Modulation of Bovine Mononuclear Cell Proliferation During Physiological Transitions of the Mammary Gland P. M. TORRE1 ,2 and S. P. OLIVER Institute of Agriculture Department of Animal Science University of Tennessee Knoxville 37901-1071 ABSTRACT
incidence of new intramammary infections at times when the mammary gland is highly susceptible. (Key words: mammary mononuclear cells, proliferation, involution)
Mammary secretions and blood were collected from five primiparous Holstein cows 14 d following cessation of milking and 14 d prior to parturition for preparation of serum and mammary secretion skim fractions. Mammary secretions and blood were collected from the same animals IS to 18 d following cessation of milking and 2 to 13 d prior to parturition for isolation of mononuclear cells. Effects of serum on mammary gland mononuclear cell proliferation and skim fractions from mammary secretions on blood mononuclear cell proliferation were evaluated. Mononuclear cell proliferation was evaluated in a mitogen-induced lymphocyte proliferation assay and in a mixed leukocyte assay. Proliferative responses of blood and mammary gland mononuclear cells did not vary significantly between the two time periods evaluated. Mammary secretion skim fractions obtained at both time periods significantly suppressed blood mononuclear cell proliferation. In contrast, exogenous serum enhanced mammary gland mononuclear cell proliferation in response to mitogens and allogeneic cells. Ability to enhance in vitro proliferation of mammary mononuclear cells isolated during physiological transitions of the mammary gland may suggest the potential for enhancing mammary mononuclear cell proliferation in vivo to reduce
Abbreviation key: IMI = intramammary infection, MGMC = mammary gland mononuclear cells, PBMC = peripheral blood mononuclear cells. INTRODUCTION
Received September 27, 1990. Accepted February 25, 1991. 1Reprint requests. 2Current address: USDA ARS, Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111. 1991 J Dairy Sci 74:2459-2466
The bovine mammary gland is most susceptible to new intramammary infection (IMI) during the first few weeks following cessation of milking and during the peripartum period (reviewed in (Il)J. In contrast, the fully involuted gland is more resistant to IMI. Mammary gland secretory tissue may sustain damage resulting in decreased milk yield during the subsequent lactation due to IMl during the nonlactating period (11). Consequently, methods of enhancing resistance of the bovine mammary gland to IMI during the nonlactating period would be beneficial to milk yield in future lactations. Ability of bovine mammary gland immune cells to respond to pathogenic challenge may affect mammary gland susceptibility to IMI. Human and bovine mammary gland mononuclear cells (MGMC) have been shown to be hyporesponsive compared with peripheral blood mononuclear cells (pBMC; 4, 5). Reasons for MGMC hyporesponsiveness are unclear although proposed causes include suppression by factors in mammary secretions (5, 17), ratio of helper:cytotoxic/suppressor Tlymphocytes (8), and suppressive soluble factors produced by bovine mammary leukocytes (6). Enhancement of MGMC function might prove beneficial to resistance of the mammary _ gland to IMI, particularly during physiological
2459
2460
TORRE AND OUVER
transitions of the gland The objective of this study was to detennine whether bovine MGMC proliferation could be enhanced during the early nonlactating period and the prepartum period. MATERIALS AND METHODS Animals
Five pregnant prumparous Holstein cows from the University of Tennessee dairy research herd were used. Cows were free from IMI at drying off based on weekly microbiological analysis of quarter foremilk samples during the last month of lactation. Cows were dried off by abrupt cessation of milking approximately 8 wk prior to calculated parturition. Mammary glands of cows were infused with a commercially available nonlactating cow antibiotic formulation (Biodry, The Upjohn Co., Kalamazoo, MI) after the last milking of lactation. Quarter samples of mammary secretion were collected weekly during the nonlactating period for microbiological analysis. Only secretions from mammary glands free from major pathogen IMI were used. Serum and Mammary secretions
Animals were bled and mammary secretions collected 14 d after cessation of milking and 14 d prior to expected parturition for preparation of serum and mammary secretion skim fractions. Blood was collected by jugular venipuncture without anticoagulant. Serum was collected by centrifugation. Mammary secretion skim fraction samples were prepared by centrifugation (37,000 x g for 30 min) to remove fat and cellular debris. Skim fraction samples were ftlter sterilized (.45-~ ftlter) prior to use. Serum and mammary secretion skims were heated (56°C for 30 min) to inactivate complement and stored at -20'C until needed Mononuclear Cell IsolatIon
