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Adherence and phagocytosis of fresh erythrocytes by glial cells in mouse cerebellum in vitro
Experimental
ADHERENCE
Cell Research 56 (1969) 92-96
AND PHAGOCYTOSIS OF FRESH ERYTHROCYTES CELLS IN MOUSE CEREBELLUM IN VITRO
BY GLIAL
G. BIRNBAUM Section in Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Blindness, National Institutes of Health, Bethesda, Md 20014, USA’
Diseases and
SUMMARY Fresh human, rat and mouse red cells, when placed onto 2-week-old cultures of newborn mouse cerebellum, adhered and were phagocytosed by basophilic, pyroninophilic cells, probably of glial origin. These cell reactions did not fix complement and were not blocked by rabbit antimouse gamma globulin. The phenomenon would thus appear to be non-immune in nature and would serve to differentiate brain erythrocyte adherence from similar adherence to cells elsewhere in the body.
Red cell adherence and phagocytosis by cells can be the result of either immune [I, 21 or nonimmune [3] phenomena. In the course of our work with immune reactions of human white cells [4] we noted that fresh red cells contaminating our white cell suspensions adhered to and were ingested by certain cells in our cultures of myelinating mouse cerebellum. It is the purpose of this paper to further define and categorize this unusual phenomenon.
METHODS Cerebellum exnlant cultures from 24 h old NIH mice were prepared-using the technique described by Bornstein & Murray [5]. Pieces of sliced cerebellum (2 x 1 x 4 mm) were placed on coverslips coated with. rat-tail collagen. One drop of medium was placed on each coverslip and the tissue then placed into Maximow double coverslip assemblies. Growth medium for the initial experiments consisted of 40% human umbilical cord serum, heat inactivated at 56°C for 30 min, 35 % Simms BSS, bovine serum ultrafiltrate (Microbiological Ass., Bethesda, Md) glucose at a final concentration of 600 mg %, and 1 % low zinc, glucagon-free insulin (Squibb Research Inst.). Later experiments were performed using 40% fetal calf serum, inactivated at 56°C for 30 min (Flow Lab., Bethesda, Md), 30% Simms BSS, 30% bovine serum ultrafiltrate, and glucose at a final concentration of 600 mg %. The complete medium did not agglutinate human erythrocytes. * Present address: Department of Neurology, New York Hospital, 525 East 68th st., New York, N.Y. 10021, USA. Expil Cell Res 56
Cultures were incubated in the lying drop position at 35.5 to 36°C. Medium was changed twice weekly. Cultures were used after 2 weeks in vitro growth. All cultures were observed during the experiments by both phase contrast and bright field microscopy.
Human cells Heparinized human blood was obtained by venipuncture using preservative-free heparin (Fisher Scientific Co.). In initial experiments [4], white cells and red cells were used. They were obtained by incubating the blood-filled syringe at an angle of approx. 80” for 45 min at 36°C. The leukocyte-rich supematant plasma was removed and the cells washed twice by centrifugation and resuspension in 0.89 % saline at 4°C. The cells were finally suspended in growth medium at a concentration of 1 x 10’ WBc’s per ml and inoculated onto the cultures. In later experiments, only red cells were used. These were freshly obtained by venipuncture, as above, washed three times in 0.89 % NaCl at 4°C and inoculated onto the cultures at a concentration of 0.2 % in growth medium. Cultures were kept at 35.5-36°C.
Mouse and rat red cells Ex-breeder NIH mice and 300 g Sprague-Dawley rats were used. Heparinized blood, using preservative-free heparin, was obtained by cardiac puncture. Cells were washed three times in 0.89 % NaCl at 4°C and inoculated into the cultures at a concentration of 0.2% in growth medium.
