Inhaled porcine pancreatic elastase causes bronchoconstriction via a bradykinin-mediated mechanism.

Neutrophil elastase has been linked to inflammatory lung diseases such as chronic obstructive pulmonary disease, adult respiratory distress syndrome, emphysema, and cystic fibrosis. In guinea pigs, aerosol challenge with human neutrophil elastase causes bronchoconstriction, but the mechanism by which this occurs is not completely understood. Our laboratory previously showed that human neutrophil elastase releases tissue kallikrein (TK) from cultured tracheal gland cells. TK has been identified as the major kininogenase of the airway and cleaves both high- and low-molecular weight kininogen to yield lysyl-bradykinin. Because inhaled bradykinin causes bronchoconstriction and airway hyperresponsiveness in asthmatic patients and allergic sheep, we hypothesized that elastase-induced bronchoconstriction could be mediated by bradykinin. To test this hypothesis, we measured lung resistance (RL) in sheep before and after inhalation of porcine pancreatic elastase (PPE) alone and after pretreatment with a bradykinin B(2) antagonist (NPC-567), the specific human elastase inhibitor ICI 200,355, the histamine H(1)-antagonist diphenhydramine hydrochloride, the cysteinyl leukotriene 1 receptor antagonist montelukast, or the cyclooxygenase inhibitor indomethacin. Inhaled PPE (125-1,000 microg) caused a dose-dependent increase in RL. Aerosol challenge with a single 500 microg dose of PPE increased RL by 132 +/- 8% over baseline. This response was blocked by pretreatment with NPC-567 and ICI-200,355 (n = 6; P < 0.001), whereas treatment with diphenhydramine hydrochloride, montelukast, or indomethacin failed to block the PPE-induced bronchoconstriction. Consistent with pharmacological data, TK activity in bronchial lavage fluid increased 134 +/- 57% over baseline (n = 5; P < 0.02). We conclude that, in sheep, PPE-induced bronchoconstriction is in part mediated by the generation of bradykinin. Our findings suggest that elastase-kinin interactions may contribute to changes in bronchial tone during inflammatory diseases of the airways.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Deletion of secretory group V phospholipase A2 attenuates cell migration and airway hyperresponsiveness in immunosensitized mice

We investigated the role of group V phospholipase A2 (gVPLA2) in OVA-induced inflammatory cell migration and airway hyperresponsiveness (AHR) in C57BL/6 mice. Repeated allergen challenge induced biosynthesis of gVPLA2 in airways. By aerosol, gVPLA2 caused dose-related increase in airway resistance in saline-treated mice; in allergic mice, gVPLA2 caused persistent airway narrowing. Neither group IIa phospholipase A2, a close homolog of gVPLA2, nor W31A, an inactive gVPLA2 mutant with reduced activity, caused airway narrowing in immune-sensitized mice. Pretreatment with MCL-3G1, a blocking Ab against gVPLA2, before OVA challenge blocked fully gVPLA2-induced cell migration and airway narrowing as marked by reduction of migrating leukocytes in bronchoalveolar lavage fluid and decreased airway resistance. We also assessed whether nonspecific AHR caused by methacholine challenge was elicited by gVPLA2 secreted from resident airway cells of immune-sensitized mice. MCL-3G1 also blocked methacholine-induced airway bronchoconstriction in allergic mice. Blockade of bronchoconstriction by MCL-3G1 was replicated in allergic pla2g5-/- mice, which lack the gene encoding gVPLA2. Bronchoconstriction caused by gVPLA2 in pla2g4-/- mice was comparable to that in pla2g4+/+ mice. Our data demonstrate that gVPLA2 is a critical messenger enzyme in the development of AHR and regulation of cell migration during immunosensitization by a pathway that is independent of group IVa phospholipase A2.     

