In Vitro and In Situ Characterization of Fish Thymic Nurse Cells

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the In Vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.

ekerle et al. (1980) iso- lated in vitro from the motlse thymus thymocyte- epithelial-cell complexes, named thymic nurse cells (TNCs), in which the lymphocytes are enclosed within vacuoles lined by the epithelial-cell mem- brane.It is generally assumed that in vitro TNCs represent the in vivo association of epithelial cells with cortical thymocytes.Some authors have never- theless reported lymphocyte-epithelial-cell com- plexes in lymphoid organs other than the thymus (Gerdes et al., 1983; Manconi et al., 1984; Wick and  Oberhuber, 1986; Tsunoda and Kojima, 1987).The epithelial nature of TNCs was established by the presence of keratin bundles (Vakharia, 1983; de Waal  Malefijt et al., 1986; Toussaint-Demylle et al., 1993), whereas thymocytes within TNCs are mainly imma- ture double-positive CD4 CD8 cells (Ritter et al.,  1981; Kyewski and Kaplan, 1982; Van Vliet et al.,  1984; Kyewski et al., 1987), although some authors have reported more mature cells in the complexes *Corresponding author. (Vakharia, 1983; Hugo et al., 1988) and others also include double-negative CD4-CD8-cells  (Singer  et al., 1986;  Kyewski, 1986; Wood et al., 1988).

Although TNCs have repeatedly been claimed to play a decisive role in T-cell maturation, their true functional significance is a matter of discussion.They have bee involved in the establishment of positive (Farr and Anderson, 1985; Kyewski, 1986; Owen  et al., 1986; Ron et al., 1986) and negative selection (Wick and Oberhuber, 1986; Lorenz and Allen, 1989;  Speiser et al., 1992), although their formation is not dependent on TCR-MHC interaction (Boyd et al.,  1993; Aguilar et al., 1994).Recently, Aguilar et al. (1994) have concluded their participation in the process of thymocyte apoptosis or in the clearing of apoptotic thymocytes.

TNCs have been found in the thymus of all higher vertebrates studied (Wick and Oberhuber, 1986;   Penninger et al., 1990, 1994; Boyd et al., 1993), but dire t evidence of their existence in ectotherms is lacking.In Rana pipiens tadpole thymi, Holtfreter and  Cohen (1987) described thymocyte-stromal-cell com- plexes with regard only to their external appearance, size, and number of internalized thymocytes, includ- ing TNCs, and also thymic rosette cells, which con- tained macrophages and presumably dendriticlike cells.In the teleost thymus, morphological stu- dies have emphasized the intimate association 18 FLANO et al.

between the epithelial cells and thymocytes (Pulsford et al., 1991; lvarez, 1993), whereas thymocyte-macrophage complexes have been re- ported in both elasmobranc es (Pulsford et al., 1984;  Navarro, 1987) and teleosts (Finge and Pulsford, 1985).

In this paper, we report for the first time the in vitro isolation of TNCs from the adult trout thymus, analyzing their cytochemical and ultra- structural ch racteristics and their presumptive relationships with thymocyte-epithelial-cell com- plexes observed in situ.


RESULTS


Light Microscopy

Isolated lymphoid-epithelial-cell complexes ap- peared as large round cells with a regular outline containing I to 11 (avera

13 showing the vesicular structures (V), a
vesicle with a fibrous content (F), and a mitochondria (M) (x39,300).( 15) High-power view of Fig. 12 showing an intracytoplasmic lymphocyte (L) surrounded by a double membrane (arrowheads) and tonofilaments (T) (x16,000).

consisted of one epithelial cell that embraced sev- eral lymphoid cells.The epithelial cell showed a euchromatic or moderately heterochromatic nucleus ovoid or irregular in shape, sometimes with inden- tations (Figs 7 and 8).The cytoplasm contained numerous mitochondria, free ribosomes, cisternae of rough endoplasmic reticulum, and large tonofil- ament bundles that frequently surrounded the vacuoles containing thymocytes (Figs 9 and 10).In addition, the epithelial cell showed numerous large electron-lucent and electron-dense membranous vesicles resembling both lysosomes and endosomes (Figs 7 to 10).Some vacuoles with internal membra- nous structures were also present throughout the cytoplasm (Figs 9 and 10).

