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Ultrastructural characterization of the different classes of lymphocytes in the peripheral blood of Mugil cephalus L. (Teleostei, Pisces)
Fish & Shellfish Immunology (1995) 5, 3-7
U l t r a s t r u c t u r a l c h a r a c t e r i z a t i o n of the different classes of l y m p h o c y t e s in the peripheral blood of Mugil cephalus L. (Teleostei, Pisces) ALBERTO LANFRANCHIAND ORTENSIOFABIANI
Department of Animal Behaviour and Human Sciences, University of Pisa, via A. Volta, 6, 1-56126 Pisa, Italy, and Department of Anatomical, Physiological Sciences and Animal Productions, University of Pisa, via Matteotti, 5, 1-56124 Pisa, Italy (Received 19 November 1993, accepted in revised form 15 March 1994) Grey mullet peripheral blood lymphocytes could be ultrastructurally separated into small cells ('resting cells'), T and B cells and plasma cells. Such cells were morphologically distinguishable independently of cell-surface antigenic markers. Key words: small lymphocytes; T and B cells; plasma cells; teleost fish; grey mullet. I. I n t r o d u c t i o n
Although lymphocytes have long been recognized as a major component of higher vertebrate blood cells, it was only relatively recently t h a t their central role in immunity has been demonstrated (Gowans & McGregor, 1965). Experiments established t h a t only lymphocytes were able to restore the immune response of irradiated animals. Since both antibody and cell-mediated responses were restored, lymphocytes were clearly responsible for both types of immune response. Subsequently, two different classes of lymphocytes were characterized: T cells, developing in the thymus and responsible for cellmediated immunity; and B cells, developing independently of the thymus and producing antibodies (Greaves et al., 1973). T and B cells look ultrastructurally very similar in the unstimulated ('resting') state (MSller, 1983), whereas they become morphologically distinguishable after activation by antigens. Activated B cells develop into antibody-secreting cells, the most mature of which are plasma cells, filled with extensive rough endoplasmic reticulum. Unlike B cells, activated T cells contain very little rough endoplasmic reticulum, and do not secrete antibodies. Data on fish lymphocytes were reviewed by Ellis (1977), who wrote "recent studies on the hapten-carrier effect in teleosts suggest the presence of two functional populations of immune-reactive cells, one producing antibody and the other performing a helper role in a similar manner as occurs in birds and mammals". The distinction between T and B lymphocytes could not be clearly made in more recent studies on fish leucocytes (Cannon et al., 1980; Temmink 1050-4648/95/010003+05 $08.00/0
~ 1995 Academic Press Limited
4
A. L A N F R A N C H I A N D O. F A B I A N I
& Bayne, 1987). The relation of plasma cells with B cells also was uncertain in another report (Morrow & Pulsford, 1980). In our investigation, lymphocytes in the peripheral blood of Mugil cephalus were studied ultrastructurally in order to verify their identity with mammalian lymphocytes. II. M a t e r i a l s a n d M e t h o d s
The peripheral blood of five grey mullets (Mugil cephalus L.), each weighing 500 g, was investigated. Leucocytes were processed as a solidified disc of the buffy coat (Anderson, 1965). Primary fixation was performed in a solution containing 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7'3). After fixation, the specimens were washed in the same buffer with 7% glucose and postfixed in 1% osmium tetroxide in 0.13 cacodylate buffer (pH 7-3). Dehydration, embedding in Epon-Araldite mixture, sectioning and double staining were all carried out according to routine methods. The sections were examined under a Siemens Elmiskop 1A electron microscope. III. R e s u l t s
