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Recombinant bovine interferon-gamma enhances expression of class I and class II bovine lymphocyte antigens
Veterinary Immunology and Immunopathology, 22 (1989) 379-383 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
379
Recombinant Bovine Interferon-Gamma Enhances Expression of Class I and Class II Bovine Lymphocyte Antigens F. WALRAND 1, F. PICARD 2, K. McCULLOUGH 3, S. MARTINOD 4 and D. LI~VY1'4
1Laboratoire Pathologie et Immunog~ndtique INRA, Ecole Nationale Vdtdrinaire, 94704 Alfort Cedex (France) ~Facultd de Mddecine Cochin, Port Royal, 75013 Paris (France) 3Biovet Unit, Centre de Recherche Ciba-Geigy S.A., 1566 Saint Aubin (FR) (Switzerland) (Accepted 26 January 1989 )
ABSTRACT Walrand, F., Picard, F., McCullough, K., Martinod, S. and L~vy, D., 1989. Recombinant bovine interferon-gamma enhances expression of class I and class II bovine lymphocyte antigens. Vet. Immunol. Immunopathol., 23: 379-383. Recombinant bovine interferon-gamma augments expression of class I and class II histocompatibility antigens on the surface membrane of bovine lymphocytes. Immunofluorescence techniques using a series of monoclonal anti-HLA antibodies demonstrate that this enhancement is detectable as early as 24 h after incubation with rBoIFN, while maximum surface expression is obtained within 3-5 days. A concentration as low as 10 units of rBoIFN is effective. Such results may be useful for characterizing the BoLA gene products.
ABBREVIATIONS BoLA, bovine lymphocyte antigens; HLA, human lymphocyte antigens; MHC, major histocompatibility complex; rBoIFN, recombinant bovine interferon-gamma; FCS, fetal calf serum.
INTRODUCTION
Cattle are an important source of food, and consequently the study of various physiological and pathological processes in cattle is important for economic reasons. Moreover, the bovine species is subject to diseases which may serve as models to human diseases (Spooner et al., 1988). The major histocompatibility complex (MHC) may influence resistance and susceptibility to diseases 4Corresponding author.
0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
380 (Dausset and Svejgaard, 1977), and certainly plays a central role in directing and controlling the immune system. The MHC comprises a series of closely linked genetic loci, for many of which there is considerable polymorphism. Recent studies with human and murine cell lines have shown that exposure to interferon-gamma both enhances HLA-specific mRNA production (Rosa and Fellous, 1984) and induces the de novo synthesis of MHC class I and class II antigens (Collins et al., 1984; Virelizier et al., 1984). In contrast to the human and the mouse, the MHC of cattle, which encodes the bovine lymphocyte antigens (BoLA), is still not well known. In particular, relatively little is known of the BoLA class II antigens (Hoang Xuan et al., 1982; Ldvy, 1988). Thus, it was important to study the effect of rBoIFN on BoLA expression. Our results demonstrate that, after exposure to rBoIFN, bovine peripheral lymphocytes express both enhanced densities of class I and detectable quantities of class II antigens. MATERIALAND METHODS
