Acidic Polyamino Acids Inhibit Human Eosinophil GranuleMajor Basic Protein ToxicityEvidence of a Functional Role for ProMBP

Robert L. Barker, Robert H. Gundel,* Gerald J. Gleich, James L. Checkel,David A. Loegenng, Larry R. Pease, and Kimm J. HamannDepartment of Immunology and the Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Foundation, and the Mayo MedicalSchool, Rochester, Minnesota 55905; and *Department of Pharmacology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield,Connecticut 06877

Abstract

Eosinophil granule major basic protein (MBP), a potent toxinfor helminths and mammalian cells in vitro, is a single polypep-tide chain rich in arginine. MBPhas been localized on damagedhelminths and tissues in hypersensitivity diseases includingbronchial asthma. The MBPcDNA indicates that MBPistranslated as a slightly acidic preproprotein with an acidic pro-part. To test the hypothesis that the acidic pro-part of proMBPinhibits the toxicity of mature MBP, acidic polyamino acids(aa) were used as antagonists of MBPtoxicity to K562 cells andguinea pig tracheal epithelium and used as antagonists of MBPairway hyperresponsiveness in primates. The acidic poly aainhibited MBPtoxicity and MBPairway hyperresponsiveness.The acidic poly aa inhibited MBPtoxicity in a charge-depen-dent manner similar to that proposed for proMBP, suggestingthat the acidic pro-part of proMBP functions to mask matureMBPtoxicity. This inhibition was not limited to MBP, but alsoapplied to polyarginine and eosinophil cationic protein. Theseacidic poly aa may be useful to inhibit the actions of a number ofcationic toxins released by the eosinophil in numerous hyper-sensitivity diseases. (J. Clin. Invest. 1991. 88:798-805.) Keywords: bronchial asthma * airway hyperresponsiveness - poly-arginine * eosinophil cationic protein - anionic polymers

Introduction

The eosinophilic leukocyte contains a number of cationic tox-ins. Major basic protein (MBP),' the predominant protein ineosinophils, comprises the crystalloid core of the eosinophilgranule (1) and in humans is a 117-amino acid (aa) residue

Address reprint requests to Kimm J. Hamann, Ph.D., Section of Pul-monary and Critical Care Medicine, University of Chicago, Box 98,5841 South Maryland Avenue, Chicago, IL 60637.

Receivedfor publication 31 January 1991 and in revisedform 13May 1991.

1. Abbreviations used in this paper: aa, amino acid; DCS, defined calfserum; e, electronic charge; ECP, eosinophil cationic protein; MBP,major basic protein; metacholine P100, the dose of inhaled metacholinerequired to cause a 100% increase in Rrs; Rrs, respiratory system resis-tance.

single polypeptide rich in arginine (2). MBPlikely damages cellmembranes (3, 4) and is a potent toxin for helminths andmammalian cells in vitro (1). It has been localized on damagedhelminths and tissues in hypersensitivity diseases includingbronchial asthma. MBPcauses extensive damage to guinea pigand human respiratory epithelium; this damage mimics thepathology of asthma, suggesting that eosinophils are a primeeffector cell for tissue damage in bronchial asthma (1). MBPinstillation into the trachea of primates results in a significantincrease in airway responsiveness, indicating a direct role forMBPin the pathogenesis of airway hyperresponsiveness (5).

The MBPcDNA from the promyelocytic cell line HL-60,which produces MBP, indicates that MBPis translated as aslightly acidic preproprotein with an acidic pro-part (6). Hu-man MBP(2) contains a high portion of aa with hydrophobicside chains, 17 arginines, only one carboxylic acid-containingresidue, and has a calculated electronic charge (e) (calculatedby Titrate program, DNAstar, Inc., Madison, WI) of 16.3 (7).The 90-aa residue acidic pro-part of proMBP contains 20 glu-tamic acids, 6 aspartic acids, 7 strongly basic aa, and has acalculated e of -19.0. The combination of the acidic and basicpeptides in proMBP has an almost neutral e of -2.58.Previously, we suggested that the acidic pro-part of proMBPmasks the toxic effects of mature MBPand protects the eosino-phil from damage while the protein is processed through theendoplasmic reticulum to its sequestered site in the eosinophilgranule as toxic MBP(6).

To test the hypothesis that the acidic pro-part of proMBPinhibits mature MBPtoxicity, acidic poly aa were used as sub-stitutes for the acidic pro-part of proMBP which has not yetbeen isolated in its native state or obtained in a recombinantform. Here, we report that acidic poly aa inhibited MBPtoxic-ity to K562 cells and guinea pig tracheal epithelium and inhib-ited MBPairway hyperresponsiveness in primates. Acidic polyaa inhibited MBPtoxicity in a charge-dependent manner simi-lar to that proposed for proMBP, providing evidence that theacidic pro-part of proMBP functions to neutralize toxic MBPand suggesting a possible therapy for bronchial asthma.

Methods

Reagents and solutionsAmino acids, amino acid polymers (Table I), RPMI 1640 mediumcontaining L-glutamine and methacholine were purchased from SigmaChemical Co., St. Louis, MO. Defined calf serum (DCS) was obtainedfrom Hyclone Laboratories, Inc., Logan, UT. Ketamine (Ketaset) andxylazine (Rompun) were purchased from Bristol Laboratories, Evans-ville, IN, and Miles Inc., West Haven, CT, respectively.

