Developmental and Comparative Immunology,Vol. 15, pp 42~430, 1991 Printed in the USA. All rights reserved.
0145-305X/91 $3.00 + .00 Copyright © 1991 Pergamon Press pie
ANALYSIS OF IMMUNOCOMPETENT CELLS IN THE BAT, Pteropus giganteus: ISOLATION AND SCANNING ELECTRON MICROSCOPIC CHARACTERIZATION Saurav K. Sarkar and Ashim K. C h a k r a v a r t y Immunology and Cell Biology Laboratory, Centre for Life Sciences, University of North Bengal, Siliguri 734430, India (Submitted May 1990; Accepted March 1991)
[~Abstract--Immunocompetent cells of the Indian fruit bat (P. giganteus) were characterized by differential surface adhesiveness and surface topography. We observed three cell types: (1) plastic adherent with pseudopodia; (2) nylon wool adherent with small microvilli and pits; and (3) a nylon wool nonadherent with comparatively smooth surfaces. These cell types resemble, respectively, macrophages, B cells and T cells of other mammals, including mice and humans. The disposition of microvilli on the B-type cell surface changes significantly after immunization, which suggests modulation of the molecular organization of the cell membrane and its fluidity. The proportions of these three cell types are not much different from those found in mice. Follicular dendritic cells, capable of retaining antigen for long periods in mice, have also been detected in bats. Further characterization of immunocompetent cells will help in understanding the mechanism of immune responses in bats.
well-organized immune system capable of mounting both antibody-mediated and cell-mediated immune responses; the onset and decay of the responses are notably delayed. A dichotomy of the lymphocytic population in bats has been suggested (9) but characterization of these cells is yet to be done. In fact, the causative m e c h a n i s m of the delayed immune response could not be defined clearly because of a lack of sufficient knowledge about the identity and nature of the bats' i m m u n o c o m p e t e n t cells. In the present study, we isolated i m m u n o c o m p e t e n t cells of the Indian fruit bat, Pteropus giganteus, on the basis of cell surface adhesiveness and cell surface topographic analyses by scanning electron microscopy before and after immunization.
[]Keywords--Immunocompetent cells; Bat; Scanning EM; Membrane fluidity; Macrophages; B cells; T cells.
Materials and Methods
Bats
Introduction The role of bats in carrying and transmitting dreaded viral and bacterial pathogens without suffering from the diseases (1-3) m a k e s them an interesting subject for immunological analyses. Recent w o r k (4-10) revealed that bats have a Address correspondence to A. K. Chakravarty.
Adult bats of both sexes, weighing about 500 g, f r o m natural populations were obtained from an animal supplier at Calcutta and maintained in our laboratory with adequate fruits and water.
Collection of Lymphocytes Spleen and l y m p h n o d e s w e r e collected a s e p t i c a l l y a n d d i s s o c i a t e d in Earle's Balanced Salt Solution (EBSS)
423
424
using a stainless steel wire mesh. Single cell suspensions were obtained by passing the initial suspension through a 27gauge hypodermic needle. RBCs were removed by 0.84% NHaCI treatment and the remaining l e u k o c y t e s were resuspended in EBSS supplemented with 10% goat serum as outlined elsewhere (11).
Immunization Sheep red blood cells (SRBC, 1.0 m L of 25% suspension) were injected intravenously in each bat as this dose was found to be most effective for generating an antibody response in bats (7-10). Bats were sacrificed using 1:3 mixture of chloroform and ether for the removal of secondary lymphoid organs after 10 days of immunization.
S K. Sarkar and A K. Chakravarty
opted for the nylon wool fibre filtration technique (16-18) because of its simplicity and rapidity, and also because the cells are not exposed to any harsh treatments. Briefly, 400 mg nylon wool was cut into small pieces, teased into loose fibres, boiled in 1N HCI and washed with triple distilled water. The fibres were packed in a 10-mL syringe and incubated in serum-free EBSS for 45 rain at 37°C. Suspensions, depleted of plastic adherent cells, were taken in 5 m L of serum supplemented EBSS, layered on the column and incubated for 45 rain at 37°C in a humidified atmosphere containing 7.5% COy. After incubation, nylon wool nonadherent cells were eluted with excess warm EBSS. The column was kept in ice for an additional 10 rain and the nylon wool adherent cells eluted with cold, H a n k ' s B a l a n c e d Salt Solution ( H B S S ) and m e c h a n i c a l agitation o f the wool.
Separation of Plastic Adherent Cells The technique of Ford (12) was followed with slight modifications. Briefly, cells from secondary lymphoid organs suspended in 5 m L of goat serum supplemented E B S S , were incubated in a plastic petri dish for 45 min at 37°C in a humidified a t m o s p h e r e containing 7.5% CO2. The supernatant was gently collected and cells adhering to the plastic were removed by scraping with a rubber policeman. Alternatively, treatment with 0.2% E D T A for 5 min also helped to remove them.
