Human coronary endothelial cell activation by endotoxin is characterized by NF-kappa B activation and TNF-alpha synthesis.

Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typically regulated by nuclear factor kappa B (NF-kappa B), plays a critical role in the development of cardiovascular dysfunction in sepsis. While several myocardial cell types synthesize TNF-alpha, the importance of the myocardial endothelium in sepsis-related cardiac cytokine production is unclear. To determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synthesis, TNF-alpha mRNA, and the associated NF-kappa B response to LPS. To determine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF-kappa B response to LPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC (12 h) that was ablated by the proteosome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF-alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappa B p50/p65 subunits were detectable in endothelial nuclear protein 60 min following LPS. In contrast, NF-kappa B subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappa B translocation, monocyte NF-kappa B translocation was completely inhibited. These data suggest endotoxin-stimulated human coronary endothelial cells express TNF-alpha, which is regulated in part by NF-kappa B activation, in a manner and degree distinct from human monocytes.

Differential regulation of myocardial NF kappa B following acute or chronic TNF-alpha exposure.

Tumor necrosis factor alpha (TNF-alpha) is a critical mediator of myocardial dysfunction during acute inflammatory states. TNF-alpha is also present in the serum of patients with chronic cardiac diseases. In monocytes, TNF-alpha stimulates cells by activating distinct signaling pathways that involve nuclear translocation of NF kappa B. Since NF kappa B may also regulate the expression of genes that could contribute to myocardial dysfunction, the cardiomyocyte NF kappa B activation following acute or chronic TNF-alpha challenges was investigated. To accomplish this, the authors either acutely administered TNF-alpha to healthy mice, or used transgenic mice which chronically overexpress TNF-alpha exclusively in cardiac myocytes. Following acute administration of TNF-alpha, cardiac NF kappa B translocation was detected from 15 min to 2 h post-challenge. The time course of I kappa B alpha degradation was consistent with the kinetics of NF kappa B translocation. I kappa B beta degradation was slower and less dramatic. In transgenic mice chronically overexpressing TNF-alpha, myocardial NF kappa B activation was detected at all ages tested (21, 40, and 75 days). In contrast to acutely challenged animals, two distinct NF kappa B proteins were activated in chronically challenged animals, p50--65 heterodimers as well as p50 homodimers. Activation of both could be transiently blocked by administration of a recombinant chimeric TNF-alpha receptor antagonist (rhTNFR:Fc). I kappa B alpha, but not I kappa B beta, levels were elevated in transgenics when compared to wild-type animals. These data indicate that following acute TNF-alpha administration, which simulates bacterial sepsis, myocardial p50-p65 translocates within minutes. Chronic TNF-alpha exposure, which is thought to occur in long-standing congestive heart failure, results in translocation of transcriptionally inactive p50 homodimers in addition to transcriptionally active p50--65 heterodimers. It is speculated that activation of p50 homodimers constitutes an adaptive response to minimize the inflammatory consequences of chronic cardiac TNF-alpha exposure.

Overexpression of cardiac I-kappaBalpha prevents endotoxin-induced myocardial dysfunction.

Nuclear factor-kappa B (NF-kappaB) is an inducible transcription factor that regulates expression of many genes, such as tumor necrosis factor-alpha (TNF-alpha), which may contribute to myocardial dysfunction. We investigated whether cardiac NF-kappaB activation is involved in the development of myocardial dysfunction after lipopolysaccharide (LPS) challenge. Mice were intraperitoneally injected with LPS, and the hearts were harvested and assayed for NF-kappaB translocation. After LPS challenge, NF-kappaB activation was detected within 30 min and remained for 8 h. In transgenic mice constitutively overexpressing a nondegradable form of I-kappaBalpha (I-kappaBalphaDeltaN) in cardiomyocytes, myocardial NF-kappaB translocation was prevented after LPS challenge. Myocytes isolated from these transgenics secreted significantly less TNF-alpha than did wild-type cardiomyocytes after LPS stimulation. When whole hearts were excised, perfused in a Langendorff preparation, and challenged with endotoxin, I-kappaBalphaDeltaN transgenic hearts displayed normal cardiac function, whereas profound contractile dysfunction was observed in wild-type hearts. These data indicate that myocardial NF-kappaB translocates within minutes after LPS administration. Inhibition of myocyte NF-kappaB activation by overexpression of myocyte I-kappaBalpha is sufficient to block cardiac TNF-alpha production and prevent cardiac dysfunction after LPS challenge.

Isolation, partial characterization, and concentration in experimental sepsis of baboon lipopolysaccharide-binding protein.

Lipopolysaccharide-binding protein (LBP) is important for mediating host responses to lipopolysaccharide (LPS). The structure and properties of human, rabbit, and murine LBP have been previously described. In this study we partially characterized baboon LBP and investigated its appearance in experimental sepsis. Recurrent bacteremia was induced in baboons by infusion of live Escherichia coli organisms over a 2-hour period at 0, 24, and 48 hours. To assay baboon plasma LBP levels, an enzyme-linked immunosorbent assay with cross-reactive antibodies to human LBP was developed. Control baboon plasma LBP concentrations were 2 to 5 microg/mL. During experimental sepsis, baboon plasma LBP levels increased to between 200 and 350 microg/mL and in parallel with the increase in C-reactive protein levels. Baboon LBP was isolated from acute phase serum by ion-exchange chromatography followed by immuno-affinity chromatography. Its NH2-terminal sequence (XNPGLVARTTNKGLEYSAQE) and its molecular weight (approximately 60 kd) were determined and were proved to be highly homologous to human LBP.

Molecular biology of septic shock.

Septic shock is a complex pathophysiologic state which often leads to multiple organ dysfunction, multiple organ failure, and death. This review summarizes current views on the molecular biology of three aspects of septic shock: recognition of bacterial invasion and induction of the cytokine response; genetic variability among humans and their predispositions toward pathologic inflammatory responses; and the signal transduction mechanisms which account for the transfer of molecular signals from cytokine receptors on the plasma membrane to cytokine-responsive genes in the nucleus. In particular, the review summarizes the pathway involved in tumor necrosis factor signaling through nuclear factor-kappaB, and elucidates the molecular signals involved in inflammatory responses and apoptosis.

Complementary DNA (cDNA) sequence of baboon tumor necrosis factor alpha.

Baboons are less LPS-sensitive than humans, even though their immune response mechanisms are similar. Since TNFalpha is a central mediator of the LPS-response we cloned and sequenced the baboon TNFalpha cDNA and compared the resulting sequence with the human TNFalpha sequence. Analysis of the TNFalpha protein coding region indicated 97% homology and of the 3' UTF 89%. The predicted baboon TNFalpha amino acid sequence differed at 10 positions from the human sequence. "TA" rich motifs within the 3' UTR were 100% homologous.