798 Barker, Gundel, Gleich, Checkel, Loegering, Pease, and Hamann

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/91/09/0798/08 $2.00Volume 88, September 1991, 798-805

Table I. Selected Properties of AA and Proteins Used in This Study

Degree of Charge (e) Concentration forMol wt polymerization* at pH 7.OW balanced charge'

L-asparticacid(A-0651)1' 155 1 -1.1 (a)7.5 X 10-5ML-glutamic acid salt (G-1626) 169 1 -1.1 (a) 7.5 X 10-s MPoly(a,#)-DL-aspartic acid (P-3418) 6,800 50 -50.1 (a) 1.6 X 10-6 MPoly (aspartic acid, glutamic acid) 1:1 (P-1408) 9,000 60 -60.0 (a) 1.4 X 10-6 MPoly-L-aspartic acid (P-5387) 11,500 84 -84.0 (a) 9.7 x 10-7 MPoly-L-glutamic acid (P-4636) 13,600 90 -89.9 (a) 9.1 X 10-7 M

(b)3.3X 10-6M

(c) 8.1 X 10-7 MPolyglutamic acid (P-4761) 36,240 240 -239.0 (a) 3.4 x 10-7 MPoly-D-glutamic acid (P-4033) 41,000 272 -271.0 (a) 3.0 x 10-7 MPoly-L-aspartic acid (P-6762) 42,500 310 -310.0 (a) 2.6 x 10-7 MPoly-D-glutamic acid (P-4637) 66,000 437 -436.0 (a) 1.9 x 10-7 MPoly-L-glutamic acid (P-4886) 77,800 515 -514.0 (a) 1.6 x 10-7 M

(b)5.9x 10-7M(c) 1.4 x 10-7 M

Poly-L-asparagine (P-8137) 10,400 91 -0.1Native MBP 13,801 117 16.3Poly-L-arginine hydrochloride (P4663) 12,000 62 60.9ECP 16,000 133 14.5

* Degree of polymerization indicates number of aa residues per molecule. $Charge (e) calculated by Titrate program (DNAstar, Inc., Madison,WI). I Concentration of acidic aa necessary to balance charge of cationic: (a) MBPat 5 x 10-6 M; (b) poly-L-arginine hydrochloride at 5 x 10-6M; or (c) ECPat 5 x 10-6 M. 1" All acids used were purchased as sodium salts; Sigma product code appears in parentheses after the product name.

Purification of MBPand eosinophil cationic proteinEosinophils from patients with hypereosinophilic syndrome were ob-tained by cytapheresis as previously described (8, 9). The proceduresfor purification of human eosinophil granules, MBP, and eosinophilcationic protein (ECP) have been described in detail elsewhere (10, 11).Briefly, after eosinophil lysis and removal of cell debris and unbrokencells by centrifugation, granule-containing supernatants were pooledand centrifuged. Granule pellets were solubilized in 0.01 MHCl (finalpH 3.0) with brief sonication and centrifuged at 40,000gfor 5 min. Thesupernatant was fractionated at 4°C on a Sephadex G-50 column equili-brated with 0.025 Msodium acetate buffer (pH 4.2) containing 0.15 MNaCI (column buffer). Fractions from the second protein peak contain-ing ECPwere pooled, dialyzed against PBS, lyophilized, and furtherpurified as described below. Fractions from the third protein peak,containing only native MBP, were pooled. MBPconcentrations weredetermined by absorbance at 277 nm using the extinction coefficientE" = 26.3. ECPwas further purified by affinity chromatography on aheparin-CL-6B column equilibrated with PBS at pH 7.4. Fractionscontaining ECPeluted from the heparin-Sepharose at high salt concen-trations (0.15 M-1.5 MNaCI gradient used) and were pooled, dialyzedagainst PBS, and concentrated by Iyophilization. ECPconcentrationswere determined by absorbance at 277 nmusing the extinction coeffi-cient E` = 15.6.

Cytotoxicity and inhibition of toxicity assaysK562 cell cultures (CCL 243; American Type Culture Collection,Rockville, MD) were maintained in RPMI 1640 supplemented withL-glutamine and 10% DCSby twice-weekly passage. 2 d before assay,cells were resuspended at 4 x 10' cells/ml of fresh RPMI 1640 with 10%DCS, pH 7.4, in 50-ml flasks. By the day of assay, cell numbers hadreached 1.5-2.0 x 105/ml. These cells were washed three times inRPMI 1640 without DCS, resuspended to 5 X 105 cells/ml, and dupli-cate 100-jAI aliquots were dispensed to flat-bottomed 96-well microtiterplates. 50 ul of various concentrations of amino acids dissolved inRPMI 1640 or RPMI 1640 alone were added followed at the appro-

priate time by 50 ,d of MBPin column buffer, poly-L-arginine in RPMI1640, ECPin RPMI 1640, or appropriate controls. MBP, poly-L-argi-nine, and ECPwere used at a final concentration of 5 X 10-6 M. Cellswere incubated for 4 h at 37°C in a 5%CO2atmosphere in a humidifiedchamber. Cell viability was determined by microscopic observationusing trypan blue exclusion and mean percent viability±I SDof dupli-cates from one or more experiments was calculated. Differences inviability between nontoxic controls and test concentrations were evalu-ated statistically using Student's t test. Nontoxic controls were used inthe statistical analysis instead of the toxic control (MBP, poly-L-argi-nine, or ECPalone) because we wished to emphasize the point at whichacidic poly aa inhibition of toxicity declined. After the initial experi-ments using all of the acidic poly aa, poly-L-glutamic acid 13,600 molwt and poly-L-glutamic acid 77,800 mol wt, the lowest and highestmolecular weight polyglutamic acid homopolymers, were used as typi-cal representatives of the acidic poly aa. Optimal assay conditions forMBPtoxicity to K562 cells have been previously determined (12).

