Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma

Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNgamma, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies.     

VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.     

Cytokines modulate endothelial cell intracellular signal transduction required for VCAM-1-dependent lymphocyte transendothelial migration

Vascular cell adhesion molecule-1 (VCAM-1) activates endothelial cell NADPH oxidase which catalyzes production of reactive oxygen species (ROS). This activity is required for VCAM-1-dependent lymphocyte migration. The focus of our study was to determine whether these VCAM-1-dependent functions are modulated by cytokines. TGF-beta1 or IFN-gamma pretreatment of mouse endothelial cell lines inhibited VCAM-1-dependent B and T cell transendothelial migration without affecting initial lymphocyte adhesion. Neutralizing anti-TGF-beta1 blocked the effects of TGF-beta1 pretreatment of endothelial cells, whereas addition of anti-TGF-beta1 after TGF-beta1 pretreatment of the endothelial cells did not block TGF-beta1-mediated inhibition. Neutralizing anti-IFN-gamma also blocked the inhibitory effects of IFN-gamma. TGF-beta1 and IFN-gamma blocked migration by inhibiting the VCAM-1-stimulated production of low levels of ROS (0.1-0.9 microM H2O2). These results demonstrate that both TGF-beta1 and IFN-gamma directly affect the endothelial cells' ability to promote lymphocyte migration. IL-4 had differing effects on T and B cells during transmigration. IL-4 augmented T cell migration across the endothelial cell lines but did not affect T cell adhesion. Conversely, IL-4 increased B cell adhesion to the endothelial cell lines without affecting migration. In summary, cytokines can directly modulate microvascular endothelial cell intracellular signaling, demonstrating a new level of cytokine regulation of lymphocyte diapedesis.     

Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase

Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0-24 h. This migration was inhibited by anti-VCAM-1 or anti-alpha4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.     

3,3'-Diindolylmethane decreases VCAM-1 expression and alleviates experimental colitis via a BRCA1-dependent antioxidant pathway

Reactive oxygen species (ROS) exhibit a key role in the pathogenesis of inflammatory bowel disease (IBD). 3,3'-Diindolylmethane (DIM) can protect against oxidative stress in a breast cancer susceptibility gene 1 (BRCA1)-dependent manner. The aim of this study was to examine the therapeutic effects of DIM in experimental colitis and investigate the possible mechanisms underlying its effects on intestinal inflammation. The therapeutic effects of DIM were studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Pathological markers of colitis severity, antioxidant activity, and ROS generation in colonic tissue were measured. The impact of DIM on ROS-induced endothelial vascular cell adhesion molecule 1 (VCAM-1) expression and leukocyte-endothelial cell interaction was further investigated in cultures of endothelial cells and in the TNBS-induced colitis model. Administration of DIM was demonstrated to attenuate experimental colitis, as judged by pathological indices. DIM could effectively stimulate the expression of BRCA1 in vitro and in vivo and reduce ROS generation, leading to the inhibition of VCAM-1 expression and leukocyte-endothelial cell adhesion, and finally resulted in an alleviation of experimental colitis. DIM has shown anti-IBD activity in animal models by inhibiting ROS-induced VCAM-1 expression and leukocyte recruitment via a BRCA1-dependent antioxidant pathway and thus may offer potential treatments for IBD patients.     

Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration

Background

During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1.

Methodology/Principal Findings

In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm² was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration.

Conclusions/Significance

VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Blockade of airway inflammation and hyperresponsiveness by HIV-TAT-dominant negative Ras

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t(1/2) = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3-10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 +/- 91 x 10(3)/ml to 288 +/- 79 x 10(3)/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 +/- 63 x 10(3)/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-gamma, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.     

VCAM-1 signals activate endothelial cell protein kinase Calpha via oxidation

Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 muM H(2)O(2). This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Calpha (PKCalpha) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm(2) laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCalpha or the PKCalpha inhibitors, R?-32-0432 or G?-6976. Phosphorylation of PKCalpha(Thr638), an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 muM H(2)O(2). The anti-VCAM-1-stimulated phosphorylation of PKCalpha(Thr638) was blocked by scavenging of H(2)O(2) and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCalpha. Oxidized PKCalpha is a transiently active form of PKCalpha that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKCalpha activation that is necessary for VCAM-1-dependent transendothelial cell migration.     

