Figure 1.
Figure 1. Identification of a novel PMN activation subset in healthy blood that has similar CD marker expression as tissue PMNs. (A) Representative histograms of CD63, CD66a, and CD11b expression are shown for human blood PMNs (CD16high/side scatter area [SSC-A]high) of healthy volunteers, compared with tissue (oral) PMNs. pPMNs (∼10% of total circulating PMNs) can be distinguished from conventional rsPMNs based on elevated tissue-level CD marker expression. FMO controls are shown. (B) A scatter dot plot shows the mean ± standard deviation (SD) pPMN percentages of total gated PMNs in blood of healthy volunteers (n = 64). (C) Representative CD66a × CD63 scatterplots of human blood and tissue (oral) PMNs and representative forward scatter area (FSC-A) × SSC-A profiles of rsPMNs, pPMNs, and tissue (oral) PMNs are shown. (D) Representative images of DiffQuik-stained, FACS-sorted human rsPMNs and pPMNs are shown. Scale bar, 10 µm. Images were acquired using a Nikon E1000 microscope with a 100×/1.3 oil-immersion Plan Fluor objective. Image analysis was performed using Image J software (National Institutes of Health) to determine nuclear area of rsPMN and pPMN PMNs from 4 independent sorts. More than 300 cells were measured for each population. Mean values ± SD are shown. (E) Mouse BM, blood, and tissue (colon) PMN expression of CD66a, CD11b, and CD63 are shown. Colon cells were pooled from 3 mice. PMNs were gated based on Ly6Ghi and F4/80low (supplemental Figure 1D). FMO controls are shown. (F) Representative CD66a × CD11b and FSC-A × SSC-A dot plots are shown for gated mouse BM, blood, and tissue (colon) PMNs. (G) DiffQuik staining of FACS-sorted mouse rsPMNs and pPMNs was performed. Image analysis was performed as described in panel D from 2 independent sorts. Approximately 85 cells were measured for each population. Mean values ± SD are shown. P values were determined by 2-tailed paired Student t test. ****P < .0001. Representative confocal images of CD66a (H) and CD11b (I) labeled mouse blood leukocytes are shown. Nuclear staining (4′,6-diamidino-2-phenylindole [DAPI]) and merged images are also shown. PMNs were identified based on nuclear morphology. Images were acquired using a Zeiss Axio Observer Z1 microscope with a Plan-Apochromat 63×/1.4 oil-immersion objective. Scale bar, 10 µm.

Identification of a novel PMN activation subset in healthy blood that has similar CD marker expression as tissue PMNs. (A) Representative histograms of CD63, CD66a, and CD11b expression are shown for human blood PMNs (CD16high/side scatter area [SSC-A]high) of healthy volunteers, compared with tissue (oral) PMNs. pPMNs (∼10% of total circulating PMNs) can be distinguished from conventional rsPMNs based on elevated tissue-level CD marker expression. FMO controls are shown. (B) A scatter dot plot shows the mean ± standard deviation (SD) pPMN percentages of total gated PMNs in blood of healthy volunteers (n = 64). (C) Representative CD66a × CD63 scatterplots of human blood and tissue (oral) PMNs and representative forward scatter area (FSC-A) × SSC-A profiles of rsPMNs, pPMNs, and tissue (oral) PMNs are shown. (D) Representative images of DiffQuik-stained, FACS-sorted human rsPMNs and pPMNs are shown. Scale bar, 10 µm. Images were acquired using a Nikon E1000 microscope with a 100×/1.3 oil-immersion Plan Fluor objective. Image analysis was performed using Image J software (National Institutes of Health) to determine nuclear area of rsPMN and pPMN PMNs from 4 independent sorts. More than 300 cells were measured for each population. Mean values ± SD are shown. (E) Mouse BM, blood, and tissue (colon) PMN expression of CD66a, CD11b, and CD63 are shown. Colon cells were pooled from 3 mice. PMNs were gated based on Ly6Ghi and F4/80low (supplemental Figure 1D). FMO controls are shown. (F) Representative CD66a × CD11b and FSC-A × SSC-A dot plots are shown for gated mouse BM, blood, and tissue (colon) PMNs. (G) DiffQuik staining of FACS-sorted mouse rsPMNs and pPMNs was performed. Image analysis was performed as described in panel D from 2 independent sorts. Approximately 85 cells were measured for each population. Mean values ± SD are shown. P values were determined by 2-tailed paired Student t test. ****P < .0001. Representative confocal images of CD66a (H) and CD11b (I) labeled mouse blood leukocytes are shown. Nuclear staining (4′,6-diamidino-2-phenylindole [DAPI]) and merged images are also shown. PMNs were identified based on nuclear morphology. Images were acquired using a Zeiss Axio Observer Z1 microscope with a Plan-Apochromat 63×/1.4 oil-immersion objective. Scale bar, 10 µm.

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