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Developmental study of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT) from Wistar rats
Developmental and Comparative Immunology 24 (2000) 683±689 www.elsevier.com/locate/devcompimm
Developmental study of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT) from Wistar rats MarõÂ a G. MaÂrquez, Gustavo A. Sosa, MarõÂ a E. Roux* Laboratory of Cellular Immunology, CONICET, Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Received 10 August 1999; accepted 13 December 1999
Abstract The aim of the present report was to study in growing Wistar rats the development of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT). We found at day 4 postpartum, a high number of TCRg/d+ T cells and very few CD8a+, CD8b+, CD5+, TCRa/b+ T cells in BALT. The latter cells and CD4+ T cells increase with age. Even though T cells expressing TCRg/d outnumber those expressing TCRa/b early in development, until 45 days of age, a/b+ predominate over g/d+ T cells only in adult rats (60 days of age). Moreover, a predominance of suppressor/cytotoxic T cells over T-helper cells was found in 60 days old rats. Surprisingly, more CD8a+ than CD8b+ T cells in BALT are observed. The number of IgA+ B and CD4+ T cells found in the BALT increases with age. The early appearance Ð 4 days of age Ð of all T-cell phenotypes in BALT especially of g/d+ T cells may imply a bene®t to respond to inhaled antigen soon after birth. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: BALT; Development of immunocompetent cells; Wistar rats
1. Introduction The notion of the lung as an immunological organ was developed in the past decade and it Abbreviations: BALT, bronchus-associated lymphoid tissue; BALUs, bronchus-associated lymphoid units; GALT, gut-associated lymphoid tissue; DAB, 3,3'-diaminobenzidin. * Corresponding author. Tel.: +5411-4801-2864; fax: +5411-4912-0989. E-mail address: mroux@huemul.yb.uba.ar (M.E. Roux).
has been described in several reviews [1,2]. The immunocompetent cells in the lung can be located in dierent compartments: the vascular pool, the interstitial pool, the bronchoalveolar space and the organized lymphoid tissue associated with the bronchi, the so called bronchus-associated lymphoid tissue (BALT) [3]. BALT is strategically located at branching points in the conducting airways Ð between an artery and a bronchus Ð where it might
0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 0 ) 0 0 0 0 3 - 3
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M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
intercept inhaled antigens. Some authors consider BALT as a functional entity and refer to the separate lymphoid aggregates as BALUs (bronchus-associated lymphoid units) [4]. Like Peyer's patches, a BALU is not separated from its surroundings by a capsule of connective tissue. Therefore, BALUs are not considered as true lymphoid organs but represent local aggregations of lymphoid and non-lymphoid cells within the connective tissue compartment of the respiratory tract [4,5]. Although B- and T-cell areas have been described in some species, in the rat, the position of these areas seems to be undetermined and varies between individual BALUs [4,5]. Most of the intraepithelial lymphocytes that in®ltrate BALU epithelium are T cells [4] and the majority have the T-helper phenotype [6]. Although BALT was recognized more than 100 years ago [7] and its structure and function have been extensively described [4,5,8], the immunocompetent cells are not completely known. It has been reported that the appearance of B-lymphocytes on day 4 precedes the presence of T cells on day 8 after birth [9,10]. Even though rat BALT T-lymphocytes have been studied showing that T-helper cells outnumber T-suppressor/cytotoxic cells [4], there is only one report indicating the presence of g/d+ T cells in the periphery of lymphoid aggregates of the BALT at day 15 of postnatal life [11]. Monoclonal antibodies that allow the dierentiation of the homodimeric and heterodimeric CD8 rat phenotypes (CD8a/a and CD8a/b T cells) were described in 1992 [12]. Commercial monoclonal antibodies detecting CD8 isoforms and rat TCRg/d T cells have been available since 1994. Since rat BALT appears only after birth and the ®rst BALUs appear 4 days postpartum [4], a sequential study of the early appearance of g/d T cells and all the other T subsets on BALT is necessary. Recently, g/d+ T cells were found not only in foetal tissues such as thymus and liver but also in gut [13]. The aim of the present study was to analyze by immunohistochemistry in the BALT of growing Wistar rats: (1) the early appearance of T
