Mast cell stabilization improves survival by preventing apoptosis in sepsis.

Inhibiting single cytokines produced modest effects in clinical trials, in part because the cytokines were not specific for sepsis, and sepsis may require cellular strategies. Previous studies reported that mast cells (MCs) fight infections in early sepsis. In this study, we report that MC stabilizers restrain serum TNF levels and improve survival in wild-type but not in MC-deficient mice. Yet, MC depletion in knockout mice attenuates serum TNF but does not improve survival in sepsis. Serum HMGB1 was the only factor correlating with survival. MC stabilizers inhibit systemic HMGB1 levels and rescue mice from established peritonitis. MC stabilizers fail to inhibit HMGB1 secretion from macrophages, but they prevent apoptosis and caspase-3 activation in sepsis. These results suggest that MC stabilization provides therapeutic benefits in sepsis by inhibiting extracellular release of HMGB1 from apoptotic cells. Our study provides the first evidence that MCs have major immunological implications regulating cell death in sepsis and represent a pharmacological target for infectious disorders in a clinically realistic time frame.

Tryptophan catabolites in mesenteric lymph may contribute to pancreatitis-associated organ failure.

BACKGROUND: Multiple organ failure (MOF) is the key determinant of mortality in acute pancreatitis (AP). Mesenteric lymph cytotoxicity contributes to organ failure in experimental models of systemic inflammation. The aim of this study was to evaluate the mesenteric lymph pathway and the lymph injury proteome in experimental AP-associated MOF, and to test the hypothesis that immunoregulatory tryptophan catabolites contribute to mesenteric lymph cytotoxicity. METHODS: Using an experimental model of AP in rats, the humoral component of mesenteric lymph in AP was compared with that from sham-operated control animals, using in vitro and in vivo cytotoxicity assays, high-throughput proteomics and high-performance liquid chromatography. The experimental findings were corroborated in a cohort of 34 patients with AP. RESULTS: Compared with biologically inactive lymph from sham-operated rats, mesenteric lymph in AP became cytotoxic 3 h after induction. Hierarchical clustering of lymph proteomic mass spectra predicted the biological behaviour of lymph. Levels of the immunoregulatory tryptophan catabolite, 3-hydroxykynurenine, were increased in cytotoxic lymph and re-created cytotoxicity in vitro. In humans with AP, plasma kynurenine concentrations correlated in real time with MOF scores and preceded a requirement for mechanical ventilation and haemodialysis. CONCLUSION: These results support the concept that mesenteric lymph-borne kynurenines may contribute to pancreatitis-associated MOF.

Inhibition of endotoxin-induced bacterial translocation in mice.

The primary functions of the gut are to absorb nutrients and exclude bacteria and their products. However, under certain circumstances the gut may lose its barrier function and serve as a reservoir for systemic microbial infections. These experiments were performed to determine the mechanisms whereby endotoxin causes bacteria to escape (translocate) from the gut. Bacteria translocated from the gut to the mesenteric lymph nodes of mice challenged with nonlethal doses of Escherichia coli 026:B6 or E. coli 0111:B4 endotoxin. Physical disruption of the gut mucosal barrier appears to be the primary mechanism whereby endotoxin promotes bacterial translocation. Mucosal injury and endotoxin-induced bacterial translocation were reduced by inhibition (allopurinol) or inactivation (tung-sten diet) of xanthine oxidase activity (P less than 0.01), but were not affected by the platelet-activation factor antagonists, SRI 63-441 or BN 52021. Because the inhibition or inactivation of xanthine oxidase activity reduced both the extent of mucosal injury and endotoxin-induced bacterial translocation, the effect of endotoxin on the gut appears to be mediated, at least to some degree, by xanthine oxidase-generated, oxygen-free radicals.

Inhibition of the Na(+)/H(+) antiporter suppresses IL-12 p40 production by mouse macrophages.

The amiloride-inhibitable Na(+)/H(+) antiporter plays an important role in macrophage activation. The intracellular pathways leading to interleukin (IL)-12 p40 production by activated macrophages are incompletely understood. In the present study, we examined the contribution of the Na(+)/H(+) antiporter to the production of IL-12 p40. Amiloride or its analogs decreased the production of IL-12 p40 in macrophages stimulated with bacterial lipopolysaccharide and interferon-gamma. The order of potency of amiloride analogs was consistent with the proposition that the effect of amiloride is mediated by the inhibition of the Na(+)/H(+) antiporter. The effect of amiloride was post-transcriptional, as IL-12 p40 mRNA levels induced by lipopolysaccharide and interferon-gamma were not affected by this inhibitor. Furthermore, the inhibitory effect of amiloride on IL-12 p40 production was not a result of interference with the activation of the p38 and p42/44 mitogen-activated protein kinases or c-Jun kinase. In summary, the production of IL-12 p40 requires a functional Na(+)/H(+) antiporter.

