Neutrophil Accumulation in the Lung in al -Antitrypsin, DeficiencySpontaneous Release of Leukotriene B4 by Alveolar MacrophagesR. C. Hubbard, G. Fells, J. Gadek, S. Pacholok,* J. Humes,* and R. G. CrystalPulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes ofHealth, Bethesda, Maryland 20892;and *Merck Institutefor Therapeutic Research, Rahway, New Jersey 07065

Abstract

The emphysema of al-antitrypsin (alAT) deficiency is concep-tualized to result from insufficient alAT allowing neutrophilelastase to destroy lung parenchyma. In addition to the defi-ciency ofalAT in these individuals resulting from mutations inthe alAT gene, it is recognized that, for unknown reasons,there are also increased numbers of neutrophils in their lungscompared with normal individuals. With the knowledge thatalveolar macrophages have surface receptors for neutrophilelastase, we hypothesized that the neutrophil accumulation inthe lower respiratory tract in alAT deficiency may result, inpart, from release ofneutrophil chemotactic activity by alveolarmacrophages as they bind uninhibited neutrophil elastase.Consistent with this hypothesis, alAT-deficient alveolar mac-rophages spontaneously released nearly threefold more neutro-phil chemotactic activity than normal alveolar macrophages.Analysis ofalAT-deficient macrophage supernates by reverse-phase HPLC, molecular sieve chromatography, radioimmuno-assay, and absorption with anti-LTB4 antibody revealed thatthe majority of the chemotactic activity was leukotriene B4(LTB4), a mediator absent from normal macrophage super-nates. Consistent with this hypothesis, incubation of normalmacrophages with human neutrophil elastase resulted in therelease of the same neutrophil chemotactic mediator. Further-more, purified human alAT was able to prevent the neutrophilelastase from stimulating the macrophages to release the che-motactic factor. Together, these findings suggest that the ab-sence of a normal antineutrophil elastase screen in the lowerrespiratory tract permits free neutrophil elastase to bind to al-veolar macrophages, resulting in the release ofLTB4, a processwhich attracts neutrophils to the alveoli ofalAT deficient indi-viduals, thus accelerating the lung destruction that character-izes this disorder. (J. Clin. Invest. 1991. 88:891-897.) Keywords: emphysema * inflammation * bronchoalveolar lavage.neutrophil elastase antiprotease

Introduction

al-Antitrypsin (alAT)I deficiency is an autosomal hereditarydisorder characterized by reduced serum levels of a 1 AT, an

Address reprint requests to Pulmonary Branch, National Institutes ofHealth, Building 10, Room 6D03, Bethesda, MD 20892.

J. Gadek's present address is Division of Pulmonary and CriticalCare Medicine, N. 325 Means Hall, 1665 Upham Drive, Columbus,OH 432 10-1228.

Receivedfor publication 30 May 1989 and in revisedform 11 April1991.

1. Abbreviations used in this paper: alAT, al-antitrypsin; LTB4, leu-kotriene B4; NE, neutrophil elastase.

The Journal of Clinical Investigation, Inc.Volume 88, September 1991, 891-897

increased risk for the development ofemphysema, and a lesser,but definite risk for hepatitis and cirrhosis ( 1-6). In the contextof the knowledge that a 1AT provides almost all of the protec-tive screen ofthe lower respiratory tract against the proteolyticenzyme neutrophil elastase (NE), the emphysema ofalAT de-ficiency is understood to result from insufficient levels ofa 1ATin the lower respiratory tract to effectively inhibit the burden ofNE in the lungs (7-9). Because NE is capable of degrading theconnective tissue matrix of the alveolar walls, the chronic bur-den of free, uninhibited elastase is associated with alveolar walldestruction, i.e., emphysema (1, 3, 5, 8-10).

While the deficiency ofa IAT in this disorder is understoodto result from various mutations in five a IAT coding exons (3,4), the destruction ofthe alveolar walls could not occur withoutthe chronic presence of neutrophils within the lung. Interest-ingly, in previous studies of a 1AT in the epithelial lining fluidof the lower respiratory tract of nonsmoking alAT deficientindividuals, we have observed that there are more neutrophilsthan observed in normals (1 1, 12). On the basis of these obser-vations, the present study is directed at evaluating the hypothe-sis that neutrophils accumulate in the alveolar structures ofthese individuals at least in part because they are attracted bychemotactic signals released by alveolar macrophages. This hy-pothesis is based on three concepts, including: (a) alveolar mac-rophages are capable, when activated by surface stimuli, of re-leasing chemotactic mediators for neutrophils (13-19); (b) al-veolar macrophages have surface receptors for neutrophilelastase (20-22); and (c) individuals with a IAT deficiency haveinsufficient alAT in the lower respiratory tract to inhibit theburden ofNE in the local milieu and thus likely have free NE inthe alveolar structures (7, 23). Putting these concepts together,they lead to a scenario of amplification of normal inflamma-tory processes, in which free NE resulting from insufficienta 1 AT binds to the NE receptors in alveolar macrophages, caus-ing the macrophages to release mediators with neutrophil che-motactic activity, thus causing an increased number ofneutro-phils to accumulate in the alveolar structures. Consequentlythis process would further add to other likely causes of alteredalveolar macrophage function in alAT-deficient patients, in-cluding cigarrette smoking, airway infection, and the presenceof matrix degradation products.

