Postshock Mesenteric Lymph Research Articles

Systemic Not Just Mesenteric Lymph Causes Neutrophil Priming After Hemorrhagic Shock

Inflammatory mediators in postshock mesenteric lymph have been causally linked to systemic polymorphonuclear cells (PMNs) priming resulting in acute lung injury (ALI) and multiple organ failure. Earlier human and animal studies demonstrated ALI after lower limb ischemia/reperfusion (I/R) injury. As hemorrhagic shock (HS) is in essence a systemic I/R insult, we postulated that systemic lymph after HS would exhibit PMN priming and this was studied in vitro. Lymph was collected at intervals from the hind limb of dogs subjected to sham or HS and crystalloid resuscitation. Human PMNs isolated from heparinized blood of normal volunteers were incubated with buffer, sham lymph, or lymph after 120 minutes of shock or resuscitation. PMN priming was indexed by CD11b expression (mean fluorescence intensity), superoxide anion (O2(-)) generation (nanomoles/mg protein), and elastase release (%) after the addition of fMLP (1 micromol). PMNs with buffer served as control. PMN priming after exposure to either shock or postshock resuscitation lymph was noted by increased expression of CD11b, superoxide generation, and elastase release after exposure to fMLP. No priming effect was noted with sham lymph. Maximal bioactivity of shock or postresuscitation shock lymph was noted at 2 hours postresuscitation. Exposure with systemic lymph after HS resulted in PMN priming. These results question the unique properties attributed to post-HS lymph from the splanchnic bed in causing PMN priming and ALI after shock. The causal agent(s) for these effects are unclear.

Arachidonic acid in postshock mesenteric lymph induces pulmonary synthesis of leukotriene B4

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.

QS388. Hemorrhagic Shock Induces the Release of 5-HETE and Thromboxane B 2 in Postshock Mesenteric Lymph

Background: Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remain elusive. Our previous work has shown that systemic administration of a phospholipase A2 (PLA2) inhibitor attenuated postshock ALI. We also identified a neutral lipid fraction within the postshock mesenteric lymph (PSML) responsible for neutrophil (PMN) priming. Arachidonic acid (AA) is metabolized by many key enzymes including 5-Lipoxygenase (5-LO) and COX 1&2.

QS378. Arachidonic Acid in Postshock Mesenteric Lymph Induces Pulmonary Synthesis of LTB 4 and PMN Accumulation

QS378. Arachidonic Acid in Postshock Mesenteric Lymph Induces Pulmonary Synthesis of LTB 4 and PMN Accumulation

Gelsolin is Depleted in Post-Shock Mesenteric Lymph

Gelsolin is a plasma protein that functions to depolymerize actin filaments preventing capillary plug formation following tissue injury. It also functions to mediate the inflammatory response by binding proinflammatory lipids such as lysophosphatidic acid, sphingosine-1-phosphate and phosphoinositides. Clinically, reduced gelsolin concentrations have been associated with increased mortality in critically ill, trauma, and burn patients. We have previously shown that following hemorrhagic shock with splanchnic hypoperfusion, mesenteric lymph contains lipid components that cause neutrophil and EC activation and that protein concentrations are severely diluted due to resuscitation. We hypothesized that lipid binding proteins such as gelsolin may be depleted after trauma/hemorrhagic shock leading to increased lipid bioactivity. Shock was induced in SD rats by controlled hemorrhage and the mesenteric duct cannulated for lymph collection. Resuscitation was performed by infusing 2x SB volume in NS over 30 min, followed by 1/2 SB volume over 30 min, then 2x SB volume in NS over 60 min. Pre and post-shock lymph was loaded at equal protein concentrations on 2D-gels, followed by trypsin digestion and identification with mass spectrometry (MS-MS). Proteomics data were confirmed with Western blotting then quantitated by densitometry. Analysis of variance was used evaluate statistical data. Gelsolin decreased in mesenteric lymph following hemorrhagic shock. Gelsolin is found at high levels (comparable to plasma) in mesenteric lymph. Following hemorrhagic shock, gelsolin levels decrease significantly, possibly due to consumption by the actin scavenging system. The magnitude of this change in concentration could release lipid bioactivity and predispose the lung and other organs to capillary injury.

