Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation

Our objective in this study was to test the hypothesis that priming of neutrophils (PMN) in vivo by trauma-hemorrhagic shock (T/HS) is mediated by factors carried in intestinal lymph that prime PMNs by enhancing their responses to inflammatory mediators. Previous studies have shown that T/HS-induced lung injury is mediated by factors contained in mesenteric lymph and that ligation of the main mesenteric lymph duct (LDL) can prevent T/HS-induced lung injury. Since T/HS-induced lung injury is associated with PMN infiltration, one mechanism underlying this protective effect may be the prevention of PMN priming and activation. Therefore, we assessed the ability of T/HS to prime PMN responses to inflammatory agonists, and the ability of mesenteric lymph duct division to protect against such T/HS-induced PMN priming in an all-rat system. PMN were collected from male rats 6 h after laparotomy (trauma) plus hemorrhagic shock (30 mmHg for 90 min; T/HS) or trauma plus sham shock (T/SS). Uninstrumented rats were used as controls (UC). In a second set of experiments, rats were subjected to T/HS with or without mesenteric lymph duct division. PMN were then stimulated with chemokine (GRO, MIP-2) and lipid (PAF) chemoattractants, and cell calcium flux was used to quantify responses to those agonists. T/SS primed PMN responses to GRO, MIP-2. and PAF in comparison to UC rats, but the addition of shock (T/HS) amplified PMN priming in a significant manner, especially in response to GRO. Mesenteric lymph duct division prior to T/HS diminished PMN priming to the levels seen in T/SS. This reversal of priming was significant for GRO and GRO/MIP-2 given sequentially, with the other agonist regimens showing similar trends. The results support the concept that trauma and hemorrhagic shock play important additive roles in inflammatory PMN priming. Entry of gut-derived inflammatory products into the circulation via mesenteric lymph seems to play a dominant role in mediating the conversion of physiologic shock insults into immunoinflammatory PMN priming. Shock-induced gut lymph priming enhances PMN responses to many important chemoattractants, most notably the chemokines, and mesenteric lymph duct division effectively reverses such priming to priming levels seen in trauma without shock.     

Lymph from a primate baboon trauma hemorrhagic shock model activates human neutrophils

We have reported that toxic factors in intestinal lymph are responsible for acute lung injury and bone marrow suppression and that they contribute to a systemic inflammatory state based on studies in rodent models of trauma-hemorrhagic shock. Rodent models may not completely reflect the responses of injured patients. Thus, it is important to confirm these findings in primates before applying them to injured human patients with trauma. Thus, we have recently established baboon trauma-hemorrhagic shock (T/HS) and trauma-sham shock (T/SS) models that showed that gut-derived factors carried in the lymph potentiates lung injury and causes human endothelial dysfunction and suppresses human bone marrow progenitor cell growth. Here, we further investigated the effects of these primate lymph samples on human neutrophils. We hypothesized that toxic factors in baboon lymph may prime and/or activate human polymorphonuclear leukocyte (PMN) leading to overproduction of superoxide, thereby contributing to the development of adult respiratory distress syndrome and multiple organ failure. To this effect, we have examined the priming effect of baboon T/HS and T/SS lymph on PMN respiratory burst and expression of adhesion molecule in human neutrophils. The results of these studies indicate that PMN treated with baboon T/HS lymph showed significantly induced respiratory burst responses compared with PMN treated with T/SS lymph or medium when phorbol myristate acetate PMA was applied after lymph pretreatment. Secondly, we found that the expression of CD11b adhesion molecule was increased by incubation with T/HS lymph. These results suggest that baboon lymph from T/HS models can increase respiratory burst and adhesion molecule expression in human PMN, thereby potentially contributing to PMN-mediated organ injury.     

