Adsorption of components of the plasma kinin-forming system on the surface of Porphyromonas gingivalis involves gingipains as the major docking platforms.

Enhanced production of proinflammatory bradykinin-related peptides, the kinins, has been suggested to contribute to the pathogenesis of periodontitis, a common inflammatory disease of human gingival tissues. In this report, we describe a plausible mechanism of activation of the kinin-generating system, also known as the contact system or kininogen-kallikrein-kinin system, by the adsorption of its plasma-derived components such as high-molecular-mass kininogen (HK), prekallikrein (PK), and Hageman factor (FXII) to the cell surface of periodontal pathogen Porphyromonas gingivalis. The adsorption characteristics of mutant strains deficient in selected proteins of the cell envelope suggested that the surface-associated cysteine proteinases, gingipains, bearing hemagglutinin/adhesin domains (RgpA and Kgp) serve as the major platforms for HK and FXII adhesion. These interactions were confirmed by direct binding tests using microplate-immobilized gingipains and biotinylated contact factors. Other bacterial cell surface components such as fimbriae and lipopolysaccharide were also found to contribute to the binding of contact factors, particularly PK. Analysis of kinin release in plasma upon contact with P. gingivalis showed that the bacterial surface-dependent mechanism is complementary to the previously described kinin generation system dependent on HK and PK proteolytic activation by the gingipains. We also found that several P. gingivalis clinical isolates differed in the relative significance of these two mechanisms of kinin production. Taken together, these data show the importance of this specific type of bacterial surface-host homeostatic system interaction in periodontal infections.

Kinin danger signals proteolytically released by gingipain induce Fimbriae-specific IFN-gamma- and IL-17-producing T cells in mice infected intramucosally with Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B(2) receptors (B(2)R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. Studies in TLR2(-/-), B(2)R(-/-), and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation of the TLR2-->PMN-->B(2)R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B(2)R-dependent generation of IFN-gamma-producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B(2)R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis.

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva.

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1ß neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.

Alpha 1-antitrypsin and alpha 2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva.

Alpha 1-Antitrypsin, alpha 2-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva. Alpha 1-antitrypsin and alpha 2-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did not inhibit or bind with urinary and salivary kallikreins.

Studies on human high molecular weight (HMW) kininogen. III. Cleavage of HMW kininogen by the action of human salivary kallikrein.

Human high molecular weight (HMW) kininogen is a single chain with a molecular weight of 120,000 and is cleaved by plasma kallikrein sequentially into a nicked kininogen, an intermediate kinin-free protein (KFP-I), and a stable KFP-II. Here we report a study into the process of cleavage of human HMW kininogen by human salivary kallikrein. On incubation with salivary kallikrein, HMW kininogen was first converted into a nicked kininogen composed of disulfide-linked chains of 62,000 and 56,000 daltons. Subsequently, the nicked kininogen was cleaved into kinin and a KFP, which was apparently of equal size to the nicked kininogen, that is, KFP-I. In contrast to plasma kallikrein, salivary kallikrein did not cleave KFP-I into KFP-II. The two chains were separated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I. The N-termini of HMW kininogen and the 62,000-daltons chain were found to be pyroglutamyl-isoleucyl, while that of the 56,000-daltons chain was found to be serine. These results indicate that the sequence of the two chains and kinin in human HMW kininogen is 62,000-daltons chain-kinin-56,000-daltons chain from the N-terminal end of HMW kininogen. Possible processes of cleavage of human HMW kininogen by human plasma and salivary kallikreins are also discussed.

Kinin generation in the gingival inflammatory response to topically applied bacterial lipopolysaccharides.

A water-soluble lipopolysaccharide from Salmonella enteritidis and a phenol-soluble lipopolysaccharide from Leptotrichia buccalis were applied topically to the healthy marginal gingivae of beagle dogs. Saline was applied to contralateral areas as an internal control. Increases in vascular permeability were monitored by measurement of gingival fluid, and the collected gingival fluid samples were assayed for kininogenase and kinin activities. Both lipopolysaccharides induced an inflammatory response, as indicated by increased gingival fluid flow. Kininogenase-kinin activities paralleled the increases in gingival fluid flow, with the highest values being associated with peak increases in gingival fluid. The results indicate that both lipopolysaccharides, although different in lipid solubility, penetrate healthy sulcular epithelium and initiate an inflammatory response which is mediated in part by the kallikrein-kinin system. Interrelationships between this system and other inflammatory mediators suggest that kinin generation not only plays a role in the early phases of acute gingival inflammation, but may also contribute to the activation of other mediators appearing later in the response and in chronic inflammatory lesions.

