Dopamine inhibition of superoxide anion production by polymorphonuclear leukocytes

pamine inhibition of superoxide reduction by polymorphonuclear

anio leuko

nehiro Yamazaki, MD, Takafumi Matsuoka, MD, Kozo Yasui, sushi Komiyama, MD, and Taro Akabane, MD Matsumoto, Japan To examine the modulatory effects of catecholamines on the respiratory burst in polymorphonuclear leukocytes (PMNs), dopamine was tested for its capacity to modify the superoxide anion (0,-J production by PMNs under their stimulation with several stimuli. Wopamine inhibited the O,- production by PMNs when PMNs were stimulated with N-formylated chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate, or opsonized zymosan,whereas dopamine did not alter the PMN mobility. The values of percentage inhibition of the O,- production by FMLP-stimulated PMNs were 57% under treatment with lo-’ mollL and 83% with, IO-” mollL of dopamine. Isoproterenol also inhibited PMN Oz- production in response to FMLP. Although a /3-adrenergic blockade, propranolol, diminished the isoproterenol-induced inhibition of the O,- production, it did not affect the inhibitory effect of dopamine. The increase in intracellular cyclic AMP levels in dopamine-treated PMNs was much smaller than the increase in isoproterenol-treated cells. Furthermore, dopamine inhibited the reduced nicotinamide-adenine dinucleotide phosphate-dependent O,- production by subcellular particles. These results indicate that dopamine inhibits PMN O,- production through its effect on PMN reduced nicotinamide-adenine dinucleotide phosphate-oxidase system rather than through its P-adrenergic action. (f ALLERGY CLIN IMMVNOL

1989;83:967-72 .)

Several leukocyte responsescan be regulated by agents that increase the cellular levels of CAMP.‘*2 Beta-adrenergic receptors are probably part of the adenylatecyclase systemand have been demonstrated in PMNs.~,4 Catecholamines stimulate membranebound adenylate cyclase to generate intracellular cAMP, which leads to inhibition of PMN functions, such as adhesiveness,chemotaxis, phagocytosis, and lysosomal enzyme release,‘.’ but it has not been established how they influence PMN respiratory burst. In this regard, Marone et al.” reported that catecholamines, such as isoproterenol, did not produce a consistent increasein the CAMP content of PMNs. Busse and Sosman’ studied the effect of isoproterenol on PMN O,- production and chemiluminescence reFrom the Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan. Supported in part by Grants-in-aid Nos. 58770656and 61570449 for Scientific Researchfrom the Ministry of Culture and Science in Japan. Received far publication April 20, 1988. Revised Oct. 21, 1988. Accepted for publication Nov. 8, 1988. Reprint requests:Munehiro Yamazaki, MD, Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032.

Abbreviations used O,- : Superoxide anion FMLP: N-formyl-methionyl-leucylphenylalanine PMA: Phorbol my&ate acetate OZ: Opsonized zymosan PMN: Polymorphonuclear leukocyte DPBS: Dulbecco’s phosphate-buffered saline SOD: Superoxide dismutase CAMP: Cyclic adenosine mo~oph~sphate LDH: Lactic dehydrogenase NADPH: Reduced nicotinamide-adenine dinmleotide phosphate PKC: Protein kinase C

sponseunder the OZ stimulation and suggestedthat certain PMN responses,as expressedby chemiluminescence, were not under the control cd cyclic nucleotides. These studies prompted the authors to examine whether and how catechol~~~es can inhibit PMN respiratory burst, such as O,- production. In the present study, we have examined the effect of dopamine, one of the most widely used catechol-

Yarrwzaki

et al.

