Journal of Cell Science 113, 2935-2940 (2000) 2935 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1421

COMMENTARY Roles of phospholipid signaling in chemoattractant-induced responses

Dianqing Wu1,*, Chi-Kuang Huang2 and Huiping Jiang3 1Department of Genetics and Developmental Biology and 2Department of Pathology, University of Connecticut, Farmington, CT 06030, USA 3Department of Pharmacology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT 06877, USA *Author for correspondence

Published on WWW 9 August 2000

SUMMARY

Chemoattractants, including chemokines, play a central and PLC-β3 to hydrolyze PtdIns(4,5)P2 and through PI3Kγ role in regulation of inflammatory reactions by attracting to phosphorylate PtdIns(4,5)P2 in mouse neutrophils. and activating leukocytes. These molecules have been found These studies also confirmed the importance and revealed to regulate metabolism of phosphatidylinositol 4,5- new roles of these signaling pathways in chemoattractant- bisphosphate (PtdIns(4,5)P2) via phospholipase C (PLC) induced responses. and phosphoinositide 3-kinase (PI3K). Recent studies of mouse lines that lack PLC-β2, PLC-β3, or PI3Kγ Key words: Chemoattractant, Chemokine, G protein, Chemotaxis, demonstrate that chemoattractants act through PLC-β2 Phospholipase, Phosphoinositide 3-kinase, Gene targeting

INTRODUCTION arthritis, ischemia-reperfusion injury, arteriosclerosis, virus- induced myocarditis, psoriasis and other inflammatory skin Many biologically active molecules function as conditions, and allergic reactions (Furie and Randolph, 1995). chemoattractants and activators of leukocytes. These include The understanding of signaling mechanisms that mediate the bacterial by-products formyl-Met-Leu-Phe (fMLP) and its actions of chemoattractants should provide more suitable related peptides, lipid derivatives such as platelet-activating targets for novel therapeutic approaches and agents that factor, complement proteolysis fragments such as C5a, and the combat inflammation. Evidence indicates that the superfamily of small (8-10 kDa), inducible, secreted, pro- chemoattractant receptors can couple to Gi proteins, a class of inflammatory cytokines called chemokines. Chemokines are G protein that is sensitive to Pertussis toxin (PTx). PTx is a produced and secreted by various cell types, usually upon bacterial toxin that specifically modifies a subset of Gα stimulation by injuries, infections, or other pro-inflammatory subunits including Gαi, Gαo and transducin α subunits, but cytokines. Members of the chemokine family share amino acid not the α subunits of the Gs, Gq or G12 classes, and prevents sequence homology and are currently divided into four classes functional coupling of receptors to modified G proteins. The (CXC, CC, C and CX3C) on the basis of the arrangement of sensitivity of chemoattractant-induced responses to PTx the conserved cysteine residues of the mature proteins (see suggests that the Gi proteins might mediate chemoattractant recent reviews for details: Baggiolini et al., 1997; Zlotnik and signal transduction. Pharmacological, biochemical and Yoshie, 2000). These chemoattractants exert their effects by transfection approaches have been used to characterize two interacting with specific cell surface receptors. To date, a large signaling pathways for chemoattractants that are mediated number of chemoattractant receptors have been cloned and by Gi proteins. Both pathways involve metabolism of sequenced (Murphy, 1994; Schall, 1994; Zlotnik and Yoshie, phosphatidylinositides, particularly phosphatidylinositol 4,5- 2000). They include the fMLP and C5a receptors, five CXC bisphosphate (PtdIns(4,5)P2). Chemoattractants were shown chemokine receptors, ten CC chemokine receptors, one C to stimulate hydrolysis and phosphorylation of PtdIns(4,5)P2 chemokine receptor and one CX3C chemokines receptor. All via phospholipase C (PLC) and phosphoinositide 3-kinase these receptors possess seven transmembrane domains, which (PI3K), respectively, in various leukocytes. The finding that are characteristic of G protein-coupled receptors. Gβγ can activate PLC-β2, PLC-β3 and PI3Kγ suggests that Great attention has been paid to these chemotactic chemoattractant receptors activate PLC and PI3K probably molecules because of their roles in inflammatory reactions through the Gβγ subunits that are released from the Gi and lymphocyte development and function. Although proteins. Pharmacological studies and studies using other inflammation plays an important role in host defense, approaches have implicated the PLC and PI3K pathways in a uncontrolled inflammatory reactions are responsible for a variety of chemoattractant-induced responses. Recent studies variety of pathological conditions, including rheumatoid of mouse lines that lack these effector proteins have provided 2936 D. Wu, C.