Receptors and Intracellular Signaling in Human Neutrophils1,2 P. DANIEL LEW
Introduction The main function of human neutrophils is to sense, approach, and destroy invading microorganisms, in particular pyogenic bacteria. The ultrastructure of a neutrophil reveals a cell extremely rich in various populations of granules, poor in mitochondria and rough endoplasmic reticulum, and with a dystrophic nucleus. Neutrophils are terminally differentiated cells, unable to replicate and are able to synthesize only low amounts of proteins. Yet they are very motile cells and are able to selectively release proteins from various populations of granules. In addition, they contain a rather unique plasma membrane enzyme (NADPH-oxidase), which, when activated, generates and releases active oxygen species into the extracellular space. The products released from granules and the active oxygen species alone or in combinations are potently bactericidal. Human neutrophils, when observed under phase-contrast microscopy,crawl rapidly over surfaces; to perform this process, they adhere and polarize into a typical asymmetrical configuration with a large anterior lamelopodim and a short tail-like uropod. The functions of adherence, perception of distant microbial or inflammatory stimuli, movement, phagocytosis, and secretion are modulated by a large variety of receptors. This review deals with receptor-generated signals in neutrophils and summarizes some of our contributions to this area of research. A comprehensive textbook on neutrophils and inflammation and several reviews have appeared recently (1-6). It is possible to divide the various classes of receptors into five main groups according to their main function: adherence receptors; chemotactic receptors; phagocytic receptors; cytokine-sensitive receptors; and receptors of unknown function. Little is known of the structure, location, and function of cytokine-sensitive receptors in neutrophils. Several substances of this group modulate and activate neutrophil functions. Thmor necrosis factor (TNF) activates the oxidative burst of adherent neutrophils. Granulocyte-macrophage colony-stimulating factor (GMCSF), in addition to its known effects in the bone marrow, primes neutrophils to subsequent stimulation (1). There are also several other receptors of unknown function, but whose presence has been welldocumented, in particular for several hormones, such as TSH. Thus, in the following sections we will discuss only the first three classes of receptors listed above: adherence, chemotaxis, and phagocytosis. Adherence Receptors A family of receptors is present in the plasma membrane of neutrophils, whose main func-
SUMMARY Adherence, chemotaxis, phagocytosis, and responses to cytokines are mediated by distinct classes of cell surface receptors in human neutrophils.lntracellular signaling by these different receptors Is a SUbject of active investigation. Observation of single neutrophils adherent to surfaces reveals the presence of spontaneous oscillations of cytosolic-free calcium, [Ca2+ll, generated by mechanisms that are presently unknown. Chemoattractant receptor activation via a specific G-regula· tory protein activates a plasma membrane phospholipase C and generates diacylglycerol and inosi· tol(1,4,5)trlsphosphate. DG activates C kinase(s).lns(1,4,5)P 3 releases Ca2+ from a specific intracellular organelle, the calciosome. C8lciosomes resemble sarcoplasmic reticulum: they contain a Ca2+-ATPase and a high capacity/lOW affinity calcium-binding, calsequestrinlike protein. Chemoattractant receptor stimulation of calcium influx across the plasma membrane in phagocytes correlates strongly with the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P. by a Ca"·calmodulin·sensitive kinase. The transduction system of phagocytosis receptors also generates DG and Ins(1,4,5)P3 and elicits [Ca"I; elevations. The Ca" signal Is an important regulator of secretion (granule exocytosis, superoxide production), whereas C klnase(s)/and other unknown mediators appear to be more important for the control of movement. Several mechanisms that could account for the specificity of cell signaling by different receptors are discussed. AM REV RESPIR DIS 1990; 141:S127-S131
