Organ specific apoptosis following polymicrobial intraperitoneal infection.

BACKGROUND: Leukocyte apoptosis allows safe removal of potentially harmful cells and facilitates resolution of inflammation. We hypothesized that the number of apoptotic cells changes in a disproportionate fashion in parenchymal organs in response to intra-abdominal infection. MATERIALS AND METHODS: The percentage of apoptotic cells in the liver, spleen, lung, and peripheral blood was evaluated following cecal ligation and puncture (CLP) in mice. Tissue myeloperoxidase (MPO) levels were measured as an index of neutrophil extravasation. RESULTS: Liver & spleen MPO continually increased, while lung MPO remained low after CLP. In parallel to the increase in MPO, liver & spleen apoptosis continually increased throughout the 9-day follow-up period, whereas lung apoptosis remained unchanged. CONCLUSIONS: The distribution of apoptotic cells during intraperitoneal infection occurs in an organ specific manner, with significant increases in the spleen and liver. This distribution likely reflected the clearance of apoptotic cells as the inflammatory focus became contained.

CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent.

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.

STAT4 is required for antibacterial defense but enhances mortality during polymicrobial sepsis.

The signal transducer and activator of transcription factor 4 (STAT4) pathway mediates the intracellular effects of interleukin-12 (IL-12), leading to the production of gamma interferon, induction of a T helper type 1 response, and increased natural killer cell cytotoxicity. The purpose of this study was to determine the role of the STAT4 pathway during polymicrobial peritonitis in the cecal ligation and puncture (CLP) model. CLP was performed on STAT4-deficient (STAT4(-/-)) and wild-type control (BALB/c) mice. At 4 h after CLP, STAT4(-/-) mice had significantly higher bacterial counts in the peritoneal lavage fluid, liver, and blood. This difference persisted for 18 h in the peritoneal lavage fluid and blood. Neutrophil migration to the site of infection and into remote tissues was unaffected. Despite higher bacterial counts locally and systemically, STAT4(-/-) mice had a lower mortality rate than BALB/c controls. In contrast, blockade of IL-12 in BALB/c mice was detrimental to host survival. A blunted serum IL-12 response at 18 h after CLP was exhibited in STAT4(-/-) mice. These results suggest several critical roles for the STAT4 pathway in the resolution of polymicrobial infections. Additionally, the disparate effects observed with IL-12 blockade and STAT4 deficiency on host survival suggest that IL-12 may activate alternate pathways promoting survival.

Immediate and delayed leukocyte apoptosis in two models of peritonitis.

Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity: (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apoptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD1lb expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.

Heparin-binding protein decreases apoptosis in human and murine neutrophils.

BACKGROUND: Heparin-binding protein (HBP), a serine protease without any known proteolytic activity, is found in human polymorphonuclear leukocyte (PMN) granules, but not in mice. HBP potentiates the endotoxin-induced release of tumor necrosis factor (TNF) alpha, interleukin (IL)-1, and IL-6 from isolated monocytes. HBP has also been shown to increase the survival of cultured monocytes and protect them from oxidative stress. However, whether HBP affects PMNs themselves is not known. MATERIALS AND METHODS: Based on our work with cultured monocytes and the survival benefit noted in experimental peritonitis, we hypothesized that HBP would have a beneficial effect on the survival of neutrophils. We evaluated the effect of HBP on apoptosis in murine peritoneal exudative cells elicited by intraperitoneal thioglycollate administration and in normal human neutrophils from volunteers. Leukocytes were isolated from the peritoneal cavity and blood of mice that underwent intraperitoneal thioglycollate instillation. The mouse peritoneal exudate cells and normal human neutrophils isolated from peripheral blood were used to study the effect of HBP on survival and apoptosis. RESULTS: HBP decreased percentage apoptosis of mouse cells in both serum-enriched (from 24.8 to 4.5%) and serum-deprived (from 23.1 to 8.2%) cultures. In human PMNs, the protective effect of HBP was seen only in the serum-deprived group, with a decrease in percentage apoptosis from 69.1 to 43.3%. CONCLUSIONS: For the first time, we have shown that HBP, in addition to its known augmentation of the proinflammatory response of monocytes, also acts as a prosurvival protein for neutrophils themselves, and thereby enhances local host defense.

Neutrophil sequestration in liver and lung is differentially regulated by C-X-C chemokines during experimental peritonitis.

