The protease inhibitor system of macaques. The identification of alleles using isoelectric focusing and family analysis

Five alpha-1-antitrypsin (alpha-1-AT) phenotypes have been revealed by isoelectrofocusing (IEF) in sera of 215 crab-eating macaques. Alpha-1-AT was monomorphic in sera of 250 Rhesus monkeys. A new allele of macaque Pi-system, designated as B' was postulated in addition to existing two (B and C) on the basis of IEF data. The above conclusion was supported by family analysis, based on 35 monkey birth cases. Alpha-1-AT phenotype frequencies were in agreement with Hardy-Weinberg equation both in wild and capture born crab-eating macaques. Alpha-1-AT was found to be microheterogeneous: several zones of the protein were revealed by IEF and Western blotting with anti human alpha-1-AT serum. The pregnancy caused a sharp increase of one band. This may lead to false identification of alpha-1-AT phenotypes, particularly when acid starch gel electrophoresis is used for alpha-1-AT identification. Such misinterpretation during alpha-1-AT phenotyping may explain the disagreement with Hardy-Weinberg equation described earlier for crab-eating macaques.     

Isolation, chemical, and physical properties of alpha-1-antitrypsin.

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.     

Role of human neutrophil peptides in lung inflammation associated with alpha1-antitrypsin deficiency

Individuals with alpha(1)-antitrypsin (alpha(1)-AT) deficiency are at risk for early-onset destructive lung disease as a result of insufficient lower respiratory tract alpha(1)-AT and an increased burden of neutrophil products such as elastase. Human neutrophil peptides (HNP), the most abundant protein component of neutrophil azurophilic granules, represent another potential inflammatory component in lung disease characterized by increased numbers of activated or deteriorating neutrophils. The purpose of this study was to determine the role of HNP in lower respiratory tract inflammation and destruction occuring in alpha(1)-AT deficiency. alpha(1)-AT-deficient individuals (n = 33) and healthy control subjects (n = 21) were evaluated by bronchoalveolar lavage. HNP concentrations were significantly higher in alpha(1)-AT-deficient individuals (1,976 +/- 692 vs. 29 +/- 12 nM, P < 0.0001), and levels correlated with markers of neutrophil-mediated lung inflammation. In vitro, HNP produced a dose-dependent cytotoxic effect on alveolar macrophages and stimulated production of the potent neutrophil chemoattractants leukotriene B(4) and interleukin-8 by alveolar macrophages, with a 6- to 10-fold increase in chemoattractant production over negative control cultures (P < 0.05). A synergistic effect was noted between HNP and neutrophil elastase with regard to leukotriene B(4) production. Importantly, the proinflammatory effects of HNP were blocked by alpha(1)-AT. HNP likely play an important role in amplifying and maintaining neutrophil-mediated inflammation in the lungs.     

Evaluation of recombinant DNA-directed E.coli produced alpha 1-antitrypsin as an anti-neutrophil elastase for potential use as replacement therapy of alpha 1-antitrypsin deficiency

alpha 1-antitrypsin (alpha 1AT) deficiency is an inherited disorder almost always associated with the development of panacinar emphysema in the fourth to fifth decades. One source of alpha 1AT for chronic replacement therapy of such individuals is that produced by E.coli directed by a cDNA coding for the human alpha 1AT molecule. Using TG1(E.coli), an alpha 1AT molecule produced by E.coli transformed with the plasmid-expressing vector pTG922, the present study shows that recombinant DNA-directed E.coli-produced alpha 1AT is as an effective inhibitor of neutrophil elastase as alpha 1AT purified from plasma. Importantly, TG1(E.coli) inhibited human neutrophil elastase with an association rate constant of 1.3 +/- 0.4X10(7) M-1 sec-1, similar to that of normal plasma alpha 1AT (1.1 +/- 0.1, p greater than 0.2). Furthermore, when TG1(E.coli) was added to alpha 1AT-deficient plasma obtained from homozygous alpha 1AT type Z individuals, the TG1(E.coli) remained functional and augmented the anti-neutrophil elastase activity of the serum proportional to the amount of TG1(E.coli) added. These observations suggest that if sufficient amounts of recombinant DNA methodology-produced alpha 1AT molecules could be safely delivered to the alveolar structures of alpha 1AT-deficient individuals, they would function to protect the alveolar walls from elastolytic attack.     

