Communicated by K. Frank Austen, February 6, 1989. ABSTRACT. The addition ..... Na+/H' exchange system, the fMet-Leu-Phe-stimulated. Na' influx is usually ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3569-3573, May 1989
Biochemistry
Granulocyte-macrophage colony-stimulating factor and human neutrophils: Role of guanine nucleotide regulatory proteins (GTPase/ions/tyrosine phosphorylation)
JULIAN GOMEZ-CAMBRONERO*, MUNEHIRO YAMAZAKI*, FATHIA METWALLY*, THADDEUS F. P. MOLSKI*, VALERIE A. BONAKt, CHI-KUANG HUANGt, ELMER L. BECKERt, AND RAMADAN I. SHA'AFI*t Departments of *Physiology and tPathology, University of Connecticut Health Center, Farmington, CT 06032
Communicated by K. Frank Austen, February 6, 1989
anced salt solution (pH 7.35) containing 10 mM Hepes, 120 mM NaC1, 5 mM KCl, 0.1% dextrose, and bovine serum albumin (1 mg/ml). The cells were incubated with the toxin [pertussis toxin (0.5 gg/ml), cholera (2 Aug/ml), or botulinum D (0.5 Ag/ml)] for 45 min before the addition of GM-CSF. The paired-sample t test was used in all the statistical analysis. In all the cases, control cells were treated exactly as cells treated with GMCSF except for the deletion of GM-CSF. Measurements of Radioactive Phosphate, Sodium Uptake, and Intracellular pH (pH1). Radioactive phosphate uptake was measured using the silicone oil method as described (13). A similar procedure was used for 22Na uptake (13). Changes in pHi were measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF) (Molecular Probes) as described (13). Fluorescence was calibrated by the method of Moolenaar et al. (14). GTPase Activity. GTPase activity in human neutrophil membrane was calculated by measuring the hydrolysis of 32p from [y-32P]GTP as described in detail (15). Briefly, membranes were prepared by nitrogen cavitation and ultracentrifugation. Cells were disrupted in buffered sucrose solution at 250 psi for 20 min at 4°C (1 psi = 6.9 kPa). Immunoblotting. Immunoblotting was carried out as described (16, 17). Approximately 5 x 106 cells per ml were incubated at 37°C in Hanks' balanced salt solution (pH 7.3) containing several protease inhibitors. Aliquots (1 ml) were removed and stimulated with 200 pM GM-CSF for 10 min. The reaction was stopped by quick centrifugation (7000 x g for 15 sec). The pellets were resuspended in 100 ,l of the same buffer and mixed with 50 ,ul of stopping solution [9% (wt/vol) SDS/6% (vol/vol) 2-mercaptoethanol/10% (vol/ vol) glycerol and a trace amount of bromophenol blue dye in 0.196 M Tris HCl (pH 6.7)]. The mixture was electrophoresed through a SDS/5%-15% polyacrylamide gradient gel. The proteins were then transferred to a nitrocellulose sheet. Tyrosine-phosphorylated products were determined with anti-phosphotyrosine antibody (17), and the antibodies bound to the nitrocellulose sheet were detected by 1251_ labeled protein A [1 uCi/ml, final concentration in 5% (wt/ vol) bovine serum albumin; 1 Ci = 37 GBq]. The specificity of the antibody has been well characterized, and the presence of free phosphotyrosine inhibits the antibody signal (16, 17). Blots were developed on Kodak X-Omat films overnight at -70°C. Molecular masses were determined using Bio-Rad standards. Chemicals. fMet-Leu-Phe, amiloride, Hepes, cholera toxin, phorbol 12-myristate 13-acetate (PMA), leupeptin, ATP, and p-nitrophenyl phosphate were obtained from Sigma (St. Louis, MO). 125I-labeled protein A [NEX-146] and other
The addition of granulocyte-macrophage colABSTRACT ony-stimulating factor (GM-CSF) to human neutrophils causes a rapid increase in the basal and fMet-Leu-Phe-stimulated Na+ influx and an increase in intracellular pH. The increase can be seen as early as 5 min after the addition of GM-CSF. Changes produced by GM-CSF are totally inhibited by amiloride and are significantly reduced in pertussis toxin-treated cells. The stimulation of the Na+/H+ exchange mechanism by GM-CSF inhibits further stimulation of this system with either fMetLeu-Phe or phorbol 12-myristate 13-acetate. In addition, membrane preparations isolated from GM-CSF-treated neutrophils have higher basal and stimulated GTPase activities. The basal and the fMet-Leu-Phe- or platelet-activating factor-stimulated GTPase activities are reduced in pertussis toxin-treated cells. Cells pretreated with GM-CSF accumulate more radioactive phosphate than control cells, and this increase is diminished by pertussis toxin treatment. In addition, GM-CSF causes a rapid increase in the tyrosine phosphorylation levels of five proteins with molecular masses of 118 kDa, 92 kDa, 78 kDa, 54 kDa, and 40 kDa. These results clearly show that GM-CSF, on its own, can initiate several changes and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein.
