EXTENDED REPORT

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

E Kunisch¹, R Fuhrmann², A Roth², R Winter², W Lungershausen³, R W Kinne¹

¹ Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany
² Clinic of Orthopaedics, Friedrich Schiller University Jena, Jena, Germany
³ Department of Traumatology, Friedrich Schiller University Jena, Jena, Germany

Correspondence to:
Correspondence to:
Dr R W Kinne
Experimental Rheumatology Unit, Friedrich Schiller University Jena, Hans-Knöll-Str 2, D-07745 Jena, Germany; raimund.w.kinne{at}rz.uni-jena.de

Objectives: To investigate the specificity of three anti-CD68 monoclonal antibodies (mAbs) for macrophages (M) 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 M 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.

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FACS, fluorescence activated cell sorter; FB, fibroblast(s); FCS, fetal calf serum; IHC, immunohistochemistry; JT, joint trauma; mAb, monoclonal antibody; M, macrophages; OA, osteoarthritis; PBS, phosphate buffered saline; PFA, paraformaldehyde; RA, rheumatoid arthritis; RT, room temperature; SSC, sodium citrate/sodium chloride; SM, synovial membrane

Keywords: CD68; synovial fibroblasts; synovial macrophages; immunohistochemistry; flow cytometry

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