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Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes
Veterinary Immunology and lmmunopathology, 36 ( 1993 ) 303-318
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Elsevier Science Publishers B.V., Amsterdam
Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes Debra C. Sellon, John M. Cullen, Linda E. Whetter, Douglas H. Gebhard, Leroy Coggins and Frederick J. Fuller Department of Microbiology, Pathology and Parasitology, North Carolina State University College of Veterinary Medicine, 4 700 Hillsborough Street, Raleigh, NC 27606, USA (Accepted 8 June 1992)
ABSTRACT Sellon, D.C., Cullen, J.M., Whetter, L.E., Gebhard, D.H., Coggins, L. and Fuller, F.J., 1993. Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes. Vet. lmmunol. Immunopathol., 36:303-318. An IgGl mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of l ipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins ( 150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes. ABBREVIATIONS EIAV, equine infectious anemia virus; FITC, fluorescein isothiocyanate; PBL, peripheral blood leukocyte; PBS, phosphate buffered saline.
Correspondence to. F.J. Fuller, Department of Microbiology, Pathology and Parasitology, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-2427/93/$06.00
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INTRODUCTION
Compared with the widespread development and use of monoclonal antibodies defining immunologic cell populations in humans and other mammalian species, definition of equine cell populations has not been widely described. The equine mononuclear phagocyte plays an important role in many equine diseases, including equine infectious anemia virus (EIAV) infection (Banks, 1975), equine monocytic ehrlichiosis (Holland et al., 1985) and equine endotoxemia (Burrows, 1981 ) To aid in the study of the pathogenesis of EIAV infection, we developed a monoclonal antibody that reacts with a cytoplasmic antigen of equine mononuclear phagocytic cells. In this paper, we describe a monoclonal antibody and the cell populations it recognizes. We were very interested in a monoclonal antibody marker for equine mononuclear phagocytes in order to address basic questions concerning the tropism of EIAV in the infected horse. Following infection with a virulent strain of EIAV, most horses become febrile and thrombocytopenic within 7-14 days (Issel and Coggins, 1979; Sellon et al., 1992). The febrile episode is associated with a high-titer viremia (Kono, 1973 ). The cellular site of viral replication during acute infection, and hence the source of the high-titer viremia, is not known. Although the virus is widely theorized to replicate in cells of the monocyte/macrophage lineage (McGuire et al., 1971 ), this has never been proven. In this paper, we describe a monoclonal antibody and the cell populations it recognizes. MATERIALS AND METHODS
Ponies and tissues Adult mixed-breed ponies were used as a source of peripheral blood mononuclear cells, peritoneal macrophages, alveolar macrophages and platelets for immunization of mice and screening of hybridoma supernatants. All ponies tested negative for EIAV by agar gel immunodiffusion and Western immunoblot. Ponies were vaccinated against equine herpesvirus I, tetanus and Streptococcus equi. Tissues from two ponies acutely infected with EIAV (Clabough et al., 1991 ) and one uninfected horse were collected at necropsy, fixed overnight in fresh 4% paraformaldehyde in phosphate-buffered saline (PBS), paraffin embedded, cut into 3 or 6 ltm sections and mounted on glass slides.
Separation of peripheral blood rnononuclear cells Colloidal silica particles (Sepracell-MN; Sepratech, Oklahoma City, OK) or Ficoll, type 400 (Histopaque 1077; Sigma Diagnostics, St. Louis, MO) were used to isolate peripheral blood mononuclear cells from blood collected
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by jugular venipuncture. After centrifugation, cells were collected at the appropriate interface and washed twice with PBS. Washed mononuclear cells were plated in 75 cm 2 flasks using RPMI- 1640 media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were allowed to adhere for at least 30 min at 37°C. Non-adherent cells were removed and adherent cells were washed twice with PBS and collected by scraping. Adherent cells were identified as predominantly mononuclear phagocytes (40-80%) based on morphology and non-specific esterase staining. Variable numbers of contaminating B lymphocytes (as determined by surface IgM) and neutrophils (as determined by morphology) were present in the adherent cell fraction, independent of the pony used, the separation m e d i u m used and the time allowed for adherence. Non-adherent cells were > 95% T lymphocytes and B lymphocytes, as determined by indirect immunofluorescence with monoclonal antibodies recognizing those subpopulations (VMRD, Pullman, WA).
Collection of peritoneal and alveolar macrophages Peritoneal fluid was collected by abdominocentesis using an 18 gauge 4 cm needle inserted at the most ventral point of the abdomen. Fluid was collected in EDTA-containing tubes and cells were centrifuged onto glass slides with a cytocentrifuge (Cytospin 2; Shandon, Pittsburgh, PA). Tracheal wash fluid was collected by transtracheal wash using 30 ml of sterile saline. Cells were centrifuged onto glass slides. Cell distribution was determined by morphologic identification of cells stained with a quick Giemsa-type stain (DifQuik; American Scientific Products, McGraw Park, IL).
Platelet isolation Blood was collected by jugular venipuncture into EDTA-containing tubes. Erythrocytes were allowed to settle briefly and plasma was collected and centrifuged at 100 × g to remove leukocytes. Aliquots of platelet-rich plasma were centrifuged at 13 000 Xg in a microcentrifuge to pellet platelets. Platelets were resuspended and washed twice in 9:1 PBS and 0.13 M Na citrate. Washed platelets were counted with an automated cell counter (Coulter).