Cows were bled and mammary secretions collected for isolation of mononuclear cells 15 to 18 d following cessation of milking and 2 to 13 d prior to parturition. Mammary secretions were diluted 1:2 to 1:4 in phosphate-buffered Journal of Dairy Science Vol. 74, No.8, 1991
saline (.15 M, pH 7.3) and centrifuged (600 x g for 20 min) to remove fat and pellet cells. The PBMC and MGMC were isolated over Ficoll-sodium diatrizoate (specific gravity = 1.083 g/mI; Histopaque 1083; Sigma Chemical Co., St. Louis, MO) as described by Torre and Oliver (18). Cells were resuspended in RPMI 1640 medium (Gibeo, Grand Island, NY) containing 25 mM HEPES buffer, 2 mM L-glutamine, 1DO U penicillin G/mI, 1DO J.Lg of streptomycin sulfate/mI, .25 J.Lg of amphotericin B/ mI, and 10% heat-inactivated fetal calf serum (Cell Culture Laboratories, Cleveland, OB). Mononuclear cell viability was detennined by hemacytometer count using trypan blue exclusion. Composition of mononuclear cell populations was detennined using cytocentrifuge smears stained with Wright-Giemsa. Mononuclear Cell Proliferative Assays
Mitogenic stimulation and the one-way mixed leukocyte reaction were used to stimulate mononuclear cell proliferation in these experiments. Mitogens used were the T-cell mitogens, Concanavalin A and phytohemagglutinin, and the T-cell-dependent B-cell mitogen, pokeweed mitogen. Optimal concentration of each mitogen was detennined to be 6.25 J.Lg/ ml in experiments (using bovine PBMC), which were conducted prior to the present study. The one-way mixed leukocyte reaction was conducted as described by Thurman et al. (16) using bovine PBMC as stimulator cells. Nonlactating Jersey cows were used as blood donors to ensure that stimulator cells came from animals genetically unrelated to the study population. Isolated Jersey PBMC were treated for 30 min with 25 J.Lg/mI mitomycin C to prevent cell proliferation, washed twice, and resuspended in RPMI 1640. Stimulator cells were added to cultures at 2 X lOS cells per well. Proliferative assays were done in 96-well microtiter plates. Well contents were 2 x lOS mononuclear cells in 50 J.Ll of RPM! 1640, 100 J.LI of mitogen or stimulator cell suspension, and 50 J.LI of autologous serum or mammary secretion skim at various dilutions. Mammary gland mononuclear cells were incubated with autologous serum. Peripheral blood mononuclear cells were incubated with autologous
2461
MAMMARY MONONUCLEAR CELL PROLIFERATION
TABLE 1. Identification of bovine mononuclear cells isolated during the dry period. Cell identity 1
Mononuclear
Day of
cell source
dry period
Blood
Mammary secretion
EarIy3 Prepartum4 Early Prepartum
Mononuclear cell viabiliry2
Lymphocyte
Monocyte or macrophage
Granulocyte
X
SEM
X
SEM
X
SEM
X
83
3 4 3 6
85 86 57
3 3 6
12 13
2 3
.1).
tios fluctuated as involution progressed, with
numbers of cytotoxicjsuppressor T-lymphocytes increasing during the peripartum period. Soluble suppressive factors produced in vitro by bovine milk lymphocytes have been suggested as a potential cause of MGMC hyporesponsiveness (6). Subiza et al. (15) found that human mammary gland macrophages were deficient in both synthesis and secretion of interleukin-1 compared with blood monocytes. Similar studies utilizing bovine mammary macrophages have not been published, however. Another likely cause of MGMC hyporesponsiveness is suppression by factors present in mammary secretions. Bovine and human mammary secretions consistently have been shown to suppress PBMC response to mitogens (5, 17). Human DISCUSSION colostrum also inhibited differentiation of The exact physiological cause of bovine blood lymphocytes into immunoglobulin-conMGMC hyporesponsiveness in proliferative taining plasma cells (5). In this study, bovine assays compared with PBMC is unknown. mammary secretions suppressed proliferation However, various potential causes have been of PBMC that had been prestimulated for 24 h. suggested. Hurley et al. (8) found that the These data suggest that suppression of PBMC helper: cytotoxicjsuppressor T-lymphocyte ra- proliferation by mammary secretions observed Journal of Dairy Science Vol. 74. No. 8, 1991
2464
TORRE AND OUVER
TABLE 4. Modulation of bovine mononuclear cell response to allogeneic cells. 1,2
Cell type
Percentage of mammary secretion or serum
Time of mononuclear cell isolation Days 15-18 Days 2-13 involution prepartum X
PBMc3
2S 1.6 .1
MGMc4
25 1.6 .1
SEM .7&.1 4.7b 1.1 19.7b 4.8 19.01' 4.4 15.6& 4.2 8.6& 2.2 3.2&.8 6.0" 2.0
o
o
X
SEM 1.5&.1 6.1 ab 2.1 14.2b 4.7 b 20.4 6.2 113.1& 40.7 191.0" 12.2 82.9" 11.4 36.7& 13.8
""Means with different superscripts within a cell type and stage of the nonlactating period differ (P
< .05). Means
between days of the dry period for individual cell types did not differ (P > .1). IData expressed as mean counts
pel'
minute x 103 ± SEM.
