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Ontogeny of IgM and IgM-bearing cells in rainbow trout
Developmental and Comparative Immunology,Vol. 17, pp. 419-424, 1993 Printed in the USA. All rights reserved.
0145-305X/93 $6.00 + .00 Copyright © 1993 PergamonPress Ltd.
ONTOGENY OF IgM AND IgM-BEARING CELLS IN RAINBOW TROUT Ana Castillo,* Carmen S&nchez,? Javier Dominguez,t Stephen L. Kaattari,:l: and Alberto J. Villena* *Departamento de Biolog[a Celular, Universidad de Le6n, 24071-L6on, Spain, 1-Departamento de Sanidad Animal, INIA-CIT, 28012-Madrid, Spain, and :[:Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804
(Submitted March 1993; Accepted June 1993)
FqAbstract--We have studied the ontogenic
salmonids (8,9), and the appearance of i m m u n o g l o b u l i n M (IgM) b e a r i n g of IgM-bearing cells in the rainbow trout, On- (IgM +) cells (10). Similar studies have corhynchus mykiss. Lymphocytes showing cy- been conducted in carp (5,11). However, toplasmic IgM were first observed in embryos there is little data delineating the differat 12 days before hatch (14"C). At this stage, no entiative pathways of T and B lymphocells positive for surface IgM were present. cytes from their hemopoietic precursors Lymphocytes bearing surface IgM were observed at 8 days before hatch (14"(;). Unfertil- in fish. Using mAbs specific for rainbow trout ized trout eggs contained detectable amounts of IgM (11.2 - 2.6 i~g/gof egg weight), indicating Oncorhynchus rnykiss s e r u m IgM that transfer of IgM from mother to embryo (1,12,13), we describe the appearance of can occur in salmonids. The levelsof IgM from cells bearing cytoplasmic or surface whole fish increase slowlyafter the appearance IgM, as well as the levels of IgM/g of of intraembryonic cells that express surface body weight, at different stages of IgM. The amount of IgM/g of tissue peaks development. around hatch, but this parameter shows lower values up to 2 months after hatch. development of immunoglobulin M (IgM) and
F1Keywords--Immunoglobulin M (IgM); B-cells; Immunocompetence; Ontogeny; Trout; Teleost.
Materials and Methods
Animals
Introduction Monoclonal antibodies (mAbs) specific for fish immunoglobulins (I-4), and leucocyte surface determinants (3,5,6) have permitted the demonstration that T- and B-lymphocyte subpopulations exist in fish. However, there is a lack of data on the ontogenetic origins and differentiation of fish lymphoid cells (7). Several studies have described the morphogenesis of the main lymphoid organs in Address correspondence to Dr. Alberto J. Villena.
Fertilized and unfertilized rainbow trout eggs were obtained from a fish farm near L e r n (Spain), which is supplied with pathogen-free well water. Incubation of fertilized eggs and maintenance of fry were performed in departmental aquaria supplied with running dechlorinated water at 14 +-- I°C, and maintained with a photoperiod of 12 h light/12 h dark. Under these environmental conditions, trout eggs hatched at 26-27 days after fertilization. Developmental stages were determined according to the Vernier table of trout development (14). The correspondence between Vernier's
419
420
A. Castillo et al.
stages and days of incubation at 14°C is shown in Table 1.
Immunodetection of IgM + Cells At different times from Vernier stage 21 (18 days before hatch at 14°C) to stage 32 (4 days after hatch), pools of 20-50 embryos or 10 fry from each stage were overanaesthetized in -0.015% MS-222. After elimination of the yolk sacs, fish were kept in phosphate buffered saline (PBS), pH 7.2, on ice. Single-cell suspensions were obtained by repeated gentle flushing of the pooled fish through a 10-mL syringe. Cell suspensions were aliquoted, twice washed and resuspended, at 106 lymphoid cell/50 txL in PBS containing 20 mM NAN3. Staining of cytoplasmic IgM ÷ cells was performed after fixation with 1%
paraformaldehyde (15 min on ice). Surface IgM staining was demonstrated in unfixed cells kept on ice. The presence of IgM in the cells was detected by the indirect immunofluorescent technique using a mouse anti-trout IgM monoclonal antibody (mAb 1-14) developed by DeLuca, Wilson, and Warr (1). As secondary antibody a rabbit anti-mouse IgG antiserum conjugated to fluorescein (Dako) was used. Several dilutions of the primary and secondary antibodies were tested to optimize results. The stained cell suspensions were mixed with equal volumes of P B S - g l y c e r o l (1:9) and mounted on slides. Controls involved omission of the primary antibody. Fluorescence was observed under a Nikon Optiphot microscope provided with epifluorescent equipment and a 100-W halogen tungsten lamp.