Immunization and development of monoclonal antibodies Mice (CByD2F1/J mice; The Jackson Laboratory, Bar Harbor, ME) were immunized ip with adherent peripheral blood mononuclear cells. Booster immunizations were given every 2 weeks. Three days after the second booster, a mouse was killed, the spleen removed and a single-cell suspension prepared. Spleen cells were fused with the myeloma line P3-X63.Ag8.653 in polyethyl-
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ene glycol and cultured in HAT m e d i u m (Carter et al., 1986 ). Positive clones were subcultured by limiting dilution and expanded into larger volumes.
Hybridoma screening Two weeks after fusion, supernatants from wells that had growth were screened using an equine peripheral blood leukocyte (PBL) preparation containing lymphocytes, monocytes and granulocytes. These cells were prepared as described above. All adherent cells were collected and counted, and nonadherent cells were added to attain the number of cells necessary to screen all samples. Cells ( 106 in 100/tl) were incubated for 1 h at 4 ° C with 100/tl of fusion supernatant. Cells were washed once with PBS and incubated for 1 h at 4°C with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody that had been pre-absorbed with normal equine lymphoid tissue. Antibody-positive supernatant fluids were identified by flow cytometric analysis with a Becton Dickinson FACScan. Clones that stained an identifiable subpopulation of equine PBL were selected for additional screening by indirect fluorescent antibody testing.
Indirect fluorescent antibody testing Adherent peripheral blood mononuclear cells, tracheal wash fluid, abdominal fluid or platelets were prepared as described above. Aliquots of approximately 10 s cells or 107 platelets were centrifuged onto glass slides with a cyto centrifuge. Cells or platelets were fixed in 4% paraformaldehyde for 10 rain and rinsed with PBS. Slides were stored at 20°C in PBS until tested. Nonspecific binding was blocked by incubation for 10 rain in 10% normal goat serum in PBS. After washing twice for 3 min in PBS, slides were incubated with 100/11 hybridoma supernatant for 30 min at 37°C. Slides were washed twice with PBS and incubated for 10 min at 37°C with a FITC-conjugated goat anti-mouse antibody that had been pre-absorbed with normal equine lymphoid tissue. After washing twice with PBS, slides were counterstained with 0.125% Evan's blue and examined by fluorescence microscopy.
Immunoperoxidase staining of tissue sections Tissue sections were deparaffinized with two changes of xylenes and rehydrated through graded ethanol to PBS. Antibody 1.646 was used as primary antibody. A mouse monoclonal antibody recognizing a canine lymphocyte surface antigen was used as a negative control. Antibody binding to tissue macrophages was detected with a biotin-labeled second antibody, and streptavidin-peroxidase conjugate, utilizing a commercial kit (Histostain SP, Zymed Lab., San Francisco, CA).
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Western immunoblot of equine monocyte/macrophage proteins After 5 days in culture, equine macrophages derived from equine peripheral blood mononuclear cells were lysed and their proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) on 10% gels. The proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and reacted with antibody 1.646 or a control IgG1 mouse monoclonal antibody (CTL3) that recognizes an activation antigen of canine and feline lymphoeytes (D. Gebhard, unpublished data, 1991 ). Reactions were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). RESULTS
Production of monoclonal antibodies to equine mononuclear phagocytes Approximately 750 hybridoma clones were initially screened by flow cytomerry. On the basis of the staining of some subpopulation of equine PBL, 100 clones were selected for indirect immunofluorescence testing. On the basis of the preferential staining of adherent peripheral blood mononuclear cells one clone, designated 1.646, was selected for further characterization. This antibody produced a diffuse granular fluorescence in the cytoplasm of adherent cells (Figs. 1 (A) and 1 (B)). Fluorescence could not be demonstrated on unfixed adherent cells, consistent with the cytoplasmic nature of the antigen recognized by this antibody.
Specificity for equine mononuclear phagocytes Cells fluorescing with antibody 1.646 were distinct from those fluorescing with either anti-equine T cell monoclonal antibodies (HT23a and EqT3, VMRD, Pullman, WA) or anti-equine IgM monoclonal antibody (1.9/3.2 and PIg45A, VMRD, Pullman, WA). Only rare cells (less than 5%) in nonadherent cell fractions fluoresced with antibody 1.646 (Figs. 1 (C) and 1 (D) ). Alveolar macrophages and peritoneal macrophages showed the same pattern of cytoplasmic fluorescence as adherent peripheral blood mononuclear cells (Figs. 2 and 3 ). In abdominal fluid, only cells with macrophage morphology fluoresced consistently (Fig. 2). Neutrophils from abdominal fluid and peripheral blood samples occasionally showed some autofluorescence and/or non-specific binding of antibody. Occasional neutrophils also exhibited specific fluorescence with antibody 1.646. The degree of neutrophil recognition appeared to vary depending on the donor animal from which target cells were collected. In all cases, however, the majority of neutrophils failed to react specifically with antibody 1.646. Respiratory epithelial cells (see the arrows in Fig. 2 ), erythrocytes and platelets did not fluoresce.
