Aq~ucu~ture, 67 (1987) 147-155 Elsevier Science Publishem B.V., Amsterdam - Printed in The Netherlands

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An Investigation of the Biliary and Intestinal Immunoglobulin and the Plasma Cell Distribution in the Gall Bladder and Liver of the Common Dogfish, Scyliorhinus canicula L.

S. HART, A.B. ~AT~~~~~, T.A. DOGGETT and J.E. HARRIS

De~a~~~e~~ of ~i~~~g~&ul Sciences, Plymouth polytechnic, Drake Circus, ~lymQuth, Devon PU 8AA (Great Britain)

(Accepted 22 January 1987)

ABSTRACT

Hart, S., Wrathmell, A.B., Doggett, T.A. and Harris, J.E., 1987. An investigation of the biliary and intestinal immunoglobulin and the plasma cell distribution in the gall bladder and liver of the common dogfish, Scyliorhinus can&la L. Aquaculture, 67: 147-155.

Local immune mechanisms were investigated in the got and gall bladder ofthe common dogfish. Administration of sonicated Vibrio anguillarum bacterin and whole sheep red blood cells (SRBC) by the oral and anal routes elicited the production of low titres of specific antibodies in the bile but not in the serum. Parenteral administration of the V. angui~~arum sonicate and SRBC elicited a strong systemic response and specific biliary antibodies were detected at higher titres than when antigens were introduced into the alimentary tract. Immunoglobulin was detected in the bile and intestine and was comparable in size to serum imm~no~~obulin. However, they had different elec- trophoretic mobilities and preliminary characterisation revealed some differences in the heavy and light chains. The origin of the bile and intestinal immunoglobulin was not elucidated although it is likely that the former contributes to the latter. A few plasma cells were found in the gall bladder and none in the liver.

INTRODUCTION

The gut of humans is equipped with a panoply of immunological and non- immunological components which work in concert to prevent the attachment, penetration and subsequent infection of this mucosal barrier (Walker, 1985). In both humans and other mammals specific adaptive immunity at mucosal surfaces is mediated mainly by polymeric IgA which enters the alimentary tract directly across the gut mucosa or is secreted by the liver into the bile (Vaerman et al., 1982). The duck, Anus ~~~~y~~y~~~os, however, has IgM as the major biliary immunoglobulin (Ng and Higgins, 1986). Although little work

0044-8486/87/$03.50 0 1987 Elsevier Science Publishers B.V.

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has been undertaken on local immunity in the gut of fishes, it seems probable that adaptive immunity is also mediated by immunoglobulin.

Immunoglobuli~ has been detected in the gut mucus of teleost fish (Brad- shaw et al., 1971; Di Conza and Halliday, 1971). Specific antibodies have been elicited in the gut by peroral administration (Fletcher and White, 1973) and demonstrated against natural infections of an intestinal helminth (Harris, 1972). Immunoglobulin has also been detected in the bile of carp, Cyprinus carpio L. ( Lamers, 1985)) and in the sheepshead, Archosargusprobatocephalus ( Lobb and Clem, 1981a, b) . In the latter species, the biliary immunoglobulin was antigenically similar to the serum immunoglobulin, although the biliary immunoglobulin existed as a dimer (320 000 daltons) in physiological buffers and the molecular weight of the heavy chain of biliary and serum immuno- globulins differed, which led the authors to postulate that this may indicate the existence of a specialised local immunoglobulin.

No comparable studies have been undertaken on non-teleost fish although immunoglobulin-containing plasma cells have been detected in the gut of lam- prey ammo~oetes (Fuji, 1982)) the skate, Raja ~enojei (Tomonaga et al., 1984) and in the dogfishes, Scy~iorhinus canicula (Hart et al., 1986a) and S. torazame (Tomonaga et al., 1986). Lymphoid aggregates or accumulations were dem- onstrated in the gut of Scyliorhinus by both groups of investigators.

MATERIALS AND METHODS

Fish

Dogfish of either sex (0.75-1.5 kg) were obtained from the Marine Biolog- ical Association, Plymouth, Great Britain, kept in recirculating seawater at 11-13” C. Blood collection has been previously described (Parish et al., 1986) and bile was aspirated from the gall bladder of freshly killed fish and dialysed against elasmobranch saline (Hale, 1965 ) .

