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Immunohistochemical study on the reactivity of Workshop monoclonal antibodies with sheep lymph nodes
Veterinary
ELSEVIER
Immunology and lmmunopathology 52 (1996) 393-401
Veterinary immunology and immunopathology
Immunohistochemical study on the reactivity of Workshop monoclonal antibodies with sheep lymph nodes P. Berthon
*,
F. Bernard, M. Olivier, S. Bernard, F. Lantier
INRA, Luhorrcrorre de Pathologic
I+xtieuse
rt Immunologir. FIYMK~
Eyuipe Ge’ne’tiyue et Immunird, 37380 Nou;illy.
Abstract
Of the 302 monoclonal antibodies submitted to the Third Workshop on Ruminant Leukocyte Antigens, 167 have been tested for their reactivity on sheep lymph node sections, according to the APAAP immunohistochemical technique. Of the 57 monoclonal antibodies able to react on ovine tissue sections, only 37 induced a clear and well-contrasted staining. The more striking results are presented in this paper.
1. Introduction
To obtain additional results to the flow cytometry data on Workshop monoclonal antibodies (mAbs), our participation in the Third Workshop on Ruminant Leukocyte Antigens consisted of an immunohistochemical analysis of the reactivity of these mAbs on sheep lymph node sections, using four of the seven antibody panels from the Workshop. Study was performed on normal lymphoid organs and on prescapular lymph nodes removed 6 days after S.C. inoculation of a virulent wild strain of Salmonella abortusouis in sheep (Bernard et al., 1996). At that day, lymph nodes showed the greatest enlargement (5-10 times), caused principally by follicular hyperplasia (P. Berthon, unpublished data, 1995).
Abbreviations: APAAP, alkaline phosphatase anti-alkaline phosphatase; APC, antigen-presenting cells; CD, cluster of differentiation; Ig, immunoglobulin: mAb, monoclonal antibody; NSS, normal sheep serum; TBS. Tris-buffered * Corresponding
saline; WC, Workshop author.
016%2427/96/$15.00 Published PII SOl65-2427(96)05592-4
cluster
by Elsevier Science B.V.
394
P. Berthon et al./
Veterinary Immunology and Immurwpathology
52 11996) 393-401
Immunohistochemical data obtained on Workshop mAbs from Salmonella-infected lymph node sections did not differ from those observed from normal tissues, suggesting that no Workshop mAb was specific for an antigen only expressed by Sulmonelluactivated leukocyte subsets. For this reason, data presented in this paper only relate to the reactivity of Workshop mAbs on normal sheep lymph node sections.
2. Materials and methods 2.1. Animals and tissues Normal prescapular lymph nodes were removed from two healthy, 6-month-old sheep of the Pre’alpes du Sud flock of the Laboratory of Infectious Pathology and Immunology (INRA, Research Centre of Tours). The structure of normal lymph nodes was defined as previously described (Arno, 1980). To simplify descriptions of tissue sections here, interfollicular cortical and paracortical areas were designated as a single lymph node area: the paracortex. 2.2. Immunohistochemicul reagents and antibodies Naphthol AS-TR phosphate (No. N-60), fast red TR salt (No. F-2768), levamisole (No. l-9756) and glycerol gelatin (No. GG-1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit immunoglobulins (Ig) to mouse Ig (2 0259) and complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase antibody (APAAP, D 0651) were obtained from DAK0 (Copenhagen, Denmark) and the Harris-type haematoxylin (3.61073) from RAL (PROLABO, Paris, France). Workshop mAbs were those from four of the seven antibody panels included in the Third Workshop, namely T and B cell, Myeloid and Activation panels. They were stored at +4”C until use and have been tested at the dilution previously defined for flow cytometry studies. Other mouse monoclonal antibodies, specific for some ovine leukocyte antigens, were also used as controls: - SBU-II (28.1, MHC Class II> (Puri et al., 1985); - SBU-Tl (25.91, CD5) and SBU-T6 (20.27, CDl) (Mackay et al., 1985; Dutia and Hopkins, 1991); . SBU-T4 (44.38 + . 97, CD4) and SBU-T8 (38.65, CD8) (Maddox et al., 1985); - SBU-T19 (19.19, y/6 TcR) (Mackay et al., 1989, Mackay et al., 1991); . SBU-~220 (20.96, CD45R) (Mackay et al., 1987; Meeusen et al., 1991); - OMl (CDllc) (Pbpin et al., 1992; Gupta et al., 1993, Gupta et al., 1995); - 0M2 (?I, 0M3 (?> and OM4 (MI-IC Class II-like) (Pe’pin et al., 1992). 2.3. Immunohistochemistry After removal, lymph nodes were wrapped in aluminium foil, frozen in liquid nitrogen and stored at - 70°C until use. Tissue sections of 5 km thickness were made