PAF-mediated Ca2+ influx in human neutrophils occurs via store-operated mechanisms.

Many inflammatory mediators activate neutrophils (PMN) partly by increasing cytosolic calcium concentration ([Ca2+]i). Modulation of PMN [Ca2+]i might therefore be useful in regulating inflammation after shock or sepsis. The hemodynamic effects of traditional Ca2+ channel blockade, however, could endanger unstable patients. Store-operated calcium influx (SOCI) is known now to contribute to Ca2+ flux in "nonexcitable" cells. Therefore, we studied the role of SOCI in human PMN responses to the proinflammatory ligand PAF. PMN [Ca2+]i was studied by spectrofluorometry with and without external calcium. We studied the effects o

Animal models of sepsis and shock: a review and lessons learned.

Over the past decade, the biotechnology/pharmaceutical industry has been diligently working on the development of immunomodulatory agents for the treatment of shock and sepsis, and the literature is rife with descriptions of novel and innovative molecules that promise to become the panacea for these conditions. Unfortunately, despite promising preclinical evidence, dozens of these new agents have failed to demonstrate clinical efficacy in controlled, randomized clinical trials, abandoning the bedside physician to the traditional armamentarium of drugs and therapeutics for the treatment of patients with these complex, progressive, and life-threatening conditions. The reasons for this quandary are controversial, complex, and multifactorial. This review focuses on the concept that the preclinical trials of many of these agents were conducted using models of sepsis and shock that do not adequately reflect the clinical realities of these conditions. As a result, it is not surprising that clinical trials of agents based on clinically flawed models failed to demonstrate clinical efficacy. The lack of clinical insight during preclinical development of these agents has contributed to the current impasse of the development of safe, efficacious, and potentially lifesaving agents for the treatment of shock and sepsis. Thus, the goal of this review article is to review the advantages and disadvantages of commonly used sepsis and shock models in light of lessons learned from these clinical trials.

Bacterial translocation across enterocytes: results of a study of bacterial-enterocyte interactions utilizing Caco-2 cells.

Due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (BT) across the intestinal epithelial barrier are poorly understood. Thus, we have utilized the Caco-2 intestinal cell line to study this process. Caco-2 cells were grown to confluence on semipermeable membranes contained in the upper compartment of a 2 compartment system. Cellular confluence and tight junction integrity was verified by measurements of the transepithelial electrical resistance in ohms cm2.BT was measured by culturing the bacteria (nonpathogenic Escherichia coli) that were able to cross the Caco-2 monolayer and were present in the bottom compartment, as well as by monitoring the passage of 1-micron fluorescent beads. Caco-2 cells were pretreated with several metabolic inhibitors: 1.0 mM sodium azide (oxidative phosphorylation), 10 micrograms/ml nocodazole (microtubule), 10 microM phalloidine (microfilament), and 5.0 micrograms/ml cytochalasin D (microfilament). To investigate the mechanisms of BT. Both bacteria and fluorescent beads crossed the Caco-2 monolayer. Azide had no effect on BT, while both nocodazole (n = 17) and phalloidine (n = 14) significantly decreased translocation of E. coli versus control monolayers (p < .05). Cytochalasin D increased BT versus control membranes, however this was associated with loss of tight junction integrity (transepithelial electrical resistance decreased from 201 +/- 79 to 87 +/- 6.4). BT across Caco-2 cells appears to be a polar process which is to some extent microtubule- and microfilament-dependent.

The gut: a cytokine-generating organ in systemic inflammation?

