IN VITRO Volume15, No. 5, 1979 Allrights reserved9
CHARACTERIZATION OF CELLS CULTURED FROM EARLY LACTATION MILKS R. L. CERIANI, ' J. TAYLOR-PAPADIMITRIOU,2J. A. PETERSON,' ANDP. BROWN
Imperial CancerResearch Fund, P.O. Box 123, Lincoln's Inn Fields, London ff'C2A 3PX, England SUMMARY Two major types of cells can be cultured from early lactation human milks: a colonyforming epithelial cell and an adherent nondividing cell referred to as a foam cell. The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differentiated epithelial cells, but tissue macrophages.
Key words: human; milk; epithelial cells; foam cells; macrophages; milk fat globule. INTRODUCTION Whereas colonies of dividing epithelial cells can be cultured from human lacteal secretions (1-4), the majority of the adherent cells do not divide and show some of the functional and morphological properties of macrophages (2, 4, 5). These cells have been variously referred to as Donn6's corpuscles, colostrum bodies and foam cells. In spite of the apparent resemblance of the adherent nondividing cells (hereafter referred to as foam cells) to macrophages, some workers hold the view that these cells are terminally differentiated epithelial cells (3) that are also phagocytic. We have attempted to resolve this problem by examining the cells in milk cultures not only for properties associated with macrophages (phagocytic ability, production of lysozyme, resistance to trypsinization, Fc receptors) but also for the presence of a marker specific for mammary epithelial cells (6). Our results show that the foam cells exhibit many properties usually associated with macrophages, and do not show a positive reaction with a specific antiserum prepared against antigenic components present in the membrane of the milk fat globule and shown to be specific for human mammary epithelial cells (6).
Company and sterilized by autoclaving before adding to cell cultures. Radioactive materials were supplied by The Radiochemical Centre, Amersham.
Preparation of cell cultures from early lactation milks. Mixed cell cultures were prepared from un-
fractionated milk cell suspensions as previously described (4) and grown on collagen-coated 3-cm Nunc (Irvine Scientific, Santa Ana, Calif.) plastic dishes (or, where indicated, on cover slips) in medium 199 supplemented with insulin (10 ktg per mlk hydrocortisone (5 /~g per ml), fetal bovine serum (FBS) (15%), and human serum (20%). Cultures of foam cells were prepared by adsorption of the milk cell suspension to glass dishes or cover slips for 10 to 20 rain. Unadsorbed cells were removed and medium (199 plus 10% fetal bovine serum) added to the foam cells. The cells were able to he maintained for 2 to 3 weeks in this medium. Homogeneous cultures of epithelial cells were prepared as previously described after separation of the foam cells by differential adhesion followed by freezing and thawing (7k The human mammary epithelial cells (HumE) were grown with feeder layers of mitomyein-treated 3T6 cells (7) in medium 199 containing insulin, hydrocortisone MATERIALSANDMETHODS and 15% fetal bovine serum. Materials. Silica particles (Partisil 5) of 5-~m Assay of lysozyme. Lysozyme was assayed by diameter were obtained from Whatman Chemical following lysis of heat-killed M. lysodeikticus (8), and assays were performed in the laboratory b y 1Permanent address: Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st Dr. Osserman, Institute of Cancer Research, College of Physicians and Surgeons of Columbia Uniand Grove Street, Oakland, California 94609. 2To whom requests for reprints should be sent. versity, New York. 356
357
CULTURED CELLS FROM MILK
Detection of Fc receptors. A 2% suspension of sheep red blood cells (SRBC) in PBS was incubated with rabbit anti-SRBC serum or normal rabbit serum, washed free of antiserum and added to 3-day cultures of foam cells. After 30 min at 37 ~ C, the SRBC were removed and the cells washed with PBS. Antiserum. The cream fractions of human milk from several donors were consolidated and made fat-free, and then injected into rabbits to produce antiserum against the fat-free milk fat globule (MFG) as already reported (6L The antiserum was precipitated with 40% ammonium sulphate at pH 7.0 and dialyzed (9L The ),-globulin preparation thus obtained was brought to the original volume of antisera and absorbed sequentially with packed human red ceils, HeLa cells, HT-29 cells (colon carcinoma cell line), and human liver. Each cell type was used at a ratio of 1"1 {volume) and absorption was for at least 4 hr at room temperature. This antiserum is hereafter referred to as anti-HME serum. Indirect immunofluorescence ~IIF). Suspensions of HumE cells were prepared from monolayers by treatment with trypsin {0.05%~ and versene (0.02%) dissolved in calcium- and magnesium-free