Oxidants Spontaneously Released by Alveolar Macrophages of CigaretteSmokers Can Inactivate the Active Site of al -Antitrypsin,Rendering It Ineffective as an Inhibitor of Neutrophil ElastaseRichard C, Hubbard, Fumitaka Ogushi, Gerald A. Fells, Andre M. Cantin,* Sophie Jallat,Michael Courtney, and Ronald G. CrystalPulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes ofHealth, Bethesda,Maryland 20892; and *Transgene SA, Strasbourg, 67000 France

AbstractCurrent concepts relating to the pathogenesis of emphysemaassociated with cigarette smoking is that an imbalance existswithin the lower respiratory tract between neutrophil elastaseand the local anti-neutrophil elastase screen, enabling uninhib-ited neutrophil elastase to destroy the alveolar structures overtime. The possible role of alveolar macrophages in contributingto this imbalance was investigated by evaluating the ability ofcigarette smokers' alveolar macrophages to inactivate alpha1-antitrypsin (alAT), the major anti-neutrophil elastase of thehuman lower respiratory tract. In vitro, alveolar macrophagesof smokers spontaneously released 2.5-fold more superoxideanion and eightfold more H202 than macrophages of nonsmok-ers (P < 0.01, both comparisons). Using a model system thatreproduced the relative amounts of alveolar macrophages andaIAT found in the epithelial lining fluid of the lower respira-tory tract, we observed that smokers' macrophages caused a60±5% reduction in the ability of alAT to inhibit neutrophilelastase. In marked contrast, under the same conditions, non-smokers' macrophages had no effect upon the anti-neutrophilelastase function of alAT. Addition of superoxide dismutase,catalase, mannitol, and methionine prevented inactivation ofalAT by smokers' macrophages, implying that the release ofoxidants mediated the inactivation of alAT. In addition, byutilizing a recombinant DNA produced modified form ofalATcontaining an active site substitution (met" -* val), the inac-tivation of alAT by smokers' alveolar macrophages was pre-vented, suggesting that the smokers' macrophages inactivatealAT by oxidizing the active site of the alAT molecule. Theseresults suggest that in cigarette smokers, the alveolar macro-phage can modulate the activity of alAT as an inhibitor ofneutrophil elastase and thus play a role in the pathogenesis ofemphysema associated with cigarette smoking.

Introduction

Alpha l-antitrypsin (alAT),' is a 52-kD glycoprotein thatserves as the major inhibitor of neutrophil elastase, a potent

Address reprint requests to Richard C. Hubbard, M.D., PulmonaryBranch, Building 10, Room 6D03, National Institutes of Health, Be-thesda, MD 20892.

Dr. Cantin's present address is Hospitalier Universitaire de Sher-brooke, Unite de Recherche, Pulmonaire, Piece 3601, Sherbrooke,P.Q. Canada J1H 5N4.

Received for publication 20 March 1987 and in revised form 30June 1987.

1. Abbreviations used in this paper: aIAT, alpha l-antitrypsin; ELF,epithelial lining fluid; MeO-SAAPV-NA, methoxy-succinyl-alanyl-

The Journal of Clinical Investigation, Inc.Volume 80, November 1987, 1289-1295

serine protease that is capable of attacking most protein com-ponents of the extracellular matrix (1-3). The critical impor-tance of al AT as an inhibitor of neutrophil elastase is high-lighted by alAT deficiency, a hereditary disorder associatedwith plasma alAT levels < 35% of normal and the develop-ment of emphysema in the third to fourth decades (4-7). Inthis context, the pathogenesis of the emphysema associatedwith alAT deficiency is conceptualized as an imbalance be-tween a 1 AT and neutrophil elastase in the alveolar structuressuch that there is insufficient alAT to provide a protectivescreen against the burden of neutrophils, and hence neutrophilelastase, in the local milieu (4, 8, 9).