Guinea pig tracheal ring assayTracheal rings were prepared from Hartley guinea pigs (Mayo InstituteHills Farm, Rochester, MN) as previously described (13). Briefly, im-mediately after CO2euthanasia tracheas were removed and placed inRPMI 1640. Adherent connective tissue was trimmed and each tracheawas cut transversely into a series of 1-mm thick rings using a previouslydescribed slicing device (14). After overnight equilibration, each ringwas carefully examined to determine that the mucosa was intact andactively beating cilia were present. Rings with damaged mucosa werenot used for experimentation.

For testing, rings were transferred to 96-well microtiter plates con-taining 100 MI RPMI 1640/well. 50 Ml of acidic poly aa at a final con-centration of 1.4 X 10-5 Mor RPMI 1640 alone were added followedwithin S min by 50 Ml of MBPat a final concentration of 1.4 X 10- Mor controls (column buffer or RPMI 1640). MBPwas not used at 5x 10-6 Mas in K562 assays, because this concentration did not causecomplete cessation of ciliary motion of tracheal ring epithelial cells

Acidic Amino Acids Inhibit Major Basic Protein Toxicity 799

over the assay period. An observer, unaware of the substance or combi-nation being tested, examined the ring cultures at 24 h and 48 h usingan inverted microscope. After final observations, specimens were fixedin 10%buffered formalin, embedded in paraffin, sectioned, and stainedwith hematoxylin and eosin.

Criteria for damage to tracheal epitheliumOur criteria for damage to the ciliated epithelium of the guinea pigtracheal rings were similar to those used previously (13). The termciliostasis is used to describe the complete cessation of ciliary motionthroughout the entire tracheal ring; "normal" tracheal rings had active,intact ciliary epithelium. At the time intervals when damage was as-sessed, ciliostasis was almost always accompanied by partial or com-plete exfoliation or "stripping" of the ciliated epithelial cells from thebasement membrane of the mucosa with precipitates of MBPon oraround the cells.

Airway hyperresponsiveness and inhibition ofhyperresponsiveness assaysAnimals. The animals used in this study were wild-caught adult malecynomolgus monkeys (Macacafascicularis) weighing 3.5-7.5 kg. Eachanimal was housed individually in a specially designed open mesh cageand provided with food twice daily and water ad lib. Animals werefasted for - 18 h before study.

Study protocol. Each animal was anesthetized with an intramuscu-lar injection of ketamine (4 mg/kg) and xylazine (1 mg/kg), intubatedwith a cuffed endotracheal tube, and seated in an upright position in aspecially designed support chair. Ketamine (4 mg/kg, i.m.) was used tosupplement anesthesia when needed. Baseline respiratory system resis-tance (Rrs) was monitored for 15 min followed by methacholine dose-response determinations (15, 16). Bronchoconstrictor response to in-haled methacholine was used to determine airway responsiveness.After completion of the methacholine dose-response, each animal re-ceived an aerosol treatment of either vehicle or polyglutamic acid (P-4886, 61,200 mol wt; Sigma; this polyglutamic acid is a different lotand molecular weight from that of the P-4886 listed in Table I). Rrs wasmonitored for an additional 10 min after which each animal receivedan intratracheal injection of native MBP. Rrs was then monitored for 1h postinstillation after which the animals were allowed to recover fromanesthesia. At 2 h postinstillation each animal was anesthetized (keta-mine/xylazine), intubated, and methacholine dose-response determina-tions were performed. The study was designed so that the polyglutamicacid treatment experiments were bracketed by control (vehicle) treat-ment studies to ensure no change in MBP-induced effects over time.

Intratracheal MBPinstillation. Native MBPwas diluted in columnbuffer to a concentration of 200 ,g/ml immediately before instillation.A total of 5 ml (1 mgMBP) was slowly infused directly into the tracheathrough the endotracheal tube via a 20-cm long piece of polyethylene240 tubing attached to a 5-ml syringe. As control fluids, PBSand col-umnbuffer were tested and produced no effect on Rrs or methacholinesensitivity.

Rrs measurements. Respiratory system impedance was measuredby discrete frequency sinusoidal forced oscillations superimposed ontidal breathing as previously described (15). The frequency range wascomputed to provide a single value representation of Rrs.

Aerosol delivery system. Aerosol inhalation treatments of vehicle orpolyglutamic acid were administered by intermittent positive pressurebreathing with a Bird Mark 7A respirator and micronebulizer (Model8158; Bird Products Corp., Palm Springs, CA). Each treatment con-sisted of 15 breaths per min (maximum inspiratory pressure 20 cmH20) for 6 min. Polyglutamic acid was dissolved in PBSat a concentra-tion of 25 mg/mi just before use. Aerosol treatment delivered - 1.5 mgof polyglutamic acid to the lungs.

Methacholine dose-response determinations. Bronchial responsive-ness was assessed by performing cumulative methacholine dose-re-sponse determinations as previously described (16). Briefly, after aninitial aerosol challenge with vehicle (PBS), increasing concentrations

of methacholine were administered until increases in Rrs of 100-200%were obtained. Aerosol challenges were separated by 5-8 min or untilRrs returned to baseline values. Linear interpolation on a logarithmicscale was used to estimate the dose of methacholine at which a 100%increase in Rrs would have occurred (methacholine PC,00).

