mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

It has been established that mammalian target of Rapamycin (mTOR) inhibitors have anti-inflammatory effects in models of experimental colitis. However, the underlying mechanism is largely unknown. In this research, we investigate the anti-inflammatory effects of AZD8055, a potent mTOR inhibitor, on T cell response in dextran sulfate sodium (DSS)-induced colitis in mice, a commonly used animal model of inflammatory bowel diseases (IBD). Severity of colitis is evaluated by changing of body weight, bloody stool, fecal consistency, histology evaluation and cytokine expression. We find that AZD8055 treatment attenuates DSS-induced body weight loss, colon length shortening and pathological damage of the colon. And AZD8055 treatment decreases colonic expression of genes encoding the pro-inflammatory cytokines interferon-γ, interleukin (IL)-17A, IL-1β,IL-6 and tumor necrosis factor(TNF)-a and increases colonic expression of anti-inflammatory cytokines IL-10. We show that AZD8055 treatment decreases the percentages of CD4+ T cells and CD8+ T cells in spleen, lymph nodes and peripheral blood of mice. We also find that AZD8055 treatment significantly reduces the number of T helper 1(TH1) cells and TH17 cells and increases regulatory T (Treg) cells in the lamina propria and mesenteric lymph nodes. Furthermore, we demonstrates that AZD8055 suppresses the proliferation of CD4+ and CD8+ T cells and the differentiation of TH1/TH17 cells and expands Treg cells in vitro. The results suggest that, in experimental colitis, AZD8055 exerts anti-inflammatory effect by regulating T helper cell polarization and proliferation.     

Diesel exhaust particle-exposed human bronchial epithelial cells induce dendritic cell maturation

Increased exposure to air pollutants such as diesel exhaust particles (DEP) has been proposed as one mechanism to explain the rise in allergic disorders. However, the immunologic mechanisms by which DEP enhance allergic sensitization and asthma remain unclear. We hypothesized that DEP act as an adjuvant for immature dendritic cell (DC) maturation via its effect on airway epithelial cell-derived microenvironment for DC. Immature monocyte-derived DC (iMDDC) failed to undergo phenotypic (CD80, CD83, CD86) or functional (T cell activation) maturation in response to exposure to DEP (0.001-100 mug/ml). In contrast, primary cultures of human bronchial epithelial cells (HBEC) treated with DEP induced iMDDC phenotypic maturation (2.6 +/- 0.1-fold increase in CD83 expression, n = 4, p < 0.05) and functional maturation (2.6 +/- 0.2-fold increase in T cell activation, n = 4, p < 0.05). Functional maturation of iMDDC was induced by conditioned medium derived from DEP-treated HBEC, and was inhibited in cultures with DEP-treated HBEC and blocking Abs against GM-CSF, or GM-CSF-targeted small interfering RNA. These data suggest that DEP induce Ag-independent DC maturation via epithelial cell-DC interactions mediated by HBEC-derived GM-CSF. Although additional signals may be required for polarization of DC, these data suggest a novel mechanism by which environmental pollutants alter airway immune responses.     

Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production by BEAS-2B cells, a bronchial epithelial cell line

Previous studies have suggested that bronchial epithelial cells may perpetuate airway inflammation. We have reported that the bronchial epithelial cell line BEAS-2B can produce interleukin (IL)-16, a potent chemoattractant for CD4+ T cells. IL-16 is thought to regulate airway inflammation in asthmatics. Recent studies showed that IL-4 induces inflammatory cytokines in bronchial epithelial cells and that IL-9 is a candidate gene for development of asthma. The present study demonstrated that BEAS-2B cells produced specifically IL-16 by synergistic effects of IL-4 + IL-16, or IL-9 + IL-16, and that the synthesized IL-16 induced migration of CD4+ T cells. This study is a first report indicating that IL-16 production may be maintained by an autocrine machinery by epithelial cell-derived IL-16 with IL-4 and IL-9 in asthma.     

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

Nitric-oxide synthase 2 interacts with CD74 and inhibits its cleavage by caspase during dendritic cell development

Dendritic cells (DC) are professional antigen-presenting cells that possess specific and efficient mechanisms to initiate immune responses. Upon encounter with pathogens, immature DC will go through a maturation process that converts them to highly immunogenic mature DC. Despite the fact that nitric oxide (NO) was produced in large amounts in maturing DC, it is still unclear whether NO is the key molecule that initiates and enhances DC maturation and T cell proliferation, respectively. Here, we report that NO donor and overexpression of either nitric-oxide synthase 2 (NOS2) or nitric-oxide synthase 3 (NOS3) alone can induce surface expression of major histocompatibility complex class II (MHC II) and both the essential co-stimulatory molecules CD80 and CD86 in immature DC. Consistently, NO donor-treated immature DC were capable of enhancing T cell proliferation in vitro in the absence of lipolysaccharide. Interestingly, NOS2 interacts with CD74 (the MHC II-associated invariant chain), and the degradation of CD74 by caspases in immature DC was inhibited upon treatment with NO donor. Because the trafficking of MHC II is CD74-dependent, the increase in cell surface localization of MHC II in maturing DC is in part due to the increase in CD74 protein expression in the presence of NOS2 and NO.     

Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention

Extracellular cyclophilins have been well described as chemotactic factors for various leukocyte subsets. This chemotactic capacity is dependent upon interaction of cyclophilins with the cell surface signaling receptor CD147. Elevated levels of extracellular cyclophilins have been documented in several inflammatory diseases. We propose that extracellular cyclophilins, via interaction with CD147, may contribute to the recruitment of leukocytes from the periphery into tissues during inflammatory responses. In this study, we examined whether extracellular cyclophilin-CD147 interactions might influence leukocyte recruitment in the inflammatory disease allergic asthma. Using a mouse model of asthmatic inflammation, we show that 1) extracellular cyclophilins are elevated in the airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) cyclophilins induce CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) in vivo treatment with anti-CD147 mAb significantly reduces (by up to 50%) the accumulation of eosinophils and effector/memory CD4+ T lymphocytes, as well as Ag-specific Th2 cytokine secretion, in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge. These findings provide a novel mechanism whereby asthmatic lung inflammation may be reduced by targeting cyclophilin-CD147 interactions.     

Ribavirin exerts differential effects on functions of cd4(+) Th1, th2, and regulatory T cell clones in hepatitis C

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10μg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that - in addition to its immunostimulatory effects on TH1 cells - ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.     

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

Dysregulated cytokine expression by CD4+ T cells from post-septic mice modulates both Th1 and Th2-mediated granulomatous lung inflammation

Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative) and TH2-(Schistosoma mansoni egg antigen) driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of T(H)2 cytokines in TH1 inflammation, and increased production of T(H)1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression.     

Anergic TH1 clones specific for hepatitis B virus (HBV) core peptides are inhibitory to other HBV core-specific CD4+ T cells in vitro.

A strong and transient hepatitis B virus core (HBc)-specific CD4+ T-cell response has been shown to be associated with viral elimination in acute self-limited hepatitis B but to be absent in chronic hepatitis B. So far, little is known about immunological mechanisms involved in the regulation of the HBc-specific CD4+ T-cell response. We studied 28 patients with acute hepatitis B, and frequently a sudden decrease in the HBc-specific CD4+ T-cell response was found between 4 and 8 weeks after disease onset. Thirty-two CD4+ T-cell clones specific for amino acids 50 to 69, 81 to 105, 117 to 131, or 141 to 165 of HBc were isolated from a patient shortly before the peripheral blood mononuclear cell response to most HBc-derived peptides abruptly disappeared. TH1 clones, but not TH0 clones, could be anergized in vitro by stimulation with specific peptides even in the presence of costimulatory cells. Moreover, when anergic cells were mixed with responsive cells, the proliferation of HBc-specific TH1 or TH0 clones was inhibited antigen specifically by anergic cells. The unusual susceptibility of HBc-specific TH1 clones to anergy induction in vitro as well as their potential to inhibit other HBc-specific TH1 and TH0 clones suggests that anergy induction may be involved in the downregulation of the virus-specific immune response during acute hepatitis B in vivo.     

Antiproliferative effect of IFN-gamma in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-gamma

Supernatants collected after primary or secondary stimulation of spleen cells contain different arrays of lymphokines. Primary supernatants from spleen cells stimulated with Con A or allogeneic spleen cells (MLC-SF) contain IL-2 but little IL-4 or IGN-gamma; in contrast, secondary MLC-SF contains IL-2 as well as substantial IL-4 and IFN-gamma. Our laboratory previously had always used secondary MLC-SF for cloning T cells, and had routinely obtained TH1 helper T lymphocyte clones. In the present study, when primary Con A-SF was used as source of growth factors, TH2 and not TH1 clones were preferentially derived. Considering the possibility that IFN-gamma may be one important factor in determining whether TH1 or TH2 clones are preferentially obtained, clone derivation was then performed either in the presence of rIL-2 or rIL-2 plus rIFN-gamma. The majority of clones derived using rIL-2 alone were TH2 cells, whereas the majority of clones derived using rIL-2 plus rIFN-gamma were TH1 cells. Using either procedure, some clones were obtained that produced IL-2, IL-4, and IFN-gamma. These data are consistent with our previous observations that IFN-gamma inhibits the proliferation of TH2 but not TH1 clones, and suggest that the presence of IFN-gamma during an immune response would result in the preferential expansion of helper T lymphocytes of the TH1 phenotype. Our procedure for the differential selection of TH1 and TH2 clones reactive with the same Ag should be useful for designing in vitro systems for studying the function of these cell subsets in specific immune responses.     