buffered saline ICMFL The suspended cells were kept for 2 days over agar in Waymouth's medium with 10% serum to allow regeneration of membrane components before testing for M F G antigens by I I F as previously described (6). HumE and foam cells on cover slips also were tested by I I F while attached to the substratum. In this case the cells were fixed with 0.2% glutaraldehyde and washed several times with C M F before adding anti-HME at a dilution of 1:5. The cover slips were incubated in a humidified atmosphere at room temperature for 20 min, washed free of serum and incubated for 20 min with fluorescent goat anti-rabbit serum (dilution 1:10) before mounting in glycerol. Binding of isotopically labeled anti-HME to cells in milk cultures. Anti-HME was labeled by the chloramine-T method to a specific activity of 7.4 mCi per mg, and the amount of [~2sI]-antiH M E binding to given samples of HumE or foam cells was determined as follows: Cells from homogeneous cultures of HumE and foam cells were scraped with a rubber policeman from their substratum and washed twice with C M F containing 0.01 M N a Azide and 0.5% bovine serum albumin {CMF-A). Foam cells were counted directly and a sample of HumE cells was trypsinized to produce single cells before estimating cell number. Sam-
pies of HumE or foam cells (2 x 104 cells per sample) were pelleted and resuspended in CMF-A, and increasing concentrations of ['2q]-anti-HME were added (final volume 100 gl). After incubation for 4 hr with intermittent agitation at room temperature, the cells were washed 3 times with C M F - A and the radioactivity bound was estimated. Empty tubes containing the different amounts of [12sI]-anti-HME were carried through the same procedure, and the amount of radioactivity carried over in the final transfer of C M F was estimated and subtracted from the experimental figures. With increasing concentrations of ['2sI]-anti-HME, increasing amounts of radioactivity were bound to the cells until a plateau was reached. The amount of ['~sI]-anti-HME required to give maximum binding then was used to test competition of M F G for the binding of anti-HME by HumE and by foam cells. HumE or foam cells were mixed with various amounts of M F G solubilized in C M F containing 0.1% Triton-X-100; 20 ~1 p~sI]-anti-HME then was added to each tube; and the mixture was made up to a volume of 100 /~1. The rest of the binding procedures, the washing and the counting were performed as given above for the binding curve of p2q]-antiHME. RESULTS
Macrophage-like Properties of Foam Cells Adherence to glass. The foam cells, which appear as single cells in milk, adhere to glass very quickly and are not removed by treatment with 0.05% trypsin after incubation for 30 min. The property of foam cells to adhere to glass has been used to separate them from the dividing epithelial cells that do not stick during the period of time required for adhesion of more than 90% of the foam cells. In this way, homogeneous cultures of either foam cells or HumE cells can be obtained although it is necessary to provide the HumE cells with other feeders (mitomycin-treated 3T6 cells) once the foam cells have been removed (TL In subsequent experiments, both mixed cultures of HumE and foam cells from unfractionated milks have been used as well as homogeneous cultures of either cell type. The preparation of these cultures is described in Materials and Methods. Production of lysozyme. The secretion of lysozyme is considered to be a property of macrophages, and fairly high levels of the enzyme are known to be present in milk. Lysozyme could be detected in supernates from unfractionated milk
358
CERIANI ET AL.
cultures even after 2 weeks in culture; and subsequent examination of supernates from homogeneous cultures of foam cells or HumE cells
100-~ -
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0 4 8 12 16 Days after addition of silica FIG. I. Killing by silica particles of foam ceils cultured from milk. Unfractionated milk cells were grown as described in Materials and Methods. The medium was changed every 3 to 4 days. When silica was used it was added for 24 hr from days 6 to 7. A, Silica was added in the concentrations indicated, and at day 8 (24 hr after removal of silica), the number of foam cells remaining attached to the dishes was estimated by counting under the microscope. The number of remaining cells is expressed as a percentage of the foam cells remaining on control plates. B, Silica (50 #g per ml) was added to a series of milk cell cultures and at various times after its removal, the number of foam cells remaining attached to the dish was estimated. At the same time, the number of foam cells was also estimated on plates untreated with silica. @--@, Untreated plates; O--C), plates treated with 50/~g per ml silica.
