Pulmonary intravascular monocytes/macrophages in a rat model of sepsis

Sepsis induces recruitment of neutrophils and monocytes/macrophages in the lung and enhances host susceptibility to a secondary bacterial challenge. The phenotype and functions of recruited pulmonary intravascular monocytes/macrophages (PIMMs) in sepsis remain largely unknown. Therefore, we characterized PIMM recruitment and functions in a rat model of E. coli-induced sepsis. Male Sprague-Dawley rats were injected intraperitoneally with saline (n=10) and 48 hr after the saline treatment treated intravenously with either saline (n=5) or E. coli lipopolysachharide (LPS; 1.5 microg/kg body weight; n=5). A second group of 10 rats was infected intraperitoneally with E. coli (2x10(7) CFU/100 g) followed by intravenous injection of either saline (n=5) or LPS (n=5) 48 hr after the first treatment. Rats were euthanized at 6 hr after LPS treatment. Immunocytochemistry showed more PIMMs stained with ED-1 antibody, which specifically reacts with rat monocytes/macrophages, in rats infected with E. coli compared with the controls (P<0.05). LPS treatment of E. coli-infected rats increased the numbers of PIMMs (P<0.05) and induced more inflammation compared to other groups. Immuno-electron microscopy localized TNF-alpha, IL-10, and TGF-beta2 in recruited PIMMs in rats challenged with both E. coli and LPS. ELISA on lung homogenates showed higher concentrations of TNF-alpha, IL-10, and TGF-beta2 in rats treated with both E. coli and LPS compared with those treated with only LPS or E. coli (P<0.05). We conclude that ED-1-positive PIMMs are recruited in this model of sepsis and contain TNF-alpha, IL-10, and TGF-beta2.     

Pathophysiology of in-vitro induced filaments, spheroplasts and rod-shaped bacteria in neutropenic mice

This study compared the in-vitro properties and in-vivo effects of Escherichia coli filaments, spheroplasts and normal cells in a murine thigh infection model. E. coli was exposed to ceftazidime, meropenem or saline to obtain filaments, spheroplasts or normal bacilli, which were then injected into neutropenic mice. After 24 h, morphology, CFUs, local and circulating endotoxin levels, cytokine levels and mortality were recorded, and correlations between bacterial and host parameters of infection were investigated. Filaments and spheroplasts contained more endotoxin/CFU than controls. Histological studies showed that morphologically altered bacteria changed into rod-shaped cells in the absence of antibiotics. Bacterial spread to the liver was significantly higher in mice challenged with rod-shaped cells, compared with antibiotic-exposed bacteria (p 0.007). Muscle endotoxin levels correlated significantly with circulating interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha, and both pro-inflammatory cytokines were correlated significantly (p 0.011). Despite a tendency toward higher local and systemic concentrations of endotoxin in the filament group, inflammatory responses and survival did not differ between groups. It was concluded that morphologically altered bacteria contain more endotoxin and can regain a rod shape after withdrawal of antibiotics, while non-antibiotic-exposed bacteria show greater spread to the liver. There was a clear intra-individual relationship between local endotoxin, systemic endotoxin, TNF-alpha and IL-6 production, but these parameters did not differ among groups.     

Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs.

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.     

Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro in humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (suPAR) was measured by enzyme-linked immunosorbent assay in plasma and supernatants from unstimulated and phytohaemagglutinin and lipopolysaccharide-stimulated peripheral blood mononuclear cell (PBMC) cultures incubated for 24 h. The spontaneous and stimulated uPAR-release from PBMC cultures was enhanced 5 h after low-dose endotoxin (both P <0.05), but coadministration of rhIL-6 during low-dose endotoxaemia abolished this enhanced uPAR release. High-dose endotoxin increased plasma suPAR levels (P <0.001) whereas low-dose endotoxin, rhIL-6 or TNF-alpha did not influence uPAR release in vivo to such degree that a systemic effect on the plasma suPAR level was detectable. Even subclinical doses of endotoxin in vivo enhance the capacity of PBMC to release uPAR after incubation in vitro. The inhibitory effect of IL-6 on endotoxin-mediated uPAR-release in vitro suggests that IL-6 has anti-inflammatory effects on endotoxin-mediated inflammation.     