J. ALLERGY

!. Effect of dopamine

opamine

Control lo-’ mol/L 10e6 m&L lo-’ mol/L 10el mol/L

-t t * 2 +

1.7 2.5 2.1 1.0” 0.8*

and isoproterenol on the O,- production FMLP-stimulated PMNs

by PMNs with

FMLP (IO-’ mol/L)

13.9 13.8 11.7 5.7 2.1

TABLE Ii. Effect of dopamine

on PMN

O,- production stimulated

O,- production incubated

oz (1 mg/ml)

32.6 32.1 29.3 13.5 6.0

r t ” z -t-

Drug

2.6 2.7 2.6 1.1” 0.6”

Control lo-’ mol/L 10e6 mol/L 10m5mol/L 1O-4 mol/L

PMNs were preincubatedwith indicated concentrationof dopamine for 10 minutes at 37” C before the addition of FMLP or OZ. Data are representedmean i SE (n = 5) of nanomolesof O,per IO6PMNs per 6 minutes for FMLP and 20 minutes for OZ. *Significantly different from control (p < 0.05).

amines, different

on the PMN O,-

production

induced

by

PMN stimuli, including a chemotactic peptide, FML,P, PMA, and OZ. We then compared the effect of dopamine on the O,- production by FMLPstimulated PMNs with that of isoproterenol, the most potent catecholamine inducing the P-adrenergic receptor-mediated response of PMNs.~ eparatiton

of PMNs

Human peripheral PMNs were isolated from healthy adult donors by dextran sedimentation and the standard Ficoll-Hypaque density-gradient centrifugation, and the cells were washed and suspended in DPBS, as previously described.“-I3 The cells with purity of >95% PMNs and viability of >98%, as determined by trypan blue dye exclusion, were used for the present experiments. Release of cytoplasmic LDH was used as an indicator of cell damage ‘ 14,I5 Rupture of cells by 0.2% Triton X-100 led to the release of total LDH.

Dopamine HCl, 1-isoproterenol HCI, theophylline, ferricytochrome c (type III), FMLP, PMA, zymosan A, SUD, xanthine, and xanthine oxidase were purchased from Sigma Chemical Co. (St. Louis, MO.), NADPH, from Boehringer Mannheim Yamanouchi (Tokyo, Japan), and propranolol HCl from Nakarai Chemicals (Kyoto, Japan). FMLP and PMA were dissolved in dimethyl sulfoxide and further diluted in DPBS before use. OZ was prepared by incubating zymosan A with fresh human serum, as previously described.”

by PMNs

O,- production was measured by determination of SOD-inhibitable reduction of ferricytochrome c. I6 Reaction mixtures, containing lo6 PMNs and 100 nmol of ferricy-

by P with

Dopamine

13.9 13.8 11.7 5.7 2.1

t r + i. +-

1.7 2.5 2.1 1.0 0.8

by

-

~sopr~tera~~~

(3) (17) (57)* (83)”

13.7 15.1 12.1 10.5 7.6

+ ? I 2 lr

1.1 0.6 1.2 0.8 0.8

(-3) (12) (23)* (44)*

PMNs (1 x IO6cells) were preincubated with the indicated concentrationof eachdrug for 10 minutesat 37” C before the addition of lo-’ mol/L of FMLP. Data are represented mean I SE (n = 5) of nanomolesof 02- per 106PMNsper 6 minutes.Vaiues in parenthesesindicate percentageinhibition. *Significantly different from control (p < 0.05).

tochrome c in a final volume of 1 ml, were incubated for 10 minutes at 37” C, and then the reaction was started by adding a stimulus. The final concentrations of stimuli were 10m7mol/L of FMLP, 1 mg/ml of OZ, and 1 ngiml or 1 kg/ml of PMA. SOD (25 p,g/ml) was added in a reference tube. After incubation for 6 minutes with FML,P, 20 minutes with OZ, and 10 minutes with PMA, cells were spun down at 4’ C, and the supematants were collected. Absorption at 550 nm was determined, as previously described.‘3 Tbe molar excitation coefficient for this change in absorption is 21,000/cm/m01/L.” To determine the effects of dopamine or isoproterenol, PMNs were incubated with dopamine or isoproterenol for 10 minutes. After the incubation with each drug, cells were stimulated with a PMN stimulus. To examine the effect of propranolol, PMNs were incubated wit!1 propranolol for 10 minutes before addition of the drug. O,- production in a simple in vitro generating system was examined by use of a xanthine-xanthine oxidase system.”