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Fig. 1. DifQuik stained cytospin of adherent (A) and non-adherent (C) peripheral blood mononuclear cells. Indirect fluorescent antibody assay of adherent (B) and non-adherent (D) peripheral blood mononuclear cells. Cytoplasmic fluorescence is present in the majority of adherent cells, but not in most non-adherent ceils. Adherent cell cytospins contain a much higher proportion of cells with monocyte/macrophage morphology.
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Fig. 2. Cytoplasmicfluorescenceof equinealveolarmacrophagescollectedby transtrachealwash. Spleen, lymph node, liver, lung and thymus were stained by immunoperoxidase methods using 1.646 as primary antibody (Fig. 4). A lace-like pattern of stain deposition was evident in the cytoplasm of positive cells. These cells were characterized by abundant cytoplasm and a vesicular central to slightly eccentric oval nucleus. Positive cells were most abundant in the spleen and lymph nodes, but were also present in the liver, lung and thymus. In the spleen, positive cells were distributed diffusely in the white pulp, in the periphery of periarteriolar lymphoid aggregates and occasionally in the center of follicles (Fig. 4 ( A ) ) In lymph nodes, positive cells were scattered throughout the interfollicular region and medulla, but were rare in follicles (Fig. 4 (B)). Positive cells found within the lung were primarily within the interstitium and occasionally within the lumen of alveoli (Fig. 4 ( C ) ) . Epithelial cells lining bronchioles and endothelial cells of blood vessels and lymphatics were negative. Staining of cells in the hepatic parenchyma was limited to a small proportion of sinusoidal lining cells with elongate cell bodies that extended into the sinusoidal space to a greater extent than endothelial cells. Owing to these histologic characteristics, positive cells were interpreted to be Kupffer cells. Granular staining of pericanalicular sites was apparent in some horses (see the arrows in Fig. 4(E) ) and much less apparent in other horses (Fig. 4(F) ). This staining was similar in distribution to that seen following the staining of
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Fig. 3. Equineperitonealmacrophagesfluoresceafter indirectfluorescenceassaywith antibody 1.646, but most neutrophilsfrom this horse do not. adjacent hepatic sections for lipofuscin. Pale cytoplasmic staining was occasionally seen in some biliary and renal tubular epithelium (data not shown ), but this was inconsistent and always of less intensity than other cellular staining.
Cross-reactivity in other species Canine, feline and human PBL were purified from venous blood as described above. Cells were plated in 75 cm 2 flasks with RPMI-1640 media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were allowed to adhere for at least 30 min at 37°C. Non-adherent cells were removed and adherent cells washed twice with PBS and collected by scraping. Paraformaldehyde-fixedcytospin samples of adherent and non-adherent cells from each species were tested for reactivity with antibody 1.646 by indirect immunofluorescence as described above. No population of feline, canine or human adherent or non-adherent PBL was found to react with antibody 1.646.
Western immunoblot of equine monocyte/macrophage proteins Monoclonal antibody 1.646 specifically recognized two proteins not recognized by a control monoclonal antibody of the same isotype. The approximate molecular weights of these proteins were 135 and 15 kDa (Fig. 5 ). No further characterization of these proteins was attempted. This demonstrates
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Fig. 5. Western immunoblot of proteins from equine monocyte/macrophages reacted with antibody 1.646 (Lanes A and D), no antibody (Lane B) and a control antibody (CTL3; Lane C) of the same isotype (IgG1). Molecular weight markers are shown in Lane E.
the specific reactivity of antibody 1.646 with proteins from these cells. We did not investigate the presence of these antigens in any other cell types. The reducing conditions of this gel did not allow us to determine whether the protein exists as a molecular complex. Additional experiments are required to more completely characterize this protein. DISCUSSION
A monoclonal antibody, designated 1.646, recognizing a 135 and a 15 kDa cytoplasmic antigen of equine mononuclear phagocytes is described. This Fig. 4. Immunoperoxidase staining of equine tissue samples with monoclonal antibody 1.646. Cell distribution is typical of tissue macrophage distribution in (A) spleen, (B) lymph node, (C) lung, (D) thymus, (E) liver (EIAV infected) and ( F ) liver (uninfected) from a second horse. Two examples of liver sections are shown in order to demonstrate the variable nature of the staining detected from horse to horse and the effect EIAV infection may have on staining properties. Storage granules (pericanalicular) between hepatocytes (see the arrows in E) also stain with this antibody.