130: Gelsolin concentrations are decreased in post-shock mesenteric lymph

130: Gelsolin concentrations are decreased in post-shock mesenteric lymph

Normal Mesenteric Lymph Blunts the Pulmonary Inflammatory Response to Endotoxin

Mesenteric lymph may provide the mechanistic link between gut ischemia and acute lung injury after hemorrhagic shock (HS). Studies have focused on the toxic mediators that develop in the post-shock mesenteric lymph (PSML). However, a complementary possibility is that there is loss of protective mediators found in pre-shock normal mesenteric lymph (NML) after HS. We hypothesize that NML protects against inflammatory insults to the pulmonary endothelium and that this effect is lost in PSML. Primary human pulmonary endothelial cells (HMVECs) were incubated with NML or PSML collected from rats subjected to HS and resuscitation and then stimulated with 20 ng/mL LPS. ICAM-1 surface expression was measured by flow cytometry. In subsequent experiments, lipoproteins were extracted from NML before incubation and LPS-induced ICAM-1 expression determined. Mean fluorescent intensity (MFI) of LPS-induced ICAM-1 in NML and PSML treated HMVECs were 10.1 +/- 2.3 versus 27.7 +/- 0.83, respectively (P < 0.05). This represented at 71% decrease in ICAM-1 expression by NML compared to ICAM-1 expression in LPS-induced controls (MFI: 34.6 +/- 6.9). Lipoprotein extraction from NML abolished this protective effect (MFI: 31.2 +/- 5.3 versus Control + LPS: 33.5 +/- 3.6, P > 0.05). Baseline ICAM-1 levels were not significantly different among control, NML, and PSML groups. Lipoproteins in NML contain anti-inflammatory properties that decrease ICAM-1 expression induced by LPS in pulmonary endothelium. Decreased protective lipoproteins after HS and resuscitation may contribute to the toxicity associated with PSML from the ischemic gut.

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.

Amiloride moderates increased gut permeability and diminishes mesenteric lymph-mediated priming of neutrophils in trauma/hemorrhagic shock

Amiloride, an inhibitor of Na+/H+ exchangers and Na+ channels has been shown recently to ameliorate both gut and lung injury in rats subjected to a combined insult of trauma and hemorrhagic shock (T/HS). We have shown previously that mesenteric lymph duct ligation prevents T/HS-induced lung endothelial injury and neutrophil activation, suggesting that toxic inflammatory factors originating from the gut and carried in the lymph are responsible for the lung injury observed after T/HS. This study investigates whether the protective effect of amiloride against T/HS-induced lung injury was associated with decreased lymph toxicity and gut permeability. Male rats subjected to trauma (laparotomy) plus hemorrhagic shock (mean arterial pressure, 30 mm Hgx90 min) (T/HS) or trauma plus sham shock (T/SS) and treated with amiloride or its vehicle had their mesenteric lymph duct catheterized. Mesenteric lymph collected before and after shock was assayed for biologic activity on endothelial cells (cytotoxicity and permeability) and neutrophils (respiratory burst activity). Gut permeability was assessed by monitoring plasma concentrations of the fluorescent dye FITC-dextran after its injection into the ileum. Amiloride administration reduced the capacity of post-shock mesenteric lymph to prime neutrophils for an increased respiratory burst. Amiloride failed to decrease the ability of mesenteric lymph to kill endothelial cells or increase their permeability. Amiloride decreased gut permeability. The mechanisms of the lung protective effect of amiloride in rats undergoing T/HS may be secondary to decreased neutrophil activation, diminished gut permeability, or an effect on the end organ.

Normal mesenteric lymph blunts the pulmonary inflammatory response to endotoxin

Alterations in mesenteric lymph may provide the mechanistic link between gut ischemia and acute lung injury after hemorrhagic shock (HS). Previous work has focused primarily on the toxic mediators that may develop in post shock mesenteric lymph (PSML). However, a complementary possibility is that there is loss of protective mediators found in normal mesenteric lymph after hemorrhagic shock and resuscitation. We hypothesize that lipoproteins in normal mesenteric lymph protect against inflammatory insults to the pulmonary endothelium and that this protective effect is lost in PSML. Methods: Normal preshock mesenteric lymph (NML) and PSML collected from rats subjected to HS (MAP 30 mmHg X 45min) and resuscitation (4X shed blood volume (SBV) in NS + 1/2 SBV in rat whole blood) were incubated (5% vol.) with primary human microvascular pulmonary endothelial cells (HMVECs). HMVECs were then stimulated with 20ng/mL LPS and mean fluorescent intensity (MFI) of ICAM-1 surface expression was measured 6 hours later by flow cytometry. In subsequent experiments, lipoproteins were extracted from NML prior to incubation with HMVECs and measurements of LPS-induced ICAM-1 expression were determined. Results: MFI of LPS-induced ICAM-1 in NML and PSML treated cells were 10.1 ± 2.3 vs. 28 ± 1.0, respectively (p < 0.05). Compared to control-LPS cells (ICAM-1 MFI: 35 ± 6.9) this represented a 71% decrease in ICAM-1 expression by NML vs. a 20% decrease in ICAM-1 expression by PSML. However, lipoprotein extraction from mesenteric lymph before incubation with HMVECs attenuated the protective effect of NML, with LPS-induced ICAM-1 expression reduced by only 11% (MFI 31 ± 5.3). In the absence of LPS stimulation, baseline ICAM-1 expressions were not significantly different among control, NML, and PSML samples. Conclusion: Lipoproteins found in normal mesenteric lymph contain anti-inflammatory properties that decrease ICAM-1 expression induced by LPS in pulmonary endothelium. Alterations of these protective lipoproteins after hemorrhagic shock and resuscitation may contribute to the toxicity associated with post shock mesenteric lymph from the ischemic gut.

