Developmental and Comparative Immunology, Vol. 17, pp. 419-424, 1993 0145-305X/93 $6.00 + .00 Printed in the USA. All rights reserved. Copyright © 1993 Pergamon Press 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 development of immunoglobulin M (IgM) and of IgM-bearing cells in the rainbow trout, On- corhynchus mykiss. Lymphocytes showing cy- toplasmic IgM were first observed in embryos at 12 days before hatch (14"C). At this stage, no cells positive for surface IgM were present. Lymphocytes bearing surface IgM were ob- served at 8 days before hatch (14"(;). Unfertil- ized trout eggs contained detectable amounts of IgM (11.2 - 2.6 i~g/g of egg weight), indicating that transfer of IgM from mother to embryo can occur in salmonids. The levels of IgM from whole fish increase slowly after the appearance of intraembryonic cells that express surface IgM. The amount of IgM/g of tissue peaks around hatch, but this parameter shows lower values up to 2 months after hatch.

salmonids (8,9), and the appearance of i m m u n o g l o b u l i n M (IgM) bear ing (IgM +) cells (10). Similar studies have been conducted in carp (5,11). However, there is little data delineating the differ- entiative pathways of T and B lympho- cytes from their hemopoietic precursors in fish.

Using mAbs specific for rainbow trout Oncorhynchus rnykiss se rum IgM (1,12,13), we describe the appearance of cells bearing cytoplasmic or surface IgM, as well as the levels of IgM/g of body weight, at different stages of development.

F1Keywords--Immunoglobulin M (IgM); B-cells; Immunocompetence; Ontogeny; Trout; Teleost.

Introduct ion

Monoclonal antibodies (mAbs) specific for fish immunoglobulins (I-4), and leu- cocyte 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 differentia- tion of fish lymphoid cells (7). Several studies have described the morphogene- sis of the main lymphoid organs in

Address correspondence to Dr. Alberto J. Villena.

Materials and Methods

Animals

Fertilized and unfertilized rainbow trout eggs were obtained from a fish farm near Lern (Spain), which is supplied with pathogen-free well water. Incuba- tion of fertilized eggs and maintenance of fry were performed in departmental aquaria supplied with running dechlori- nated water at 14 +-- I°C, and maintained with a photoperiod of 12 h light/12 h dark. Under these environmental condi- tions, trout eggs hatched at 26-27 days after fertilization. Developmental stages were determined according to the Ver- nier table of trout development (14). The co r r e spondence be tween 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 sus- pensions were obtained by repeated gen- tle flushing of the pooled fish through a 10-mL syringe. Cell suspensions were al- iquoted, twice washed and resuspended, at 106 lymphoid cell/50 txL in PBS con- taining 20 mM NAN3.

Staining of cytoplasmic IgM ÷ cells was performed after fixation with 1%

Table 1. Levels of IgM/g of Body Weight During Development of Rainbow Trout.

N u m b e r o f S tage o f Samp les ixg IgM/g~t

D e v e l o p m e n t * (n)l" (Mean -+ SD)

Unfer t . eggs 4 (50) 11.2 -+ 2.6 a 8 days

pos t - fe r t i l i za t ion 4 (30) 14.7 -+ 1.6 a 9 d.b.h. (St. 26) 10 (30) 25.7 +- 5.8 b 4 d.b.h. (St. 28) 9 (30) 39.0 -+ 10.8 b Hatch (St. 30) 10 (10) 153.1 - 40.6 c 2 d.a.h. 9 (10) 102.6 -+ 41.0 ° 4 d.a.h. (St. 32) 9 (10) 30.8 + 18.7 b 7 d.a.h. 5 (10) 49.1 +- 12.4 b 14 d.a.h. (St, 34) 10 (10) 11.5 -+ 4.3 b 21 d.a.h. (St. 35) 10 (10) 13.9 -+ 3.9 b 30 d.a.h. 7 (10) 6.6 -+ 1.6 b 45 d.a.h. 5 (4) 10.1 -+ 3.1 ~ 60 d.a.h. 4 (4) 14.7 +- 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 val- ues 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,

paraformaldehyde (15 min on ice). Sur- face 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 De- Luca, Wilson, and Warr (1). As second- ary antibody a rabbit anti-mouse IgG an- tiserum conjugated to fluorescein (Dako) was used. Several dilutions of the pri- mary and secondary antibodies were tested to optimize results. The stained cell suspensions were mixed with equal volumes of PBS-glycerol (1:9) and mounted on slides. Controls involved omission of the primary antibody. Fluo- rescence was observed under a Nikon Optiphot microscope provided with epi- fluorescent equipment and a 100-W halo- gen tungsten lamp.

