Bacterial endotoxin [lipopolysaccharide (LPS)] stimulates macrophages to sequentially release early [tumor necrosis factor (TNF)] and late [high mobility group box 1 (HMGB1)] proinflammatory cytokines. The requirement of CD14 and mitogen-activated protein kinases [MAPK; e.g., p38 and extracellular signal-regulated kinase (ERK)1/2] for endotoxin-induced TNF production has been demonstrated previously, but little is known about their involvement in endotoxin-mediated HMGB1 release. Here, we demonstrated that genetic disruption of CD14 expression abrogated LPS-induced TNF production but only partially attenuated LPS-induced HMGB1 release in cultures of primary murine peritoneal macrophages. Pharmacological suppression of p38 or ERK1/2 MAPK with specific inhibitors (SB203580, SB202190, U0126, or PD98059) significantly attenuated LPS-induced TNF production but failed to inhibit LPS-induced HMGB1 release. Consistently, an endogenous, immunosuppressive molecule, spermine, failed to inhibit LPS-induced activation of p38 MAPK and yet, still significantly attenuated LPS-mediated HMGB1 release. Direct suppression of TNF activity with neutralizing antibodies or genetic disruption of TNF expression partially attenuated HMGB1 release from macrophages induced by LPS at lower concentrations (e.g., 10 ng/ml). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

To defi ne the roles of endothelial-intrinsic nuclear factor B (NF-B) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-B ␣ mt) that conditionally overexpress a degradation-resistant form of the NF-B inhibitor I-B ␣ (I-B ␣ mt) selectively on vascular endothelium. The ECrtTA/I-B ␣ mt mice had no basal, but a relatively high level of doxycycline-inducible, I-B ␣ mt expression. I-B ␣ mt expression was detected in endothelial cells, but not in fi broblasts, macrophages, and whole blood cells, confi rming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-B ␣ mt mice showed endothelial-selective blockade of NF-B activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infi ltration 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-B ␣ mt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-B activity is an essential mediator of septic multiple organ infl ammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.