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Antigens expressed predominantly on monocytes and granulocytes: identification of bovine CD11b and CD11c
Veterinary Immunology and hnmunopathology, 27 ( 1991 ) 87-90
87
Elsevier Science Publishers B.V., Amsterdam
4.9 Antigens expressed predominantly on rnonocytes and granulocytes: identification of bovine CD 11 b and CD 11 c G. Splitter~ and W.I. Morrison b aUniversity of ~I'isconsin, Madison, Wi 53706, USA bILRAD, P.O. Box 30709, Nairobi, Kenya
INTRODUCTION
The mononuclear phagocytic system comprises a broad group of cells found in the circulation or fixed within various tissues throughout the body. Cells of this lineage include blood monocytes, the Langerhans cells of skin epidermis, Kupffer cells of the liver, pulmonary alveolar macrophages, osteoclasts and interdigitating or dendritic cells in solid lymphoid tissues. Differentiation in particular microenvironments may result in the expression of certain antigens and not others. Further, antigens present on cells of this lineage may be expressed on other cell types suggesting a common functional importance of the molecules in myeloid or both myeloid and lymphoid differentiation. Nine monoclonal antibodies (mAbs) which react predominantly with monocytes and granulocytes were identified. Only four of these reagents gave results in immunoprecipitation studies. Moreover, since cell lines of the myelomonocytic lineage were not available, the clustering data for these mAbs were less definitive than for mAbs which recognised lymphocyte antigens. Nevertheless, the results of the workshop studies indicated that one mAb was specific for the bovine equivalent of CD 11b and three mAbs were specific for the homologue of CD l I c. FLOW CYTOMETRIC ANALYSES
The lineage-specificity of the mAbs was evaluated by immunoflucrescence staining and flow cytometric analysis of whole peripheral blood mononuclear cells (PBM), monocyte depleted PBM, purified monocytes and granulocytes. Monocyte-depleted PBM and purified monocytes were prepared as described previously (Goddeeris et al., 1986 ). In addition, as shown in Appendix 9.3a, the antibodies were tested on a number of antigen-specific or transformed 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
88
G. SPLITTER AND W.l. MORRISON
TABLE l Specificities of monoclonal antibodies which react predominantly with monocytes and granulocytes Monoclonai antibody
Isotype Reactivity with different cell types Monocytes Granulocytes Lymphocytes
Molecule immunoprecipitated
Proposed specificity
95/160 95/150 95/150 95/150 -
C D I lb CDIIc ~llc C D I lc
Resting Activated IL-AI5 C5B6 IL-AI6 NAM4 I L-A46 IL-A24 46-66 CH 16A CH 137A
G~ G~ G~ Gt M Gt Gi M M
+ + + + + + + + +
+ + + + + + . . .
+/.
. .
. .
-~ +/+/+/+ /+/. .
.
. .
t Reacted with one lymphoblastoid cell line derived from a leukaemic animal.
lymphoblastoid cell lines. The results of these analyses revealed four different patterns of staining (Table l ). One mAb, IL-A 15, reacted strongly with monocytes and granulocytes, and, in some animals, with a small subpopulation oflymphocytes. With the exception of one cell line derived from a BLV-infected animal, IL-A 15 did not react with activated lymphocytes. Independent studies (N.D. MacHugh and J. Naessens, unpublished data) have shown that the population of lymphocytes in PBM recognised by IL-AI5 are B cells. A further four mAbs, C5B6 (Eskra et al_, !99! )~ !L-A!6, NAM 4 and llJ-A4fi, reacted with manaeytes~ less strongly with granulocytes and were negative on resting blood lymphocytes. These antibodies reacted with activated antigen-specific T lymphocytes. It is of note that IL-A16 did not react with any cells in the PBM of one animal, suggesting that it may recognise a polymorphic epitope on the target antigen. Only mAb, IL-A24, showed similar specificity to the abov~"group of four mAbs when tested on resting leukocyte populations, but on the activated lymphocytic cell lines reacted predominantly with cell lines derived from BLVinfected cattle. The remaining four mAbs reacted only with monocytes but did not react with the cells of all cattle and in many animals gave weak staining. TISSUE DISTRIBUTION