cells bearing CD5 and its subsets such as CD4, CD8a, CD8b, TCRa/b y TCRg/d, and (2) the variation of IgA B cells from weaning up to 60 days of age. 2. Materials and methods Suckling rats 4, 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, 24.6% protein) up to 60 days of age. Water and diet were given ad libitum. During all the experimental time animals were submitted to a 12 h light±darkness cycle, room temperature was kept at 218C 2 1. Experiments were performed using 4±11 animals per group. Rats were sacri®ced at 4, 7 and 12 days old, and after 4 h of fasting at weaning (21 days), 39, 45 and 60 days of age. The lower respiratory tract was removed and placed in ethanol at 48C in order to be processed by Sainte-Marie's technique [14]. Brie¯y, tissues were ®xed in 958C ethanol pre-cooled at 48C, dehydrated in four changes of pre-cooled absolute ethanol, cleared by passing through three consecutive baths of xylene and embedded in paran at 568C. Tissue sections (4±5 mm thick) were placed on glass slides. Paran 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 958C ethanol and three baths of saline solution (0.9% NaCl wt/vol in distilled water). Tissue sections of the lower respiratory tract were studied with an Olympus ¯uorescence microscope. Lymphoid cells were detected by an indirect immuno¯uorescence technique with the following antibodies: (1) IgA+ B cells were labeled with anity-puri®ed goat IgG to rat IgA (a speci®c) (Bethyl) followed by ¯uorescein-conjugated F(ab)2' fraction of rabbit against goat IgG (H and L) (Organon Teknika), and (2) T cells were labeled with the xenogeneic monoclonal antibodies against rat CD5, CD4,
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
CD8a, CD8b, TCRa/b and TCRg/d (Pharmingen, San Diego, CA) followed by the ¯uoresceinconjugated goat F(ab)2' fragment to mouse IgG (whole molecule) (Organon Teknika). Results are expressed as number of cells per ®ve ®elds (7 and 12 days old rats) and per 15 ®elds (21, 39, 45 and 60 days old rats) in BALT and BALT epithelium in each tissue section; and two or three sections per animal were recorded. Two blinded investigators reviewed all slides. Controls for the labeling procedure described include deletion of the primary antibody step. To con®rm the presence of certain T-cell phenotypes at 4 days of age, we utilised the avidin± biotin±peroxidase±DAB that is the most sensitive immunohistochemistry technique. Statistical analysis was performed using Student's t test or Tukey±Kramer, taking p < 0.05 as signi®cant.
685
3. Results The early appearance of T-lymphocytes was studied on day 4 postpartum, since in the rat BALT appears only after birth. We observed at day 4 an aggregation of reticulum and lymphoid cells in the bronchial submucosal connective tissue. In this aggregation called BALU, we found a high number of TCRg/d+ T cells (Fig. 1) and very few isolated lymphoid cells bearing the CD8a, CD8b, CD5 and TCRa/b phenotypes. On successive days, BALUs appear further along the bronchial tree and the number of T cells gradually increases with age (Figs. 2 and 3). We observed that the number of CD5, CD4, CD8a and CD8b T cells in BALT was signi®cantly increased at 12 days of age, when compared with the number found at day 7 (Fig. 2). Even though T cells expressing TCRg/d outnumber those expressing TCRa/b, there was not a
Fig. 1. Paran section of rat BALU 4 days after birth stained with anti TCRg/d monoclonal antibody (arrows) (avidin±biotin±peroxidase±DAB), C: cartilage (magni®cation 100).
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Fig. 2. Number of CD5+, CD4+, CD8a+ and CD8b+ Tcells in BALT of 7 and 12 days old rats. The bars represent the mean (2SE) of the number of cells from 5 animals. Student t-test: () p < 0.0001; (#) p < 0.0002; (&) p < 0.02 compared to 7 days old rats.
signi®cant dierence between 7 and 12 days of age (Fig. 3). Occasional lymphocytes expressing the phenotypes shown in Figs. 2 and 3 were found in BALT epithelium at both ages. The number of CD5+ T cells in BALT increases with age and there is a signi®cant increase at 60 days of age compared with day 21 (Fig. 4). CD5+ T cells in BALT epithelium increases signi®cantly from 45 days of age (Fig. 4). Although CD8+ T-cell subpopulations increase with age, T cells bearing the CD8a+ phenotype outnumber those bearing the CD8b+ phenotype at 60 days of age in BALT and BALT epithelium (Figs. 5 and 6, respectively).