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antibody does not react with equine T or B cells, platelets, red blood cells or alveolar epithelial cells. Most horses had a few strongly positive neutrophils, but the percentage of the neutrophil population that stained with antibody 1.646 appeared to vary between animals. This limited specific cross-reactivity with some neutrophils is not surprising. Most monoclonal antibodies recognizing human or other mammalian monocyte/macrophages also react with granulocytes (Ugolini et al., 1980; Springer, 1981; Todd et al., 1981; Bernstein et al., 1982; Dimitriu-Bona et al., 1983), probably owing to their common lineage. Other anti-mononuclear phagocyte antibodies cross-react with various other cells of the immune system (Springer, 1981; Hancock et al., 1983 ). At least one monoclonal antibody (Ki-M6) specifically recognizing a cytoplasmic antigen expressed only in human monocyte/macrophages has been previously described (Parwaresch et al., 1986). The intensity of staining with 1.646 was markedly greater in mature tissue macrophages than in peripheral blood monocyte/macrophages. This is most obvious in comparing the intensity of fluorescence of peripheral blood adherent mononuclear cells, peritoneal macrophages and alveolar macrophages. The differential intensity of staining is possibly due to increased expression of the antigen recognized by 1.646 during the process of macrophage differentiation and/or activation. This theory is supported by the more intense staining seen in tissue sections from two ponies infected with EIAV than in one uninfected pony (Fig. 4 ( F ) ) . This lentivirus replicates to a high titer in cells that stain with antibody 1.646 (Sellon et al., 1992) and EIAV infection results in in vivo monocyte/macrophage activation (Banks, 1975 ). The pattern of tissue staining seen following immunoperoxidase staining with antibody 1.646 is typical of mature tissue macrophage distribution. The antibody appears to stain mononuclear phagocytes of lymph nodes and spleen most consistently. While staining of hepatic Kupffer cells was seen in liver sections from all horses examined, the intensity of staining varied markedly between horses. The same was true for staining of pulmonary macrophages. The granular pericanalicular stair is seen in liver sections was typical of lipofuscin distribution in that tissue. Lipofuscin is oxidized lipid that is stored in hepatic lysosomes. Renal tubular and biliary epithelium are also tissues that contain a high concentration of lysosomes. The cross-reactivity of antibody 1.646 with these cell types may reflect recognition of a lysosomal membrane antigen that is most strongly expressed in mature tissue macrophages. Increased expression of the antigen which antibody 1.646 recognizes with macrophage maturation parallels findings with some enzyme-cytochemical characterizations of mononuclear phagocytes. Specifically, staining for different acid hydrolases increases with cell differentiation. Alveolar macrophages show higher activity for most enzymes localized to lysosomes than do peripheral blood monocytes or peritoneal macrophages (Parwaresch et al., 1981; Radzun et al., 1983). As with 1.646, the human monocyte/macrophage
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marker Ki-M6 shows a pattern of increased reactivity as cells differentiate from blood monocytes into tissue macrophages, both in vivo and in vitro. Electron microscopic studies with monoclonal antibody Ki-M6 demonstrated recognition of a 60 kDa antigen closely associated with lysosomal structures (Parwaresch et al., 1986). The similarities in antigen distribution between antibody Ki-M6 and antibody 1.646 may indicate a common lysosomal origin. This hypothesis could only be confirmed by ultrastructural studies. Antibody 1.646 recognizes a cytoplasmic antigen specific to equine mononuclear phagocytes. The lack of cross-reactivity with other equine cell types makes this antibody particularly useful in the investigation of immunologic phenomena in the horse. It is already proving useful in identifying the in vivo cell tropism of EIAV. Similar studies may also be helpful in elucidating the pathogenesis of other important equine disorders. ACKNOWLEDGMENTS
This work was supported by Public Health Service grant 1K11-AI00963 (DLC), R01-AI24904 from the National Institutes of Health and 91-372046732 from the Department of Agriculture.
REFERENCES Banks, K.L., 1975. Monocyte activation in horses persistently infected with equine infectious anemia virus. Infect. Immun., 12: 1219-1221. Bernstein, I.D., Andrews, R.G., Cohen, S.F. and McMaster, B.E., 1982. Normal and malignant human myelocytic and monocytic cells identified by monoclonal antibodies. J. Immunol., 128: 876-881. Burrows, G.E., 1981. Endotoxemia in the horse. Equine Vet. J., 13: 89-94. Carter P.B., Beegle, K.H. and Gebhard, D.H., 1986. Monoclonal antibodies: clinical uses and potential. In: R. Ford (Editor), Veterinary Clinics of North America, Vol. 16 (6) Saunders, pp. 1171-1179. Dimitriu-Bona, A., Burmester, G.R., Waters, S.J. and Winchester, R.J., 1983. Human mononuclear phagocyte differentiation antigens: I. Patterns of antigenic expression on the surface of human monocytes and macrophages defined by monoclonal antibodies J. Immunol., 130: 145-152. Hancock, W.W., Zola, H. and Atkins, R.C., 1983. Antigenic heterogeneity of human mononuclear phagocytes: Immunohistologic analysis using monoclonal antibodies. Blood, 62:12711279. Holland, C.J., Ristic, M., Cole, A.I., Johnson, P., Baker, G. and Goetz, T., 1985. Isolation, experimental transmission, and characterization of causative agent of Potamac horse fever. Science, 227: 522-524. Issel, C.J. and Coggins, L., 1979. Equine infectious anemia: current knowledge. J. Am. Vet. Med. Assoc., 174: 727-733. Kono, Y.,1973. Recurrences of equine infectious anemia. In: Proc. 3rd Int. Conf. Equine Infectious Diseases, Karger, Basel, pp. 175-186.