The circulating lymphocytes of M. cephalus are numerous (about 25 for 100 leucocytes), variable in size and appearance from very small (2.5/1m) to relatively large (4-6/1m) cells. They can be classified on the basis of their ultrastructural features in: SMALL LYMPHOCYTES
The small lymphocytes are spherical with an average diameter of about 2.5 ~tm [Fig. l(a)]. Generally the plasma membrane possesses some microvilli. The nucleus, round in shape with varying degrees in indentation, is located centrally and mostly heterochromatic, with a scarcely-developed nucleolus. The cytoplasm, of moderate density, is limited to a narrow rim, where scattered free ribosomes, large mitochondria with tubular cristae, light vesicles, few long profile of rough endoplasmic reticulum, scarce electron-dense granules and multilamellar bodies are visible. The nuclear indentation holds the Golgi apparatus composed of numerous flattened cisternae arranged near the centriole pair. The small lymphocyte could be a T cell or a B cell, for these 'resting' cells are difficult to distinguish morphologically until they have been activated by antigens. LARGE LYMPHOCYTES
The relatively large lymphocytes vary between 4 and 6/~m in diameter on account of the wider cytoplasmic volume. They can be subdivided into two classes: 1. Lymphocytes of slightly larger size (6/Lm) t h a n the subsequent, possessing an indented or horseshoe-shaped nucleus [Fig. l(b)]. Condensed chromatin
CHARACTERIZATION OF MUGIL PERIPHERAL LYMPHOCYTES
5
a
Fig. 1. (a) Small lymphocyte. The nucleus is round and mostly heterochromatic. The nuclear indentation holds the Golgi apparatus and the centriole pair. The cytoplasm, limited to a narrow rim, contains a few organelles. The plasma membrane possesses some microvilli. The small lymphocyte could be a T cell or a B cell, undistinguishable until they have been activated. Magnification 60 000 x . (b) T lymphocyte. This cell has relatively little rough endoplasmic reticulum but is filled with free ribosomes. Magnification 28 000 x .
l o c a t e d in clumps is p r e s e n t n e a r t h e n u c l e a r envelope. T h e n u c l e o l u s is u s u a l l y visible. T h e c y t o p l a s m is c h a r a c t e r i z e d by v e r y n u m e r o u s free ribosomes, e l e c t r o n d e n s e g r a n u l e s s u r r o u n d i n g a lipid d r o p l e t (Gall body), m i t o c h o n d r i a , t r a n s p a r e n t vesicles a n d Golgi a p p a r a t u s .
A. LANFRANCHIAND O. FABIANI
6
r Fig. 2. (a) B lymphocyte. The cytoplasm contains abundant rough endoplasmic reticulum. Magnification 28 000 ×. (b) Plasma cell. The cytoplasm is filled with an extensive rough endoplasmic reticulum (distended with antibody molecules). This cell is an activated B cell. Magnification 28 000 x.
Such cells can be considered as T lymphocytes for their u l t r a s t r u c t u r a l morphology. 2. Lymphocytes having an ovoid shape, provided with a nucleolus characterized by a b u n d a n t condensed chromatin located adjacent to the nuclear envelope and clumped towards the interior like wheel spokes [Fig. 2(a)]. The nucleolus is well developed. Amounts of rough endoplasmic reticulum are present in the cytoplasm and often appear in a loosely-stacked
CHARACTERIZATION OF MUGIL PERIPHERAL LYMPHOCYTES
7
configuration. Small a n d m e d i u m size vesicles, free ribosomes, electron-dense g r a n u l e s and e l o n g a t e d m i t o c h o n d r i a w i t h t u b u l a r cristae are also visible. S u c h cells can be c o n s i d e r e d as B l y m p h o c y t e s in the process of being t r a n s f o r m e d into plasma cells [Fig. 2(b)]; t h e y are filled with dilated c i s t e r n a e of r o u g h endoplasmic r e t i c u l u m w i t h a fluffy c o n t e n t ( a n t i b o d y molecules).
