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CD18 panel report for swine CD workshop
Veterinary immunology
.
~ ~~ - ~ : ~
E LS EV l E R
~d immunopatholo~
Veterinary Immunology and Immunopathology 43 (1994) 289-291
CD 11/CD 18 panel report for swine CD workshop Y o o n Berm K i m a'*, Jie Zhang a, William C. Davis b, Joan K. L u n n e y c aDepartment of Microbiology and lmmunology, University of Health Sciences~The Chicago Medical School, North Chicago, IL 60064, USA b Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA CHelminthic Diseases Laboratory, LPSI, ARS, USDA, Building 1040, Room 2, Beltsville, MD 20705, USA
Abstract
Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W ~078), MUC93A (W ~079) and MHM23 (W ~ 136) of CD 11 / 18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding o f mAb by flow cytometric analysis based on PNK-I as CD 18 epitope defining antibody resuited H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD 18a mAbs.
1. Introduction The human leucocyte adhesion molecules: LFA-1 ( C D l l a / C D 1 8 ) , Mac-I (CDI l b / C D 1 8 ) , and P150, 95 (CDI I c / C D I 8 ) are a group of three non-covalently linked ol/fl heterodimer glycoprotein molecules composed of distinct asubunits with molecular weights of 180 kDa (CD 11 a, LFA- I c~ ), 170 kDa (CD 11 b, Mac-l~ or CR3a) and 150 kDa (CDI lc, P 1 5 0 / 9 5 a or CR4~) and a common fl-subunit (CD18) of 95 kDa and they are a part of a larger group of intergins superfamily (Sanchez-Madrid et al., 1983; Tamkun et al., 1986; Kishimoto et al., 1987 ). The biologic importance of these CD 11/CD 18 molecules is demonstrated by the fact that patients having a genetic deficiency of these molecules succumb. to serious illness related to leucocyte impairment (Anderson and Springer, 1987 ) Porcine natural killer (NK) inhibitory mAb (PNK-I, W#037; MHM23, * Corresponding author 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-2427 (94) 06038-2
290
Y.B. Kim et al. / Veterinary Immunology and Immunopathology 43 (1994) 289-291
W # 136 ) have been reported (Pescovitz et al., 1988; D ato and Kim, 1990). PNKI was shown to inhibit porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) (Dato and Kim, 1990). These CD-18 mAb bind virtually all PBL showing a bimodal distribution with 74% 'dim' and 15% 'bright', and monocytes and granulocytes stain with an intermediate intensity with > 90% staining positively. By cell sorting, PNK-I ÷ 'bright' lymphocytes contain all detectable and inducible NK and ADCC activities in porcine PBL that are large granular lymphocytes, and PNK-I + 'dim' cells were devoid of all baseline as well as inducible NK and ADCC activities (Dato and Kim, 1990). PNK-I immunoprecipitates molecules of 166, 155, 95 kDa under non-reducing and reducing conditions. PNKoI recognizes an epitope on CD 18 molecules (fl chain of CD 11 a, b, c) as determined by epitope mapping with competition experiment with a wellcharacterized cross-reactive anti-CD 18 mAb MHM23 (W#136 ) that recognizes the common//-chain (CD18 ) on human leucocytes (Hildreth et al., 1989; Dato and Kim, 1990). Another mAb, H20A (W#077) reported to be pan leucocyte antigen, crossreacts with pig, cow, horse and human PBL (Davis et al., 1984), and two other mAbs MUC76A (W#078) and MUC93A (W#079) showed CDI 1/CD18 type reactivity in the first round analysis.
2. Results and conclusion
All five mAbs (PNK-I, H20A, MUC76A, MUC93A and MHM23) o f C D 1 1 / 18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies were performed on CD 18 by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD 18 epitope defining antibody. PBMC or PMN were preincubated with a saturating amount of each mAb ( 100#1 of culture supernatant per 1 × l 0 6 cells per tube) for 45 min on ice, washed with PBS twice and incubated with 1/tg of biotinylated PNK-I (BioPNK-I) for 30 min on ice, washed, incubated with streptavidin-phycoerythrin (SPE) for 30 min on ice and washed and analyzed for PNK-I binding. Results in Table 1 showed that H20A and MHM23 almost completely blocked binding of PNK-I indicating that H20A and MHM23 bind to the same shared epitope as PNK-I which will now be assigned the epitope CD18a (Table 1 ). However, MUC76A and MUC93A did not block binding of PNK-I indicating that MUC76A and MUC93A may be recognizing distinct epitopes on CD 18, recognizing CD 11 molecules, or reacting with other molecules. Indeed, the pattern of labeling obtained by flow cytometry with MUC93A is clearly different from the pattern obtained with PNK-I; this indicates it does not recognize CD 18. Further studies with immunoprecipitation experiments with non-reduced/reduced SDS-PAGE analysis are needed to define whether MUC76A and MUC93A recognizes CD 11 a, b, c or CD 18 molecules. In conclusion, of five CD 11/CD 18 panel mAbs analyzed, three (PNK-I, H20A and MHM23 ) were designated as CD18a.
