© 1990 Oxford University Press 0953-8178/90 $3.00
International Immunology, Vol. 2, No. 10
Distribution of the CD68 macrophage/myeloid associated antigen Karen A. F. Pulford, Adrien Sipos1, Jacqueline L. Cordell, W. Paul Stross, and David Y. Mason The Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, UK 1 3rd Department of Medicine, University Medical School, Debrecen 4004, Hungary Key words: macrophage, granulocytes, immunohistochemistry, antigens, CD68
The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoletlc cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences In glycosylatlon. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphold and non-haemopoletlc cells means that care should be taken when using them for the diagnosis of tumours of unknown origin. Introduction
Methods
A number of monoclonal antibodies have been raised which share the common characteristic of reacting with human monocytes and macrophages. Studies reported at the Fourth International Workshop on Human Leucocyte Differentiation Antigens indicated that six monoclonal antibodies with this reactivity pattern (antibodies Y1/82A, Y2/131, KP1, Ki-M6, Ki-M7 and EBM11) are all directed against the same molecule, CD68, a 110 kd glycoprotein (1). This specificity was demonstrated both by immunoprecipitation and immunoaffinity purification studies, and also by staining cells transfected with a cDNA clone encoding the antigen. The CD68 antigen is found within the cytoplasm of human macrophages and monocytes, and several laboratories have used these antibodies as selective markers of this cell type (2 - 4). However, their reactivity with other cell types has not been explored in detail and in the present paper we report a immunocytochemical study of this sort. We show that the CD68 antigen is present not only in several other white cell types (e.g. large granular lymphocytes, basophils), but also in non-haemopoietic tissues (e.g. renal glomeruli) and in serum and urine. These experiments showed that the antibodies which define the CD68 antigen, although clearly recognizing the same molecule, differ subtly in their immunocytochemical staining patterns. This suggests that the CD68 antigen may be a more heterogeneous entity than has previously been suspected, possibly because of variations in glycosylation between different cell types.
Haematological samples Normal leucocytes from heparinized peripheral blood were separated on Lymphoprep (Nycomed, Oslo). T cells were removed from the mononuclear cell preparation as described by Gelsthorpe and Doughty (5). The remaining mononuclear cells were then incubated on fibronectin coated surfaces (6). The nonadherent B cell enriched population was poured off and the adherent monocytes were removed with EDTA (0.02 g/1). Neutrophils were obtained from normal peripheral blood by dextran sedimentation (6% wt/v, Fisons). Cytocentrifuge preparations were made of the original mononuclear cells and of the other cell preparations. In some experiments mononuclear cells were cultured on Formvar coated slides for 24 h in RPMI 1640 containing 10% FCS (Gibco Biocult Ltd). Buffy coat smears were prepared from the peripheral blood of normal donors and patents with chronic myeloid leukaemia attending the Haematology Department, John Radcliffe Hospital, Oxford. All cytocentrifuge preparations and smears were air-dried, wrapped in aluminium foil, stored at -20°C, and fixed prior to use as previously described (7). Cell lines The following cell lines were obtained from the Sir William Dunn School of Pathology, Oxford: K562 (erythroleukaemia), HeLa
Correspondence to. K. A. F. Pulford Transmitting editor: E. Simpson
Received 21 May 1990, accepted 16 July 1990
Downloaded from http://intimm.oxfordjournals.org/ at University of Arizona on December 17, 2014
Abstract
974
Distribution of the CD68 antigen
