The Effect of Lipopolysaccharide on Bovine Mammary Macrophage Function I. Politis, X. Zhao, B.W. McBride and J.H. Burton
ABSTRACT
The effect of Escherichia coli lipopolysaccharide (LPS) on the expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages was examined. The ability of LPS-treated mammary macrophages to support antigen-specific T-cell proliferation, as a measure of their antigen presentation ability, was also evaluated. For this purpose, control and LPS-treated macrophages were pulsed with heatkilled Staphylococcus aureus and then cultured with S. aureus-sensitized T-cells. Our data show that LPS had no significant effect on the expression of MHC class II molecules on the surface of mammary macrophages. Furthermore, LPS-induced macrophages were no more active in supporting T-cell proliferation on a per cell basis than unstimulated macrophages. The lack of macrophage response to LPS with respect to expression of MHC class II molecules and the antigen presentation ability is another example of the hyporesponsive nature of macrophages isolated from the bovine mammary gland.
RESUME
L'effet du lipopolysaccharide (LPS), extrait d'Escherichia coli, sur l'expression des antigenes d'histocompatibilite (CMH) de classe II, par des macrophages isoles de glandes mammaires de bovins a ete evalue et la capacite que possdent ces macrophages, traites avec le LPS, a presenter un antigene a des lymphocytes T specifiques, favorisant ainsi leur proliferation, a aussi ete
analysee. Pour ce faire, des macrophages temoins et traites au LPS ont ete mis en contact avec un antigene soit une souche inactivee par la chaleur de Staphylococcus aureus. Puis, ces macrophages ont e mis en culture avec des lymphocytes sensibilises par S. aureus. Les resultats demontrent que le LPS n'a eu aucun effet significatif sur l'expression a la surface des macrophages d'antigenes de classe II du CMH. De plus, les macrophages trait6s avec le LPS n'ont pas favorise davantage la proliferation des lymphocytes, que ne l'ont fait les macrophages temoins. L'incapacite des macrophages provenant de glandes mammaires de bovins a repondre au LPS, afin de favoriser l'expression a leur surface des antigenes de classe II du CMH et leur capacite a presenter un antigene au lymphocyte, est un autre exemple du caractere d'hyporeactivite de ces macrophages. (Traduit par Dr Pauline Brousseau)
INTRODUCTION Infections of the bovine mammary gland giving rise to mastitis have adverse effects on the production and the composition of the milk, even when the mastitis is subclinical (1). The manipulation of the natural defence mechanisms of the gland has not yet provided a useful means of controlling this costly disease. It is imperative that a better understanding of the immune system of the bovine mammary gland be acquired. Macrophages are the predominant cells in bovine milk during the course
of a healthy lactation (2). Expression of class II major histocompatibility complex (MHC) molecules on the surface of macrophages is a strict requirement for effective antigen presentation resulting in lymphocyte activation (3). Our previous work has demonstrated that mammary macrophages are not very effective antigen presenting cells. Furthermore, mammary macrophages expressed low levels of MHC class II molecules (4). Because of the critical role of MHC expression in immune induction, it is important to understand in detail the regulation of mammary macrophages MHC class II expression. Lipopolysaccharide (LPS), a major outer membrane component of gramnegative bacteria, interacts with macrophages to bring about a variety of changes regarding macrophage function (5). Lipopolysaccharide augments the expression of MHC class II molecules on the surfaces of murine macrophages (6,7). In contrast, others suggested that LPS suppresses expression of class II MHC antigens and inhibits both interferon-gamma (IFN--y) induction and maintenance of class II molecules on murine macrophages (8,9). The effect of LPS on the expression of MHC class II antigens by bovine mammary macrophages has not yet been investigated. The present study was undertaken to investigate the effect of LPS on expression of MHC class II antigens by bovine mammary macrophages. Furthermore, the ability of LPS-treated macrophages to interact with T-cells was assessed.
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1. Present address of I. Politis: Department of Animal Science, Morrison Hall, Cornell University, Ithaca, New York 14853. Reprint requests to B.W. McBride. Supported by a grant from Natural Sciences and Engineering Research Council of Canada and Agriculture Canada. Submitted October 2, 1990.
220
Can J Vet Res 1991; 55: 220-223
MATERIALS AND METHODS
ISOLATION OF BLOOD LEUKOCYTES
6-Dichlorotriarin-2yl) amino] fluorescein (Jackson Immunoresearch, Avondale, Pennsylvania). Percentage of positively staining cells was determined by counting approximately 500 cells. T- and B-cells always represented less than 2% of the total adherent population. The purity of the antigen-specific T-cells was assessed using the same set of monoclonal antibodies. The purity of this T-cell population was always greater than 907o.
