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Macrophages from human colostrum
Experimental Cell Research 66 (1971) 317-320
MACROPHAGES Multinucleated
FROM HUMAN
Giant Cell Formation
COLOSTRUM
by Phytohemagglutinin
and Concanavalin
A
C. WAYNE SMITH and A. S. GOLDMAN Division of Immunology, Department of Pediatrics, Department of Human Genetics, University of Texas Medical Branch and Shriner’s Burns Institute, Galveston, Tex. 775S0, USA
SUMMARY Macrophages obtained from human colostrum were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PHA caused multinucleated giant cell formation which could be inhibited by the addition of N-acetyl-o-galactosamine. Con A caused multinucleated giant cell formation and was cytotoxic in higher concentrations. Both effects could be inhibited by addition of cc-methyl-n-mannoside and cc-methylo-glucoside. PWM did not cause multinucleated giant cell formation but was cytotoxic in high concentrations. In contrast to colostral macrophages, mouse peritoneal macrophages did not undergo multinucleated giant cell formation in response to these mitogens. Some cell interlinkages were observed with exposure to PHA. Con A was not cytotoxic at the levels used while PWM was cytotoxic. These results indicate that human colostral macrophages interact with certain plant agglutinins in a manner distinct from mouse peritoneal macrophages.
Human colostrum is a rich source of macrophages [l]. This report concerns a study of the interaction of these macrophages in vitro with certain phytomitogens. METHODS Colostrum was collected from 12 human volunteers with a suction breast pump during the last month of pregnancy and the first 3 days postpartum. None of these individuals exhibited signs of mastitis. The colostrum was diluted 1:3 with cold (4°C) Hanks solution and centrifuged at 300 g for 5 min. The cells were washed once in cold Hanks solution and the cell button was resuspended in medium 199 (BBL, Cockeyville, Md) containing 20 % autologous serum or fetal calf serum (Microbiological Assoc., Bethesda, Md) and antibiotics (100 pg/ml streptomycin and 100 units/ml penicillin). The concentration of macrophages was adjusted to 1 x loo/ml and 0.5 ml of the cell suspension was overlayed on glass coverslips 18 mm in diameter in 50 mm plastic Petri
dishes (Falcon Plastics, Los Angeles). After 30 min incubation at 37°C in an atmosphere of 5 % COz in air, the coverslips were flooded with an additional 5 ml of medium and incubation was continued. Mouse oeritoneal macroohages were obtained bv washing ihe peritoneal davi;y of 6-lo-week-old female CFW mice (Carworth Farms. New York) with 5 ml cold Hanks solution. The cells were washed once in cold Hanks solution and resuspended in medium 199 with 20 % fetal calf serum and antibiotics. The macrophages were adjusted to a concentration of 1 x 106/ml and overlayed on glass coverslins 18 mm in diameter in 50 mm plastic-Petri dishes.After 15 min incubation at room temperature the coverslius were washed in Hanks solution and placed into Petri dishes containing 5 ml of medium and incubated at 37°C in an atmosphere of 5 % CO, in air. Phytohemagglutinin (PHA-P; Difco, Detroit) was added to some cultures in a concentration of 0.05 ml/5 ml of medium. Concanavalin A (Con A), prepared by the method Sumner & Howell [2], was added in concentrations of 10-50 pg/ml. Pokeweed mitogen (PWM; Grand Island Biological Co., New York) was added in concentrations of 0.05-0.3 ml/5 ml of medium. The sugars, N-acetyl-o-galactoExptl Cell Res 66
318
C. Wayne Smith & A. S. Goldman
samine, N-acetyl-D-glucosamine, cc-methyt-o-mannoside, a-methyl-D-glucoside and arabinose obtained from Sigma (St Louis, MO.), were added to some cultures in a concentration of 0.05 M. After various periods of incubation the following cytological preparations were made: (1) Cells were fixed in 1 %, glutaraldehyde in phosphate buffer (pH 7.2) at 4°C for 30 min. The coverslips were mounted in buffer and examined with a phase contrast microscope. (2) 3H-Thymidine (New England Nuclear, Boston, Mass.) was added to some cultures at a concentration of 1 Ci/ml at the beginning of the incubation period. These coverslips were coated with NBT3 emulsion (Kodak) for autoradiography [l] and exposed for 7 days. These preparations were stained with Giemsa. (3) Some coverslips were stained supravitally with acridine orange and examined under blue light (BG 12 filter) from a xenon lamp. (4) Some preparations were stained for acid phosphatase [3]. Phagocytosis was tested by the addition of heatkilled Sruphylococcous uureus, zymosan (Sigma), latex (0.8 pm, Difco) or neutral red stained, heatkilled Candida albicuns to the monolayer cultures. The cells were examined at various times, thereafter, for intracellular localization of these particles.