The aim of this study was to test the hypothesis that the gut is capable of becoming a cytokine-generating organ following either a lethal or nonlethal inflammatory insult. Adult male rats were given an intraperitoneal challenge with saline, or with a nonlethal (.1 mg/g) or LD50 (.5 mg/g) dose of zymosan. Mesenteric lymph nodes, efferent mesenteric lymph, liver, spleen, and blood (portal and systemic) were obtained at 2, 4, 6, 8, or 10 h post challenge. Organs, lymph, and blood were tested for bacterial translocation (BT); blood and lymph were assayed for tumor necrosis factor (TNF) and IL-6. After .1 mg/g zymosan, BT was limited to the mesenteric lymph node complex only; .5 mg/g zymosan promoted BT to blood, mesenteric lymph, and organs (p < .05 vs. control or .1 mg/g zymosan). The magnitude of portal bacteremia was greater than systemic bacteremia (p < .003). Serum TNF peaked at 2 h (p < .05 vs. control), and serum IL-6 peaked at 4-6 h (p < .05 vs. control) post zymosan challenge. Portal and systemic bioactivity was similar for either cytokine, and serum bioactivity did not correlate with zymosan dose. TNF bioactivity was increased in the mesenteric lymph at 2 h post challenge with .5 mg/g zymosan only (p < .05 vs. control or .1 mg/g zymosan). IL-6 bioactivity was increased in the mesenteric lymph at 4 through 10 h post zymosan challenge (p < .05 vs. control), but was similar with either dosage of zymosan. In conclusion, the gut may be capable of producing cytokines in response to an inflammatory stimulus, even in the absence of portal or systemic spread of bacteria. The magnitude of the cytokine response does not correlate with the magnitude of bacterial translocation.

Chemokine stimulation of human neutrophil [Ca2+]i signaling in biologic environments.

Clinical neutrophil (PMN) priming is the net result of multiple stimuli, with intracellular calcium ([Ca2+]i) being a key second messenger for PMN agonists such as the chemokines. Thus, [Ca2+]i measurement may be a robust tool for the assessment of global PMN activation. [Ca2+]i is difficult to measure in complex biologic environments, however, so data in this area are limited. We therefore developed an in vitro system to measure the effects of chemokines on PMN [Ca2+]i. PMN were isolated from volunteer blood. PMN [Ca2+]i responses to interleukin (IL)-8 and Growth-Related Oncogene (GRO)-alpha were studied by fura-2-acetoxymethyl ester fluorescence with or without reincubation in autologous plasma just prior to study. The effects of IL-8 and GRO-alpha on PMN [Ca2+]i at ascending doses, with or without plasma reincubation, given sequentially and in the presence or absence of extracellular calcium, were studied. PMN basal [Ca2+]i was increased by plasma, as were low-dose priming and higher-dose spike responses to IL-8. GRO-alpha caused a more pronounced priming of PMN [Ca2+]i than IL-8 at low doses, although significantly lower peak responses were observed with GRO-alpha than IL-8 at higher doses. Plasma suppressed both priming and spike responses to GRO-alpha. When given serially at clinically relevant agonist doses, GRO-alpha was permissive of IL-8 signaling, whereas IL-8 blocked GRO-alpha signaling. IL-8 generates high [Ca2+]i spikes using intracellular calcium stores only. GRO-alpha produces lower [Ca2+]i spikes despite using both intra- and extracellular stores. Plasma preincubation has profound effects on PMN [Ca2+]i responses to chemokines. These can be measured accurately, as described. In clinically relevant environments, IL-8 and GRO-alpha interact in a regulatory fashion. GRO-alpha may act as a priming agent, with IL-8 activating PMN functions requiring high [Ca2+]i. This cross-cooperation is probably terminated by IL-8 regulation of GRO-alpha activity at the C-X-C chemokine receptor 2.

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.

Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma.

Polyamines are endogenous immunomodulatory molecules. Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide. In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions. Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages. The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected. In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production. The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice. The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase. In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody. In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10.

Role of mucin, mannose, and beta-1 integrin receptors in Escherichia coli translocation across Caco-2 cell monolayers.

Our previous work suggests that Caco-2 cells play an active role in bacterial translocation (BT). Since bacterial enterocyte interactions may be receptor-mediated, the current study was performed to investigate the role of beta 1 integrin and mannose receptors as well as the general protective effect of the mucous layer in this process. Caco-2 cells grown to confluence on semipermeable membranes contained in the upper compartment of a two compartment system were utilized. BT was assessed by quantitating the number of Escherichia coli crossing the monolayers after challenge with 10(8) E. coli C25. Pretreatment of the Caco-2 cells with the beta 1 integrin receptor competitive inhibitors fibronectin or RGD did not inhibit BT; while pretreatment of Caco-2 cells with the LFA-1 (lectin) receptor competitive inhibitor mannose (12 mg/ml) or purified mucin (8 mg/ml) decreased BT compared to control membranes (p < .001). Transepithelial resistance was similar among all the groups indicating maintenance of tight junction integrity. These studies suggest that E. coli BT in the Caco-2 system can be reduced by mannose and that intestinal mucin contributes to the barrier function of the monolayer.

