Poxviruses encode a large number of proteins that attenuate the inflammatory and immune responses to infection. In this report we demonstrate that a number of orthopoxviruses express a type I interferon (IFN)-binding protein, which is encoded by the B18R open reading frame in the WR strain of vaccinia virus. The B18R protein has significant regions of homology with the alpha subunits of the mouse, human, and bovine type I IFN receptors, bound human IFN alpha 2 with high affinity, and inhibited transmembrane signaling as demonstrated by inhibition of Fc receptor factor gamma 1/gamma 2 and interferon-stimulated gene factor-3 formation as well as inhibition of the IFN alpha antiviral response. Among viral host response modifiers, the B18R protein is unique inasmuch as it exists as a soluble extracellular as well as a cell surface protein and thus should effectively block both autocrine and paracrine functions of IFN.

The host immune response to Helicobacter pylori infection might be of importance with regard to the outcome of infection by this organism, e.g., to explain why only a proportion of infected subjects develop peptic ulcers. In this study we have analyzed the local response of different cytokines-i.e., the proinflammatory interleukin-1beta, (IL-1beta), IL-6, tumor necrosis factor alpha, and IL-8; the immunoregulatory gamma interferon (IFN-gamma); and IL-4; and the anti-inflammatory transforming growth factor beta (TGF-beta)-in antral biopsy specimens from H. pylori-infected duodenal ulcer (DU) patients and asymptomatic (AS) carriers (i.e., with chronic gastritis only). For comparison, biopsy specimens from uninfected healthy individuals were also analyzed. An immunohistochemical technique was used to allow quantification of the cytokine responses as well as identification of the cell types associated with the cytokine expression. We found that the levels of all of the studied cytokines except IL-4 were increased in the H. pylori-infected subjects compared to the levels in the healthy individuals. Our results indicate that the antral cytokine response is of the Th1 type since IFN-gamma, but not IL-4, was up-regulated both in H. pylori-infected DU patients and in AS carriers. However, there were no significant differences in either proinflammatory or immunoregulatory cytokine levels when H. pylori-infected subjects with and without peptic ulcers were compared. Some of the cytokines, particularly IL-1beta and TGF-beta, were also found in the gastric mucosae of healthy, uninfected subjects. We also showed that the gastric epithelium contributes substantially to the antral cytokine response of the proinflammatory cytokines IL-1beta and IL-6 in addition to IL-8.

The farnesoid X receptor (FXR) is a bile acid-regulated nuclear receptor expressed in enterohepatic tissues. In this study we investigated whether FXR is expressed by cells of innate immunity and regulates inflammation in animal models of colitis. Acute (7 days) and chronic (8 wk) colitis were induced in wild-type and FXR(-/-) mice by intrarectal administration of trinitrobenzensulfonic acid or by 7-day administration of 5% dextran sulfate in drinking water. The results of this experiment demonstrate that FXR is expressed by and exerts counterregulatory effects on cells of innate immunity. Exposure of LPS-activated macrophages to 6-ethyl chenodeoxycholic acid (6E-CDCA; INT-747) a synthetic FXR ligand, results in a reciprocal regulation of NF-kappaB dependent-genes (TNF-alpha, IL-1beta, IL-6, COX-1, COX-2, and iNOS) and induction of SHP, a FXR-regulated gene. FXR activation stabilizes the nuclear corepressor NCoR on the NF-kappaB responsive element on the IL-1beta promoter. Colon inflammation in Crohn's disease patients and in rodent models of colitis is associated with a reduced expression of FXR mRNA. Using two rodent models of colon inflammation, we show that progression of these immune-mediated disorders is exacerbated in FXR(-/-) mice (p < 0.01). In vivo treatment with INT-747 attenuates organ injury and immune cell activation. FXR activation increased the colon expression of I-BABP, FXR, and SHP while reducing IL-1beta, IL-2, IL-6, TNF-alpha, and IFN-gamma mRNA expression and attenuating disease severity. In aggregate, these findings provide evidence that FXR is an essential component of a network of nuclear receptors that regulate intestinal innate immunity and homeostasis.