RESULTS Multinucleated giant cells appeared in colostral cell cultures containing PHA-P or Con A (lo-20 pg/ml) as early as 6-18 h. These cells had 3-6 nuclei at this time (fig. 1). By 48 h numerous multinucleated cells with as many as 50 nuclei were found. These cells appeared to be large syncythia of macrophages (fig. 2), and lymphocytes were frequently seen migrating over their surfaces. These giant cells contained abundant acid phosphatase and a large number of acridine orange positive granules. They were phagocytic as shown by the uptake of particles and their outer border was often ruffled and exhibited extensive pinocytosis. Less than 1 % (mean of 0.2%) of the nuclei of the macrophages were labeled with 3H-TdR and no mitoses were seen. Control cultures and those treated with PWM had no multinucleated cells at 6 or 24 h and such cultures at 5 days had less than 2% of the number of multinucleated cells in 24 h PHA- or Con A treated cultures. Also, giant cells arising in Exptl Cell Res 66
the control cultures were small as compared to PHA-P or Con A treated preparations. Increasing concentrations of PWM were cytotoxic but failed to produce multinucleated cells. Concentrations of Con A, greater than 30 pug/ml, were also cytotoxic. No change in cell motility, pinocytosis, phagocytosis, acid phosphatase content or acridine orange staining was observed in cultures containing the additional sugars but no mitogens. The number of giant cells formed by the action of PHA and Con A was greatly reduced by adding certain sugars to the medium. N-acetyl-D-galactosamine inhibited the formation of multinucleated cells in cultures containing PHA-P, while Nacetyl-D-glucosamine, a-methyl-o-mannoside, cr-methyl-D-glucoside and arabinose did not. In contrast, a-methyl-D-mannoside and CImethyl-D-glucoside prevented the appearance of multinucleated cells in cultures containing Con A, while N-acetyl-o-galactosamine, N-acetyl-D-glucosamine and arabinose did not. Alpha-methyl-D-mannoside and a-methyl-D-glucoside also prevented the cytotoxicity of Con A. In contrast to the colostral cells, cultures of mouse macrophages did not develop multinucleated cells with the mitogens. The macrophage interlinkage reaction [4] was seen 24 h after addition of PHA-P and this change was markedly reduced by the addition of N-acetyl-D-galactosamine. Con A in a concentration of 50 pg/ml produced no interlinkages and no cytotoxicity. PWM produced no interlinkages but rounding up of macrophages and the release of cells from the coverslips was found when the PWM concentrations were 0.2 ml/5 ml or greater. DISCUSSION These results are consistent with the interpretation that human colostral macrophages
Macrophages from human colostrum
3 19
Fig. 1. Multinucleated cell in a culture of human colostral macrophages 18 h after addition of PHA-P. Phase contrast, x 530. Fig. 2. Multinucleated cell in a culture of human colostral macrophages 48 h after addition of PHA-P. Phase contrast, x 380.