tion is to allow adherence (figure 1)(1). They are glycoproteins with two different subunits: an alpha subunit with an apparent molecular mass of about 160 kD and a beta subunit with a molecular mass of about 95 kD. The beta subunit is common to three of these proteins, Mol, LFAl, and p150,95(leukocyte adhesion proteins, LEUCAM). The best known of these proteins, Mol, mediates adhesion to surfaces or particles coated with iC3b or to activated endothelium. The Mol receptor is also the receptor involved in C3bi-mediated phagocytosis. The biologic importance of these receptors is underscored by the finding that their inherited deficiency in humans impairs leukocyteadhesion-dependent inflammatory functions and predisposes to life-threatening bacterial infections. LEU CAM are also members of a large family of cell adhesion heterodimeric receptors, the integrins, which include the fibronectins and possibly the Iamilin receptor. In addition to mediating adherence to specific proteins present in the basement membrane of the endothelium (lamilin) or in interstitial tissues (fibronectin), these receptors have been shown to modulate other important functions. In particular, the fibronectin receptor has been shown, in the presence of other stimuli, to activate complementmediated phagocytosis. Little is known about the signals generated by activation of adherence receptors. Recently, the development of the techniques that enable investigators to measure directly cytosolic free Ca 2 + ([Ca2+Ji) in single cells has allowed insight into the levels and fluctuations of this ion during neutrophil adherence and movement. Initially it was shown that the adherence step of neutrophils is associated with a single large [Ca"], spike. More recently, spontaneous oscillations of [Ca'"]; have been observed in single neutrophils adherent to nude or fibronectin-coated surfaces (figure 1) (7). The amplitude of these [Ca2+] i peaks
averaged approximately 100nm above basal, and their mean duration was around 30 s; oscillatory bursts of [Ca 2 +] i could last up to 15 min. These experiments, however, do not indicate if the [Ca2+li oscillations are "spontaneous," or occur secondarily to activation of adherence or other classes of receptors. In general, the evidence indicates that adherent neutrophils are much more active than cells in suspension. Thus, adherence primes or activates neutrophils, and this effect is possibly mediated through adherence receptors.
Chemoattractant Receptors This is the class of receptors where most information has been collected about the signaling network of neutrophils (1-6). Various chemoattractant receptors have been characterized and extensiveeffort is being performed to purify them. Among these receptors the three most known are: the receptors for formylated peptides derived from bacterial cell products [the most widely used compound in research is N-formylmethionine-Ieucylphenylalanine, (fMLP)]; the receptor for leukotriene B. (LTB.); and for the small complement fragment C5a (figure 2). These receptors share the property of being able to trigger directional movement in the presence of very low concentrations ofligand (10-'0 to 10-8 M), whereas at higher ligand concentration there is an arrest of cell movement and activation of secretory processes (10-7 to 10-6 M). Bindingstudies of fMLP to intact cellsdemonstrated the presence of receptors for this chemoattractant with approximately 55,000 binding sites per cell and an average Kd of 20 nM. The action of fMLP depends upon
I From the Infectious Diseases Division, Geneva University Hospital, Geneva, Switzerland. 2 Supported by a grant from the Swiss National Foundation (3.829.0.87).
S127
5128
P. DANIEL LEW
TUT'l8 (lTIIn)
Fig. 1 (Left). The adherence receptors on human neutrophils include the leukocyte adhesion proteins Mol, LFA1, and p150,95as well as the fibronectin and laminin receptors. (Right, top) Fluorescence of fura-z loaded neutrophil monitored at excitation wavelengths 350 and 380 nm simultaneously with a time resolution of 200 ms. (Right, bottom) Ratio of fura-2 fluorescence is converted into [Ca'+]i.
the hydrolysis of membrane phosphoinositides. Receptor activation (figure 2), via a specific G regulatory protein, activates a plasma membrane phospholipase C, inducing the cleavage of phosphatidyl-inositol-bisphosphate (PtdIns(4,5)P,), resulting in the formation of diacylglycerol (DG) and inositol(1,4, 5)trisphosphate (Ins(I,4,5)PJ ) (8-11). These two products then function as second messengers to activate two independent but parallel signal pathways. DG functions within the plane of the membrane to increase protein phosphorylation by activation of C kinase. Ins(I,4,5)P J is released in the cytosol to function as a second messenger to mobilize Ca2+ from intracellular stores. In addition, there are metabolic pathways that rapidly remove these internal signals or generate additional second messengers, when the external signal is withdrawn. An inositoltrisphosphatase converts Ins(I,4,5)P 3 into Ins(1,4)P" whereas DG is either phosphorylated to phosphatidic acid by a DG kinase or dephosphorylated to monoacyIglycerol by a DG lipase. Another pathway of Ins(1,4,5)PJ metabolism has become of
major interest more recently: it involves the conversion of this compound via a Ca-t-calmodulin-sensitive kinase to Ins(I,3,4,5)P., which in turn appears also to be a second messenger (9). The levels of these signals will depend upon the balance of their rates of formation and degradation.