The same concepts have been extended to conceptualizethe pathogenesis of the emphysema associated with cigarettesmoking (10-15). Despite the fact that most of these individ-uals have normal levels of a 1 AT in blood and lung (16, 17),the knowledge that alAT can be rendered impotent by oxida-tion has led to the concept that cigarette smoking may lead toan inactivation of a 1AT in the lung, leaving the lower respira-tory tract without its normal anti-neutrophil elastase defenses(1, 12-15, 18). This concept is supported by in vitro studiesshowing that cigarette smoke will inactivate alAT by oxida-tive mechanisms (12, 19-21) and that the alAT recoveredfrom the epithelial surface of the lower respiratory tract ofcigarette smokers has a reduced association rate constant forneutrophil elastase (22). Furthermore, the alAT recoveredfrom the lung ofcigarette smokers has been observed to have adecreased ability to inhibit porcine pancreatic elastase com-pared with the lung alAT of nonsmokers (13, 23, 24), al-though this observation has been disputed (25, 26).

Although it is compelling to invoke cigarette smoke, withits high concentration of oxidants (20), as a source of the oxi-dant burden that potentially could inactivate a lAT in thelower respiratory tract, another potential source of oxidants inthe lower respiratory tract of cigarette smokers are alveolarmacrophages. In this regard: (a) when activated, alveolar mac-rophages are capable of releasing oxidants, including superox-ide anion, hydrogen peroxide, and hydroxyl radical (27, 28);(b) the number of alveolar macrophages in the lower respira-tory tract of cigarette smokers is increased severalfold (17, 29,30); (c) when activated in vitro, alveolar macrophages can re-lease sufficient oxidants to render a 1 AT an ineffective inhibi-tor of neutrophil elastase (31, 32); and (d) alveolar macro-phages recovered from the lungs ofcigarette smokers are spon-taneously releasing oxidants (29, 33). In the context of theseobservations it is reasonable to hypothesize that alveolar mac-rophages in the lower respiratory tract of cigarette smokers

alanyl-prolyl-valine nitroanilide; O°, superoxide anion; SOD, super-oxide dismutase.

Macrophage Inactivation ofal-Antitrypsin 1289

may be spontaneously releasing sufficient oxidants to inacti-vate aIAT in the local milieu.

To evaluate this hypothesis, we recovered alveolar macro-phages from the lower respiratory tract of nonsmokers andsmokers, and evaluated the ability of these cells to modulatethe activity of alAT as an inhibitor of neutrophil elastase. Inaddition, by utilizing a recombinant DNA-produced modifiedform of a 1 AT containing an active site substitution (met358val) rendering it resistant to oxidation but still active as aninhibitor of neutrophil elastase, we have demonstrated that thespontaneous inactivation ofa IAT by macrophages of cigarettesmokers occurs, at least in part, by oxidation of the active siteof the a IAT.

Methods

Study population. The study population consisted of 5 healthy non-smoking individuals (three male, two female; age 31±7 yr) and 10healthy smoking individuals (six male, four female; age 32±6 yr) withan average cigarette smoking history of 27±11 pack years. (Data ispresented as mean±SE of the mean; all statistical comparisons werecarried out using the two-tailed Student's t test.) All individuals werefree of lung disease as determined by the combined criteria of havingnormal histories, physical examinations, chest x rays, and lung func-tion tests (34).

Lower respiratory tract inflammatory cells. Inflammatory cellswere recovered from the lower respiratory tract of nonsmokers andsmokers by bronchoalveolar lavage as previously described (35). Al-veolar macrophages were purified by adherence (1 h, 370) at 5 X 105cells in 1 ml in 24-well tissue culture plates (Falcon Labware, Oxnard,CA) in RPMI medium (Gibco, Grand Island, NY) containing 10% calfserum (Whitaker M. A. Bioproducts, Walkersville, MD). In all casesthe alveolar macrophages were > 95% pure and > 90% viable (as as-sessed by trypan blue). The volume of epithelial lining fluid (ELF)recovered, quantified using the urea method (36), was 2.0±1.0 ml fornonsmokers and 2.7±1.1 ml for smokers (P> 0.2). Concentrations ofalAT in ELF were quantified using an enzyme-linked" immunoassay(in quadruplicate) with a standard aIAT that had been quantified byamino acid analysis (37).

