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 ϳ1,000 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.Administration of specific pathogens has proven to be a powerful tool for investigating the host's inflammatory response. Infusion of endotoxin, a cell wall component of gramnegative bacteria, is a well-established model for studying the acute inflammatory response (11,17,35). The murine endotoxin model has provided basic information resulting in dramatic improvements in understanding the inflammatory response. As a specific example, injecting endotoxin into mice led to the discovery of tumor necrosis factor (TNF) (6). Further investigation of this critical mediator of inflammation demonstrated that TNF also played a role in chronic inflammatory conditions, such as arthritis and inflammatory bowel disease. At this time, TNF inhibitors are therapies for the treatment of patients with rheumatoid arthritis (32) and Crohn's disease (23) approved by the Food and Drug Administration.Complementary studies of endotoxin administration in humans are often limited due to the risk of toxicity. On the basis of the pyrogenic response to endotoxin, humans are considered the most sensitive of all models to the effects of endotoxin (38). Despite the differences in sensitivity, the human model is useful in measuring the responses that are common to the acute inflammatory response and those responses that are specific to the endotoxin (17). Linking the murine and human models into one definitive comparison proves to be more difficult, if not impossible. A recent review article has highlighted the differences between murine and human imm...

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.

The use of proteomics for efficient, accurate, and complete analysis of clinical samples poses a variety of technical challenges. The presence of higher abundance proteins in the plasma, such as albumin, may mask the detection of lower abundance proteins such as the cytokines. Methods have been proposed to deplete the sample of these higher abundance proteins to facilitate detection of those with lower abundance. In this study, a commercially available albumin depletion kit was used to determine if removal of albumin would measurably reduce detection of lower abundance cytokine proteins in human plasma. The Montage Albumin Deplete Kit (Millipore) was used to deplete albumin from LPS-stimulated whole blood from 15 normal human donors. Albumin depletion was measured using the BCG reagent and SDS-PAGE, and cytokine recovery was determined by a microassay immunoassay that measures both pro- and anti-inflammatory cytokines. Average albumin depletion from the samples was 72%. However, several cytokines were also significantly reduced when the albumin was removed from the plasma. Additionally, there was a variable reduction in cytokine recovery from a known mixture of cytokines in a minimal amount of plasma that were loaded onto the columns. These data demonstrate that there may be a non-specific loss of cytokines following albumin depletion, which may confound subsequent proteomic analysis.

Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs.

In a kinetic study, TNF-alpha levels within the bronchoalveolar lavage (BAL) fluid increased quickly peaking at 2 h while BAL levels of IL-4, IL-5, and IL-13 peaked at later time-points. Mch hyper-responsiveness was measured 24 h after the last challenge, and mice were killed 24 h later. TNF inhibition resulted in an augmentation of these Th2 cytokines. However, the allergic pulmonary inflammation was significantly reduced by anti-TNF-alpha antibody treatment as demonstrated by a substantial reduction in the number of BAL eosinophils, lymphocytes, macrophages, and neutrophils compared with rat IgG-treated mice. Mch hyper-responsiveness was also significantly reduced in anti-TNF-alpha antibody-treated mice and the pulmonary histology was also significantly improved. Inhibition of TNF significantly reduced eotaxin levels within the lung, suggesting a potential mechanism for the beneficial effects. These data indicate that anti-TNF-alpha antibody can reduce the inflammation and pathophysiology of asthma in a murine model of asthma induced by a house dust extract.