Kegg Pathway: Leukocyte transendothelial migration

Int J Biol Sci. 2009; 5(7): 695-705
van Buul JD, van Alphen FP, Hordijk PL

The majority of the Leukocytes cross the endothelial lining of the vessels through cell-cell junctions. The junctional protein Vascular Endothelial (VE)-cadherin is transiently re-distributed from sites of cell-cell contacts during passage of Leukocytes. VE-cadherin is part of a protein complex comprising p120-catenin and beta-catenin as intracellular partners. Beta-catenin connects VE-cadherin to alpha-catenin. This VE-cadherin-catenin complex is believed to dynamically control endothelial cell-cell junctions and to regulate the passage of Leukocytes, although not much is known about the role of alpha- and beta-catenin during the process of transendothelial migration (TEM). In order to study the importance of the interaction between alpha- and beta-catenin in TEM, we used a cell-permeable version of the peptide encoding the binding site of alpha-catenin for beta-catenin (S27D). The data show that S27D interferes with the interaction between alpha- and beta-catenin and induces a reversible decrease in electrical resistance of the endothelial monolayer. In addition, S27D co-localized with beta-catenin at cell-cell junctions. Surprisingly, transmigration of neutrophils across endothelial monolayers was blocked in the presence of S27D. In conclusion, our results show for the first time that the association of alpha-catenin with the cadherin-catenin complex is required for efficient Leukocyte TEM.

Eur J Immunol. 2009 Oct 28;
Nakhaei-Nejad M, Zhang QX, Murray AG

Leukocyte movement from the blood to the tissues is a fundamental process in acute and chronic inflammatory diseases. While the role of endothelial cells (EC) to recruit Leukocytes to sites of inflammation is well known, the mechanisms that control remodeling of EC shape and adhesive contacts during Leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of IQGAP1, an adaptor protein that binds to F-actin and microtubules (MT) at interendothelial junctions, during lymphocyte TEM. EC IQGAP1 knockdown decreases MT tethered to the adherens junction (AJ), and decreases lymphocyte TEM to approximately 70% (p<0.05) vs. control. Similarly, loss of AJ-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM to approximately 65% of control (p<0.01). Confocal microscopy imaging indicates that EC IQGAP1 knockdown and MT depolymerization both result in lymphocyte accumulation above the VE-cadherin junctions and reduces the fraction of adherent lymphocytes that complete diapedesis across interendothelial cell junctions. However, we observe no change in VE-cadherin gap formation underlying adherent lymphocytes among control, IQGAP1 knockdown, or MT deploymerized EC monolayers. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions. Further, IQGAP1 is involved in junction remodeling required for efficient lymphocyte diapedesis, independent of VE-cadherin gap formation.

Cell Mol Bioeng. 2008 Sep 1; 1(2-3): 189-200
Liang S, Hoskins M, Khanna P, Kunz RF, Dong C

The primary cause of cancer mortality is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites. Tumor cell adhesion to blood vessel endothelium (EC) and subsequent transendothelial migration within the circulation are critical components of the metastasis cascade. Previous studies have shown polymorphonuclear neutrophils (PMNs) may facilitate melanoma cell adhesion to the EC and subsequent extravasation under flow conditions. The melanoma cell-PMN interactions are found to be mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells and beta(2) integrin on PMNs and by endogenously secreted interleukin 8 (IL-8) within the tumor-Leukocyte microenvironment. In this study, the effects of fluid convection on the IL-8-mediated activation of PMNs and the binding kinetics between PMNs and melanoma cells were investigated. Results indicate that the shear rate dependence of PMN-melanoma cell adhesion and melanoma cell extravasation is due, at least partly, to the convection of tumor-secreted proinflammatory cytokine IL-8.

Curr Opin Hematol. 2009 Oct 27;
Woodfin A, Voisin MB, Nourshargh S

PURPOSE OF REVIEW: As the migration of neutrophils from blood to inflamed tissues is an essential component of innate immunity and a key contributing factor to the pathogenesis of inflammatory disorders, this aspect of Leukocyte biology continues to be a highly dynamic field of research. This review summarizes recent findings in this area, focusing on the mechanisms that mediate neutrophil transmigration, an area where significant progress has been made. RECENT FINDINGS: The topics to be covered will include responses that are prerequisite to neutrophil migration through venular walls, such as Leukocyte luminal crawling and cellular and molecular changes in Leukocytes and endothelial cells (e.g. formation of protrusions) that collectively support Leukocyte transendothelial cell migration. Advances in both paracellular and transcellular neutrophil migration through endothelial cells will be discussed, addressing the associated roles and regulation of expression of endothelial cell luminal and junctional adhesion molecules. Beyond the endothelium, migration through the vascular pericyte coverage and basement membrane will be reviewed. SUMMARY: The unquestionable role of neutrophils in the development and progression of inflammatory conditions suggests that a better understanding of the tissue-specific and stimulus-specific mechanisms that mediate this response may identify novel pathways that could be exploited for the development of more specific anti-inflammatory interventions.