CXCR2 stimulation primes CXCR1 [Ca2+]i responses to IL-8 in human neutrophils

Neutrophil (PMN) priming and subsequent responses to the IL-8 presented on pulmonary endothelial surfaces may be crucial determinants of the development of adult respiratory distress syndrome after injury. Elevated plasma ELR+ C-X-C chemokine (CXC) levels might contribute to PMN priming after trauma, but the role of CXCs in priming circulating PMNs is unstudied. We evaluated the interactions of IL-8 and GRO-alpha in priming human PMN calcium fluxes [Ca2+]i within circulatory environments. At physiologic concentrations, GRO-alpha primes PMN for IL-8 mediated [Ca2+]i mobilization, whereas IL-8 abolishes GRO-alpha responses. Repeated GRO-alpha exposures further enhance IL-8 responses. PMN priming for IL-8 responses in normal plasma was CXCR2 dependent. CXCR2 was more responsive than CXCR1 to low levels of IL-8, together suggesting that CXCR2 is the important CXC receptor at circulating (i.e., low) agonist concentrations. CXCR1 stimulation down-regulated CXCR2 surface expression, whereas CXCR2 stimulation upregulated CXCR1 expression. GRO-alpha/ CXCR2 signaling enhanced post-receptor IL-8 initiated PMN [Ca2+]i influx as well as efflux. Sufficient stimulation of the CXCR1 terminated this cooperative relationship by downregulating surface expression of CXCR2. This study is the first to report that at physiologic concentrations, C-X-C chemokines can act on circulating human PMNs as an integrated system where CXCR2 agonists, rather than cross-desensitizing CXCR1, act to enhance signaling of IL-8 at CXCR1 both by receptor and post-receptor mechanisms. Such CXCR2 mediated priming of CXCR1/ IL-8 interaction may enhance PMN attack on the lung after injury.     

Hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock

There is evidence suggesting that the ischemic gut is a major source of factors that lead to neutrophil activation, and that neutrophil activation can be reduced by hypertonic saline resuscitation. Thus, we tested whether trauma-hemorrhagic shock-induced neutrophil activation can be reduced by hypertonic saline resuscitation, as well as whether hypertonic saline reduces the ability of mesenteric lymph from shocked animals to activate neutrophils. Male Sprague-Dawley rats subjected to trauma (laparotomy), plus 90 min of shock [mean arterial pressure (MAP) MAP = 30 mmHg] or sham shock were resuscitated with Ringer's lactate or 7.5% hypertonic saline at an equivalent sodium load. Whole blood samples were collected before shock as well as at 1 and 2 h after the end of the shock period for neutrophil CD11b and CD18 expression measurements. In a second set of experiments, mesenteric lymph samples collected from rats subjected to trauma plus hemorrhagic shock (T/HS) or trauma plus sham-shock (T/SS) and resuscitated with Ringer's lactate or hypertonic saline were tested for their ability to modulate PMN CD11b, CD18, or L-selectin expression, as well as prime PMN for an augmented respiratory burst. To avoid confounding results due to interspecies differences, while at the same time looking at potential human responses, both naive rat and human PMN were tested. Both CD11b and CD18 expression were increased in PMN harvested from rats subjected to T/HS and resuscitated with Ringer's lactate solution, but not in T/HS rats resuscitated with hypertonic saline. These results indicate that PMN activation is increased to a greater extent in Ringer's lactate-resuscitated than hypertonic saline-resuscitated animals. Likewise, mesenteric lymph from the T/HS rats resuscitated with Ringer's lactate increased naive rat and human PMN CD11b and CD18 expression to a greater extent than did T/HS lymph from the hypertonic saline-treated rats. Additionally, T/HS lymph from the Ringer's lactate- but not the hypertonic saline-treated rats induced PMN L-selectin shedding. Lastly, T/HS lymph from the Ringer's lactate-treated rats induced the greatest PMN respiratory burst. These results indicate that resuscitation from T/HS with hypertonic saline is associated with less PMN activation than resuscitation with Ringer's lactate, and that factors produced or released by the postischemic intestine and carried in the mesenteric lymph contribute to neutrophil activation after an episode of T/HS.     

Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury

The objective of this work was to test the hypothesis that Intraluminal serine proteases are involved in trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury. Male Sprague-Dawley rats were administrated the serine protease inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, Nafamostat) either intraluminally into the gut or intravenously after a laparotomy (trauma) and then subjected to 90 min of hemorrhagic shock (T/HS) or sham shock (T/SS). Intestinal and lung injury was assessed at 3 h after resuscitation with Ringer's lactate solution. In a second set of experiments, mesenteric lymph was collected from the groups of rats subjected to T/HS or T/SS and its ability to activate normal neutrophils was tested. Lung permeability, pulmonary myeloperoxidase levels, and the bronchoalveolar lavage fluid protein to plasma protein ratio were increased after T/HS but were significantly decreased in the T/HS rats receiving intraluminal (P < 0.05), but not intravenous, nafamostat. Likewise, T/HS-induced intestinal villus injury was less in the nafamostat-treated shock rats (P < 0.05). Last, the ability of T/HS mesenteric lymph to increase PMN CD11b expression or prime neutrophils for an augmented respiratory burst was significantly reduced by the intraluminal administration of nafamostat. Because intraluminal nafamostat reduced T/HS-induced gut and lung injury as well as the neutrophil activating ability of intestinal T/HS lymph, the presence of serine proteases in the ischemic gut may play an important role in T/HS-induced gut and hence lung injury.     

Hyperosmolarity abrogates neutrophil cytotoxicity provoked by post-shock mesenteric lymph

Hypertonic saline (HTS) resuscitation inhibits acute lung injury in animal models of shock, but some argue this may simply represent more efficient fluid resuscitation. Inflammatory mediators within mesenteric lymph have been identified as a link between splanchnic hypoperfusion and acute respiratory distress syndrome (ARDS). We hypothesize that HTS resuscitation abrogates post-shock lymph-mediated neutrophil (PMN) priming and PMN-mediated human endothelial cell cytotoxicity. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock, defined as a mean arterial pressure (MAP) of 40 mmHg for 30 min, and after resuscitation (shed blood + 2 x lactated Ringers (LR) versus 7.5% NaCl, 4 cc/kg, over 5 min). Isolated human PMNs were primed with physiologic concentrations (5% v:v) of lymph either from animals resuscitated with LR or HTS and activated with either PMA or fMLP. In a separate set of experiments, human PMNs were primed with LR lymph after incubation with HTS (180 mM NaCl). The maximal rate of superoxide production was measured by reduction of cytochrome C. In addition, the effect of HTS pretreatment on PMN adherence to human pulmonary microvascular endothelial cells (HMVEC) and PMN-mediated cytotoxicity was determined after lymph-mediated PMN priming. PHSML primed isolated PMNs above buffer controls and pre-shock lymph in a normotonic environment; HTS resuscitation abrogated this effect. HTS preincubation of isolated PMNs inhibited PHSML-induced PMN priming, adherence to HMVECs, and PMN-mediated HMVEC cytotoxicity. Hypertonic resuscitation (HTS) abrogates PHSML pniming of the PMN and PMN-mediated HMVEC cytotoxicity. Furthermore, incubation of PMNs in clinically relevant HTS (180 mM NaCl) prevents PHSML PMN priming and PMN:HMVEC interactions. These studies suggest inhibition of PMN signal transduction is a mechanism whereby HTS resuscitation abrogates acute lung injury.     

The lipid fraction of post-hemorrhagic shock mesenteric lymph (PHSML) inhibits neutrophil apoptosis and enhances cytotoxic potential

Dysfunctional neutrophil (PMN) apoptosis facilitates hyperinflammatory tissue injury. Previous work has demonstrated that post-hemorrhagic shock mesenteric lymph (PHSML) provokes PMN-mediated acute lung injury in animal models, but the mechanism remains unclear. We have documented that the lipid fraction of PHSML is responsible for PMN priming of the respiratory burst. In this study, we hypothesized that PHSML lipids delay PMN apoptosis and thereby further enhance PMN cytotoxic potential. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock (MAP 40 mmHg, 30 min), and during resuscitation (shed blood + 2x crystalloid). Human PMNs were incubated with control, PHSML, PHSML lipid extracts, and heat-treated PHSML (60 degrees C, 30 min.) at 1-10% (v:v) in RPMI 1640 for 24 h. Apoptosis was assessed using acridine orange/ethidium bromide staining and fluorescence microscopy. Priming of the respiratory burst was evaluated by incubating PMNs with (a) control PHSML or (b) PHSML lipid extracts for 24 h and by activating with fMLP (1 micromol/L). PHSML and PHSML lipid extracts (5-10%) inhibited PMN apoptosis. Heat denaturing the PHSML (to eliminate cytokines and complement) had no effect on the inhibition of PMN apoptosis. Similarly, incubation with polymixin B at a concentration that binds endotoxin had no effect. Both the PHSML and PHSML lipids (5%) following 24-h incubation primed the fMLP-activated oxidase. At physiologic concentrations, both PHSML and the lipid fraction of PHSML delay PMN apoptosis and prime the NADPH oxidase. These data further implicate the lipid components of mesenteric lymph as central in the pathogenesis of hemorrhagic shock induced PMN-mediated acute lung injury.     