Peripheral blood leukocytes (PBL) were isolated following the procedure Seven Holstein cows (third to described elsewhere (11). Briefly, seventh month in lactation) were used 30 mL of heparinized (10 IU of throughout this investigation for heparin per mL) venous blood were obtaining peripheral blood monocytes layered onto 20 mL of Lymphoprep and milk macrophages. These cows (specific gravity 1.077 g/cm3) and were free from disease or signs of then centrifuged at 500 x g for 45 min mammary gland inflammation, and at 8°C. Cells at the interface were colhad somatic cell counts (SCC) in milk lected, assessed for viability by trypan less than 50,000/mL. A Fossomatic blue exclusion (typically > 95% cell counter (Foss Electric, Hillerod, viable), washed twice in RPMI-1640, Denmark) was used to determine SCC and resuspended at appropriate cell TEST FOR ANTIGEN PRESENTATION Cultures were performed in in milk. The guidelines of the Guide to concentrations. quadruplicates in 96-well flat bottomed the Care and Use of Experimental culture plates (Flow Laboratories) in Animals of the Canadian Council on GENERATION OF ANTIGEN SPECIFIC 200 $L of RPMI-1640 + 10% FBS. Animal Care were followed throughout. T-CELLS Antigen specific T-cells (1.2 x 105 were Peripheral blood leukocytes ISOLATION OF MILK MACROPHAGES adjusted to 106 cells/mL and cultured cells/well) were cultured with 1.8 x Enriched milk macrophages were in RPMI-1640 + 10% FBS containing 104 mammary macrophages (control isolated following the procedure 60 ug (dry weight)/mL of heat-killed or LPS-treated) in the presence or described elsewhere (10). Milk samples Staphylococcus aureus (100°C, absence of S. aureus (60 1g/mL). Pre(8 L) were collected during the morn- 30 min). Staphylococcus aureus was liminary experiments had shown that ing milking. Approximately 6-8 L of kindly provided by Prof. S. Rosendal the above concentration of S. aureus milk were centrifuged at 500 x g for (Ontario Veterinary College). After induced optimal responses under the 20 min. Pelleted somatic cells were five days, the nonadherent cells current culture conditions. Cultures washed three times with RPMI-1640 were removed and washed twice in were incubated at 37°C in 5 % CO2 containing 10% fetal bovine serum RPMI-1640. T-cells were further and cell proliferation was assessed (FBS) (Sigma Chemical Co., St. Louis, purified by the procedure described by after three or five days. Eighteen hours Missouri), penicillin (100 IU/mL) and Splitter and Everlith (12) using the before termination of the cultures, streptomycin (100 Ag/mL). Cells were anti-MHC class II monoclonal anti- 0.5 ltCi [3H] thymidine (Amersham, resuspended in 16 mL of RPMI-1640 body (Mab) H4 (One Lambda, Oakville, Ontario) was added per well. and layered on top of 9 mL Lympho- Los Angeles, California) and a mix- The plates were harvested using a prep (Nycomed AS, Oslo, Norway) ture of Mabs DAS-9 and DAS-10 multiple cell harvester (Cambridge (specific gravity 1.077 g/cm3) and (Ultimate Conception, Amherst, Techn., Cambridge, Massachusetts) centrifuged at 500 x g for 45 min at Massachusetts) which recognize the and radioactivity was counted in a 8°C. Cells at the interface were col- light and the heavy chain of bovine liquid scintillation counter (Searle, lected and viability was assessed by immunoglobulin (IgG), respectively. Des Plaines, Illinois). Results are trypan blue exclusion (typically > T-cells were used immediately or expressed as mean counts per minute 90%o viable). Cells were washed twice briefly maintained in culture (less than (CPM) ± SD. in RPMI-1640 and plated at 5 x 107 3 h) and processed further as described EXPRESSION OF MHC CLASS II cells/mL of 2 mL RPMI-1640 + 10% later. ANTIGENS BY BOVINE MACROPHAGES FBS/well in a 12-well plastic plate IDENTIFICATION OF MACROPHAGES An enzyme-linked immunosorbent (Flow Laboratories, McLean, Virginia) and were incubated in 5%To CO2 at AND T-CELLS assay procedure was employed for The presence of a-naphthyl acetate measuring MHC class II determinants 37°C. Following a 24 h incubation, the cells adhering to the plastic dishes were esterase activity was used as a means expression on the surface of mammary washed thoroughly in RPMI-1640 to of identifying monocytes and macro- macrophages (12). The first antibody remove weakly adherent cells and then phages (13). Only cells with diffuse was the HLA-DR specific Mab H4. further incubated in RPMI-1640 + cytoplasmic staining, as opposed to the The secondary antibody was a perox10% FBS in the presence of various dot-like staining of lymphocytes, were idase conjugated F(ab,)2 goat antilevels (0-30 ug/mL) of Escherichia coli considered as monocytes. The adherent mouse IgG (Sigma). Results are LPS (Sigma). Following one, three or cell population isolated from milk con- expressed as change in absorbance