(cervical epithelial carcinoma), U937 (malignant histiocytosis), HL60 (promyelocytic leukaemia), RVH421 (melanoma), A431 (vulval carcinoma), Daudi and Raji (both Burkitt's lymphoma), and Molt-4 (Tcell acute lymphoblastic leukaemia). The cell lines L428 (Hodgkin's disease), SU-DHL-1 (a T cell lymphoma with a 2;5 chromosomal translocation), and KATO (gastric carcinoma), and umbilical endothelial cells were obtained from Dr V. Diehl, Dr M. Clearly, Prof. P. M. Comoglio, and the Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, respectively. All lines were cutured in RPMI 1640 and 10% FCS at 37°C. Cytocentrifuge preparations were made and treated as previously described. Tissue sections
Immunocytochemical labelling Antibodies. The CD68 monoclonal antibodies used in the present study comprised Y1/82A, Y2/131, KP1 (all raised in the authors' own laboratory; 3,9), EBM11 (4), and Ki-M6 and Ki-M7 (both obtained via the Fourth Workshop on Leucocyte Differentiation Antigens; 2,10). A monoclonal anti-myeloperoxidase antibody MPO-7 (Dakopatts) was also used. All antibodies were used, unless otherwise stated, as undiluted tissue culture supernatants, with the exception of Ki-M6 and Ki-M7, which were obtained in the form of ascitic fluid and were diluted 1/100 and 1/10,000 in TBS (0.5 M Tns, pH 7.6, diluted 1:10 in 0.15 M saline) respectively. Monoclonal APAAP immune complexes were prepared in the authors' own laboratory (8). Immunoenzymatic technique. Cytocentrifuge preparations were fixed for 30 s in BFA (11). These slides and the tissue sections were then stained using the APAAP technique (7,8). The endogenous myeloperoxidase activity of monocytes and neutrophils was demonstrated in some experiments by incubation in 0.6 mg/ml DAB (Sigma) and 0.001% H2O2 for 8 min immediately prior to APAAP staining. For double immunocytochemical labelling, cells were incubated with the monoclonal antibody MPO-7 followed by peroxidase-conjugated anti-mouse Ig. The peroxidase reaction was then developed using 0.6 mg/ml DAB and 0.01% H2O2 in TBS before APAAP staining was carried out (12). Neurammidase treatment. Cryostat sections of tonsil were incubated for 30 mm at room temperature in neuraminidase Type X (0.05 units/ml in phosphate buffered saline containing 3% bovine serum albumin, Sigma Chemical Co.). The slides were then washed in TBS and stained with the CD68 panel using the APAAP technique. Immunoaffinity purification and Western blotting Supernatants and cells were collected from the cell lines U937, A431, and RVH421. Cell pellets were lysed in 20 mM Tris (pH 7.5) containing 1 % Nonidet P40 (NP40), 1 mM iodoacetamide, 0.2 mM phenylmethyl sulphoryl fluoride, 0.5% sodium azide,
Results CD68 antigen in cells of haemopoietic origin The immunocytochemical staining patterns of CD68 monoclonal antibodies on different tissues are summarized in Table 1. The results were all obtained using a range of dilutions of each antibody to exclude differences due to sub-saturating concentrations of antibody. All of the antibodies reacted strongly with macrophages in various tissues, as shown in Figure 1(a and b) Antibody Ki-M6 appeared to label fewer macrophages than other CD68 antibodies in tissues such as thymus, kidney, and pancreas. The HL60 histiocytic cell line was also strongly positive for CD68 (Fig. 1c). The reactivity patterns of four of the CD68 monoclonal antibodies on normal peripheral blood white cells are summarized in Table 2. Monocytes. All of the antibodies labelled the cytoplasm of monocytes in a granular pattern (Fig. 2a). In cytospin preparations labelling tended to be strongest close to the concave border of the nucleus. Neutrophils. Antibody KP1 stained neutrophils very strongly (Fig. 2a) while Y2/131 and EBM11 reacted only weakly with these cells (Fig. 2b). No neutrophils were stained by antibody Y1/82A and none of the CD68 antibodies labelled eosinophils. Lymphocytes. Each of the four antibodies labelled a few intracytoplasmic granules in the majority of large lymphocytes (Fig. 2c). This staining was not detectable, however, in preparations of mononuclear cell suspensions which had been kept overnight at 4°C before cytocentrifugatjon. Basophils. A small population (