Cells. The mononuclear cells (lymphocytes and monocytes) from a normal bovine donor were obtained by centrifugation of heparinized venous blood on a Ficoll-Hypaque (Pharmacia) gradient. The bovine BL-3 cell line, kindly provided by Dr. H. Lewin, University of Illinois, U.S.A., was maintained in Leibovitz L. 15 medium supplemented with 10% fetal calf serum. Antibodies. Two monoclonal anti-HLA antibodies were used: W6/32, a monomorphic anti-class I antibody and 2.06, a gift from Prof. D. Charron, recognizing HLA-DR antigens. Both were demonstrated to cross react with either BoLA class I (Chardon et al., 1983) or class II antigens (L~vy, 1988). Immunofluorescence assay. For the experiments, the fresh mononuclear cells or BL-3 cells were incubated at a density of either 106 or 10~ cells/ml, respectively, in flat-bottomed 16 mm diameter multiwell plastic plates (Nunclon) containing either control medium (RPMI 1640 + 10% FCS) or the same medium supplemented with rBoIFN at various concentrations. At different time intervals, aliquots were collected, washed and resuspended cells stained in 50 /A of a 1/100 dilution of monoclonal anti-class I or anti-class II mouse antibodies for 30 min at 4 ° C. After three washings in phosphate-buffered saline, the cells were then incubated in a 1/50 dilution of FITC-labeled antiserum raised against mouse immunoglobulin G (Nordic Laboratories) for 30 min at 4 ° C. The cells were then washed again, and fluorescence was determined with a cytofluorograph. Interferon. Recombinant bovine interferon gamma (rBoIFN), supplied by Ciba-Geigy Limited, Basle, was more than 95% pure as determined by electro-
381 phoresis. I F N titers were determined by virus-induced cytopathic effect inhibition assays in microtiter dishes using M D B K cells challenged with vesicular stomatitis virus (VSV) and expressed in laboratory units based on standards. RESULTS As shown in Table 1, rBoIFN had a pronounced enhancing effect on the membrane expression of BoLA. Under basal conditions (incubation in control medium), only 63 to 77% of normal lymphocytes expressed M H C class I antigens detectable by the broad reactive anti-HLA class I monoclonal antibody, whereas the a n t i - H L A - D R monoclonal antibody poorly reacted with the cells. After incubation with rBoIFN, almost all cells bore detectable class I antigens within 48 h. The kinetics of enhancement of BoLA class II expression was slower, taking 3 to 4 days to reach a maximum of 57%. Table 1 also illustrates that spontaneous expression of BoLA class II antigens was moderate or low but the enhancement of their expression was clearly observed after r B o I F N treatment in repeated experiments. In addition, dose response studies showed that concentrations of r B o I F N as low as 10 u n i t s / m l induced expression of the BoLA up to 98% (class I) or 57% (class II) within 4 days of culture. A r B o I F N concentration of 100 and 1000 u n i t s / m l did not induce obvious increase in maximum intensity of B o L A expression nor in the maximum proportion of antigen-positive cells. The role of r B o I F N was further examined on the bovine continuous cell line BL-3. The expression of BoLA class I and class II antigens was studied with various r B o I F N concentrations and different incubation times. Results of a representative experiment are presented in Table 2. It appeared that rBoIFN induced antigen expression that could be detected as early as day 1 of incubation. The effect of r B o I F N reached its maximum at day 2 and remained at the same level on day 3. This effect was not only noticed on the percentage of positive cells, b u t also on the level of fluorescence intensity (see various peak channels). TABLE 1 Effects of rBoIFN on bovine lymphocyte antigens in normal peripheral blood lymphocytes IFN concentration W6/32 binding (U/ml) 24a 48
72
96
24
48
72
96
0 10 100 1000
75 (78) 98 (124) 98 (119) 95 (124)
73 (76) 91 (125) 89 (117) 86 (115)
13 (29) 26 (43) 27 (46) 27 (41)
35 (28) 49 (53) 45 (52) 43 (33)
32 (29) 57 (48) 49 (42) 44 (36)
31 (25) 53 (49) 45 (41) 43 (43)
63b (58) c 85 (71) 81 (67) 79 (69)
77 (73) 99 (151) 99 (139) 96 (92)
2.06 binding
aCulture time in hours, bpercent positive cells, cPeak channel.