Bronchoconstrictor and antibronchoconstrictor properties of inhaled prostacyclin in asthma

Prostacyclin (PGI2) is generated in appreciable amounts during allergic reactions in human lung tissue. To define its activity on human airways we have studied the effects of doubling concentrations of inhaled PGI2 and its hydrolysis product 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha) on specific airway conductance (sGaw), maximum expiratory flow at 30% vital capacity (Vmax30), forced expiratory volume in 1 s (FEV1), and static lung volumes in subjects with mild allergic asthma. In a second study the effect of inhaled PGI2 on bronchoconstriction provoked by increasing concentrations of inhaled prostaglandin (PG) D2 and methacholine was observed. Inhalation of PGI2 up to a concentration of 500 micrograms/ml had no significant effect on sGaw but produced a concentration-related decrease in FEV1 and Vmax30 in all subjects. In two of four subjects inhalation of PGI2 also increased residual volume and decreased vital capacity but had no effect on total lung capacity. PGI2, but not 6-oxo-PGF1 alpha, protected against bronchoconstriction provoked by either PGD2 or methacholine whether airway caliber was measured as sGaw, FEV1, or Vmax30. The apparent disparity between the bronchoconstrictor and antibronchoconstrictor effects of PGI2 might be explained by its potent vasodilator effect in causing airway narrowing through mucosal engorgement and reducing the spasmogenic effects of other inhaled mediators by increasing their clearance from the airways.     

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model.Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways.These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.     

Influence of plasma proteinase inhibitors and aprotinin on trypsin-induced bradykinin release in vitro in man

The consumption of kininogen (measured as kinin-releasable material) was studied in an experimental model in vitro. Analyses were made following the addition of increasing amounts of human cationic trypsin to human serum and plasma. The consumption of kininogen was correlated with the degree of saturation of the plasma proteinase inhibitors alpha 2-macroglobulin (alpha 2-M) and alpha 1-proteinase inhibitor (alpha 1-PI) with trypsin in the presence and absence of aprotinin (Trasylol). The level of kininogen fell dramatically when alpha 2-M was saturated to 70% in spite of 90% free alpha 1-PI. Trypsin-alpha 2-M complexes had no effect on kininogen levels. 60 mumol/l of aprotinin, i.e. approximately 3 X 10(6) KIU/l, blocked only 60% of the trypsin-induced kininogen consumption in serum, while 15 mumol/l of aprotinin blocked 100% of this consumption in plasma. With increasing concentration of aprotinin in serum, a decreasing consumption of alpha 2-M and especially of alpha 1-PI was observed on the addition of trypsin. The high aprotinin concentration needed to block trypsin-induced kininogen cleavage in human serum or plasma may explain the poor clinical effect of aprotinin to date in human acute pancreatitis.     

Elastase is a constituent product of T cells

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.     

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase.     

Antibody-antigen interaction in the airway drives early granulocyte recruitment through BLT1

Antibody-antigen interactions in the airway initiate inflammation in acute asthma exacerbations. This inflammatory response is characterized by the recruitment of granulocytes into the airways. In murine models of asthma, granulocyte recruitment into the lung contributes to the development of airway hyperresponsiveness (AHR), mucus production, and airway remodeling. Leukotriene B4 is a mediator released following antigen challenge that has chemotactic activity for granulocytes, mediated through its receptor, BLT1. We investigated the role of BLT1 in granulocyte recruitment following antigen challenge. Wild-type mice and BLT1-/- mice were sensitized and challenged with ovalbumin (OVA) to induce acute allergic airway inflammation. In addition, to explore the relevance to antibody-antigen interactions, we injected OVA bound to anti-OVA IgG1 or anti-OVA IgE intratracheally into na?ve wild-type and BLT1-/- mice. Cell composition of the lungs, cytokine levels, histology, and AHR were determined. After sensitization and challenge with ovalbumin, there was significantly reduced neutrophil and eosinophil recruitment into the airways of BLT1-/- mice compared with wild-type animals after one or two daily antigen challenges, but this difference was not seen after three or four daily antigen challenges. Mucus production and AHR were not affected. Intratracheal injection of OVA bound to IgG1 or IgE induced neutrophil recruitment into the airways in wild-type mice but not in the BLT1-/- mice. We conclude that BLT1 mediates early recruitment of granulocytes into the airway in response to antigen-antibody interactions in a murine model of acute asthma.     

Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease

Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.     