In sections of lymphoid tissues stained by immunoperoxidase, mAb IL-A 15 gave intense staining of cells in the splenic marginal zones and in the intestinal lamina propria. Many of these cells appeared round in profile and were medium to small in size. In addition, IL-A 15 stained large dendritic-shaped
89
IDENTIFICATION OF BOVINE CDI lb AND CD! lc
cells in the T dependent areas of secondary lymphoid tissues, namely the periarteriolar regions of the spleen, the paracortex of the lymph nodes and the interfollicular areas of the Peyer's patches and tonsil. Similar staining was also observed in germinal centres. In the thymus, staining of large irregular cells was observed, predominantly in the medulla but with a few positive cells also in the cortex. MAb C5B6, IL-A16, NAM4 and IL-A46, stained large dendritic-like cells found mainly in the T-dependent areas of lyr~lphoid tissues. Numerous positive cells were observed in the splenic periarteriolar regions, paracortical areas of lymph nodes, interfollicular areas of Peyer's patches and intestinal lamina propria. There was no staining of B cell follicles. In the thymus the large positive cells were confined entirely to the medulla. The pattern of staining with mAb IL-A24 was similar to that observed with the above group of four mAbs except that positive cells were less numerous and were more focally distributed in the T dependent areas of lymph nodes, spleen and Peyer's patches. The remaining three mAbs gave weak or undetectable staining of the lymphoid tissues examined and thus the tissue distribution of positive cells could not be interpreted.
123456 -
200
-
92
/
68
-
46
m
30
Fig. 1. Autoradiograph of immunopredpitates analysed by SDS-PAGE under reducing conditions. Immunoprecipitation was carried out on detergent-solubilised lysates of purified blood monocytes surface-labelled with '251. Lane l contains precipitates obtained with a control IgG~, antibody. Lanes 2 to 6 contain precipitates obtained with mAbs IL-Al 5, IL-AI 6, IL-A24, C5B6 and NAM4, respectively.
90
G. SPLITTER AND W.I. MORRISON
IMMUNOPRECIPITATION
The antigens detected by four of the mAbs were identified by immunoprecipitation from surface-labelled cells (Fig. 1 ). MAb IL-AI 5 detected molecules of 95 kDa and 160 kDa when precipitates were run on SDS-PAGE under reducing conditions. A further three mAbs, C5B6, IL-AI6 and NAM4 detected an antigen composed of 95 kDa and 150 kDa components. CONCLUSIONS
The results of flow cytometric analyses, immunohistology and immunoprecipitation indicate that the antigen identified by IL-A 15 is the bovine homologue ofCD1 lb and that identified by C5B6, IL-AI6 and NAM4 is the homologue of CD 1 lc (for reviews of human CD 11 antigens see Hogg, 1989; Kishimoto et al., 1989). MAb IL-A46 may also be specific for CD 11c but it failed to immunoprecipitate. The tissue distribution and expression on activated lymphocytes of the antigens identified IL-A24 was different from that of the above five mAbs. Although the target antigen was not identified by immunoprecipitation, previous studies indicate that IL-A24 recognises a 74/ 100 kDa dimeric molecule (Ellis et al., 1988 ). This antigen does not have an obvious homologue in man. The remaining three mAbs, although they were apparently specific for monocytes, varied markedly in their reactivity with cells from different cattle, and in many instances were venq weak or completely negative. These observations may reflect polymorphi=m of the antigens. Immunoprecipitation of the antigens and definition of their tissue distribution were also unsuccessful. REFERENCES Ellis, J.A., Davis, W.C., MacHugh, N.D., Emery, D.L., Kaushal, A. and Morrison, W.I., 1988. Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies. Vet. Immunol. Immunopathol., 19: 325-340. Eskra, L., O'Reilly, K. and Splitter, G.A., 1991. The bovine p 150/95 molecule (CD 11c/CD 18 ) functions in primary cell-cell interaction. Vet. Immunol. Immunopathol., in press. Goddeeris, B.M., Baldwin, C.L., ole MoiYoi, O. and Morrison, W.I., 1986. Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. Functional evaluation of monocytes in responses to lectins. J. Immunol. Methods, 89:165173. Hogg, N., 1989. The leukocyte integrins. Immunol. Today, 10: I I l-114. Kishimoto, T.K., Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E. and Springer, T.A., 1989. The leukocyte integrins. Adv. Immunol., 46:149-182.