C-X-C chemokines play an important role in the migration and activation of neutrophils (PMNs) during an inflammatory event. We measured mRNA and protein expression of the murine C-X-C chemokines macrophage inflammatory protein-2 (MIP-2) and KC in the lungs, liver, blood, and peritoneal cavity of Swiss Webster mice after cecal ligation and puncture (CLP). Neutralizing antibodies to MIP-2 and KC were also used to determine the biological effects of these chemokines on neutrophil sequestration and organ injury in the CLP model. The data showed that early after CLP, MIP-2 mRNA and protein were expressed predominantly by the lung, whereas KC mRNA and protein were expressed by the liver. Inhibition of MIP-2 reduced both lung neutrophil sequestration and peritoneal neutrophil migration. Inhibition of KC had no effect on overall neutrophil sequestration in liver but reduced injury as measured by serum transaminases. An early survival benefit was found with anti-KC treatment, although overall survival was not different. Our study showed a differential expression by organs of C-X-C chemokines during sepsis and suggested that such chemokine effects are tissue-specific.

Regulation of early peritoneal neutrophil migration by macrophage inflammatory protein-2 and mast cells in experimental peritonitis.

Neutrophil (PMN) migration into the peritoneal cavity in response to fecal peritonitis is an important mechanism of host defense against bacterial invasion. We show that the murine C-X-C (PMN-specific) chemokine, macrophage inflammatory protein-2 (MIP-2), on intraperitoneal injection in mice, causes PMN migration into the peritoneum. MIP-2 mRNA and protein were expressed by peritoneal leukocytes after cecal ligation and puncture (CLP) in mice and neutralization of MIP-2 reduced peritoneal PMN migration. A prerequisite for neutrophil-endothelial adhesion and subsequent migration from the circulation is selectin-mediated rolling. Pretreatment of mice with an anti-P-selectin antibody before intraperitoneal injection of MIP-2 significantly reduced peritoneal PMN migration. However, there are no reports that a C-X-C chemokine can up-regulate endothelial selectins. We postulated that MIP-2, when injected intraperitoneally, interacts with a cell that is known to release factors that up-regulate endothelial selectins. A likely candidate is the mast cell, which contains histamine and tumor necrosis factor alpha (TNF-alpha), and both of these factors induce selectins. Intraperitoneally injected MIP-2 caused an early significant increase in peritoneal TNF-alpha, whereas histamine levels were unaffected. In a subsequent experiment, mast cell-deficient mice and their normal controls were then injected intraperitoneally with MIP-2 or underwent CLP. Significantly fewer PMNs migrated into the peritoneal cavity in the mast cell-deficient mice after MIP-2 injection or CLP. Thus, our findings indicate that mast cells and MIP-2 are necessary for PMN migration into the peritoneum in response to intra-abdominal infection, and that MIP-2 appears to facilitate this through an increase in TNF-alpha release.

Neutrophil migration into the peritoneum is P-selectin dependent, but sequestration in lungs is selectin independent during peritonitis.

Neutrophil (PMN) influx into the peritoneal cavity in response to bacterial peritonitis is an indispensable aspect of host defense. PMNs also are responsible for the remote organ injury observed after major abdominal infection. The aim of this study was to examine the effect of selectin blockade on PMN migration into the peritoneum and on PMN sequestration in the lungs, early in the course of peritonitis. Cecal ligation and puncture (CLP) was performed on P-selectin-deficient (P-def) mice and their genetic controls (C57). Both groups were treated with anti-E-selectin antibody, anti-L-selectin, or isotypic control immunoglobulin G at the time of CLP. 6 h after CLP, mice were sacrificed. Peritoneal PMN migration decreased in P-def mice compared with C57 controls after CLP. Blockade of E- or L-selectin alone in controls did not alter peritoneal PMN influx or circulating PMNs after CLP. In the P-def mice, treatment with anti-E-antibody or anti-L-antibody nearly eliminated PMN influx into the peritoneum. In contrast, circulating PMNs markedly increased after CLP in P-def mice when compared with baseline values. Lung myeloperoxidase increased in all groups of mice following CLP. Blockade of P-selectin with anti-P-selectin antibody elicited a response similar to that observed in the P-def mice. In conclusion, P-selectin mediates PMN influx into the peritoneal cavity, while E- and L-selectins do not appear to play any significant role in the 6 h time period following CLP. Lung PMN sequestration, after CLP, occurred independent of P-, E-, or L-selectin expression. Blockade of P-selectin during peritonitis appears to be potentially deleterious by preventing early PMN influx into the compartment containing the septic focus.