Acute endotoxin-induced lymphocyte subset sequestration in sheep lungs

Endotoxemia is associated with an early phase of pulmonary hypertension and a later increase in microvascular permeability. These physiologic changes are attended by peripheral blood and lung lymph leukopenia and a rapid accumulation of both granulocytes and lymphocytes in the peripheral lung. In the present study, the numbers of lymphocytes in blood, lung lymph, and lung tissue after infusion of endotoxin were determined by fluorescent labeling of lymphocyte populations with monoclonal antibodies to sheep T1, T4, T8, or leukocyte common antigen and with rabbit anti-sheep immunoglobulins (B cells). Peripheral blood, lung lymph, and lung tissue samples were collected at baseline and 15, 30, 60, 120, 180, and 240 minutes after the start of intravenous infusion of E. coli endotoxin (1.25 micrograms/kg, N = 6) or saline (N = 4) from open-chest anesthetized sheep. Pulmonary artery pressure and lung lymph flow were also monitored at these times. Endotoxin caused marked reductions in the number of T and B lymphocytes in blood and lung lymph. As compared with baseline, total blood leukocytes and granulocytes were significantly reduced below control levels from 30 minutes of endotoxin, and lymphocyte numbers were reduced from 60 minutes. T1, T4, T8 and B lymphocyte subsets contributed to the fall in blood lymphocytes. Endotoxin caused a significant fall in number of lung lymph leukocytes (T1, T4 and T8 cells) from 120 minutes; numbers of B lymphocytes were also reduced. Counts of the number of lymphocytes in the biopsy tissue revealed a significant rise in T lymphocytes sequestered in the lung. We conclude endotoxemia in sheep causes a reduction in lymphocytes in blood and lung lymph and an increase in these cell types in peripheral lung tissue. These findings suggest that lymphocyte subpopulations accumulate in the lungs during periods of pulmonary hypertension and increased permeability and may participate in endotoxin-induced lung injury.     

Roles of tumor necrosis factor receptor signaling during murine Escherichia coli pneumonia

We hypothesized that tumor necrosis factor (TNF)-alpha signaling is essential to inflammation and host defense during Escherichia coli pneumonia. We tested this hypothesis by instilling E. coli into the lungs of wild-type (WT) mice and gene-targeted mice that lack both p55 and p75 receptors for TNF-alpha. The emigration of neutrophils 6 h after instillation of E. coli was not decreased, but rather was significantly increased (167% of WT), in TNF receptor (TNFR)-deficient mice. This increased neutrophil emigration did not result from peripheral blood neutrophilia or enhanced neutrophil sequestration, inasmuch as the numbers of neutrophils in the circulating blood and in the pulmonary capillaries did not differ between TNFR-deficient and WT mice. The accumulation of pulmonary edema fluid was not inhibited in TNFR-deficient compared with WT mice. Nuclear factor-kappaB (NF-kappaB) translocation in the lungs was not prevented in TNFR-deficient mice. Thus, signaling pathways independent of TNFRs can mediate the acute inflammatory response during E. coli pneumonia. However, despite this inflammatory response, bacterial clearance was impaired in TNFR-deficient mice (109 +/- 8% versus 51 +/- 14% of the original inoculum viable after 6 h in TNFR-deficient and WT mice, respectively). Increased neutrophil emigration during E. coli pneumonia in TNFR-deficient mice may thus result from an increased bacterial burden in the lungs. During acute E. coli pneumonia, the absence of TNFR signaling compromised bacterial killing, but did not prevent inflammation, as measured by the accumulation of edema fluid and neutrophils.     