Serology

Fish were exposed to 10’ formalin-killed, sonicated Vibrio anguillarum and 10’ SRBC by oral and anal intubation and intraperitoneal (IP) injection at weekly intervals for 1 month. The antibody levels in serum and bile were meas- ured 60 days after the final administration of antigen by direct agglutination. The second protocol, in which fish were immunized with SRBC as above, was followed by an IP injection of lo9 SRBC and an equal volume of Freunds com- plete adjuvant (FCA) some 60 days later. Fish were examined after a further 60 days.

Antiserum to whole dogfish serum and to dogfish IgM and subsequent im- munoelectrophoresis has been previously described (Hart et al., 1986c).

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Protein sepuru~ion

The IgM-containing fraction of dogfish anti-SRBC serum was separated by gel filtration on a sepharose 6B column and by agarose block electrophoresis ( Morrow et al., 1983 ) . Biliary immunoglobulin-containing fractions were ex- amined by immunoelectrophoresis with a rabbit antiserum to dogfish IgM.

Polyu~~lamide gel e~~~rop~ores~s (PAGE)

Two-layer PAGE was performed according to Laemmli (1970) using a 13% separating and a 4.5% stacking gel. Samples were prepared by boiling in an equal volume of SDS and 2-mercaptopethanol loading buffer and 20-40 ,~l sam- ples were run for 30 min at 20 mA and then at 30 mA until the run was com- plete. Appropriate marker molecules were separated simultaneously and the gels finally stained in Coomassie brilliant blue, R.

One-layer PAGE was used to analyse unreduced immunoglobulin. A 3% gel was cast with 1% agarose to provide rigidity. They were loaded, run and stained as above.

Histology

Appropriate samples of liver and gall bladder were examined histolo~cally as previously described (Hart et al., 1986a).

RESULTS

Humoral response

Biliary antibodies against V. ung~~ZZarum and SRBC were detected after per- oral and peranal exposure to these antigens (Table 1) , no systemic response was elicited. Parenteral challenge of both antigens elicited a systemic and bil- iary response, the latter being more significant than that occurring after intro- duction of antigens in the alimentary tract. Injection of SRBC with adjuvants enhanced the agglutinin titre in both bile and serum irrespective of the initial mode of exposure.

distribution of immunog~b~~in

I_Jsing the rabbit antisera, immunoglobulin was detected in the bile and in- testine but not in the anterior region of the gut (Fig. 1). The immunoglobulin was the only protein detected in the bile with the rabbit anti-whole dogfish serum (Fig. 2a), compared to the number of proteins detected in whole serum (Fig. 2b).

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TABLE 1

Agglutination titres of dogfish serum and bile to orally, anally and intraperitoneally administered Vibrio and SRBC antigens”

Route of administration Antigen

Vibrio

Serum Bile

SRBC

Serum Bile

SRBCj’FCA

Serum Bile

Oral Anal Intraperitoneal (IP)

I/4 - l/2 l/512 l/256 l/8 - l/2 l/l024 l/256

l/512 l/16 l/1024 l/16 l/4096 l/256

‘Details of antigen administration regimes are given in the Materials and Methods.

CARDIAC

OESOPHAOUS SToMACH SPLEEN

BUCCAL CAVITY

STOMACH SPIRAL INTESTlNE

RECTUM

Fig. 1. Diagrammatic representation of the major regions of the alimentary canal of the common dogfish.

Isolation, partial characterization and comparison of serum and biliary immunoglobulin

Serum and biliary immunoglobulins were the first proteins from serum and bile, respectively, to be eluted by gel filtration. The fraction containing serum immunoglobulin also contained a protein with a fast cathodic migration, which was removed by agarose block electrophoresis. Bile contained only a single serum protein, i.e., immunoglobulin, and after collection of the immunoglob- ulin-containing fraction from the column no further purification was neces- sary. After concentration by dialysis in Aquacide (Morrow et al., 1983) and by Amicon microconcentration (Danvers, MA) immunoglobulins were compared.

(1 ,I ~oZe~u~ar weight. Samples migrated an equally short distance into a 3% I?AGE gel suggesting the proteins were of similar molecular weight.

(2) Electrophoretic mobility. Bile Ig migrated cathodically (Fig. 2a) whereas serum had a bimigrational movement ( Fig. 2c ) .

(3) M. W. of Eight CL) and heavy (H) chains. Analysis of the constituent chains

_ ,.. “; ” -.

‘;’ 1 ..J., / _ -/

Pig. 2. (a) Immunoelectrophoreto~am of dogfish bile against rabbit anti-whole dogfish serum. (b) Immunoelectrophoreto~am of whole dogfish serum against rabbit anti-whole dogfish serum. (c ) Immunoele~trophoreto~am of whole dogfish serum against rabbit anti-dogfish serum IgM.

Fig. 3. Electrophoreto~am of immunoglobulin separation on 13% PAGE. B, Bile; S, serum; H, heavy chain; L, light chain; Kd, kilodaltons.

of bile and serum immunoglobulin revealed that the L chain of bile Ig and H chain of serum Ig appeared to migrate as more than one band (Fig. 3 ) .

Intraepithelial leucocytes (Fig. 4 ) and intralaminal plasma cells (Fig. 5 )

Fig. 4. Intraepithelial cells in the gall bladder of the dogfish (scale bar = 10 pm). IEL, Intraepi- thelial leucocyte; E, epithelial cell; C, cilia.

Fig. 5. Plasma cells in the lamina propria of the dogfish gall bladder (scale bar = 10 m) . P, Plasma cell.

were detected in the gall bladder. Leucocytes were, however, absent from the liver.

DISCUSSION

Bile was found to contain ~mmunoglobulin and specific antibodies and was much easier to collect and handle than intestinal mucus. The bile, however,

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had to be initially dialysed against elasmobranch saline to remove detergent activity.

Peroral and peranal administration of the Vibrio and SRBC antigens elicited a biliary response, but did not stimulate the production of serum antibodies. This may be evidence of a local immune response in the gut, possibly mediated by the gut-associated lymphoid tissue recently described in S. canicuEa (Hart et al., 1986a, b, c) and S. torture (Tomonaga et al., 1986). The latter authors suggested that the lymphoid accumulations detected in the intestine of elas- mobranchs may represent a precursor of mammalian Peyers patches. The ab- sence of a systemic response may, however, have been influenced by the choice of antigen: in teleosts whole soluble protein antigens are rapidly absorbed by the gut and released into the circulation (McLean and Ash, 1986) while par- ticulate antigens appear not to move further than the mucosa (Nelson et al., 1985). As evidence derived from teleosts cannot necessarily be applied to elas- mobranchs, a study of antigen processing by the gut and especially the role of the lymphoid accumulations in the spiral valve is needed.

The demonstration that IP injections of Vibrio and SRBC elicited a stronger biliary response indicates that antigens do not necessarily have to cross the gut epithelium to elicit the production of biliary Ig. A similar phenomenon was recognised in rats following intravenous administration of K cholercze antigens (Jackson and Cooper, 1981),

When examined by immunoelectrophoresis with a rabbit antiserum to dog- fish IgM a stronger reaction was seen in the bile than in the intestinal mucus indicating that more IgM was present in the former. The origin of the biliary immunoglobulin is unclear, most plasma cells are found in the spiral valve and in this study none was found in the liver and few in the gall bladder mucosa. It is possible that some of the biliary Ig may be derived from the serum in a manner similar to mammals (Vaerman et al., 1982). Lobb and Clem (1981b) proposed that the biliary Ig of the teleost sheepshead was not derived from the serum but from local sources of production; the liver or gall bladder was not examined immunohistochemically in this study.

The mechanism(s) by which Ig enters the gall bladder or intestine of Scy- Ziorhinus is unclear. The presence of Ig as the only serum protein in the bile indicates that a specific secretory mechanism probably exists. A secretory pro- tein component was not found in the bile of A~cho~urg~ (Lobb and Clem, 1981a) but may be present in the cutaneous mucus (Lobb and Clem, 1981c),

When biliary and serum Ig were compared, the molecular weight of the two proteins appeared to be the same, unlike Dhe sheepshead which had a dimeric form of Ig in the bile (Lobb and Clem, Y981a). It is unclear, in the dogfish, whether the differences in electrophoretic mobility and heterogeneity in light and heavy chain molecular weights represent the characteristics of two sepa- rate immunoglobulin populations. Lobb and Clem (1981a) proposed that the biliary immunoglobulin of the sheepshead was indeed a separate population.

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More recently, one of these authors ( Lobb, 1986) proposed that phylogenetic ~vers~~cation of immunoglobulin occurred in the teleosts and Kobayashi et al. (1985) and Tomonaga et al. (1986) have established that there are two populations of immunoglobulin producing cells in the skates.

Evidence is now beginning to accumulate to suggest that adaptive immunity may be mediated by immunoglobulins in the gut of fish. The degree of auton- omy from the systemic system is unclear as are the precise functions of intes- tinal immunoglobulin.

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