Nuclear factor-kappa B (NFkappaB) component p50 in blood mononuclear cells regulates endothelial tissue factor expression in sickle transgenic mice: implications for the coagulopathy of sickle cell disease

Abstract

Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease-but not the normal-mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFkappaB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFkappaB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFkappaB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFalpha], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFkappaB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFkappaB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFkappaB(p50) in peripheral blood mononuclear cells-but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFkappaB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.

Figure 1

Effect of NFκB(p50) knockout state. TF expression is indicated as percentage of pulmonary veins with TF positive endothelium, and is shown as mean ± SD for wild-type (p50+/+) NY1DD sickle mice in ambient air (“air”) or after exposure to H/R (“H/R”). Compared to the TF response of wild-type NY1DD sickle mice to H/R (two bars on left), NY1DD NFκB(p50)−/− mice have lower TF at ambient air (third bar) and abrogated TF response to H/R (fourth bar). Remaining bars are from cross-transplantation experiments, NY1DD NFκB(p50)−/− to NY1DD NFκB(p50)+/+ and vice versa, and demonstrate that the TF response to H/R requires blood cell NFκB(p50) but not vessel wall/tissue NFκB(p50). * = P<.001, and # = P<.01, compared to air control (bar on far left). ** = P<.001 compared to respective air control (the fifth bar from left). And + = P<.01 and ++ = P<.001, compared to +/+ blood cells post H/R (the second bar from left). From left to right, number of animals used = 3,3,6,6,4,4,4,3.