Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Two simian virus 40 transcriptional promoter elements, the 72-base pair (bp) repeat and the 21-bp repeat region, induce chromatin structures of increased DNase I sensitivity when transposed elsewhere in the viral genome. The induction of a sufficiently long stretch of DNase I-sensitive chromatin leads to the appearance of a visible nucleosome-free region.

In intact cells, mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 is rapidly activated by various cytokines, stresses, and chemotactic factors. The small heat shock protein p27 has been shown to be a substrate for MAPKAP kinase 2. Recently, we identified a novel substrate, designated p60, for MAPKAP kinase 2 in human neutrophils (Zu, Y.-L., Ai, Y., Gilchrist, A., Labadia, M. E., Sha'afi, R. I., and Huang, C.-K. (1996) Blood 87, 5287-5296). To further understand the signaling pathway of MAPKAP kinase 2, we have purified p60 from a heat-treated neutrophil lysate by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis. Microsequencing of five peptides derived from purified p60 indicates that p60 is lymphocyte-specific protein 1 (LSP1). Furthermore antibodies specific for human and mouse LSP1 react with human and mouse p60. The sequence of human LSP1 indicates two serine residues at positions 204 and 252 as potential phosphorylation sites. The amino acid sequences surrounding these two sites are in agreement with the consensus sequence (Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa) for phosphorylation by MAPKAP kinase 2. Both serine residues in human LSP1 and the corresponding conserved serine residues in mouse LSP1 are in the basic C-terminal F-actin binding domain. Various fusion proteins of wild type and truncated mouse LSP1 with glutathione S-transferase were tested for their capacity to be phosphorylated by MAPKAP kinase 2. The results indicate that LSP1 is a substrate for MAPKAP kinase 2 in vitro and that the phosphorylation sites are located in the basic C-terminal domain of LSP1. Because both the small heat shock proteins and LSP1 are F-actin binding proteins, these results suggest a role for MAPKAP kinase 2 in the regulation of cytoskeletal structure or function.A variety of extracellular stimuli activate mitogen-activated protein kinases (MAPK) 1 through an intracellular kinase cascade, which includes MAP kinase kinase kinase and MAP kinase kinase. The MAPK family includes members of the p42/p44 MAPKs (ERK1 and 2), the p54 MAPKs (SAPKs or JNKs), and the p38 MAPKs (1-7).MAP kinase-activated protein (MAPKAP) kinase 2 was originally identified as a substrate for the p42/p44 MAPKs in vitro (8). However, recent data indicate that in intact cells, the upstream kinase that regulates MAPKAP kinase 2 is p38 MAPK (9 -12). Treatment of cells with endotoxin, interleukin-1, tumor necrosis factor, or various stress stimuli activate p38 MAPK and MAPKAP kinase 2 (9 -12). In human neutrophils MAPKAP kinase 2 is also activated by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate 13-acetate (13). The function of MAPKAP kinase 2 is not known, but its upstream kinase p38 MAPK has been proposed to be involved in the biosynthesis of inflammatory cytokines (14), apoptosis (15), and platelet aggregation (16). MAPKAP kinase 2 contains a C-terminal autoinhibitory domain (17)(18)(19) and is activated by phosphorylation at multiple sites (18,19).MAPKAP kinase 2 can phosphorylate several ...

The study of T cell activation, proliferation, and effector function has been in vitro cultures of clonal T cell lines (1-3). Growth of such cell lines depends on periodic stimulation with growth factors, including IL-2, and with antigen that is recognized by the Ti/CD3 cell surface complex. It is believed that these IL-2-dependent, antigen-specific T cell lines, which function in vitro as helper or cytolytic cells, are normal counterparts of the T lymphocytes that perform these important effector and regulatory roles in vivo .To identify genes and gene products involved in the molecular mechanisms of T cell activation and effector function, we have begun a systematic search for cDNA clones expressed in functional cytolytic and Th cell lines but not in cell lines of other lineages . Here we report the structure of two overlapping cDNA clones that represent a gene, designated 519, that is expressed in 10 functional T cell lines but not in 14 established tumor lines, including 6 T cell tumor lines. We also show that the expression of 519 mRNA in PBL is increased more than 10-fold by mitogenic or antigenic stimulation. This increase is first detected 3-5 d after initiation of the culture. It is thus possible that the 519 gene product is involved in the activation and subsequent proliferation and/or differentiation of resting T cells. The data also show that functional T cell lines express gene products that are not readily detected in long term T cell tumor lines. Materials and MethodsCell Lines. The characterization and growth of the funct have been described (4, 5).Construction of cDNA Libraries. Constructi specific sequences was as described (6, 7) with minor modifications. Briefly, total cytoplasmic RNA was extracted from the CD8+, HLA-A2-specific functional cytolytic T cell line AH2 (4) by NP-40 lysis, and poly(A)+ mRNA was prepared by oligo(dT) chromatography (8). Single-stranded cDNA was synthesized using reverse transcriptase primed with i T cell lines and clones