Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expression

Immunobiology. 2011 Aug;216(8):882-90. doi: 10.1016/j.imbio.2011.02.001. Epub 2011 Feb 22.


Background: CD163 is expressed exclusively on cells of the monocyte/macrophage lineage and is widely used as a marker of human macrophages. Further, it has been suggested as a diagnostic marker of monocyte/macrophage activity in inflammatory conditions and as a therapeutic target. However, studies continue to exhibit great discrepancy in the measured percentage of CD163-expressing blood monocytes in healthy individuals. In this study we sought to clarify this inconsistency in reported levels of CD163 surface expression by a detailed analysis of a panel of CD163 antibodies used in previous studies.

Materials and methods: The cellular distribution of CD163 on human peripheral blood monocytes in freshly drawn blood and peripheral blood mononuclear cells isolated from buffy-coats was investigated by flow cytometry using CD163 monoclonal antibodies recognizing scavenger receptor cysteine-rich (SRCR) domain 1 (MAC2-158), domain 4 (R-20), domain 7 (GHI/61), and domain 9 (RM3/1). The CD163 monoclonal antibodies were characterized in binding and endocytosis experiments in human macrophages and CD163-transfected Flp-In CHO cells. Calcium-dependent ligand binding was assessed using surface plasmon resonance, and the specificity of the CD163 monoclonal antibodies was analyzed by western blotting.

Results and discussion: Flow cytometric analysis revealed that the estimated proportion of CD163-expressing human peripheral blood monocytes increased when using CD163 monoclonal antibodies recognizing epitopes in the N-terminal part of CD163, remote from the membrane surface. Moreover, the proportion of CD163 positive monocytes observed was highly dependent on free calcium. GHI/61 did not exhibit CD163 binding in the presence of calcium as measured by surface plasmon resonance, which was in agreement with the concordant loss of binding in heparin-stabilized whole blood observed by flow cytometry. In contrast, RM3/1 exhibited weak binding to CD163 in the absence of calcium but high affinity binding to CD163 in the presence of calcium. R-20 and MAC2-158 were unaffected by extracellular calcium levels. The latter SRCR domain 1mAb consistently recognized more than 80% CD163-positive monocytes in human peripheral blood.

Conclusion: Epitope accessibility and extracellular calcium dependence elucidate discrepancies in reported levels of monocytic CD163 expression. Utilizing monoclonal antibodies to the N-terminal part of CD163 more than 80% monocytes in human peripheral blood could be identified as CD163 positive, indicating that most, and conceivably all, human peripheral blood monocytes do express CD163.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / analysis
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism
  • Antigens, CD / blood
  • Antigens, CD / chemistry
  • Antigens, CD / genetics
  • Antigens, CD / immunology*
  • Antigens, Differentiation, Myelomonocytic / blood
  • Antigens, Differentiation, Myelomonocytic / chemistry
  • Antigens, Differentiation, Myelomonocytic / genetics
  • Antigens, Differentiation, Myelomonocytic / immunology*
  • Biomarkers / analysis*
  • Biomarkers / chemistry
  • Blotting, Western
  • CHO Cells
  • Calcium / chemistry
  • Calcium / metabolism
  • Cell Membrane / chemistry
  • Cell Membrane / immunology*
  • Cell Membrane / metabolism
  • Cricetinae
  • Cricetulus
  • Epitopes / blood
  • Epitopes / chemistry
  • Epitopes / genetics
  • Epitopes / immunology*
  • Flow Cytometry
  • Gene Expression
  • Humans
  • Macrophages / cytology
  • Macrophages / immunology*
  • Macrophages / metabolism
  • Monocytes / cytology
  • Monocytes / immunology*
  • Monocytes / metabolism
  • Observer Variation
  • Plasmids
  • Protein Structure, Tertiary
  • Receptors, Cell Surface / blood
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / immunology*
  • Surface Plasmon Resonance
  • Transfection


  • Antibodies, Monoclonal
  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Biomarkers
  • CD163 antigen
  • Epitopes
  • Receptors, Cell Surface
  • Calcium