DR-alpha mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1alpha and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-gamma signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-gamma signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-gamma induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4(+) T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation.

IL-18 binding protein increases spontaneous and IL-1-induced prostaglandin production via inhibition of IFN-gamma.

IL-18 shares with IL-1 the same family of receptors and several identical signal transduction pathways. Because of these similarities, IL-18 was investigated for its ability to induce prostaglandin E(2) (PGE(2)) synthesis in human peripheral blood mononuclear cells (PBMC), a prominent, proinflammatory property of IL-1. IL-18 was highly active in PBMC by inducing the synthesis of the chemokine IL-8; however, no induction of PGE(2) synthesis nor cyclooxygenase type-2 gene expression was observed in PBMC stimulated with IL-18. In the same cultures, IL-1beta induced a 12-fold increase in PGE(2). Although IL-1beta-induced IL-8 synthesis was augmented 3-fold by IL-18, IL-18 suppressed IL-1beta-induced PGE(2) production by 40%. The suppressive effect of IL-18 on PGE(2) production was mediated by interferon (IFN)-gamma because anti-human IFN-gamma-antibody prevented IL-18-induced reduction in PGE(2). Consistent with these observations, IL-12, a known inducer of IFN-gamma, augmented IL-1beta-induced IFN-gamma but suppressed IL-1beta-induced PGE(2) by 75%. IL-18 binding protein (IL-18BP) is a naturally occurring and specific inhibitor of IL-18. When recombinant IL-18BP was added to PBMC cultures, unexpectedly, spontaneous PGE(2) production increased. PGE(2) production was also increased by the addition of IL-18BP to PBMC stimulated with either IL-1beta or IL-12 and also in whole blood cultures stimulated with Staphylococcus epidermidis. These studies demonstrate that IL-18BP decreases endogenous IL-18 activity by reducing IFN-gamma-mediated responses.

Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen.

BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.