Distinct and additive effects of elastase and endotoxin on expression of alpha 1 proteinase inhibitor in mononuclear phagocytes

Expression of alpha 1 proteinase inhibitor (alpha 1-PI) in human mononuclear phagocytes may provide a local mechanism for inactivation of serine proteases at sites of tissue injury, thereby preventing incidental damage to surrounding tissue and allowing for orderly initiation of repair. We have previously shown that serine (neutrophilic or pancreatic) elastase and lipopolysaccharide (LPS) each mediate an increase in the expression of alpha 1-PI in human peripheral blood monocytes and bronchoalveolar macrophages. In this study we demonstrate that elastase and LPS have an additive positive regulatory effect on alpha 1-PI expression. Distinct pretranslational and translational mechanisms of action for elastase and LPS, respectively, account for the additive effect. The possibility that translational regulation of alpha 1-PI by LPS involves a mechanism analogous to that of the yeast gene GCN4 during amino acid starvation and that of the human ferritin gene in response to iron is discussed.     

Differential inhibitory properties of dog and human alpha 1-proteinase inhibitors

Dog alpha 1-proteinase inhibitor (alpha 1-PI) was found to be an effective inhibitor of bovine chymotrypsin and also of porcine pancreatic elastase as in the case of human inhibitor. The dog inhibitor inactivated both proteinases at a molar ratio of 1:1. However, compared to the human inhibitor, dog alpha 1-PI was a relatively poor inhibitor of bovine trypsin. The association rate constants (kass) of the interactions of dog alpha 1-PI with bovine chymotrypsin and with porcine elastase were determined to be 6.9 +/- 0.3 X 10(6) M-1 s-1 and 6.4 +/- 0.1 X 10(5) M-1 s-1, respectively. These values are 1.3- and 2.7-fold higher than the corresponding values for the human inhibitor. On the other hand, kass for the dog inhibitor with bovine trypsin (2.6 +/- 0.3 X 10(4)M-1 s-1) was found to be about 5 times smaller than that of the human inhibitor.     

Airway closure on imaging relates to airway hyperresponsiveness and peripheral airway disease in asthma

The regional pattern and extent of airway closure measured by three-dimensional ventilation imaging may relate to airway hyperresponsiveness (AHR) and peripheral airways disease in asthmatic subjects. We hypothesized that asthmatic airways are predisposed to closure during bronchoconstriction in the presence of ventilation heterogeneity and AHR. Fourteen asthmatic subjects (6 women) underwent combined ventilation single photon emission computed tomography/computed tomography scans before and after methacholine challenge. Regional airway closure was determined by complete loss of ventilation following methacholine challenge. Peripheral airway disease was measured by multiple-breath nitrogen washout from which S(cond) (index of peripheral conductive airway abnormality) was derived. Relationships between airway closure and lung function were examined by multiple-linear regression. Forced expiratory volume in 1 s was 87.5 ± 15.8% predicted, and seven subjects had AHR. Methacholine challenge decreased forced expiratory volume in 1 s by 23 ± 5% and increased nonventilated volume from 16 ± 4 to 29 ± 13% of computed tomography lung volume. The increase in airway closure measured by nonventilated volume correlated independently with both S(cond) (partial R(2) = 0.22) and with AHR (partial R(2) = 0.38). The extent of airway closure induced by methacholine inhalation in asthmatic subjects is greater with increasing peripheral airways disease, as measured by ventilation heterogeneity, and with worse AHR.     

Role of disulfide exchange in alpha 1-protease inhibitor.

