Development of T Lymphocytes in the Nasal-associated Lymphoid Tissue (NALT) from Growing Wistar Rats

The aim of the present report was to study the development of several T-lymphocyte subsets in the nasal-associated lymphoid tissue (NALT) of growing Wistar rats. CD5+ and CD4+ lymphocytes gradually increased with age. A predominance of CD8α+ over CD4+ T cells was found from 7 to 45 days but from 45 to 60 days of age T helper cells outnumbered the cytotoxic subpopulation. The majority of CD8+ T lymphocytes expressed the heterodimeric isoform. The most relevant findings by immunohistochemistry are: (1) the predominance of TCRγδ+ and CD8α+ cells at 7 days postpartum over all the other T-cell subpopulations; and (2) that TCRγβ+ outnumbered TCRαβ+ T cells from 7 to 45 days postpartum whereas αβ T cells predominated in 45- and 60-day-old rats. Besides, cytometric studies have shown that the percentages of TCRγ+, CD8+, as well as the population coexpressing both phenotypes (TCRγδ+CD8α+), were significantly higher in rats at 7 days postpartum when compared to 60 day-old rats. In the present study, the finding of a high number of γδ+ and CD8+ T cells early in NALT development may indicate the importance of these subpopulations in the protection of the nasal mucosa in suckling and weaning Wistar rats.


INTRODUCTION
The mucosal membranes of vertebrates are weak mechanical barriers that possess extensive innate and specific mechanisms of defence. The innate mechanisms have been extensively reviewed elsewhere (Pruitt et al., 1999;Diamond et al., 2000).
A morphologically and functionally distinct line of protection characteristic of mucosal surfaces is the so-called mucosa-associated lymphoid tissue (MALT). MALT refers to solitary lymphoid nodules and large organised collections of lymphoid tissues located in the lamina propria of the mucosal membranes. The tissues that are part of the MALT include the middle ear, parts of the urogenital tract, the mammary gland, the conjunctiva, the salivary glands, the nasopharyngeal lymphoid tissue (NALT), as well as bronchus-associated lymphoid tissue (BALT) and gut-associated lymphoid tissue (GALT) (Croitoru and Bienenstock, 1994).
MALT is characterised by the predominance of local dimeric IgA production, secreted as secretory IgA (sIgA) that is responsible for the immune exclusion of bacteria and viruses (Mazanec et al., 1993). The term common mucosal immune system (CMIS) implies that activated lymphocytes derived from one mucosal surface can recirculate and localise selectively in other mucosal surfaces. This connection between different mucosal surfaces permits immunity initiated at one anatomical site to protect other mucosal sites (McDermott and Bienenstock, 1979).
In the upper respiratory tract, MALT is represented by nasal-associated lymphoid tissue (NALT) and the term was coined by different authors (Mair et al., 1987;Spit et al., 1989). In humans, NALT is known as Waldeyer's ring and it consists of the adenoids or nasopharyngeal tonsil, the bilateral pharyngeal lymphoid bands, the bilateral tubal tonsils around the opening of the two Eustachian tubes, the bilateral palatine tonsils at the lateral opening of the oropharynx, and the lingual tonsil.
Massive lymphoid accumulations have been described in the mucosa of the opposite lateral walls near the anterior orifice of the nasopharyngeal duct when studying the rat upper respiratory tract (Kelemen, 1947). This paired and rod-shaped tissue, situated in the floor of the nasal cavity, is the only well-organised MALT with a fixed location in the nasopharynx of rodents (Spit et al., 1989;Hameleers et al., 1989;Kuper et al., 1990;van der Ven and Sminia, 1993). Therefore, this tissue can be regarded as the nasopharyngeal tonsil and as an equivalent of Waldeyer's ring because structures similar to the palatine and lingual tonsils have not been observed in rodents.
In rats, NALT is present at birth, earlier than BALT and this is probably due to its strategic position with respect to the incoming air. As rats are obligate nasal breathers, the inspired air, laden with environmental antigens, passes the nasal cavities before it reaches the lungs. Some immunohistochemical studies regarding the ontogeny of lymphoid and non-lymphoid cells have been performed in rats only from birth up to weaning (21 days of age). Furthermore, those concerning the phenotypic characterisation of T lymphocytes were semiquantitative and incomplete due to the lack of some monoclonal antibodies (Hameleers et al., 1989).
Therefore, the aim of the present report was to perform a complete phenotypic study of T-cell subpopulations in the NALT of growing Wistar rats from 7 to 60 days of age.

