We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
AHR; dendritic cells; eosinophils; mice; Syk
Mast cells are the main effector cells of immediate hypersensitivity and anaphylaxis. Their role in the development of allergen-induced airway hyperresponsiveness (AHR) is controversial and based on indirect evidence. To address these issues, mast cell–deficient mice (W/W v) and their congenic littermates were sensitized to ovalbumin (OVA) by intraperitoneal injection and subsequently challenged with OVA via the airways. Comparison of OVA-specific immunoglobulin E (IgE) levels in the serum and numbers of eosinophils in bronchoalveolar lavage fluid or lung digests showed no differences between the two groups of mice. Further, measurements of airway resistance and dynamic compliance at baseline and after inhalation of methacholine were similar. These data indicate that mast cells or IgE–mast cell activation is not required for the development of eosinophilic inflammation and AHR in mice sensitized to allergen via the intraperitoneal route and challenged via the airways.
The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcɛRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcɛRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cγ1 (PLCγ1) and PLCγ2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCγ1 and PLCγ2 and calcium responses. Thus, Vav1 is essential to FcɛRI-mediated activation of PLCγ and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCγ-activated calcium signals.
Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcɛ receptor I (FcɛRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT−/− NTAL−/− bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT−/− NTAL−/− BMMCs. Sos was preferentially required for FcɛRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT−/− NTAL−/− BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cγ (PLCγ) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT−/− NTAL−/− BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCγ and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.
Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.
BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.
Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.
These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3–/– mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3–/– mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3–/– mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3–/– mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3–/– mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.
Astragali radix Antiasthmatic Decoction (AAD), a traditional Chinese medication, is found effective in treating allergic diseases and chronic cough. The purpose of this study is to determine whether this medication could suppress allergen-induced airway hyperresponsiveness (AHR) and remodeling in mice, and its possible mechanisms.
A mouse model of chronic asthma was used to investigate the effects of AAD on the airway lesions. Mice were sensitized and challenged with ovalbumin (OVA), and the extent of AHR and airway remodeling were characterized. Cells and cytokines in the bronchoalveolar lavage fluid (BALF) were examined.
AAD treatment effectively decreased OVA-induced AHR, eosinophilic airway inflammation, and collagen deposition around the airway. It significantly reduced the levels of IL-13 and TGF-β1, but exerted inconsiderable effect on INF-γ and IL-10.
AAD greatly improves the symptoms of allergic airway remodeling probably through inhibition of Th2 cytokines and TGF-β1.
Astragalus Antiasthmatic Decoction; Airway hyperresponsiveness; Airway remodeling
The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell–deficient WBB6F1-W/Wv mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F1-+/+ normal mice. However, W/Wv mice that had undergone selective reconstitution of tissue mast cells with in vitro–derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/Wv mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/Wv mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the “asthma model” investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice.
eosinophil; epithelium; hyperresponsiveness; tissue remodeling; T lymphocyte
Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.
Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.
Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.
Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.
airway hyperresponsiveness; chronic asthma; cytokine; dendritic cells; eosinophil
H89 is a potent inhibitor of Protein Kinase A (PKA) and Mitogen- and Stress-Activated protein Kinase 1 (MSK1) with some inhibitory activity on other members of the AGC kinase family. H89 has been extensively used in vitro but its anti-inflammatory potential in vivo has not been reported to date. To assess the anti-inflammatory properties of H89 in mouse models of asthma.
Mice were sensitized intraperitoneally (i.p.) to ovalbumin (OVA) with or without alum, and challenged intranasally with OVA. H89 (10 mg/kg) or vehicle was given i.p. two hours before each OVA challenge. Airway hyperresponsiveness (AHR) was assessed by whole-body barometric plethysmography. Inflammation was assessed by the total and differential cell counts and IL-4 and IL-5 levels in bronchoalveolar lavage (BAL) fluid. Lung inflammation, mucus production and mast cell numbers were analyzed after histochemistry. We show that treatment with H89 reduces AHR, lung inflammation, mast cell numbers and mucus production. H89 also inhibits IL-4 and IL-5 production and infiltration of eosinophils, neutrophils and lymphocytes in BAL fluid.
Taken together, our findings implicate that blockade of AGC kinases may have therapeutic potential for the treatment of allergic airway inflammation.
Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcɛRI. Diacylglycerol kinase (DGK)ζ is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKζ−/− mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKζ−/− bone marrow–derived mast cells (BMMCs) demonstrate impaired degranulation after FcɛRI cross-linking, associated with diminished phospholipase Cγ activity, calcium flux, and protein kinase C–βII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of FcɛRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of FcɛRI signals.
