Neutrophil Accumulation in the Lung in Alpha 1-Antitrypsin Deficiency

Home , Alveolar macrophage

Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. Spontaneous release of leukotriene B4 by alveolar macrophages.

R C Hubbard, … , J Humes, R G Crystal

J Clin Invest. 1991;88(3):891-897. https://doi.org/10.1172/JCI115391.

Research Article

The emphysema of alpha 1-antitrypsin (alpha 1AT) deficiency is conceptualized to result from insufficient alpha 1AT allowing neutrophil elastase to destroy lung parenchyma. In addition to the deficiency of alpha 1AT in these individuals resulting from mutations in the alpha 1AT gene, it is recognized that, for unknown reasons, there are also increased numbers of neutrophils in their lungs compared with normal individuals. With the knowledge that alveolar macrophages have surface receptors for neutrophil elastase, we hypothesized that the neutrophil accumulation in the lower respiratory tract in alpha 1AT deficiency may result, in part, from release of neutrophil chemotactic activity by alveolar macrophages as they bind uninhibited neutrophil elastase. Consistent with this hypothesis, alpha 1AT-deficient alveolar macrophages spontaneously released nearly threefold more neutrophil chemotactic activity than normal alveolar macrophages. Analysis of alpha 1AT-deficient macrophage supernates by reverse-phase HPLC, molecular sieve chromatography, radioimmunoassay, and absorption with anti-LTB4 antibody revealed that the majority of the chemotactic activity was leukotriene B4 (LTB4), a mediator absent from normal macrophage supernates. Consistent with this hypothesis, incubation of normal macrophages with human neutrophil elastase resulted in the release of the same neutrophil chemotactic mediator. Furthermore, purified human alpha 1AT was able to prevent the neutrophil elastase from stimulating the macrophages to release the chemotactic factor. Together, these findings suggest that the absence of a normal antineutrophil […]

Find the latest version: https://jci.me/115391/pdf Neutrophil Accumulation in the Lung in al -Antitrypsin, Deficiency Spontaneous Release of Leukotriene B4 by Alveolar Macrophages R. C. Hubbard, G. Fells, J. Gadek, S. Pacholok,* J. Humes,* and R. G. Crystal Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes ofHealth, Bethesda, Maryland 20892; and *Merck Institute for Therapeutic Research, Rahway, New Jersey 07065

