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Roles of PLC-β2 and -β3 and PI3Kγ in Chemoattractant-Mediated Signal Transduction

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Science  11 Feb 2000:
Vol. 287, Issue 5455, pp. 1046-1049
DOI: 10.1126/science.287.5455.1046

Abstract

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kγ was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell–independent antigen-specific antibodies composed of the immunoglobulin λ light chain (TI-IgλL). The study of the mice lacking PLC-β2 and -β3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IgλL production.

Chemoattractants have important roles in inflammatory reactions. Their receptors couple to the inhibitory heterotrimeric guanine nucleotide–binding proteins (Gi proteins) and elicit a wide range of responses in leukocytes (1–3). It is thought that two signaling pathways mediated by PLC (4) and PI3K (5, 6) are activated by chemoattractant receptors. To investigate the role of the PI3K-linked pathway in chemoattractant-mediated responses, we generated a mouse line that lacks PI3Kγ. A gene-targeting vector was constructed so that a green fluorescence protein (GFP) coding sequence was fused with the coding frame of PI3Kγ (Fig. 1A). Thus, the expression of GFP is under the control of the endogenous PI3Kγ promoter in the transgenic mice. The mice heterozygous and homozygous for the disrupted PI3Kγ genes were generated as described (7), and the genotypes were verified with Southern cDNA and Western blot analyses (Fig. 1A).

Figure 1

Generation and characterization of the mouse lines that lack PI3Kγ, PLC-β2, and PLC-β3. (A) Southern cDNA and Western blot analyses. Mouse tail DNA was digested with Eco RI and hybridized with a probe from the PI3K gene outside the gene-targeting construct. Equal amounts of neutrophil extracts were analyzed by Western blot with an antibody to PI3Kγ. (B)fMLP-induced PtdIns (3,4,5)P3 (PIP3) production. The levels of PtdIns (3,4,5)P3 were determined as described (8). wt, wild type. (C) Expression of GFP in tissue samples from a wild-type mouse (+/+) and a mouse homozygous for the disrupted PI3Kγ genes (−/−). Tissue extracts were analyzed by Western blot with an antibody to GFP. Tissue sample abbreviations are as follows: Spl, spleen; Bm, bone marrow; Neu, neutrophil; Int, intestine; Kid, kidney. (D) Western blot analysis of PLC-deficient mice. Neutrophil extracts from wt, PLC-β2 (p2)–null mice, PI3Kγ (pi3kγ)–null mice, PLC-β3 (p3)–null mice, and PLC-β2/-β3 (p2/3)–null mice were analyzed with antibodies to PLC-β2 (lower panel) or PLC-β3 (upper panel). (E) fMLP (1 μM)–induced production of IP3. Neutrophils were treated with 1 μMfMLP for 45 s and IP3 concentration was determined with a radioreceptor assay (NEN, Boston, MA). (F) fMLP-induced Ca2+ efflux. Neutrophils were labeled with fura-2/AM and treated with 1 μMfMLP. Excitation ratios of 340/380 nm were measured in a fluorimeter.

To determine the contribution of PI3Kγ to chemoattractant-induced PtdIns (3,4,5)P3 production in mouse neutrophils, we compared formyl peptideN-formyl-Met-Leu-Phe (fMLP)–induced PtdIns (3,4,5)P3production in neutrophils from either wild-type or PI3Kγ-deficient mice (8). The neutrophils were prepared from the peritonea of mice treated with 2% casein (9). Although fMLP-elicited production of PtdIns (3,4,5)P3 was clearly detected in the wild-type neutrophils, no production of PtdIns (3,4,5)P3 was detected in response to fMLP in cells lacking PI3Kγ (Fig. 1B). This result indicates that PI3Kγ is the predominant PI3K isoform that mediates fMLP-induced PtdIns (3,4,5)P3 production in mouse neutrophils.

The expression of PI3Kγ in mice was examined by detecting expression of GFP in the transgenic mice. GFP was detected in the spleen cells, bone marrow cells, and neutrophils isolated from mice homozygous for the disrupted PI3Kγ genes, but not those from wild-type mice (Fig. 1C). No GFP proteins were detected in other tested tissue samples of the PI3Kγ-deficient mice. These results suggest that the expression of PI3Kγ may be restricted to hematopoietic cells. GFP was detected with flow cytometry in over 90% of Mac1+ cells in the peritoneum, and about 82% of CD45R+ and 70% of CD3+ cells from the spleens of transgenic mice also expressed GFP (10).

Chemoattractants can also activate PLC, leading to transient increases in intracellular Ca2+concentrations. PLC-deficient mouse lines were generated to investigate the roles of PLC-β isoforms in leukocyte functions (Fig. 1D) (11). fMLP-induced inositol trisphosphate (IP3) production and Ca2+ efflux were not detected in neutrophils lacking PLC-β2 and PLC-β3 (PLC-β2/-β3), while cells lacking only PLC-β2 also show clear reduction in IP3 production and Ca2+ efflux (Fig. 1, E and F). Similar results were also observed with the other chemoattractants interleukin-8 (IL-8) and macrophage inflammatory protein (MIP)–1α (10). All these results support the conclusion that PLC-β2 and PLC-β3 are the sole PLC isoforms that are activated by chemoattractants in mouse neutrophils.

