IL-18 has IL-12-independent effects in delayed-type hypersensitivity: studies in cell-mediated crescentic glomerulonephritis

IL-18 (formerly known as IFN-gamma-inducing factor) enhances Th1 responses via effects that are thought to be dependent on and synergistic with IL-12. The potential for IL-18 to exert IL-12-independent effects in delayed-type hypersensitivity (DTH) responses was studied in a model of Th1-directed, DTH-mediated crescentic glomerulonephritis induced by planting an Ag in glomeruli of sensitized mice as well as in cutaneous DTH. Sensitized genetically normal (IL-12(+/+)) mice developed proteinuria and crescentic glomerulonephritis with a glomerular influx of DTH effectors (CD4(+) T cells, macrophages, and fibrin deposition) in response to the planted glomerular Ag. IL-12p40-deficient (IL-12(-/-)) mice showed significant reductions in crescent formation, proteinuria, and glomerular DTH effectors. Administration of IL-18 to IL-12(-/-) mice restored the development of histological (including effectors of DTH) and functional glomerular injury in IL-12(-/-) mice to levels equivalent to those in IL-12(+/+) mice. IL-18 administration to IL-12(-/-) mice increased glomerular ICAM-1 protein expression, but did not restore Ag-stimulated splenocyte IFN-gamma, GM-CSF, IL-2, or TNF-alpha production. Sensitized IL-12(+/+) mice also developed cutaneous DTH following intradermal challenge with the nephritogenic Ag. Cutaneous DTH was inhibited in IL-12(-/-) mice, but was restored by administration of IL-18. IL-12(+/+) mice given IL-18 developed augmented injury, with enhanced glomerular and cutaneous DTH, demonstrating the synergistic effects of IL-18 and IL-12 in DTH responses. These studies demonstrate that even in the absence of IL-12, IL-18 can induce in vivo DTH responses and up-regulate ICAM-1 without inducing IFN-gamma, GM-CSF, or TNF-alpha production.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

Crescentic glomerulonephritis--a manifestation of a nephritogenic Th1 response?

Crescentic glomerulonephritis (GN) is the histopathological correlate of the clinical syndrome of rapidly progressive glomerulonephritis. Glomerular crescent formation complicates proliferative forms of GN and indicates severe disease with a poor renal prognosis. In the past 10 years evidence from experimental models of GN and from human disease has accumulated suggesting that crescentic glomerulonephritis is a manifestation of a delayed type hypersensitivity (DTH)-like response to nephritogenic antigens. The elucidation of T helper 1 (Th1) and Th2 subsets in mice and in humans has led to the hypothesis that crescentic GN is a manifestation of a Th1 predominant DTH mediated immune response. Recent experiments performed mainly in a murine model of crescentic glomerulonephritis have tested this hypothesis. Crescent formation in this model is substantially interleukin (IL)-12 and interferon-gamma (IFN-gamma) dependent. Administration of IL-12, deletion of endogenous IL-4 or IL-10 results in enhanced disease, while administration of exogenous IL-4 and/or IL-10 reduces crescentic injury. These findings, together with the available evidence from human studies (examining the pattern of immune effectors in glomeruli, data on cytokine production by peripheral blood mononuclear cells and case reports of the induction of proliferative and/or crescentic GN by administration of IFN-gamma or IL-2) suggest that human crescentic GN is manifestation of a Th1 mediated DTH-like nephritogenic immune response.     

Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.     

STAT6 expression in multiple cell types mediates the cooperative development of allergic airway disease

Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6(-/-) mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6(-/-) mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6(-/-) mice even when STAT6(+/+) BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2(-/-) mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4(+)CD25(+)Foxp3(+) T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6(-/-) mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6(-/-) mice were equally efficient in suppressing CD4(+) T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.     

Effect of Angiotensin II Type 2 Receptor-Interacting Protein on Adipose Tissue Function via Modulation of Macrophage Polarization

We demonstrated that angiotensin II type 2 (AT₂) receptor-interacting protein (ATIP) 1 ameliorates inflammation-mediated vascular remodeling independent of the AT₂ receptor, leading us to explore the possibility of whether ATIP1 could exert anti-inflammatory effects and play a role in other pathophysiological conditions. We examined the possible anti-inflammatory effects of ATIP1 in adipose tissue associated with amelioration of insulin resistance. In mice fed a high-cholesterol diet, adipose tissue macrophage (ATM) infiltration and M1-to-M2 ratio were decreased in ATIP1 transgenic mice (ATIP1-Tg) compared with wild-type mice (WT), with decreased expression of inflammatory cytokines such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in white adipose tissue (WAT), but an increase in interleukin-10, an anti-inflammatory cytokine. Moreover, 2-[³H]deoxy-d-glucose (2-[³H]DG) uptake was significantly increased in ATIP1-Tg compared with WT. Next, we examined the roles of ATIP1 in BM-derived hematopoietic cells, employing chimeric mice produced by BM transplantation into irradiated type 2 diabetic mice with obesity, KKAy, as recipients. ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 chimera (ATIP1-tg as BM donor), with improvement of insulin-mediated 2-[³H]DG uptake and amelioration of inflammation in WAT. Moreover, serum adiponectin concentration in ATIP1 chimera was significantly higher than that in WT chimera (WT as BM donor) and KKAy chimera (KKAy as BM donor). These results indicate that ATIP1 could exert anti-inflammatory effects in adipose tissue via macrophage polarization associated with improvement of insulin resistance, and ATIP1 in hematopoietic cells may contribute to these beneficial effects on adipose tissue functions in type 2 diabetes.     