Post-hemorrhagic shock mesenteric lymph lipids prime neutrophils for enhanced cytotoxicity via phospholipase A2.

Hemorrhagic shock induced mesenteric hypoperfusion has long been implicated as a key event in the pathogenesis of the adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Previous work links post-hemorrhagic shock mesenteric lymph (PHSML) lipids and neutrophil (PMN) priming in the pathogenesis of ARDS. We hypothesize that gut phospholipase A2 (PLA2) liberates proinflammatory lipids following hemorrhagic shock, which are responsible for enhanced PMN cytotoxicity. Mesenteric lymph was collected from rats (n > or = 5) before hemorrhagic shock, during hemorrhagic shock (MAP 40 mm Hg x 30 min), and after resuscitation (shed blood + 2x lactated Ringers). PMNs were incubated with physiologic concentrations (1-5%, v:v) of (a) buffer control, (b) sham (c) pre-shock lymph, (c) PHSML, (d) PHSML lipid extracts, (e) heat-denatured PSHML, and (f) PHSML harvested after i.v. pretreatment with a known PLA2 inhibitor (quinacrine, 10 mg/kg). PMNs were activated with fMLP (1 micromol), and the maximal rate of superoxide production measured by reduction of cytochrome c. Gut morphology was assessed histologically using hematoxalin and eosin (HE) staining. PHSML and PHSML lipid extracts (5%, v:v) primed for enhanced superoxide production compared to buffer controls (2.5-fold and 3.6-fold), sham (2.5-fold) and pre-shock lymph (2.0-fold). Lymph collected after systemic PLA2 inhibition, in contrast, abrogated the PMN priming response. Gut mucosal morphology, at end-resuscitation, was intact on HE staining both with and without PLA2 inhibition. Heat denaturing the PHSML (eliminating cytokines and complement), on the other hand, did not reduce PMN priming. Physiologic concentrations of PHSML lipids prime the PMN respiratory burst. Lymph priming is diminished with systemic PLA2 inhibition, implicating gut PLA2 as a source of proinflammatory lipids that may be central in the pathogenesis of hemorrhagic shock induced ARDS/MOF.     

Inflammatory roles of P-selectin.

Polymorphonuclear leukocytes (PMNs) bind rapidly and reversibly to endothelial cells induced to express P-selectin, a glycoprotein that mediates adhesive intercellular interactions. In addition, PMNs adherent to endothelium expressing P-selectin demonstrate an intracellular Ca2+ transient, functionally up-regulate beta-2-integrins (CD11/CD18 glycoproteins), become polarized in shape, and are primed for enhanced degranulation when subsequently stimulated with chemotactic factors. However, P-selectin induces none of these responses directly when used alone, when incorporated into model membranes, or when expressed by transfected cells. The absence of direct activation of the PMNs is not due to competing antiinflammatory effects of P-selectin; instead, purified P-selectin and P-selectin in membranes support agonist-stimulated PMN responses. Furthermore, tethering of PMNs to endothelial surfaces by P-selectin is required for priming to occur efficiently, as shown by experiments with blocking monoclonal antibodies. The priming event is directly mediated by the signaling molecule, platelet-activating factor (PAF), and is inhibited by blocking the PAF receptor on PMNs. Thus, P-selectin and PAF are components of an adhesion and activation cascade, but have distinct roles: P-selectin tethers and captures the PMN, whereas PAF mediates juxtacrine activation. In vivo, selectins may facilitate interaction of target cells with membrane-bound molecules that send intercellular signals, in addition to mediating rolling of leukocytes and other adhesive functions.     