382 TABLE 2 Effects of rBoIFN on bovine lymphocyte antigens in the BL-3 cell line IFN concentration (U/m[)
W6/32 binding 24~
0 10 100 1000
715 87 89 91
(62) c (71) (67) (74)
2.06 binding
48
72
87 (78) 94(105) 93 (122) 95 (124)
91 98 98 99
(92) (123) (125) (128)
24
48
72
56 (51) 61(65) 60 (63) 65 (64)
62 (64) 95 {124) 98(119) 97 (117)
67 99 99 98
(71) {139) (129) (133)
aCulture time in hours, bPercent positive cells, cPeak channel. DISCUSSION
In the present work, rBoIFN has been used to study the expression of bovine MHC antigens in cultured lymphoid cells, rBoIFN increases class I BoLA molecule levels at concentrations far lower than those needed to induce the antiviral state in fibroblast cells (Czarniecki et al., 1986). This enhancement was observed not only in terms of percent of positive cells but also in terms of fluorescence intensity. Class II MHC antigens show a limited tissue distribution in vivo and are involved in the presentation of antigen to T-helper cells. Interferon gamma has been shown to increase the expression of class II genes in many human and murine cells types. In the results reported here, rBoIFN also enhanced BoLA class II antigens as revealed by an anti-HLA-DR monoclonal antibody. Time course studies determined the appropriate time lengths of IFN incubation. BoLA expression on normal peripheral lymphocytes as well as on the BL-3 cell line was fully induced in 48 h by rBoIFN. All inductions were stable for up to 5 days (data not shown). Therefore, 3-day IFN incubation periods were chosen for all experiments, rBoIFN increase of BoLA cell surface expression was less than 2-fold, both in terms of positive cells and fluorescence intensity. This is in accordance with results in the murine H-2 system where a discrepancy was reported between the level of H-2 messenger RNA enhancement by IFN (15-fold) and H-2 cell surface expression (2-fold) {Wong et al., 1984). It would be expected that all bovine cells would express MHC class I antigens. The lower level of normal bovine lymphocytes reacting with W6/32 antibody in the absence of interferon is not due to non-optimal experimental conditions as demonstrated by the level of reactivity with the BL-3 cells, but may reflect a lower density of class I antigens at the surface of some normal cells. On the other hand, the differences observed in the magnitude of response in normal bovine lymphocytes and in the BL-3 cell line may reflect the proportion of cells responding to IFN in the population, or a difference in the IFNreceptor expression.
383
Finally, the use of rBoIFN may provide a useful system for characterizing BoLA class II gene products and for analyzing the mechanism of regulation of the bovine MHC. Certainly the rBoIFN can induce or enhance the expression of BoLA class II antigens. Since this expression is essential for leukocyte communication during antigen presentation by monocytes and B lymphocytes, rBoIFN may prove effective at enhancing antigen-specific immune response. This appears to be the case for human and murine IFN.
REFERENCES Chardon, P., Kalil, J., Leveziel, H., Colombani, J. and Vaiman, M., 1983. Monoclonal antibodies to HLA recognize monomorphic and polymorphic epitopes on BoLA T. Antigens, 22: 62-71. Collins, T., Korman, A.J., Wake, C.T., Boss, J.M., Kappes, D.J., Fiefs, W., Ault, K.A., Gimbrone, M.A., Strominger, J.L. and Pober, J.S., 1984. Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 81: 4917-4921. Czarniecki, C., Hamilton, E.B., Fennie, C. and Wolf, R.L., 1986. In vitro biological activities of Escherichia coli derived bovine interferon. J. Interferon. Res., 6: 29-37. Dausset, J. and Svejgaard, A., 1977. HLA and Disease. Munksgaard, Copenhagen. Hoang-Xuan, M., Charron, D., Zilber, M.T. and Levy, D., 1982. Biochemical characterization of class II bovine major histocompatibility complex antigens using cross-species reactive antibodies. Immunogenetics, 15: 621-624. L~vy, D., 1988. BoLA class II antigens: structure, biosynthesis and polymorphism. Anim. Genet., 19: 26-28. Rosa, F. and Fellous, M., 1984. The effect of gamma-interferon on MHC antigens. Immunol. Today, 5: 261-263. Spooner, R.L., Teale, A.J. and Cullen, P., 1988. The MHC of cattle and sheep. In: R. Pandey (Editor), Moving Frontiers in Veterinary Immunology. Karger, Basle, pp. 88-107. Virelizier, J.L., Perez, N., Arenzana-Seisdedos, F. and Devos, R., 1984. Pure interferon gamma enhances class II HLA antigens on human monocyte cell lines. Eur. J. Immunol., 14: 106-108. Wong, G.H.W., Bartlett, P.F., Clark-Lewis, I., Battye, F. and Schrader, J.W., 1984. Inducible expression of H-2 and Ia-antigens on brain cells. Nature, 310: 688-691.