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Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1971. Immunofluorescent localization of equine infectious anemia virus in tissue. Am. J. Pathol., 62: 283-292. Parwaresch, M.R., Radzun, H.J. and Dommes, H., 198 I. The homogeneity and monocytic origin of human peritoneal macrophages evidence by comparison of esterase polymorphism. Am. J. Pathol., 102:209-218. Parwaresch, M.R., Radzun, H.J., Kreipe, H., Hansmann, M.L. and Barth, J., 1986. Monocyte/ macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am. J. Pathol., 124: 141-151. Radzun, H.J., Parwaresch, M.R. and Kreipe, H., 1983. Monocytic origin of human alveolar macrophages. J. Histochem. Cytochem., 31: 318-324. Sellon, D.C., Coggins, L. and Fuller, F.J., 1992. Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages but not in peripheral blood monocytes. J. Virol., 66: 5906-5913. Springer, T.A., 1981. Monoclonal antibodies as tools for the study of mononuclear phagocytes. In: Methods for Studying Mononuclear Phagocytes. Academic Press, London, pp. 305-313. Todd, R.F., Nadler, UM. and Schlossman, S.F., 1981. Antigens on human monocytes identified by monocional antibodies. J. Immunol., 126:1435-1442. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D., 1980. Initial characterization of monoclonal antibodies against human monocytes. Proc Natl. Acad. Sci. USA, 77: 67646768.
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Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes Debra C. Sellon, John M. Cullen, Linda E. Whetter, Douglas H. Gebhard, Leroy Coggins and Frederick J. Fuller Department of Microbiology, Pathology and Parasitology, North Carolina State University College of Veterinary Medicine, 4 700 Hillsborough Street, Raleigh, NC 27606, USA (Accepted 8 June 1992)
ABSTRACT Sellon, D.C., Cullen, J.M., Whetter, L.E., Gebhard, D.H., Coggins, L. and Fuller, F.J., 1993. Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes. Vet. lmmunol. Immunopathol., 36:303-318. An IgGl mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of l ipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins ( 150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes. ABBREVIATIONS EIAV, equine infectious anemia virus; FITC, fluorescein isothiocyanate; PBL, peripheral blood leukocyte; PBS, phosphate buffered saline.
Correspondence to. F.J. Fuller, Department of Microbiology, Pathology and Parasitology, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-2427/93/$06.00
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INTRODUCTION
Compared with the widespread development and use of monoclonal antibodies defining immunologic cell populations in humans and other mammalian species, definition of equine cell populations has not been widely described. The equine mononuclear phagocyte plays an important role in many equine diseases, including equine infectious anemia virus (EIAV) infection (Banks, 1975), equine monocytic ehrlichiosis (Holland et al., 1985) and equine endotoxemia (Burrows, 1981 ) To aid in the study of the pathogenesis of EIAV infection, we developed a monoclonal antibody that reacts with a cytoplasmic antigen of equine mononuclear phagocytic cells. In this paper, we describe a monoclonal antibody and the cell populations it recognizes. We were very interested in a monoclonal antibody marker for equine mononuclear phagocytes in order to address basic questions concerning the tropism of EIAV in the infected horse. Following infection with a virulent strain of EIAV, most horses become febrile and thrombocytopenic within 7-14 days (Issel and Coggins, 1979; Sellon et al., 1992). The febrile episode is associated with a high-titer viremia (Kono, 1973 ). The cellular site of viral replication during acute infection, and hence the source of the high-titer viremia, is not known. Although the virus is widely theorized to replicate in cells of the monocyte/macrophage lineage (McGuire et al., 1971 ), this has never been proven. In this paper, we describe a monoclonal antibody and the cell populations it recognizes. MATERIALS AND METHODS
Ponies and tissues Adult mixed-breed ponies were used as a source of peripheral blood mononuclear cells, peritoneal macrophages, alveolar macrophages and platelets for immunization of mice and screening of hybridoma supernatants. All ponies tested negative for EIAV by agar gel immunodiffusion and Western immunoblot. Ponies were vaccinated against equine herpesvirus I, tetanus and Streptococcus equi. Tissues from two ponies acutely infected with EIAV (Clabough et al., 1991 ) and one uninfected horse were collected at necropsy, fixed overnight in fresh 4% paraformaldehyde in phosphate-buffered saline (PBS), paraffin embedded, cut into 3 or 6 ltm sections and mounted on glass slides.
Separation of peripheral blood rnononuclear cells Colloidal silica particles (Sepracell-MN; Sepratech, Oklahoma City, OK) or Ficoll, type 400 (Histopaque 1077; Sigma Diagnostics, St. Louis, MO) were used to isolate peripheral blood mononuclear cells from blood collected
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by jugular venipuncture. After centrifugation, cells were collected at the appropriate interface and washed twice with PBS. Washed mononuclear cells were plated in 75 cm 2 flasks using RPMI- 1640 media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were allowed to adhere for at least 30 min at 37°C. Non-adherent cells were removed and adherent cells were washed twice with PBS and collected by scraping. Adherent cells were identified as predominantly mononuclear phagocytes (40-80%) based on morphology and non-specific esterase staining. Variable numbers of contaminating B lymphocytes (as determined by surface IgM) and neutrophils (as determined by morphology) were present in the adherent cell fraction, independent of the pony used, the separation m e d i u m used and the time allowed for adherence. Non-adherent cells were > 95% T lymphocytes and B lymphocytes, as determined by indirect immunofluorescence with monoclonal antibodies recognizing those subpopulations (VMRD, Pullman, WA).
