3rd Annual BMRP Investigator Meeting - Abstract
High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β Through Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Monocytes
Robert W. Colman1,a, Mohammad M. Khan1, Bo Liu2 and R. Balfour Sartor2
1Sol Sherry Thrombosis Research Center, Temple University (Philadelphia, Pennsylvania, U.S.A.) and 2Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, U.S.A.)
Kallikrein cleavage of high molecular weight kininogen (HK) to a two chain form (HKa) with release of bradykinin (BK) occurs in inflammatory bowel disease (IBD). We have shown in rat experimental IBD mimicking Crohn’s disease that plasma kallikrein inhibitors or rats deficient in kininogen modulate the clinical and pathological signs. HK is known to bind to Mac-1 and uPAR on leukocytes. We hypothesized that HKa would stimulate secretion of inflammatory cytokines.
Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We expressed HK domain 3 (D3) and a fragment of D3 coded for by exon 7, E7P (aaG235-Q292), and HK domain 5 (D5) in E. coli GST fusion proteins. All proteins contained <0.01 EU/ml endotoxin. Mononuclear cells were preincubated with monoclonal antibodies or murine IgG (1.8 mM). HK, HKa, GST-D3, GST-E7P, GST-D5 or GST, all at 600 nM, were added for 30 min at 37°C. After centrifugation, the supernatant suspension was assayed for interleukin-1β (IL-1β). All HK fragments tested significantly stimulated secretion of IL-1β when incubated with mononuclear cells. Fractionation of mononuclear cells by negative selection using antibody-coated magnetic beads identified the responsible cell as a monocyte.
Anti-Mac-1 antibody inhibited IL-1β secretion by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HKa 77, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 and a mAb to HK D3 both inhibited IL-1β released by HKa, GST-D5 and GST-D3, which indicates that both D3 and D5 are important in cytokine release. We have shown similar effects in monocytes isolated from rat spleen. Cleaved kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood monocytes.
aPrincipal Investigator