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Background —The inability to inhibit multiple mediators of septic shock represents a major hurdle in the treatment of septic shock. In vivo inhibition of nuclear factor (NF)-κB activation, a transcription factor regulating expression of many proinflammatory genes, could provide a useful strategy for the treatment of septic shock. Methods and Results —In rats challenged with lipopolysaccharide (LPS) 8 mg/kg IV, we determined the time course of NF-κB activation and expression of multiple inflammatory signals: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM)-1. We studied the effects of in vivo inhibition of NF-κB activation using pyrrolidine dithiocarbamate (PDTC) on the expression of these mediators. NF-κB activation preceded the induction of TNF-α, COX-2, CINC, and ICAM-1 mRNAs. PDTC prevented the LPS-induced NF-κB activation but did not inhibit activation of the transcription factors AP-1, Sp-1, and CREB. PDTC inhibited the LPS-induced expression of TNF-α, COX-2, CINC, and ICAM-1 mRNA and proteins and reduced the LPS-induced increases in plasma TNF-α, 6-keto-prostaglandin F 1α , and CINC concentrations. Inhibition of expression of these mediators prevented the increases in myeloperoxidase activity (a measure of neutrophil sequestration) in the heart, lungs, and liver. Conclusions —NF-κB activation correlates with LPS-induced expression of TNF-α, COX-2, CINC, and ICAM-1 genes in vivo. PDTC inhibits NF-κB activation and expression of these proinflammatory genes and their products. Thus, blocking NF-κB activation may be an effective strategy in the treatment of septic shock.

Prostaglandin (PG) synthesis during inflammation occurs mainly via the transcriptionally regulated cyclooxygenase, COX-2. In pulmonary type II A549 cells, Northern analysis identified multiple IL-1 beta-inducible COX-2 mRNA transcripts. Amplification of 3'-cDNA ends by anchored PCR revealed products corresponding to the predominant 4.5 and 2.7 kb transcripts. Sequence analysis of amplification products indicated that these transcripts arose by alternate consensus and non-consensus polyadenylation site usage. The predominant 4.5 kb transcript showed a half-life in excess of two hours that was further stabilized by IL-1 beta. In addition, the COX-2 3'-untranslated region (UTR), which contains 22 copies of the putative RNA instability motif, AUUUA, when cloned downstream of a constitutively expressed luciferase gene, was found to confer partial IL-1 beta responsiveness in LA-4 cells. Finally, in vivo in LPS-treated rats, differential expression of similar COX-2 mRNA isoforms was also observed. Taken together these data suggest a functional role for post-transcriptional mechanisms, including alternate polyadenylation, in the control of COX-2.

We studied the effects of ozone exposure and dexamethasone on inducible nitric oxide synthase (iNOS) gene expression in Brown Norway rats in vivo. Using a murine iNOS cDNA probe, we detected a 4.4 kb iNOS mRNA by Northern analysis in rat lung. The iNOS signal was weak in control lungs, but increased in lungs exposed to ozone (3 ppm, 6 h). Ozone-induced iNOS mRNA expression was time-dependent, with maximal expression at 2 h, declining by 8 and increasing again at 24 h postexposure. Dexamethasone significantly reduced the iNOS mRNA expression in the lungs of both controls and ozone-exposed rats. These results demonstrate that ozone inhalation induces iNOS expression in vivo, thus providing evidence at the molecular level for the possible involvement of nitric oxide generation in ozone-induced pulmonary inflammation or lung damage.

The properties of endothelin-3 (ET-3) were investigated in isolated pulmonary artery rings and isolated blood-perfused lungs of the rat. ET-3 elicited a concentration-dependent relaxation of pulmonary artery rings, and effect inhibited by the nitric oxide synthesis inhibitor L-NG-monomethyl-L-arginine. At 0.1 microM, the response to ET-3 was biphasic, resulting in a sustained contraction. In the isolated lung, ET-3 caused a dose-dependent increase in pulmonary arterial pressure. In lungs ventilated with 3% oxygen, 10 nM ET-3 completely reversed the resultant hypoxic vasoconstriction (HPV) by 100 +/- 8%, an effect unchanged by either indomethacin (1 microM) or glibenclamide (10 microM). L-NG-monomethyl-L-arginine attenuated both the ET-3 dilation in prostaglandin F2 alpha-constricted lungs and the dose-dependent vasodilation of HPV by acetylcholine. ET-3 (10 nM) showed the response time to peak pulmonary arterial pressure generation by hypoxia, the size of the response being unchanged. These results demonstrate that ET-3 has both vasodilator and constrictor actions in the rat lung and that, like acetylcholine, the former is mediated in part via the release of nitric oxide. ET-3 also has the ability to modulate HPV.

The effects of tumor necrosis factor (TNF) on hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent relaxation were examined in a blood-perfused rat lung preparation. Lungs from TNF-treated rats (0.26 mg/kg iv 12 h before experimentation) had a significantly greater HPV and a reduced vasorelaxant response to the endothelium-dependent vasodilator acetylcholine (ACh) but a similar vasorelaxant response to the endothelium-independent vasodilator nitroprusside compared with lungs from control rats (pretreated with 0.1 ml saline iv). Pentoxifylline (20 mg/kg iv and ip 20 min before administration of TNF) had no detectable effect on either HPV or ACh-induced relaxation but completely negated the augmentation on HPV and the inhibiting action on ACh-induced relaxation caused by TNF. The TNF effect on ACh relaxation was unaffected by pretreatment with L-arginine. These results indicate that TNF induces endothelial dysfunction and enhances HPV, effects that are inhibited by pentoxifylline.

To define the roles of endothelial-intrinsic nuclear factor kappaB (NF-kappaB) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-kappaB alpha mt) that conditionally overexpress a degradation-resistant form of the NF-kappaB inhibitor I-kappaB alpha (I-kappaB alpha mt) selectively on vascular endothelium. The EC-rtTA/I-kappaB alpha mt mice had no basal, but a relatively high level of doxycycline-inducible, I-kappaB alpha mt expression. I-kappaB alpha mt expression was detected in endothelial cells, but not in fibroblasts, macrophages, and whole blood cells, confirming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-kappaB alpha mt mice showed endothelial-selective blockade of NF-kappaB activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture-induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-kappaB alpha mt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-kappaB activity is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.

We have previously demonstrated that challenge of rat or mice with lipopolysaccharide (LPS) in vivo promotes Sp1 protein degradation. The protease responsible for the LPS-induced Sp1 degradation has not been identified. In this study, we have identified, characterized and partially purified an LPS-inducible Sp1-degrading enzyme (LISPDE) activity from rat lungs. LISPDE activity selectively degraded Sp1, but not nuclear protein, C-fos, p65, I-kappaBalpha and protein actin. Nuclear extract contains approximately 14-fold of the LISPDE activity as that detected in cytoplasmic extract, suggesting that LISPDE is predominantly a nuclear protease. Using biochemical reagents, protease inhibitors and peptide substrates, we have characterized the LISPDE activity. Based on biochemical characteristics, inhibitor profile, and substrate specificity, we have shown that LISPDE activity is not 26S proteasome, caspase or cathepsin-like activity, but is a trypsin-like serine protease activity. Using soybean trypsin inhibitor (SBTI)-sepharose affinity column, we have partially purified the LISPDE protein, which has an estimated molecular mass of 33 kDa and selectively degrades native Sp1 protein. We mapped the initial site for proteolytic cleavage of Sp1 by LISPDE to be located within the region between amino acids 181-328. We conclude that LPS causes Sp1 degradation by inducing a unique trypsin-like serine protease, LISPDE.