Statistical analysis. Changes in Rrs and methacholine PC1oOvalueswere evaluated statistically with the use of Fischer's paired t test. Ineach case, statistical significance was accepted when P < 0.05.

Inhibition of blood clottingTo determine whether acidic poly aa inhibit blood clotting, stock con-centrations of heparin, poly-L-glutamic acid 13,600 mol wt, poly-L-glu-tamic acid 77,800 mol wt, and/or MBPeach at 1 X 10-' Mwere addedto 500 ul whole blood from a normal human volunteer to final concen-trations of 10-4 Mto 10-6 Min 10 X 75-mm glass tubes and mixed.PBSand column buffer were used as controls. The tubes were checkedat 1-min intervals and clotting time for each sample was recorded.After 30 min, samples were checked every 30 min to a maximum of15 h.

Results

Inhibition of MBPtoxicity to K562 cells by acidic poly aa.Various polyglutamic and polyaspartic aa were added to K562cells followed by addition of MBPand cell viability was deter-mined after 4 h (Table II). The poly aa ranged from 6,800 to77,800 in mol wt (Table I) and were tested at concentrationsfrom 1 x l0-` Mto 5 x 10-6 Mfor their ability to inhibit MBPtoxicity at 5 X 10-6 M. All of the acidic poly aa inhibited MBPtoxicity at equimolar MBPconcentrations (Table II). This inhi-bition was related to the acidic or anionic nature of the poly-mers because poly-L-asparagine at 5 X 10-6 Mdid not inhibitMBPtoxicity. Also, some degree of polymerization was neces-sary for this effect because acidic amino acid monomers didnot inhibit MBPtoxicity. The acidic amino acids themselveswere not toxic to the K562 cells at the concentrations tested.

As the concentration of the acidic poly aa was lowered be-low that of MBP, inhibition of MBPtoxicity to the K562 cellswas reduced (Table II). This reduction was not due to the typeof acidic poly aa (glutamic versus aspartic or D versus Lisomers) but was dependent upon the molecular weight andthus the degree of polymerization of the acidic aa. Because thedegree of polymerization directly affects the total e of the acidicpolymers, a comparison between the acidic poly aa concentra-tions necessary to balance the e of MBPat 5 X 10-6 Mand thereduction in acidic poly aa inhibition of MBPtoxicity to K562cells was made (Tables I and II). At acidic polymer concentra-tions greater than or equal to those necessary to balance e ofMBPonly poly-L-aspartic acid (1 1,500 mol wt) and poly-L-glu-tamic acid (13,600 mol wt), both at 1 x 10-6 M, were signifi-cantly different from controls; however, at acidic polymer con-centrations less than those necessary to balance e of MBP, allpolymers exhibited a drastic reduction in their ability to inhibitMBPtoxicity. These data suggest that the e of an acidic poly aais a critical determinant of its ability to inhibit MBPtoxicity.

To better define the point at which acidic poly aa begin toeffectively inhibit MBPtoxicity, several of the acidic polymerswere tested at 2.0, 1.0, and 0.5 times the concentration neces-sary to balance e of MBPat 5 x 10-6 M (Fig. 1). All fourpolymers tested inhibited MBPtoxicity to K562 cells at a con-centration of 2.0 times e and were not significantly differentfrom the medium control, although poly-DL-aspartic acid, thesmallest polymer used in the study, was the least effective. At a

800 Barker, Gundel, Gleich, Checkel, Loegering, Pease, and Hamann

Table II. Inhibition of MBPToxicity by Acidic AA

Percent viability of K562 cells*

Native MPB(5 X 10-6 M) added to aa concentration

Mol wt Controls* I x 10-3M 5 x 10-6 M I X 10-6M 5 X 10-7 M I X 10-7M

L-aspartic acid 155 98.5±0.2 3.4±4.7§ ND ND ND NDL-glutamic acid 169 98.8±0.2 0.0±0.0§ ND ND ND NDPoly-(a,fl)-DL-aspartic acid 6,800 97.0±2.7 ND 90.1±6.71" 1.6±1.5 6.0±3.9§ 2.9±0.4§Poly (aspartic acid, glutamic acid) 1:1 9,000 98.4±1.4 ND 95.5±0.9 22.0±19.7§ 4.9±2.3§ 0.0±0.00Poly-L-aspartic acid 11,500 98.0±2.4 ND 87.5±7.9 84.3±1.6§ 22.7±7.2§ 2.7±3.8§Poly-L-glutamic acid 13,600 98.9±1.0 ND 89.5±2.0 72.9±0.7§ 4.9±5.9§ 0.0±0.0§Poly-L-glutamic acid 36,240 94.7±2.6 ND 96.2±6.5 96.9±1.8 67.5±20.4 i.3±1.8§Poly-D-glutamic acid 41,000 100.0±0.0 ND 98.0±1.8 99.0±1.7 96.2±1.4 1 1.2±0.9§Poly-L-aspartic acid 42,500 97.4±2.3 ND 100.0±0.0 99.4±0.9 100.0±0.0 8.7±1.4§Poly-D-glutamic acid 66,000 93.4±6.1 ND 96.4±1.7 97.3±1.0 90.6±3.4 0.0±0.0§Poly-L-glutamic acid 77,800 95.1±3.8 ND 93.9±8.0 95.5±6.4 95.4±3.2 2.6±3.5§Poly-L-asparagine 10,400 85.3±5.1 ND 5.9±9.5§ 1.2±2.1§ 0.0±0.0' O.O+O.O§Native MBP 13,801 1.0±3.2Column buffer 96.7±1.6Medium 96.5±3.7