Methods

Study population. The study population consisted of 14 individualswith the Z homozygous form of alAT deficiency, verified by serumalAT levels, isoelectric focusing of serum, and family analysis (24).There were five females and nine males with an average age of40±5 yr(all data presented as mean±standard error ofthe mean, and all statisti-cal comparisons were made using the two-tailed Student's t test). Theaverage serum alAT level was 4.6 uM (normal 20-48 MM; based on atrue laboratory standard; see reference 23 for details). There were sixlifelong nonsmokers, and eight former cigarette smokers; the formersmokers had stopped an average of 3.5±2.6 yr before the study, with a

Lung Neutrophil Accumulation ofal-Antitrypsin Deficiency 891

minimum of 1 yr previously. All individuals had clinical evidence ofemphysema based on physical examination, chest x-ray, xenon-133ventilation scans, technetium-99m-labeled macroaggregated albuminscans and lung function studies (25) (vital capacity 74±15% predicted,forced expiratory volume in 1 s 59±19% predicted, and diffusing capac-ity for carbon monoxide 65±15% predicted [corrected for hemoglobinand volume]).

For comparison, a population of 11 normal individuals includedseven men and four women with an average age of 29±5 yr. All werenonsmokers and none had evidence oflung disease by history, physicalexamination, chest x-ray, and pulmonary function tests. None weretaking any medications. Serum alAT phenotyping demonstrated allwere of the MM phenotype (i.e., various combinations of the normalMl(Val213), Ml(Ala213), M2, and M3 alleles) (3, 4). As a group, thenormals had a serum a lAT level of 39±4 1M.

Quantification of neutrophils in epithelial lining fluid. Bronchoal-veolar lavage and cell analysis was carried out in each individual aspreviously described (26). The volume of epithelial lining fluid (ELF)recovered was quantified by the urea method and the total number ofneutrophils recovered was calculated and expressed per unit volume ofELF recovered (27).

Release ofneutrophil chemotactic activity by alveolar macrophages.Alveolar macrophages obtained by bronchoalveolar lavage were incu-bated (106 cells/ml, 370) in RPMI-1640 media (Whittaker M. A. Bio-products, Walkersville, MD) in sterile 24-well culture plates (FalconLabware, Oxnard, CA). After a 1-h incubation period to permit adher-ence, the cells were washed three times with iced RPMI-1640, afterwhich additional RPMI-1640 (1 ml) was added to each well. Afterincubation (3 h, 370), the supernate was evaluated for neutrophil che-motactic activity (see below). The viability of macrophages at the endof the incubation period was > 90% in all cases, as determined bytrypan blue exclusion ( 14).

Neutrophil chemotactic activity was quantified using a 48-well mi-crochemnotaxis chamber (Neuro Probe, Inc., Cabin John, MD) and3-MM pore size filters (Nuclepore, Pleasanton, CA) (28). Macrophagesupernate or RPMI- 1640 media alone (as a negative control) wasplaced in the lower portion ofthe chamber, and I0O human neutrophils(prepared from a single donor by Plasmagel [Cellular Products, Buf-falo, NY] sedimentation and hypotonic lysis of residual erythrocytes,neutrophil purity > 90% and viability > 95% as assessed by trypan blueexclusion [29]) in RPMI-1640 were added to the upper chamber. Afterincubation (30 min, 37°), the filters were removed, wiped, and stainedwith Diff Quik (American Scientific Products, McGraw Park, IL) andthe number ofneutrophils per 10 randomly selected high-power micro-scope fields which had reached the lower surface ofthe micropore filterwas determined. Each supernate was assayed in triplicate, for a totalnumber of 30 high-power fields (hpf), and the neutrophil chemotacticactivity of each supernate determined as the mean number of cellscounted per 10 hpf. The neutrophil chemotactic activity was expressedas the mean number of cells counted per 10 hpfofa sample supernate,minus the mean number of cells responding to a simultaneously as-sayed control consisting ofRPMI- 1640 media alone.

