lization of CD11b to the sites of interaction of APECs in aggregates. It is important to point out that although CD11b appeared enriched at the sites of cell-cell interaction in fluorescence imaging of a single Z plane, overall CD11b amounts on the cell surface of APECs were reduced upon Ifn treatment. Overall, the expression of several adhesion molecules was marginally reduced and is accompanied by preferential localization of CD11b to the sites of interaction in aggregated APECs. To study the functional contribution of CD11b, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698151 studies with Reopro, a purified Fab against glycoprotein GP IIb/IIIa that also blocks CD11b, were performed. The addition of Reopro reduced CD11b, but not E-Selectin, detection on the cell surface after 36 h. Importantly, Reopro treatment in a dose dependent manner reduced Ifn induced aggregation of APECs, but not nitrite production. These data were confirmed using siRNA to Cd11b. As oligonucleotides are known to affect immune responses, initial experiments were performed to determine the role of the scrambled control in modulating functions of APECs. As seen in Fig. A in S1 File, transfections with the scrambled siRNA did not alter any major AIC316 site responses in APECs with respect to nitrite, CD11b expression and aggregation. However, CD11b expression, but not E-Selectin, is lowered with siRNA to Cd11b but not the scrambled control. Although Ifn-induced nitrite amounts remained unaffected, the number of aggregates was significantly reduced upon knockdown of CD11b. Therefore, CD11b on the cell surface aids in the formation of aggregates of APECs in response to Ifn. Nos2 derived nitric oxide promotes the aggregation response of APECs to Ifn Next, we investigated the intracellular signaling molecules that might contribute to the phenomenon of Ifn mediated aggregation of APECs. Ifn is a potent inducer of reactive oxygen species and NO in macrophages and several Ifn induced responses are dependent on these molecules. Ifn induced ROS in a kinetic manner, which could be quenched PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19699128 by exogenously added Polyethyleneglycol-Catalase; however, the 
aggregation of APECs remained unaffected. On the other hand, Ifn treatment also induced the production of nitrite in a kinetic manner, which was inhibited by the Nos inhibitor, LNMA. Importantly, addition of LNMA inhibited Ifn induced aggregation of APECs in a dose dependent manner. As the predominant isoform of Nos expressed in macrophages is Nos2, the possible role of Nos2 generated NO in mediating Ifn induced APECs aggregation was investigated. First, Ifn treatment did not affect the viability of APECs derived from either C57BL/6 or Nos2-/- mice. Second, Ifn induced nitrite in APECs from C57BL/6, but not from Nos2-/-, mice. Third and most importantly, the lack of Nos2 completely abrogated the Ifn induced aggregation response of APECs. To study the direct contribution of NO, exogenous supplementation experiments were performed with NO donor, SNAP. It is important to point out that that the concentrations of SNAP used were ones that produced nitrite amounts similar to that seen with Ifn stimulation of C57BL/6 APECs. Notably, Nos2-/- APECs do not form aggregates with SNAP alone; however, aggregates of Nos2-/- APECs were observed with the combination of Ifn and SNAP. These results were confirmed using another NO donor, DETA/NO, which has a longer half life compared to SNAP. As seen in Fig. B in S1 File, DETA/NO, in a dose-dependent manner, induced nitrite. However, the aggregation of Nos2-/-
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