The spontaneous release of superoxide anion (O-) and H202 by thepurified alveolar macrophages of the nonsmokers and smokers wasquantified in vitro (38, 39). Briefly, for O-, the alveolar macrophages(5 X 105/well) in 24-well plates were incubated in 0.5 ml of Hanks'balanced salt solution (HBSS; Gibco) containing 80 nm ferricy-tochrome c (Type III, Sigma Chemical Co., St. Louis, MO). Afterincubation for 30 min at 370, the amount of O" released into themedium was quantified at 550 nm. For the measurement of the spon-taneous release of H202, HBSS containing 0.28 mM phenol red, 8.5U/ml horseradish peroxidase (Type II, Sigma Chemical Co.) was addedand the macrophages incubated for 30 min at 37°. The amount ofH202 in the supernatant was then determined by the addition of 10 Mlof 1 N NaOH and the absorbance read at 61'0 nm. The concentra-tion of H202 was based on a standard curve using various concentra-tions of reagent grade H202.

Source of aJAT. Normal human alAT was purified from theplasma of an individual homozygous for the M l(va1213) form ofalATas identified by'the combined criteria of isoelectric focusing, seruma 1 AT levels, family studies, and BstEII restriction endonuclease map-ping ofgenomic DNA (40, 41). Purification ofalAT from plasma wasaccomplished by positive selection affinity chromatography (42). Thepurified alAT was' concentrated by pressure filtration (YM-10 mem-brane, Amicon Corp., Danvers, MA) and stored in aliquots in liquidnitrogen vapor until use. The final preparation was > 95% pureas assessed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis.

Recombinant aIAT was produced in Escherichia coli as previously

described (43, 44), using an aIAT complementary DNA (cDNA) ob-tained from translation of human liver RNA. Competent TGE900 E.coli cells were transformed with an expression plasmid containing thealAT cDNA and the recombinant alAT purified from bacterial ly-sates. Two forms of recombinant alAT were produced. The normalform (met358 ra I AT) has the identical sequence of the Ml (val213) formofhuman a 1AT present in plasma except for an additional NH2-termi-nal methionine and the absence of carbohydrates. It is designatedmet358 ralAT to indicate that it contains a methionine in the activeinhibitory site, as does the normal M l(vaI2I3) protein. The val358ra 1AT form was identical to the met358 ra 1AT except for the substitu-tion of val358 at the active site. The val358 ra IAT has been shown to besimilar to the met358 ralAT in terms of its ability to inhibit humanneutrophil elastase, but the va1358 ra1AT is much more resistant tooxidation (44-48).

Evaluation ofability ofthe alA Tpreparations to inhibit neutrophilelastase. The ability ofa 1AT to inhibit neutrophil elastase was evalu-ated using two assays: (a) the time-dependent inhibition of equimolarconcentration of neutrophil elastase and a 1 AT; and (b) the time-inde-pendent inhibition of neutrophil elastase by increasing concentrationsof alAT. Both assays were carried out using a neutrophil elastasestandard for which the activity had been determined (37).

The time-dependent inhibition of purified alAT with neutrophilelastase was carried out by the method of Beatty et al. ( 11) with minormodifications described by Straus et al. (37). In brief, the alAT wastitrated using neutrophil elastase to determine the percentage of activealAT in the preparation. Equimolar amounts of elastase and activea 1AT (I nM each) were then reacted at 230 in a 1 -ml reaction volumecontaining 100 mM Hepes, pH 7.5, 0.5 M NaCl, and 0.1% Brij-35.Residual elastase activity at various times was determined by terminat-ing the reaction by the addition of the specific neutrophil elastasesubstrate methoxy-succinyl-alanyl-alanyl-prolyl-valine nitroanilide(MeO-SAAPV-NA; 1 mM; Sigma Chemical Co.) and the residualelastase activity measured at 410 nm as a change in optical density perminute using a DU-7 spectrophotometer (Beckman Instruments, Ful-lerton, CA). At each time point the percent inhibition of elastase wasthen quantified.