P. Berthon et al./ Veterinary Immunology and Immunopathology 52 (19%) 393-401
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using an RUA cryostat, mounted on clean slides, air-dried and fixed in acetone at - 20°C for 20 min. Then, after wrapping in aluminium foil, slides were stored at - 70°C until use. After thawing, frozen tissue sections were rehydrated for 15 min with 0.05 M Tris-buffered saline (TBS; pH 7.6). Sections were incubated with the appropriate dilution of monoclonal antibody (100 p,l per section) for 45 min in a humid chamber at room temperature. Then the immunoenzymatic procedure was slightly modified from the APAAP technique previously described (Cordell et al., 1984; Pepin et al., 1993). After three washes in TBS, sections were incubated with a 1:30 dilution of rabbit anti-mouse Ig antibody (lOOp,l per section) in RPMI-1640 (GIBCO, Grand Island, NY, USA; 041-01870) supplemented with 20% normal sheep serum (NSS) (20% NSS-RPM11 for 45 min at room temperature. After three washes in TBS, slides were incubated with a 1:60 dilution of APAAP complexes in 20% NSS-RPM1 (100 pl per section) for 45 min at room temperature. After one wash in TBS (pH 7.6) and one wash in 0.1 M TBS (pH 8.21, the alkaline phosphatase activity was revealed using as enzyme substrate 20mg naphthol AS-TR phosphate, 2ml dimethylformamide, 98ml TBS (pH 8.21, 30mg levamisole and 100 mg fast red TR salt during a 30 min incubation at room temperature in the dark. After washes in tap-water, slides were counter-stained with haematoxylin and mounted in glycerol gelatin. Negative controls were performed using 10% fetal calf serum-RPM1 instead of the monoclonal antibody samples, and no endogenous alkaline phosphatase was detected on sheep lymph node sections. Slides were observed in light microscopy (Leitz Aristoplan microscope, 40 X to 400 X magnification), and blinded descriptions of labelling patterns on tissue sections were given for all Workshop monoclonal antibodies.
3. Results and discussion The immunohistochemical studies have involved 167 Workshop mAbs, including 18 Workshop control Abs. Of the 149 mAbs with an unknown specificity, 43 appeared negative on sheep lymph node sections, 28 showed a very low specificity for ovine leukocytes and 21 labelled all the cells from the tissue sections. Of the remaining 57 mAbs which reacted on sheep lymph node sections, only 37 were able to induce a clear and well-contrasted labelling. Most of these 37 antibodies recognized an antigen expressed by a large number of lymph node leukocytes, such as lymphocytes, antigen-presenting cells (APC: monocytes, macrophages and dendritic cells) and neutrophils, making the establishment of their specificity difficult. Nevertheless, some of them, able to react with a well-defined leukocyte subset and/or showing an identical labelling pattern on tissue sections, appeared noteworthy even in the absence of CD clustering. 3.1. Antibodies specific for a T cell subset: y/ S T cells? Seven mAbs showed a labelling pattern on sheep lymph node sections which could be related to the staining observed with SBU-T19 (y/6 TcR). The mAb BAQ89
3%
P. Berthon et al./ Veterinary Immunology
and Immunopathology 52 (19%) 393-401
Fig. 1. Cryostat sections of a sheep lymph node stained with BAQ89 (A, B), CC188 (C, D) and SC-6 (E. F), at the paracortical (A, C, E) and medullary (B, D, F) levels. B, B follicles in the paracortex; T, paracortical T area(X100).