five days incubation, the cells were sisted of 88% (84-92Oo) macrophages. (A A) per 106 cells. In an attempt to account for a T-cell thoroughly washed in RPMI-1640 and The presence of contaminating T- and then detached by incubating them with B-cells in the above cell population was dependent increase in MHC expres0.35% ethylenediaminetetraacetic acid assessed using Mab 8-1E3 (antipan sion, we questioned whether LPS (EDTA). Recovered cells were washed T cell) (14) and DAS-9 (anti-IgG light could have an indirect effect on three times in RPMI-1640 and pro- chain) by indirect immunofluoresence macrophage MHC class II expression. using goat antimouse-IgG-[5-(4, All the details of this experiment were cessed further as described later. ANIMALS
221
TABLE I. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with various levels of Escherichia coli lipopolysacchride (LPS) LPS (ug/mL) MHC expressiona (A A/106 cells) 0 0.28 ± 0.032 15 0.26 ± 0.039 30 0.31 ± 0.029 aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD of seven independent samples TABLE II. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with Escherichia coli Iipopolysacchride (15 Ag/mL) in the presence or absence of T-cells
Cells MHC expressiona (A A/106 cells) 0.32 ± 0.029 Macrophages alone 0.36 + 0.036 Macrophages + T-cells aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD of seven independent samples
as above, the only difference being that macrophages plus T-cells (1: 1 cell ratio) and not macrophages alone were treated overnight with various levels of LPS. All data are expressed as means + SD. Differences between the means were evaluated using Student's t-test (p < 0.01).
fer markedly (p > 0.05) in mammary macrophages treated with LPS for one, three or five days. ANTIGEN-PRESENTATION FUNCTION OF LPS-INDUCED MACROPHAGES
Furthermore, it was observed that LPS-induced macrophages were no more active on a per cell basis in supporting T-cell proliferation than unstimulated macrophages. DISCUSSION The novel finding emerging from this study was that E. coli LPS had no significant effect on the expression of MHC class II molecules by bovine mammary macrophages. Even when very high doses of LPS (30 Ag/mL) were used, expression of MHC class II molecules was unaffected. This concentration of LPS is known to be optimal for production of interleukin-l by mammary macrophages (15). To rule out the possibility that the kinetics of MHC expression are different in LPS-treated macrophages, we showed that MHC class II expression was similar in stimulated and unstimulated macrophages at one, three or five days of culture. Our findings are in agreement with those of others (9) who were unable to demonstrate significant effects of LPS on MHC expression in the absence of IFN-'y. In contrast, others were capable of demonstrating both positive (7,12) and negative effects (8) of LPS on MHC expression. These differences could be explained by known differences in macrophages isolated from different tissues and species. We considered it possible that MHC class II expression may be a T-cell
The ability of LPS-induced macrophages to support antigen-specific T-cell proliferation may be taken as a measure of their antigen presentation ability (7). Control and LPS-treated macrophages were pulsed with heatRESULTS killed S. aureus and then cultured with LPS-DOSE DEPENDENCE S. aureus-sensitized T-cells. The results The effect of various levels of LPS of this experiment are presented in (0-30 Ag/mL) on the expression of Table IV. There was a 5.3-fold increase MHC class II molecules on the surface in T-cell proliferation in the presence of bovine mammary macrophages was of macrophages when compared to investigated and the results are pre- that in the absence of macrophages. sented in Table I. As shown, addition of LPS had no significant (p > 0.05) effect on the expression of MHC class II antigens on the surface of mammary TABLE III. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with Escherichia coil lipopolysacchride (15 ug/mL) for different macrophages. In attempts to account culture times for a T-cell dependent effect of LPS on MHC expression we questioned Culture time (days) MHC expressiona (A A/106 cells) whether LPS could affect MHC 1 0.31 + 0.029 3 0.37 ± 0.034 expression only in the presence of 5 0.32 ± 0.041 T-cells. Table II reveals no significant differences in MHC class II expression aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD in macrophages cocultured with T-cells of seven independent samples relative to macrophages cultured in medium alone. TABLE IV. Proliferative responses of T-cells (1.2 x 105 cells/well) cultured with unstimulated or stimulated with 30 Ag/mL Escherichia coil lipopolysaccharide mammary macrophages (1.8 x 104 cells/well) in the presence of 60 Ag/mL S. aureus KINETICS OF MHC EXPRESSION Cells T-cell proliferation (mean CPMa + SD) Mammary macrophages were treated with LPS and were removed T-cells alone 615 ± 115 from the cultured medium at the + unstimulated macrophages 3,430 ± 393 3,217 ± 423 indicated times. Table III shows that + stimulated macrophages MHC class II expression did not dif- aCounts per minute