Evidence favoring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock.

There is increasing evidence of an association between intestinal injury and the development of a septic state and distant organ failure. Since this phenomenon can occur in the absence of detectable systemic bacterial translocation (BT), we tested the hypothesis that shock-induced intestinal injury will result in the gut becoming a cytokine-generating organ by measuring interleukin 6 (IL-6) and tumor necrosis factor (TNF) levels in the portal blood, cardiac blood, and intestinal lymph of rats subjected to sham, 30, 60, or 90 min of hemorrhagic shock (30 mm Hg). These blood and lymph samples, as well as the mesenteric lymph nodes (MLN), spleens, and livers, were cultured for translocating bacteria. Although all the portal and cardiac blood samples were sterile, the portal blood levels of TNF and IL-6 were increased to a greater extent than simultaneously obtained cardiac blood samples in rats subjected to 60 or 90 min of shock (p < .05). The lymph IL-6 levels increased but were similar between the groups. BT was limited to the MLN and occurred in a dose-dependent fashion with 38, 63, and 100% of the animals having culture-positive MLNs after 30, 60, or 90 min of shock, respectively. In conclusion, after hemorrhagic shock, the gut appears to become a cytokine liberating organ even in the absence of detectable bacteria in the portal circulation.

Comparison of plasma cytokine levels in rats subjected to superior mesenteric artery occlusion or hemorrhagic shock.

The overall goal of this study was to compare the effects of systemic hypotension (hemorrhagic shock) versus local gut ischemia (superior mesenteric artery (SMA) occlusion) on cytokine production and bacteria/endotoxin translocation. Sham or actual SMA occlusion led to an increase in tumor necrosis factor (TNF), which was greatest at the end of the occlusion period, while the IL-6 response peaked 3 h post-SMA occlusion. The TNF and IL-6 response after hemorrhagic shock differed from that observed after SMA occlusion in that the peak response occurred later and was of lower magnitude (p < .05). Although the animals subjected to SMA occlusion had a significantly increased incidence of bacterial translocation to both the mesenteric lymph nodes and systemic organs compared to rats subjected to hemorrhagic shock, in neither group was the blood level of endotoxin elevated and there was no association between bacterial translocation and cytokine levels. These results suggest that different models of intestinal ischemia have different cytokine profiles and that the early TNF response associated with SMA occlusion model is primarily due to the laparotomy.

Caco-2 and IEC-18 intestinal epithelial cells exert bactericidal activity through an oxidant-dependent pathway.

Intestinal epithelial cells have receptors that recognize bacterial antigens and in some circumstances are actively involved in bacterial internalization. To test the hypothesis that intestinal epithelial cells possess bactericidal capabilities, the bactericidal activity of two intestinal cell lines (IEC-18 and Caco-2) was measured using Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli as test organisms. The relative bactericidal efficacy of these two intestinal cell lines to kill these bacteria was compared against neutrophils (PMN) using a standard in vitro bactericidal assay. The IEC-18 and Caco-2 cells as well as the PMNs killed S. aureus and P. aeruginosa but not E. coli (p < .05). In fact, when tested in serum-free medium, the IEC-18 and Caco-2 cells killed a greater percentage of bacteria than the PMNs (p < .05). The addition of the antioxidant, superoxide dismutase, significantly reversed the bactericidal activity of both Caco-2 cells and neutrophils for P. aeruginosa and S. aureus, while catalase had no effect. Nitric oxide inhibition by NG-nitro-L-arginine methyl ester (L-NAME) had no effect on bactericidal activity of Caco-2 cells. These results indicate that intestinal epithelial cells can kill certain strains of bacteria and may function as "nonprofessional" phagocytes. Additionally, the mechanisms involved in the killing of P. aeruginosa and S. aureus by the Caco-2 and IEC-18 cells appear similar to the PMNs to the extent that bactericidal activity appeared to be oxidant-mediated but not nitric oxide-mediated in both the Caco-2 cell line and in the neutrophils.