Fig. 3. Number of TCRa/b+ and TCRg/d+ T-cells in BALT of 7 and 12 days old rats. The bars represent the mean (2SE) of the number of cells from 5 animals. Student t-test: p values are not signi®cant.
Fig. 4. Number of CD5+ T-cells in BALT and BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 10 animals. Tukey±Kramer: BALT: (#) 21, 39, 45 vs. 60, p < 0.001; BALT epithelium: () 21 vs. 45, 60, p < 0.001 and 39 vs. 45, 60, p < 0.05.
The number of IgA+ B and CD4+ T cells found in BALT increases with age (Fig. 7). CD4+ T cells in the BALT epithelium remain constant from 21 up to 60 days of age (average 8 CD4+ intraepithelial lymphocytes per 15 ®elds). The development of TCRg/d T cells in BALT of Wistar rats from 7 to 60 days of age has been recently published [15] and it is interesting to compare these data with those obtained studying the development of TCRa/b+ T cells. The number of T cells expressing TCRg/d outnumbers T cells expressing TCRa/b from 21 up to 45 days in BALT and up to 39 days in BALT
Fig. 5. Number of CD8a+ and CD8b+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 9 animals. Tukey± Kramer: CD8a: () 21, 39, 45 vs. 60, p < 0.001 and (#) 21 vs. 45, p < 0.01; CD8b: (&) 21 vs. 39, p < 0.01 and (.) 45 vs. 21, 60 p < 0.001.
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
Fig. 6. Number of CD8a+ and CD8b+ T-cells in BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 9 animals. Tukey±Kramer: CD8a: () 60 vs. 21, 39, 45, p < 0.001; CD8b: (#): 45 vs. 21, 60, p < 0.001 and (&) 39 vs. 60, p < 0.05.
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Fig. 8. Number of TCRa/b+ and TCRg/d+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 6 animals. Tukey±Kramer: TCRa/b: (#) 21 vs. 45, 60, p < 0.01 and (.) 39 vs. 45, p < 0.01 and 39 vs. 60, p < 0.001; TCRg/d: () 21 vs. 39, p < 0.05; 21 vs. 45, p < 0.001 and 21 vs. 60, p < 0.01.
Even though several authors have studied rat BALT development, there are few reports
about the early appearance of immunocompetent cells. The gut-associated lymphoid tissue (GALT) has often been regarded as analogous to BALT [5,8] but it is interesting to note the dierences between the development of BALT and GALT. It is known that on day 20 of gestation the rat gut wall shows special spots in which villi are absent, pointing to the developing Peyer's patches where T-lymphocytes are detectable for the ®rst time around birth followed by B cells a few days later [9,16]. Peyer's patches reach their adult structure only 4 weeks after birth with the appearance of
Fig. 7. Number of IgA+ B and CD4+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 4 to 8 animals. Tukey± Kramer: () IgA: 21, 39, 45 vs. 60, p < 0.001; CD4: (#) 21 vs. 39, p < 0.01 and 21 vs. 45, p < 0.001; (.) 21, 39, 45 vs. 60, p < 0.001.
Fig. 9. Number of TCRa/b+ and TCRg/d+ T-cells in BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 11 animals. Tukey±Kramer: TCRa/b: () 60 vs. 21, p < 0.001 and 60 vs. 39, 45, p < 0.01; TCRg/d: (#) 39 vs. 45, 60, p < 0.05.