Complement Freshly reconstituted guinea pig complement (Flow Lab.) was used at a dilution of 1: 50. The complement wa- adsorbed with human red cells for 20 min in an ice bath. A 1: 65 dilution of this batch of complement contained 2 hemolytic units per 0.2 ml. The complement was added
Erythrocyte
adherence and phagocytosis
:o the cultures & h after the addition of red cells. The :ultures were incubated at 35.S36°C and observed at half-hourly intervals. Rabbit anti-mouse gamma globulin Anti-mouse gamma globulin was prepared by immunizaiion of a rabbit with gamma globulin isolated by zone :lectrophoresis. The antiserum reacted with all classes of mouse immunoglobulin as well as with mouse K-chains [a. The antiserum was adsorbed with human red cells for 20 min in an ice bath. Cultures were treated with dilutions of antiserum for 4 h at 35%36°C. The antiserum was then removed and red cells inoculated onto the explants.
RESULTS Cultures
After two weeks of in vitro growth, explants of 24 h old mouse cerebellum had sizeable outgrowths of cells measuring up to 4 mm in diameter. The outgrowths consisted mainly of glial cells, both astrocytes and oligodendroglial cells, which formed a sheet around the explant. Neurons were seen within the explants and frequent myelin formation was noted. In almost all cultures after two weeksgrowthone could see numerous macrophages, characterized by their amoeboid shape and a cytoplasm filled with large numbers of highly refractable fat droplets. These cells were seen over the entire culture and were never noted to adhere to one another. In addition, varying numbers of iounded cells with clear, finely granular cytoplasm and large vesicular nuclei were noted. Prominent
nucleoli were present in the cells in
numbers ranging from 1 to 4. These cells were of varying size and appeared gradually to increase in numbers in the explant. Such cells usually lay on top of the glial cell sheet but were occasionally seen within the cell outgrowth. Groups of such cells would occasionally cluster together though numerous cells lay free. It was these superficially located cells that exhibited the adherence phenomenon when fresh red cells were added to the supernate. No alterations in the explants were noted with change in the media containing
human cord serum to that with
fetal calf serum other than a slight decrease in the degree of myelination. The presence or ab‘sence of myelin in vitro had no effect on the observed red cell phenomena.
in mouse cerebellum
93
Human red cells
In the initial experiments combinations of red cells and white cells were used. Later only red cell suspensions were added to the explant. The results in both series were identical. After inoculation of the red cells, the cultures were kept at 35.5-36°C and examined periodically. Within Q h after the introduction of red cells, marked adherence of the erythrocytes to many but not all of the cells with large, open nuclei, resting on the glial sheet were seen (fig. 1). Rosettes were formed and occasionally red cells were several layers thick around the “adherence” cells. No adherence was noted to neurons or macrophages. After several hours at 35.5-36°C no change in red cell adherence was noted. Only very rarely was there any phagocytosis by the “adherence” cells. The cells that were more obviously phagocytic in the cultures and seemed to be typical macrophages on the basis of their shape and cytoplasmic characteristics had, after several hours, no red cells adherent to their surfaces and did not show erythrophagocytosis at this point. After 24 h in vitro erythrocytes were still adherent in large numbers to the cells noted above. There was a more ruffled appearance to the plasma membrane of some of the “adherence” cells due to the appearance of numerous fine pseudopodia. Granules could occasionally be seen within the cytoplasm and ingestion of erythrocytes by these cells was now more prominent (fig. 2). In addition amoeboid macrophages noted early to be devoid of red cells now had erythrocytes adherent to their surfaces and often demonstrated phagocytosed red cells within their cytoplasm. After 48 h and thereafter for periods up to 10 days little further change occurred. RBC adherence persisted and more pronounced erythrophagocytosis was noted. Rat and mouse red cells
Addition of 0.2 % fresh rat or mouse red cells to the mouse cerebellum explants resulted in an identical course of events as those seen with human red cells. Adherence occurred within 4 h and involved the same type of brain cells as those Exptl Cell Res 56
94
G. Birnbaum
Fig. 1. Clusters and rosettes of human fresh red cells on top of “adherent’‘-type cells. Living cells. x 512.
