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Figure 2. Expression of CCR5, CXCR4, and galactosylceramide (GalCer) in human gingiva. (A) Low expression of CCR5 in healthy gingiva and increased expression in inflammation: Representative sections of (A1) healthy (n = 8) and (A2) inflamed gingiva (n = 12) obtained from surgical crown lengthening and during periodontal surgery, respectively, were subjected to single immunoenzyme labeling with CCR5 antibodies and 20x fields scanned by image analysis, as described in MATERIALS & METHODS. Final magnification shown is approximately 250X. (A3) H&E staining of healthy gingiva shown in A1. (A4) H&E staining of inflamed gingiva shown in A2. (B,C,D) Double-immunofluorescence staining of representative inflamed gingival tissues showing expression of CCR5 by T-cells (Fig. 2B), macrophages (Fig. 2C), and dermal dendritic cells (Fig. 2D): In all fluorescent Figs., the green channel (panel 1), red channel (panel 2), and green/red merged channel (panel 3) are shown. Yellow cells in panel 3 denote co-localization of the two markers. In panel 4, the images merging from the green/red channel (panel 3) are overlaid with the image from differential interference microscopy (DIC), an optical variant of phase-contrast microscopy, for better documentation of co-localizations. Images were taken with the use of a 40X objective at a final magnification of 400X. Also included in all the images (B,C,D) is the enlarged inset image, with arrows demonstrating co-localization. (B) Several CD4+ T-cells (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (C) Several CD68+ macrophages (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (D) Several DC-SIGN+ dermal dendritic cells (red channel, panel 2) in the lamina propria co-express CCR5 (panels 3 and 4). (E1–4) Shown is the expression of CXCR4 and GalCer in the same healthy gingival tissue by single immunoenzyme staining: (E 1) expression of CXCR4 (arrows), (E 2) expression of GalCer (bracket), (E 3) H&E staining, (E 4) isotype control. (E 5–6) Shown is the the CXCR4 and CCR5 staining in the same inflamed gingival tissue: (E 5) expression of CXCR4 (arrows), (E 6) expression of CCR5 (arrows). (F) Shown are the mean numbers (± SE) of CCR5 cells in the healthy and inflamed gingiva. *Statistically significant difference (p < 0.05, Student’s t test). (G) Linear regression analysis was performed on the number of inflammatory cells (H&E-positive/20x field in lamina propria) (Y-axes) and the number of CCR5+cells (x-axis) per 20x field. Light and fluorescent microscopy images were acquired with a Nikon Eclipse E600 microscope equipped with a color high-resolution CCD camera and a PC running Image-Pro software (Media Cybernetics, Inc., Silver Spring, MD, USA).
