Mouse alpha 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.     

alpha 1-Antichymotrypsin is the human plasma inhibitor of macrophage ectoenzymes that cleave pro-macrophage stimulating protein

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.     

Cigarette smoke components are not very effective in directly inactivating alpha 1-proteinase inhibitor

Cigarette smoke was found to be rather ineffective in inactivating alpha 1-proteinase inhibitor (alpha 1-PI) in aqueous solution, whereas a slow inactivation of alpha 1-PI by a dimethyl sulfoxide extract of whole cigarette smoke condensate was observed. However, this inactivation could only partially be prevented by antioxidants indicating that it is not, or at least not exclusively, due to oxidation. The bulk of inactive alpha 1-PI found in lung lavage fluids from smokers is thus probably generated through endogenous mechanisms and not through smoke components directly.     

The assessment of alpha 1 proteinase inhibitor form and function in lung lavage fluid from healthy subjects

The form and function of alpha 1 proteinase inhibitor in lung lavage fluid from healthy smoking and non smoking individuals has been accurately assessed using critically appraised techniques. The present study demonstrated that it is possible to accurately assess alpha 1 PI function in unconcentrated lavage fluid but that sample collection, storage and subsequent processing may all affect the results. Absolute levels of alpha 1 PI were elevated in subjects who smoke and a substantial quantity of inactive protein was found in both smokers and non smokers. The proportion of inactive alpha 1 PI was similar for both groups, which by inference implies that normal smoking subjects do not have decreased protection by this inhibitor at the bronchoalveolar level. Physicochemical analysis of the alpha 1 PI in these normal subjects showed that it was different from alpha 1 PI previously reported from patients with established disease and this may have important implications regarding the pathogenesis of their condition. Western immunoblotting of bronchoalveolar lavage fluid (BALF) showed that all of the alpha 1 PI was present in the native molecular mass form (54,000 Da). Pre-incubation of samples with methionine sulphoxide peptide reductase restored alpha 1 PI function only by approximately 10% suggesting the presence of little reversibly oxidised alpha 1 PI in either group. Anion exchange HPLC of BALF revealed the presence of two alpha 1 PI species, one of which co-eluted with native, oxidised or proteolyzed forms and the other which was more cationic and did not inhibit porcine pancreatic elastase. Finally, thirteen out of sixteen BALF samples inhibited more neutrophil elastase than could be accounted for by the amounts of functional alpha 1 PI present, suggesting that the presence of other inhibitors is a feature of normal lavage fluids.     

Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

Expression of an active proteinase inhibitor, alpha 1-antichymotrypsin, by human breast epithelial cells

The synthesis of an active proteinase inhibitor, gp 66, by human breast epithelial cells is reported. This glycoprotein is identical to serum alpha 1-antichymotrypsin, which inhibits proteinases that cleave at hydrophobic residues. Immunohistological studies show the in vivo expression on normal secretory and ductal epithelial cells and on primary and metastatic adenocarcinomas. Immunoaffinity-purified gp 66 from MCF-7 culture supernatants is an active inhibitor of chymotrypsin as determined in a fluorogenic enzyme assay and can form stable 88 kDa enzyme-inhibitor complexes. The synthesis of a functional inhibitor may represent the epithelial cell's attempt to stabilize its extracellular milieu.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

Granzyme activity in the inflamed lung is not controlled by endogenous serine proteinase inhibitors

Numerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0-37) and 0 (0-6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0-1,889) and 10 (0-78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely alpha1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP.     

Ornithodoros moubata complement inhibitor is an equally effective C5 inhibitor in pigs and humans

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.     

Elafin is a potent inhibitor of proteinase 3

Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C.     

High-molecular-mass proteinases in rabbit reticulocytes: the multicatalytic proteinase is an ATP-independent enzyme and ATP-activated proteolysis is in part associated with a cysteine proteinase complexed to alpha 1-macroglobulin

We have investigated the proteolytic degradation of [14C]methylcasein and 125I-labeled bovine serum albumin at pH 7.8 and 37 degrees C by lysates of rabbit reticulocytes purified from rabbit blood by two different procedures. (I) Lysates obtained from reticulocytes after removal of plasma and buffy coat as well as after washing of cells, degraded casein and albumin, and released from the two substrates 1.3%/h and 0.4%/h, respectively, of acid-soluble radioactivity. The activity towards both substrates was stimulated about 4-fold by ATP/Mg2+. Chromatography of whole blood on a column of cellulose prior to washing and lysis of cells had profound but differential effects on these activities in that stimulation of casein-degradation by ATP/Mg2+ was almost completely lost, whereas degradation of albumin, albeit at a low rate, was measurable in the presence of ATP/Mg2+ only. (II) Degradation of casein by these lysates is largely inhibited by a monospecific antibody against rabbit multicatalytic proteinase, whereas digestion of albumin is not affected by the antibody, either in the presence or absence of ATP/Mg2+. The latter activity is partially inhibited by a specific antibody against rabbit alpha 1-macroglobulin. (III) The immunoreactive amount of multicatalytic proteinase is about 1.2 micrograms per mg of lysate protein and almost identical in the two lysates. In contrast, the immunologically detectable levels of alpha 1-macroglobulin vary and are much lower in reticulocyte-lysates following chromatography on cellulose than in lysates from washed reticulocytes. (IV) Caseinolytic activity of multicatalytic proteinase, purified from rabbit reticulocyte lysate, is not activated by ATP/Mg2+ and the enzyme is proteolytically inactive towards albumin. On the other hand, a complex consisting of the proteinase inhibitor alpha 1-macroglobulin and the cysteine proteinase, cathepsin B, does degrade both substrates at pH 7.8, in an ATP/Mg2+-activated fashion. From these results it is concluded that the multicatalytic proteinase is an ATP-independent enzyme and a cellular constituent of rabbit reticulocytes whereas the activity stimulated by ATP/Mg2+ appears to be associated, at least in part, with a cysteine proteinase complexed to alpha 1-macroglobulin.     

Rat lung tissue is a site of alpha 1-proteinase inhibitor synthesis: evidence by cell-free translation

Poly(A) +RNA isolated from lungs of normal rats and of rats suffering from experimental inflammation was translated in a cell-free translation mixture from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-proteinase inhibitor and alpha 2-macroglobulin. Comparable levels of mRNA for alpha 1-proteinase inhibitor were found in rat lung tissue from control and experimentally inflamed animals. alpha 2-Macroglobulin mRNA could not be detected in rat lung tissue.