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Derangements in tissue perfusion occur during critical illness, and the resulting deficit in oxygen delivery may play an important role in the pathogenesis of hemorrhagic and septic shock. Cells and organisms have developed a variety of adaptive strategies to maintain adequate energy production to maintain normal cellular function under hypoxic conditions. Recent studies from our laboratory suggest that certain proinflammatory cytokines, which are likely to be elaborated during or after shock, can interfere with the ability of cells to adapt to hypoxia, and thereby contribute to the development of organ system dysfunction.

Background & Aims: In both clinical and experimental studies, intestinal epithelial barrier function is routinely assessed by measuring mucosal permeability to various hydrophilic compounds. We performed experiments to determine whether permeation of several hydrophilic compounds across rat colonic mucosa is polarized., Methods: Sheets of colonic mucosa, stripped of the underlying seromuscular coats, were mounted in Ussing chambers., Results: The rates of permeation across colonic mucosa by numerous hydrophilic compounds (fluorescein isothiocyanate [FITC]-dextrans with molecular weights of 4000 [FD4] and 70,000 [FD70] daltons, fluorescein disulfonic acid [FS], lucifer yellow [LY], lactulose, and mannitol) were several times greater in the serosal-to-mucosal (S-->M) direction than in the opposite direction. Increased S-->M permeation by FD4, lactulose, and mannitol was evident at 37 degrees C, but not at 4 degrees C. Efflux of FD4 and FS in the S-->M direction was dose-dependently inhibited by verapamil, an inhibitor of the P-glycoprotein efflux system. Indomethacin, an anion transporter inhibitor, showed no effect on the S-->M permeation of FD4, FD70, FS, or LY. Adding an excess of unlabeled dextran (mol wt, 10,000 daltons) dose-dependently decreased the S-->M efflux of FD4, but not FS or LY., Conclusions: The transport across rat colonic mucosa of a number of hydrophilic substances, including some compounds that are commonly used to measure intestinal permeability in clinical practice, is greater in the S-->M than in the M-->S direction. S-->M transport of these hydrophilic solutes is temperature dependent, suggesting that the process is an active one. S-->M transport of FD4 may occur via a process that manifests some degree of substrate specificity for polysaccharides.

Objective: We sought to test the hypothesis that ileal mucosal oxygen consumption is impaired in endotoxemic rats., Methods: Male Sprague-Dawley rats were injected intravenously with either Escherichia coli lipopolysaccharide (5 mg/kg) or a similar volume of vehicle. A segment of ileum was excised 8 hrs later, and the serosal and muscular layers of the bowel were stripped away from the mucosa. A strip of mucosa was mounted in a polarographic chamber containing air-saturated Krebs-Henseleit buffer plus 20 mM glucose, PO2 being monitored during a 10-min period. Some rats were injected intraperitoneally with the inducible nitric oxide synthase inhibitor, aminoguanidine (30 mg/kg per dose), or a similar volume of vehicle, at 1, 3 and 6 hrs after injection of lipopolysaccharide., Results: In an initial experiment, the rate of oxygen consumption was significantly lower for mucosal samples from endotoxemic rats as compared with control rats (0.76+/-0.11 ng-atoms vs. 1.42+/-0.22 ng-atoms of 0/min per microg dry weight, respectively; n = 8 per group; p<.05). The rate of mucosal oxygen consumption was higher in aminoguanidine-treated as compared with vehicle-treated endotoxemic rats (1.25+/-0.11 ng-atoms and 0.73+/-0.07 ng-atoms of 0/min per microg, respectively; n = 7 and n = 6, respectively; p<.05)., Conclusion: Endotoxemia is associated with diminished intestinal mucosal oxygen utilization due to an intrinsic acquired derangement in cellular respiration that is caused, at least in part, by an aminoguanidine-inhibitable mechanism.

In a reductionist in vitro system, intestinal epithelial permeability appears to be dependent on cellular ATP content. In order to extend these prior observations, we used rat models of mesenteric ischemia/reperfusion (I/R) and pressure-controlled hemorrhagic shock to test the hypothesis that intestinal barrier function is directly proportional to tissue ATP content. I/R was induced by clamping the mesenteric vessels for 60 min followed by 60 min of reperfusion. Normal, ischemic, and reperfused ileal segments were prepared from each rat (n = 12). Hemorrhagic shock was induced by bleeding rats (n = 9) to a mean arterial pressure of 30 mmHg and maintaining this pressure for 4 h by infusing Ringer's lactate solution as necessary. Ileal segments were harvested before induction of hemorrhage and at 1 h intervals thereafter. Everted gut sacs were prepared to measure the mucosal-to-serosal passage of fluorescein-conjugated dextran (FD4; M.W. = 4 kDa). Tissue ATP levels were determined using a luciferase assay. FD4 clearance rates were plotted as a function of tissue ATP content. Linear regression analyses showed a strong inverse relationship between intestinal permeability and tissue ATP level in rats subjected to I/R (r2 = 0.78; P < 0.001) or hemorrhage (r2 = 0.82; P < 0.001). These data support the idea that ATP content is a determinant of intestinal epithelial barrier function in vivo.

In an effort to determine whether treatment with lisofylline (LSF) ameliorates intestinal barrier dysfunction in rats subjected to mesenteric ischemia and reperfusion (I/R), regional mesenteric vessels were occluded for 60 min and then unclamped for 60 min more. In Protocol 1, intravenous LSF (15 mg/kg bolus then 10 mg/kg/h) was administered 5 min before ischemia. In Protocol 2, LSF (same dose) was given 1 min before reperfusion. Controls received an equivalent volume of Ringer's lactate solution. Permeability was assessed by determining the mucosal-to-serosal clearance of fluorescein isothiocyanate-conjugated dextran (4 kDa) in everted ileal gut sacs incubated ex vivo. In Protocol 1, LSF treatment during ischemia ameliorated mucosal barrier dysfunction; mean +/- SEM clearances for the LSF and Ringer's lactate solution groups after 60 min of ischemia were 34.4+/-6.1 and 64+/-7.1 nL/min/cm2, respectively; p = .007. Clearances after reperfusion were the same in the two groups. In Protocol 2, LSF treatment just before reperfusion ameliorated barrier dysfunction measured 60 min after the restoration of blood flow; clearances for the LSF and Ringer's lactate solution groups were 23.1+/-3.8 and 40.2+/-4.5 nL/min/cm2, respectively; p = .012. Treatment with LSF did not affect intestinal levels of reduced glutathione or adenosine triphosphate or the extent of histological damage to the mucosa after I/R. Nevertheless, villus height was greater in animals treated with LSF than RLS prior to ischemia in Protocol 1 (250+/-37 and 160+/-15 microm, respectively; p = .04) and during reperfusion in Protocol 2 (170+/-21 and 82+/-7 microm, respectively; p = .002). We conclude that LSF is effective in reducing both ischemia- and I/R-induced gut barrier dysfunction, possibly due to a mechanism related to preservation of villus height.

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.

Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-alpha. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-alpha levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.

During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. CD14, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which CD14, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that CD14-mediated signaling is effected through a receptor-associated tyrosine kinase (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human CD14 (CHO/CD14). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/CD14 cells and to elucidate the relationship of NF-kappa B activation to the CD14 receptor system. LPS-stimulated translocation of NF-kappa B in CHO/CD14 cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, demonstrating that NF-kappa B translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for CD14-mediated signal transduction to occur in response to LPS.

We have examined the expression and structure of vegetative specific genes belonging to the V and H gene classes. Both classes of genes are deactivated at the onset of development by a reduction in the rate of transcription. Thus, the genes must be reactivated when the terminally differentiated spores germinate and the resulting amebae return to the vegetative state. During germination, activation of expression of most members of the V gene class was found to parallel the emergence of amoebae from the spore coats. The activation of the V genes did not occur when protein synthesis was inhibited. The timing of activation of the H genes was more heterogeneous and did not parallel emergence. H gene activation occurred even when protein synthesis was inhibited. V4 was found to be the only vegetative specific gene that was responsive to the presence of bacteria. V4 expression was induced by 25-100 fold via transcriptional activation when bacteria were added to amebae growing axenically. Isolation and sequence analysis of the corresponding genomic clones revealed that two V genes, V18 and V1, encode ribosomal proteins. Promoter analysis has delineated the sequences necessary for expression and regulation for several of the V and H genes. In all cases, expression was determined by sequences within the first several hundred base pairs of the transcription start site. For V18 and V14, a positive constitutive element was identified in addition to the sequences involved in regulation. Finally, all of the characterizations and findings are discussed in terms of postulated models for V and H gene expression and regulation.

We have identified and begun characterizations of the differential expression of 15 genes whose corresponding mRNA levels decrease during the preaggregative period of the developmental program of Dictyostelium discoideum. Upon the onset of development, the mRNAs decrease from 5- to 1000-fold over the first 8 hr. The rates of loss of each mRNA were similar to one another but distinct, and the decreases were dependent on progress through the developmental program. One exception to this dependency was observed, and the decrease in this mRNA was dependent on the absolute time after initiation of development instead of progress through development. With two exceptions, the decreases in mRNA levels were dependent on developmental conditions and were not seen when cells were shaken in starvation buffer. When the polysomal distributions of each species were examined, three classes were found: most showed no significant shifts off of polysomes upon initiation of development, two were characterized by a 20% shift to nonpolysomal RNA fractions upon development, and two gave a 40-50% shift. Collectively, these characterizations reveal differences in behavior which suggest that deactivation of genes upon initiation of development in Dictyostelium involves more than one regulatory pathway.