showed that the enzyme is secreted by the foam cells and not by the epithelial cells. Levels of 15 to 20 ~g were detected in medium from dishes containing approximately 10 x 105 foam cells, indicating that at least a fraction of these cells was actively secreting lysozyme. Killing by silica. The phagocytic ability of foam cells in lacteal secretions has been reported (5, 6, 10, 11). Many cells, including fibroblasts, are phagocytic to some degree, and a more specific test for the abnormally high phagocytic ability of macrophages is their sensitivity to the cytotoxic action of silica particles (12). The mechanism of the cell killing is not fully understood but does depend on the ingestion of the silica particles. By using the mixed cell cultures from unfractionated milks, we were able to show that most of the foam cells could be killed by silica particles, whereas the growth of the HumE colonies was unaffected. The number of foam cells per plate could be estimated directly by microscopic counting; Fig. 1A shows that incubation with silica at a concentration of 50/~g per ml or greater for 24 hr effectively kills 90% of the foam cells. The killing effect is manifest 2 days after addition of the silica as is illustrated in Fig. l B . To assess the effect of silica treatment on the growth of epithelial cell colonies, the labeling index of control and silica-treated colonies was determined after incubation with [SH]dT thymidine. Fig. 2,4 shows that treatment for 24 hr with up to 100 pg per ml of silica has no dfect on the labeling index of epithelial cell colonies measured 1 day after silica treatment. Furthermore, subsequent development of HumE colonies is the same in silica-treated cultures as it is in controls; Fig. 2B shows that the number of cells per H u m E colony increases in the same way in control and silica-treated plates, clearly demonstrating that the HumE cells are not killed by silica. Our conclusions from the above data is that more than 95% of the single nondividing cells in cultures from early lactation milks can be killed by silica and in this respect resemble macrophages. Fc receptors. Macrophages have receptors on their surface for the Fc portion of IgG. We tested for the presence of Fc receptors on foam cells by treating them with sheep red blood cells previously incubated with rabbit anti-SRBC serum as described in Materials and Methods. Antibodycoated erythrocytes adhere to, and were phagocytosed by, the foam cells (Fig. 3A), whereas uncoated erythrocytes did not attach (Fig. 3B). This result is in agreement with the data of Lascelles,
CULTURED CELLS FROM MILK Gurner and Coombs (5) who found Fc receptors on the adherent cells from colostrum.
Mammary EpitheliM Cell Markers From the above observations, it seems clear that several properties of macrophages are exhibited by the foam cells but not by the epithelial cells in monolayer cultures. If the foam cells represent terminally differentiated epithelial cells that also
100
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Days after addition of silica FIG. 2. Growth of epithelial ceils from milk is unaffected by silica..4, Cultures of unfractionated milk ceils were treated with different concentrations of silica as described in Fig. ]A. When silica was removed at 7 days, [3H]methyl thymidine (5 ~Ci per ml) was added; after 24 hr, the cultures were fixed and processed for autoradiography. The points represent the average labeling index of five different HumE colonies. B, Cultures of unfractionated milk cells were prepared and some treated with 50 ~g per ml of silica as described in Fig. 1.4. At the times indicated, the average number of ceils per HumE colony was estimated in control and silica-treated plates. Each point is an average of the cells in five colonies. 9 - - 9 Control; O--C), silica-treated plates.
