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Culture Conditions for Blastogenic Responses of Bovine Mammary Mononuclear Cells
Veterinary Immunology and Immunopathology, 24 (1990) 323-330 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
323
Culture Conditions for Blastogenic Responses of B o v i n e M a m m a r y Mononuclear Cells* MARGARET H. KENSINGER 1, DAVID J. HURLEY 1'2 and RICHARD A. WILSON 1
The Departments of 1Veterinary Science and 2Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802 (U.S.A.) (Accepted 17 October 1989)
ABSTRACT Kensinger, M.H. Hurley, D.J. and Wilson, R.A., 1990. Culture conditions for blastogenic responses of bovine mammary mononuclear cells. Vet. Immunol. Immunopathol., 24: 323-330. Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 #g/ml) and phytohemagglutinin (PHA; 1, 5, 10/~g/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantlyhigher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ. ABBREVIATIONS MMC, mammary mononuclear cells; ConA, concanavalinA; PHA, phytohemagglutinin;RPMI, Rosewell Park Memorial Park Institute; L-15, Liebovitz-15; PBL, peripheral blood lymphocytes; FBS, fetal bovine serum; PBS, phosphate-buffered saline; cpm, counts per minute. *This research was funded by USDA grant # 87 CRSR23124, Paper No. 8093 in the Journal Series of the Pennsylvania Agricultural Experiment Station.
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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INTRODUCTION There have been several reports concerning the hypofunctionality of bovine mammary mononuclear cells (Concha et al., 1980; Shore et al., 1981; Collins and Oldham, 1986; Harp and Nonnecke, 1986) when compared to the blastogenic potential of peripheral blood mononuclear cells. These reports are based on the ability of mammary and blood lymphocytes in the presence of monocytes to proliferate in response to such lectins as ConA, PHA, and pokeweed mitogen. The in vitro studies are most often performed in RPMI 1640 medium with supplemented fetal ~)ovine serum, antibiotic, L-glutamine and a buffering agent. Our laboratory is also interested in the functional capacity of the mammary immune system, and we have recently characterized the populations of cells in the nonlactating bovine mammary gland (Hurley et al., 1990). Secretions obtained from cows at a mid-point in their nonlactating (dry) period showed a predominance of lymphocytes and macrophages with few neutrophils (Hurley et al., 1990). We postulated that mononuclear cells move from the peripheral circulation into the gland during the dry period in a predetermined differentiation state, or are altered by cell activation factors in their new environment. As a result, it may be that their in vitro requirements for optimal responses to mitogenic stimulation differ from peripheral blood mononuclear cells. To test this hypothesis we designed an experiment to examine several aspects of the mitogen culture. The parameters measured were pre-incubation of cells in medium before assay, mitogen concentration, duration of mitogen exposure, and type of culture medium. MATERIALSAND METHODS
Animals Six Holstein cows from the Pennsylvania State University dairy herd were used in experiments evaluating blastogenic responses of mononuclear cells from nonlactating secretion. All animals had at least one previous lactation, were clinically healthy, and were sampled at a mid-point (approximately 30 days after last milking) in their respective dry periods. The four cows used in the peripheral blood study were from the Pennsylvania State University research herd, and also had at least one previous lactation. At time of sampling, three animals were lactating and one was nonlactating. Mammary mononuclear cells Mammary secretions were obtained from quarters negative for microbes on culture, and pooled from each cow. Secretions were diluted 1: 4 in phosphatebuffered saline (PBS) pH 7.2, 0.15 M with 5% fetal bovine serum (FBS, GIBCO) and centrifuged at 6 0 0 x g for 20 min at 4°C. Cell pellets were recovered, washed and resuspended in either RPMI 1640 (GIBCO), or a mixture
BLASTOGENIC RESPONSES OF BOVINE MAMMARY MONONUCLEAR CELLS
325
of 50% RPMI and 50% L-15 (GIBCO). RPMI was supplemented with 0.2% (w/v) sodium bicarbonate and 0.03% (w/v) L-glutamine. Both media were supplemented with 10% heat inactivated FBS and 1% gentamicin (10 mg/ml, Sigma). Cell concentrations were adjusted to 2.0 × 106/ml, and 0.2 ml of cells were added to appropriate wells in a 96-well round-bottom microtiter plate (Corning 25850). In addition 20 ml of each cell suspension were added to a 75 cm 2 tissue culture flask (Falcon 3072) and incubated overnight at 37 °C in a 5% CO2 atmosphere before addition to microtiter wells as described for fresh cells.
