Sera from hMPO and HSA-immunized WKY rats bind to WKY and human neutrophils. (A) Human and WKY rat PMNs were indirectly stained with sera (1:20) from hMPO (EAV) or HSA-immunized (control) rats, and sera from patients with AASV and observed by confocal microscopy (× 63; UplanApo 63×/1.20 water dipping). Representative of 5 experiments involving 5 to 13 separate sera. (B) Cells were stained as in panel A, and fluorescence intensity was quantified using Image-Pro plus software. Circles indicate anti-hMPO-positive sera; triangles, anti-hMPO-negative sera. Statistically significant differences are shown by asterisks, ^**P < .01 and ^***P < .001. AFU indicates arbitrary fluorescence unit. (C) Rat leukocytes were incubated with Ig prepared from EAV rats (open curves) or control rats (filled curves). Both surface and intracellular antigens were stained sequentially, and rat PMNs were identified based on side scatter profile and binding of anti-HIS48 monoclonal antibody (mAb). Panels Ci and Cii represent cells stained with control Ig (closed curves) or EAV Ig (open curves) on rat and human PMNs, respectively. Results are representative of 4 separate experiments. (D) Lysates of WKY rat and human leukocytes were prepared and analyzed by Western blotting. (Lane 1) Purified hMPO incubated with pooled Ig from rats with EAV. (Lane 2) WKY rat leukocyte lysate incubated with pooled Ig from rats with EAV. (Lane 3) WKY rat leukocyte lysate incubated with pooled Ig from control rats. (Lane 4) Human PMN lysate incubated with pooled Ig from rats with EAV. Separate blots are indicated by vertical lines.