Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4(+) Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25(+)CD4(+) Tr cells in humans has not been reported. Here we show that human CD25(+)CD4(+) Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor-mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte-associated antigen (CTLA)-4. Human CD25(+)CD4(+) Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-beta, low levels of interferon (IFN)-gamma, and no IL-4 or IL-2. Importantly, CD25(+)CD4(+) Tr cells strongly inhibited the proliferative responses of both naive and memory CD4(+) T cells to alloantigens, but neither IL-10, TGF-beta, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25(+)CD4(+) Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25(+)CD4(+) Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell-mediated diseases.

Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.

Naturally occurring CD4(+)CD25(+) regulatory T cells (Treg) suppress in vitro the proliferation of other T cells in a cell-contact-dependent manner. Dendritic cells (DCs) appear to be a target of Treg-mediated immune suppression. We show here that, in coculture of dye-labeled Treg cells and CD4(+)CD25(-) naïve T cells in the presence of T cell receptor stimulation, Treg cells, which are more mobile than naïve T cells in vitro, out-compete the latter in aggregating around DCs. Deficiency or blockade of leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) abrogates Treg aggregation, whereas that of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152) does not. After forming aggregates, Treg cells specifically down-regulate the expression of CD80/86, but not CD40 or class II MHC, on DCs in both a CTLA-4- and LFA-1-dependent manner. Notably, Treg exerts this CD80/86-down-modulating effect even in the presence of strong DC-maturating stimuli, such as GM-CSF, TNF-alpha, IFN-gamma, type I IFN, and lipopolysaccharide. Taken together, as a possible mechanism of in vitro Treg-mediated cell contact-dependent suppression, we propose that antigen-activated Treg cells exert suppression by two distinct steps: initial LFA-1-dependent formation of Treg aggregates on immature DCs and subsequent LFA-1- and CTLA-4-dependent active down-modulation of CD80/86 expression on DCs. Both steps prevent antigen-reactive naïve T cells from being activated by antigen-presenting DCs, resulting in specific immune suppression and tolerance.

Listeriosis in mice with the severe combined immunodeficiency (SCID) mutation is an established model in vivo and in vitro of interferon gamma (IFN-gamma)-dependent macrophage activation by natural killer (NK) cells during the development of natural immunity. We demonstrate that IFN-gamma production from SCID splenocytes is stimulated by interleukin (IL) 12, tumor necrosis factor alpha (TNF-alpha), and IL-2 but is inhibited by IL-10, IL-10, IL-12, and TNF are induced by heat-killed Listeria monocytogenes (hk-LM) from SCID splenocytes and peritoneal macrophages. IL-12 production is necessary for hk-LM to stimulate IFN-gamma production by SCID splenocytes since neutralization of IL-12 totally blocks IFN-gamma production in this system. TNF-alpha and IL-2 act synergistically with IL-12 to augment IFN-gamma production. Also, exogenous IL-2 increases the response of NK cells to hk-LM or to IL-12 and TNF-alpha. In contrast, IL-10 inhibits hk-LM-induced IFN-gamma production at two levels: (i) by inhibiting TNF and IL-12 production from these cultures (presumably from the macrophage) and (ii) by inhibiting the stimulatory effects of IL-12 and TNF-alpha on NK-cell IFN-gamma production. Thus, these data indicate that macrophage production of TNF-alpha and IL-12 stimulates the release of IFN-gamma by NK cells and that IL-10 produced in response to hk-LM inhibits this response at the level of the macrophage and the NK cell.

Interleukin (IL)-23 is a heterodimeric cytokine that shares the identical p40 subunit as IL-12 but exhibits a unique p19 subunit similar to IL-12 p35. IL-12/23 p40, interferon gamma (IFN-gamma), and IL-17 are critical for host defense against Klebsiella pneumoniae. In vitro, K. pneumoniae-pulsed dendritic cell culture supernatants elicit T cell IL-17 production in a IL-23-dependent manner. However, the importance of IL-23 during in vivo pulmonary challenge is unknown. We show that IL-12/23 p40-deficient mice are exquisitely sensitive to intrapulmonary K. pneumoniae inoculation and that IL-23 p19-/-, IL-17R-/-, and IL-12 p35-/- mice also show increased susceptibility to infection. p40-/- mice fail to generate pulmonary IFN-gamma, IL-17, or IL-17F responses to infection, whereas p35-/- mice show normal IL-17 and IL-17F induction but reduced IFN-gamma. Lung IL-17 and IL-17F production in p19-/- mice was dramatically reduced, and this strain showed substantial mortality from a sublethal dose of bacteria (10(3) CFU), despite normal IFN-gamma induction. Administration of IL-17 restored bacterial control in p19-/- mice and to a lesser degree in p40-/- mice, suggesting an additional host defense requirement for IFN-gamma in this strain. Together, these data demonstrate independent requirements for IL-12 and IL-23 in pulmonary host defense against K. pneumoniae, the former of which is required for IFN-gamma expression and the latter of which is required for IL-17 production.

