Junichi Aiboshi

Circulating Postinjury neutrophils are primed for the release of proinflammatory cytokines

BACKGROUND Postinjury neutrophil (PMN) priming identifies the injured patient at risk for the subsequent development of multiple organ failure (MOF). PMN priming has previously been shown to cause enhanced release of proteases and superoxide. PMNs, however, are a rich source of proinflammatory cytokines, such as interleukin (IL)-8 and tumor necrosis factor (TNF), which have been implicated in the development of MOF. PMNs also make IL-1ra, which is an anti-inflammatory cytokine that inhibits IL-1. It is our hypothesis that postinjury PMNs are primed for increased stimulated release of the proinflammatory cytokines IL-8 and TNF but not the anti-inflammatory cytokine IL-1ra. METHODS Twelve trauma patients with a mean Injury Severity Score of 24 (+/-4.6) and 10 elective surgical patients were studied. Postinjury PMNs were isolated from blood obtained at presentation (within 2 hours after injury) and 24 hours after trauma. PMNs from elective surgical patients were obtained preoperatively, immediately postoperatively, and at 24 hours. The PMNs were stimulated with platelet-activating factor (200 nM)/N-formyl-methionyl-leucyl-phenylalanine (1 micromol/L) or lipopolysaccharide (100 ng/mL) incubated for 24 hours in RPMI-1640, and release of IL-8, TNF, and IL-1ra were measured. RESULTS Postinjury PMNs were primed for both platelet-activating factor/N-formyl-methionyl-leucyl-phenylalanine-stimulated and lipopolysaccharide-stimulated IL-8 and TNF release at 2 hours after injury (fourfold increase of IL-8 release and fivefold increase of TNF release), whereas elective surgical patients demonstrated no priming. In contrast, postinjury patients were not primed for increased release of the counterinflammatory cytokine IL-1ra, suggesting a specific postinjury up-regulation of IL-8 and TNF. CONCLUSION After injury, PMNs are primed for proinflammatory cytokine release in addition to superoxide and elastase. This augmented release of IL-8 and TNF may be involved in the subsequent development of organ dysfunction and ultimately MOF.

Lipidomics analysis of mesenteric lymph after trauma and hemorrhagic shock.

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-phenylalanine–activated 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.

Group VIB Ca(2+)-independent phospholipase A(2γ) is associated with acute lung injury following trauma and hemorrhagic shock.

BACKGROUND Gut-derived mediators are carried via mesenteric lymph duct into systemic circulation after trauma/hemorrhagic shock (T/HS), thus leading to acute lung injury (ALI)/multiple-organ dysfunction syndrome. Phospholipase A2 (PLA2) is a key enzyme for the production of lipid mediators in posthemorrhagic shock mesenteric lymph (PHSML). However, the precise functions of PLA2 subtype, such as cytosolic PLA2, secretory PLA2, and Ca2+-independent PLA2, in the acute phase of inflammation have remained unclear. Our previous study has suggested that the activation of Group VIB Ca2+-independent PLA2&ggr; (iPLA2&ggr;) may be associated with increased lyso-phosphatidylcholines (LPCs) in the PHSML. Therefore, our purpose was to verify the role of iPLA2&ggr; on the production of 2-polyunsaturated LPC species and the pathogenesis of T/HS-induced ALI using an iPLA2&ggr;-specific inhibitor, R-(E)-6-(bromoethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (R-BEL). METHODS Male Sprague-Dawley rats were anesthetized and cannulated in blood vessels and mesenteric lymph duct. Animals in the T/HS group underwent a midline laparotomy plus hemorrhagic shock (mean arterial pressure, 35 mm Hg, 30 minutes) and 2-hour resuscitation with shed blood and 2× normal saline. Trauma/sham shock rats were performed the identical procedure without hemorrhage. R-BEL or DMSO was administered 30 minutes before T/HS or trauma/sham shock. Polyunsaturated LPCs and arachidonic acid in the PHSML were analyzed with a liquid chromatography/electrospray ionization–mass spectrometry. Furthermore, ALI was assessed by lung vascular permeability, myeloperoxidase activity, and histology. RESULTS T/HS increased 2-polyunsaturated LPCs and arachidonic acid in the PHSML. The R-BEL pretreatment significantly decreased these lipids and also inhibited ALI. CONCLUSION The iPLA2&ggr; enzyme is possibly involved in the pathogenesis of ALI following T/HS through the mesenteric lymph pathway.