involved in human erythrocyte adherence (fig. 3). Phagocytosis occurred after approx. 12 h inoculation and increasedin frequency with time. The differences noted were mainly quantitative ones. Although not all the expected cells bound human erythrocytes, the number was smaller using rat RBC’s and smaller yet with mouse RBS’s. Effect of complement Addition of fresh guinea pig complement to cerebellum cultures after inoculation of human red cells did not result in lysis of the adhering erythrocytes. The effect on rat and mouse cells was not tested. Effect of rabbit anti-mousegamma globulin Dilutions of rabbit anti-mouse gamma globulin ranging from 1: 2 to 1: 50 were not capable of inhibiting adherence and phagocytosis by cells Expti
Cell
Res 56
2. Several “adherence” cells, one of which has two ingested fresh human red cells within its cytoplasm. Liv ing cells. x 640. Fig.
Erythrocyte
adherence andphagocytosis
in mouse cerebellum
95
Fig. 3. A group of “adherence” cells to which numerous small, fresh mouse erythrocytes have become adherent. Living cells. x 512.
in mouse cerebellum explants. At low dilutions the antiserum appeared to be toxic to some of the “adherence” cells. In such cases the extent of adherence was greatly reduced. Similar experiments with a rabbit anti-mouse alpha, globulin intended as a control for the above also had no effect on adherence or phagocytosis.
phagocytosed by them. In some cultures mitosis were prominent. Vacuoles were occasionally present at the periphery of the cytoplasm. Methyl green-pyronine staining showed these cells to be very pyroninophilic.
Staining
We have observed the adherence and phagocytosis of fresh human, rat, and mouse erythrocytes to cells derived from 24 h old mouse cerebellum explants. On the basis of their several characteristics such cells are probably not macrophages or of neuronal origin. These cells are rounded, and usually lie on top of the glial cell sheet. They stain intensely with methyl green-pyronine, probably have the ability to divide, are able to cause adherence of erythrocytes to their cell surfaces, and can be activated into erythrophagocytosis. The most probable
At various time intervals after red cell inoculation cultures were fixed in either Zenker’s acid formalin or Zenker’s formalin solutions and stained with hematoxylin-eosin, May-Grunwald Giemsa, or methyl green-pyronine stains. The “adherence” cells were prominently seen in all cultures. The nuclei were usually eccentric and very basophilic with ‘H-E and Giemsa. The cytoplasm was basophilic with Giemsa and slightly eosinophilic with H-E. Red cells were seen to be both adherent to these cells and
DISCUSSION
Exptl
Cell Res 56
96
G. Birnbaum
analogous cell type found in brain in vivo would be the microglial cell. It has long been noted for its phagocytic potential [7, 8, 91 and in wounds of the brain such cells accumulate around the necrotic areas becoming rounded and filled with phagocytosed particles 18, 91. Although the origin of microglia is not definitely established, they are most probably derived from mesodermal tissue, either the circulating blood cells or from endothelial cells [7]. It is also possible that these unusually reactive cells are neuroglial cells, either astrocytes or oligodendroglia, that have become dislodged from the outgrowth’s monolayer and then have re-attached to its surface. Such cells with a rounded appearance and phagocytic capacity have been produced by addition of polyvinyl pyrrolidone to the supernatant medium of similar cultures [13]. In such instances the dislodged neuroglial cells acquired microglial morphology and activity. In contrast to the immediate adherence of erythrocytes to glial cells the adherence and phagocytosis of RBC’s by macrophages occurred approx. 12 to 24 h after the inoculation of the red cells. This is probably a similar phenomenon to that seen to occur with macrophages in the presence of effete red cells [12]. It appears relatively unlikely that the red cell adherence and phagocytosis observed in our cultures are mediated by immunoglobulins. No complement dependent red cell lysis was noted [2] and no blockage of the reaction by rabbit anti-mouse gamma globulin occurred. In the cultures inoculated with high concentrations of anti-mouse gamma globulin toxicity and death of “adherence” cells was noted. In such cases a reduction in erythrocyte adherence was observed. Although further work must be done, this observation may imply that red cell adherence requires an active metabolism on the part of these cells. This may serve further to differentiate the process from that seen with cytophilic antibody. As noted by others [IO] the process of ad-
Exptl Cell Res 56
herence of red cells appeared to be quire separate from that of erythrophagocytosis. Whereas RBC adherence to brain cells occurred almost immediately phagocytosis was observed to any extent only after a lag period of about 12 h. Macrophages that were most active in phagocytosing red cells were usually those that were already active in the cultures as shown by their amoeboid shape and the granularity of their cytoplasm. “Adherence” cells phagocytosed red cells less frequently although it is entirely pos-’ sible that a conversion to amoeboid forms could occur in such rounded cells. One cannot say on the basis of this preliminary work what role this phenomenon of non-immune adherence and phagocytosis of fresh red cells by. some glial cells in mouse cerebellum plays in vivo. Previous workers have noted red cell adherence and phagocytosis only in cells from animals immunized to red cells [l, 21 or in experiments with altered red cells [3, 11, 121. On the basis of our observations it would thus’ appear that mouse glial cells in vitro possess surface characteristics different from that of mouse cells in other organs. I would like to acknowledge the great assistance of Dr Richard Asofsky and the valuable help in staining our cultures given by Miss Betty Sanders.