87
Elsevier Science Publishers B.V., Amsterdam
4.9 Antigens expressed predominantly on rnonocytes and granulocytes: identification of bovine CD 11 b and CD 11 c G. Splitter~ and W.I. Morrison b aUniversity of ~I'isconsin, Madison, Wi 53706, USA bILRAD, P.O. Box 30709, Nairobi, Kenya
INTRODUCTION
The mononuclear phagocytic system comprises a broad group of cells found in the circulation or fixed within various tissues throughout the body. Cells of this lineage include blood monocytes, the Langerhans cells of skin epidermis, Kupffer cells of the liver, pulmonary alveolar macrophages, osteoclasts and interdigitating or dendritic cells in solid lymphoid tissues. Differentiation in particular microenvironments may result in the expression of certain antigens and not others. Further, antigens present on cells of this lineage may be expressed on other cell types suggesting a common functional importance of the molecules in myeloid or both myeloid and lymphoid differentiation. Nine monoclonal antibodies (mAbs) which react predominantly with monocytes and granulocytes were identified. Only four of these reagents gave results in immunoprecipitation studies. Moreover, since cell lines of the myelomonocytic lineage were not available, the clustering data for these mAbs were less definitive than for mAbs which recognised lymphocyte antigens. Nevertheless, the results of the workshop studies indicated that one mAb was specific for the bovine equivalent of CD 11b and three mAbs were specific for the homologue of CD l I c. FLOW CYTOMETRIC ANALYSES
The lineage-specificity of the mAbs was evaluated by immunoflucrescence staining and flow cytometric analysis of whole peripheral blood mononuclear cells (PBM), monocyte depleted PBM, purified monocytes and granulocytes. Monocyte-depleted PBM and purified monocytes were prepared as described previously (Goddeeris et al., 1986 ). In addition, as shown in Appendix 9.3a, the antibodies were tested on a number of antigen-specific or transformed 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
88
G. SPLITTER AND W.l. MORRISON
TABLE l Specificities of monoclonal antibodies which react predominantly with monocytes and granulocytes Monoclonai antibody
Isotype Reactivity with different cell types Monocytes Granulocytes Lymphocytes
Molecule immunoprecipitated
Proposed specificity
95/160 95/150 95/150 95/150 -
C D I lb CDIIc ~llc C D I lc
Resting Activated IL-AI5 C5B6 IL-AI6 NAM4 I L-A46 IL-A24 46-66 CH 16A CH 137A
G~ G~ G~ Gt M Gt Gi M M
+ + + + + + + + +
+ + + + + + . . .
+/.
. .
. .
-~ +/+/+/+ /+/. .
.
. .
t Reacted with one lymphoblastoid cell line derived from a leukaemic animal.
lymphoblastoid cell lines. The results of these analyses revealed four different patterns of staining (Table l ). One mAb, IL-A 15, reacted strongly with monocytes and granulocytes, and, in some animals, with a small subpopulation oflymphocytes. With the exception of one cell line derived from a BLV-infected animal, IL-A 15 did not react with activated lymphocytes. Independent studies (N.D. MacHugh and J. Naessens, unpublished data) have shown that the population of lymphocytes in PBM recognised by IL-AI5 are B cells. A further four mAbs, C5B6 (Eskra et al_, !99! )~ !L-A!6, NAM 4 and llJ-A4fi, reacted with manaeytes~ less strongly with granulocytes and were negative on resting blood lymphocytes. These antibodies reacted with activated antigen-specific T lymphocytes. It is of note that IL-A16 did not react with any cells in the PBM of one animal, suggesting that it may recognise a polymorphic epitope on the target antigen. Only mAb, IL-A24, showed similar specificity to the abov~"group of four mAbs when tested on resting leukocyte populations, but on the activated lymphocytic cell lines reacted predominantly with cell lines derived from BLVinfected cattle. The remaining four mAbs reacted only with monocytes but did not react with the cells of all cattle and in many animals gave weak staining. TISSUE DISTRIBUTION
In sections of lymphoid tissues stained by immunoperoxidase, mAb IL-A 15 gave intense staining of cells in the splenic marginal zones and in the intestinal lamina propria. Many of these cells appeared round in profile and were medium to small in size. In addition, IL-A 15 stained large dendritic-shaped