III. D i s c u s s i o n In the p r e s e n t s t u d y on M. cephalus, the u l t r a s t r u c t u r a l m o r p h o l o g y of the l y m p h o c y t e s is f o u n d to be similar to t h a t of mammals: this teleost possesses the e q u i v a l e n t s of m a m m a l i a n T lymphocytes, B l y m p h o c y t e s and plasma cells (cfr. Fig. 17.4 p. 955 Alberts et al., 1983; Grossi et al., 1988; R o w l e y et al., 1988). Our o b s e r v a t i o n s confirm the a s s u m p t i o n s of M o r r o w & Pulsford (1980), i.e. these cells h a v e c h a n g e d v e r y little d u r i n g the e v o l u t i o n and d e v e l o p m e n t of a n i m a l species, since t h e y are f o u n d in such diverse phyla. This work was supported by the MURST (60%)
References Alberts, B., Bray, D., Lewis, J., Raft, M., Roberts, K. & Watson, J. D. (1983). Molecular Biology of the Cell. Garland Pub., New York & London. Anderson, D. R. (1965). A method of preparing peripheral leucocytes for electron microscopy. Journal of Ultrastructural Research, 13, 263-268. Cannon, M. S., Mollenhauer, H. H., Eurell, T. E., Lewis, D. H., Cannon, A. M. & Tompkins, C. (1980). An ultrastructural study of the leukocytes of the channel catfish, Ictalurus punctatus. Journal of Morphology, 164, 1-20. Ellis, A. E. (1977). The leucocytes of fish: a review. Journal of Fish Biology 11,453-491. Gowans, J. L. & McGregor, D. D. (1965). The immunological activities of lymphocytes. Progress in Allergy 9, 1-78. Greaves, M. F., Owen, J. J. T. & Raft, M. C. (1973). T and B lymphocytes: origins, properties and roles in immune responses. In Excerpta Medica, Amsterdam. Grossi, C. E., Ferrarini, M. & Greaves, M. F. (1988). Linfociti normali. In: Le Cellule del Sangue. Funzioni e Patologia. Atlante. (D. Zucker-Franklin, M. F. Greaves, C. E. Grossi & A. M. Marmont, eds) pp. 383-443. Edi. Ermes, Milano. MSller, G. (1983). Functional T cell subsets defined by monoclonal antibodies. In Immunological Review, 74. Morrow, W. J. W. & Pulsford, A. (1980). Identification of peripheral blood leucocytes of the dogfish (Scyliorhinus canicula L.) by electron microscopy. Journal of Fish Biology 17, 461-475. Rowley, A. F., Hunt, T. C., Page, M. & Mainwaring, G. (1988). Fish. In Vertebrate blood cells. (A. F. Rowley & N. A. Ratcliffe, eds) pp. 19-127. Cambridge University Press. Temmink, J. H. M. & Bayne, C. J. (1987). Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio L.). Development and Comparative Immunology, 11, 125-137.
U l t r a s t r u c t u r a l c h a r a c t e r i z a t i o n of the different classes of l y m p h o c y t e s in the peripheral blood of Mugil cephalus L. (Teleostei, Pisces) ALBERTO LANFRANCHIAND ORTENSIOFABIANI
Department of Animal Behaviour and Human Sciences, University of Pisa, via A. Volta, 6, 1-56126 Pisa, Italy, and Department of Anatomical, Physiological Sciences and Animal Productions, University of Pisa, via Matteotti, 5, 1-56124 Pisa, Italy (Received 19 November 1993, accepted in revised form 15 March 1994) Grey mullet peripheral blood lymphocytes could be ultrastructurally separated into small cells ('resting cells'), T and B cells and plasma cells. Such cells were morphologically distinguishable independently of cell-surface antigenic markers. Key words: small lymphocytes; T and B cells; plasma cells; teleost fish; grey mullet. I. I n t r o d u c t i o n
Although lymphocytes have long been recognized as a major component of higher vertebrate blood cells, it was only relatively recently t h a t their central role in immunity has been demonstrated (Gowans & McGregor, 1965). Experiments established t h a t only lymphocytes were able to restore the immune response of irradiated animals. Since both antibody and cell-mediated responses were restored, lymphocytes were clearly responsible for both types of immune response. Subsequently, two different classes of lymphocytes were characterized: T cells, developing in the thymus and responsible for cellmediated immunity; and B cells, developing independently of the thymus and producing antibodies (Greaves et al., 1973). T and B cells look ultrastructurally very similar in the unstimulated ('resting') state (MSller, 1983), whereas they become morphologically distinguishable after activation by antigens. Activated B cells develop into antibody-secreting cells, the most mature of which are plasma cells, filled with extensive rough endoplasmic reticulum. Unlike B cells, activated T cells contain very little rough endoplasmic reticulum, and do not secrete antibodies. Data on fish lymphocytes were reviewed by Ellis (1977), who wrote "recent studies on the hapten-carrier effect in teleosts suggest the presence of two functional populations of immune-reactive cells, one producing antibody and the other performing a helper role in a similar manner as occurs in birds and mammals". The distinction between T and B lymphocytes could not be clearly made in more recent studies on fish leucocytes (Cannon et al., 1980; Temmink 1050-4648/95/010003+05 $08.00/0
