VLA-4 blockade suppresses endotoxin-induced uveitis: in vivo evidence for functional integrin up-regulation.

Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammatory diseases and is mediated in part by the leukocyte integrin, VLA-4, which binds to endothelial vascular cell adhesion molecule (VCAM) -1. Here, we investigate VLA-4's role in endotoxin-induced uveitis (EIU). At various time points (6-48 h) after EIU induction, the severity of the inflammation was evaluated by quantifying cell and protein content in the aqueous fluid, firm leukocyte adhesion in the retinal vessels, and the number of extravasated leukocytes into the vitreous. Functional activation of VLA-4 in vivo was investigated in our previously introduced autoperfused micro flow chamber assay. Firm adhesion of EIU leukocytes to immobilized VCAM-1 under physiological blood flow conditions was significantly increased compared with normal controls (P<0.05), suggesting an important role for VLA-4 in EIU. VLA-4 blockade in vivo significantly suppressed all uveitis-related inflammatory parameters studied, decreasing the clinical score by 45% (P<0.01), protein content in the aqueous fluid by 21% (P<0.01), retinal leukostasis by 68% (P<0.01), and leukocyte accumulation in the vitreous by 75% (P<0.01). Our data provide novel evidence for functional up-regulation of VLA-4 during EIU and suggest VLA-4 blockade as a promising therapeutic strategy for treatment of acute inflammatory eye diseases.

L-selectin shedding regulates leukocyte recruitment.

The physiologic role of L-selectin shedding is unknown. Here, we investigate the effect of L-selectin shedding on firm adhesion and transmigration. In a tumor necrosis factor alpha-induced model of inflammation, inhibition of L-selectin shedding significantly increased firm adhesion and transmigration by a lymphocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1-dependent mechanism. We examined the quality of leukocyte rolling and L-selectin-mediated signaling. Blockade of L-selectin shedding significantly reduced the "jerkiness" of leukocyte rolling, defined as the variability of velocity over time. A low level of jerkiness was also observed in the rolling of microbeads conjugated with L-selectin, a model system lacking the mechanism for L-selectin shedding. Inhibition of L-selectin shedding potentiated activation of LFA-1 and Mac-1 induced by L-selectin cross-linking as shown by activation epitope expression and binding of ICAM-1-conjugated beads. We conclude that inhibition of L-selectin shedding increases leukocyte adhesion and transmigration by (a) increasing leukocyte exposure to the inflamed endothelium by decreasing jerkiness and (b) promoting leukocyte activation by outside-in signaling. These observations help to resolve the apparent discrepancy between the minor contribution of L-selectin to rolling and the significant leukocyte recruitment defect in L-selectin knockout mice.

ApoE deficiency compromises the blood brain barrier especially after injury.

BACKGROUND: Apolipoprotein E (apoE) mediates lipoprotein uptake by receptors such as the LDL receptor (LDLR). The isoform apoE4 has been linked to Alzheimer's disease and to poor outcomes after brain injury. Astrocytes that induce blood brain barrier (BBB) properties in endothelium also produce apoE. We decided to investigate the role of apoE in BBB function and in the restoration of BBB after brain injury. MATERIALS AND METHODS: Wild-type (WT) mice and mice deficient in apoE or LDLR were fed normal chow or diets rich in fat and cholesterol. The BBB leakage was determined through injection of Evans blue dye and measurement of the amount of dye extravasated in the brains 3 hours later. Brain injury was induced by applying dry ice directly onto the excised parietal region of the brain. The mice were given 7 days to recover. In some experiments, peroxidase was infused to observe the site of leakage by histology. RESULTS: We found 70% more spontaneous leakage of injected Evans blue dye in the brains of apoE-/- mice than in wild type. This increase in permeability appeared selective for the brain. The leaky BBB in apoE-/- mice may provide an explanation for the neurological deficits seen in these animals. In an established model of BBB leakage induced by trauma (cold injury), the apoE-/- mice showed even more compromised BBB function, compared with WT mice, suggesting that apoE is important for BBB recovery. No deficit in BBB was observed in injured LDLR-/- mice, even on Western Diet. In contrast, higher plasma cholesterol levels in apoE-/- mice further increased BBB leakage after injury. We extracted 5x more Evans blue from these brains than from WT. In the injury model, injection of peroxidase resulted in prominent retention of this protein in the cortex of apoE-/- but not in WT. CONCLUSIONS: Our results show that the combination of loss of apoE function with high plasma cholesterol and especially brain injury results in dramatic BBB defects in the cortex and may explain in part the importance of apoE in Alzheimer's disease and in successful recovery from brain injury.

The CD24/P-selectin binding pathway initiates lung arrest of human A125 adenocarcinoma cells.

Carbohydrates on tumor cells have been shown to play an important role in tumor metastasis. We demonstrated before that CD24, a Mr 35,000-60,000 mucine-type glycosylphosphatidylinositol-linked cell surface molecule, can function as ligand for P-selectin and that the sialylLex carbohydrate is essential for CD24-mediated rolling of tumor cells on P-selectin. To investigate the role of both antigens more closely, we transfected human A125 adenocarcinoma cells with CD24 and/or fucosyltransferase VII (Fuc TVII) cDNAs. Stable transfectants expressed CD24 and/or sialylLex. Biochemical analysis confirmed that in A125-CD24/FucTVII double transfectants, CD24 was modified with sialylLex. Only double transfectants showed rolling on P-selectin in vivo. When injected into mice, double transfectants arrested in the lungs, and this step was P-selectin dependent because it was strongly enhanced in lipopolysaccharide (LPS) pretreated wild-type mice but not in P-selectin knockout mice. CD24 modified by sialylLex was required on the tumor cells because the LPS-induced lung arrest was abolished by removal of CD24 from the cell surface by phosphatidylinositol-specific phospholipase C. A125-FucTVII single transfectants expressing sialylLex but not CD24 did not show P-selectin-mediated lung arrest. The sialylLex epitope is abundantly expressed on human carcinomas, and significant correlations between sialylLex expression and clinical prognosis exist. Our data suggest an important role for sialylLex-modified CD24 in the lung colonization of human tumors.

