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ROLE OF ION MOVEMENTS IN Annu. Rev. Physiol. 1990.52:365-379. Downloaded from www.annualreviews.org by Brown University on 07/03/12. For personal use only.
NEUTROPHIL ACTIVATION Ramadan
I.
Sha'afi and Thaddeus
F. P.
Molski
Department of Physiology, University of Connecticut Health Center, Fannington Connecticut, 06032 KEY
WORDS:
motility, degranulation, oxidative burst, GM-eSF, calcium
INTRODUCTION The neutrophils represent the first line of defense against foreign pathogens. They are highly specialized for the performance of this primary function, the phagocytosis and destruction of microorganisms. The microbial invasion elicits several neutrophil responses. They include: chemotaxis, phagocytosis, oxidative burst, digestion, extracellular release, and aggregation. In this chapter we will restrict our discussion to the possible role of ion (Na+, K+, Ca2+) movements in these cell responses. Discussion of the methods for measuring ion movements will not be presented since this subject has been reviewed recently (27). Other aspects of the excitation-response coupling of neutrophil activation will not be dealt with since this subject has been discussed extensively (7, 8, 12, 29, 32-35,42,43, 46).
SODIUM AND POTASSIUM IONS The intracellular concentration of K+ in neutrophils, like that in other mammalian cells, is much higher than the corresponding value in the ex tracellular fluid, whereas the reverse is true for Na+. The plasma membrane of the neutrophil is permeable to these ions. Depending on the species studied, the value of the unidirectional flux for Na+ is 1-5 meq/liter cell water/min; the corresponding value for K+ is 1-7 meqlliter cell water/min. 365
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These values are at least 50 times faster than those obtained with mammalian red cells. The experimentally determined values for the intracellular con centrations of Na+ and K+ vary considerably among different investigators and most likely reflect the experimental differences (temperature, pH) under which these values were determined. The physiologic values should be in the range of 20 mM for Na+ and 120 mM for K+. These concentration gradients are maintained by the Na+, K+ pump, which is driven by metabolic energy derived from the hydrolysis of ATP by mem brane-associated (Mg2+ + Na+ + K+) activated adenosine triphosphatase (ATPase). This view is based on the following observations: (a) ouabain inhibits the rates of K+ influx and Na+ cfflux; (b) the rate of Na+ efflux is significantly reduced when extracellular K+ is removed, and (c) a Na+, K+ activated, ouabain-inhibited ATPase activity is present in the plasma mem brane of neutrophils (6, 10, 28, 33, 35, 38-41). Sodium ions should be able to enter the cell by at least one or more of three known distinct pathways (Figure 1). They include the Na+JH+ antiport, the Na+/Ca2+ exchange, and the Na+ channel. The plasma membrane of the neutrophil contains a 1 : 1 tightly coupled antiport that exchanges extracellular Na+ for internal H+, and it has an affinity for Li + and NH+4 in addition to Na+ (17, 34). The interaction of external Na+ with the antiport follows Michaelis-Menten kinetics and suggests a single binding site. This component of Na+ inward movement is inhibited by amiloride and its analogues and is stimulated by, among other things, protein kinase C activation. A second mechanism by which Na+ can enter the cell is through the Na+/Ca2+ ex change system. This low affinity high capacity transport system for regulating the free concentration of intracellular calcium in which Na-influx is coupled to Ca-efflux is quite common in muscle and probably non-muscle cells. Although such a system most likely is found in the plasma membrane of the neutrophil, its presence has been difficult to demonstrate experimentally (44). Unlike the Na+/H+ antiport, there are no known specific inhibitors for the Na+/Ca2+ exchange system. A third possible mechanism for Na+ entry is through channels. Again, because of the lack of specific inhibitors, the presence of such a transport mechanism is difficult to demonstrate experimentally. The main mechanism for Na+ -efflux is through the well-known Na+, K+ pump that exchanges 3Na+ for 2K+, and it is driven by metabolic energy derived from the hydrolysis of ATP (28, 35, 41). This transport system is inhibited by ouabain and requires the presence of K+ in the bathing medium. A second pathway for Na+ -efflux has been demonstrated recently in human neutrophils (40). This system transports 3Na + out of the cell in exchange for 1 Ca2+. This counter exchange system is noncompetitively inhibited by benza mil and by some other amiloride analogues bearing a substituent on the terminal nitrogen atom of the guanidine group (40).
