MHC class I Abs bind to the PMN, not the endothelium, and interact with antigens on the PMN surface. (A) Lungs were taken from rats injected with either NS (i) or LPS and infused with 0.15 mg/kg OX27 (ii) and snap frozen, fixed, and sectioned. The lung sections were stained with bis-benzamide (blue, a nuclear stain), WGA linked to Cy-3 (red, a membrane-specific stain) and probed with donkey anti–mouse antibody labeled with Alexa 488 (green). Lungs from rats injected with NS and infused with OX27 had OX27 on the PMN membrane as demonstrated by the colocalization (yellow) of the red membrane WGA and the green Alexa 488 antibody (i, arrow), with no colocalization on the endothelium. Moreover, when the rat was pretreated with LPS and transfused with OX27, which caused ALI, there is more intense colocalization of OX27 with the PMN membrane (arrow) with little to no antibody present on the endothelium or on the lung parenchyma (ii; n = 3). (B) Rat PMNs were isolated from whole blood and 3.5 × 10⁵ PMNs used in each group. PMNs were incubated with 1.25 to 10 μg/mL (equal to 0.075-0.60 mg/kg antibody transfused; in parentheses next to in vitro dose) of either OX18 (▧) or OX27 () for 5 minutes and stimulated with fMLP, and the release of superoxide was measured (nmol O₂⁻/minute). Both OX18 and OX27 caused significant priming (P < .05 from fMLP alone) at the higher concentrations (5-10 μg/mL), although only OX27 primed at 2.5 μg/mL; whereas neither Ab primed at 1.25 μg/mL (*P < .05 from fMLP alone, n = 5). (C) Rat PMNs were isolated and incubated for 10 minutes at 4°C with either buffer or Fc block (Fcγ receptor blocker; Accurate Chemical) followed by a 30-minute incubation at 4°C with PE-labeled OX27 and fixed with paraformaldehyde. PMNs incubated with fluorescent OX27 antibodies demonstrated an increase in mean fluorescent intensity compared with the PMNs incubated with the isotypic controls. The Fc block had little effect on the OX27-mediated fluorescent shift (n = 3).