epithelium. At day 60 TCRa/b+ T cells predominate in BALT and BALT epithelium (Figs. 8 and 9, respectively). At 60 days of age lymphoid cells appeared to be closely packed adjacent to blood vessels and the overlying epithelium was in®ltrated with lymphocytes as it can be seen in Fig. 10. 4. Discussion
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germinal centers [9,16], indicating that young rats are not able to generate an IgA memory until 4 weeks of age [9]. Recent studies indicate that gd+ T cells are found in foetal gut as well as in foetal thymus and liver [13]. In birds and mammals T-cell dierentiation proceeds along two discrete pathways characterized by the expression of a/b or g/d TCRs. The conservation of these T-cell lineages during vertebrate evolution is further indicated by identi®cation of a, b, g and d TCR genes among amphibians, bony ®shes and cartilaginous ®shes [17]. In the chicken and in the ruminant immune system the gd T cells comprise a much higher proportion that the one found in humans and mice [18]. It has been reported that at 15 days of age g/ d+ T cells appear in the periphery of lymphoid aggregates of the rat BALT [11] and also in the epithelium of the chicken caecal tonsil [19]. In the present report, we ascertain the appearance of a high number of g/d+ T cells at 4 days
of age with the ®rst BALU, increasing up to 45 days of age. Besides, at 60 days of age TCRa/ b+ T cells outnumber TCRg/d+ cells both in rat BALT and BALT epithelium. The number of the dierent T-cell subsets also increases with age, up to 60 days of age implying a maturity allowing the response to inhaled antigens as a consequence of the very early appearance of T cells with adult phenotypes. However, in contrast with a previous report [4], we found in adult rats (60 days of age) a predominance of the suppressor/cytotoxic T cells (CD8+) over T-helper cells in BALT and BALT epithelium. Besides, due to the possibility of detecting both CD8 subsets we found that the CD8a T-cell population outnumbers the CD8b one in BALT and BALT epithelium. The present observation diers from the data obtained from gut lamina propria and intraepithelium where heterodimeric CD8a/b isoform predominates [20]. Such a disparity may be explained by microenvironmental dierences in the antigenic repertoire between the gut and the respiratory tract.
Fig. 10. Haematoxylin±eosin paran section of rat BALU 60 days of age. Arrows indicate intraepithelial lymphocytes (magni®cation 1000).
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From weaning up to 60 days of age the appearance of IgA+ B cells in BALT was followed and we found a rapid increase of these cells accompanied by an increase of CD4+ T cells. This coincides with the appearance of germinal centers in Peyer's patches [9,16]. The early appearance of g/d+ T cells in BALT may indicate that this population contributes to the mucosae immunosurveillance at an early stage of development. Little information is available regarding the development of T-cell function in the early postnatal period of the rat but our phenotypic study shows that T cells in BALT and BALT epithelium are predominantly of the suppressor/ cytotoxic phenotype since they appear very early in BALT development.
[7] [8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements The authors thank the Department of Nutrition for the use of animal facilities. This investigation was supported by grant PIP 4147 from CONICET and grant TB 072 from UBA. This paper is part of the Thesis of MarõÂ a Gabriela MaÂrquez.
[14]
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[1] Bienenstock J. Bronchus-associated lymphoid tissue. In: Bienenstock J, editor. Immunology of the lung and upper respiratory tract. New York: McGraw-Hill, 1984. p. 96±118. [2] Reynolds HY. Immunologic system in the respiratory tract. Physiol Rev 1991;71:1117±33. [3] Pabst R. Compartmentalization and kinetics of lymphoid cells in the lung. Reg Immunol 1990;3:62±71. [4] Sminia T, van der Brugge-Gamelkoorn GJ, Jeurissen SHM. Structure and function of bronchus-associated lymphoid tissue (BALT). Crit Rev Immunol 1989;9:119± 50. [5] Bienenstock J, Johnston N, Perey DYE. Bronchial lymphoid tissue. I. Morphologic characteristics. Lab Invest 1973;28:686±92. [6] van der Brugge-Gamelkoorn GJ, van de Ende M, Sminia T. Changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intra-