-K. Huang and H. Jiang novel insights into the roles of these pathways in CHEMOATTRACTANT-INDUCED PtdIns(3,4,5)P3 chemoattractant-induced responses. Here, we focus on these PRODUCTION IN MOUSE NEUTROPHILS recent advances. Chemoattractant receptors stimulate the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in CHEMOATTRACTANTS ACTIVATE PLC-β2 AND PLC- leukocytes. The finding (Stephens et al., 1997) that Gβγ β3 IN MOUSE NEUTROPHILS potently activates PI3Kγ suggests that chemoattractant receptors may activate PI3Kγ through the Gβγ subunits Many chemoattractants induce rapid elevation of cytosolic released from Gi proteins. However, the results from two Ca2+ levels, which presumably results from activation of PLC studies using tyrosine kinase inhibitors contradicted this (Murphy, 1994). PLC hydrolyzes PtdIns(4,5)P2 to generate hypothesis (Ptasznik et al., 1996; Thelen and Didichenko, two important second messengers, inositol 1,3,4-triphosphate 1997). These studies suggested that PI3Kγ is not the major (Ins(1,4,5)P3) and diacylglycerol (DAG; Berridge, 1989). The PI3K isoform responsible for fMLP-induced production of known phosphatidylinositide-specific PLC-molecules can be PtdIns(3,4,5)P3 in human neutrophils. The gene targeting divided into three families: β, γ and δ (Rhee and Bae, 1997). experiments, however, clearly demonstrated that PI3Kγ is the The β family, which consists of four isoforms, PLC-β1-PLC- major isoform in mouse neutrophils. Three groups have β4, is regulated by G proteins (Rhee and Bae, 1997). PLC-β2 independently shown that mouse neutrophils lacking PI3Kγ do has been detected primarily in hematopoietic cells thus far, not produce detectable levels of PtdIns(3,4,5)P3 in response to whereas PLC-β3 and PLC-β1 are found in a wide range of fMLP (Hirsch et al., 2000; Li et al., 2000; Sasaki et al., 2000), cells and tissues (Rhee and Bae, 1997). PLC-β4 is IL-8 and C5a (Hirsch et al., 2000). The apparent discrepancy predominantly expressed in certain neuronal cells (Jiang et al., between the results from human and mouse neutrophils may 1996b; Kano et al., 1998). We have demonstrated using the be due to the specie difference, lack of specificity of the cotransfection assays that many chemoattractant receptors, inhibitors, or possibility that other PI3K isoforms might be including CXCR-1 and CXCR-2 (Wu et al., 1993), CCR-1 and involved in amplification of the signal initiated by PI3Kγ. CCR-2 (Kuang et al., 1996) CCR-5, CXCR-4 (D. Wu et al., unpublished data), and the fMLP receptor and C5a receptor (Jiang et al., 1996a) can couple to the Gi proteins to stimulate ROLES OF PHOSPHOLIPID SIGNALING IN PLC activities. Although the α subunits of Gi proteins cannot CHEMOATTRACTANT-INDUCED CHEMOTAXIS regulate PLC activity directly (Wu et al., 1992), the βγ subunits released from activated Gi proteins were shown to Chemoattractants induce a wide range of responses in activate PLC-β2 (Jiang et al., 1996a; Katz et al., 1992; Kuang leukocytes, including chemotaxis, superoxide production, et al., 1996; Wu et al., 1993). This was further confirmed by cytoskeleton reorganization, and upregulation of cell surface the observations that purified Gβγ proteins activated adhesion molecules. The PLC-β2/PLC-β3-null and PI3Kγ-null recombinant PLC-β2 as well as PLC-β3 (Camps et al., 1992; mouse lines, in which two prominent phospholipid signaling Carozzi et al., 1993; Smrcka and Sternweis, 1993). Since pathways are largely abrogated in neutrophils, allowed us to responses to many chemoattractants in mature leukocytes examine genetically the roles of these pathways in these are sensitive to PTx, the Gi-Gβγ-PLC-β2/β3 pathway was chemoattractant-elicited responses. Chemotaxis attracts the postulated to occur in leukocytes. most attention because this process plays a key role in We investigated the significance of the β2 and β3 isoforms inflammatory responses. Previous studies have shown that of PLC in chemoattractant-induced PLC activation in mouse clamping intracellular Ca2+ with Ca2+ chelators does not neutrophils, using gene targeting. Neutrophils isolated from appear to affect chemoattractant-induced chemotaxis of mice lacking PLC-β2 showed significant reduction in PLC neutrophils in the absence of adhesive molecules (Alteraifi and activation and Ca2+ efflux in response to fMLP and IL-8 Zhelev, 1997; Elferink et al., 1992; Fabbri et al., 1997; Kuijpers (Jiang et al., 1997), which suggests that the PLC-β2 isoform et al., 1992; Marks et al., 1991). However, some of these can mediate chemoattractant-induced PLC activation. studies also indicated that chemoattractant-induced chemotaxis However, this study also suggested that PLC-β2 is not the of neutrophils on surfaces coated with adhesive molecules only PLC isoform active in neutrophils. We observed residual appears to depend on intracellular Ca2+ (Alteraifi and Zhelev, levels of PLC activity and Ca2+ efflux in response to 1997; Hofman et al., 