Cytosolic Phospholipase A2 Translocates to Forming Phagosomes during Phagocytosis of Zymosan in Macrophages

Abstract

Resident tissue macrophages mediate early innate immune responses to microbial infection. Cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) is activated in macrophages during phagocytosis of non-opsonized yeast (zymosan) triggering arachidonic acid release and eicosanoid production. cPLA 2 ␣ translocates from cytosol to membrane in response to intracellular calcium concentration ([Ca 2؉ ] i ) increases. Enhanced green fluorescent protein (EGFP)-cPLA 2 ␣ translocated to forming phagosomes, surrounding the zymosan particle by 5 min and completely overlapping with early endosome (Rab5) and plasma membrane (F4/80) markers but only partially overlapping with resident endoplasmic reticulum proteins (GRP78 and cyclooxygenase 2). EGFP-cPLA 2 ␣ also localized to membrane ruffles during phagocytosis. Zymosan induced an initial high amplitude calcium transient that preceded particle uptake followed by a low amplitude sustained calcium increase. Both phases were required for optimal phagocytosis. Extracellular calcium chelation prevented only the sustained phase but allowed a limited number of phagocytic events, which were accompanied by translocation of cPLA 2 ␣ to the phagosome although [Ca 2؉ ] i remained at resting levels. The results demonstrate that cPLA 2 ␣ targets the phagosome membrane, which may serve as a source of arachidonic acid for eicosanoid production.

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

“…Taking into account the known dependency of several NADPH oxidase subunits (including gp91 phox ) on AA, we suggest that LBs may be used by phagocytes to locally release AA for NADPH oxidase activation near the phagosome. This hypothesis is strengthened by reports showing the presence of AA-releasing cPLA 2 on LBs (9) and the translocation of cPLA 2 to the phagosome in PLB-985 cells and macrophages incubated with zymosan particles (10,11). We expect that future studies with cPLA 2 -deficient PLB-985 cells, which have become available (23), may provide insight related to this hypothesis.…”

“…Arachidonic acid (AA) is the precursor for the synthesis of eicosanoids, and the enzyme that liberates AA from phospholipids upon stimulation of neutrophils and macrophages, cytosolic phospholipase A 2 (cPLA 2 ), has been colocalized with LBs (9). Interestingly, cPLA 2 recently was shown also to translocate to phagosomes in phagocytes that had ingested zymosan particles (10,11). It was found that gp91 phox (in which ''phox'' is phagocyte oxidase) is responsible for binding cPLA 2 to the phagosomal membrane (10).…”

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…Surprisingly, human cPLA 2 -deficient cells show normal translocation of phox proteins (Dana et al, 1994;Dana et al, 1998;Shmelzer et al, 2003), suggesting cPLA 2 serves other roles in oxidase activation during Nox2 assembly. Interestingly, both cPLA 2 and secretory PLA 2 accumulate on newly formed phagosomes (Shmelzer et al, 2003;Girotti et al, 2004;Balestrieri et al, 2006). Furthermore, cells lacking Nox2 show no translocation of cPLA 2 to membranes, and it seems that human cPLA 2 associates directly with the cytoplasmic phox proteins and the membrane-bound Nox2 complex after stimulation by several agonists that activate the oxidase (Shmelzer et al, 2003).…”

A C2 domain protein kinase initiates phagocytosis in the protozoan parasite Entamoeba histolytica

“…A number of C2-domain-containing proteins, such as PKCs, phospholipases and PI3K are known to participate in phagocytosis [37][38][39] . Though their role in phagosome closure is well documented, it is not clear whether these are also involved in the initiation.…”