P. Berthon et al./ Veterinary Immunology and lmmurwpathology 52 (19%) 393-401
Fig. 2. Cryostat sections of a sheep lymph node stained with the anti-CD45R SBVp220 (A) and Ce38D3 at the paracortical level. B, B follicles in the paracortex; T, paracortical T area (X 100).
397
(B),
labelled some T lymphocytes with a similar frequency to SBU-T19: some positive isolated cells in the paracortical T area, most of them being located in the medulla (Fig. l(A), Fig. l(B)). BAQ89 also induced a slight background staining in the medullary area, B follicle germinal centres and on endothelial cells. In comparison with SBU-T19, CC187 stained a lower number of T cells and could be specific for a y/8 T cell subpopulation (data not shown), whereas CC188 bound to a greater cell subpopulation including some neutrophils in both the medulla and paracortex (Fig. l(C), Fig. l(D)). Four mAbs (SC-6, SC-12, BAQ90 and SC-29) showed an identical staining pattern on tissue sections, slightly different from that of SBU-T19. They labelled a slightly
Fig. 3. Cryostat section of a sheep lymph node labelled with DM7. (A) Paracortex; GC, B follicle germinal centre (X 100). (B) Medulla with two positive macrophage-like cells (arrowheads) (X 250).
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P. Berthon et al./ Veterinary Immunology and Immurwpathology 52 (1996) 393-401
higher number of lymphocytes in the paracortex T area and fewer cells in the medulla (Fig. l(E), Fig. l(F)). They also induced a weak background staining in the medulla, B follicle germinal centres and on endothelial cells. 3.2. Antibodies specific for B cells and a T cell subset Two mAbs, Co-46D5 and Co-38D3, showed an identical labelling pattern on sheep lymph node sections. They induced a clear and very specific cellular staining of some isolated lymphocytes located near the subcapsular sinus, in the paracortical T area and in the medulla, and of all the B follicle cells (germinal centre and mantle). The density of positive cells was higher than with the mAb 20.96 (SBU-~220, CD45R) (Fig. 2(A), Fig. 2(B)).
3.3. Antibodies specific for B cells and an APC subset A clear and very specific staining of the B follicle cells (germinal centre and mantle), of few scattered small lymphocytes in the paracortical T area, of isolated lymphocytes and APCs near the subcapsular sinus and in the medullary area was observed with five mAbs: DM7, GB25, Du2-128, DM6 and Du2-87 (Fig. 3(A), Fig. 3(B)). Two others mAbs, Du2-74 and LCT28, showed a similar labelling pattern with a much lower reactivity to sheep lymph node leukocytes, resulting in a weaker and more diffuse staining of tissue sections (data not shown). The spot-pattern labelling observed in the light zone of B follicle germinal centres (Fig. 3(A)) suggested that these mAbs would be able to react with an antigen also possibly expressed by follicular dendritic cells (Lindhout and de Groot, 1995; Maeda et al., 1995).
Fig. 4. Cryostat section of a sheep lymph node stained with 36HlO. (A) Isolated labelled cells in the paracortical T area. B, B follicles in the pamcortex; T, pamcortical T area (X 100). (B) Detail of (A) showing the neutrophil-like cells ( X 250).
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Immunology and Immurwpathology 52 (19%) 393-401
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3.4. Antibodies speci$c for neutrophils The antibody 36HlO induced a clear and well-contrasted staining of a specific sheep lymph node leukocyte subpopulation. Some positive cells were located near the subcapsular sinus and in the medulla, but most of them were gathered in the paracortical T area surrounding the B follicles (Fig. 4(A)). Those cells were characterized by a very refringent and multilobate nucleus, looking like neutrophils (Fig. 4(B)).
3.5. CD14 Three mAbs, VPM65, VPM66 and VPM67, have been raised against ovine CD14
((Iv CD14, Gupta et al., 1996; Hopkins and Gupta, 1996) and were also able to
Fig. 5. Cryostat sections of a sheep lymph node stained with the anti-CD14 VPM65 (A-C) and the related Ab IL-A97 CD). (A) VPM65 staining in the paracortex; GC, B follicle germinal centre ( X 100). (B) Detail of (A) ( X 250). (C) VPM65 stained macrophage-like cells in the medulla (arrowheads) ( X 250). (D) IL-A97 labclled medullq macrophages (arrowhead) (X 400).