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tracheal challenge with horseradish peroxidase. Cell Tissue Res 1986;245:439±44. Klein E. The anatomy of the lymphatic system. II. The lung. London: Smith, Elder and Co, 1875. Bienenstock J, Johnston N, Perey DYE. Bronchial lymphoid tissue. II. Functional characteristics. Lab Invest 1973;28:693±8. Van Rees EP, Dijkstra ChD, Sminia T. Ontogeny of the rat immune system: an immunological approach. Dev Comp Immunol 1990;14:9±18. Plesch BEC, Gamelkoorn GJ, van de Ende M. Development of bronchus-associated lymphoid tissue (BALT) in the rat, with special reference to T and B cells. Dev Comp Immunol 1983;7:179±88. Kuhnlein P, Vicente A, Varas A, HuÈnig T, Zapata A. g/ d T cells in fetal neonatal and adult rat lymphoid organs. Develop Immunol 1995;4:181±5. Torres-Nagel N, Kraus E, Brown MH, Tiefenthaler G, Mitnacht R, Williams AF, HuÈnig T. Dierential thymus dependence of rat CD8 isoform expression. Eur J Immunol 1992;22:2841±8. Mc Vay LD, Jaswal ShS, Kennedy Ch, Hayday A, Carding SR. The generation of human g/d T-cell repertoires during fetal development. J Immunol 1998;160:5851±6. Sainte-Marie G. A paran embedding technique for studies employing immuno¯uorescence. J Histochem Cytochem 1962;10:250±6. MaÂrquez MG, Roux ME. g/d T cells development in the bronchus-associated lymphoid tissue (BALT) of Wistar rats. In: Talwar GP, Nath I, Ganguly NK, Rao KVS, editors. Proceedings of the 10th International Congress of Immunology. Bologna, Italy: Monduzzi Editore S.p.A, 1998. p. 1085±8. Sminia T, Janse EM, Plesch BEC. Ontogeny of Peyer's patches. Anat Rec 1983;207:309±16. Kubota T, Wang J, GoÈbel TWF, Hockett RD, Cooper MD, Chen CH. Characterization of an avian (Gallus gallus domesticus ) TCR ad gene locus. J Immunol 1999;163:3858±66. Hein WR, Mackay CR. Prominence of gd T cells in the ruminant immune system. Immunol Today 1991;12(1):30±4. Gomez del Moral M, Fonfria J, Varas A, Jimenez E, Moreno J, Zapata AG. Appearance and development of lymphoid cells in the chicken (Gallus gallus ) caecal tonsil. Anat Rec 1998;250(2):182±9. Roux MEB, MaÂrquez MG, Sosa GA, Florin-Christensen A. Intestinal mucosae T cells and delayed-type hypersensitivity (DTH) to dextrin in immunode®cient rats orally treated with thymomodulin (TmB). In: Talwar GP, Nath I, Ganguly NK, Rao KVS, editors. Proceedings of the 10th International Congress of Immunology. Bologna, Italy: Monduzzi Editore S.p.A, 1998. p. 1461±4.
Developmental study of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT) from Wistar rats MarõÂ a G. MaÂrquez, Gustavo A. Sosa, MarõÂ a E. Roux* Laboratory of Cellular Immunology, CONICET, Department of Biological Sciences, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Received 10 August 1999; accepted 13 December 1999
Abstract The aim of the present report was to study in growing Wistar rats the development of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT). We found at day 4 postpartum, a high number of TCRg/d+ T cells and very few CD8a+, CD8b+, CD5+, TCRa/b+ T cells in BALT. The latter cells and CD4+ T cells increase with age. Even though T cells expressing TCRg/d outnumber those expressing TCRa/b early in development, until 45 days of age, a/b+ predominate over g/d+ T cells only in adult rats (60 days of age). Moreover, a predominance of suppressor/cytotoxic T cells over T-helper cells was found in 60 days old rats. Surprisingly, more CD8a+ than CD8b+ T cells in BALT are observed. The number of IgA+ B and CD4+ T cells found in the BALT increases with age. The early appearance Ð 4 days of age Ð of all T-cell phenotypes in BALT especially of g/d+ T cells may imply a bene®t to respond to inhaled antigen soon after birth. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: BALT; Development of immunocompetent cells; Wistar rats
1. Introduction The notion of the lung as an immunological organ was developed in the past decade and it Abbreviations: BALT, bronchus-associated lymphoid tissue; BALUs, bronchus-associated lymphoid units; GALT, gut-associated lymphoid tissue; DAB, 3,3'-diaminobenzidin. * Corresponding author. Tel.: +5411-4801-2864; fax: +5411-4912-0989. E-mail address: mroux@huemul.yb.uba.ar (M.E. Roux).