Abstract increased risk for the development ofemphysema, and a lesser, The emphysema of al-antitrypsin (alAT) deficiency is concep- but definite risk for hepatitis and cirrhosis ( 1-6). In the context tualized to result from insufficient alAT allowing neutrophil of the knowledge that a 1AT provides almost all of the protec- elastase to destroy lung parenchyma. In addition to the defi- tive screen of the lower respiratory tract against the proteolytic ciency of alAT in these individuals resulting from mutations in enzyme neutrophil elastase (NE), the emphysema of alAT de- the alAT gene, it is recognized that, for unknown reasons, ficiency is understood to result from insufficient levels of a 1AT there are also increased numbers of neutrophils in their lungs in the lower respiratory tract to effectively inhibit the burden of compared with normal individuals. With the knowledge that NE in the lungs (7-9). Because NE is capable of degrading the alveolar macrophages have surface receptors for neutrophil connective tissue matrix of the alveolar walls, the chronic bur- elastase, we hypothesized that the neutrophil accumulation in den of free, uninhibited elastase is associated with alveolar wall the lower respiratory tract in alAT deficiency may result, in destruction, i.e., emphysema (1, 3, 5, 8-10). part, from release of neutrophil chemotactic activity by alveolar While the deficiency of a I AT in this disorder is understood macrophages as they bind uninhibited neutrophil elastase. to result from various mutations in five a I AT coding exons (3, Consistent with this hypothesis, alAT-deficient alveolar mac- 4), the destruction ofthe alveolar walls could not occur without rophages spontaneously released nearly threefold more neutro- the chronic presence of neutrophils within the lung. Interest- phil chemotactic activity than normal alveolar macrophages. ingly, in previous studies of a 1AT in the epithelial lining fluid Analysis of alAT-deficient macrophage supernates by reverse- of the lower respiratory tract of nonsmoking alAT deficient phase HPLC, molecular sieve chromatography, radioimmuno- individuals, we have observed that there are more neutrophils assay, and absorption with anti-LTB4 antibody revealed that than observed in normals (1 1, 12). On the basis of these obser- the majority of the chemotactic activity was leukotriene B4 vations, the present study is directed at evaluating the hypothe- (LTB4), a mediator absent from normal macrophage super- sis that neutrophils accumulate in the alveolar structures of nates. Consistent with this hypothesis, incubation of normal these individuals at least in part because they are attracted by macrophages with human neutrophil elastase resulted in the chemotactic signals released by alveolar macrophages. This hy- release of the same neutrophil chemotactic mediator. Further- pothesis is based on three concepts, including: (a) alveolar mac- more, purified human al AT was able to prevent the neutrophil rophages are capable, when activated by surface stimuli, of re- elastase from stimulating the macrophages to release the che- leasing chemotactic mediators for neutrophils (13-19); (b) al- motactic factor. Together, these findings suggest that the ab- veolar macrophages have surface receptors for neutrophil sence of a normal antineutrophil elastase screen in the lower elastase (20-22); and (c) individuals with a I AT deficiency have respiratory tract permits free neutrophil elastase to bind to al- insufficient alAT in the lower respiratory tract to inhibit the veolar macrophages, resulting in the release of LTB4, a process burden of NE in the local milieu and thus likely have free NE in which attracts neutrophils to the alveoli of alAT deficient indi- the alveolar structures (7, 23). Putting these concepts together, viduals, thus accelerating the lung destruction that character- they lead to a scenario of amplification of normal inflamma- izes this disorder. (J. Clin. Invest. 1991. 88:891-897.) Key tory processes, in which free NE resulting from insufficient words: emphysema * inflammation * bronchoalveolar lavage. a 1 AT binds to the NE receptors in alveolar macrophages, caus- neutrophil elastase antiprotease ing the macrophages to release mediators with neutrophil chemotactic activity, thus causing an increased number of neutro- Introduction phils to accumulate in the alveolar structures. Consequently this process would further add to other likely causes of altered al-Antitrypsin (alAT)I deficiency is an autosomal hereditary alveolar macrophage function in a lAT-deficient patients, in- disorder characterized by reduced serum levels of a 1 AT, an cluding cigarrette smoking, airway infection, and the presence of matrix degradation products. Address reprint requests to Pulmonary Branch, National Institutes of Health, Building 10, Room 6D03, Bethesda, MD 20892. Methods J. Gadek's present address is Division of Pulmonary and Critical Care Medicine, N. 325 Means Hall, 1665 Upham Drive, Columbus, Study population. The study population consisted of 14 individuals OH 432 10-1228. with the Z homozygous form of alAT deficiency, verified by serum Receivedfor publication 30 May 1989 and in revisedform 11 April alAT levels, isoelectric focusing of serum, and family analysis (24). 1991. 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- 1. Abbreviations used in this paper: alAT, al-antitrypsin; LTB4, leu- cal comparisons were made using the two-tailed Student's t test). The kotriene B4; NE, neutrophil elastase. average serum alAT level was 4.6 uM (normal 20-48 MM; based on a true laboratory standard; see reference 23 for details). There were six The Journal of Clinical Investigation, Inc. lifelong nonsmokers, and eight former cigarette smokers; the former Volume 88, September 1991, 891-897 smokers 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 of nol/0.08% acetic acid; solvent B, 100% methanol. Step 1 (0-27 min), emphysema based on physical examination, chest x-ray, xenon-133 45% solvent B; step 2 (27-70 min), 67% solvent B; step 3 (70-100 min), ventilation scans, technetium-99m-labeled macroaggregated albumin 100% solvent B. Identification of LTB4 in supernate assays was estab- scans and lung function studies (25) (vital capacity 74±15% predicted, lished by coelution with a LTB4 standard (provided by Rokach, J., forced expiratory volume in 1 s 59±19% predicted, and diffusing capac- Merck Frosst, Canada) with the column eluate monitored at 280 nm. ity for carbon monoxide 65±15% predicted [corrected for hemoglobin Separate chromatographic analyses were performed on five ZZ and five and volume]). MM individuals. For comparison, a population of 11 normal individuals included Direct measurement of the concentration of LTB4 in alveolar mac- seven men and four women with an average age of 29±5 yr. All were rophage supernates was performed using a radioimmunoassay specific nonsmokers and none had evidence of lung disease by history, physical for LTB4 with a lower limit of detectability of 0.08 ng/ml (31, 32). examination, chest x-ray, and pulmonary function tests. None were Alveolar macrophages (2 X 106) were cultured as described above, and taking any medications. Serum alAT phenotyping demonstrated all the supernates directly analyzed without purification for the presence were of the MM phenotype (i.e., various combinations of the normal of immunoreactive LTB4. Ml(Val213), Ml(Ala213), M2, and M3 alleles) (3, 4). As a group, the Determination that LTB is responsiblefor the chemotactic activity normals had a serum a lAT level of 39±4 1M. ofalveolar macrophage supernates ofZ homozygotes. To demonstrate Quantification of neutrophils in epithelial lining fluid. Bronchoal- that LTB4 was responsible for the majority ofthe neutrophil chemotac- veolar lavage and cell analysis was carried out in each individual as tic activity present in supernates from ZZ alveolar macrophages, molec- previously described (26). The volume of epithelial lining fluid (ELF) ular sieve chromatographic analysis was carried out and the peak neu- recovered was quantified by the urea method and the total number of trophil chemotactic activity evaluated by reverse phase HPLC in super- neutrophils recovered was calculated and expressed per unit volume of nates of three ZZ individuals. Supernate (500 ,ul) from 3 X 106 ZZ ELF recovered (27). alveolar macrophages cultured in HBSS was applied to Superose 12 Release ofneutrophil chemotactic activity by alveolar macrophages. and Superose 6 molecular sieves (Pharmacia Fine Chemicals, Piscata- Alveolar macrophages obtained by bronchoalveolar lavage were incu- way, NJ) connected in series. The entire column eluate was collected in bated (106 cells/ml, 370) in RPMI-1640 media (Whittaker M. A. Bio- l-ml fractions, and each fraction was separately assayed for its chemo- products, Walkersville, MD) in sterile 24-well culture plates (Falcon tactic activity in filter chambers as described above. The peak fractions Labware, Oxnard, CA). After a 1-h incubation period to permit adher- demonstrating significant chemotactic activity were collected and sub- ence, the cells were washed three times with iced RPMI-1640, after sequently analyzed by reverse-phase HPLC as described above. which additional RPMI-1640 (1 ml) was added to each well. After To positively identify the chemotactic factor induced by elastase, incubation (3 h, 370), the supernate was evaluated for neutrophil che- supernates (n = 3) from elastase exposed MM alveolar macrophages motactic activity (see below). The viability of macrophages at the end and alveolar macrophages from ZZ individuals (n = 3) were individu- of the incubation period was > 90% in all cases, as determined by ally applied to an affinity chromatography column containing rabbit trypan blue exclusion ( 14). anti-human LTB4 antibody (supplied by Ford-Hutchinson, A., Merck Neutrophil chemotactic activity was quantified using a 48-well mi- Frosst, Canada). As a negative control a column containing rabbit crochemnotaxis chamber (Neuro Probe, Inc., Cabin John, MD) and anti-human lysozyme was used. Before and after chromatography, the 3-MM pore size filters (Nuclepore, Pleasanton, CA) (28). Macrophage supernates were evaluated for chemotactic activity in filter chambers as supernate or RPMI- 1640 media alone (as a negative control) was described above. placed in the lower portion ofthe chamber, and I0O human neutrophils Assessment ofthe ability ofneutrophil elastase to stimulate the re- (prepared from a single donor by Plasmagel [Cellular Products, Buf- lease ofLTB by alveolar macrophages. To evaluate the concept that falo, NY] sedimentation and hypotonic lysis of residual erythrocytes, neutrophil elastase might be responsible for the stimulation of alveolar neutrophil purity > 90% and viability > 95% as assessed by trypan blue macrophages to release neutrophil chemotactic activity, purified hu- exclusion [29]) in RPMI-1640 were added to the upper chamber. After man neutrophil elastase (100 nM), in RPMI- 1640 was added to normal incubation (30 min, 37°), the filters were removed, wiped, and stained (phenotype MM) alveolar macrophages (30 min, 370) and the superna- with Diff Quik (American Scientific Products, McGraw Park, IL) and tants evaluated for neutrophil chemotactic activity as described above. the number of neutrophils per 10 randomly selected high-power micro- To determine whether alAT had a modulating effect upon the scope fields which had reached the lower surface of the micropore filter macrophage elaboration of chemotactic activity in response to neutro- was determined. Each supernate was assayed in triplicate, for a total phil elastase, aclAT purified from MM human plasma (33) was incu- number of 30 high-power fields (hpf), and the neutrophil chemotactic bated at concentrations ranging from 50 to 500 nM with 100 nM neu- activity of each supernate determined as the mean number of cells trophil elastase before addition to the MM alveolar macrophages. After counted per 10 hpf. The neutrophil chemotactic activity was expressed a 30-min incubation, the macrophage supernates were collected and as the mean number of cells counted per 10 hpf of a sample supernate, assayed for neutrophil chemotactic activity. minus the mean number of cells responding to a simultaneously assayed control consisting of RPMI- 1640 media alone. Results To insure that the migration of neutrophils across the membrane in response to macrophage supernates was due to directed migration (che- Neutrophils in the lower respiratory tract in al-antitrypsin defi- motaxis) and not to increased random migration (chemokinesis), a ciency. Quantification of the lung neutrophil burden by bron- checkerboard analysis was performed in which varying amounts of choalveolar lavage demonstrated that ZZ individuals had a alveolar macrophage supernate were added to both the upper and lower greater number of neutrophils in their alveolar epithelial lining compartments of the chemotaxis chamber. fluid than did normal MM individuals. In this regard, ZZ indi- Identification ofLTB4 in alveolar macrophage supernates. Reverse- viduals contained almost threefold more neutrophils in ELF phase HPLC was performed in the manner of Kouzan et al. (30) with than normals (P < 0.05) (Fig. 1). In contrast the numbers of all minor modifications. Supernates from 2 x 106 alveolar macrophages in other cell types (macrophages, lymphocytes, and eosinophils) RPMI-1640, were diluted with 4 vol ice-cold HPLC grade ethanol (Al- was similar in both groups, (P > 0.5 all comparisons). Impor- drich Chemical Co., Milwaukee, WI), vortexed, and evaporated to dry- ness at 370 under a stream of dry nitrogen. The residual material was tantly, the increased number of neutrophils was present with- dissolved in 250 ,l 30% methanol and applied to a C18 Ultrasphere out evidence of infection, or overt inflammation in the form of column (4.6 x 25 cm, Beckman Instruments, Inc., Fullerton, CA). hyperemia of the airways or increased bronchial mucous. Fur- Successive isocratic elution steps were performed at a flow rate of 1 thermore, there was no significantly increased number ofbron- ml/min using two solvent mixtures: solvent A, 90% water/ I0% metha- chial cells in the ZZ lavage fluid compared to normal.

892 Hubbard, Fells, Gadek, Pacholok, Humes, and Crystal 10 r Table L Checkerboard Analysis of Neutrophil Chemotactic U- Activity Spontaneously Released by Alveolar Macrophages w-J 81- ofIndividuals with the Z Homozygous Form -a ofal-A ntitrypsin Deficiency 0 61 Figure 1. Characterization of x Relative concentration of AM supernate in upper En Relative concentration chamber ._ 4 the lung neutrophil burden in of supernate 2 epithelial fluid from MM in lower chamber 0% 50% 100% and individuals.lining Shown is z 2 ZZ Li the number of neutrophils per ll microliter epithelial lining fluid 0 80±11 89±14 66±9 MM Z (ELF) recovered by bronchoal- 50 29±24 202±28 169±21 veolar lavage of nonsmoking 100 541±29 312±31 173±26 Phenotype MM and ZZ individuals. Alveolar macrophages recovered from the lower respiratory tract of Spontaneous release of neutrophil chemotactic activity. individuals with alAT deficiency were incubated (3 h, 370) and the Consistent with the observed increase in numbers of neutro- supernates evaluated for neutrophil chemotactic activity in a chemo- phils in ELF of the ZZ individuals, when alveolar macrophages tactic chamber (see Methods). The data is presented as the mean number of cells 10 recovered from these individuals were cultured in vitro, they per high-power fields reaching the lower surface of a filter. spontaneously released significantly greater amounts ofneutro- micropore phil chemotactic activity than MM alveolar macrophages, (P < 0.01) (Fig. 2). Checkerboard analysis of ZZ macrophage su- gerated amounts of LTB4 compared with MM alveolar macro- pernate demonstrated that the neutrophil migration was due to phages. In this regard, the ZZ alveolar macrophages released true chemotactic activity, not chemokinesis (Table I). Further- threefold more LTB4 than unstimulated MM macrophages (P more, the spontaneous release of neutrophil chemotactic activ- < 0.01) (Fig. 4), an observation consistent with the relative ity by ZZ alveolar macrophages equaled the neutrophil chemo- amount of neutrophil chemotactic activity observed in the su- tactic factor released by MM alveolar macrophages stimulated pernates of the ZZ and MM macrophages (Fig. 2). in vitro by zymosan (P > 0.5 compared to spontaneous release by ZZ macrophages). Analysis of alveolar macrophage supernates for LTB4. In A _ the context ofthe knowledge that LTB4 is the major neutrophil 0.02 chemotactic factor released by human alveolar macrophages, the supernates of ZZ and MM alveolar macrophages cultured LTD4 in vitro were analyzed for the presence of LTB4. Using a C18 reverse-phase column, we identified an LTB4 standard by its 0.0 elution time (Fig. 3 A). Consistent with the comparative lack of neutrophil chemotactic activity present in the supernates of MM alveolar macrophages, no LTB4 was evident on the chro- matogram of the MM alveolar macrophage supernates (Fig. 3 B). In contrast, supernates of ZZ alveolar macrophages con- 0.02 B tained LTB4, as determined by the presence of a chromato- E graphic peak eluting at the same time as the LTB4 standard c 3 C). (Fig. Consistent with the chromatographic analysis, cm quantitative analysis of LTB4 production by MM and ZZ al- 0 veolar macrophages by radioimmunoassay also demonstrated c 0.01 that the ZZ alveolar macrophages spontaneously released exag- DmE

600 Figure 2. Spontaneous re- Figure 3. C18 reverse-phase lease of neutrophil chemo- HPLC analysis of super- tactic activity by alveolar 0.02 C nates form MM and ZZ al- macrophages from MM veolar macrophages. (A) and ZZ individuals. For LTB4 standard. (B) Super- comparison, also shown is nate generated by 2 x 106 200 the neutrophil chemotactic unstimulated MM alveolar CD activity released by MM 0.01 macrophages. (C) Super- alveolar macrophages stim- nate from 2 x 106 unstimu- l I I ulated in vitro with zymo- lated ZZ alveolar macro- MM MM + Z san (100 ,g/ml). Neutrophil phages. Note material with Zymosan chemotactic activity is ex- i an elution time identical Phenotype pressed as the number of L I --A to the LTB4 standard. Or- neutrophils per 10 high- 15 30 45 60 dinate, absorbance at 280 power fields reaching the lower surface of a micropore filter. Elution time (min) nm; abscissa, elution time.

Lung Neutrophil Accumulation ofal-A ntitrypsin Deficiency 893 ity chromatography column containing antibody against LTB4 the neutrophil chemotactic activity of the supernates was lost. I- No such loss of chemotactic activity was seen following super-

(00 nate exposure to a column containing antibody against human 0 lysozyme (not shown). Similarly, passage of supernates from _ _ _ Figure 4. Amount of LTB4 spon- PiZZ alveolar macrophages through the anti-LTB4 affinity col- 78 taneously released by MM (n umn resulted in loss of the majority of the neutrophil chemo- c 0.2 1) and ZZ (n = I1) alveolar tactic activity (Fig. 6). a) macrophages. Quantification was Neutrophil elastase stimulation of neutrophil chemotactic 0.1 performed by radioimmunoassay; factor release by MM alveolar macrophages. Consistent with the data is expressed as the amount the possibility that it is uninhibited neutrophil elastase in the of LTB4 released by 2 x lO alveo- lower respiratory tract that provides the activation signal to the MM = lar macrophages form each indi- were vidual over a 3-h period. The dif- alveolar macrophages, when MM alveolar macrophages Phenotype ference is significant (P < 0.01). incubated in vitro with neutrophil elastase, neutrophil chemotactic factor was released in amounts similar to that observed after zymosan incubation (Fig. 7 A). Furthermore, purified human alAT was able to modulate the neutrophil elastase stimu- Demonstration that LTB4 is the neutrophil chemotacticfac- lated release of neutrophil chemotactic factor from MM alveo- tor in ZZ alveolar macrophage supernates. To directly demon- lar macrophages in a dose-dependent manner. In this regard, strate that LTB4 was the major chemotactic mediator responsi- incubation ofal AT with neutrophil elastase before its addition ble for the neutrophil chemotactic factor activity found in ZZ to the macrophages resulted in a nearly complete reduction in alveolar macrophage supernates, combined molecular sieve the stimulation of neutrophil chemotactic factor release by and reverse-phase HPLC was performed on supernates of un- MM alveolar macrophages (Fig. 7 B). The dose-dependent abil- stimulated ZZ macrophages cultured in vitro. When fractions ity of a l AT to suppress the effect of elastase on the MM macro- of ZZ macrophage supernate that had been separated accord- phages is consistent with the known function of alAT to bind ing to size on a molecular sieve were analyzed for their neutro- to and inactivate neutrophil elastase. phil chemotactic activity, the major peak of chemotactic activity occurred in the size range between 1,000 and 200 D, a size consistent with that of LTB4 (336 D) (Fig. 5 A). When these Discussion pooled low-molecular weight fractions containing neutrophil The present study suggests that one process which may contrib- chemotactic activity were then analyzed by Cl 8 reverse-phase ute to the accelerated lung destruction that characterizes alAT chromatography, LTB4 was observed (Fig. 5 B). deficiency is a chronically increased neutrophil burden in the Antibody absorption studies definitively identified the che- lower respiratory tract caused by alveolar macrophages releas- motactic factor as LTB4. When supernates from elastase ex- ing neutrophil chemotactic factor LTB4. A likely mechanism posed MM alveolar macrophages were passed through an affin- which explains this increased release of LTB4 is the binding of free neutrophil elastase by ZZ alveolar macrophages. Consis- tent with this hypothesis, the ability of elastase to mediate the A Figure 5. Correlation of the release of LTB4 does not occur if sufficient alAT is present. 670 128 144 17 1.4 0.2 These observations suggest a pathologic scenario in which the 400 f 4 neutrophil chemotactic activity [ * * f in the supernates of ZZ alveo- deficiency ofa lAT in the lungs of ZZ individuals results in the re- la lar macrophages with LTB4 presence of free elastase, which then interacts with surface spontaneously released by the 0 1 same alveolar macrophages. 200 (A) Molecular sieve chromatographic analysis of supernate Figure 6. Identification of of unstimulated ZZ alveolar 500 neutrophil chemotactic fac- macrophages demonstrating tor activity in supernates the neutrophil chemotactic ac- of neutrophil elastase (100 20 30 40 tivity of fractions separated ac- 400 nM) exposed MM alveolar Elution volume (ml) cording to their relative molec- macrophages and super- ular weights. Neutrophil che- IL nates of ZZ alveolar mac- a. rophage as LTB4. Macro- B motactic activity is expressed I 300 Q phage supernates were as- 12 on the ordinate as the number 0.C of neutrophils per 10 high- sayed for neutrophil 0 FE power fields reaching the lower chemotactic activity before c ,(0 200 surface of a micropore filter. z and after application to an C (B) C18 reverse-phase HPLC affinity column containing O.1 analysis of the fractions dem- anti-LTB4 antibody. Neu- LTBL onstrating neutrophil chemo- 100 trophil chemotactic activity tactic activity (panel A frac- is expressed as the number tions 41-45). The position of of neutrophils per 10 high- elution of an LTB4 standard power fields reaching the 15 30 45 60 is indicated. Numbers at top Anti-LTB4 Anti-LTB4 lower surface of a micro- Elution time (min) indicate molecular weight. MM + NE zz pore filter.

894 Hubbard, Fells, Gadek, Pacholok, Humes, and Crystal A Figure 7. Demonstration that trophil elastase, mainly via a low-affinity, high-volume surface 600 - neutrophil elastase can stimu- receptor (20-22). This receptor is not elastase-specific; cathep- late normal MM alveolar mac- sin G and lactoferrin are also bound (21). Bound elastase is rophages to release neutrophil internalized the cell via and is a 400 - chemotactic factor activity. (A) by endocytosis, slowly degraded, but under some circumstances may be released in active form CL Neutrophil chemotactic activ- O2 ity spontaneously released into from the macrophage (22, 49). Binding of elastase by its recep- <, 200 the supernate by MM alveolar tor occurs in the absence of and a z a2-macroglobulin 1 -antitryp- macrophages after exposure of sin, and is distinct from the macrophage uptake of a2-macro- the macrophages to NE (100 globulin-protease complexes, which is thought to contribute to MM NE MM nM). NE by itself had no che- the removal of proteases from the extracellular space (20, 50). Only + motactic activity. (B) Ability Interestingly, after exposure to a variety of neutral B NE proteases 600- of a I -antitrypsin (a I AT) to including pancreatic elastase and collagenase, mouse perito- prevent neutrophil elastase neal macrophages demonstrate increased prostaglandin synthe- from stimulating normal MM sis and release of arachidonic acid metabolites (51). Therefore, = 400 - alveolar macrophages to release neutrophil chemotactic the finding in the present study that alveolar macrophages spe- Z N cifically react to neutrophil elastase by the release of LTB4 is 0. activity. Shown is the neutro- 2 200 phil chemotactic activity of su- consistent with what is known of alveolar macrophage binding pernates of MM alveolar mac- of elastase via a surface receptor, and the subsequent activation z rophages exposed to neutrophil of macrophage arachidonic acid metabolic pathways. 0 100 200 300 400 500 elastase which had been prein- alAT as a complexfactor in modulating inflammation. By ajAT (nM) cubated with various concen- virtue of its principal physiologic role as an inhibitor of neutro- trations of al-antitrypsin. phil elastase, a 1AT plays an important role in modulating in- Neutrophil chemotaxis is expressed as the number of neutrophils per flammation. By directly inhibiting elastase through the 10 high-power fields reaching the lower surface of a binding micropore filter. of elastase to its active site on a stressed, external loop with methionine358 at its center, a I AT specifically acts to limit proteolytic activity of neutrophil elastase, thereby protecting bys- ceptors on alveolar macrophages, stimulating the release of tander parenchymal tissues from proteolysis (52). The active LTB4 by the macrophages and consequently the recruitment of site methionine358 residue of a 1 AT can be oxidized by the sec- still more neutrophils with their burden of elastase, thus estab- tion of endogenous or exogenously produced oxidants, result- lishing a cycle characterized by a chronically increased lower ing in the inactivation of alAT as an effective antiprotease, respiratory tract proteolytic burden. In this context, the imbal- with the result that elastase may act without the normal degree ance between neutrophil elastase and a 1 AT in the lower respi- of restraint offered by active alAT (53). This "switching off" of ratory tract of a I AT-deficient individuals is beyond that attri- alAT by oxidants may be beneficial in certain pathologic butable to the alAT deficiency itself, but is further increased states such as infection, where elastolytic attack of a foreign by exaggeration of the neutrophil, and hence neutrophil elas- organism is desirable, but in other circumstances, such as in tase, burden. cigarette smoking, adult respiratory distress syndrome, cystic Alveolar macrophage interaction with neutrophil elastase. fibrosis, or asbestos lung disease, the oxidative inactivation of Neutrophils are present in the lungs of normal individuals in alAT likely leads to deleterious lung damage (54-60). small numbers. A variety of mediators have been identified Another role played by alAT in modulating inflammation which likely play a role in neutrophil recruitment into the is its ability to directly act as a chemoattractant after interac- lungs, including bacterial endotoxin, complement fragments, tion with proteases. After proteolysis by mouse macrophage platelet-activating factor, interleukin-8, the bacterial equiva- elastase, a 1 AT undergoes cleavage and structural reorganiza- lent of formyl-methionyl-leucyl-phenylalanine, other arachi- tion, with the generation of a 4.2-kD fragment which is che- donic acid metabolites, and one or more polypeptides (13, 16- moattractive for neutrophils (61). A similar fragment can be 19, 34-41). The central role of the alveolar macrophage as a generated after inactivation of human neutrophil elastase by mediator of neutrophil accumulation has been clearly docu- alAT, which also possesses neutrophil chemotactic activity mented (42-44). Importantly, as the initial cell to encounter (62). Therefore, alAT contains latent intrinsic neutrophil che- foreign substances in the lower respiratory tract, the macro- moattractant properties which become manifest after interac- phage is capable of amplifying the defenses ofthe lower respiration with its target protease, neutrophil elastase. This intrinsic tory tract by recruiting neutrophils. It is recognized that LTB4 chemotactic activity of alAT unmasked after interaction with is the predominant neutrophil chemotactic mediator released neutrophil elastase may also play a role in recruiting some of by alveolar macrophages (16-18, 39, 40, 42-44). In this regard, the neutrophils observed in the lungs of Z homozygotes, and alveolar macrophages synthesize and release LTB4 in response may also be responsible for the observation in the present study to a number of naturally occurring stimuli including immune that incubation of fivefold excess alAT with elastase did not complexes, IgE, bacteria, and other phagocytic stimuli such as completely eliminate chemotactic activity in macrophage su- asbestos (16-18, 35, 39, 44-47). Neutrophil elastase can now pernates, i.e., the residual chemotactic activity may have been be added to the list of agents which result in release of LTB4 by due to release of chemotactic fragments from the a 1 AT mole- alveolar macrophages. cule after its cleavage by elastase. The finding of the present Although it was not the purpose ofthe present investigation study, that a deficiency of alAT serves as the basis for an exag- to examine in detail the manner in which neutrophil elastase gerated, prolonged inflammatory response characterized by interacts with the alveolar macrophage to effect LTB4 release, it LTB4-mediated neutrophil recruitment, represents a new con- is known that alveolar macrophages bind and internalize neu- cept of the role of a lAT in modulating inflammation. In this

