Regulation of Eosinophil Recruitment and Allergic Airway Inflammation by Tropomyosin Receptor Kinase A.

Eosinophilia is a hallmark of allergic airway inflammation (AAI). Identifying key molecules and specific signaling pathways that regulate eosinophilic inflammation is critical for development of novel therapeutics. Tropomycin receptor kinase A (TrkA) is the high-affinity receptor for nerve growth factor. AAI is associated with increased expression of TrkA by eosinophils; however, the functional role of TrkA in regulating eosinophil recruitment and contributing to AAI is poorly understood. This study identifies, to our knowledge, a novel mechanism of eotaxin-mediated activation of TrkA and its role in regulating eosinophil recruitment by using a chemical-genetic approach to specifically inhibit TrkA kinase activity with 1-NM-PP1 in TrkAF592A-knock-in (TrkA-KI) eosinophils. Blockade of TrkA by 1-NM-PP1 enhanced eosinophil spreading on VCAM-1 but inhibited eotaxin-1 (CCL11)-mediated eosinophil migration, calcium flux, cell polarization, and ERK1/2 activation, suggesting that TrkA is an important player in the signaling pathway activated by eotaxin-1 during eosinophil migration. Further, blockade of matrix metalloprotease with BB-94 inhibited eotaxin-1-induced TrkA activation and eosinophil migration, additively with 1-NM-PP1, indicating a role for matrix metalloproteases in TrkA activation. TrkA inhibition in Alternaria alternata-challenged TrkA-KI mice markedly inhibited eosinophilia and attenuated various features of AAI. These findings are indicative of a distinctive eotaxin-mediated TrkA-dependent signaling pathway, which, in addition to other TrkA-activating mediators, contributes to eosinophil recruitment during AAI and suggests that targeting the TrkA signaling pathway to inhibit eosinophil recruitment may serve as a therapeutic strategy for management of eosinophilic inflammation in allergic airway disease, including asthma.

Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

Chronic exposure of tubular renal cells to high glucose contributes to tubulointerstitial changes in diabetic nephropathy. In the present study, we identified a new fibrosis gene called galectin-1 (Gal-1), which is highly expressed in tubular cells of kidneys of type 1 and type 2 diabetic mouse models. Gal-1 protein and mRNA expression showed significant increase in kidney cortex of heterozygous Akita+/- and db/db mice compared with wild-type mice. Mouse proximal tubular cells exposed to high glucose showed significant increase in phosphorylation of Akt and Gal-1. We cloned Gal-1 promoter and identified the transcription factor AP4 as binding to the Gal-1 promoter to up-regulate its function. Transfection of cells with plasmid carrying mutations in the binding sites of AP4 to Gal-1 promoter resulted in decreased protein function of Gal-1. In addition, inhibition of Gal-1 by OTX-008 showed significant decrease in p-Akt/AP4 and protein-promoter activity of Gal-1 and fibronectin. Moreover, down-regulation of AP4 by small interfering RNA resulted in a significant decrease in protein expression and promoter activity of Gal-1. We found that kidney of Gal-1-/- mice express very low levels of fibronectin protein. In summary, Gal-1 is highly expressed in kidneys of type 1 and 2 diabetic mice, and AP4 is a major transcription factor that activates Gal-1 under hyperglycemia. Inhibition of Gal-1 by OTX-008 blocks activation of Akt and prevents accumulation of Gal-1, suggesting a novel role of Gal-1 inhibitor as a possible therapeutic target to treat renal fibrosis in diabetes.-Al-Obaidi, N., Mohan, S., Liang, S., Zhao, Z., Nayak, B. K., Li, B., Sriramarao, P., Habib, S. L. Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

Pulmonary delivery of ORMDL3 short hairpin RNA - a potential tool to regulate allergen-induced airway inflammation.

