Fish & Shellfish Immunology (1997) 7, 235–245

Immunohistochemistry of gut-associated lymphoid tissueof the sea bass Dicentrarchus labrax (L.)

LUIGI ABELLI*, SIMONA PICCHIETTI, NICLA ROMANO, LUCIA MASTROLIA AND

GIUSEPPE SCAPIGLIATI

Department of Environmental Sciences, Faculty of Sciences,Tuscia University, Viterbo 01100, Italy

(Received 20 August 1996, accepted in revised form 9 December 1996)

Histology revealed scattered lymphoid cells at all levels of the digestive tractof the sea bass Dicentrarchus labrax (L.), notably in the mucosa and sub-mucosa. Immunohistochemistry with monoclonal antibodies against T cells(DLT15) and immunoglobulin-bearing cells (DLIg3 and DLIg13) demonstratedthe presence of a gut-associated lymphoid tissue. Quantitative analysisshowed that the concentration of DLT15-positive cells largely exceeded that ofIg-bearing cells, while the number and the distribution of DLIg3- and DLIg13-immunoreactive cells were superimposable. An apparent gradient in thenumber of lymphoid cells was present, concentrating them towards the anus.This suggests that, as in other fish species, the posterior gut acquired higherimmunological relevance. ? 1997 Academic Press Limited

Key words: gut-associated lymphoid tissue (GALT), T-lymphocytes,immunoglobulins (Ig), monoclonal antibodies (MAbs), fish,Dicentrarchus labrax.

I. Introduction

The mucosal surfaces of fish skin, gills and gut are protected by both humoraland cellular mechanisms (Peleteiro & Richards, 1985; Hart et al., 1988; Rowleyet al., 1988; Roberts, 1989). Leucocytes occur in all parts of the teleost digestivesystem, most extensively in the intestine, where lymphocytes, plasma cells,granulocytes and macrophages are present in and under the epithelium.Although large lymphoid centres are lacking, many lymphoid cells, eitherscattered or in small groups, were reported to be present in the epithelium andlamina propria (Zapata, 1979; Davina et al., 1980; Temkin & McMillan, 1986;Hart et al., 1988).Various studies indicated that regional di#erences occur in organisation of

the gut-associated lymphoid tissue (GALT) at di#erent levels of the digestivetract, possibly reflecting specialised immunological functions. Uptake andtransport of antigens have been shown to occur mainly in the second gutsegment of carp, Cyprinus carpio L. (Rombout et al., 1985, 1986; Rombout &Van den Berg, 1989), and trout, Oncorhynchus mykiss (Georgopoulou et al.,

*Author to whom all correspondence should be addressed at: Department of EnvironmentalSciences, Faculty of Sciences, Tuscia University, Via San Camillo de Lellis, block D, 01100Viterbo, Italy.

2351050-4648/97/040235+11 $25.00/0/fi960079 ? 1997 Academic Press Limited

236 L. ABELLI ET AL.

1986), where antigens are transported from the lumen to large intraepithelialmacrophages, which can exert an antigen-presenting function (Rombout et al.,1985, 1986). The GALT of carp has been studied with monoclonal antibodies(MAbs) raised against carp immunoglobulins (Ig) or leucocytes, whichrevealed the presence of putative B and T cells, plasma cells and Ig-bindingmacrophages; i.e. all cells necessary for a local or mucosal immune response(Rombout et al., 1993a).Recently, MAbs have been produced and characterised that recognise the

light chain (DLIg3) and the heavy chain (DLIg13), respectively, of sea bassserum Ig (Scapigliati et al., 1996), as well as MAb DLT15, recognisingantigenic determinants expressed by the majority of thymocytes and byperipheral T cells (Scapigliati et al., 1995; Abelli et al., 1996). This panel ofMAbs was employed in the present work to define: (i) the existence of GALT inthe sea bass Dicentrarchus labrax (L.); (ii) variations in the GALT at di#erentlevels of the digestive system; and (iii) the relative abundance of Ig-bearingcells and Ig-negative lymphoid elements throughout the digestive system.