International Immunology, Vol. 2, No. 10
Distribution of the CD68 macrophage/myeloid associated antigen Karen A. F. Pulford, Adrien Sipos1, Jacqueline L. Cordell, W. Paul Stross, and David Y. Mason The Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, UK 1 3rd Department of Medicine, University Medical School, Debrecen 4004, Hungary Key words: macrophage, granulocytes, immunohistochemistry, antigens, CD68
The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoletlc cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences In glycosylatlon. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphold and non-haemopoletlc cells means that care should be taken when using them for the diagnosis of tumours of unknown origin. Introduction
Methods
A number of monoclonal antibodies have been raised which share the common characteristic of reacting with human monocytes and macrophages. Studies reported at the Fourth International Workshop on Human Leucocyte Differentiation Antigens indicated that six monoclonal antibodies with this reactivity pattern (antibodies Y1/82A, Y2/131, KP1, Ki-M6, Ki-M7 and EBM11) are all directed against the same molecule, CD68, a 110 kd glycoprotein (1). This specificity was demonstrated both by immunoprecipitation and immunoaffinity purification studies, and also by staining cells transfected with a cDNA clone encoding the antigen. The CD68 antigen is found within the cytoplasm of human macrophages and monocytes, and several laboratories have used these antibodies as selective markers of this cell type (2 - 4). However, their reactivity with other cell types has not been explored in detail and in the present paper we report a immunocytochemical study of this sort. We show that the CD68 antigen is present not only in several other white cell types (e.g. large granular lymphocytes, basophils), but also in non-haemopoietic tissues (e.g. renal glomeruli) and in serum and urine. These experiments showed that the antibodies which define the CD68 antigen, although clearly recognizing the same molecule, differ subtly in their immunocytochemical staining patterns. This suggests that the CD68 antigen may be a more heterogeneous entity than has previously been suspected, possibly because of variations in glycosylation between different cell types.
Haematological samples Normal leucocytes from heparinized peripheral blood were separated on Lymphoprep (Nycomed, Oslo). T cells were removed from the mononuclear cell preparation as described by Gelsthorpe and Doughty (5). The remaining mononuclear cells were then incubated on fibronectin coated surfaces (6). The nonadherent B cell enriched population was poured off and the adherent monocytes were removed with EDTA (0.02 g/1). Neutrophils were obtained from normal peripheral blood by dextran sedimentation (6% wt/v, Fisons). Cytocentrifuge preparations were made of the original mononuclear cells and of the other cell preparations. In some experiments mononuclear cells were cultured on Formvar coated slides for 24 h in RPMI 1640 containing 10% FCS (Gibco Biocult Ltd). Buffy coat smears were prepared from the peripheral blood of normal donors and patents with chronic myeloid leukaemia attending the Haematology Department, John Radcliffe Hospital, Oxford. All cytocentrifuge preparations and smears were air-dried, wrapped in aluminium foil, stored at -20°C, and fixed prior to use as previously described (7). Cell lines The following cell lines were obtained from the Sir William Dunn School of Pathology, Oxford: K562 (erythroleukaemia), HeLa
Correspondence to. K. A. F. Pulford Transmitting editor: E. Simpson
Received 21 May 1990, accepted 16 July 1990
Downloaded from http://intimm.oxfordjournals.org/ at University of Arizona on December 17, 2014
Abstract
974
Distribution of the CD68 antigen