Generation of plasma kinin by three types of protease isolated from Porphyromonas gingivalis 381.

Three types of protease (A, B and C) isolated from the culture supernatant of Porphyromonas gingivalis 381 had peculiar activities on kinin generation from high molecular-weight kininogen in vitro. Protease C released bradykinin from the kininogen in a reaction mixture containing 2 mM dithiothreitol, but A and B did not. However, the activity of degrading bradykinin was much stronger in protease A and B than in C. These findings suggest that only protease C shows plasma kallikrein activity.

Kinin generation by hemodialysis membranes as a possible cause of anaphylactoid reactions.

1. Severe anaphylactoid reactions have been reported in some patients treated with angiotensin converting enzyme (ACE) inhibitors during hemodialysis with a polyacrylonitrile (PAN) membrane. Generation of bradykinin via contact activation at the negatively charged membrane surface and reduced bradykinin breakdown due to ACE inhibition have been suggested as possible causes. This hypothesis was evaluated in the present study. 2. PAN or cellulose dialyzer membranes were incubated with plasma at different concentrations of ACE inhibitor. The rate and extent of kinin accumulation was dependent on the ACE inhibitor concentration. 3. Bradykinin levels were determined in "historical" plasma samples drawn from patients treated with ACE inhibitor at the onset of anaphylactoid reactions during hemodialysis with PAN dialyzers. The kinin levels were significantly higher (2.4 +/- 0.05 pmol/ml) than in samples from a group of control patients (0.29 +/- 0.02 pmol/ml). 4. Plasma kinin levels were measured in patients who developed anaphylactoid reactions during dialysis with a PAN membrane though not being treated with ACE inhibitor. At the onset of the reaction, kinin levels increased to 2.1 pmol/ml in the line entering the dialyzer and to 10.5 pmol/ml in the line leaving the dialyzer compared to not more than 0.12 pmol/ml upon dialysis with other membranes. 5. These in vitro and in vivo results demonstrate that contact of blood with a polyacrylonitrile membrane leads to the generation of kinins which accumulate if ACE is inhibited. It is very likely that kinin accumulation in the circulation is the cause of anaphylactoid reactions during hemodialysis with PAN membranes in patients treated with ACE inhibitors and, in some cases, in patients not receiving ACE inhibitor medication.

The role of bacterial and host proteinases in periodontal disease.

It is abundantly obvious that the uncontrolled degradation and/or activation of host defense pathways is the major pathway by which the periodontal pathogen P. gingivalis promotes its growth and proliferation. By being able to shed host receptors, degrade cytokines, and activate coagulation, complement, and kallikrein/kinin pathways it is clear that this organism has found a mechanism(s) to evade host defense and at the same time develop a system for cannibalizing host proteins for its own nutritional usage (Fig 2). Thus, it seems only logical that the development of inhibitors against these bacterial proteinases would be a useful method for negating their activities and making such pathogens more susceptible to attack by host phagocyte cells. In this respect, the structure of the truncated form of RGP has just been elucidated. Thus, it should only be a question of time before inhibitors to this enzyme will be developed and, hopefully, be used to reduce the pathologies associated with the development of periodontitis and/or eliminate the disease altogether.

Interaction of hemoglobin vesicles, a cellular-type artificial oxygen carrier, with human plasma: effects on coagulation, kallikrein-kinin, and complement systems.

Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro. Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

Kinin-mediated anaphylactoid reaction implicated in acute intra-operative pulseless electrical activity.

A 65-year-old patient undergoing total hip replacement under general anaesthesia suffered acute pulseless electrical activity with a fatal outcome. A kinin-mediated analphylactoid reaction following administration of a polygeline plasma expander (Haemaccel) was implicated by in vitro testing. This case report illustrates the diagnostic difficulties posed by non-histaminoid anaphylactoid reactions and the resistance to epinephrine of kinin-mediated hypotension.