Tissue Transglutaminase-2 Modifies Fibrin(ogen) Stimulated Macrophage Cytokine Expression

The blood clotting protein fibrin(ogen) acts as a bridge between the hemostatic system and the inflammatory response. Specifically, conversion of fibrinogen to its clotted form (i.e., fibrin) enables fibrin(ogen) engagement of leukocyte β2 integrins. Interestingly, we found that cross-linking of fibrin(ogen) by tissue transglutaminase-2 (TG2), a process distinct from blood clotting, inhibited lipopolysaccharide (LPS)- mediated induction of the anti-inflammatory cytokine IL-10 in cultured macrophages. Thus, we hypothesized that TG2 cross-linking enhances fibrin(ogen)-directed macrophage pro-inflammatory activity. Methods: Bone marrow-derived macrophages from wild-type mice were cultured on uncoated tissue culture plates or plates coated with fibrinogen or TG2-cross-linked fibrin(ogen) (10 μg/ml) for 4 hours. The cells were then stimulated with LPS (1 ng/mL) for 4 additional hours and levels of mRNAs encoding the pro-inflammatory cytokines TNF and IL-6 were measured using a qRT-PCR. Results: LPS stimulation significantly increased TNF and IL-6 mRNA levels compared to unstimulated cells. Unmodified surface-adhered fibrin(ogen) had minimal effect on basal and LPS-stimulated expression of TNF and IL-6 mRNA. In contrast, TG2-crossilinked fibrin(ogen) caused a reproducible enhancement of LPS-stimulated TNF and IL-6 mRNA induction. Conclusion: The results indicate that LPS activation of macrophages in culture is enhanced by exposure to TG2 cross-linked fibrin(ogen). This suggests that the pro-inflammatory activity of fibrin(ogen) in vivo may be influenced by changes in fibrin(ogen) structure mediated by processes independent of blood coagulation. Funding: Student support was provided by NIH grant R25 HL103156