Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo
Matthew J. Flick, … , Edward F. Plow, Jay L. Degen
Matthew J. Flick, … , Edward F. Plow, Jay L. Degen
Published June 1, 2004
Citation Information: J Clin Invest. 2004;113(11):1596-1606. https://doi.org/10.1172/JCI20741.
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Article Hematology

Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo

Abstract

The leukocyte integrin αMβ2/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-αMβ2 interaction in vivo, gene-targeted mice were generated in which the αMβ2-binding motif within the fibrinogen γ chain (N390RLSIGE396) was converted to a series of alanine residues. Mice carrying the Fibγ390–396A allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support αMβ2-mediated adhesion of primary neutrophils, macrophages, and αMβ2-expressing cell lines. The elimination of the αMβ2-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for αMβ2, integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.

Authors

Matthew J. Flick, XinLi Du, David P. Witte, Markéta Jiroušková, Dmitry A. Soloviev, Steven J. Busuttil, Edward F. Plow, Jay L. Degen

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Figure 1

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Modification of the fibrinogen γ chain gene. (A) ...
Modification of the fibrinogen γ chain gene. (A) Overall structure of the Fibγ390–396A gene-targeting vector, the WT fibrinogen γ chain gene, and the targeted Fibγ390–396A allele. Exons are depicted as solid areas, and the introduced nucleotide substitutions and PvuII site are indicated in text boxes as 390–396A and PvuII, respectively. The PvuII fragments that were diagnostic for the WT and targeted alleles by Southern blot assays are indicated by thin lines. Arrowheads indicate the position of diagnostic PCR primers used for detecting homologous recombination events in ES cells. Routine animal genotyping was done using primers 1 and 2 followed by PvuII digestion. (B) Partial nucleotide sequence of exons 9 and exon 10 of the WT and γ390–396A genes. Asterisks indicate nucleotides that were mutated in the Fibγ390–396A allele. A vertical arrow indicates the position of the exon 9 to 10 splice junction. Amino acids altered by the nucleotide substitutions are in italics. Underlined amino acids indicate the glutamine and lysine that participate in transglutaminase-mediated cross-linking. Amino acids that are known to be critical for platelet integrin receptor αIIbβ3 binding (23) are bracketed. (C) Representative PCR analyses to establish animal genotypes using DNA template from ear biopsies of WT, hemizygous, and homozygous mutant Fibγ390–396A mice. Primers 1 and 2 were used to amplify a 527-bp fragment, which was subsequently digested with PvuII to yield the diagnostic fragments of 304 bp and 223 bp. ddH2O, double distilled water.