Karen Y. Larbi

PECAM-1 (CD31) Homophilic Interaction Up-Regulates α6β1 on Transmigrated Neutrophils In Vivo and Plays a Functional Role in the Ability of α6 Integrins to Mediate Leukocyte Migration through the Perivascular Basement Membrane

Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of α6 integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1–mediated increased expression of α6β1 on transmigrating neutrophils in vivo. An anti-α6 integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1β–stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1–deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1β (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS® analysis of neutrophils demonstrated increased expression of α6β1 on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of α6 as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of α6β1 on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of α6β1 on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1–mediated neutrophil migration through the PBM.

Matrix Metalloproteinase-9 Deficiency Results in Enhanced Allergen-Induced Airway Inflammation

Matrix metalloproteinases (MMPs) are a large family of endopeptidases that proteolytically degrade extracellular matrix. Many different cells produce MMP-9, and levels have been shown to be up-regulated in patients with allergic asthma. The aim of this study was to investigate the in vivo role of MMP-9 during allergen-induced airway inflammation. Acute allergic pulmonary eosinophilia was established in MMP-9 knockout (KO) and wild-type (WT) control mice by sensitization and challenge with OVA. Cell recruitment was significantly increased in both bronchoalveolar lavage (BAL) and lung tissue compartments in MMP-9 KO mice compared with WT mice. This heightened cell recruitment was primarily due to increased eosinophils and Th2 cells in the BAL and lung tissue of MMP-9 KO mice in comparison with WT controls. Moreover, levels of the Th2 cytokines, IL-4 and IL-13, and the chemokines eotaxin/CCL11 and macrophage-derived chemokine/CCL22 were substantially increased in MMP-9 KO mice compared with WT after OVA challenge. Resolution of eosinophilia was similar between MMP-9 KO and WT mice, but Th2 cells persisted in BAL and lungs of MMP-9 KO mice for longer than in WT mice. Our results indicate that MMP-9 is critically involved in the recruitment of eosinophils and Th2 cells to the lung following allergen challenge, and suggest that MMP-9 plays a role in the development of Th2 responses to allergen.

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo.

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE−/−) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE−/− mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1β and TNF-α and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE−/− mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1β, KC, and macrophage inflammatory protein-1α in NE−/− mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.

Migration of leukocytes through the vessel wall and beyond

The migration of leukocytes from the vascular lumen to sites of infection and/or injury in the extravascular tissue involves a series of sequential and coordinated molecular and cellular events with the resultant primary response being that of reduced leukocyte velocity within the blood stream, followed by leukocyte firm adhesion to endothelial cells lining the vessel wall and eventually migration through the vessel wall. Despite the growing knowledge of the mechanisms that mediate initial interaction of leukocytes with the endothelium, very little is known about the mechanisms that mediate and regulate leukocyte migration through the venular wall, the endothelium and its associated perivascular basement membrane. This review, whilst giving a brief outline of the stepwise cascade of molecular interactions involved in this process and the methods employed to investigate leukocyte migration in vivo, focuses primarily on mechanisms of leukocyte transmigration, the final step in the process of leukocyte emigration. Furthermore, special emphasis is placed on discussing the process and the mechanisms involved in leukocyte migration through the basement membrane, a structure that presents significant impedance to transmigrating leukocytes but is seldom investigated in the context of leukocyte transmigration in vivo. The review also discusses the growing evidence supporting the concept that leukocyte transmigration is not only a response that describes the passage of leukocytes through the venular wall, but also acts as a means of regulating leukocyte responsiveness beyond the vessel wall, i.e. within the extravascular tissue.

Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo.

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin αvβ3 have been implicated in this process, and in vitro studies have identified αvβ3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and αvβ3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and β3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1β in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-β3 integrin mAbs, 7E3 F(ab′)2 (5 mg/kg) and F11 F(ab′)2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1β-induced leukocyte responses. These findings indicate roles for both PECAM-1 and β3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.

P-selectin mediates IL-13-induced eosinophil transmigration but not eotaxin generation in vivo: a comparative study with IL-4-elicited responses

The study investigated the role of P‐selectin in the responses of eosinophil transmigration and eotaxin generation in vivo elicited by interleukin (IL)‐13, as compared with IL‐4. Two murine models of leukocyte transmigration were used, migration into cytokine‐stimulated peritoneal cavities and through stimulated cremasteric venules, as observed by intravital microscopy. In mice lacking P‐selectin, eosinophil infiltration elicited by the cytokines in the peritonitis model was totally inhibited. In the cremaster muscle, however, although spontaneous leukocyte‐rolling flux and stimulated leukocyte firm adhesion were inhibited by ∼97% and ∼48%, respectively, stimulated transmigration was unaffected. However, IL‐13‐induced leukocyte transmigration was totally blocked in P‐selectin‐deficient mice treated with an anti‐α4 integrin monoclonal antibody (mAb; PS/2). In comparison, treatment of wild‐type mice with the anti‐α4 integrin mAb resulted in only partial suppression of IL‐13‐induced leukocyte transmigration. Significant levels of eotaxin were detected in response to IL‐13/IL‐4 in both tissues in P‐selectin‐deficient animals. In conclusion, the regulatory role of P‐selectin in leukocyte transmigration elicited by IL‐13 appears to be tissue‐specific, a phenomenon that is independent of the ability of the cytokine to stimulate eotaxin generation.

VCAM-1 contributes to rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4: evidence for basal expression of functional VCAM-1 in rat skin

The aim of the present study was to investigate the role of the adhesion pathway α4 integrins/vascular cell adhesion molecule type 1 (VCAM‐1) in rapid eosinophil accumulation induced by the chemoattractants PAF and LTB4. For this purpose we have used an in vivo model of local 111In‐eosinophil accumulation to quantify eosinophil accumulation induced by intradermal administration of platelet‐activating factor (PAF) and leukotriene B4 (LTB4) in rats. Initial experiments carried out over 4 hr demonstrated that intravenous administration of an anti‐VCAM‐1 monoclonal antibody (mAb; 5F10) or an anti‐α4 integrin mAb (TA2) caused a significant reduction in PAF‐ or LTB4‐induced 111In‐labelled eosinophil accumulation. Time–course experiments demonstrated that the anti‐VCAM‐1 mAb was effective at suppressing early phases of the 111In‐labelled eosinophil accumulation induced by PAF and LTB4 (e.g. within the first 60 min). In contrast, 111In‐labelled eosinophil accumulation induced by these chemoattractantswas unaffected by the local administration of the transcriptional inhibitor actinomycin D, suggesting a role for basally expressed VCAM‐1. Indeed, basal expression of VCAM‐1 in rat skin sites was demonstrated by the localization of intravenously administered radiolabelled mAb. The localization of the radiolabelled antibody was not altered in skin sites injected with PAF or LTB4. Finally, the inhibitory effects seen with the anti‐VCAM‐1 mAb were enhanced when the antibody was co‐injected into rats with an anti‐intercellular adhesion molecule‐1 (ICAM‐1) mAb (1A29). The combination of these two mAb also caused a significant inhibition of PAF‐induced oedema, as quantified by the local accumulation of 125I‐labelled human serum albumin. The results indicate a role for α4 integrins/VCAM‐1 and ICAM‐1, in PAF‐ and LTB4‐induced eosinophil accumulation in vivo and suggest that basally expressed VCAM‐1 may have a functional role in rapid accumulation of eosinophils induced by chemoattractants.

Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils

Wang et al. 2006. J. Exp. Med. doi:10.1084/jem.20051210 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20051210%26rft_id%253Dinfo%253Apmid%252F16754715%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%