1999; Mandeville and Maxfield, 1997; chemoattractants in neutrophils lacking PLC-β2 (Jiang et al., Marks et al., 1991). The idea that chemoattractant-induced 1997). Another Gβγ-regulated PLC-β isoform, PLC-β3, increases in cytosolic Ca++ levels are not required for might be responsible for the residual activities. Subsequent chemotaxis in the absence of adhesive molecules is consistent study of PLC-β3-deficient mice and those that lack both PLC- with the finding that neutrophils lacking both PLC-β2 and β2 and PLC-β3 confirmed this idea, showing that neutrophils PLC-β3, despite showing no fMLP-induced Ca2+ efflux, lacking both PLC-β2 and PLC-β3 cannot respond to respond to fMLP as well as do wild-type cells in the modified chemoattractants in PLC and Ca2+ efflux assays (Li et al., Boyden-chamber assay (Jiang et al., 1997; Li et al., 2000). We 2000). This result indicates that PLC-β2 and PLC-β3 are the have also examined two important chemotactic parameters – only isoforms that are responsible for chemoattractant- speed and orientation – using Zigmond chambers containing induced PLC activation in mouse neutrophils. The fact that gradients of fMLP and time-lapsed video microscopy. We PLC-β3-deficiency alone does not markedly affect the PLC did not observe significant differences between wild-type activities suggests that PLC-β2 is the major PLC isoform in and PLC-β2/PLC-β3-deficient neutrophils in these two neutrophils (Li et al., 2000). chemotactic parameters (Huang et al., unpublished data). Roles of phospholipid signaling in chemoattractant-induced responses 2937

These results further confirm the conclusion above. Studies of PtdIns(3,4,5)P3 plays a regulatory role in chemotaxis, rather PLC-deficient neutrophils also suggest that chemoattractant- than functioning as a primary mediator for chemoattractant- induced changes in intracellular Ca2+ levels are not required mediated motilty. for neutrophil chemotaxis in vivo, because PLC-deficiency What is the precise role of PI3Kγ in neutrophil chemotaxis? does not impair the infiltration of neutrophils into peritonea in Recent findings (Jin et al., 2000; Servant et al., 2000) that a bacteria-induced peritonitis model (Li et al., 2000). This revealed the polarized nature of neutrophils have implicated result appears to contradict those from the Ca2+-clamping PI3K in polarization. They showed that pleckstrin homology studies (Alteraifi and Zhelev, 1997; Hofman et al., 1999; (PH)-domain-containing proteins relocate to the leading edges Mandeville and Maxfield, 1997; Marks et al., 1991). Knowing of stimulated chemotactic cells (Parent et al., 1998; Servant et that Ca2+ chelators used in the Ca2+-clamping studies may not al., 2000). Since PH domains are believed to be involved in 2+ only block ligand-induced increases in intracellular Ca binding to PtdIns(3,4,5)P3, it is reasonable to hypothesize that concentrations, but also reduce the intracellular Ca2+ activation of PI3K has a role in translocation of these PH- concentrations below the basal levels (those in the absence of domain proteins to the leading edges of polarized cells. Our ligands), one may interpret the apparent discrepancy to suggest recent observations support the idea that PI3Kγ is involved in that the basal, but not chemoattractant-elevated, levels of Ca2+ neutrophil polarization. We examined, using confocal are critical for adhesion-dependent chemotaxis. microscopy with serial optical sectioning, the subcellular In contrast to lack of impairment of IL-8- or fMLP-induced localization of Akt and F-actin in neutrophils lacking PI3Kγ chemotaxis in cells lacking PLC-β isoforms, MIP1α-induced undergoing chemotaxis in Zigmond chambers containing chemotaxis of neutrophils lacking PLC-β2 is enhanced (Jiang gradients of fMLP. In wildtype cells, Akt and cortical actin et al., 1997), suggesting that the PLC-β2 pathway selectively were colocalized at the leading edges of chemotactic cells at downregulates chemoattractant-induced chemotactic activities. both the top and bottom parts of the cells. However, in the Similar results were also observed for neutrophils lacking both PI3Kγ-null cells, although Akt and cortical actin are still PLC-β2 and PLC-β3 (Li et al., 2000). In addition, PLC-β2- colocalized at the leading edges following the gradient at the deficiency also enhances chemotaxis of eosenophils in bottom optical section that is close to the cell attachment site, response to Eotaxin and of T cells in response to MIP1α and these two proteins no longer locate at the leading edges of the RANTES (Jiang et al., 1997). The finding that PLC deficiency top part of cells that follow the fMLP gradient. It seems that results in an enhanced response is not restricted to more than one factor regulates cell polarization: something hematopoietic cells; PLC-β3 appears to inhibit the effects of related to cell attachment and PI3Kγ are required for the