Time-independent inhibition of neutrophil elastase by alAT wasdetermined by incubating a fixed amount (2 nM) of active neutrophilelastase with various concentrations (0-8 nM) ofalAT at 230 for 2 h, atime at which the time-dependent assays demonstrated were at leasttwofold longer than necessary for reaction of neutrophil elastase withthe a IAT (1 1, 49). After the incubation, the neutrophil elastase-spe-cific substrate MeO-SAAPV-NA was added and percent residual elas-tase activity was determined as previously described.

Evaluation ofability ofsmoker alveolar macrophages to spontane-ously inactivate alAT as an inhibitor of elastase. The ability ofsmokers' alveolar macrophages to spontaneously inactivate a IAT wasevaluated using an in vitro assay that mimicked the numbers ofmacro-phages and concentrations ofalAT in the epithelial lining fluid of thelower respiratory tract. To accomplish this, alveolar macrophages re-covered from the lower respiratory tract of smokers and nonsmokerswere exposed in vitro to purified plasma alAT (type Ml[val213], seeprevious section) in a chamber designed to permit only low-molecularweight products of the macrophages to reach the a 1 AT. The chamberwas divided in two sections, a lower alAT-containing section and anupper macrophage chamber. The total volume of the chamber was1,400 Id (upper section, 1,000 ,ul + lower section, 400 Ml). Separatingthe chambers was a dialysis membrane (Union Carbide Corp., Dan-bury, CT) with a molecular weight cutoff of 12,000-14,000 kD. Al-veolar macrophages (2 X 106) from a nonsmoker or a smoker wereplaced in HBSS containing 0.1% glucose in the upper chamber andalAT (IIMM in HBSS containing 0.1% glucose) was placed in the lowerchamber. The numbers of alveolar macrophages and the amounts ofa1AT used were always in the ratio of 4.65 X 109 macrophages/MmolalAT. This value was based on the average ratio of macrophages toa1AT recovered in the ELF of nonsmokers, and is approximatelythreefold less than that observed in smokers, i.e., the design was based

1290 Hubbard, Ogushi, Fells, Cantin, Jallat, Courtney, and Crystal

on that found in normals and was biased against the increased ratio ofmacrophages to alAT present in the lower respiratory tract ofsmokers. Control samples contained alAT in the lower chamber butno added alveolar macrophages. After the chambers were incubatedfor 18 h at 370, alAT was retrieved from the lower portion of thechamber and then evaluated for its ability to inhibit neutrophil elas-tase, as previously described.

To demonstrate that low-molecular weight mediators of smokeralveolar macrophages that inactivated the a I AT were oxidants, paral-lel incubations were carried out using alveolar macrophages fromsmokers and purified plasma alAT but with the addition of antioxi-dants to the chambers. Superoxide dismutase (SOD; 400 U/ml, TypeIII, Sigma Chemical Co.), catalase (1,000 U/ml, bovine liver, SigmaChemical Co.), or a combination of both antioxidants were added toboth the macrophage and alAT portions of the chambers and thechambers incubated as previously described. In selected experimentsthe hydroxyl radical (OW) scavenger mannitol (I mM) or methionine(1 mM) (which inactivates HOCI) was added. After incubation, thea IAT was retrieved and its ability to inhibit porcine pancreatic elastase(Calbiochem-Behring Corp., La Jolla, CA) was measured. Pancreaticelastase was specifically used in this experiment because when theactive site met358 residue of alAT is oxidized, the alAT moleculecompletely loses its ability to inhibit porcine pancreatic elastase. (Incontrast, it is still able to inhibit neutrophil elastase, albeit with amarkedly reduced association rate constant [ 1 1]). Thus, although por-cine pancreatic elastase is not the normal physiologic target ofhumana 1AT in vivo, it offers a convenient method to analyze the protectiveeffect of antioxidants. The reaction between the aIAT recovered fromthe chambers and the porcine pancreatic elastase was carried out in a1-ml reaction volume containing 100 mM Tris-HCI, pH 8.3, 0.5 MNaCl, 0.1% Brij-35, and 8 nM porcine pancreatic elastase (I 1). Varyingconcentrations of a 1AT were added and the reaction mixture incu-bated for 3 h at 230. After incubation, the porcine pancreatic elastase-specific substrate (n-succinyl-alanyl-alanyl-alanyl-NA, I mM, Calbio-chem-Behring Corp.) was added to the incubation. The residual pan-creatic elastase activity was then quantified by measuring the change inoptical density at 410 nm, and percent inhibition of elastase was cal-culated as previously described.