400
P. Berthon et al./ Veterinary Immunology and Immurwpathology 52 (1996) 393-401
recognize bovine CD14 (Bo CD14, Sopp et al., 1996). These three mAbs showed an identical labelling pattern on sheep lymph node sections. They mainly stained macrophage-like cells and some neutrophils near the subcapsular sinus, in the paracortical T area surrounding the B follicles (Fig. .5(A), Fig. 5(B)) and in the medulla (Fig. 5(C)). They also cross-reacted weakly with endothelial cells (Fig. 5(A), Fig. 5(B)). The slight spot-pattern labelling seen in the light zone of B follicle germinal centres (Fig. 5(A) suggested that follicular dendritic cells could possibly express a small amount of the CD14 antigen (Lindhout and de Groot, 1995). Related to these mAbs, but not specific for the CD14 molecule (Sopp et al., 1996), IL-A97 also stained many isolated macrophage-like cells and some neutrophils, mainly located in the medullary area of the lymph node (Fig. 5(D)). 3.6. CD71 From our study, no result was obtained with CD71-specific mAbs. IL-A77 showed a low specificity for sheep lymph node leukocytes, resulting in a very weak staining of lymphoblast- and macrophage-like cells located near the medullary sinuses. IL-Al65 appeared as a negative antibody on tissue samples. 3.7. WC12 According to the labelling pattern induced by mAbs from this cluster on ovine tissue sections, no further data was obtained. IL-A81 reacted weakly with ovine lymph node leukocytes, inducing a diffuse cytoplasmic staining of some medullary macrophages and lymphoblasts. CACTl14 was considered as a negative antibody on sheep lymph node sections. 3.8. WC14 It has not been possible to define this cluster for ovine cells. In our study, no staining on tissue sections was observed with either BT3/8.12 or IL-A155.
References Arno J. Atlas of Lymph Node Pathology. Current Histopathology Series, Vol. 1 1980 MTP Press Lancaster, UK. Bernard, S., Olivier, M., Bernard, F., Berthon, P. and Lantier, F., 1996. Flow cytometric analysis of the reactivity of workshop monoclonal antibodies on sheep lymph node cells, after infection with Salmonella abortusouis. Vet. Immunol. Immunopathol., 52: 403-409. Cordell, J.L., Falini, B., Erber, W.N., Ghosh, A.K., Abdulaziz, Z., McDonald, S., Pulford, K.A.F., Stein, H. and Mason, D.Y., 1984. lmmunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem., 32: 219-229. Dutia, B. and Hopkins, J., 1991. Analysis of the CD1 cluster in sheep. Vet. Immunol. Immunopathol., 27: 189- 194.
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Gupta, V.K., McConnell, I. and Hopkins, J., 1993. Reactivity of the CDll/CDlS Workshop monoclonal antibodies in the sheep. Vet. Immunol. Immunopathol., 39: 93-102. Gupta, V.K., McConnell, I., Pgpin, M., Davis, W.C., Dalziel, R.G. and Hopkins, J., 1995. Biochemical and phenotypic characterization of the ovine b2 (leukocyte) integrins. J. Comp. Pathol., 112: 339-349. Gupta, V.K., McConnell, I., Dalziel, R.G. and Hopkins. J., 1996. Identification of the sheep homologue of the monocyte cell surface molecule-CD14. Vet. Immunol. lmmunopathol., 52: 329-339. Hopkins, J. and Gupta, V.K., 1996. Identification of three myeloid-specific differentiation antigens in sheep. Vet. Immunol. lmmunopathol., 00: 000-000. Lindhout. E. and de Groot, C., 1995. Follicular dendritic cells and apoptosis: life and death in the germinal centm. Histochem. J., 27: 167-183. Mackay, C.R., Maddox, J.F., Gogolin-Ewens, K.J. and Brandon, M.R., 1985. Characterization of two sheep lymphocyte differentiation antigens, SBU-Tl and SBUT6. Immunology, 55: 729-737. Mackay, C.R., Maddox, J.F. and