As an acute phase protein, alpha-antitrypsin (AAT) is thought to play an important role in limiting host tissue injury by proteases released from activated myeloid cells, specifically neutrophil elastase (NE). The clinical importance of AAT is highlighted in individuals with inherited deficiency in circulating AAT who have an increased susceptibility to early onset pulmonary emphysema, chronic bronchitis, bronchiectasis, and liver diseases. Administration of exogenous human plasma-derived AAT is used in various animal models to test the value of AAT augmentation therapy. Unrelated to emphysema models, administration of exogenous AAT inhibits HIV 1 expression, prevents murine islet cell allografts from rejection, blocks cell apoptosis and increases survival in an allogeneic marrow transplantation models. The mechanism of the above mentioned anti-inflammatory activities of AAT has been assumed to be due to its anti-proteolytic activity; however this has not been studied. We hypothesized that the anti-elastase activity of AAT was not required for its anti-inflammatory properties. To investigate this prediction, effects of native human plasma derived (nAAT, inhibitory form) and recombinant (rAAT, non-inhibitory form) on lipopolysaccharide (LPS)-induced IL-8 and TNFα release from neutrophils isolated from human blood or the bone marrow from wild type (WT) and elastase deficient mice (NE-/-) were examined. When WT or NE deficient neutrophils were treated with LPS plus rAAT (50 nM), release of TNFα (42%, p=0.002 and 24%, p=0.011, respectively) as well as of IL-8 was decreased (57%, p=0.004 and 40%, p=0.01, respectively) relative to LPS challenged cells. By comparison, to achieve the same reduction in TNFα from either WT or NE deficient neutrophils, a concentration of nAAT of 2000 nM was required. Next, human blood neutrophils were incubated with LPS (10ng/ml) alone or LPS plus increasing concentrations of nAAT or rAAT. After 8 and 18 hours, cytokines were measured in the supernatants. At 20.000 nM, nAAT reduced TNFα release by 46% (p=0.002) and IL-8 by 29%, (p=0.024). However, markedly lower concentrations of rAAT (5nM) reduced TNFα release by 41% (p=0.004) and IL-8 by 40% (p=0.05). We conclude that anti-proteolytic activity of AAT may be required but less important for its anti-inflammatory function. Acknowledgments: This study was supported by Soohyun Kim who provided the rAAT and our collaborator Charles A. Dinarello.