To directly demonstrate the involvement of the a 1AT met358 resi-due as the target for the smoker alveolar macrophage-released oxi-dants as the mechanism for inactivating the a I AT, we carried outidentical experiments as described for the smoker macrophages andthe plasma a 1 AT, but recombinant forms ofa 1 AT, met358 ra 1 AT, andval358 ra 1 AT were substituted for the plasma a1AT. Briefly, smokers'alveolar macrophages were added to the upper portion of a chamberacross a dialysis membrane from either met358 a I AT or val358 a IAT inthe same ratios used previously. After incubation for 18 h at 370 therecombinant a 1AT was retrieved and evaluated for its ability to inhibitneutrophil elastase as previously described.

Results

Relationship between alveolar macrophages and alAT on thealveolar epithelial surface. Evaluation of lavage fluid demon-strated that, relative to the amounts ofa 1AT present, the envi-ronment of the epithelial lining fluid of the lower respiratorytract of nonsmokers and cigarette smokers were substantiallydifferent both in number of alveolar macrophages and amountof oxidants released per hour. In this regard, nonsmokers hadan average of 21.4±14.2 X 103 alveolar macrophages per ,l oflower respiratory tract epithelial lining fluid, whereas thesmokers had 43.2±26.3 X 103/pl ELF (P < 0.01). However,the concentration of alAT in ELF was similar in the twogroups (nonsmokers, 4.5±0.8 gM; smokers, 3.21 ± 1.0 AM; P> 0.1). The ELF of smokers contained nearly threefold morealveolar macrophages per mole of a 1 AT than did nonsmokers

(Fig. 1 A; P < 0.01). Not only did the smokers have relativelymore alveolar macrophages, but the macrophages were acti-vated and spontaneously releasing more oxidants than themacrophages of nonsmokers. In this context nonsmoker al-veolar macrophages were releasing 18.2±8.3 nmol 07/106 al-veolar macrophages per h and 1.6±0.6 nmol H202/ 106 macro-phages per h, whereas smoker macrophages were releasing42.4±13.3 nmol 07/106 macrophages per h and 13.1±4.2nmol H202/ 106 macrophages per h (nonsmokers vs. smokers,P < 0.01 for 07 and H202, respectively). When considered interms ofamounts ofalAT present in ELF, it was apparent thatsmokers' alveolar macrophages present a burden of oxidantsmarkedly greater than do nonsmoker macrophages. For exam-ple, nonsmoker macrophages spontaneously released 84±12mol/h' 07 per mol aIAT in ELF, whereas smoker macro-phages released 570±120 mol/h- °2 per mol alAT (P< 0.005). The burden of H202 released by the alveolar macro-phages was even greater. Smoker alveolar macrophages re-leased 28-fold more H202 per mol of alAT in ELF than didnonsmoker alveolar macrophages (Fig. 1 B, P < 0.001). Thus,on the average, the oxidant burden from alveolar macrophagesfaced by a molecule of a 1 AT within the smokers' alveolus isfar greater than that within the nonsmokers' alveolus, andconsequently the likelihood ofoxidative alteration ofthe prop-erties of the a1AT molecule is correspondingly much greaterwithin the smokers' lung than in the lung of the nonsmoker.