* Values are mean percent viability± 1 SDof one to three 4-h experiments, each consisting of duplicate wells. t Controls consist of aa, acidic polyaa, poly-L-asparagine, native MBP, column buffer, or medium tested alone. All controls at S x 10-6 Mexcept L-aspartic and L-glutamic acidmonomers which were tested at 1 x 10-3 M. § P < 0.001 for values tested against appropriate nontoxic aa control using Student's t test; MBPadded within 5 min after addition of acidic aa. II Values underlined are from acidic poly aa test concentrations closest to but not less thanconcentration for balanced charge (Table I) for a particular acidic poly aa.

concentration of 1.0 times e the polyaspartic acids were signifi-cantly different from the controls while the polyglutamic acidswere not; mean percent viability was lowest for poly-DL-aspar-tic acid (23.9±4.2) and highest for poly-L-glutamic acid 77,800mol wt (89.1±2.3). All of the polymers were significantly differ-ent from the medium control at a concentration of 0.5 times eand afforded little protection against MBP. Thus, acidic polyaa begin to effectively protect K562 cells against MBPtoxicityat concentrations between 1.0 and 2.0 times e.

In the first set of experiments, the acidic poly aa were addedto the K562 cells before MBP. The ability of the acidic poly aa

to inhibit MBPtoxicity after MBPhad already been added tothe cells was tested and the data are shown in Fig. 2. Acidic polyaa concentrations which were effective in inhibiting MBPtoxic-ity previously were not as effective in inhibiting MBPtoxicitywhen MBPwas added to the cells before the aa polymers.There is some protection when the acidic poly aa are addedwithin 15 min after MBPaddition. However, little protection isseen when the polymers are added one or more hours afterMBPaddition because - 90% of cells are killed within 30 minof incubation with MBP. Only poly-L-glutamic acid (77,800mol wt) at equimolar MBPconcentration of 5 X 1o-6 Madded

100

80

0-~60

20

0Controls

Controls

MediumColumn buffer

MBP5 x 106M

Poly-DL-aspartic acid (6,800)Poly-L-glutamic acid (1 3,600)

Poly-L-aspartic acid (42,500)Poly-L-glutamic acid (77.800)

-., \..;

.^\

i. o

t \./ . s\

*r

;'\o.... ... : . ._ .... _ _ .

2xe 1 xe 0.5 x e

Acidic polyamino acid concentration

Figure 1. Acidic poly aa inhibition of MBPtoxicity to K562 cells at 2,1, and 0.5 times acidic poly aa concentration necessary to balancee of MBPat 5.0 x 10-6 M. Values are mean percent± I SDof oneexperiment consisting of duplicate wells. *P < 0.01 for values testedagainst appropriate controls using Student's t test.

100 --

80 - .

60 "\

40

20

0Controls

X K~ ~

1

ControlsMedium

Column buffer

> MBP5xITO6MPoly-L-glutamic acid (1 3,600)X1x O-6MlX10

5 x 10 6MPoly-L-glutarnic acid (77,800)5x 10-7M

x 10-6M'K 5x106M -

1 hour 2 hours

Elapsed time before addition of acidic po'yamino acids

Figure 2. Acidic poly aa inhibition of MBPtoxicity to K562 cells overtime. Acidic polymers were added before MBP(0 min) or 15 min,1 h, or 2 h after addition of MBPto cell cultures. Values are meanpercent viability± I SDof one to two experiments consisting of du-plicate wells. *P < 0.05 values tested against acidic poly aa added at0 min using Student's t test.

Acidic Amino Acids Inhibit Major Basic Protein Toxicity 801

Table III. Inhibition of ECPand Poly-L-arginine Toxicity by Acidic Poly AA

Percent viability of K562 celis*

Controlst Poly-iarginine or ECP(both at 5 x 10-6 M) added to acidic poly aa concentrations of

Molwt 5x 10-6M 5 X10-6M I x 10-6M 5x 10-7M I x 10-7M

Poly-L-glutamic acid 13,600 96.9±4.5 99.1±1.3§ 4.9 X 0.311 0.0±0.0"1 0.0±0.0"1Poly-L-glutamic acid 77,800 97.2±2.5 97.9±3.0 97.2 X 4.0 22.9±3.0" 2.0±2.91Poly-L-arginine hydrochloride 12,000 0.0±0.0Medium 97.8±1.0Poly-L-glutamic acid 13,600 100.0±0.0 77.2±4.911 73.7 X 5.811 50.2±1.311 59.8±20.6Poly-L-glutamic acid 77,800 100.0±0.0 98.3±0.5 96.4 X 5.2 91.2±3.3 58.4±11.8ECP 16,000 35.7±6.2Medium 86.8±2.1

* Values are mean percent viability± 1 SDof one 4-h experiment consisting of duplicate wells for poly-L-arginine hydrochloride and a secondexperiment consisting of duplicate wells for ECP. t Controls consist of acidic poly aa, poly-L-arginine, ECP, or medium tested alone. § Valuesunderlined are from acidic poly aa test concentrations closest to but not less than concentration for balanced charge (Table I) for a particularacidic poly aa. 1l P < 0.05 for values tested against appropriate acidic poly aa controls using Student's t test; poly-L-arginine hydrochloride or ECPadded within 5 min after addition of acidic poly aa.