have binding sites for PHA-P and Con A and that mouse peritoneal macrophages have binding sites for PHA-P. The observations that support this interpretation are as follows: (1) PHA-P and Con A caused the formation of multinucleated giant cells in cultures of human colostral macrophages although the multiplication of cells by cell division was minimal. (2) Con A was cytotoxic for human colostral macrophages. (3) PHA-P caused the ‘macrophage interlinkage reaction’ in cultures of mouse peritoneal macrophages. (4) These reactions were prevented by the addition of sugars known to inhibit binding of Con A [5] and leukoagglutination due to PHA-P [6]. Con A produced no apparent effect on mouse peritoneal macrophages in vitro, suggesting a lack of binding sites for Con A. The appearance of giant cells in cultures of human colostral macrophages upon addition of PHA-P or Con A reveals a striking distinction between these cells and mouse peritoneal macrophages. Human macrophages derived from 5-lo-day-old cultures of blood leucocytes respond to PHA in the same way as mouse peritoneal macrophages [4]. However, after 10-25 days in vitro human
macrophages form giant cells in response to PHA [7]. This suggeststhat the blood macrophages apparently differentiate in culture into cells with an enhanced potential for transformation into giant cells. Human colostral macrophages appear to be at such a stage of differentiation. The occurrence of Con A receptors on human colostral macrophages may represent an unusual feature of these cells since such binding sites appear to be hidden in other cell types [8]. Whether surface binding sites for Con A are acquired through differentiation of macrophage precursors or are present but masked on such precursors is undetermined. The authors wish to thank Mrs Beth Rudloff and Mrs Nora L. Taylor MacDougall for their technical assistance. This work was supported by a grant from the National Institute of Child Health and Human Development, no. DHEW 5ROl HD02966.
REFERENCES 1. Smith, C W & Goldman, A S, Pediat res 2 (1968) 103. 2. Sumner, J B & Howell, S F, J bacterial 32 (1935) 227. Exptt Cell Res 66
320 C. Wayne Smith & A. S. Goldman 3. Humason, G L, Animal tissue techniques, p. 408. 2nd edn. Freeman, San Francisco (1967). 4. Maclaurin, B P, Aust j exptl med sci 47 (1969) 105. 5. Goldstein, I J, Hollerman, C E & Smith, E E, Biochemistry 4 (1965) 876. 6. Borberg, H, Woodruff, J, Hirschhorn, R, Gisner, B, Miescher, P & Silber, R, Science 154 (1966) 1019.
Exptl Cell Res 66
7. Berman, L & Stulberg, C S, Lab invest 11 (1962) 1322. 8. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418.
Received December 22, 1970
MACROPHAGES Multinucleated
FROM HUMAN
Giant Cell Formation
COLOSTRUM
by Phytohemagglutinin
and Concanavalin
A
C. WAYNE SMITH and A. S. GOLDMAN Division of Immunology, Department of Pediatrics, Department of Human Genetics, University of Texas Medical Branch and Shriner’s Burns Institute, Galveston, Tex. 775S0, USA
SUMMARY Macrophages obtained from human colostrum were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PHA caused multinucleated giant cell formation which could be inhibited by the addition of N-acetyl-o-galactosamine. Con A caused multinucleated giant cell formation and was cytotoxic in higher concentrations. Both effects could be inhibited by addition of cc-methyl-n-mannoside and cc-methylo-glucoside. PWM did not cause multinucleated giant cell formation but was cytotoxic in high concentrations. In contrast to colostral macrophages, mouse peritoneal macrophages did not undergo multinucleated giant cell formation in response to these mitogens. Some cell interlinkages were observed with exposure to PHA. Con A was not cytotoxic at the levels used while PWM was cytotoxic. These results indicate that human colostral macrophages interact with certain plant agglutinins in a manner distinct from mouse peritoneal macrophages.