REFERENCES 1, Greenhyke, R M et al, Blood 22 (1963) 295. 2. Berken, A & Benacerraf, B, J exptl med 123 (1966) 119. 3. Rabinovitch, M, Proc sot exptl biol med 124 (1967) 396. 4. Birnbaum, G. In preparation. 5. Bornstein, M B & Murray, M R, J biophys biochem cytol4 (1958) 499. 6. Asofsky, R. Personal communication. 7. Mihalik, P V, Arch exptl Zellforsch 17 (1935) 119. 8. de1 Rio-Hortega & Penfield, W, Bull Johns Hopkins hosp 41 (1927) 278. 9. Russel, D S, Am j path01 5 (1929) 451. 10. Rabinovitch, M, Exptl cell res 46 (1967) 19. 11. - J immuno199 (1967) 232. 12. Vaughan, R B, Immunology 8 (1965) 245. 13. Bornstein, M B, J neuropathol exptl neurol 22 (1963) 353. Received November 14, 1968
ADHERENCE
Cell Research 56 (1969) 92-96
AND PHAGOCYTOSIS OF FRESH ERYTHROCYTES CELLS IN MOUSE CEREBELLUM IN VITRO
BY GLIAL
G. BIRNBAUM Section in Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Blindness, National Institutes of Health, Bethesda, Md 20014, USA’
Diseases and
SUMMARY Fresh human, rat and mouse red cells, when placed onto 2-week-old cultures of newborn mouse cerebellum, adhered and were phagocytosed by basophilic, pyroninophilic cells, probably of glial origin. These cell reactions did not fix complement and were not blocked by rabbit antimouse gamma globulin. The phenomenon would thus appear to be non-immune in nature and would serve to differentiate brain erythrocyte adherence from similar adherence to cells elsewhere in the body.
Red cell adherence and phagocytosis by cells can be the result of either immune [I, 21 or nonimmune [3] phenomena. In the course of our work with immune reactions of human white cells [4] we noted that fresh red cells contaminating our white cell suspensions adhered to and were ingested by certain cells in our cultures of myelinating mouse cerebellum. It is the purpose of this paper to further define and categorize this unusual phenomenon.
METHODS Cerebellum exnlant cultures from 24 h old NIH mice were prepared-using the technique described by Bornstein & Murray [5]. Pieces of sliced cerebellum (2 x 1 x 4 mm) were placed on coverslips coated with. rat-tail collagen. One drop of medium was placed on each coverslip and the tissue then placed into Maximow double coverslip assemblies. Growth medium for the initial experiments consisted of 40% human umbilical cord serum, heat inactivated at 56°C for 30 min, 35 % Simms BSS, bovine serum ultrafiltrate (Microbiological Ass., Bethesda, Md) glucose at a final concentration of 600 mg %, and 1 % low zinc, glucagon-free insulin (Squibb Research Inst.). Later experiments were performed using 40% fetal calf serum, inactivated at 56°C for 30 min (Flow Lab., Bethesda, Md), 30% Simms BSS, 30% bovine serum ultrafiltrate, and glucose at a final concentration of 600 mg %. The complete medium did not agglutinate human erythrocytes. * Present address: Department of Neurology, New York Hospital, 525 East 68th st., New York, N.Y. 10021, USA. Expil Cell Res 56
Cultures were incubated in the lying drop position at 35.5 to 36°C. Medium was changed twice weekly. Cultures were used after 2 weeks in vitro growth. All cultures were observed during the experiments by both phase contrast and bright field microscopy.