Acute inflammatory response to endotoxin in mice and humans

Endotoxin injection has been widely used to study the acute inflammatory response. In this study, we directly compared the inflammatory responses to endotoxin in mice and humans. Escherichia coli type O113 endotoxin was prepared under identical conditions, verified to be of equal biological potency, and used for both mice and humans. The dose of endotoxin needed to induce an interleukin-6 (IL-6) concentration in plasma of approximately 1,000 pg/ml 2 h after injection was 2 ng/kg of body weight in humans and 500 ng/kg in mice. Healthy adult volunteers were injected intravenously with endotoxin, and male C57BL/6 mice (n=4 to 12) were injected intraperitoneally with endotoxin. Physiological, hematological, and cytokine responses were determined. Endotoxin induced a rapid physiological response in humans (fever, tachycardia, and slight hypotension) but not in mice. Both mice and humans exhibited lymphopenia with a nadir at 4 h and recovery by 24 h. The levels of tumor necrosis factor (TNF) and IL-6 in plasma peaked at 2 h and returned to baseline levels by 4 to 6 h. IL-1 receptor antagonist RA and TNF soluble receptor I were upregulated in both mice and humans but were upregulated more strongly in humans. Mice produced greater levels of CXC chemokines, and both mice and humans exhibited peak production at 2 h. These studies demonstrate that although differences exist and a higher endotoxin challenge is necessary in mice, there are several similarities in the inflammatory response to endotoxin in mice and humans.     

Promotion of bacterial translocation by major liver resection in obstructive jaundice in rats colonised predominantly with indigenous Escherichia coli

The influence of major liver resection in obstructive jaundice on bacterial translocation was evaluated in rats that were colonised predominantly with a genetically labelled strain of Escherichia coli. The strain, JNW14, originally isolated from rat faeces, was labelled with bacitracin, neomycin and streptomycin resistance markers. Fifty-two specific-pathogen-free male Wistar rats were divided into three experimental groups and were treated as follows: group 1 (n = 8), sham ligation of common bile duct; group 2 (n = 7), common bile duct ligation (CBDL); and group 3 (n = 37), 70% hepatectomy 7 days after CBDL. The rats were treated with the above antibiotics and then given E. coli strain JNW14 in their drinking water. Translocation of E. coli JNW14 from the gastrointestinal tract to the mesenteric lymph nodes (MLNs), lungs, liver, spleen and portal vein was evaluated in each group. In group 3 (CBDL plus hepatectomy), the incidence of translocation of E. coli JNW14 to the liver and spleen after hepatectomy was significantly higher than in groups 1 and 2. This result indicates that major liver resection in obstructive jaundice promotes bacterial translocation to systemic organs. Furthermore, the numbers of viable E. coli JNW14 in the MLNs in the lung culture-positive rats were significantly higher than those in the lung culture-negative rats, suggesting that lymphatic-thoracic duct systemic circulation is a major route of bacterial translocation.     

Augmentation of the resistance against Escherichia coli by oral administration of a hot water extract of Chlorella vulgaris in rats

In previous studies, we demonstrated that a hot water extract of Chlorella vulgaris (CVE) augmented the resistance against an intraperitoneal infection with Escherichia coli by its intraperitoneal, intravenous or subcutaneous administration. The augmented resistance appeared to be attributable to the enhanced activity of polymorphonuclear leukocytes (PMN). In this study, the effect of oral administration of CVE against Escherichia coli infection was examined. Male Fisher rats (F344/DuCrj) were administered 1000 mg/kg of CVE orally for 14 days and challenged with 2.7 x 10(8) Escherichia coli intraperitoneally. The numbers of living bacteria in the peritoneal cavity, blood, spleen and liver at 1, 6, and 24 h after the inoculation were counted. The bacterial numbers increased during 1-6 h and reached the peak at 6 h in both control and CVE-administered groups. The bacterial numbers decreased to an undetectable level at 24 h in both groups. In a CVE-administered group, the numbers of viable bacteria in each organ were remarkably lower than those in a control group in all organs so far tested. Whereas, the leukocyte numbers, especially PMN numbers, in the peritoneal cavity and peripheral blood maintained higher levels in the CVE-administered group at 6 h after E. coli inoculation. Chemiluminescent responses of peritoneal exudate cells induced by casein or E. coli were higher in a CVE-administered group. These results form the basis for the judgment that the degree of effectiveness of bacteria clearance from the peritoneal cavity shown by oral CVE administration may be strong enough to warrant developing this material as a new type of biological response modifier.     

