Upregulation of tissue factor in monocytes by cleaved high molecular weight kininogen is dependent on TNF-alpha and IL-1beta

Abstract

Inflammatory bowel disease and arthritis are associated with contact activation that results in cleavage of kininogen to form high molecular weight kininogen (HKa) and bradykinin. We have previously demonstrated that HKa can stimulate inflammatory cytokine and chemokine secretion from human monocytes. We now show that HKa can upregulate tissue factor antigen and procoagulant activity on human monocytes as a function of time (1-4 h) and HKa concentration (75-900 nM). The amino acid sequence responsible to block HKa effects is G440-H455. The HKa receptor macrophage-1 (Mac-1; CD11b18) is the binding site as shown by inhibition by a monoclonal antibody to CD11b/18. Chemical inhibitors of JNK, ERK, and p38 signaling pathways block cell signaling, as does an inhibitor to the transcription factor NF-kappaB. A combination of monoclonal antibodies to TNF-alpha and IL-1beta but neither alone inhibited the HKa induction of tissue factor. These results suggest that HKa mimics LPS by triggering a paracrine pathway in monocytes that depends on TNF-alpha and IL-1beta. Antibodies to kininogen or peptidomimetics might be a useful and safe therapy in inflammatory diseases or sepsis involving cytokines.

Fig. 1.

Effect of high molecular weight kininogen (HKa) on monocyte tissue factor (TF) expression. A: immunofluorescence analysis of human mononuclear cells incubated with HKa for 3 h. The cells were stained with anti-TF MAb-Alexa Fluor 647 (i) or IgG-Alexa Fluor 647 (ii) as a control. Another aliquot of cells was stained with IgG-PE (iii) as a control. B: the antibody to TF inhibited TF activity. Mononuclear cells were preincubated 10 min with or without the anti-TF antibody. The cells were treated for 3 h with or without HKa (600 nM) or LPS. TF activity was measured by TF procoagulant assay. ★P < 0.05.

Fig. 2.

Effects of antibodies to G486–K502 and G440–H455 peptides of HKa on TF expression. Mononuclear cells were preincubated with anti-peptide antibodies at a concentration of 1.8 μM [3 times higher than uncleaved one-chain kininogen (HK) plasma concentration 600 nM]. Anti-peptide antibodies to G486–K502 or G440–H455 were each incubated for 30 min and then challenged with HKa (600 nM) or D5 (600 nM) for 3 h. Data are plotted as means ± SE. *P < 0.05. Experiments were performed in triplicate.

Fig. 3.

Effects of antibodies to macrophage-1 (Mac-1; CD11b18), urokinase plasminogen activator receptor (uPAR), leukocyte function-associated antigen-1 (CD11a18), and gC1qR on TF expression. A: the antibody to Mac-1 inhibited TF expression induced by HKa. Mononuclear cells were preincubated with the MAb (0.1, 0.5, or 5.0 μg/ml) against Mac-1 for 30 min before stimulation with HKa (600 nM). Data are represented as means ± SE (n = 3 experiments). Comparisons were made with the control. ★P < 0.05. B: antibody to uPAR had no effects on TF expression. Mononuclear cells were preincubated without or with a variety of concentration of the MAbs (0.1, 0.5, or 5.0 μg/ml) against uPAR for 30 min at 37°C and then treated with HKa (600 nM) for additional 180 min at 37°C. Data are presented as in A. None of the differences is significant.

Fig. 4.

Tissue factor expression was greatly attenuated by selective inhibitors of p38, JNK, ERK, and NF-κB. Mononuclear cells were preincubated 1 h without or with 10 μM SB-202190 (a p38 inhibitor), 10 μM SP-600125 (a JNK inhibitor), 10 μM U-0126 (an ERK inhibitor), or 10 μM MG-132 (an NF-κB inhibitor). The stock concentration was 10 mM in DMSO vehicle for all inhibitors, except the JNK inhibitor was 25 mM. The dilution was 1:1,000 in all cases. Cells incubated with DMSO alone showed no inhibition of TF at 1:1,000 dilution. The cultures were then incubated for 3 h without or with HKa (600 nM). Bars represent means of TF expression ± SE (n = 3 experiments).

Fig. 5.

Combination of antibodies to IL-1β and TNF-α completely blocked TF expression. Mononuclear cells were preincubated with the neutralizing MAb against IL-1β, TNF-α, or IL-1β plus TNF-α for 10 min before stimulation with HKa (600 nM) for an additional 180 min at 37°C. Data are plotted as means ± SE. Comparisons were made vs. control. ★P < 0.05.

Fig. 6.

HK stimulated mRNA synthesis of TF in mononuclear cells. Mononuclear cells were incubated with HKa or LPS for the indicated time. Total RNA of the cells was isolated by TRIzol reagent. RT-PCR was carried out as described in materials and methods. PCR products were separated in 1% agarose gel. The detection of the 588-bp DNA bands indicated TF mRNA synthesis from corresponding background (0 time point).