To insure that the migration ofneutrophils across the membrane inresponse to macrophage supernates was due to directed migration (che-motaxis) and not to increased random migration (chemokinesis), acheckerboard analysis was performed in which varying amounts ofalveolar macrophage supernate were added to both the upper and lowercompartments of the chemotaxis chamber.

Identification ofLTB4 in alveolar macrophage supernates. Reverse-phase HPLC was performed in the manner of Kouzan et al. (30) withminor modifications. Supernates from 2 x 106 alveolar macrophages inRPMI-1640, were diluted with 4 vol ice-cold HPLC grade ethanol (Al-drich Chemical Co., Milwaukee, WI), vortexed, and evaporated to dry-ness at 370 under a stream of dry nitrogen. The residual material wasdissolved in 250 ,l 30% methanol and applied to a C18 Ultraspherecolumn (4.6 x 25 cm, Beckman Instruments, Inc., Fullerton, CA).Successive isocratic elution steps were performed at a flow rate of 1ml/min using two solvent mixtures: solvent A, 90% water/ I0% metha-

nol/0.08% acetic acid; solvent B, 100% methanol. Step 1 (0-27 min),45% solvent B; step 2 (27-70 min), 67% solvent B; step 3 (70-100 min),100% solvent B. Identification of LTB4 in supernate assays was estab-lished by coelution with a LTB4 standard (provided by Rokach, J.,Merck Frosst, Canada) with the column eluate monitored at 280 nm.Separate chromatographic analyses were performed on five ZZ and fiveMM individuals.

Direct measurement ofthe concentration ofLTB4 in alveolar mac-rophage supernates was performed using a radioimmunoassay specificfor LTB4 with a lower limit of detectability of 0.08 ng/ml (31, 32).Alveolar macrophages (2 X 106) were cultured as described above, andthe supernates directly analyzed without purification for the presenceof immunoreactive LTB4.

Determination that LTB is responsiblefor the chemotactic activityofalveolar macrophage supernates ofZ homozygotes. To demonstratethat LTB4 was responsible for the majority ofthe neutrophil chemotac-tic activity present in supernates fromZZ alveolar macrophages, molec-ular sieve chromatographic analysis was carried out and the peak neu-trophil chemotactic activity evaluated by reverse phase HPLC in super-nates of three ZZ individuals. Supernate (500 ,ul) from 3 X 106 ZZalveolar macrophages cultured in HBSS was applied to Superose 12and Superose 6 molecular sieves (Pharmacia Fine Chemicals, Piscata-way, NJ) connected in series. The entire column eluate was collected inl-ml fractions, and each fraction was separately assayed for its chemo-tactic activity in filter chambers as described above. The peak fractionsdemonstrating significant chemotactic activity were collected and sub-sequently analyzed by reverse-phase HPLC as described above.

To positively identify the chemotactic factor induced by elastase,supernates (n = 3) from elastase exposed MM alveolar macrophagesand alveolar macrophages from ZZ individuals (n = 3) were individu-ally applied to an affinity chromatography column containing rabbitanti-human LTB4 antibody (supplied by Ford-Hutchinson, A., MerckFrosst, Canada). As a negative control a column containing rabbitanti-human lysozyme was used. Before and after chromatography, thesupernates were evaluated for chemotactic activity in filter chambers asdescribed above.

Assessment ofthe ability ofneutrophil elastase to stimulate the re-lease ofLTB by alveolar macrophages. To evaluate the concept thatneutrophil elastase might be responsible for the stimulation ofalveolarmacrophages to release neutrophil chemotactic activity, purified hu-man neutrophil elastase (100 nM), in RPMI- 1640 was added to normal(phenotype MM) alveolar macrophages (30 min, 370) and the superna-tants evaluated for neutrophil chemotactic activity as described above.

To determine whether alAT had a modulating effect upon themacrophage elaboration ofchemotactic activity in response to neutro-phil elastase, aclAT purified from MM human plasma (33) was incu-bated at concentrations ranging from 50 to 500 nM with 100 nM neu-trophil elastase before addition to theMM alveolar macrophages. Aftera 30-min incubation, the macrophage supernates were collected andassayed for neutrophil chemotactic activity.

Results

Neutrophils in the lower respiratory tract in al-antitrypsin defi-ciency. Quantification of the lung neutrophil burden by bron-choalveolar lavage demonstrated that ZZ individuals had agreater number ofneutrophils in their alveolar epithelial liningfluid than did normalMM individuals. In this regard, ZZ indi-viduals contained almost threefold more neutrophils in ELFthan normals (P < 0.05) (Fig. 1). In contrast the numbers of allother cell types (macrophages, lymphocytes, and eosinophils)was similar in both groups, (P > 0.5 all comparisons). Impor-tantly, the increased number of neutrophils was present with-out evidence ofinfection, or overt inflammation in the form ofhyperemia ofthe airways or increased bronchial mucous. Fur-thermore, there was no significantly increased number ofbron-chial cells in the ZZ lavage fluid compared to normal.