Heat shock factor 1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of bone marrow stem/progenitor cells

Bone marrow (BM)-derived stem/progenitor cells play an important role in ischemia-induced angiogenesis in cardiovascular diseases. Heat shock factor 1 (HSF1) is known to be induced in response to hypoxia and ischemia. We examined whether HSF1 contributes to ischemia-induced angiogenesis through the mobilization and recruitment of BM-derived stem/progenitor cells using HSF1-knockout (KO) mice. After the induction of ischemia, blood flow and microvessel density in the ischemic hindlimb were significantly lower in the HSF1-KO mice than in the wild-type (WT) mice. The mobilization of BM-derived Sca-1- and c-kit-positive cells in peripheral blood after ischemia was significantly lower in the HSF1-KO mice than in the WT mice. BM stem/progenitor cells from HSF1-KO mice showed a significant decrease in their recruitment to ischemic tissue and in migration, adhesion, and survival when compared with WT mice. Blood flow recovery in the ischemic hindlimb significantly decreased in WT mice receiving BM reconstitution with donor cells from HSF1-KO mice. Conversely, blood flow recovery in the ischemic hindlimb significantly increased in HSF1-KO mice receiving BM reconstitution with donor cells from WT mice. These findings suggest that HSF1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of BM-derived stem/progenitor cells.     

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells

IL-10-producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10-producing B cells (IL-10(-/-)B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10(-/-) B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (μMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10(-/-) mice increased Foxp3(+) Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10(+/+) B cells established longer contact times with arthritogenic CD4(+)CD25(-) T cells compared with IL-10(-/-) B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4(+) T cells. Thus, IL-10-producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.     

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.     

A novel role of CD30L/CD30 signaling by T-T cell interaction in Th1 response against mycobacterial infection

A CD30 ligand (CD30L, CD153) is a type II membrane-associated glycoprotein belonging to the TNF family. To illustrate the potential role of CD30L in CD4(+) Th1 cell responses, we investigated the fate of Ag-specific CD4(+) T cells in CD30L-deficient (CD30L(-/-)) mice after Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. The number of bacteria was significantly higher in organs of CD30L(-/-) mice than in wild-type (WT) mice 4 wk postinfection. The numbers of purified protein derivative- or Ag85B-specific-IFN-gamma-producing-CD4(+) T cells in spleen, lung, or peritoneal exudate cells were significantly fewer in CD30L(-/-) mice than in WT mice. During the infection, CD30L was expressed mainly by CD44(+)CD3(+)CD4(+) T cells but not by CD3(+)CD8(+) T cells, B cells, dendritic cells, or macrophages. Costimulation with agonistic anti-CD30 mAb or coculturing with CD30L-transfected P815 cells restored IFN-gamma production by CD4(+) T cells from BCG-infected CD30L(-/-) mice. Coculturing with CD30L(+/+)CD4(+) T cells from BCG-infected WT mice also restored the number of IFN-gamma(+)CD30L(-/-)CD4(+) T cells. When transferred into the CD30L(+/+) mice, Ag-specific donor CD30L(-/-) CD4(+) T cells capable of producing IFN-gamma were restored to the compared level seen in CD30L(+/+) CD4(+) T cells on day 10 after BCG infection. When naive CD30L(+/+) T cells were transferred into CD30L(-/-) mice, IFN-gamma-producing-CD4(+) Th1 cells of donor origin were normally generated following BCG infection, and IFN-gamma-producing-CD30L(-/-)CD4(+) Th1 cells of host origin were partly restored. These results suggest that CD30L/CD30 signaling executed by CD30(+) T-CD30L(+) T cell interaction partly play a critical role in augmentation of Th1 response capable of producing IFN-gamma against BCG infection.     

A mandatory role for STAT4 in IL-12 induction of mouse T cell CCR5.

