Attenuated kinin release from human neutrophil elastase-pretreated kininogens by tissue and plasma kallikreins

Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.     

Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents.     

Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen.

1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.     

A new kinin moiety in human plasma kininogens

Recently, we isolated a new kinin from human urine and tentatively identified it as [Ala3]-Lys-bradykinin. However, there were inconsistencies between the properties of the naturally occurring new kinin and synthetic [Ala3]-Lys-bradykinin. In the present work, we determined whether the new kinin was released from human plasma kininogen, and further investigated the structure of the new kinin. After incubation of plasma (n = 6) with human urinary kallikrein, kinins were separated by HPLC and measured by RIA. The new kinin and Lys-bradykinin were found representing 23 +/- 3 and 76 +/- 6%, respectively, of total kinins released (2.0 +/- 0.4 micrograms/ml). The new kinin was also released from both purified low- and high-molecular-weight kininogens, representing 40-42% of total kinins released. Amino acid sequencing and composition analysis indicated that the structure of the new kinin was [Hyp3]-Lys-bradykinin (Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and not [Ala3]-Lys-bradykinin. We conclude that an important proportion of human kininogens contain hydroxyproline instead of proline in position three of the bradykinin moiety.     

In vitro proteolysis of human plasma low density lipoproteins by an elastase released from human blood polymorphonuclear cells

In vitro incubation of human plasma low density lipoproteins (LDL) with human blood polymorphonuclear cells (PMN) for 1 h at 37 degrees C resulted in an increased (2-4-fold) release into the medium of an enzymatic activity which co-eluted with LDL by column chromatography at physiological ionic strength but dissociated from it in high salt media in an ultracentrifugal field. The release of this enzymatic activity increased with increasing concentration of LDL in the medium and caused the hydrolysis of the LDL apoprotein B100 as indicated by the appearance of 7-8 low molecular weight bands (immunoreactive with anti-LDL) which were not present in the electropherogram of control LDL. The proteolytic activity was identified as an elastase by the following criteria: 1) capacity to hydrolyze the synthetic substrate methoxysuccinyl-Ala-Ala-Pro-Val-4-methylcoumaryl-7-amide known to be specific for the PMN elastase, 2) pattern of apo-B proteolysis identical to that exhibited by pure PMN elastase, 3) inhibition of the proteolysis by the elastase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-CH2Cl, 4) identity in molecular weight (28,000-30,000) of this activity with a pure preparation of PMN elastase labeled with [3H]diisopropylfluorophosphate. Based on thiobarbituric acid analyses and the lack of effect by vitamin E, oxidative events appeared to play no detectable role in apo-B proteolysis. Since we previously reported (Byrne, R. E., Polacek, D., Gordon, J. I., and Scanu, A. M. (1984) J. Biol. Chem. 259, 14531-14543) that high density lipoprotein-3 promotes the in vitro release of PMN elastase which cleaves apo-A-II, it is apparent that in vitro, both LDL and high density lipoprotein, two of the major plasma lipoprotein classes, can affect the export from PMN of an elastase which exhibits proteolytic action on apo-B and apo-A-II.     

Limited proteolysis of T-kininogen (thiostatin). Release of comparable fragments by different endopeptidases

Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.     

Kallikrein activates bradykinin B2 receptors in absence of kininogen

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.     

Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors.

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.     

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.     

Quantification of kininogen in human renal medulla

Renal kininogen was detected in human medullary tissue as well as human medullary tubule suspensions. After treatment with pig pancreatic kallikrein or human renal cortical homogenate liberated kinin was measured by bradykinin radioimmunoassay. In the absence of inhibitors kinins were degraded by kininases located in the same part of the kidney. Several known inhibitors of kininase I and II did not inhibit this activity. Endogenous medullary kininase was inhibited by preincubation of homogenates at 56 degrees C for one hour or by addition of 0.25 mmol/l HgCl2. Under these conditions endogenous medullary kinin release amounted to 9-26 nmol/g protein. The action of renal cortical kininogenase on kinin formation from papillary kininogen was completely inhibited by addition of 1 mumol/l aprotinin. Kininogen examined in renal tubule suspensions revealed an increase in amount per g protein compared to homogenates, confirming the tubular localization of renal kininogen.     

Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.     

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.     

Tissue kallikrein of human seminal plasma is secreted by the prostate gland

Samples of human seminal plasma were subjected to gel filtration, and the eluted fractions were analysed for their contents of tissue kallikrein-like antigen, arginine esterase activity and kininogenase activity. Two peaks of tissue kallikrein-like antigen were detected with apparent molecular masses of about 72 and 48 kDa. As judged by the criteria of molecular mass, immunoreactivity, kininogenase activity, identification of the released kinin as kallidin and inhibition studies, a genuine tissue kallikrein has been identified in the 48-kDa peak. In addition, this peak contains one or more species of immunoreactive tissue kallikrein which differ in molecular mass and enzymatic activities. The 72-kDa peak probably represents the complex of tissue kallikrein with alpha 1-proteinase inhibitor rather than a true high molecular mass tissue kallikrein. The prostate gland was identified as the site of origin of the tissue kallikrein in the seminal fluid by indirect methods and by demonstrating immunoreactive tissue kallikrein in prostatic tissue and secretion.     

