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Firm neutrophil (PMN)-endothelial (EC) adhesion is crucial to the PMN-mediated hyperinflammation observed in acute lung injury. Hypertonic saline (HTS) used for resuscitation of hemorrhagic shock has been associated with a decreased incidence of PMN-mediated lung injuryacute respiratory distress syndrome. We hypothesize that physiologically accessible hypertonic incubation (170 vs. 140 mM, osmolarity ranging from 360 to 300 mOsmL) inhibits proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs). Proinflammatory activation of HMVECs was investigated in response to tumor necrosis factor- (TNF-), including interleukin 8 (IL-8) release, intercellular adhesion molecule 1 (ICAM-1) surface expression, PMN adhesion, and signaling mechanisms under both isotonic (control) and hypertonic conditions. Hyperosmolarity alone had no effect on either basal IL-8 release or ICAM-1 surface expression but did lead to concentration-dependent decreases in TNF-–induced IL-8 release, ICAM-1 surface expression, and PMN-HMVEC adhesion. Conversely, HTS activated p38 mitogen-activated protein kinase (MAPK) and enhanced TNF- activation of p38 MAPK. Despite this basal activation, hyperosmolar incubation attenuated TNF-–stimulated IL-8 release and ICAM-1 surface expression and subsequent PMN adherence, while p38 MAPK inhibition did not further influence the effects of hyperosmolar conditions on ICAM-1 surface expression. In addition, TNF- induced nuclear factor-B DNA binding, but HTS conditions attenuated this by 31 (P< 0.01). In conclusion, HTS reduces PMN-HMVEC adhesion and TNF-–induced proinflammatory activation of primary HMVECs via attenuation of nuclear factor-B signaling.