Animals
Suckling rats 7 and 12 days old, and weaning rats of the Wistar strain (closed colony from the breeding unit kept at the animal facilities of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Argentina) of either sex were used. Weaning rats (21 days old) were fed a stock diet (Cargill, Argentina) up to 60 days of age. Water and diet were given ad libitum. During all the experimental time animals were subjected to a 12-h light period and room temperature was kept at 21^18C.
Experiments were performed using 5 or 6 animals per group.

Isolation of NALT
Rats were killed by decapitation under anaesthesia with diethyl ether at 7, 12, at weaning (21 days), and at 45 and 60 days of age.
The lower jaws and the tongues were removed and the palates were excised with a scalpel blade from behind the incisor teeth to the molar teeth. Then, palates were gently pulled from front to rear with fine forceps, carefully dissecting them from the underlying bone tissue with the scalpel blade.

Indirect Immunofluorescence
Palates, containing NALT on their nasal sides, were immediately placed in 958 ethanol at 48C in order to be processed by Sainte-Marie's technique (Sainte-Marie, 1962). Briefly, tissues were fixed in 958 ethanol precooled at 48C, dehydrated in four changes of pre-cooled absolute ethanol, cleared by passing through three consecutive baths of xylene and embedded in paraffin at 568C. Tissue sections (4 -5 mm thick) were placed on glass microscope slides. Paraffin was removed by gently swirling the slides in two consecutive baths of xylene, which was removed in two baths of absolute ethanol, two baths of 958 ethanol and three baths of saline solution (0.9% NaCl w/v in distilled water).
T cells were labelled with the xenogeneic monoclonal antibodies against rat CD5 (clone OX-19), CD4 (clone OX-38), CD8a (clone OX-8), CD8b (clone 341), TCRab (clone R73) and TCRgd (clone V65) (BD PharMingen, San Diego, CA, USA). After an overnight incubation at 48C, the fluorescein-conjugated goat F(ab) 0 2 fragment to mouse IgG (whole molecule) (ICN Cappel, Aurora, OH, USA) was added. All tissue incubations were performed in a moist chamber, and sections were washed three times in saline after each incubation step. Control staining was carried out simultaneously omitting the primary antibody step. Tissue sections of NALT were viewed at 1,000 £ magnification with an Olympus epifluorescence microscope. Cell counts were performed by the authors in a blind fashion.

Avidin-biotin-peroxidase
To confirm the presence of certain T-cell phenotypes at 7 days of age we used avidin-biotin-peroxidase-DAB (Vectastain w ABC Kit, Vector Laboratories, Inc, Burlingame, CA, USA) that is the most sensitive immunohistochemical technique.
In brief, tissue sections were deparaffinized and hydrated as explained above. Then the sections were incubated in 0.3% H 2 O 2 in methanol and, after rinsing in PBS, incubated with diluted horse normal serum. T cells were labelled with the xenogeneic monoclonal antibodies against rat CD8a, CD8b, TCRab and TCRgd. After an overnight incubation at 48C, the sections were incubated with biotinylated horse anti mouse IgG and then with Vectastain w ABC reagent. Finally, sections were immersed in 1 mg/ml DAB solution in Tris -HCl pH 7.2, rinsed in tap water, counterstained with Harris' haematoxylin, cleared and mounted.

Preparation of Cell Suspensions
Palates, containing NALT, were immediately placed in a Petri dish containing ice-cold HBSS supplemented with 5% heat-inactivated foetal bovine serum (HBSS/FBS) and teased gently against a stainless steel mesh immersed in the medium in order to release cells. NALT cell suspensions from individual animals were pooled and washed three times with HBSS/FBS by centrifugation. Then, erythrocytes were eliminated by the addition of 0.83% NH 4 Cl solution. The dissociated cell suspensions were filtered through nylon wool to remove large cellular aggregates and detritus and then washed three times with HBSS/FBS by centrifugation. Viability of cell preparations routinely exceeded 90% as judged by Tripan Blue staining.
Results are expressed as the percentage of positively stained cells in the total cell population exceeding the background-staining signal.

Statistical Analysis
Results are expressed as mean^standard errorðSEÞ of the absolute number of cells in NALT lamina propria. Due to the very small size of NALT in 7 day-old rats, 2 fields were recorded and 5 fields for 12, 21, 45 and 60 day-old rats.
Statistical evaluation of results was performed with GraphPad InStat version 3.01 (GraphPad Software, San Diego, CA, USA) using 2-tailed Student's t-test and one-way ANOVA with Tukey-Kramer's post test, taking p , 0:05 as significant.

