Expression, recognition and usage of laminin-8 (a4b1g1, Lm-411) by monocytes and neutrophils

Laminins (LNs) are a family of large αβγ heterotrimeric glycoproteins found in all basement membranes (BMs). They are expressed in a tissueand developmental stage-specific manner and implicated in vital cellular functions, including cell adhesion, migration and signaling. So far, eleven laminin chains (5α, 3β and 3γ) that assemble into 15 LN isoforms (LN-1 to 15, Lm-111 to Lm-523) have been identified. LN-8 (α4β1γ1, Lm-411) is a major LN isoform of vascular endothelial BM. Its expression and functional importance in blood platelets and lymphocytes have been documented. However, expression, recognition and utilization of LNs by blood monocytes and neutrophils are poorly understood. Monocytes and neutrophils originate from a common myeloid progenitor cell and are crucial cellular elements in both innate and adaptive immunity. In response to inflammatory signals, these leukocytes extravasate and migrate to the affected tissue. Leukocyte extravasation is a multi-step process involving sequential participation of various adhesion molecules. While the initial steps, such as leukocyte interaction with endothelium, are well characterized, the subsequent steps of leukocyte extravasation, such as migration through the vascular BM, are not well defined. In this thesis, efforts have been made to detect, isolate and functionally characterize LN-8 in monocytes and neutrophils, and to define the role of α4 LNs in leukocyte migration and extravasation. First, we determined the chain specificity of 16 commonly used monoclonal antibodies (mAbs) to human LN by ELISA, Western blotting, and immunoprecipitation using recombinant (r) LNβ1 and LNγ1 chains. In addition, eight novel mAbs to LNα4 chain were generated and characterized. By immunohistochemistry, differential distribution of LNα4 chain in developing and adult human tissues was found. LNα4 was mainly localized in tissues of mesodermal origin, such as endothelial BM. By indirect immunofluorescence, LN-8 chains were detected in permeabilized monocytes and neutrophils, and intact LN-8 was isolated from these cells. This LN isoform was synthesized by monoblastoid cells and secreted by stimulated neutrophils. mAbs to LNα4 chain inhibited neutrophil migration through human serum albumin coated inserts, suggesting participation of the endogenous LN-8 in the cell migration. Monoblastoid JOSK-I cells adhered constitutively to rhLN-8 via α6β1 and, to a lower extent, β2 integrins, whereas stimulated neutrophils adhered to rhLN-8, rhLN-10 (α5β1γ1, Lm-511), and mouse LN-1 (α1β1γ1, Lm-111) via αMβ2 integrin. rhLN-8 strongly promoted monocyte and neutrophil migration both in the absence and presence of chemoattractants, and the migration-promoting activity on neutrophils was mediated by αMβ2 and, to a lower extent, β1 integrins. Compared to rhLN-8, several commercial LN preparations isolated from human placenta displayed lower migrationpromoting activity on neutrophils, in general. These preparations contained fragmented LN chains, a mixture of LN isoforms, and/or contaminating fibronectin. In LNα4 deficient mice, neutrophil recruitment to inflamed tissue was significantly impaired. rhLN-8 also protected neutrophils against spontaneous apoptosis. Altogether, the results from these studies indicate that both monocytes and neutrophils express LN-8, and that this laminin isoform can be secreted by the cells. In addition, LN-8 plays a major role in the physiology of myeloid cells, including their adhesion, migration, extravasation and survival. The results also indicate that αMβ2 integrin may constitute a novel receptor for LN-8 and other LN isoforms.