Peripheral blood lymphocytes Approximately 40 ml of blood were obtained by jugular venipuncture. Blood was diluted 1:4 in PBS pH 7.2, 0.15 M, and layered on Ficoll-Hypaque density 1077 (1.077 g/ml, Sigma) at a ratio of 4:1. Tubes were centrifuged for 35 min at 800 × g, 4 ° C. Cells at the interface were collected, washed and resuspended in either routine RPMI 1640 or RPMI/L-15 both supplemented with 10% heat inactivated FBS and 1% gentamicin. Cell concentrations were adjusted to 2.0 × 106/ml, and 0.2 ml of cells per well were added to a round-bottom microtiter plate.
Viability measurements Cell viability assays were performed using trypan blue exclusion. Estimates of cell viability were made on day of collection and daily thereafter for the duration of culture. At each time point, cell viability was determined on cells which were not exposed to mitogen.
MMC stimulation assay The lectins used in this study were ConA (Miles Scientific ), and PHA (Burroughs Wellcome Co. ). ConA was added to cell suspensions at 3.3 and 6.6/~g/ ml, while PHA was added at 1, 5 and 10/~g/ml. Mitogens were added to mammary mononuclear cells (2.0 × 106/ml), and 200/tl aliquots of the suspension added to microtiter wells. Control wells received cells only. Microtiter plates were incubated at 37 ° C, in a 5% CO2 atmosphere. Wells were pulsed with 0.2 /2Ci 3H-thymidine (specific activity 6.7 Ci/mmol, New England Nuclear) for 6 h on days 2, 3 or 4 of culture. Cultures were harvested on glass fiber filtermats by means of a cell harvester (Skatron). Filtermats were placed in 10.0 ml scintillation fluid (Econofluor, New England Nuclear), and counts per minute (cpm) were determined in a Beckman (model LS-3105T) scintillation counter. Means were calculated from quadruplicate or triplicate wells for each treatment, logarithmically (base 10) transformed, and used for statistical analysis.
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P B L stimulation assay The mitogens ConA (3.3 z g / m l ) and P H A (5.0/~g/ml) were added to peripheral blood mononuclear cells (2.0 X 106/ml). Aliquots of 200/~l were added to appropriate microtiter wells, and plates incubated at 37°C, in a 5% CO2 atmosphere. Wells were pulsed with 0.2/ICi 3H-thymidine for 6 h and cells were harvested on day 3 of culture. Cultures were harvested, counted and analyzed as for MMC.
Statistical analyses A multifactor analysis of variance for mitogen and viability means was performed using the G L M option of SAS (SAS Institute) according to a completely randomized design. T r e a t m e n t means within a main effect were tested for significant differences using Scheffe's method of multiple comparisons. RESULTS
Effect of culture conditions on M M C viability M a m m a r y cell viability was high ( > 77% ) in both media on all days of culture. M a m m a r y cell viability was significantly higher (P < 0.05) in the R P M I / L-15 medium (86.2%) t h a n in the R P M I alone (81.5%).
Effect of culture conditions on M M C stimulation Day of culture harvest (length of incubation) had a significant effect on the proliferative responses of M M C to mitogens ConA and P H A (Table 1). ProTABLE 1 Analysis of variance for the proliferative response of mammary mononuclearcells to mitogenic lectins and culture conditions Variable1
Mean square
D T M P D XT DXM DXP TXM T×P MXP DXTXM
1.52" 6.29** 59.73** 0.44 0.03 0.08 0.17 1.75"* O.O7 0.00 O.08
1D=day of harvest, T--treatment (control, ConA levels, or PHA levels), M=media, P=preincubation. *P<0.05; **P<0.01.