Analysis ofability ofnonsmoker and smoker alveolar mac-rophages to inactivate alA T as an inhibitor ofneutrophil elas-tase. When alveolar macrophages were incubated with a 1 ATin the in vitro system designed to reproduce the microenvi-ronment of ELF, very different effects were observed betweensmokers' and nonsmokers' alveolar macrophages on the an-tielastase function of the a 1 AT present. The nonsmokers'macrophages caused almost no loss of function ofthe alAT asan inhibitor of neutrophil elastase (Fig. 2). In contrast, smokeralveolar macrophages caused a significant loss of the antielas-tase function of alAT. The loss of antielastase function waspresent at all elastase-a 1 AT incubation intervals measured.Lack in increase in antielastase function with time suggeststhat a significant proportion of the population of alAT mole-cules were rendered completely impotent as inhibitors of neu-trophil elastase. Strikingly, the ability of smokers' macro-

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Figure 1. Relationship of alveolar macrophages and alAT in the epi-thelial lining fluid of the lower respiratory tract of nonsmokers andsmokers. (A) Ratio of number of alveolar macrophages recovered bylavage to amount of aI AT in the same lavage sample. (B) Ratio ofamount of H202 spontaneously released by alveolar macrophages/h-' to the amount ofalAT in ELF.

Macrophage Inactivation ofal-Antitrypsin 1291

100 Control Figure 2. Effect of low-Expo to

molecular weight medi-non smokers' AM ators spontaneously re-

80 hi leased by alveolar mac-rophages of nonsmokersand smokers to sup-

60 - press the ability ofoS alAT to inhibit neutro-

phil elastase. aIAT (1n 40 - umol) was exposed to

Expos to alveolar macrophagessmoker's'AM

(106/ml; 4.65 X 10920 macrophages/ismol

a IlAT) in chambers for18 h, 37°. Dialysis tub-

30 60 90 120 ing (mol wt cutoff,Time (min) 12-14 kD) separated

the macrophages fromthe alAT. The a lAT was recovered from the chamber and evalu-ated for its ability to inhibit neutrophil elastase over time (2 nM elas-tase, 0-8 nM alAT). Shown is the elastase inhibitory capacity ofa lAT incubated in the chamber without alveolar macrophages onthe other side of the membrane (a); a IAT exposed to alveolar mac-rophages of nonsmokers (o); and a 1AT exposed to alveolar macro-phages of smokers (A).

phages to inactivate a IAT occurred despite that fact that con-ditions within the assay mimicked those within thenonsmokers' alveolus rather than those of the smoker al-veolus. In this regard, when the number of smokers' alveolarmacrophages was increased beyond the ratio of alveolar mac-rophages to a1AT ELF found in nonsmokers, the loss of an-tineutrophil activity was even greater, with the loss of activityproportional to numbers ofmacrophages in the chamber (datanot shown). When put in the context ofthe microenvironmentof the ELF of the smoker, where the ratio of macrophages permicromole alAT ELF is threefold greater than that used inthis study, the observed effect upon alAT (Fig. 2) probablyrepresents the minimum reduction ofa 1 AT antielastase activ-ity ongoing in the lower respiratory tract of smokers.

Effect of antioxidants on ability of mediators released bysmoker alveolar macrophages to interfere with alAT antielas-tase function. When antioxidants were added to the incuba-tions containing smoker alveolar macrophages and a 1 AT, thesuppression of a1AT antielastase function caused by thesmoker macrophages was not observed. In this regard, addi-tion of SOD or catalase to the chambers before the addition ofsmoker macrophages resulted in a suppression of the effectsmoker macrophages have on the ability of the a IAT to sub-sequently inhibit elastase (P < 0.01 for each vs. control; TableI). Importantly, when both SOD and catalase were added, thesubsequent ability of the a lAT to inhibit elastase was fullypreserved (P < 0.005, compared with control). In contrast,when SOD and catalase were heat-inactivated, their protectiveeffect (either alone or in combination) was not observed. Ad-dition of mannitol or methionine (scavengers of OH' andHOCl, respectively) also resulted in partial protection ofa IATantielastase capacity (mannitol, 64 + 4, P < 0.05 vs. control;methionine, 63 + 7, P < 0.05 vs. control). These findingsindicate that low-molecular weight oxidants were the media-tors responsible for the observed loss of the elastase inhibitoryfunction of a 1AT after its incubation with smokers' alveolarmacrophages. This is consistent with the observation that