µ-opioid receptor, another Gi/o-coupled receptor, as establishing cell polarity and each factor appear to have determined by both behavioral studies and electrophysiological predominant effect on different part of cells. Thus, it is not study of dorsal root ganglion neurons (Xie et al., 1999). All difficult to image that PI3K deficiency should cause problems these results suggest that PLC-β2 and PLC-β3 may underlie in direction sensing. In fact, when cell movement was physiological pathways that inhibit certain responses induced examined in the Zygmond chamber, the PI3Kγ-null cells clear by some Gi-coupled receptors. The molecular mechanisms of lack a sense of direction, i.e. PI3K-null cells moved in all these PLC-mediated inhibitory effects, especially those directions while wild-type cells generally move following the involved in chemotaxis, will be particular interesting. The gradient of chemoattractants. In addition, we found that the selective effect of the PLC-deficiency on some of the mutant cells moved at approximately half of the speed of wild- chemokines suggests that different chemoattractant receptors type cells (Huang et al., unpublished data). The slower have different sensitivities to PLC-mediated modification. A movement speed of the mutant cells may be caused by plausible hypothesis is that the PLC-pathway leads, via PKC, conflicting polarization signals or modulation of motility by to phosphorylation of chemoattractant receptors, resulting in PI3Kγ. The idea that multiple factors regulate cell polarization enhanced desensitization of some of the receptors (Ali et al., is supported by the finding that RhoA G proteins might also be 1999). This hypothesis needs to be investigated experimentally. involved in cell polarization (Servant et al., 2000). With regard to the involvement of PI3K in chemotaxis, there It is also worth noting that the importance of PI3Kγ in seem to be contradictory results from various pharmacological chemotaxis appears to be restricted to the myeloid lineage, studies. Treatment with PI3K inhibitors, including wortmaninn because PI3Kγ deficiency does not alter chemotactic activities and LY-294002, inhibited chemotaxis in some studies (Coffer of spleen B cells in response to SDF-1, MIP3β or fractalkine et al., 1998; Harakawa et al., 1997; Knall et al., 1997) but not (D. Wu et al., unpublished results). There are two possible in others (Neptune and Bourne, 1997; Thelen et al., 1995). explanations for the lack of effects on B cells: (1) there might Studies of mice lacking PI3Kγ showed that PI3Kγ appears to be PI3Kγ homologs in B cells that compensate for the lack of play an important role in neutrophil chemotaxis (Hirsch et al., PI3Kγ (determination of SDF-1-induced PtdIns(3,4,5)P3 2000; Li et al., 2000; Sasaki et al., 2000). Neutrophils purified formation in B cells would provide an answer); and (2) PI3K from either the bone marrow or the peritoneal cavity of PI3Kγ- might not be essential for B cell chemotaxis. null mice showed a approximately 50% reduction in activities measured in the modified Boyden assay in response to fMLP, MIP1α, IL-8 and C5a in comparison with those from wild-type ROLES OF PHOSPHOLIPID SIGNALING IN animals. The activities of neutrophils were also impaired in in CHEMOATTRACTANT-INDUCED SUPEROXIDE vivo infiltration assays. In addition, macrophages from the FORMATION PI3Kγ-deficient mice appear less chemotactic (Hirsch et al., 2000). Interestingly, in all these assays, cells lacking PI3Kγ Superoxide production plays an important role in innate still show chemotactic motility, although reduced. It seems that immunity. Many stimuli, including chemoattractants, can 2938 D. Wu, C.-K. Huang and H. Jiang stimulate its production (Babior, 1999; Baggiolini et al., 1993; protein via a more direct pathway in neutrophils. These results, Bokoch, 1995). Pharmacological studies have implicated both however, appear to be in disparity with those obtained from PI3K and PLC-PKC pathways in regulation of superoxide studies of human neutrophils using PI3K inhibitors (Akasaki production (Lew, 1990; Park and Babior, 1992; Thelen et al., et al., 1999; Benard et al., 1999). Benard et al. also showed that 1994; Vlahos et al., 1995). These results were in fact confirmed a tyrosine inhibitor could block fMLP-induced Rac activation. by the studies of the knockout mice. Chemoattractant-induced These discrepancies reminisce those described above for which production of superoxides is blocked in neutrophils isolated PI3K isoforms are predominantly involved in fMLP-induced from PI3Kγ-deficient (Hirsch et al., 2000; Li et al., 2000; production of PtdIns(3,4,5)P3, and may be explained by the Sasaki et al., 2000) or PLC-β2/PLC-β3-deficient mice (Jiang same possibilities. et al., 1997; Li et al., 2000). However, cells from these mice Although the mechanisms by which PI3Kγ regulates can still produce superoxides in