Aim/Purpose: Exposure to various allergens has been shown to increase expression of ORMDL3 in the lung in models of allergic asthma. Studies using genetically modified (transgenic or knock out) mice have revealed some of the functions of ORMDL3 in asthma pathogenesis, although amid debate. The goal of this study was to use targeted post-transcriptional downregulation of ORMDL3 in allergen-challenged wild-type (WT) mice by RNA interference to further elucidate the functional role of ORMDL3 in asthma pathogenesis and evaluate a potential therapeutic option.Methods: Allergen (ovalbumin [OVA])-challenged WT mice were administered intranasally (i.n) with a single dose of five short hairpin RNA (shRNA) constructs with different target sequence for murine ORMDL3 cloned in a lentiviral vector or with the empty vector (control). Mice were evaluated for allergen-induced airway hyperresponsiveness (AHR) and various features of airway inflammation after 72 hours.Results: I.n administration of a single dose of ORMDL3 shRNAs to OVA-challenged mice resulted in reduction of ORMDL3 gene expression in the lungs associated with a significant reduction in AHR to inhaled methacholine and in the number of inflammatory cells recruited in the airways, specifically eosinophils, as well as in airway mucus secretion compared to OVA-challenged mice that received the empty vector. Administration of ORMDL3 shRNAs also significantly inhibited levels of IL-13, eotaxin-2 and sphingosine in the lungs. Additionally, ORMDL3 shRNAs significantly inhibited the allergen-mediated increase in monohexyl ceramides C22:0 and C24:0.Conclusions: Post-transcriptional down regulation of ORMDL3 in allergic lungs using i.n-delivered ORMDL3 shRNA (akin to inhaled therapy) attenuates development of key features of airway allergic disease, confirming the involvement of ORMDL3 in allergic asthma pathogenesis and serving as a model for a potential therapeutic strategy.

Regulation of eosinophilia and allergic airway inflammation by the glycan-binding protein galectin-1.

Galectin-1 (Gal-1), a glycan-binding protein with broad antiinflammatory activities, functions as a proresolving mediator in autoimmune and chronic inflammatory disorders. However, its role in allergic airway inflammation has not yet been elucidated. We evaluated the effects of Gal-1 on eosinophil function and its role in a mouse model of allergic asthma. Allergen exposure resulted in airway recruitment of Gal-1-expressing inflammatory cells, including eosinophils, as well as increased Gal-1 in extracellular spaces in the lungs. In vitro, extracellular Gal-1 exerted divergent effects on eosinophils that were N-glycan- and dose-dependent. At concentrations ≤0.25 µM, Gal-1 increased eosinophil adhesion to vascular cell adhesion molecule-1, caused redistribution of integrin CD49d to the periphery and cell clustering, but inhibited ERK(1/2) activation and eotaxin-1-induced migration. Exposure to concentrations ≥1 µM resulted in ERK(1/2)-dependent apoptosis and disruption of the F-actin cytoskeleton. At lower concentrations, Gal-1 did not alter expression of adhesion molecules (CD49d, CD18, CD11a, CD11b, L-selectin) or of the chemokine receptor CCR3, but decreased CD49d and CCR3 was observed in eosinophils treated with higher concentrations of this lectin. In vivo, allergen-challenged Gal-1-deficient mice exhibited increased recruitment of eosinophils and CD3(+) T lymphocytes in the airways as well as elevated peripheral blood and bone marrow eosinophils relative to corresponding WT mice. Further, these mice had an increased propensity to develop airway hyperresponsiveness and displayed significantly elevated levels of TNF-α in lung tissue. This study suggests that Gal-1 can limit eosinophil recruitment to allergic airways and suppresses airway inflammation by inhibiting cell migration and promoting eosinophil apoptosis.

Knob protein enhances epithelial barrier integrity and attenuates airway inflammation.

BACKGROUND: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. OBJECTIVE: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. METHODS: Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. RESULTS: Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. CONCLUSION: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced ß2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Regulation of eosinophil recruitment and allergic airway inflammation by heparan sulfate proteoglycan (HSPG) modifying enzymes.