Thyroid hormones increase inducible nitric oxide synthase gene expression downstream from PKC-zeta in murine tumor T lymphocytes

Regulation of cell proliferation by thyroid hormone (TH) has been demonstrated, but the effect of THs and the mechanisms involved in lymphocyte activity have not been elucidated. Differential expression of PKC isoenzymes and high nitric oxide synthase (NOS) activity have been described in tumor T lymphocytes. We have analyzed the direct actions of TH on normal T lymphocytes and BW5147 T lymphoma cells in relation to PKC and NOS activities. THs increased tumor and mitogen-induced normal T lymphocyte proliferation. PKC isoenzyme-selective blockers impaired these effects in both cell types, indicating the participation of Ca2+-dependent and -independent isoenzymes in normal and tumor cells, respectively. TH actions were blunted by extra- and intracellular Ca2+ blockers only in normal T lymphocytes, whereas NOS blockers impaired TH-induced proliferation in T lymphoma cells. Incubation for 24 h with TH induced a rise in total and membrane-associated PKC activities in both cell types and led to a rapid and transient effect only in tumor cells. THs increased atypical PKC-zeta expression in BW5147 cells and classical PKC isoenzymes in mitogen-stimulated normal T cells. TH augmented NOS activity and inducible NOS protein and gene expression only in tumor cells. Blockade of PKC and the atypical PKC-zeta isoform inhibited TH-mediated stimulation of inducible NOS and cell proliferation. These results show, for the first time, that differential intracellular signals are involved in TH modulation of lymphocyte physiology and pathophysiology.     

Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.     

Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4+ T cells

CD4+ murine T cell clones, TH1 and TH2, can be distinguished by both functional responses and by their patterns of lymphokine secretion. Recently, a mAb, 23G2, which reacts with a subset of CD45 molecules (CD45R), has been reported to bind differentially to clones of TH1 and TH2 cells. In the present study, normal splenic T cells were analyzed for differences in 23G2-reactivity and were separated into two populations based on their density of CD45R (CD45Rhi and CD45Rlo). The CD45Rhi cells secrete more IL-2 than IL-4 after stimulation in vitro; the reverse is true for the CD45Rlo cells. Because neither population secretes only IL-2 or IL-4, we were unable to classify cells as TH1 or TH2. In vivo and in vitro analyses of the CD45Rhi and CD45Rlo cells suggest a lineage relationship between the two subsets that correlates with the degree of Ag exposure and the state of maturation of the mice. In newborn mice and mice raised under sterile conditions, splenic CD4+ T cells are predominantly CD45Rhi. Under conditions of increased antigenic exposure and maturation of the mice, CD45Rlo cells develop; after long term priming in vivo, the majority of specific Ag-reactive cells are CD45Rlo. Adoptive transfer studies using BALB/c nu/nu recipients demonstrate that CD45Rhi cells become CD45Rlo cells and that the recall response (IgG) to specific Ag is mediated by CD45Rlo cells. Taken together, these data indicate that the level of expression of CD45R on CD4+ T cells distinguishes virgin (CD45Rhi) from primed/memory (CD45Rlo) T cells in normal mice.     

Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major

CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.     

Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2.

On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diesel exhaust particle-treated human bronchial epithelial cells upregulate Jagged-1 and OX40 ligand in myeloid dendritic cells via thymic stromal lymphopoietin

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)-dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.     

Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production

PD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family. B7-H1 (PD-L1) and B7-DC (PD-L2), which belong to the B7 family, have been identified as ligands for PD-1. Paradoxically, it has been reported that both B7-H1 and B7-DC co-stimulate or inhibit T cell proliferation and cytokine production. To determine the role of B7-H1 and B7-DC in T cell-APC interactions, we examined the contribution of B7-H1 and B7-DC to CD4+ T cell activation by B cells, dendritic cells, and macrophages using anti-B7-H1, anti-B7-DC, and anti-PD-1 blocking mAbs. Anti-B7-H1 mAb and its Fab markedly inhibited the proliferation of anti-CD3-stimulated naive CD4+ T cells, but enhanced IL-2 and IFN-gamma production in the presence of macrophages. The inhibition of T cell proliferation by anti-B7-H1 mAb was abolished by neutralizing anti-IFN-gamma mAb. Coculture of CD4+ T cells and macrophages from IFN-gamma-deficient or wild-type mice showed that CD4+ T cell-derived IFN-gamma was mainly responsible for the inhibition of CD4+ T cell proliferation. Anti-B7-H1 mAb induced IFN-gamma-mediated production of NO by macrophages, and inducible NO synthase inhibitors abrogated the inhibition of CD4+ T cell proliferation by anti-B7-H1 mAb. These results indicated that the inhibition of T cell proliferation by anti-B7-H1 mAb was due to enhanced IFN-gamma production, which augmented NO production by macrophages, suggesting a critical role for B7-H1 on macrophages in regulating IFN-gamma production by naive CD4+ T cells and, hence, NO production by macrophages.     

Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression

This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.