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

In vitro and in vivo studies were performed to assess the involvement of IL-12 in resistance to acute and chronic infection with an avirulent strain of Toxoplasma gondii. Our previous findings implicated macrophages as a major source of parasite-induced IL-12. This finding was confirmed by showing that peritoneal macrophages exposed to either live parasites or soluble tachyzoite Ags produce IL-12 protein. In mice, increased expression of IL-12 (p40) mRNA in both spleen and peritoneal cells was detected as early as 2 days postinfection. Treatment with neutralizing mAbs against IL-12 increased the susceptibility of C57BL/6, BALB/c, and severe combined immunodeficient (SCID) mice to acute infection, which resulted in 100% mortality within the first 15 days after parasite inoculation. In contrast, neutralization of endogenously produced IL-12 had no effect when given during chronic infection. In agreement with the survival data, treatment with anti-IL-12 resulted in decreased IFN-gamma and enhanced Th2 (IL-4 and IL-10) cytokine synthesis by splenocytes when given during acute, but not chronic, toxoplasmosis. Sorting experiments on spleen cells from acutely infected mice indicated that both CD4+ lymphocytes and NK1.1+/CD3- cells contribute to the early IFN-gamma response. In contrast, CD4+ cells were found to be the major source of the cytokine during chronic disease. Together, these results suggest that the stimulation of macrophage-derived IL-12 plays a major role in both the induction of resistance and Th1 cell subset selection in acute T. gondii infection, but may not be required to maintain established immunity.

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.

NY-ESO-1 is a "cancer-testis" antigen frequently expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8(+) T cells derived from peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), and tumor-associated lymphocytes (TALs) of EOC patients with NY-ESO-1-expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1-specific CD8(+) T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBLs following in vitro stimulation. Compared with PBLs, tumor-derived NY-ESO-1-specific CD8(+) T cells demonstrated impaired effector function, preferential usage of dominant T-cell receptor, and enriched coexpression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8(+) T cells was up-regulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived antigen-presenting cells. Functionally, CD8(+)LAG-3(+)PD-1(+) T cells were more impaired in IFN-gamma/TNF-alpha production compared with LAG-3(+)PD-1(-) or LAG-3(-)PD-1(-) subsets. Dual blockade of LAG-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8(+) T cells, indicating that antitumor function of NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Natural killer (NK) and NK T cells are tissue lymphocytes that secrete cytokines rapidly upon stimulation. Here, we show that these cells maintain distinct patterns of constitutive cytokine mRNAs. Unlike conventional T cells, NK T cells activate interleukin (IL)-4 and interferon (IFN)-gamma transcription during thymic development and populate the periphery with both cytokine loci previously modified by histone acetylation. Similarly, NK cells transcribe and modify the IFN-gamma gene, but not IL-4, during developmental maturation in the bone marrow. Lineage-specific patterns of cytokine transcripts predate infection and suggest evolutionary selection for invariant but distinct types of effector responses among the earliest responding lymphocytes.

BACKGROUND

Although lymphocyte recruitment and activation are associated with cerebral ischemia-reperfusion (I/R) injury, the contributions of specific lymphocyte subpopulations and lymphocyte-derived interferon-gamma (IFN-gamma) to stroke remain unknown. The objectives of this study were to define the contribution of specific populations of lymphocytes to the inflammatory and prothrombogenic responses elicited in the cerebral microvasculature by I/R and to investigate the role of T-cell-associated IFN-gamma in the pathogenesis of ischemic stroke.

RESULTS

Middle cerebral artery occlusion was induced for 1 hour (followed by 4 or 24 hours of reperfusion) in wild-type mice and mice deficient in lymphocytes (Rag1(-/-)), CD4+ T cells, CD8+ T cells, B cells, or IFN-gamma. Platelet and leukocyte adhesion was assessed in cortical venules with intravital video microscopy. Neurological deficit and infarct volume were determined 24 hours after reperfusion. Rag1(-/-), CD4+ T-cell(-/-), CD8+ T-cell(-/-), and IFN-gamma(-/-) mice exhibited comparable significant reductions in I/R-induced leukocyte and platelet adhesion compared with wild-type mice exposed to I/R. Infarct volume was reduced and I/R-induced neurological deficit was improved in immunodeficient Rag1(-/-) mice. These protective responses were reversed in Rag1(-/-) mice reconstituted with either wild-type or, to a lesser extent, IFN-gamma(-/-) splenocytes. B-cell-deficient mice failed to show improvement against ischemic stroke injury.