Brandon, M.R., 1987. A monoclonal antibody to the ~220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol., 110: 46-55. Mackay, C.R., Beya, M.F. and Matzinger, P., 1989. y/6 T cells express a unique surface molecule appearing late during thymic development. Eur. J. Immunol., 19: 1477- 1483. Mackay, CR., Marston, W.L., Dudler, L. and Hem, W.R., 1991. Expression of the ‘T19’ and ‘null cell’ markers on y6 T cells of the sheep. Vet. Immunol. lmmunopathol., 27: 183-188. Maddox, J.F., Mackay, C.R. and Brandon, M.R., 1985. Surface antigens, SBUT4 and SBU-T8, of sheep T lymphocyte subsets defmed by monoclonal antibodies. Immunology, 55: 739-748. Maeda, K., Kosco-Vilbois, M.H., Burton, G.F., Szakal, A.K. and Tew, J.G., 1995. Expression of the intercellular adhesion molecule- 1 on high endothelial venules and on non-lymphoid antigen handling cells: interdigitating cells, antigen transporting cells and follicular dendritic cells. Cell Tissue Res., 279: 47-54. Meeusen, E., Lee, C.S. and Brandon, M., 1991. Differential migration of T and B cells during an acute inflammatory response. Eur. J. Immunol., 21: 2269-2272. Pepin, M., Cannella, D., Fontaine, J.J., Pittet, J.C. and Le Pape, A., 1992. Ovine mononuclear phagocytes in situ: identification by monoclonal antibodies and involvement in experimental pyogranulomas. J. Leuko. Biol., 51: 188-198. Pepin, M., Bernard, S., Menanteau, P., Olivier, M., Berthon, P. and Lantier, F., 1993. Reactivity of Workshop monoclonal antibodies with normal and pathological ovine lymph nodes. Vet. Immunol. Immunopatbol., 39: 249-267. Puti, N.K., Mackay, C.R. and Brandon, M.R., 1985. Sheep lymphocyte antigens COLA): II-Major histocompatibility complex class II molecules. Immunology, 56: 725-733. Sopp, P., Kwong, L.S. and Howard, C.J., 1996. Identification of bovine CD14. Vet. Immunol. Immunopathol., 52: 323-328.
ELSEVIER
Immunology and lmmunopathology 52 (1996) 393-401
Veterinary immunology and immunopathology
Immunohistochemical study on the reactivity of Workshop monoclonal antibodies with sheep lymph nodes P. Berthon
*,
F. Bernard, M. Olivier, S. Bernard, F. Lantier
INRA, Luhorrcrorre de Pathologic
I+xtieuse
rt Immunologir. FIYMK~
Eyuipe Ge’ne’tiyue et Immunird, 37380 Nou;illy.
Abstract
Of the 302 monoclonal antibodies submitted to the Third Workshop on Ruminant Leukocyte Antigens, 167 have been tested for their reactivity on sheep lymph node sections, according to the APAAP immunohistochemical technique. Of the 57 monoclonal antibodies able to react on ovine tissue sections, only 37 induced a clear and well-contrasted staining. The more striking results are presented in this paper.
1. Introduction
To obtain additional results to the flow cytometry data on Workshop monoclonal antibodies (mAbs), our participation in the Third Workshop on Ruminant Leukocyte Antigens consisted of an immunohistochemical analysis of the reactivity of these mAbs on sheep lymph node sections, using four of the seven antibody panels from the Workshop. Study was performed on normal lymphoid organs and on prescapular lymph nodes removed 6 days after S.C. inoculation of a virulent wild strain of Salmonella abortusouis in sheep (Bernard et al., 1996). At that day, lymph nodes showed the greatest enlargement (5-10 times), caused principally by follicular hyperplasia (P. Berthon, unpublished data, 1995).
Abbreviations: APAAP, alkaline phosphatase anti-alkaline phosphatase; APC, antigen-presenting cells; CD, cluster of differentiation; Ig, immunoglobulin: mAb, monoclonal antibody; NSS, normal sheep serum; TBS. Tris-buffered * Corresponding
saline; WC, Workshop author.