Mesenteric lymph from rats subjected to trauma-hemorrhagic shock are injurious to rat pulmonary microvascular endothelial cells as well as human umbilical vein endothelial cells

Previously, we have documented that gut-derived lymph from rats subjected to trauma/hemorrhagic shock (T/HS) is injurious to human umbilical vein endothelial cells (HUVEC). To verify these findings in an all rat systems, the ability of T/HS lymph to increase rat pulmonary microvascular endothelial cell (RPMVEC) monolayer permeability and kill RPMVEC was compared with that observed with HUVECs. RPMVEcs isolated from male rats or HUVECs were grown in 24-well plates for the cytotoxicity assays or on permeable filters in a two-chamber system for permeability assays. Mesenteric lymph was collected from male rats subjected to trauma (laparotomy) plus hemorrhagic shock (T/HS group) or to a laparotomy plus sham-shock (T/SS group). The T/HS group had their mean arterial pressure decreased to 30 mmHg and kept there for 90 min. Lymph samples centrifuged to remove the cellular component were incubated with the RPMVECs or HUVECs at a 10% concentration. Neither T/SS lymph nor post-T/HS portal vein plasma was toxic to or increased the permeability of the RPMVECs or HUVECs. The pattern of cytotoxicity observed in the HUVECs incubated with T/HS mesenteric lymph was similar to that observed in the RPMVECs, as reflected by trypan blue dye exclusion, with more than 95% of the HUVECs and RPMVECs being killed after a 16-h incubation with T/HS mesenteric lymph. However, at earlier time points the amount of LDH released from the HUVEC cells incubated with T/HS lymph was greater than that observed with the PRMVEC, although trypan blue dye exclusion was similar. Similarly, incubation with 10% T/HS lymph increased the permeability of both HUVEC and RPMVEC monolayers more than 2-fold, even with an incubation period as short as 1 h. In conclusion, these results provide further evidence that T/HS lymph, but not T/SS lymph or post-T/HS portal vein plasma, is injurious to endothelial cells and that RPMVECs are as susceptible to injury as HUVECs. Additionally, these studies support the emerging concept that gut-induced distant organ injury is mediated by factors contained in mesenteric lymph.     

Trauma-shock-induced gut injury and the production of biologically active intestinal lymph is abrogated by castration in a large animal porcine model

Although small animal rodent studies indicate that there is a sexual dimorphism in the resistance to organ injury after trauma-hemorrhagic shock (T/HS), confirmatory studies are largely lacking in more clinically relevant large animal species. Thus, we tested the hypothesis that castration would reduce the susceptibility of adult minipigs to gut injury and abrogate the production of biologically active intestinal (mesenteric) lymph after T/HS. The hemodynamic response to T/HS was similar between castrated and noncastrated minipigs. Mesenteric lymph collected during the preshock period and in the trauma-sham shock (T/SS) animals did not have increased biological activity. However, T/HS-lymph from the noncastrated males increased the respiratory burst of normal neutrophils, increased endothelial cell monolayer permeability, and was cytotoxic for endothelial cells. Castration abrogated the T/HS-induced neutrophil-activating and endothelial-injurious activities of mesenteric lymph, and the biological activity of the T/HS-lymph from the castrated minipigs was not different from the T/SS animals. As compared with the T/SS minipigs, T/HS increased ileal mucosal injury and intestinal permeability. This increase in gut permeability after T/HS was manifest by in vivo bacterial translocation and by the increased passage of bacteria as well as permeability probes across intestinal segments when tested in the Ussing chamber system. In contrast, neither mucosal injury nor increased intestinal permeability was observed in the castrated minipigs subjected to T/HS. In summary, this large animal porcine study validates the notion that castration limits gut injury and the production of biologically active intestinal lymph after T/HS.     