Measurement

rugs and reagents

production

CLIN. IMMUNOL. MAY 1989

of intracellular

c

PMNs, 2 x 106, in 0.1 ml of DPBS, containing 10-j mol/L of theophylline, were preincubated for 10 minutes and then incubated with the individual drug for an additional 10 minutes. After the incubation, the reaction was terminated by boiling the cell suspension for 5 minutes. The samples were acetylated, and CAMP was measured by radioimmunoassay. I9

Chemotaxis

assay

PMN mobility was assayed by the agarose-plate method, as previously described. 2o,21Cells were allowed to migrate toward the stimulus of lo-’ mol/L of FMLP for 3 hours in a CO, incubator at 37” C. The linear distance of migration toward FMLP (chemotaxis) and toward control medium (random mobility) was determined. The PMN chemotaxis

VOLUME NUMBER

Dopamine

33 5

111.Effect

-r

of dopamine

~so~ro~erencl~

on the O,-

P&l&stimulated

PMi’ds

and superoxide

pro~~ct~o~

and

production

by

PMA ation tions

Buffer Dopamine (10-S) Buffer Isoproterenol

1 nglml

1 pglml

11.6 t 1.0 (7)

58.1 ? 3.1 (5)

6.6 + 0.5 (7)* 12.0 k 1.3 (6)

8.1 ? 1.3 (6)*

25.3 t

1.2 (5)”

60.2 4 1.9 (5) 49.3 2 2.7 (5)”

(10-X) PM& were preincubatedwith 10e5mol/L of dopamine or isoproterenol for 10 minutes at 37” C before the addition of PMA. The number in parenthesesrepresentthe number of experiments and the values represent mean i- SE (nanomoles per O,- per lo6 PMNs per 10 minutes). *Significantly different from control (p < 0.05).

assay was also done by the filter method with the use of a 3 p,rn pore polycarbonate filter and lo-’ mol/L of FMLP as a chemoattractant. After the 30-minute incubation, total numbers of the cells that had migrated through the filter to its lower surface were counted in five randomly selected

fields.” H-dependent

O,- production

To obtain subcellular particles from stimulated PMNs,‘~

PMNs (2 x 10’ cells) were incubated for 5 minutes at 37” C with 1 pg/ ml of PMA in 2 ml of DPBS. The reaction was terminated by addition of an equal volume of cold DPBS. The cells recovered by centrifugation at 400 g for 10 minutes at 4” C were suspended in 2 ml of 0.34 mol/L of sucrosethat was buffered with 10 mmol/L of Tris HCl (pH 7.1; 0.34 mol/L of sucrose-Tris), and homogenized with a nonstick-coated pestle for 5 minutes at 4” C. The

supernatant was harvested for subcellular particles and cen-

trifuged at 27,000 g for 20 minutes. The pellet was suspended in 0.34 mol/L of sucrose-Tris. The protein concentration was determined according to the method of Lowry

et aLz3The NADPH-dependent O,- production was measured continuously by the reduction of cytochrome c, as described by Babior and Cohen.16 Briefly, 0.9 ml of 0.1 mol/L of potassium phosphate buffer (pH 7.4) containing 100 ~mol/L of cytochrome c, 200 to 300 pg protein of subcellular particles, and with or without dopamine (final concentration of lo-’ mol/L) was put in a cuvette and kept at 30” C. The reference cuvette contained 25 pg/ml of

SOD. After 5 minutes, 0.1 ml of 4 mmol/L of NADPH was added, and the absorbance at 550 nm was continuously monitored.