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+ +
Na+/Ca2+ Activation
+
Phosphorylation of 60 KD Protein
Increose Inward Na+- Movement Figure
1
Schematic representation of the various pathways for Na + -influx. The symbols are as
follows: PLC, phospholipase C; PIP2 phosphatidylinositoI4,5-bis-phosphate; PIP, phosphatidyli nositol 4-phosphate; PI, phosphatidylinositol; DO, diacylglycerol; IPz inositol 1,4-bisphosphate; IP, inositol I-phosphate; IP), inositol 1,4,5-trisphosphate.
Potassium can enter the cell by at least two transport mechanisms. The first transport system involves the above mentioned ouabain inhibited Na+, K+ pump (28, 41). A second mechanism for K+ entry is through K+ channels. The presence of K+ channels and their specificity is difficult to demonstrate experimentally because of the lack of suitable inhibitors. There are several channels through which K+ can leave the cells: those that are present when the cells are under resting conditions and those that are normally quiescent and become evident following cell activation. The most widely known system is the calcium-activated potassium channel. This system, which can be in hibited by oligomycin, furosemide, quinine, and quinidine, was first de scribed in red cells, and it is known as the Gardos effect (21). The rise in Ca2+ also activates other non-selective cation channels as measured by the patch clamp method (45). Na T -influx is enhanced by agonists that are known to activate the neutro phils. These agonists include fMet-Leu-Phe, leukotriene B4, platelet activating factor, arachidonic acid, phorbol 12-myristate, 13-acetate (but not inactive phorbol esters), and others (25, 28, 40, 41; for review see 34, 35). These stimuli activate one or more of the pathways through which Na+ can enter the cell (Figure 1). This enhancement of Na T -influx can be inhibited by
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1-(5(H-7) or by the
amiloride and its analogues but not by the protein kinase C inhibitor isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride calmodulin inhibitor trifluoperazine (TFP)
(25, R. Sha'afi, unpublished data).
Among the various agonists, the effect of the chemotactic factor fMet-Leu Phe on cation transport has been the most studied. The chemotactic factor produces a rapid and concentration-dependent increase in Na+ -influx. Small er enhancements of K+ -influx and Na + -efflux are also produced by fMet Leu-Phe. The chemotactic factor-sensitive increase in K+ -influx and Na +
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efflux are inhibited by ouabain. In addition, the observed increase in Na+ efflux is abolished in the absence of extracellular K+. Unlike Na + -efflux the efflux of K+ is not stimulated by fMet-Leu-Phe. In considering the role of Na+ and K+ in neutrophil responses elicited by soluble and particulate agents, we would like to distinguish between the two ways in which they may be involved. First, they may modulate cell responses indirectly by affecting one or more steps in the main sequence of events in the excitation response mechanism (receptor binding, guanine nucleotide binding
C activity), or they may modify the biochemical or (pH, actin polymerization, levels of cyclic nucleotides)
protein, or phospholinase biophysical c hanges
that are elicited by agonists. Such a role implies that Na+ and K+ are not necessary to elicit the response by the stimulus, but they could be necessary for optimal stimulation. Second, Na+ and/or K+ may play a direct role in some or all of the neutrophil responses elicited by various stimuli. Such a role implies that Na+ and/or K+ are either necessary and sufficient, necessary but not sufficient, or sufficient but not necessary to elicit the response. The role of univalent cations in the physiologic responses of neutrophils has been thor oughly investigated using various experimental manipulations (3, 4 , 18, 20,
25, 30, 31, 33, 35-37, 46-48). These studies can be conveniently divided into substitution experiments in which one or more ionic species is removed and/or replaced by another and investigations into the effects of ionophores. It is noteworthy that a change in the concentration of any one of the ions in the