Quantification of IgM Table 1. Levels of IgM/g of Body Weight During Development of Rainbow Trout. Stage of Development* Unfert. e g g s 8 days post-fertilization 9 d.b.h. (St. 26) 4 d.b.h. (St. 28) H a t c h (St. 30) 2 d.a.h. 4 d.a.h. (St. 32) 7 d.a.h. 14 d.a.h. (St, 34) 21 d.a.h. (St. 35) 30 d.a.h. 45 d.a.h. 60 d.a.h.
Number of Samples (n)l" 4 (50) 4 10 9 10 9 9 5 10 10 7 5 4
(30) (30) (30) (10) (10) (10) (10) (10) (10) (10) (4) (4)
ixg IgM/g~t ( M e a n -+ SD) 11.2 -+ 2.6 a 14.7 25.7 39.0 153.1 102.6 30.8 49.1 11.5 13.9 6.6 10.1 14.7
-+ 1.6 a +- 5.8 b -+ 10.8 b - 40.6 c -+ 41.0 ° + 18.7 b +- 12.4 b -+ 4.3 b -+ 3.9 b -+ 1.6 b -+ 3.1 ~ +- 8.5
St.: Vernier's stages of trout development. * At 14°C; d.b.h.: Days before hatch; d.a.h.: Days after hatch. "i Number of pooled individuals (n) in each sample in
parentheses. ~: The concentration of IgM was determined by ELISA, using a mAb against rainbow trout IgM. a Statistically significant (t9 > 0.01) compared with values from St. 26 to 7 d.a.h. b Statistically significant (/9 > 0.01) compared with the previous stage. c Statistically significant (p > 0.05) compared with 30 d.a.h,
Measurements of the IgM tissue levels were performed with homogenized samples of unfertilized eggs, embryos, and fry. As indicated in Table 1, 4-10 samples were used for each stage analyzed. Before homogenization, unfertilized eggs were washed five times in a large volume of PBS to remove any external IgM from maternal body fluids. Eggs, embryos, and fry were weighed and homogenized in cold PBS by the use of a tissue grinder (Braun). Tissues were diluted in PBS and centrifuged (20 min at 700×g, 4°C). The supernatants were collected, aliquoted, and kept at -80°C until use. The concentrations of IgM in the samples were determined by an ELISA procedure, using the mAB 1G7 against the rainbow trout IgM, as described by S~mchez et al. (12,13). In this assay, the minimum detectable IgM concentration was 0.20 ~g/mL (12). Data were expressed as ~g of IgM/g of egg or body
Ontogeny of IgM and IgM-bearing cells in rainbow trout
421
weight. Differences between the means obtained for each stage of development were analyzed using Student's t-test (p < 0.01 or <0.05).
Results
IgM-Positive Cells Control experiments without primary antibody were negative. Lymphoid cells bearing cytoplasmic IgM were first observed at Vernier stage 23-24 (12 days before hatch at 14°C, Fig. 1). At this stage, no cells positive for surface IgM were present. Surface IgM + lymphocytes were first observed in cell suspensions from embryos at Vernier stage 26 (8 days before hatch, Fig. 2).
Figure 2. Expression of surface IgM. (A) Micrograph showing three small lymphocytes expressing surface igM, from a pool of 40 trout embryos at Vernier stage 26 (8 days before hatch, at 14°C). (B) Micrograph showing the same field under phase contrast microscopy. ×968.
onward, the parameter showed decreasing values until 2 months after hatch, when a slow recovery was observed (Table 1).
Quantification of Levels of IgM From Whole Trout Discussion Unfertilized eggs contained small amounts of IgM (11.2 ~g/g of egg), which did not increase significantly by 8 days post-fertilization. The amount of IgM/g of fry body weight increased from Vernier stage 26 until hatch, when a significant peak was reached. From this stage
Figure 1. Expression of cytoplasmic IgM. (A) Immunofluorescence staining of cytoplasmic IgM on a lymphoid cell from a pool of 50 trout embryos at Vernier stage 24 (12 days before hatch, at 14°C). IgM was detected with mAb 1-14 antitrout IgM. (B) Micrograph showing the same field observed under phase contrast microscopy. The arrow indicates the IgM + cell. ×990.