15 min after MBPwas not significantly different from its con-trol. Increasing acidic poly aa concentrations 10-fold to 5x 10-5 M(data not shown) at all addition times had no signifi-cant effect. Apparently, the acidic poly aa have little effect onK562 viability unless they are added before or immediatelyafter addition of 5 X 10-6 MMBP.

Inhibition ofpoly-L-arginine and ECPtoxicity to K562 cellsby acidic poly aa. To determine if this toxic inhibition by acidicpoly aa is limited only to MBPor may apply to other cationictoxins, acidic poly aa were tested for their ability to inhibitpoly-L-arginine and ECPtoxicity to K562 cells. Both poly-L-ar-ginine and ECPtoxicity were inhibited by acidic poly aa (TableIII). Generally, there were no significant differences betweentest concentrations and controls when acidic poly aa test con-centrations were at least equal to the concentration needed fora balanced e for poly-L-arginine or ECPexcept for poly-L-gluta-mic acid 13,600 mol wt tested against ECP. Below acidic polyaa concentrations necessary for a balanced e, inhibition of tox-icity was greatly reduced for all acidic poly aa tested. Becausethese acidic poly aa inhibit poly-L-arginine and ECP toxicity,this mechanism is not specific to MBPand mayapply to numer-ous cationic toxins.

Inhibition of MBPtoxicity to tracheal ring epithelium byacidic poly aa. Acidic poly aa were used as antagonists of MBPtoxicity to guinea pig tracheal epithelium as a model for theirrole as an inhibitor of MBPtoxicity in bronchial asthma. Poly-glutamic acids inhibited MBPtoxicity to the tracheal epithe-lium (Fig. 3). Tracheal rings incubated with MBPalone exhib-ited gross morphologic damage at 24 h. The damage includedciliostasis and exfoliation of mucosal cells. Precipitates of MBPwere apparent on and around the cells. After 48 h of incubationonly the MBPalone-treated tracheal rings showed damage(Fig. 3, A and B). The tracheal rings treated with acidic polyglu-tamic acids or a combination of polyglutamic acids and MBPappeared normal with active cilia (Fig. 3, Cand D). Someof thecilia of the rings treated with a combination of polyglutamicacids and MBPappeared to have a small amount of precipitateon them which may be polyglutamic acid and/or MBP(Fig. 3D). There was no detectable damage to the cilia associated withthis precipitate.

Inhibition ofMBP-induced effects on the airway by polyglu-tamic acid. MBPinstillation resulted in an immediate increasein Rrs that peaked between 5 and 10 min postinstillation andresolved by 1 h (Fig. 4). Pretreatment with polyglutamic acidsignificantly inhibited the MBP-induced increase in Rrs. Poly-glutamic acid treatment by itself did not alter Rrs (data notshown).

The effects of MBPinstillation on airway responsiveness toinhaled methacholine during control and polyglutamic acidtreatment studies are shown in Fig. 5. MBPadministrationalone resulted in a dramatic increase in airway responsivenessas indicated by a decrease in calculated methacholine PC,10values. Polyglutamic acid pretreatment significantly inhibitedthe MBP-induced increase in airway responsiveness in eachanimal studied.

Effect of acidic poly aa on blood clotting. Although heparinis an effective antagonist of MBPtoxicity (1, 17), its possibleclinical use in bronchial asthma could be limited by its abilityto inhibit blood clotting. To determine if acidic poly aa inhibitblood clotting, whole blood was incubated with heparin, poly-glutamic acids, and/or MBP, and the clotting time was ob-served. These data are shown in Fig. 6. At a concentration of 1X 10-5 M, twice the highest acidic poly aa concentration usedin the K562 assays and comparable to that used in the trachealring assays, there was no significant difference between poly-glutamic acids alone or polyglutamic acids + MBPwhen com-pared to controls in their ability to inhibit clotting. However,poly-L-glutamic acid 77,800 mol wt was significantly differentfrom controls at 1 X 10-0 M. Heparin was significantly differ-ent from controls at all test concentrations and increased clot-ting time at least to 45 times that of poly-L-glutamic acid77,800 molwtat l x 10-4M.

Discussion

The mechanism by which MBPkills cells is unknown. It hasbeen suggested that the cationic charge of MBPmay attract itto the anionic charged cell surface of targets, whereupon theapolar residues insert into and perturb the lipid milieu (2).MBPlikely damages cell membranes (3, 4) which increases

802 Barker, Gundel, Gleich, Checkel, Loegering, Pease, and Hamann

Figure 3. Effect of acidic poly aa on MBPtoxicity to guinea pig tracheal rings by histologic examination. Photomicrographs of fixed tissue,stained with hematoxylin-eosin, and examined by light microscopy. (A) Treatment with MBPalone. Arrows indicate exfoliation and destructionof ciliated epithelial cells. (B) Treatment with MBPalone. Straight arrow indicates exfoliated cells. Note MBPprecipitate on and around exfo-liated cells (curved arrow). (C) Treatment with poly-L-glutamic acid mol wt 77,800 alone. Ciliated epithelium (arrow) appears intact. (D) Treat-ment with poly-L-glutamic acid mol wt 77,800 and MBP. Ciliated epithelium appears intact. Note particulate matter (arrow) on some ciliawhich may be precipitates of polyglutamic acid and/or MBP. (A) X225; (B-D) X560.

their permeability without concurrent formation of transmem-brane channels (3).

