Barbara Balestrieri

Group V Secretory Phospholipase A2 Translocates to the Phagosome after Zymosan Stimulation of Mouse Peritoneal Macrophages and Regulates Phagocytosis

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.

GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT1R) 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) D4 with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT1R, the robust CysLT1R-mediated calcium response to LTD4 was abolished. The membrane expression of the CysLT1R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD4-elicited ERK phosphorylation and the specific binding of [3H]LTD4 to microsomal membranes were fully inhibited. CysLT1R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT1R 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 CysLT1R protein by FACS analysis and the LTD4-elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT1R 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 CysLT1R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT1R that suppresses CysLT1R-mediated function at the cell membrane.

A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis

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.

Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans

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.

Secretory phospholipases A2 activate selective functions in human eosinophils.

Secretory phospholipases A2 (sPLA2s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA2. We have examined the effects of two structurally different sPLA2s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA2 induced a concentration-dependent release of β-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA2s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA2s examined induced the production of IL-5, the de novo synthesis of leukotriene C4 and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA2s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes’ effects. In addition, inactivation of sPLA2 enzymatic activity by bromophenacyl bromide did not influence the release of β-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA2s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA2s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.

Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF‐α, IL‐6 and IL‐12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration‐dependent release of TNF‐α and IL‐6 from bloodmonocytes. These sPLA2 activated IL‐12 production only in monocytes preincubated with IFN‐γ. Group IA and IIA sPLA2 also induced TNF‐α and IL‐6 release from synovial fluid monocytes. TNF‐α and IL‐6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in theproinflammatory activity of sPLA2 released during inflammatory diseases.

Secretory Phospholipases A2 as Multivalent Mediators of Inflammatory and Allergic Disorders

Phospholipases A2 (PLA2s) are enzymes responsible for mobilization of fatty acids, including arachidonic acid (AA), from phospholipids. These enzymes are classified as high-molecular-weight cytosolic PLA2s (cPLA2s) and low-molecular-weight secretory PLA2s (sPLA2s). There is increasing evidence that large quantities of sPLA2s are released in the plasma of patients with systemic inflammatory and autoimmune diseases. In addition, high levels of sPLA2s can be detected at sites of allergic inflammation including the upper airways of patients with rhinitis and the lower airways of patients with asthma. These extracellular enzymes play an important role in inflammation by releasing AA, which can be subsequently converted to proinflammatory prostaglandins and leukotrienes. Generation of AA mediated by sPLA2s occurs through different mechanisms, including (1) the direct hydrolysis of outer cell membrane phospholipids, (2) internalization and transfer of sPLA2s to intracellular pools of phospholipids enriched in AA, and (3) activation of cPLA2s. In addition, sPLA2s induce degranulation and production of cytokines and chemokines from a variety of cells involved in inflammatory and immune responses. These effects are exerted by mechanisms that are independent of the enzymatic activity and are mediated by the interaction of sPLA2s with specific or promiscuous membrane receptors. Therefore, sPLA2s may have an important role in inflammatory and allergic reactions by activating multiple mechanisms within inflammatory and immune cells, leading to the production of eicosanoids, cytokines and chemokines.

Group V Secretory Phospholipase A2 Reveals Its Role in House Dust Mite-Induced Allergic Pulmonary Inflammation by Regulation of Dendritic Cell Function

We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by D. farinae, and whose function is also regulated by group V sPLA2.

Defective surface expression of attractin on T cells in patients with common variable immunodeficiency (CVID)

The proliferative responses of T lymphocytes of a subset of patients with CVID are abnormally low. This may be due to abnormalities in extracellular interactions or signalling defects downstream from membrane‐associated receptors. Demonstrating that the T cell receptor signalling was normal, we observed no abnormal pattern of activation‐induced tyrosine phosphorylation in cells from CVID patients. Moreover, the addition of exogenous IL‐2 increased the low proliferation to mitogens, thus indicating the integrity of the IL‐2R signalling apparatus. Attractin is a rapidly expressed T cell activation antigen involved in forming an association between T cells and monocytes. Twenty‐four to 48 h after activation by CD3 cross‐linking, attractin expression was not up‐regulated on the cells of CVID patients despite normal up‐regulation of CD25 and CD26. On control cells, however, attractin expression was up‐regulated together with CD25 and CD26. The addition of the purified 175‐kD attractin was capable of restoring the proliferative response of peripheral blood mononuclear cells following CD3 X‐L in the presence of suboptimal concentrations of rIL‐2 (10 and 20 U/ml). The effect was dose‐dependent with the maximal effect at a concentration of 500 ng/ml, and present at a concentration as low as 50 ng/ml. Due to the likely role of attractin in cell guidance and amplification of the immune response, our results indicate that the lack of up‐regulation of the molecule in patients with CVID may in turn affect any further step of productive immune response. Our finding may also imply a potential therapeutic role for this novel molecule.

Group V Secretory Phospholipase A2 Is Involved in Macrophage Activation and Is Sufficient for Macrophage Effector Functions in Allergic Pulmonary Inflammation

We reported that Pla2g5-null mice lacking group V secretory phospholipase A2 (gV-sPLA2) showed reduced eosinophilic pulmonary inflammation and Th2 cytokine generation when challenged with an extract from house dust mite Dermatophagoides farinae, compared with wild-type (WT) controls. Adoptive transfer studies suggested that gV-sPLA2 in dendritic cells was necessary for sensitization of Pla2g5-null mice, but was not sufficient to induce the effector phase of pulmonary inflammation. In this study, we demonstrate that gV-sPLA2 is inducibly expressed in mouse and human macrophages (Mϕ) activated by IL-4 and is required for the acquisition of Mϕ effector functions that facilitate the effector phase of pulmonary inflammation. We demonstrate that gV-sPLA2 expression in Mϕ is sufficient for the development of pulmonary inflammation, even when inflammation is induced by intrapulmonary administration of IL-4. The concentrations of CCL22/CCL17 and effector T cell recruitment are severely impaired in Pla2g5-null mice. Intratracheal transfers of enriched CD68+ cells isolated from the lungs of D. farinae–challenged WT donor mice induce eosinophilia, chemokine production, and recruitment of T cells into the lungs of Pla2g5-null recipients previously sensitized by WT D. farinae–loaded dendritic cells. Our studies identified a unique function of gV-sPLA2 in activation of Mϕ and in their capacity to recruit T cells to amplify the effector phase of pulmonary inflammation.