Human colostrum is a rich source of macrophages [l]. This report concerns a study of the interaction of these macrophages in vitro with certain phytomitogens. METHODS Colostrum was collected from 12 human volunteers with a suction breast pump during the last month of pregnancy and the first 3 days postpartum. None of these individuals exhibited signs of mastitis. The colostrum was diluted 1:3 with cold (4°C) Hanks solution and centrifuged at 300 g for 5 min. The cells were washed once in cold Hanks solution and the cell button was resuspended in medium 199 (BBL, Cockeyville, Md) containing 20 % autologous serum or fetal calf serum (Microbiological Assoc., Bethesda, Md) and antibiotics (100 pg/ml streptomycin and 100 units/ml penicillin). The concentration of macrophages was adjusted to 1 x loo/ml and 0.5 ml of the cell suspension was overlayed on glass coverslips 18 mm in diameter in 50 mm plastic Petri
dishes (Falcon Plastics, Los Angeles). After 30 min incubation at 37°C in an atmosphere of 5 % COz in air, the coverslips were flooded with an additional 5 ml of medium and incubation was continued. Mouse oeritoneal macroohages were obtained bv washing ihe peritoneal davi;y of 6-lo-week-old female CFW mice (Carworth Farms. New York) with 5 ml cold Hanks solution. The cells were washed once in cold Hanks solution and resuspended in medium 199 with 20 % fetal calf serum and antibiotics. The macrophages were adjusted to a concentration of 1 x 106/ml and overlayed on glass coverslins 18 mm in diameter in 50 mm plastic-Petri dishes.After 15 min incubation at room temperature the coverslius were washed in Hanks solution and placed into Petri dishes containing 5 ml of medium and incubated at 37°C in an atmosphere of 5 % CO, in air. Phytohemagglutinin (PHA-P; Difco, Detroit) was added to some cultures in a concentration of 0.05 ml/5 ml of medium. Concanavalin A (Con A), prepared by the method Sumner & Howell [2], was added in concentrations of 10-50 pg/ml. Pokeweed mitogen (PWM; Grand Island Biological Co., New York) was added in concentrations of 0.05-0.3 ml/5 ml of medium. The sugars, N-acetyl-o-galactoExptl Cell Res 66
318
C. Wayne Smith & A. S. Goldman
samine, N-acetyl-D-glucosamine, cc-methyt-o-mannoside, a-methyl-D-glucoside and arabinose obtained from Sigma (St Louis, MO.), were added to some cultures in a concentration of 0.05 M. After various periods of incubation the following cytological preparations were made: (1) Cells were fixed in 1 %, glutaraldehyde in phosphate buffer (pH 7.2) at 4°C for 30 min. The coverslips were mounted in buffer and examined with a phase contrast microscope. (2) 3H-Thymidine (New England Nuclear, Boston, Mass.) was added to some cultures at a concentration of 1 Ci/ml at the beginning of the incubation period. These coverslips were coated with NBT3 emulsion (Kodak) for autoradiography [l] and exposed for 7 days. These preparations were stained with Giemsa. (3) Some coverslips were stained supravitally with acridine orange and examined under blue light (BG 12 filter) from a xenon lamp. (4) Some preparations were stained for acid phosphatase [3]. Phagocytosis was tested by the addition of heatkilled Sruphylococcous uureus, zymosan (Sigma), latex (0.8 pm, Difco) or neutral red stained, heatkilled Candida albicuns to the monolayer cultures. The cells were examined at various times, thereafter, for intracellular localization of these particles.
RESULTS Multinucleated giant cells appeared in colostral cell cultures containing PHA-P or Con A (lo-20 pg/ml) as early as 6-18 h. These cells had 3-6 nuclei at this time (fig. 1). By 48 h numerous multinucleated cells with as many as 50 nuclei were found. These cells appeared to be large syncythia of macrophages (fig. 2), and lymphocytes were frequently seen migrating over their surfaces. These giant cells contained abundant acid phosphatase and a large number of acridine orange positive granules. They were phagocytic as shown by the uptake of particles and their outer border was often ruffled and exhibited extensive pinocytosis. Less than 1 % (mean of 0.2%) of the nuclei of the macrophages were labeled with 3H-TdR and no mitoses were seen. Control cultures and those treated with PWM had no multinucleated cells at 6 or 24 h and such cultures at 5 days had less than 2% of the number of multinucleated cells in 24 h PHA- or Con A treated cultures. Also, giant cells arising in Exptl Cell Res 66