~ 1995 Academic Press Limited
4
A. L A N F R A N C H I A N D O. F A B I A N I
& Bayne, 1987). The relation of plasma cells with B cells also was uncertain in another report (Morrow & Pulsford, 1980). In our investigation, lymphocytes in the peripheral blood of Mugil cephalus were studied ultrastructurally in order to verify their identity with mammalian lymphocytes. II. M a t e r i a l s a n d M e t h o d s
The peripheral blood of five grey mullets (Mugil cephalus L.), each weighing 500 g, was investigated. Leucocytes were processed as a solidified disc of the buffy coat (Anderson, 1965). Primary fixation was performed in a solution containing 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7'3). After fixation, the specimens were washed in the same buffer with 7% glucose and postfixed in 1% osmium tetroxide in 0.13 cacodylate buffer (pH 7-3). Dehydration, embedding in Epon-Araldite mixture, sectioning and double staining were all carried out according to routine methods. The sections were examined under a Siemens Elmiskop 1A electron microscope. III. R e s u l t s
The circulating lymphocytes of M. cephalus are numerous (about 25 for 100 leucocytes), variable in size and appearance from very small (2.5/1m) to relatively large (4-6/1m) cells. They can be classified on the basis of their ultrastructural features in: SMALL LYMPHOCYTES
The small lymphocytes are spherical with an average diameter of about 2.5 ~tm [Fig. l(a)]. Generally the plasma membrane possesses some microvilli. The nucleus, round in shape with varying degrees in indentation, is located centrally and mostly heterochromatic, with a scarcely-developed nucleolus. The cytoplasm, of moderate density, is limited to a narrow rim, where scattered free ribosomes, large mitochondria with tubular cristae, light vesicles, few long profile of rough endoplasmic reticulum, scarce electron-dense granules and multilamellar bodies are visible. The nuclear indentation holds the Golgi apparatus composed of numerous flattened cisternae arranged near the centriole pair. The small lymphocyte could be a T cell or a B cell, for these 'resting' cells are difficult to distinguish morphologically until they have been activated by antigens. LARGE LYMPHOCYTES
The relatively large lymphocytes vary between 4 and 6/~m in diameter on account of the wider cytoplasmic volume. They can be subdivided into two classes: 1. Lymphocytes of slightly larger size (6/Lm) t h a n the subsequent, possessing an indented or horseshoe-shaped nucleus [Fig. l(b)]. Condensed chromatin
CHARACTERIZATION OF MUGIL PERIPHERAL LYMPHOCYTES
5
a
Fig. 1. (a) Small lymphocyte. The nucleus is round and mostly heterochromatic. The nuclear indentation holds the Golgi apparatus and the centriole pair. The cytoplasm, limited to a narrow rim, contains a few organelles. The plasma membrane possesses some microvilli. The small lymphocyte could be a T cell or a B cell, undistinguishable until they have been activated. Magnification 60 000 x . (b) T lymphocyte. This cell has relatively little rough endoplasmic reticulum but is filled with free ribosomes. Magnification 28 000 x .
l o c a t e d in clumps is p r e s e n t n e a r t h e n u c l e a r envelope. T h e n u c l e o l u s is u s u a l l y visible. T h e c y t o p l a s m is c h a r a c t e r i z e d by v e r y n u m e r o u s free ribosomes, e l e c t r o n d e n s e g r a n u l e s s u r r o u n d i n g a lipid d r o p l e t (Gall body), m i t o c h o n d r i a , t r a n s p a r e n t vesicles a n d Golgi a p p a r a t u s .
A. LANFRANCHIAND O. FABIANI
6
r Fig. 2. (a) B lymphocyte. The cytoplasm contains abundant rough endoplasmic reticulum. Magnification 28 000 ×. (b) Plasma cell. The cytoplasm is filled with an extensive rough endoplasmic reticulum (distended with antibody molecules). This cell is an activated B cell. Magnification 28 000 x.