892 Hubbard, Fells, Gadek, Pacholok, Humes, and Crystal

10 r

81-

61

4

2

Figure 1. Characterization of

the lung neutrophil burden in

Liepithelial lining fluid from MMand ZZ individuals. Shown isthe number of neutrophils per

ll microliter epithelial lining fluidMM Z (ELF) recovered by bronchoal-

veolar lavage ofnonsmokingPhenotype MM and ZZ individuals.

Spontaneous release of neutrophil chemotactic activity.Consistent with the observed increase in numbers of neutro-phils in ELF ofthe ZZ individuals, when alveolar macrophagesrecovered from these individuals were cultured in vitro, theyspontaneously released significantly greater amounts ofneutro-phil chemotactic activity than MM alveolar macrophages, (P< 0.01) (Fig. 2). Checkerboard analysis ofZZ macrophage su-pernate demonstrated that the neutrophil migration was due totrue chemotactic activity, not chemokinesis (Table I). Further-more, the spontaneous release ofneutrophil chemotactic activ-ity by ZZ alveolar macrophages equaled the neutrophil chemo-tactic factor released byMM alveolar macrophages stimulatedin vitro by zymosan (P > 0.5 compared to spontaneous releaseby ZZ macrophages).

Analysis of alveolar macrophage supernates for LTB4. Inthe context ofthe knowledge that LTB4 is the major neutrophilchemotactic factor released by human alveolar macrophages,the supernates of ZZ and MM alveolar macrophages culturedin vitro were analyzed for the presence of LTB4. Using a C18reverse-phase column, we identified an LTB4 standard by itselution time (Fig. 3 A). Consistent with the comparative lack ofneutrophil chemotactic activity present in the supernates ofMM alveolar macrophages, no LTB4 was evident on the chro-matogram of the MM alveolar macrophage supernates (Fig. 3B). In contrast, supernates of ZZ alveolar macrophages con-

tained LTB4, as determined by the presence of a chromato-graphic peak eluting at the same time as the LTB4 standard(Fig. 3 C). Consistent with the chromatographic analysis,quantitative analysis of LTB4 production by MM and ZZ al-veolar macrophages by radioimmunoassay also demonstratedthat the ZZ alveolar macrophages spontaneously released exag-

Table L Checkerboard Analysis ofNeutrophil ChemotacticActivity Spontaneously Released by Alveolar MacrophagesofIndividuals with the Z Homozygous Formofal-A ntitrypsin Deficiency

Relative concentration ofAM supernate in upperRelative concentration chamber

of supernatein lower chamber 0% 50% 100%

0 80±11 89±14 66±950 29±24 202±28 169±21100 541±29 312±31 173±26

Alveolar macrophages recovered from the lower respiratory tract ofindividuals with alAT deficiency were incubated (3 h, 370) and thesupernates evaluated for neutrophil chemotactic activity in a chemo-tactic chamber (see Methods). The data is presented as the meannumber of cells per 10 high-power fields reaching the lower surfaceof a micropore filter.

gerated amounts ofLTB4 compared with MM alveolar macro-phages. In this regard, the ZZ alveolar macrophages releasedthreefold more LTB4 than unstimulated MM macrophages (P< 0.01) (Fig. 4), an observation consistent with the relativeamount of neutrophil chemotactic activity observed in the su-pernates of the ZZ and MM macrophages (Fig. 2).

0.0

0.0

Ec

cm0

c

DmE

600 Figure 2. Spontaneous re-

lease of neutrophil chemo-tactic activity by alveolarmacrophages from MMand ZZ individuals. For

comparison, also shown is200 the neutrophil chemotactic

CD activity released by MMalveolar macrophages stim-

l I I ulated in vitro with zymo-MM MM + Z san (100 ,g/ml). Neutrophil

Zymosan chemotactic activity is ex-

Phenotype pressed as the number of

neutrophils per 10 high-power fields reaching the lower surface of a micropore filter.

0.02

0.01

0.02

0.01

2

A

LTD4

B

C

15 30 45 60

Elution time (min)

Figure 3. C18 reverse-phaseHPLC analysis of super-nates form MM and ZZ al-veolar macrophages. (A)LTB4 standard. (B) Super-nate generated by 2 x 106unstimulated MM alveolarmacrophages. (C) Super-nate from 2 x 106 unstimu-lated ZZ alveolar macro-

phages. Note material withan elution time identicalto the LTB4 standard. Or-dinate, absorbance at 280nm; abscissa, elution time.