Chemoattractants induce various responses in leukocytes, one of which is chemotaxis. Neutrophils purified from casein-treated PLC-β2/-β3–null mice did not show differences from wild-type cells in fMLP-induced (Fig. 2A) or IL-8–induced (10) chemotactic activities. Thus, we conclude that the PLC pathway is not required for chemotaxis in neutrophils. Consistent with our previous report (9), PLC-β2/-β3–null neutrophils showed enhanced chemotactic activities in response to the CC chemokine MIP-1α (Fig. 2B). PI3Kγ deficiency impaired but did not eliminate the migration of neutrophils in response tofMLP or MIP-1α in an in vitro migration assay (Fig. 2, A and B). PI3Kγ deficiency also significantly impaired infiltration of neutrophils into the peritoneal cavity in Escherichia coli–induced peritonitis (P< 0.01), whereas PLC-β2/-β3–null mice showed enhanced infiltration of leukocytes (Fig. 2C). These results suggest that PI3Kγ has an important but not essential role in chemoattractant-induced chemotaxis of neutrophils.

Figure 2

Chemoattractant-induced responses in leukocytes. (A and B) Chemotaxis of purified casein-elicited neutrophils.fMLP-mediated (A) or MIP-1α–mediated (B) chemotaxis was determined in a 48-well modified Boyden chamber. The index represents the ratio of the number of migrated cells in the presence of ligand to those in the absence of ligand. (C) E. coli–induced peritonitis. E. coli (107) were injected into mice, and the number of cells in the peritonea was determined after 16 hours. Error bars represent SD. (D) Superoxide production determined in the presence or absence of 1 μM fMLP for 10 min at 37°C as described (12). The assay was performed in triplicate with six different sets of mice. Error bars represent SD. (E) F-actin formation. Neutrophils were treated with 1 μM fMLP for 45 s, and the levels of F-actin were determined in a flow cytometer after binding of rhodamime phalloidin. Experiments were repeated three times with three different sets of mice. The data shown are from a representative set. (F) Rac activation. The amount of GTP-bound Rac in the presence or absence of 1 μM fMLP was assayed by determining the levels of Rac that bound to the PBD of p21-activated protein kinase by Western blot with a Rac-specific antibody (26). Each experiment was repeated three times and used cells pooled from three to five mice. (G) Translocation of p47phox. Neutrophils (5 × 107) pooled from four mice were treated with 1 μM of fMLP for 45 s. Cytosolic and particulate fractions were prepared and analyzed with an antibody to p47phox. This experiment was performed twice. Abbreviations are the same as in Fig. 1.

Pharmacological studies suggest that PI3K may be required for chemoattractant-induced superoxide formation (12,13). Consistently, fMLP-induced superoxide production was insignificant in cells lacking PI3Kγ (Fig. 2D). PLC-β2/-β3–null cells also completely lost the ability to produce superoxide in response tofMLP (Fig. 2D). Thus, the PI3Kγ- and PLC-β–linked pathways are required for superoxide production. Regulation of Rac activity and p47phox translocation are two important steps in regulation of superoxide production, and both steps are influenced by various signaling pathways (14–16). PI3K has been implicated in regulation of Rac (17,18). However, neither PI3Kγ deficiency nor the PLC-β2/-β3 double deficiency had an effect onfMLP-induced activation of Rac in murine neutrophils (Fig. 2F). Thus, neither the PLC- nor the PI3K-linked pathway is required for activation of Rac. This conclusion is further supported by the lack of effect of these deficiencies on actin polymerization (Fig. 2E). Although fMLP-stimulated translocation of p47phox was observed in cells lacking PI3Kγ, the process did not occur in PLC-β2/-β3–null cells (Fig. 2G). These results indicate that the PLC-linked pathway rather than the PI3Kγ-linked pathway is required for p47phox translocation, and this may account for the failure to produce superoxide in response to fMLP in cells lacking PLC.

Protein kinase C (PKC) has been implicated in regulation of p47phox translocation (19). fMLP stimulates PKC activity, manifested as increases in autophosphorylation of PKC (20) (Fig. 3A). PLC deficiency blockedfMLP-induced autophosphorylation of PKC, but PI3Kγ deficiency did not (Fig. 3A). This result appears to contradict the finding that PKC activation requires the phosphorylation of the activation loop by a PtdIns (3,4,5)P3–dependent mechanism (21). A possible explanation for this may be that the PKC molecules may already have been phosphorylated by basal PI3Kγ activity or by other PI3K isoforms.