response to other stimuli such superoxide production remain to be characterized, the as lipopolysaccharide (LPS; D. Wu et al., unpublished data) involvement of the PLC-pathway in production of superoxides and phorbol esters (Sasaki et al., 2000). The apparent lack of appears to involve at least the translocation of p47phox, one of any increase in fatality due to spontaneous microbial infection the components of the NADPH oxidase. The PLC-β2/PLC-β3 of PLC-β2/PLC-β3- or PI3Kγ-deficient mice that are housed deficiency, but not the PI3Kγ deficiency, impairs the in non-germ-free environments suggests that chemoattractant- translocation of the protein from the cytosol to membranes elicited superoxide production plays an auxiliary role in host (Li et al., 2000). Because PKC-mediated phosphorylation of defense. p47phox regulates this translocation process (Park and Babior, The small GTP-binding protein Rac regulates superoxide 1992) and PLC deficiency blocks chemoattractant-induced production by directly interacting with the NADPH oxidase PKC activation (Li et al., 2000), it is reasonable to believe that (Dagher et al., 1995; Knaus et al., 1991; Umeki, 1994). Some the PLC pathway regulates p47phox translocation via PKC. studies using transfection and pharmacological reagents suggests that the PI3K pathway may be involved in regulation of small GTP-binding proteins including Rac (Akasaki et al., INVOLVEMENT OF PHOSPHOLIPID SIGNALING IN 1999; Bacon, 1999; Parker, 1995). However, PI3Kγ deficiency OTHER CHEMOATTRACTANT-ELICITED does not significantly block fMLP-induced activation of Rac RESPONSES in mouse neutrophils. In addition, the time course for ligand- induced Rac activation, which peaks within 5 seconds (Li et Chemoattractants regulate many protein kinases, including al., 2000), is faster than that for ligand-induced increases in the members of the MAPK superfamily – ERK, JNK and p38 PtdIns(3,4,5)P3 levels, which peaks at about 45-60 seconds kinase. The PLC-pathway appears to play a more significant (Hirsch et al., 2000). The short time course for Rac regulation role in mediating the regulation of these kinases by suggests that chemoattractant receptors may regulate the small chemoattractants, because neutrophils lacking PLC-β2 and

Fig. 1. Roles of PLC and PI3K pathways in chemoattractant- Chemoattractants induced responses. This figure is intended for illustration of only the pathways that were characterized in these recent transgenic studies. γ Chemoattractants stimulate γ γ γ α Ins(3,4,5)P3 production and β i Y increase intracellular Ca2+ β β PLC-β2/3

concentrations by activating PI3K β2 β PLC- and PLC- 3 and Actin ? X stimulate PtdIns(3,4,5)P3 polymerization CD11b γ production by activating PI3K Small G in mouse neutrophils. PI3Kγ Chemotaxis Ca2+, DAG plays a significant role in proteins chemotaxis and CD4+ T cell Cell polarization survival and is required for PtdIns PKC chemoattractant–induced TI-Igλ production superoxide production and for (3,4,5)P3 production of antibodies containing the λ light chains in Superoxide Phox47 JNK response to T-cell-independent CD4+ cell production translocation ? antigens. The PLC-pathway p38 plays a major role in survival MAPK chemoattractant-mediated regulation of protein kinases including PKC, ERK, JNK and p38. The PLC-pathway is also required for upregulation of CD11b and superoxide production in response to chemoattractants. In addition, the PLC-pathway inhibits chemotaxis induced by certain chemoattractants and the production of antibodies containing the λ light chains in response to T cell-independent antigens. Thick lines represent significant pathways, and the thin lines represent minor pathways or those whose significance need to be further investigated. Roles of phospholipid signaling in chemoattractant-induced responses 2939

PLC-β3 showed a more significant reduction in fMLP-induced known to regulate cytoskeleton reorganization, which is activation of JNK (Li et al., 2000) as well as ERK and p38 essential for cell movement. Recent study of Rac2-null mice kinase (D. Wu et al., unpublished data) than did those lacking further confirmed the importance of small G proteins in PI3Kγ. The PI3Kγ pathway might also be involved in chemoattractant-induced responses including chemotaxis, regulation of ERK, because neutrophils lacking PI3Kγ showed superoxide production, and actin polymerization (Roberts et some reduction in fMLP-induced ERK activation (Sasaki et al., al., 1999). 