Gerald Bolgos

Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture.

Sepsis remains a serious clinical problem despite intense efforts to improve survival. Experimental animal models of sepsis have responded dramatically to immunotherapy blocking the activity of cytokines. Despite these preclinical successes, human clinical trials have not demonstrated any improvement in survival. We directly compared the mortality, morbidity, and immunopathology in two models of sepsis, one due to lipopolysaccharide (LPS) and the other to cecal ligation and puncture (CLP). BALB/c mice were injected intraperitoneally with 250 microg of LPS or subjected to CLP with an 18-gauge needle. Both models yielded similar mortality (> 85%) and morbidity. Additionally, neutropenia and lymphopenia developed in both groups. Plasma and peritoneal levels of cytokines (TNF, IL-1, IL-6, and the chemokines KC and MIP-2) were measured at 1.5, 4, and 8 h after challenge. LPS induced substantially higher levels of cytokines in both compartments with peak levels between 1.5 and 4 h that began to decline at 8 h. In contrast, cytokine levels in the CLP model were continuing to increase at the 8 h-time point and often exceeded the LPS-induced values at this time. Our data demonstrate that the LPS and CLP models have similar mortality but significant differences in the kinetics and magnitude of cytokine production. Immunotherapy for sepsis based on cytokine production after LPS challenge is misdirected because the LPS model does not accurately reproduce the cytokine profile of sepsis.

Blockade of tumor necrosis factor reduces lipopolysaccharide lethality, but not the lethality of cecal ligation and puncture.

ABSTRACT Inhibition of tumor necrosis factor (TNF) bioactivity has afforded protection in several animal models of sepsis. We examined whether inhibition of TNF could improve survival after lethal lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) in CD-1 or BALB\c mice. Neutralizing rabbit anti-TNF antisera were evaluated in CD-1 mice by injecting the antisera 3 h before intravenous (i.v.) LPS (600 μ g). Implantable radiotransmitters were used for continuous monitoring of temperature. No decrease in mortality was observed, and the anti-TNF failed to prevent the drop in temperature. In BALB\c mice injected with antisera before LPS (200 μ sg) mortality was reduced (dead/total: control sera, 14/14; anti-TNF, 4/12; p = .007 control sera vs. anti-TNF). CD-1 mice were pretreated with anti-TNF or control sera; CLP was performed followed by administration of antibiotics. Anti-TNF did not decrease pulmonary neutrophil sequestration, improve survival, or prevent the decrease in temperature observed as sepsis developed. CLP was performed in the BALB\c mice using antibiotics plus anti-TNF antisera, but no protection was observed. Our results demonstrate that anti-TNF treatment prevents LPS mortality only when using certain strains of mice and inhibition of TNF fails to reduce mortality in a more clinically relevant model of sepsis.

Role of Interleukin-6 in Mortality from and Physiologic Response to Sepsis

ABSTRACT Previous studies have suggested that interleukin-6 (IL-6) serves as both a marker and a mediator for the severity of sepsis. We tested whether interleukin 6 knockout (IL-6KO) mice were more susceptible to sepsis mortality induced by cecal ligation and puncture. IL-6KO and wild-type (WT) mice were subjected to increasing degrees of sepsis severity. Physiologic support was given with fluids and appropriate antibiotics. Plasma IL-6 levels were determined 6 h after the onset of sepsis, and a complete hematologic profile was performed on day 2. As expected, increasing sepsis severity resulted in greater and more rapid mortality. However, the mortality was nearly identical in the IL-6KO and WT mice. All WT septic mice had high plasma levels of IL-6 6 h after the onset of sepsis, while IL-6KO were near or below the lower limit of detection. Among the WT mice, mortality was significantly higher in mice with plasma IL-6 >3,000 pg/ml. Both IL-6KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 h. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 h of severe sepsis routinely observed in the WT mice. These data demonstrate that IL-6 serves as a marker of disease severity in sepsis and does modulate some physiologic responses, but complete lack of IL-6 does not does not alter mortality due to sepsis.

Humane endpoints in shock research.