Within the TNCs, several thymocytes were indi- vidually enclosed in vacuoles lined by the epithelial- cell membrane.Membranes of both cell types were in close contact, forming a narrow intercellular space (Fig. 9).Intra-TNC thymocytes, without signs of apoptosis or degeneration, had round or avoid heterochromatic nuclei and homogeneous, electron- dense cytoplasm containing numerous free ribo- somes and mitochondria (Figs 7 and 9).

In Situ Lymphoid-Epithelial Cell Complexes Previously, we had defined in situ seven different types of epithelial cells in the trout thymus accord- ing to their loc tion in the organ, morphology, histochemical reactivity, and ontogenetical development (Castillo et al., 1990, 1991).Two of these epithelial cell types, pale and intermediate electron-lucent, which occupied the so-called

nner zone o
the thymus, appeared frequently in close association with thymocytes apparently enclosed in their cytoplasm (Figs 11 and 12).The intermediate electron-lucent epithelial cells had numerous cell processes extend- ing between neighboring thymocytes and other epithelial cells (Fig. 11).Their cytoplasm contained microfilaments, elements of the Golgi apparatus, profiles of rough endoplasmic reticulum, a few elec- tron-dense vesicles, as well as numerous vesicles with a heterogeneous electron-pale content (Fig. 11).The nucleus, irregular in shape, was moderately heterochromatic, with a prominent nucleolus.Engulfed thymocytes frequently appeared in the epi- thelial cytoplasm in close contact with pale vesicles (Fig. 11).On the other hand, pale epithelial cells had euchromatic nuclei and ovoid electron-lucent cytoplasm.They contained free ribosomes, mitochondria, some groups of dictyos mes, rough endoplasmic reticulum, and large vesicles with a heterogeneous content (Figs 12 to 15).In addition, bundles of tonofilaments, sometimes closely apposed to thymocyte-containing vacuoles (Fig. 15), and vesi- cles with a fibrous content, appeared in these cells (Fig. 14).

Intraepithelial thymocytes were individually en- closed in vacuoles lined by the epithelial-cell mem- brane (Fig. 15).They showed no signs of cell degeneration, containing a large number of free ribosomes and mitochondria.


DISCUSSION

Our results demonstrate for the first time that the trout thymus contains thymocyte-epithelial cell com- plexes, which can be isolated in vitro, resembling morphologically the TNCs described in higher ver- tebrates.As previously reported in both mammals and birds (Wekerle et al., 1980; Vakharia, 1983;  Penninger et al., 1994), the teleost TNCs consist of epithelial cells with numerous mitochondria, profiles of rough endoplasmic reticulum, large bundles of tonofilaments, and membranous organelles re- sembling endoand lysosomes.In addition, they are keratin-positive and exhibit acid and alkaline phosphatase and nonspecific esterase activities.These morpho-cytochemical features suggest that trout TNCs have a high metabolic activity and con- tain presumably acidic cytoplasmic compartments, important for antigen processing, as recently dem- onstrated in chicken TNCs (Penninger et al., 1994).

On the other hand, our in situ electron microscopy study confirms previous results (Pulsford et al.,   1991; ,lvarez, 1993) concerning the occurrence of thymocyte-epithelial-cell complexes in the teleost thymus.They correspond to both pale and inter- mediate electron-lucent epithelial cells of the inner zone of trout thymus.Indeed, they resemble mor- phologically the so-called type 2 (pale) and type 3 (intermediate) epithelial cells of the human thymus (Wijngaert et al., 1984), which have been proposed as the TNCs found in suspensions (Van de Wijngaert  et al., 1984; Boyd et al., 1993).Thus, because mam- malian TNCs seem to corr spond mainly to cortical epithelial cells (Ritter et al., 1981; Kyewski and  Kaplan, 1982; Van Vlie