Inhibition of airway inflammation and hyperreactivity by an antioxidant mimetic

A catalytic antioxidant, AEOL 10113, was used in a murine model of asthma to test the hypothesis that oxidants contribute to the pathogenesis of asthma. Balb/c mice were immunized and challenged with ovalbumin. AEOL 10113 was administered to the mice by intratracheal instillation during ovalbumin challenges. Enhanced pause as an indicator of airway reactivity and differential cell count of lavage cells as an indicator of airway inflammation were assessed. Lung expressions of the adhesion molecules VCAM-1 and ICAM-1 were measured. We found that treatment of ovalbumin-challenged mice with AEOL 10113 drastically reduced the severity of airway inflammation as evidenced by the reduced numbers of eosinophils, neutrophils, and lymphocytes found in bronchoalveolar lavage fluid. Inhibition of ovalbumin-induced airway inflammation is associated with inhibited expression of VCAM-1, which is a key adhesion molecule responsible for the recruitment of inflammatory cells into the lungs of ovalbumin-challenged mice. In addition, treatment with AEOL 10113 reduced the magnitude of ovalbumin-induced airway hyperreactivity to methacholine. These results suggest that oxidative stress is an important factor in the pathogenesis of asthma and that a synthetic catalytic antioxidant could be effective in the treatment of asthma.     

IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production

IL-11 is a pleiotropic cytokine that induces tissue remodeling with subepithelial fibrosis when expressed in the airway. Its effects on the Th2-dominated airway inflammation that is characteristic of asthma, however, are poorly understood. To characterize the effects of IL-11 on Th2 tissue inflammation, we compared the inflammatory responses elicited by OVA in sensitized mice in which IL-11 is overexpressed in a lung-specific fashion (CC10-IL-11) with that in transgene- wild-type littermate controls. Transgene- and CC10-IL-11 transgene+ mice had comparable levels of circulating Ag-specific IgE after sensitization. OVA challenge of sensitized transgene- mice caused airway and parenchymal eosinophilic inflammation, Th2 cell accumulation, and mucus hypersecretion with mucus metaplasia. Exaggerated levels of immunoreactive endothelial cell VCAM-1, mucin (Muc) 5ac gene expression and bronchoalveolar lavage and lung IL-4, IL-5, and IL-13 protein and mRNA were also noted. In contrast, OVA challenge in CC10-IL-11 animals elicited impressively lower levels of tissue and bronchoalveolar lavage inflammation, eosinophilia, and Th2 cell accumulation, and significantly lower levels of VCAM-1 and IL-4, IL-5, and IL-13 mRNA and protein. IL-11 did not cause a comparable decrease in mucus hypersecretion, Muc 5ac gene expression, or the level of expression of RANTES, monocyte chemoattractant protein-2, or monocyte chemoattractant protein-3. In addition, IL-11 did not augment IFN-gamma production demonstrating that the inhibitory effects of IL-11 were not due to a shift toward Th1 inflammation. These studies demonstrate that IL-11 selectively inhibits Ag-induced eosinophilia, Th2 inflammation, and VCAM-1 gene expression in pulmonary tissues.     

Regulation of cockroach antigen-induced allergic airway hyperreactivity by the CXCR3 ligand CXCL9

Allergic airway disease is characterized by a robust lymphocytic infiltrate, elaboration of Th2-type inflammatory mediators, pulmonary eosinophil accumulation, and airway hyperreactivity. The CXCR3 ligands, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-inducible protein, 10 kDa), are IFN-gamma-inducible, Th1-type chemokines. As CXCL10 has been previously shown to participate in the modulation of allergic inflammation, we were interested in investigating the possible role that CXCL9 may play in this inflammatory response. Expression of CXCL9 was primarily identified in airway epithelial cells by immunohistochemical staining. Airway neutralization of CXCL9 at the time of allergen challenge significantly increased airway hyperreactivity, airway eosinophil accumulation, and IL-4 levels in the bronchoalveolar lavage while significantly decreasing airway levels of IL-12. In contrast, introduction of exogenous CXCL9 into the airway at the time of allergen challenge dramatically reduced airway hyper-reactivity and eosinophil accumulation. Moreover, pulmonary levels of IL-4 were significantly reduced, whereas levels of IL-12 were significantly increased, with exogenous CXCL9 treatment. In lymphocytes restimulated with CXCL9 and allergen in vitro, CXCL9 down-regulated IL-4 expression and up-regulated IFN-gamma expression, suggesting that CXCL9 is able to direct activated lymphocytes toward a Th1-type phenotype. Additionally, CXCL9 was shown to inhibit CC chemokine ligand 11-induced eosinophil chemotaxis in in vitro assays. Taken together, our results demonstrate that the CXCR3 ligand CXCL9 is involved in regulation of the allergic response in the lung by regulation of lymphocyte activation and eosinophil recruitment.     

