Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2

Abstract

We have investigated whether the signaling protein phospholipase D is implicated in leukocyte cell motility. Treating differentiated HL-60 cells with small interfering RNAs (siRNAs), to deplete endogenous expression of the PLD1 isoform, led to an abolishment of basal chemokinesis that could not be rescued with chemoattractants ENA-78, FMLP, and IL-8. Transient overexpression of PLD1 increased both chemokinesis and chemotaxis toward IL-8 and FMLP but not toward ENA-78. Chemokinesis was not mediated by the enzymatic activity of PLD1, but the chemotactic response was, because a lipase-inactive mutant (PLD1-K830R) negated all chemokineinduced potentiating actions and because IL-8 and FMLP increased activity in vitro. Gene expression silencing of the other mammalian isoform, PLD2, also led to cell migration arrest, whereas ENA-78 selectively increased endogenous PLD2 activity and chemotaxis of HL-60 cells overexpressing a mycpcDNA-PLD2 construct. Thus, PLD1 is differentially activated by CXCR-1, whereas CXCR-2 (and possibly CXCR-1) mediates PLD2 activation. Finally, immunofluorescence microscopy showed that both isoforms were associated with cell polarity and directionality concomitantly with adhesion and F-actin polymerization in response to IL-8. These data represent the first demonstration of the involvement of PLD and its enzymatic activity toward chemokines in the key physiologic process of leukocyte migration. ( IntroductionInflammation, wound repair, and angiogenesis have in common an initial physiologic event of cell migration or chemotaxis. Related pathologic processes, such as chronic inflammation, atherosclerosis, and cancer metastasis, are also heavily dependent on cell chemotaxis. In the case of normal leukocyte function, cell migration begins with the reorientation and alignment of the cells (polarization) in the direction of the inflammation site followed by the directional migration (chemotaxis) toward host-or pathogenderived chemical stimuli (chemoattractants).Known major neutrophil chemoattractants are the tripeptide FMLP, the lipids LTB 4 and PAF, the activated complement protein C5a, 1,2 and a group of cytokines collectively known as ELR ϩ CXC chemokines. [3][4][5][6] These chemokines are characterized by the invariable presence of the Cys-X-Cys (CXC) consensus motif in the N-terminus of the protein, which is preceded by the amino acid sequence Glu-Leu-Arg (ELR). Classical neutrophil ELR ϩ CXC chemokines are IL-8 (CXCL8); ENA-78 (CXCL5); GRO␣, GRO␤, and GRI␥; NAP-2; and GCP-2. They all induce cytosolic calcium changes, chemotaxis, and exocytosis 3 and recruit neutrophils by binding and activating a specific class of receptors, called CXCR-2. 7 Apart from binding to CXCR-2, IL-8 and GCP-2 also bind to another class of receptors, the CXCR-1. 4,8 Neutrophil chemotaxis depends on PI3K␥ and Akt/PKB, 9-11 whereas FMLP fails to induce cell directionality in PI3K␥-deprived neutrophils. 12 Pharmacologic studies, however, do not indicate an absolute requirement for PIP 3 . 13,14 However, not all neutrophil...

A Novel Phospholipase D2-Grb2-WASp Heterotrimer Regulates Leukocyte Phagocytosis in a Two-Step Mechanism

“…It is known that PLD2 plays an important role during phagocytosis, yet the mechanism for how this is accomplished is far from being understood (13). Our laboratory has shown previously that PLD2 is a key protein during chemotaxis mediated by ancillary proteins such as Grb2 (15,16). We began this study by hypothesizing that Grb2 could also mediate the effect of PLD2 on phagocytosis.…”

The cytokine response in THP-1 (monocyte) and HL-60 (neutrophil-differentiated) cells infected with different genotypes of Helicobacter pylori strains

“…In addition, HL-60 cells can differentiate into neutrophils in response to various stimuli like dimethyl sulfoxide, retinoic acid, and dibutyryl cyclic adenosine monophosphate (Bt 2 -cAMP). Due to the differentiation potential of these cells, neutrophil differentiated HL-60 cell lines were obtained through incubating them with 1.75% dimethyl sulfoxide and then were selected as a neutrophil model in this study (18,19).…”

Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

“…and Rac small GTPases are integral to cell signaling (37,41), and PLD activity and Rac-GTP loading are both activated by the same stimuli and to approximately the same extent. Fig.…”

“…In neutrophils, PLD activation has been implicated in agonist-induced cell migration, exocytosis, phagocytosis of opsonized particles, and activation of NADPH oxidase (36,37). In Madin-Darby canine kidney epithelial cells, expression of ARNO (a guanine nucleotide exchange factor for ARF) GTPases leads to increased activation of endogenous Rac1 and that Rac activation is required for ARNO-induced cell motility.…”