et al., 1984; N
barra and  Adrianarison, 1987), although their relationship to subcapsular (Kyewski and Kaplan, 1982; Kaneshima   et al., 1987; Mizutami et al., 1987) and medullar epithelium has also been mentioned (Hugo et al., 1988), our results indirectly demonstrate the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates, a fact repeatedly suggested by our group (Zapata, 1981;   Castillo et al., 1990, 1991).It is, however, difficult, from a merely morphological study, to establish cor- relations between the in vitro isolated TNCs and these lymphocyte-epithelial-cell clusters identified in situ.Moreover, some authors have mentioned a certain heterogeneity in enriched TNCs from the human thymus (Ritter et al., 1981).

All phenotypical studies remark on the expression of class I and class II MHC on higher-vertebrate TNCs (Wekerle et al., 1980; Ritter et al., 1981; Boyd  et al., 1984), although the use of trypsine treatment during the isolation of cell clusters modifies this expression (Kyewski and Kaplan, 1982).Unfortu- nately, there are no available reagents to detect MH molecules in teleost fish, although recent molecular data demonstrated the existence of both class I and class II antigens in them (Kromenberg et al., 1994).

Ltd., Bale, Switzerland), blood was extracted from the caudal sinus, and the thymi were dissected.


Cell Isolation

The thymi were mechanically minced in ice-cold phosphate-buffered saline (PBS), and the super- natant containing free cells was discarded.The re- maining tissue fragments were incubated in PBS for 10 min at 15C with stirring, and the supernatant discarded again.After this mechanical diss

ents wer
digested with 0.6 mg/ml type Ia collagenase (Sigma, St. Louis) in PBS for 15 min at 15C under agitation.The supernatant was discarded and this step was repeated three times, the supernatants of the two last steps being har- vested.After collagenase digestion, the remaining tissue fragments were trypsinized (1% trypsin-EDTA in PBS; Boehringer-Mannheim) for 30 min at 15C under gentle agitation.Collection of TNC was achieved using a modification of the method of By using cross-reactive monoclonal antibodies, Wekerle et al. (1980).TNC-containing fractions wer

Kaufman et al. (19
0) detected MHC-like molecules in some nonmammalian vertebrates.On this same basis, we have demonstrated a slight expression of MHC class II antigens in trout TNCs using a anti- chicken class II MHC molecule monoclonal antibody.

The expression of class II molecules and the ex- istence of an acidic cytoplasmic compartment, as indicated by our enzyme-histochemical and ultra- structural results, stggest some capacity of teleost TNCs for antigenic pr cessing and presentation, as recently reported for chicken TNCs (Penninger et al.,  1994).Nevertheless, the TCR-MHC interactions are apparently not necessary for the formation of mouse TNCs (Boyd et al., 1993) and, although most authors emphasize a role for TNCs in intra-thymic T-cell maturation, their functional significance is really obscure.


MATERIALS AND METHODS


Animals

Thymi from rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were used for cell isola- tion and histological examination.Fish were aged by scalimetry and were between 1 and 3 years.No differences were seen between the two species stud- ied in any of the experimental systems used.Fish were anesthesized with MS-222 (Sandoz Pharma enriched for TNC by one 1-g sedimentation over FCS at 4C for 15 min.After sedimentation, the top layer was discarded, and the rest of the suspension was centrifuged at 250 g for 10 min at 4C and resus- pended in PBS.


Routine Histology

The isolated cells were placed in drop

onto slides, air dried, and stain
d with haematoxylin-eosin for routine examination and identification of multi- cellular complexes.


Enzymeand Immuno-Cytochemical Analysis

The assayed enzymatic activities and the substrates used are summarized in Table 1.Negative controls for the specificity of the enzymatic reactions were established using incubation media in which the cor- responding substrates were lacking.

For immunodetection, cells were fixed in acetone for 10 min at room temperature, air dried, and processed by the indirect immunoperoxidase techni- que using 3,3-diaminobenzidine-tetrahydrochloride (Sigma) as a coupling reagent.Controls were sys- tematically performed by omission of the first anti- body, and endogenou