Chemoattractant-induced Changes in Intracellular Ca2 +: The Role of Calciosomes and Receptor-triggered Ca2 + Influx The [Ca2+]i of a resting neutrophil is around 100nM, approximately 20,OOO-fold lowerthan the extracellular free Ca2+ concentration. To obtain and maintain this low [Ca'+li, intracellular Ca'" pumps sequester Ca2+ into intracellular Ca" stores and a plasma membrane Ca2+ pump transports Ca" to the extracellular space (12-14). During fMLP activation, there is a biphasic rise in Ca'" resulting from a transient Ca2+ release from internal stores and a more sustained Ca2+ influx from the extracellular space (11). There is increasing evidence that the intracellular store in a wide
variety of cellsis an organelle that werecently identified and termed the calciosome (14-18). Calciosomes contain two proteins similar to those present in muscle sarcoplasmic reticulum: calsequestrin (CS), a calsequestrinlike protein (Ca2+ storage protein), and the Ca"ATPase.Byseveralcell fractionation methods, the'Cs-like protein copurifies with the markers of the Ins(I,4,5)P J releasable Ca2+ store. In HL60 cells the CS-like protein was shown to be localized in small vacuoles with a diameter of between 50 and 250 nM. Both biochemical and morphologic approaches indicate that calciosomes are distinct from other organelles such as endoplasmic reticulum, Golgi, lysosomes, or endosomes. In phagocytes, receptor-triggered Ca2+ influx appears to involve the opening of a nonselective cation channel (11, 19). How Ca2+ entry is regulated remains to be ascertained. Studies in neutrophils, the HL60 human leukemia cell line, support the concept that activation of Ca2+ influx is dependent both on a rise in [Ca2+]i and a sustained elevation in Ins(I,3,4,5)P. (20). Single cell measurement of Ca2+ in adherent neutrophils reveals that low concentrations of fMLP induce sustained Ca2+ oscillations (10-10 to 10-9 M). By contrast, higher agonist concentrations (10-6 M) induced a singlelarge Ca" transient followedby a sustained non-oscillatory phase of Ca2+ elevation.
Ongoing Studies Although much has been learned about the signaling pathway of chemoattractant receptors, extensive efforts by several laboratories interested in neutrophils are presently being dedicated to the pursuit of the following objectives: (1) to purify and obtain structural information on the various chemoattractant receptors; (2)to characterize the chemoattractant receptor-linked G proteins - this has been
PlASMA
MEMBRANI!
chemota xissecretion
t. ,.. %(F )40)
Fig. 2 (Left). The chemoattractant receptors in human neutrophils. At low concentrations, chemoattractants trigger directional movement, whereas at high concentrations they trigger secretion. (Right) Schematic representation of the signal transduction pathway of chemoattractant receptors. IP3 (inositol 1,4,5 trisphosphate) releases Ca'+ from intracellular stores, the calciosomes.
5129
INTRACELWLAR SIGNALING IN NEUTROPHILS
helped by the fact that the G protein(s) may be ribosylated and inhibited by pertussis toxin (1-6, 8,21); (3) to study the role of the different inositol phosphates in neutrophil activation - in particular it is becoming increasingly clear that there is a large variety of highly phosphorylated inositol phosphates isomers, including various InsP 4, InsP 5, and InsP 6 (20, 22); and (4) to understand the exact role of C kinase enzymes in neutrophil activation. The molecular cloning of protein kinase C has revealed the existence of various C kinase subtypes whose presence and functions need to be assessed in different tissues (23).
