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

Laminins (LNs) are a family of large alphabetagamma heterotrimeric glycoproteins found in all basement membranes (BMs). They are expressed in a tissue- and developmental stagespecific manner and implicated in vital cellular functions, including cell adhesion, migration and signaling. So far, eleven laminin chains (5alpha, 3beta and 3gamma) that assemble into 15 isoforms (LN-1 to 15, Lm-111 to Lm-523) have been identified. LN-8 (alpha4beta1gamma1, 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 alpha4 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) LNbeta1 and LNgamma1 chains. In addition, eight novel mAbs to LNalpha4 chain were generated and characterized. By immunohistochemistry, differential distribution of LNalpha4 chain in developing and adult human tissues was found. LNalpha4 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 LNalpha4 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 alpha6beta1 and, to a lower extent, beta2 integrins, whereas stimulated neutrophils adhered to rhLN-8, rhLN-10 (alpha5beta1gamma1, Lm-511), and mouse LN-1 (alpha1beta1gamma1, Lm-111) via alphaMbeta2 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 via alphaMbeta2 and, to a lower extent, 01 integrins. Compared to rhLN-8, several commercial LN preparations isolated from human placenta displayed in general lower migration-promoting activity on neutrophils. These preparations contained fragmented LN chains, a mixture of LN isoforms, and/or containing fibronectin. In LNalpha4 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 alphaMbeta2 integrin may constitute a novel receptor for LN-8 and other LN isoforms.

List of scientific papers

I. Geberhiwot T, Wondimu Z, Salo S, Pikkarainen T, Kortesmaa J, Tryggvason K, Virtanen I, Patarroyo M (2000). Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin beta1 and gamma1 chains. Matrix Biol. 19(2): 163-7.
https://pubmed.ncbi.nlm.nih.gov/10842099

II. Petajaniemi N, Korhonen M, Kortesmaa J, Tryggvason K, Sekiguchi K, Fujiwara H, Sorokin L, Thornell LE, Wondimu Z, Assefa D, Patarroyo M, Virtanen I (2002). Localization of laminin alpha4-chain in developing and adult human tissues. J Histochem Cytochem. 50(8): 1113-30.
https://pubmed.ncbi.nlm.nih.gov/12133914

III. Pedraza C, Geberhiwot T, Ingerpuu S, Assefa D, Wondimu Z, Kortesmaa J, Tryggvason K, Virtanen I, Patarroyo M (2000). Monocytic cells synthesize, adhere to, and migrate on laminin-8 (alpha 4 beta 1 gamma 1). J Immunol. 165(10): 5831-8.
https://pubmed.ncbi.nlm.nih.gov/11067943

IV. Wondimu Z, Geberhiwot T, Ingerpuu S, Juronen E, Xie X, Lindbom L, Doi M, Kortesmaa J, Thyboll J, Tryggvason K, Fadeel B, Patarroyo M (2004). An endothelial laminin isoform, laminin 8 ({alpha}4{beta}1{gamma}1), is secreted by blood neutrophils, promotes neutrophil migration and extravasation, and protects neutrophils from apoptosis. Blood. 104(6): 1859-1866. Epub 2004 Jun 01
https://pubmed.ncbi.nlm.nih.gov/15172971

V. Wondimu Z, Gorfu G, Tryggvason K, Patarroyo M (2004). Immunochemical and functional characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). [Manuscript]