Human cells Heparinized human blood was obtained by venipuncture using preservative-free heparin (Fisher Scientific Co.). In initial experiments [4], white cells and red cells were used. They were obtained by incubating the blood-filled syringe at an angle of approx. 80” for 45 min at 36°C. The leukocyte-rich supematant plasma was removed and the cells washed twice by centrifugation and resuspension in 0.89 % saline at 4°C. The cells were finally suspended in growth medium at a concentration of 1 x 10’ WBc’s per ml and inoculated onto the cultures. In later experiments, only red cells were used. These were freshly obtained by venipuncture, as above, washed three times in 0.89 % NaCl at 4°C and inoculated onto the cultures at a concentration of 0.2 % in growth medium. Cultures were kept at 35.5-36°C.
Mouse and rat red cells Ex-breeder NIH mice and 300 g Sprague-Dawley rats were used. Heparinized blood, using preservative-free heparin, was obtained by cardiac puncture. Cells were washed three times in 0.89 % NaCl at 4°C and inoculated into the cultures at a concentration of 0.2% in growth medium.
Complement Freshly reconstituted guinea pig complement (Flow Lab.) was used at a dilution of 1: 50. The complement wa- adsorbed with human red cells for 20 min in an ice bath. A 1: 65 dilution of this batch of complement contained 2 hemolytic units per 0.2 ml. The complement was added
Erythrocyte
adherence and phagocytosis
:o the cultures & h after the addition of red cells. The :ultures were incubated at 35.S36°C and observed at half-hourly intervals. Rabbit anti-mouse gamma globulin Anti-mouse gamma globulin was prepared by immunizaiion of a rabbit with gamma globulin isolated by zone :lectrophoresis. The antiserum reacted with all classes of mouse immunoglobulin as well as with mouse K-chains [a. The antiserum was adsorbed with human red cells for 20 min in an ice bath. Cultures were treated with dilutions of antiserum for 4 h at 35%36°C. The antiserum was then removed and red cells inoculated onto the explants.
RESULTS Cultures
After two weeks of in vitro growth, explants of 24 h old mouse cerebellum had sizeable outgrowths of cells measuring up to 4 mm in diameter. The outgrowths consisted mainly of glial cells, both astrocytes and oligodendroglial cells, which formed a sheet around the explant. Neurons were seen within the explants and frequent myelin formation was noted. In almost all cultures after two weeksgrowthone could see numerous macrophages, characterized by their amoeboid shape and a cytoplasm filled with large numbers of highly refractable fat droplets. These cells were seen over the entire culture and were never noted to adhere to one another. In addition, varying numbers of iounded cells with clear, finely granular cytoplasm and large vesicular nuclei were noted. Prominent
nucleoli were present in the cells in
numbers ranging from 1 to 4. These cells were of varying size and appeared gradually to increase in numbers in the explant. Such cells usually lay on top of the glial cell sheet but were occasionally seen within the cell outgrowth. Groups of such cells would occasionally cluster together though numerous cells lay free. It was these superficially located cells that exhibited the adherence phenomenon when fresh red cells were added to the supernate. No alterations in the explants were noted with change in the media containing
human cord serum to that with
fetal calf serum other than a slight decrease in the degree of myelination. The presence or ab‘sence of myelin in vitro had no effect on the observed red cell phenomena.