222
dependent phenomenon. Therefore, we questioned whether LPS could modulate the expression of MHC class II molecules only in the presence of mature T-cells. Our data revealed no significant differences in MHC class II expression when macrophages were cultured with T-cells or in the medium alone. We concluded that MHC expression is either completely independent of T-cells or requires very low numbers of T-cells. The latter possibility cannot be eliminated because it is practically impossible to obtain macrophages completely free of T-cells. The present observations document that mammary macrophages can act as antigen presenting cells as judged by their ability to induce (fivefold) S. aureus specific T-cell proliferation (Table IV). This is in agreement with earlier findings (4). The ability of macrophages to present antigens to T-cells is directly related to the levels of MHC class II expression by the macrophages (3). In keeping with this idea, our data show that LPS, which was unable to affect MHC class II expression, was also unable to enhance the proliferation of antigen-specific T-cells. The present study shows an inability of LPS to modulate the expression of MHC class II molecules and to enhance the antigen presentation function of mammary macrophages. The lack of macrophage response to LPS is another example of the hyporesponsive nature of macrophages isolated from the bovine mammary gland. In
a previous study we have shown that despite the fact that mammary macrophages could act as antigen presenting cells, they were much less efficient than their autologous blood monocytes (4). This might explain why the mammary gland is most susceptible to infection at times that macrophages are the predominant cells in milk (16). In summary, the present observations document the absence of effects of LPS on MHC class II expression by bovine mammary macrophages. Furthermore, LPS could not enhance the antigen presentation ability of mammary macrophages. REFERENCES 1. KITCHEN BJ. Review of the progress of dairy science. Bovine mastitis: milk compositional changes and related diagnostic tests. J Dairy Res 1981; 48: 162-178. 2. LEE CS, WOODING FBP, KEMP P. Identification, properties and differential counts of cell populations using electron microscopy of dry cows secretions, colostrum and milk from normal cows. J Dairy Res 1980; 47: 39-50. 3. UNANUE ER, BELLER DI, LU CY, ALLEN PM. Antigen presentation: comments on its regulation and mechanism. J Immunol 1984; 132: 1-5. 4. POLITIS I, ZHAO X, McBRIDE BW, BURTON JH. Function of bovine mammary macrophages as antigen presenting cells. Vet Immunol Immunopathol (in press). 5. MORRISON DC, RYAN JL. Bacterial endotoxins and host immune responses. Adv Immunol 1979; 28: 293-442. 6. WENTWORTH PA, ZIEGLER HK. Induction of macrophage Ia expression by lipopolysaccharide and listeria monocytogenes in congenitally athymic nude mice. J Immunol 1987; 138: 3167-3173.
7. ZIEGLER HK, STAFFILENO LK, WENTWORTH P. Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia in vivo. J Immunol 1984; 133: 1825-1835. 8. KOERNER TJ, HAMILTON TA, ADAMS DO. Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA. J Immunol 1987; 139: 239-243. 9. STEEG PS, JOHNSON HM, OPPENHEIM JJ. Regulation of murine macrophage Ia antigen expression by an interferon-like lymphokine: inhibitory effect of endotoxin. J Immunol 1982; 129: 2402-2406. 10. LEYVA-COBIAN F, CLEMENTE J. Phenotypic characterization and functional activity of human milk macrophages. Immunol Lett 1984; 8: 249-256. 11. GODDEERIS BM, BALDWIN CL, OLEMOI YOI 0, MORRISON WI. Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. J Immunol Methods 1985; 89: 165-173. 12. SPLITER GA, EVERLITH KM. Brucela abortus regulates macrophage-T-cell interaction by major histocompatibility complex class II and interleukin 1 expression. Infect Immun 1989; 57: 1151-1157. 13. YANG TC, JANTZEN PA, WILLIAMS LF. Acid a-naphthyl acetate esterase: presence of activity in bovine and human T-lymphocytes and B-lymphocytes. Immunology 1979; 38: 85-93. 14. O'REILLY KL, SPLITTER GA. Monoclonal antibodies to ovine SBU-T8 and SBU-T6 bind analogous molecules on bovine lymphocytes. Immunology 1989; 67: 176-180. 15. POLITIS I, ZHAO X, McBRIDE BW, BURTON JH. Limited secretion of interleukin 1 by bovine mammary macrophages. Am J Vet Res 1991; 52: (in press). 16. CRAVEN N, WILLIAMS MR. Defenses of the bovine mammary gland against infection and prospects for their enhancement. Vet Immunol Immunopathol 1985; 10: 71-127.
