Flow cytometric analysis of anti-MPO binding by circulating leukocytes from naive mice or mice pretreated with LPS. Mice were treated with fluorochrome-conjugated anti-MPO or anti-OVA and whole blood was removed either 5 or 60 minutes later. Neutrophils in blood samples were identified by initially gating on cells of high forward scatter/side scatter characteristics, and examining staining for Gr-1 and M1/70. (A) Neutrophils in mice treated with anti-OVA or anti-MPO alone, showing the rapid removal of neutrophils from the circulation in response to anti-MPO, but not anti-OVA (representative of n = 3). (B) Time course of circulating leukocyte counts of mice that received either anti-OVA or anti-MPO (50 μg, n = 6/gp). (*P < .05 vs anti-OVA at designated time points.) Following treatment with anti–β2-integrin mAb (2E6; C,D,F), sufficient leukocytes were retained in the circulation to allow assessment of anti-MPO binding. (C-F) Gates shown in the left-hand panels define the leukocytes analyzed for binding of anti-MPO/OVA in the right-hand panels. (C) Binding of anti-OVA or anti-MPO by circulating neutrophils in otherwise untreated wild-type C57BL/6 mice. In mice treated with anti-OVA (n = 8), neutrophils stain homogeneously for M1/70, although minimal binding of anti-OVA is observed. In contrast, in wild-type mice treated with anti-MPO (n = 8), 2 neutrophil populations are apparent: cells that stain moderately (M1/70^int) or strongly (M1/70^hi) for M1/70. Approximately 50% of M1/70^int cells stain positively with anti-MPO, whereas the majority of M1/70^hi cells stain positively for anti-MPO. Also shown is the anti-MPO staining pattern of neutrophils from MPO^−/− mice treated with anti-MPO (n = 4), showing negligible binding. (D) Monocyte binding of anti-MPO. In the blood of mice treated as in panel C, monocytes were identified as cells positive for both CD115 and F4/80 (n = 5). These cells were not found in the high forward scatter/side scatter gate that contained the neutrophils. Approximately 40% of circulating monocytes stained positively for anti-MPO. Monocyte binding of anti-OVA (n = 4) is shown on the same trace. (E) Binding of anti-MPO by circulating neutrophils 60 minutes after anti-MPO administration (in the absence of β2-integrin blockade, n = 3). At this point, Gr1⁺/M170^int cells were again present in the circulation, and the majority of cells stained strongly with anti-MPO. Also shown is neutrophil binding of anti-OVA at the corresponding time point. (F) Binding of anti-OVA or anti-MPO by circulating neutrophils in LPS (0.1 μg, 4 hours)–pretreated mice (n = 6). In mice treated with anti-OVA, neutrophil M1/70 staining is unaltered from that seen in the absence of LPS and minimal binding of anti-OVA is observed. However, in mice treated with LPS and anti-MPO, a wide spread of M1/70 staining is apparent within the granulocyte gate. The 2 major populations are as follows: Gr-1^hi/M1/70^hi cells, of which the majority stain strongly with anti-MPO; and cells of lower Gr-1 staining, but greater M1/70 staining (Gr-1^lo/M1/70^hi+), of which 70% are stained by anti-MPO.