II. Materials and Methods

HISTOLOGY AND IMMUNOHISTOCHEMISTRY

Two-year-old sea bass (N=10) were killed with tricaine methanesulphonate(1 mg ml"1) and di#erent segments of the digestive system dissected asillustrated in a schematic drawing (Fig. 1). Considering the big cranio-caudalextension of the stomach fundus, a descending anterior portion (length,10 mm) and a caecal portion (length, 8 mm) were sampled. Di#erent segmentsof the intestine were taken as follows: anterior intestine at the opening of bileduct (length, 10 mm), middle intestine (length, 10 mm) and posterior intestine(length, 10 mm) 5 mm away from the anus.

Fig. 1. Schematic drawing of digestive system of the sea bass (ventral view). Areassampled for histology and immunohistochemistry are numbered as follows: 1–2,anterior and caecal portions of the stomach, 3–5, anterior, middle and posteriorintestine. Broken lines show the liver, while the gall bladder and spleen aresketched. Bar=1 cm.

GALT OF THE SEA BASS 237

Bouin’s fixative and para$n embedding were utilised for histology. Serialsections of 7 ìm thickness were stained with haematoxylin and eosin, May-Grünwald/Giemsa (Pappenheim method) and Mallory’s trichrome.Bouin’s fixative (7 h at 4) C) and para$n embedding were utilised for

immunohistochemistry, as previously described (Scapigliati et al., 1995). Serialtransverse sections (7 ìm) were incubated for 18 h at 25) C with the MAbDLT15 (diluted 1:10 in phosphate-bu#ered saline (PBS) containing 5% normalhorse serum, 5% normal sea bass serum and 0·1% sodium azide) or the MAbsDLIg3 and DLIg13 (undiluted culture media). Normal mouse serum or themyeloma culture medium substituted for primary antibodies in controlsections.Following rinses in PBS, sections were incubated for 1 h at 25) C with

biotinylated horse anti-mouse IgG serum (Vector Labs., Burlingame, U.S.A.)diluted 1:1000 with PBS containing 0·1% sodium azide and 1% bovine serumalbumin, followed by incubation for 1 h with avidin-biotinylated peroxidasecomplex (ABC, Vectastain> Elite, Vector) with the avidin and biotinylatedhorseradish peroxidase solutions diluted 1:2000 in Tris-bu#ered saline (TBS;0·05 M, pH 7·6). Following rinses in TBS, sections were incubated in 0·05 M Trisbu#er containing 0·4% nickel ammonium sulphate, 0·02% diaminobenzidineand 0·015% hydrogen peroxide. After rinsing in Tris bu#er, sections weredehydrated, mounted and examined under bright-field illumination.In each animal, four sets of three consecutive sections (12 sections per

tissue) of anterior and caecal stomach, and di#erent levels of the intestinewere di#erentially immunostained with DLT15, DLIg3 and DLIg13, respect-ively. Counts of immunoreactive cells (nucleated only) in 10 000 ìm2 areas ofmucosa and submucosa (N=10 per section) were performed with a computer-assisted image analysis system by an observer unaware of treatments. Esti-mates of the number of immunoreactive cells present in the di#erent tissueswere then calculated by averaging the cell numbers from three animals, andcalculating the mean and standard error of the mean.Cell measurements were performed in three animals and pooled to obtain

cumulative values of the mean and standard error of the mean. Homogeneityof variances was tested before data processing and numerical results wereanalysed by one-way ANOVA test followed by Tukey test.

III. Results

HISTOLOGY AND IMMUNOHISTOCHEMISTRY OF DIGESTIVE SYSTEM

Immunohistochemistry allowed the localisation of cells recognised byDLT15, DLIg3 and DLIg13, respectively, to be studied in descending (anterior)and caecal fundus of the stomach [Fig. 2(a) & (b)] and di#erent levels of theintestine [Fig. 2(c) & (d) and Fig. 3]. Scattered lymphoid cells were found at alllevels of the digestive system, notably in the mucosa (epithelium and laminapropria) and submucosa. The number of positive cells is shown in Table 1.The anterior portion of the stomach was rectilinear and large mucosal folds

almost filled the lumen. The sacciform caecal portion had lesser extension ofmucosal folds and conspicuous smooth muscle. The mucosa of the fundus was

238 L. ABELLI ET AL.

formed of both columnar epithelial cells and secretory cells organised intolong tubular glands. Numerous lymphoid cells were present in the mucosa andsubmucosa; in the latter, a significant number of eosinophilic granulocytesalso occurred. DLT15-immunoreactive cells were inside the mucosa andsubmucosa of anterior [Fig. 2(a)] and caecal portion of the stomach fundus[Fig. 2(b)]. Their number did not change significantly at the two segmentallevels, and it was similar in mucosa and submucosa (Table 1). Ig-bearing cellswere found occasionally in the mucosa (epithelium and glands) and muscularlayer. Very infrequent isolated cells were in the submucosa.The anterior intestine had a larger diameter than the parts which followed.