016%2427/96/$15.00 Published PII SOl65-2427(96)05592-4
cluster
by Elsevier Science B.V.
394
P. Berthon et al./
Veterinary Immunology and Immurwpathology
52 11996) 393-401
Immunohistochemical data obtained on Workshop mAbs from Salmonella-infected lymph node sections did not differ from those observed from normal tissues, suggesting that no Workshop mAb was specific for an antigen only expressed by Sulmonelluactivated leukocyte subsets. For this reason, data presented in this paper only relate to the reactivity of Workshop mAbs on normal sheep lymph node sections.
2. Materials and methods 2.1. Animals and tissues Normal prescapular lymph nodes were removed from two healthy, 6-month-old sheep of the Pre’alpes du Sud flock of the Laboratory of Infectious Pathology and Immunology (INRA, Research Centre of Tours). The structure of normal lymph nodes was defined as previously described (Arno, 1980). To simplify descriptions of tissue sections here, interfollicular cortical and paracortical areas were designated as a single lymph node area: the paracortex. 2.2. Immunohistochemicul reagents and antibodies Naphthol AS-TR phosphate (No. N-60), fast red TR salt (No. F-2768), levamisole (No. l-9756) and glycerol gelatin (No. GG-1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit immunoglobulins (Ig) to mouse Ig (2 0259) and complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase antibody (APAAP, D 0651) were obtained from DAK0 (Copenhagen, Denmark) and the Harris-type haematoxylin (3.61073) from RAL (PROLABO, Paris, France). Workshop mAbs were those from four of the seven antibody panels included in the Third Workshop, namely T and B cell, Myeloid and Activation panels. They were stored at +4”C until use and have been tested at the dilution previously defined for flow cytometry studies. Other mouse monoclonal antibodies, specific for some ovine leukocyte antigens, were also used as controls: - SBU-II (28.1, MHC Class II> (Puri et al., 1985); - SBU-Tl (25.91, CD5) and SBU-T6 (20.27, CDl) (Mackay et al., 1985; Dutia and Hopkins, 1991); . SBU-T4 (44.38 + . 97, CD4) and SBU-T8 (38.65, CD8) (Maddox et al., 1985); - SBU-T19 (19.19, y/6 TcR) (Mackay et al., 1989, Mackay et al., 1991); . SBU-~220 (20.96, CD45R) (Mackay et al., 1987; Meeusen et al., 1991); - OMl (CDllc) (Pbpin et al., 1992; Gupta et al., 1993, Gupta et al., 1995); - 0M2 (?I, 0M3 (?> and OM4 (MI-IC Class II-like) (Pe’pin et al., 1992). 2.3. Immunohistochemistry After removal, lymph nodes were wrapped in aluminium foil, frozen in liquid nitrogen and stored at - 70°C until use. Tissue sections of 5 km thickness were made
P. Berthon et al./ Veterinary Immunology and Immunopathology 52 (19%) 393-401
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using an RUA cryostat, mounted on clean slides, air-dried and fixed in acetone at - 20°C for 20 min. Then, after wrapping in aluminium foil, slides were stored at - 70°C until use. After thawing, frozen tissue sections were rehydrated for 15 min with 0.05 M Tris-buffered saline (TBS; pH 7.6). Sections were incubated with the appropriate dilution of monoclonal antibody (100 p,l per section) for 45 min in a humid chamber at room temperature. Then the immunoenzymatic procedure was slightly modified from the APAAP technique previously described (Cordell et al., 1984; Pepin et al., 1993). After three washes in TBS, sections were incubated with a 1:30 dilution of rabbit anti-mouse Ig antibody (lOOp,l per section) in RPMI-1640 (GIBCO, Grand Island, NY, USA; 041-01870) supplemented with 20% normal sheep serum (NSS) (20% NSS-RPM11 for 45 min at room temperature. After three washes in TBS, slides were incubated with a 1:60 dilution of APAAP complexes in 20% NSS-RPM1 (100 pl per section) for 45 min at room temperature. After one wash in TBS (pH 7.6) and one wash in 0.1 M TBS (pH 8.21, the alkaline phosphatase activity was revealed using as enzyme substrate 20mg naphthol AS-TR phosphate, 2ml dimethylformamide, 98ml TBS (pH 8.21, 30mg levamisole and 100 mg fast red TR salt during a 30 min incubation at room temperature in the dark. After washes in tap-water, slides were counter-stained with haematoxylin and mounted in glycerol gelatin. Negative controls were performed using 10% fetal calf serum-RPM1 instead of the monoclonal antibody samples, and no endogenous alkaline phosphatase was detected on sheep lymph node sections. Slides were observed in light microscopy (Leitz Aristoplan microscope, 40 X to 400 X magnification), and blinded descriptions of labelling patterns on tissue sections were given for all Workshop monoclonal antibodies.