Presence of the stress-inducible form of hsp-70 (hsp-72) in normal rat colon.

The expression of heat shock proteins (hsp) is probably one of the most primitive mechanisms of cellular protection from stress. Pathogens such as viruses and bacteria have recently been found to induce the heat shock gene expression. In the present study hsp-72, the stress-inducible form of hsp-70, was detected by Western blotting in samples from rat distal colon (DC), proximal colon (PC), and terminal ileum (TI), but was not found in proximal small bowel (PSB) or other organs (liver, kidney, spleen, heart, and brain) of unstressed animals. The signal intensity of hsp-72 in colon (DC > PC > TI > PSB) correlates qualitatively with the presence of normal gut microflora. hsp-72 was also observed in DC, to a lesser extent in PC, but not in TI or PSB of bacteria-free or antibiotic-treated rats. Inflammatory states induced by the intravenous administration of endotoxin (1 mg/kg), the subcutaneous injection of zymosan (1 g/kg) or by cecal ligation and puncture (sepsis) failed to increase the hsp-72 levels in rat colon or other organs. These results demonstrate that hsp-72 is expressed in normal rat colon. However, the induction of hsp-72 expression may not be due solely to the presence of resident bacteria in the gut, but instead, may be the result of a more complex process.

Interleukin-6 and tumor necrosis factor production in an enterocyte cell model (Caco-2) during exposure to Escherichia coli.

Data linking interactions between bacteria and the intestine with elevated serum cytokine levels has led to the concept of the gut as a cytokine-producing organ. An in vitro cell culture model was used to investigate the potential role of intestinal mucosa within this paradigm. Polarized monolayers of human enterocytes (Caco-2) were grown in a two compartment system where the apical and basal aspects of the membrane could be studied. Supernatant was collected at 0, 1, 3, 6, and 24 h after the monolayer was exposed (apically or basally) to 10(2), 10(5), or 10(8) colony-forming units of Escherichia coli C25/mL and saved for interleukin (IL)-6 and tumor necrosis factor (TNF) bioassay analysis. Caco-2 cells (not bacterially challenged) secreted significant amounts of constitutive IL-6, but not TNF, into the apical and basal chambers. Both cytokines levels were increased in a dose-dependent fashion (p < .05) after the E. coli challenge. This stimulated cytokine response was polar, in that the highest cytokine levels were at the side of the bacterial challenge and were most notable at the highest dose (10(8) colony-forming units/mL) of E. coli C25 tested. Caco-2 cells produce IL-6 and TNF in a dose-dependent fashion in response to E. coli C25 and the magnitude of this response is maximal on the side of the bacterial challenge. This data supports the hypothesis that bacterially challenged human enterocytes may be important producers of cytokines.

Endotoxin-induced mesenteric microvascular changes involve iNOS-derived nitric oxide: results from a study using iNOS knock out mice.

The administration of endotoxin alters intestinal blood flow, increases nitric oxide (NO) production, and induces gut barrier dysfunction. Thus, we investigated the hypothesis that microvascular reactivity and permeability of the mesenteric vascular bed are altered to a lesser degree in iNOS knock out (iNOS -/-) mice than their wild-type (iNOS +/+) litter mates after an endotoxin challenge. To test this hypothesis, we compared the microvascular response of iNOS knockout (iNOS -/-) mice after a topical or systemic endotoxin challenge against that of their wild-type litter mates (iNOS +/+). Intravital microscopy was used to measure arteriolar diameter and postcapillary venular permeability in the mouse ileum. Both parameters were determined by computer-assisted image analysis. Diameter was measured in A1, A2, and A3 arterioles (1, 2, 3 = rank of deployment). Changes in microvascular permeability were measured from changes in interstitial fluorescence caused by extravasation of fluorescein isothiocyanate (FITC)-dextran 150 (molecular weight = 150 kDa) and expressed as changes in integrated optical intensity (IOI). In the first series of experiments, endotoxin (100 microg/mL) was applied topically to the ileal segment. In the second series, endotoxin (10 mg/kg) was administered intraperitoneally (i.p.). Administration of topical or i.p.. endotoxin caused vasoconstriction and was associated with an early increase in permeability in both iNOS +/+ and -/- mice, although over time the responses of the iNOS -/- and iNOS +/+ mice diverged. Twenty minutes after topical endotoxin, the increase in permeability in iNOS -/- mice had reached a plateau whereas it continued to increase in the iNOS +/+ mice, such that at 80 min post-topical endotoxin, IOI was 27+/-7 in iNOS -/- vs. 39+/-5 in iNOS +/+ (P < 0.05). A similar permeability response was observed after i.p.. endotoxin, where the increase in post-capillary venular permeability was greater in the iNOS +/+ mice (P < 0.05). Both iNOS -/- and iNOS +/+ mice had a similar transient vasoconstrictive response after topical endotoxin challenge (reduction in A2 arteriolar diameters by -17+/-4% vs. -24+/-7%), with return to baseline values by 60-80 min post-endotoxin challenge. The iNOS +/+ but not the iNOS -/- mice manifested a secondary vasodilatory response that persisted throughout the experimental period. The arteriolar vasoreactive response of the iNOS -/- and iNOS +/+ mice to i.p.. endotoxin was similar to that of topical endotoxin, but of a lesser magnitude. In conclusion, the similarity in effects between topical and systemic endotoxin indicates that endotoxin causes microvascular dysfunction in the gut by directly on the microcirculation. In addition, our data suggest that NO production by iNOS is involved in the microvascular alterations associated with gut barrier dysfunction.