bathing medium results in rapid and significant changes in the concentrations of other intracellular ionic species. This is true because the permeability of the plasma membrane of the neutrophils to Na+ and K+ is relatively high, and the movements of the various cations are coupled. Rarely can the effects of the removal of one ionic species from the extracellular fluid be pinpointed precisely. Therefore the r�sults from substitution experiments, though in formative, must be interpreted with caution. The basic assumption in the use of ionophores to investigate the role of a given ion in a specific event is that the basal permeability of the plasma membrane of the cell under study is very low and that the addition of the ionophore significantly increases the membrane permeability to this specific ion. This assumption holds true in many but not necessarily all cases. Studies
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in which ionophores specific to K+ or Na+ are used in neutrophils must be viewed with reservation and interpreted cautiously unless the ionophoretic ability is directly demonstrated. Modulatory influences of extracellular Na+ and K+ on chemotaxis have been studied, and it has been found that physiologically balanced salt solu tions are necessary for optimal cell locomotion. If the concentration of Na + or K+ is varied quite widely, a locomotor response can still be obtained though the strength of the response is significantly diminished (3, 35,46, for recent review see reference 11). For example, removal of K+ from the bathing medium and/or the addition of ouabain depress the chemotactic responsive ness of neutrophils by about 50%, i.e. the concentration of the chemotactic factor required to elicit a specific level of responsiveness is doubled in the presence of ouabain. Under these conditions chemotaxis is not abolished. The dose-response curve is simply displaced to the right. In addition, increasing the concentration of K+ can overcome the ouabain inhibition of chemotaxis. Furthermore, increasing the extracellular K+ concentration causes a polariz ing response in neutrophils similar to the response produced by fMet-Leu Phe, and this response is not inhibited by ouabain (18). It is worth noting that the K+ channel blocker tetraethylammonium chloride does not inhibit cell polarization produced by fMet-Leu-Phe. The effects of the known K+ ionophores valinomycin and nigericin on chemotaxis in neutrophils have been thoroughly studied. Valinomycin at low concentrations (l0-7M) slightly enhances chemotaxis in the presence of extracellular K+ but has no effect in its absence. Nigericin at the same concentration has no effect on chemotaxis in the presence of K+ and slightly inhibits chemotaxis in its absence. The differential effects of these two K+ ionophores suggest that the potentiating action of valinomycin on chemotaxis may not be mediated by its K+ transporting ability. Brief removal of extracellular sodium leads to an increase in spontaneous (i.e. in the absence of the chemotactic factor) motility (3, 18, 35, 36). This enhancement in spontaneous activity is dependent on the presence of ex tracellular Ca2+ and may result from the inhibition of a Na + influx, Ca2+ efflux exchange mechanism. On the other hand, replacing sodium by choline significantly inhibits cell polarization induced by fMet-Leu-Phe (18). The number of polarized cells decreases with decreasing Na+ concentration in the extracellular solution. One likely interpretation of these results is that the stimulated influx of sodium produced by fMet-Leu-Phe is required for cell polarization and subsequent locomotion. The observed increase in spontaneous activity in the absence of Na+ may account for this decrease in cell polarization. Since the increased spontaneous activity is dependent on the presence of Ca2+ , it will be interesting to examine the effect of this ion on the observed decrease in cell polarization produced by the removal of Na+.
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The roles of extracellular Na+ and K+ in degranulation induced by various stimuli have been examined (4,20,33,35). The effect of extracellular K+ on degranulation by cytochalasin B-treated neutrophils stimulated by fMet-Leu Phe is essentially similar to that observed for chemotaxis. Removal of K + from the bathing medium shifts the dose-response curve to the right, and ouabain is essentially without any effect. The two ionophores tested, val inomycin and nigericin, have no effect on chemotactic factor-induced de granulation from cytochalasin B-treated cells. However, valinomycin at con centrations greater than 10-7 M causes rabbit neutrophils to degranulate to a small but significant extent. Its secretory action requires the presence of extracellular Ca2+ and is enhanced by cytochalasin B. The most likely explanation for this finding is that valinomycin may act as a weak Ca + ionophore. Replacing extracellular sodium with choline inhibits the release of lyso zyme and {3-glucuronidase