=
2Unstimulated control values for peripheral blood mononuclear cells (PBMC) isolated 15 to 18 d of involution 2.5 ± .6 cpm x 103. Unstimulated control values for PBMC isolated 2 to 13 d prepartum 2.3 ± .s cpm x 103. Unstimulated control values for mammary gland mononuclear cells (MGMC) isolated 15 to 18 d of involution 1.1 ± .3 cpm x 103. Unstimulated control values for MGMC isolated 2 to 13 d prepartum 1.8 ± .4 cpm x 103.
=
=
=
3Mammary secretions added to PBMC 24 h after initiation of cultwes. Mammary secretions obtained at 14 d of involution were added to PBMC isolated at 15 to 18 d of involution. Mammary secretions obtained at 14 d prepartum were added to PBMC isolated 2 to 13 d prepartum. 4Serum added to MGMC 24 b after initiation of cultures. Serum obtained at 14 d of involution was added to MGMC isolated at 15 to 18 d of involution. Serum obtained at 14 d prepartum was added to MGMC isolated 2 to 13 d prepartum.
in this study and in prior studies (3, 17) was not due to nonspecific binding of mitogens by mammary secretion proteins but to actual suppression of proliferation. Schechter (13) reported that complete removal of mitogens 20 to 24 h following initiation of cultures had limited effects on subsequent lymphocyte proliferation. The source of inununosuppressive activity in mammary secretions is unknown. However, proteins such as lactoferrin (12), lysozyme (19), and free secretory component (5) are present in mammary secretions and suppressed PBMC proliferation in vitro. Other factors, such as free fatty acids (3), are found also in the mammary gland and may be suppressive. Consequently, MGMC hyporesponsiveness may be a result of suppression by factors present in mammary secretions. Freshly isolated bovine MGMC were hyporesponsive to mitogenic and allogeneic cell stimulation compared with PBMC in the present study, corroborating results of Concha et al. (4). Viability of isolated PBMC and MGMC was comparable and should not have contributed to differences in responsiveness. Journal of Dairy Science Vol. 74, No.8, 1991
Human neutrophils have been shown to release substances inhibitory to lymphocyte mitogenesis (20), suggesting that increased granulocytic contamination of MGMC preparations compared with contamination of PBMC preparations may have contributed to MGMC hyporesponsiveness. Another potential contributing factor to MGMC hyporesponsiveness in this study was differences in overall composition of mononuclear cell populations. On average, MGMC preparations contained 25% fewer lymphocytes than PBMC preparations. Consequently, the possibility exists that MGMC hyporesponsiveness was due to fewer numbers of lymphocytes available to respond to proliferative stimuli. However, MGMC hyporesponsiveness could be reversed without removing granulocytes or adjusting MGMC preparationswcontainsimilarconce~atwns
of lymphocytes as PBMC preparations. Therefore, lack of responsiveness of MGMC to proliferative stimuli compared with PBMe in this study appears to be due to more than differences in granulocyte contamination or lymphocyte numbers in mononuclear cell preparations.
