4th Annual BMRP Investigator Meeting - Abstract
Cleavage Products of Kininogen Variably Induce Proinflammatory Cytokines by Fisher Rat Splenocytes
Bo Liu1, Fengling Li1, Irma Isordia-Salas2, Harlan N. Bradford2, Robert W. Colman2,a, Robin A. Pixley2 and R. Balfour Sartor1,b
1The University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, U.S.A.); 2Temple University (Philadelphia, Pennsylvania, U.S.A.)
Introduction: Cleavage of high molecular weight kininogen (HK) by plasma kallikrein releases bradykinin (BK) and produces HKa, a cleaved protein with additional biological activity. Studies in two separate rat models demonstrate that chronic experimental enterocolitis and systemic inflammation are in part due to the production of kallikrein and cleavage of HK.
Aim: To determine which component of cleaved HK mediates inflammation through induction of proinflammatory cytokines.
Methods: Human HK (9-1450nM), HKa (9-730nM), bradykinin (1-100nM), kallikrein (45-455 nM), GST-fusion proteins of human HKa domains {GST-D5(aa 420-513) and GST-D5-D6(aa 420-626)}(100-1000nM), LPS(1μg/ml), GST (500nM) and media alone were incubated with unfractionated Fisher 344 rat splenocytes (1x107cell/ml) in triplicate for six hours in serum free medium. TNF and IL-1β levels in the supernatants were measured by ELISAs. All proteins contained <0.1 EU/ml endotoxin.
Results: HKa stimulated TNF and IL-1β secretion by rat splenocytes. No cytokine inductive activity was observed by the addition of HK, BK, or kallikrein. HKa stimulated TNF secretion in a concentration and time dependent manner. Maximum TNF secretion (1200pg/ml) was reached at four hours using 730nM HKa, equivalent to TNF levels induced by 1μg/ml LPS. HK (730nM) incubated with kallikrein (455nM) induced comparable levels of TNF secretion (1446±108pg/ml vs. HK alone 315±51pg/ml, p<0.001). Cell subset depletion studies demonstrated that macrophages were responsible for HKa- induced cytokine secretion. GST-D5 increased TNF secretion (2017±261pg/ml vs. GST-D5-6 106±8pg/ml or GST alone 184±10pg/ml, p<0.002). Blocking antibody studies confirmed the D5 region of HKa as the active region.
Conclusions: HKa and cleavage of HK by kallikrein induces TNF and IL-1β secretion by rat magrophages. However, bradykinin, HK and kallikrein alone have no effect, suggesting that generation of HKa by kallikrein stimulates inflammatory responses. Domain 5 of HKa, which contains binding regions for αmβ2, uPAR, gC1qr and cytokeratin-1, mediates inflammatory cytokine secretion. HKa may provide a novel molecular target for inflammatory bowel disease treatment.
aPrincipal Investigator; bCo-Investigator and Presenter