Blood transfusion and the two-insult model of post-injury multiple organ failure

Neutrophils (PMNs) have been implicated in the pathogenesis of multiple organ failure (MOF). The two-insult model of MOF is based on the fundamental concept that two sequential and independent insults that are individually innocuous against the host can cause overwhelming inflammation. The in vitro PMN priming/activation sequence simulates the two-insult model. Our work has demonstrated that transfusion is an early consistent risk factor for post-injury MOF and lysophosphatidylcholines (lyso-PCs) are generated in stored blood components. Additionally, platelet-activating factor (PAF) is a key inflammatory agent produced in severely injured patients. We therefore hypothesize that two events, trauma and transfusion, enhance PMN cytotoxicity irrespective of the sequence. Superoxide (O2-) production was measured by reduction of cytochrome c, adherence to fibrinogen was assessed by the radioactivity of adherent Na2(51)CrO4 (51Cr)-labeled PMNs, and endothelial cell (EC) damage by measuring the radioactivity released from 51Cr-labeled human umbilical vein endothelial cells monolayers. Isolated PMNs were primed with buffer, PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM) followed by activation with buffer, N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM), PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM). Neither PAF nor lyso-PCs alone stimulated O2- production. While PAF alone caused PMN adherence, lyso-PCs alone did not allowed PMNs to adhere to fibrinogen. However, both combinations of PAF/lyso-PCs and lyso-PCs/PAF significantly augmented O2- production and PMN adherence. Furthermore, these enhanced PMN cytotoxic responses significantly caused EC damage. These findings suggest that in the scenario of the two-insult model, early or late transfusion administered following trauma can provoke PMN cytotoxicity via priming or activation, thereby increasing the risk of post-injury MOF.     

Pancreatic duct ligation abrogates the trauma hemorrhage-induced gut barrier failure and the subsequent production of biologically active intestinal lymph

The studies of the mechanisms by which trauma-hemorrhagic shock leads to gut injury and dysfunction have largely ignored the nonbacterial factors contained within the lumen of the intestine. Yet, there is increasing evidence suggesting that intraluminal pancreatic proteases may be involved in this process. Thus, we tested the hypothesis that pancreatic proteases are necessary for the trauma-hemorrhagic shock-induced gut injury and the production of biologically active mesenteric lymph by determining the extent to which pancreatic duct ligation (PDL) would limit gut injury and mesenteric lymph bioactivity. To assess the effect of PDL on gut injury and dysfunction gut morphology, the mucus layer structure and the gut permeability were measured in the following four groups of male rats subjected to laparotomy (trauma) and hemorrhagic shock (pressure, 30 mmHg for 90 min): (1) rats subjected to trauma plus sham-shock (T/SS), (2) T/SS rats undergoing PDL (T/SS + PDL), (3) rats subjected to trauma and hemorrhagic shock (T/HS), and (4) rats subjected to T/HS + PDL. The ability of mesenteric lymph from these four rat groups to kill endothelial cells and activate neutrophils was tested in vitro. The PDL did not affect any of the parameters studied because there were no differences between the T/SS and the T/SS + PDL groups. However, PDL protected the gut from injury and dysfunction because PDL significantly abrogated T/HS-induced mucosal villous injury, loss of the intestinal mucus layer, and gut permeability. Likewise, PDL totally reversed the endothelial cell cytotoxic activity of T/HS lymph and reduced the ability of T/HS lymph to prime naive neutrophils for an augmented respiratory burst. Thus, it seems that intraluminal pancreatic proteases are necessary for the T/HS-induced gut injury and the production of bioactive mesenteric lymph.     

库存血小板血浆中sCD40L含量变化及其对PMNs呼吸爆发的作用

目的探讨单采血小板(A-PLTs)和白膜法浓缩血小板(BC-PLTs)库存过程中,其乏血小板血浆(简称血浆)中可溶性CD40配体(sCD40L)含量变化及对中性粒细胞(PMNs)呼吸爆发的作用。方法取库存d1、d3、d5的A-PLTs和BC-PLTs,离心分离获得血浆;ELISA检测血浆中sCD40L的含量;用活性氧特异性荧光探针二氢若丹明123(DHR),流式细胞术检测PMNs呼吸爆发;以小鼠抗人CD154抗体抑制试验去除sCD40L。结果 sCD40L含量(pg/mL),在血小板库存d1、d3、d5时,A-PLTs血浆分别为1 341.62±279.92、2 589.69±856.83及3 250±1 170.44(P<0.05);BC-PLTs血浆分别为3 342.35±1 322.30、3 827.12±1 714.43及3 691.87±1 631.03(P>0.05)。血浆引发fMLP(formyl-Met-Leu-Phe)活化的PMNs呼吸爆发程度(PMNs的引发活性),库存d1、d5 A-PLTs血浆处理组相对于单独加入fMLP阴性对照组其MFI比值相应为1.25±0.1、1.76±0.41(P<0.05)。用小鼠抗人CD154抗体特异性去除血浆中的sCD40L后血浆引发fMLP活化的PMNs呼吸爆发程度相应降低41.84%。以0.1μm滤器滤除血小板微粒后血浆引发fMLP活化的PMNs呼吸爆发程度相应降低69.56%。结论常规库存的血小板随库存时间延长,其血浆中的sCD40L水平升高,并对fMLP活化的PMNs的呼吸爆发具有引发作用。     

