Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils.
We have generated mice with a cell type-specific disruption of the Stat3 gene in macrophages and neutrophils. The mutant mice are highly susceptible to endotoxin shock with increased production of inflammatory cytokines such as TNF alpha, IL-1, IFN gamma, and IL-6. Endotoxin-induced production of inflammatory cytokines is augmented because the suppressive effects of IL-10 on inflammatory cytokine production from macrophages and neutrophils are completely abolished. The mice show a polarized immune response toward the Th1 type and develop chronic enterocolitis with age. Taken together, Stat3 plays a critical role in deactivation of macrophages and neutrophils mainly exerted by IL-10. (+info
Role of nitric oxide in lipopolysaccharide-induced hepatic injury in D-galactosamine-sensitized mice as an experimental endotoxic shock model.
The role of nitric oxide (NO) in lipopolysaccharide (LPS)-induced hepatic injury was studied in D-galactosamine (D-GalN)-sensitized mice. The inducible isoform of NO synthase (iNOS) was immunohistochemically detected on hepatocytes around blood vessels in livers of mice injected with D-GalN and LPS not on hepatocytes in mice injected with D-GalN or LPS alone, although mRNA for iNOS was found in those mice. Nitrotyrosine (NT) was also found in livers of mice injected with D-GalN and LPS. The localization of NT was consistent with that of iNOS, and the time courses of NT and iNOS expression were almost the same. Expression of iNOS and NT was detected exclusively in the hepatic lesions of mice injected with D-GalN and LPS. Anti-tumor necrosis factor alpha neutralizing antibody inhibited iNOS and NT expression and hepatic injury. The results suggested that NO from iNOS may play a role in LPS-induced hepatic injury on D-GalN-sensitized mice as an experimental endotoxic shock model. (+info
Neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide.
Endotoxin (lipopolysaccharide [LPS]) is the major pathogenic factor of gram-negative septic shock, and endotoxin-induced death is associated with the host overproduction of tumor necrosis factor alpha (TNF-alpha). In the search for new antiendotoxin molecules, we studied the endotoxin-neutralizing capacity of a human lactoferrin-derived 33-mer synthetic peptide (GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP; designated LF-33) representing the minimal sequence for lactoferrin binding to glycosaminoglycans. LF-33 inhibited the coagulation of the Limulus amebocyte lysate and the secretion of TNF-alpha by RAW 264.7 cells induced by lipid A and four different endotoxins with a potency comparable to that of polymyxin B. The first six residues at the N terminus of LF-33 were critical for its antiendotoxin activity. The endotoxin-neutralizing capacity of LF-33 and polymyxin B was attenuated by human serum. Coinjection of Escherichia coli LPS (125 ng) with LF-33 (2.5 microg) dramatically reduced the lethality of LPS in the galactosamine-sensitized mouse model. Significant protection of the mice against the lethal LPS challenge was also observed when LF-33 (100 microg) was given intravenously after intraperitoneal injection of LPS. Protection was correlated with a reduction in TNF-alpha levels in the mouse serum. These results demonstrate the endotoxin-neutralizing capability of LF-33 in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock. (+info
In vivo isolated kidney perfusion with tumour necrosis factor alpha (TNF-alpha) in tumour-bearing rats.
Isolated perfusion of the extremities with high-dose tumour necrosis factor alpha (TNF-alpha) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-alpha and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 microg TNF-alpha. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-alpha and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-alpha alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-alpha, the minimal threshold concentration of TNF-alpha to exert its anti-tumour effects was not reached. The applicability of TNF-alpha in isolated kidney perfusion for human tumours seems, therefore, questionable. (+info
Resistance of CD7-deficient mice to lipopolysaccharide-induced shock syndromes.