Comparison of the chemical, physical, and survival properties of normal and Z-variant alpha-1-antitrypsins.

A procedure has been developed for the purification of Z-type alpha-1-antitrypsin (alpha-1-AT) which is rapid, gentle, and results in good yields. From 4 units (750 ml) of fresh human plasma, obtained from two individuals possessing the Pizz phenotype, 53 mg of pure Z-type alpha-1-AT was obtained. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulfate, and by analytical ultracentrifugation. When compared to pure alpha-1-AT from plasma of individuals possessing the normal PiMM phenotype, the two proteins were indistinguishable with respect to amino acid composition, sedimentation coefficient (s20w of 3.33 for both M and Z), molecular weight (51,000 by sodium dodecyl sulfate gel electrophoresis and 47,000 by sedimentation equilibrium for both M and Z), and trypsin-combining ratio (0.91 for Z and 0.99 for M). The only difference which was observed between the variant forms of alpha-1-AT was in the carbohydrate composition. The Z-type alpha1-AT contains between 20 and 25% less carbohydrate than the M-type alpha-1-AT. Specifically, the Z-type alpha-1-AT is deficient in 1 glucosamine residue, 3 neutral sugar residues (1 mannose and 2 galactose), and 2 sialic acid residues. Although the Z-variant is deficient in sialic acid, its survival time in the serum of a rabbit was not significantly different from that of M-type alpha-1-AT.     

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

The degradation of bovine parathyroid hormone by leukocytes

Human peripheral leukocytes catalyse an elastase-like cleavage of bovine parathyroid hormone, with an identical specificity to that previously observed with a neutral proteinase (EC 3.4.21-) isolated from the outer surface of plasma membranes from human leukocytes. Parathyroid hormone is not hydrolysed by human erythrocytes, and polymorphonuclear leukocytes are much more effective in hormone degradation than lymphocytes. Despite the fact that purified human leukocyte granular elastase (EC 3.4.21.37) catalyses an identical cleavage reaction, the hydrolysis observed with intact cells is clearly not due to proteinases secreted by the cells. The reaction is inhibited by human serum and by purified human alpha-1-antitrypsin, but not by antibodies to human leukocyte granular elastase. The possible significance of these phenomena to the in vivo metabolism of parathyroid hormone are discussed.     

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use

Summary. Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area¹. ¹ The opinions expressed in this paper reflect the authors’ personal views, based on published data and the information available from the public domains, and have no relation to the official statements, if any, held by the US FDA, National Institutes of Health, or the US Department of Health and Human Services. FDA official recommendations for plasma protein therapeutics and recombinant proteins regarding safety, purity, and potency of new drugs and biologics produced by recombinant DNA technology are referred below as the US FDA Guidances.     

Radioimmunoassay of somatomedin-C in the baboon (Papio cynocephalus): A comparison of multiple techniques of measurement

This study was undertaken to assess the nature of somatomedin-C (SM-C) in baboon (Papio cynocephalus) blood and to compare various methods for estimating SM-C concentrations. Parallel dose-response curves were obtained with normal baboon serum, normal human serum, and purified SM-C. Recovery of purified SM-C added to baboon serum over a wide dosage range (n = 17) was 111 ± 12%, with slightly better recovery at higher potencies. Chromatography of normal baboon serum on Sephadex G-200 at neutral pH produced a profile similar to that observed in the human, as did samples chromatographed on Sephadex G-50 in acid. Although the SM-C content in acid chromatographed plasma was approximately 2.5 times higher than in native unprocessed plasma, there was excellent correlation between the values (r = 0.9143, p < 0.0001). The SM-C in baboon plasma which had been preincubated with glycine HCl was approximately twice that of unprocessed plasma, but the correlation between the two methods was excellent (r = 0.9593, p < 0.0001). The correlation between values obtained after simple acid-ethanol extraction and those observed in unextracted plasma were also significant (r = 0.7689, p < 0.0001). Following a series of four injections of human growth hormone (hGH) to a normal baboon, plasma SM-C rose approximately sevenfold above the initial concentration and returned to basal levels five days after the final injection. These studies show that although the radioimmunoassay (RIA) for SM-C in unprocessed baboon plasma does not measure all of the SM-C present, it provides a reliable index of the total SM-C concentration and reflects GH status in the baboon.     

Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin

The addition of either cathepsin-G or leukocyte elastase to endotoxin-stimulated human peripheral blood monocytes decreased the immunoreactive tumor necrosis factor (TNF) detected in culture supernatants in a concentration-dependent manner. Both enzymes also induced a loss of supernatant cytolytic activity as determined on the WEHI-164 target cell line. Incubation of recombinant human TNF and lymphotoxin (LT) with either cathepsin-G or leukocyte elastase resulted in a loss of cytokine bioactivity. Examination of enzyme-treated recombinant cytokines by gel electrophoresis revealed that cathepsin-G cleaved LT into a 12.6-kDa fragment and leukocyte elastase fragmented LT into a 14.1-kDa product. On Western blots cathepsin-G and leukocyte elastase degraded TNF into 11- and 7.6-kDa fragments, respectively. Incubating leukocyte elastase with plasma elastase inhibitor alpha-1-antitrypsin prevented the loss of recombinant TNF bioactivity and blocked the degradation of this cytokine. This study suggests that two of the most abundant neutrophil proteases, cathepsin-G and leukocyte elastase, may be important regulators of TNF and LT bioactivity.     

Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes

Both normal and chronic granulomatous disease (CGD) neutrophils were able to degrade the subendothelial matrix secreted by human endothelial cells via an elastase-dependent process. In the absence of the plasma antiproteinase, alpha-1-proteinase inhibitor (alpha-1-PI), normal neutrophils protect their released elastase from inactivation by using the chlorinated oxidants hypochlorous acid and endogenous N-chloroamines to suppress the antiproteinase's activity. In contrast, CGD neutrophils were unable to generate either class of chlorinated oxidant or to inactivate the porcine pancreatic elastase inhibitory capacity of alpha-1-PI unless the cells were supplemented with exogenous hydrogen peroxide. Despite the reliance of normal neutrophils on chlorinated oxidants to inactivate alpha-1-PI, neutrophils triggered in the presence of agents that block the generation of these reactive species continued to degrade the subendothelial matrix at a suppressed but significant rate in the presence of a 50-fold excess of the antiproteinase. The continued solubilization of the matrix by normal neutrophils was not due to the incomplete inhibition of oxidant generation because triggered CGD neutrophils were also able to degrade the matrix in the presence of excess alpha-1-PI. If CGD neutrophils were stimulated in the presence of an exogenous source of H2O2 and alpha-1-PI, the proteolytic potential of the cells was identical to that observed with normal stimulated neutrophils. We conclude that normal neutrophils can enhance their ability to degrade the subendothelial matrix by oxidatively protecting elastase from inactivation by alpha-1-PI but both normal and CGD neutrophils possess non-oxidatively linked mechanisms for sequestering and using elastase to mediate proteolytic effects in the presence of native antiproteinase.     