Quantitative assessment of microvascular permeability in endotoxin-induced lung injury in anaesthetized dogs

We quantitatively assessed the change of pulmonary microvascular permeability following E. coli endotoxin infusion (1 mg/kg) in anaesthetized dogs. We used mathematical analysis to estimate membrane parameters from lung lymph data. Lung lymph was collected from the afferent lymphatic connecting to the left tracheobronchial lymph node whose lymph could be considered to represent an average sample of lung tissue fluid. To separate the effects of changes in the driving pressures and surface area on lymph fluid and protein flux from those in membrane permeability, lymph flow (Jv) was increased greater than 6 times baseline by left atrial pressure (Pla) elevation until lymph protein concentration (CL) approached to a constant value independent of Jv. Membrane parameters for plasma total protein, i.e., osmotic reflection coefficient (sigma d), solvent-drag reflection coefficient (sigma f), permeability surface area product (PS), filtration coefficient (Kfc), were calculated from lung lymphatic data in control (Pla elevation alone (n = 10), and endotoxin group (n = 7). Among these parameters, osmotic reflection coefficient (sigma d) decreased significantly to 0.61 in endotoxin group from the value of 0.71 in control group. This result indicates a moderate increase in pulmonary microvascular permeability following E. coli endotoxin infusion. However, there was no significant difference in the other membrane parameters (sigma f, PS, Kfc) between control and endotoxin group. Based on these results, we conclude that sigma d could quantitatively represent the moderate change of the microvascular permeability in endotoxin-induced lung injury in anaesthetized dogs.     

Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep structure:function relationships

Infusion of Escherichia coli endotoxin into sheep causes a syndrome analogous to the adult respiratory distress syndrome. Physiologic measurements show an initial phase of marked pulmonary hypertension followed by a phase characterized by the production of large quantities of protein-rich lung lymph. The present study relates the structural changes that occur during endotoxemia to concomitant functional changes. In five anesthetized open-chest sheep, we monitored pulmonary and systemic artery pressure for a 1 hour baseline period and for 4 hours after the start of E. coli endotoxin infusion (1.25 microgram/kg, intravenously). We also measured cardiac output, arterial blood gases and pH, and number of circulating leukocytes. In addition, we sequentially biopsied random lobes from the lungs of each sheep at baseline and at 15, 30, 60, 120, 180, and 240 minutes after the start of endotoxin. Five control sheep were treated identically except that they received saline instead of endotoxin. By 15 minutes after the start of endotoxin infusion, light microscopy revealed margination and accumulation of leukocytes in the lungs' microcirculation. Counts of the number of peripheral lung granulocytes in biopsy specimens showed a 3-fold increase above baseline by 15 minutes and a 6-fold increase by 4 hours. By electron microscopy, the leukocytes were identified as both granulocytes and lymphocytes, present in approximately equal numbers. Some granulocytes were fragmented, and specific granules were found free in the vessel lumen. By 30 minutes, some leukocytes were migrating into the interstitium. By 60 minutes, interstitial edema was seen, and there was focal endothelial cell damage. Correlation of the structural with the physiologic changes shows that the initial accumulation of leukocytes in the microcirculation occurs when pulmonary hypertension develops. The migration of leukocytes into the interstitium and endothelial cell damage precedes the physiologic changes that we interpret as increased pulmonary vascular permeability. Since gram negative septicemia is a frequent occurrence in the adult respiratory distress syndrome the changes described here may be similar to the alterations that occur early in the development of the syndrome in man.     

Human models of low-grade inflammation: bolus versus continuous infusion of endotoxin

Systemic low-grade inflammation is recognized in an increasing number of chronic diseases. With the aim of establishing an experimental human in vivo model of systemic low-grade inflammation, we measured circulating inflammatory mediators after intravenous administration of Escherichia coli endotoxin (0.3 ng/kg of body weight) either as a bolus injection or as a 4-h continuous intravenous infusion, as well as after saline administration, in 10 healthy male subjects on three separate study days. Only bolus endotoxin caused an increase in heart rate, whereas a slight increase in rectal temperature was observed in both endotoxin groups. Tumor necrosis factor alpha (TNF-alpha), interleukin-6, and neutrophil responses were earlier and more pronounced in the bolus trial compared with the infusion trial results, whereas lymphocytes increased after endotoxin bolus injection as well as infusion without any difference between groups. Finally, endotoxin activated the hypothalamo-pituitary-adrenal axis slightly earlier in the bolus compared to the infusion trial. The continuous endotoxin infusion model may be more representative of human low-grade inflammation than the bolus injection model due to a less dynamic and more sustained increase in circulating levels of inflammatory mediators over time. In conclusion, low-dose endotoxin infusion elicits an inflammatory response, as assessed by a rise in TNF-alpha, and the responses are significantly different according to whether low-dose endotoxin is applied as a bolus injection or as a continuous infusion.     

Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin

Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids. Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7. The inflammatory eell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present. TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor. In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose ofEscherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle. However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.     

Circulating adiponectin levels during human endotoxaemia

Adiponectin, an adipocytokine secreted by fat tissue, may prevent development of diabetes type II, as high adiponectin levels are linked with insulin sensitivity. In contrast, tumour necrosis factor (TNF)-alpha, which is also produced by fat tissue, leads to insulin resistance and furthermore inhibits adiponectin mRNA production and secretion of the protein. However, adiponectin also negatively regulates TNF-alpha levels. Therefore, we set out to test whether an infusion of endotoxin would influence circulating adiponectin levels in healthy human subjects. Twenty-three healthy human subjects were injected with endotoxin (2 ng/kg body weight); eight of these subjects were also injected with saline and served as controls. Plasma levels of adiponectin, TNF-alpha and interleukin-6 were measured at 0, 1.5, 2, 4, 8 and 24 h. TNF-alpha and interleukin-6 levels peaked at 1.5 h and 2 h, respectively. Control subjects injected with saline showed a decrease in adiponectin plasma levels with time (P < 0.05) presumably owing to the effect of fasting or physical inactivity. However, there was no change in adiponectin plasma levels in endotoxin injected subjects, thus the effect of fasting was opposed. In conclusion, circulating adiponectin levels are reduced during a resting and fasting state, an effect reversed by endotoxin injection.     

Protective features of monoclonal antibodies to Escherichia coli during experimental infection of mice with homologous and heterologous serotypes of E. coli

Murine monoclonal antibodies (MAbs) MT1F and ARM1-4, recognising proteins on the surface of untreated Escherichia coli O6:K-, protected 100% of mice challenged intraperitoneally with 2 x LD50 of the same strain. MAb MT1F protected 70% of animals challenged with 2 x LD50 of E. coli O111:B4, whereas ARM1-4 gave complete protection. Lower survival was observed in mice given either MAb and challenged with E. coli O128:K-, with values ranging from 30 to 42%. However, the protection afforded against E. coli O111:B4 and E. coli O128:K- was significantly improved when the mice were pre-treated with a mixture of the two MAbs. Control mice, pre-treated with unrelated ascitic fluid and challenged with any of the E. coli serotypes, showed 100% mortality and organ histological lesions resembling those of the early stages of septic shock. The mice had high levels of circulating endotoxin and tumour necrosis factor-alpha (TNF-alpha) at 90 min after challenge. In contrast, mice treated with MAbs and surviving the infection displayed moderate histological lesions, enhanced bacterial clearance and lower serum levels of TNF-alpha, despite circulating endotoxin levels that were higher than in the control group. Protection by the MAbs was probably due to the prevention of the bacterial spread to organs and of the cascade of events leading to septic shock. This occurred in spite of the presence of high levels of circulating endotoxin.     

Morphological and morphometrical analysis of the acute response of the bovine alveolar wall to Pasteurella haemolytica A1-derived endotoxin and leucotoxin

Endotoxin or leucotoxin derived from Pasteurella haemolytica biotype A serotype 1 or saline was deposited by fibreoptic bronchoscopy into the caudal segment of the right anterior lung lobe of calves, and the lesions were characterized by light and transmission electron microscopy. Morphometric techniques were used to determine if changes in the arithmetic mean thickness of the alveolar wall occurred. Group 1 calves (n = 2) were inoculated with 6 ml saline, groups 2 calves (n = 3) received 6 ml of a partially purified leucotoxin preparation, group 3 calves (n = 3) received 96 micrograms of endotoxin in 6 ml of saline and group 4 calves (n = 3) received 2.5 mg of endotoxin in 6 ml of saline. Calves were killed 4 h after inoculation. Lesions in groups 2, 3 and 4 were similar and we found that (a) endotoxin alone is capable of initiating an inflammatory response in the bovine lung, (b) leucotoxin causes cytotoxic changes in alveolar macrophages but not in parenchymal cells of the lung, (c) neutrophil sequestration and platelet aggregation occur in alveolar capillaries in association with pulmonary intravascular macrophages, (d) neutrophils and fibrin were found in the alveolus in close association with alveolar macrophages, (e) disruption of the alveolar epithelial layer occurred in association with neutrophils and (f) there were no significant increases in the arithmetic mean thickness of the alveolar wall.     