The human hormone granulocyte-macrophage colonystimulating factor (GM-CSF), which is released by several activated cells, such as T lymphocytes, is an important stimulus for the proliferation of erythroid and myelomonocytic stem cells in vitro (1-7). The addition of GM-CSF to mature human neutrophils primes these cells to subsequent stimulation by the chemotactic factor fMet-Leu-Phe (8-11). Thus, GM-CSF plays an important role in the host defense. In spite of its importance, the mechanism of GM-CSF action is totally unknown. In this regard, one main unanswered question is the role, if any, played by the known guanine nucleotide regulatory proteins in the effects of GMCSF on neutrophils. The present studies were undertaken to investigate two questions. (i) Are there changes that can be elicited by GM-CSF alone? (ii) Are the effects of GM-CSF mediated by one or more of the known guanine nucleotide regulatory proteins?
MATERIALS AND METHODS Cell Preparation. Human neutrophils were isolated from peripheral blood on Ficoll/Hypaque gradients as described by English and Andersen (12). The remaining erythrocytes were lysed by hypotonic shock (30-sec exposure). The neutrophils were resuspended in modified buffered Hanks' bal-
Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; pHi, intracellular pH; PMA, phorbol 12-myristate 13acetate; PAF, platelet-activating factor. STo whom reprint requests should be addressed.
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Proc. Natl. Acad Sci. USA 86 (1989)
Biochemistry: Gomez-Cambronero et al.
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Table 1. Basal and stimulated GTPase activities in membrane preparations isolated from control and GM-CSF-treated human neutrophils GTPase activity, pmol per mg of protein per min GM-CSF-treated Control cells cells Stimulus 43 ± 4 (20) 34 ± 2 (40) No addition 32 ± 4 (6) 28 ± 5 (6) Pertussis toxin (17 Ag/ml) 92 ± 6 (15) 59 ± 4 (15) fMet-Leu-Phe (10-7M) Pertussis toxin and 52 ± 6 (3) 36 ± 5 (3) fMet-Leu-Phe 56 ± 5 (12) 45 ± 5 (12) PAF (106M) 41 ± 5 (4) 33 ± 5 (4) Pertussis toxin and PAF Cells were incubated with 200 pM GM-CSF for 45 min before membranes were prepared. Control cells were treated the same. When pertussis toxin was used, the cells were incubated with the toxin before the addition of GM-CSF. All reactions were carried out for 5 min; PAF and fMet-Leu-Phe were present for the 5-min duration. Each value represents the mean ± SEM, and the numbers in parentheses refers to number of experiments done.
Table 2. Effect of GM-CSF on the uptake of radioactive phosphate in control, pertussis, cholera, and botulinum D toxin-treated human neutrophils 32p uptake (relative to control) + GM-CSF Basal Cells 1.95 ± 0.15 1.0 Control 1.45 ± 0.10 0.95 ± 0.05 Pertussis toxin-treated 1.88 ± 0.14 1.03 ± 0.06 Cholera toxin-treated 1.88 ± 0.13 1.05 ± 0.08 Botulinum D toxin-treated The method used was as in Fig. 1 except that the cells were incubated with GM-CSF (200 pM) for 45 min before the radioactive phosphate was added, and the uptake was measured after a 20-min incubation with the radioactivity. Each value represents the mean ± SEM of at least three experiments; each done in triplicate. The neutrophils were incubated first with the toxin for 45 min and then with GM-CSF (200 pM) for an additional 45 min. The incubation was carried out at 37°C, and control cells were treated in the same manner. The value 1.45 is significantly (P < 0.05) less than 1.95, and the latter is significantly (P < 0.001) greater than 1.0.
radioactive compounds were purchased from New England Nuclear. Pertussis toxin was a gift from J. J. Munoz (National Institutes of Health, Rocky Mountain Laboratory, Hamilton, MT). Botulinum D toxin was obtained from Wako Chemicals (Dallas, TX). Antibodies against phosphotyrosine were prepared and characterized as described (17). They were used at a 1:1000 dilution for immunoblotting. Diisopropyl fluorophosphate was purchased from Aldrich, and nitrocellulose sheets were obtained from Schleicher & Schuell. Electrophoresis chemicals were supplied by Bio-Rad. Platelet-activating factor (PAF) was purchased from Calbiochem. Recombinant GM-CSF was obtained from Amgen Biologicals.
GTPAse Activity. The basal and stimulated GTPase activity in membrane preparation from control and GM-CSF-treated cells were determined (Table 1). The basal activity is significantly (P < 0.05, paired-sample t test) higher (27%) in GM-CSF-treated cells. Both fMet-Leu-Phe (74%) and PAF (32%) stimulate the GTPase activity in control cells. These stimulations are greatly enhanced in cells pretreated with 200 pM GM-CSF for 45 min. Pertussis toxin reduced significantly the increase in the basal (P < 0.01) and stimulated activities in GM-CSF-treated cells (P < 0.001 for fMet-Leu-Phe and P < 0.05 for PAF) as well as the GTPase activities in control cells (P < 0.001 for both fMet-Leu-Phe and PAF) stimulated by PAF or fMet-Leu-Phe. GM-CSF added after the membranes were prepared showed small increases in both the basal and stimulated GTPase activities, but the increases were within experimental errors. Radioactive Phosphate Uptake. The time course of radioactive phosphate uptake in control and GM-CSF-treated human neutrophils has been investigated and the results are summarized in Fig. 1. The data clearly show that phosphate
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