In biomedical research using animal models, the phrase “humane endpoints” refers to predetermined criteria used to judge when the research animals should be humanely euthanized. The intended goal of humane endpoints is to minimize the distress or suffering of research animals; however, if applied incorrectly, this well-intended concept could lead to premature decisions and inaccurate data, resulting in a waste of animal life. A concensus on specific endpoints for shock and inflammation research is not available but several biochemical, physical and behavioral parameters have been suggested for other research models. In addition, the authors have found, in the studies presented here, that increasing body weight, decreased body temperature, and inability to ambulate are important parameters in a model of cecal ligation and puncture. However, it is clear that the applicability of these endpoints may change with the model of disease, intensity of insults, experimental treatments and other factors. Consequently, humane endpoints should be assigned cautiously and preferably after preliminary studies to prevent aberrant research results. In order to accomplish this, investigators must become aware of certain concepts including: when to implement endpoints, what endpoints to consider, and how to establish the endpoints for their studies. Equipped with the basic principles of humane endpoints, investigators can make informed decisions that meet current standards of animal care while still achieving the scientific goals of their research studies.

Antibiotic treatment influences outcome in murine sepsis : Mediators of increased morbidity

Different antibiotic treatments may affect the survival and physiological responses of Balb/c mice following cecal ligation and puncture (CLP). The broad spectrum imipenem (IMP) was compared with a triple antibiotic mixture of gentamicin, clindamycin, and ciprofloxacin (3AB). Control mice received injections of 5% dextrose (D5W). After CLP with a 25 gauge needle, Mini-Mitters were implanted to measure temperature and activity. Therapy began with 1 mL injections of antibiotics or D5W 2 h post-CLP and continued every 12 h for 3 days. Survival was higher in IMP mice than in 3AB or D5W mice. Starting with the injection 12 h after CLP, 3AB always induced a profound hypothermic response not observed with D5W or IMP. Nocturnal activity levels were higher in IMP mice compared with 3AB or D5W mice during the first night following CLP. To determine the cause of this hypothermic response and to further investigate the acute effects that antibiotic choice may have on murine physiology, the kinetic appearance of IL-1, IL-6, TNF, and KG as well as lipopolysaccharide (LPS), were measured in the plasma and peritoneum of mice sacrificed at 0, .5, and 1.5 h after antibiotic injection at 24 h post-CLP. Cytokine and LPS concentrations in 3AB mice were not significantly different at any of the three time points when compared with IMP or D5W mice. Our data demonstrate that antibiotic therapy consisting of 3AB produces greater morbidity and mortality compared with therapy consisting of IMP. However, the mechanism of these alterations is not due to elevated systemic levels of cytokines or LPS.

Ratio of Local to Systemic Chemokine Concentrations Regulates Neutrophil Recruitment

CXC chemokines are important regulators of local neutrophil recruitment. In this study, we examined the role of the ratio of local to systemic chemokine concentrations as a significant factor determining local neutrophil recruitment. Thioglycollate was injected intraperitoneally into BALB/c mice resulting in a dose-dependent increase in neutrophil recruitment and local inflammation, as measured by peritoneal levels of interleukin 6. At the high dose of 3% thioglycollate, antibody inhibition of the murine chemokines KC and macrophage inflammatory protein-2 caused a reduction in peritoneal neutrophil recruitment by as much as 93%. A paradoxical effect was observed with a 0.3% thioglycollate intraperitoneal challenge. In this situation, inhibition of KC resulted in a significant increase in peritoneal neutrophils, and inhibition of macrophage inflammatory protein-2 also resulted in increased peritoneal neutrophils. These results were consistent with a reverse chemotactic gradient as described by the ratio of peritoneal to plasma KC levels. A higher ratio (ie, increased peritoneal chemokines compared to plasma) resulted in increased neutrophil recruitment after either the 3% or 0.3% thioglycollate challenge. Our results demonstrate that whereas sufficient local concentrations of chemokines are necessary, a critical factor dictating local neutrophil recruitment is the ratio of the local to the systemic chemokine concentrations.

CXC Chemokine Redundancy Ensures Local Neutrophil Recruitment during Acute Inflammation