Lipopolysaccharide-induced non-specific resistance to systemic Escherichia coli infection in mice

A high degree of non-specific resistance to a lethal systemic Escherichia coli infection was induced in mice by pretreatment with a small dose (less than 5 micrograms/mouse) of the homologous lipopolysaccharide (LPS) or with heterologous rough-type LPS from E. coli K-12. The route of LPS administration, intraperitoneally or subcutaneously, did not influence the development of resistance, suggesting that a systemic cell activation was responsible for the effect. The enhanced elimination of bacteria was similar to that in mice recovering from a sublethal E. coli infection. In the LPS-treated mice, elimination of the challenge bacteria from the peritoneal cavity and the blood started 3-4 h after challenge whereas, in controls, the bacterial numbers continued to increase until the mice died. The detoxified LPS derivative, monophosphoryl lipid A (MPL), also increased the survival of mice infected with E. coli O18:K1. However, the dose of MPL required for optimal infection resistance was 100-fold greater than that of native, E. coli K-12 LPS, corresponding to the 100-fold reduced toxicity of MPL for mice and rabbits in lethality and pyrogenicity assays.     

Lack of endotoxin tolerance with respect to TNF alpha production in the subarachnoid space

To study endotoxin tolerance in the subarachnoid space 0.1 mg of endotoxin derived from Neisseria meningitidis was injected intracisternally into rabbits on 2 consecutive days. On day 1 the maximum peak level of TNF alpha was 7 ng/ml 2 h after injection, whereas on day 2 the highest levels were 3.6 ng/ml and 3.7 ng/ml, respectively, 1 and 2 h after injection. Pretreatment with intravenous endotoxin 5 or 21 h before consecutive intracisternal endotoxin did not affect the cerebrospinal fluid (CSF) levels of TNF alpha. In contrast, there was a marked endotoxin tolerance with respect to TNF alpha in the systemic circulation. Cells appearing in the CSF 5, 12 and 20 h after intracisternal injection of endotoxin were harvested, cultured, and then stimulated with 0.1 mg/ml of endotoxin. In 10 experiments a marked TNF alpha production in the range 10-70 ng/ml was detected in the supernatants, whereas unstimulated cells did not produce TNF alpha. We conclude that tolerance to endotoxin does not develop in the subarachnoid space as evaluated by the present experimental design. The pattern of TNF alpha production and endotoxin tolerance is distinctly different in the subarachnoid space and systemic circulation.     

Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 microg of LPS, 0.3 microg of LPS plus 100 micro g of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 microg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.     

Chylomicrons enhance endotoxin excretion in bile.

Chylomicrons prevent endotoxin toxicity and increase endotoxin uptake by hepatocytes. As a consequence, less endotoxin is available to activate macrophages, thereby reducing tumor necrosis factor secretion. To determine whether the chylomicron-mediated increase in hepatocellular uptake of endotoxin results in increased endotoxin excretion into bile, we examined bile after endotoxin administration. A sublethal dose (7 micrograms/kg) of 125I-endotoxin was incubated with either rat mesenteric lymph containing nascent chylomicrons (500 mg of chylomicron triglyceride per kg of body weight) or an equal volume of normal saline (controls) for 3 h and then infused into male Sprague-Dawley rats. Bile samples were collected via a common bile duct catheter for 24 h. Infusion of endotoxin incubated with chylomicrons increased biliary excretion of endotoxin by 67% at 3 h (P < or = 0.006) and by 20% at 24 h (P < or = 0.01) compared with infusion of endotoxin incubated in saline. Endotoxin activity, as measured by the Limulus assay, was not detected in the bile of test animals. However, endotoxin activity was detected after hot phenol-water extraction of bile, demonstrating that endotoxin is inactive in the presence of bile but retains bioactivity after hepatic processing. Since the majority of an intravenous endotoxin load has been shown to be cleared by the liver, acceleration of hepatocyte clearance and biliary excretion of endotoxin may represent a component of the mechanism by which chylomicrons protect against endotoxin-induced lethality.