Using an immunofluorescent technique on cell suspensions from pooled trout embryos, IgM-bearing lymphocytes are present by 12 days before hatch (at 14°C). In a previous immunohistochemical study on cryostat sections (10) using the same monoclonal antibody anti-trout IgM, we reported that IgMbearing cells first appeared at day 4 after hatching in the kidney of rainbow trout. This difference in the appearance of IgM + cells may be attributable to the low number and scattered distribution of these cells in embryonic tissues. Thus, IgM + cells are more easily observed in cell suspensions of pooled embryos than in tissue sections. Our results indicated that lymphocytes showing cytoplasmic +, surfaceexpression of IgM (pre-B cells) appeared before surface IgM + (mature B cells) could be detected. This is in contrast to previous studies (15,16) describing the
422
simultaneous presence of cytoplasmic and surface IgM in immature B cells during fish ontogeny. However, polyclonal anti-IgM antisera were used in those studies, and the presumed lower affinities and specificities of these reagents may explain the different staining patterns. On the other hand, our data may be interpreted as agreeing with those reported for amphibians (17,18) and mammals (19). However, as the mAb 1-14 reacts only with trout p,-chains, and no antibodies were available for trout light chains, the observation of pre-B cells in trout has not been accomplished. Hence, the earliest ontogenetic origin of the B cell precursors in trout is still obscure. Although the adult fish pronephros seems to be the equivalent to mammalian bone marrow (10,20-22), the first IgM ÷ lymphocytes observed in this study appeared before the pronephros becomes lymphopoietic (9,10). In most vertebrates, the yolk sac and fetal liver seem to be major sites for early B-cell differentiation (17,23-26). Also, other embryonic tissues have been considered as a s o u r c e for l y m p h o c y t e p r e c u r s o r s (27,28). In fish, there is little information on the contribution of the yolk sac to hemopoiesis (29). Parenthetically, the yolk sac was not included in the present study, because it was impractical to obtain cell suspensions from this organ, due to the large amount of yolk droplets remaining in the cell suspensions. However, attempts to stain IgM + cells in frozen sections of the trout yolk sac were negative (Castillo, unpublished observations). On the other hand, the liver of embryonic or adult teleosts appear to have no hemopoietic capacities (7), nor IgM-bearing cells (Castillo, unpublished observations). Few studies have reported serum IgM levels during development of salmonids (30,31), and quantitative levels of IgM have only been investigated after hatching (32). In our results, unfertilized eggs
A. Castillo et al.
contained low but detectable amounts of IgM. This fact was not seen in previous studies (32,33), which probably reflects the higher sensitivity of the ELISA assay with monoclonal antibodies. The presence of lgM in the unfertilized trout eggs indicates that vertical transmission of this molecule can occur in trout, as described in other teleosts (30,34). Embryonic levels of IgM/g of embryo remained similar to those of unfertilized eggs until the Vernier's stage 26, when surface IgM + cells (mature B cells) appeared. From this stage onward, this parameter progressively increased during embryonic development, reaching a peak at hatch. The appearance of B cells should correlate with the development of humoral immunity. However, our study cannot ascertain the source of the increased levels of IgM during embryonic development because the cells were disrupted by homogenization. Thus, the IgM observed may stem from cytoplasmic and membrane fragments of pre-B and B cells, rather than plasma cells. The greatly increased concentration of IgM at hatch is an intriguing phenomenon. It is of particular interest in that it is temporally next to the colonization of the pronephros by IgM + cells. As indicated above, the first IgM-bearing cells in the propnephros were detected at day 4 after hatching (10), but the colonization of this organ by presumed pre-B cells and some B cells may probably occur some time earlier. The interaction of such putative pre-B and B cells with a specific m i c r o e n v i r o n m e n t advantageous for their differentiation, might play an important role in the amplification of these populations, and subsequently be responsible for high levels of IgM. Alternatively, the high IgM tissue levels observed at hatch might also represent a nonspecific stimulation related to hatching, influenced by: An imbalance between the T-cell and B-cell compartments in the embryos; and/or the hot-
Ontogeny of IgM and IgM-bearing cells in rainbow trout
monal and stress factors involved in fish hatching (35), which in turn may dramatically influence immune function (3638). The decreasing levels of IgM after hatch may also be related to the increase in body weight after first feeding. In any case, the high IgM levels found at hatch may play a protective role, when fry are exposed to aquatic pathogens, at a developmental stage in which
423
their lymphoid organs show poor organization (8-10). Acknowledgements--This research was supported by the Spanish CICYT Grant MAR88/ 0565. Cooperation between the University of Le6n (Dr. A. Villena) and the Oregon State University (Dr. S. Kaattari) was funded by a grant from the D.G.I.C.Y.T. (Ministerio Espafiol de Educacirn y Ciencia).