In this study MBPtoxicity to K562 cells and guinea pigtracheal epithelium was inhibited by acidic poly aa. This inhibi-tion was directly related to the ability of negatively chargedacidic poly aa to balance and thus neutralize the positivelycharged MBP. The ratio of the absolute values of the predictede for the acidic pro-part of proMBP to the predicted e of cat-ionic MBPis 1.2. Inhibition of MBPtoxicity by acidic poly aaat concentrations above that necessary to balance charge (ratioof 1.0) but not significantly at concentrations below that neces-sary to balance charge (Table II; Fig. 1) suggests that the acidic

pro-portion of proMBP functions to mask MBPtoxicity. Thus,it is likely that proMBP is a nontoxic molecule that protects theeosinophil from damage while the protein is processed throughthe endoplasmic reticulum to its sequestered site in the eosino-phil granule as toxic MBP.

These data also show that the acidic poly aa must be addedbefore or immediately after addition of 5 x 10-6 MMBPtoinhibit its toxicity (Fig. 2). Presumably, when MBPis addedbefore the acidic polymers and allowed to incubate with thetarget for a short period of time, MBPwill bind to the targetmembranes and will not be effectively inhibited by the acidicpolymers after that time; addition of acidic poly aa before MBP

Acidic Amino Acids Inhibit Major Basic Protein Toxicity 803

At-

Figure 4. Changes inRrs after MBPinstilla-tion alone or MBPin-stillation 10 min afterpolyglutamic acid inha-lation treatment. Poly-glutamic acid treatmentsignificantly inhibited

MBP-induced increases in Rrs. Circles represent MBPalone instilla-tion. Squares indicate polyglutamic acid inhalation followed in 10min by MBPinstillation. Values are mean+SEM, n = 5. *P < 0.05.

(0

E

E

>900

200

100

20

0~ ~~~~~~~~~..1

Con'rols 1 x 1'C6M

Controls

Saline

Acetate buffer

Poly-L-gluLamic acid 113 60C)Poly-L-glutamic acid (77.800(Haparpn

MBP

Poty-L-glutamic acia 03 600)+ MBP

Poly-L-glutamic acid (77.800)MBPMOM

M l x104M

may allow them to bind to MBPpreventing it from binding tothe target membranes.

This has implications in the processing of proMBP. Thepresumed enzyme that cleaves proMBP is unknown. It is un-likely that this enzyme cleaves proMBP only at one point intopro-part and toxic MBPfor several reasons. First, if proMBPwere cleaved at only one point, it appears that at neutral ornearly neutral pH, the intact pro-part would still continue to beassociated with mature MBPas are the acidic poly aa in thisstudy and MBPwould still be nontoxic while bound to thepro-part. Second, repeated attempts by Ponz et al. to identify asmall acidic protein with the characteristics expected of thepro-part of BTAH, a cationic toxin similar to MBP, using gelfiltration, preparative electrophoresis at alkaline pH, andHPLC were unsuccessful (18). Third, the acidic pro-part ofpromellitin, a cationic toxin from bee venom that damages cellmembranes, is cleaved every second residue by a dipeptidylpeptidase (19). These data are in keeping with the hypothesisthat the acidic pro-part of proMBP is cleaved in a stepwisefashion that results in toxic MBPand a number of smallerpeptides which do not bind sufficiently to MBP, if at all, toinhibit its toxicity.

Polyarginine and ECPare potent toxins and although theymay kill targets differently from MBP(3, 20), polyarginine andECPshare with MBPthe property of a highly cationic nature.Acidic poly aa inhibition of polyarginine and ECPtoxicity toK562 cells (Table III) may reflect the binding of polyarginine

Concentration

Figure 6. Effects of acidic poly aa on blood coagulation. Values aremean coagulation time± 1 SDof one to two experiments in triplicate.*P < 0.001 for values tested against saline control using Student's ttest.

and ECPto the acidic poly aa, precluding the binding of thesetoxins to target cells. Because this inhibition is not restricted toMBPbut also applies to ECPand polyarginine, treatment oftarget cells with acidic poly aa may perturb the toxic activitiesof numerous cationic toxins including those from eosinophils.

The ability of acidic poly aa (which were nontoxic and didnot inhibit coagulation [Fig. 6]) to inhibit MBPtoxicity to tra-cheal ring epithelium (Fig. 3) and inhibit MBPairway hyper-responsiveness (Figs. 4 and 5) suggests a possible role for theacidic poly aa as therapy for many of the hypersensitivity dis-eases associated with eosinophil degranulation, especially bron-chial asthma. Presently, it is not known whether these acidicpoly aa would inhibit MBPand other cationic eosinophil de-granulation products in vivo. Heparin, which has been shownto inhibit MBPtoxicity (1, 17), does inhibit whole eosinophiltoxicity to Trypanosoma cruzi trypomastigotes (21). Studiesare underway to test the effectiveness of acidic poly aa to in-hibit airway hyperresponsiveness associated with antigen in-duced asthma in the primate model.

Acknowledgments

Figure 5. Changes inmethacholine PCOOval-ues 2 h post-MBP instil-

C) lation during controlstudies (MBP alone;EL

05 shaded circles) and0 polyglutamic acid pre-

treatment studies10 -,G(empty circles) for each

cm of five animals. Bars1>15 represent mean±SEM,

in n = 5, for each treat-ment group. Polygluta-

2.0 mic acid treatment ex-periments were brack-eted by control (MBP

-2.5

MBP MBP MBP alone) treatment studies+ to ensure no change inPolyglutamic acid MBP-induced effects

over time. Animals were rested for a minimum of 3 wk betweentreatments. MBP-induced increases in airway responsiveness weresignificantly inhibited in each animal.

