Adenosine deaminase is not required for the generation of superoxide anion

CLINICAL

IMMUNOLOGY

Adenosine

AND

IMMUNOPATHOLOGY

30, 495-499 (19841

Deaminase Is Not Required for the Generation of Superoxide Anion’

BRUCE N. CRONSTEIN, SARA B. KRAMER, GERALD WEISSMANN, AND

ROCHELLE HIRSCHHORN Department

qf Medicine.

Division

of Rheumatology, New York, New

New York York 10016

University

School

of Medicine

Neutrophils and macrophages generate superoxide anion during the respiratory burst in response to various stimuli, including microorganisms. It has recently been proposed that an important source of superoxide anion during the respiratory burst that stimulates murine macrophages is the sequential metabolism of adenosine via adenosine deaminase and xanthine oxidase to uric acid. Thus, the immunodeficiency state associated with adenosine deaminase deficiency may be caused at least in part by a defect in superoxide anion generation. The ability to generate superoxide anion of stimulated neutrophils isolated from three children with adenosine deaminase deficiency and associated severe combined immunodeficiency was tested. Neutrophils from all three patients were able to generate superoxide anion. One of these generated 19.1 nmol cytochrome c reduced/ 10” cells (normals = 5.3-33.0, mean 18.4 * 7.1) while the other two generated low normal levels. Neutrophils from all three children also generated more superoxide anion after addition of exogenous adenosine deaminase. Thus, no evidence to support a role for cellular adenosine deaminase in the release of superoxide anion by stimulated neutrophils was found. Although neutrophils from patients deficient in adenosine deaminase appear to have no inherent defect in the generation of superoxide anion, the abnormally high concentrations of adenosine found in the plasma of these patients could, in Go, secondarily, inhibit superoxide anion release.

INTRODUCTION

Tritsch and Niswander have recently proposed that an important source of superoxide anion during the respiratory burst of stimulated murine macrophages is the sequential metabolism of adenosine to inosine and hypoxanthine then to uric acid by xanthine oxidase (l-3). This hypothesis rests upon their demonstration that superoxide anion release by murine macrophages treated with a variety of stimuli is proportional to total cellular adenosine deaminase activity. Adenosine deaminase, the first enzyme in the degradative pathway of adenosine to uric acid, catalyzes the irreversible metabolism of adenosine to inosine. Inosine is then further metabolized to hypoxanthine and xanthine which are necessary, according to this hypothesis, for the generation of superoxide anion (l-3). These investigators have further hypothesized that the immunodeficiency state associated with i This work was supported by grants from the Kroc Foundation to Dr. Hirschhorn and Dr. Weissmann; from the New York Arthritis Foundation to Dr. Cronstein; from the National Institutes of Health to Dr. Hirschhom (Grant AI 10343) and to Dr. Weissmann (Grants AM 11949, HL 19721, and AI 17365); and from the March of Dimes Birth Defects Foundation (Grant 6). 495 0090-1229/84 $1.50 Copyright 8 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

496

HKIHI. COMMCINI(~‘A’I IOhS

inherited deficiency of adenosine deaminase is caused, at least in part, by a defccl in the generation of superoxide anion ( I-3). In this communication we report that neutrophils from three children suffering from inherited deficiency of adenosine deaminase with associated severe combined immunodeficiency are capable of generating superoxide anion if1 ~sifro ;ii levels within the normal range. MATERIALS