Nylon Wool Fibre Separation of Remaining Cells There are several techniques for separating B and T lymphocytes depending on their differential ability to bind antiimmunoglobulin molecules, a d h e r e to nylon wool fibres or their mobility in an electrical field (13-19). H o w e v e r , we
Staining with Vital Dyes Viable cells were counted by the trypan blue exclusion technique. The cells were also stained with 1% neutral red for counting the plastic adherent cells.
Scanning Electron Microscopy The three categories of cells were smeared on clean, grease-free glass coverslips, fixed with 2% gluteraldehyde for 2V2 h and dehydrated by passing them through a graded series of ethyl alcohol. Cells were then critical point dried using amyl alcohol and liquid CO2 in a Polaron Critical Point Dryer and sputter coated with 150 to 200 A layer of gold as per standard techniques (20-22). Cells were examined in a Philips SEM using an accelerating voltage of 20 KV at Bose Institute, Calcutta.
Immunocompetent cells of bats
425
positive. The total yield of viable cells from the column was more than 40%.
Results Plastic Adherent Cells Figure 1 shows the recovery of plastic adherent (PA) cells. A fairly constant percentage of cells, about 3 to 4% of the total population, always adhered to plastic. About 90% of these cells were neutral red positive, while the plastic nonadherent cells did not take up the stain (Table 1).
Nylon Wool Adherent and Nonadherent Cells The plastic nonadherent cells could be further divided into two categories--the nylon wool adherent (NA) and the nylon wool nonadherent (NNA) cells depending on their surface adhesiveness to nylon wool fibres (Table 2). The recovered NA cells represented about 9% and the recovered NNA cells about 33% of the plastic nonadherent population. None of these two populations were neutral red
Scanning Electron Microscopy of Different Cell Types of Normal and Immunized Bat Most cells in the PA cell population revealed a highly irregular shape, having different pseudopodial projections [Fig. 2(a)]. The cell size was between 4 to 5 ~m. Occasionally, some cells were found whose surface projections did not resemble the pseudopodial extensions of most others: these projections were much broader and bulbuous in shape [Fig. 2(b)]. The size of these cells was also larger than the other plastic adherent cells--in the range of 6 to 8 p+m. These cells appeared in a low frequency and were grouped as a separate category of plastic adherent cells. The NNA cells generally showed a diameter of 6 to 7 ~m and a round shape; the cell surface was relatively smooth without any projections [Fig. 2(c)].
160
I r =o_120 x .,.a --I ua U
MEAN NOOF
0145-305X/91 $3.00 + .00 Copyright © 1991 Pergamon Press pie
ANALYSIS OF IMMUNOCOMPETENT CELLS IN THE BAT, Pteropus giganteus: ISOLATION AND SCANNING ELECTRON MICROSCOPIC CHARACTERIZATION Saurav K. Sarkar and Ashim K. C h a k r a v a r t y Immunology and Cell Biology Laboratory, Centre for Life Sciences, University of North Bengal, Siliguri 734430, India (Submitted May 1990; Accepted March 1991)
[~Abstract--Immunocompetent cells of the Indian fruit bat (P. giganteus) were characterized by differential surface adhesiveness and surface topography. We observed three cell types: (1) plastic adherent with pseudopodia; (2) nylon wool adherent with small microvilli and pits; and (3) a nylon wool nonadherent with comparatively smooth surfaces. These cell types resemble, respectively, macrophages, B cells and T cells of other mammals, including mice and humans. The disposition of microvilli on the B-type cell surface changes significantly after immunization, which suggests modulation of the molecular organization of the cell membrane and its fluidity. The proportions of these three cell types are not much different from those found in mice. Follicular dendritic cells, capable of retaining antigen for long periods in mice, have also been detected in bats. Further characterization of immunocompetent cells will help in understanding the mechanism of immune responses in bats.
well-organized immune system capable of mounting both antibody-mediated and cell-mediated immune responses; the onset and decay of the responses are notably delayed. A dichotomy of the lymphocytic population in bats has been suggested (9) but characterization of these cells is yet to be done. In fact, the causative m e c h a n i s m of the delayed immune response could not be defined clearly because of a lack of sufficient knowledge about the identity and nature of the bats' i m m u n o c o m p e t e n t cells. In the present study, we isolated i m m u n o c o m p e t e n t cells of the Indian fruit bat, Pteropus giganteus, on the basis of cell surface adhesiveness and cell surface topographic analyses by scanning electron microscopy before and after immunization.
[]Keywords--Immunocompetent cells; Bat; Scanning EM; Membrane fluidity; Macrophages; B cells; T cells.
Materials and Methods
Bats
Introduction The role of bats in carrying and transmitting dreaded viral and bacterial pathogens without suffering from the diseases (1-3) m a k e s them an interesting subject for immunological analyses. Recent w o r k (4-10) revealed that bats have a Address correspondence to A. K. Chakravarty.