in human neutrophils stimulated with immune complexes fMet-Leu-Phe or the calcium ionophore A23187 (20, 35). These results suggest that a Na+ -influx may be necessary for degranulation. On the other hand, amiloride, which inhibits most of the stimulated Na+ -influx, has little, if any, inhibitory action on the release of lysozyme or {3-glucuronidase in neutrophils stimulated with several agonists (4). These agonists include fMet-Leu-Phe, PMA, and A23187. This argues against a necessary role for Na+-influx in degranulation. Moreover the Na + ionophore monensin, which has been shown to increase Na+ -influx, does not elicit {3-glucuronidase release (20). These data strongly suggest that Na+ -influx is not sufficient to stimulate degranulation. Replacement of extracellular Na + by other univalent cations such as cho line significantly inhibits (>50%) superoxide generation stimulated with various agonists including fMet-Leu-Phe (20, 33, 38). Furthermore, pro longed incubation of human neutrophils with amiloride (l0-3M) greatly reduces superoxide generation produced by fMet-Leu-Phe, but it does not affect the corresponding increase produced by the protein kinase activator PMA (4). On the other hand, monensin does not elicit superoxide generation (20), and protein kinase C inhibitors,though they block PMA-induced super oxide production, do not alter the stimulation of Na+ -influx generated by PMA. These data strongly suggest that the presence of Na+ and/or Na+ -influx are necessary for fMet-Leu-Phe but not PMA-produced stimulation of the oxidative burst, and that Na+ -influx is not sufficient for this response. It has been suggested that the apparent requirement for Na + for a stimulated oxidative burst in neutrophils is due to exposing the cells to an erythrocyte lysis medium that contains NH+ 4 (30). We tested this point by examining the effect of replacing sodium chloride with choline chloride on superoxide production in rabbit peritoneal neutrophils stimulated with fMet-Leu-Phe and
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PMA. We found that at the lower concentration of the stimulus, removal of Na+ has no effect on the magnitude of superoxide production. At the higher concentration of the stimulus, however, removal of Na+ significantly de presses (>25%) superoxide production by the two stimuli. The role, if any, of the univalent cations Na+ and K+ in stimulated neutrophil responses appears to be modulatory in nature, and these cations are not an essential part of the basic sequence of the excitation-response coupling. Having the proper intracellular concentration of Na+ and K+ is important for the optimum responsiveness of the cells. The main points concerning the role of Na+ and K+ in neutrophil response are summarized in Table 1.
CALCIUM MOVEMENT The regulation of the intracellular concentration of free calcium, Ca?+ , in the neutrophils is achieved by pump-leak systems at the plasma membrane and by binding of Ca2+ by cytoplasmic constituents and plasma membrane (35). There are probably two separate channels by which Ca2+ could leak: into the cells. The first channel is independent of membrane potential, whereas the second channel could be controlled by the membrane potential. In addition, there are at least two different energy-dependent mechanisms for the control of Ca2+ efflux: a specific calcium pump driven by the hydrolysis of ATP by a Mg2+, Ca2+ -activated ATPase, the presence of which has been demonstrated in the plasma membrane of neutrophils, and a Na+ influx, Ca2+ efflux Table 1
The effects of changing Na+ and K+ movements by several experimental manipula
tions on the neutrophil responses produced by the chemotatic factor fMet-Leu-Phe
1. Removal of extracellular Na+ inhibits cell polarization, reduces chemotaxis, and significantly inhibits (>50%) degranulation and superoxide production. The effect on chemotaxis and degranulation is to shift the dose-response curve to the right. 2. Amiloride (l0-3M), which inhibits Na+-influx. slightly inhibits «25%) degranulation and greatly (>50%) reduces superoxide generation. The PMA-induced superoxide production is
not inhibited by arniloride.
3. Ouabain, which inhibits the Na +, K+ pump, reduces chemotaxis but has no effect on either degranulation or the oxidative burst.
4. The Na+ ionophore monensin, which stimulates Na+-influx in neutrophils, does not by itself elicit superoxide generation or significant degranulation (J3-glucuronidase). 5. The potassium ionophores valinomycin and nigericin do not significantly enhance chemotax is. and they do not by themselves produce significant degranulation.
