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Background Intestinal ischemia caused by hemorrhagic shock is known to induce systemic inflammatory responses. Previous studies have shown that mesenteric lymph (ML) plays a crucial role in gut-mediated inflammation. Lipid mediators, such as lysophosphatidylcholines (LPCs) which contain polyunsaturated fatty acids (PUFAs), are present in the post-shock ML. Exosomes are also present in the ML and act as transcellular carriers of lipids; however, their role in post-shock systemic inflammation has not been revealed. Here, we aimed to identify changes in lipid mediators in ML exosomes after intestinal ischemia. Methods Male Sprague-Dawley rats underwent laparotomy, followed by ML duct cannulation. Animals were subjected to 60 min of intestinal ischemia by superior mesenteric artery clamping, followed by 120 min of reperfusion. ML was obtained before and after intestinal ischemia and exosomes were isolated from ML by ultracentrifugation. The biological activity of ML exosomes was determined using the monocyte NF-κB activation assay. Lipids of ML exosomes were extracted and quantified by liquid chromatography/electrospray ionization mass spectrometry. Results ML exosomes-induced NF-κB activation significantly increased after intestinal ischemia and lipid analysis revealed a significant increase in the concentration of PUFA-containing LPCs. Additionally, PUFA-containing LPCs also induced NF-κB activation. Conclusion Our results suggest that biologically active lipid mediators in ML exosomes may be involved in the inflammatory response after intestinal ischemia. Level of evidence Not applicable (Basic Science paper).

BACKGROUND Phospholipase A2 (PLA2) is associated with a variety of inflammatory processes related to polymorphonuclear neutrophil (PMN)-endothelial cell interactions. However, the cellular and molecular mechanisms underlying the interactions and the causative isoform(s) of PLA2 remain elusive. In addition, we recently showed that calcium-independent PLA2γ (iPLA2γ), but not cytosolic PLA2 (cPLA2), is responsible for the cytotoxic functions of human PMN including respiratory bursts, degranulation, and chemotaxis. We therefore hypothesized that iPLA2γ is a prerequisite for the PMN recruitment cascade into the site of inflammation. The aim of this study was to elucidate the roles of the three major phospholipases A2, iPLA2, cPLA2 and secretory PLA2, in leukocyte rolling and adherence and in the surface expression of β2-integrins in vivo and in vitro in response to well-defined stimuli. METHODS Male Wistar rats were pretreated with PLA2 inhibitors selective for iPLA2β, iPLA2γ, cPLA2, or secretory PLA2. Leukocyte rolling/adherence in the mesenteric venules superfused with platelet-activating factor (PAF) were quantified by intravital microscopy. Furthermore, isolated human PMNs or whole blood were incubated with each PLA2 inhibitor and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or PAF. PMN adherence was assessed by counting cells bound to purified fibrinogen, and the surface expression of lymphocyte function-associated antigen 1 and macrophage antigen 1 (Mac-1) was measured by flow cytometry. RESULTS The iPLA2γ-specific inhibitor almost completely inhibited the fMLP/PAF-induced leukocyte adherence in vivo and in vitro and also decreased the fMLP/PAF-stimulated surface expression of Mac-1 by 60% and 95%, respectively. In contrast, the other inhibitors did not affect these cellular functions. CONCLUSION iPLA2γ seems to be involved in leukocyte/PMN adherence in vivo and in vitro as well as in the up-regulation of Mac-1 in vitro in response to PAF/fMLP. This enzyme is therefore likely to be a major regulator in the PMN recruitment cascade.

The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA.Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system.The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis.iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

BACKGROUND: After trauma and hemorrhagic shock (T/HS), a variety of inflammatory mediators enter the systemic circulation through mesenteric lymph ducts, leading to acute lung injury and multiple-organ dysfunction syndrome. Recent studies have demonstrated that post-HS mesenteric lymph (PHSML) activates polymorphonuclear leukocytes (PMNs) and causes vascular endothelial cell and red blood cell dysfunction. Furthermore, PHSML contains proinflammatory mediators, such as biologically active lipids. The purpose of this study was to identify the lipid mediators in PHSML and plasma by liquid chromatography/electrospray ionization mass spectrometry and then estimate the biologic activities of the identified lipids on PMNs. METHODS: PHSML was collected from male Sprague-Dawley rats undergoing trauma (laparotomy) plus HS (40 mm Hg, 30 minutes) or sham shock (SS). The lipids in PHSML and plasma were extracted using the methods of Bligh and Dyer, and liquid chromatography/ electrospray ionization mass spectrometry was performed. The biologic activities (superoxide production and elastase release) of identified lipids on human PMNs were tested. RESULTS: Phosphatidylcholine, lysophosphatidylcholine (LPC), phosphatidylethanolamine, lysophosphatidylethanolamine (LPE), and sphingomyelin were detected in the PHSML. Furthermore, linoleoyl, arachidonoyl, and docosahexaenoyl LPCs and LPEs significantly increased in the PHSML of the T/HS group as compared with those of the T/SS group. In the plasma, arachidonoyl and docosahexaenoyl LPCs of the T/HS group also significantly increased in comparison with that of the T/SS group. Linoleoyl and arachidonoyl LPCs and LPEs showed the priming activity on N-formyl-methionyl-leucyl-phenylalanineYactivated PMNs. The elastase release was also induced by linoleoyl and arachidonoyl LPCs. CONCLUSION: Mesenteric lymph after T/HS contains biologically active lipids, such as LPCs and LPEs with polyunsaturated fatty acids, which may be involved in the pathogenesis of acute lung injury/multiple-organ dysfunction syndrome. (J Trauma Acute Care Surg. 2012;72: 1541Y1547.