References 1. DeLuca, D.; Wilson, M; Warr, G. W. Lymphocyte heterogeneity in the trout, Salrno gairdneri, defined with monoclonal antibodies to IgM. Eur. J. Immunol. 13:546-551; 1983. 2. Egberts, E.; Secombes, C. J.; Wellink, J. E.; van Groningen, J. J. M.; van Muiswinkel W. B. Analysis of lymphocyte heterogeneity in carp, Cyprinus carpio L., using monoclonal antibodies, Dev. Comp. Immunol. 7:749-754; 1983. 3. Secombes, C. J.; Van Groningen, J. J. M.; Egberts, E. Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: Immunohistochemical studies. Immunology 48:165-175; 1983. 4. Lobb, C. J.; Clem, L. W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev. Comp. Immunol. 6:473-479; 1982. 5. Van Loon, J. J. A.; Secombes, C. J.; Egberts, E.; van Muiswinkel, W. B. Ontogeny of the immune system in fish--Role of the thymus. In: Nieuwenhuis, P.; van den Broek, A. A.; Hanna, M. G., eds. In vivo immunology, Histophysiology of the lymphoid system. Advances in Experimental and Medical Biology, Vol. 149. New York: Plenum Press; 1982:335341. 6. Miller, N. W.; Bly, J. E.; van Ginkel, E; Ellsaesser, C. E; Clem, L. W. Phylogeny of lymphocyte heterogeneity: Identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies. Dev. Comp. Immunol. 11:739747; 1987. 7. Zapata, A.; Cooper, E. L., Eds. The immune system: Comparative histophysiology. Chichester: John Wiley & Son; 1990. 8. Ellis, A. E. Ontogeny of the immune response in Salmo salar. Histogenesis of lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. In: Solomon, J. B.; Horton, J. D., eds. Developmental immunobiology. Amsterdam: ElsevierNorth Holland; 1977:225-231. 9. Grace, M. E; Manning, M. J. Histogenesis of the lymphoid organs in rainbow trout, Salmo
gairdneri Rich. 1836. Dev. Comp. Immunol. 4:255-264; 1980. 10. Razquin, B. E.; Castillo, A.; L6pez-Fierro, P.; Alvarez, E; Zapata, A.; Villena, A. J. Ontogeny of IgM-producing cells in the lymphoid organs of Salmo gairdneri: An immuno and enzyme-histochemical study. J. Fish Biol. 36;159-173; 1990. 11. Secombes, C. J.; Van Groningen, J. J. M.; van Muiswinkel, J. B.; Egberts, E. Ontogeny of the immune system in carp (Cyprinus carpio L,). The appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocyte monoclonal antibodies. Dev. Comp. Immunol. 7:455-464; 1983. 12. S~inchez, C. An~ilisis estructural y antigrnico de las inmunoglobulinas de la trucha arco iris, Oncorhynchus mykiss. Ph. Thesis. Universidad Complutense de Madrid, 1992. 13. S~tnchez, C.; Coil, J.; Dominguez, J. One step purification of trout immunoglobulin. Vet. Immunol. Immunopathol. 27:383-392; 1991. 14. Vernier, J. M. Table chronologique du drveloppement embryonnaire de la truite arc-en-ciel, Salmo gairdneri Rich. 1836. Ann. d'Embryol. Morphogen. 2:495-520; 1969. 15. Grossi, C. E.; Lyard, P. M.; Cooper, M. D. Changing patterns of cytoplasmic IgM expression and of modulation requirements of surface IgM by anti-M antibodies. J. Immunol. 119: 749-755; 1977. 16. Lassila, O. Embryonic differentiation of lymphoid stem cells: A review. Dev. Comp. Immunol. 5:403-404; 1981. 17. Zettergren, L. D. Ontogeny and distribution of cells in B lineage in the American leopard frog (Rana pipiens). Dev. Comp. Immunol. 6:311320; 1982. 18. Hadji-Azimi, I.; Schwager, J.; Thiebaud, C. B lymphocyte differentiation in Xenopus laevis larvae. Dev. Biol. 90:253-258; 1982. 19. Landreth, K. S.; Kinkade, P. W. Mammalian B lymphocyte precursors. Dev. Comp. Immunol. 8:773-790; 1984. 20. Zapata, A. Ultrastructural study of the teleosts fish kidney. Dev. Comp. Immunol. 3:55-65; 1979.