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ABSTRACT
The effect of Escherichia coli lipopolysaccharide (LPS) on the expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages was examined. The ability of LPS-treated mammary macrophages to support antigen-specific T-cell proliferation, as a measure of their antigen presentation ability, was also evaluated. For this purpose, control and LPS-treated macrophages were pulsed with heatkilled Staphylococcus aureus and then cultured with S. aureus-sensitized T-cells. Our data show that LPS had no significant effect on the expression of MHC class II molecules on the surface of mammary macrophages. Furthermore, LPS-induced macrophages were no more active in supporting T-cell proliferation on a per cell basis than unstimulated macrophages. The lack of macrophage response to LPS with respect to expression of MHC class II molecules and the antigen presentation ability is another example of the hyporesponsive nature of macrophages isolated from the bovine mammary gland.
RESUME
L'effet du lipopolysaccharide (LPS), extrait d'Escherichia coli, sur l'expression des antigenes d'histocompatibilite (CMH) de classe II, par des macrophages isoles de glandes mammaires de bovins a ete evalue et la capacite que possdent ces macrophages, traites avec le LPS, a presenter un antigene a des lymphocytes T specifiques, favorisant ainsi leur proliferation, a aussi ete
analysee. Pour ce faire, des macrophages temoins et traites au LPS ont ete mis en contact avec un antigene soit une souche inactivee par la chaleur de Staphylococcus aureus. Puis, ces macrophages ont e mis en culture avec des lymphocytes sensibilises par S. aureus. Les resultats demontrent que le LPS n'a eu aucun effet significatif sur l'expression a la surface des macrophages d'antigenes de classe II du CMH. De plus, les macrophages trait6s avec le LPS n'ont pas favorise davantage la proliferation des lymphocytes, que ne l'ont fait les macrophages temoins. L'incapacite des macrophages provenant de glandes mammaires de bovins a repondre au LPS, afin de favoriser l'expression a leur surface des antigenes de classe II du CMH et leur capacite a presenter un antigene au lymphocyte, est un autre exemple du caractere d'hyporeactivite de ces macrophages. (Traduit par Dr Pauline Brousseau)
INTRODUCTION Infections of the bovine mammary gland giving rise to mastitis have adverse effects on the production and the composition of the milk, even when the mastitis is subclinical (1). The manipulation of the natural defence mechanisms of the gland has not yet provided a useful means of controlling this costly disease. It is imperative that a better understanding of the immune system of the bovine mammary gland be acquired. Macrophages are the predominant cells in bovine milk during the course
of a healthy lactation (2). Expression of class II major histocompatibility complex (MHC) molecules on the surface of macrophages is a strict requirement for effective antigen presentation resulting in lymphocyte activation (3). Our previous work has demonstrated that mammary macrophages are not very effective antigen presenting cells. Furthermore, mammary macrophages expressed low levels of MHC class II molecules (4). Because of the critical role of MHC expression in immune induction, it is important to understand in detail the regulation of mammary macrophages MHC class II expression. Lipopolysaccharide (LPS), a major outer membrane component of gramnegative bacteria, interacts with macrophages to bring about a variety of changes regarding macrophage function (5). Lipopolysaccharide augments the expression of MHC class II molecules on the surfaces of murine macrophages (6,7). In contrast, others suggested that LPS suppresses expression of class II MHC antigens and inhibits both interferon-gamma (IFN--y) induction and maintenance of class II molecules on murine macrophages (8,9). The effect of LPS on the expression of MHC class II antigens by bovine mammary macrophages has not yet been investigated. The present study was undertaken to investigate the effect of LPS on expression of MHC class II antigens by bovine mammary macrophages. Furthermore, the ability of LPS-treated macrophages to interact with T-cells was assessed.
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1. Present address of I. Politis: Department of Animal Science, Morrison Hall, Cornell University, Ithaca, New York 14853. Reprint requests to B.W. McBride. Supported by a grant from Natural Sciences and Engineering Research Council of Canada and Agriculture Canada. Submitted October 2, 1990.
220
Can J Vet Res 1991; 55: 220-223
MATERIALS AND METHODS
ISOLATION OF BLOOD LEUKOCYTES
6-Dichlorotriarin-2yl) amino] fluorescein (Jackson Immunoresearch, Avondale, Pennsylvania). Percentage of positively staining cells was determined by counting approximately 500 cells. T- and B-cells always represented less than 2% of the total adherent population. The purity of the antigen-specific T-cells was assessed using the same set of monoclonal antibodies. The purity of this T-cell population was always greater than 907o.