Phagocytic Receptors Phagocytic leukocytes express on their surfaces a large variety of phagocytic receptors: these are the receptors for IgG, complement, mannose-terminated oligosaccharides, galactose-terminated oligosaccharides, and for advanced glycosylation products. Among those, the most comprehensively studied are the IgG and the complement receptors (figure 3) (1 and 4). Human cells have three different Fc receptors, which differ in sensitivity to trypsin, affinities for human immunoglobulin isotypes, ability to recognize monovalent or aggregated immunoglobulins, and expression on different types of phagocytic cells.Among Fc receptors present in neutrophils, the Fc gamma RIll has recently stirred considerable interest. In fact, the Fc gamma RIll is not a transmembrane protein; rather it is anchored to the plasma membrane by a glycosyl phosphatidylinositollinkage. It might thus be cleaved from the plasma membrane by a phospholipase C and be liberated in a soluble form (25). Three distinct receptors for the third component of complement have been identified; two of these receptors, CRI and CR3, mediate binding the phagocytosis of complementcoated particles and are important in immune clearance of microorganisms. Optimal ligand binding by the CR3 receptor requires divalent cations, while CRI function is not dependent on divalent cations. CRI (C3b receptor) preferentially binds C3b, but also recognizes iC3b and C4b. CR3 (Mo-l, a member of the LEU CAM family) is the receptor for C3bi. CRI and CR3 mediate binding and, when appropriately stimulated, phagocytosis. By contrast to Fc receptors that are constitutively active, the complement receptors must be rendered "phagocytosis competent" by an additional stimulus, and this is the case for the CRI receptor in human neutrophils where its activation appears to require both the presence of fibronectin and a chemotactic stimulus. Fc receptors generate several signals upon binding to ligands: severalinvestigations have documented changes in plasma membrane potential and ionic fluxes; additionally, there is clearly a [Ca'·1i transient due to release of Ca" from intracellular stores, followed by influx of Ca" across the plasma membrane. The phosphorylation of several proteins has also been detected and ongoing experiments indi-
Ph agocytosis -sec retio n
a
60
30
100
11M quin2/AM
Fig. 3 (Left). The phagocytosis receptors in human neutrophils include several receptors for IgG and the third component of complement. (Right) Phagocytosis of C3bi or IgG-coated particles by neutrophils incubated with various concentrations of quin2/AM in the absence of extracellular Ca" + 1 mM EGTA([Ca"]i out = 10-· M). This procedure depletes the cells of intracellular calcium and reveals that C3bi-mediated phagocytosis isa [Ca"li independent process, whereas IgG-mediated phagocytosis is modulated by [Ca"]; in human neutrophils.
cate that phagocytosis is associated with enhanced phosphoinositide turnover. In contrast to the situation with chemoattractant receptors, phagocytic receptor activation leads to a marked stimulation of DG production, which appears to be more sustained and generated in larger amounts than InsP •. This indicates sources for DG in addition to PdtIns(4,5)P" possibly phosphatidylcholine. The more potent and persistent levels of DG also suggest a role for protein kinase C in the control of phagocytosis. Additional differences with chemoattractant receptors are the absence of an inhibitory effect of pertussis toxin both on phagocytosis and phosphoinositide turnover triggered by phagocytic receptors, suggesting an involvement of a different G protein (24). More recently, we could demonstrate that [Ca'·]i elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils (36).
The Role of Ca'· and Protein Kinase C in the Control of Neutrophil Functions Various studies have shown that chemoattractant-triggered secretion requires an elevation of [Ca2+]i: indeed, when [Ca'·]i transients were specifically buffered by the intracellu-
larly trapped Ca'" chelator quin2, there was a profound inhibition of both granule content releaseand NADPH oxidase activity (figure 4). However, additional experiments indicated that for the control of these processes there is a necessary synergism of Ca2+ with other signals, and the most obvious candidate appears to be protein kinase C (26-29). Each secretory activity has a different Ca2+ sensitivity. This has been recently demonstrated in quin2-loaded neutrophils stimulated with ionomycin. While the Ca2+ threshold for ionomycin-induced secretion is similar (around 300 nM) for all types of granules, the [Ca'"]: giving half-maximal release (EC 5o ) is very different for the different granules, i.e.,about fivefold higher for azurophilic compared to specific and tertiary granules. Similar conclusions concerning the Ca" dependency of secretion have been also reached in permeabilized neutrophils (26-29). Although Ca2+ appears to be an important signal for the control of secretion, this does not appear to be the case for phagocytosis (30). We and others have reported that very drastic intracellular calcium depletion does not impair complement-mediated phagocytosis by human neutrophils (figure 3) or Femediated phagocytosis by macrophages (31).
FMLP 10-7 M FMLP
~
80
s
::!!