Y.B. Kim et al. / Veterinary Immunology and Immunopathology 43 (1994) 289-291
291
Table 1 Flow cytometric analysis of mAb epitope binding competition experiments on porcine leucocytes Workshop #
1st mAb
2nd mAb
Percent binding
Results (epitope defined)
037 077 078 079 136
MOPC (IgGl.x) MOPC PNK-I H20A MUC76A MUC93A MHM23
MOPC BioPNK-I/SPE BioPNK-I/SPE BioPNK-1/SPE BioPNK-1/SPE BioPNK-I/SPE BioPNK-I/SPE
0.78 96.04 7.10 1.92 94.84 94.40 6.70
(Isotype control) (Positive control) CD 18a CD18a No a Distinct b CD 18a
NO, no inhibition o f C D l 8a mAb. b Different pattern from CDI 1/CD18 and no inhibition ofCD18a mAb. a
References D.C. Anderson and T.A. Springer (1987), Ann. Rev. Med. 38, 175. M.E. Dato and Y.B. Kim (1990), J. Immunol. 144, 4452. W.C. Davis, L.E. Perryman, T.C. McGuire (1984), in Hybridoma Technology in Agricultural and Veterinary Research, N.J. Stern and H.R. Gamble, Eds., (Rowman and Allanheld, Lanham, MD), pp. 121-150. J.E.K. Hildreth, V. Holt, J.T. August, M.D. Pescovitz, Mol. Immunol. (1989) 26, 883. T.K. Kishimoto, K. O'Conner, A. Lee, T.M. Roberts, T.A. Springer (1987), Cell 48, 681. M.D. Pescovitz, M.A. Lowman, D.H. Sachs ( 1988 ), Immunology 65, 267. F. Sanchez-Madrid, J.A. Nagy, E. Robbins, P. Simon, T.A. Springer (1983), J. Exp. Med. 158, 1785. J.W. Tamkun et al. (1986), Cell 46, 271.
.
~ ~~ - ~ : ~
E LS EV l E R
~d immunopatholo~
Veterinary Immunology and Immunopathology 43 (1994) 289-291
CD 11/CD 18 panel report for swine CD workshop Y o o n Berm K i m a'*, Jie Zhang a, William C. Davis b, Joan K. L u n n e y c aDepartment of Microbiology and lmmunology, University of Health Sciences~The Chicago Medical School, North Chicago, IL 60064, USA b Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA CHelminthic Diseases Laboratory, LPSI, ARS, USDA, Building 1040, Room 2, Beltsville, MD 20705, USA
Abstract
Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W ~078), MUC93A (W ~079) and MHM23 (W ~ 136) of CD 11 / 18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding o f mAb by flow cytometric analysis based on PNK-I as CD 18 epitope defining antibody resuited H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD 18a mAbs.
1. Introduction The human leucocyte adhesion molecules: LFA-1 ( C D l l a / C D 1 8 ) , Mac-I (CDI l b / C D 1 8 ) , and P150, 95 (CDI I c / C D I 8 ) are a group of three non-covalently linked ol/fl heterodimer glycoprotein molecules composed of distinct asubunits with molecular weights of 180 kDa (CD 11 a, LFA- I c~ ), 170 kDa (CD 11 b, Mac-l~ or CR3a) and 150 kDa (CDI lc, P 1 5 0 / 9 5 a or CR4~) and a common fl-subunit (CD18) of 95 kDa and they are a part of a larger group of intergins superfamily (Sanchez-Madrid et al., 1983; Tamkun et al., 1986; Kishimoto et al., 1987 ). The biologic importance of these CD 11/CD 18 molecules is demonstrated by the fact that patients having a genetic deficiency of these molecules succumb. to serious illness related to leucocyte impairment (Anderson and Springer, 1987 ) Porcine natural killer (NK) inhibitory mAb (PNK-I, W#037; MHM23, * Corresponding author 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-2427 (94) 06038-2
290
Y.B. Kim et al. / Veterinary Immunology and Immunopathology 43 (1994) 289-291
W # 136 ) have been reported (Pescovitz et al., 1988; D ato and Kim, 1990). PNKI was shown to inhibit porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) (Dato and Kim, 1990). These CD-18 mAb bind virtually all PBL showing a bimodal distribution with 74% 'dim' and 15% 'bright', and monocytes and granulocytes stain with an intermediate intensity with > 90% staining positively. By cell sorting, PNK-I ÷ 'bright' lymphocytes contain all detectable and inducible NK and ADCC activities in porcine PBL that are large granular lymphocytes, and PNK-I + 'dim' cells were devoid of all baseline as well as inducible NK and ADCC activities (Dato and Kim, 1990). PNK-I immunoprecipitates molecules of 166, 155, 95 kDa under non-reducing and reducing conditions. PNKoI recognizes an epitope on CD 18 molecules (fl chain of CD 11 a, b, c) as determined by epitope mapping with competition experiment with a wellcharacterized cross-reactive anti-CD 18 mAb MHM23 (W#136 ) that recognizes the common//-chain (CD18 ) on human leucocytes (Hildreth et al., 1989; Dato and Kim, 1990). Another mAb, H20A (W#077) reported to be pan leucocyte antigen, crossreacts with pig, cow, horse and human PBL (Davis et al., 1984), and two other mAbs MUC76A (W#078) and MUC93A (W#079) showed CDI 1/CD18 type reactivity in the first round analysis.
