Role of FKN in apoptotic germinal center B cells and other lymphocytes. (A) Expression of FKN in normal lymphoid tissue in situ. Immunohistochemical staining was performed on archive paraffin sections of human tonsil. DAB chromogen product (brown) represents FKN staining (hematoxylin counterstain). Primary anti-FKN mAb (clone 81513); isotype control mAb (mouse IgG1) produced negative results, not shown. GC indicates germinal center of lymphoid follicle. Arrows indicate examples of tingible body macrophages. Bar represents 52 μm (left panel); bar represents 27 μm (right panel). Stained tissue was analyzed using a Zeiss Axioskop 2 microscope (Zeiss) at 20°C with objective lenses Plan-NEOFLUAR 20× (numerical aperture 0.50) and Plan-NEOFLUAR 40× magnification (numerical aperture 0.75) and histomount medium (Raymond A. Lamb), and images were taken using Jenoptik ProgRes C14 camera (Jenoplite) with Openlab 4.0.2 software (Improvision/PerkinElmer). (B) Release of FKN by murine germinal center B cells. Immunoblots using rabbit antimouse FKN polyclonal Ab (Torrey Pines Biolabs, East Orange, NJ) of cell-free supernatants (equal volumes of TCA/acetone-precipitated supernatants per lane) showing changes in FKN in purified germinal center B cells (GC) and follicular B cells (fol) from mouse spleens cultured in vitro for 2 or 18 hours. (C) Effects of loss of CX3CR1 on macrophage migration and of clearance of apoptotic cells in lymphoid tissue in vivo. Quantitative immunohistochemical analyses were performed on splenic follicles and extrafollicular areas of mice (wt, CX3CR1^−/−, and CD14^−/−) immunized with SRBCs. Numbers of macrophages and apoptotic cells in situ were determined by counting CD68⁺ and TUNEL⁺ cells per millimeter-squared, respectively. CD14^−/− mice were used as positive control animals that are known to show defective apoptotic cell clearance in vivo. Significant reduction in numbers of macrophages in follicles was observed in CX3CR1^−/− mice, but apoptotic cells did not persist in these animals. Results shown are mean plus or minus SEM (n = 3-6 spleens); ANOVA, wt vs CX3CR1^−/−, ***P = .009; wt vs CD14^−/−, **P = .018. (D) Modulation and release of FKN during apoptosis of activated primary lymphocytes (CD19-depleted). Flow cytometric analyses showing loss of surface FKN labeling of CD19-depleted lymphocytes after induction of apoptosis. Cells were preactivated by culture with 2 μg/mL concanavalin A to obtain surface FKN labeling (freshly isolated cells were surface FKN-negative, data not shown). Cells were labeled by indirect immunofluorescence using anti-FKN mAb (clone 81513) at the indicated times after induction of apoptosis by staurosporine treatment (1 μM). Histograms of viable zone cells are as follows: black represents time 0; white, 1 hour; coarse stippling, 2 hours; fine stippling, 4.5 hours after treatment; pale gray, isotype control immunostaining. Percentages of “FKN-bright” cells lying within the indicated gate are shown. Levels of apoptosis (assessed using annexin V) were 8%, 17%, 18%, and 28% at 0, 1, 2, and 4.5 hours after treatment, respectively. Representative experiment from 3.