The mucosa formed large folds which almost filled the lumen and it housednumerous scattered lymphoid cells and some granulocytes. The epitheliumwas of the simple columnar type, with a conspicuous brush border and withmucous cells. DLT15-immunoreactive cells were very numerous between

Fig. 2. Immunohistochemistry of GALT in the stomach and anterior intestine. DLT15immunoreactive cells (arrows) were in the mucosa of anterior stomach (a). Theasterisk indicates the lumen. Bar=10 ìm. DLT15 positive cells around a blood vesselof the submucosa are shown in the inset. Bar=20 ìm. DLT15 immunoreactive cells(arrows) were in the mucosa of caecal stomach fundus (b). Asterisk and bar asbefore. Scattered positive cells in the submucosa are shown in the inset. Bar=50 ìm.Interferential contrast. Numerous DLT15 immunoreactive cells were in the baso-lateral epithelium and lamina propria (lp) of the anterior intestine (c), whileinfrequent positive cells were detected by DLIg3 immunostaining (d). Asterisksagain indicate the lumen. Bar=50 ìm.

GALT OF THE SEA BASS 239

epithelial cells, mainly localised in the basolateral epithelium, and in thelamina propria [Fig. 2(c)]. Scarce cells were in the submucosa and muscularlayers. Ig-bearing cells were infrequent in the mucosa [Fig. 2(d)] and veryscarce in the submucosa and muscular layers.The mucosa of the middle intestine was less folded, and the lamina propria

was filled with many scattered cells, mainly lymphocytes, infrequentbasophilic granulocytes and some eosinophilic granulocytes, which alsooccurred intraepithelially. Very numerous DLT15-immunoreactive cells(diameter 3·1&0·1 ìm, range 2·7–3·9 ìm, N=100) were in the basolateral

Fig. 3. Immunohistochemistry of GALT in the middle and posterior segments of theintestine. Numerous DLT15-immunoreactive cells were in the mucosa of middleintestine: in the basolateral epithelium, intraepithelially and in the lamina propria(a). Sections immunostained with DLIg3 (b) and DLIg13 (c), respectively, show thatthe GALT contained low numbers of Ig-bearing cells, while it was mostly composedof Ig-negative cells recognised by the MAb DLT15. The mucosa of posterior intestinewas densely infiltrated by DLT15-immunoreactive cells (d), while only scatteredDLIg3 positive cells were detected (e). Interferential contrast; bars=20 ìm.

Table1.Relativedensity

(numberof

cells/10000

ìm2)of

DLT15-andIg-immunoreactivecellsin

sectionsfrom

di#erentsegm

entsof

digestivesystem

oftheseabass

MAb

Segmentsofdigestivesystem

Stomachfundus

Intestine

Anterior

Caecal

Anterior

Middle

Posterior

Mucosa

Subm

ucosa

Mucosa

Subm

ucosa

Mucosa

Subm

ucosa

Mucosa

Subm

ucosa

Mucosa

Subm

ucosa

DLT15

12·9

&1·7

6·0&

0·6

12·3

&1·7

14·7

&1·4

32·8

&3·1a

1·0&

0·6*

54·5

&9·3b

4·0&

0·4*

62·3

&8·2b

7·5&

0·9*

DLIg3

0·6&

0·2

<0·01

0·8&0·2

<0·01

1·5&0·4c

<0·01*

0·9&

0·2c

<0·01

8·3&

1·2c

<0·01*

DLIg13

0·3&

0·1

<0·01

1·1&0·3

<0·01*

1·7&0·4c

<0·01*

2·3&

0·6c

<0·01*

9·7&

1·8c

<0·01*

Eachnumberisthemean

&S.E.M.ofresultsobtained

inthreeanimals.

aSignificantlydi

#erentfrom

mucosaoftheanteriorandcaecalstom

achfundus(P<0·01).

bSignificantlydi

#erentfrom

mucosaofanteriorandcaecalstom

achfundus,andanteriorintestine(P<0·01).

cSignificantlydi

#erentfrom

DLT15-immunoreactivecells(P<0·01).