359
exhibit macrophage functions, they would be expected to have membrane components similar to those found in the milk fat globule formed by budding from the secretory epithelial cell. A specific antiserum against components of the M F G membrane has been prepared and shown to react exclusively with human mammary epithelial cells. We have used this antiserum to determine which of the cells cultured from milk have membrane components specific for M F G . The epithelial cells could be suspended by trypsin treatment and showed a clear positive reaction indicated by ring-type fluorescence when tested by I I F with the antiserum (see Fig. 4A, B). Because of the fragility of the foam cells, they could not be examined in suspension; the cells could be scraped off the glass surface but seemed dead when kept in suspension over agar. Accordingly, the foam cells were tested in monolayer culture for reaction with the specific antiserum both in homogeneous culture and in mixed culture with HumE cells. Fig. 4 C - F shows phase and fluorescent photographs of foam cells and a colony of HumE cells, respectively. From Fig. 4D it can be seen that although there are some fluorescent inclusions in the foam cells mainly associated with vacuoles, and probably representing ingested material, the membranes show no positive fluorescence such as is seen on cells in the H u m E colony shown in Fig. 4F. The foam cells showed a negative reaction with the antiserum whether they were cultured alone or with HumE cells, and when examined at days 1 through 14 after seeding. Similar results were obtained when cells from postweaning milk were used both for the foam and HumE cells. The positive reaction of the HumE cells with the specific antiserum and the negative reaction of the foam cells were confirmed using radioactively labeled anti-HME as described in Materials and Methods. Fig. 5 shows that the HumE cells bound I0 times more radioactivity than the same number of foam cells, and, furthermore, that the amount of radioactivity bound by the HumE cells was effectively competed by solubilized M F G protein when it was added together with the [12sI]anti-HME. The fact that M F G protein can compete with the HumE cells for the [~2Sl]-anti-HME indicates that the reaction of the antiserum with the HumE cells is specific. The amount of radioactivity bound to the foam cells is low and is not competed out by solubilized M F G , suggesting that the low level of binding observed is nonspecific.
360
CERIANI ET AL.
FIG. 3. Fc receptors on foam cells. Foam cells were adsorbed to cover slips from a suspension of unfractionated milk cells and kept for 5 days in culture in medium 199 with 10% fetal bovine serum. Cells were incubated for 30 mln at room temperature with a 2% suspension of SRBC which had been pretreated with goat anti-SRBC serum (A) or with untreated SRBC (B).)
CHARACTERIZATION OF CELLS CULTURED FROM EARLY LACTATION MILKS R. L. CERIANI, ' J. TAYLOR-PAPADIMITRIOU,2J. A. PETERSON,' ANDP. BROWN
Imperial CancerResearch Fund, P.O. Box 123, Lincoln's Inn Fields, London ff'C2A 3PX, England SUMMARY Two major types of cells can be cultured from early lactation human milks: a colonyforming epithelial cell and an adherent nondividing cell referred to as a foam cell. The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differentiated epithelial cells, but tissue macrophages.
Key words: human; milk; epithelial cells; foam cells; macrophages; milk fat globule. INTRODUCTION Whereas colonies of dividing epithelial cells can be cultured from human lacteal secretions (1-4), the majority of the adherent cells do not divide and show some of the functional and morphological properties of macrophages (2, 4, 5). These cells have been variously referred to as Donn6's corpuscles, colostrum bodies and foam cells. In spite of the apparent resemblance of the adherent nondividing cells (hereafter referred to as foam cells) to macrophages, some workers hold the view that these cells are terminally differentiated epithelial cells (3) that are also phagocytic. We have attempted to resolve this problem by examining the cells in milk cultures not only for properties associated with macrophages (phagocytic ability, production of lysozyme, resistance to trypsinization, Fc receptors) but also for the presence of a marker specific for mammary epithelial cells (6). Our results show that the foam cells exhibit many properties usually associated with macrophages, and do not show a positive reaction with a specific antiserum prepared against antigenic components present in the membrane of the milk fat globule and shown to be specific for human mammary epithelial cells (6).
Company and sterilized by autoclaving before adding to cell cultures. Radioactive materials were supplied by The Radiochemical Centre, Amersham.
