Final Progress Report
Proposal No. IBD-0080R
Principal Investigator: Robert W. Colman, M.D.
Applicant Organization: Temple University (Philadelphia, Pennsylvania, U.S.A.)
Project Title: Kallikrein-kinin system in inflammatory bowel disease
Period of Award: January 1, 2004 – December 31, 2006
i. Summary of Specific Aims
This project explored the novel hypothesis that cleaved high molecular weight kininogen (HKa) is a key mediator of inflammation through induction of proinflammatory cytokines. We postulated that inflammation is variably mediated by the multiple components of high molecular weight kininogen (HK) cleavage, with each product having a unique contribution relative to other products. Plasma and tissue kallikrein cleave HK and LK, respectively, resulting in different products. Both molecules release bradykinin (BK), which stimulates physiologic and pathophysiologic processes by binding to the constitutive BK-2-receptor (B2R) and/or the inducible BK-1 receptor (B1R). In some systems, stimulation of B1R and B2R leads to counterbalancing activities. Plasma kallikrein releases HKa in addition to BK. We determined the molecular mechanisms by which HKa preferentially leads to increased production of inflammatory cytokines by human monocytes. HKa binds to the urokinase receptor (uPAR) as well as to MAC-1 and gC1q receptors on monocytes. We tested the C1q relative role of each receptor by in vitro blocking and in vivo by using uPAR, MAC-1, B1R and B2R knockout mice to determine which HKa product and which receptors preferentially induce leukocytes to produce inflammatory cytokines. These novel studies explored a new mechanism of physiologic and pathophysiologic stimulation of innate immune cells and identified new targets for IBD treatment.
1. Specific Aim 1: Determine the molecular and cellular mechanisms by which HKa stimulates proinflammatory cytokines.
a. Rationale. HK and HKa binds to MAC-1, a β2 integrin present on monocytes, neutrophils and NK cells. HKa also binds to uPAR, a glycerol phosphatidyl-anchored protein present on monocytes and endothelial cells and to the gC1q receptor. Preliminary data indicate that HKa or physiologic plasma concentrations of HK and plasma kallikrein, when incubated together, stimulate TNF release from splenocytes and monocytes. BK, kallikrein or HK alone has no effect on TNF secretion.
b. Hypotheses. We will test three closely linked hypotheses: (a) domain 5 of HKa is the active region of HK and acts by binding to uPAR; (b) bradykinin does not affect the formation of inflammatory cytokines even after kininases and aminopeptidases are inhibited to enhance its stability; (c) macrophages are primarily responsible for producing proinflammatory cytokines in response to HKa
2. Specific Aim 2: Determine which receptor (uPAR, MAC-1 or both) transduces signals from HKa to stimulate inflammatory cytokines and induce colitis. HKa binds to uPAR and MAC-1. uPAR has no cytoplasmic tail so probably does not transducer signals. We will determine which receptors (single or in combination) are required for cytokine stimulation by HKa by antibody blockage. We will also test which signaling pathways lead to cytokine synthesis.
ii. and iii. Accomplishments toward meeting these aims and list of significant results
These are summarized under each of the papers published
iv. List of publications resulting from the grant.
a. Devani M, Vecchi M, Ferrero S, Avesani EC, Arizzi C, Chao L, Colman RW, Cugno M. Kallikrein-kinin system in inflammatory bowel diseases: intestinal involvement and correlation with the degree of tissue inflammation. Digestive and Liver Disease 2005;37:665-73.