Needs for disaster medicine: lessons from the field of the Great East Japan Earthquake.

PROBLEM The Great East Japan Earthquake, which occurred in Tohoku, Japan on 11 March 2011, was followed by a devastating tsunami and damage to nuclear power plants that resulted in radiation leakage. CONTEXT The medical care, equipment and communication needs of four Disaster Medical Assistance Teams (DMAT) during four missions are discussed. DMATs are medically trained mobile teams used in the acute phase of disasters. ACTION The DMATs conducted four missions in devastated areas from the day of the earthquake to day 10. The first and second missions were to triage, resuscitate and treat trauma victims in Tokyo and Miyagi, respectively. The third mission was to conduct emergency medicine and primary care in Iwate. The fourth was to assist with the evacuation and screening of inpatients with radiation exposure in Fukushima. OUTCOME Triage, resuscitation and trauma expertise and equipment were required in Missions 1 and 2. Emergency medicine in hospitals and primary care in first-aid stations and evacuation areas were required for Mission 3. In Mission 4, the DMAT assisted with evacuation by ambulances and buses and screened people for radiation exposure. Only land phones and transceivers were available for Missions 1 to 3 although they were ineffective for urgent purposes. DISCUSSION These DMAT missions showed that there are new needs for DMATs in primary care, radiation screening and evacuation after the acute phase of a disaster. Alternative methods for communication infrastructure post-disaster need to be investigated with telecommunication experts.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

BACKGROUND 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 iPLA2&ggr;-specific inhibitor attenuates trauma/hemorrhagic shock–induced lung injury. Therefore, iPLA2&ggr; 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 iPLA2&ggr; 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. METHODS Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2&bgr;, iPLA2&ggr;, 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. RESULTS The iPLA2&ggr;-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 iPLA2&ggr;, but not iPLA2&bgr; or cPLA2, completely inhibited the platelet activating factor/fMLP–induced chemotaxis. CONCLUSION 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.

Novel role of group VIB Ca2+-independent phospholipase A2γ in leukocyte-endothelial cell interactions: An intravital microscopic study in rat mesentery.

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 PLA2&ggr; (iPLA2&ggr;), but not cytosolic PLA2 (cPLA2), is responsible for the cytotoxic functions of human PMN including respiratory bursts, degranulation, and chemotaxis. We therefore hypothesized that iPLA2&ggr; 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 &bgr;2-integrins in vivo and in vitro in response to well-defined stimuli. METHODS Male Wistar rats were pretreated with PLA2 inhibitors selective for iPLA2&bgr;, iPLA2&ggr;, 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 iPLA2&ggr;-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 iPLA2&ggr; 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.

Encapsulating Peritoneal Sclerosis Complicated by an Intra-abdominal Abscess

We present a case of a 68-year-old woman who developed encapsulating peritoneal sclerosis (EPS) with an intra-abdominal abscess. The patient was referred to our hospital with abdominal pain, nausea, and vomiting. She had end-stage kidney disease secondary to diabetes mellitus that had been treated with continuous ambulatory peritoneal dialysis for 9 years. EPS had been diagnosed 1 year ago, and she had been treated with prednisone daily. On presentation, a computed tomographic scan showed a calcified peritoneum and intra-abdominal abscess, and surgery showed that the abscess was caused by a bowel perforation. The perforated bowel could not be sutured or resected because of the presence of fibrotic tissue and peritoneal calcification. She was treated with bowel rest with total parenteral nutrition, as well as general antibiotic therapy and drainage for 8 months. However, the site of perforation did not heal, and she died of septic shock. Because treatment of EPS complicated by bowel perforation is very difficult, it is necessary to diagnose and treat the early stages of EPS to prevent bowel perforation. Imaging techniques are important in making an early diagnosis and successfully managing EPS.