Preparation of cell cultures from early lactation milks. Mixed cell cultures were prepared from un-
fractionated milk cell suspensions as previously described (4) and grown on collagen-coated 3-cm Nunc (Irvine Scientific, Santa Ana, Calif.) plastic dishes (or, where indicated, on cover slips) in medium 199 supplemented with insulin (10 ktg per mlk hydrocortisone (5 /~g per ml), fetal bovine serum (FBS) (15%), and human serum (20%). Cultures of foam cells were prepared by adsorption of the milk cell suspension to glass dishes or cover slips for 10 to 20 rain. Unadsorbed cells were removed and medium (199 plus 10% fetal bovine serum) added to the foam cells. The cells were able to he maintained for 2 to 3 weeks in this medium. Homogeneous cultures of epithelial cells were prepared as previously described after separation of the foam cells by differential adhesion followed by freezing and thawing (7k The human mammary epithelial cells (HumE) were grown with feeder layers of mitomyein-treated 3T6 cells (7) in medium 199 containing insulin, hydrocortisone MATERIALSANDMETHODS and 15% fetal bovine serum. Materials. Silica particles (Partisil 5) of 5-~m Assay of lysozyme. Lysozyme was assayed by diameter were obtained from Whatman Chemical following lysis of heat-killed M. lysodeikticus (8), and assays were performed in the laboratory b y 1Permanent address: Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st Dr. Osserman, Institute of Cancer Research, College of Physicians and Surgeons of Columbia Uniand Grove Street, Oakland, California 94609. 2To whom requests for reprints should be sent. versity, New York. 356
357
CULTURED CELLS FROM MILK
Detection of Fc receptors. A 2% suspension of sheep red blood cells (SRBC) in PBS was incubated with rabbit anti-SRBC serum or normal rabbit serum, washed free of antiserum and added to 3-day cultures of foam cells. After 30 min at 37 ~ C, the SRBC were removed and the cells washed with PBS. Antiserum. The cream fractions of human milk from several donors were consolidated and made fat-free, and then injected into rabbits to produce antiserum against the fat-free milk fat globule (MFG) as already reported (6L The antiserum was precipitated with 40% ammonium sulphate at pH 7.0 and dialyzed (9L The ),-globulin preparation thus obtained was brought to the original volume of antisera and absorbed sequentially with packed human red ceils, HeLa cells, HT-29 cells (colon carcinoma cell line), and human liver. Each cell type was used at a ratio of 1"1 {volume) and absorption was for at least 4 hr at room temperature. This antiserum is hereafter referred to as anti-HME serum. Indirect immunofluorescence ~IIF). Suspensions of HumE cells were prepared from monolayers by treatment with trypsin {0.05%~ and versene (0.02%) dissolved in calcium- and magnesium-free buffered saline ICMFL The suspended cells were kept for 2 days over agar in Waymouth's medium with 10% serum to allow regeneration of membrane components before testing for M F G antigens by I I F as previously described (6). HumE and foam cells on cover slips also were tested by I I F while attached to the substratum. In this case the cells were fixed with 0.2% glutaraldehyde and washed several times with C M F before adding anti-HME at a dilution of 1:5. The cover slips were incubated in a humidified atmosphere at room temperature for 20 min, washed free of serum and incubated for 20 min with fluorescent goat anti-rabbit serum (dilution 1:10) before mounting in glycerol. Binding of isotopically labeled anti-HME to cells in milk cultures. Anti-HME was labeled by the chloramine-T method to a specific activity of 7.4 mCi per mg, and the amount of [~2sI]-antiH M E binding to given samples of HumE or foam cells was determined as follows: Cells from homogeneous cultures of HumE and foam cells were scraped with a rubber policeman from their substratum and washed twice with C M F containing 0.01 M N a Azide and 0.5% bovine serum albumin {CMF-A). Foam cells were counted directly and a sample of HumE cells was trypsinized to produce single cells before estimating cell number. Sam-
pies of HumE or foam cells (2 x 104 cells per sample) were pelleted and resuspended in CMF-A, and increasing concentrations of ['2q]-anti-HME were added (final volume 100 gl). After incubation for 4 hr with intermittent agitation at room temperature, the cells were washed 3 times with C M F - A and the radioactivity bound was estimated. Empty tubes containing the different amounts of [12sI]-anti-HME were carried through the same procedure, and the amount of radioactivity carried over in the final transfer of C M F was estimated and subtracted from the experimental figures. With increasing concentrations of ['2sI]-anti-HME, increasing amounts of radioactivity were bound to the cells until a plateau was reached. The amount of ['~sI]-anti-HME required to give maximum binding then was used to test competition of M F G for the binding of anti-HME by HumE and by foam cells. HumE or foam cells were mixed with various amounts of M F G solubilized in C M F containing 0.1% Triton-X-100; 20 ~1 p~sI]-anti-HME then was added to each tube; and the mixture was made up to a volume of 100 /~1. The rest of the binding procedures, the washing and the counting were performed as given above for the binding curve of p2q]-antiHME. RESULTS