RESULTS
At day 7 postpartum NALT consisted of two very small structures in which we observed few lymphocytes (Fig. 1A). All CD5þ T-cell subpopulations were present and there was a predominance of CD8aþ and TCRgdþ over CD4þ and TCRabþ T cells, respectively. Due to the very small size of NALT at this age, data are expressed as number of cells in 2 fields as shown in Table I. TCRgdþ cells are indicated with arrows in Fig. 1B.
We observed that the absolute number of CD5þ T cells gradually increased with age and that there are only significant differences when we compare the 60-day-old group with 12-, 21-and 45-day-old rats (Fig. 2).
The study performed on CD5þ T-cell subpopulations showed that: (1) CD4þ T cells increased with age but significant differences were only observed between the 45-and 60-day-old rats with respect to the other groups (Fig. 3); and there were no significant differences in the CD8aþ T lymphocytes, with the majority expressing the heterodimeric isoform (CD8ab), as it can be seen in Fig. 3. The CD8aþ /CD4þ ratio was high at 7 days postpartum,   diminished with age and in 45-day-old rats reversed up to 60 days of age (Table II). We found significant differences in TCRgd only between 12 and 21 day-old rats (two-tailed Student's t test, p ¼ 0:0338Þ and in the TCRabþ subset significant differences were observed from 12 to 60 days postpartum as shown in Fig. 4. gdþ T cells outnumber those expressing TCRab from 7 to 45 days of age whereas in 45-and 60-day-old rats, abþ cells slightly outnumber gdþ T cells. Consequently, at these ages the TCRgdþ / TCRabþ ratio reversed (Table II).
Lymphoid cells in NALT from 60 day-old rats are closely packed (Fig. 1C) and the overlying epithelium is infiltrated with intraepithelial lymphocytes. Many gdþ lymphocytes in the lamina propria are shown in Fig. 1D.

DISCUSSION
NALT is present at birth and some findings in mice concerning the mechanisms of organogenesis have recently been published (Harmsen et al., 2002;Fukuyama et al., 2002). There is only one report in mice regarding the postnatal development and structure up to 28 days of age (van der Ven and Sminia, 1993). Moreover, the early appearance of immunocompetent cells in Wistar rats has been described but only up to 21 days of age (Hameleers et al., 1989). The latter is a semi-quantitative study indicating the existence, from day 4 to 10, of a predominance of the T helper subpopulation over the suppressor/cytotoxic one. An immunohistochemistry study in 15 week-old Wistar rats (Kuper et al., 1990) and a cytometric analysis in 60 day-old Lewis rats have shown a predominance of T helper cells (Koornstra et al., 1993). However, none of them has referred to TCRab and TCRgd subpopulations during the postnatal development.
In the present study, the number of CD5þ T cells, as well as all its subpopulations, increases gradually with age, in parallel with the increase in size of this organ. A predominance of CD8aþ over CD4þ T cells was found from 7 to 45 days of age but from 45 to 60 days of age T helper cells outnumber the cytotoxic subpopulation. This finding is in agreement with the study performed in 60 day-old Lewis rats (Koornstra et al., 1993). Similarly, NALT of adult chickens contains numerous CD4þ and scattered CD8þ T cells only in the large lymphoid accumulations present in the lamina propria under the respiratory epithelium (Ohshima and Hiramatsu, 2000).
Due to the lack of monoclonal antibodies there was no previous information on CD8 isoforms, as well as TCRab and TCRgd. Therefore, the early appearance-from 7 to 60 days of age-of CD8aþ and CD8bþ , TCRgdþ and TCRabþ subpopulations was studied and is discussed in the present report.
In our study by immunohistochemistry, the most relevant finding is the predominance of gdþ over abþ T cells from 7 to 45 days postpartum whereas in 45-and 60-day-old rats, ab slightly outnumber gd T cells. Moreover, in the present report cytometric studies have shown that the percentage of CD8aþ and gdþ T cells, as  well as the population coexpressing both phenotypes (TCRgdþ CD8aþ ), are significantly higher at 7 days of age when compared to 60 day-old rats. Another relevant finding is that the majority of CD8þ T cells belong to the heterodimeric subset. This observation is in agreement with results obtained in mice (Heritage et al., 1997).
Therefore, the present study shows that the pattern of development of T-cell subpopulations in NALT is similar to the one found previously in our laboratory when studying BALT (Márquez et al., 2000).
It is well known that gd T cells are involved in the immune surveillance and repair of damaged epithelia (Boismenu and Havran, 1994). Besides, it was suggested that gd T cells provide immunoprotective functions in instances where ab T cells do not, especially in young animals (Hayday, 2000). Therefore, the high number of gd and CD8 T cells early in NALT development may indicate the importance of these subpopulations in the protection of the nasal mucosa in suckling and weaning Wistar rats.