The following antibodies were used for flow cytometry studies and FACS: mouse anti-CD11b Alexa Fluor 488 -conjugated (clone M1/70; isotype, rat IgG 2b -488); mouse anti-F4/80 (clone 6F12; isotype, rat IgG 2a ); mouse anti-NK1.1 PE-conjugated (clone PK136; isotype, mouse IgG 2a -PE); mouse anti-Gr1 PE-conjugated (clone RB6-8C5; isotype, mouse IgG 2a -PE); rat anti-mouse IFN-␥ (clone XMG 1.2; isotype, rat IgG 1 ); rat anti-mouse IL-10 (clone JES5-16E3; isotype, rat IgG 2b ); biotin mouse anti-human TLR4 (clone HTA 125; isotype, mouse IgG 2a -biotin); and biotin anti-Annexin V (all from BD Biosciences Pharmingen, San Di-ego, CA); mouse anti-KDR PE-conjugated (isotype, mouse IgG 1 -PE) and mouse anti-ACE fluorescein isothiocyanateconjugated (isotype, goat IgG-fluorescein isothiocyanate) (both from R&D Systems, Inc.); rat anti-mouse TNF-␣ (clone MP6-XT22; isotype, rat IgG 1 ; Invitrogen Corp.); rabbit polyclonal anti-NOS 2 , (clone N-20; isotype, goat IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and NO-Cu-Fl, final concentration 10 mol/L (Intracellular Nitric Oxide Sensor Kit, catalog No. 96 -0293; Strem Chemicals, Inc., Newburyport, MA). For intracellular staining, mouse lung cells were first fixed via incubation using 100 L of fixation buffer (eBioscience, Inc., San Diego, CA) for 20 minutes at room temperature, and washed twice with 1 ml of permeabilization buffer (eBioscience, Inc.). Consequently, 1 to 5 g/10 6 cells of antibodies against TNF-␣, IL-10, IFN-␥, or inducible NO synthase (iNOS) were then added in 100-L volume of permeabilization buffer, and cells were incubated for 30 minutes at 4°C. After an additional washing with 1 ml of permeabilization buffer, the cells were stained with secondary anti-rat-fluorescein isothiocyanate or anti-rabbit-PE (iNOS) conjugated antibody for 20 minutes at 4°C. The cells were resuspended in flow cytometry buffer and analyzed immediately. Intracellular NO staining with fluorescentbound copper was performed per the manufacturer's instructions and in accordance with published protocols. 17, 18 Surface and intracellular marker expression were analyzed using software (LSR and CellQuest Pro; Becton Dickinson & Co., San Jose, CA).