Biomaterials. 2010 Jan; 31(2): 366-74
Valois CR, Braz JM, Nunes ES, Vinolo MA, Lima EC, Curi R, Kuebler WM, Azevedo RB

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. transendothelial migration of Leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of Leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 x 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in Leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications.

Regul Pept. 2009 Oct 1;
Helle KB

Chromogranin A (CgA) and secretogranin II (SgII) of the granin family of uniquely acidic proteins secreted from elements of the diffuse neuroendocrine system are also produced by cells involved in inflammation. CgA and the CgA-derived peptides vasostatin-I and catestatin are products of polymorphonuclear neutrophils accumulating at sites of injury or infections while SgII and the Sg II-derived secretoneurin may contribute to neurogenic inflammation when released from sensory nerve terminals. This review is directed towards vasostatin-I, catestatin and secretoneurin as modulators of cells and tissues associated with inflammatory conditions. The accumulated literature indicates that concerted effects of vasostatin-I and catestatin may be relevant for the first-line host-defence against invading microorganisms, contrasting the apparent lack of antibacterial potencies in secretoneurin. Oppositely directed effects of vasostatin-I and secretoneurin on endothelial permeability and transendothelial extravasation are particularly striking. While vasostatin-I protects the integrity of the endothelial barrier against the disruptive effects of proinflammatory agents, secretoneurin activates transendothelial extravasation, chemotaxis and migration of Leukocytes. Oppositely directed effects of vasostatin-I and secretoneurin on formation of blood vessels are also indicated, vasostatin-I inhibiting angiogenetic parameters while secretoneurin activates not only angiogenesis but also vascularization.

J Neuroimmunol. 2009 Oct 30; 215(1-2): 49-64
Liu KK, Dorovini-Zis K

Chemokines have emerged as important mediators of Leukocyte recruitment to the CNS across the normally restrictive blood-brain barrier (BBB). In the present study we investigated the regulation of CXCL12 and its receptor, CXCR4, expression in human brain microvessel endothelial cells (HBMEC) and the effects of CXCL12 on the adhesion and migration of CD4+ and CD8+ T lymphocytes across HBMEC monolayers. Resting HBMEC constitutively expressed CXCL12 and CXCR4. Treatment with TNF-alpha, IFN-gamma, IL-1beta and LPS downregulated CXCL12 and CXCR4 expression and CXCL12 ligation induced internalization of CXCR4. The minimal adhesion and migration of CD4+ and CD8+ T lymphocytes across resting HBMEC were increased following cytokine treatment of HBMEC. CXCL12 gradients further enhanced adhesion of both T cell subsets to activated HBMEC and migration across resting monolayers. A greater number of CD8+ T lymphocytes adhered and migrated across activated HBMEC compared to CD4+ T cells. These studies provide insight into the regulation of CXCL12 and CXCR4 expression in cerebral EC and indicate an important role for CXCL12 in T cell subset recruitment across the BBB in CNS inflammation.

Circulation. 2009 Sep 1; 120(9): 774-84
Seehaus S, Shahzad K, Kashif M, Vinnikov IA, Schiller M, Wang H, Madhusudhan T, Eckstein V, Bierhaus A, Bea F, Blessing E, Weiler H, Frommhold D, Nawroth PP, Isermann B

BACKGROUND: Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. METHODS AND RESULTS: De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM(Pro/Pro)ApoE(-/-) and FVL(Q/Q)ApoE(-/-) mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. CONCLUSIONS: Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.

Cell Mol Life Sci. 2009 Oct; 66(20): 3375-85
Myrsky E, Caja S, Simon-Vecsei Z, Korponay-Szabo IR, Nadalutti C, Collighan R, Mongeot A, Griffin M, Mäki M, Kaukinen K, Lindfors K

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.