Such cells can be considered as T lymphocytes for their u l t r a s t r u c t u r a l morphology. 2. Lymphocytes having an ovoid shape, provided with a nucleolus characterized by a b u n d a n t condensed chromatin located adjacent to the nuclear envelope and clumped towards the interior like wheel spokes [Fig. 2(a)]. The nucleolus is well developed. Amounts of rough endoplasmic reticulum are present in the cytoplasm and often appear in a loosely-stacked
CHARACTERIZATION OF MUGIL PERIPHERAL LYMPHOCYTES
7
configuration. Small a n d m e d i u m size vesicles, free ribosomes, electron-dense g r a n u l e s and e l o n g a t e d m i t o c h o n d r i a w i t h t u b u l a r cristae are also visible. S u c h cells can be c o n s i d e r e d as B l y m p h o c y t e s in the process of being t r a n s f o r m e d into plasma cells [Fig. 2(b)]; t h e y are filled with dilated c i s t e r n a e of r o u g h endoplasmic r e t i c u l u m w i t h a fluffy c o n t e n t ( a n t i b o d y molecules).
III. D i s c u s s i o n In the p r e s e n t s t u d y on M. cephalus, the u l t r a s t r u c t u r a l m o r p h o l o g y of the l y m p h o c y t e s is f o u n d to be similar to t h a t of mammals: this teleost possesses the e q u i v a l e n t s of m a m m a l i a n T lymphocytes, B l y m p h o c y t e s and plasma cells (cfr. Fig. 17.4 p. 955 Alberts et al., 1983; Grossi et al., 1988; R o w l e y et al., 1988). Our o b s e r v a t i o n s confirm the a s s u m p t i o n s of M o r r o w & Pulsford (1980), i.e. these cells h a v e c h a n g e d v e r y little d u r i n g the e v o l u t i o n and d e v e l o p m e n t of a n i m a l species, since t h e y are f o u n d in such diverse phyla. This work was supported by the MURST (60%)
References Alberts, B., Bray, D., Lewis, J., Raft, M., Roberts, K. & Watson, J. D. (1983). Molecular Biology of the Cell. Garland Pub., New York & London. Anderson, D. R. (1965). A method of preparing peripheral leucocytes for electron microscopy. Journal of Ultrastructural Research, 13, 263-268. Cannon, M. S., Mollenhauer, H. H., Eurell, T. E., Lewis, D. H., Cannon, A. M. & Tompkins, C. (1980). An ultrastructural study of the leukocytes of the channel catfish, Ictalurus punctatus. Journal of Morphology, 164, 1-20. Ellis, A. E. (1977). The leucocytes of fish: a review. Journal of Fish Biology 11,453-491. Gowans, J. L. & McGregor, D. D. (1965). The immunological activities of lymphocytes. Progress in Allergy 9, 1-78. Greaves, M. F., Owen, J. J. T. & Raft, M. C. (1973). T and B lymphocytes: origins, properties and roles in immune responses. In Excerpta Medica, Amsterdam. Grossi, C. E., Ferrarini, M. & Greaves, M. F. (1988). Linfociti normali. In: Le Cellule del Sangue. Funzioni e Patologia. Atlante. (D. Zucker-Franklin, M. F. Greaves, C. E. Grossi & A. M. Marmont, eds) pp. 383-443. Edi. Ermes, Milano. MSller, G. (1983). Functional T cell subsets defined by monoclonal antibodies. In Immunological Review, 74. Morrow, W. J. W. & Pulsford, A. (1980). Identification of peripheral blood leucocytes of the dogfish (Scyliorhinus canicula L.) by electron microscopy. Journal of Fish Biology 17, 461-475. Rowley, A. F., Hunt, T. C., Page, M. & Mainwaring, G. (1988). Fish. In Vertebrate blood cells. (A. F. Rowley & N. A. Ratcliffe, eds) pp. 19-127. Cambridge University Press. Temmink, J. H. M. & Bayne, C. J. (1987). Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio L.). Development and Comparative Immunology, 11, 125-137.