Figure 3

Regulation of kinase activities. Casein-elicited neutrophils were treated with 1 μM offMLP for varying times, and the same amount of cell extracts were subjected to Western analysis with an antibody that detects autophosphorylated PKC-α, -βI, -βII, -γ, and -δ (Anti-pan-P-PKC) and antibodies specific to PKC-α, and PKC-δ (A) and phospho-JNK and JNK (B). Representative experiments are shown. The experiments were repeated at least three times with three different sets of mice. Abbreviations are the same as in Fig. 1.

Chemoattractant receptors activate mitogen-activated protein kinases (MAPKs), and PI3K has been implicated in mediating this activation. However, PI3Kγ deficiency did not obviously affectfMLP-induced activation of JNK (Fig. 3B) in mouse neutrophils. In contrast, PLC deficiency caused a reduction in chemoattractant-induced phosphorylation of JNK (Fig. 3B) and MAPK (10). Thus, the PLC pathway has a more important role than the PI3Kγ pathway in fMLP-induced JNK and MAPK activation in murine neutrophils. Our findings that PLC deficiencies impair JNK activation but not Rac activation suggest that Rac is not sufficient to activate JNK in mouse neutrophils.

PI3Kα regulates B cell development (22,23). We investigated whether PI3Kγ also influences B cell functions. Analyses with flow cytometry did not reveal differences between the wild-type and PI3Kγ-deficient mice in the populations of CD45R+cells in spleens or of CD45R+ and CD43+ cells in bone marrow (10). Unlike wild-type mice, however, mice lacking PI3Kγ produced few antibodies containing the λ light chain when immunized with T cell–independent (TI) antigen hydroxylnitrophenyl (NP)–Ficoll (Fig. 4A). By contrast, mice lacking both PLC-β2 and PLC-β3 consistently produced larger amount of TI antigen-specific antibodies composed of the immunoglobulin λ light chain (TI-IgλL) than did wild-type mice (Fig. 4A). It appears that the PLC pathway, in this case, opposes the PI3K pathway. Enhancement in TI-IgλL production appeared to be primarily dependent on the PLC-β3 deficiency (Fig. 4A). Neither PLC nor PI3K deficiency affected the production of TI-Igκ (Fig. 4B) or of T cell–dependent (TD) antigen NP–chicken gamma globulin (NP-CCG)–specific antibodies composed of either λ or κ light chains (10). Together these data suggest that the production of TI-IgλL may be subjected to regulation by G protein–mediated signaling pathways. Because no differences were detected between wild-type and PI3Kγ-deficient mice in the amount of total serum IgλL and in the number of B cells carrying cell surface λL (10), we think that PI3Kγ deficiency is more likely to affect antigen-dependent processes than early development of B cells.

Figure 4

Systemic phenotypes. (A andB) Production of TI-IgλL in mice. Mice were immunized with TI antigen NP-Ficoll, and the sera were collected 7 days after immunization (27). The amount of NP-specific λ chains (A) and κ chains (B) was determined by ELISA. The data presented are subtracted by the measurements of the corresponding pre-immune sera. In (A), n = 18 for p2/3, wt, and pi3kγ; n = 6 for p2 and p3. In (B),n = 18 for p2/3, wt, and pi3kγ. (C throughF) Spontaneous skin ulcers in PLC-β3–deficient mice. A representative lesion area of a PLC-β3–deficient mouse is shown in (C). Histological sections of the tissue lesions (D) and (E) and normal tissue (F) are shown. Abbreviations are the same as inFig. 1.

Mice lacking PLC-β3 developed spontaneous multifocal skin ulcers usually starting at the age of 6 months or older (Fig. 4C). The lesions were localized mainly behind ears or on the neck, but sometimes also appeared on the face. Similar phenotypes were observed with mice lacking both PLC-β2 and PLC-β3. Histological examination of the lesion tissues revealed hyperinfiltration of leukocytes in the lesion tissues (Fig. 4, D and E). Most of the infiltrated leukocytes had morphological characteristics of macrophages and lymphocytes. No ulcerative lesions were observed in wild-type mice, mice heterozygous for the disrupted PLC-β3 genes, or other transgenic lines including PLC-β2– and PI3Kγ-null mice that were housed in the same rooms under the same conditions. This ulcerative phenotype is consistent with the idea that the PLC pathways act to inhibit some important responses mediated by chemoattractants.

In summary, this study with mouse lines deficient in two prominent chemoattractant-activated signaling pathways confirms that both PI3Kγ and PLC-β2/-β3 have important roles in chemoattractant-induced responses. The study also revealed roles for these proteins in leukocyte functions, including the involvement of PI3Kγ in the production of TI-IgλL and the PLC pathway in down-modulation of chemotaxis and production of TI-IgλL and in hyperinflammatory conditions.

  • * These authors contributed equally to this work.

  • Present address: Department of Pharmacology, Boehringer Inglheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.

  • To whom correspondence should be addressed. E-mail: dwu{at}neuron.uchc.edu

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