*Significantlydi

#erentfrom

themucosa(P<0·01).

GALT OF THE SEA BASS 241

epithelium and in the lamina propria [Fig. 3(a)]. Their number decreased inthe submucosa, where they were around blood vessels, and it was scarce in thetunica muscularis. Both DLIg3- (diameter 5·3&0·2 ìm, range 3·4–7·5 ìm,N=100) and DLIg13-immunoreactive cells (diameter 4·7&0·3 ìm, range 3·2–8·3 ìm, N=100) were infrequent in the mucosa [Figs 3(b) & (c)] and very scarcein the submucosa, inside blood vessels only.The posterior segment of the intestine was straight, the filiform mucosa was

extensively folded and the epithelium contained a large number of mucouscells. The mucosa housed many lymphoid cells and very scarce eosinophilicgranulocytes. DLT15-immunoreactive cells (diameter 4·1&0·2 ìm, range 2·7–5·4 ìm, N=100) were very numerous between epithelial cells and in the laminapropria [Fig. 3(d)]. Although less numerous, positive cells were found in thesubmucosa. Ig-bearing cells (diameter 4·1&0·2 ìm, range 3·2–5·5 ìm, N=100)were in significant number both intraepithelially and in the lamina propria[Fig. 3(e)]. Very scarce cells were in the submucosa, inside blood vessels only.The number of DLT15-positive cells was higher in the mucosa than in the

submucosa at all levels of the intestine (P<0·01) and largely exceeded that ofIg-bearing cells (P<0·01) (Table 1). The relative density of DLT15-positive cellsin the mucosa of middle and posterior intestine was not di#erent, while it wassignificantly higher compared with upper levels of the digestive tract (Table1). In each segment analysed, Ig-bearing cells recognised either by DLIg3 orDLIg13 had similar distribution and concentration. A gradually increasingnumber of Ig-bearing cells was established from the anterior to the posteriorpart of the gut (Table 1).

IV. Discussion

The development of MAbs against homologous Ig and Ig-bearing cells inconjunction with in vitro assays have proven that teleosts possess functionalequivalents of T and B lymphocytes (Sizemore et al., 1984; Clem et al., 1985,1991). However, the lack of specific antigen markers has not allowed un-equivocal identification of T cell (sub)populations, thus dampening knowledgeabout their ontogeny and distribution.Recently a MAb (DLT15) recognising antigenic determinants expressed by

the majority of thymocytes and by peripheral T cells of the sea bass has beenproduced and characterised (Scapigliati et al., 1995). DLT15 allowed identifi-cation of T cells in head kidney, spleen and blood of juvenile fish and detectionof antigenic determinants in developing lymphoid organs (Scapigliati et al.,1995; Abelli et al., 1996). The MAb DLT15 in conjunction with MAbs (DLIg3and DLIg13) against serum Ig of the sea bass (Scapigliati et al., 1996) were usedin this study to prove the existence of a GALT in this species and to definethe distribution and concentration in the GALT of identifiable lymphocytesubpopulations.Histology revealed many scattered lymphoid cells at all levels of digestive

tract of the sea bass, whereas no aggregates were found, as in other fishspecies (Zapata et al., 1979; Davina et al., 1980; Temkin &McMillan, 1986; Hartet al., 1988). The existence of a GALT, notably present in mucosa andsubmucosa of digestive tube, was demonstrated by immunohistochemistry.

242 L. ABELLI ET AL.

The intestine was significantly enriched with DLT15-positive cells comparedwith upper gut levels, and a gradient was apparently present to concentratethe GALT towards the anus. This arrangement is reminiscent of that pre-viously described in other fish species (Iida & Yamamoto, 1985; Georgopoulou& Vernier, 1986; Fujino et al., 1987; Rombout & Van den Berg, 1989), where thesecond gut segment acquired the most important immunological function. Thepinocytotic capability of enterocytes in the second gut segment of teleostsresembles that of mammalian M cells, allowing rapid release of macromol-ecules in the intercellular space, where they may contact antigen-processingcells and lymphoid cells (Rombout & Van den Berg, 1989).The gut immune system has to protect the epithelium from infection and