89
IDENTIFICATION OF BOVINE CDI lb AND CD! lc
cells in the T dependent areas of secondary lymphoid tissues, namely the periarteriolar regions of the spleen, the paracortex of the lymph nodes and the interfollicular areas of the Peyer's patches and tonsil. Similar staining was also observed in germinal centres. In the thymus, staining of large irregular cells was observed, predominantly in the medulla but with a few positive cells also in the cortex. MAb C5B6, IL-A16, NAM4 and IL-A46, stained large dendritic-like cells found mainly in the T-dependent areas of lyr~lphoid tissues. Numerous positive cells were observed in the splenic periarteriolar regions, paracortical areas of lymph nodes, interfollicular areas of Peyer's patches and intestinal lamina propria. There was no staining of B cell follicles. In the thymus the large positive cells were confined entirely to the medulla. The pattern of staining with mAb IL-A24 was similar to that observed with the above group of four mAbs except that positive cells were less numerous and were more focally distributed in the T dependent areas of lymph nodes, spleen and Peyer's patches. The remaining three mAbs gave weak or undetectable staining of the lymphoid tissues examined and thus the tissue distribution of positive cells could not be interpreted.
123456 -
200
-
92
/
68
-
46
m
30
Fig. 1. Autoradiograph of immunopredpitates analysed by SDS-PAGE under reducing conditions. Immunoprecipitation was carried out on detergent-solubilised lysates of purified blood monocytes surface-labelled with '251. Lane l contains precipitates obtained with a control IgG~, antibody. Lanes 2 to 6 contain precipitates obtained with mAbs IL-Al 5, IL-AI 6, IL-A24, C5B6 and NAM4, respectively.
90
G. SPLITTER AND W.I. MORRISON
IMMUNOPRECIPITATION
The antigens detected by four of the mAbs were identified by immunoprecipitation from surface-labelled cells (Fig. 1 ). MAb IL-AI 5 detected molecules of 95 kDa and 160 kDa when precipitates were run on SDS-PAGE under reducing conditions. A further three mAbs, C5B6, IL-AI6 and NAM4 detected an antigen composed of 95 kDa and 150 kDa components. CONCLUSIONS
The results of flow cytometric analyses, immunohistology and immunoprecipitation indicate that the antigen identified by IL-A 15 is the bovine homologue ofCD1 lb and that identified by C5B6, IL-AI6 and NAM4 is the homologue of CD 1 lc (for reviews of human CD 11 antigens see Hogg, 1989; Kishimoto et al., 1989). MAb IL-A46 may also be specific for CD 11c but it failed to immunoprecipitate. The tissue distribution and expression on activated lymphocytes of the antigens identified IL-A24 was different from that of the above five mAbs. Although the target antigen was not identified by immunoprecipitation, previous studies indicate that IL-A24 recognises a 74/ 100 kDa dimeric molecule (Ellis et al., 1988 ). This antigen does not have an obvious homologue in man. The remaining three mAbs, although they were apparently specific for monocytes, varied markedly in their reactivity with cells from different cattle, and in many instances were venq weak or completely negative. These observations may reflect polymorphi=m of the antigens. Immunoprecipitation of the antigens and definition of their tissue distribution were also unsuccessful. REFERENCES Ellis, J.A., Davis, W.C., MacHugh, N.D., Emery, D.L., Kaushal, A. and Morrison, W.I., 1988. Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies. Vet. Immunol. Immunopathol., 19: 325-340. Eskra, L., O'Reilly, K. and Splitter, G.A., 1991. The bovine p 150/95 molecule (CD 11c/CD 18 ) functions in primary cell-cell interaction. Vet. Immunol. Immunopathol., in press. Goddeeris, B.M., Baldwin, C.L., ole MoiYoi, O. and Morrison, W.I., 1986. Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. Functional evaluation of monocytes in responses to lectins. J. Immunol. Methods, 89:165173. Hogg, N., 1989. The leukocyte integrins. Immunol. Today, 10: I I l-114. Kishimoto, T.K., Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E. and Springer, T.A., 1989. The leukocyte integrins. Adv. Immunol., 46:149-182.