The leukocyte integrin α M β 2 /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)-α M β 2 interaction in vivo, gene-targeted mice were generated in which the α M β 2 -binding motif within the fibrinogen γ chain (N 390 RLSIGE 396 ) was converted to a series of alanine residues. Mice carrying the Fibγ 390-396A 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 α M β 2 -mediated adhesion of primary neutrophils, macrophages, and α M β 2 -expressing cell lines. The elimination of the α M β 2 -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 α M β 2 , 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.

Fibrinogen can support host antimicrobial containment/clearance mechanisms, yet selected pathogens appear to benefit from host procoagulants to drive bacterial virulence. Here, we explored the hypothesis that host fibrin(ogen), on balance, supports Staphylococcus aureus infection in the context of septicemia. Survival studies following intravenous infection in control and fibrinogen-deficient mice established the overall utility of host fibrin(ogen) to S. aureus virulence. Complementary studies in mice expressing mutant forms of fibrinogen-retaining clotting function, but lacking either the bacterial ClfA (Fibγ(Δ5)) binding motif or the host leukocyte integrin receptor αMβ2 (Fibγ(390-396A)) binding motif, revealed the preeminent importance of the bacterial ClfA-fibrin(ogen) interaction in determining host survival. Studies of mice lacking platelets or the platelet integrin receptor subunit αIIb established that the survival benefits observed in Fibγ(Δ5) mice were largely independent of platelet αIIbβ3-mediated engagement of fibrinogen. Fibγ(Δ5) mice exhibited reduced bacterial burdens in the hearts and kidneys, a blunted host proinflammatory cytokine response, diminished microscopic tissue damage, and significantly diminished plasma markers of cardiac and other organ damage. These findings indicate that host fibrin(ogen) and bacterial ClfA are dual determinants of virulence and that therapeutic interventions at the level of fibrinogen could be advantageous in S. aureus septicemia.

In addition to its well-characterized role in hemostasis, fibrin(ogen) has been proposed to be a central regulator of the inflammatory response. Multiple in vitro studies have demonstrated that this hemostatic factor can alter leukocyte function, including cell adhesion, migration, cytokine and chemokine expression, degranulation, and other specialized processes. One important link between fibrin(ogen) and leukocyte biology appears to be the integrin receptor alpha(M)beta(2)/Mac-1, which binds to immobilized fibrin(ogen) and regulates leukocyte activities. Although it is well established that fibrin(ogen) is a ligand for alpha(M)beta(2), the precise molecular determinants that govern this interaction are only now becoming clear. A novel line of mice expressing a mutant form of fibrinogen (Fib gamma(390-396A)) has revealed that gamma chain residues 390-396 are important for the high-affinity engagement of fibrinogen by alpha(M)beta(2) and leukocyte function in vivo. Fibrinogen gamma(390-396A) failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, monocytes, and macrophages, and mice expressing this fibrinogen variant were found to exhibit a major defect in the host inflammatory response following acute challenges. Most notably, Fib gamma(390-396A) mice display a profound impediment in Staphylococcus aureus elimination by leukocytes following intraperitoneal inoculation. These findings have positively established the physiological importance of fibrin(ogen) as a ligand for alpha(M)beta(2) and illustrate that the fibrin(ogen) gamma chain residues 390-396 constitute a critical feature of the alpha(M)beta(2) binding motif. Finally, the Fib gamma(390-396A) mice represent a valuable system for better defining the contribution of fibrin(ogen) to the inflammatory response in the absence of any confounding alteration in clotting function.

Blood genomic profiling has been applied to disorders of the blood and various organ systems including brain to elucidate disease mechanisms and identify surrogate disease markers. Since most studies have not examined specific cell types, we performed a preliminary genomic survey of major blood cell types from normal individuals using microarrays. CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ natural killer cells, and CD14+ monocytes were negatively selected using the RosetteSep antibody cocktail, while polymorphonuclear leukocytes were separated with density gradient media. Genes differentially expressed by each cell type were identified. To demonstrate the potential use of such cell subtype-specific genomic expression data, a number of the major genes previously reported to be regulated in ischemic stroke, migraine, and Tourette syndrome are shown to be associated with distinct cell populations in blood. These specific gene expression, cell-type-related profiles will need to be confirmed in larger data sets and could be used to study these and many other neurological diseases.

The blood-clotting protein fibrinogen has been implicated in host defense followingStaphylococcus aureusinfection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly clearedS. aureusfollowing intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib–) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib– mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activatorvWbpwas identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminateS. aureusdeficient invWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.