AND METHODS

Materials. Cytochalasin B was purchased from Aldrich Chemical Company (Milwaukee, Wise.). Cytochrome c (Type III). adenosine, N-formylmethionylleucyl-phenylalanine (FMLP),’ and calf intestinal adenosine deaminase (Type 1 in ammonium sulfate) were obtained from Sigma Chemical Company (St. Louis, MO.). Superoxide dismutase was obtained from Miles Laboratory (Elkhart. Ind. 1. [8-14C]Adenosine (sp act 52 mCi/mmol) was obtained from ICN Pharmaceuticals Company (Irvine, Calif.). Patients. Patients D.D. and L.D. are siblings known to be deficient for adenosine deaminase activity with associated immunodeficiency (4). They have been treated with regular and frequent partial exchange transfusions of irradiated packed red blood cells (5). At the time of study these patients were 7 and 5 years old, respectively. Patient C.R. is a classic severe combined immunodeficient with adenosine deaminase deficiency (6). At the time of study the patient was 6 weeks old and had been treated with partial exchange transfusions of irradiated packed red blood cells. Prepurution of cell suspensions. Heparinized blood was obtained from patients or normal volunteers. Purified preparations of neutrophils were isolated by means of Hypaque-Ficoll gradients (7) followed by dextrdn sedimentation and hypotonic lysis of erythrocytes (8). This procedure allowed studies of cell suspensions containing 98 ? 2% neutrophils with few contaminating erythrocytes or platelets. The cells were suspended in a buffered salt solution consisting of Na+ ( 150 mM), K+ (5 mM), Ca?’ (1.3 mM), Mg’+ (1.2 mM), Cl- (155 mm, and Hepes (10 mM), pH 7.45. Slrperoxide anion generutiorz. Superoxide anion generation was monitored by determination of superoxide dismutase-inhibitable reduction of cytochrome c in the presence or absence of cytochalasin B. Duplicate reaction mixtures containing 2 x lo6 neutrophils, 75 nmol horse heart ferricytochrome c (Type III), and buffer or either adenosine or adenosine deaminase, at designated concentrations in a final volume of 1 ml, were incubated for 5 min at 37°C. Cytochalasin B (5 pgiml) was added and cells were incubated at 37°C for 5 additional min before exposure to stimuli (e.g., FMLP). Five minutes following stimulation, cells were spun down at 4°C at 1OOOgin a Sorvall RC-3 centrifuge and the supernatants were collected. Absorption at 550 nm was determined in a Beckman Mode1 25 spectrophotometer and the nanomoles of superoxide anion generated was calculated as previously 2 Abbreviations nine.

used: ADA. adenosine deaminase; FMLP. N-formylmethionyl-leucyl-phenylala-

497

BRIEF COMMUNICATIONS

described (9). Adenosine deaminase was dialyzed against phosphate-buffered saline for at least 3 hr before use in assays. Adenosinr deaminase activity. The conversion of [8-t4C]adenosine to [S-t4C]inosine and [8-14C]hypoxanthine by lysates of neutrophils was measured by a modification of the method of Coleman and Hutton (IO) and is described in detail elsewhere (11). RESULTS

Upon stimulation with FMLP (0.1 pJ4), in the presence of the fungal metabolite cytochalasin B, neutrophils from normal adult donors generate superoxide anion (18.4 2 7.1 (SD) nmol cytochrome c reduced/IO6 neutrophils, n = 12). We have found a wide variation in superoxide anion generation by normals with a range of 5.3 to 33.0 nmol of cytochrome c reduced/IO6 cells. Neutrophils from the three patients all generate superoxide anion (Table 1). One child generates normal levels of superoxide anion (D.D.), while his sibling (L.D.) and the third child (C.R.) generate low normal levels of superoxide anion. Adenosine is markedly inhibitory for the release of superoxide anion by normal neutrophils stimulated with FMLP (12) and this is also the case for neutrophils from one of the two children tested. We have previously shown that exogenous adenosine deaminase dramatically increases the generation of superoxide anion by stimulated neutrophils (12). Exogenous adenosine deaminase metabolizes the adenosine present in the extra cellular milieu of neutrophil suspensions. The extracellular adenosine present in these suspensions causes baseline inhibition of superoxide anion generation; thus elimination of this adenosine enhances superoxide anion generation. Exogenous adenosine deaminase also enhances superoxide anion release by neutrophils from the deficient children. The enhancement of superoxide release by cells from deficient children is very similar in magnitude to that found for normal neutrophils. Neutrophils from patient D.D., who has the highest superoxide anion release, show only modest enhancement of superoxide anion generation by exogenous adenosine deaminase whereas inhibition by adenosine is much more striking. The converse is true for patient L.D. Thus, there appears to be an inverse ratio between the enhancement by exogenous adenosine deaminase and the inhibition TABLE Patient __D.D. L.D. C.R. Control t t-SD)

I

Buffer

ADA

19.1 5.2 8.3 18.4 r 7.1 tn = 12)

22.5 12.3 15.4 27.0 k 9.4 (n = 12)

Adenosine 6.5 4.6 ND 10.3 2 5.2 (n = 7)

Note. Neutrophils (2 x 10Vml) were incubated with buffer alone, ADA (0.125 mu). or adenosine (100 p.M) for 5 min at 37°C before stimulation with FMLP (0.1 pM) in the presence of cytochalasin B (50 ug/ml). Control values are derived from data previously published t 12). Superoxide dismutase inhibitable reduction of cytochrome c was measured and is expressed as nanomoles of cytochrome c reduced/IO6 neutrophils.

\I)A 1nniol Patient

~.onv~iteJifO”

D.D.

elsewhere

(n

= 3) _____--

Lysates

of neutrophils

activity

of adenosine

(IO.