Natural killer-22 (NK-22) cells are a human NK cell subset situated in mucosal-associated lymphoid tissues that specialize in IL-22 secretion in response to IL-23. Here we investigated the cytokine requirements for NK-22 cell expansion. IL-7 maintained the survival of NK-22 cells and IL-22 production in response to IL-23 but was insufficient to induce robust expansion. Proliferation of NK-22 cells was increased markedly by adding either IL-1beta or IL-2 to IL-7 and was even stronger in the presence of IL-1beta plus IL-2. In contrast to IL-7, continuous culture in IL-1beta and IL-2 modified NK-22 cytokine profiles. IL-1beta promoted constitutive IL-22 secretion rather than acute IL-22 production in response to IL-23 and induced IL-17 in some cells. IL-2 reduced secretion of IL-22 and IL-17, increasing production of IFN-gamma and leukemia inhibitory factor. Functional deviation toward IFN-gamma production also was induced by continuous culture in IL-23. These results demonstrate the functional plasticity of NK-22 cells, which may allow flexible responses to different pathogens. Finally, we found that NK-22 cells released the B-cell survival factor, B-cell activating factor belonging to the TNF family (BAFF), suggesting a potential role of NK-22 cells in promoting B-cell-mediated mucosal immunity.

Schistosomiasis continues to be a significant cause of parasitic morbidity and mortality worldwide. This review considers the basic features of the pathology and clinical outcomes of hepatointestinal and genitourinary schistosomiasis, presents an overview of the numerous studies on animal models that have clarified many of the immunopathological features, and provides insight into our current understanding of the immunopathogenesis and genetic control of human schistosomiasis. In murine schistosomiasis, pathology is induced by a CD4(+) Th2 driven granulomatous response directed against schistosome eggs lodged in the host liver. The Th2 cytokines IL-4 and IL-13 drive this response, whereas IL-10, IL13Ralpha2, IFN-gamma and a subset of regulatory T-cells act to limit schistosome induced pathology. A variety of cell types including hepatic stellate cells, alternatively activated macrophages and regulatory T-cells have also been implicated in the pathogenesis of schistosomiasis. Current knowledge suggests the immunopathogenic mechanisms underlying human schistosomiasis are likely to be similar. The review also considers the future development of anti-pathology schistosome vaccines. As fibrosis is an important feature of many other diseases such as Crohn's disease and sarcoidosis, a comprehensive understanding of the cellular and molecular mechanisms involved in schistosomiasis may also ultimately contribute to the development an effective disease intervention strategy for other granulofibrotic diseases.

We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-gamma (MIG/CXCL9), IFN-gamma-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), members of the IFN-gamma-inducible tripeptide motif Glu-Leu-Arg (ELR)(-) CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-gamma-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-gamma-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-gamma-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.

CD4 help for CD8(+) T lymphocytes prevents tolerance and promotes the survival of effector and memory CD8(+) T cells. Here, we describe additional helper functions that require CD4(+) T cells within the tumor environment. CD8(+) T-cell recruitment, proliferation, and effector function within the tumor were greatly enhanced by tumor-specific CD4(+) T cells. Recruitment of CD8(+) T cells was accelerated by IFN-γ-dependent production of chemokines. Production of interleukin-2 by tumor resident CD4(+) T cells enhanced CD8(+) T-cell proliferation and upregulated expression of granzyme B. These results highlight a novel role for tumor-specific CD4(+) T cells in promoting CD8(+) T-cell recruitment and cytolytic function, two previously unappreciated aspects of tumor-specific CD4 help.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-gamma-secreting CD4(+) and CD8(+) T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8(+) T cells in infected tissues of challenged mice. In contrast, specific IFN-gamma production by CD4(+) T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-gamma secretion by CD4(+) T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.

Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-gammaR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (gamma(c)) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15.