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exchange system. This component depends on maintaining the Na+ gradient across the cell membrane by the classic Na+, K+ pump. In addition to these membrane events, control of cytosol Ca2+ is also dependent on binding to the membrane, buffering by cytosol constituents such as soluble proteins and others, and accumulating into intracellular organelles such as mitochondria, endoplasmic reticulum, granules, and others. With the exception of PMA, all agonists that have been tested that stimulate neutrophils cause an increase in the intracellular concentration of free cal cium, Cai2+ (7,8,9, 12, 19,24,26,29,32,33). This rise in Ca?+ is brought about by a net influx from the outside because of an increase in plasma membrane permeability to calcium and a release of bound calcium from 'internal stores. Evidence that supports an increase in the plasma membrane permeability to calcium includes (7-9, 12, 29, 3 2, 33): (a) an increase in Ca2+ influx, and in the presence of extracellular Ca2+,an increase in steady state levels of Ca2+; (b) chemotactic factors increase the specific activity of neutrophil Ca2+ and increase the pool of exchangeable Ca?+; (c) the cytoplasmic Ca2+ signals monitored by quin2 and other calcium-sensitive dyes that are produced by fMet-Leu-Phe are reduced in the presence of EGTA; and (d) a rise in the Ca?+ seems to open nonspecific ion channels as measured by patch-clamp methods. The main evidence supporting a release of calcium from intracellular compartments following stimulation of neutrophils include (7-9, 12, 29, 32,33): (a) chemotactic factors cause an efflux ofCa2+ from pre10aded neutrophils,and in the absence of extracellular Ca2+ cause a transient decrease in steady-state levels of cell-associated Ca2+; (b) these agents cause an early transient decrease in Ca2+ specific activity in the neutrophil; and (c) chemotactic factors cause a transient decrease in the fluorescence of chlorotetracycline-treated neutrophils, and an increase in quin2 and other calcium-sensitive dye signals. The intracellular messenger responsible for calcium release is inositol 1,4,5-trisphosphate,IP3 (1, 5). The increase in membrane permeability to calcium is probably initiated by the depletion of calcium from the IP3-sensitive pool. In discussing the role of Ca2+ in various cell responses, one must distin guish between the basal level of Ca2+ and the stimulus-induced rise in Ca2+. In some responses a rise in Ca?+ may be necessary while in others the basal level of Ca?+ may be sufficient. While it is easier to determine if a rise in Caj2+ is necessary for a given cell response, it is extremely difficult to determine if calcium per se is required (it is experimentally difficult to re duce Cai2+ to zero). The role of calcium in locomotion, degranulation, oxi dative burst, and other cell responses will be discussed with this point in mind. The migration of the neutrophil into inflamed tissue is important in fulfill ing its vital function. Neutrophil locomotion, whether random or directed, depends on the displacement of the cell. Such displacement requires the
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conversion of chemical energy to mechanical energy, and the motile appara tus, with actin and myosin as the major components, must be involved in the locomotive activities of the cell. Regardless of whether the motility of the neutrophil is actin-based or actin-myosin based, it is most likely that calcium is closely related to this activity. For example, calcium and gelsolin may regulate the length of the actin filaments andlor activate the actin-myosin ATPase. Although there is no one single experiment that establishes un equivocally a role for calcium in cell motility, it is generally thought that Ca2+ is important for motility (3, 11, 35, 42, 46, 47). The available experimental data concerning calcium and cell motility can be summarized in several points. (a) In spite of early observations, ex tracellular calcium is not necessary for fMet-Leu-Phe-induced chemotaxis as assayed by migration through micropore filters and by time-lapse videomi croscopy (48). (b) Increasing Cai2+ excessively (this is normally achieved by incubating the cells with a calcium ionophore in the presence of calcium) inhibits locomotion of neutrophils stimulated with fMet-Leu-Phe (23, 48). (e) Binding of intracellular free calcium by quin2 or chlorotetracycline or inhibit ing the release of calcium by TMB-8 inhibits chemotaxis toward fMet-Leu Phe (l3, 22; see original citation 34). The effects of these agents are probably not due to their actions on Ca2+ alone, since they are likely to have other effects as well. Consistent with this conclusion is the observation that reduc ing Ca?+ by incubating the cells with the calcium ionophore A23l87 and EGTA has no effect on fMet-Leu-Phe-induced chemotaxis (48). (d) Inhibitors of calmodulin reduce chemotaxis toward fMet-Leu-Phe (22). Since calmodu lin is known to mediate certain calcium-dependent enzymatic activities, it appears that Ca2+ is necessary for chemotaxis. Again the inhibitory effect of these compounds is probably not due solely to their action on calcium. A reasonable conclusion, based on the available experimental data, is that a very low concentration of Ca2+ is sufficient, if Ca2+ is indeed necessary, for chemotaxis. The addition of chemotactic factors and other soluble and insoluble ago nists to neutrophils cause, under the appropriate conditions, the release of both the azurophil and specific granule contents. Since secretion requires the fusion of intracellular granules with the plasma membrane,it is reasonable to hypothesize that chemotactic factor-induced degranulation in neutrophils in volves Ca2+ and that the level of Ca2+ i necessary for degranulation is most likely higher than that needed for locomotion. Different requirements are needed for the release from the azurophil and specific granules (2). With certain stimuli such as PMA, a rise in cellular Ca2+ does not seem to be necessary for the small, slow lysosomal enzyme release. But even in this case, a role for calcium cannot be completely ruled out. It is expected that a reduction in the basal level of Cai2+ will inhibit PMA-induced lysosomal enzyme release.