Mesenteric lymph duct ligation provides long term protection against hemorrhagic shock-induced lung injury.

Recently we have shown that ligation of the main mesenteric lymph (MLN) duct prior to an episode of hemorrhagic shock (HS) prevents shock-induced lung injury. Yet, ligation or diversion of intestinal lymph immediately prior to injury is not clinically feasible. Diversion of intestinally derived lymph after injury to protect against secondary insults is possible, but it is not known how long the protective effects of lymph ligation would last. Thus, we tested whether ligation of the MLN duct seven days prior to HS would still be protective. Male Sprague-Dawley rats were subjected to laparotomy with or without MLN duct ligation. Seven days later, half of the sham and actual MLN duct ligated animals randomly were selected to undergo HS (30 mmHG for 90 min). The other half of the animals was subjected to sham shock. Lung permeability, pulmonary myeloperoxidase (MPO) activity, and bronchoalveolar fluid (BALF) protein content were used to determine lung injury. Lymphatic division 7 days prior to HS continued to prevent shock induced lung injury as assessed by a lower Evans Blue dye concentration, BALF protein and MPO activity. In addition, there was no evidence of Patent Blue dye in the previously ligated MLN duct. Since ligation of the main mesenteric lymphatic duct continues to protect against shock-induced lung injury 1 week after duct ligation, it is feasible that lymphatic ligation performed after an injury remains protective against certain secondary insults for at least 1 week.

Post-hemorrhagic shock mesenteric lymph is cytotoxic to endothelial cells and activates neutrophils.

The goal of these experiments was to test the hypothesis that after a nonlethal episode of hemorrhagic shock, factors carried in the mesenteric lymph would promote endothelial cell injury and activate neutrophils to a greater extent than portal vein plasma. Catheters were placed in the efferent lymphatic duct draining the mesenteric lymph node complex, after which male rats were subjected to sham or actual shock (30 mmHg for 90 min), and lymph was collected. Portal vein plasma was collected from the sham-shock and shocked rats at 6 h post-shock or sham-shock. When the effect of lymph or portal blood plasma was tested on endothelial cell (HUVEC) monolayer permeability, it was found that post-shock lymph, but not post-shock portal vein plasma, increased HUVEC permeability to both 10 kDa and 40 kDa permeability probes. Subsequent experiments documented that only post-shock lymph was cytotoxic to endothelial cells as manifest both by decreased trypan blue dye exclusion and the increased release of Chromium-51 from chromium-loaded endothelial cells. Furthermore post-shock lymph induced a greater increase in neutrophil superoxide formation than pre-shock lymph, pre-shock, or post-shock portal vein plasma. Lastly, neutrophil-mediated endothelial cell injury was potentiated by the presence of post-shock lymph, and the magnitude of HUVEC injury was greater in endothelial cells incubated with post-shock lymph plus neutrophils than in monolayers incubated with post-shock lymph or neutrophils alone. These results suggest that post-shock lymph is cytotoxic to endothelial cells and activates neutrophils. Since the lung is the first organ that is exposed to mesenteric lymph, lung injury after hemorrhagic shock may be mediated by factors contained in mesenteric lymph.