Macrophage deactivating factor (MDF) and three members of the transforming growth factor-beta family (TGF-beta 1, -beta 2, and -beta 3) blocked the ability of IFN-gamma to induce release of reactive nitrogen intermediates from mouse peritoneal macrophages. Raising the concentration of IFN-gamma did not diminish the potency of the inhibitors (50% inhibition by approximately 7 nM MDF, 2 pM TGF-beta 1, 4 pM TGF-beta 2, and 8 pM TGF-beta 3). These inhibitors partially blocked induction of nitrite release in macrophages activated with the combination of IFN-gamma plus TNF-alpha, but were incapable of inhibiting when macrophages were activated by the combination of IFN-gamma plus LPS. MDF and TGF-beta 1, -beta 2, and -beta 3 inhibited IFN-gamma-induced nitrite release only if present during the induction phase; once IFN-gamma-nitrite release had commenced, addition of the same cytokines was no longer inhibitory. Maximum inhibition of synthesis over a 48-h period required that the inhibitors be present during the first 3 h of induction. Thus, cytokines can suppress as well as induce macrophage synthesis of reactive nitrogen intermediates, products with cytotoxic, antimicrobial, and vasodilatory properties.

Macrophages can become activated to kill both tumor cells and a variety of microbes. Results here show that synthesis of nitric oxide (NO), a mediator of many macrophage cytotoxic functions, was greatly increased when cells of the mouse macrophage cell line RAW 264.7 were costimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), compared to LPS alone. This increase paralleled increases in cytotoxicity. Northern analysis showed that increased production of NO was preceded by markedly enhanced expression of mRNA for the inducible form of macrophage NO synthase (mac-NOS), which catalyzes the synthesis of NO. Cycloheximide inhibited the induction of mac-NOS mRNA by IFN-gamma and LPS, indicating that expression of this mRNA required de novo protein synthesis. Elevated expression of mac-NOS mRNA was not due to an increase in its stability. Rather, the combination of IFN-gamma and LPS induced a much higher rate of transcription of the mac-NOS gene than did stimulation with LPS alone. These results provide one explanation of why priming and triggering stimuli, such as IFN-gamma and LPS, respectively, synergistically activate macrophages and may be applicable to explaining how IFN-gamma augments NO-dependent microbicidal activity in macrophages as well.

Murine gamma-herpesvirus 68 (MHV-68) is a natural pathogen of small rodents and insectivores (mice, voles and shrews). The primary infection is characterized by virus replication in lung epithelial cells and the establishment of a latent infection in B lymphocytes. The virus is also observed to persist in lung epithelial cells, dendritic cells and macrophages. Splenomegaly is observed two weeks after infection, in which there is a CD4+ T-cell-mediated expansion of B and T cells in the spleen. At three weeks post-infection an infectious mononucleosis-like syndrome is observed involving a major expansion of Vbeta4+CD8+ T cells. Later in the course of persistent infection, ca. 10% of mice develop lymphoproliferative disease characterized as lymphomas of B-cell origin. The genome from MHV-68 strain g2.4 has been sequenced and contains ca. 73 genes, the majority of which are collinear and homologous to other gamma-herpesviruses. The genome includes cellular homologues for a complement-regulatory protein, Bcl-2, cyclin D and interleukin-8 receptor and a set of novel genes M1 to M4. The function of these genes in the context of latent infections, evasion of immune responses and virus-mediated pathologies is discussed. Both innate and adaptive immune responses play an active role in limiting virus infection. The absence of type I interferon (IFN) results in a lethal MHV-68 infection, emphasizing the central role of these cytokines at the initial stages of infection. In contrast, type II IFN is not essential for the recovery from infection in the lung, but a failure of type II IFN receptor signalling results in the atrophy of lymphoid tissue associated with virus persistence. Splenic atrophy appears to be the result of immunopathology, since in the absence of CD8+ T cells no pathology occurs. CD8+ T cells play a major role in recovery from the primary infection, and also in regulating latently infected cells expressing the M2 gene product. CD4+ T cells have a key role in surveillance against virus recurrences in the lung, in part mediated through 'help' in the genesis of neutralizing antibodies. In the absence of CD4+ T cells, virus-specific CD8+ T cells are able to control the primary infection in the respiratory tract, yet surprisingly the memory CD8+ T cells generated are unable to inhibit virus recurrences in the lung. This could be explained in part by the observations that this virus can downregulate major histocompatibility complex class I expression and also restrict inflammatory cell responses by producing a chemokine-binding protein (M3 gene product). MHV-68 provides an excellent model to explore methods for controlling gamma-herpesvirus infection through vaccination and chemotherapy. Vaccination with gp150 (a homologue of gp350 of Epstein-Barr virus) results in a reduction in splenomegaly and virus latency but does not block replication in the lung, nor the establishment of a latent infection. Even when lung virus infection is greatly reduced following the action of CD8+ T cells, induced via a prime-boost vaccination strategy, a latent infection is established. Potent antiviral compounds such as the nucleoside analogue 2'deoxy-5-ethyl-beta-4'-thiouridine, which disrupts virus replication in vivo, cannot inhibit the establishment of a latent infection. Clearly, devising strategies to interrupt the establishment of latent virus infections may well prove impossible with existing methods.