Adult bats of both sexes, weighing about 500 g, f r o m natural populations were obtained from an animal supplier at Calcutta and maintained in our laboratory with adequate fruits and water.
Collection of Lymphocytes Spleen and l y m p h n o d e s w e r e collected a s e p t i c a l l y a n d d i s s o c i a t e d in Earle's Balanced Salt Solution (EBSS)
423
424
using a stainless steel wire mesh. Single cell suspensions were obtained by passing the initial suspension through a 27gauge hypodermic needle. RBCs were removed by 0.84% NHaCI treatment and the remaining l e u k o c y t e s were resuspended in EBSS supplemented with 10% goat serum as outlined elsewhere (11).
Immunization Sheep red blood cells (SRBC, 1.0 m L of 25% suspension) were injected intravenously in each bat as this dose was found to be most effective for generating an antibody response in bats (7-10). Bats were sacrificed using 1:3 mixture of chloroform and ether for the removal of secondary lymphoid organs after 10 days of immunization.
S K. Sarkar and A K. Chakravarty
opted for the nylon wool fibre filtration technique (16-18) because of its simplicity and rapidity, and also because the cells are not exposed to any harsh treatments. Briefly, 400 mg nylon wool was cut into small pieces, teased into loose fibres, boiled in 1N HCI and washed with triple distilled water. The fibres were packed in a 10-mL syringe and incubated in serum-free EBSS for 45 rain at 37°C. Suspensions, depleted of plastic adherent cells, were taken in 5 m L of serum supplemented EBSS, layered on the column and incubated for 45 rain at 37°C in a humidified atmosphere containing 7.5% COy. After incubation, nylon wool nonadherent cells were eluted with excess warm EBSS. The column was kept in ice for an additional 10 rain and the nylon wool adherent cells eluted with cold, H a n k ' s B a l a n c e d Salt Solution ( H B S S ) and m e c h a n i c a l agitation o f the wool.
Separation of Plastic Adherent Cells The technique of Ford (12) was followed with slight modifications. Briefly, cells from secondary lymphoid organs suspended in 5 m L of goat serum supplemented E B S S , were incubated in a plastic petri dish for 45 min at 37°C in a humidified a t m o s p h e r e containing 7.5% CO2. The supernatant was gently collected and cells adhering to the plastic were removed by scraping with a rubber policeman. Alternatively, treatment with 0.2% E D T A for 5 min also helped to remove them.
Nylon Wool Fibre Separation of Remaining Cells There are several techniques for separating B and T lymphocytes depending on their differential ability to bind antiimmunoglobulin molecules, a d h e r e to nylon wool fibres or their mobility in an electrical field (13-19). H o w e v e r , we
Staining with Vital Dyes Viable cells were counted by the trypan blue exclusion technique. The cells were also stained with 1% neutral red for counting the plastic adherent cells.
Scanning Electron Microscopy The three categories of cells were smeared on clean, grease-free glass coverslips, fixed with 2% gluteraldehyde for 2V2 h and dehydrated by passing them through a graded series of ethyl alcohol. Cells were then critical point dried using amyl alcohol and liquid CO2 in a Polaron Critical Point Dryer and sputter coated with 150 to 200 A layer of gold as per standard techniques (20-22). Cells were examined in a Philips SEM using an accelerating voltage of 20 KV at Bose Institute, Calcutta.
Immunocompetent cells of bats
425
positive. The total yield of viable cells from the column was more than 40%.
Results Plastic Adherent Cells Figure 1 shows the recovery of plastic adherent (PA) cells. A fairly constant percentage of cells, about 3 to 4% of the total population, always adhered to plastic. About 90% of these cells were neutral red positive, while the plastic nonadherent cells did not take up the stain (Table 1).
Nylon Wool Adherent and Nonadherent Cells The plastic nonadherent cells could be further divided into two categories--the nylon wool adherent (NA) and the nylon wool nonadherent (NNA) cells depending on their surface adhesiveness to nylon wool fibres (Table 2). The recovered NA cells represented about 9% and the recovered NNA cells about 33% of the plastic nonadherent population. None of these two populations were neutral red
Scanning Electron Microscopy of Different Cell Types of Normal and Immunized Bat Most cells in the PA cell population revealed a highly irregular shape, having different pseudopodial projections [Fig. 2(a)]. The cell size was between 4 to 5 ~m. Occasionally, some cells were found whose surface projections did not resemble the pseudopodial extensions of most others: these projections were much broader and bulbuous in shape [Fig. 2(b)]. The size of these cells was also larger than the other plastic adherent cells--in the range of 6 to 8 p+m. These cells appeared in a low frequency and were grouped as a separate category of plastic adherent cells. The NNA cells generally showed a diameter of 6 to 7 ~m and a round shape; the cell surface was relatively smooth without any projections [Fig. 2(c)].
160
I r =o_120 x .,.a --I ua U
MEAN NOOF