379
Recombinant Bovine Interferon-Gamma Enhances Expression of Class I and Class II Bovine Lymphocyte Antigens F. WALRAND 1, F. PICARD 2, K. McCULLOUGH 3, S. MARTINOD 4 and D. LI~VY1'4
1Laboratoire Pathologie et Immunog~ndtique INRA, Ecole Nationale Vdtdrinaire, 94704 Alfort Cedex (France) ~Facultd de Mddecine Cochin, Port Royal, 75013 Paris (France) 3Biovet Unit, Centre de Recherche Ciba-Geigy S.A., 1566 Saint Aubin (FR) (Switzerland) (Accepted 26 January 1989 )
ABSTRACT Walrand, F., Picard, F., McCullough, K., Martinod, S. and L~vy, D., 1989. Recombinant bovine interferon-gamma enhances expression of class I and class II bovine lymphocyte antigens. Vet. Immunol. Immunopathol., 23: 379-383. Recombinant bovine interferon-gamma augments expression of class I and class II histocompatibility antigens on the surface membrane of bovine lymphocytes. Immunofluorescence techniques using a series of monoclonal anti-HLA antibodies demonstrate that this enhancement is detectable as early as 24 h after incubation with rBoIFN, while maximum surface expression is obtained within 3-5 days. A concentration as low as 10 units of rBoIFN is effective. Such results may be useful for characterizing the BoLA gene products.
ABBREVIATIONS BoLA, bovine lymphocyte antigens; HLA, human lymphocyte antigens; MHC, major histocompatibility complex; rBoIFN, recombinant bovine interferon-gamma; FCS, fetal calf serum.
INTRODUCTION
Cattle are an important source of food, and consequently the study of various physiological and pathological processes in cattle is important for economic reasons. Moreover, the bovine species is subject to diseases which may serve as models to human diseases (Spooner et al., 1988). The major histocompatibility complex (MHC) may influence resistance and susceptibility to diseases 4Corresponding author.
0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
380 (Dausset and Svejgaard, 1977), and certainly plays a central role in directing and controlling the immune system. The MHC comprises a series of closely linked genetic loci, for many of which there is considerable polymorphism. Recent studies with human and murine cell lines have shown that exposure to interferon-gamma both enhances HLA-specific mRNA production (Rosa and Fellous, 1984) and induces the de novo synthesis of MHC class I and class II antigens (Collins et al., 1984; Virelizier et al., 1984). In contrast to the human and the mouse, the MHC of cattle, which encodes the bovine lymphocyte antigens (BoLA), is still not well known. In particular, relatively little is known of the BoLA class II antigens (Hoang Xuan et al., 1982; Ldvy, 1988). Thus, it was important to study the effect of rBoIFN on BoLA expression. Our results demonstrate that, after exposure to rBoIFN, bovine peripheral lymphocytes express both enhanced densities of class I and detectable quantities of class II antigens. MATERIALAND METHODS
Cells. The mononuclear cells (lymphocytes and monocytes) from a normal bovine donor were obtained by centrifugation of heparinized venous blood on a Ficoll-Hypaque (Pharmacia) gradient. The bovine BL-3 cell line, kindly provided by Dr. H. Lewin, University of Illinois, U.S.A., was maintained in Leibovitz L. 15 medium supplemented with 10% fetal calf serum. Antibodies. Two monoclonal anti-HLA antibodies were used: W6/32, a monomorphic anti-class I antibody and 2.06, a gift from Prof. D. Charron, recognizing HLA-DR antigens. Both were demonstrated to cross react with either BoLA class I (Chardon et al., 1983) or class II antigens (L~vy, 1988). Immunofluorescence