lipid-a and Necrosis

The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation.

Topics: Animals; Caspases; Caspases, Initiator; Cell Death; Cells, Cultured; Enzyme Activation; Epithelial Cells; Genetic Complementation Test; Humans; Immunity, Innate; Inflammation; Keratinocytes; Lipid A; Lipopolysaccharides; Macrophages; Mice; Mutant Proteins; Necrosis; Protein Binding; Protein Multimerization; Rhodobacter sphaeroides; Substrate Specificity; Surface Plasmon Resonance

Intrauterine infection plays a pivotal role in preterm birth (PTB) and is characterized by inflammation. Currently, there is no effective therapy available to treat or prevent bacterial-induced PTB. Using Fusobacterium nucleatum, a Gram-negative anaerobe frequently associated with PTB, as a model organism, the mechanism of intrauterine infection was investigated. Previously, it was shown that F. nucleatum induced preterm and term stillbirth in mice. Fusobacterial-induced placental infection was characterized by localized bacterial colonization, inflammation, and necrosis. In this study, F. nucleatum was shown to activate both TLR2 and TLR4 in vitro. In vivo, the fetal death rate was significantly reduced in TLR4-deficient mice (C57BL/6 TLR4(-/-) and C3H/HeJ (TLR4(d/d))), but not in TLR2-deficient mice (C57BL/6 TLR2(-/-)), following F. nucleatum infection. The reduced fetal death in TLR4-deficient mice was accompanied by decreased placental necroinflammatory responses in both C57BL/6 TLR4(-/-) and C3H/HeJ. Decreased bacterial colonization in the placenta was observed in C3H/HeJ, but not in C57BL/6 TLR4(-/-). These results suggest that inflammation, rather than the bacteria per se, was the likely cause of fetal loss. TLR2 did not appear to be critically involved, as no difference in bacterial colonization, inflammation, or necrosis was observed between C57BL/6 and C57BL/6 TLR2(-/-) mice. A synthetic TLR4 antagonist, TLR4A, significantly reduced fusobacterial-induced fetal death and decidual necrosis without affecting the bacterial colonization in the placentas. TLR4A had no bactericidal activity nor did it affect the birth outcome in sham-infected mice. TLR4A could have promise as an anti-inflammatory agent for the treatment or prevention of bacterial-induced preterm birth.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomimetic Materials; Female; Fetal Death; Fusobacterium Infections; Fusobacterium nucleatum; Humans; Inflammation; Lipid A; Mice; Mice, Knockout; Necrosis; Placenta; Placenta Diseases; Pregnancy; Pregnancy Complications, Infectious; Premature Birth; Stillbirth; Toll-Like Receptor 2; Toll-Like Receptor 4; Uterine Diseases

The use of dynamic myoplasty to restore function to failing organs is an exciting new application of skeletal muscle flaps. A complication of large flap elevation that can compromise flap function is ischemia-induced necrosis; one approach to minimizing this is to pretreat tissues with ischemic preconditioning. The purpose of this study was to determine whether systemic administration of monophosphoryl lipid A, a drug known to mimic late-phase ischemic preconditioning in the heart, could reduce ischemia-induced necrosis in latissimus dorsi muscle flaps. Forty latissimus dorsi muscle flaps from 20 Sprague-Dawley rats were allocated into four groups. In group I (n = 10), flaps were not preconditioned and served as controls. In group II (n = 10), flaps received ischemic preconditioning with two 30-minute periods of ischemia interspersed by 10 minutes of reperfusion. In group III (n = 10), rats received an intravenous bolus of approximately 0.3 ml of monophosphoryl lipid A vehicle only. In group IV (n = 10), rats received an intravenous bolus of 450 microg/kg of monophosphoryl lipid A and vehicle. Twenty-four hours after treatment, all latissimus dorsi muscle flaps were elevated on a single neurovascular pedicle and subjected to 4 hours of ischemia. After 72 hours of reperfusion, latissimus dorsi muscles were harvested, weighed, stained with nitroblue tetrazolium, and assessed for percent necrosis using digitized images of muscle sections and computerized planimetry. The percent necrosis in ischemic preconditioning-treated flaps (group II) was significantly reduced by 57 percent (p < 0.05) compared with control flaps (group I). The percent necrosis in flaps treated with monophosphoryl lipid A (group IV) was significantly reduced by 58 percent (p < 0.05) compared with vehicle-control flaps (group III). There was no difference in mean percent necrosis between ischemic preconditioning (group II) and monophosphoryl lipid A-treated (group IV) flaps or between ischemic preconditioning-control (group I) and monophosphoryl lipid A vehicle-control (group III) flaps. Intravenous administration of systemic monophosphoryl lipid A mimics the late-phase protective effect of ischemic preconditioning in the authors' rat latissimus dorsi muscle flap model.