S - production The effect of dopamine on the O,- production by PMNs is presented in Table I. Incubations of PMNs

L

I

1

PRO DOP

IS0 DiP

I

PRO +

IS0

FIG. 1. Effect of propranoiol on dopamine and isoproterenol-induced inhibition of the O,- production by FMLP-stimulated PMNs. PMNs (1 x IO” cells) were incubated with propranolol (10e4 mol/L) for 10 minutes at 37” C before adding the catecholamine (IV mol/L). Percentage control of O,- production was determined by comparison with O,- production from u~treeted ceils.

with dopamine resulted in a dose-dependent

~~~~~itio~

of the O,- production in response to Dopamine also inhibited PMA-induced Cl,- production by PMNs in a dose-dependent manner; 0,’ production by nontreated and dopamine (IO-’ mol/L)treated PMNs stimulated with 1 rig/ml of PMA were 10.6 t 0.9 and 6.4 k 0.6, respectively. These inhibitory effects on the O,- production were recovered when the cells were washed four times in DPBS to remove the drug. The O,- production of the cells treated by lo-’ mol/L of dopamine was recovered to 107% of the activity of the control cells. There was no comparable release of the cyto~lasmic enzyme LDH (<6% of total cell LDH), excluding the cell damage, as a cause of the diminished O,production. Dopamine did not inhibit the reduction of cytochrome c in the xanthine and xanthineoxidase system, indicating that it does not scavenge O,-. For comparison, we examined the effect of a P-adrenergic agonist, isoproterenol, on the 02- production by FMLP-stimulated PMNs. As presented in Table II, although isoproterenol also inhibited the

Yamazaki et al.

cAMP concentrations (pm&2

.I. ALLERGY

x lo6 PMNs)

1.48 c 0.12

2.08 t 0.27

CLIN. IMMUMOL. MA\’ 1989

3.47 ” 0.28*

The values are mean I SE of five separate experiments. Cells were preincubated for 10 minutes with lo-’ mol/L of theophylline. Dopamine or isaproterenol was then added, and the cells were incubated for an additional 10 minutes. *Sig&icantly different from control (p < 0.001).

FMLP-induced O,- production by PMNs, the levels of suppression by dopamine were higher than levels by isoproterenol. The values of percentage inhibition with 10d5 mol/L agonists were 57% and 23%, respectively, and similarly the values with 10m4mol/L were 83% and 44%, respectively (p < 0.01, Mannitney IJ test). Similar results were obtained when the cells were stimulated with PMA (Table III). To determine whether the dopamine- or isoproterenol-induced inhibition of the OZ- production by PMNs could be affected by a /3-adrenergic blockade, we next examined the effect of propranolol on the inhibition of the FMLP-stimulated O,- production by dopamine or isoproterenol (Fig. 1). Propranolol by itself did not alter the O,- production by LP-stimulated PMNs. Propranolol diminished the inhibitory effect of the O,- production by isoproterenol. In contrast, propranolol did not significantly affect the inhibition of O,- production by dopamine, although some blocking effects by propranolol were observed in two of the six studies, suggesting that the dopamine-induced inhibition of PMN O,- production may occur independently of the P-adrenergic receptormediated response.

We further examined whether dopamine could increase intracellular CAMP levels, and the results are presented in Table IV. The lo-minute incubation with 10e5 mol/L of dopamine stimulated PMN CAMP generation. However, the increase in PMN CAMP levels by dopamine was much smaller than the increase by isoproterenol. When PMNs were incubated with 1O-4 mol/L of catecholamine, the increase by dopamine was also small, compared to the increase by isoproterenol. The CAMP concentrations were 2.02 t 0.2 in dopamine-treated cells, and 3.27 ? 0.37 in isoproterenol-treated cells (n = 4).

The effect of dopamine on the mobility of PMNs was studied with FMLP. PMNs were incubated with or without lo-’ mol/L of dopamine during chemotaxis assay. In the agarose method, dopamine did not alter the PMN mobility. Random mobility