I

0. I7

C.K. Control Note.

crll\ihl

Il.Oh Il.OX

L.D.

Specific

activit! adenos,nr

3i.xo (20

x

deaminase

106/ml)

were

in these

incubated lysates

with was

2 3.20 [S’T]adenosine

measured

and

for calculated

Z hr at 37°C‘. as described

I I).

by exogenous adenosine of the neutrophils of these children. although further studies must be done to clarify this relationship. We demonstrate here that adenosine deaminase activity is indeed deficient in neutrophils from these children (Table 2). Contamination of neutrophil preparations by the normal red blood cells with which these children have been transfused is sufficient to account for the low adenosine deaminase activity remaining in these preparations. DISCUSSION Our data demonstrate that adenosine deaminase activity is not necessary for release of superoxide anion by human neutrophils. The ability of neutrophils from adenosine deaminase deficient children to release superoxide anion in response to FMLP is clear evidence against this hypothesis. Data from a previous study also strongly mitigate against a role for tluxes through the purine salvage pathway in the generation of superoxide anion by human neutrophils. Deoxycoformycin, an irreversible inhibitor of adenosine deaminase, has no effect on the release of superoxide anion by FMLP-stimulated neutrophils. Additionally, exogenous adenosine. the initial substrate for the purine salvage pathway, inhibits superoxide anion generation; this is directly contrary to the expected effect if metabolism of adenosine is crucial for superoxide anion generation. In addition, neither inosine, the product of adenosine deamination, nor hypoxanthine, the substrate for xanthine oxidase, has any effect upon the release of superoxide anion by stimulated neutrophils (12). The enhancement of superoxide anion release found in the presence of exogenously added adenosine deaminase is due to the elimination of endogenously generated adenosine (12). The discordance between our data and those of Tritsch and Niswander may arise from the different cell types used. In the studies mentioned above murine macrophages from peritoneai exudates were used whereas we have examined both normal and deficient human neutrophils. Thus, we find no evidence to support a role for cellular adenosine deaminase in the release of superoxide anion by stimulated neutrophils. Although we can not demonstrate a primary defect of superoxide anion generation in deficient patients, the abnormally high concentrations of adenosine found in the plasma of

499

BRlEF COMMUNICATIONS

these patients (13) could, secondarily, functionally intact neutrophils.

inhibit

superoxide

anion release by these

ACKNOWLEDGMENTS We are indebted to Dr. Arye Rubinstein, Department of Pediatrics. Albert Einstein College of Medicine. for providing samples from patients L.D. and D.D. We also appreciate the assistance of Ms. Vivien Roegner-Maniscalco who performed the adenosine deaminase assays.

REFERENCES I. 2. 3. 4.

Tritsch. G. L.. and Niswander, P. W., Biochem. Med. 26. 185. 1981. Tiitsch. G. L.. and Niswander, P. W., Immunol. Commun. 10, I. 1981. Tritsch, G. L., and Niswander, P. W.. Mol. Cell. Biockm. 49, 49. 1982. Rubinstein, A., Hirschhorn, R.. Sicklick. M.. and Murphy, R. A., N. Eng/. J. Med. 300, 387. 1979. 5. Polmar, S. H., Stern, R. C., Schwartz, A. L.. Wetzler. E. M., Chase. P. A.. and Hirschhorn. R.. N. EngI.

.I. Med.

295,

1337,

1976.

6. Hirschhorn, R.. Borkowsky, W.. and Ratech, H.. submitted for publication. 7. Boyum, A.. Stand. J. C/in. Lub. Inwsr. 21, 77. 1968. 8. Zurier. R. B.. Hoffstein, S., and Weissmann, G.. I. Cell Biol. 58, 27. 1973. 9. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissmann, G.. J. Clir~. In~euf. 56, 1155, 1975. 10. Coleman, M. S., and Hutton, J. J.. Biochen~. Med. 13, 46. 1975. I I. Hirschhorn, R.. Roegner. V.. Jenkins. T., Seaman, C.. Piomelli. S.. and Borkowsky, W., J. C/jr]. Inl’esl. 64, 1130. 1979. I?. Cronstein. B. N.. Kramer, S. B.. Weissmann, G.. and Hirschhorn. R.. J. Exp. Med. 158, 1160. 19x3.

13. Hirschhorn.

R.. Roegner-Maniscalco,

V., Kuritnky. L., and Rosen. F. S.. J. C/in.

13X7. 1981.

Received September 20, 1983; accepted October 19. 1983.

Invest.

68,