has been described in several reviews [1,2]. The immunocompetent cells in the lung can be located in dierent compartments: the vascular pool, the interstitial pool, the bronchoalveolar space and the organized lymphoid tissue associated with the bronchi, the so called bronchus-associated lymphoid tissue (BALT) [3]. BALT is strategically located at branching points in the conducting airways Ð between an artery and a bronchus Ð where it might
0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 0 ) 0 0 0 0 3 - 3
684
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
intercept inhaled antigens. Some authors consider BALT as a functional entity and refer to the separate lymphoid aggregates as BALUs (bronchus-associated lymphoid units) [4]. Like Peyer's patches, a BALU is not separated from its surroundings by a capsule of connective tissue. Therefore, BALUs are not considered as true lymphoid organs but represent local aggregations of lymphoid and non-lymphoid cells within the connective tissue compartment of the respiratory tract [4,5]. Although B- and T-cell areas have been described in some species, in the rat, the position of these areas seems to be undetermined and varies between individual BALUs [4,5]. Most of the intraepithelial lymphocytes that in®ltrate BALU epithelium are T cells [4] and the majority have the T-helper phenotype [6]. Although BALT was recognized more than 100 years ago [7] and its structure and function have been extensively described [4,5,8], the immunocompetent cells are not completely known. It has been reported that the appearance of B-lymphocytes on day 4 precedes the presence of T cells on day 8 after birth [9,10]. Even though rat BALT T-lymphocytes have been studied showing that T-helper cells outnumber T-suppressor/cytotoxic cells [4], there is only one report indicating the presence of g/d+ T cells in the periphery of lymphoid aggregates of the BALT at day 15 of postnatal life [11]. Monoclonal antibodies that allow the dierentiation of the homodimeric and heterodimeric CD8 rat phenotypes (CD8a/a and CD8a/b T cells) were described in 1992 [12]. Commercial monoclonal antibodies detecting CD8 isoforms and rat TCRg/d T cells have been available since 1994. Since rat BALT appears only after birth and the ®rst BALUs appear 4 days postpartum [4], a sequential study of the early appearance of g/d T cells and all the other T subsets on BALT is necessary. Recently, g/d+ T cells were found not only in foetal tissues such as thymus and liver but also in gut [13]. The aim of the present study was to analyze by immunohistochemistry in the BALT of growing Wistar rats: (1) the early appearance of T
cells bearing CD5 and its subsets such as CD4, CD8a, CD8b, TCRa/b y TCRg/d, and (2) the variation of IgA B cells from weaning up to 60 days of age. 2. Materials and methods Suckling rats 4, 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, 24.6% protein) up to 60 days of age. Water and diet were given ad libitum. During all the experimental time animals were submitted to a 12 h light±darkness cycle, room temperature was kept at 218C 2 1. Experiments were performed using 4±11 animals per group. Rats were sacri®ced at 4, 7 and 12 days old, and after 4 h of fasting at weaning (21 days), 39, 45 and 60 days of age. The lower respiratory tract was removed and placed in ethanol at 48C in order to be processed by Sainte-Marie's technique [14]. Brie¯y, tissues were ®xed in 958C ethanol pre-cooled at 48C, dehydrated in four changes of pre-cooled absolute ethanol, cleared by passing through three consecutive baths of xylene and embedded in paran at 568C. Tissue sections (4±5 mm thick) were placed on glass slides. Paran 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 958C ethanol and three baths of saline solution (0.9% NaCl wt/vol in distilled water). Tissue sections of the lower respiratory tract were studied with an Olympus ¯uorescence microscope. Lymphoid cells were detected by an indirect immuno¯uorescence technique with the following antibodies: (1) IgA+ B cells were labeled with anity-puri®ed goat IgG to rat IgA (a speci®c) (Bethyl) followed by ¯uorescein-conjugated F(ab)2' fraction of rabbit against goat IgG (H and L) (Organon Teknika), and (2) T cells were labeled with the xenogeneic monoclonal antibodies against rat CD5, CD4,
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
CD8a, CD8b, TCRa/b and TCRg/d (Pharmingen, San Diego, CA) followed by the ¯uoresceinconjugated goat F(ab)2' fragment to mouse IgG (whole molecule) (Organon Teknika). Results are expressed as number of cells per ®ve ®elds (7 and 12 days old rats) and per 15 ®elds (21, 39, 45 and 60 days old rats) in BALT and BALT epithelium in each tissue section; and two or three sections per animal were recorded. Two blinded investigators reviewed all slides. Controls for the labeling procedure described include deletion of the primary antibody step. To con®rm the presence of certain T-cell phenotypes at 4 days of age, we utilised the avidin± biotin±peroxidase±DAB that is the most sensitive immunohistochemistry technique. Statistical analysis was performed using Student's t test or Tukey±Kramer, taking p < 0.05 as signi®cant.