MAMMARY MONONUCLEAR CELL PROLIFERAnON
All mononuclear cell cultures were conducted using 10% fetal calf serum. as a source of nutrients and growth factors. Under these conditions, MGMC were substantially less responsive to mitogenic and allogeneic cell stimulation than PBMC. Addition of autologous adult bovine serum at concentrations as low as 1.6% of culture volume enhanced MGMC responsiveness. These data suggest that MGMC may possess nutritional or growth factor requirements for optimal proliferation that are not provided by fetal calf serum but can be supplied by adult serum. All factors in serum that support mononuclear cell proliferation have not been fully characterized, although albumin, transferrin, glucose, and various growth factors are serum components known to be required for mononuclear cell proliferation (2). 1bese factors should have been provided by fetal calf serum. Consequently, adult serum may have contained additional growth factors or immunostimulatory cytokines that were required to overcome lack of responsiveness of MGMC. In support of this hypothesis, Manak (9) showed that serum from lo-d-old calves was more supportive of cord blood lymphocyte proliferation than serum from l-d-old calves. These data also suggest that bovine MGMC may require additional stimuli for optimal proliferation compared with PBMC. High concentrations (25% of culture volume) of autologous adult bovine serum added to MGMC isolated during the prepartum period restored MGMC proliferative capacities to levels comparable with those of stimulated PBMC. Proliferation of MGMC isolated during the early dry period and cultured with adult serum did not reach levels comparable with PBMC. However, proliferation of MGMC is0lated during the early dry period was significantly enhanced by addition of adult bovine serum to cultures. These data suggest that enhancement of MGMC function during times of greatest susceptibility to IMI of the bovine mammary gland may be feasible. The bovine mammary gland is most susceptible to IMI during physiological transitions from lactation to involution and from involution to colostrogenesis. Hyporesponsiveness of MGMC could contribute to or exacerbate this situation. Data presented in this study suggest that bovine MGMC proliferative activity can
2465
be enhanced at times when the gland is most
susceptible to IMI. Shing and Klagsbnm (14) found growth factor activity in human and bovine mammary secretions. Growth factor activity was detected in human milk throughout lactation. In contrast, bovine mammary secretions contained growth factor activity only during the colostral phase. Head (7) suggested that a function of colostrum may be passive transfer of cell-mediated as well as humoral immunity to the neonate. These data may suggest that the prepartum period could be a likely target for attempted enhancement of MGMC function in vitro. However, results of this study suggest that in vitro enhancement of MGMC function could be achieved during the early nonlactating period as well. At present, methodologies for in vivo immunoenhancement are unavailable. However, availability of a number of recombinant bovine cytokines makes use of these factors as in vivo enhancers of MGMC function a logical choice. Studies using recombinant bovine interferons in respiratory disease models have shown promising results (l), and similar studies utilizing recombinant interleukin-2 in a bovine mastitis model have been initiated (10). Such techniques may prove useful in enhancing resistance of the bovine mammary gland to IMI during the nonlactating period. ACKNOWLEDGMENTS
This work was supported by the Tennessee Agricultural Experiment Station and The University of Tennessee College of Veterinary Medicine Center of Excellence Research Pr0gram in Livestock Diseases and Human Health. Authors express their appreciation to Arlene Stewart for clerical assistance. REFERENCES 1 Babiuk, L. A, II. Bielcfeldt-OhmaDn, G. Gifford, C. W. Czamiecki, V. T. Scia11i, and E. B. Hamilton. 1985. Effect of bovine al interferon on bovine herpes virus typo-l-induccd respiratory disease. J. Gen. ViroL 66:2383. 2 Barta, O. 1983. Serums lympbocyte immunoreguIa!Dry factors (SLIF). Vet. Immunol. Immunopatbol. 4: 279. 3 Buttke, T. M, and M A. Cucbeos. 1984. Inhibition of lymphocyte proliferation by free fany acids. n. Toxicity of stearic acid towards pbytobaemagglutinin-activated T ccl1s. Immunology 53:507. 4 Concha, C., O. Holmberg, and B. Morein. 1980. CharJoumal of Dairy Science Vol. 74, No.8, 1991
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TORRE AND OLIVER