~ Q)
CJl
Ca~+ _ 10. 9 M
60
Introduction The main function of human neutrophils is to sense, approach, and destroy invading microorganisms, in particular pyogenic bacteria. The ultrastructure of a neutrophil reveals a cell extremely rich in various populations of granules, poor in mitochondria and rough endoplasmic reticulum, and with a dystrophic nucleus. Neutrophils are terminally differentiated cells, unable to replicate and are able to synthesize only low amounts of proteins. Yet they are very motile cells and are able to selectively release proteins from various populations of granules. In addition, they contain a rather unique plasma membrane enzyme (NADPH-oxidase), which, when activated, generates and releases active oxygen species into the extracellular space. The products released from granules and the active oxygen species alone or in combinations are potently bactericidal. Human neutrophils, when observed under phase-contrast microscopy,crawl rapidly over surfaces; to perform this process, they adhere and polarize into a typical asymmetrical configuration with a large anterior lamelopodim and a short tail-like uropod. The functions of adherence, perception of distant microbial or inflammatory stimuli, movement, phagocytosis, and secretion are modulated by a large variety of receptors. This review deals with receptor-generated signals in neutrophils and summarizes some of our contributions to this area of research. A comprehensive textbook on neutrophils and inflammation and several reviews have appeared recently (1-6). It is possible to divide the various classes of receptors into five main groups according to their main function: adherence receptors; chemotactic receptors; phagocytic receptors; cytokine-sensitive receptors; and receptors of unknown function. Little is known of the structure, location, and function of cytokine-sensitive receptors in neutrophils. Several substances of this group modulate and activate neutrophil functions. Thmor necrosis factor (TNF) activates the oxidative burst of adherent neutrophils. Granulocyte-macrophage colony-stimulating factor (GMCSF), in addition to its known effects in the bone marrow, primes neutrophils to subsequent stimulation (1). There are also several other receptors of unknown function, but whose presence has been welldocumented, in particular for several hormones, such as TSH. Thus, in the following sections we will discuss only the first three classes of receptors listed above: adherence, chemotaxis, and phagocytosis. Adherence Receptors A family of receptors is present in the plasma membrane of neutrophils, whose main func-
SUMMARY Adherence, chemotaxis, phagocytosis, and responses to cytokines are mediated by distinct classes of cell surface receptors in human neutrophils.lntracellular signaling by these different receptors Is a SUbject of active investigation. Observation of single neutrophils adherent to surfaces reveals the presence of spontaneous oscillations of cytosolic-free calcium, [Ca2+ll, generated by mechanisms that are presently unknown. Chemoattractant receptor activation via a specific G-regula· tory protein activates a plasma membrane phospholipase C and generates diacylglycerol and inosi· tol(1,4,5)trlsphosphate. DG activates C kinase(s).lns(1,4,5)P 3 releases Ca2+ from a specific intracellular organelle, the calciosome. C8lciosomes resemble sarcoplasmic reticulum: they contain a Ca2+-ATPase and a high capacity/lOW affinity calcium-binding, calsequestrinlike protein. Chemoattractant receptor stimulation of calcium influx across the plasma membrane in phagocytes correlates strongly with the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P. by a Ca"·calmodulin·sensitive kinase. The transduction system of phagocytosis receptors also generates DG and Ins(1,4,5)P3 and elicits [Ca"I; elevations. The Ca" signal Is an important regulator of secretion (granule exocytosis, superoxide production), whereas C klnase(s)/and other unknown mediators appear to be more important for the control of movement. Several mechanisms that could account for the specificity of cell signaling by different receptors are discussed. AM REV RESPIR DIS 1990; 141:S127-S131