assay. For the experiments, the fresh mononuclear cells or BL-3 cells were incubated at a density of either 106 or 10~ cells/ml, respectively, in flat-bottomed 16 mm diameter multiwell plastic plates (Nunclon) containing either control medium (RPMI 1640 + 10% FCS) or the same medium supplemented with rBoIFN at various concentrations. At different time intervals, aliquots were collected, washed and resuspended cells stained in 50 /A of a 1/100 dilution of monoclonal anti-class I or anti-class II mouse antibodies for 30 min at 4 ° C. After three washings in phosphate-buffered saline, the cells were then incubated in a 1/50 dilution of FITC-labeled antiserum raised against mouse immunoglobulin G (Nordic Laboratories) for 30 min at 4 ° C. The cells were then washed again, and fluorescence was determined with a cytofluorograph. Interferon. Recombinant bovine interferon gamma (rBoIFN), supplied by Ciba-Geigy Limited, Basle, was more than 95% pure as determined by electro-
381 phoresis. I F N titers were determined by virus-induced cytopathic effect inhibition assays in microtiter dishes using M D B K cells challenged with vesicular stomatitis virus (VSV) and expressed in laboratory units based on standards. RESULTS As shown in Table 1, rBoIFN had a pronounced enhancing effect on the membrane expression of BoLA. Under basal conditions (incubation in control medium), only 63 to 77% of normal lymphocytes expressed M H C class I antigens detectable by the broad reactive anti-HLA class I monoclonal antibody, whereas the a n t i - H L A - D R monoclonal antibody poorly reacted with the cells. After incubation with rBoIFN, almost all cells bore detectable class I antigens within 48 h. The kinetics of enhancement of BoLA class II expression was slower, taking 3 to 4 days to reach a maximum of 57%. Table 1 also illustrates that spontaneous expression of BoLA class II antigens was moderate or low but the enhancement of their expression was clearly observed after r B o I F N treatment in repeated experiments. In addition, dose response studies showed that concentrations of r B o I F N as low as 10 u n i t s / m l induced expression of the BoLA up to 98% (class I) or 57% (class II) within 4 days of culture. A r B o I F N concentration of 100 and 1000 u n i t s / m l did not induce obvious increase in maximum intensity of B o L A expression nor in the maximum proportion of antigen-positive cells. The role of r B o I F N was further examined on the bovine continuous cell line BL-3. The expression of BoLA class I and class II antigens was studied with various r B o I F N concentrations and different incubation times. Results of a representative experiment are presented in Table 2. It appeared that rBoIFN induced antigen expression that could be detected as early as day 1 of incubation. The effect of r B o I F N reached its maximum at day 2 and remained at the same level on day 3. This effect was not only noticed on the percentage of positive cells, b u t also on the level of fluorescence intensity (see various peak channels). TABLE 1 Effects of rBoIFN on bovine lymphocyte antigens in normal peripheral blood lymphocytes IFN concentration W6/32 binding (U/ml) 24a 48
72
96
24
48
72
96
0 10 100 1000
75 (78) 98 (124) 98 (119) 95 (124)
73 (76) 91 (125) 89 (117) 86 (115)
13 (29) 26 (43) 27 (46) 27 (41)
35 (28) 49 (53) 45 (52) 43 (33)
32 (29) 57 (48) 49 (42) 44 (36)
31 (25) 53 (49) 45 (41) 43 (43)
63b (58) c 85 (71) 81 (67) 79 (69)
77 (73) 99 (151) 99 (139) 96 (92)
2.06 binding
aCulture time in hours, bpercent positive cells, cPeak channel.