Lung Neutrophil Accumulation ofal-Antitrypsin Deficiency 893

U--Jw-a0

xEn

._2

z

iI --AL

_

I-

0(00

_ _ _ Figure 4. Amount of LTB4 spon-

78 taneously released by MM (nc 0.2 1) and ZZ (n = I1) alveolara) macrophages. Quantification was

0.1 performed by radioimmunoassay;the data is expressed as the amountofLTB4 released by 2 x lO alveo-

MM = lar macrophages form each indi-vidual over a 3-h period. The dif-

Phenotype ference is significant (P < 0.01).

Demonstration that LTB4 is the neutrophil chemotacticfac-tor in ZZ alveolar macrophage supernates. To directly demon-strate that LTB4 was the major chemotactic mediator responsi-ble for the neutrophil chemotactic factor activity found in ZZalveolar macrophage supernates, combined molecular sieveand reverse-phase HPLC was performed on supernates of un-stimulated ZZ macrophages cultured in vitro. When fractionsof ZZ macrophage supernate that had been separated accord-ing to size on a molecular sieve were analyzed for their neutro-phil chemotactic activity, the major peak ofchemotactic activ-ity occurred in the size range between 1,000 and 200 D, a sizeconsistent with that of LTB4 (336 D) (Fig. 5 A). When thesepooled low-molecular weight fractions containing neutrophilchemotactic activity were then analyzed by Cl 8 reverse-phasechromatography, LTB4 was observed (Fig. 5 B).

Antibody absorption studies definitively identified the che-motactic factor as LTB4. When supernates from elastase ex-posedMM alveolar macrophages were passed through an affin-

A670 128 144 17 1.4 0.2[ * *f f 4

20 30 40Elution volume (ml)

B

12

LTBL

15 30 45 60

Figure 5. Correlation of theneutrophil chemotactic activityin the supernates ofZZ alveo-lar macrophages with LTB4spontaneously released by thesame alveolar macrophages.(A) Molecular sieve chromato-graphic analysis of supernateof unstimulated ZZ alveolarmacrophages demonstratingthe neutrophil chemotactic ac-tivity of fractions separated ac-cording to their relative molec-ular weights. Neutrophil che-motactic activity is expressedon the ordinate as the numberof neutrophils per 10 high-power fields reaching the lowersurface ofa micropore filter.(B) C18 reverse-phase HPLCanalysis ofthe fractions dem-onstrating neutrophil chemo-tactic activity (panel A frac-tions 41-45). The position ofelution of an LTB4 standardis indicated. Numbers at topindicate molecular weight.

ity chromatography column containing antibody against LTB4the neutrophil chemotactic activity of the supernates was lost.No such loss of chemotactic activity was seen following super-nate exposure to a column containing antibody against humanlysozyme (not shown). Similarly, passage of supernates fromPiZZ alveolar macrophages through the anti-LTB4 affinity col-umn resulted in loss of the majority of the neutrophil chemo-tactic activity (Fig. 6).

Neutrophil elastase stimulation of neutrophil chemotacticfactor release by MM alveolar macrophages. Consistent withthe possibility that it is uninhibited neutrophil elastase in thelower respiratory tract that provides the activation signal to thealveolar macrophages, when MM alveolar macrophages wereincubated in vitro with neutrophil elastase, neutrophil chemo-tactic factor was released in amounts similar to that observedafter zymosan incubation (Fig. 7 A). Furthermore, purified hu-man alAT was able to modulate the neutrophil elastase stimu-lated release of neutrophil chemotactic factor fromMM alveo-lar macrophages in a dose-dependent manner. In this regard,incubation ofalAT with neutrophil elastase before its additionto the macrophages resulted in a nearly complete reduction inthe stimulation of neutrophil chemotactic factor release byMM alveolar macrophages (Fig. 7 B). The dose-dependent abil-ity ofal AT to suppress the effect ofelastase on theMM macro-phages is consistent with the known function ofalAT to bindto and inactivate neutrophil elastase.

Discussion

The present study suggests that one process which may contrib-ute to the accelerated lung destruction that characterizes alATdeficiency is a chronically increased neutrophil burden in thelower respiratory tract caused by alveolar macrophages releas-ing neutrophil chemotactic factor LTB4. A likely mechanismwhich explains this increased release ofLTB4 is the binding offree neutrophil elastase by ZZ alveolar macrophages. Consis-tent with this hypothesis, the ability of elastase to mediate therelease of LTB4 does not occur if sufficient alAT is present.These observations suggest a pathologic scenario in which thedeficiency ofa lAT in the lungs ofZZ individuals results in thepresence of free elastase, which then interacts with surface re-

500

400

ILa.I 300Q

0

, 200(0z

100

Anti-LTB4 Anti-LTB4MM + NE zz

Figure 6. Identification ofneutrophil chemotactic fac-tor activity in supernatesof neutrophil elastase (100nM) exposedMM alveolarmacrophages and super-nates of ZZ alveolar mac-rophage as LTB4. Macro-phage supernates were as-sayed for neutrophilchemotactic activity beforeand after application to anaffinity column containinganti-LTB4 antibody. Neu-trophil chemotactic activityis expressed as the numberof neutrophils per 10 high-power fields reaching thelower surface of a micro-pore filter.