acterization and response to mitogens of mammary lymphocytes from the bovine dry-period secretion. J. Dairy Res. 47:305. 5 Crago, S. S., R Kulbavy, S. J. Prince. and J. Mestecky. 1981. Inhibition of the pokeweed mitogeninduced response of normal peripheral blood lymphocytes by humoral components of colostrum. Coo. Exp. Immunol. 45:386. 6 Harp, J. A., and B. J. Nonnecke. 1986. Regulation of mitogenic responses by bovine milk leukocytes. Vet. Immunol. Immunopathol. 11:215. 7 Head, J. R. 1977. Immunobiology of lactation. Semin. Perinatol. New York 1:195. 8 Hurley, D. J., M H. Kensinger, A. M. Mastro, and R. A. Wilson. 1990. An evaluation of the mononuclear cells derived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties, and nonspecific esterase staining. Vet. Immunol. Immunopathol. 25: 177. 9 Manak, R. C. 1986. Neonatal calf serum immunoregulation of lymphocyte blastogenic responses. J. Anim. Sci. 62:759. 10 Nickerson, S. C., P. A. Baker, and P. Trinidad. 1989. Local immunostimulation of the bovine mammary gland with interluekin-2. J. Dairy Sci. 72:1764. 11 Oliver, S. P., and L. M. Sordillo. 1989. Approaches to the manipulation of mammary involution. J. Dairy Sci. 72:1647. 12 Richie, E. R., J. R. Hilliard, R. Gilmore, and D. J. Gillespie. 1987. Human milk-derived lactoferrin inhibits mitogen and alloantigen induced human lymphocyte proliferation. J. Reprod. Immunol. 12:137. 13 Schechter, B. 1980. Lymphocyte stimulation by non-
Journal of Dairy Science Vol. 74, No.8, 1991
specific mitogens. Page 1 in Lymphocyte stimulation. Differential sensitivity to radiation. A. Castellani, ed. Plenum Press, New York, NY. 14 Shing, Y. W., and M Klagsbrun. 1984. Human and bovine milk contain different sets of growth factors. Endocrinology 115:273. 15 Subiza, J. L., C. Rodriguey, A. Figueredo. P. Mateos, R. Alvarey, and E. G. de la Concha. 1988. Impaired production and lack of secretion of interleukin-I by human breast milk macrophages. Coo. Exp. Immunol. 71:493. 16 Thurman, G. B., D. M. Strong, A. Ahmed, S. S. Green, K. W. Sell, R. J. Hartzman, and F. H. Bach. 1973. Human mixed lymphocyte cultures. Evaluation of a microculture technique utilizing the multiple automated sample harvester (MASH). Clin. Exp. Immunol. 15:289. 17 Torre, P. M, and S. P. Oliver. 1989. Suppression of mitogenic response of peripheral blood mononuclear cells by bovine mammary secretions. J. Dairy Sci. 72: 219. 18 Torre, P. M., and S. P. Oliver. 1989. Enhancement of bovine mammary mononuclear cell proliferation. Page 126 in Proc. Int. Conf. Mastitis, S1. Georgen/Langsee, Carinthia, Austria. 19 Varaldo, P. E., S. Valisena, M. C. Mingari, and G. Satta. 1989. Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins. Proc. Soc. Exp. BioI. Med. 190:54. 20 Zoschke, D. L., and R. P. Messner. 1984. Suppression of human lymphocyte mitogenesis mediated by phagocyte-released inactive oxygen radicals: comparative activities in normal and in chronic granulomatous disease. Clin. Immunol. Immunopathol. 32:29.
incidence of new intramammary infections at times when the mammary gland is highly susceptible. (Key words: mammary mononuclear cells, proliferation, involution)
Mammary secretions and blood were collected from five primiparous Holstein cows 14 d following cessation of milking and 14 d prior to parturition for preparation of serum and mammary secretion skim fractions. Mammary secretions and blood were collected from the same animals IS to 18 d following cessation of milking and 2 to 13 d prior to parturition for isolation of mononuclear cells. Effects of serum on mammary gland mononuclear cell proliferation and skim fractions from mammary secretions on blood mononuclear cell proliferation were evaluated. Mononuclear cell proliferation was evaluated in a mitogen-induced lymphocyte proliferation assay and in a mixed leukocyte assay. Proliferative responses of blood and mammary gland mononuclear cells did not vary significantly between the two time periods evaluated. Mammary secretion skim fractions obtained at both time periods significantly suppressed blood mononuclear cell proliferation. In contrast, exogenous serum enhanced mammary gland mononuclear cell proliferation in response to mitogens and allogeneic cells. Ability to enhance in vitro proliferation of mammary mononuclear cells isolated during physiological transitions of the mammary gland may suggest the potential for enhancing mammary mononuclear cell proliferation in vivo to reduce
Abbreviation key: IMI = intramammary infection, MGMC = mammary gland mononuclear cells, PBMC = peripheral blood mononuclear cells. INTRODUCTION
Received September 27, 1990. Accepted February 25, 1991. 1Reprint requests. 2Current address: USDA ARS, Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111. 1991 J Dairy Sci 74:2459-2466
The bovine mammary gland is most susceptible to new intramammary infection (IMI) during the first few weeks following cessation of milking and during the peripartum period (reviewed in (Il)J. In contrast, the fully involuted gland is more resistant to IMI. Mammary gland secretory tissue may sustain damage resulting in decreased milk yield during the subsequent lactation due to IMl during the nonlactating period (11). Consequently, methods of enhancing resistance of the bovine mammary gland to IMI during the nonlactating period would be beneficial to milk yield in future lactations. Ability of bovine mammary gland immune cells to respond to pathogenic challenge may affect mammary gland susceptibility to IMI. Human and bovine mammary gland mononuclear cells (MGMC) have been shown to be hyporesponsive compared with peripheral blood mononuclear cells (pBMC; 4, 5). Reasons for MGMC hyporesponsiveness are unclear although proposed causes include suppression by factors in mammary secretions (5, 17), ratio of helper:cytotoxic/suppressor Tlymphocytes (8), and suppressive soluble factors produced by bovine mammary leukocytes (6). Enhancement of MGMC function might prove beneficial to resistance of the mammary _ gland to IMI, particularly during physiological
2459
2460
TORRE AND OUVER
transitions of the gland The objective of this study was to detennine whether bovine MGMC proliferation could be enhanced during the early nonlactating period and the prepartum period. MATERIALS AND METHODS Animals
Five pregnant prumparous Holstein cows from the University of Tennessee dairy research herd were used. Cows were free from IMI at drying off based on weekly microbiological analysis of quarter foremilk samples during the last month of lactation. Cows were dried off by abrupt cessation of milking approximately 8 wk prior to calculated parturition. Mammary glands of cows were infused with a commercially available nonlactating cow antibiotic formulation (Biodry, The Upjohn Co., Kalamazoo, MI) after the last milking of lactation. Quarter samples of mammary secretion were collected weekly during the nonlactating period for microbiological analysis. Only secretions from mammary glands free from major pathogen IMI were used. Serum and Mammary secretions
Animals were bled and mammary secretions collected 14 d after cessation of milking and 14 d prior to expected parturition for preparation of serum and mammary secretion skim fractions. Blood was collected by jugular venipuncture without anticoagulant. Serum was collected by centrifugation. Mammary secretion skim fraction samples were prepared by centrifugation (37,000 x g for 30 min) to remove fat and cellular debris. Skim fraction samples were ftlter sterilized (.45-~ ftlter) prior to use. Serum and mammary secretion skims were heated (56°C for 30 min) to inactivate complement and stored at -20'C until needed Mononuclear Cell IsolatIon
Cows were bled and mammary secretions collected for isolation of mononuclear cells 15 to 18 d following cessation of milking and 2 to 13 d prior to parturition. Mammary secretions were diluted 1:2 to 1:4 in phosphate-buffered Journal of Dairy Science Vol. 74, No.8, 1991
saline (.15 M, pH 7.3) and centrifuged (600 x g for 20 min) to remove fat and pellet cells. The PBMC and MGMC were isolated over Ficoll-sodium diatrizoate (specific gravity = 1.083 g/mI; Histopaque 1083; Sigma Chemical Co., St. Louis, MO) as described by Torre and Oliver (18). Cells were resuspended in RPMI 1640 medium (Gibeo, Grand Island, NY) containing 25 mM HEPES buffer, 2 mM L-glutamine, 1DO U penicillin G/mI, 1DO J.Lg of streptomycin sulfate/mI, .25 J.Lg of amphotericin B/ mI, and 10% heat-inactivated fetal calf serum (Cell Culture Laboratories, Cleveland, OB). Mononuclear cell viability was detennined by hemacytometer count using trypan blue exclusion. Composition of mononuclear cell populations was detennined using cytocentrifuge smears stained with Wright-Giemsa. Mononuclear Cell Proliferative Assays
Mitogenic stimulation and the one-way mixed leukocyte reaction were used to stimulate mononuclear cell proliferation in these experiments. Mitogens used were the T-cell mitogens, Concanavalin A and phytohemagglutinin, and the T-cell-dependent B-cell mitogen, pokeweed mitogen. Optimal concentration of each mitogen was detennined to be 6.25 J.Lg/ ml in experiments (using bovine PBMC), which were conducted prior to the present study. The one-way mixed leukocyte reaction was conducted as described by Thurman et al. (16) using bovine PBMC as stimulator cells. Nonlactating Jersey cows were used as blood donors to ensure that stimulator cells came from animals genetically unrelated to the study population. Isolated Jersey PBMC were treated for 30 min with 25 J.Lg/mI mitomycin C to prevent cell proliferation, washed twice, and resuspended in RPMI 1640. Stimulator cells were added to cultures at 2 X lOS cells per well. Proliferative assays were done in 96-well microtiter plates. Well contents were 2 x lOS mononuclear cells in 50 J.Ll of RPM! 1640, 100 J.LI of mitogen or stimulator cell suspension, and 50 J.LI of autologous serum or mammary secretion skim at various dilutions. Mammary gland mononuclear cells were incubated with autologous serum. Peripheral blood mononuclear cells were incubated with autologous
2461
MAMMARY MONONUCLEAR CELL PROLIFERATION
TABLE 1. Identification of bovine mononuclear cells isolated during the dry period. Cell identity 1
Mononuclear
Day of
cell source
dry period
Blood
Mammary secretion
EarIy3 Prepartum4 Early Prepartum
Mononuclear cell viabiliry2
Lymphocyte
Monocyte or macrophage
Granulocyte
X
SEM
X
SEM
X
SEM
X
83
3 4 3 6
85 86 57
3 3 6
12 13
2 3
.1).