Delayed onset of inflammation in protease-activated receptor-2-deficient mice.

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 +/- 11 to 71 +/- 24% (p < 0.05) and decreased mean rolling velocity from 63 +/- 29 to 32 +/- 2 micrometer/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 +/- 8 to 30 +/- 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0-15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 +/- 3 vs 30 +/- 6%, p < 0.05) and mean rolling velocity was higher (67 +/- 46 vs 52 +/- 36 micrometer/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase.

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes. Controversy exists, however, concerning whether ER has a role outside the nucleus, particularly in mediating the cardiovascular protective effects of oestrogen. Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ER alpha are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ER alpha with PI(3)K.

Role of vascular cell adhesion molecule-1 and fibronectin connecting segment-1 in monocyte rolling and adhesion on early atherosclerotic lesions.

Atherosclerotic lesion development seems to be inflammatory in nature and involves the recruitment of monocytes to the vessel wall. In this study, we investigated the role of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) connecting segment-1 containing the amino acid sequence ILDV as functional ligands for alpha(4)beta(1) integrin (VLA-4) in monocyte rolling and adherence to early atherosclerotic lesions. Carotid arteries of apolipoprotein E-deficient mice were isolated and perfused with monocytes or U937 cells. Cell adhesion was reduced 95+/-4% by monoclonal antibodies HP1/2 and HP2/1, which block VLA-4 binding to both VCAM-1 and FN connecting segment-1. mAb HP1/3 preferentially blocked interaction of VLA-4 with FN but not VCAM-1 and decreased adhesion by 30+/-8%. In contrast, blocking VCAM-1 by perfusing the isolated carotid artery with mAb MK-2.7 reduced adhesion by 75+/-12%. Mononuclear cell adhesion to the early atherosclerotic endothelium was inhibited by 68+/-10% in the presence of EILDVPST but not in the presence of control peptide EIDVLPST. When VLA-4 or VCAM-1 was blocked, more mononuclear cells rolled on early lesions at significantly higher (approximately doubled) rolling velocities. These data demonstrate that (1) blockade of VCAM-1 can abrogate the majority (75+/-12%) of VLA-4-dependent monocyte adhesion on early atherosclerotic endothelia and (2) ILDV peptide interferes with VLA-4 binding to both VCAM-1 and FN and may be useful in limiting monocyte adhesion to atherosclerotic lesions.

Relevance of L-selectin shedding for leukocyte rolling in vivo.

The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 micrometer/seconds in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-alpha treatment 2.5-3 h before the experiment), rolling velocity was 4 micrometer/seconds and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 micrometer/seconds in E-selectin-deficient mice treated with TNF-alpha. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.

CD24 mediates rolling of breast carcinoma cells on P-selectin.

P-selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P-selectin glycoprotein ligand-1 (PSGL-1). Certain PSGL-1 negative tumor cell lines can bind P-selectin under static conditions through the GPI-linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL-1- KS breast carcinoma cells attach to and roll on recombinant P-selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL-1+ cells, such as HL-60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol-phospholipase C (PI-PLC) significantly reduced KS cell rolling on P-selectin. The ability of KS cells to roll on P-selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl-Lewis(x) antigen was also necessary for CD24-mediated rolling on P-selectin. CD24 purified from KS cells supported rolling of P-selectin transfectants, but not L-selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P-selectin-dependent manner. Together our data show that CD24 serves as a ligand for P-selectin under physiological flow conditions. Interaction of tumor cells with P-selectin via CD24 may be an important adhesion pathway in cancer metastasis.

The leukocyte integrin Mac-1 (CD11b/CD18) contributes to binding of human granulocytes to collagen.

Adhesion of polymorphonuclear granulocytes (PMN) to extracellular matrix proteins has been shown to be important for their migration in vitro and is thought to participate in PMN recruitment to sites of inflammation. Isolated human PMN stimulated with PMA were found to adhere best to microtiter wells coated with the novel ECM glycoprotein undulin (27 +/- 3% of PMNs added), followed by fibrinogen (25 +/- 2%), collagen type VI (18 +/- 2%), fibronectin (16 +/- 2%), and laminin (15 +/- 3%). PMN adhesion to other collagens ranged between 3 and 11%. Monoclonal antibodies recognizing CD18 and CD11b subunits of Mac-1 inhibited adhesion of PMN to collagens by an order of magnitude more effectively than to all noncollagenous substrates. F(ab')2 fragments of the anti-CD18 antibody were also able to block adhesion to collagens. Anti-LFA-1 (CD11a) and anti-CD44 antibodies did not significantly reduce adhesion. PMN adhesion was also inhibited by soluble collagens type II and VI (ID50 approximately 75 micrograms/ml). Binding of soluble radiolabeled collagens type II and VI to PMNs was specific and saturable with apparent dissociation constants of 2.2 and 1.9 nM, respectively, and specific binding of collagens type II and VI was almost completely inhibited by anti-CD18, but not by control antibodies. These data indicate that Mac-1 function is required for binding of human PMN to collagens.