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The available data in the neutrophil system strongly suggest that the calcium ion is required for degranulation and that in most cases a rise in Ca?+ is also necessary. This conclusion is supported by several experimental findings (7, 8, 12, 29, 33). (a) The calcium ionophore A23187 stimulates degranulation, and the addition of EGTA significantly reduces lysozyme release and abolishes f3-glucuronidase release. Removal of extracellular cal cium diminishes, but does not totally abolish, degranulation produced by chemotactic factors such as Csa• The pattern of granule fusion as visualized in freeze-fracture replicas is influenced by calcium in the suspending medium during stimulation by fMet-Leu-Phe or CSa' It has been found that the directed pattern of fusion is initiated by release of intracellular calcium or a calcium independent pathway, and the nondirected convoluted pattern of fusion is initiated by entry of extracellular calcium. (b) Incubation of neutrophils for 20 min or longer in calcium-free medium reduces lysosomal enzyme re lease produced by fMet-Leu-Phe, and this inhibition can be restored by the addition of calcium. The addition of EGTA totally inhibits N-acetyl-f3glucosaminidase release induced by platelet-activating factor. (c) Calcium causes degranulation in permeabilized cells. Calmodulin inhibitors and the intracellular calcium release antagonist TMB-8 reduce degranulation pro duced by various stimuli. However, the effects of these inhibitors may be unrelated to their actions on calcium or calcium-calmodulin. (d) The extent of exocytosis from specific granules (vitamin BJ2 binding protein), azurophil granules (f3-glucuronidase), and secretory vesicles (gelatinase) is a function of the intracellular concentration of free calcium. Although the minimum con centration of Ca2+ that causes significant release from the three granule populations is similar, the ECso values are significantly different for the three compartments. The rank order of increasing ECso values is specific as fol lows: < secretory < azurophil (33). Rabbit neutrophils maintained, washed, and reacted in the absence of extracellular Ca2+ give a dose-response curve that is shifted to the right and in which the maximal level of release is significantly decreased. Depletion of intracellular calcium by exposure to the calcium ionophore blocks the ability of fMet-Leu-Phe to stimulate enzyme release. A number of soluble and particulate agents stimulate the oxidative metabo lism in the neutrophil, and this activation increases the production of superox ide radicals (02-), Although a rise in intracellular concentration of free calcium is likely to be necessary for optimal stimulation of the oxidative burst, it is not absolutely required. Moreover, a rise in Cai2+ alone is not sufficient since several soluble and insoluble stimuli can produce a rise in Cai2+, but do not stimulate superoxide generation. Based on the available data, it is most likely that, although a rise in intracellular calcium may not be
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absolutely necessary, a rise in Ca?+ is closely involved in this response to certain stimuli. This hypothesis is supported by numerous studies (7, 8, 12, 16,29,32,33,43). (a) The calcium ionophore A23187 stimulates superoxide release, and this release is abolished in the presence of EGTA. The action of A23187 may be mediated through the generation of one or more lipid mediators. These mediators are produced by activation of phospholipase A2, which is critically dependent on Ca2+ . (b) There is a good correlation between the rise in Catt- and O2- generation produced by fMet-Leu-Phe. (c) The addition of EGTA reduces superoxide generation by most stimuli tested. (d) Although outside calcium may not be required for O2- generation, cells depleted of calcium show no release of O2- on stimulation with fMet-Leu Phe when calcium is absent in the outside medium, and this inhibition can be overcome by the addition of calcium. It must be pointed out, however, that under calcium depletion conditions, the level of Ca?+ may be too low to sustain phospholipase C activation by fMet-Leu-Phe. This depressed state will interfere with the hydrolysis of PIP2 and the generation of diacylglycerol, which is important for O2- production. The role of Ca2+ in the stimulated oxidative burst can be best summarized as follows: First, with some but not all stimuli, a rise in Ca?+ is neither necessary nor sufficient. Second, although a rise in Ca?+ is not necessary, some calcium ion is most likely required. The basal, or somewhat lower, concentration of Caj 2+ is probably sufficient to fulfill this requirement. Third, the involvement of calcium is most likely in the translocation andlor activa tion of protein kinase C to the membrane.