CONCLUSIONS

These findings indicate that CD4+ and CD8+ T lymphocytes, but not B lymphocytes, contribute to the inflammatory and thrombogenic responses, brain injury, and neurological deficit associated with experimental stroke. Although IFN-gamma plays a pivotal role in stroke-induced inflammatory responses, T lymphocytes appear to be a minor source of this cytokine.

Cancer patients can harbor significant numbers of CD8 and CD4 T cells with specificities to tumor antigens (Ags). Yet, in most cases, such T cells fail to eradicate the tumor in vivo. Here, we investigated the interference of Ag-specific CD4(+)CD25(+) regulatory T cells (Treg) with the tumor-specific CD8 T cell immune response in vivo, by monitoring the homing, expansion, and effector function of both subsets in draining and nondraining lymph nodes. The results show that CD8 cells expand to the same extent and produce similar levels of IFN-gamma in the presence or absence of Ag-specific Treg. Nevertheless, these Treg abrogate CD8 T cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8 cells. The molecular mechanism of suppression involves TGF-beta because expression of a dominant-negative TGF-beta receptor by tumor-specific CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

IL-10 gene transcription and IL-10 protein production was assessed in both type 1 (Th1) and type 2 (Th2) CD4+ human T cell clones by polymerase chain reaction and ELISA, respectively. Although Th2 clones apparently showed higher IL-10 mRNA levels, IL-10 mRNA expression was consistently found in Th1 clones, as well. Likewise, measurable IL-10 levels were found in the supernatants of both Th1 and Th2 clones. The effect of human IL-10 (h-IL-10) and viral IL-10 (v-IL-10) on the proliferative response and cytokine production by Th1 and Th2 human clones was also investigated. Addition in culture of h-IL-10 and v-IL-10 significantly reduced the proliferation of both Th1 and Th2 clones in response to the specific Ag and to PHA, but it had no inhibitory effect on the proliferative response of Th1 and Th2 clones to IL-2. h-IL-10 and v-IL-10 also inhibited the Ag-induced production of gamma-interferon (IFN-gamma) by Th1 clones and the production of IL-4 and IL-5 by Th2 clones, whereas they had no effect on the cytokine synthesis by the same clones stimulated with PMA plus anti-CD3 antibody. Preincubation of APC, but not of clonal T blasts, with h-IL-10 resulted in the inhibition of Ag-induced proliferation of both Th1 and Th2 clones, supporting the view that h-IL-10 primarily affects APC. These data demonstrate that, unlike the murine system where IL-10 is a product of Th2 (but not Th1) cells and seems to mainly down-regulate the Th1 response, in the human system, IL-10 is produced by, and down-regulates the function of, both Th1 and Th2 cells.

The intracellular protozoan Toxoplasma gondii is a widespread opportunistic parasite of humans and animals. Normally, T. gondii establishes itself within brain and skeletal muscle tissues, persisting for the life of the host. Initiating and sustaining strong T-cell-mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. The parasite induces high levels of gamma interferon (IFN-gamma) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFN-gamma synthesis. The latter cytokine, in addition to promoting the differentiation of Th1 effectors, is important in macrophage activation and acquisition of microbicidal functions, such as nitric oxide release. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-gamma, which is required to prevent cyst reactivation. T-cell-mediated cytolytic activity against infected cells, while easily demonstrable, plays a secondary role to inflammatory cytokine production. While part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression.

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.

The mammalian immune response to infection is mediated by 2 broad arms, the innate and adaptive immune systems. Innate immune cells are a first-line defense against pathogens and are thought to respond consistently to infection, regardless of previous exposure, i.e., they do not exhibit memory of prior activation. By contrast, adaptive immune cells display immunologic memory that has 2 basic characteristics, antigen specificity and an amplified response upon subsequent exposure. Whereas adaptive immune cells have rearranged receptor genes to recognize the universe of antigens, natural killer (NK) cells are innate immune lymphocytes with a limited repertoire of germ-line encoded receptors for target recognition. NK cells also produce cytokines such as IFN-gamma (IFN-gamma) to protect the host during the innate response to infection. Herein, we show that cytokine-activated NK cells transferred into naïve hosts can be specifically detected 7-22 days later when they are phenotypically similar to naïve cells and are not constitutively producing IFN-gamma. However, they produce significantly more IFN-gamma when restimulated. This memory-like property is intrinsic to the NK cell. By contrast, memory-like NK cells do not express granzyme B protein and kill targets similarly to naïve NK cells. Thus, these experiments identify an ability of innate immune cells to retain an intrinsic memory of prior activation, a function until now attributed only to antigen-specific adaptive immune cells.

Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Mice with a null mutation of ICSBP exhibit two prominent phenotypes related to previously described activities of the IRF family. The first is enhanced susceptibility to virus infections associated with impaired production of IFN(gamma). The second is deregulated hematopoiesis in both ICSBP-/- and ICSBP+/- mice that manifests as a syndrome similar to human chronic myelogenous leukemia. The chronic period of the disease progresses to a fatal blast crisis characterized by a clonal expansion of undifferentiated cells. Normal mice injected with cells from mice in blast crisis developed acute leukemia within 6 weeks of transfer. These results suggest a novel role for ICSBP in regulating the proliferation and differentiation of hematopoietic progenitor cells.

BALB/c mice vaccinated with a temperature-sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to lethal challenge with a highly virulent toxoplasma strain (RH). This immunity is known to be dependent on IFN-gamma synthesis. In vitro and in vivo T cell depletions were performed in order to identify the subsets responsible for both protective immunity and IFN-gamma production. When stimulated with crude tachyzoite Ag in vitro, CD4+ cells from vaccinated mice produced high levels of TH1 cytokines (IL-2 and IFN-gamma) but not TH2 cytokines (IL-4 and IL-5). CD8+ cells, in contrast, produced less IFN-gamma and no detectable IL-2. Nevertheless, they could be induced to synthesize IFN-gamma when exposed in culture to exogenous IL-2. In vivo treatment with anti-CD4 plus anti-CD8 or anti-IFN-gamma antibodies during challenge infection completely abrogated resistance to T. gondii. In contrast, treatment with anti-CD4 alone failed to reduce immunity, whereas anti-CD8 treatment partially decreased vaccine-induced resistance. These results suggest that although IFN-gamma and IL-2-producing CD4+ lymphocytes are induced by vaccination, IFN-gamma-producing CD8+ T cells are the major effectors of immunity in vivo. Nevertheless, CD4+ lymphocytes appear to play a synergistic role in vaccine-induced immunity, probably through the augmentation of IFN-gamma synthesis by the CD8+ effector cells. This hypothesis is supported by the observation that when giving during vaccination, as opposed to after challenge, anti-CD4 antibodies are capable of blocking protective immunity.

The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

Interferons (IFNs) induce antiviral activity in many cell types. The ability of IFN-gamma to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells' production of nitric oxide (NO). Viral replication was restored in IFN-gamma-treated macrophages exposed to inhibitors of NO synthase. Conversely, epithelial cells with no detectable NO synthesis restricted viral replication when transfected with a complementary DNA encoding inducible NO synthase or treated with organic compounds that generate NO. In mice, an inhibitor of NO synthase converted resolving ectromelia virus infection into fulminant mousepox. Thus, induction of NO synthase can be necessary and sufficient for a substantial antiviral effect of IFN-gamma.

Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1(neg)) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-gamma and IL-4. NK1.1(neg) iNKT cells produce IL-17 upon synthetic (alpha-galactosylceramide [alpha-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1(neg) iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by alpha-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell-deficient Jalpha18(-/-) mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before alpha-GalCer administration. Collectively, our findings reveal that NK1.1(neg) iNKT lymphocytes represent a new population of IL-17-producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion.

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-gamma (IFN-gamma), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40(-/-) mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40(-/-) mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-gamma. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40(-/-) mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-gamma and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.

Idiopathic pulmonary fibrosis (IPF) is a destructive inflammatory disease with limited therapeutic options. To better understand the inflammatory responses that precede and concur with collagen deposition, we used three models of pulmonary fibrosis and identify a critical mechanistic role for IL-17A. After exposure to bleomycin (BLM), but not Schistosoma mansoni eggs, IL-17A produced by CD4(+) and gammadelta(+) T cells induced significant neutrophilia and pulmonary fibrosis. Studies conducted with C57BL/6 il17a(-/-) mice confirmed an essential role for IL-17A. Mechanistically, using ifngamma(-/-), il10(-/-), il10(-/-)il12p40(-/-), and il10(-/-)il17a(-/-) mice and TGF-beta blockade, we demonstrate that IL-17A-driven fibrosis is suppressed by IL-10 and facilitated by IFN-gamma and IL-12/23p40. BLM-induced IL-17A production was also TGF-beta dependent, and recombinant IL-17A-mediated fibrosis required TGF-beta, suggesting cooperative roles for IL-17A and TGF-beta in the development of fibrosis. Finally, we show that fibrosis induced by IL-1beta, which mimics BLM-induced fibrosis, is also highly dependent on IL-17A. IL-17A and IL-1beta were also increased in the bronchoalveolar lavage fluid of patients with IPF. Together, these studies identify a critical role for IL-17A in fibrosis, illustrating the potential utility of targeting IL-17A in the treatment of drug and inflammation-induced fibrosis.