Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.
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BACKGROUND: The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents. RESULTS: In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements. CONCLUSIONS: These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur. (+info)
Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1.
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The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo. (+info)
Severely reduced neutrophil adhesion and impaired host defense against fecal and commensal bacteria in CD18-/-P-selectin-/- double null mice.
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Leukocyte recruitment to sites of inflammation requires the functions of selectins and integrins. P-selectin null (CD62P-/-) mice show a mild and CD18 null (CD18-/-) mice a more severe neutrophil recruitment defect in some inflammatory models. To investigate the possible cooperative interactions between CD18 integrins and P-selectin in mediating neutrophil recruitment, we generated CD18-/-CD62P-/- double null mice. CD18-/-CD62P-/- mice were apparently normal at weaning and fertile but later failed to gain weight, showed increased susceptibility to infection by fecal and commensal bacteria, and survived only 5-6 months. Some CD18-/-CD62P-/- mice showed severe spontaneous skin lesions; most showed neutrophil infiltration in the lungs and liver, and positive bacterial cultures from internal organs. The number and velocity of rolling leukocytes in tumor necrosis factor alpha treated venules of CD18-/-CD62P-/- mice was similar to those in wild-type mice, but neutrophil adhesion was severely reduced. Only 25% of adhered leukocytes were neutrophils in CD18-/-CD62P-/- mice vs. >90% in wild-type, CD62P-/-, and CD18-/- single mutants. Our data show that removing both P-selectin and CD18 integrins from mice leads to severe neutrophil recruitment defects and spontaneous pathology. (+info)
ITAM-based interaction of ERM proteins with Syk mediates signaling by the leukocyte adhesion receptor PSGL-1.
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P-selectin glycoprotein ligand 1 (PSGL-1) is a leukocyte adhesion molecule involved in cell tether and rolling on activated endothelium. Our work shows that PSGL-1 associates with Syk. This association is mediated by the actin-linking proteins moesin and ezrin, which directly interact with Syk in an ITAM-dependent manner. PSGL-1 engagement induces tyrosine phosphorylation of Syk and SRE-dependent transcriptional activity. Treatment of cells with the Syk inhibitor piceatannol and overexpression of either a Syk dead kinase mutant or an ITAM-mutated moesin abrogated PSGL-1-induced transcriptional activation. These data unveil a new functional role for the ERMs (ezrin/radixin/moesin) as adaptor molecules in the interactions of adhesion receptors and intracellular tyrosine kinases and show that PSGL-1 is a signaling molecule in leukocytes. (+info)
Glycosylation-dependent inhibition of cutaneous lymphocyte-associated antigen expression: implications in modulating lymphocyte migration to skin.
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Constitutive E-selectin expression on dermal microvascular endothelial cells plays a critical role in mediating rolling adhesive interactions of human skin-homing T cells and in pathologic accumulation of lymphocytes in skin. The major E-selectin ligand on human skin-homing T cells is cutaneous lymphocyte-associated antigen (CLA), a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1) defined by monoclonal antibody HECA-452. Since HECA-452 reactivity, and not PSGL-1 polypeptide itself, confers the specificity of human T cells to enter dermal tissue, inhibition of HECA-452 expression is a potential strategy for modulating lymphocyte migration to skin. In this study, we examined the efficacy of several well-characterized metabolic inhibitors of glycosylation and of a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose [4-F-GlcNAc]) to alter HECA-452 expression on human CLA(+) T cells and prevent cell tethering and rolling on selectins under shear stress. At concentrations that did not affect PSGL-1 expression, we found that swainsonine (inhibitor of complex-type N-glycan synthesis) had no effect on HECA-452 expression or selectin ligand activity, whereas benzyl-O-N-acetylgalactosamide (BAG; inhibitor of O-glycan biosynthesis) ablated HECA-452 expression on PSGL-1 and significantly lowered selectin ligand activity. We found that 4-F-GlcNAc (putative inhibitor of poly-N-acetyllactosamine biosynthesis) was more potent than BAG at lowering HECA-452 expression and selectin binding. In addition, we show that 4-F-GlcNAc was directly incorporated into native CLA expressed on T cells, indicating direct inhibition on poly-N-acetyllactosamine elongation and selectin-binding determinants on PSGL-1 O-glycans. These observations establish a potential treatment approach for targeting pathologic lymphocyte trafficking to skin and indicate that 4-F-GlcNAc may be a promising agent for treatment of dermal tropism associated with malignancies and inflammatory disorders. (+info)
Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium.
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Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking postfixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere with the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of dissected human umbilical vein tissue. In either experimental model system, both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites. VE-cadherin moved away to different ends of the transmigration site, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 minutes. The relative positions of some junction proteins might rearrange to form a new interendothelial contour after PMNs had transmigrated through multicellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, whereas PECAM-1-containing junctions were opened during PMN transendothelial migration. (+info)
Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.
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Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair. (+info)
CCR4 versus CCR10 in human cutaneous TH lymphocyte trafficking.
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The chemokine receptors (CCRs) CCR4 and CCR10, and the cutaneous lymphocyte antigen (CLA), have each been proposed as critical mediators of skin-specific TH lymphocyte homing in mice and humans. CLA initiates skin homing by mediating E-selectin-dependent tethering and rolling within cutaneous venules, but the specific roles of CCR4 and CCR10 are unclear. We have generated an antihuman CCR10 monoclonal antibody (mAb; 1B5) to illuminate the individual contributions of these molecules. This mAb allows us to compare CCR10, CCR4, and CLA expression within human TH populations. The mAb 1B5 recognizes functional CCR10 expression, as chemotactic responsiveness to cutaneous T-cell-attracting chemokine (CTACK)/CCL27 (a CCR10 ligand) parallels the staining of TH subsets. We find CCR10 expressed by only a minority (approximately 30%) of blood-borne, skin-homing (CLA+/CCR4+) TH cells. However, essentially all members of the relatively small "effector" (CLA+/CCR4+/CD27-/CCR7-) skin-homing TH population express CCR10. Most skin-infiltrating lymphocytes in allergic delayed-type hypersensitivity (DTH) and bacterial chancroid skin lesions express both CCR4 and CLA, but only about 10% express CCR10. This suggests for the 2 models of TH skin homing studied here that CCR10+ TH cells have no advantage over other CLA+/CCR4+ TH cells in homing to cutaneous sites. We conclude that the skin-homing TH compartment is itself divided into distinct subpopulations, the smaller of which expresses both CCR4 and CCR10, and the larger of which expresses only CCR4. Thus, CCR10 is unlikely to be necessary for cutaneous homing of TH cells in the models studied here. CCR10 may instead play a role in the movement of specialized "effector" cutaneous TH cells to and/or within epidermal microenvironments. (+info)