CD7 is an immunoglobulin superfamily molecule involved in T and natural killer (NK) cell activation and cytokine production. CD7-deficient animals develop normally but have antigen-specific defects in interferon (IFN)-gamma production and CD8(+) CTL generation. To determine the in vivo role of CD7 in systems dependent on IFN-gamma, the response of CD7-deficient mice to lipopolysaccharide (LPS)-induced shock syndromes was studied. In the high-dose LPS-induced shock model, 67% of CD7-deficient mice survived LPS injection, whereas 19% of control C57BL/6 mice survived LPS challenge (P < 0.001). CD7-deficient or C57BL/6 control mice were next injected with low-dose LPS (1 microgram plus 8 mg D-galactosamine [D-gal] per mouse) and monitored for survival. All CD7-deficient mice were alive 72 h after injection of LPS compared with 20% of C57BL/6 control mice (P < 0.001). After injection of LPS and D-gal, CD7-deficient mice had decreased serum IFN-gamma and tumor necrosis factor (TNF)-alpha levels compared with control C57BL/6 mice (P < 0.001). Steady-state mRNA levels for IFN-gamma and TNF-alpha in liver tissue were also significantly decreased in CD7-deficient mice compared with controls (P < 0.05). In contrast, CD7-deficient animals had normal liver interleukin (IL)-12, IL-18, and interleukin 1 converting enzyme (ICE) mRNA levels, and CD7-deficient splenocytes had normal IFN-gamma responses when stimulated with IL-12 and IL-18 in vitro. NK1.1(+)/ CD3(+) T cells are known to be key effector cells in the pathogenesis of toxic shock. Phenotypic analysis of liver mononuclear cells revealed that CD7-deficient mice had fewer numbers of liver NK1.1(+)/CD3(+) T cells (1.5 +/- 0.3 x 10(5)) versus C57BL/6 control mice (3.7 +/- 0.8 x 10(5); P < 0.05), whereas numbers of liver NK1.1(+)/CD3(-) NK cells were not different from controls. Thus, targeted disruption of CD7 leads to a selective deficiency of liver NK1.1(+)/ CD3(+) T cells, and is associated with resistance to LPS shock. These data suggest that CD7 is a key molecule in the inflammatory response leading to LPS-induced shock. (+info
The flesh-eating bacterium: what's next?
Since the 1980s, there has been a marked increase in the recognition and reporting of highly invasive group A streptococcal (GAS) infections associated with shock and organ failure, with or without necrotizing fasciitis. Such dramatic cases have been defined as streptococcal toxic shock syndrome (StrepTSS). Strains of GAS isolated from patients with invasive disease have been predominantly M types 1 and 3, which produce either pyrogenic exotoxin A or B or both. The clinical and demographic features of streptococcal bacteremia, myositis, and necrotizing fasciitis are presented and compared with those of StrepTSS. Current concepts in the pathogenesis of invasive streptococcal infection will be presented, with emphasis on the interaction between GAS virulence factors and host defense mechanisms. Finally, new concepts in the treatment of StrepTSS will be discussed. (+info
A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock.
The regulation of monocyte function and the inhibition of TNF-alpha production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-alpha found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-alpha Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-alpha production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-kappa B nuclear translocation resulting in greater TNF-alpha production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-alpha secretion and attenuating endotoxic shock. (+info
Pyrrolidine dithiocarbamate prevents I-kappaB degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs.
Lipopolysaccharide (LPS) is a key mediator of multiple organ injury observed in septic shock. The mechanisms responsible for LPS-induced multiple organ injury remain obscure. In the present study, we tested the hypothesis that the LPS-induced injury occurs through activation of the transcription factor, nuclear factor-kappaB (NF-kappaB). We examined the effects of inhibiting NF-kappaB activation in vivo in the rat on LPS-induced: 1) gene and protein expression of the cytokine-inducible neutrophil chemoattractant (CINC) and intercellular adhesion molecule-1 (ICAM-1); b) neutrophil influx into lungs, heart, and liver; and c) increase in microvascular permeability induced by LPS in these organs. LPS (8 mg/kg, i.v.) challenge of rats activated NF-kappaB and induced CINC and ICAM-1 mRNA and protein expression. Pretreatment of rats with pyrrolidine dithiocarbamate (50, 100, and 200 mg/kg, i.p.), an inhibitor of NF-kappaB activation, prevented LPS-induced I-kappaBalpha degradation and the resultant NF-kappaB activation and inhibited, in a dose-related manner, the LPS-induced CINC and ICAM-1 mRNA and protein expression. Pyrrolidine dithiocarbamate also markedly reduced the LPS-induced tissue myeloperoxidase activity (an indicator of tissue neutrophil retention) and the LPS-induced increase in microvascular permeability in these organs. These results demonstrate that NF-kappaB activation is an important in vivo mechanism mediating LPS-induced CINC and ICAM-1 expression, as well as neutrophil recruitment, and the subsequent organ injury. Thus, inhibition of NF-kappaB activation may be an important strategy for the treatment of sepsis-induced multiple organ injury. (+info