Bacterial products primarily mediate fibroblast inhibition in biomaterial infection.

PURPOSE: The stimulation of fibroblast growth is essential for the normal healing and tissue integration of biomaterials. The local elevation of proinflammatory mediators in infected perigraft fluid (PGF) may inhibit this growth. We sought to determine whether infected PGF inhibited fibroblast growth, and, if so, whether this was primarily dependent on the biomaterial, bacteria, or host. METHODS: In vivo Dacron or expandable polytetra-fluoroethylene (ePTFE) grafts, sterile or colonized with slime-producing (RP-62A, viable or formalin-killed) or nonslime-producing (RP-62NA) Staphylococcus epidermidis (1 x 10(7) CFU/cm2), were implanted in Swiss Webster mice, and the PGF was harvested at 7 and 28 days. Antibodies to tumor necrosis factor alpha, interleukin 1 alpha, interferon gamma (7 micrograms/day), and indomethacin (50 micrograms/day) were administered by microinfusion pumps for 7 days and the PGF was harvested. Inhibition of the proinflammatory mediators was confirmed by enzyme-linked immunosorbant assay. The nontreated, heat-treated, or trypsin-digested in vivo PGF was incubated with an in vitro [3H]thymidine murine fibroblast (ATCC CCL-12) proliferation assay. RESULTS: Fibroblast inhibition was significant at 7 and 28 days with infected PGF incubation compared with sterile and was not dependent on bacterial slime production or viability. Dacron sterile PGF did not significantly inhibit fibroblasts compared with control, whereas sterile ePTFE stimulated (P < 0.05) fibroblasts. Treatment of the PGF with proinflammatory cytokines, heat, and trypsin failed to reverse fibroblast inhibition in the infected state. CONCLUSION: Biomaterial infection is associated with fibroblast inhibition that is dependent primarily on bacterial products and not the host or biomaterial. Conservative intervention strategies for graft infection need to address the problem of poor healing as well as bacterial clearance.

Inhibition of neutrophil migration at the site of infection increases remote organ neutrophil sequestration and injury.

Up-regulation of the leukocyte beta 2 integrin, CD18, is a key event in neutrophil-endothelial adhesion and neutrophil-mediated organ injury. Inhibition of CD18 with monoclonal antibodies reduces lung and liver neutrophil sequestration in animal models of Gram-negative bacteremia or endotoxemia. However, with a persistent septic challenge, interference with host leukocyte phagocytic defense could adversely affect outcome. To assess the effects of inhibiting CD18 on organ neutrophil responses, bacteremia, and organ injury after fecal peritonitis, mice underwent cecal ligation and puncture (CLP). At the time of CLP and 12 h later, mice received intravenous anti-CD18 antibody or control IgG. At 3, 6, and 18 h after CLP, lung and liver tissue neutrophil content were measured by myeloperoxidase (MPO) assay, peritoneal cells and blood leukocytes were differentially counted, blood was cultured, and serum aspartate aminotransferase was measured. There was a significant reduction in peritoneal neutrophil migration and an increase in blood neutrophils after anti-CD18 treatment compared with results from treatment with the control antibody. In the anti-CD18-treated group, liver MPO was increased fivefold at 6 and 18 h, while lung MPO was increased two-fold at 18 h when compared with the control antibody-treated group. The anti-CD18-treated group also had an increase in bacteria cultured from the blood at 6 and 18 h and an increase in serum aminotransferase at 18 h. Our data demonstrate that peritoneal neutrophil migration in response to an endogenous fecal challenge is CD18-dependent, and that this mechanism forms a vital part of host defense. Inhibition of CD18 increased neutrophil sequestration in the liver and lung and increased liver injury. This study demonstrates a paradoxical increase in organ neutrophil sequestration using a leukocyte anti-adhesion therapy during sepsis and suggests that anti-adhesion therapies targeted towards neutrophil may worsen outcome if given during an ongoing, localized infection.

Staphylococcus epidermidis graft infection is associated with locally suppressed major histocompatibility complex class II and elevated MAC-1 expression.