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21. Zapata, A. Phylogeny of the fish immune system. Bull. Inst. Pasteur 81:175-186; 1983. 22. Irwin, M. J.; Kaattari, S. L. Salmonid B-lymphocytes demonstrated organ dependent functional heterogeneity. Vet. Immunol. Immunopathol. 12:39-45; 1986. 23. Le Douarin, N. M.; Dieterlen-Lievre, E ; Oliver, P. D. Ontogeny of primary lymphoid organs and lymphoid stem cells. Am. J. Anat. 170:261-299; 1981. 24. Natarajan, K.; Muthukkaruppan, V. R. Distribution and ontogeny of B cells in the garden lizard, Calotes versicolor. Dev. Comp. Immunol. 9:301-310; 1985. 25. E1 Deeb, S.; Zada, S.; El Ridi, R. Ontogeny of hemopoietic tissues in the lizard, Chalcides ocellatus (Reptilia, Sauria, Scincidae). J. Morphol. 185;241-253; 1985. 26. Maeno, M.; Tochinai, S.; Katagiti, C. Differential participation of ventral and dorsolateral mesoderm in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimaeras. Dev. Biol. 110:503-508; 1985. 27. Dieterlen-Lievre, E; Martin, C. Diffuse intraembryonic hemopoiesis in normal and chimeric avian development. Dev. Biol. 88:i80190; 1981. 28. Kubai, L.; Auerbach, R. A new source of embryonic lymphocytes in the mouse. Nature 301:154-156; 1983. 29. Ellis, A. E. The leucocytes offish: A review. J. Fish Biol. 11:453-491 ; 1977. 30. Van Loon, J. J. A.; van Oosterom, R.; van Muiswinkel, W. B. Development of immune system in carp. In: Solomon, J. B., ed. Aspects of developmental and comparative ira-
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31.
32. 33. 34.
35.
36.
37.
38.
munology, Vol. 1. Oxford: Pergamon Press; 1981:469-470. Fuda, H.; Soyano, K.; Yamazaki, E ; Hara, A. Serum immunoglobulin M (IgM) during early development of masu salmon (Oncorhynchus rnasou). Comp. Biochem. Physiol. 99A:637643; 1991. Dorson, M. Role and characterization of fish antibody. Dev. Biol. Standard 49:307-319; 1981. Ellis, A. E. Ontogeny of the immune system in teleost fish. In: Ellis, A. E., ed. Fish vaccination. London: Academic Press; 1988:20-31. Bly, J. E.; Grimm, A. S.; Morris, I. G. Transfer of passive immunity from mother to young in a teleost fish: Haemagglutinating activity in the serum and eggs of plaice, Pleuronectes platessa L. Comp. Biochem. Physiol. 84A:309314; 1986. Yamagami K. Mechanism of hatching in fish. In: Hoar, W. S.; Randal, D. J.; eds. Fish physiology, Vol. X1, Part A. San Diego: Academic Press; 1988:447-499. Besedovsky, H. O.; Del Ray, A. E.; Sorkin, E. Immunological-neuroendocrine feedback circuits. In: Guillemin, R.; Cohn, M.; Melnechuk, T., eds. Neural modulation of immunity. New York: Raven Press; 1985;165-177. Ellis, A. E. Stress and the modulation of defence mechanisms in fish. In: Picketing, A. D. ed. Stress and fish. London: Academic Press; 1981:147-169. Flory, M. C. Phylogeny of neuroimmunoregulation: Effects of adrenergic and cholinergic agents on the in vitro antibody response of the rainbow trout, Oncorhynchus rnykiss. Dev. Comp. lmmunol. 14:283-294; 1990.
0145-305X/93 $6.00 + .00 Copyright © 1993 PergamonPress Ltd.