values (times 0.3 mm) of PMNs treated with and without dopamine were 6.9 t 0.3 and 4.3 rt 0.3, respectively, and similarly, chemotaxis values were 11.5 t 0.5 and 11.8 -t 0.3, respectively (mean t SE; n = 5). When assayed by the filter method, random mobility and chemotactic responsiveness to FMLP were not affected by dopamine. Random mobility values were 90 & 6 in control cells and 88 i 8 in dopamine-treated cells. Chemotaxis values in control cells, low4 mol/L, 10m5 mol/L, and low6 mol/L of dopamine-treated cells, were 202 k 8, 219 -+ 8, 202 It 15, and 201 + 15, respectively (n = 4). NADPH-dependent

Q,- produc

To determine the effect of dopamine on the O,production by subcellular particles from PMA-stimulated cells, dopamine was added to assay cuvettes containing the subcellular particles before addition of NADPH. As illustrated by the tracing in Fig. 2, dopamine inhibited the O,- production by the subcellular particles. NADPH-dependent O,- production with and without low5 mol/L of do~ami~e was 0.47 i- 0.07 and 1.11 rt 0.11 nmol of 02per minute per milligram of protein (n = 4), respectively. DISCUSSION Our data demonstrate that dopamine can lead to the dose-dependent inhibition of the O,- production by PMNs in response to a soluble stimulus of FMLP or PMA and to a particulate stimulus of OZ. The disappearance of the dopamine-induced inhibition of the O,- production after washing indicates that the inhibitory effect of dopamine is reversible. Busse and Sosman’ reported that neutrophil c~emilumi~esce~ce and O,- production in response to OZ were suppressed by a catecholamine, isoproterenol. Our present study provides new information that another catecholamine, dopamine, also suppresses the O,-producing ability of PMNs in response to several different stimuli. It has been demonstrated that the inhibitor actions of the catecholamines, such as isoproterenol on P lysosomal enzyme release, are associated with their

VOLUME NUMBER

83 5

P-adrenergic action5. 6 Isoproterenol is the most potent stimulus of P-adrenergic receptor-mediated response of PMNs.~ In the present study, we compared the effect of dopamine on the O,- production by PMNs with effect of isoproterenol with FMLP as a MN-stimulus. Although both dopamine and isoproterenol inhibited PMN O,- production, levels of the inhibitory effect on the O,- production of dopamine were higher than levels of the effect of isoproterenol under every concentration of the agents studied. The observed inhibitory effects by the two techolamines did not correspond to the potency of -adrenergic receptor-mediated response of PMNs. If dopamine inhibited the O,- production through its padrenergic action, the inhibitory effect might be blocked by a P-adrenergic blockade, propranolol. We thus studied the effect of propranolol on the dopamineor isoproterenol-induced inhibition of the O,- production. Propranolol blocked the inhibitory effect of isoproterenol on the 02- production, which indicates that isoproterenol inhibits the O,- production through the P-adrenergic action. In contrast, propranolol did not block the inhibitory effect of dopamine on the O,production. These findings suggest that the dopamineinduced inhibition of PMN O,- production can occur independently of the p-adrenergic receptor-mediated response. The inverse relationship between CAMP content and PMN respiratory burst has been demonstrated.24 However, Marone et al.” found that catecholamines did not produce consistent increases in the CAMP content of PMNs. Busse and Sosman’ also suggested that certain PMN responses, as expressed by chemiluminescence, were not controled by cyclic nucleotides. We thus studied the effect of dopamine on the CAMP content of PMNs and compared with the effect of isoproterenol. Although dopamine stimulated PMN CAMP generation, the increase by dopamine was much smaller than increase by isoproterenol. The observed inhibitory effects on PMN O,- production by the two catecholamines therefore did not correspond to the increases in PMN CAMP levels in the present study. Based on these results, it is difficult to explain the mechanism of the inhibitory effect of dopamine on PMN O,- production by the increase in PMN cAMP content. It appears that, although the isoproterenol-induced inhibition of PMN O,- production occurs with an increase in the intracellular P level in PMNs, dopamine inhibits the O,- production through another mechanism different from an adenylate cyclase system. The failure of inhibition of PMN mobility by dopamine may be another evidence to support this notion. It has been demonstrated that the mechanism of activation of the respiratory burst is not always the