3. Results and discussion The immunohistochemical studies have involved 167 Workshop mAbs, including 18 Workshop control Abs. Of the 149 mAbs with an unknown specificity, 43 appeared negative on sheep lymph node sections, 28 showed a very low specificity for ovine leukocytes and 21 labelled all the cells from the tissue sections. Of the remaining 57 mAbs which reacted on sheep lymph node sections, only 37 were able to induce a clear and well-contrasted labelling. Most of these 37 antibodies recognized an antigen expressed by a large number of lymph node leukocytes, such as lymphocytes, antigen-presenting cells (APC: monocytes, macrophages and dendritic cells) and neutrophils, making the establishment of their specificity difficult. Nevertheless, some of them, able to react with a well-defined leukocyte subset and/or showing an identical labelling pattern on tissue sections, appeared noteworthy even in the absence of CD clustering. 3.1. Antibodies specific for a T cell subset: y/ S T cells? Seven mAbs showed a labelling pattern on sheep lymph node sections which could be related to the staining observed with SBU-T19 (y/6 TcR). The mAb BAQ89
3%
P. Berthon et al./ Veterinary Immunology
and Immunopathology 52 (19%) 393-401
Fig. 1. Cryostat sections of a sheep lymph node stained with BAQ89 (A, B), CC188 (C, D) and SC-6 (E. F), at the paracortical (A, C, E) and medullary (B, D, F) levels. B, B follicles in the paracortex; T, paracortical T area(X100).
P. Berthon et al./ Veterinary Immunology and lmmurwpathology 52 (19%) 393-401
Fig. 2. Cryostat sections of a sheep lymph node stained with the anti-CD45R SBVp220 (A) and Ce38D3 at the paracortical level. B, B follicles in the paracortex; T, paracortical T area (X 100).
397
(B),
labelled some T lymphocytes with a similar frequency to SBU-T19: some positive isolated cells in the paracortical T area, most of them being located in the medulla (Fig. l(A), Fig. l(B)). BAQ89 also induced a slight background staining in the medullary area, B follicle germinal centres and on endothelial cells. In comparison with SBU-T19, CC187 stained a lower number of T cells and could be specific for a y/8 T cell subpopulation (data not shown), whereas CC188 bound to a greater cell subpopulation including some neutrophils in both the medulla and paracortex (Fig. l(C), Fig. l(D)). Four mAbs (SC-6, SC-12, BAQ90 and SC-29) showed an identical staining pattern on tissue sections, slightly different from that of SBU-T19. They labelled a slightly
Fig. 3. Cryostat section of a sheep lymph node labelled with DM7. (A) Paracortex; GC, B follicle germinal centre (X 100). (B) Medulla with two positive macrophage-like cells (arrowheads) (X 250).
398
P. Berthon et al./ Veterinary Immunology and Immurwpathology 52 (1996) 393-401
higher number of lymphocytes in the paracortex T area and fewer cells in the medulla (Fig. l(E), Fig. l(F)). They also induced a weak background staining in the medulla, B follicle germinal centres and on endothelial cells. 3.2. Antibodies specific for B cells and a T cell subset Two mAbs, Co-46D5 and Co-38D3, showed an identical labelling pattern on sheep lymph node sections. They induced a clear and very specific cellular staining of some isolated lymphocytes located near the subcapsular sinus, in the paracortical T area and in the medulla, and of all the B follicle cells (germinal centre and mantle). The density of positive cells was higher than with the mAb 20.96 (SBU-~220, CD45R) (Fig. 2(A), Fig. 2(B)).