BACKGROUND: HSPGs are glycoproteins containing covalently attached heparan sulfate (HS) chains which bind to growth factors, chemokines, etc., and regulate various aspects of inflammation including cell recruitment. We previously showed that deletion of endothelial N-acetylglucosamine N-deacetylase-N-sulfotransferase-1 (Ndst1), an enzyme responsible for N-sulfation during HS biosynthesis, reduces allergic airway inflammation (AAI). Here, we investigated the importance of O-sulfation mediated by uronyl 2-O-sulfotransferase (Hs2st) in development of AAI relative to N-sulfation. METHODS: Mice deficient in endothelial and leukocyte Hs2st (Hs2stf/fTie2Cre+) or Ndst1 (Ndst1f/fTie2Cre+) and WT mice were challenged with Alternaria alternata and evaluated for airway inflammation. Trafficking of murine eosinophils on lung endothelial cells was examined in vitro under conditions of flow. RESULTS: Exposure to Alternaria decreased expression level of Hs2st in WT mice while level of Ndst1 remained unchanged. Compared to WT mice, Alternaria-challenged Hs2stf/fTie2Cre+ mice exhibited significantly increased eosinophils in the bone marrow, bronchoalveolar lavage fluid [BALF] and lung tissue associated with persistent airway hyperresponsiveness, airway mucus hypersecretion and elevated Th2 cytokines. In contrast, Alternaria-challenged Ndst1f/fTie2Cre+ mice exhibited a marked reduction in airway eosinophilia, mucus secretion and smooth muscle mass compared to WT counterparts. While BALF eotaxins were lower in Alternaria-challenged Hs2stf/fTie2Cre+ relative to WT mice, they were not reduced to background levels as in allergen-challenged Ndst1f/fTie2Cre+ mice. Trafficking of murine eosinophils under conditions of flow in vitro was similar on Hs2st-deficient and WT endothelial cells. Expression of ZO-1 in Hs2st-deficient lung blood vessels in control and allergen-challenged mice was significantly lower than in WT counterparts. CONCLUSIONS: Our study demonstrates that allergen exposure reduces expression of Hs2st; loss of uronyl 2-O-sulfation in endothelial and leukocyte HSPG amplifies recruitment of eosinophils likely due to a compromised vascular endothelium resulting in persistent inflammation whereas loss of N-sulfation limits eosinophilia and attenuates inflammation underscoring the importance of site-specific sulfation in HSPG to their role in AAI.

Tumor eosinophil infiltration and improved survival of colorectal cancer patients: Iowa Women's Health Study.

The role of the innate immune response in colorectal cancer is understudied. We examined the survival of colorectal cancer patients in relation to eosinophils, innate immune cells, infiltrating the tumor. Tissue microarrays were constructed from paraffin-embedded tumor tissues collected between 1986 and 2002 from 441 post-menopausal women diagnosed with colorectal cancer in the Iowa Women's Health Study. Tissue microarrays were stained with an eosinophil peroxidase antibody. Eosinophils in epithelial and stromal tissues within the tumor (called epithelial and stromal eosinophils, hereafter) were counted and scored into three and four categories, respectively. In addition, the degree of eosinophil degranulation (across epithelial and stromal tissues combined) was quantified and similarly categorized. We used Cox regression to estimate the hazard ratios and 95% confidence interval for all-cause and colorectal cancer death during 5-year follow-up after diagnosis and during follow-up through 2011 ('total follow-up'). The hazard ratios associated with eosinophil scores were adjusted for age of diagnosis, SEER (Surveillance, Epidemiology, and End Results) stage, tumor grade, body mass, and smoking history. High tumor stromal eosinophil score was inversely correlated with age and stage, and was associated with a decreased risk for all-cause and colorectal cancer death: hazard ratios (95% confidence intervals) were 0.61 (0.36-1.02; P-trend=0.02) and 0.48 (0.24-0.93; P-trend=0.01), respectively, during the 5-year follow-up for the highest vs lowest category. The inverse associations also existed for total follow-up for all-cause and colorectal cancer death for the highest vs lowest stromal eosinophil score: hazard ratios (95% confidence intervals) were 0.72 (0.48-1.08; P-trend=0.04) and 0.61 (0.34-1.12; P-trend=0.04), respectively. Further adjustment for treatment, comorbidities, additional lifestyle factors, tumor location, or molecular markers did not markedly change the associations, while adjustment for cytotoxic T cells slightly attenuated all associations. The infiltration of tumors with eosinophils, especially in stromal tissue, may be an important prognostic factor in colorectal cancer.