in mouse cerebellum
93
Human red cells
In the initial experiments combinations of red cells and white cells were used. Later only red cell suspensions were added to the explant. The results in both series were identical. After inoculation of the red cells, the cultures were kept at 35.5-36°C and examined periodically. Within Q h after the introduction of red cells, marked adherence of the erythrocytes to many but not all of the cells with large, open nuclei, resting on the glial sheet were seen (fig. 1). Rosettes were formed and occasionally red cells were several layers thick around the “adherence” cells. No adherence was noted to neurons or macrophages. After several hours at 35.5-36°C no change in red cell adherence was noted. Only very rarely was there any phagocytosis by the “adherence” cells. The cells that were more obviously phagocytic in the cultures and seemed to be typical macrophages on the basis of their shape and cytoplasmic characteristics had, after several hours, no red cells adherent to their surfaces and did not show erythrophagocytosis at this point. After 24 h in vitro erythrocytes were still adherent in large numbers to the cells noted above. There was a more ruffled appearance to the plasma membrane of some of the “adherence” cells due to the appearance of numerous fine pseudopodia. Granules could occasionally be seen within the cytoplasm and ingestion of erythrocytes by these cells was now more prominent (fig. 2). In addition amoeboid macrophages noted early to be devoid of red cells now had erythrocytes adherent to their surfaces and often demonstrated phagocytosed red cells within their cytoplasm. After 48 h and thereafter for periods up to 10 days little further change occurred. RBC adherence persisted and more pronounced erythrophagocytosis was noted. Rat and mouse red cells
Addition of 0.2 % fresh rat or mouse red cells to the mouse cerebellum explants resulted in an identical course of events as those seen with human red cells. Adherence occurred within 4 h and involved the same type of brain cells as those Exptl Cell Res 56
94
G. Birnbaum
Fig. 1. Clusters and rosettes of human fresh red cells on top of “adherent’‘-type cells. Living cells. x 512.
involved in human erythrocyte adherence (fig. 3). Phagocytosis occurred after approx. 12 h inoculation and increasedin frequency with time. The differences noted were mainly quantitative ones. Although not all the expected cells bound human erythrocytes, the number was smaller using rat RBC’s and smaller yet with mouse RBS’s. Effect of complement Addition of fresh guinea pig complement to cerebellum cultures after inoculation of human red cells did not result in lysis of the adhering erythrocytes. The effect on rat and mouse cells was not tested. Effect of rabbit anti-mousegamma globulin Dilutions of rabbit anti-mouse gamma globulin ranging from 1: 2 to 1: 50 were not capable of inhibiting adherence and phagocytosis by cells Expti
Cell
Res 56
2. Several “adherence” cells, one of which has two ingested fresh human red cells within its cytoplasm. Liv ing cells. x 640. Fig.
Erythrocyte
adherence andphagocytosis
in mouse cerebellum
95
Fig. 3. A group of “adherence” cells to which numerous small, fresh mouse erythrocytes have become adherent. Living cells. x 512.
in mouse cerebellum explants. At low dilutions the antiserum appeared to be toxic to some of the “adherence” cells. In such cases the extent of adherence was greatly reduced. Similar experiments with a rabbit anti-mouse alpha, globulin intended as a control for the above also had no effect on adherence or phagocytosis.
phagocytosed by them. In some cultures mitosis were prominent. Vacuoles were occasionally present at the periphery of the cytoplasm. Methyl green-pyronine staining showed these cells to be very pyroninophilic.