IL-12 was recently shown to induce CCR5 on TCR-triggered mouse T cells. Considering that STAT4 is the most critical of IL-12 signaling molecules, this study investigated the role for STAT4 in the induction of CCR5 expression. IL-12R was induced by stimulation with anti-CD3 plus anti-CD28 mAb similarly on T cells from wild-type (WT) and STAT4-deficient (STAT4(-/-)) mice, but the levels of IL-12R induced on IFN-gamma-deficient (IFN-gamma(-/-)) T cells were lower compared with WT T cells. Exposure of TCR-triggered WT T cells to IL-12 induced CCR5 expression. In contrast, TCR-triggered STAT4(-/-) T cells failed to express CCR5 in response to IL-12. IL-12 stimulation induced detectable albeit reduced levels of CCR5 expression on IFN-gamma(-/-) T cells. Addition of rIFN-gamma to cultures of IFN-gamma(-/-) T cells, particularly to cultures during TCR triggering resulted in restoration of CCR5 expression. However, CCR5 expression was not induced in STAT4(-/-) T cells by supplementation of rIFN-gamma. These results indicate that for the induction of CCR5 on T cells, 1) STAT4 plays an indispensable role; 2) such a role is not substituted by simply supplementing rIFN-gamma; and 3) IFN-gamma amplifies CCR5 induction depending on the presence of STAT4.     

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R-/-) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R-/- mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R-/- mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R-/- BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R-/- macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R-/- lymphocyte population and correlated with reduced levels of IFN-gamma and NO and apoptosis of lymphocytes. mRNA and protein for IFN-gamma were also significantly reduced in the CNS of P2X7R-/- mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R-/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R-/- mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.     

Specific loss of toll-like receptor 2 on bone marrow derived cells decreases atherosclerosis in LDL receptor null mice

Innate immunity and, notably, Toll-like receptors (TLR), have an important role in atherogenesis. We have tested the hypothesis that the selective loss of TLR-2 by cells of bone marrow (BM) origin will protect low-density receptor-deficient (Ldlr (-/-)) mice from both early- and late-stage atherosclerosis. BM cells from Tlr2(+/+) and Tlr2(-/-) littermates were used to reconstitute lethally irradiated Ldlr(-/-) mice. Following a recovery period, mice were placed either on a diet containing 21% saturated fat - 0.15% cholesterol for 8?weeks to study early-stage atherosclerosis, or on a diet richer in cholesterol (1.5%) for 16?weeks to study late-stage atherosclerosis. Donor cell Tlr2 genotype did not alter serum cholesterol levels or lipoprotein profiles in recipient animals. After 8?weeks on the 0.15% cholesterol diet, deficiency of TLR-2 expression on cells of BM origin reduced atherosclerosis in the aortic root and the aortic arch in both genders of mice. In contrast, the BM recipients who received the 1.5% cholesterol diet for 16?weeks showed much larger lesions in the aortic root, and TLR-2 deficiency in BM cells failed to provide protection. Thus, TLR-2 expression in BM-derived cells contributes primarily to early stage atherosclerosis.     

B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

IL-18 is redundant in T-cell responses and in joint inflammation in antigen-induced arthritis

IL-18 is an important cofactor in Th1 immune responses and it has additional roles in inflammation. Recent reports suggest the contribution of IL-18 to immune responses may vary between mouse strains and immune contexts. We investigated the contribution of IL-18 to T-cell activation and joint inflammation in Ag-induced arthritis (AIA) in C57Bl/6 mice. AIA and cutaneous delayed-type hypersensitivity (DTH) reactions were induced in wild-type (WT) and IL-18-/- C57Bl/6 mice, and Ag-specific T-cell proliferation and IFN-gamma and IL-4 production were measured. The humoral immune response was measured as serum antibody to the disease-initiating Ag, methylated BSA (mBSA). Splenocyte production of IL-6 was measured by ELISA. To confirm the dependence of this model on Th1-cell-mediated immunity, IL-12p40-/- mice were similarly studied. WT mice developed synovitis, joint effusion, cartilage destruction and bone damage associated with induction of DTH, and in vitro Ag-specific T-cell proliferation and IFN-gamma production. Unexpectedly, IL-18-/- mice developed AIA and indices of T-cell activation were similar to those of WT mice. In contrast, IL-12p40-/- mice did not develop AIA, DTH or T-cell activation. WT and IL-18-/- mice, but not IL-12p40-/- mice, developed significantly increased serum antibody to mBSA compared with naive controls. WT and IL-18-/- splenocytes produced high levels of IL-6, whereas IL-12p40-/- cells had significantly lower IL-6 production compared with both. In conclusion, IL-18 is redundant both as a Th1 response cofactor and inflammatory cytokine, whereas IL-12p40-/- is a key cytokine, in AIA in C57Bl/6 mice.     