Persistent activation of nuclear factor κB (NF-κB) has been associated with the development of asthma. Galangin, the active pharmacological ingredient from Alpinia galanga, is reported to have a variety of anti-inflammatory properties in vitro via negative regulation of NF-κB. This study aimed to investigate whether galangin can abrogate ovalbumin- (OVA-) induced airway inflammation by negative regulation of NF-κB. BALB/c mice sensitized and challenged with OVA developed airway hyperresponsiveness (AHR) and inflammation. Galangin dose dependently inhibited OVA-induced increases in total cell counts, eosinophil counts, and interleukin-(IL-) 4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. Galangin also attenuated AHR, reduced eosinophil infiltration and goblet cell hyperplasia, and reduced expression of inducible nitric oxide synthase and vascular cell adhesion protein-1 (VCAM-1) levels in lung tissue. Additionally, galangin blocked inhibitor of κB degradation, phosphorylation of the p65 subunit of NF-κB, and p65 nuclear translocation from lung tissues of OVA-sensitized mice. Similarly, in normal human airway smooth muscle cells, galangin blocked tumor necrosis factor-α induced p65 nuclear translocation and expression of monocyte chemoattractant protein-1, eotaxin, CXCL10, and VCAM-1. These results suggest that galangin can attenuate ovalbumin-induced airway inflammation by inhibiting the NF-κB pathway.
The Tec family tyrosine kinase Itk is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired TH2 cytokine secretion; however, they have increased circulating serum IgE but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcεRI signaling. We report here that Itk null mice have reduced allergen/IgE induced histamine release as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, since the transfer of Itk null BMMC into mast cell deficient W/Wv animals is able to fully rescue histamine release in the W/Wv mice. Further analysis of Itk null BMMC in vitro revealed that while they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-α, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcεRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses, however, these cells express elevated levels of NFAT which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.
“This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.”
Tec; kinase/Airway; Hyperresponsiveness/IgE/Allergic; Asthma/cytokine
SWAP-70, an unusual phosphatidylinositol-3-kinase-dependent protein that interacts with the RhoGTPase Rac, is highly expressed in mast cells. Cultured bone marrow mast cells (BMMC) from SWAP-70−/− mice are reduced in FcɛRI-triggered degranulation. This report describes the hitherto-unknown role of SWAP-70 in c-kit receptor signaling, a key proliferation and differentiation pathway in mast cells. Consistent with the role of Rac in cell motility and regulation of the actin cytoskeleton, mutant cells show abnormal actin rearrangements and are deficient in migration in vitro and in vivo. SWAP-70−/− BMMC are impaired in calcium flux, in proper translocation and activity of Akt kinase (required for mast cell activation and survival), and in translocation of Rac1 and Rac2 upon c-kit stimulation. Adhesion to fibronectin is reduced, but homotypic cell association induced through c-kit is strongly increased in SWAP-70−/− BMMC. Homotypic association requires extracellular Ca2+ and depends on the integrin αLβ2 (LFA-1). ERK is hyperactivated upon c-kit signaling in adherent and dispersed mutant cells. Together, we suggest that SWAP-70 is an important regulator of specific effector pathways in c-kit signaling, including mast cell activation, migration, and cell adhesion.
We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcɛRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcɛRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcɛRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcɛRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcɛRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcɛRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcɛRI-induced mast cell responses.
CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.
Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.
Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.
We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.
chemokine receptor; CXCR3; CD8+ T lymphocyte; airway inflammation; airway hyperresponsiveness
Engagement of the Fcɛ receptor I (FcɛRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non–T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow–derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcɛRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C γ1 and phospholipase C γ2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain–containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.
mast cell; signal transduction; Fcɛ receptor; calcium mobilization; adapter molecules
The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl4 and butylated hydroxytoluene (BHT) injury models. Foxf1+/− mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation after injury. Pulmonary inflammation in Foxf1+/− mice was associated with diminished expression of Foxf1, increased mast cell tryptase, and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of CXCL12 were observed in Foxf1+/− mice. During lung development, Foxf1+/− embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of a doxycycline-inducible Foxf1 dominant-negative transgene in primary cultures of lung endothelial cells increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation after chemically induced or allergen-mediated lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.
Foxf1; CXCL12; SDF-1; mast cells; lung injury
Dendritic cells (DCs) are considered to be the most efficient antigen-presenting cells. Intratracheal administration of allergen-pulsed bone marrow–derived dendritic cells (BMDCs) before allergen challenge induces airway hyperresponsiveness (AHR) and inflammation. Ovalbumin (OVA)-pulsed BMDCs from wild-type (WT) mice were transferred into naive WT, CD4−/−, CD8−/−, or IL-13−/− mice. Two days (short protocol) or 10 days (long protocol) after BMDC transfer, mice were challenged with 1% OVA for 3 days and assayed 2 days later. Transfer of OVA-primed BMDCs into BALB/c or C57BL/6 mice elicited AHR in both protocols. Airway eosinophilia, Th2 cytokines, or goblet cell metaplasia were increased in the long but not short protocol. Lung T cells from both protocols produced Th2 cytokines in response to OVA in vitro. Carboxyfluorescein diacetate succinimidylester–labeled BMDCs were observed in bronchoalveolar lavage (BAL) fluid and lung parenchyma at early time points, and were detected in draining lymph nodes 48 hours after transfer. CD8−/− mice developed AHR comparable to WT mice in the short protocol, but decreased levels of AHR, airway eosinophilia, Th2 cytokines in BAL fluid, and goblet cell metaplasia compared with WT mice in the long protocol. CD4−/− or IL-13−/− mice did not develop AHR or airway inflammation in either protocol. These data suggest that allergen-pulsed BMDCs initiate development of AHR that is dependent initially on CD4+ T cells, and at later time periods on CD8+ T cells and IL-13. Thus, the timing of allergen challenge after transfer of allergen-pulsed BMDC affects the development of AHR and airway inflammation.
