lipid-a and Peritonitis

To investigate the therapeutic effect of E5564 (a clinically used TLR4 inhibitor) in murine abdominal sepsis elicited by intraperitoneal infection with a highly virulent Escherichia coli in the context of concurrent antibiotic therapy.. Mice were infected with different doses (~2 × 10(4)-2 × 10(6) CFU) of E. coli O18:K1 and treated after 8 h with ceftriaxone 20 mg/kg i.p. combined with either E5564 10 mg/kg i.v. or vehicle. For survival studies this treatment was repeated every 12 h. Bacterial loads and inflammatory parameters were determined after 20 h in peritoneal lavage fluid, blood, liver and lung tissue. Plasma creatinin, AST, ALT and LDH were determined to assess organ injury.. E5564 impaired bacterial clearance under the antibiotic regime after infection with a low dose E. coli (1.7 × 10(4) CFU) while renal function was slightly preserved. No differences were observed in bacterial load and organ damage after infection with a tenfold higher (1.7 × 10(5) E. coli) bacterial dose. While treatment with E5564 slightly attenuated inflammatory markers provoked by the sublethal doses of 104-105 E. coli under the antibiotic regime, it did not affect lethality evoked by infection with 1.7 × 106 E. coli.. The impact of TLR4 inhibition during abdominal sepsis by virulent E. coli bacteria is only beneficial at low infection grade at cost of bactericidal activity.

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Ascitic Fluid; Bacterial Load; Ceftriaxone; Cytokines; Escherichia coli; Escherichia coli Infections; Female; Lipid A; Liver; Lung; Mice, Inbred C57BL; Peritoneal Lavage; Peritonitis; Sepsis; Toll-Like Receptor 4

Acute peritonitis, in which peritoneal mesothelial cells are directly exposed to bacterial components, is a major cause of peritoneal dysfunction in continuous ambulatory peritoneal dialysis patients. We have previously shown that Toll-like receptors (TLR) are expressed in kidney cells, and LPS induces TLR4-dependent chemokine production in tubular epithelial cells. The present work was designed to investigate the involvement of TLR, especially TLR4, in the lipid A-mediated chemokine production by murine peritoneal mesothelial cells (MPMC). A primary cell culture of MPMC from C3H/HeN mice (wild-type mice; LPS sensitive) and from C3H/HeJ mice (containing a point mutation of TLR4; LPS hyposensitive) was established. The expression profile of the TLR family and their accessory molecules, CD14 and MD-2, which are requisite for the LPS signaling pathway, was examined by RT-PCR, Northern blot test, and immunohistochemical staining. Synthetic lipid A-mediated chemokine production by MPMC was studied. The involvement of MAP kinase family (ERK, JNK, and p38 mitogen-activated protein kinase) and nuclear factor (NF)-kappaB in these processes was also studied. MPMC constitutively express TLR4, CD14, and MD-2. A prominent induction of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein (MIP)-2 by MPMC was detected after lipid A stimulation and was strictly dependent on TLR4. Furthermore, TLR4-dependent chemokine production followed by leukocyte influx into the peritoneal cavity was also confirmed in vivo after stimulation with LPS. mRNA expression of MCP-1 was abolished by NF-kappaB inhibition, but were not affected by the inhibition of ERK, JNK, or p38. As compared with MCP-1, MIP-2 mRNA expression was inhibited by a high dose of curcumin but not by NF-kappaB decoy oligodeoxynucleotide and individual inhibitions of MAP kinase, suggesting that the additional signaling pathway with NF-kappaB might be involved in mRNA expression of MIP-2. These show that TLR4 is directly involved in the production of MCP-1 and MIP-2 by MPMC in a NF-kappaB-dependent manner, but the process does not require any MAP kinase activation. The results provide a candidate molecular target in prevention of it.