Therapeutic anti-inflammatory effects of myeloid cell adenosine receptor A2a stimulation in lipopolysaccharide-induced lung injury

To determine the role of the adenosine receptor A2a in a murine model of LPS-induced lung injury, migration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lung was determined by flow cytometry, microvascular permeability was assessed by the extravasation of Evans blue, and the release of chemotactic cytokines into the alveolar airspace was determined by ELISA. Measurements were performed in wild-type and A2a gene-deficient mice (A2a(-/-)). To differentiate the role of A2a on hemopoietic and nonhemopoietic cells, we created chimeric mice by transfer of bone marrow (BM) between wild-type and A2a(-/-) mice and used mice that lacked A2a expression selectively on myeloid cells (A2a(flox/flox) x LysM-cre). A specific A2a receptor agonist (ATL202) was used to evaluate its potential to reduce lung injury in vivo. In wild-type mice, therapeutic treatment with ATL202 reduced LPS-induced PMN recruitment, and release of cytokines. Pretreatment, but not posttreatment, also reduced Evans blue extravasation. In the BM chimeric mice lacking A2a on BM-derived cells, PMN migration into the alveolar space was increased by approximately 50%. These findings were confirmed in A2a(flox/flox) x LysM-cre mice. ATL202 was only effective when A2a was present on BM-derived cells. A2a agonists may be effective at curbing inflammatory lung tissue damage.     

CD4+ T lymphocytes mediate hypercholesterolemia-induced endothelial dysfunction via a NAD(P)H oxidase-dependent mechanism

Although hypercholesterolemia is known to impair endothelium-dependent vasodilation (EDV) long before the appearance of atherosclerotic plaques, it remains unclear whether the immune mechanisms that have been implicated in atherogenesis also contribute to the early oxidative stress and endothelial cell dysfunction elicited by hypercholesterolemia. EDV (wire myography), superoxide generation (cytochrome c reduction), and NAD(P)H oxidase mRNA expression were monitored in aortic rings from wild-type (WT) and mutant mice placed on either a normal diet or a cholesterol-enriched diet (HC) for 2 wk. WT mice on HC exhibited impaired EDV, enhanced superoxide generation, and increased expression of NAD(P)H oxidase subunit Nox-2 mRNA. The impaired EDV and increased superoxide generation induced by HC were significantly blunted in severe combined immunodeficient (SCID) mice and CD4+ T lymphocyte-deficient mice. These responses were also attenuated in HC mice genetically deficient in IFN-gamma; however, adoptive transfer of WT-HC CD4+ T lymphocytes to IFN-gamma-deficient recipients restored HC-induced responses. The HC-induced impaired EDV and oxidative stress were also attenuated in HC mice genetically deficient in Nox-2 (gp91(phox-/-)) and in WT-->gp91(phox-/-)-HC chimeras. HC-induced gp91(phox) mRNA expression was significantly blunted in mice deficient in CD4+ T cells or IFN-gamma and was restored with adoptive transfer of WT-HC CD4+ T cells to IFN-gamma-deficient recipients. These findings implicate the immune system in the early endothelial cell dysfunction associated with hypercholesterolemia and are consistent with a mechanism of impaired EDV that is mediated by CD4+ T cells and IFN-gamma, acting through the generation of superoxide from vascular NAD(P)H oxidase.     

Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells

The regulatory roles of Th1 and Th2 cells in immune protection against Helicobacter infection are not clearly understood. In this study, we report that a primary H. pylori infection can be established in the absence of IL-12 or IFN-gamma. However, IFN-gamma, but not IL-12, was involved in the development of gastritis because IFN-gamma(-/-) (GKO) mice exhibited significantly less inflammation as compared with IL-12(-/-) or wild-type (WT) mice. Both IL-12(-/-) and GKO mice failed to develop protection following oral immunization with H. pylori lysate and cholera toxin adjuvant. By contrast, Th2-deficient, IL-4(-/-), and WT mice were equally well protected. Mucosal immunization in the presence of coadministered rIL-12 in WT mice increased Ag-specific IFN-gamma-producing T cells by 5-fold and gave an additional 4-fold reduction in colonizing bacteria, confirming a key role of Th1 cells in protection. Importantly, only protected IL-4(-/-) and WT mice demonstrated substantial influx of CD4(+) T cells in the gastric mucosa. The extent of inflammation in challenged IL-12(-/-) and GKO mice was much reduced compared with that in WT mice, indicating that IFN-gamma/Th1 cells also play a major role in postimmunization gastritis. Of note, postimmunization gastritis in IL-4(-/-) mice was significantly milder than WT mice, despite a similar level of protection, indicating that immune protection is not directly linked to the degree of gastric inflammation. Only protected mice had T cells that produced high levels of IFN-gamma to recall Ag, whereas both protected and unprotected mice produced high levels of IL-13. We conclude that IL-12 and Th1 responses are crucial for H. pylori-specific protective immunity.