Table I. Effect ofAntioxidants on Ability ofLow-MolecularWeight Mediators Spontaneously Released by AlveolarMacrophages ofCigarette Smokers to Suppress the Abilityofal-Antitrypsin to Inhibit Elastase

InhibitionCondition of Elastase*

a 1 AT exposed to smoker macrophages 27±12+ in the presence of SOD4 68±9+ in the presence of catalase* 71±2+ in the presence ofSOD + catalase 98±2+ in the presence of mannitolli 64±4+ in the presence of methionine' 63±7

* All assays contained 8 nM active porcine pancreatic elastase and0-20 nM a1AT; 100% inhibition = all of the elastase was inhibited.$400 U/ml. Heat-inactivated SOD gave no protection (26±1 1% inhi-bition of elastase). §1,000 U/ml. Heat-inactivated catalase gave noprotection (30±15% inhibition of elastase). III mM. '1 mM.

smoker macrophages release exaggerated amounts of H202,O2 and OH', and can utilize myeloperoxidase (either their ownendogenous enzyme or myeloperoxidase scavenged from neu-trophils) to generate the strong oxidant HOCi.

The met358 active site ofalAT as a targetfor smoker mac-rophages. The ability to produce recombinant forms ofaIATthat are identical except for a single amino acid substitution atthe active inhibitory site provides a means to confirm that atleast part of the mechanism by which alveolar macrophagesinactive a lAT as an inhibitor of elastase is by oxidizing themet358 residue at the site that is central to the elastase-inhibi-tory function of the alAT molecule. In this regard, cigarettesmokers' alveolar macrophages caused a nearly total loss oftheability of met358 ra IAT to inhibit neutrophil elastase (Fig. 3 A).In contrast, the ability of val358 ralAT, to inhibit neutrophilelastase was not significantly affected by exposure to cigarettesmokers' macrophages (Fig. 3 B). On the average, when com-pared with control met358 ra 1AT not exposed to macrophages,the met358 ra 1 AT exposed to smoker macrophages was only14±6% as active (Fig. 3 C). In contrast, val358 ralAT retained84±8% of its activity (P < 0.005). Because the only differencebetween met358 ra 1AT and val1358 ra 1AT is the substitution ofvaline for methionine at the active site of the aIAT molecule,this differential effect strongly suggests that the mechanism ofsuppression of a 1AT antielastase activity by smokers' macro-phages is associated with the action ofoxidants upon the activesite methionine residue.

Discussion

The finding that cigarette smokers' alveolar macrophagesspontaneously are releasing sufficient amounts of oxidants tomodulate the neutrophil elastase-inhibitory function ofalATprovides insight into the mechanisms through which cigarettesmoking is linked to a markedly increased risk for the develop-ment of emphysema. In cigarette smokers, alveolar macro-phages are present in the lower respiratory tract in numbersseveralfold greater than in nonsmokers (17, 29, 30). Charac-teristically they accumulate in terminal bronchioles, wherethey represent an early pathologic lesion in asymptomatic cig-

1292 Hubbard, Ogushi, Fells, Cantin, Jallat, Courtney, and Crystal

A B C Figure 3. Effect of low-molecular weight10 100l mediators spontaneously released by alveo-

\ t xp o s e 0o lar macrophages of cigarette smokers onso SM'AM so the ability of recombinant forms ofalAT

0 \ I \\s1 x to inhibit neutrophil elastase. Recombi-P 60 R \KK ° 60 nant alAT (I gM) was separated from* \ smokers' alveolar macrophages as in Fig. 2.