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BLASTOGENICRESPONSESOFBOVINEMAMMARYMONONUCLEARCELLS TABLE 2 Effect of day of harvest on mammary mononuclear cell mitogenesis1 Day of harvest
Tritiated thymidine incorporation (log10 cpm)
2
3.58 ± 0.06 a
3 4
3.54 ± 0.05a 3.37 ± 0.055
1Data expressed as least squares means ± s.e.m. a,bMeans within a column are different at P < 0.05. TABLE3 Effects of mitogens and culture medium on proliferative responses of mammary mononuclear cells 1 Medium
RPMI RPMI/L-15
Control
ConA (#g/ml)
PHA (pg/ml)
0
3.3
6.6
1
5
10
2.78 2.91
3.17 4.08
3.12 4.10
3.12 4.04
3.19 4.15
3.18 4.11
1Values in the table represent least squares means of tritiated thymidine (loglo) incorporated into mammary mononuclear cells after 3 days in vitro exposure to no lectin {control), ConA or PHA. TABLE4 Viability of peripheral blood lymphocytes after various days in culture in RPMI or RPMI/L-151 Day of culture
0 1 2 3
Viability (%) RPMI
RPMI/L-15
97.8 ± 0.3a 61.5 ___2.7b 60.5 ± 3.35 58.3 ± 4.45
99.3 ± 0.5a 63.0 ± 2.25 58.3 _+3.85 57.0 + 2.25
1Values in table represent means _+s.e.m. a'5Means within a column are different at P < 0.01. liferative r e s p o n s e s of M M C were s i g n i f i c a n t l y h i g h e r w h e n cells were harv e s t e d o n d a y s 2 a n d 3 o f c u l t u r e (3.58 a n d 3.54 log c p m ) a s o p p o s e d t o d a y 4 ( T a b l e 2 ). A s t h e r e w a s n o d i f f e r e n c e i n t h e r e s p o n s e s o n d a y s 2 a n d 3, c u l t u r e s were harvested on day 3 in subsequent experiments. M i t o g e n i c r e s p o n s e s to C o n A a n d P H A were c o n s i s t e n t l y higher in the R P M I / L - 1 5 m e d i u m ( m e a n r e s p o n s e o f 4.10 log c p m ) , t h a n i n R P M I ( m e a n
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TABLE 5 Tritiated thymidine incorporation by peripheral blood lymphocytes after control, ConA or P H A treatment 1 Mitogen
Control (no mitogen) ConA (3.3 lzg/ml) P H A (5/zg/ml)
Tritiated thymidine incorporation (loglo cpm) RPMI
RPMI/L-15
3.69_+0.08 ~ 4.62 ± 0.05 b 4.62 ± 0.05 b
3.69_+0.15 a 4.62 ± 0.02 b 4.65 ± 0.01 b
Walues in table represent means ± s,e.m. a'bMeans within a column are different at P < 0.01.
response of 3.16 log cpm; see Table 3 ) as evidenced by a significant mitogen by medium interaction (Table 1 ). Further statistical examination of the mitogen by medium interaction using Scheffe's multiple comparisons provided the following information: ( 1 ) control (cells + medium) responses were not different in the two media, (2) MMC were stimulated by lectins to proliferate in both the RPMI and the RPMI/L-15, (3) there was not a significant dose response in either medium at the levels of mitogen tested, and (4) the proliferative responses of MMC in the RPMI/L-15 were higher at all doses of mitogens used, as compared to t h e responses in the RPMI alone. As there was no difference in the response of MMC to different mitogen doses (Table 3), concentrations of 3.3 and 5.0 #g/ml of ConA and PHA, respectively, were used in subsequent mitogen assays. Pre-incubation of cells in either medium for 24 h prior to exposure to mitogen had no effect on the proliferative responses to mitogens (Table 1 ).
Effect of culture medium on P B L viability and stimulation Viability of peripheral blood lymphocytes was quite high on day of collection (Table 4), decreased significantly in both RPMI (61.5%) and RPMI/L-15 (63.0%) the following day, then remained constant in each medium for the duration of the culture. In contrast to mammary mononuclear cells, peripheral blood lymphocytes were stimulated by lectins to an equivalent degree in both RPMI and R P M I / L-15 (Table 5). Furthermore, the response to lectins in each medium was one order of magnitude higher than control responses. DISCUSSION
Viability of PBL and MMC would suggest that both RPMI and RPMI/L15 support the survival of these cell populations in culture. The number of live
BLASTOGENIC RESPONSES OF BOVINE MAMMARY MONONUCLEAR CELLS
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PBL determined on each day of culture was nearly identical in both media tested. In contrast, on each day of culture, a higher percentage of mammary cells survived in R P M I / L - 1 5 than in RPMI. As mammary cell viability was greater than 77% in each medium at all times measured, it appears that cell viability is not the critical factor responsible for the observed increase in proliferative responses of MMC when cultured in RPMI/L-15. One parameter of the mitogen assay examined was pre-incubation of cells overnight in media. Several authors (Collins and Oldham, 1986; Harp and Nonnecke, 1986) have suggested that secretions from nonlactating cows contain soluble suppressive factors which when co-cultured with PBL inhibit the proliferative response of PBL. Attempts to reverse the potential effects of these suppressive factors in the present study by removing mammary cells from secretion and holding t h e m in media for 24 h, did not result in an increased proliferative response. The results of the determinations of the optimal day for pulse and harvest of mammary cultures (day 3), and the optimal concentrations of ConA and PHA (3.3 and 5.0/tg/ml) to use in the mitogen assay, are in line with those conditions defined by Mastro and Pepin (1982) for bovine lymph node cells, and by Torre and Oliver (1988) and Pearson et al. ( 1979 ) for bovine peripheral blood lymphocytes. Mammary mononuclear cells exhibited a greater proliferative response to lectins in RPMI/L-15 than in RPMI alone. Peripheral blood mononuclear cells on the other hand demonstrated the equivalent ability to respond significantly to lectins in both media. One possible explanation for this difference may be the calcium ion requirement for in vitro lymphocyte stimulation. Calcium ion has been proposed as a potential second messenger in lymphocyte mitogenensis for the following reasons: extracellular calcium is required for DNA synthesis (Alford, 1970; Whitney and Sutherland, 1972), and mobilization of calcium ion stores to free cytosolic calcium occurs shortly after binding of a ligand to cell surface receptors (Allwood et al., 1971; Freedman et al., 1975). We have measured calcium ion concentrations in nonlactating mammary secretions in a preliminary study. Results suggested that calcium ion concentrations may vary depending on the length and stage of the dry period, but levels are higher than those reported in serum (Stout et al., 1976). Lymphocytes transported from the blood into the nonlactating mammary gland are likely to experience an increase in extracellular calcium ion concentration. Interestingly, R P M I / L-15 medium contains twice the amount of calcium ion (0.84 mM/1) than does R P M I (0.42 mM/1). It may be that the in vitro CA 2+ requirements for mammary cell proliferation parallel those which exist in the gland, and to which the mammary cell has adapted. Another possible explanation for the enhanced mammary lymphocyte function in R P M I / L - 1 5 involves the pH requirements for lymphocyte division in mitogen-stimulated cultures. Gerson and Kiefer (1983) estimated the intra-
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cellular pH for murine ConA lymphoblasts during the DNA synthesis phase of the cell cycle, to be between 7.40 and 7.45. The extracellular pH during the experiment, was carefully maintained at a constant 7.30 in order that ultimately, intracellular regulation of the cells' biosynthetic functions not be disturbed. In our assay system, RPMI is buffered with sodium bicarbonate and CO2. L-15 is a phosphate-buffered medium. We have observed (by color change) a subtle, but greater increase in media pH after 3 days culture in wells containing RPMI alone than in wells containing RPMI/L-15. It may be that maintenance of a certain extracellular pH is more critical for the biosynthetic functions of mammary mononuclear cells than for peripheral blood lymphocytes. REFERENCES Alford, R.H., 1970. Metal cation requirements for phytohemagglutinin-induced transformation of human peripheral blood lymphocytes. J. Immunol., 104: 698-703. Allwood, G., Asherson, G.L., Davey, M.J. and Goodford, P.J., 1971. The early uptake of radioactive calcium by human lymphocytes treated with phytohemagglutinin. Immunology, 21: 509516. Collins, R.A. and Oldham, G., 1986. Proliferative responses and IL-2 production by mononuclear cells from bovine mammary secretions, and the effect of mammary secretions on peripheral blood lymphocytes. Immunology, 58: 647-651. Concha, C., Homberg, 0. and Morein, B., 1980. Characterization and response to mitogens of mammary lymphocytes from the bovine dry period secretion. J. Dairy Res., 47:305-311. Freedman, M.H., Raft, M.C. and Gomperts, B., 1975. Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides, nature, 255: 378-382. Gerson, D.F. and Kiefer, H., 1983. Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes. J. Cell. Physiol., 114: 132-136. Harp, J.A. and Nonnecke, B.J., 1986. Regulation of mitogenic responses by bovine milk leukocytes. Vet. Immunol. Immunopathol., 11: 215-224. Hurley, D.J., Kensinger, M.H. and Wilson, R.A., 1990. An evaluation of the mononuclear cells derived from bovine mammary dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties, and non-specific esterase staining. Submitted. Mastro, A.M. and Pepin, K.G., 1982. Effects of 12-o-tetradecanoylphorbol-13-acetateon lymphocyte proliferation. In: E. Hecker et al. (Editors), Carcinogenesis, Vol. 7. Raven Press, New York, NY, pp. 439-442. Pearson, T.W., Roelants, G.E., Lundin, L.B. and Mayor-Withey, K.S., 1979. The bovine lymphoid system: binding and stimulation of peripheral blood lymphocytes by lectins. J. Immunol. Methods, 26: 271-282. Shore, C.E., Osburn, B.I., Jasper, D.E. and Tyler, D.E., 1981. B and T lymphocytes in the bovine mammary gland: rosette formation and mitogen response. Vet. Immunol. Immunopathol., 2: 561-569. Stout, W.L., Kradel, D.C., Jung, G.A. and Smiley, C.G., 1976. Blood composition of well-managed high-producing Holsteincows in Pennsylvania. The Pennsylvania State University Agricultural Experiment Station, Progress Report 358, pp. 2-11. Torre, P.M. and Oliver, S.P., 1988. Changes in blastogenic activity of bovine blood mononuclear cells throughout the nonlactating period. J. Dairy Sci., 71: 1078-1084. Whitney, R.B. and Sutherland, R.M., 1972. Enhanced uptake of calcium by transforming lymphocytes. Cell. Immunol., 5: 137-147.
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Culture Conditions for Blastogenic Responses of B o v i n e M a m m a r y Mononuclear Cells* MARGARET H. KENSINGER 1, DAVID J. HURLEY 1'2 and RICHARD A. WILSON 1
The Departments of 1Veterinary Science and 2Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802 (U.S.A.) (Accepted 17 October 1989)
ABSTRACT Kensinger, M.H. Hurley, D.J. and Wilson, R.A., 1990. Culture conditions for blastogenic responses of bovine mammary mononuclear cells. Vet. Immunol. Immunopathol., 24: 323-330. Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 #g/ml) and phytohemagglutinin (PHA; 1, 5, 10/~g/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantlyhigher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ. ABBREVIATIONS MMC, mammary mononuclear cells; ConA, concanavalinA; PHA, phytohemagglutinin;RPMI, Rosewell Park Memorial Park Institute; L-15, Liebovitz-15; PBL, peripheral blood lymphocytes; FBS, fetal bovine serum; PBS, phosphate-buffered saline; cpm, counts per minute. *This research was funded by USDA grant # 87 CRSR23124, Paper No. 8093 in the Journal Series of the Pennsylvania Agricultural Experiment Station.
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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M.H. KENSINGER ET AL.
INTRODUCTION There have been several reports concerning the hypofunctionality of bovine mammary mononuclear cells (Concha et al., 1980; Shore et al., 1981; Collins and Oldham, 1986; Harp and Nonnecke, 1986) when compared to the blastogenic potential of peripheral blood mononuclear cells. These reports are based on the ability of mammary and blood lymphocytes in the presence of monocytes to proliferate in response to such lectins as ConA, PHA, and pokeweed mitogen. The in vitro studies are most often performed in RPMI 1640 medium with supplemented fetal ~)ovine serum, antibiotic, L-glutamine and a buffering agent. Our laboratory is also interested in the functional capacity of the mammary immune system, and we have recently characterized the populations of cells in the nonlactating bovine mammary gland (Hurley et al., 1990). Secretions obtained from cows at a mid-point in their nonlactating (dry) period showed a predominance of lymphocytes and macrophages with few neutrophils (Hurley et al., 1990). We postulated that mononuclear cells move from the peripheral circulation into the gland during the dry period in a predetermined differentiation state, or are altered by cell activation factors in their new environment. As a result, it may be that their in vitro requirements for optimal responses to mitogenic stimulation differ from peripheral blood mononuclear cells. To test this hypothesis we designed an experiment to examine several aspects of the mitogen culture. The parameters measured were pre-incubation of cells in medium before assay, mitogen concentration, duration of mitogen exposure, and type of culture medium. MATERIALSAND METHODS
Animals Six Holstein cows from the Pennsylvania State University dairy herd were used in experiments evaluating blastogenic responses of mononuclear cells from nonlactating secretion. All animals had at least one previous lactation, were clinically healthy, and were sampled at a mid-point (approximately 30 days after last milking) in their respective dry periods. The four cows used in the peripheral blood study were from the Pennsylvania State University research herd, and also had at least one previous lactation. At time of sampling, three animals were lactating and one was nonlactating. Mammary mononuclear cells Mammary secretions were obtained from quarters negative for microbes on culture, and pooled from each cow. Secretions were diluted 1: 4 in phosphatebuffered saline (PBS) pH 7.2, 0.15 M with 5% fetal bovine serum (FBS, GIBCO) and centrifuged at 6 0 0 x g for 20 min at 4°C. Cell pellets were recovered, washed and resuspended in either RPMI 1640 (GIBCO), or a mixture
BLASTOGENIC RESPONSES OF BOVINE MAMMARY MONONUCLEAR CELLS
325
of 50% RPMI and 50% L-15 (GIBCO). RPMI was supplemented with 0.2% (w/v) sodium bicarbonate and 0.03% (w/v) L-glutamine. Both media were supplemented with 10% heat inactivated FBS and 1% gentamicin (10 mg/ml, Sigma). Cell concentrations were adjusted to 2.0 × 106/ml, and 0.2 ml of cells were added to appropriate wells in a 96-well round-bottom microtiter plate (Corning 25850). In addition 20 ml of each cell suspension were added to a 75 cm 2 tissue culture flask (Falcon 3072) and incubated overnight at 37 °C in a 5% CO2 atmosphere before addition to microtiter wells as described for fresh cells.
Peripheral blood lymphocytes Approximately 40 ml of blood were obtained by jugular venipuncture. Blood was diluted 1:4 in PBS pH 7.2, 0.15 M, and layered on Ficoll-Hypaque density 1077 (1.077 g/ml, Sigma) at a ratio of 4:1. Tubes were centrifuged for 35 min at 800 × g, 4 ° C. Cells at the interface were collected, washed and resuspended in either routine RPMI 1640 or RPMI/L-15 both supplemented with 10% heat inactivated FBS and 1% gentamicin. Cell concentrations were adjusted to 2.0 × 106/ml, and 0.2 ml of cells per well were added to a round-bottom microtiter plate.
Viability measurements Cell viability assays were performed using trypan blue exclusion. Estimates of cell viability were made on day of collection and daily thereafter for the duration of culture. At each time point, cell viability was determined on cells which were not exposed to mitogen.
MMC stimulation assay The lectins used in this study were ConA (Miles Scientific ), and PHA (Burroughs Wellcome Co. ). ConA was added to cell suspensions at 3.3 and 6.6/~g/ ml, while PHA was added at 1, 5 and 10/~g/ml. Mitogens were added to mammary mononuclear cells (2.0 × 106/ml), and 200/tl aliquots of the suspension added to microtiter wells. Control wells received cells only. Microtiter plates were incubated at 37 ° C, in a 5% CO2 atmosphere. Wells were pulsed with 0.2 /2Ci 3H-thymidine (specific activity 6.7 Ci/mmol, New England Nuclear) for 6 h on days 2, 3 or 4 of culture. Cultures were harvested on glass fiber filtermats by means of a cell harvester (Skatron). Filtermats were placed in 10.0 ml scintillation fluid (Econofluor, New England Nuclear), and counts per minute (cpm) were determined in a Beckman (model LS-3105T) scintillation counter. Means were calculated from quadruplicate or triplicate wells for each treatment, logarithmically (base 10) transformed, and used for statistical analysis.
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P B L stimulation assay The mitogens ConA (3.3 z g / m l ) and P H A (5.0/~g/ml) were added to peripheral blood mononuclear cells (2.0 X 106/ml). Aliquots of 200/~l were added to appropriate microtiter wells, and plates incubated at 37°C, in a 5% CO2 atmosphere. Wells were pulsed with 0.2/ICi 3H-thymidine for 6 h and cells were harvested on day 3 of culture. Cultures were harvested, counted and analyzed as for MMC.
Statistical analyses A multifactor analysis of variance for mitogen and viability means was performed using the G L M option of SAS (SAS Institute) according to a completely randomized design. T r e a t m e n t means within a main effect were tested for significant differences using Scheffe's method of multiple comparisons. RESULTS
Effect of culture conditions on M M C viability M a m m a r y cell viability was high ( > 77% ) in both media on all days of culture. M a m m a r y cell viability was significantly higher (P < 0.05) in the R P M I / L-15 medium (86.2%) t h a n in the R P M I alone (81.5%).
Effect of culture conditions on M M C stimulation Day of culture harvest (length of incubation) had a significant effect on the proliferative responses of M M C to mitogens ConA and P H A (Table 1). ProTABLE 1 Analysis of variance for the proliferative response of mammary mononuclearcells to mitogenic lectins and culture conditions Variable1
Mean square
D T M P D XT DXM DXP TXM T×P MXP DXTXM
1.52" 6.29** 59.73** 0.44 0.03 0.08 0.17 1.75"* O.O7 0.00 O.08
1D=day of harvest, T--treatment (control, ConA levels, or PHA levels), M=media, P=preincubation. *P<0.05; **P<0.01.
327
BLASTOGENICRESPONSESOFBOVINEMAMMARYMONONUCLEARCELLS TABLE 2 Effect of day of harvest on mammary mononuclear cell mitogenesis1 Day of harvest
Tritiated thymidine incorporation (log10 cpm)
2
3.58 ± 0.06 a
3 4
3.54 ± 0.05a 3.37 ± 0.055
1Data expressed as least squares means ± s.e.m. a,bMeans within a column are different at P < 0.05. TABLE3 Effects of mitogens and culture medium on proliferative responses of mammary mononuclear cells 1 Medium
RPMI RPMI/L-15
Control
ConA (#g/ml)
PHA (pg/ml)
0
3.3
6.6
1
5
10
2.78 2.91
3.17 4.08
3.12 4.10
3.12 4.04
3.19 4.15
3.18 4.11
1Values in the table represent least squares means of tritiated thymidine (loglo) incorporated into mammary mononuclear cells after 3 days in vitro exposure to no lectin {control), ConA or PHA. TABLE4 Viability of peripheral blood lymphocytes after various days in culture in RPMI or RPMI/L-151 Day of culture
0 1 2 3
Viability (%) RPMI
RPMI/L-15
97.8 ± 0.3a 61.5 ___2.7b 60.5 ± 3.35 58.3 ± 4.45
99.3 ± 0.5a 63.0 ± 2.25 58.3 _+3.85 57.0 + 2.25
1Values in table represent means _+s.e.m. a'5Means within a column are different at P < 0.01. liferative r e s p o n s e s of M M C were s i g n i f i c a n t l y h i g h e r w h e n cells were harv e s t e d o n d a y s 2 a n d 3 o f c u l t u r e (3.58 a n d 3.54 log c p m ) a s o p p o s e d t o d a y 4 ( T a b l e 2 ). A s t h e r e w a s n o d i f f e r e n c e i n t h e r e s p o n s e s o n d a y s 2 a n d 3, c u l t u r e s were harvested on day 3 in subsequent experiments. M i t o g e n i c r e s p o n s e s to C o n A a n d P H A were c o n s i s t e n t l y higher in the R P M I / L - 1 5 m e d i u m ( m e a n r e s p o n s e o f 4.10 log c p m ) , t h a n i n R P M I ( m e a n
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TABLE 5 Tritiated thymidine incorporation by peripheral blood lymphocytes after control, ConA or P H A treatment 1 Mitogen
Control (no mitogen) ConA (3.3 lzg/ml) P H A (5/zg/ml)
Tritiated thymidine incorporation (loglo cpm) RPMI
RPMI/L-15
3.69_+0.08 ~ 4.62 ± 0.05 b 4.62 ± 0.05 b
3.69_+0.15 a 4.62 ± 0.02 b 4.65 ± 0.01 b
Walues in table represent means ± s,e.m. a'bMeans within a column are different at P < 0.01.
response of 3.16 log cpm; see Table 3 ) as evidenced by a significant mitogen by medium interaction (Table 1 ). Further statistical examination of the mitogen by medium interaction using Scheffe's multiple comparisons provided the following information: ( 1 ) control (cells + medium) responses were not different in the two media, (2) MMC were stimulated by lectins to proliferate in both the RPMI and the RPMI/L-15, (3) there was not a significant dose response in either medium at the levels of mitogen tested, and (4) the proliferative responses of MMC in the RPMI/L-15 were higher at all doses of mitogens used, as compared to t h e responses in the RPMI alone. As there was no difference in the response of MMC to different mitogen doses (Table 3), concentrations of 3.3 and 5.0 #g/ml of ConA and PHA, respectively, were used in subsequent mitogen assays. Pre-incubation of cells in either medium for 24 h prior to exposure to mitogen had no effect on the proliferative responses to mitogens (Table 1 ).
Effect of culture medium on P B L viability and stimulation Viability of peripheral blood lymphocytes was quite high on day of collection (Table 4), decreased significantly in both RPMI (61.5%) and RPMI/L-15 (63.0%) the following day, then remained constant in each medium for the duration of the culture. In contrast to mammary mononuclear cells, peripheral blood lymphocytes were stimulated by lectins to an equivalent degree in both RPMI and R P M I / L-15 (Table 5). Furthermore, the response to lectins in each medium was one order of magnitude higher than control responses. DISCUSSION
Viability of PBL and MMC would suggest that both RPMI and RPMI/L15 support the survival of these cell populations in culture. The number of live
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PBL determined on each day of culture was nearly identical in both media tested. In contrast, on each day of culture, a higher percentage of mammary cells survived in R P M I / L - 1 5 than in RPMI. As mammary cell viability was greater than 77% in each medium at all times measured, it appears that cell viability is not the critical factor responsible for the observed increase in proliferative responses of MMC when cultured in RPMI/L-15. One parameter of the mitogen assay examined was pre-incubation of cells overnight in media. Several authors (Collins and Oldham, 1986; Harp and Nonnecke, 1986) have suggested that secretions from nonlactating cows contain soluble suppressive factors which when co-cultured with PBL inhibit the proliferative response of PBL. Attempts to reverse the potential effects of these suppressive factors in the present study by removing mammary cells from secretion and holding t h e m in media for 24 h, did not result in an increased proliferative response. The results of the determinations of the optimal day for pulse and harvest of mammary cultures (day 3), and the optimal concentrations of ConA and PHA (3.3 and 5.0/tg/ml) to use in the mitogen assay, are in line with those conditions