ONTOGENY OF IgM AND IgM-BEARING CELLS IN RAINBOW TROUT Ana Castillo,* Carmen S&nchez,? Javier Dominguez,t Stephen L. Kaattari,:l: and Alberto J. Villena* *Departamento de Biolog[a Celular, Universidad de Le6n, 24071-L6on, Spain, 1-Departamento de Sanidad Animal, INIA-CIT, 28012-Madrid, Spain, and :[:Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804
(Submitted March 1993; Accepted June 1993)
FqAbstract--We have studied the ontogenic
salmonids (8,9), and the appearance of i m m u n o g l o b u l i n M (IgM) b e a r i n g of IgM-bearing cells in the rainbow trout, On- (IgM +) cells (10). Similar studies have corhynchus mykiss. Lymphocytes showing cy- been conducted in carp (5,11). However, toplasmic IgM were first observed in embryos there is little data delineating the differat 12 days before hatch (14"C). At this stage, no entiative pathways of T and B lymphocells positive for surface IgM were present. cytes from their hemopoietic precursors Lymphocytes bearing surface IgM were observed at 8 days before hatch (14"(;). Unfertil- in fish. Using mAbs specific for rainbow trout ized trout eggs contained detectable amounts of IgM (11.2 - 2.6 i~g/gof egg weight), indicating Oncorhynchus rnykiss s e r u m IgM that transfer of IgM from mother to embryo (1,12,13), we describe the appearance of can occur in salmonids. The levelsof IgM from cells bearing cytoplasmic or surface whole fish increase slowlyafter the appearance IgM, as well as the levels of IgM/g of of intraembryonic cells that express surface body weight, at different stages of IgM. The amount of IgM/g of tissue peaks development. around hatch, but this parameter shows lower values up to 2 months after hatch. development of immunoglobulin M (IgM) and
F1Keywords--Immunoglobulin M (IgM); B-cells; Immunocompetence; Ontogeny; Trout; Teleost.
Materials and Methods
Animals
Introduction Monoclonal antibodies (mAbs) specific for fish immunoglobulins (I-4), and leucocyte surface determinants (3,5,6) have permitted the demonstration that T- and B-lymphocyte subpopulations exist in fish. However, there is a lack of data on the ontogenetic origins and differentiation of fish lymphoid cells (7). Several studies have described the morphogenesis of the main lymphoid organs in Address correspondence to Dr. Alberto J. Villena.
Fertilized and unfertilized rainbow trout eggs were obtained from a fish farm near L e r n (Spain), which is supplied with pathogen-free well water. Incubation of fertilized eggs and maintenance of fry were performed in departmental aquaria supplied with running dechlorinated water at 14 +-- I°C, and maintained with a photoperiod of 12 h light/12 h dark. Under these environmental conditions, trout eggs hatched at 26-27 days after fertilization. Developmental stages were determined according to the Vernier table of trout development (14). The correspondence between Vernier's
419
420
A. Castillo et al.
stages and days of incubation at 14°C is shown in Table 1.
Immunodetection of IgM + Cells At different times from Vernier stage 21 (18 days before hatch at 14°C) to stage 32 (4 days after hatch), pools of 20-50 embryos or 10 fry from each stage were overanaesthetized in -0.015% MS-222. After elimination of the yolk sacs, fish were kept in phosphate buffered saline (PBS), pH 7.2, on ice. Single-cell suspensions were obtained by repeated gentle flushing of the pooled fish through a 10-mL syringe. Cell suspensions were aliquoted, twice washed and resuspended, at 106 lymphoid cell/50 txL in PBS containing 20 mM NAN3. Staining of cytoplasmic IgM ÷ cells was performed after fixation with 1%
paraformaldehyde (15 min on ice). Surface IgM staining was demonstrated in unfixed cells kept on ice. The presence of IgM in the cells was detected by the indirect immunofluorescent technique using a mouse anti-trout IgM monoclonal antibody (mAb 1-14) developed by DeLuca, Wilson, and Warr (1). As secondary antibody a rabbit anti-mouse IgG antiserum conjugated to fluorescein (Dako) was used. Several dilutions of the primary and secondary antibodies were tested to optimize results. The stained cell suspensions were mixed with equal volumes of P B S - g l y c e r o l (1:9) and mounted on slides. Controls involved omission of the primary antibody. Fluorescence was observed under a Nikon Optiphot microscope provided with epifluorescent equipment and a 100-W halogen tungsten lamp.
