Mechanisms of Acute Eosinophil Mobilization from the Bone Marrow Stimulated by Interleukin 5: The Role of Specific Adhesion Molecules and Phosphatidylinositol 3-Kinase

Mobilization of bone marrow eosinophils is a critical early step in their trafficking to the lung during allergic inflammatory reactions. We have shown previously that the cytokine interleukin (IL)-5, generated during an allergic inflammatory reaction in the guinea pig, acts systemically to mobilize eosinophils from the bone marrow. Here, we have investigated the mechanisms underlying this release process. Examination by light and electron microscopy revealed the rapid migration of eosinophils from the hematopoietic compartment and across the bone marrow sinus endothelium in response to IL-5. Using an in situ perfusion system of the guinea pig hind limb, we showed that IL-5 stimulated a dose-dependent selective release of eosinophils from the bone marrow. Eosinophils released from the bone marrow in response to IL-5 expressed increased levels of β2 integrin and a decrease in L-selectin, but no change in α4 integrin levels. A β2 integrin–blocking antibody markedly inhibited the mobilization of eosinophils from the bone marrow stimulated by IL-5. In contrast, an α4 integrin blocking antibody increased the rate of eosinophil mobilization induced by IL-5. In vitro we demonstrated that IL-5 stimulates the selective chemokinesis of bone marrow eosinophils, a process markedly inhibited by two structurally distinct inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002. Wortmannin was also shown to block eosinophil release induced by IL-5 in the perfused bone marrow system. The parallel observations on the bone marrow eosinophil release process and responses in isolated eosinophils in vitro suggest that eosinophil chemokinesis is the driving force for release in vivo and that this release process is regulated by α4 and β2 integrins acting in opposite directions

A Novel Endothelial L-Selectin Ligand Activity in Lymph Node Medulla That Is Regulated by α(1,3)-Fucosyltransferase-IV

Lymphocytes home to peripheral lymph nodes (PLNs) via high endothelial venules (HEVs) in the subcortex and incrementally larger collecting venules in the medulla. HEVs express ligands for L-selectin, which mediates lymphocyte rolling. L-selectin counterreceptors in HEVs are recognized by mAb MECA-79, a surrogate marker for molecularly heterogeneous glycans termed peripheral node addressin. By contrast, we find that medullary venules express L-selectin ligands not recognized by MECA-79. Both L-selectin ligands must be fucosylated by α(1,3)-fucosyltransferase (FucT)-IV or FucT-VII as rolling is absent in FucT-IV+VII−/− mice. Intravital microscopy experiments revealed that MECA-79–reactive ligands depend primarily on FucT-VII, whereas MECA-79–independent medullary L-selectin ligands are regulated by FucT-IV. Expression levels of both enzymes paralleled these anatomical distinctions. The relative mRNA level of FucT-IV was higher in medullary venules than in HEVs, whereas FucT-VII was most prominent in HEVs and weak in medullary venules. Thus, two distinct L-selectin ligands are segmentally confined to contiguous microvascular domains in PLNs. Although MECA-79–reactive species predominate in HEVs, medullary venules express another ligand that is spatially, antigenically, and biosynthetically unique. Physiologic relevance for this novel activity in medullary microvessels is suggested by the finding that L-selectin–dependent T cell homing to PLNs was partly insensitive to MECA-79 inhibition

Brief Definitive Report The CCR7 Ligand ELC (CCL19) Is Transcytosed in High Endothelial Venules and Mediates T Cell Recruitment

Abstract Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEVderived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (EpsteinBarr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC ( plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest

Kinetics of Eotaxin Generation and Its Relationship to Eosinophil Accumulation in Allergic Airways Disease: Analysis in a Guinea Pig Model In Vivo

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12–24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge

Neutralisation of interleukin-13 in mice prevents airway pathology caused by chronic exposure to house dust mite.

BACKGROUND: Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM). METHODOLOGY/PRINCIPAL FINDINGS: Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R(L)) and dynamic compliance (C(dyn)). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R(L) and the decrease in baseline C(dyn) caused by chronic exposure to inhaled HDM. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction