inhibitors of elastase and cathepsin g in chediak-higashi (beige

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, InC.

VOl. 264, No. 13, Issue of May 5 , pp. 7431-7436,1989 Printed in U. S. A.

Inhibitors of Elastase and Cathepsin G in Chediak-Higashi (Beige) Neutrophils*

(Received for publication, August 29, 1988)

Kiyoshi H. Takeuchil and Richard T. Swanks From the Roswell Park Memorial Institute, Department of Molecular and Cellular Biology, Buffalo, New York 14263

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chediak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G . There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G activities. ‘251-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-lZ6I- elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G . No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus repre- sent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactiva- tion of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chediak-Higashi patients to infection.

In humans, Ch6diak-Higashi syndrome (CHS)’ is a reces- sively inherited mutation causing several clinical symptoms including lowered neutral proteinase activity in neutrophils (1, 2), increased susceptibility to bacterial infection (3, 4), platelet storage pool deficiency (5), and deficiency of func- tional natural killer cells (6). Human CHS patients are weak- ened by repeated infection and usually die before or during

* This work was supported by National Institutes of Health Grant HL-31698. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Ralph Lowell Laboratories, McLean Hospital, Harvard Medical School, Belmont, MA 02178.

§ To whom correspondence should be sent: Roswell Park Memorial Institute, Dept. of Molecular and Cell Biology, 666 Elm St., Buffalo, NY 14263.

The abbreviations used are: CHS, Ch6diak-Higashi syndrome; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.

their teen years. A likely cause of the increased susceptibility to infection is an inability of neutrophils to kill invading bacteria (3). Neutrophils contain lysosomal neutral proteinases such as elastase and cathepsin G (2, 7, 8). Both proteinases are thought to contribute to the killing of invading microorganisms in neutrophils (7, 9, 29). It is therefore plau- sible that a deficiency of these proteinases contributes to the increased susceptibility to infection and eventual death of CHS patients. Other factors such as defective degranulation of abnormal neutrophil granules may also contribute (4).

In beige mice, which express most of the characteristic features of human CHS, we have recently found that mature neutrophils isolated from either blood or the peritoneal cavity are highly deficient in both elastase and cathepsin G activities (2). Of equal interest, bone marrow neutrophils of beige mice maintain 60% of normal levels of both enzymes (10). Early neutrophil precursors in beige mice express near normal levels of enzyme activity in bone marrow. Mature neutrophils, in contrast, including some metamyelocytes, have no enzyme activities in beige marrow (10). Marrow neutrophils from normal mice, however, have high levels of these enzyme activities through all developmental stages. These findings indicated that the major mechanism for the lowered neutrophil elastase and cathepsin G activities in mature neutrophils of beige mice is not due to an absence of enzyme synthesis at early neutrophil developmental stages in bone marrow.

These facts prompted us to investigate the mechanism(s) of inactivation or degradation of enzymatically active neutral proteinases in mature neutrophils of beige mice. In this study, we report endogenous inhibitors of the two neutral proteinases, elastase and cathepsin G, in neutrophils of beige mice.

MATERIALS AND METHODS

Materials and their sources were as follows: Hanks’ Caz+- and MF-free balanced saline solution (Gibco); Ficoll-Paque (Pharmacia LKB Biotechnology Inc.); methoxysuccinyl-alanyl-alanyl-prolyl- valine-4-methoxycoumarinyl-7-amide (MeO-Suc-Ala-Ala-Pro-Val- MCA), methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethyl ketone (MeO-Suc-Ala-Ala-Pro-Val-CHzCl), and Z-glycyl-leucyl- phenylalanine-chloromethyl ketone (Z-Gly-Leu-Phe-CH&l) (En- zyme Systems Products); succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA), phenylmethanesulfonyl fluoride (PMSF), dimethyl sulfoxide, &isopropyl fluorophosphate, soybean trypsin inhibitor, a-1-antitrypsin, proteinase K, and 4-chloro-1-naphthol (Sigma); sodium [’251]iodide (Amersham Corp.); human neutrophil elastase and cathepsin G purified from human sputum (Elastin Products). This elastase is purified by two affinity chromatography procedures and is an essentially homogeneous product on SDS gels. It is free of cathepsin G activity. The cathepsin G was chromatographically purified and was free of elastase activity.

Animals-C57BL/GJ (+/+I (normal) and C57BL/6J (bgJ/bgJ) (beige) male and female mice were originally purchased from the Jackson Laboratories and were subsequently bred at the animal facilities of Roswell Park Memorial Institute.

Induction and Preparation of Elicited Peritoneal Neutrophils- Peritoneal neutrophils were elicited by the copper rod implantation

7431

7432 Inhibitors of Elastase and Cathepsin G technique originally described by McGarry (11). Harvesting and purification of peritoneal neutrophils were described (2). We routinely obtain about lo7 cells/mouse, >95% neutrophils.

Purified neutrophils were resuspended with cold 0.2% Triton X- 100, 0.2 M sucrose, 0.15 M NaCl, 5 mM EDTA in 20 mM imidazole- HC1, pH 7.4 (homogenization buffer), and extended according to the methods in Ref. 2. The homogenate was centrifuged at 13,000 X g for 10 min at 4 "C, and the supernatant was used as a neutrophil extract.

Isolation of Neutrophils from Peripheral Blood-Blood samples were collected by cardiac puncture, and red blood cells were removed by dextran sedimentation (10). Lymphocytes, monocytes, and plate- lets were removed by centrifugation using a Ficoll-Paque gradient, and purified neutrophils were prepared as described (10).

Preparation of Bone Marrow Cells-Bone marrow was prepared according to the method of Watt et al. (12) with a minor modification (10) and was extracted as above.

Enzyme Assays-Elastase was assayed with the substrate MeO- Suc-Ala-Ala-Pro-Val-MCA (13, 14), and cathepsin G was measured using Suc-Ala-Ala-Pro-Phe-pNA (15) as substrate. One unit of elastase activity corresponds to the release of 1 nmol of AMC/min. Elastase activities in normal and beige neutrophils are about 4.2 X

units/neutrophil and 0.1 X lo-' units/neutrophil, respectively (2). Cathepsin G activity is expressed as the change in absorbance/ min at 410 nm (X lo3). Cathepsin G activities in normal and beige neutrophils are 4.1 X units/neutrophil and 0.3 X units/ neutrophil, respectively (2).

Protein was determined using the Bio-Rad protein assay with ovalbumin as a standard protein.

Polyacrylamide Gel Electrophoresis-Samples were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide (SDS- PAGE) slab gels by the method of Laemmli (16). The standard proteins, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme of molecular weights 92,500, 68,000, 45,000, 31,000, 21,500, and 14,400, respectively, were used. The sample buffer (pH 6.8) contained 50 mM Tris-HC1, 2% SDS, 10% glycerol, 5 mM EDTA, and 10 mM dithiothreitol. After electrophoresis, gels were stained for protein with Coomassie Brilliant Blue.

Radiolabeling of Human Neutrophil Elastase and Cathepsin G with Na'25Z-Purified human neutrophil elastase (100 pg in 100 pl of iodination buffer (see below)) was radiolabeled by incubating with 0.5 mCi of Na'''I in a polypropylene tube coated with 10 rg of IODO- GEN (17). The iodination buffer was 50 mM potassium phosphate, pH 7.0,300 mM NaCl containing 0.5 mg/ml benzamidine (to protect against proteolysis by trypsin-like contaminants). Free lZ5I was removed from iodinated proteins on a Sephadex G-25 column (0.8 X 20 cm) eluted with iodination buffer. The products, '2SI-labeled elastase and lZ5I-labeled cathepsin G, were stored at -20 "C in small aliquots until used.

Formation of '25Z-Humn Neutrophil Elastase-Znhibitor Com- plexes-'261-Elastase or lBI-cathepsin G (lo6 cpm) was incubated 10 min at 4 "C with peritoneal neutrophil or bone marrow extracts containing 10-100 pg of protein in 20 mM imidazole-HC1, pH 7.4, 0.15 M NaCl, 5 mM EDTA, and 0.2% Triton X-100. Samples were subsequently boiled 2 min in 1.0% SDS plus 50 mM dithiothreitol and electrophoresed under denaturing conditions on SDS-polyacrylamide gels. Gels were dried, and autoradiography was performed on Kodak XAR-5 film with Du Pont Lighting Plus intensifier screens at -70 "C.

Proteinase K Digestion of Beige Extracts-Neutrophils (5.5 X lo6) of beige mice were extracted and prepared at 5 mg of protein/ml with 0.2% Triton X-100, 0.2 M sucrose, 0.15 M NaCl, 5 mM EDTA in 20 mM imidazole-HC1, pH 7.4 at 0 "C in the presence of 5 mg/ml proteinase K (Boehringer Mannheim). After 10-min incubation at 0 "C, proteinase K was inactivated with 2 mM PMSF. All samples were then applied to a Sephadex G-25 column (0.8 X 20 cm) to remove free PMSF. The column was equilibrated and eluted with 20 mM Tris-HC1, pH 7.4, 0.15 M NaCl at 4 "C. Fractions of 150 pl were collected at a flow rate of 0.6 ml/min. 150 p1 (235 pg of protein) of the void volume of the column was collected for assay of inhibitory activity to either elastase or cathepsin G according to the regular procedure.

Determination of the Apparent Molecular Weight of Elastase Znhib- itors in Beige Neutrophil Extracts-Beige neutrophil extracts were prepared in 20 mM imidazole-HC1, pH 7.4, 0.15 M NaCl, 0.2% Triton X-100,0.2 M sucrose, and 5 mM EDTA by described methods (2, 10). This extract was centrifuged at 13,000 X g for 10 min at 4 "C and the resulting clear supernatant (50-100 pl) was injected onto Superose

12 HR 10/30 for gel filtration by fast protein liquid chromatography (Pharmacia). The column was equilibrated and eluted with 20 mM Tris-HC1, pH 7.4, 0.15 M NaCl, and 0.02% NaN3 at 4 "C. The flow rate was maintained at 0.5 ml/min, and 0.5-ml fractions were collected. Each fraction was assayed for human neutrophil elastase or cathepsin G inhibitory activity. The molecular weight standards used were blue dextran, aldolase, bovine serum albumin, ovalbumin, carbonic anhydrase, chymotrypsinogen A, and aprotinin.

Assay for Inhibitors-The assay utilized 0.02 pg of pure human neutrophil elastase and 0.5 mM fluorogenic neutrophil elastase substrate, MeO-Suc-Ala-Ala-Pro-Val-MCA, in 100 pl of assay mixture. Human neutrophil elastase was incubated with cell extracts containing the inhibitor in 50 pl of 0.15 M NaCl, 20 mM Tris-HC1, pH 7.5, 0.2% Triton X-100, 5 mM EDTA, and 1 mg/ml BSA at 4 "C for 10 min. BSA was found to prevent self-digestion of human neutrophil elastase. Following the preincubation, the complete reaction mixture was assayed 60 min at 37 "C for elastase activity, and the reaction was stopped by the addition of 1 ml of 5 M formic acid. The fluorescent product, aminomethylcoumarin, was measured as described (13, 14). A unit of inhibitor is defined as the amount which inhibits 50% of human elastase activity in the above standard assay conditions. The assay for inhibitors of human neutrophil cathepsin G utilized 0.2 pg of pure human neutrophil cathepsin G and 2 mM substrate, Suc-Ala- Ala-Pro-Phe-pNA, in 500 pl. The cathepsin G and extracts containing the inhibitor were preincubated 10 min at 4 "C in 0.15 M NaCl, 20 mM Tris-HC1, pH 7.5, 0.2% Triton X-100, 5 mM EDTA in 50 pl. Following the preincubation, the complete reaction mixture (500 p l ) (2, 15) was incubated 20 min a t 50 "C to assay cathepsin G activity, and the reaction was stopped by the addition of 50 pg of soybean trypsin inhibitor. The released 4-nitroaniline was measured at 410 nm as described (15). A unit of inhibitor is defined as the amount which inhibits 50% human cathepsin G activity under the described conditions.

RESULTS

Evidence for Inhibitors of Elastase and Cathepsin G in Beige (Chidiak-Higashi) Neutrophil Extracts-Positive indication of inhibitors of human neutrophil elastase (Fig. l.4) and human cathepsin G (Fig. 1B) in beige neutrophils was ob- tained in mixing experiments. Human neutrophil elastase activity was 50% inhibited when 1.2 pg of beige extract was preincubated with 0.02 pg of purified human neutrophil elastase (Fig. U), and 90% inhibition occurred at about 20 pg of beige extract. Furthermore, 80% inhibition of human cathepsin G activity was demonstrated when about 30 pg of beige neutrophil extract was preincubated with 0.2 kg of purified human cathepsin G (Fig. 1B). However, these inhibitions were nearly completely abolished if beige neutrophil extracts were boiled 3 min prior to mixing with human neutrophil elastase or cathepsin G, suggesting that the inhibitor is a protein. The lack of an inhibitory effect of BSA on either enzyme activity suggested a specific protein inhibitor.

From the above data (i.e. that 1.2 pg of beige neutrophil extract inhibits 50% of the activity of 0.02 pg of human elastase) and assuming a 1:l interaction between inhibitor and elastase and taking a mean molecular mass of 30 kDa for the inhibitors, it can be calculated there is about 10 pg of inhibitors per mg of beige neutrophil protein or per lo7 neutrophils.

The activities of both elastase (Fig. 2 A ) and cathepsin G (Fig. 2B) of normal mouse neutrophils were likewise dimin- ished when excess extract from beige neutrophils was added to extract from normal neutrophils. Significant inhibition was observed when equal amounts of normal and beige extracts were preincubated. Only 10% of elastase activity remained when 100 pg of beige protein was preincubated with 18 pg of normal extract protein (Fig. 2 A ) , and 40% of cathepsin G activity remained at 90 pg of beige protein/l7 pg of normal extract protein (Fig. 2B). The somewhat less efficient inhibition of cathepsin G may be due in part to the fact that two inhibitors to elastase and only one inhibitor to cathepsin G

Inhibitors of Elastase and Cathepsin G 7433

A

"- "" Boiled Beige Exlroct

BSA

F A Normal Extract

t Boiled Beige Extract

7 l & * U

50

0

t

Beige Extract ( p g protein)

Normal Extract t

Boiled Beige Extract - B

Beige Neutrophil Extract (pg)

B Human Cathepain G

e t Beige Extract - .- 9 c 0 ' ~ " " " " " " " " " ' ' Q) 50 N

400

Beige Neutrophil Extract ( p g )

FIG. 1. Human neutrophil elastase and cathepsin G inhibition by beige neutrophil extracts. Human neutrophil elastase (0.02 pg) or cathepsin G (0.2 pg) was treated with the indicated amounts of beige neutrophil extract in 20 mM imidazole-HC1, pH 7.4, 0.15 M NaCl, 0.2% Triton X-100,0.2 M sucrose, and 5 mM EDTA. In one case, beige extract was boiled 3 min before addition to elastase or cathepsin G, and in another case, control bovine serum albumin (50 pg of BSA for elastase and 90 pg for cathepsin G assays) was substituted for the beige extract. In each case, the mixture was preincubated for 10 min at 4 "C before assaying for either elastase or cathepsin G activity. A, elastase activity; B, cathepsin G activity.

are present in beige extracts (see Fig. 5). Inhibition of both elastase and cathepsin G was nearly completely eliminated when beige extracts were boiled prior to incubation with normal extracts (Fig. 2).

Formation of Inhibitor-Human Neutrophil Elastase and Ca- thepsin G Complexes-The discovery of elastase and cathepsin G inhibitors in beige neutrophil extracts (Figs. 1 and 2) encouraged us to test for the enzyme-inhibitor complexes by SDS-PAGE. Purified human neutrophil elastase and human cathepsin G were iodinated with NalZ5I, mixed with various amounts of normal and beige extracts, and these mixtures were separated by denaturing electrophoresis on SDS-polyacrylamide gels. High molecular weight lZ5I-labeled protein complexes were detected by autoradiography of dried gels.

It was found (Fig. 3) that '2SI-elastase forms significant levels of complexes at 64 and 52 kDa when added to beige peritoneal neutrophil extracts. The beige neutrophil proteins complexed with '251-elastase were estimated to be 36 and 24 kDa since the molecular mass of human elastase is known to

U

Beige Extract (pg protein)

FIG. 2. Beige neutrophil extracts inhibit both cathepsin G and elastase of normal neutrophils. Neutrophil extracts of normal and beige mice were mixed and preincubated for 10 min at 4 "C. After incubation, enzyme activities were assayed for elastase or cathepsin G. In some experiments, beige extracts were boiled 3 min prior to the preincubation step. A, elastase activity; nonboiled beige extract added (U), boiled beige extract added (0). 18 pg of normal extract protein was used. B, cathepsin G activity; nonboiled beige extract added (A), boiled beige extract added (A). 16.5 pg of normal extract protein was used.

.. . "- 0 Origin

[1251]-HNE-+ 1

FIG. 3. Extracts of elicited neutrophils of beige mice form complexes with '"1-human neutrophil elastase ('251-HNE). '251-elastase (lo5 cpm) was incubated with peritoneal neutrophil extracts (10 min at 4 "C) containing 20 pg of protein in 20 mM imidazole- HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 0.2 M sucrose, and 0.2% Triton X-100. Samples were subsequently boiled 2 min with 1.0% SDS + 5 mM dithiothreitol and electrophoresed under denaturing conditions on 10% SDS-polyacrylamide gels. Gels were dried and exposed to x-ray film with intensifying screens at -80 "C. The control lane represents the 1251-elastase preparation alone.

7434 Inhibitors of Elastase and Cathepsin G

be 28 kDa (14). The complexes were not detectable in peritoneal neutrophil extracts of normal mice (Fig. 3). Boiled neutrophil extracts from normal and beige mice also failed to form complexes. The large amount of radioactivity at the front of the gel in all lanes resulted from the excess of 1251- elastase used. Of the high molecular mass label, 60% was in the 64-kDa component, and 40% was at 52 kDa by densito- metric analysis of autoradiograms. The complexes of 1251- elastase and beige neutrophil inhibitor(s) were stable to heat- ing in 1% SDS at 100 "C, indicating covalent association.

A preliminary experiment indicated that '251-cathepsin G also formed protein complex(es) at approximately 64 kDa with beige neutrophil extracts (not shown). However, the interpretation of these gels was difficult because several iodinated bands, presumably protein impurities in the cathepsin G preparation, were present in this molecular mass region.

Involvement of the Active Site of Elastase in Complex For- mation-Formation of complex(es) with '251-ela~ta~e by the beige neutrophil extracts was inhibited by serine proteinase active site inhibitors such as 0.5 mM PMSF, 0.2 mM diisopropyl fluorophosphate, or 1.0 mM soybean trypsin inhibitor (Fig. 4). More significantly, the synthetic chloromethyl ketone MeO-Suc-Ala-Ala-Pro-Val-CH2C1, which is a specific active site inhibitor of human neutrophil elastase (18), completely prevented the formation of human neutrophil elastase-inhibitor complexes. On the other hand, neither bovine serum albumin nor 2-Gly-Leu-Phe-CH2C1, which is a cathepsin G inhibitor (15), inhibited the formation of complexes (Fig. 4). These findings indicate that the integrity of the active site of human neutrophil elastase is required for the formation of complexes with beige neutrophil inhibitor(s).

Gel Permeation Chromatography Analyses of Inhibitors of Human Neutrophil Elastase in Beige Neutrophil Extracts- To investigate the nature of the inhibitors from beige neutrophils under nondenaturing conditions, we analyzed beige extracts on Superose 12 gel filtration columns (Fig. 5). The inhibitory activity of each fraction was tested against human neutrophil elastase and human cathepsin G with MeO-Suc- Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively, as substrates. No measurable inhibitory activity was present in normal extracts (Fig. 5A) . However, two frac-

['251]-HNE + + + + + + + + Beige Extract - + + + + + + +

Origin- 1 . - 3

64kDa - 52kDa-

- -

FIG. 4. Inhibition of formation of '261-human neutrophil elastase ( '261-HNE)-beige neutrophil inhibitor complex by proteinase inhibitors. In some cases 1251-elastase was preincubated with 20 pg of beige neutrophil extract for 10 min at 4 "C. In the other cases, 1Z51-elastase was preincubated with 0.5 mM PMSF, 0.2 mM diisopropyl fluorophosphate (DFP) , 1 mM soybean trypsin inhibitor (SBTZ), 0.2 mM MeO-Suc-Ala-Ala-Pro-Val-CH2Cl (AAPV), 20 pg of BSA, or 0.2 mM 2-Gly-Leu-Phe-CH2C1 (GLF) for 10 min at 4 "C prior to incubation with 20 pg of beige neutrophil extracts. Samples were subsequently analyzed on 10% SDS-PAGE followed by autoradiography. Arrows indicate the lZ5I-inhibitor complexes of M, = 64,000 and 52,000.

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FIG. 5. Analysis of elastase and cathepsin G inhibitors in peritoneal neutrophil extracts by gel filtration. Peritoneal neutrophil extracts were prepared in 20 mM imidazole-HC1, pH 7.4,0.15 M NaC1, 0.2% Triton X-100, 0.2 M sucrose, and 5 mM EDTA. 50 pl of normal ( A ) and beige ( B ) extracts containing 370 pg of protein was subsequently injected onto a Superose 12 column and eluted at 0.5 ml/min with a fast protein liquid chromatography apparatus (Pharmacia). The elution buffer was 20 mM Tris-HC1, pH 7.4, 0.15 M NaCI, and 0.02% NaN3. Fractions were monitored at 280 nm for protein, and 0.5-ml fractions were collected and analyzed for their ability to inhibit the activities of either human neutrophil elastase (o"-o) or cathepsin G (u). The elution positions of standard proteins (bovine serum albumin, 68 kDa; ovalbumin, 45; carbonic anhydrase, 30; chymotrypsinogen A, 25; and aprotinin, 7) were determined in separate experiments.

tions inhibitory to human neutrophil elastase appeared in beige extracts at 36 and 24 kDa (Fig. 5B). 60% of inhibitory activity was recovered in the 36-kDa fraction and 40% in the 24-kDa fraction. The apparent masses of 36 and 24 kDa are in agreement with the molecular masses of the beige inhibitors estimated with '251-elastase-inhibitor complexes on SDS- PAGE (Fig. 3).

The inhibitory activity to human cathepsin G, in contrast,

Inhibitors of Elastase and Cathepsin G 7435

was present only at the 36-kDa region in beige extracts (Fig. 5B).

Presence of Inhibitors in Circulating Neutrophiki and Ab- sence in Eone Marrow-Mature neutrophils circulating in the blood were examined for the presence of inhibitor(s) to human neutrophil elastase and cathepsin G. Beige neutrophils had inhibitor(s) to both enzymes by enzyme assays (data not shown). The addition of extracts from circulating neutrophils of normal mice, however, did not cause inhibition of enzyme activities. Extracts from circulating neutrophil of beige mice formed complexes with 1251-elastase of the same molecular masses (Fig. 6) as that formed by elicitedperitoneal neutrophil extracts (Fig. 3).

Bone marrow extracts of beige mice, however, produced no 1251-elastase-inhibitor complexes (Fig. 6). These results dem- onstrate that only mature neutrophils released from beige bone marrow contain detectable endogenous inhibitors to neutral proteinases.

Comparison with al-Proteinase Inhibitors-The inhibitors are not identical to the classical serum inhibitor of neutrophil elastase, al-proteinase inhibitor (19-21). When 1251-elastase was incubated with either normal or beige plasma, a major complex at 78 kDa was formed (Fig. 7) which is presumably due to mouse plasma al-proteinase inhibitor complexing with elastase. This mass is consistent with the 50-kDa size of the al-proteinase inhibitor (20, 21). A minor component was consistently visible at 60 kDa, and a trace component was inconsistently visible at 52 kDa. No complex was visible at the 64-kDa position observed ~hen'~~I-elastase was incubated with beige peritoneal neutrophils. The 64-kDa complex, com- monly observed when 1251-elastase is incubated with beige peritoneal neutrophils, was not seen when 1251-elastase was incubated with beige or normal plasma.

It is also unlikely that normal neutrophils contain significant amounts of al-proteinase inhibitor since no complexes were observed when normal neutrophil extracts were incu-

r B e i g e 1 r N o r m a l 7

Origin -

64kDa - 52kDa-

FIG. 6. Comparison of the '2sI-human neutrophil elastase ('2s1-HNE) inhibitor complexes in three neutrophil developmental stages. Bone marrow extracts (50 pg) from normal and beige mice were prepared by the methods described elsewhere (10) and incubated with 1251-elastase (lo5 cpm in 20 mM imidazole-HC1, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 0.2 M sucrose, and 0.2% Triton X- 100). Blood neutrophils (PMNs) (20 pg) and peritoneal neutrophils (PMNs) (20 pg) were also extracted and incubated with '251-elastase. After completion of incubation (10 min at 4 "C), '251-elastase-inhibitor complexes were analyzed on SDS-PAGE followed by autoradiography. The control autoradiogram demonstrates the results of incubation of the '251-elastase without the various extracts.

64 kDa "--* 6- 78kDa

O r I I I r- c251] -HNE

FIG. 7. Comparison of the 12'I-human neutrophil elastase ('''I-HNE) complexes formed by plasma and by extracts of beige neutrophils. '251-Elastase (lo5 cpm) was incubated without (as control) or with beige plasma (30 pg), normal plasma (30 pg), or extracts of mature beige neutrophils (20 pg). The reaction mixtures of 1251-elastase-inhibitor complexes were reduced and analyzed on SDS-PAGE followed by autoradiography.

bated with 1251-elastase (Fig. 6). Other Characteristics of the Beige Inhibitor-Proteinase K

digestion caused complete loss of inhibitory activity against human neutrophil elastase. The inhibitor activity is heat- labile and nondialyzable. After 24-h dialysis of beige extracts against 0.15 M NaC1, 20 mM Tris-HC1,0.2 mM NaN3 (pH 7.4) at 0 "C, 90% of the inhibitory activity against human elastase was maintained. 50% of inhibitory activity to human elastase remained after one cycle of lyophilization of beige neutrophil extracts. Under acidic conditions (pH 4.0) for 24 h at 0 "C, inhibitory activity was stable.

In other experiments (not shown), we have observed that '251-trypsin, like 1251-elastase, formed two complexes on SDS gels after mixing with beige but not normal extracts. In contrast, previous studies (2) have revealed that activity of another proteinase, endogenous plasminogen activator, is not inhibited in beige neutrophils. Further specificity studies will require separation and purification of the inhibitors.

DISCUSSION

Mature neutrophils of beige mice have profoundly decreased activity levels of elastase and cathepsin G (2). Decreased neutral proteinase activities have also been reported in neutrophils from human CHS patients (1). Our studies indicate that a likely and very unusual cause of this enzyme deficiency state is the appearance of specific proteinase inhibitors. A developmental stage-specific appearance of inhibitors is consistent with previous data (10) indicating that early stages in the neutrophil bone marrow lineage have normal activities of these enzymes in beige mice, while later stages including metamyelocytes and mature neutrophils have little or no activity. It is uncertain if inhibition leads to an increased rate of degradation of elastase and cathepsin G in beige neutrophils. We were unable to answer this question since available antibodies to human elastase and cathepsin G do not cross- react with the mouse enzymes. However, Ganz et al. (30) have found that neutrophils of three CHS patients have reductions, as monitored by Western blots, in cathepsin G and elastase antigens in proportion to the decrease in activity of these enzymes. Proteinase-inhibitor complexes or other aberrant forms of these enzymes were not observed in the Western blots. These forms could be rapidly degraded, could be present in small amounts, or could be nonreactive with the antisera.