Topics: Animals; Cell Line; Cell Movement; Chemokine CCL2; Chemokine CXCL2; Chemokines; Epithelium; Gene Expression; Lipid A; Macrophages, Peritoneal; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Mitogen-Activated Protein Kinases; NF-kappa B; Peritoneum; Peritonitis; Receptors, Cell Surface; RNA, Messenger; Toll-Like Receptor 4; Toll-Like Receptors; Up-Regulation

Endotoxin is a principle mediator of septic shock during peritonitis. Induction of endotoxin tolerance with monophosphoryl lipid A (MPL), a nontoxic derivative of lipid A, improves survival from peritonitis. The induction of tolerance with intravenous versus intraperitoneal administration of MPL before peritonitis was compared. Mice were pretreated with varying doses of MPL (intravenously) and MPL (intraperitoneally) 48 hours before peritonitis was induced by cecal ligation and perforation. Survival was determined at 72 hours, and serum and peritoneal levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) were assayed at 24 hours. Survival was 0% in control animals, 20% in MPL (100 micrograms intravenously) animals, and 70% in MPL (100 micrograms intraperitoneally) animals (p < 0.05 versus control, MPL [intravenously]). Cytokine release was compared in control animals and animals receiving MPL 100 micrograms (intraperitoneally) or MPL 100 micrograms (intravenously). In MPL (intraperitoneally)-treated animals, serum and peritoneal TNF-alpha levels, 160 +/- 7 pg/ml and 204 +/- 25 pg/ml, were significantly lower than those in control animals, 429 +/- 34 pg/ml and 642 +/- 108 pg/ml, and MPL (intravenously)-treated animals, 302 +/- 68 pg/ml and 495 +/- 97 pg/ml, (p < 0.05). Similarly, IL-1 alpha levels were significantly lower in MPL (intraperitoneally)-treated animals than in control animals. Because the development of tolerance appears to be a cytokine-mediated process, a subsequent experiment compared peritoneal and serum TNF-alpha and IL-1 alpha levels at 2 hours after MPL (intraperitoneally) or MPL (intravenously). Peritoneal TNF-alpha and IL-1 alpha release was greatest after MPL (intraperitoneally); serum levels were greatest after MPL (intravenously).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cytokines; Drug Tolerance; Endotoxins; Injections, Intraperitoneal; Injections, Intravenous; Lipid A; Mice; Mice, Inbred Strains; Peritonitis; Survival Analysis

Failure of the immune system to successfully prevent organ failure after injury or infection may be associated with a shift in macrophage function from antigen recognition and presentation to overexpression of inflammatory cytokines. Regulation may be due to changes in macrophage gene expression. Levels of mRNA for tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), and the Ia subunit, I-A alpha, in peritoneal macrophages, liver, spleen, kidney, and lung were measured following cecal ligation and puncture (CLP) in Swiss Webster mice. Northern blot analysis was performed using 32P-labeled mouse cDNA probes. Peritoneal macrophage TNF alpha and IL-1 beta mRNA expression increased 2.5- and 2-fold, respectively, by 6 hr after CLP and remained elevated at 24 hr. Peritoneal macrophage I-A alpha mRNA levels decreased 8-fold by 24 hr after CLP. I-A alpha mRNA expression in liver, spleen, kidney, and lung decreased following CLP, with a return toward normal levels by 8 days in all tissues except spleen. IL-1 beta and TNF alpha mRNA were barely detectable in liver and kidney. IL-1 beta mRNA tended to increase over time in lung and spleen, whereas TNF alpha mRNA in these tissues did not vary greatly after CLP. Muramyl dipeptide or monophosphoryl lipid A pretreatment of animals prior to CLP was ineffective in altering the expression of TNF alpha and I-A alpha mRNA. We conclude that peritonitis is associated with an early increase in peritoneal macrophage TNF alpha and IL-1 beta mRNA levels and a sharp decline in macrophage I-A alpha mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Gene Expression Regulation; Histocompatibility Antigens Class II; Interleukin-1; Lipid A; Macrophages; Male; Mice; Peritonitis; RNA, Messenger; Tumor Necrosis Factor-alpha