The major endogenous inhibitor of neutrophil elastase in the plasma, alpha 1-protease inhibitor (alpha 1-PI), has a single cysteine residue which has been shown to form mixed disulfides with a number of thiols in vitro. Under normal physiological conditions, the plasma concentrations of reduced and oxidized thiols are such that a major fraction of alpha 1-PI in the circulation in vivo is in the form of mixed disulfides [Laurell, C.-B. (1979) in The Chemistry and Physiology of Human Plasma Proteins (Bing, D. H., Ed.) pp 329-341, Pergamon, New York]. We show here that the mixed disulfide between glutathione or cysteine and alpha 1-PI (alpha 1-PI-SSG or alpha 1-PI-SScys) has an intrinsic fluorescence which distinguishes it from the reduced form of alpha 1-PI. By employing the fluorescence difference, we have measured the ratio of alpha 1-PI-SH to mixed disulfide alpha 1-PI in redox buffers of different ratios of reduced to oxidized glutathione (GSH to GSSG) or reduced to oxidized cysteine (cys to cysSScys) and have calculated an equilibrium constant and redox potential of 0.74 +/- 0.08 and 8 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SSG couple and of 0.32 +/- 0.02 and 29 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SScys couple. We are unable to detect any change in Trp fluorescence in the complex of alpha 1-PI and elastase when the preformed complex is added to the same GSH/GSSG or cys/cysSScys redox buffers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor

We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.     

Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase

Mouse macrophage elastase, a metalloproteinase, catalytically inactivates human alpha 1-proteinase inhibitor (alpha 1-PI) by attacking a single peptide bond between Pro357 and Met358, resulting in Mr = 4,200 and 47,800 fragments. We show here that this proteolytically inactivated alpha 1-PI is a potent chemotactic factor for human neutrophils at a concentration of 1 nM. The chemotactic response is equivalent to that elicited by formyl-methionyl-leucyl-phenylalanine. Native alpha 1-PI does not stimulate chemotaxis. Purification of the two fragments of alpha 1-PI that result from proteolysis by macrophage elastase indicated that the Mr = 4,200 fragment is responsible for the chemotactic activity. However, the two proteolysis fragments do not dissociate from each other under physiologic conditions. Therefore, the ability of proteolytically inactivated alpha 1-PI to act as a mediator of inflammation is due to rearrangement of the alpha 1-PI molecule rather than to release of a cleavage fragment.     

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.     

Study of the effect of high molecular weight kininogen upon the fluid-phase inactivation of kallikrein by C1 inhibitor

Plasma kallikrein and factor XIa circulate bound to high molecular weight kininogen, and such binding has been reported to protect these enzymes from inactivation by their respective inhibitors. However, this observation is controversial, and the effect of high molecular weight kininogen upon the interaction between kallikrein and C1 inhibitor (C1-INH) has been questioned. We have re-evaluated this reaction and studied the rate of inhibition of kallikrein by C1-INH in the presence and absence of high molecular weight kininogen. The second-order rate constant of inhibition of kallikrein by C1-INH was unaffected by saturating concentrations of high molecular weight kininogen. Our results suggest that although high molecular weight kininogen clearly augments the rate of formation of kallikrein and other enzymes of the contact activation pathway, it has no effect on the rate of enzyme inhibition by C1-INH.     

Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products

Previously we demonstrated that in vivo exposure of humans to NO2 resulted in significant inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the bronchoalveolar lavage fluid. However, alpha 1-PI retains its elastase inhibitory activity in vitro when exposed to 10 times the concentration of NO2 used in vivo. We suggested exogenous oxidants such as O2 and NO2 exert their effect in vivo in part through lipid peroxidation. We investigated the mechanism of inactivation of alpha 1-PI in the presence or absence of lipids under oxidant atmosphere. alpha 1-PI in solutions containing phosphate buffer (control), 0.1 mM stearic acid (saturated fatty acid, 18:0), or 0.1 mM linoleic acid (polyunsaturated fatty acid, 18:2) was exposed to either N2 or NO2 (50 ppm for 4 h). Elastase inhibitory capacity of alpha 1-PI was significantly diminished in the presence of 0.1 mM linoleic acid and under NO2 atmosphere (75 +/- 8% of control, P less than 0.01), whereas there was no change in elastase inhibitory capacity of alpha 1-PI in the presence or absence (buffer only) of 0.1 mM stearic acid under a similar condition (109 +/- 11 and 94 +/- 6%, respectively). The inactivated alpha 1-PI as the result of peroxidized lipid could be reactivated by dithiothreitol and methionine sulfoxide peptide reductase, suggesting oxidation of methionine residue at the elastase inhibitory site. Furthermore the inhibitory effect of peroxidized lipid on alpha 1-PI could be prevented by glutathione and glutathione peroxidase and to some extent by alpha-tocopherol.