Peripheral blood leukocytes (PBL) were isolated following the procedure Seven Holstein cows (third to described elsewhere (11). Briefly, seventh month in lactation) were used 30 mL of heparinized (10 IU of throughout this investigation for heparin per mL) venous blood were obtaining peripheral blood monocytes layered onto 20 mL of Lymphoprep and milk macrophages. These cows (specific gravity 1.077 g/cm3) and were free from disease or signs of then centrifuged at 500 x g for 45 min mammary gland inflammation, and at 8°C. Cells at the interface were colhad somatic cell counts (SCC) in milk lected, assessed for viability by trypan less than 50,000/mL. A Fossomatic blue exclusion (typically > 95% cell counter (Foss Electric, Hillerod, viable), washed twice in RPMI-1640, Denmark) was used to determine SCC and resuspended at appropriate cell TEST FOR ANTIGEN PRESENTATION Cultures were performed in in milk. The guidelines of the Guide to concentrations. quadruplicates in 96-well flat bottomed the Care and Use of Experimental culture plates (Flow Laboratories) in Animals of the Canadian Council on GENERATION OF ANTIGEN SPECIFIC 200 $L of RPMI-1640 + 10% FBS. Animal Care were followed throughout. T-CELLS Antigen specific T-cells (1.2 x 105 were Peripheral blood leukocytes ISOLATION OF MILK MACROPHAGES adjusted to 106 cells/mL and cultured cells/well) were cultured with 1.8 x Enriched milk macrophages were in RPMI-1640 + 10% FBS containing 104 mammary macrophages (control isolated following the procedure 60 ug (dry weight)/mL of heat-killed or LPS-treated) in the presence or described elsewhere (10). Milk samples Staphylococcus aureus (100°C, absence of S. aureus (60 1g/mL). Pre(8 L) were collected during the morn- 30 min). Staphylococcus aureus was liminary experiments had shown that ing milking. Approximately 6-8 L of kindly provided by Prof. S. Rosendal the above concentration of S. aureus milk were centrifuged at 500 x g for (Ontario Veterinary College). After induced optimal responses under the 20 min. Pelleted somatic cells were five days, the nonadherent cells current culture conditions. Cultures washed three times with RPMI-1640 were removed and washed twice in were incubated at 37°C in 5 % CO2 containing 10% fetal bovine serum RPMI-1640. T-cells were further and cell proliferation was assessed (FBS) (Sigma Chemical Co., St. Louis, purified by the procedure described by after three or five days. Eighteen hours Missouri), penicillin (100 IU/mL) and Splitter and Everlith (12) using the before termination of the cultures, streptomycin (100 Ag/mL). Cells were anti-MHC class II monoclonal anti- 0.5 ltCi [3H] thymidine (Amersham, resuspended in 16 mL of RPMI-1640 body (Mab) H4 (One Lambda, Oakville, Ontario) was added per well. and layered on top of 9 mL Lympho- Los Angeles, California) and a mix- The plates were harvested using a prep (Nycomed AS, Oslo, Norway) ture of Mabs DAS-9 and DAS-10 multiple cell harvester (Cambridge (specific gravity 1.077 g/cm3) and (Ultimate Conception, Amherst, Techn., Cambridge, Massachusetts) centrifuged at 500 x g for 45 min at Massachusetts) which recognize the and radioactivity was counted in a 8°C. Cells at the interface were col- light and the heavy chain of bovine liquid scintillation counter (Searle, lected and viability was assessed by immunoglobulin (IgG), respectively. Des Plaines, Illinois). Results are trypan blue exclusion (typically > T-cells were used immediately or expressed as mean counts per minute 90%o viable). Cells were washed twice briefly maintained in culture (less than (CPM) ± SD. in RPMI-1640 and plated at 5 x 107 3 h) and processed further as described EXPRESSION OF MHC CLASS II cells/mL of 2 mL RPMI-1640 + 10% later. ANTIGENS BY BOVINE MACROPHAGES FBS/well in a 12-well plastic plate IDENTIFICATION OF MACROPHAGES An enzyme-linked immunosorbent (Flow Laboratories, McLean, Virginia) and were incubated in 5%To CO2 at AND T-CELLS assay procedure was employed for The presence of a-naphthyl acetate measuring MHC class II determinants 37°C. Following a 24 h incubation, the cells adhering to the plastic dishes were esterase activity was used as a means expression on the surface of mammary washed thoroughly in RPMI-1640 to of identifying monocytes and macro- macrophages (12). The first antibody remove weakly adherent cells and then phages (13). Only cells with diffuse was the HLA-DR specific Mab H4. further incubated in RPMI-1640 + cytoplasmic staining, as opposed to the The secondary antibody was a perox10% FBS in the presence of various dot-like staining of lymphocytes, were idase conjugated F(ab,)2 goat antilevels (0-30 ug/mL) of Escherichia coli considered as monocytes. The adherent mouse IgG (Sigma). Results are LPS (Sigma). Following one, three or cell population isolated from milk con- expressed as change in absorbance five days incubation, the cells were sisted of 88% (84-92Oo) macrophages. (A A) per 106 cells. In an attempt to account for a T-cell thoroughly washed in RPMI-1640 and The presence of contaminating T- and then detached by incubating them with B-cells in the above cell population was dependent increase in MHC expres0.35% ethylenediaminetetraacetic acid assessed using Mab 8-1E3 (antipan sion, we questioned whether LPS (EDTA). Recovered cells were washed T cell) (14) and DAS-9 (anti-IgG light could have an indirect effect on three times in RPMI-1640 and pro- chain) by indirect immunofluoresence macrophage MHC class II expression. using goat antimouse-IgG-[5-(4, All the details of this experiment were cessed further as described later. ANIMALS