tion is to allow adherence (figure 1)(1). They are glycoproteins with two different subunits: an alpha subunit with an apparent molecular mass of about 160 kD and a beta subunit with a molecular mass of about 95 kD. The beta subunit is common to three of these proteins, Mol, LFAl, and p150,95(leukocyte adhesion proteins, LEUCAM). The best known of these proteins, Mol, mediates adhesion to surfaces or particles coated with iC3b or to activated endothelium. The Mol receptor is also the receptor involved in C3bi-mediated phagocytosis. The biologic importance of these receptors is underscored by the finding that their inherited deficiency in humans impairs leukocyteadhesion-dependent inflammatory functions and predisposes to life-threatening bacterial infections. LEU CAM are also members of a large family of cell adhesion heterodimeric receptors, the integrins, which include the fibronectins and possibly the Iamilin receptor. In addition to mediating adherence to specific proteins present in the basement membrane of the endothelium (lamilin) or in interstitial tissues (fibronectin), these receptors have been shown to modulate other important functions. In particular, the fibronectin receptor has been shown, in the presence of other stimuli, to activate complementmediated phagocytosis. Little is known about the signals generated by activation of adherence receptors. Recently, the development of the techniques that enable investigators to measure directly cytosolic free Ca 2 + ([Ca2+Ji) in single cells has allowed insight into the levels and fluctuations of this ion during neutrophil adherence and movement. Initially it was shown that the adherence step of neutrophils is associated with a single large [Ca"], spike. More recently, spontaneous oscillations of [Ca'"]; have been observed in single neutrophils adherent to nude or fibronectin-coated surfaces (figure 1) (7). The amplitude of these [Ca2+] i peaks
averaged approximately 100nm above basal, and their mean duration was around 30 s; oscillatory bursts of [Ca 2 +] i could last up to 15 min. These experiments, however, do not indicate if the [Ca2+li oscillations are "spontaneous," or occur secondarily to activation of adherence or other classes of receptors. In general, the evidence indicates that adherent neutrophils are much more active than cells in suspension. Thus, adherence primes or activates neutrophils, and this effect is possibly mediated through adherence receptors.
Chemoattractant Receptors This is the class of receptors where most information has been collected about the signaling network of neutrophils (1-6). Various chemoattractant receptors have been characterized and extensiveeffort is being performed to purify them. Among these receptors the three most known are: the receptors for formylated peptides derived from bacterial cell products [the most widely used compound in research is N-formylmethionine-Ieucylphenylalanine, (fMLP)]; the receptor for leukotriene B. (LTB.); and for the small complement fragment C5a (figure 2). These receptors share the property of being able to trigger directional movement in the presence of very low concentrations ofligand (10-'0 to 10-8 M), whereas at higher ligand concentration there is an arrest of cell movement and activation of secretory processes (10-7 to 10-6 M). Bindingstudies of fMLP to intact cellsdemonstrated the presence of receptors for this chemoattractant with approximately 55,000 binding sites per cell and an average Kd of 20 nM. The action of fMLP depends upon
I From the Infectious Diseases Division, Geneva University Hospital, Geneva, Switzerland. 2 Supported by a grant from the Swiss National Foundation (3.829.0.87).
S127
5128
P. DANIEL LEW
TUT'l8 (lTIIn)
Fig. 1 (Left). The adherence receptors on human neutrophils include the leukocyte adhesion proteins Mol, LFA1, and p150,95as well as the fibronectin and laminin receptors. (Right, top) Fluorescence of fura-z loaded neutrophil monitored at excitation wavelengths 350 and 380 nm simultaneously with a time resolution of 200 ms. (Right, bottom) Ratio of fura-2 fluorescence is converted into [Ca'+]i.
the hydrolysis of membrane phosphoinositides. Receptor activation (figure 2), via a specific G regulatory protein, activates a plasma membrane phospholipase C, inducing the cleavage of phosphatidyl-inositol-bisphosphate (PtdIns(4,5)P,), resulting in the formation of diacylglycerol (DG) and inositol(1,4, 5)trisphosphate (Ins(I,4,5)PJ ) (8-11). These two products then function as second messengers to activate two independent but parallel signal pathways. DG functions within the plane of the membrane to increase protein phosphorylation by activation of C kinase. Ins(I,4,5)P J is released in the cytosol to function as a second messenger to mobilize Ca2+ from intracellular stores. In addition, there are metabolic pathways that rapidly remove these internal signals or generate additional second messengers, when the external signal is withdrawn. An inositoltrisphosphatase converts Ins(I,4,5)P 3 into Ins(1,4)P" whereas DG is either phosphorylated to phosphatidic acid by a DG kinase or dephosphorylated to monoacyIglycerol by a DG lipase. Another pathway of Ins(1,4,5)PJ metabolism has become of
major interest more recently: it involves the conversion of this compound via a Ca-t-calmodulin-sensitive kinase to Ins(I,3,4,5)P., which in turn appears also to be a second messenger (9). The levels of these signals will depend upon the balance of their rates of formation and degradation.
