Conclusions regarding the contribution of low molecular weight secretory phospholipase A₂ (sPLA₂) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA₂ enzymes. To elucidate the role of group V sPLA₂, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C₄ and prostaglandin E₂ was attenuated ∼50% in peritoneal macrophages from group V sPLA₂-null mice compared with macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA₂-null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA₂ in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.

Phospholipase A₂ (PLA₂) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA₂ enzymes (sPLA₂), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA₂ in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene‐deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA₂ isoform plays a novel anti‐inflammatory role that opposes the pro‐inflammatory activity of group IIA sPLA₂. Mechanistically, group V sPLA₂ counter‐regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti‐inflammatory function for a PLA₂ and identify group V sPLA₂ as a potential biotherapeutic for treatment of immune‐complex‐mediated inflammation.

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT ₁ R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D ₄ with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT ₁ R, the robust CysLT ₁ R-mediated calcium response to LTD ₄ was abolished. The membrane expression of the CysLT ₁ R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD ₄ -elicited ERK phosphorylation and the specific binding of [ ³ H]LTD ₄ to microsomal membranes were fully inhibited. CysLT ₁ R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT ₁ R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMΦs) increased both the membrane expression of CysLT ₁ R protein by FACS analysis and the LTD ₄ -elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT ₁ R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT ₁ R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT ₁ R that suppresses CysLT ₁ R-mediated function at the cell membrane.

We have previously reported that group V secretory phospholipase A₂ (sPLA₂) amplifies the action of cytosolic phospholipase A₂(cPLA₂) α in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA₂, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA₂ on phagocytosis of zymosan. We report that group V sPLA₂ is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA₂ is recruited to the phagosome. There it co-localizes with cPLA ₂ α, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C₄ synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA₂-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA₂ but IIA not group sPLA₂. These data demonstrate that group V sPLA₂ contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.

Phospholipase A₂ (PLA₂) hydrolyzes the sn-2 position of cell membrane phospholipids to release fatty acids and lysophospholipids. We have previously reported that group V secretory PLA₂ (sPLA₂) translocates from the Golgi and recycling endosomes of mouse peritoneal macrophages to newly formed phagosomes and regulates the phagocytosis of zymosan, suggesting a role in innate immunity. Here we report that in macrophages lacking group V sPLA₂, phagosome maturation was reduced 50–60% at early time points while the binding of zymosan was unimpaired. The ability of group V sPLA₂ to regulate phagocytosis extended to phagocytosis of IgG- and complement-opsonized sheep RBC. Moreover, macrophages lacking group V sPLA₂ had delays in phagocytosis, phagosome maturation, and killing of Candida albicans. Cytokine production and eicosanoid generation were not impaired by the lack of group V sPLA₂. Furthermore, in a model of systemic candidiasis, mice lacking group V sPLA₂ had an increased fungal burden in the kidney, liver, and spleen at day 7 postinfection and increased mortality. Thus, group V sPLA₂ regulates phagocytosis through major phagocytic receptors and contributes to the innate immune response against C. albicans by regulating phagocytosis and killing through a mechanism that is likely dependent on phagolysosome fusion.

We show that the P2Y₆ receptor, a purinergic G protein-coupled receptor with a high affinity for the nucleotide uridine diphosphate, is an important endogenous inhibitor of T cell function in allergic pulmonary inflammation. Mice conditionally deficient in P2Y₆ receptors [p2ry6 (flox/flox);cre/+ mice] exhibited severe airway and tissue pathology relative to P2Y₆-sufficient [p2ry6 (flox/flox)] littermates (+/+ mice) when treated intranasally with an extract of the dust mite Dermatophagoides farinae (Df). P2Y₆ receptors were inducibly expressed by lung, lymph node, and splenic CD4⁺ and CD8⁺ T cells of Df-treated +/+ mice. Df-restimulated P2Y₆-deficient lymph node cells produced higher levels of Th1 and Th2 cytokines, and polyclonally stimulated P2Y₆-deficient CD4⁺ T cells proliferated faster than comparably stimulated P2Y₆-sufficient cells. The absence of P2Y₆ receptors on CD4⁺ cells, but not APCs, was sufficient to amplify cytokine generation. Thus, P2Y₆ receptors protect the lung against exuberant allergen-induced pulmonary inflammation by inhibiting the activation of effector T cells.