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.

Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-gamma, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.

The importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis was studied in mice chronically infected with Toxoplasma gondii by using a mAb to this lymphokine. Control mice chronically infected with the ME49 strain that received saline or normal IgG had slight inflammation in their brains whereas those that received the mAb developed severe encephalitis. In contrast to control mice, the mAb-treated mice had many areas of acute focal inflammation and infiltration of large numbers of inflammatory cells in the meninges and parenchyma of their brains. In the areas of acute focal inflammation, tachyzoites and Toxoplasma Ag were demonstrated by immunoperoxidase staining with the use of rabbit anti-Toxoplasma antibody, suggesting that the focal inflammation was induced by Toxoplasma organisms. Acute inflammation was also observed around cysts of Toxoplasma. Immunohistologic staining revealed tachyzoites and Toxoplasma Ag surrounding the periphery of these cysts suggesting cyst disruption had occurred. Mice treated with mAb against IFN-gamma had five times the numbers of cysts in their brains as did control mice. These results clearly indicate that endogenous IFN-gamma plays a significant and important role in prevention of encephalitis in mice chronically infected with Toxoplasma. The mAb-treated mice had the same Toxoplasma antibody titers and the same degree of macrophage killing of Toxoplasma as did untreated controls. These results suggest that IFN-gamma may have a direct role in preventing cyst rupture and toxoplasmic encephalitis.

OBJECTIVE

Sepsis is a major cause of death for intensive care patients. High concentrations of inflammatory cytokines are characteristic of severe systemic inflammation and activated monocytes are their predominant cellular source. To identify targets for antiinflammatory intervention, we investigated the response of human macrophages to inflammatory and antiinflammatory mediators.

RESULTS

We profiled gene expression in human macrophages exposed to lipopolysaccharide (LPS) and interferon (IFN)-gamma in the presence or absence of recombinant activated protein C (APC) or IL-10 and identified Wnt5A as one of the transcripts most highly induced by LPS/IFN-gamma and suppressed by APC and IL-10. We confirmed regulation of Wnt5A protein in macrophages and detected it in sera and bone marrow macrophages of patients with severe sepsis. We established that a functional Wnt5A/frizzled-5/CaMKII signaling pathway was essential for macrophage inflammatory activation. To prove the essential contribution of Wnt5A we measured inflammatory cytokines after stimulation with Wnt5A, silenced Wnt5A by siRNA, and blocked receptor binding with soluble Frizzled-related peptide-1 (sFRP1).

CONCLUSIONS

Wnt5A is critically involved in inflammatory macrophage signaling in sepsis and is a target for antiinflammatory mediators like APC or antagonists like sFRP1.

BACKGROUND

Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes.

METHODS

We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA-NP+M1) and conducted a phase I clinical trial in healthy adults.

RESULTS

The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8(+).