894 Hubbard, Fells, Gadek, Pacholok, Humes, and Crystal

400

2001

la

0

FEc

C

0.C

O.1

Elution time (min)

A Figure 7. Demonstration that600 - neutrophil elastase can stimu-

late normal MM alveolar mac-

rophages to release neutrophila 400 - chemotactic factor activity. (A)CL Neutrophil chemotactic activ-O2 ity spontaneously released into<, 200 the supernate by MM alveolarz

macrophages after exposure ofthe macrophages to NE (100

MM NE MM nM). NE by itself had no che-Only + motactic activity. (B) Ability

B NE of a I -antitrypsin (a IAT) to600-prevent neutrophil elastasefrom stimulating normal MM

= 400 - alveolar macrophages to re-lease neutrophil chemotactic

Z N activity. Shown is the neutro-0.2 200 phil chemotactic activity of su-

pernates ofMM alveolar mac-z rophages exposed to neutrophil

0 100 200 300 400 500 elastase which had been prein-ajAT (nM) cubated with various concen-

trations of al-antitrypsin.Neutrophil chemotaxis is expressed as the number of neutrophils per10 high-power fields reaching the lower surface of a micropore filter.

ceptors on alveolar macrophages, stimulating the release ofLTB4 by the macrophages and consequently the recruitment ofstill more neutrophils with their burden of elastase, thus estab-lishing a cycle characterized by a chronically increased lowerrespiratory tract proteolytic burden. In this context, the imbal-ance between neutrophil elastase and a 1AT in the lower respi-ratory tract of a I AT-deficient individuals is beyond that attri-butable to the alAT deficiency itself, but is further increasedby exaggeration of the neutrophil, and hence neutrophil elas-tase, burden.

Alveolar macrophage interaction with neutrophil elastase.Neutrophils are present in the lungs of normal individuals insmall numbers. A variety of mediators have been identifiedwhich likely play a role in neutrophil recruitment into thelungs, including bacterial endotoxin, complement fragments,platelet-activating factor, interleukin-8, the bacterial equiva-lent of formyl-methionyl-leucyl-phenylalanine, other arachi-donic acid metabolites, and one or more polypeptides (13, 16-19, 34-41). The central role of the alveolar macrophage as amediator of neutrophil accumulation has been clearly docu-mented (42-44). Importantly, as the initial cell to encounterforeign substances in the lower respiratory tract, the macro-phage is capable ofamplifying the defenses ofthe lower respira-tory tract by recruiting neutrophils. It is recognized that LTB4is the predominant neutrophil chemotactic mediator releasedby alveolar macrophages (16-18, 39, 40, 42-44). In this regard,alveolar macrophages synthesize and release LTB4 in responseto a number of naturally occurring stimuli including immunecomplexes, IgE, bacteria, and other phagocytic stimuli such asasbestos (16-18, 35, 39, 44-47). Neutrophil elastase can nowbe added to the list ofagents which result in release ofLTB4 byalveolar macrophages.

Although it was not the purpose ofthe present investigationto examine in detail the manner in which neutrophil elastaseinteracts with the alveolar macrophage to effect LTB4 release, itis known that alveolar macrophages bind and internalize neu-

trophil elastase, mainly via a low-affinity, high-volume surfacereceptor (20-22). This receptor is not elastase-specific; cathep-sin G and lactoferrin are also bound (21). Bound elastase isinternalized by the cell via endocytosis, and is slowly degraded,but under some circumstances may be released in active formfrom the macrophage (22, 49). Binding of elastase by its recep-tor occurs in the absence ofa2-macroglobulin and a 1 -antitryp-sin, and is distinct from the macrophage uptake of a2-macro-globulin-protease complexes, which is thought to contribute tothe removal of proteases from the extracellular space (20, 50).Interestingly, after exposure to a variety of neutral proteasesincluding pancreatic elastase and collagenase, mouse perito-neal macrophages demonstrate increased prostaglandin synthe-sis and release ofarachidonic acid metabolites (51). Therefore,the finding in the present study that alveolar macrophages spe-cifically react to neutrophil elastase by the release of LTB4 isconsistent with what is known ofalveolar macrophage bindingofelastase via a surface receptor, and the subsequent activationof macrophage arachidonic acid metabolic pathways.