Macrophage-like Properties of Foam Cells Adherence to glass. The foam cells, which appear as single cells in milk, adhere to glass very quickly and are not removed by treatment with 0.05% trypsin after incubation for 30 min. The property of foam cells to adhere to glass has been used to separate them from the dividing epithelial cells that do not stick during the period of time required for adhesion of more than 90% of the foam cells. In this way, homogeneous cultures of either foam cells or HumE cells can be obtained although it is necessary to provide the HumE cells with other feeders (mitomycin-treated 3T6 cells) once the foam cells have been removed (TL In subsequent experiments, both mixed cultures of HumE and foam cells from unfractionated milks have been used as well as homogeneous cultures of either cell type. The preparation of these cultures is described in Materials and Methods. Production of lysozyme. The secretion of lysozyme is considered to be a property of macrophages, and fairly high levels of the enzyme are known to be present in milk. Lysozyme could be detected in supernates from unfractionated milk
358
CERIANI ET AL.
cultures even after 2 weeks in culture; and subsequent examination of supernates from homogeneous cultures of foam cells or HumE cells
100-~ -
80
~
40-
A
2o
0 0
i 20
i 50 /~g/ml silica
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x
2-
0
1
I
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I
I
0 4 8 12 16 Days after addition of silica FIG. I. Killing by silica particles of foam ceils cultured from milk. Unfractionated milk cells were grown as described in Materials and Methods. The medium was changed every 3 to 4 days. When silica was used it was added for 24 hr from days 6 to 7. A, Silica was added in the concentrations indicated, and at day 8 (24 hr after removal of silica), the number of foam cells remaining attached to the dishes was estimated by counting under the microscope. The number of remaining cells is expressed as a percentage of the foam cells remaining on control plates. B, Silica (50 #g per ml) was added to a series of milk cell cultures and at various times after its removal, the number of foam cells remaining attached to the dish was estimated. At the same time, the number of foam cells was also estimated on plates untreated with silica. @--@, Untreated plates; O--C), plates treated with 50/~g per ml silica.
showed that the enzyme is secreted by the foam cells and not by the epithelial cells. Levels of 15 to 20 ~g were detected in medium from dishes containing approximately 10 x 105 foam cells, indicating that at least a fraction of these cells was actively secreting lysozyme. Killing by silica. The phagocytic ability of foam cells in lacteal secretions has been reported (5, 6, 10, 11). Many cells, including fibroblasts, are phagocytic to some degree, and a more specific test for the abnormally high phagocytic ability of macrophages is their sensitivity to the cytotoxic action of silica particles (12). The mechanism of the cell killing is not fully understood but does depend on the ingestion of the silica particles. By using the mixed cell cultures from unfractionated milks, we were able to show that most of the foam cells could be killed by silica particles, whereas the growth of the HumE colonies was unaffected. The number of foam cells per plate could be estimated directly by microscopic counting; Fig. 1A shows that incubation with silica at a concentration of 50/~g per ml or greater for 24 hr effectively kills 90% of the foam cells. The killing effect is manifest 2 days after addition of the silica as is illustrated in Fig. l B . To assess the effect of silica treatment on the growth of epithelial cell colonies, the labeling index of control and silica-treated colonies was determined after incubation with [SH]dT thymidine. Fig. 2,4 shows that treatment for 24 hr with up to 100 pg per ml of silica has no dfect on the labeling index of epithelial cell colonies measured 1 day after silica treatment. Furthermore, subsequent development of HumE colonies is the same in silica-treated cultures as it is in controls; Fig. 2B shows that the number of cells per H u m E colony increases in the same way in control and silica-treated plates, clearly demonstrating that the HumE cells are not killed by silica. Our conclusions from the above data is that more than 95% of the single nondividing cells in cultures from early lactation milks can be killed by silica and in this respect resemble macrophages. Fc receptors. Macrophages have receptors on their surface for the Fc portion of IgG. We tested for the presence of Fc receptors on foam cells by treating them with sheep red blood cells previously incubated with rabbit anti-SRBC serum as described in Materials and Methods. Antibodycoated erythrocytes adhere to, and were phagocytosed by, the foam cells (Fig. 3A), whereas uncoated erythrocytes did not attach (Fig. 3B). This result is in agreement with the data of Lascelles,
CULTURED CELLS FROM MILK Gurner and Coombs (5) who found Fc receptors on the adherent cells from colostrum.