382 TABLE 2 Effects of rBoIFN on bovine lymphocyte antigens in the BL-3 cell line IFN concentration (U/m[)
W6/32 binding 24~
0 10 100 1000
715 87 89 91
(62) c (71) (67) (74)
2.06 binding
48
72
87 (78) 94(105) 93 (122) 95 (124)
91 98 98 99
(92) (123) (125) (128)
24
48
72
56 (51) 61(65) 60 (63) 65 (64)
62 (64) 95 {124) 98(119) 97 (117)
67 99 99 98
(71) {139) (129) (133)
aCulture time in hours, bPercent positive cells, cPeak channel. DISCUSSION
In the present work, rBoIFN has been used to study the expression of bovine MHC antigens in cultured lymphoid cells, rBoIFN increases class I BoLA molecule levels at concentrations far lower than those needed to induce the antiviral state in fibroblast cells (Czarniecki et al., 1986). This enhancement was observed not only in terms of percent of positive cells but also in terms of fluorescence intensity. Class II MHC antigens show a limited tissue distribution in vivo and are involved in the presentation of antigen to T-helper cells. Interferon gamma has been shown to increase the expression of class II genes in many human and murine cells types. In the results reported here, rBoIFN also enhanced BoLA class II antigens as revealed by an anti-HLA-DR monoclonal antibody. Time course studies determined the appropriate time lengths of IFN incubation. BoLA expression on normal peripheral lymphocytes as well as on the BL-3 cell line was fully induced in 48 h by rBoIFN. All inductions were stable for up to 5 days (data not shown). Therefore, 3-day IFN incubation periods were chosen for all experiments, rBoIFN increase of BoLA cell surface expression was less than 2-fold, both in terms of positive cells and fluorescence intensity. This is in accordance with results in the murine H-2 system where a discrepancy was reported between the level of H-2 messenger RNA enhancement by IFN (15-fold) and H-2 cell surface expression (2-fold) {Wong et al., 1984). It would be expected that all bovine cells would express MHC class I antigens. The lower level of normal bovine lymphocytes reacting with W6/32 antibody in the absence of interferon is not due to non-optimal experimental conditions as demonstrated by the level of reactivity with the BL-3 cells, but may reflect a lower density of class I antigens at the surface of some normal cells. On the other hand, the differences observed in the magnitude of response in normal bovine lymphocytes and in the BL-3 cell line may reflect the proportion of cells responding to IFN in the population, or a difference in the IFNreceptor expression.
383
Finally, the use of rBoIFN may provide a useful system for characterizing BoLA class II gene products and for analyzing the mechanism of regulation of the bovine MHC. Certainly the rBoIFN can induce or enhance the expression of BoLA class II antigens. Since this expression is essential for leukocyte communication during antigen presentation by monocytes and B lymphocytes, rBoIFN may prove effective at enhancing antigen-specific immune response. This appears to be the case for human and murine IFN.
REFERENCES Chardon, P., Kalil, J., Leveziel, H., Colombani, J. and Vaiman, M., 1983. Monoclonal antibodies to HLA recognize monomorphic and polymorphic epitopes on BoLA T. Antigens, 22: 62-71. Collins, T., Korman, A.J., Wake, C.T., Boss, J.M., Kappes, D.J., Fiefs, W., Ault, K.A., Gimbrone, M.A., Strominger, J.L. and Pober, J.S., 1984. Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 81: 4917-4921. Czarniecki, C., Hamilton, E.B., Fennie, C. and Wolf, R.L., 1986. In vitro biological activities of Escherichia coli derived bovine interferon. J. Interferon. Res., 6: 29-37. Dausset, J. and Svejgaard, A., 1977. HLA and Disease. Munksgaard, Copenhagen. Hoang-Xuan, M., Charron, D., Zilber, M.T. and Levy, D., 1982. Biochemical characterization of class II bovine major histocompatibility complex antigens using cross-species reactive antibodies. Immunogenetics, 15: 621-624. L~vy, D., 1988. BoLA class II antigens: structure, biosynthesis and polymorphism. Anim. Genet., 19: 26-28. Rosa, F. and Fellous, M., 1984. The effect of gamma-interferon on MHC antigens. Immunol. Today, 5: 261-263. Spooner, R.L., Teale, A.J. and Cullen, P., 1988. The MHC of cattle and sheep. In: R. Pandey (Editor), Moving Frontiers in Veterinary Immunology. Karger, Basle, pp. 88-107. Virelizier, J.L., Perez, N., Arenzana-Seisdedos, F. and Devos, R., 1984. Pure interferon gamma enhances class II HLA antigens on human monocyte cell lines. Eur. J. Immunol., 14: 106-108. Wong, G.H.W., Bartlett, P.F., Clark-Lewis, I., Battye, F. and Schrader, J.W., 1984. Inducible expression of H-2 and Ia-antigens on brain cells. Nature, 310: 688-691.