Systemic Neutrophil Priming by Lipid Mediators in Post-Shock Mesenteric Lymph Exists Across Species

Post-hemorrhagic shock mesenteric lymph (PHSML) has been linked with neutrophil (PMN) priming, endothelial cell (EC) activation, and acute lung injury (ALI) in rodent models. We have previously identified the lipid fraction of PHSML as containing the causative agent(s). Due to the lesson learned from the rodent gut bacterial translocation experience, we sought to confirm this phenomenon using a large animal model; hypothesizing that lymph collected from the porcine gut following ischemia/reperfusion (I/R) would cause PMN priming. Mesenteric lymph was collected from adult pigs before, during, and for 2 hours after non-lethal hemorrhagic shock (mean arterial pressure = 30 mm Hg x 45 minutes). Whole lymph and the extracted lipid fractions of the lymph were then added to isolated human and porcine PMNs and superoxide production was measured by cytochrome C reduction. Hemorrhagic shock profoundly affected mesenteric lymph flow from baseline (pre-shock) flow rates of 75.63 +/- 8.86 mL/hr to 49.38 +/- 5.76 mL/hr during shock and increasing to 253.38 +/- 27.62 mL/hr after 2 hours of resuscitation. Human PMNs exposed to both whole lymph (PHSML) and its extracted lipids (PHSML Lipid) collected 2 hours after shock exhibited more than a two-fold increase in superoxide release upon activation compared with pre-shock samples: PHSML- 6.27 +/- 0.83 versus 2.56 +/- 0.60 nmolO2(-)/ 3.75 cells/mL/min, respectively (p = 0.007), PHSML Lipid- 4.93 +/- 0.34 versus 2.49 +/- 0.11 nmolO2(-)/ 3.75 cells/mL/min (p < 0.001). Similarly, porcine PMNs exhibited close to a two-fold activation when exposed to the lymph and lipid fraction: PHSML- 4.51 +/- 0.42 versus 1.06 +/- 0.28 nmolO2(-)/ 3.75 cells/mL/min (p = 0.008), PHSML Lipid-4.80 +/- 0.81 versus 1.55 +/- 0.23 nmolO2(-)/ 3.75 cells/mL/min (p = 0.002). Mesenteric lymphatics serve as the conduit for inflammatory mediators elaborated by the post-ischemic gut in both small and large animal models. Further, the causal agent(s) exist in the lipid fraction of the lymph and are active on both human and animal PMNs.

Neutrophil priming by postshock mesenteric lymph is biphasic

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury following hemorrhagic shock (HS). Postshock mesenteric lymph (PSML) primes neutrophils (PMNs) for enhanced superoxide release. Recently, the bioactivity of lipid (rapid priming) and aqueous (slower priming) factors within the lymph has been questioned. We sought to quantify early and late effects of PSML on PMNs. Methods. Adult male Sprague-Dawley rats (325–375 g) underwent cannulation of the mesenteric lymphatics, femoral artery, and vein. HS to a MAP of 30 mmHg for 45 min was induced. PSML collected in the resuscitation phase was incubated with human PMNs for 5, 15, 30, and 60 min. Priming effect was assessed by stimulated superoxide release. Results. Five minutes of PSML incubation primed PMNs substantially compared to preshock lymph 10.47 ± 1.01 versus 6.25 ± 0.54 nmol O 2 −/3.75 × 10 5 cells/mL/min ( P = 0.06). PSML had no appreciable effect at 15 min, 4.29 ± 0.64 nmol O 2 −/3.75 × 10 5 cells/mL/min, but demonstrated increased priming following 30 min, 7.45 ± 0.04 versus 4.19 ± 0.84 nmol O 2 −/3.75 × 10 5 cells/mL/min ( P < 0.05) increasing to 10.11 ± 0.13 nmol O 2 −/3.75 × 10 5 cells/mL/min following 60 min. Conclusions. The biphasic priming response shown by these data suggest that PSML may exert its effects by both fast and slow priming agents, supporting potential roles for both lipid and aqueous factors.