Chemoattractant-induced Changes in Intracellular Ca2 +: The Role of Calciosomes and Receptor-triggered Ca2 + Influx The [Ca2+]i of a resting neutrophil is around 100nM, approximately 20,OOO-fold lowerthan the extracellular free Ca2+ concentration. To obtain and maintain this low [Ca'+li, intracellular Ca'" pumps sequester Ca2+ into intracellular Ca" stores and a plasma membrane Ca2+ pump transports Ca" to the extracellular space (12-14). During fMLP activation, there is a biphasic rise in Ca'" resulting from a transient Ca2+ release from internal stores and a more sustained Ca2+ influx from the extracellular space (11). There is increasing evidence that the intracellular store in a wide
variety of cellsis an organelle that werecently identified and termed the calciosome (14-18). Calciosomes contain two proteins similar to those present in muscle sarcoplasmic reticulum: calsequestrin (CS), a calsequestrinlike protein (Ca2+ storage protein), and the Ca"ATPase.Byseveralcell fractionation methods, the'Cs-like protein copurifies with the markers of the Ins(I,4,5)P J releasable Ca2+ store. In HL60 cells the CS-like protein was shown to be localized in small vacuoles with a diameter of between 50 and 250 nM. Both biochemical and morphologic approaches indicate that calciosomes are distinct from other organelles such as endoplasmic reticulum, Golgi, lysosomes, or endosomes. In phagocytes, receptor-triggered Ca2+ influx appears to involve the opening of a nonselective cation channel (11, 19). How Ca2+ entry is regulated remains to be ascertained. Studies in neutrophils, the HL60 human leukemia cell line, support the concept that activation of Ca2+ influx is dependent both on a rise in [Ca2+]i and a sustained elevation in Ins(I,3,4,5)P. (20). Single cell measurement of Ca2+ in adherent neutrophils reveals that low concentrations of fMLP induce sustained Ca2+ oscillations (10-10 to 10-9 M). By contrast, higher agonist concentrations (10-6 M) induced a singlelarge Ca" transient followedby a sustained non-oscillatory phase of Ca2+ elevation.
Ongoing Studies Although much has been learned about the signaling pathway of chemoattractant receptors, extensive efforts by several laboratories interested in neutrophils are presently being dedicated to the pursuit of the following objectives: (1) to purify and obtain structural information on the various chemoattractant receptors; (2)to characterize the chemoattractant receptor-linked G proteins - this has been
PlASMA
MEMBRANI!
chemota xissecretion
t. ,.. %(F )40)
Fig. 2 (Left). The chemoattractant receptors in human neutrophils. At low concentrations, chemoattractants trigger directional movement, whereas at high concentrations they trigger secretion. (Right) Schematic representation of the signal transduction pathway of chemoattractant receptors. IP3 (inositol 1,4,5 trisphosphate) releases Ca'+ from intracellular stores, the calciosomes.
5129
INTRACELWLAR SIGNALING IN NEUTROPHILS
helped by the fact that the G protein(s) may be ribosylated and inhibited by pertussis toxin (1-6, 8,21); (3) to study the role of the different inositol phosphates in neutrophil activation - in particular it is becoming increasingly clear that there is a large variety of highly phosphorylated inositol phosphates isomers, including various InsP 4, InsP 5, and InsP 6 (20, 22); and (4) to understand the exact role of C kinase enzymes in neutrophil activation. The molecular cloning of protein kinase C has revealed the existence of various C kinase subtypes whose presence and functions need to be assessed in different tissues (23).