tios fluctuated as involution progressed, with
numbers of cytotoxicjsuppressor T-lymphocytes increasing during the peripartum period. Soluble suppressive factors produced in vitro by bovine milk lymphocytes have been suggested as a potential cause of MGMC hyporesponsiveness (6). Subiza et al. (15) found that human mammary gland macrophages were deficient in both synthesis and secretion of interleukin-1 compared with blood monocytes. Similar studies utilizing bovine mammary macrophages have not been published, however. Another likely cause of MGMC hyporesponsiveness is suppression by factors present in mammary secretions. Bovine and human mammary secretions consistently have been shown to suppress PBMC response to mitogens (5, 17). Human DISCUSSION colostrum also inhibited differentiation of The exact physiological cause of bovine blood lymphocytes into immunoglobulin-conMGMC hyporesponsiveness in proliferative taining plasma cells (5). In this study, bovine assays compared with PBMC is unknown. mammary secretions suppressed proliferation However, various potential causes have been of PBMC that had been prestimulated for 24 h. suggested. Hurley et al. (8) found that the These data suggest that suppression of PBMC helper: cytotoxicjsuppressor T-lymphocyte ra- proliferation by mammary secretions observed Journal of Dairy Science Vol. 74. No. 8, 1991
2464
TORRE AND OUVER
TABLE 4. Modulation of bovine mononuclear cell response to allogeneic cells. 1,2
Cell type
Percentage of mammary secretion or serum
Time of mononuclear cell isolation Days 15-18 Days 2-13 involution prepartum X
PBMc3
2S 1.6 .1
MGMc4
25 1.6 .1
SEM .7&.1 4.7b 1.1 19.7b 4.8 19.01' 4.4 15.6& 4.2 8.6& 2.2 3.2&.8 6.0" 2.0
o
o
X
SEM 1.5&.1 6.1 ab 2.1 14.2b 4.7 b 20.4 6.2 113.1& 40.7 191.0" 12.2 82.9" 11.4 36.7& 13.8
""Means with different superscripts within a cell type and stage of the nonlactating period differ (P
< .05). Means
between days of the dry period for individual cell types did not differ (P > .1). IData expressed as mean counts
pel'
minute x 103 ± SEM.
=
2Unstimulated control values for peripheral blood mononuclear cells (PBMC) isolated 15 to 18 d of involution 2.5 ± .6 cpm x 103. Unstimulated control values for PBMC isolated 2 to 13 d prepartum 2.3 ± .s cpm x 103. Unstimulated control values for mammary gland mononuclear cells (MGMC) isolated 15 to 18 d of involution 1.1 ± .3 cpm x 103. Unstimulated control values for MGMC isolated 2 to 13 d prepartum 1.8 ± .4 cpm x 103.
=
=
=
3Mammary secretions added to PBMC 24 h after initiation of cultwes. Mammary secretions obtained at 14 d of involution were added to PBMC isolated at 15 to 18 d of involution. Mammary secretions obtained at 14 d prepartum were added to PBMC isolated 2 to 13 d prepartum. 4Serum added to MGMC 24 b after initiation of cultures. Serum obtained at 14 d of involution was added to MGMC isolated at 15 to 18 d of involution. Serum obtained at 14 d prepartum was added to MGMC isolated 2 to 13 d prepartum.
in this study and in prior studies (3, 17) was not due to nonspecific binding of mitogens by mammary secretion proteins but to actual suppression of proliferation. Schechter (13) reported that complete removal of mitogens 20 to 24 h following initiation of cultures had limited effects on subsequent lymphocyte proliferation. The source of inununosuppressive activity in mammary secretions is unknown. However, proteins such as lactoferrin (12), lysozyme (19), and free secretory component (5) are present in mammary secretions and suppressed PBMC proliferation in vitro. Other factors, such as free fatty acids (3), are found also in the mammary gland and may be suppressive. Consequently, MGMC hyporesponsiveness may be a result of suppression by factors present in mammary secretions. Freshly isolated bovine MGMC were hyporesponsive to mitogenic and allogeneic cell stimulation compared with PBMC in the present study, corroborating results of Concha et al. (4). Viability of isolated PBMC and MGMC was comparable and should not have contributed to differences in responsiveness. Journal of Dairy Science Vol. 74, No.8, 1991
Human neutrophils have been shown to release substances inhibitory to lymphocyte mitogenesis (20), suggesting that increased granulocytic contamination of MGMC preparations compared with contamination of PBMC preparations may have contributed to MGMC hyporesponsiveness. Another potential contributing factor to MGMC hyporesponsiveness in this study was differences in overall composition of mononuclear cell populations. On average, MGMC preparations contained 25% fewer lymphocytes than PBMC preparations. Consequently, the possibility exists that MGMC hyporesponsiveness was due to fewer numbers of lymphocytes available to respond to proliferative stimuli. However, MGMC hyporesponsiveness could be reversed without removing granulocytes or adjusting MGMC preparationswcontainsimilarconce~atwns
of lymphocytes as PBMC preparations. Therefore, lack of responsiveness of MGMC to proliferative stimuli compared with PBMe in this study appears to be due to more than differences in granulocyte contamination or lymphocyte numbers in mononuclear cell preparations.