Topics: Animals; Ischemia; Ischemic Preconditioning; Lipid A; Muscle, Skeletal; Necrosis; Rats; Rats, Sprague-Dawley; Surgical Flaps

Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 microg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.

Topics: Alginates; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Biocompatible Materials; Blood Proteins; Capsules; Cell Death; Fibrosarcoma; Fibrosis; Glucuronic Acid; Hexuronic Acids; Humans; Islets of Langerhans Transplantation; Jurkat Cells; Lipid A; Membrane Proteins; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Monocytes; Necrosis; Polylysine; Tumor Necrosis Factor-alpha

Monophosphoryl lipid-A (MLA) has a late window (24 hours) of cardioprotection against acute myocardial infarction. It is not known whether MLA, administered, 24 hours before surgery, attenuates intraoperative ventricular dysfunction "stunning" associated with aortic cross-clamping and reperfusion during elective cardiac surgery. We determined the dose-response relationship between MLA and ventricular function in a canine model of global myocardial stunning in the absence of necrosis. The role of expression of inducible heat shock protein 70 (HSP 70i) was also investigated.. Mongrel dogs (n = 32) were intravenously injected with either a vehicle solution or 3, 5, 10, 35 ug/kg MLA. Twenty four hours later, dogs were anesthetized and instrumented, in situ, to monitor the left ventricular performance (the slope of regression between stroke-work and end diastolic length). Tissue samples were obtained to determine HSP70i using immunoblot analysis. After a period of equilibration on cardiopulmonary bypass, the aortic cross-clamp was applied at normothermia for 30 minutes followed by 60 minutes of reperfusion. ATP and catabolites were determined in transmural myocardial biopsies. Triphenyl-tetrazolium chloride (TTC) staining was used to determine myocardial necrosis.. MLA treatment did not alter myocardial contractility or ATP metabolism. Global ischemia resulted in about 50% depletion of ATP and remained depressed during reperfusion in all groups. MLA-treated hearts had improved functional recovery in a dose dependent-manner. Significant recovery was observed at the highest dose (35 ug/kg) compared to the control group. Immunoblot analysis demonstrated significant increase in HSP 70i in the MLA-treated hearts.. MLA exhibits a delayed (24 hours) window of protection against myocardial stunning associated with aortic cross-clamping. HSP70i expression may play a role in MLA-mediated cardioprotection.

Topics: Adjuvants, Immunologic; Animals; Dogs; Female; HSP70 Heat-Shock Proteins; Injections, Intravenous; Lipid A; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardial Stunning; Myocardium; Necrosis

A novel experimental model of submassive liver necrosis with impaired regeneration has been established. A novel lipid A analogue, FS-112, was injected intravenously into male BALB/c mice, followed 2 days later by a 70% partial hepatectomy. Over the next 9 days, mice became severely jaundiced, with a peak total bilirubin (TBil) concentration of (mean +/- s.d.) 12.9 +/- 2.1 mg/dL 7 days postoperatively. In contrast, the TBil concentration in vehicle-treated mice remained less than 2 mg/dL. Significant elevations of L-alanine:2-oxoglutarate aminotransferase (ALT) were also observed 3-7 days after the operation in mice pretreated with FS-112, compared with mice pretreated with the vehicle. Submassive liver necrosis was observed with extensive mononuclear cell infiltration in mice treated with FS-112 and subjected to partial hepatectomy. Furthermore, both the BrdU and the proliferating cell nuclear antigen (PCNA) labelling index (LI) 1 day following partial hepatectomy in mice pretreated with FS-112 (8.6 +/- 4.3 and 7.9 +/- 4.2%, respectively) were significantly lower than levels in vehicle-treated mice (25.8 +/- 3.8 and 26.5 +/- 10.5%, respectively). The time course of changes in the BrdU LI in liver specimens from mice treated with both FS-112 and partial hepatectomy did not increase, even 3, 5, and 7 days postoperatively. Excellent liver regeneration with a PCNA LI 10-fold higher than the resting level was observed in mice treated with D-galactosamine hydrochloride. These results strongly suggest that this animal model of submassive liver necrosis may be suitable for clarifying the mechanisms of impaired liver cell regeneration often seen in fulminant hepatitis.

Topics: Animals; Bilirubin; Cell Division; Disease Models, Animal; Hepatectomy; Hepatic Encephalopathy; Lipid A; Liver; Liver Regeneration; Male; Mice; Mice, Inbred BALB C; Necrosis; Time Factors

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.