CONCLUSIONS

We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

The tryptophan (trp)-catabolizing enzyme indolamine 2,3-dioxygenase (IDO) is induced by the T helper 1 (Th 1) cytokine IFN-gamma during infections in various tissues including the brain. Recent studies demonstrated an immune modulatory function of this enzyme, since IDO-mediated depletion of trp hinders T cell proliferation, while its inhibition by 1-methyl-tryptophan (1-Mt) induces breakdown of immune tolerance in the placenta, leading to rejection of allogeneic concepti. Here, we tested IDO expression and function during experimental autoimmune encephalomyelitis (EAE) actively induced in adult SJL mice by immunization with PLP139-151. IDO activity (determined by HPLC analysis of the kynurenine/tryptophan ratio) was increased in the spleen during the preclinical phase, and within the brain and spinal cord at the onset of symptoms. Immunocytochemistry revealed macrophages/activated microglia expressing IDO during EAE and in vitro experiments confirmed IDO induction in microglia upon IFN-gamma treatment with synergistic effects of TNF-alpha. Inhibition of IDO by systemic administration of 1-Mt at clinical onset significantly exacerbated disease scores. From these data, it is tempting to speculate that IFN-gamma from encephalitogenic Th 1 cells induces local IDO expression, thereby initiating a negative feedback loop which may underlie the self-limitation of autoimmune inflammation during EAE and multiple sclerosis.

C57BL/6 mice infected with the ME-49 strain of Toxoplasma gondii develop a progressive encephalitis culminating in 100% mortality between 12 and 15 wk after intraperitoneal inoculation of the parasite. Moreover, when injected at 4 wk after infection with anti-IFN-gamma mAb, progression of toxoplasmic encephalitis is markedly accelerated, resulting in death of the animals by 9 to 12 days posttreatment. In this study, we investigated the expression of mRNAs encoding cytokines as well as lymphocyte and macrophage markers during the development of toxoplasmic encephalitis. High levels of lymphocyte CD4 and CD8 surface Ag transcript were detected in the brains of mice throughout the infection. In addition from 2 to 4 wk we found elevations of Th1 (IFN-gamma and IL-2) but not of Th2 (IL-4 and IL-5) cytokine mRNAs. The elevation in Th1 cytokines was accompanied by increases in the expression of monokine (IL-1, IL-6, IL-10, granulocyte macrophage-colony stimulating factor [GM-CSF], and TNF-alpha) mRNAs, as well as markers expressed by activated macrophages (major histocompatibility class II [Ia], inducible nitric oxide synthase [iNOS] and macrophage activation gene 1 [Mag-1]). Interestingly, after 8 wk of infection with T. gondii we observed a dramatic decrease of Th1 cytokine and most monokine (IL-1, IL-6, GM-CSF, and TNF-alpha) as well as Mag-1 and iNOS mRNA levels. This down-regulation was associated with enhanced necrosis and neutrophilic infiltrates in the brain accompanied by increased expression of genes expressed specifically by the tachyzoite stage of T. gondii (T. gondii surface antigen 1 [SAG-1] and T. gondii surface antigen 2 [SAG-2]). Similarly, in mice chronically infected with T. gondii and treated with anti-IFN-gamma mAb the resulting pathology was associated with decreased expression of TNF-alpha and iNOS and increased expression of SAG-1 and SAG-2. Moreover, treatment with anti-TNF-alpha mAb also resulted in enhanced pathology, which correlated with low levels of iNOS mRNA and high levels of tachyzoite-specific mRNAs. Together these results suggest that reactivation of T. gondii results from a down-regulation of IFN-gamma and TNF-alpha expression leading to decreased macrophage or microglial cell activation, release of parasite growth, and subsequent tissue damage.