Dopamine

and superoxide

production

971

O.OI ODunits

NADPH FIG. 2. Effect of dopamine on NA~PH-dependent 0,- production by subcellular particles from P~A”stim~late~ PMNs. Subcellular particles were added to cuvettes in the presence and absence of 10e5 mol/L of dopamine and assayed for the Cl- production, es described in Methods.

same for each PMN stimu1us.‘4~2sIn the present study, we used three different PMN stimuli: chemotactic factor PMLP, PMA, and OZ. Binding of FMLP to its specific membrane receptors causes an intracellular calcium increase and activation of PKC, leading to the NADPH-oxidase activation, the enzyme responsible for the production of O,- , but PMA directly activates the kinase without a rise in ~~tracell~~~~calcium and stimulates the enzyme.X-27 contrast, OZ stimulation of the NADPH-oxidase d rs from PMA in that the particulate stimulus is phos AZ-mediated calcium dependent and in PKC .28-30Therefore, it is of great imp dopamine inhibits O,- production induc different stimuli. It may be therefore di plain the mechanism of the i~ibito~ pamine on PMN O,- generation by its effect on calcium mobilization or PKC. Indeed, dopami~e did not inhibit the calcium release from the intracellular storage sites of PMNs by the fluorescence change of chlortetracycline (data not presented). Since it has been reported that the same ox&se is activated by different transductional mechanisms,‘5, 31 we tested the effect of dopamine on the NADPH-dependent tion. Dopamine inhibited the NADPH-dependent 8,production by subcellular particles) indicating that dopamine modulates the PMN respiratory burst at a. site of the expression of NADPH-oxidase system. Finally, the present study suggests that catecholamines modulate PMN respiratory burst in more than one way.

72

Yamazaki

et al.

1. Boume FIR, Lichtenstein LM, Melmon KL, Henney CS, Weinstein Y, Shearer GM. Modulation of inflammation and immunity by cyclic AMP. Science 1974; 184: 19. 2. Melmon KL, Rocklin RS, Rosenkranz RP. Autacoids as modulators of the inflammatory and immune response. Am J Med 1981;71:100. 3. Galant SP, Allred SJ. Demonstration of beta,-adrenergic receptors of high coupling efficiency in human neutrophil sonmates. J Lab Clin Med 1980;96:15. 4. Davis PB, Dieckman L, Boat TF, Stem RC, Doershuk CF. Beta-adrenergic receptors in lymphocytes and granulocytes from patients with cystic fibrosis. J Clin Invest 1983;71:1787. 5. Ignarro LJ, Lint TF, George WJ. Hormonal control of lysosomal enzyme release from human neutrophils: effects of autonomic agents on enzyme release, phagocytosis, and cyclic nucleotide levels. J Exp Med 1974; 139: 1395. 6. Zurier RB, Weissmann G, Hoffstein S, Kammemran S, Tai H-H. Mechanisms of lysosomal enzyme release from human leukocytes. II. Effects of CAMP and cGMP, autonomic agonists, arid agents which affect microtubule function. J Clin Invest 1974;53:297. 7. Rivkin I, Rosenblatt J, Becker EL. The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils: the inhibition of neutrophil movement and the elevation of cyclic AMP levels by catecholamines, prostaglandins, theophylline, and cholera toxin. J Immunol 1975;115:1126. 8. Boxer L.A, Allen JM, Baehner RL, Amick V. Diminished polymorphonuclear leukocyte adherence: function dependent on release of cyclic AMP by endothelial cells after stimulation of B-receptors by epinephrine. J Clin Invest 1980;66:268. 9. Busse WW, Sosman JM. Isoproterenol inhibition of isolated human neutrophil function. J ALLERGYCLIN IMMUNOL1984; 73:404. 