685
3. Results The early appearance of T-lymphocytes was studied on day 4 postpartum, since in the rat BALT appears only after birth. We observed at day 4 an aggregation of reticulum and lymphoid cells in the bronchial submucosal connective tissue. In this aggregation called BALU, we found a high number of TCRg/d+ T cells (Fig. 1) and very few isolated lymphoid cells bearing the CD8a, CD8b, CD5 and TCRa/b phenotypes. On successive days, BALUs appear further along the bronchial tree and the number of T cells gradually increases with age (Figs. 2 and 3). We observed that the number of CD5, CD4, CD8a and CD8b T cells in BALT was signi®cantly increased at 12 days of age, when compared with the number found at day 7 (Fig. 2). Even though T cells expressing TCRg/d outnumber those expressing TCRa/b, there was not a
Fig. 1. Paran section of rat BALU 4 days after birth stained with anti TCRg/d monoclonal antibody (arrows) (avidin±biotin±peroxidase±DAB), C: cartilage (magni®cation 100).
686
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
Fig. 2. Number of CD5+, CD4+, CD8a+ and CD8b+ Tcells in BALT of 7 and 12 days old rats. The bars represent the mean (2SE) of the number of cells from 5 animals. Student t-test: () p < 0.0001; (#) p < 0.0002; (&) p < 0.02 compared to 7 days old rats.
signi®cant dierence between 7 and 12 days of age (Fig. 3). Occasional lymphocytes expressing the phenotypes shown in Figs. 2 and 3 were found in BALT epithelium at both ages. The number of CD5+ T cells in BALT increases with age and there is a signi®cant increase at 60 days of age compared with day 21 (Fig. 4). CD5+ T cells in BALT epithelium increases signi®cantly from 45 days of age (Fig. 4). Although CD8+ T-cell subpopulations increase with age, T cells bearing the CD8a+ phenotype outnumber those bearing the CD8b+ phenotype at 60 days of age in BALT and BALT epithelium (Figs. 5 and 6, respectively).
Fig. 3. Number of TCRa/b+ and TCRg/d+ T-cells in BALT of 7 and 12 days old rats. The bars represent the mean (2SE) of the number of cells from 5 animals. Student t-test: p values are not signi®cant.
Fig. 4. Number of CD5+ T-cells in BALT and BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 10 animals. Tukey±Kramer: BALT: (#) 21, 39, 45 vs. 60, p < 0.001; BALT epithelium: () 21 vs. 45, 60, p < 0.001 and 39 vs. 45, 60, p < 0.05.
The number of IgA+ B and CD4+ T cells found in BALT increases with age (Fig. 7). CD4+ T cells in the BALT epithelium remain constant from 21 up to 60 days of age (average 8 CD4+ intraepithelial lymphocytes per 15 ®elds). The development of TCRg/d T cells in BALT of Wistar rats from 7 to 60 days of age has been recently published [15] and it is interesting to compare these data with those obtained studying the development of TCRa/b+ T cells. The number of T cells expressing TCRg/d outnumbers T cells expressing TCRa/b from 21 up to 45 days in BALT and up to 39 days in BALT
Fig. 5. Number of CD8a+ and CD8b+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 9 animals. Tukey± Kramer: CD8a: () 21, 39, 45 vs. 60, p < 0.001 and (#) 21 vs. 45, p < 0.01; CD8b: (&) 21 vs. 39, p < 0.01 and (.) 45 vs. 21, 60 p < 0.001.
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Fig. 6. Number of CD8a+ and CD8b+ T-cells in BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 9 animals. Tukey±Kramer: CD8a: () 60 vs. 21, 39, 45, p < 0.001; CD8b: (#): 45 vs. 21, 60, p < 0.001 and (&) 39 vs. 60, p < 0.05.
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Fig. 8. Number of TCRa/b+ and TCRg/d+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 6 animals. Tukey±Kramer: TCRa/b: (#) 21 vs. 45, 60, p < 0.01 and (.) 39 vs. 45, p < 0.01 and 39 vs. 60, p < 0.001; TCRg/d: () 21 vs. 39, p < 0.05; 21 vs. 45, p < 0.001 and 21 vs. 60, p < 0.01.
Even though several authors have studied rat BALT development, there are few reports
about the early appearance of immunocompetent cells. The gut-associated lymphoid tissue (GALT) has often been regarded as analogous to BALT [5,8] but it is interesting to note the dierences between the development of BALT and GALT. It is known that on day 20 of gestation the rat gut wall shows special spots in which villi are absent, pointing to the developing Peyer's patches where T-lymphocytes are detectable for the ®rst time around birth followed by B cells a few days later [9,16]. Peyer's patches reach their adult structure only 4 weeks after birth with the appearance of
Fig. 7. Number of IgA+ B and CD4+ T-cells in BALT of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 4 to 8 animals. Tukey± Kramer: () IgA: 21, 39, 45 vs. 60, p < 0.001; CD4: (#) 21 vs. 39, p < 0.01 and 21 vs. 45, p < 0.001; (.) 21, 39, 45 vs. 60, p < 0.001.
Fig. 9. Number of TCRa/b+ and TCRg/d+ T-cells in BALT epithelium of 21, 39, 45 and 60 days old rats. The bars represent the mean (2SE) of the number of cells from 5 to 11 animals. Tukey±Kramer: TCRa/b: () 60 vs. 21, p < 0.001 and 60 vs. 39, 45, p < 0.01; TCRg/d: (#) 39 vs. 45, 60, p < 0.05.
epithelium. At day 60 TCRa/b+ T cells predominate in BALT and BALT epithelium (Figs. 8 and 9, respectively). At 60 days of age lymphoid cells appeared to be closely packed adjacent to blood vessels and the overlying epithelium was in®ltrated with lymphocytes as it can be seen in Fig. 10. 4. Discussion
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germinal centers [9,16], indicating that young rats are not able to generate an IgA memory until 4 weeks of age [9]. Recent studies indicate that gd+ T cells are found in foetal gut as well as in foetal thymus and liver [13]. In birds and mammals T-cell dierentiation proceeds along two discrete pathways characterized by the expression of a/b or g/d TCRs. The conservation of these T-cell lineages during vertebrate evolution is further indicated by identi®cation of a, b, g and d TCR genes among amphibians, bony ®shes and cartilaginous ®shes [17]. In the chicken and in the ruminant immune system the gd T cells comprise a much higher proportion that the one found in humans and mice [18]. It has been reported that at 15 days of age g/ d+ T cells appear in the periphery of lymphoid aggregates of the rat BALT [11] and also in the epithelium of the chicken caecal tonsil [19]. In the present report, we ascertain the appearance of a high number of g/d+ T cells at 4 days
of age with the ®rst BALU, increasing up to 45 days of age. Besides, at 60 days of age TCRa/ b+ T cells outnumber TCRg/d+ cells both in rat BALT and BALT epithelium. The number of the dierent T-cell subsets also increases with age, up to 60 days of age implying a maturity allowing the response to inhaled antigens as a consequence of the very early appearance of T cells with adult phenotypes. However, in contrast with a previous report [4], we found in adult rats (60 days of age) a predominance of the suppressor/cytotoxic T cells (CD8+) over T-helper cells in BALT and BALT epithelium. Besides, due to the possibility of detecting both CD8 subsets we found that the CD8a T-cell population outnumbers the CD8b one in BALT and BALT epithelium. The present observation diers from the data obtained from gut lamina propria and intraepithelium where heterodimeric CD8a/b isoform predominates [20]. Such a disparity may be explained by microenvironmental dierences in the antigenic repertoire between the gut and the respiratory tract.
Fig. 10. Haematoxylin±eosin paran section of rat BALU 60 days of age. Arrows indicate intraepithelial lymphocytes (magni®cation 1000).
M.G. MaÂrquez et al. / Developmental and Comparative Immunology 24 (2000) 683±689
From weaning up to 60 days of age the appearance of IgA+ B cells in BALT was followed and we found a rapid increase of these cells accompanied by an increase of CD4+ T cells. This coincides with the appearance of germinal centers in Peyer's patches [9,16]. The early appearance of g/d+ T cells in BALT may indicate that this population contributes to the mucosae immunosurveillance at an early stage of development. Little information is available regarding the development of T-cell function in the early postnatal period of the rat but our phenotypic study shows that T cells in BALT and BALT epithelium are predominantly of the suppressor/ cytotoxic phenotype since they appear very early in BALT development.
[7] [8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements The authors thank the Department of Nutrition for the use of animal facilities. This investigation was supported by grant PIP 4147 from CONICET and grant TB 072 from UBA. This paper is part of the Thesis of MarõÂ a Gabriela MaÂrquez.
[14]
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