Bioactivity of postshock mesenteric lymph depends on the depth and duration of hemorrhagic shock

Mesenteric hypoperfusion due to circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Postshock mesenteric lymph (PSML) has been shown to contain proinflammatory mediators elaborated from the ischemic gut. We hypothesize that the relative bioactivity of PSML depends on the depth and duration of circulatory shock. To first determine the timing of PSML bioactivity, we subjected rats to hemorrhagic shock (30 mm Hg x 45 min) and then resuscitation with 50 vol% of shed blood and normal saline (4x shed blood) over 2 h. Mesenteric lymph was collected hourly up to 6 h after shock. Superoxide release was measured from human neutrophils (polymorphonuclear neutrophils [PMNs]) incubated with lymph fractions collected from each of the hourly time points. Rats were then subjected to four different shock variations: (1) 30 mm Hg x 45 min, (2) 30 mm Hg x 15 min, (3) 45 mm Hg x 45 min, and (4) 45 mm Hg x 15 min, and were resuscitated. PSML flow depends on depth of shock, but not duration of shock or resuscitation volume. Maximal PSML bioactivity, as measured by PMN priming for the respiratory burst, occurred during the third postshock hour, which correlated with peak lymph flow rate. PSML bioactivity was greatest with 30 mm Hg x 45 min, followed by 30 mm Hg x 15 min, 45 mm Hg x 45 min, and 45 mm Hg x 15 min. Hemorrhagic shock provokes the release of bioactive agents in PSML that is dependent on both depth and duration of shock.     

Hypertonic saline inhibits neutrophil (PMN) priming via attenuation of p38 MAPK signaling

Priming of the neutrophil cytotoxic response is central to the pathogenesis of early postinjury multiple organ failure (MOF). Platelet-activating factor (PAF) has been implicated as a key inflammatory mediator in postinjury neutrophil priming and requires p38 MAPK signaling to produce its biologic effects. Hypertonic saline (HTS) resuscitation decreases the postinjury inflammatory response following shock in animals and decreases receptor-mediated neutrophil (PMN) cytotoxic functions in vitro. We hypothesized that HTS attenuates PAF priming of the PMN cytotoxic response by interfering with PAF-mediated p38 MAPK signal transduction. Isolated PMNs were preincubated in isotonic buffer or HTS (Na+ = 180 mM), then primed with PAF. Neutrophil CD11b/CD18 expression was measured by flow cytometry. Receptor-dependent (fMLP), N-formyl-methionyl-leucyl-phenylalanine, fMLP) and receptor-independent (PMA) O2- production was measured by reduction of cytochrome c in resting and PAF primed PMNs. Total p38 MAPK protein PAF-mediated p38 MAPK activation was assessed by western blot of PMN lysates. Clinically relevant levels of HTS attenuated PAF-mediated beta2-integrin expression. While HTS attenuated receptor-dependent (fMLP and PAF/fMLP) O2- production, receptor-independent (PMA) O2- production was unaffected. Conversely, HTS attenuated PAF priming of PMA-mediated O2- production. PAF and HTS did not alter total cellular p38 MAPK content. Clinically relevant levels of HTS alone did not activate p38 MAPK but inhibited PAF mediated p38 MAPK activation. HTS attenuates PAF priming of the PMN cytotoxic response by altering intracellular signal transduction. Therefore, HTS resuscitation may attenuate postinjury PMN priming and ultimately the risk of developing MOF.     

Activation of toll-like receptor 4 is necessary for trauma hemorrhagic shock-induced gut injury and polymorphonuclear neutrophil priming

Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)-induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4 mut) mice would be more resistant to T/HS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4 mut mice. In addition, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the wild-type mice was abrogated in TLR4 mut mice as well the TRIF mut-deficient mice and partially attenuated in Myd88 mice, suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph-induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that T/HS lymph-induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these "danger" proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph-induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph-associated effects of TLR4 on PMN priming.     