alAT as a complexfactor in modulating inflammation. Byvirtue of its principal physiologic role as an inhibitor ofneutro-phil elastase, a1AT plays an important role in modulating in-flammation. By directly inhibiting elastase through the bindingof elastase to its active site on a stressed, external loop withmethionine358 at its center, a IAT specifically acts to limit pro-teolytic activity of neutrophil elastase, thereby protecting bys-tander parenchymal tissues from proteolysis (52). The activesite methionine358 residue ofa 1AT can be oxidized by the sec-tion of endogenous or exogenously produced oxidants, result-ing in the inactivation of alAT as an effective antiprotease,with the result that elastase may act without the normal degreeof restraint offered by active alAT (53). This "switching off" ofalAT by oxidants may be beneficial in certain pathologicstates such as infection, where elastolytic attack of a foreignorganism is desirable, but in other circumstances, such as incigarette smoking, adult respiratory distress syndrome, cysticfibrosis, or asbestos lung disease, the oxidative inactivation ofalAT likely leads to deleterious lung damage (54-60).

Another role played by alAT in modulating inflammationis its ability to directly act as a chemoattractant after interac-tion with proteases. After proteolysis by mouse macrophageelastase, a 1AT undergoes cleavage and structural reorganiza-tion, with the generation of a 4.2-kD fragment which is che-moattractive for neutrophils (61). A similar fragment can begenerated after inactivation of human neutrophil elastase byalAT, which also possesses neutrophil chemotactic activity(62). Therefore, alAT contains latent intrinsic neutrophil che-moattractant properties which become manifest after interac-tion with its target protease, neutrophil elastase. This intrinsicchemotactic activity of alAT unmasked after interaction withneutrophil elastase may also play a role in recruiting some ofthe neutrophils observed in the lungs of Z homozygotes, andmay also be responsible for the observation in the present studythat incubation of fivefold excess alAT with elastase did notcompletely eliminate chemotactic activity in macrophage su-pernates, i.e., the residual chemotactic activity may have beendue to release of chemotactic fragments from the a 1AT mole-cule after its cleavage by elastase. The finding of the presentstudy, that a deficiency of alAT serves as the basis for an exag-gerated, prolonged inflammatory response characterized byLTB4-mediated neutrophil recruitment, represents a new con-cept of the role of a lAT in modulating inflammation. In this

Lung NeutrophilAccumulation ofal-A ntitrypsin Deficiency 895

regard, a lAT is not directly active as an inflammatory modula-tor, aside from its elastase-inhibitory properties. Rather, alATacts to interrupt a potential feedback cycle in which free elas-tase results in the recruitment ofmore neutrophils, hence moreelastase, via the interaction of free elastase with macrophageswhich leads to elaboration of LTB4. By inactivating free elas-tase, a IAT breaks the cycle; conversely deficiency of alATmay permit amplification and perpetuation of the inflamma-tory reaction. As with oxidative "switching off" of alAT,a IAT deficiency may be a "two-edged sword"-beneficial inthe face ofmassive infection or other major threat to the organ-ism when the deficiency is temporary and localized, but damag-ing in the chronic situation such as a lAT deficiency.

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tion therapy for alpha 1-antitrypsin deficiency. Am. J. Med. 84:52-62.6. Morse, J. 0. 1978. Alpha 1-antitrypsin deficiency. N. Engl. J. Med.

299:1045-1048; 1099-1105.7. Gadek, J. E., G. A. Fells, R. L. Zimmerman, S. L. Rennard, and R. G.

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12. Keogh, B. A., J. E. Gadek, G. W. Hunninghake, and R. G. Crystal. 1982.Determinants of disease expression in PiZZ phenotype alpha 1-antitrypsin defi-ciency. Clin. Res. 30:432A. (Abstr.)

13. Kazmierowski, J. A., J. I. Gallin, and H. Y. Reynolds. 1977. Mechanismfor the inflammatory response in primate lungs. Demonstration and partial char-acterization ofan alveolar macrophage-derived chemotactic factor with preferen-tial activity for polymorphonuclear leukocytes. J. Clin. Invest. 59:273-281.

14. Hunninghake, G. W., J. E. Gadek, T. J. Lawley, and R. G. Crystal. 1981.Mechanisms of neutrophil accumulation in the lungs of patients with idiopathicpulmonary fibrosis. J. Clin. Invest. 68:259-269.

15. Gadek, J. E., G. W. Hunninghake, R. L. Zimmerman, and R. G. Crystal.1980. Regulation of the release of alveolar macrophage-derived neutrophil che-motactic factor. Am. Rev. Respir. Dis. 121:723-733.