Staining
We have observed the adherence and phagocytosis of fresh human, rat, and mouse erythrocytes to cells derived from 24 h old mouse cerebellum explants. On the basis of their several characteristics such cells are probably not macrophages or of neuronal origin. These cells are rounded, and usually lie on top of the glial cell sheet. They stain intensely with methyl green-pyronine, probably have the ability to divide, are able to cause adherence of erythrocytes to their cell surfaces, and can be activated into erythrophagocytosis. The most probable
At various time intervals after red cell inoculation cultures were fixed in either Zenker’s acid formalin or Zenker’s formalin solutions and stained with hematoxylin-eosin, May-Grunwald Giemsa, or methyl green-pyronine stains. The “adherence” cells were prominently seen in all cultures. The nuclei were usually eccentric and very basophilic with ‘H-E and Giemsa. The cytoplasm was basophilic with Giemsa and slightly eosinophilic with H-E. Red cells were seen to be both adherent to these cells and
DISCUSSION
Exptl
Cell Res 56
96
G. Birnbaum
analogous cell type found in brain in vivo would be the microglial cell. It has long been noted for its phagocytic potential [7, 8, 91 and in wounds of the brain such cells accumulate around the necrotic areas becoming rounded and filled with phagocytosed particles 18, 91. Although the origin of microglia is not definitely established, they are most probably derived from mesodermal tissue, either the circulating blood cells or from endothelial cells [7]. It is also possible that these unusually reactive cells are neuroglial cells, either astrocytes or oligodendroglia, that have become dislodged from the outgrowth’s monolayer and then have re-attached to its surface. Such cells with a rounded appearance and phagocytic capacity have been produced by addition of polyvinyl pyrrolidone to the supernatant medium of similar cultures [13]. In such instances the dislodged neuroglial cells acquired microglial morphology and activity. In contrast to the immediate adherence of erythrocytes to glial cells the adherence and phagocytosis of RBC’s by macrophages occurred approx. 12 to 24 h after the inoculation of the red cells. This is probably a similar phenomenon to that seen to occur with macrophages in the presence of effete red cells [12]. It appears relatively unlikely that the red cell adherence and phagocytosis observed in our cultures are mediated by immunoglobulins. No complement dependent red cell lysis was noted [2] and no blockage of the reaction by rabbit anti-mouse gamma globulin occurred. In the cultures inoculated with high concentrations of anti-mouse gamma globulin toxicity and death of “adherence” cells was noted. In such cases a reduction in erythrocyte adherence was observed. Although further work must be done, this observation may imply that red cell adherence requires an active metabolism on the part of these cells. This may serve further to differentiate the process from that seen with cytophilic antibody. As noted by others [IO] the process of ad-
Exptl Cell Res 56
herence of red cells appeared to be quire separate from that of erythrophagocytosis. Whereas RBC adherence to brain cells occurred almost immediately phagocytosis was observed to any extent only after a lag period of about 12 h. Macrophages that were most active in phagocytosing red cells were usually those that were already active in the cultures as shown by their amoeboid shape and the granularity of their cytoplasm. “Adherence” cells phagocytosed red cells less frequently although it is entirely pos-’ sible that a conversion to amoeboid forms could occur in such rounded cells. One cannot say on the basis of this preliminary work what role this phenomenon of non-immune adherence and phagocytosis of fresh red cells by. some glial cells in mouse cerebellum plays in vivo. Previous workers have noted red cell adherence and phagocytosis only in cells from animals immunized to red cells [l, 21 or in experiments with altered red cells [3, 11, 121. On the basis of our observations it would thus’ appear that mouse glial cells in vitro possess surface characteristics different from that of mouse cells in other organs. I would like to acknowledge the great assistance of Dr Richard Asofsky and the valuable help in staining our cultures given by Miss Betty Sanders.
REFERENCES 1, Greenhyke, R M et al, Blood 22 (1963) 295. 2. Berken, A & Benacerraf, B, J exptl med 123 (1966) 119. 3. Rabinovitch, M, Proc sot exptl biol med 124 (1967) 396. 4. Birnbaum, G. In preparation. 5. Bornstein, M B & Murray, M R, J biophys biochem cytol4 (1958) 499. 6. Asofsky, R. Personal communication. 7. Mihalik, P V, Arch exptl Zellforsch 17 (1935) 119. 8. de1 Rio-Hortega & Penfield, W, Bull Johns Hopkins hosp 41 (1927) 278. 9. Russel, D S, Am j path01 5 (1929) 451. 10. Rabinovitch, M, Exptl cell res 46 (1967) 19. 11. - J immuno199 (1967) 232. 12. Vaughan, R B, Immunology 8 (1965) 245. 13. Bornstein, M B, J neuropathol exptl neurol 22 (1963) 353. Received November 14, 1968