Lung NeutrophilAccumulation of al-A ntitrypsin Deficiency 895 regard, a lAT is not directly active as an inflammatory modula- 1979. Receptor-mediated binding and internalization of leukocyte elastase by tor, aside from its elastase-inhibitory properties. Rather, a lAT alveolar macrophages in vitro. J. Clin. Invest. 64:824-833. 21. Campbell, E. J. 1982. Human leukocyte elastase, cathespin G, and lacto- acts to interrupt a potential feedback cycle in which free elas- ferrin: family of neutrophil granule glycoproteins that bind to an alveolar macro- tase results in the recruitment of more neutrophils, hence more phage receptor. Proc. Natl. Acad. Sci. USA. 79:6941-6945. elastase, via the interaction of free elastase with macrophages 22. McGowan, S. E., R. D. Arbeit, P. J. Stone, and G. L. Snider. 1983. A to comparison of the binding and fate of internalized neutrophil elastase in human which leads elaboration of LTB4. By inactivating free elas- monocytes and alveolar macrophages. Am. Rev. Respir. Dis. 128:688-694. tase, a IAT breaks the cycle; conversely deficiency of alAT 23. Wewers, M. D., M. A. Casolaro, S. E. Sellers, S. C. Swayze, K. M. may permit amplification and perpetuation of the inflamma- McPhaul, J. T. Wittes, and R. G. Crystal. 1987. Replacement therapy for alpha tory reaction. As with oxidative "switching off" of a lAT, I-antitrypsin deficiency associated with emphysema. N. Engl. J. Med. 316:1055- 1062. a IAT deficiency may be a "two-edged sword"-beneficial in 24. Cox, D. W., A. M. Johnson, and M. K. Fagerhol. 1980. Report of nomen- the face ofmassive infection or other major threat to the organ- clature meeting for al-antitrypsin. Hum. Genet. 53:429-433. ism when the deficiency is temporary and localized, but damag- 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 of cellu- ing in the chronic situation such as a lAT deficiency. 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- References vage. Am. Rev. Respir. Dis. 130:650-658. 27. Rennard, S. I., G. Basset, D. Lecossier, K. M. O'Donnell, P. G. Martin, 1. Gadek, J. E., and R. G. Crystal. 1982. al-antitrypsin deficiency. In The and R. G. Crystal. 1986. Estimation of the apparent volume of epithelial lining Metabolic Basis of Inherited Disease. J. B. Stanbury, J. B. Wyngaarden, D. S. fluid recovered by bronchoalveolar lavage using urea as an endogenous marker of Fredrickson, J. L. Goldstein, and M. S. Brown, editors. McGraw-Hill Book Co., dilution. J. Appl. Physiol. 60:532-658. New York. 1450-1467. 28. Harvath, L., W. Falk, and E. J. Leonard. 1980. Rapid quantitation of 2. Keuppers, R., and L. F. Black. 1974. a I-Antitrypsin and its deficiency. Am. neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate mem- Rev. Respir. Dis. 1 10: 176-194. brane in a multiwell assembly. J. Immunol. Methods. 37:39-45. 3. Crystal, R. G., M. L. Brantly, R. C. Hubbard, D. T. Curiel, D. J. States, and 29. Boynum, A. 1968. Isolation of mononuclear cells and granulocytes from M. D. Holmes. 1989. The al-antitrypsin gene and its mutations: clinical conse- human blood. J. Clin. Invest. 21:77-89. quences and strategies for therapy. Chest. 95:196-208. 30. Henke, D. C., S. Kouzan, and T. E. Eling. 1984. Analysis of leukotrienes, 4. Brantly, M., T. Nukiwa, and R. G. Crystal. 1988. Molecular basis ofal-an- prostaglandins, and other oxygenated metabolites of arachidonic acid by high- titrypsin deficiency. Am. J. Med. 84:13-31. performance liquid chromatography. Anal. Biochem. 140:87-94. 5. Hubbard, R. C., and R. G. Crystal. 1988. Alpha 1-antitrypsin augmenta- 31. Humes, J. L. 1981. Prostaglandins. In Methods for Studying Mononu- tion therapy for alpha 1-antitrypsin deficiency. Am. J. Med. 84:52-62. clear Phagocytes. D. A. Adams, H. Kuren, and P. Edelson, editors. Academic 6. Morse, J. 0. 1978. Alpha 1-antitrypsin deficiency. N. Engl. J. Med. Press, Inc., New York. 541-654. 299:1045-1048; 1099-1105. 32. Rokach, J., E. C. Hayes, Y. Girard, D. Lombardo, A. L. Maycock, A. S. 7. Gadek, J. E., G. A. Fells, R. L. Zimmerman, S. L. Rennard, and R. G. Rosental, R. N. Young, R. Zamboni, and H. J. Zweerink. 1984. The develop- Crystal. 1981. Antielastases of the human alveolar structures. J. Clin. Invest. ment of sensitive and specific radioimmunoassays for leukotrienes. Prostaglan- 68:889-898. dins Leukotrienes Med. 13:21-25. 8. Janoff, A. 1985. Elastases and emphysema: current assessment of the pro- 33. Ogushi, F., G. A. Fells, R. C. Hubbard, S. D. Straus, and R. G. Crystal. tease-antiprotease hypothesis. Am. Rev. Respir. Dis. 132:417-433. 1987. Z-Type al-antitrypsin is less competent than MI-type al-antitrypsin asan 9. Gadek, J. E., G. W. Hunninghake, G. A. Fells, R. L. Zimmerman, B. A. inhibitor of neutrophil elastase. J. Clin. Invest. 80:1366-1374. Keogh, and R. G. Crystal. 1980. Evaluation ofthe protease-antiprotease theory of 34. Henson, P. M., G. L. Larsen, and R. V. Webster. 1982. Pulmonary micro- human destructive lung disease. Bull. Eur. Physiopathol. Respir. 16:27-40. vascular alterations and injury induced by complement fragments. Synergistic 10. Bieth, J. G. 1986. Elastases: catalytic and biological properties. In Regula- effects of complement fragments: complement activation, neutrophil sequestration of Matrix Accumulation. R. Mechan, editor. Academic Press, Inc., New tion and prostaglandins. Ann. NYAcad. Sci. 384:287-300. York. 217-320. 35. Gosset, P., A. B. Tonnel, M. Joseph, L. Prin, A. Mallart, J. Charon, and A. 11. Gadek, J. E., G. W. Hunninghake, G. A. Fells, R. L. Zimmerman, B. A. Capron. 1984. Secretion of a chemotactic factor for neutrophils and eosinophils Keogh, and R. G. Crystal. 198 1. Validation ofthe alpha 1-antitrypsin recovery of by alveolar macrophages from asthmatic patients. J. Allergy Clin. Immunol. active, connective tissue-specific proteases from the lung of PiZ patients and 74:827-834. reversal with alpha 1-antitrypsin replacement therapy. Clin. Res. 49:550A. 36. Fowler, A. A., and L. B. Schwartz. 1984. Macrophage-derived chemotac- (Abstr.) tic factors and allergic asthma. J. Allergy Clin. Immunol. 74:777-780. 12. Keogh, B. A., J. E. Gadek, G. W. Hunninghake, and R. G. Crystal. 1982. 37. Kew, R. R., B. Ghebrehiwet, and A. Janoff. 1986. The role ofcomplement Determinants of disease expression in PiZZ phenotype alpha 1-antitrypsin defi- in cigarette smoke-induced chemotactic activity of lung fluids. Am. Rev. Respir. ciency. Clin. Res. 30:432A. (Abstr.) Dis. 133:478-481. 13. Kazmierowski, J. A., J. I. Gallin, and H. Y. Reynolds. 1977. Mechanism 38. Laskin, D. L., T. Kimura, S. Sakakibara, D. J. Riley, and R. A. Berg. 1986. for the inflammatory response in primate lungs. Demonstration and partial char- Chemotactic activity of collagen-like polypeptides for human peripheral blood acterization ofan alveolar macrophage-derived chemotactic factor with preferen- neutrophils. J. Leukocyte Biol. 39:255-266. tial activity for polymorphonuclear leukocytes. J. Clin. Invest. 59:273-281. 39. Martin, T. R., G. Raugi, T. L. Merritt, and W. R. Henderson, Jr. 1987. 14. Hunninghake, G. W., J. E. Gadek, T. J. Lawley, and R. G. Crystal. 1981. Relative contribution of leukotriene B4 to the neutrophil chemotactic activity Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic produced by the resident human alveolar macrophage. J. Clin. Invest. 80:1114- pulmonary fibrosis. J. Clin. Invest. 68:259-269. 1124. 15. Gadek, J. E., G. W. Hunninghake, R. L. Zimmerman, and R. G. Crystal. 40. Worthen, G. S., C. Haslett, A. J. Rees, R. S. Gumbay, J. E. Henson, and 1980. Regulation of the release of alveolar macrophage-derived neutrophil che- P. M. Henson. 1987. Neutrophil-mediated pulmonary vascular injury. Synergis- motactic factor. Am. Rev. Respir. Dis. 121:723-733. tic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascu- 16. Fels, A. 0. S., N. A. Pawlowski, E. B. Cramer, T. K. C. King, Z. A. Cohn, lar permeability and neutrophil sequestration in the lung. Am. Rev. Respir. Dis. and W. A. Scott. 1982. Human alveolar macrophages produce leukotriene B4. 136:19-28. Proc. Natl. Acad. Sci. USA. 79:7866-7870. 41. Sylvester, I., J. A. Rankin, T. Yoshimura, S. Tanaka, and E. J. Leonard. 17. Martin, T. R., L. C. Altman, R. C. Albert, and W. R. Henderson. 1984. 1990. Secretion ofneutrophil attractant/activation protein (NAP-1/IL-8) by lipo- Leukotriene B4 production by the human alveolar macrophage: a potential mech- polysaccharide-stimulated lung macrophages. Am. Rev. Respir. Dis. 141:683- anism for amplifying inflammation in the lung. Am. Rev. Respir. Dis. 129:106- 688. 111. 42. Pierce, J. A., and R. M. Senior. 1981. Alveolar macrophage secrets. J. Lab. 18. Rankin, J. A., C. E. Schrader, S. M. Smith, and R. A. Lewis. 1989. Recom- Clin. Med. 97:463-466. binant interferon-gamma primes alveolar macrophages cultured in vitro for the 43. Reiko, T., and Z. Werb. 1984. Secretory products of macrophages and release of leukotriene B4 in response to IgG stimulation. J. Clin. Invest. 83:1691- their physiological functions. Am. Physiol. Soc. 246(Cell Physiol. 15):ClI-9. 1700. 44. Nathan, C. F. 1987. Secretory products of macrophages. J. Clin. Invest. 19. Merrill, W. W., G. P. Nagel, R. A. Matthay, and H. Y. Reynolds. 1980. 79:319-326. Alveolar macrophage-derived chemotactic factor. Kinetics and in vitro produc- 45. Laviolette, M., R. Coulombe, S. Picard, P. Braquet, and P. Borgeat. 1986. tion and partial characterization. J. Clin. Invest. 65:268-276. Decreased leukotriene B4 synthesis in smokers' alveolar macrophages in vitro. J. 20. Campbell, E. J., R. R. White, R. M. Senior, R. J. Rodriguez, and C. Kuhn. Clin. Invest. 77:54-60.