OBJECTIVES: To determine the local cellular immune response in a series of human patients with Staphylococcus epidermidis prosthetic graft infection and to use a murine model to investigate the response in polytef (PTFE) and in a nonslime-producing S epidermidis variant. METHODS: Externally supported Dacron and PTFE grafts, either sterile or colonized with slime (RP-62A)- or nonslime (RP-62NA)-producing S epidermidis (10(7) colony forming units/cm2) were implanted in a dorsal subcutaneous pocket of Swiss Webster mice (Taconic, Germantown, NY). The grafts were harvested at 7, 10, 14, and 28 days with local bacterial and leukocyte counts obtained. Perigraft and blood monocyte major histocompatibility complex class II (MHC-II) (immune antigen) and membrane attack complex type 1 (MAC-1) (glycoprotein) expression were analyzed by flow cytometry in the murine model and in 3 patients representing 4 Dacron graft infections. RESULTS: The human infected Dacron perigraft monocytes revealed a suppressed MHC-II and elevated MAC-1 expression, and early correlation with the murine model was seen. No notable perigraft monocyte MHC-II suppression occurred in the infected PTFE graft. The reciprocal relationship in Dacron between monocyte MAC-1 and MHC-II expression was exaggerated with the lack of slime production. Bacterial clearance was variable. Supranormal expression was observed at 1 month in sterile Dacron but not in PTFE grafts. CONCLUSIONS: Staphylococcus epidermidis infection is associated with local cellular immune suppression in Dacron but not PTFE grafts. Slime-producing S epidermidis induced a lesser cytotoxic-phagocytic response than the nonslime variant. The reduced immunologic response to slime-producing S epidermidis may explain, in part, its indolent nature and resistance to eradication.

The pulmonary inflammatory response to experimental fecal peritonitis: relative roles of tumor necrosis factor-alpha and endotoxin.

The roles of endotoxin (LPS) and tumor necrosis factor-alpha (TNF-alpha) in the causation of organ injury during sepsis are unclear. To study LPS and TNF-alpha in the genesis of lung inflammation after cecal ligation and puncture (CLP), we used endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive mice (C3H/HeOuJ). We examined lung neutrophil sequestration, interleukin 1 (IL-1)beta mRNA expression, IL-1 beta protein expression, and injury. We also determined the expression of two C-X-C chemokine mRNAs, macrophage inflammatory protein-2 (MIP-2) and KC, in the lung to determine whether in vivo, endotoxin, or TNF-alpha are significant modulators of MIP-2 and KC mRNA expression. After CLP, increased neutrophils sequestrated in the lungs of both strains of mice and coincided with an increase in expression of IL-1 beta, MIP-2 and KC mRNAs, and IL-1 beta protein. Lung and serum TNF-alpha were significantly increased in the C3H/HeOuJ strain but not in the C3H/HeJ strain. Histologic studies of the lung revealed similar injury in both strains. Our results suggest that bacterial factors other than endotoxin cause lung neutrophil sequestration and injury after CLP and, further, that TNF-alpha production is not a prerequisite. Our findings also suggest a potential role for local pulmonary chemokine production in the control of neutrophil sequestration after CLP.

In vitro and in vivo effects of rifampin on Staphylococcus epidermidis graft infections.

Rifampin, bound in high concentrations to prosthetic grafts, has been proposed for the treatment of vascular graft infections. The optimum antibiotic concentration and duration of treatment for infected grafts is not known. This study compared the in vitro and in vivo efficacy of varying concentrations of rifampin against three different strains of slime producing Staphylococcus epidermidis (RP62A, KC2, and KB1) bound to the knitted Dacron at high and low concentrations at 10(4) and 10(7) CFU/cm2 of prosthetic. Time kill experiments were performed at 4, 18, and 42 hr, in which each Dacron bound bacterial strain was exposed in vitro to 4X, 64X, 100X, and 1,000X minimum inhibitory concentration (MIC) of rifampin. In vitro, the Dacron bound laboratory strain RP62A was implanted subcutaneously in the backs of male Swiss-Webster mice and exposed to 4X, 100X, and 1,000X the MIC of rifampin for similar time periods. In addition, systemic vancomycin (10 mg/kg) was assessed for synergy and prevention of rifampin resistance. In vitro, all concentrations of rifampin showed near total killing (< 1 log) of all bacterial strains at low initial concentrations (10(4) CFU/cm2) but not high (10(7) CFU/cm2) to 42 hr. Importantly, resistance was shown to develop in all three strains of S. epidermidis with high initial bacterial biofilm concentrations. In vivo, rifampin concentrations between 4X MIC and 100X MIC achieved a balance between optimal killing and prevention of resistance. Systemic vancomycin slightly improved bacterial clearance but did not alter the development of rifampin resistance at high local concentrations. Caution is advised with the use of antibiotic bonded grafts because resistance may develop even with the addition of systemic antibiotics.

