Anti-PLA2G10 monoclonal antibody, clone 6H22

Background: Phospholipase A(2)s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA(2)alpha (cPLA(2)alpha) is thought to be the dominant PLA(2) in eosinophils; however, eosinophils also have secreted PLA2 (sPLA(2)) activity that has not been fully defined. Objectives: To examine the expression of sPLA(2) group X (sPLA(2)-X) in eosinophils, the participation of sPLA(2)-X in the formation of CysLTs, and the mechanism by which sPLA(2)-X initiates the synthesis of CysLTs in eosinophils. Methods: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA(2)-X antibody identified sPLA(2)-X by Western blot. We used confocal microscopy to colocalize the sPLA(2)-X to intracellular structures. Aninhibitor of sPLA(2)-X (ROC-0929) that does not inhibit other mammalian sPLA(2)s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA(2)alpha, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Results: Eosinophils express the mammalian sPLA(2)-X gene (PLA2G10). The sPLA(2)-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA(2)alpha also attenuated the fMLP-mediated formation of CysLT. The sPLA(2)-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA(2)a during cellular activation, indicating that sPLA(2)-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA(2)alpha. We further demonstrate that sPLA(2)-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1 beta increased the expression of PLA2G10 by eosinophils. Conclusions: These results demonstrate that sPLA(2)-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA(2)alpha. The sPLA(2)-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

An appropriate female reproductive environment is essential for pregnancy success. In several species, including mice, pigs and horses, seminal plasma (SP) components have been shown to modulate this environment, leading to increased embryo viability and implantation. Due to the characteristics of mating in the aforementioned species, SP comes into direct contact with the uterus. However, it is questionable whether any SP reaches the uterus in species that ejaculate inside the vagina, such as humans and cattle. Hence, we hypothesized that sperm, perhaps acting as a vehicle for SP factors, play a more important role in the modulation of the maternal uterine environment in these species. In addition, changes elicited by SP and/or sperm may originate in the vagina and propagate to more distal regions of the female reproductive tract. To test these hypotheses, a bovine model in which heifers were mated to intact or vasectomized bulls or were left unmated was used. RNA-sequencing of endometrial samples collected 24 h after mating with a vasectomized bull did not reveal any differentially expressed genes (DEGs) in comparison with control samples. However, the endometrium of heifers mated with intact bulls exhibited 24 DEGs when compared to heifers mated with vasectomized bulls, and 22 DEGs when compared to unmated control heifers. The expression of a set of cytokines (IL6,IL1A,IL8, andTNFA) and candidate genes identified in the endometrial RNA-sequencing (PLA2G10,CX3CL1,C4BPA,PRSS2,BLA-DQB, andCEBPD) were assessed by RT-qPCR in the vagina and oviductal ampulla. No differences in expression of these genes were observed between treatments in any region. However, mating to both intact and vasectomized bulls induced an increase inIL1AandTNFAexpression in the vagina compared to the oviduct. These data indicate that sperm, but not secretions from the accessory glands alone, induce modest changes in endometrial gene expression after natural mating in cattle. However, it is not clear whether this effect is triggered by inherent sperm proteins or SP proteins bound to sperm surface at the time of ejaculation.