P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes.

Lymphocyte homing to peripheral lymph nodes (LNs) requires L-selectin. Previous studies, however, suggest that there are L-selectin-independent mechanisms of lymphocyte homing. P-selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed in a selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-selectin interaction, since infusion of an anti-L-selectin mAb together with an anti-PSGL-1 mAb or anti-P-selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.

Matrix metalloproteinase-1 produced by human CXCL12-stimulated natural killer cells.

Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood. In this study, we show that CXCL12 significantly enhanced CD16(+)CD56(+) human peripheral NK-cell invasion into type I collagen by the catalytic activity of matrix metalloproteinase-1 (MMP-1). Confocal immunofluorescence and co-immunoprecipitation studies suggest that MMP-1 colocalized with alpha(2)beta(1) integrin on CXCL-12-stimulated NK-cell surface. The binding of pro-MMP-1 with alpha(2)beta(1) integrin required activation of G(i)-coupled pathway. However, the production of MMP-1 from CXCL12-stimulated NK cells was mediated by p38 and mitogen-activated or extracellular signal-regulation protein kinase kinase 1/2 in a manner independent of the G(i)-coupled pathway. These results suggest that CXCL12/CXCR4 interaction transduces the two signaling pathways to promote NK-cell invasion, which stimulates pericellular degradation of extracellular matrix proteins by membrane-associated MMP-1. The mechanisms would thus play a role in facilitating lymphocyte trafficking and accumulation in tissues during physiological and pathological processes.

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase.

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.