prevent penetration by pathogens. At the same time, over-reaction againstfood antigens and normal microbial flora must be avoided. Intraepitheliallymphocytes (IEL) present in the gut epithelium probably play important rolesin these phenomena. Given the di#erent conditions prevailing along thedigestive tract, it is conceivable that IEL have to meet special demands atdi#erent levels of the intestine. Uptake of particulate antigens (Vibrioanguillarum bacterin) could be detected in the posterior intestine of sea bassafter oral or anal administration (Vigneulle & Baudin-Laurencin, 1981).Immunohistochemistry showed that the vast majority of lymphoid cells in

the sea bass GALT were T cells, recognised by the MAb DLT15. Theimmunostaining was seen on the cell membrane of most cells, and membranelocalisation of antigenic determinants recognised by DLT15 was confirmed byindirect immunofluorescence, FACS and pre-embedding immuno-electronmicroscopy (unpublished data). However, the presence of immunoreactivemolecules within the cytoplasm cannot be excluded, and therefore post-embedding studies are in progress to define the subcellular localisation ofDLT15 immunoreaction.Immunohistochemical studies in carp also revealed that more than 90% of

gut leucocytes were Ig-negative lymphoid cells recognised by the anti-leucocyte MAbWCT23 (Rombout et al., 1993a). The teleost GALT thus appearsto predominantly use cell-mediated immune defences against pathogen pen-etration, and functional studies are in progress to yield more conclusiveevidence. Specific cellular immune responses in which fish T lymphocytes arethe e#ector cells have been demonstrated by the mixed lymphocyte reaction(Kaattari & Holland, 1990), allograft reactivity (Botham &Manning, 1981) anddelayed-type hypersensitivity (Stevenson & Raymond, 1990).To date, no evidence has been provided about the existence of secretory IgA

in teleosts, whereas antigen-specific IgM were detected in bile and mucus, andhardly in serum, after repeated oral immunisation or anal intubation withparticulate antigens (Rombout et al., 1986, 1989), indicating that antibodieswould form a first-line of defence against antigens in the gut. Di#erencesbetween mucosal and serum Ig were revealed in the sheepshead Archosargusprobatocephalus (Lobb & Clem, 1981) and in the carp (Rombout et al., 1993b).This study provides evidence about localisation of Ig-positive cells in the

mucosa of sea bass digestive tract. The immunostaining was seen on the cellmembrane of most cells. Indirect immunofluorescence and FACS analysis withDLIg3 have already shown the membrane localisation of the antigenic

GALT OF THE SEA BASS 243

determinant in midgut lymphoid cells (Scapigliati et al., 1996). Cytoplasmicstaining with DLIg3 or DLIg13 was seen in a limited number of cells, mostlikely plasma cells.Western blot analysis demonstrated that DLIg3 and DLIg13 recognised

di#erent epitopes (Scapigliati et al., 1996), however, no significant di#erencewas found concerning the distribution and concentration of immunoreactivecells. Although it has not been fully proven that both MAbs stained the samecell population, the combined application of the antibodies did not changesignificantly the number of positive cells detected (data not shown). Further-more, the use of DLIg3 on cryosections of paraformaldehyde-fixed intestineshowed similar number and distribution of immunoreactive cells (data notshown).The number of Ig-bearing cells found in the GALT of the sea bass was

apparently lower compared with earlier data in the carp (Rombout et al.,1993a), possibly reflecting species di#erences in GALT organisation, as well asthe existence of subpopulations of Ig-bearing cells in the sea bass. This has notyet been proven, although apparent heterogeneity of light chain of sea bassserum IgM has been shown (Palenzuela et al., 1996).In conclusion, MAbs specific for distinct lymphocyte subpopulations of the

sea bass defined the existence of GALT, variations in the GALT throughoutthe digestive system and the predominance of T cells. Future studies willdefine GALT ontogeny and the functional and molecular characterisation ofintestinal lymphocytes.

The authors are indebted to ENEL/CRTN, Torre Valdaliga (Roma) and to ‘ Itticol-tura La Rosa ’, Orbetello (Grosseto), for the generous supply of experimental animals.This work was supported in part by grants from the Italian MRAAF (IV Plan, project4.C.047) and the Italian Ministry of University and Scientific Research (60% and 40%).

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