Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry

Abstract

Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (Mphi) in immunohistochemistry (IHC) and flow cytometry (FACS).

Methods: IHC was performed on cryostat sections of rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes using the anti-CD68 mAbs KP1, EBM11, and PGM1, and the fibroblast (FB) markers CD90 and prolyl 4-hydroxylase. Expression of CD68 was also analysed by FACS on the monocytic cell lines THP-1 and U937, as well as on synovial fibroblasts (SFB), skin FB, and gingival FB (both surface and intracellular staining).

Results: In IHC, there was an overlap between CD68 (mAbs KP1 and EBM11) and the FB markers CD90/prolyl 4-hydroxylase in the lining layer, diffuse infiltrates, and stroma of RA and OA synovial membranes. In FACS analysis of THP-1 and U937 cells, the percentage of cells positive for the anti-CD68 mAbs KP1 and EBM11 progressively increased from surface staining of unfixed cells, to surface staining of pre-fixed cells, to intracellular staining of the cells. Upon intracellular FACS of different FB, nearly all cells were positive for KP1 and EBM11, but only a small percentage for PGM1. In surface staining FACS, a small percentage of FB were positive for all three anti-CD68 mAbs.

Conclusion: An overlap between CD68 (mAbs KP1 or EBM11) and the FB markers CD90 or prolyl 4-hydroxylase may prevent unequivocal identification of Mphi in synovial tissue by IHC or in monocytic cells and FB upon intracellular FACS. This may be due to sharing of common markers by completely different cell lineages.

Figure 1

Immunohistochemical analysis of synovial tissue from one representative patient with RA for CD68 (mAbs KP1, EBM11, and PGM1), the FB marker CD90 (mAb AS02), and the monocyte/ Mφ marker CD14 (mAb TÜK4). Control isotype matched mAbs showed no positive reaction (fig 1A). The anti-CD68 mAbs KP1 (B) and EBM11 (C) strongly stained cells in the lining layer (filled arrowhead in fig 1F), diffuse infiltrates, and stroma. The mAb KP1 also detected endothelial cells (B; arrow in fig 1J). The anti-CD90 mAb AS02 very faintly stained cells in the lining layer, whereas this mAb strongly stained endothelial cells (E; arrow in fig 1F). Cells in diffuse infiltrates and stroma were weakly stained by this mAb. The anti-CD14 mAb TÜK4 faintly stained cells in the lining layer (I). This mAb strongly stained cells in the diffuse infiltrates and the stroma. After double staining experiments with the anti-CD68 mAb KP1 or EBM11 (blue colour) and the anti-CD90 mAb AS02 (brown colour), double-positive cells in diffuse infiltrates were seen (F and G; open arrowheads figs 1F and G). After double staining experiments with the anti-CD68 KP1 or EBM11 (blue colour) and the anti-CD14 mAb TüK4 (brown colour), double-positive cells in diffuse infiltrates were seen (J and K; open arrowheads in figs 1J and K). The anti-CD68 mAb PGM1 strongly stained cells in the lining layer and diffuse infiltrates (D, H, and L). This strong staining was only seen after fixation of the tissue sections with PFA followed by heating in SSC.

Figure 2

Flow cytometry analysis of the human monocytic cell lines THP-1 and U937 for CD68 (mAbs KP1, EBM11, and PGM1) using unfixed cells, cells after pre-fixation with 4% PFA, or cells after pre-fixation and permeabilisation with 0.25% saponin (in each case, data from one representative experiment are shown). In both cell lines, the percentages of positive cells for the anti-CD68 mAbs KP1 and EBM11 increased from surface staining of unfixed cells over surface staining of pre-fixed cells to intracellular staining of pre-fixed and permeabilised cells. In contrast, the percentages of positive cells for the anti-CD68 mAb PGM1 was highest on unfixed cells and decreased after pre-fixation with PFA or after pre-fixation with PFA and permeabilisation with saponin (isotype control: shaded curve; specific antibodies: black line).

Figure 3

Flow cytometry analysis of RA SFB, OA SFB, and JT SFB, skin FB, and gingival FB for intracellular expression of prolyl 4-hydroxylase or CD68 (mAbs KP1, EBM11, and PGM1) and surface expression of the fibroblast marker CD90 (in each case, data from one representative experiment are shown). The anti-CD68 mAbs KP1 and EBM11 showed a positive intracellular staining in a high percentage of SFB (RA, OA, and JT), skin FB, and gingival FB. In contrast, the anti-CD68 mAb PGM1 detected almost no cells in any of the FB populations. In all FB populations, nearly all cells stained positively for the FB marker prolyl 4-hydroxylase, whereas the percentage of positive cells for the FB marker CD90 varied depending on the FB population (isotype control: shaded curve; specific antibodies: black line).

Figure 4

Flow cytometry analysis of RA SFB, OA SFB, and JT SFB, skin FB, and gingival FB for surface expression of CD14, CD68 (mAbs KP1, EBM11, and PGM1) and the FB marker CD90 (in each case, data from one representative experiment are shown). In surface staining experiments with unfixed RA SFB, OA SFB, skin FB, and gingival FB, only a small percentage of the cells stained positively for the three anti-CD68 mAbs KP1, EBM11, and PGM1. Almost no cells stained positively for the macrophage marker CD14, whereas the percentage of positive cells for the FB marker CD90 varied depending on the FB population (isotype control: shaded curve; specific antibodies: black line).