defined by Mastro and Pepin (1982) for bovine lymph node cells, and by Torre and Oliver (1988) and Pearson et al. ( 1979 ) for bovine peripheral blood lymphocytes. Mammary mononuclear cells exhibited a greater proliferative response to lectins in RPMI/L-15 than in RPMI alone. Peripheral blood mononuclear cells on the other hand demonstrated the equivalent ability to respond significantly to lectins in both media. One possible explanation for this difference may be the calcium ion requirement for in vitro lymphocyte stimulation. Calcium ion has been proposed as a potential second messenger in lymphocyte mitogenensis for the following reasons: extracellular calcium is required for DNA synthesis (Alford, 1970; Whitney and Sutherland, 1972), and mobilization of calcium ion stores to free cytosolic calcium occurs shortly after binding of a ligand to cell surface receptors (Allwood et al., 1971; Freedman et al., 1975). We have measured calcium ion concentrations in nonlactating mammary secretions in a preliminary study. Results suggested that calcium ion concentrations may vary depending on the length and stage of the dry period, but levels are higher than those reported in serum (Stout et al., 1976). Lymphocytes transported from the blood into the nonlactating mammary gland are likely to experience an increase in extracellular calcium ion concentration. Interestingly, R P M I / L-15 medium contains twice the amount of calcium ion (0.84 mM/1) than does R P M I (0.42 mM/1). It may be that the in vitro CA 2+ requirements for mammary cell proliferation parallel those which exist in the gland, and to which the mammary cell has adapted. Another possible explanation for the enhanced mammary lymphocyte function in R P M I / L - 1 5 involves the pH requirements for lymphocyte division in mitogen-stimulated cultures. Gerson and Kiefer (1983) estimated the intra-
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cellular pH for murine ConA lymphoblasts during the DNA synthesis phase of the cell cycle, to be between 7.40 and 7.45. The extracellular pH during the experiment, was carefully maintained at a constant 7.30 in order that ultimately, intracellular regulation of the cells' biosynthetic functions not be disturbed. In our assay system, RPMI is buffered with sodium bicarbonate and CO2. L-15 is a phosphate-buffered medium. We have observed (by color change) a subtle, but greater increase in media pH after 3 days culture in wells containing RPMI alone than in wells containing RPMI/L-15. It may be that maintenance of a certain extracellular pH is more critical for the biosynthetic functions of mammary mononuclear cells than for peripheral blood lymphocytes. REFERENCES Alford, R.H., 1970. Metal cation requirements for phytohemagglutinin-induced transformation of human peripheral blood lymphocytes. J. Immunol., 104: 698-703. Allwood, G., Asherson, G.L., Davey, M.J. and Goodford, P.J., 1971. The early uptake of radioactive calcium by human lymphocytes treated with phytohemagglutinin. Immunology, 21: 509516. Collins, R.A. and Oldham, G., 1986. Proliferative responses and IL-2 production by mononuclear cells from bovine mammary secretions, and the effect of mammary secretions on peripheral blood lymphocytes. Immunology, 58: 647-651. Concha, C., Homberg, 0. and Morein, B., 1980. Characterization and response to mitogens of mammary lymphocytes from the bovine dry period secretion. J. Dairy Res., 47:305-311. Freedman, M.H., Raft, M.C. and Gomperts, B., 1975. Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides, nature, 255: 378-382. Gerson, D.F. and Kiefer, H., 1983. Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes. J. Cell. Physiol., 114: 132-136. Harp, J.A. and Nonnecke, B.J., 1986. Regulation of mitogenic responses by bovine milk leukocytes. Vet. Immunol. Immunopathol., 11: 215-224. Hurley, D.J., Kensinger, M.H. and Wilson, R.A., 1990. An evaluation of the mononuclear cells derived from bovine mammary dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties, and non-specific esterase staining. Submitted. Mastro, A.M. and Pepin, K.G., 1982. Effects of 12-o-tetradecanoylphorbol-13-acetateon lymphocyte proliferation. In: E. Hecker et al. (Editors), Carcinogenesis, Vol. 7. Raven Press, New York, NY, pp. 439-442. Pearson, T.W., Roelants, G.E., Lundin, L.B. and Mayor-Withey, K.S., 1979. The bovine lymphoid system: binding and stimulation of peripheral blood lymphocytes by lectins. J. Immunol. Methods, 26: 271-282. Shore, C.E., Osburn, B.I., Jasper, D.E. and Tyler, D.E., 1981. B and T lymphocytes in the bovine mammary gland: rosette formation and mitogen response. Vet. Immunol. Immunopathol., 2: 561-569. Stout, W.L., Kradel, D.C., Jung, G.A. and Smiley, C.G., 1976. Blood composition of well-managed high-producing Holsteincows in Pennsylvania. The Pennsylvania State University Agricultural Experiment Station, Progress Report 358, pp. 2-11. Torre, P.M. and Oliver, S.P., 1988. Changes in blastogenic activity of bovine blood mononuclear cells throughout the nonlactating period. J. Dairy Sci., 71: 1078-1084. Whitney, R.B. and Sutherland, R.M., 1972. Enhanced uptake of calcium by transforming lymphocytes. Cell. Immunol., 5: 137-147.