Compartmentalization of macrophage inflammatory protein-2, but not cytokine-induced neutrophil chemoattractant, in rats challenged with intratracheal endotoxin

An important feature of the pulmonary inflammatory response is that the production of certain cytokines and chemokines is largely confined to the lung. This study investigated the local and systemic responses of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) in rats administered with either intratracheal or intravenous lipopolysaccharide (LPS). Intratracheal LPS induced a significant increase in MIP-2 in bronchoalveolar lavage (BAL) fluid with no detectable MIP-2 in the plasma. In contrast, CINC was significantly increased in both BAL fluid and the plasma after intratracheal LPS challenge. Cell-associated MIP-2 was increased in the pulmonary-recruited neutrophils (PMNs) but not in the circulating PMNs in rats given intratracheal LPS. Cell-associated CINC was increased in both the recruited and circulating PMNs in these animals. Intravenous LPS caused a marked increase in plasma MIP-2 and CINC, whereas only a small elevation of both MIP-2 and CINC concentrations in BAL fluid was observed. The lack of CINC compartmentalization compared to MIP-2 implies that these C-X-C chemokines are regulated differentially and may have different effects upon polymorphonuclear leukocyte (PMN) recruitment into the alveolar space in response to intrapulmonary LPS or bacterial challenge.     

Calcium entry inhibition during resuscitation from shock attenuates inflammatory lung injury

Trauma and hemorrhagic shock (T/HS) induce a systemic inflammatory response syndrome (SIRS). Neutrophils (polymorphonuclear leukocytes [PMN]) and other cells involved in acute lung injury (ALI) are activated by Ca2+ entry. Thus, inhibiting Ca2+ entry might attenuate post-traumatic lung injury. Inhibiting voltage-operated (L-type) Ca2+ channels during shock could cause cardiovascular collapse, but PMN are "nonexcitable" cells, lack L-type channels, and mobilize Ca2+ via nonspecific channels. We previously showed that PMN Ca2+ entry requires sphingosine 1-phosphate synthesis by sphingosine kinase and that both sphingosine kinase inhibition and blockade of nonspecific channels attenuate ALI when begun before shock. Pretreatment for clinical injuries, however, is impractical. Therefore, we now studied whether Ca2+ entry inhibition that begun during resuscitation from T/HS could attenuate SIRS and ALI without causing hemodynamic compromise. Male Sprague-Dawley rats underwent laparotomy and fixed-pressure shock (mean arterial pressure, 35 +/- 5 mmHg; 90 min). Sphingosine kinase inhibition or nonspecific Ca2+ channel inhibition was begun after resuscitation with 10% of shed blood. We then studied in vivo PMN activation and associated lung injury in the presence or absence of Ca2+ entry inhibition. Neither treatment worsened shock. Each treatment decreased CD11b expression, respiratory burst, PMN p38 MAP-kinase phosphorylation, PMN sequestration, and lung capillary leak in vivo. The similar results seen with two different forms of inhibition strengthen the conclusion that the biological effects seen were specific for calcium entry inhibition. Ca2+ entry inhibition is a candidate therapy for management of lung injury after shock.     

Hypertonic saline activation of p38 MAPK primes the PMN respiratory burst

Investigation of hypertonic saline (HTS) modulation of neutrophils (PMN) cytotoxic responses has generated seemingly contradictory results. Clinically relevant levels of HTS attenuate receptor-mediated p38 MAPK signaling, whereas higher levels activate p38 MAPK. Concurrently, HTS exerts a dose-dependent attenuation of the PMN respiratory burst, most notably at concentrations where p38 MAPK is activated. We hypothesized that HTS-mediated p38 MAPK activation augments the PMN respiratory burst on return to normotonicity. We found that although clinically relevant levels of HTS (Na+ > or = 200 mM) did not activate p38 MAPK, higher concentrations (Na+ > or = 300 mM) resulted in activation comparable with that after PAF stimulation. Transient stimulation with high levels of HTS primed the PMN respiratory burst in response to fMLP and PMA. This effect was attenuated by pretreatment with SB 203580, a p38 MAPK specific inhibitor. We conclude that severe osmotic shock primes the respiratory burst via p38 MAPK signaling, further supporting the role of this signaling cascade in PMN priming.