Quantification of IgM Table 1. Levels of IgM/g of Body Weight During Development of Rainbow Trout. Stage of Development* Unfert. e g g s 8 days post-fertilization 9 d.b.h. (St. 26) 4 d.b.h. (St. 28) H a t c h (St. 30) 2 d.a.h. 4 d.a.h. (St. 32) 7 d.a.h. 14 d.a.h. (St, 34) 21 d.a.h. (St. 35) 30 d.a.h. 45 d.a.h. 60 d.a.h.
Number of Samples (n)l" 4 (50) 4 10 9 10 9 9 5 10 10 7 5 4
(30) (30) (30) (10) (10) (10) (10) (10) (10) (10) (4) (4)
ixg IgM/g~t ( M e a n -+ SD) 11.2 -+ 2.6 a 14.7 25.7 39.0 153.1 102.6 30.8 49.1 11.5 13.9 6.6 10.1 14.7
-+ 1.6 a +- 5.8 b -+ 10.8 b - 40.6 c -+ 41.0 ° + 18.7 b +- 12.4 b -+ 4.3 b -+ 3.9 b -+ 1.6 b -+ 3.1 ~ +- 8.5
St.: Vernier's stages of trout development. * At 14°C; d.b.h.: Days before hatch; d.a.h.: Days after hatch. "i Number of pooled individuals (n) in each sample in
parentheses. ~: The concentration of IgM was determined by ELISA, using a mAb against rainbow trout IgM. a Statistically significant (t9 > 0.01) compared with values from St. 26 to 7 d.a.h. b Statistically significant (/9 > 0.01) compared with the previous stage. c Statistically significant (p > 0.05) compared with 30 d.a.h,
Measurements of the IgM tissue levels were performed with homogenized samples of unfertilized eggs, embryos, and fry. As indicated in Table 1, 4-10 samples were used for each stage analyzed. Before homogenization, unfertilized eggs were washed five times in a large volume of PBS to remove any external IgM from maternal body fluids. Eggs, embryos, and fry were weighed and homogenized in cold PBS by the use of a tissue grinder (Braun). Tissues were diluted in PBS and centrifuged (20 min at 700×g, 4°C). The supernatants were collected, aliquoted, and kept at -80°C until use. The concentrations of IgM in the samples were determined by an ELISA procedure, using the mAB 1G7 against the rainbow trout IgM, as described by S~mchez et al. (12,13). In this assay, the minimum detectable IgM concentration was 0.20 ~g/mL (12). Data were expressed as ~g of IgM/g of egg or body
Ontogeny of IgM and IgM-bearing cells in rainbow trout
421
weight. Differences between the means obtained for each stage of development were analyzed using Student's t-test (p < 0.01 or <0.05).
Results
IgM-Positive Cells Control experiments without primary antibody were negative. Lymphoid cells bearing cytoplasmic IgM were first observed at Vernier stage 23-24 (12 days before hatch at 14°C, Fig. 1). At this stage, no cells positive for surface IgM were present. Surface IgM + lymphocytes were first observed in cell suspensions from embryos at Vernier stage 26 (8 days before hatch, Fig. 2).
Figure 2. Expression of surface IgM. (A) Micrograph showing three small lymphocytes expressing surface igM, from a pool of 40 trout embryos at Vernier stage 26 (8 days before hatch, at 14°C). (B) Micrograph showing the same field under phase contrast microscopy. ×968.
onward, the parameter showed decreasing values until 2 months after hatch, when a slow recovery was observed (Table 1).
Quantification of Levels of IgM From Whole Trout Discussion Unfertilized eggs contained small amounts of IgM (11.2 ~g/g of egg), which did not increase significantly by 8 days post-fertilization. The amount of IgM/g of fry body weight increased from Vernier stage 26 until hatch, when a significant peak was reached. From this stage
Figure 1. Expression of cytoplasmic IgM. (A) Immunofluorescence staining of cytoplasmic IgM on a lymphoid cell from a pool of 50 trout embryos at Vernier stage 24 (12 days before hatch, at 14°C). IgM was detected with mAb 1-14 antitrout IgM. (B) Micrograph showing the same field observed under phase contrast microscopy. The arrow indicates the IgM + cell. ×990.