10. Marone 6, Thomas LL, Lichtenstein LM. The role of agonists that activate adenylate cyclase in the control of CAMP metabolism and enzyme release by human polymorphonuclear leukocytes. J Immunol 1980;125:2277. 11. Komiyama A, Kawai H, Yamada S, Aoyama K, Yamazaki M, Saitoh H, Miyagawa Y, Akabane T, Uehara Y. Impaired natural killer cell recycling in childhood chronic neutropenia with morphological abnormalities and defective chemotaxis of neutrophils. Blood 1985;66:99. 12. Komiyama A, Saitoh H, Yamazaki M, Kawai H, Miyagawa Y, Akabane T, Ichikawa M, Shigematsu H. Hyperactive phagocytosis by circulating neutrophils and monocytes in ChediakHigashi syndrome. Stand J Haematol 1986;37:162. 13. Yamazaki M, Matsuoka T, Yasui K, Komiyama A, Akabane T. Increased production of superoxide anion by neonatal polymorphonuclear leukocytes stimulated with a chemotactic peptide. Am J Hematol 1988;27:169. 14. Wacker WEC, Ulmer DD, Vallee BL. Metalloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. N Engl J Med 1956;255:449. 15. Cronstein BN, Kramer SB, Weissmann G, Hirschhom R. Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983;158:1160. 16. Babior BMI Cohen HJ. Measurement of neutrophil function:

S. ALLERGY

CLIN. IMMLINGL. MAY i989

phagocytosis, degranuiation, the respiratory burst, and bacterial killing. In: Cline MJ, ed. Leukocyte function (Methods in hematology, vol 3). New York: Churchill Livingstone, 1981:l. 17. Massey V. The microestimation of succinate and the extinction coefficient of cytochrome c. Biochim Biophys Acta 1959; 34:255. 18. Cohen HJ, Chovaniec ME, Ellis SE. Chlorpromazine inhibition of granulocyte superoxide production. Blood 1980;56:23. 19. Mittal CK. Measurements of cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in polymorphonuclear leukocytes, macrophages, and lymphocytes. Methods Enzymol 1986;132:428. 20. Yamazaki M, Yasui K, Kawai H, Miyagawa Y, Komiyama A, Akabane T. A monocyte disorder in siblings with chronic candidiasis: a combined abnormality of monocyte mobility and phagocytosis-killing ability. Am J Dis Child 1984;138: 192. 21. Yamazaki M, Komiyama A, Yamazaki T, Yasui K, Morohashi F, Miyagawa Y, Akabane T. Leukaemia cell mobility in childhood acute myeloid leukaemia based on the FAB classification. Stand J Haematol 1985;35:481, 22. Tauber AI, Goetzl EJ. Structural and catalytic properties of the solubilized superoxide-generating activity of human polymorphonuclear leukocytes: solubilization, stabilization in solution, and partial characterization. Biochemistry 1979; 18:5577. 23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951;193:265. 24. McPhail LC, Snyderman R. Mechanisms of regulating the respiratory burst in leukocytes. In: Snyderman R, ed. Regulation of leukocyte function (Contemporary topics in immunology, vol 14). New York: Plenum Press, 1984247. 25. Tauber AI. Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 1987;69:711_ 26. Sha’afi RI, White JR, Molski TFP, Shefcyk J, Volpi M, Naccache PH, Feinstein MB. Phobol 12-myristate 13”acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium. Biochem Biophys Res Commun 1983;114:638. 27. Becker EL, Kermode JC, Naccache PH, Yassin R, Munoz JJ, Marsh ML, Huang CK, Sha’afi RI. Pertussis toxin as a probe of neutrophil activation. Fed Proc 1986;45:2151. 28. Korchak HM, Vienne K, Rutherford LE, Wilkenfeld C, Finkelstein MC, Weissmann G. Stimulus response coupling in the human neutrophil. II. Temporal analysis of changes in cytosolic calcium and calcium efflux. J Biol Chem 1984;259:4076. 29. Christiansen NO, Larsen CS, Juhl H. Ca2’ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8. Biochim Biophys Acta 1986;882:57. 30. Maridonneau-Parini I, Tauber AI. Activation of NADPHoxidase by arachidonic acid involves phospholipase A, in intact human neutrophils but not in the cell-free system. Biochem Biophys Res Commun 1986;138:1099. 31. McPhail LC, Snyderman R. Activation of the respiratory burst enzyme in human polymorphonuclear leukocytes by chemoattractants and other soluble stimuli: evidence that the same oxidase is activated by different transductionai mechanisms. J Clin Invest 1983;72: 192.