Topics: Animals; Cornea; Endotoxins; Female; Freund's Adjuvant; Guinea Pigs; Hypersensitivity, Delayed; Immunization; Inflammation; Lipid A; Lipopolysaccharides; Mycobacterium tuberculosis; Necrosis; Skin; Tuberculin

We investigated the ability of various tumour-necrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity in Propionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic.polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum from P.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Blood Physiological Phenomena; Cells, Cultured; DNA; Female; Immune Sera; Lipid A; Male; Mice; Mice, Inbred BALB C; Necrosis; Neoplasms, Experimental; Poly A-U; Recombinant Proteins; Tumor Necrosis Factor-alpha

Injection of sonic extracts of Bordetella parapertussis into the shaved backs of guinea pigs produced hemorrhagic necrosis, which previously has been attributed to the action of heat-labile toxin. As heat-labile toxin was purified from this crude mixture, its ability to induce hemorrhagic lesions decreased significantly. However, ischemic lesions were apparent after injection of the purified toxin. These lesions, while not hemorrhagic in nature, were marked by erythema surrounded by a region in which the ischemia was apparent. Exogenous agents were found to alter the nature of the skin lesion induced by heat-labile toxin. The lipid A portion of endotoxin in combination with heat-labile toxin caused hemorrhagic lesions surrounded by a ring of ischemia, whereas bovine serum albumin increased the area of erythema. While the nature of lesions induced by heat-labile toxin was affected by exogenous agents, the diameter of ischemia produced by the toxin was found to be independent of the presence of these agents and was linear with toxin dose. These results indicate that induction of hemorrhagic necrosis may not be a reliable indicator of heat-labile toxin activity. Instead, measurement of the ischemic lesion produced by heat-labile toxin may be a useful assay for the toxin.

Topics: Animals; Bacterial Toxins; Bordetella; Endotoxins; Guinea Pigs; Ischemia; Lipid A; Necrosis; Skin; Time Factors; Transglutaminases; Virulence Factors, Bordetella

The pathogenesis of toxic shock syndrome (TSS) remains unknown. On the basis of experimental data, it has been hypothesized that staphylococcal TSS Toxin 1 (TSST-1) may interact synergistically with low levels of endotoxin and give rise to many of the clinical findings. We have demonstrated previously that lipid A, the biologically active component of lipopolysaccharide (LPS), or endotoxin, induces dose-dependent necrosis of isolated rat renal tubular cells (RTCs). In the present studies, the authors investigated whether this injury could be augmented by TSST-1. The viability of RTCs was assessed by vital dye exclusion. Incubation of freshly isolated rat RTCs with either 1 ng/ml of TSST-1 or 0.1 ng/ml LPS or lipid A had minimal cytotoxicity (less than 6%). Exposure of RTCs to 1 ng/ml TSST-1 for 20 minutes, followed by washing, resulted in a significant enhancement of cytotoxicity when RTCs were exposed to 0.1 ng/ml LPS or lipid A. The sensitization of RTCs by TSST-1 to LPS- or lipid-A-induced injury was prevented by methylamine and chloroquine, two inhibitors of receptor-mediated endocytosis (RME). Chelation of extracellular calcium by 2 mM EGTA also blocked the TSST-1-induced sensitization of RTCs to LPS or lipid A. Inhibition of RTC arachidonic acid metabolism by methylprednisolone, indomethacin, ibuprofen, and piriprost significantly inhibited RTC necrosis induced by TSST-1 and LPS or lipid A by 33-62%. Thiourea and deferoxamine, agents which ameliorate oxidant injury, also inhibited this synergistic injury by 34-67%. Thus, TSST-1 enhanced the cytotoxic effects of LPS/lipid A, and the sensitization of RTCs appeared to involve RME or TSST-1. Oxidative metabolism of arachidonic acid and generation of reactive oxygen species appeared to participate in LPS/lipid-A-mediated RTC death.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bacterial Toxins; Cell Separation; Cell Survival; Chloroquine; Deferoxamine; Endocytosis; Endotoxins; Enterotoxins; Hydroxides; In Vitro Techniques; Kidney Tubules; Lipid A; Lipopolysaccharides; Male; Methylamines; Necrosis; Rats; Rats, Inbred Strains; Superantigens; Thiourea

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.

Topics: Animals; Cell Survival; Drug Tolerance; Endotoxins; Epitopes; Escherichia coli; In Vitro Techniques; Lethal Dose 50; Limulus Test; Lipid A; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mitogens; Necrosis; Prostaglandins; Pyrogens; Rabbits; Sarcoma, Experimental; Shwartzman Phenomenon