221
TABLE I. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with various levels of Escherichia coli lipopolysacchride (LPS) LPS (ug/mL) MHC expressiona (A A/106 cells) 0 0.28 ± 0.032 15 0.26 ± 0.039 30 0.31 ± 0.029 aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD of seven independent samples TABLE II. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with Escherichia coli Iipopolysacchride (15 Ag/mL) in the presence or absence of T-cells
Cells MHC expressiona (A A/106 cells) 0.32 ± 0.029 Macrophages alone 0.36 + 0.036 Macrophages + T-cells aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD of seven independent samples
as above, the only difference being that macrophages plus T-cells (1: 1 cell ratio) and not macrophages alone were treated overnight with various levels of LPS. All data are expressed as means + SD. Differences between the means were evaluated using Student's t-test (p < 0.01).
fer markedly (p > 0.05) in mammary macrophages treated with LPS for one, three or five days. ANTIGEN-PRESENTATION FUNCTION OF LPS-INDUCED MACROPHAGES
Furthermore, it was observed that LPS-induced macrophages were no more active on a per cell basis in supporting T-cell proliferation than unstimulated macrophages. DISCUSSION The novel finding emerging from this study was that E. coli LPS had no significant effect on the expression of MHC class II molecules by bovine mammary macrophages. Even when very high doses of LPS (30 Ag/mL) were used, expression of MHC class II molecules was unaffected. This concentration of LPS is known to be optimal for production of interleukin-l by mammary macrophages (15). To rule out the possibility that the kinetics of MHC expression are different in LPS-treated macrophages, we showed that MHC class II expression was similar in stimulated and unstimulated macrophages at one, three or five days of culture. Our findings are in agreement with those of others (9) who were unable to demonstrate significant effects of LPS on MHC expression in the absence of IFN-'y. In contrast, others were capable of demonstrating both positive (7,12) and negative effects (8) of LPS on MHC expression. These differences could be explained by known differences in macrophages isolated from different tissues and species. We considered it possible that MHC class II expression may be a T-cell
The ability of LPS-induced macrophages to support antigen-specific T-cell proliferation may be taken as a measure of their antigen presentation ability (7). Control and LPS-treated macrophages were pulsed with heatRESULTS killed S. aureus and then cultured with LPS-DOSE DEPENDENCE S. aureus-sensitized T-cells. The results The effect of various levels of LPS of this experiment are presented in (0-30 Ag/mL) on the expression of Table IV. There was a 5.3-fold increase MHC class II molecules on the surface in T-cell proliferation in the presence of bovine mammary macrophages was of macrophages when compared to investigated and the results are pre- that in the absence of macrophages. sented in Table I. As shown, addition of LPS had no significant (p > 0.05) effect on the expression of MHC class II antigens on the surface of mammary TABLE III. Expression of major histocompatibility complex (MHC) class II molecules by bovine mammary macrophages treated with Escherichia coil lipopolysacchride (15 ug/mL) for different macrophages. In attempts to account culture times for a T-cell dependent effect of LPS on MHC expression we questioned Culture time (days) MHC expressiona (A A/106 cells) whether LPS could affect MHC 1 0.31 + 0.029 3 0.37 ± 0.034 expression only in the presence of 5 0.32 ± 0.041 T-cells. Table II reveals no significant differences in MHC class II expression aMHC expression was measured as change in absorbance per 106 cells. Results are the means ± SD in macrophages cocultured with T-cells of seven independent samples relative to macrophages cultured in medium alone. TABLE IV. Proliferative responses of T-cells (1.2 x 105 cells/well) cultured with unstimulated or stimulated with 30 Ag/mL Escherichia coil lipopolysaccharide mammary macrophages (1.8 x 104 cells/well) in the presence of 60 Ag/mL S. aureus KINETICS OF MHC EXPRESSION Cells T-cell proliferation (mean CPMa + SD) Mammary macrophages were treated with LPS and were removed T-cells alone 615 ± 115 from the cultured medium at the + unstimulated macrophages 3,430 ± 393 3,217 ± 423 indicated times. Table III shows that + stimulated macrophages MHC class II expression did not dif- aCounts per minute