Previous publications demonstrated that elevated systemic levels of interleukin (IL)-8 decrease local neutrophil recruitment. We tested whether sustained, high plasma levels of IL-8 would prevent local inflammation after inflammatory insults. Mice carrying the transgene for human IL-8 were separated on the basis of their plasma levels of IL-8 into IL-8-positive (plasma levels >90 ng/ml) and IL-8-negative (IL-8 below detection). Presence of the IL-8 transgene did not improve survival or morbidity nor did it alter peritoneal neutrophil recruitment induced by the cecal ligation and puncture model of sepsis. In an acute lung injury model created by intratracheal injection of acid, IL-8-positive mice showed no reduction in alveolar neutrophil recruitment. There was no difference in the local recruitment of neutrophils when either thioglycollate or glycogen was injected intraperitoneally. We examined the chemotactic response to murine chemokines to test how neutrophil recruitment occurs in the setting of elevated plasma IL-8 and found that neutrophils from both IL-8-positive and -negative mice respond equally well to recombinant KC or macrophage inflammatory protein (MIP)-2. We measured KC and MIP-2 in the peritoneum after thioglycollate injection and demonstrated that IL-8-positive mice have significantly higher levels of the chemokines compared to the IL-8-negative mice. Antibody inhibition of KC and MIP-2 in the IL-8-positive mice significantly decreased peritoneal neutrophil recruitment in response to thioglycollate, clarifying their important role in the local neutrophil recruitment. Our data demonstrate that despite the presence of high plasma levels of IL-8, neutrophils may still be recruited to sites of local inflammation because of chemokine redundancy.

Keratinocyte growth factor pretreatment is associated with decreased macrophage inflammatory protein-2α concentrations and reduced neutrophil recruitment in acid aspiration lung injury

A two-hit model of acid aspiration was used to examine the effect of keratinocyte growth factor (KGF) on chemokine levels and neutrophil recruitment into the lung. Mice were subjected to cecal ligation and puncture and then either KGF or saline, intratracheally (IT). Forty-eight hours later, the mice were given IT acid. After 8 h, neutrophil counts in bronchoalveolar lavage (BAL) fluid were significantly decreased in animals pretreated with KGF (23 ± 4 × 103/mouse) compared with saline (74 ± 2 × 103/mouse). In addition, the BAL fluid IL-6 levels were decreased in the KGF-treated group (88 ± 44 pg/mL) compared with the saline group (166 ± 34 pg/mL). To examine the mechanism behind the KGF-induced reduction in neutrophil influx, the murine chemokines KC and macrophage inflammatory protein (MIP)-2&agr; were measured. KC levels in plasma and BAL fluid were not significantly different between the treatment groups. Likewise, levels of MIP-2&agr; in plasma were not affected by KGF treatment. However, 8 h after acid aspiration, MIP-2&agr; concentrations were significantly lower in the KGF-treated group. The ratio of MIP-2&agr; in BAL fluid versus plasma was lower in the KGF group (0.72 ± 0.28) than in the saline group at 3 h (2.23 ± 0.93) and also significantly lower in the KGF group (3.02 ± 0.78) compared with the saline group (6.23 ± 1.19) at 8 h. In this study, KGF pretreatment after acid aspiration was associated with reduced neutrophil recruitment into the lung and a decrease in MIP-2&agr; gradients between BAL fluid and plasma.

Critical role of CD14 for production of proinflammatory cytokines and cytokine inhibitors during sepsis with failure to alter morbidity or mortality.

ABSTRACT We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1β [IL-1β], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2α and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.

Allergens induce enhanced bronchoconstriction and leukotriene production in C5 deficient mice

BackgroundPrevious genetic analysis has shown that a deletion in the complement component 5 gene-coding region renders mice more susceptible to allergen-induced airway hyperresponsiveness (AHR) due to reduced IL-12 production. We investigated the role of complement in a murine model of asthma-like pulmonary inflammation.MethodsIn order to evaluate the role of complement B10 mice either sufficient or deficient in C5 were studied. Both groups of mice immunized and challenged with a house dust extract (HDE) containing high levels of cockroach allergens. Airways hyper-reactivity was determined with whole-body plesthysmography. Bronchoalveolar lavage (BAL) was performed to determine pulmonary cellular recruitment and measure inflammatory mediators. Lung homogenates were assayed for mediators and plasma levels of IgE determined. Pulmonary histology was also evaluated.ResultsC5-deficient mice showed enhanced AHR to methylcholine challenge, 474% and 91% increase above baseline Penh in C5-deficient and C5-sufficient mice respectively, p < 0.001. IL-12 levels in the lung homogenate (LH) were only slightly reduced and BAL IL-12 was comparable in C5-sufficient and C5-deficient mice. However, C5-deficient mice had significantly higher cysteinyl-leukotriene levels in the BAL fluid, 1913 +/- 246 pg/ml in C5d and 756 +/- 232 pg/ml in C5-sufficient, p = 0.003.ConclusionThese data demonstrate that C5-deficient mice show enhanced AHR due to increased production of cysteinyl-leukotrienes.