Expression, regulation, and function of atypical chemerin receptor CCRL2 on endothelial cells

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)β(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.     

The affinity of integrin alpha(4)beta(1) governs lymphocyte migration

The interaction of integrin alpha(4)beta(1) with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of alpha(4)beta(1) for VCAM-1 (activation). To investigate the biological function of alpha(4)beta(1) activation, we isolated Jurkat T cell lines with defective alpha(4)beta(1) activation. Using these cells, we found that alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent cell migration was dramatically reduced in cells with defects in alpha(4)beta(1) activation. These cells required 20 times more VCAM-1 to promote alpha(L)beta(2)-dependent cell migration. This defect was at the level of alpha(4)beta(1) affinity as an activating alpha(4)beta(1) Ab rescued alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent migration. In contrast, migration of alpha(4)beta(1) activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, alpha(4)beta(1) activation determines a set point or threshold at which VCAM-1 can regulate alpha(L)beta(2)-dependent as well as alpha(4)beta(1)-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.     

Various adhesion molecules impair microvascular leukocyte kinetics in ventilator-induced lung injury

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.     

A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture

Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.     

Protectin D1 is generated in asthma and dampens airway inflammation and hyperresponsiveness

Protectins are newly identified natural chemical mediators that counter leukocyte activation to promote resolution of inflammation. In this study, we provide the first evidence for protectin D1 (PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) formation from docosahexaenoic acid in human asthma in vivo and PD1 counterregulatory actions in allergic airway inflammation. PD1 and 17S-hydroxy-docosahexaenoic acid were present in exhaled breath condensates from healthy subjects. Of interest, levels of PD1 were significantly lower in exhaled breath condensates from subjects with asthma exacerbations. PD1 was also present in extracts of murine lungs from both control animals and those sensitized and aerosol challenged with allergen. When PD1 was administered before aeroallergen challenge, airway eosinophil and T lymphocyte recruitment were decreased, as were airway mucus, levels of specific proinflammatory mediators, including IL-13, cysteinyl leukotrienes, and PGD(2), and airway hyperresponsiveness to inhaled methacholine. Of interest, PD1 treatment after aeroallergen challenge markedly accelerated the resolution of airway inflammation. Together, these findings provide evidence for endogenous PD1 as a pivotal counterregulatory signal in allergic airway inflammation and point to new therapeutic strategies for modulating inflammation in asthmatic lung.     

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.     

Inhibition of Th1- and Th2-mediated airway inflammation by the sphingosine 1-phosphate receptor agonist FTY720

The sphingosine 1-phosphate receptor agonist FTY720 is a novel immunomodulator that sequesters lymphocytes in secondary lymphoid organs and thereby prevents their migration to sites of inflammation. However, there is currently no information available on whether this drug affects Th1 or Th2 cell-mediated lung-inflammatory responses. The effect of FTY720 was therefore investigated in a murine airway inflammation model using OVA-specific, in vitro differentiated, and adoptively transferred Th1 and Th2 cells. Both Th1 and Th2 cells express a similar pattern of FTY720-targeted sphingosine 1-phosphate receptors. The OVA-induced Th1-mediated airway inflammation characterized by increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage fluid was significantly inhibited by oral FTY720 treatment. Similarly, FTY720 suppressed the Th2 cell-induced bronchoalveolar lavage fluid eosinophilia and the infiltration of T lymphocytes and eosinophils into the bronchial tissue. Moreover, the Ag-induced bronchial hyperresponsiveness to inhaled metacholine was almost completely blocked. The inhibitory effect of FTY720 on airway inflammation, induction of bronchial hyperresponsiveness, and goblet cell hyperplasia could be confirmed in an actively Ag-sensitized murine asthma model, clearly indicating that Th2 cell-driven allergic diseases such as asthma could benefit from such treatment.