2000). The fact that the PLC-deficiency blocked JNK Thus far, the studies of these mouse lines have largely activation, but not Rac activation, suggest that Rac is not the focused on neutrophils. Chemoattractants, especially many primary mediator for chemoattractant-induced JNK activation, chemokines, also play significant roles in lymphocyte although Rac activates JNK in some transfection assays (Coso development and function. Although there is no profound et al., 1995; Minden et al., 1995). Thus, the signaling pathways effects of PLC-β2/PLC-β3 deficiency or PI3Kγ deficiency on that mediate the regulation of MAPK kinases by extracellular lymphocyte development and function, differences in the stimuli might be cell-type dependent. number of CD4+ T cells in spleens from the mice lacking Studies of PLC-β2/PLCβ3 and PI3Kγ knockout mice also PI3Kγ (Sasaki et al., 2000) and in production of antibodies revealed that the PLC-pathway, but not the PI3K pathway, containing the λ light chains in response of immunization of T mediates chemoattractant-induced upregulation of cell surface cell-independent antigens in mice lacking PI3Kγ or PLC-β3 adhesion molecule CD11b (Jiang et al., 1997; D. Wu et al., (Li et al., 2000) have been observed. However, the unpublished data). In addition, neither pathway appears to be physiological significance and mechanistic basis for the involved in regulation of actin polymerization, because the involvement of PI3Kγ and/or PLC-molecules in these fMLP-stimulated binding of F-actin-binding phalloidin to processes need to be studied further. neutrophils isolated from mutant mice is similar to that of wild- type cells (Li et al., 2000). This observation is consistent with the assertion that neither the PLC-β- nor PI3Kγ-pathway is the REFERENCES primary mediator for regulation of Rac, which is known to regulate actin polymerization. Akasaki, T., Koga, H. and Sumimoto, H. (1999). Phosphoinositide 3-kinase- dependent and -independent activation of the small GTPase Rac2 in human neutrophils. J. Biol. Chem. 274, 18055-18059. Ali, H., Richardson, R. M., Haribabu, B. and Snyderman, R. (1999). FUTURE DIRECTIONS Chemoattractant receptor cross-desensitization. J. Biol. Chem. 274, 6027- 6030. Studies of knockout mice have clearly confirmed the previous Alteraifi, A. M. and Zhelev, D. V. (1997). Transient increase of free cytosolic belief that phospholipid signaling mediated by PLC and PI3K calcium during neutrophil motility responses. J. Cell Sci. 110, 1967-1977. Arai, H., Tsou, C. L. and Charo, I. F. (1997). Chemotaxis in a lymphocyte has important roles in chemoattractant-induced responses. Fig. cell line transfected with C-C chemokine receptor 2B: evidence that directed 1 summarizes the roles of these two prominent phospholipid- migration is mediated by betagamma dimers released by activation of Gαi- signaling pathways that these recent studies have revealed or coupled receptors. Proc. Nat. Acad. Sci. USA 94, 14495-14499. confirmed. Although many results from the transgenic studies Babior, B. M. (1999). NADPH oxidase: an update. Blood 93, 1464-1476. are in agreement with those obtained from pharmacological Bacon, K. B. (1999). Chemokine-induced signal transduction in leukocytes. Eur. Cytokine Network 10, 260-261. studies, which mainly use human leukocytes, there are a few Baggiolini, M., Boulay, F., Badwey, J. A. and Curnutte, J. T. (1993). apparent discrepancies. Further studies are warranted to clarify Activation of neutrophil leukocytes: chemoattractant receptors and these differences. respiratory burst. FASEB J. 7, 1004-1010. Despite the impairment of chemotaxis by PI3Kγ deficiency, Baggiolini, M., Dewald, B. and Moser, B. (1997). Human chemokines: an update. Annu. Rev. Immunol. 15, 675-705. the PI3K pathway appears to play a more important role in Benard, V., Bohl, B. P. and Bokoch, G. M. (1999). Characterization of rac determining the direction of the movement than in movement and cdc42 activation in chemoattractant-stimulated human neutrophils using itself. Thus, primary mechanisms by which chemoattractants a novel assay for active GTPases. J. Biol. Chem. 274, 13198-13204. induce increase in motility still remain elusive, and the Berridge, M. J. (1989). Inositol phosphates and cell signaling. Nature 341, understanding of these mechanisms will be undoubtedly of 197-205. Bokoch, G. M. (1995). Chemoattractant signaling and leukocyte activation. great interest and could provide additional therapeutic targets Blood 86, 1649-1660. for anti-inflammatory drugs. The fact that chemoattractant- Camps, M., Carozzi, A., scheer, A., Park, P. J. and Giershik, P. (1992). mediated chemotaxis, like other chemoattractant-induced Isozyme-selective stimulation of phospholipase Cβ2 by G protein βγ- subunits. Nature 360, 683-686. responses, is sensitive to PTx suggests that Gi proteins is Carozzi, A., Camps, M., Gierschik, P. and Parker, P. J. (1993). Activation involved. Two recent studies demonstrated that of phosphatidylinositol lipid-specific phospholipase C-beta 3 by G-protein chemoattractant receptors might use the Gβγ subunits, rather beta gamma subunits. FEBS Lett. 315, 340-342. than the Gαi subunits, to activate chemotaxis (Arai et al., 1997; Coffer, P. J., Geijsen, N., M’Rabet, L., Schweizer, R. C., Maikoe, T., Neptune and Bourne, 1997). In addition, our studies of the Raaijmakers, J. A., Lammers, J. W. and Koenderman, L. (1998). PLC-β2/PLC-β3-deficient mice have eliminated any direct Comparison of the roles of mitogen-activated protein kinase kinase and β2 β phosphatidylinositol 3-kinase signal transduction in neutrophil effector involvement of PLC- or PLC- 3 in mediation of function. Biochem. J. 329, 121-130. chemoattractant-induced chemotaxis. Thus, it is reasonable to Coso, O. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P., Xu, N., hypothesize that chemoattractant receptors use yet-to-be- Miki, T. and Gutkind, J. S. (1995). The small GTP-binding proteins Rac1 characterized Gβγ-mediated pathways in stimulating motility. and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell βγ 81, 1137-1146. The missing link might lie in the mechanism by which G Dagher, M. C., Fuchs, A., Bourmeyster, N., Jouan, A. and Vignais, P. V. regulates the RhoA family of small GTP-binding proteins, (1995). Small G proteins and the neutrophil NADPH oxidase. Biochim. 77, which includes Rac, Cdc42 and RhoA. These proteins are 651-560. 2940 D. Wu, C.-K. Huang and H. Jiang

Elferink, J. G., Boonen, G. J. and de Koster, B. M. (1992). The role of activation of the JNK signaling cascade and c-Jun transcriptional activity by calcium in neutrophil migration: the effect of calcium and calcium- the small GTPase Rac and Cdc42Hs. Cell 81, 1147-1157. antagonists in electroporated neutrophils. Biochem. Biophys. Res. Commun. Murphy, P. M. (1994). The molecular biology of leukocyte chemoattractant 182, 864-869. receptors. Annu. Rev. Immunol. 12, 593-633. Fabbri, E., Spisani, S., Biondi, C., Barbin, L., Colamussi, M. L., Cariani, Neptune, E. R. and Bourne, H. R. (1997). Receptors induce chemotaxis by A., Traniello, S., Torrini, I. and Ferretti, M. E. (1997). Two for-Met-Leu- releasing the betagamma subunit of Gi, not by activating Gq or Gs. Proc. Phe-OMe analogues trigger selective neutrophil responses. A differential Nat. Acad. Sci. USA 94, 14489-14494. effect on cytosolic free Ca2+. Biochim. Biophys. Acta 1359, 233-240. Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B. and Furie, M. B. and Randolph, G. J. (1995). Chemokines and Tissue Injury. Devreotes, P. N. (1998). G protein signaling events are activated at the Am. J. Pathol. 146, 1287-1301. leading edge of chemotactic cells. Cell 95, 81-91. Harakawa, N., Sasada, M., Maeda, A., Asagoe, K., Nohgawa, M., Takano, Park, J. W. and Babior, B. M. (1992). The translocation of respiratory burst K., Matsuda, Y., Yamamoto, K. and Okuma, M. (1997). Random oxidase components from cytosol to plasma membrane is regulated by migration of polymorphonuclear leukocytes induced by GM-CSF involving guanine nucleotides and diacylglycerol. J. Biol. Chem. 267, 19901-19906. a signal transduction pathway different from that of fMLP. J. Leuk. Biol. 61, Parker, P. J. (1995). Intracellular signalling. PI 3-kinase puts GTP on the Rac. 500-506. Curr. Biol. 5, 577-579. Hirsch, E., Katanaev, V. L., Garlanda, C., Azzolino, O., Pirola, L., Silengo, Ptasznik, A., Prossnitz, E. R., Yoshikawa, D., Smrcka, A., Traynor- L., Sozzani, S., Mantovani, A., Altruda, F. and Wymann, M. P. (2000). Kaplan, A. E. and Bokoch, G. M. (1996). A tyrosine kinase signaling Central role for G protein-coupled phosphoinositide 3-kinase gamma in pathway accounts for the majority of phosphatidylinositol 3, 4, 5- inflammation. Science 287, 1049-1053. trisphosphate formation in chemoattractant-stimulated human neutrophils. Hofman, P., d’Andrea, L., Guzman, E., Selva, E., Le Negrate, G., Far, D. J. Biol. Chem. 271, 25204-25207. F., Lemichez, E., Boquet, P. and Rossi, B. (1999). Neutrophil F-actin and Rhee, S. G. and Bae, Y. S. (1997). Regulation of phosphoinositide-specific myosin but not microtubules functionally regulate transepithelial migration phospholipase C isozymes. J. Biol. Chem. 272, 15045-15048. induced by interleukin 8 across a cultured intestinal epithelial monolayer. Roberts, A. W., Kim, C., Zhen, L., Lowe, J. B., Kapur, R., Petryniak, B., Eur. Cytokine Network 10, 227-236. Spaetti, A., Pollock, J. D., Borneo, J. B., Bradford, G. B. et al. (1999). Jiang, H. P., Kuang, Y. N., Wu, Y. P., Smrcka, A., Simon, M. I. and Wu, Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is D. Q. (1996a). Pertussis toxin-sensitive activation of phospholipase C by the characterized by abnormalities in neutrophil function and host defense. C5a and fMet-Leu-Phe receptors. J. Biol. Chem. 271, 13430-13434. Immunity 10, 183-196. Jiang, H. P., Lyubarsky, A., Dodd, R., Vardi, N., Pugh, E., Baylor, D., Sasaki, T., Irie-Sasaki, J., Jones, R. G., Oliveira-dos-Santos, A. J., Stanford, Simon, M. I. and Wu, D. (1996b). Phospholipase C β4 is involved in W. L., Bolon, B., Wakeham, A., Itie, A., Bouchard, D., Kozieradzki, I. et modulating the visual response in mice. Proc. Nat. Acad. Sci. USA 93, al. (2000). Function of PI3Kgamma in thymocyte development, T cell 14598-14601. activation, and neutrophil migration. Science 287, 1040-1046. Jiang, H., Kuang, Y., Simon, M. I. and Wu, D. (1997). The roles of Schall, T. J. (1994). The chemokines. In The Cytokine Handbook (ed. A. phospholipase Cβ2 in chemoattractant-induced responses. Proc. Nat. Acad. Thomson), pp. 419-460. New York: Academic Press. Sci USA 94, 7971-7975. Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W. and Jin, T., Zhang, N., Long, Y., Parent, C. A. and Devreotes, P. N. (2000). Bourne, H. R. (2000). Polarization of chemoattractant receptor signaling Localization of the G protein betagamma complex in living cells during during neutrophil chemotaxis. Science 287, 1037-1040. chemotaxis. Science 287, 1034-1036. Smrcka, A. V. and Sternweis, P. C. (1993). Regulation of purified subtypes Kano, M., Hashimoto, K., Watanabe, M., Kurihara, H., Offermanns, S., of PLC β by G protein α and βγ subunits. J. Biol. Chem. 268, 9663-9674. Jiang, H., Wu, Y., Simon, M. I. and Wu, D. (1998). Phospholipase Cbeta4 Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., is specifically involved in climbing fiber synapse elimination in the Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P. et developing cerebellum. Proc. Nat. Acad. Sci. USA 95, 15724-15729. al. (1997). The G beta-gamma sensitivity of a PI3K is dependent upon a Katz, A., Wu, D. and Simon, M. I. (1992). Subunits βγ of heterotrimeric G tightly associated adaptor, p101. Cell 89, 105-114. protein activate β2 isoform of phospholipase C. Nature 360, 686-689. Thelen, M., Wymann, M. P. and Langen, H. (1994). Wortmannin binds Knall, C., Worthen, G. S. and Johnson, G. L. (1997). Interleukin 8- specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine stimulated phosphatidylinositol-3-kinase activity regulates the migration of nucleotide-binding protein-coupled receptor signaling in neutrophil human neutrophils independent of extracellular signal-regulated kinase and leukocytes. Proc. Nat. Acad. Sci. USA 91, 4960-4964. p38 mitogen-activated protein kinases. Proc. Nat. Acad. Sci. USA 94, 3052- Thelen, M., Uguccioni, M. and Bosiger, J. (1995). PI 3-kinase-dependent and 3057. independent chemotaxis of human neutrophil leukocytes. Biochem. Biophys. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T. and Bokoch, G. Res. Commun. 217, 1255-1262. M. (1991). Regulation of phagocyte oxygen radical production by the GTP- Thelen, M. and Didichenko, S. A. (1997). G-protein coupled receptor- binding protein Rac 2. Science 254, 1512-1515. mediated activation of PI 3-kinase in neutrophils. Ann. NY Acad. Sci. 832, Kuang, Y., Wu, Y., Jiang, H. and Wu, D. (1996). Selective coupling of C-C 368-382. chemokine receptor to G proteins. J. Biol. Chem. 271, 3975-3978. Umeki, S. (1994). Activation factors of neutrophil NADPH oxidase complex. Kuijpers, T. W., Hoogerwerf, M. and Roos, D. (1992). Neutrophil migration Life Sci. 55, 1-13. across monolayers of resting or cytokine-activated endothelial cells. Role of Vlahos, C. J., Matter, W. F., Brown, R. F., Traynor-Kaplan, A. E., intracellular calcium changes and fusion of specific granules with the plasma Heyworth, P. G., Prossnitz, E. R., Ye, R. D., Marder, P., Schelm, J. A., membrane. J. Immunol. 148, 72-77. Rothfuss, K. J. et al. (1995). Investigation of neutrophil signal transduction Lew, P. D. (1990). Receptors and intracellular signaling in human neutrophils. using a specific inhibitor of phosphatidylinositol 3-kinase. J. Immunol. 154, Am. Rev. Respir. Dis. 141, S127-131. 2413-2422. Li, Z., Jiang, H., Xie, W., Zhang, Z., Smrcka, A. V. and Wu, D. (2000). Wu, D., Lee, C.-H., Rhee, S. G. and Simon, M. I. (1992). Activation of Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant- phospholipase C by the α subunits of the Gq and G11 proteins on transfected mediated signal transduction. Science 287, 1046-1049. Cos-7 cells. J. Biol. Chem. 267, 1811-1817. Mandeville, J. T. and Maxfield, F. R. (1997). Effects of buffering intracellular Wu, D., LaRosa, G. J. and Simon, M. I. (1993). G protein-mediated signal free calcium on neutrophil migration through three-dimensional matrices. J. transduction pathways for Interleukin-8. Science 261, 101-103. Cell. Physiol. 171, 168-178. Xie, W., McLaughlin, J. P., Samorisky, G., Gross, R., Bidlack, J. M., Jiang, Marks, P. W., Hendey, B. and Maxfield, F. R. (1991). Attachment to H. and Wu, D. (1999). Genetic alteration of phospholipase C beta3 fibronectin or vitronectin makes human neutrophil migration sensitive to expression modulates behavioral and cellular responses to µ opioids. Proc. alterations in cytosolic free calcium concentration. J. Cell Biol. 112, 149- Nat. Acad. Sci. USA 96, 10385-10390. 158. Zlotnik, A. and Yoshie, O. (2000). Chemokines: a new classification system Minden, A., Lin, A., Claret, F., Abo, A. and Karin, M. (1995). Selective and their role in immunity. Immunity 12, 121-127.