the control cultures were small as compared to PHA-P or Con A treated preparations. Increasing concentrations of PWM were cytotoxic but failed to produce multinucleated cells. Concentrations of Con A, greater than 30 pug/ml, were also cytotoxic. No change in cell motility, pinocytosis, phagocytosis, acid phosphatase content or acridine orange staining was observed in cultures containing the additional sugars but no mitogens. The number of giant cells formed by the action of PHA and Con A was greatly reduced by adding certain sugars to the medium. N-acetyl-D-galactosamine inhibited the formation of multinucleated cells in cultures containing PHA-P, while Nacetyl-D-glucosamine, a-methyl-o-mannoside, cr-methyl-D-glucoside and arabinose did not. In contrast, a-methyl-D-mannoside and CImethyl-D-glucoside prevented the appearance of multinucleated cells in cultures containing Con A, while N-acetyl-o-galactosamine, N-acetyl-D-glucosamine and arabinose did not. Alpha-methyl-D-mannoside and a-methyl-D-glucoside also prevented the cytotoxicity of Con A. In contrast to the colostral cells, cultures of mouse macrophages did not develop multinucleated cells with the mitogens. The macrophage interlinkage reaction [4] was seen 24 h after addition of PHA-P and this change was markedly reduced by the addition of N-acetyl-D-galactosamine. Con A in a concentration of 50 pg/ml produced no interlinkages and no cytotoxicity. PWM produced no interlinkages but rounding up of macrophages and the release of cells from the coverslips was found when the PWM concentrations were 0.2 ml/5 ml or greater. DISCUSSION These results are consistent with the interpretation that human colostral macrophages
Macrophages from human colostrum
3 19
Fig. 1. Multinucleated cell in a culture of human colostral macrophages 18 h after addition of PHA-P. Phase contrast, x 530. Fig. 2. Multinucleated cell in a culture of human colostral macrophages 48 h after addition of PHA-P. Phase contrast, x 380.
have binding sites for PHA-P and Con A and that mouse peritoneal macrophages have binding sites for PHA-P. The observations that support this interpretation are as follows: (1) PHA-P and Con A caused the formation of multinucleated giant cells in cultures of human colostral macrophages although the multiplication of cells by cell division was minimal. (2) Con A was cytotoxic for human colostral macrophages. (3) PHA-P caused the ‘macrophage interlinkage reaction’ in cultures of mouse peritoneal macrophages. (4) These reactions were prevented by the addition of sugars known to inhibit binding of Con A [5] and leukoagglutination due to PHA-P [6]. Con A produced no apparent effect on mouse peritoneal macrophages in vitro, suggesting a lack of binding sites for Con A. The appearance of giant cells in cultures of human colostral macrophages upon addition of PHA-P or Con A reveals a striking distinction between these cells and mouse peritoneal macrophages. Human macrophages derived from 5-lo-day-old cultures of blood leucocytes respond to PHA in the same way as mouse peritoneal macrophages [4]. However, after 10-25 days in vitro human
macrophages form giant cells in response to PHA [7]. This suggeststhat the blood macrophages apparently differentiate in culture into cells with an enhanced potential for transformation into giant cells. Human colostral macrophages appear to be at such a stage of differentiation. The occurrence of Con A receptors on human colostral macrophages may represent an unusual feature of these cells since such binding sites appear to be hidden in other cell types [8]. Whether surface binding sites for Con A are acquired through differentiation of macrophage precursors or are present but masked on such precursors is undetermined. The authors wish to thank Mrs Beth Rudloff and Mrs Nora L. Taylor MacDougall for their technical assistance. This work was supported by a grant from the National Institute of Child Health and Human Development, no. DHEW 5ROl HD02966.
REFERENCES 1. Smith, C W & Goldman, A S, Pediat res 2 (1968) 103. 2. Sumner, J B & Howell, S F, J bacterial 32 (1935) 227. Exptt Cell Res 66
320 C. Wayne Smith & A. S. Goldman 3. Humason, G L, Animal tissue techniques, p. 408. 2nd edn. Freeman, San Francisco (1967). 4. Maclaurin, B P, Aust j exptl med sci 47 (1969) 105. 5. Goldstein, I J, Hollerman, C E & Smith, E E, Biochemistry 4 (1965) 876. 6. Borberg, H, Woodruff, J, Hirschhorn, R, Gisner, B, Miescher, P & Silber, R, Science 154 (1966) 1019.
Exptl Cell Res 66
7. Berman, L & Stulberg, C S, Lab invest 11 (1962) 1322. 8. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418.
Received December 22, 1970