Cytotoxicity of post shock mesenteric lymph depends on the depth and duration of hemorrhagic shock

Mesenteric hypoperfusion of circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Postshock mesenteric lymph (PSML) has been shown to contain cytotoxic mediators elaborated from the ischemic gut. We hypothesize that the cytotoxicity of PSML is dependent on the depth and/or duration of circulatory shock. Method. Exp. 1: To first determine the timing of PSML cytotoxicity, we subjected rats to hemorrhagic shock (30 mmHg × 45 min) and then resuscitation with 50 vol % of shed blood and normal saline (4× shed blood) over 2 h. Mesenteric lymph was collected hourly up to 6 h after shock. Superoxide release was measured from human neutrophils incubated with lymph fractions collected from each of the hourly time points. Exp. 2: Rats were subjected to three different shock variations: (1) 30 mmHg × 45 min; (2) 30 mmHg × 15 min; (3) 45 mmHg × 45 min; and were resuscitated. Superoxide production was measured with neutrophils incubated with PSML collected from the third postshock hour in each group. Statistics: ANOVA. Results. Maximal PSML cytotoxicity was noted during the third postshock hour (Fig. 1). Varying the depth of shock increased PSML cytotoxicity more than its duration (Fig. 2). ∗ indicates that superoxide production in each of three groups is significantly different from one another ( P < 0.05). Conclusion. Hemorrhagic shock provokes the release of bioactive agents in PSML that is dependent on both depth and duration of shock. The depth of shock is a more important factor for generating toxic lymph than the duration of shock.

Postshock mesenteric lymph is a significant source of systemic inflammation

Mesenteric hypoperfusion of circulatory shock is a key event in the pathogenesis of subsequent distant organ injury. Previous studies have shown that postshock mesenteric lymph (PSML) contains cytotoxic mediators elaborated from the ischemic gut and that lymph diversion abrogates postshock acute lung injury. In this study we observed that maximal PSML cytotoxicity occurred during the period of greatest PSML volume contribution into the systemic circulation. Method. Sprague-Dawley rats were subjected to hemorrhagic shock (30 mmHg × 45 min) and then resuscitated over 2 h in a clinically relevant manner: 2× volume of shed blood using normal saline (NS) in the first 30 min; 50% volume shed blood in the next 30 min; another 2× volume shed blood using NS over the remaining 60 min. Mesenteric lymph was collected hourly and flow rates were recorded up to 6 h postshock. Superoxide was measured from isolated human neutrophils incubated with 5% (v/v) lymph taken from different time points. Results. Shocked rats recovered to baseline hemodynamics within 60 min of resuscitation. Despite blood pressure recovery, mesenteric lymph flow at 2–4 h postshock increased to 400% above the preshock level (Fig. 1). Mesenteric lymph fractions collected during the peak flow had the greatest cytotoxicity (Fig. 2). Conclusion. With clinically relevant resuscitation of shock, maximal PSML cytotoxicity and flow into the systemic circulation occurs 2–4h after shock. This finding suggests that the systemic contribution of gut-derived cytotoxic lymph after hemorrhagic shock may be more significant than previously realized.

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms.

To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1.

Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukey's correction. Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.

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.

Mesenteric lymph is responsible for post-hemorrhagic shock systemic neutrophil priming.