Tissue kallikrein and its natural inhibitor, kallistatin, play opposite roles in the generation of bradykinin, a potent mediator of inflammation. Observations on experimental models and humans with ulcerative colitis suggest a pathogenetic role of the kallikrein-kinin system in inflammatory bowel diseases. To evaluate tissue kallikrein and kallistatin in intestinal tissue samples from Crohn's disease and ulcerative colitis patients with different degrees of disease involvement. Full-thickness surgical intestinal samples were obtained from 144 subjects (38 normal controls, 32 inflammatory controls, 38 Crohn's disease, 36 ulcerative colitis) and tested for kallikrein and kallistatin by immunoperoxidase techniques. Compared with controls, kallikrein immunoreactivity was significantly weaker in goblet cells (p=0.0001) and significantly stronger in interstitium (p=0.0001) of the Crohn's disease and ulcerative colitis samples. Kallistatin colocalised with kallikrein, with almost no reactivity in goblet cells but strong reactivity in interstitium of inflammatory bowel disease patients (p=0.0001 versus controls). The kallikrein and kallistatin depletion of goblet cells and the increased interstitial kallikrein and kallistatin reactivity correlated with the degree of tissue inflammation (p=0.0001). Disease-free samples had normal kallikrein and kallistatin patterns. Kallikrein-kinin system is actively involved in inflammatory bowel disease as a result of the release of kallikrein in the intestinal extracellular space; this involvement correlates with the degree of tissue inflammation. The normal pattern observed in the disease-free samples seems to rule out a genetic defect of kallikrein and kallistatin in inflammatory bowel diseases.
b. Isordia-Salas I, Pixley RA, Sainz IM, Martinez-Murillo C, Colman RW. The role of plasma high molecular weight kininogen in experimental intestinal and systemic inflammation. Archives of Medical Research 2004;35:369-77.
Inflammation is accompanied by activation of the plasma kallikrein-kinin system (KKS). KKS activation has been demonstrated in a variety of inflammatory human diseases. To further explore the participation of KKS in arthritis and inflammatory bowel disease, we used two experimental animal models in arthritis and enterocolitis. We found that activation of KKS is associated with arthritis induced by intraperitoneal injection of peptidoglycan-polysaccharide polymers (PG-PS) as well as the enterocolitis and systemic inflammation induced also by PG-PS when injected into the intestinal wall of genetically susceptible Lewis rats. We postulated that KKS participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion, and release of proteases. We demonstrated that therapy with a specific plasma kallikrein inhibitor modulated the experimental enterocolitis, arthritis, and systemic inflammation. The fact that deficiency of plasma high molecular weight kininogen in the genetically susceptible Lewis rat results in decreased chronic enterocolitis and systemic inflammation also supports our hypothesis. We suggest that KKS plays a similar role in idiopathic human intestinal inflammatory disease and arthritis, making kallikrein-kinin system proteins appealing targets for drug therapy in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease.
c. Keith JC Jr, Sainz IM, Isordia-Salas I, Pixley RA, Leathurby Y, Albert LM, Colman RW. A monoclonal antibody against kininogen reduces inflammation in the HLA-B27 transgenic rat. Arthritis Research & Therapy 2005;7:R769-76.
The human leukocyte antigen B27 (HLA-B27) transgenic rat is a model of human inflammatory bowel disease, rheumatoid arthritis and psoriasis. Studies of chronic inflammation in other rat models have demonstrated activation of the kallikrein-kinin system as well as modulation by a plasma kallikrein inhibitor initiated before the onset of clinicopathologic changes or a deficiency in high-molecular-mass kininogen. Here we study the effects of monoclonal antibody C11C1, an antibody against high-molecular-mass kininogen that inhibits the binding of high-molecular-mass kininogen to leukocytes and endothelial cells in the HLA-B27 rat, which was administered after the onset of the inflammatory changes. Thrice-weekly intraperitoneal injections of monoclonal antibody C11C1 or isotype IgG1 were given to male 23-week-old rats for 16 days. Stool character as a measure of intestinal inflammation, and the rear limbs for clinical signs of arthritis (tarsal joint swelling and erythema) were scored daily. The animals were killed and the histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Administration of monoclonal C11C1 rapidly decreased the clinical scores of pre-existing inflammatory bowel disease (P < 0.005) and arthritis (P < 0.001). Histological analyses confirmed significant reductions in colonic lesions (P = 0.004) and synovitis (P = 0.009). Decreased concentrations of plasma prekallikrein and high-molecular-mass kininogen were found, providing evidence of activation of the kallikrein-kinin system. The levels of these biomarkers were reversed by monoclonal antibody C11C1, which may have therapeutic potential in human inflammatory bowel disease and arthritis.
d. Stadnicki A, Pastucha E, Nowaczyk G, Mazurek U, Plewka D, Machnik G, Wilczok T, Colman RW. Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease. American Journal of Physiology - Gastrointestinal and Liver Physiology 2005;289:G361-6.
Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R) are G-protein coupled receptors which mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD) - namely, ulcerative colitis (UC) and Crohn's disease (CD). Both B2R and B1R proteins are expressed in the epithelial cells of normal and IBD intestines. B1R protein is visualized in macrophages at the center of granulomas in CD. B2R protein is normally present in the apices of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B1R protein is found in the basal area of enterocytes in normal intestine, but in the apical portion of enterocytes in inflamed tissue. B1R protein is significantly increased in both active UC and CD intestines compared to controls. In patients with active UC, B1R mRNA is significantly higher than B2R mRNA. However, in inactive UC patients, the B1R and B2R mRNA did not differ significantly. Thus, bradykinin receptors in IBD may reflect intestinal inflammation. Increased B1R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.
e. Khan M, Bradford H, Isordia-Salas I, Liu Y, Wu Y, Espinola R, Ghebrehiwet B, Colman R, High-Molecular-Weight Kininogen Fragments Stimulate the Secretion of Cytokines and Chemokines Through uPAR, Mac-1, and gC1qR in Monocytes. Arterioscel Thromb Vasc Biol 26; 2260-2266 (2006)
Plasma high-molecular-weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to HKa with release of bradykinin (BK). We postulated a direct link between HKa and cytokine/chemokine release. HKa, but not BK, releases cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-S-transferase (GST) fusion proteins of kininogen domain 3 (D3), a fragment of domain 3, E7P (aaG255-Q292), HK domain 5 (D5), the D5 recombinant peptides HG (aa K420-D474) and HGK (aa H475-S626) stimulated secretion of IL-1beta from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1beta by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1beta secretion induced by 98%, 89%, 85%, and 62%, respectively. Fractionation of mononuclear cells identified the responsible cell, a blood monocyte. Inhibitors of signaling pathways NFkB, JNK, and p38 but not extracellular signal-regulated kinase (ERK) decreased cytokine release from mononuclear cells. HKa increased the synthesis of IL-1b as deduced by an increase of IL-1b mRNA at 1 to 2 hours. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1beta from human monocytes using intracellular signaling pathways initiated by uPAR, beta2 integrins and gC1qR.
v. Lay Summary
This application further explores the role of blood plasma enzymes in releasing mediators of the inflammation process which is responsible for many of the manifestations of Crohn’s disease. It has long been appreciated that an enzyme formed in plasma “kallikrein” can cleave a target protein-kininogen (HK) and yield a small peptide composed of 9 amino acids, bradykinin, and a larger fragment (HKa). Bradykinin is known to be responsible for the swelling (permeability) and redness (blood vessel widening) characteristic of inflammation. Recent basic research shows that HKa also had biologic activity. The novel hypothesis of this research is that HKa can trigger certain white blood cells (monocytes) to synthesize and liberate certain molecules “cytokines” known to be central in the mechanism of inflammation. We have shown that HKa can stimulate monocytes to secrete key inflammatory cytokines including tumor necrosis factor (TNFα) and interleukin-1β (IL-1B). In this study we also found that interleukin 6 (IL-6) which causes many of the systemic responses seen in Crohn’s disease. We also found that two key chemokines (substances which affect white cells) – IL-8 and MCP-1 are also released by HKa. We have also determined which part of HKa was responsible and found the domain 5 could reproduce all of the responses of the larger molecule HKa. Since HKa is a novel stimulator of monocytes, we explored what protein (receptor) binds HKa to the monocytes and identify three major receptors. Having determined which receptors were involved we then could map the pathway for signaling (intracellular changes caused by binding of HKa). Knowledge of both receptors and the signaling pathways provide opportunities for development of new drugs which can inhibit there response and thus decrease the inflammation which characterizes Crohn’s disease.