Antibiotic efficacy against Staphylococcus epidermidis adherent to vascular grafts.

Antibiotic bonded grafts may improve the treatment results of vascular graft infections. The purpose of this study was to determine the antibiotic and antibiotic concentration needed to be effective against Staphylococcus epidermidis-infected vascular grafts. The efficacy of four antibiotics (minocycline, cefazolin, vancomycin, and rifampin) against S. epidermidis adherent to Dacron or Teflon vascular grafts was studied in vitro. Kill kinetic studies were performed with 18 and 42 hr of exposure of Dacron-adherent and Teflon-adherent S. epidermidis at 1, 4, 16, and 64 times the minimum inhibitory concentration (MIC) of each antibiotic. Antibiotic efficacy against graft-adherent S. epidermidis at 42 hr was best at concentrations 64x MIC for minocycline, cefazolin, and vancomycin and 4x MIC for rifampin. None of the antibiotics totally eradicated the graft-adherent bacteria. Antibiotics were equally effective for S. epidermidis adherent to Dacron and Teflon grafts. Antibiotic concentrations several times that predicted by the MIC were needed for all antibiotics to achieve significant killing of graft-adherent bacteria, with rifampin the most effective at the lowest concentration.

Immunosuppression augments growth of graft-adherent Staphylococcus epidermidis.

OBJECTIVE: To determine if systemic suppression of host defenses during graft implantation alters the initial adherence and subsequent growth of Staphylococcus epidermidis on vascular prostheses. DESIGN: Dacron grafts 1 cm2 were implanted in the back subcutaneous tissue of Swiss-Webster mice (n = 247), followed by topical inoculation with 2 x 10(7), 2 x 10(5), 2 x 10(3), or 2 x 10(1) colony-forming units of S epidermidis. Half of the mice were immunosuppressed with cyclophosphamide (150 mg/kg intraperitoneally), to achieve a consistent, significant decrease in the white blood cell count and major histocompatibility complex class II (Ia) expression. Control mice received an equal volume of saline solution. Graft bacterial biofilm concentrations were determined at 1 day for adherence and within 2 weeks for bacterial growth, by using sonication and quantitative agar culture. RESULTS: Immunosuppression did not significantly alter the initial adherence of bacteria to vascular grafts. Immunosuppressed animals that were inoculated with 2 x 10(7) and 2 x 10(5) colony-forming units of S epidermidis had significantly higher bacterial biofilm concentrations as compared with those in control animals. Graft infection persisted at 14 days in all animals, with and without immunosuppression. CONCLUSIONS: Suppression of immune function during graft implantation augmented growth of adherent bacteria. The effect of short-term perioperative immunosuppression on late-appearing S epidermidis graft infection needs further study.

Passive immunization against tumor necrosis factor and interleukin-1 fails to reduce lung neutrophil sequestration in chronic sepsis.

The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are produced within the lung during sepsis, and may induce neutrophil sequestration resulting in neutrophil-mediated lung injury. We hypothesized that, if there is a cause and effect between TNF alpha or IL-1 production and lung neutrophil sequestration during chronic sepsis, TNF alpha mRNA and IL-1 mRNA levels in the lung after cecal ligation and puncture should correlate with the number of sequestered neutrophils as measured by the myeloperoxidase (MPO) content of the lung. To test this hypothesis, Swiss Webster mice were subjected to varying degrees of infectious challenge by single and double-puncture cecal ligation and puncture, or simultaneous antibiotic treatment, and their lungs and blood were harvested at 24 h. Lung TNF alpha and IL-1 beta mRNAs were measured by the reverse-transcription differential polymerase chain reaction, and MPO was measured by colorimetric assay. TNF alpha serum levels showed no correlation with the MPO content of the lung, whereas IL-1 levels were undetectable. Lung TNF alpha mRNA correlated weakly, and IL-1 beta mRNA exhibited a strong correlation with lung MPO (r = .9, p < .01), but administration of anti-TNF alpha- or anti-IL-1-neutralizing antibodies did not prevent a rise in lung MPO. IL-1 beta mRNA in bronchoalveolar macrophages correlated well with whole lung tissue IL-1 beta mRNA levels (r = .91, p < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin and tumour necrosis factor do not cause mortality from caecal ligation and puncture.