Hemorrhagic shock-induced splanchnic hypoperfusion has been implicated as a priming event in the two event model of multiple organ failure (MOF). We have previously shown that early postinjury neutrophil (PMN) priming identifies the injured patient at risk for MOF. Recent in vitro studies have demonstrated that postshock mesenteric lymph primes isolated human neutrophils. We hypothesize that lymphatic diversion before hemorrhagic shock abrogates systemic PMN priming and subsequent lung injury. Sprague-Dawley rats (n >or= 5 per group) underwent hemorrhagic shock (MAP 40 mm Hg x 30 min) and resuscitation (shed blood + 2x crystalloid) with and without mesenteric lymphatic duct diversion. Sham animals underwent anesthesia and laparotomy. Whole blood was taken 2 hours after resuscitation, heparinized, and incubated for 5 min at 37 degrees C. Surface expression of CD11b (a marker for PMN priming) was determined by flow-cytometry compared with isotype controls. In addition, lung myeloperoxidase (MPO) was measured for PMN sequestration, and Evans blue lung leak was assessed in the bronchoalveolar lavage fluid in sham, and shock +/- lymph diversion animals. Hemorrhagic shock resulted in increased surface expression of PMN CD11b relative to sham (23.8 +/- 6.7 vs. 9.9 +/- 0.6). Mesenteric lymphatic diversion before hemorrhagic shock abrogated this effect (8.0 +/- 2.6). Lung PMN accumulation, as assessed by MPO, was greater in the lungs of nondiverted (113 +/- 14 MPO/mg lung) versus sham (55 +/- 4 MPO/mg lung, p < 0.05); lymph diversion reduced lung PMNs to control levels (71 +/- 6.5 MPO/mg lung, p < 0.05). Evans blue lung leak was 1.6 times sham in the hemorrhagic shock group; this was returned to sham levels after lymph diversion (p < 0.05). Post-hemorrhagic shock mesenteric lymph primes circulating PMNs, promotes lung PMN accumulation, and provokes acute lung injury. Lymphatic diversion abrogates these pathologic events. These observations further implicate the central role of mesenteric lymph in hemorrhagic shock-induced lung injury. Characterizing the PMN priming agents could provide insight into the pathogenesis of postinjury MOF and ultimately new therapeutic strategies.

Factors larger than 100 kd in post-hemorrhagic shock mesenteric lymph are toxic for endothelial cells

Background. Post-shock mesenteric lymph kills and injures endothelial cells (ECs), but neither the mechanism nor the mediators of lymph's toxic effect are known. Thus, in these studies we investigated and characterized potential factors that may be involved in lymph's toxic effect on ECs. Methods. Lymph was collected hourly from rats before shock, during the shock period, and for 6 hours post-shock and processed in several ways—including removal of cellular elements, freezing, heating, or separation by molecular weight—after which they were tested for toxicity (lactate dehydrogenase as a marker of cell injury and trypan blue as a marker of cell viability). Results. Controls consisting of medium, pre-shock lymph, and post-shock portal vein plasma had no EC toxicity. Lymph collected 1 to 3 hours post-shock resulted in the death of 90% to 95% of ECs and caused an 8- to 10-fold increase in lactate dehydrogenase release; however, this toxic effect waned by 4 hours post-shock. Endotoxin neutralization and immune cell removal did not decrease lymph cytotoxicity but complement inactivation did. By fractionating the toxic lymph samples by size, it appears that the putative EC cytotoxic mediator(s) is larger than 100,000 d. Conclusions. Mesenteric lymph collected 1 to 3 hours after hemorrhagic shock is toxic to ECs, but this effect is lost by 4- to 5-hours post-shock and is not dependent on the presence of immune cells or endotoxin but does involve complement and other putative mediators of greater than 100,000 d. (Surgery 2001;129:351-62.)

Hypertonic saline resuscitation abrogates neutrophil priming by mesenteric lymph.

Neutrophil (PMN) priming after hemorrhagic shock is predictive of the subsequent development of multiple organ failure, but the mechanism remains unknown. Recently, we and others have demonstrated that mesenteric lymph from shock animals resuscitated with lactated Ringer's solution (LR) is not only a potent PMN priming agent but also causes lung injury. Work by others has shown that resuscitation with hypertonic saline (HTS) protects animals from lung injury after hemorrhagic shock. Therefore, we hypothesize that resuscitation with HTS will abolish PMN priming by postshock mesenteric lymph. After mesenteric lymph duct catheterization, male rats underwent hemorrhagic shock (mean arterial pressure of 40 mm Hg for 90 minutes) and resuscitation with shed blood plus either LR (2x volume of shed blood) or 4 mL/kg of 7% HTS (isonatremic). Priming for superoxide by PMN was measured after fMLP (1 microM) activation. Shock significantly decreased mesenteric lymph flow from preshock levels in both groups. LR resuscitation produced significantly more mesenteric lymph than HTS resuscitation. Mesenteric lymph from LR animals primed PMN for superoxide production, whereas, HTS eliminated this priming. HTS not only decreases postshock mesenteric lymph production, it eliminates PMN priming by mesenteric lymph, suggesting a mechanism for the beneficial effects of HTS resuscitation.