896 Hubbard, Fells, Gadek, Pacholok, Humes, and Crystal 46. Schoenberger, C. I., G. W. Hunninghake, 0. Kawanami, V. J. Ferrans, smokers contains oxidized methionine and has decreased elastase inhibitory ca- and R. G. Crystal. 1982. Role of alveolar macrophages in asbestosis: modulation pacity. Proc. Nat!. Acad. Sci. USA. 79:2041-2045. of neutrophil migration to the lung after acute asbestos exposure. Thorax. 55. Gadek, J. E., G. A. Fells, and R G. Crystal. 1979. Cigarette smoking 37:803-809. induces functional antiprotease deficiency in the lower respiratory tract of hu- 47. Rola-Pleszynski, M., S. Gouin, and R. Begin. 1984. Asbestos-induced mans. Science (Wash. DC). 206:1315-1316. lung inflammation: role of local macrophage-derived chemotactic factors in ac- 56. Cohen, A. B. 1979. The effects of in vivo and in vitro oxidative damage to cumulation of neutrophils in the lungs. Inflammation. 8:53-61. purified al -antitrypsin and to the enzyme-inhibiting activity ofplasma. Am. Rev. 48. Wesselius, L. J., A. Catanzaro, and S. I. Wasserman. 1984. Neutrophil Respir. Dis. 119:953-960. chemotactic activity generation by alveolar macrophages after bleomycin injury. 57. Cochrane, C. G., R. Spragg, and S. D. Revak. 1983. Pathogenesis of the Am. Rev. Respir. Dis. 129:485-490. adult respiratory distress syndrome: evidence of oxidant activity in bronchoal- 49. Campbell, E. J., and M. S. Wald. 1983. Hypoxic injury to human alveolar veolar lavage fluid. J. Clin. Invest. 71:754-761. macrophages accelerates release ofpreviously bound neutrophil elastase: implica- 58. Goldstein, W., and G. Doring. 1986. Lysosomal enzymes from polymor- tions for lung connective tissue injury including pulmonary emphysema. Am. phonuclear leukocytes and proteinase inhibitors in patients with cystic fibrosis. Rev. Respir. Dis. 127:631-635. Am. Rev. Respir. Dis. 134:49-56. 50. Kaplan, J., and M. L. Nielsen. 1979. Analysis of macrophage surface 59. Hubbard, R. C., and R. G. Crystal. 1986. Antiproteases and antioxidants: receptors. I. Binding of a2-macroglobulin-protease complexes to rabbit alveolar strategies for the pharmacologic prevention of lung destruction. Respiration. macrophages. J. Biol. Chem. 254:7323-7328. 50:56-73. 60. Hubbard, R. C., F. Ogushi, G. A. Fells, A. M. Cantin, S. Jallat, M. Court- 51. Chang, J., F. Wigley, and D. Newcombe. 1980. Neutral protease activa- ney, and R. G. Crystal. 1987. Oxidants spontaneously released by alveolarmacro- tion of peritoneal macrophage prostaglandin synthesis. Proc. Nat!. Acad. Sci. phages of cigarette smokers can inactivate the active site of al-antitrypsin, ren- USA. 77:4736-4740. dering it ineffective as an inhibitor ofneutrophil elastase. J. Clin. Invest. 80:1289- 52. Travis, J., and G. S. Salveson. 1983. Human plasma proteinase inhibitors. 1295. Annu. Rev. Biochem. 52:655-709. 61. Banda, M. J., A. G. Rice, G. L. Griffin, and R. M. Senior. 1988. al-Pro- 53. Beatty, K., J. Bieth, and J. Travis. 1980. Kinetics of association of serine teinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by proteinase with native and oxidized a- I -proteinase inhibitor and a- 1-antichymo- macrophage elastase. J. Biol. Chem. 263:4481-4484. trypsin. J. Biol. Chem. 255:3931-3934. 62. Banda, M. J., A. G. Rice, G. L. Griffin, and R. M. Senior. 1988. The 54. Carp, H., F. Miller, J. R. Hoidal, and A. Janoff. 1982. Potential mecha- inhibitory complex of human al-proteinase inhibitor and human leukocyte elas- nism of emphysema: al-proteinase inhibitor recovered from lungs of cigarette tase is a neutrophil chemoattractant. J. Exp. Med. 167:1608-1615.

Lung Neutrophil Accumulation ofal-A ntitrypsin Deficiency 897