dendritic cells; CD8+ T cells; airway hyperresponsiveness
Arginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.
To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3), or HEPA-filtered air (FA), for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.
Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models.
This study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.
Apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen –activated protein (MAP) kinase kinase kinases (MAP3Ks) protein family, plays a crucial role in the induction of apoptosis and inflammation in some cell types. Allergic asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammatory cell infiltration, and airway remodeling. In the present study, we examined whether ASK1 is involved in the induction of bronchial asthma using a mouse model of airway inflammation.
ASK1-deficient (ASK1−/−) and wild-type (WT) control mice were sensitized with ovalbumin (OVA) in saline intraperitoneally on consecutive 7 days. Eighteen days later, mice received intranasal administration of OVA aerosol and were assayed for AHR, cytokine production, cell proliferation, antibody (Ab) production, and lung tissue histopathology at 24 hours after the last serial OVA administration. Levels of Ab and cytokines were determined by enzyme-linked immunosorbent assay (ELISA).
Control WT mice showed inflammatory infiltrates in airways in response to OVA to a greater extent than ASK1−/− mice. The number of cells, especially eosinophils accumulating in airways, was reduced in ASK1−/− mice relative to control mice. OVA-induced AHR is also compromised in ASK1−/− mice. Anti-OVA IgE Ab production in ASK1−/− mice was substantially reduced, although levels of other isotypes were comparable to those in control mice. Levels of some Th2 cytokines (IL-5 and IL-13) and pro-inflammatory cytokine TNF-a in BAL fluid from ASK1−/− mice were substantially diminished relative to control, although a comparable level of a typical Th2 cytokine IL-4 and anti-inflammatory cytokine IL-10 was produced. Although the BAL fluid TNF-a levels from ASK1−/− mice were severely diminished, lymph node cells from ASK1−/− mice produced comparable levels of TNF-a to WT in vitro. Intranasal administration of recombinant TNF-a caused a comparable increase in AHR between ASK1−/− and WT mice, whereas the TNF-a -induced accumulation of inflammatory cells was severely reduced in ASK1−/− mice.
ASK1 appears to be involved in the induction of OVA-induced bronchial asthma, probably through cytokine production such as TNF-a and IL-13. Moreover, TNF-a sensitivity in response to OVA is also regulated by ASK1.
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.
Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR.
Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge.
These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.
Asthma; Dendritic cells; Endotoxin; Notch ligands
Murine models have highlighted the importance of T-cells and TH2 cytokines in development of allergen-induced airway disease. In contrast, the role of mast cells for the development of allergic airway disease has been controversial. Recent studies in murine models demonstrate a significant contribution of mast cells during the development of airway hyperresponsiveness and airway inflammation. Furthermore these models have allowed identifying certain mast cell-produced mediators (e.g. histamine and leukotriene B4) to be involved in the recruitment of effector T-cells into the lung. Additionally, mast cell-produced TNF can directly activate TH2 cells and contribute to the development of allergic airway disease. These new findings demonstrate a complex role of mast cells and their mediators, not only as effector cells, but also during sensitization and development of allergic airway disease. Therefore mast cells and certain mast cell-produced mediators might be an interesting target for the prevention and treatment of allergic asthma.
Airway hyperresponsiveness (AHR), a hallmark of asthma and several other diseases, can be modulated by γδ T cells. In mice sensitized and challenged with ovalbumin, AHR depends on allergen-specific αβ T cells, but Vγδ1+ γδ T cells spontaneously enhance AHR, whereas Vγ4+ γδ T cells after being induced by airway challenge suppress AHR. The activity of these γδ T cell modulators is allergen-nonspecific, and how they develop is unclear. We now show that CD8 is essential for the development of both the AHR-suppressor and enhancer γδ T cells although neither type needs to express CD8 itself. Both cell types encounter CD8-expressing non-T cells in the spleen, and their functional development in an otherwise CD8-negative environment can be restored with transferred spleen cell preparations containing CD8+ DC, but not CD8+ T cells or CD8− DC. Our findings suggest that CD8+ DC in the lymphoid tissues enable an early step in the development of γδ T cells, through direct cell-contact. DC-expressed CD8 might take part in this interaction.
Rodent; dendritic cells; T cells; cell differentiation; spleen and lymph nodes