2. Results and conclusion
All five mAbs (PNK-I, H20A, MUC76A, MUC93A and MHM23) o f C D 1 1 / 18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies were performed on CD 18 by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD 18 epitope defining antibody. PBMC or PMN were preincubated with a saturating amount of each mAb ( 100#1 of culture supernatant per 1 × l 0 6 cells per tube) for 45 min on ice, washed with PBS twice and incubated with 1/tg of biotinylated PNK-I (BioPNK-I) for 30 min on ice, washed, incubated with streptavidin-phycoerythrin (SPE) for 30 min on ice and washed and analyzed for PNK-I binding. Results in Table 1 showed that H20A and MHM23 almost completely blocked binding of PNK-I indicating that H20A and MHM23 bind to the same shared epitope as PNK-I which will now be assigned the epitope CD18a (Table 1 ). However, MUC76A and MUC93A did not block binding of PNK-I indicating that MUC76A and MUC93A may be recognizing distinct epitopes on CD 18, recognizing CD 11 molecules, or reacting with other molecules. Indeed, the pattern of labeling obtained by flow cytometry with MUC93A is clearly different from the pattern obtained with PNK-I; this indicates it does not recognize CD 18. Further studies with immunoprecipitation experiments with non-reduced/reduced SDS-PAGE analysis are needed to define whether MUC76A and MUC93A recognizes CD 11 a, b, c or CD 18 molecules. In conclusion, of five CD 11/CD 18 panel mAbs analyzed, three (PNK-I, H20A and MHM23 ) were designated as CD18a.
Y.B. Kim et al. / Veterinary Immunology and Immunopathology 43 (1994) 289-291
291
Table 1 Flow cytometric analysis of mAb epitope binding competition experiments on porcine leucocytes Workshop #
1st mAb
2nd mAb
Percent binding
Results (epitope defined)
037 077 078 079 136
MOPC (IgGl.x) MOPC PNK-I H20A MUC76A MUC93A MHM23
MOPC BioPNK-I/SPE BioPNK-I/SPE BioPNK-1/SPE BioPNK-1/SPE BioPNK-I/SPE BioPNK-I/SPE
0.78 96.04 7.10 1.92 94.84 94.40 6.70
(Isotype control) (Positive control) CD 18a CD18a No a Distinct b CD 18a
NO, no inhibition o f C D l 8a mAb. b Different pattern from CDI 1/CD18 and no inhibition ofCD18a mAb. a
References D.C. Anderson and T.A. Springer (1987), Ann. Rev. Med. 38, 175. M.E. Dato and Y.B. Kim (1990), J. Immunol. 144, 4452. W.C. Davis, L.E. Perryman, T.C. McGuire (1984), in Hybridoma Technology in Agricultural and Veterinary Research, N.J. Stern and H.R. Gamble, Eds., (Rowman and Allanheld, Lanham, MD), pp. 121-150. J.E.K. Hildreth, V. Holt, J.T. August, M.D. Pescovitz, Mol. Immunol. (1989) 26, 883. T.K. Kishimoto, K. O'Conner, A. Lee, T.M. Roberts, T.A. Springer (1987), Cell 48, 681. M.D. Pescovitz, M.A. Lowman, D.H. Sachs ( 1988 ), Immunology 65, 267. F. Sanchez-Madrid, J.A. Nagy, E. Robbins, P. Simon, T.A. Springer (1983), J. Exp. Med. 158, 1785. J.W. Tamkun et al. (1986), Cell 46, 271.