Mammary EpitheliM Cell Markers From the above observations, it seems clear that several properties of macrophages are exhibited by the foam cells but not by the epithelial cells in monolayer cultures. If the foam cells represent terminally differentiated epithelial cells that also
100
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A
riO-
= 40 20 0 0
I
I
I
20
50
100
/~glml silica
B
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_
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0
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Days after addition of silica FIG. 2. Growth of epithelial ceils from milk is unaffected by silica..4, Cultures of unfractionated milk ceils were treated with different concentrations of silica as described in Fig. ]A. When silica was removed at 7 days, [3H]methyl thymidine (5 ~Ci per ml) was added; after 24 hr, the cultures were fixed and processed for autoradiography. The points represent the average labeling index of five different HumE colonies. B, Cultures of unfractionated milk cells were prepared and some treated with 50 ~g per ml of silica as described in Fig. 1.4. At the times indicated, the average number of ceils per HumE colony was estimated in control and silica-treated plates. Each point is an average of the cells in five colonies. 9 - - 9 Control; O--C), silica-treated plates.
359
exhibit macrophage functions, they would be expected to have membrane components similar to those found in the milk fat globule formed by budding from the secretory epithelial cell. A specific antiserum against components of the M F G membrane has been prepared and shown to react exclusively with human mammary epithelial cells. We have used this antiserum to determine which of the cells cultured from milk have membrane components specific for M F G . The epithelial cells could be suspended by trypsin treatment and showed a clear positive reaction indicated by ring-type fluorescence when tested by I I F with the antiserum (see Fig. 4A, B). Because of the fragility of the foam cells, they could not be examined in suspension; the cells could be scraped off the glass surface but seemed dead when kept in suspension over agar. Accordingly, the foam cells were tested in monolayer culture for reaction with the specific antiserum both in homogeneous culture and in mixed culture with HumE cells. Fig. 4 C - F shows phase and fluorescent photographs of foam cells and a colony of HumE cells, respectively. From Fig. 4D it can be seen that although there are some fluorescent inclusions in the foam cells mainly associated with vacuoles, and probably representing ingested material, the membranes show no positive fluorescence such as is seen on cells in the H u m E colony shown in Fig. 4F. The foam cells showed a negative reaction with the antiserum whether they were cultured alone or with HumE cells, and when examined at days 1 through 14 after seeding. Similar results were obtained when cells from postweaning milk were used both for the foam and HumE cells. The positive reaction of the HumE cells with the specific antiserum and the negative reaction of the foam cells were confirmed using radioactively labeled anti-HME as described in Materials and Methods. Fig. 5 shows that the HumE cells bound I0 times more radioactivity than the same number of foam cells, and, furthermore, that the amount of radioactivity bound by the HumE cells was effectively competed by solubilized M F G protein when it was added together with the [12sI]anti-HME. The fact that M F G protein can compete with the HumE cells for the [~2Sl]-anti-HME indicates that the reaction of the antiserum with the HumE cells is specific. The amount of radioactivity bound to the foam cells is low and is not competed out by solubilized M F G , suggesting that the low level of binding observed is nonspecific.
360
CERIANI ET AL.
FIG. 3. Fc receptors on foam cells. Foam cells were adsorbed to cover slips from a suspension of unfractionated milk cells and kept for 5 days in culture in medium 199 with 10% fetal bovine serum. Cells were incubated for 30 mln at room temperature with a 2% suspension of SRBC which had been pretreated with goat anti-SRBC serum (A) or with untreated SRBC (B).)