3.3. Antibodies specific for B cells and an APC subset A clear and very specific staining of the B follicle cells (germinal centre and mantle), of few scattered small lymphocytes in the paracortical T area, of isolated lymphocytes and APCs near the subcapsular sinus and in the medullary area was observed with five mAbs: DM7, GB25, Du2-128, DM6 and Du2-87 (Fig. 3(A), Fig. 3(B)). Two others mAbs, Du2-74 and LCT28, showed a similar labelling pattern with a much lower reactivity to sheep lymph node leukocytes, resulting in a weaker and more diffuse staining of tissue sections (data not shown). The spot-pattern labelling observed in the light zone of B follicle germinal centres (Fig. 3(A)) suggested that these mAbs would be able to react with an antigen also possibly expressed by follicular dendritic cells (Lindhout and de Groot, 1995; Maeda et al., 1995).
Fig. 4. Cryostat section of a sheep lymph node stained with 36HlO. (A) Isolated labelled cells in the paracortical T area. B, B follicles in the pamcortex; T, pamcortical T area (X 100). (B) Detail of (A) showing the neutrophil-like cells ( X 250).
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Immunology and Immurwpathology 52 (19%) 393-401
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3.4. Antibodies speci$c for neutrophils The antibody 36HlO induced a clear and well-contrasted staining of a specific sheep lymph node leukocyte subpopulation. Some positive cells were located near the subcapsular sinus and in the medulla, but most of them were gathered in the paracortical T area surrounding the B follicles (Fig. 4(A)). Those cells were characterized by a very refringent and multilobate nucleus, looking like neutrophils (Fig. 4(B)).
3.5. CD14 Three mAbs, VPM65, VPM66 and VPM67, have been raised against ovine CD14
((Iv CD14, Gupta et al., 1996; Hopkins and Gupta, 1996) and were also able to
Fig. 5. Cryostat sections of a sheep lymph node stained with the anti-CD14 VPM65 (A-C) and the related Ab IL-A97 CD). (A) VPM65 staining in the paracortex; GC, B follicle germinal centre ( X 100). (B) Detail of (A) ( X 250). (C) VPM65 stained macrophage-like cells in the medulla (arrowheads) ( X 250). (D) IL-A97 labclled medullq macrophages (arrowhead) (X 400).
400
P. Berthon et al./ Veterinary Immunology and Immurwpathology 52 (1996) 393-401
recognize bovine CD14 (Bo CD14, Sopp et al., 1996). These three mAbs showed an identical labelling pattern on sheep lymph node sections. They mainly stained macrophage-like cells and some neutrophils near the subcapsular sinus, in the paracortical T area surrounding the B follicles (Fig. .5(A), Fig. 5(B)) and in the medulla (Fig. 5(C)). They also cross-reacted weakly with endothelial cells (Fig. 5(A), Fig. 5(B)). The slight spot-pattern labelling seen in the light zone of B follicle germinal centres (Fig. 5(A) suggested that follicular dendritic cells could possibly express a small amount of the CD14 antigen (Lindhout and de Groot, 1995). Related to these mAbs, but not specific for the CD14 molecule (Sopp et al., 1996), IL-A97 also stained many isolated macrophage-like cells and some neutrophils, mainly located in the medullary area of the lymph node (Fig. 5(D)). 3.6. CD71 From our study, no result was obtained with CD71-specific mAbs. IL-A77 showed a low specificity for sheep lymph node leukocytes, resulting in a very weak staining of lymphoblast- and macrophage-like cells located near the medullary sinuses. IL-Al65 appeared as a negative antibody on tissue samples. 3.7. WC12 According to the labelling pattern induced by mAbs from this cluster on ovine tissue sections, no further data was obtained. IL-A81 reacted weakly with ovine lymph node leukocytes, inducing a diffuse cytoplasmic staining of some medullary macrophages and lymphoblasts. CACTl14 was considered as a negative antibody on sheep lymph node sections. 3.8. WC14 It has not been possible to define this cluster for ovine cells. In our study, no staining on tissue sections was observed with either BT3/8.12 or IL-A155.
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