The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via α-actinin: Receptor positioning in microvilli does not require interaction with α-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein α-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified α-actinin to L-selectin (K(d) = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of α-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of α-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including α-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that α-actinin binds directly to L-selectin and that vinculin associates by binding to α-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with α-actinin or vinculin. Surprisingly, this mutant L- selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L- selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of α-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton

Human Spinal Cord Injury Causes Specific Increases in Surface Expression of Beta Integrins on Leukocytes

Spinal cord injury (SCI) activates circulating leukocytes that migrate into the injured cord and bystander organs using adhesion molecule-mediated mechanisms. These cells cause oxidative damage, resulting in secondary injury to the spinal cord, as well as injury to bystander organs. This study was designed to examine, over a 6-h to 2-week period, changes in adhesion molecule surface expression on human peripheral leukocytes after SCI (9 subjects), using as controls 10 uninjured subjects and 6 general trauma patients (trauma controls, TC). Both the percentage of cells expressing a given adhesion molecule and the average level of its expression was quantified for both circulating neutrophils and monocytes. The percentage of neutrophils and monocytes expressing the selectin CD62L was unchanged in TC and SCI patients after injury compared to uninjured subjects. Concurrently, the amount of surface CD62L on neutrophils was decreased in SCI and TC subjects, and on monocytes after SCI. The percentage of neutrophils expressing α4 decreased in TC, but not in SCI, subjects. Likewise, the percentage of neutrophils and monocytes expressing CD11d decreased markedly in TC subjects, but not after SCI. In contrast, the mean surface expression of α4 and CD11d by neutrophils and monocytes increased after SCI compared with uninjured and TC subjects. The percentage of cells and surface expression of CD11b were similar in neutrophils of all three groups, whereas CD11b surface expression increased after SCI in monocytes. In summary, unlike changes found after general trauma, the proinflammatory stimulation induced by SCI increases the surface expression of adhesion molecules on circulating neutrophils and monocytes before they infiltrate the injured spinal cord and multiple organs of patients. Integrins may be excellent targets for anti-inflammatory treatment after human SCI

The distribution of CD45R, CD29 and CD45RO (UCHL1) antigens in mature CD4 positive T-cell leukaemias.

We have studied the expression of antigens characterizing functional T-cell subsets in 32 CD4+ mature T-cell leukaemias. In this analysis we used two monoclonal antibodies (McAb) of the CD45R group (2H4 and GRT22) which have been shown to identify the 'native/virgin' T-cell population that functions as 'suppressor-inducer' cells in vitro, and two McAb, CD29 (4B4) and CD45RO (UCHL1), which characterize non-identical 'memory' cells that proliferate in response to soluble recall antigens and provide help in antigen-specific IgG synthesis. Four groups of CD4+ cases were identified according to this reactivity: (a) 15 CD45R+, CD29+; (b) 13 CD45R-, CD29+; (c) three CD45R-, CD29-; and (d) one case only CD45+, CD29-. The high incidence of coexpression of CD45R and CD29 (47% of cases) is a new finding which contrasts with the mutual exclusion of these antigens on normal CD4+ T-lymphocytes. There was no correlation between subset phenotypes and pathological disease entities. None of the six cases of adult T-cell leukaemia/lymphoma (ATLL), which is known as a disorder of activated 'suppressor-inducer' cells, had the 'expected' CD45R+, CD29- phenotype. Reactivity with UCHL1 showed a good correlation with CD29 in the CD45R- CD29+ cases which included three with ATLL. These results may help in the further characterization of T-cell malignancies according to functional subgroups and may clarify further the role of T-differentiation antigens in health and disease