HYDROGEN ION MOVEMENT The presence in the plasma membrane of the neutrophil of an antiport that exchanges outside sodium for intracellular hydrogen has been demonstrated (17, 34). The exchanger is a Ubiquitous transport system with a tightly coupled 1 : 1 stoichiometry, and it has affinity for Li+ and NH+ 4 in addition to Na+ and H+. The energy required for this exchange is derived from the inwardly directed Na+ concentration gradient, which is generated by the ouabain sensitive Na+, K+ pump. In neutrophils this Na+/H+ exchange system is largely quiescent near the physiologic pH. It is found that the addition of the chemotactic factor tMet-Leu-Phe to neturophils causes rapid and biphasic changes in intracellular pH. Initially there is a rapid drop followed by a slower and larger increase. The importance of the Na+/H+ antiport in relation to maintaining ionic and pH equilibrium during normal cell metabolism is obvious. The importance of changes in
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Table 2
Effect of GM-CSF on the basal and stimulated level of intracellular
concentration of free calcium in human neutrophils Intracellular concentrations of free calcium, nM Stimulus
Control cells
GM-CSF-treated cells I
100 276 318
110 700 430
No addition fMet-Leu-Phe Platelet
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1
activating factor (5 nM)
The cells were incubated with GM-CSF (200 pM) for 30 min (Similar results were
first reported in reference 26).
intracellular pH in cell function or as a signal in the activation and/or regulation of cell activation has been reviewed recently (34). EFFECT OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR ON ION MOVEMENTS IN HUMAN NEUTROPHILS
The human hormone granulocyte-macrophage colony stimulating 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 (14). Although the addition of GM-CSF to mature human neutrophils does not activate cells responses, it primes these cells to subse quent stimulation by the chemotactic factor fMet-Leu-Phe. 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. The effects of GM-CSF on the basal and stimulated ion (Na+, Ca2+, pH, PO= 4) movements in human neutrophils stimulated wth fMet-Leu-Phe have been studied, and the results are summarized in Tables 2 and 3 (15), Several Table 3
GM-CSF-induced stimulation of ion movements in control and pertussis toxin-treated human neutrophils GM-CSF-induced change (relative
to
control) - pertussis toxin
Ion
+
pertussis toxin2
PAF-induced
[Ca?+] rise 22Na+ -influx P04 uptake 2
1.35 2.3 1.90
1.20 1.60 1.50
Cells were incubated with 0.5 /-Lg/ml pertussis toxin for 45 min
before the addition of GM-CSF (20OpM). The remaining con ditions are the same as those for Table 2.
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points can be made from these results. Both the basal and fMet-Leu-Phe
stimulated Na+ -influx are increased in GM-CSF-treated neutrophils. GM CSF causes a rise in the intracellular pH (3). The ch anges in Na+ and H+ movements produced by GM-CSF are rapid, and they can be abolished by amiloride, protein kinase C inhibitors, and they are significantly reduced by pertussis toxin. The stimulation of the Na+/H+ antiport by GM-CSF de
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activates this system to further activation by PMA or fMet-Leu-Phe. While
the basal concentration of Ca?+ is not affected, the rise in Ca?+ produced by fMet-Leu-Phe or platelet activating factor (PAF) is greatly increased in GM-CSF treated cells. GM-CSF stimulates the upt ake of radioactive phos phate. Unlike Na+ -influx, the action of GM-CSF on phosphate uptake is much slower. The GM-CSF-induced increase in cell-associated radioactive
phosp ha te reflects several biochemical changes such as increased ATP generation, phosphorylation of proteins by tyrosine and other kinases, and other cellular mod ificat ion s (R. Sha afi unpublished data). The observed '
,
stimulation of phosphate uptake is inhibited by pertussis toxin, but not by cholera toxin or botulinum D toxin.
ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health grants AI-24937 and GM-37694.
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