40 \{\\ oControl a IAT was placed in identicala \ \\{ Expowd to = chambers but not exposed to alveolar mac-

contm control smokers'AM rophages. After 18 h, 370, alAT was re-20 trieved and tested for its ability to inhibit

neutrophil elastase (8 nM) over a 1-h incu-2 4 6 8 2 4 6 8 bation period. (A) Recombinant met358

Met3W Val alAT. Shown is an example from one in-Met3" ralAT (nM) Valse ralAT (nM) ralAT ralAT dividual; data are presented as percent ac-

tivity of the neutrophil elastase remaining. (B) Recombinant val358 a IAT. Shown are data from one individual presented as in panel A. (C)Comparison of proportion of recombinant met358 alAT and recombinant val358 alAT capable of inhibiting neutrophil elastase after exposureto low-molecular weight products spontaneously released by alveolar macrophages of cigarette smokers. Recombinant met358 alAT and val358alAT were exposed to smokers' alveolar macrophages in chambers as described in panels A and B. The incubation was continued for 18 h at370, after which the elastase-inhibitory capacity ofthe recombinant alAT was determined. Each point represents a single individual.

arette smokers (50). Alveolar macrophages, by virtue of theirincreased numbers and exaggerated production of oxidants(29, 32, 33), are ideal candidates to effect the functional integ-rity of the antielastase screen of the lower respiratory tract. Inthis regard, in the context ofthe present study, it is reasonableto conceptualize that macrophages create local areas withinthe alveolus in which there is deficient antineutrophil elastaseprotection due to oxidation of alAT. In this scenario, lack ofantielastase screen makes these areas of the lung vulnerable toelastase released by neutrophils or by macrophages that haveingested neutrophil elastase (51, 52). The result would be de-velopment of localized tissue destruction that ultimately maybecome the clinical disorder emphysema.

Oxidation of alAT by smokers' alveolar macrophages isalso consistent with the study by Carp et al. (23) that observedoxidized alAT in bronchoalveolar lavage fluid obtained fromsmokers' lungs. These observations are consistent with theknowledge that oxidized alAT has a reduced association rateconstant for neutrophil elastase (1 1) and that smoker lowerrespiratory tract a IAT has a lower association rate constant forneutrophil elastase than does nonsmoker lower respiratorytract alAT (22). However, the demonstration that alAT canbe oxidized by smokers' macrophages does not necessarilyimply that a significant proportion of the alAT in all regionswithin the lower respiratory tract is oxidized. A more reason-able conceptualization based upon this model is that somealAT is inactivated by oxidants released by the macrophagesin areas ofmacrophage accumulation. This process may not beoccurring to the same degree everywhere within the lung, dueto local differences in macrophage concentration, availabilityof antioxidants, transudation of plasma, or other factors thatcould have the effect ofdecreasing the local intensity of alveo-lar macrophage exposure to a 1 AT. Furthermore, because ciga-rette smoke itselfcan oxidize alAT, the extent, type, and tech-nique of smoking may also influence the status of alAT indifferent areas of the lower respiratory tract (19, 24). Hence,a 1AT recovered by bronchoalveolar lavage may contain activeand inactive aIAT in varying proportions. This heterogeneitymay explain, at least in part, why some studies have shownthat the population of alAT molecules recovered from thelower respiratory tract of cigarette smokers is relatively unable

to inhibit pancreatic elastase compared with that recoveredfrom nonsmokers (13, 23, 24), whereas other studies have notshown such a difference (25, 26).

The fact that alveolar macrophages in the lower respiratorytract of cigarette smokers are capable of modifying aIAT inthe local milieu by oxidant mechanisms has implications forfuture approaches to investigating therapeutic strategies forpreventing emphysema associated with cigarette smoking.First, because alveolar macrophages are replaced slowly (53,54), this may explain why some individuals continue to de-velop lung destruction even after discontinuing cigarettesmoking. Therefore, therapeutic modalities directed at sup-pressing the exaggerated oxidant release by the macrophagesmay be useful. Second, antioxidants may provide an impor-tant means of preserving the antielastase screen of the lowerrespiratory tract. Finally, while there are theoretical dangers inproviding a "super" form ofalAT that cannot be inactivated,the val358 form of recombinant alAT may be one means toprotect the lower respiratory tract against a burden of neutro-phil elastase when there is a concomitant burden of oxidantssuch as those released by alveolar macrophages of cigarettesmokers.

References

1. Travis, J., and G. Salvesen. 1983. Human plasma proteinaseinhibitors. Ann. Rev. Biochem. 52:655-709.

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