Endothelial and leukocyte heparan sulfates regulate the development of allergen-induced airway remodeling in a mouse model.

Heparan sulfate (HS) proteoglycans (HSPGs) participate in several aspects of inflammation because of their ability to bind to growth factors, chemokines, interleukins and extracellular matrix proteins as well as promote inflammatory cell trafficking and migration. We investigated whether HSPGs play a role in the development of airway remodeling during chronic allergic asthma using mice deficient in endothelial- and leukocyte-expressed N-deacetylase/N-sulfotransferase-1 (Ndst1), an enzyme involved in modification reactions during HS biosynthesis. Ndst1-deficient and wild-type (WT) mice exposed to repetitive allergen (ovalbumin [OVA]) challenge were evaluated for the development of airway remodeling. Chronic OVA-challenged WT mice exhibited increased HS expression in the lungs along with airway eosinophilia, mucus hypersecretion, peribronchial fibrosis, increased airway epithelial thickness and smooth muscle mass. In OVA-challenged Ndst1-deficient mice, lung eosinophil and macrophage infiltration as well as airway mucus accumulation, peribronchial fibrosis and airway epithelial thickness were significantly lower than in allergen-challenged WT mice along with a trend toward decreased airway smooth muscle mass. Leukocyte and endothelial Ndst 1 deficiency also resulted in significantly decreased expression of IL-13 as well as remodeling-associated mediators such as VEGF, FGF-2 and TGF-ß1 in the lung tissue. At a cellular level, exposure to eotaxin-1 failed to induce TGF-ß1 expression by Ndst1-deficient eosinophils relative to WT eosinophils. These studies suggest that leukocyte and endothelial Ndst1-modified HS contribute to the development of allergen-induced airway remodeling by promoting recruitment of inflammatory cells as well as regulating expression of pro-remodeling factors such as IL-13, VEGF, TGF-ß1 and FGF-2 in the lung.

Inactivation of heparan sulfate 2-O-sulfotransferase accentuates neutrophil infiltration during acute inflammation in mice.

Neutrophil recruitment and extravasation at sites of inflammation provide a mechanism for host defense. We showed previously that heparan sulfate, a type of sulfated glycosaminoglycan, facilitates neutrophil recruitment based on the reduction of neutrophil infiltration in mice in which the overall sulfation of the chains was reduced by selective inactivation of N-acetylglucosamine N-deacetylase-N-sulfotransferase (Ndst1) in endothelial cells. Here we show that inactivation of uronyl 2-O-sulfotransferase in endothelial cells (Hs2st), an enzyme that acts downstream from Ndst1, results in enhanced neutrophil recruitment in several models of acute inflammation. Enhanced neutrophil infiltration resulted in part from reduced rolling velocity under flow both in vivo and in vitro, which correlated with stronger binding of neutrophil L-selectin to mutant endothelial cells. Hs2st-deficient endothelial cells also displayed a striking increase in binding of IL-8 and macrophage inflammatory protein-2. The enhanced binding of these mediators of neutrophil recruitment resulted from a change in heparan sulfate structure caused by increased N-sulfation and 6-O-sulfation of glucosamine units in response to the decrease in 2-O-sulfation of uronic acid residues. This gain-of-function phenotype provides formidable evidence demonstrating the importance of endothelial heparan sulfate in inflammation and suggests a novel enzyme target for enhancing the innate immune response.

Regulation of eosinophil trafficking by SWAP-70 and its role in allergic airway inflammation.

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70-deficient (Swap-70(-/-)) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70(-/-) eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70(-/-) eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70(-/-) eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70(-/-) mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

High-fat diet promotes lung fibrosis and attenuates airway eosinophilia after exposure to cockroach allergen in mice.