Using an immunofluorescent technique on cell suspensions from pooled trout embryos, IgM-bearing lymphocytes are present by 12 days before hatch (at 14°C). In a previous immunohistochemical study on cryostat sections (10) using the same monoclonal antibody anti-trout IgM, we reported that IgMbearing cells first appeared at day 4 after hatching in the kidney of rainbow trout. This difference in the appearance of IgM + cells may be attributable to the low number and scattered distribution of these cells in embryonic tissues. Thus, IgM + cells are more easily observed in cell suspensions of pooled embryos than in tissue sections. Our results indicated that lymphocytes showing cytoplasmic +, surfaceexpression of IgM (pre-B cells) appeared before surface IgM + (mature B cells) could be detected. This is in contrast to previous studies (15,16) describing the
422
simultaneous presence of cytoplasmic and surface IgM in immature B cells during fish ontogeny. However, polyclonal anti-IgM antisera were used in those studies, and the presumed lower affinities and specificities of these reagents may explain the different staining patterns. On the other hand, our data may be interpreted as agreeing with those reported for amphibians (17,18) and mammals (19). However, as the mAb 1-14 reacts only with trout p,-chains, and no antibodies were available for trout light chains, the observation of pre-B cells in trout has not been accomplished. Hence, the earliest ontogenetic origin of the B cell precursors in trout is still obscure. Although the adult fish pronephros seems to be the equivalent to mammalian bone marrow (10,20-22), the first IgM ÷ lymphocytes observed in this study appeared before the pronephros becomes lymphopoietic (9,10). In most vertebrates, the yolk sac and fetal liver seem to be major sites for early B-cell differentiation (17,23-26). Also, other embryonic tissues have been considered as a s o u r c e for l y m p h o c y t e p r e c u r s o r s (27,28). In fish, there is little information on the contribution of the yolk sac to hemopoiesis (29). Parenthetically, the yolk sac was not included in the present study, because it was impractical to obtain cell suspensions from this organ, due to the large amount of yolk droplets remaining in the cell suspensions. However, attempts to stain IgM + cells in frozen sections of the trout yolk sac were negative (Castillo, unpublished observations). On the other hand, the liver of embryonic or adult teleosts appear to have no hemopoietic capacities (7), nor IgM-bearing cells (Castillo, unpublished observations). Few studies have reported serum IgM levels during development of salmonids (30,31), and quantitative levels of IgM have only been investigated after hatching (32). In our results, unfertilized eggs
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contained low but detectable amounts of IgM. This fact was not seen in previous studies (32,33), which probably reflects the higher sensitivity of the ELISA assay with monoclonal antibodies. The presence of lgM in the unfertilized trout eggs indicates that vertical transmission of this molecule can occur in trout, as described in other teleosts (30,34). Embryonic levels of IgM/g of embryo remained similar to those of unfertilized eggs until the Vernier's stage 26, when surface IgM + cells (mature B cells) appeared. From this stage onward, this parameter progressively increased during embryonic development, reaching a peak at hatch. The appearance of B cells should correlate with the development of humoral immunity. However, our study cannot ascertain the source of the increased levels of IgM during embryonic development because the cells were disrupted by homogenization. Thus, the IgM observed may stem from cytoplasmic and membrane fragments of pre-B and B cells, rather than plasma cells. The greatly increased concentration of IgM at hatch is an intriguing phenomenon. It is of particular interest in that it is temporally next to the colonization of the pronephros by IgM + cells. As indicated above, the first IgM-bearing cells in the propnephros were detected at day 4 after hatching (10), but the colonization of this organ by presumed pre-B cells and some B cells may probably occur some time earlier. The interaction of such putative pre-B and B cells with a specific m i c r o e n v i r o n m e n t advantageous for their differentiation, might play an important role in the amplification of these populations, and subsequently be responsible for high levels of IgM. Alternatively, the high IgM tissue levels observed at hatch might also represent a nonspecific stimulation related to hatching, influenced by: An imbalance between the T-cell and B-cell compartments in the embryos; and/or the hot-
Ontogeny of IgM and IgM-bearing cells in rainbow trout
monal and stress factors involved in fish hatching (35), which in turn may dramatically influence immune function (3638). The decreasing levels of IgM after hatch may also be related to the increase in body weight after first feeding. In any case, the high IgM levels found at hatch may play a protective role, when fry are exposed to aquatic pathogens, at a developmental stage in which
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their lymphoid organs show poor organization (8-10). Acknowledgements--This research was supported by the Spanish CICYT Grant MAR88/ 0565. Cooperation between the University of Le6n (Dr. A. Villena) and the Oregon State University (Dr. S. Kaattari) was funded by a grant from the D.G.I.C.Y.T. (Ministerio Espafiol de Educacirn y Ciencia).
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