16. Fels, A. 0. S., N. A. Pawlowski, E. B. Cramer, T. K. C. King, Z. A. Cohn,and W. A. Scott. 1982. Human alveolar macrophages produce leukotriene B4.Proc. Natl. Acad. Sci. USA. 79:7866-7870.

17. Martin, T. R., L. C. Altman, R. C. Albert, and W. R. Henderson. 1984.Leukotriene B4 production by the human alveolar macrophage: a potential mech-anism for amplifying inflammation in the lung. Am. Rev. Respir. Dis. 129:106-111.

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22. McGowan, S. E., R. D. Arbeit, P. J. Stone, and G. L. Snider. 1983. Acomparison of the binding and fate of internalized neutrophil elastase in humanmonocytes and alveolar macrophages. Am. Rev. Respir. Dis. 128:688-694.

23. Wewers, M. D., M. A. Casolaro, S. E. Sellers, S. C. Swayze, K. M.McPhaul, J. T. Wittes, and R. G. Crystal. 1987. Replacement therapy for alphaI-antitrypsin deficiency associated with emphysema. N. Engl. J. Med. 316:1055-1062.

24. Cox, D. W., A. M. Johnson, and M. K. Fagerhol. 1980. Report ofnomen-clature meeting for al-antitrypsin. Hum. Genet. 53:429-433.

25. Fulmer, J. D., W. C. Roberts, E. R. Von Gal, and R. G. Crystal. 1979.Morphologic-physiologic correlates of the severity of fibrosis and degree ofcellu-larity in idiopathic pulmonary fibrosis. J. Clin. Invest. 63:665-676.

26. Saltini, C., A. J. Hance, V. J. Ferrans, F. Basset, P. B. Bitterman, and R. G.Crystal. 1984. Accurate quantification of cells recovered by bronchoalveolar la-vage. Am. Rev. Respir. Dis. 130:650-658.

27. Rennard, S. I., G. Basset, D. Lecossier, K. M. O'Donnell, P. G. Martin,and R. G. Crystal. 1986. Estimation of the apparent volume of epithelial liningfluid recovered by bronchoalveolar lavage using urea as an endogenous marker ofdilution. J. Appl. Physiol. 60:532-658.

28. Harvath, L., W. Falk, and E. J. Leonard. 1980. Rapid quantitation ofneutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate mem-brane in a multiwell assembly. J. Immunol. Methods. 37:39-45.

29. Boynum, A. 1968. Isolation of mononuclear cells and granulocytes fromhuman blood. J. Clin. Invest. 21:77-89.

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31. Humes, J. L. 1981. Prostaglandins. In Methods for Studying Mononu-clear Phagocytes. D. A. Adams, H. Kuren, and P. Edelson, editors. AcademicPress, Inc., New York. 541-654.

32. Rokach, J., E. C. Hayes, Y. Girard, D. Lombardo, A. L. Maycock, A. S.Rosental, R. N. Young, R. Zamboni, and H. J. Zweerink. 1984. The develop-ment of sensitive and specific radioimmunoassays for leukotrienes. Prostaglan-dins Leukotrienes Med. 13:21-25.

33. Ogushi, F., G. A. Fells, R. C. Hubbard, S. D. Straus, and R. G. Crystal.1987. Z-Type al-antitrypsin is less competent than MI-type al-antitrypsin asaninhibitor of neutrophil elastase. J. Clin. Invest. 80:1366-1374.

34. Henson, P. M., G. L. Larsen, and R. V. Webster. 1982. Pulmonary micro-vascular alterations and injury induced by complement fragments. Synergisticeffects of complement fragments: complement activation, neutrophil sequestra-tion and prostaglandins. Ann. NYAcad. Sci. 384:287-300.

35. Gosset, P., A. B. Tonnel, M. Joseph, L. Prin, A. Mallart, J. Charon, and A.Capron. 1984. Secretion of a chemotactic factor for neutrophils and eosinophilsby alveolar macrophages from asthmatic patients. J. Allergy Clin. Immunol.74:827-834.

36. Fowler, A. A., and L. B. Schwartz. 1984. Macrophage-derived chemotac-tic factors and allergic asthma. J. Allergy Clin. Immunol. 74:777-780.

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46. Schoenberger, C. I., G. W. Hunninghake, 0. Kawanami, V. J. Ferrans,and R. G. Crystal. 1982. Role ofalveolar macrophages in asbestosis: modulationof neutrophil migration to the lung after acute asbestos exposure. Thorax.37:803-809.

47. Rola-Pleszynski, M., S. Gouin, and R. Begin. 1984. Asbestos-inducedlung inflammation: role of local macrophage-derived chemotactic factors in ac-cumulation of neutrophils in the lungs. Inflammation. 8:53-61.

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