Collection of peritoneal and alveolar macrophages Peritoneal fluid was collected by abdominocentesis using an 18 gauge 4 cm needle inserted at the most ventral point of the abdomen. Fluid was collected in EDTA-containing tubes and cells were centrifuged onto glass slides with a cytocentrifuge (Cytospin 2; Shandon, Pittsburgh, PA). Tracheal wash fluid was collected by transtracheal wash using 30 ml of sterile saline. Cells were centrifuged onto glass slides. Cell distribution was determined by morphologic identification of cells stained with a quick Giemsa-type stain (DifQuik; American Scientific Products, McGraw Park, IL).
Platelet isolation Blood was collected by jugular venipuncture into EDTA-containing tubes. Erythrocytes were allowed to settle briefly and plasma was collected and centrifuged at 100 × g to remove leukocytes. Aliquots of platelet-rich plasma were centrifuged at 13 000 Xg in a microcentrifuge to pellet platelets. Platelets were resuspended and washed twice in 9:1 PBS and 0.13 M Na citrate. Washed platelets were counted with an automated cell counter (Coulter).
Immunization and development of monoclonal antibodies Mice (CByD2F1/J mice; The Jackson Laboratory, Bar Harbor, ME) were immunized ip with adherent peripheral blood mononuclear cells. Booster immunizations were given every 2 weeks. Three days after the second booster, a mouse was killed, the spleen removed and a single-cell suspension prepared. Spleen cells were fused with the myeloma line P3-X63.Ag8.653 in polyethyl-
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ene glycol and cultured in HAT m e d i u m (Carter et al., 1986 ). Positive clones were subcultured by limiting dilution and expanded into larger volumes.
Hybridoma screening Two weeks after fusion, supernatants from wells that had growth were screened using an equine peripheral blood leukocyte (PBL) preparation containing lymphocytes, monocytes and granulocytes. These cells were prepared as described above. All adherent cells were collected and counted, and nonadherent cells were added to attain the number of cells necessary to screen all samples. Cells ( 106 in 100/tl) were incubated for 1 h at 4 ° C with 100/tl of fusion supernatant. Cells were washed once with PBS and incubated for 1 h at 4°C with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody that had been pre-absorbed with normal equine lymphoid tissue. Antibody-positive supernatant fluids were identified by flow cytometric analysis with a Becton Dickinson FACScan. Clones that stained an identifiable subpopulation of equine PBL were selected for additional screening by indirect fluorescent antibody testing.
Indirect fluorescent antibody testing Adherent peripheral blood mononuclear cells, tracheal wash fluid, abdominal fluid or platelets were prepared as described above. Aliquots of approximately 10 s cells or 107 platelets were centrifuged onto glass slides with a cyto centrifuge. Cells or platelets were fixed in 4% paraformaldehyde for 10 rain and rinsed with PBS. Slides were stored at 20°C in PBS until tested. Nonspecific binding was blocked by incubation for 10 rain in 10% normal goat serum in PBS. After washing twice for 3 min in PBS, slides were incubated with 100/11 hybridoma supernatant for 30 min at 37°C. Slides were washed twice with PBS and incubated for 10 min at 37°C with a FITC-conjugated goat anti-mouse antibody that had been pre-absorbed with normal equine lymphoid tissue. After washing twice with PBS, slides were counterstained with 0.125% Evan's blue and examined by fluorescence microscopy.
Immunoperoxidase staining of tissue sections Tissue sections were deparaffinized with two changes of xylenes and rehydrated through graded ethanol to PBS. Antibody 1.646 was used as primary antibody. A mouse monoclonal antibody recognizing a canine lymphocyte surface antigen was used as a negative control. Antibody binding to tissue macrophages was detected with a biotin-labeled second antibody, and streptavidin-peroxidase conjugate, utilizing a commercial kit (Histostain SP, Zymed Lab., San Francisco, CA).
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Western immunoblot of equine monocyte/macrophage proteins After 5 days in culture, equine macrophages derived from equine peripheral blood mononuclear cells were lysed and their proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) on 10% gels. The proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and reacted with antibody 1.646 or a control IgG1 mouse monoclonal antibody (CTL3) that recognizes an activation antigen of canine and feline lymphoeytes (D. Gebhard, unpublished data, 1991 ). Reactions were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). RESULTS
Production of monoclonal antibodies to equine mononuclear phagocytes Approximately 750 hybridoma clones were initially screened by flow cytomerry. On the basis of the staining of some subpopulation of equine PBL, 100 clones were selected for indirect immunofluorescence testing. On the basis of the preferential staining of adherent peripheral blood mononuclear cells one clone, designated 1.646, was selected for further characterization. This antibody produced a diffuse granular fluorescence in the cytoplasm of adherent cells (Figs. 1 (A) and 1 (B)). Fluorescence could not be demonstrated on unfixed adherent cells, consistent with the cytoplasmic nature of the antigen recognized by this antibody.