Ectosomes released by human neutrophils are specialized functional units.

Here we show that human polymorphonuclear leukocytes (PMN) release ectosomes independently of complement attack during their activation both in vitro and at the site of inflammation in vivo. Patterns of biotinylated proteins on the surface of PMN and on PMN-derived ectosomes indicated a specific sorting of cell surface proteins into and out of ectosomes. Ectosomes expressed clusters of complement receptor 1 (CR1), which allowed them to bind efficiently to opsonized bacteria. Myeloperoxidase and human leukocyte elastase, both stored within the azurophilic granules of PMN, were found to colocalize on ectosomes with CR1. Furthermore, myeloperoxidase colocalized with human leukocyte elastase. In contrast, not present on CR1-expressing ectosomes were CD63, a selective marker for the azurophilic granules, and CD14, which is located within the same granules and the secretory vesicles as CR1. Of the other complement regulatory proteins expressed by PMN, only CD59 colocalized with CR1, while CD55 and CD46 were almost absent. Ectosomes released by activated PMN at the site of inflammation may function as a well organized element (ecto-organelle), designed to focus antimicrobial activity onto opsonized surfaces.     

Induction of vascular leakage and blood pressure lowering through kinin release by a serine proteinase from Aeromonas sobria

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B(2) receptor-, and partially in a histamine-H(1) receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B(2) receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B(2) receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.     

Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.     

Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells

Cultured endothelial cells isolated from bovine carotid aorta produce prostacyclin (prostaglandin I2) and a small amount of prostaglandin E2. The effects of kallikrein (EC 3.4.21.8) on the release of prostacyclin from the cells were studied with the radioimmunoassay technique. Kallikrein stimulated the release of prostacyclin in a dose-dependent manner. The maximal stimulation reached up to 9.2-fold at 0.1 micrograms/ml of kallikrein. The effect was not associated with the activation of the fatty acid cyclooxygenase, but with the stimulation of arachidonic acid release. But kallikrein itself did not have phospholipase activity. On the other hand, at the same doses, kallikrein failed to induce platelet aggregation or enhance platelet aggregation induced by collagen. Our findings suggest that the vasodilator effect of kallikrein is mediated in part by prostacyclin production. Furthermore, we investigated the possibility that the stimulatory effect of kallikrein on prostacyclin production in endothelial cells is associated with kinin formation. Bradykinin and lysylbradykinin (kallidin) also stimulated the release of prostacyclin, but the effects were far less than that of kallikrein. And the stimulation due to the addition of both kallikrein and bradykinin on prostacyclin and arachidonic acid release was not competitive or additive, but synergistic. Moreover, even if fetal calf serum was incubated with kallikrein, bradykinin was not detected at all. When kallikrein was pre-incubated with aporotinin, which is an inactivator of kallikrein, the effect of kallikrein was completely abolished. These findings suggest that the stimulatory effect of kallikrein on the release of prostacyclin from vascular cells is possibly not due to kinin formation, but to other substance(s) produced by this serine proteinase.     

Tissue kallikrein KLK1 is expressed de novo in endothelial cells and mediates relaxation of human umbilical veins.

Bradykinin released by the endothelium is thought to play an important local role in cardiovascular regulation. However, the molecular identity of endothelial proteases liberating bradykinin from its precursors remained unclear. Using RT-PCR and Southern blotting techniques we detected mRNA for tissue kallikrein (KLK1) in human umbilical vein endothelial cells and in bovine aortic endothelial cells. Protein expression was confirmed by precipitation of KLK1 from lysates of endothelial cells pre-labeled with [35S]-cysteine/methionine. Partial purification of tissue kallikrein from total endothelial cell extracts resulted in a protein triplet of about 50 kDa in Western blots using specific anti-KLK1 antibodies. The immunodetection of tissue kallikrein antigen in the fractions from ion exchange chromatography correlated with the presence of amidolytic tissue kallikrein activity. Stimulation of endothelial cells with angiotensin II (ANG-II), which recently has been shown to activate the vascular kinin system and to cause vasodilation, resulted in the release of bradykinin and kallidin. ANG-II-dependent relaxation of pre-constricted rings from human umbilical veins was abolished in the presence of a specific tissue kallikrein inhibitor. We conclude that endothelial cells de novo express significant amounts of tissue kallikrein, which likely serves in the local generation of vasoactive kinins.