Anticytokine therapies for acute inflammation and the systemic inflammatory response syndrome: IL-10 and ischemia/reperfusion injury as a new paradigm.

The results of recent anticytokine trials for sepsis syndrome have been disappointing. Several Phase II and Phase III clinical trials have shown a modest benefit in various subsets of patients; however, there has been no reported benefit in the primary endpoint of 28-day all-cause mortality. The failure of these trials is clearly multifactorial, and causes include the overall complexity of the inflammatory response, heterogeneity of the patient populations, absence of a hypercytokine response at the time of drug treatment, and the relatively short half-life of the administered drugs. The failure of anticytokine therapies may represent inadequate application of the treatment modality rather than any inherent weakness of the treatment itself. We have recently initiated a Phase I clinical trial examining the role of the anti-inflammatory cytokine IL-10 during surgical repair of a thoracoabdominal aortic aneurysm. This study may overcome some of the-design limitations of previous anticytokine trials in sepsis, and serve as a paradigm for future anticytokine therapy trials. Although the incidence of thoracoabdominal aortic aneurysms is relatively low, the patient population is homogeneous and the surgical injury associated with its repair reproducible. Additionally, postoperative mortality and morbidity rates are significant. Most importantly, the operative repair is associated with an obligatory visceral ischemia and reperfusion injury that appears to be associated with a proinflammatory cytokine response and postoperative organ dysfunction. IL-10 is a pleuripotent anti-inflammatory cytokine that both inhibits TNFalpha and IL-1 synthesis, and antagonizes their actions through upregulation of cytokine antagonists. Furthermore, IL-10 administration has been associated with only minimal adverse side effects during Phase I and Phase II trials.

Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.

Attenuation of skeletal muscle ischemia/reperfusion injury by inhibition of tumor necrosis factor.

PURPOSE: Tumor necrosis factor alpha (TNF-alpha) has been shown to play a role in pulmonary injury after lower-extremity ischemia/reperfusion (I/R). However, its role in direct skeletal muscle injury is poorly understood. The hypothesis that endogenous TNF production contributes to skeletal muscle injury after hindlimb I/R in rats was tested. METHODS: Juvenile male Sprague-Dawley rats underwent 4 hours of bilateral hindlimb ischemia and 4 hours of reperfusion (IR) or sham operation (SHAM). A subset was treated with a soluble TNF receptor I construct (STNFRI, 10 mg/kg) 1 hour before ischemia (PRE) or at reperfusion (POST). Direct skeletal muscle injury (SMII) and muscle endothelial capillary permeability (MPI) were quantified by means of Tc99 pyrophosphate and I125 albumin uptake. Pulmonary neutrophil infiltration and hepatocellular injury were assessed by means of myeloperoxidase content (MPO) and aspartate aminotransferase (AST) concentrations, respectively. Serum TNF bioactivity was measured with the WEHI bioassay. RESULTS: Hindlimb I/R (IR vs SHAM) resulted in a significant (P <.05) increase in the SMII (0.52 +/- 0.06 vs 0.07 +/- 0.01) and MPI (0.35 +/-.04 vs 0.06 +/- 0.01). Pretreatment with STNFRI (PRE vs IR) significantly ameliorated both SMII (0.30 +/- 0.05 vs 0.52 +/- 0.06) and MPI (0.23 +/- 0.02 vs 0.35 +/- 0.04), whereas treatment at reperfusion (POST vs IR) had no effect. Hindlimb I/R (IR vs SHAM) resulted in both significant pulmonary neutrophil infiltration (MPO 16.4 +/- 1.06 U/g vs 11.3 +/- 1.4 U/g) and hepatocellular injury (AST 286 +/- 45 U/mL vs 108 +/- 30 U/mL), but neither was inhibited by pretreatment with STNFRI before ischemia. Detectable levels of TNF were measured during ischemia in a significantly higher percentage of the IR group compared with SHAM (9 of 12 vs 3 of 12), and the maximal TNF values were also significantly greater (51.1 +/- 12.6 pg/mL vs 5.5 +/- 2.9 pg/mL). No TNF was detected in any treatment group during reperfusion nor after administration of the STNFRI. CONCLUSION: Acute hindlimb IR initiates a systemic TNF response during the ischemic period that is partly responsible for the associated skeletal muscle injury.

LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor.

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P

Caspase-3-dependent organ apoptosis early after burn injury.

OBJECTIVE: To examine the role played by endotoxin, tumor necrosis factor-alpha (TNF-alpha), and caspase-3 in the increased apoptosis seen in solid organs in the early period after a burn injury. SUMMARY BACKGROUND DATA: Burn injury is often associated with immune suppression. Bacterial translocation and systemic endotoxemia have been reported after a burn injury, and caspase-3 activation due to TNF-alpha and Fas ligand (FasL) are presumed to initiate apoptosis. We hypothesized that endotoxin-induced TNF-alpha expression and caspase-3 activation could be the stimulus for the apoptosis after burn injury. METHODS: A 20% full-thickness scald burn was used in C57BL/6 mice. Three hours after burn injury, tissue samples were obtained from the thymus, lung, liver, and spleen. Lipopolysaccharide-nonresponsive (C3H/HeJ) and TNFalpha null B6x129tnf-/- mice were also used. To detect apoptosis, hematoxylin and eosin stain, in situ TUNEL, DNA extraction, and gel electrophoresis were all performed. Caspase-3 activity and TNF-alpha and FasL mRNA were also measured. RESULTS: Increased apoptosis and caspase-3 activity were observed in the thymus and spleen 3 hours after burn injury but were not seen in liver or lung. In the thymus and spleen, increased expression of FasL mRNA was also observed, whereas increased TNF-alpha mRNA was not. Increased apoptosis in thymus and spleen were also observed in C3H/HeJ and B6x129tnf-/- mice after burn injury. An inhibitor of the caspase-3 (Z-VAD-fmk) reduced apoptosis in both thymus and spleen. CONCLUSIONS: In the early period after a burn injury, increased apoptosis is observed primarily in the lymphoid organs and is independent of endotoxin or TNF-alpha. The increased caspase-3 activity in thymus and spleen contributes to apoptosis in these organs.