Phagocytic Receptors Phagocytic leukocytes express on their surfaces a large variety of phagocytic receptors: these are the receptors for IgG, complement, mannose-terminated oligosaccharides, galactose-terminated oligosaccharides, and for advanced glycosylation products. Among those, the most comprehensively studied are the IgG and the complement receptors (figure 3) (1 and 4). Human cells have three different Fc receptors, which differ in sensitivity to trypsin, affinities for human immunoglobulin isotypes, ability to recognize monovalent or aggregated immunoglobulins, and expression on different types of phagocytic cells.Among Fc receptors present in neutrophils, the Fc gamma RIll has recently stirred considerable interest. In fact, the Fc gamma RIll is not a transmembrane protein; rather it is anchored to the plasma membrane by a glycosyl phosphatidylinositollinkage. It might thus be cleaved from the plasma membrane by a phospholipase C and be liberated in a soluble form (25). Three distinct receptors for the third component of complement have been identified; two of these receptors, CRI and CR3, mediate binding the phagocytosis of complementcoated particles and are important in immune clearance of microorganisms. Optimal ligand binding by the CR3 receptor requires divalent cations, while CRI function is not dependent on divalent cations. CRI (C3b receptor) preferentially binds C3b, but also recognizes iC3b and C4b. CR3 (Mo-l, a member of the LEU CAM family) is the receptor for C3bi. CRI and CR3 mediate binding and, when appropriately stimulated, phagocytosis. By contrast to Fc receptors that are constitutively active, the complement receptors must be rendered "phagocytosis competent" by an additional stimulus, and this is the case for the CRI receptor in human neutrophils where its activation appears to require both the presence of fibronectin and a chemotactic stimulus. Fc receptors generate several signals upon binding to ligands: severalinvestigations have documented changes in plasma membrane potential and ionic fluxes; additionally, there is clearly a [Ca'·1i transient due to release of Ca" from intracellular stores, followed by influx of Ca" across the plasma membrane. The phosphorylation of several proteins has also been detected and ongoing experiments indi-
Ph agocytosis -sec retio n
a
60
30
100
11M quin2/AM
Fig. 3 (Left). The phagocytosis receptors in human neutrophils include several receptors for IgG and the third component of complement. (Right) Phagocytosis of C3bi or IgG-coated particles by neutrophils incubated with various concentrations of quin2/AM in the absence of extracellular Ca" + 1 mM EGTA([Ca"]i out = 10-· M). This procedure depletes the cells of intracellular calcium and reveals that C3bi-mediated phagocytosis isa [Ca"li independent process, whereas IgG-mediated phagocytosis is modulated by [Ca"]; in human neutrophils.
cate that phagocytosis is associated with enhanced phosphoinositide turnover. In contrast to the situation with chemoattractant receptors, phagocytic receptor activation leads to a marked stimulation of DG production, which appears to be more sustained and generated in larger amounts than InsP •. This indicates sources for DG in addition to PdtIns(4,5)P" possibly phosphatidylcholine. The more potent and persistent levels of DG also suggest a role for protein kinase C in the control of phagocytosis. Additional differences with chemoattractant receptors are the absence of an inhibitory effect of pertussis toxin both on phagocytosis and phosphoinositide turnover triggered by phagocytic receptors, suggesting an involvement of a different G protein (24). More recently, we could demonstrate that [Ca'·]i elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils (36).
The Role of Ca'· and Protein Kinase C in the Control of Neutrophil Functions Various studies have shown that chemoattractant-triggered secretion requires an elevation of [Ca2+]i: indeed, when [Ca'·]i transients were specifically buffered by the intracellu-
larly trapped Ca'" chelator quin2, there was a profound inhibition of both granule content releaseand NADPH oxidase activity (figure 4). However, additional experiments indicated that for the control of these processes there is a necessary synergism of Ca2+ with other signals, and the most obvious candidate appears to be protein kinase C (26-29). Each secretory activity has a different Ca2+ sensitivity. This has been recently demonstrated in quin2-loaded neutrophils stimulated with ionomycin. While the Ca2+ threshold for ionomycin-induced secretion is similar (around 300 nM) for all types of granules, the [Ca'"]: giving half-maximal release (EC 5o ) is very different for the different granules, i.e.,about fivefold higher for azurophilic compared to specific and tertiary granules. Similar conclusions concerning the Ca" dependency of secretion have been also reached in permeabilized neutrophils (26-29). Although Ca2+ appears to be an important signal for the control of secretion, this does not appear to be the case for phagocytosis (30). We and others have reported that very drastic intracellular calcium depletion does not impair complement-mediated phagocytosis by human neutrophils (figure 3) or Femediated phagocytosis by macrophages (31).
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Ca~+ _ 10. 9 M
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