(cervical epithelial carcinoma), U937 (malignant histiocytosis), HL60 (promyelocytic leukaemia), RVH421 (melanoma), A431 (vulval carcinoma), Daudi and Raji (both Burkitt's lymphoma), and Molt-4 (Tcell acute lymphoblastic leukaemia). The cell lines L428 (Hodgkin's disease), SU-DHL-1 (a T cell lymphoma with a 2;5 chromosomal translocation), and KATO (gastric carcinoma), and umbilical endothelial cells were obtained from Dr V. Diehl, Dr M. Clearly, Prof. P. M. Comoglio, and the Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, respectively. All lines were cutured in RPMI 1640 and 10% FCS at 37°C. Cytocentrifuge preparations were made and treated as previously described. Tissue sections
Immunocytochemical labelling Antibodies. The CD68 monoclonal antibodies used in the present study comprised Y1/82A, Y2/131, KP1 (all raised in the authors' own laboratory; 3,9), EBM11 (4), and Ki-M6 and Ki-M7 (both obtained via the Fourth Workshop on Leucocyte Differentiation Antigens; 2,10). A monoclonal anti-myeloperoxidase antibody MPO-7 (Dakopatts) was also used. All antibodies were used, unless otherwise stated, as undiluted tissue culture supernatants, with the exception of Ki-M6 and Ki-M7, which were obtained in the form of ascitic fluid and were diluted 1/100 and 1/10,000 in TBS (0.5 M Tns, pH 7.6, diluted 1:10 in 0.15 M saline) respectively. Monoclonal APAAP immune complexes were prepared in the authors' own laboratory (8). Immunoenzymatic technique. Cytocentrifuge preparations were fixed for 30 s in BFA (11). These slides and the tissue sections were then stained using the APAAP technique (7,8). The endogenous myeloperoxidase activity of monocytes and neutrophils was demonstrated in some experiments by incubation in 0.6 mg/ml DAB (Sigma) and 0.001% H2O2 for 8 min immediately prior to APAAP staining. For double immunocytochemical labelling, cells were incubated with the monoclonal antibody MPO-7 followed by peroxidase-conjugated anti-mouse Ig. The peroxidase reaction was then developed using 0.6 mg/ml DAB and 0.01% H2O2 in TBS before APAAP staining was carried out (12). Neurammidase treatment. Cryostat sections of tonsil were incubated for 30 mm at room temperature in neuraminidase Type X (0.05 units/ml in phosphate buffered saline containing 3% bovine serum albumin, Sigma Chemical Co.). The slides were then washed in TBS and stained with the CD68 panel using the APAAP technique. Immunoaffinity purification and Western blotting Supernatants and cells were collected from the cell lines U937, A431, and RVH421. Cell pellets were lysed in 20 mM Tris (pH 7.5) containing 1 % Nonidet P40 (NP40), 1 mM iodoacetamide, 0.2 mM phenylmethyl sulphoryl fluoride, 0.5% sodium azide,
Results CD68 antigen in cells of haemopoietic origin The immunocytochemical staining patterns of CD68 monoclonal antibodies on different tissues are summarized in Table 1. The results were all obtained using a range of dilutions of each antibody to exclude differences due to sub-saturating concentrations of antibody. All of the antibodies reacted strongly with macrophages in various tissues, as shown in Figure 1(a and b) Antibody Ki-M6 appeared to label fewer macrophages than other CD68 antibodies in tissues such as thymus, kidney, and pancreas. The HL60 histiocytic cell line was also strongly positive for CD68 (Fig. 1c). The reactivity patterns of four of the CD68 monoclonal antibodies on normal peripheral blood white cells are summarized in Table 2. Monocytes. All of the antibodies labelled the cytoplasm of monocytes in a granular pattern (Fig. 2a). In cytospin preparations labelling tended to be strongest close to the concave border of the nucleus. Neutrophils. Antibody KP1 stained neutrophils very strongly (Fig. 2a) while Y2/131 and EBM11 reacted only weakly with these cells (Fig. 2b). No neutrophils were stained by antibody Y1/82A and none of the CD68 antibodies labelled eosinophils. Lymphocytes. Each of the four antibodies labelled a few intracytoplasmic granules in the majority of large lymphocytes (Fig. 2c). This staining was not detectable, however, in preparations of mononuclear cell suspensions which had been kept overnight at 4°C before cytocentrifugatjon. Basophils. A small population (