Specificity for equine mononuclear phagocytes Cells fluorescing with antibody 1.646 were distinct from those fluorescing with either anti-equine T cell monoclonal antibodies (HT23a and EqT3, VMRD, Pullman, WA) or anti-equine IgM monoclonal antibody (1.9/3.2 and PIg45A, VMRD, Pullman, WA). Only rare cells (less than 5%) in nonadherent cell fractions fluoresced with antibody 1.646 (Figs. 1 (C) and 1 (D) ). Alveolar macrophages and peritoneal macrophages showed the same pattern of cytoplasmic fluorescence as adherent peripheral blood mononuclear cells (Figs. 2 and 3 ). In abdominal fluid, only cells with macrophage morphology fluoresced consistently (Fig. 2). Neutrophils from abdominal fluid and peripheral blood samples occasionally showed some autofluorescence and/or non-specific binding of antibody. Occasional neutrophils also exhibited specific fluorescence with antibody 1.646. The degree of neutrophil recognition appeared to vary depending on the donor animal from which target cells were collected. In all cases, however, the majority of neutrophils failed to react specifically with antibody 1.646. Respiratory epithelial cells (see the arrows in Fig. 2 ), erythrocytes and platelets did not fluoresce.
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Fig. 1. DifQuik stained cytospin of adherent (A) and non-adherent (C) peripheral blood mononuclear cells. Indirect fluorescent antibody assay of adherent (B) and non-adherent (D) peripheral blood mononuclear cells. Cytoplasmic fluorescence is present in the majority of adherent cells, but not in most non-adherent ceils. Adherent cell cytospins contain a much higher proportion of cells with monocyte/macrophage morphology.
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Fig. 2. Cytoplasmicfluorescenceof equinealveolarmacrophagescollectedby transtrachealwash. Spleen, lymph node, liver, lung and thymus were stained by immunoperoxidase methods using 1.646 as primary antibody (Fig. 4). A lace-like pattern of stain deposition was evident in the cytoplasm of positive cells. These cells were characterized by abundant cytoplasm and a vesicular central to slightly eccentric oval nucleus. Positive cells were most abundant in the spleen and lymph nodes, but were also present in the liver, lung and thymus. In the spleen, positive cells were distributed diffusely in the white pulp, in the periphery of periarteriolar lymphoid aggregates and occasionally in the center of follicles (Fig. 4 ( A ) ) In lymph nodes, positive cells were scattered throughout the interfollicular region and medulla, but were rare in follicles (Fig. 4 (B)). Positive cells found within the lung were primarily within the interstitium and occasionally within the lumen of alveoli (Fig. 4 ( C ) ) . Epithelial cells lining bronchioles and endothelial cells of blood vessels and lymphatics were negative. Staining of cells in the hepatic parenchyma was limited to a small proportion of sinusoidal lining cells with elongate cell bodies that extended into the sinusoidal space to a greater extent than endothelial cells. Owing to these histologic characteristics, positive cells were interpreted to be Kupffer cells. Granular staining of pericanalicular sites was apparent in some horses (see the arrows in Fig. 4(E) ) and much less apparent in other horses (Fig. 4(F) ). This staining was similar in distribution to that seen following the staining of
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Fig. 3. Equineperitonealmacrophagesfluoresceafter indirectfluorescenceassaywith antibody 1.646, but most neutrophilsfrom this horse do not. adjacent hepatic sections for lipofuscin. Pale cytoplasmic staining was occasionally seen in some biliary and renal tubular epithelium (data not shown ), but this was inconsistent and always of less intensity than other cellular staining.
Cross-reactivity in other species Canine, feline and human PBL were purified from venous blood as described above. Cells were plated in 75 cm 2 flasks with RPMI-1640 media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were allowed to adhere for at least 30 min at 37°C. Non-adherent cells were removed and adherent cells washed twice with PBS and collected by scraping. Paraformaldehyde-fixedcytospin samples of adherent and non-adherent cells from each species were tested for reactivity with antibody 1.646 by indirect immunofluorescence as described above. No population of feline, canine or human adherent or non-adherent PBL was found to react with antibody 1.646.
Western immunoblot of equine monocyte/macrophage proteins Monoclonal antibody 1.646 specifically recognized two proteins not recognized by a control monoclonal antibody of the same isotype. The approximate molecular weights of these proteins were 135 and 15 kDa (Fig. 5 ). No further characterization of these proteins was attempted. This demonstrates
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Fig. 5. Western immunoblot of proteins from equine monocyte/macrophages reacted with antibody 1.646 (Lanes A and D), no antibody (Lane B) and a control antibody (CTL3; Lane C) of the same isotype (IgG1). Molecular weight markers are shown in Lane E.
the specific reactivity of antibody 1.646 with proteins from these cells. We did not investigate the presence of these antigens in any other cell types. The reducing conditions of this gel did not allow us to determine whether the protein exists as a molecular complex. Additional experiments are required to more completely characterize this protein. DISCUSSION
A monoclonal antibody, designated 1.646, recognizing a 135 and a 15 kDa cytoplasmic antigen of equine mononuclear phagocytes is described. This Fig. 4. Immunoperoxidase staining of equine tissue samples with monoclonal antibody 1.646. Cell distribution is typical of tissue macrophage distribution in (A) spleen, (B) lymph node, (C) lung, (D) thymus, (E) liver (EIAV infected) and ( F ) liver (uninfected) from a second horse. Two examples of liver sections are shown in order to demonstrate the variable nature of the staining detected from horse to horse and the effect EIAV infection may have on staining properties. Storage granules (pericanalicular) between hepatocytes (see the arrows in E) also stain with this antibody.