7436 Inhibitors of Elastase and Cathepsin G

Rausch et al. (22) reported that CHS neutrophils contain cross-reacting materials to elastase and cathepsin G, as detected by immunohistochemical procedures, although quan- titation was not possible by this method.

Two inhibitors of 36 and 24 kDa are present in mature beige neutrophils and are absent in neutrophils from normal mice. The finding of identical molecular masses by both denaturing and nondenaturing conditions suggests that these are the monomeric molecular sizes. The inhibitors are specific in their interaction in that only the 36-kDa form inhibits cathepsin G, while both inhibit elastase. The stability of the inhibitor-proteinase complexes after boiling in SDS indicates that covalent complexes are formed. Blocking of enzyme- inhibitor complex formation by pretreatment of extracts with synthetic low molecular mass active site inhibitors of the proteinases shows that the enzyme-inhibitor complex is formed via the proteinase active sites. These results also suggest that the inhibitors and proteinases form a 1:l complex since each proteinase has only one active site.

The inhibitors are only detectable in circulating and peritoneal neutrophils of beige mice but not in bone marrow neutrophils (10). The reason for the lack of detectable inhibitor in bone marrow is uncertain. At this maturation state there may be limiting levels of inhibitor which are completely complexed with endogenous elastase and cathepsin G.

Ganz et al. (30) have reported no evidence for inhibitors of elastase or cathepsin G when normal and CHS neutrophil extracts were mixed. The reason(s) for this discrepancy with our findings is not certain. One possible cause is a difference between the beige mouse model and human CHS. Another consideration is that especially for cathepsin G (see Fig. 2B) , it is necessary to mix relatively large amounts of mutant extracts with normal extracts to detect significant inhibition.

The source of the inhibitors is uncertain. There is very little protein synthesis in neutrophils at the late stages (late metamyelocyte/early polymorphonuclear neutrophils) (23) of neutrophil maturation when inhibition of elastase and cathepsin G activities is first observed (10) so that de nouo synthesis of inhibitors, while possible, may not occur at these late stages. Post-translational activation of pre-existing inhibitors or abnormal endocytosis of inhibitors in mature beige cells could account for their appearance. If inhibitors are derived by endocytosis of plasma, additional inhibitor modification must occur within the beige neutrophil since the molecular masses of the neutrophil and major plasma inhibitors are considerably different. It is, however, possible that the trace inhibitor-elastase complex detected in plasma at 52 kDa is the source of the corresponding complex in beige neutrophils. It is also possible that by unknown mechanisms the inhibitors accumulate in beige neutrophils as a secondary result of formation of the characteristic giant lysosomes of CHS since the giant granules have been observed at very early (promyelocyte or myelocyte) stages of neutrophil maturation (24).

Intracellular proteinase inhibitors with properties at least partially similar to those found in beige neutrophils have been reported in a few cases. For example, an inhibitor forming a 66-kDa complex with radioiodinated pancreatic elastase was found in guinea pig peritoneal macrophages (25), and inhibitor of similar size was detected in human monocytes (26). It is of

interest that this latter inhibitor appeared only after several days in culture, thus resembling the maturation-dependent appearance of inhibitor in beige neutrophils. Several cytosolic inhibitors of elastase have been found in neutrophils of the horse (27). One of these inhibitors of 35.2 kDa has been highly characterized and, like the 36-kDa inhibitor of beige neutrophils, inhibits cathepsin G (28). Junger et al. (31) have purified an elastase inhibitor of 40 kDa from ovine granulocytes.

The absence of active elastase and cathepsin G in circulating neutrophils may contribute to the higher susceptibility of CHS patients to repeated infections. Our findings suggest that strategies to prevent inactivation of both enzymes by the 36- and 24-kDa inhibitors are clinically relevant.

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inhibitors of elastase and cathepsin g in chediak-higashi (beige

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