FASEB J. 2009 Nov; 23(11): 3906-16
O'Boyle G, Mellor P, Kirby JA, Ali S

Interaction between chemokines and heparan sulfate (HS) is essential for Leukocyte recruitment during inflammation. Previous studies have shown that a non-HS-binding mutant form of the inflammatory chemokine CCL7 can block inflammation produced by wild-type chemokines. This study examined the anti-inflammatory mechanism of a non-HS-binding mutant of the homeostatic chemokine CXCL12. Initial experiments demonstrated that mutant CXCL12 was an effective CXCR4 agonist. However, this mutant chemokine failed to promote transendothelial migration in vitro and inhibited the haptotactic response to wild-type CCL7, CXCL12, and CXCL8, and naturally occurring chemoattractants in synovial fluid from the rheumatoid synovium, including CCL2, CCL7, and CXCL8. Notably, intravenous administration of mutant CXCL12 also inhibited the recruitment of Leukocytes to murine air pouches filled with wild-type CXCL12. Following intravenous administration, wild-type CXCL12 was cleared from the circulation rapidly, while the mutant chemokine persisted for >24 h. Chronic exposure to mutant CXCL12 in the circulation reduced Leukocyte-surface expression of CXCR4, reduced the chemotactic response of these cells to CXCL12, and inhibited normal chemokine-mediated induction of adhesion between the alpha4beta1 integrin, VLA-4, and VCAM-1. These data demonstrate that systemic administration of non-HS-binding variants of CXCL12 can mediate a powerful anti-inflammatory effect through chemokine receptor desensitization.

Blood. 2009 Oct 8; 114(15): 3335-42
Urbinati C, Nicoli S, Giacca M, David G, Fiorentini S, Caruso A, Alfano M, Cassetta L, Presta M, Rusnati M

The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV(+) ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.

Am J Physiol Renal Physiol. 2009 Aug 5;
Sheikh-Hamad D

The mammalian homologue of the fish calcium regulatory hormone, stanniocalcin-1 (STC1) is ubiquitously expressed and likely functions in an autocrine/paracrine fashion. Mammalian STC1 does not appear to exert significant effects on serum calcium and its physiologic role remains to be determined. In macrophages, STC1: decreases intracellular calcium and cell mobility; attenuates the response to chemoattractants; and diminishes superoxide generation through induction of UCP2. In cytokine-treated endothelial cells, STC1: attenuates superoxide generation and the activation of inflammatory pathways (JNK and NF-kappaB); it maintains the expression of tight junction proteins, preserving endothelial monolayer seal; and decreases transendothelial migration of Leukocytes. Combined, the effects of STC1 on endothelial cells and macrophages predict potent anti-inflammatory action. Indeed, applying the anti-glomerular basement membrane (GBM) glomerulonephritis model to STC1 transgenic mice that display increased expression of STC1 transgene in endothelial cells and macrophages yields renal protection. Our data suggest that STC1 activates anti-oxidant pathways in endothelial cells and macrophages and displays cytoprotective and anti-inflammatory actions. Key words: monocytes/macrophages, endothelium, uncoupling proteins, free radicals.

Thromb Haemost. 2009 Aug; 102(2): 191-7
Chavakis E, Choi EY, Chavakis T

Leukocyte recruitment plays a major role in the immune response to infectious pathogens and during inflammatory and autoimmune disorders. The process of Leukocyte extravasation from the blood into the inflamed tissue requires a complex cascade of adhesive events between the Leukocytes and the endothelium including Leukocyte rolling, adhesion and transendothelial migration. Leukocyte-endothelial interactions are mediated by tightly regulated binding interactions between adhesion receptors on both cells. In this regard, Leukocyte adhesion onto the endothelium is governed by Leukocyte integrins and their endothelial counter-receptors of the immunoglobulin superfamily. The present review will focus on novel aspects with respect to the modulation of the Leukocyte adhesion cascade.

Circ Res. 2009 Jul 31; 105(3): 223-30
Muller WA

A great deal of progress has been made recently in understanding the molecules and mechanisms that regulate transendothelial migration of Leukocytes, or diapedesis, a critical step in the inflammatory response. This review focuses mainly on the active role of the endothelial cell in this process as it occurs at endothelial cell borders. It discusses some of the many molecules that have been reported to play a role in transendothelial migration and asks why so many molecules seem to be involved. The concept is emerging that diapedesis itself can be dissected into sequential steps controlled by specific molecule(s) at the endothelial cell border. Several mechanisms have been shown to play a critical role in transendothelial migration including signals derived from clustering of apically disposed intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. A hypothesis that integrates the various known mechanisms is proposed.