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antibody does not react with equine T or B cells, platelets, red blood cells or alveolar epithelial cells. Most horses had a few strongly positive neutrophils, but the percentage of the neutrophil population that stained with antibody 1.646 appeared to vary between animals. This limited specific cross-reactivity with some neutrophils is not surprising. Most monoclonal antibodies recognizing human or other mammalian monocyte/macrophages also react with granulocytes (Ugolini et al., 1980; Springer, 1981; Todd et al., 1981; Bernstein et al., 1982; Dimitriu-Bona et al., 1983), probably owing to their common lineage. Other anti-mononuclear phagocyte antibodies cross-react with various other cells of the immune system (Springer, 1981; Hancock et al., 1983 ). At least one monoclonal antibody (Ki-M6) specifically recognizing a cytoplasmic antigen expressed only in human monocyte/macrophages has been previously described (Parwaresch et al., 1986). The intensity of staining with 1.646 was markedly greater in mature tissue macrophages than in peripheral blood monocyte/macrophages. This is most obvious in comparing the intensity of fluorescence of peripheral blood adherent mononuclear cells, peritoneal macrophages and alveolar macrophages. The differential intensity of staining is possibly due to increased expression of the antigen recognized by 1.646 during the process of macrophage differentiation and/or activation. This theory is supported by the more intense staining seen in tissue sections from two ponies infected with EIAV than in one uninfected pony (Fig. 4 ( F ) ) . This lentivirus replicates to a high titer in cells that stain with antibody 1.646 (Sellon et al., 1992) and EIAV infection results in in vivo monocyte/macrophage activation (Banks, 1975 ). The pattern of tissue staining seen following immunoperoxidase staining with antibody 1.646 is typical of mature tissue macrophage distribution. The antibody appears to stain mononuclear phagocytes of lymph nodes and spleen most consistently. While staining of hepatic Kupffer cells was seen in liver sections from all horses examined, the intensity of staining varied markedly between horses. The same was true for staining of pulmonary macrophages. The granular pericanalicular stair is seen in liver sections was typical of lipofuscin distribution in that tissue. Lipofuscin is oxidized lipid that is stored in hepatic lysosomes. Renal tubular and biliary epithelium are also tissues that contain a high concentration of lysosomes. The cross-reactivity of antibody 1.646 with these cell types may reflect recognition of a lysosomal membrane antigen that is most strongly expressed in mature tissue macrophages. Increased expression of the antigen which antibody 1.646 recognizes with macrophage maturation parallels findings with some enzyme-cytochemical characterizations of mononuclear phagocytes. Specifically, staining for different acid hydrolases increases with cell differentiation. Alveolar macrophages show higher activity for most enzymes localized to lysosomes than do peripheral blood monocytes or peritoneal macrophages (Parwaresch et al., 1981; Radzun et al., 1983). As with 1.646, the human monocyte/macrophage
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marker Ki-M6 shows a pattern of increased reactivity as cells differentiate from blood monocytes into tissue macrophages, both in vivo and in vitro. Electron microscopic studies with monoclonal antibody Ki-M6 demonstrated recognition of a 60 kDa antigen closely associated with lysosomal structures (Parwaresch et al., 1986). The similarities in antigen distribution between antibody Ki-M6 and antibody 1.646 may indicate a common lysosomal origin. This hypothesis could only be confirmed by ultrastructural studies. Antibody 1.646 recognizes a cytoplasmic antigen specific to equine mononuclear phagocytes. The lack of cross-reactivity with other equine cell types makes this antibody particularly useful in the investigation of immunologic phenomena in the horse. It is already proving useful in identifying the in vivo cell tropism of EIAV. Similar studies may also be helpful in elucidating the pathogenesis of other important equine disorders. ACKNOWLEDGMENTS
This work was supported by Public Health Service grant 1K11-AI00963 (DLC), R01-AI24904 from the National Institutes of Health and 91-372046732 from the Department of Agriculture.
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Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1971. Immunofluorescent localization of equine infectious anemia virus in tissue. Am. J. Pathol., 62: 283-292. Parwaresch, M.R., Radzun, H.J. and Dommes, H., 198 I. The homogeneity and monocytic origin of human peritoneal macrophages evidence by comparison of esterase polymorphism. Am. J. Pathol., 102:209-218. Parwaresch, M.R., Radzun, H.J., Kreipe, H., Hansmann, M.L. and Barth, J., 1986. Monocyte/ macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am. J. Pathol., 124: 141-151. Radzun, H.J., Parwaresch, M.R. and Kreipe, H., 1983. Monocytic origin of human alveolar macrophages. J. Histochem. Cytochem., 31: 318-324. Sellon, D.C., Coggins, L. and Fuller, F.J., 1992. Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages but not in peripheral blood monocytes. J. Virol., 66: 5906-5913. Springer, T.A., 1981. Monoclonal antibodies as tools for the study of mononuclear phagocytes. In: Methods for Studying Mononuclear Phagocytes. Academic Press, London, pp. 305-313. Todd, R.F., Nadler, UM. and Schlossman, S.F., 1981. Antigens on human monocytes identified by monocional antibodies. J. Immunol., 126:1435-1442. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D., 1980. Initial characterization of monoclonal antibodies against human monocytes. Proc Natl. Acad. Sci. USA, 77: 67646768.