We compared the induction of endotoxin tolerance with Salmonella minnesota monophosphoryl lipid A (MPL), a nontoxic derivative of lipid A, and S. minnesota endotoxin (LPS) in lethal endotoxemia and peritonitis. Lethal endotoxemia was induced by injecting 750 micrograms/mouse LPS intravenously. Cecal ligation and perforation was used to induce peritonitis. Tumor necrosis factor (TNF) was measured by immunoassay at 2 hr after lethal endotoxin infusion and 24 hr after peritonitis. A dose of 0.1 micrograms/mouse of MPL or LPS significantly reduced endotoxin mortality from 100% to 50% and 27%, respectively (P < 0.05). The LD50 for a 0.1 micrograms dose of MPL was 750 micrograms of LPS and the LD50 for a 0.1 micrograms dose of LPS was 1150 micrograms of endotoxin (P < 0.05). TNF levels decreased linearly when increasing doses of MPL and LPS were used to induce tolerance. At higher pretreatment doses of LPS, survival benefits were attenuated despite the reduction in TNF levels. A 25 micrograms dose of LPS reduced mortality from peritonitis from 93% to 45% (P < 0.05). Although MPL reduced short-term mortality, overall mortality was not significantly reduced despite using large doses of MPL. TNF levels peaked at 24 hr and were significantly lower than those following lethal endotoxemia. The induction of endotoxin tolerance by LPS and MPL is dose dependent, and LPS is modestly more effective in inducing endotoxin tolerance than MPL. Both LPS and MPL are significantly less effective in protecting against lethality from peritonitis.

Topics: Animals; Dose-Response Relationship, Drug; Drug Tolerance; Endotoxins; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred ICR; Peritonitis; Survival Analysis; Time Factors; Tumor Necrosis Factor-alpha

We analyzed the in vitro effects of monophosphoryl lipid A (MPLA), a nontoxic bacterial endotoxin-derived immunomodulant, on the depressed immune functions of peritoneal lymphocytes (PLy) and macrophages (PMO) of 6 CAPD patients with relapsing bacterial peritonitis. MPLA was also tested for its capacity to stimulate the peritoneal fibroblast proliferation as determined by 3H-thymidine incorporation. In vitro incubation of PLy and PMO with escalating doses of MPLA up to 5 micrograms/ml, resulted in a dose-dependent enhancement of Gamma-Interferon (Gamma-IFN) and Interleukin-2 (IL-2) production by PLy, and Interleukin-1 (IL-1) by PMO. There was also an increase in PMO bacterial killing and membrane Fc receptor number, while no change in peritoneal fibroblast proliferation was seen with any of the MPLA concentrations tested. These results suggest that the peritoneal leukocyte abnormalities observed in some high peritonitis rate CAPD patients may be reversed, to some degree, by MPLA, without directly inducing a potentially deleterious peritoneal fibrosis.

Topics: Adult; Cell Division; Female; Fibroblasts; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-1; Interleukin-2; Lipid A; Lymphocyte Activation; Lymphocytes; Macrophage Activation; Macrophages; Male; Middle Aged; Peritoneal Cavity; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Receptors, Fc; Recurrence; Staphylococcus epidermidis

To study the role of antibodies in promoting survival during gram-negative bacterial sepsis, we have developed several murine monoclonal antibodies (MAbs). One MAb (5B10) reacted in an enzyme-linked immunosorbent assay with only a single organism (Escherichia coli 0111:B4), while the other (8A1) reacted to all gram-negative whole-cell and lipopolysaccharide (LPS) antigens examined. Either 5B10 MAb, 8A1 MAb, or sterile saline solution was administered intravenously to outbred male Swiss-Webster mice immediately before one of three challenges: (1) viable bacteria intravenously, (2) viable bacteria with hemoglobin intraperitoneally, or (3) intravenous actinomycin D plus LPS. 5B10 MAb provided significant protection against either an E. coli 0111:B4 bacterial or LPS challenge but not against any other organism or type of LPS. 8A1 MAb provided protection against several challenge bacteria (intravenously or intraperitoneally) and against all types of LPS studied except Pseudomonas aeruginosa LPS. A higher dose (2 mg) of cross-reactive antibody (8A1 MAb) was required to produce protection when compared with the type-specific protection produced with 5B10 MAb (0.1 mg). Although ideal antibody therapy would consist of directing a specific MAb against a single microorganism, the acute nature of the disease process and time required to prepare reagents may preclude the use of type-specific MAbs. We believe that the cross-reactive and cross-protective capacity of 8A1 MAb or a similar MAb may be useful in averting the lethal effects of clinical gram-negative bacterial sepsis and warrants testing in the clinical setting.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cross Reactions; Disease Models, Animal; Endotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Lipid A; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Peritonitis; Sepsis