Wethank Linda H. Arneson for preparation of the manuscript.This work was supported in part by grants from the National Insti-

tutes of Health, AI-07047, AI-15231 and AI-09728, and by the MayoFoundation.

References

1. Gleich, G. J., and C. R. Adolphson. 1986. The eosinophilic leukocyte:structure and function. Adv. Immunol. 39:177-253.

2. Wasmoen, T. L., M. P. Bell, D. A. Loegering, G. J. Gleich, F. G. Prender-gast, and D. J. McKean. 1988. Biochemical and amino acid sequence analysis ofhuman eosinophil granule major basic protein. J. Biol. Chem. 263:12559-12563.

3. Young, J. D.-E., C. G. B. Peterson, P. Venge, and Z. A. Cohn. 1986.Mechanism of membrane damage mediated by human eosinophil cationic pro-tein. Nature (Lond.). 321:613-616.

4. Kroegel, C., U. Costabel, and H. Matthys. 1987. Mechanism of membranedamage mediated by eosinophil major basic protein. Lancet. i: 1380-1381.

5. Gundel, R. H., L. G. Letts, and G. J. Gleich. 1991. Human eosinophilmajor basic protein induces airway constriction and airway hyperresponsivenessin primates. J. Clin. Invest. 87:1470-1473.

6. Barker, R. L., G. J. Gleich, and L. R. Pease. 1988. Acidic precursorrevealedin human eosinophil granule major basic protein cDNA. J. Exp. Med. 168:1493-1498.

804 Barker, Gundel, Gleich, Checkel, Loegering, Pease, and Hamann

7. Barker, R. L., D. A. Loegering, R. M. Ten, K. J. Hamann, L. R. Pease, andG. J. Gleich. 1989. Eosinophil cationic protein cDNA: comparison with othertoxic cationic proteins and ribonucleases. J. Immunol. 143:952-955.

8. Pineda, A. A., S. M. Brzica, Jr., and H. F. Taswell. 1977. Continuous- andsemicontinuous flow blood centrifugation systems: therapeutic applications, withplasma-, platelet-, lympha-, and eosinoapheresis. Transfusion (Phila.). 17:407-416.

9. Gleich, G. J., A. A. Pineda, G. 0. Solley, and H. F. Taswell. 1981. Cytapher-esis for eosinophilia. In Proceedings of the Workshop on Therapeutic Plasma-pheresis and Cytapheresis. G. J. Nemoand H. Taswell, editors. U. S. Departmentof Health and HumanServices, NIH Publication No. 82-1665. 73-79.

10. Gleich, G. J., D. A. Loegering, K. G. Mann, and J. E. Maldonado. 1976.Comparative properties of the Charcot-Leyden crystal protein and the majorbasic protein from human eosinophils. J. Clin. Invest. 57:633-40.

11. Ackerman, S. J., D. A. Loegering, P. Venge, I. Olsson, J. B. Harley, A. S.Fauci, and G. J. Gleich. 1983. Distinctive cationic proteins of the human eosino-phil granule: major basic protein, eosinophil cationic protein, and eosinophil-der-ived neurotoxin. J. Immunol. 131:2977-2982.

12. Nakajima, H., D. A. Loegering, and G. J. Gleich. 1988. Cytotoxicity ofeosinophil granule proteins for tumor cells. FASEB(Fed. Am. Soc. Exp. Biol.). J.2:A81 1. (Abstr.)

13. Motojima, S., E. Frigas, D. A. Loegering, and G. J. Gleich. 1989. Toxicityof eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am.Rev. Respir. Dis. 139:801-805.

14. Lane, B. P., S. L. Miller, and E. J. Drummond. 1976. Use oftracheal organcultures in toxicity testing. Environ. Health Perspect. 16:89-98.

15. Wegner, C. D., A. C. Jackson, J. D. Berry, and J. R. Gillespie. 1984.Dynamic respiratory mechanics in monkeys measured by forced oscillations.Respir. Physiol. 55:47-61.

16. Gundel, R. H., M. E. Gerritsen, G. J. Gleich, and C. D. Wegner. 1990.Repeated antigen inhalation results in a prolonged airway eosinophilia and air-way hyperresponsiveness in primates. J. Appl. Physiol. 68:779-786.

17. Butterworth, A. E., D. L. Wassom, G. J. Gleich, D. A. Loegering, and J. R.David. 1979. Damage to schistosomula of Schistosoma mansoni induced directlyby eosinophil major basic protein. J. Immunol. 122:221-229.

18. Ponz, F., J. Paz-Ares, C. Hernandez-Lucas, F. Garcia-Olmedo, and P.Carbonero. 1986. Cloning and nucleotide sequence of a cDNA encoding theprecursor of the barley toxin a-hordothionin. Eur. J. Biochem. 156:131-135.

19. Kreil, G., L. Haiml, and G. Suchanek. 1980. Stepwise cleavage of the propart of promelittin by dipeptidylpeptidase IV. Eur. J. Biochem. 11 1:49-58.

20. Kierszenbaum, F., S. J. Ackerman, and G. J. Gleich. 1981. Destruction ofbloodstream forms of Trypanosoma cruzi by eosinophil granule major basic pro-tein. Am. J. Trop. Med. Hyg. 30:775-779.

21. Kierszenbaum, F., S. J. Ackerman, and G. J. Gleich. 1982. Inhibition ofantibody-dependent eosinophil-mediated cytotoxicity by heparin. J. Immunol.128:515-5 17.

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