Quantification of IgM

Measurements of the IgM tissue levels were performed with homogenized sam- ples of unfertilized eggs, embryos, and fry. As indicated in Table 1, 4-10 sam- ples were used for each stage analyzed. Before homogenizat ion, unferti l ized 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 ho- mogenized in cold PBS by the use of a tissue grinder (Braun). Tissues were di- luted in PBS and centrifuged (20 min at 700×g, 4°C). The supernatants were col- lected, aliquoted, and kept at -80°C un- til use.

The concentrations of IgM in the sam- ples were determined by an ELISA pro- cedure, 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 ex- pressed 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 ob- served 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 + lympho- cytes were first observed in cell suspen- sions from embryos at Vernier stage 26 (8 days before hatch, Fig. 2).

Quantification of Levels of IgM From Whole Trout

Unfert i l ized 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 Ver- nier stage 26 until hatch, when a signifi- cant peak was reached. From this stage

Figure 1. Expression of cytoplasmic IgM. (A) Im- munofluorescence staining of cytoplasmic IgM on a lymphoid cell from a pool of 50 trout em- bryos at Vernier stage 24 (12 days before hatch, at 14°C). IgM was detected with mAb 1-14 anti- trout IgM. (B) Micrograph showing the same field observed under phase contrast micros- copy. The arrow indicates the IgM + cell. ×990.

Figure 2. Expression of surface IgM. (A) Micro- graph showing three small lymphocytes ex- pressing 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 decreas- ing values until 2 months after hatch, when a slow recovery was observed (Ta- ble 1).

Discussion

Using an immunofluorescent tech- nique on cell suspensions from pooled trout embryos, IgM-bearing lympho- cytes are present by 12 days before hatch (at 14°C). In a previous immunohisto- chemical study on cryostat sections (10) using the same monoclonal antibody anti-trout IgM, we reported that IgM- bearing 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 lympho- cytes showing cytoplasmic +, surface- expression 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 A. Castillo et al.

simultaneous presence of cytoplasmic and surface IgM in immature B cells dur- ing fish ontogeny. However, polyclonal anti-IgM antisera were used in those studies, and the presumed lower affini- ties and specificities of these reagents may explain the different staining pat- terns. On the other hand, our data may be interpreted as agreeing with those re- ported for amphibians (17,18) and mam- mals (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 ap- peared before the pronephros becomes lymphopoietic (9,10). In most verte- brates, the yolk sac and fetal liver seem to be major sites for early B-cell differ- entiation (17,23-26). Also, other embry- onic tissues have been considered as a source for l y m p h o c y t e p recurso r s (27,28). In fish, there is little information on the contribution of the yolk sac to he- mopoiesis (29). Parenthetically, the yolk sac was not included in the present study, because it was impractical to ob- tain cell suspensions from this organ, due to the large amount of yolk droplets remaining in the cell suspensions. How- ever, attempts to stain IgM + cells in fro- zen sections of the trout yolk sac were negative (Castillo, unpublished observa- tions). 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 hatch- ing (32). In our results, unfertilized eggs

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 pres- ence of lgM in the unfertilized trout eggs indicates that vertical transmission of this molecule can occur in trout, as de- scribed 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) ap- peared. From this stage onward, this pa- rameter 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 in- creased levels of IgM during embryonic development because the cells were dis- rupted by homogenization. Thus, the IgM observed may stem from cytoplas- mic and membrane fragments of pre-B and B cells, rather than plasma cells.

The greatly increased concentration of IgM at hatch is an intriguing phenom- enon. It is of particular interest in that it is temporally next to the colonization of the pronephros by IgM + cells. As indi- cated 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 microenvironment advanta- geous for their differentiation, might play an important role in the amplification of these populations, and subsequently be responsible for high levels of IgM. Alter- natively, the high IgM tissue levels ob- served at hatch might also represent a nonspecific stimulation related to hatch- ing, influenced by: An imbalance be- tween the T-cell and B-cell compart- ments in the embryos; and/or the hot-

Ontogeny of IgM and IgM-bearing cells in rainbow trout 423

monal and stress factors involved in fish hatching (35), which in turn may dramat- ically influence immune function (36- 38). 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 patho- gens, at a developmental stage in which

their lymphoid organs show poor organi- zation (8-10).

Acknowledgements--This research was sup- ported 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 Es- pafiol de Educacirn y Ciencia).

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