MAMMARY MONONUCLEAR CELL PROLIFERAnON
All mononuclear cell cultures were conducted using 10% fetal calf serum. as a source of nutrients and growth factors. Under these conditions, MGMC were substantially less responsive to mitogenic and allogeneic cell stimulation than PBMC. Addition of autologous adult bovine serum at concentrations as low as 1.6% of culture volume enhanced MGMC responsiveness. These data suggest that MGMC may possess nutritional or growth factor requirements for optimal proliferation that are not provided by fetal calf serum but can be supplied by adult serum. All factors in serum that support mononuclear cell proliferation have not been fully characterized, although albumin, transferrin, glucose, and various growth factors are serum components known to be required for mononuclear cell proliferation (2). 1bese factors should have been provided by fetal calf serum. Consequently, adult serum may have contained additional growth factors or immunostimulatory cytokines that were required to overcome lack of responsiveness of MGMC. In support of this hypothesis, Manak (9) showed that serum from lo-d-old calves was more supportive of cord blood lymphocyte proliferation than serum from l-d-old calves. These data also suggest that bovine MGMC may require additional stimuli for optimal proliferation compared with PBMC. High concentrations (25% of culture volume) of autologous adult bovine serum added to MGMC isolated during the prepartum period restored MGMC proliferative capacities to levels comparable with those of stimulated PBMC. Proliferation of MGMC isolated during the early dry period and cultured with adult serum did not reach levels comparable with PBMC. However, proliferation of MGMC is0lated during the early dry period was significantly enhanced by addition of adult bovine serum to cultures. These data suggest that enhancement of MGMC function during times of greatest susceptibility to IMI of the bovine mammary gland may be feasible. The bovine mammary gland is most susceptible to IMI during physiological transitions from lactation to involution and from involution to colostrogenesis. Hyporesponsiveness of MGMC could contribute to or exacerbate this situation. Data presented in this study suggest that bovine MGMC proliferative activity can
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be enhanced at times when the gland is most
susceptible to IMI. Shing and Klagsbnm (14) found growth factor activity in human and bovine mammary secretions. Growth factor activity was detected in human milk throughout lactation. In contrast, bovine mammary secretions contained growth factor activity only during the colostral phase. Head (7) suggested that a function of colostrum may be passive transfer of cell-mediated as well as humoral immunity to the neonate. These data may suggest that the prepartum period could be a likely target for attempted enhancement of MGMC function in vitro. However, results of this study suggest that in vitro enhancement of MGMC function could be achieved during the early nonlactating period as well. At present, methodologies for in vivo immunoenhancement are unavailable. However, availability of a number of recombinant bovine cytokines makes use of these factors as in vivo enhancers of MGMC function a logical choice. Studies using recombinant bovine interferons in respiratory disease models have shown promising results (l), and similar studies utilizing recombinant interleukin-2 in a bovine mastitis model have been initiated (10). Such techniques may prove useful in enhancing resistance of the bovine mammary gland to IMI during the nonlactating period. ACKNOWLEDGMENTS
This work was supported by the Tennessee Agricultural Experiment Station and The University of Tennessee College of Veterinary Medicine Center of Excellence Research Pr0gram in Livestock Diseases and Human Health. Authors express their appreciation to Arlene Stewart for clerical assistance. REFERENCES 1 Babiuk, L. A, II. Bielcfeldt-OhmaDn, G. Gifford, C. W. Czamiecki, V. T. Scia11i, and E. B. Hamilton. 1985. Effect of bovine al interferon on bovine herpes virus typo-l-induccd respiratory disease. J. Gen. ViroL 66:2383. 2 Barta, O. 1983. Serums lympbocyte immunoreguIa!Dry factors (SLIF). Vet. Immunol. Immunopatbol. 4: 279. 3 Buttke, T. M, and M A. Cucbeos. 1984. Inhibition of lymphocyte proliferation by free fany acids. n. Toxicity of stearic acid towards pbytobaemagglutinin-activated T ccl1s. Immunology 53:507. 4 Concha, C., O. Holmberg, and B. Morein. 1980. CharJoumal of Dairy Science Vol. 74, No.8, 1991
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