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dependent phenomenon. Therefore, we questioned whether LPS could modulate the expression of MHC class II molecules only in the presence of mature T-cells. Our data revealed no significant differences in MHC class II expression when macrophages were cultured with T-cells or in the medium alone. We concluded that MHC expression is either completely independent of T-cells or requires very low numbers of T-cells. The latter possibility cannot be eliminated because it is practically impossible to obtain macrophages completely free of T-cells. The present observations document that mammary macrophages can act as antigen presenting cells as judged by their ability to induce (fivefold) S. aureus specific T-cell proliferation (Table IV). This is in agreement with earlier findings (4). The ability of macrophages to present antigens to T-cells is directly related to the levels of MHC class II expression by the macrophages (3). In keeping with this idea, our data show that LPS, which was unable to affect MHC class II expression, was also unable to enhance the proliferation of antigen-specific T-cells. The present study shows an inability of LPS to modulate the expression of MHC class II molecules and to enhance the antigen presentation function of mammary macrophages. The lack of macrophage response to LPS is another example of the hyporesponsive nature of macrophages isolated from the bovine mammary gland. In
a previous study we have shown that despite the fact that mammary macrophages could act as antigen presenting cells, they were much less efficient than their autologous blood monocytes (4). This might explain why the mammary gland is most susceptible to infection at times that macrophages are the predominant cells in milk (16). In summary, the present observations document the absence of effects of LPS on MHC class II expression by bovine mammary macrophages. Furthermore, LPS could not enhance the antigen presentation ability of mammary macrophages. REFERENCES 1. KITCHEN BJ. Review of the progress of dairy science. Bovine mastitis: milk compositional changes and related diagnostic tests. J Dairy Res 1981; 48: 162-178. 2. LEE CS, WOODING FBP, KEMP P. Identification, properties and differential counts of cell populations using electron microscopy of dry cows secretions, colostrum and milk from normal cows. J Dairy Res 1980; 47: 39-50. 3. UNANUE ER, BELLER DI, LU CY, ALLEN PM. Antigen presentation: comments on its regulation and mechanism. J Immunol 1984; 132: 1-5. 4. POLITIS I, ZHAO X, McBRIDE BW, BURTON JH. Function of bovine mammary macrophages as antigen presenting cells. Vet Immunol Immunopathol (in press). 5. MORRISON DC, RYAN JL. Bacterial endotoxins and host immune responses. Adv Immunol 1979; 28: 293-442. 6. WENTWORTH PA, ZIEGLER HK. Induction of macrophage Ia expression by lipopolysaccharide and listeria monocytogenes in congenitally athymic nude mice. J Immunol 1987; 138: 3167-3173.
7. ZIEGLER HK, STAFFILENO LK, WENTWORTH P. Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia in vivo. J Immunol 1984; 133: 1825-1835. 8. KOERNER TJ, HAMILTON TA, ADAMS DO. Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA. J Immunol 1987; 139: 239-243. 9. STEEG PS, JOHNSON HM, OPPENHEIM JJ. Regulation of murine macrophage Ia antigen expression by an interferon-like lymphokine: inhibitory effect of endotoxin. J Immunol 1982; 129: 2402-2406. 10. LEYVA-COBIAN F, CLEMENTE J. Phenotypic characterization and functional activity of human milk macrophages. Immunol Lett 1984; 8: 249-256. 11. GODDEERIS BM, BALDWIN CL, OLEMOI YOI 0, MORRISON WI. Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. J Immunol Methods 1985; 89: 165-173. 12. SPLITER GA, EVERLITH KM. Brucela abortus regulates macrophage-T-cell interaction by major histocompatibility complex class II and interleukin 1 expression. Infect Immun 1989; 57: 1151-1157. 13. YANG TC, JANTZEN PA, WILLIAMS LF. Acid a-naphthyl acetate esterase: presence of activity in bovine and human T-lymphocytes and B-lymphocytes. Immunology 1979; 38: 85-93. 14. O'REILLY KL, SPLITTER GA. Monoclonal antibodies to ovine SBU-T8 and SBU-T6 bind analogous molecules on bovine lymphocytes. Immunology 1989; 67: 176-180. 15. POLITIS I, ZHAO X, McBRIDE BW, BURTON JH. Limited secretion of interleukin 1 by bovine mammary macrophages. Am J Vet Res 1991; 52: (in press). 16. CRAVEN N, WILLIAMS MR. Defenses of the bovine mammary gland against infection and prospects for their enhancement. Vet Immunol Immunopathol 1985; 10: 71-127.
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