Roles of phospholipase C β2 in chemoattractant-elicited responses

Abstract

The physiological roles of phospholipase C (PLC) ␤2 in hematopoiesis, leukocyte function, and host defense against infection were investigated using a mouse line that lacks PLC ␤2. PLC ␤2 deficiency did not affect hematopoiesis, but it blocked chemoattractant-induced Ca 2؉ release, superoxide production, and MAC-1 up-regulation in neutrophils. In view of these effects, it was surprising that the absence of PLC ␤2 enhanced chemotaxis of different leukocyte populations and sensitized the in vivo response of the PLC ␤2-deficient mice to bacteria, viruses, and immune complexes. These data raise questions about the roles that PLC ␤2 may play in signal transduction induced by chemoattractants in leukocytes.Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce two important second messengers, inositol trisphosphates and diacylglycerol (1). There are four different PLC ␤ isoforms that have been cloned. They are all regulated by heterotrimeric G proteins, and there is evidence suggesting that different isoforms may be involved in a variety of signaling circuits. The ␤2 isoform is found primarily in hematopoietic cells (2, 3), and it can be activated by both the G␣ subunits of the Gq class and by the ␤␥ subunits generated by a number of different heterotrimeric G proteins (3-9). Cotransfection experiments in COS-7 and HEK cells suggest that PLC ␤2 may function downstream of chemoattractant receptors. Transfection of receptors for complement component C5a and fMet-Leu-Phe (fMLP) (10), interleukin (IL)-8 receptors a and b (11), and CKR-1 and -2 (12) demonstrated that each of the receptors activates PLC ␤2 through the pertussis toxin (PTx)-sensitive release of ␤␥ from the G i class of heterotrimeric G proteins. In addition, this may be a primary signaling pathway in neutrophils, because much of the PLC activity elicited through chemoattractant receptors also appears to function through the G i -mediated release of ␤␥ (13-17).To confirm the existence of the G␤␥-PLC ␤2 pathway in vivo and to investigate the function of the pathway in hematopoiesis and leukocyte function, we generated a mouse line that lacks PLC ␤2. We found that PLC ␤2 is the major isoform that mediates PTx-sensitive PLC activation induced by chemoattractants and that PLC ␤2 is critical to many chemoattractant-elicited responses, including Ca 2ϩ efflux, superoxide production, and up-regulation of MAC-1. However, PLC ␤2 deficiency does not attenuate chemoattractant-induced chemotaxis; surprisingly, it was found to enhance the process. MATERIALS AND METHODS Generation of PLC ␤2-NullMice. An 8-kb genomic DNA from a 129SV agouti mouse strain library contains two exons of the PLC ␤2 gene, and it was used to make the gene-targeting construct. The exons encoded residues 378-464, which are located in the C terminus of the X box. Parts of the exons were replaced with a neomycin-resistance gene. The gene-targeting construct was transfected into embryonic stem (ES) cells (CJ7 clone) by electroporation. After selection with Geneticin, e...

Gαi2-mediated signaling events in the endothelium are involved in controlling leukocyte extravasation

“…17). Interestingly, in vitro MIP-2-mediated chemotaxis of G␣ i2 -deficient neutrophils appeared to be enhanced relative to wild-type controls, consistent with our observations of eosinophils and previous studies by others investigating leukocytes deficient in genes mediating G proteincoupled receptor signal transduction (18). The similarity of the data examining both neutrophil and eosinophil recruitment again supports the leukocyte-independent character of the G␣ i2 deficiency in knockout mice.…”

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

“…The PLC-β2/β3 knockout mice showed a more dramatic increase in the number of thoracic leukocytes in comparison to wild-type mice. 54 Thus, this result also supports the hypothesis that Ins(1,3,4,5)P4 is a negative regulator for chemotaxis, and thereby for leukocyte recruitment. counts (neutrophilia), which is often associated with bacterial infection, myeloid leukemia and acute myocardial infarction.…”

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA

“…Only subsets of these genes are expressed in immune cells; however, it is difficult to determine the precise roles of the isoforms that are expressed because they have very similar biochemical activity (7). With respect to chemotaxis, G␤␥ has been shown to interact with and to activate a variety of proteins, including phospholipase C (PLC)-␤2, PLC-␤3, PLC-, p21-activated kinase 1, phosphatidylinositol 3-kinase, and isoforms of adenylyl cyclase (8)(9)(10)(11). In addition, ␤␥ has been shown to modulate the activity of a various ion channels (12,13).…”