The degradation of human lung elastin by neutrophil proteinases

Human lung elastin has been isolated by both a degradative and nondegradative procedure and the products obtained found to have amino acid compositions comparable to published results. These elastin preparations, when utilized as substrates for various mammalian proteinases, were solubilized by porcine elastase at a rate six times faster than human leukocyte elastase. Leukocyte cathepsin G also solubilized lung elastin but only at 12% of the rate of the leukocyte elastase. In all cases the elastin prepared by nondegradative techniques proved to be the best substrate in these studies. The differences in the rate of digestion of elastin of the two elastolytic proteinases was readily attributed to the specificity differences of each enzyme as judged by carboxyterminal analysis of solubilized elastin peptides. The plasma proteinase inhibitors, alpha-1-proteinase inhibitor and alpha-2-macroglobulin abolished the elastolytic activity of both leukocyte enzymes, while alpha-1-antichymotrypsin specifically inactivated cathespsin G. Two synthetic inhibitors, Me-O-Suc-Ala-Ala-Pro-Val-CH2Cl (for elastase and Z-Gly-Leu-Phe-CH2Cl (for cathepsin G) were equally effective in abolishing the elastolytic activity of the two neutrophil enzymes. However, inhibition of leukocyte elastase by alpha-1-proteinase inhibitor was significantly suppressed if the enzyme was preincubated with elastin prior to addition of the inhibitor.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

Isolation and Characterization of Alpha-1-Antitrrpsin from the Cohn Fraction Iv-1 of Human Plasma

Cohn Fraction IV-I from pooled human plasma was used as a starting material in the large-scale purification of alpha-1-antitrypsin (alpha-1-AT). Following ion-exchange, blospecific affinity and gel exclusion chromatographic procedures, material of high biological activity was obtained in 307percnt; overall yield. Homogeneity was demonstrated by acrylamide gel electrophoresis, immunoelectrophoresis, ultracentrifugation, gel filtration and end-group determination. The present preparation should be applicable to large scale industrial processing of alpha-1-AT with the potential for use in protein replacement therapy.     

Purification, characterization, and acute phase response of plasma alpha-1-antiproteinase in the hamster, Mesacricetus auratus.

1. alpha-1-Antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) with a molecular mass of 60 kDa was purified to apparent homogeneity from hamster plasma. 2. It inhibited elastase, chymotrypsin and trypsin, but did not significantly affect pancreatic kallikrein, plasma kallikrein or plasmin. 3. It has the same N-terminal heptapeptide sequence as that of rat alpha-1-antiproteinase. 4. Its plasma level decreased after injection of bacterial lipopolysaccharide.     

Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein.

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.     

Differences in acute lung response to elastase instillation in two rodent species may determine differences in severity of emphysema development

Elastase intratracheal instillation induces early emphysema in rodents. However, Syrian Golden hamsters develop more severe emphysema than Sprague-Dawley rats. We have reported species differences in oxidant/antioxidant balance modulating antiprotease function early after instillation. We now hypothesize that other components of the initial lung response to elastase might also be species-dependent. Sprague-Dawley rats and Syrian Golden hamsters received a single dose of pancreatic elastase (0.55 U/100 g body wt) to study acute lung injury biomarkers. Using serum, lung, and bronchoalveolar lavage fluid (BALF) samples, we evaluated changes in alveolar-capillary permeability, alpha 1-antitrypsin (α(1)-AT) concentration and activity, glutathione content, and proinflammatory cytokines. Rats showed a large increase in alveolar-capillary permeability and few hemorrhagic changes, whereas hamsters exhibited large hemorrhagic changes (P < 0.01) and mild transendothelial passage of proteins. Western blots showed a 30-fold increase in BALF α(1)-AT concentration in rats and only a 7-fold increase in hamsters (P < 0.001), with [α(1)-AT-elastase] complexes only in rats, suggesting differences in antiprotease function. This was confirmed by the α(1)-AT bioassay showing 20-fold increase in α(1)-AT activity in rats and only twofold increase in hamsters (P < 0.001). In rats, results were preceded by a 3-, 60-, and 20-fold increase in IL-6, IL-1β, and TNF-α respectively (P < 0.001). In hamsters, only IL-1β and TNF-α showed mild increases. All parameters studied were back to baseline by 4 days. In conclusion, several components of the initial lung response showed species differences. Cytokine release pattern and functional inhibition of α(1)-AT were the most significant components differing among species and could account for differences in susceptibility to elastase.