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)

The tick-borne encephalitis (TBE) complex of viruses, genus Flavivirus, can cause severe encephalitis, meningitis, and/or hemorrhagic fevers. Effective interferon (IFN) responses are critical to recovery from infection with flaviviruses, and the mosquito-borne flaviviruses can inhibit this response. However, little is known about interactions between IFN signaling and TBE viruses. Langat virus (LGTV), a member of the TBE complex of viruses, was found to be highly sensitive to the antiviral effects of IFN. However, LGTV infection inhibited IFN-induced expression of a reporter gene driven by either IFN-alpha/beta- or IFN-gamma-responsive promoters. This indicated that LGTV can inhibit the IFN-mediated JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway of signal transduction. The mechanism of inhibition was due to blocks in the phosphorylation of both Janus kinases, Jak1 and Tyk2, during IFN-alpha signaling and at least a failure of Jak1 phosphorylation following IFN-gamma stimulation. To determine the viral protein(s) responsible, we individually expressed all nonstructural (NS) proteins and examined their ability to inhibit signal transduction. Expression of NS5 alone inhibited STAT1 phosphorylation in response to IFN, thus identifying NS5 as a potential IFN antagonist. Examination of interactions between NS5 and cellular proteins revealed that NS5 associated with IFN-alpha/beta and -gamma receptor complexes. Importantly, inhibition of JAK-STAT signaling and NS5-IFN receptor interactions were demonstrated in LGTV-infected human monocyte-derived dendritic cells, important target cells for early virus replication. Because NS5 may interfere with both innate and acquired immune responses to virus infection, this protein may have a significant role in viral pathogenesis.

The role of nocturnal sleep for normal immune regulation and its relation to circadian rhythm was examined in 10 men participating in two 51-h sessions. One session included two regular wake-sleep cycles; the other included a night of sustained wakefulness followed by a night of recovery sleep. Blood was collected every 3 h to determine PBMC counts, including the enumeration of monocytes, NK cells, and lymphocyte subsets (CD19+, CD3+, CD4+, CD8+, HLA-DR+). Production of IL-1beta, TNF-alpha, IL-2, and IFN-gamma was determined after stimulation of whole blood samples with LPS and PHA, respectively. Concentrations of IL-6 and cortisol were assessed in plasma. Enumeration of cells indicated significant circadian rhythms for all PBMC subsets under conditions of sustained wakefulness. Compared with sustained wakefulness, nocturnal sleep acutely reduced the numbers of monocytes, NK cells, and counts of all lymphocyte subsets. However, in the afternoon and evening of the day following sleep, counts of NK cells and lymphocytes were significantly higher than after nocturnal wakefulness, indicating that effects of sleep interacted with those of the circadian pacemaker. Sleep markedly enhanced production of IL-2 by T cells (CD3+) but did not influence production of IL-1beta and TNF-alpha, or IL-6 concentrations. Effects of sleep were not mediated by changes in cortisol. The decrease in monocytes, NK cells, and lymphocytes, together with an increased production of IL-2 during sleep, may serve to support ongoing immune defense in extravascular lymphoid tissue during a time of diminished acute Ag challenge.

Professional antigen-presenting dendritic cells (DCs) are critical in regulating T cell immune responses at both systemic and mucosal sites. Many Lactobacillus species are normal members of the human gut microflora and most are regarded as safe when administered as probiotics. Because DCs can naturally or therapeutically encounter lactobacilli, we investigated the effects of several well defined strains, representing three species of Lactobacillus on human myeloid DCs (MDCs) and found that they modulated the phenotype and functions of human MDCs. Lactobacillus-exposed MDCs up-regulated HLA-DR, CD83, CD40, CD80, and CD86 and secreted high levels of IL-12 and IL-18, but not IL-10. IL-12 was sustained in MDCs exposed to all three Lactobacillus species in the presence of LPS from Escherichia coli, whereas LPS-induced IL-10 was greatly inhibited. MDCs activated with lactobacilli clearly skewed CD4(+) and CD8(+) T cells to T helper 1 and Tc1 polarization, as evidenced by secretion of IFN-gamma, but not IL-4 or IL-13. These results emphasize a potentially important role for lactobacilli in modulating immunological functions of DCs and suggest that certain strains could be particularly advantageous as vaccine adjuvants, by promoting DCs to regulate T cell responses toward T helper 1 and Tc1 pathways.