Macrophage tumour necrosis factor-alpha (TNF-alpha) production is thought to represent an important pathogenic mechanism by which Gram-negative sepsis is mediated. We compared the effects of caecal ligation and puncture (CLP) on endotoxin-sensitive (C3H/HeSnJ) and endotoxin-resistant (C3H/HeJ) mice. Mortality after CLP for C3H/HeSnJ mice compared with C3H/HeJ mice was not significantly different (32% and 55%, respectively). When survivors were injected with lipopolysaccharide intraperitoneally on the 7th day after CLP, the mortality rate was 82% for C3H/HeSnJ mice versus 0% for C3H/HeJ mice (P < 0.0001). Serum endotoxin levels at 24 h after CLP were only slightly elevated. Serum TNF levels and peritoneal macrophage TNF production were undetectable in C3H/HeJ mice and were only slightly elevated in C3H/HeSnJ mice by 24 h after CLP. Peritoneal macrophage mRNA levels for TNF-alpha, IL-1 beta, and I-A alpha displayed a similar pattern in the two strains of mice, with a 2- to 3-fold increase in TNF-alpha and IL-1 beta mRNA levels by 24 h and a sharp decrease in I-A alpha mRNA by 24 h. The cause of mortality in mice that undergo CLP cannot be attributed to overwhelming endotoxemia and/or TNF production.

Tissue tumor necrosis factor mRNA expression following cecal ligation and puncture or intraperitoneal injection of endotoxin.

Tumor necrosis factor (TNF) has been implicated as a key mediator of the septic response. Although very high serum levels of TNF are detected in animal models of endotoxemia or gram-negative bacteremia, human patients with sepsis rarely have greatly elevated TNF serum levels. It has therefore been postulated that TNF may act in a paracrine fashion to cause local injury. In order to examine the role of locally produced TNF in sepsis, we compared serum TNF levels and TNF messenger RNA (mRNA) expression in various tissues following cecal ligation and puncture (CLP) or intraperitoneal injection of a sublethal dose of endotoxin. TNF mRNA expression was determined by the reverse transcription differential polymerase chain reaction using beta-actin as an internal standard. Serum levels of TNF were threefold higher after endotoxin administration compared to CLP. TNF mRNA in peritoneal macrophages rose fourfold after both endotoxin injection and CLP, with rapid return to normal in endotoxin-treated animals. There was a significant increase in TNF mRNA in the liver and lung, but not the spleen, during the first 24 hr after CLP. An increase in TNF mRNA was seen in all three tissues after injection of endotoxin. These results support the concept of locally produced TNF as a contributing factor in tissue damage and multiple organ failure during sepsis.

The natural history of bacterial biofilm graft infection.

A mouse model was developed to study the natural history of vascular prosthetic graft infection due to Staphylococcus epidermidis. Graft infections were established in the back subcutaneous tissue of 46 mice by implantation of Dacron prostheses colonized in vitro with slime-producing S. epidermidis to form an adherent bacterial biofilm [1.7 x 10(7) colony forming units (CFU)/cm2 graft]. Control animals (n = 16) had implantation of sterile Dacron prostheses. None of the control animals developed a graft infection or graft-cutaneous sinus tract. All study animals developed a biofilm graft infection with typical anatomic (perigraft abscess), microbiologic (low bacterial concentration in surface biofilm), and immunologic (normal white blood count) characteristics. A graft-cutaneous sinus tract developed in a significantly higher number of mice with infected grafts by 8-10 weeks (9 of 21) compared to infected grafts explanted at 2 and 4-6 weeks (1 of 25, P < 0.01) and controls (0 of 16, P < 0.03). By 8-10 weeks, 2 animals had no signs of graft infection and the S. epidermidis study strain was not recoverable from 7 grafts. The natural history of bacterial biofilm vascular prostheses infection in the mouse model was similar to that in man, provoking a chronic inflammatory process curiously presenting as a perigraft abscess or graft-cutaneous sinus tract.