Obesity is an important risk factor for asthma but the mechanistic basis for this association is not well understood. In the current study, the impact of obesity on lung inflammatory responses after allergen exposure was investigated. C57BL/6 mice maintained on a high-fat diet (HFD) or a normal diet (ND) after weaning were sensitized and challenged with cockroach allergen (CRA). Airway inflammation was assessed based on inflammatory cell recruitment, measurement of lung Th1-Th2 cytokines, chemokines, eicosanoids, and other proinflammatory mediators as well as airway hyperresponsiveness (AHR). CRA-challenged mice fed a HFD exhibited significantly decreased allergen-induced airway eosinophilia along with reduced lung IL-5, IL-13, LTC4, CCL11, and CCL2 levels as well as reduced mucus secretion and smooth muscle mass compared to ND fed mice. However, allergen-challenged HFD fed mice demonstrated significantly increased PAI-1 and reduced PGE2 levels in the lung relative to corresponding ND fed mice. Interestingly, saline-exposed HFD fed mice demonstrated elevated baseline levels of TGF-ß1, arginase-1, hypoxia-inducible factor-1α, and lung collagen expression associated with decreased lung function compared to corresponding ND fed mice. These studies indicate that a HFD inhibits airway eosinophilia while altering levels of PAI-1 and PGE2 in response to CRA in mice. Further, a HFD can lead to the development of lung fibrosis even in the absence of allergen exposure which could be due to innate elevated levels of specific profibrotic factors, potentially affecting lung function during asthma.

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation.

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside ß1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice.

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis.

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF(164) to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF(164), reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

Allergen-induced airway remodeling is impaired in galectin-3-deficient mice.

The role played by the beta-galactoside-binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-beta were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.

Deficiency of endothelial heparan sulfates attenuates allergic airway inflammation.

The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1(f/f)TekCre(+) (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-beta1, and eotaxin levels, as well as decreased expression of TGF-beta1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1(f/f)TekCre(-) wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-beta1.

Chronic allergen challenge induces pulmonary extramedullary hematopoiesis.

Allergic inflammation is associated with increased generation and trafficking of inflammatory cells, especially eosinophils, to sites of inflammation. The effect of acute versus chronic airway allergen challenge on hematopoietic activity in the bone marrow (BM) and lungs was investigated using murine models of allergic airway inflammation. Acute allergen challenge induced proliferation of BM cells and significantly increased generation of eosinophil, but not multipotent, granulocyte-macrophage (GM), or B-lymphocyte progenitor cells. However, no hematopoietic activity was observed in the lungs. With chronic challenge, BM cells failed to proliferate, but exhibited increased capacity to generate multipotent as well as eosinophil, GM, and B-lymphocyte progenitors. In addition, increased generation of eosinophil- and GM-specific progenitors was observed in the lungs. Although no differences were observed in their ability to roll on BM endothelium in vitro or in vivo, CD34-enriched hematopoietic/stem progenitor cells (HSPCs) from chronic-, but not acute-, challenged mice demonstrated reduced migration across BM endothelial cells associated with decreased CXCR4 expression. Overall, these studies demonstrate that chronic allergen exposure can alter BM homing due to decreased transendothelial migration enabling noninteracting HSPCs to egress out of the BM and recruit to sites of inflammation such as the airways, resulting in extramedullary hematopoiesis.

Procedures to Evaluate Inflammatory and Pathological Changes During Allergic Airway Inflammation.

Cellular inflammation, with elevated levels of Th1/Th2 cytokines, airway mucus hypersecretion, and thickening of the airway smooth muscle, are characteristic features of the allergic lung. Assessment of pathophysiological changes in allergic lungs serves as an important tool to determine disease progression and understand the underlying mechanisms of pathogenesis. This can be achieved by evaluating the lung tissue for inflammation and airway structural changes along with the measurement of important pro-inflammatory mediators such as Th1/Th2 cytokines and eotaxins. This chapter describes procedures to histologically evaluate inflammatory and pathological changes observed during allergic airway inflammation using lung tissue from mice.

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin.

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation-dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.