J Immunol. 2009 Sep 1; 183(5): 3204-11
Zabel BA, Wang Y, Lewén S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ

CXCR7 binds chemokines CXCL11 (I-TAC) and CXCL12 (SDF-1) but does not act as a classical chemoattractant receptor. Using CCX771, a novel small molecule with high affinity and selectivity for CXCR7, we found that, although CXCR7 is dispensable for "bare filter" in vitro chemotaxis, CXCR7 plays an essential role in the CXCL12/CXCR4-mediated transendothelial migration (TEM) of CXCR4(+)CXCR7(+) human tumor cells. Importantly, although CXCL11 is unable to stimulate directly the migration of these cells, it acts as a potent antagonist of their CXCL12-induced TEM. Furthermore, even though this TEM is driven by CXCR4, the CXCR7 ligand CCX771 is substantially more potent at inhibiting it than the CXCR4 antagonist AMD3100, which is more than 100 times weaker at inhibiting TEM when compared with its ability to block bare filter chemotaxis. Far from being a "silent" receptor, we show that CXCR7 displays early hallmark events associated with intracellular signaling. Upon cognate chemokine binding, CXCR7 associates with beta-arrestin2, an interaction that can be blocked by CXCR7-specific mAbs. Remarkably, the synthetic CXCR7 ligand CCX771 also potently stimulates beta-arrestin2 recruitment to CXCR7, with greater potency and efficacy than the endogenous chemokine ligands. These results indicate that CXCR7 can regulate CXCL12-mediated migratory cues, and thus may play a critical role in driving CXCR4(+)CXCR7(+) tumor cell metastasis and tissue invasion. CXCR7 ligands, such as the chemokine CXCL11 and the newly described synthetic molecule CCX771, may represent novel therapeutic opportunities for the control of such cells.

Proc Natl Acad Sci U S A. 2009 Aug 25; 106(34): 14552-7
Di Lorenzo A, Fernández-Hernando C, Cirino G, Sessa WC

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1(-/-) mice showed a markedly reduced edema versus Akt2(-/-) and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect Leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates Leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1(-/-) versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and Leukocyte extravasation.

BMC Genomics. 2009; 10: 298
Rush C, Nyara M, Moxon JV, Trollope A, Cullen B, Golledge J

BACKGROUND: An animal model commonly used to investigate pathways and potential therapeutic interventions relevant to abdominal aortic aneurysm (AAA) involves subcutaneous infusion of angiotensin II within the apolipoprotein E deficient mouse. The aim of this study was to investigate genes differentially expressed in aneurysms forming within this mouse model in order to assess the relevance of this model to human AAA. RESULTS: Using microarrays we identified genes relevant to aneurysm formation within apolipoprotein E deficient mice. Firstly we investigated genes differentially expressed in the aneurysm prone segment of the suprarenal aorta in these mice. Secondly we investigated genes that were differentially expressed in the aortas of mice developing aneurysms relative to those that did not develop aneurysms in response to angiotensin II infusion. Our findings suggest that a host of inflammation and extracellular matrix remodelling pathways are upregulated within the aorta in mice developing aneurysms. Kyoto Encyclopedia of Genes and Genome categories enriched in the aortas of mice with aneurysms included cytokine-cytokine receptor interaction, Leukocyte transendothelial migration, natural killer cell mediated cytotoxicity and hematopoietic cell lineage. Genes associated with extracellular matrix remodelling, such as a range of matrix metalloproteinases were also differentially expressed in relation to aneurysm formation. CONCLUSION: This study is the first report describing whole genome expression arrays in the apolipoprotein E deficient mice in relation to aneurysm formation. The findings suggest that the pathways believed to be critical in human AAA are also relevant to aneurysm formation in this mouse model. The findings therefore support the value of this model to investigate interventions and mechanisms of human AAA.

Am J Physiol Heart Circ Physiol. 2009 Sep; 297(3): H920-9
Mizuguchi S, Stephen J, Bihari R, Markovic N, Suehiro S, Capretta A, Potter RF, Cepinskas G

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear Leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

J Mol Med. 2009 Aug; 87(8): 765-74
Dodelet-Devillers A, Cayrol R, van Horssen J, Haqqani AS, de Vries HE, Engelhardt B, Greenwood J, Prat A

The blood-brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during Leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).

Methods Enzymol. 2009; 461: 311-32
Shulman Z, Alon R

The mechanisms underlying Leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of Leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, Leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of Leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in Leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow Leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of Leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the Leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of Leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.