VETERINARSKI ARHIV 68 (5), 183-189, 1998

ISSN 0372-5480
Printed in Croatia

Development of T-cell sub-populations in postnatal chicken lymphoid organs

Mohammad Zahirul Islam Khan¹, Yoshiharu Hashimoto², and Mohammad Asaduzzman¹

¹Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Bangladesh

²Laboratory of Anatomy, Department of Biomedical Science, Postgraduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

* Contact address:
Prof. Dr. Mohammad Zahirul Islam Khan,
Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh,
Phone: 88 91 5596 2161; Fax: 88 91 558 10

KHAN, M. Z. I., Y. HASHIMOTO, M. ASADUZZMAN: Development of T-cell sub-populations in postnatal chicken lymphoid organs. Vet. arhiv 68, 183-189, 1998.

ABSTRACT

Postnatal development of T-cell sub-populations was studied in the lymphoid organs of White Leghorn chickens, using an immunohistochemical method. The chicken lymphoid organs were very small at hatchery. Their further growth was correlated with the advancement of age of the chicken. CD3, CD4, CD8, gd-TCR, ab-Vb1-TCR and ab-Vb2-TCR-positive cells were stained in all age groups of chickens, being highest in frequencies of occurrence at 15 weeks. A significant decline was observed at 78 weeks of age. These results indicated that the postnatal development of T-cell sub-populations in the lymphoid organs of chickens is age-dependent.

Key words: T-cells, lymphoid organs, immunohistochemistry, postnatal chicken, White Leghorn/Dekalb chicken

Introduction

Chickens have been used as experimental animals for studies of the immune system because chicken T and B cells maturate in the thymus and bursa of Fabricius, respectively, and because these organs are easily manipulated. The development, differentiation, histological distribution and function of lymphocytes in these tissues, including cecal tonsils and spleen of chicken, have been investigated by using surgical thymectomy, bursectomy, X-ray irradiation, colloidal carbon uptake method, and administration of immunosuppressive drugs (PAPERMASTER and GOOD, 1962; GRAETZER et al., 1963; COOPER et al., 1965; HOSHI and MORI, 1973; SUGIMURA and HASHIMOTO, 1980; BEFUS et al., 1980). In addition, the conventional hematoxylin and eosin method was also used for the study of lymphoid organs in chicken (BEFUS et al., 1980). Recently, KON-OGURA et al. (1993) used monoclonal antibodies Lc-6 and Lc-4, specific chicken CD4⁺ and CD8⁺ lymphocytes, respectively, to study the T-cell subsets in chicken lymphoid tissues.

In the present study, a variety of novel monoclonal antibodies (mAbs) (CHEN et al., 1986; CHEN et al., 1988; CHAN et al., 1988; SOWDER et al., 1988; CIHAK et al., 1988; CHAR et al., 1990) specific for chicken T-cell sub-population were used to evaluate the age-related development of these cells in the postnatal lymphoid organs.

Materials and methods

One-day-old White Leghorn chickens (Dekalb strain) were purchased from a local poultry farm (Uni-cho, Hokkaido, Japan). The chickens were reared in the departmental poultry house in hygienic conditions, with food and water ad libitum. Five chickens were killed by the subluxation method after proper anaesthesia on day one, and thereafter a further five at weeks 1, 4, 5, 9, 15, 19, 21, 32 and 78.

The normal lymphoid organs (spleen, thymus and cecal tonsil) were collected and snap frozen in liquid nitrogen. Cryosections 4 µm thick were placed on 0.5% Neopren W (Nisshin EM Co. Ltd., Japan) coated glass slides and stained with various mAbs (CT3, CT4, CT8, TCR₁, TCR₂ and TCR₃) as previously reported (KHAN et al., 1996a, 1996b, 1996c). The sections were fixed immediately in ice-cold 100% acetone for 15 min and kept at -20 °C until staining. The sections were placed in a moisture chamber and pre-incubated with 2% normal goat serum in 0.01 M phosphate-buffered saline (PBS) for 30 min, followed by incubation with mAbs (CT3, CT4, and CT8 mAbs were used at a dilution of 1:200, 1:100, and 1:100 respectively; TCR₁, TCR₂ and TCR₃ mAbs were all diluted in 1:500) for 3 h at room temperature. The sections were then treated with 1% biotin conjugated goat anti-mouse Igs (Tago Immunologicals, USA) for 1 h, followed by incubation with ABC (Avidin-Biotin-Complex) solution (Vector Lab., USA) for 1 h. The incubated sections were finally coloured with 0.2 mg 3,3-diamino-benzidine-tetrahydrochloride dihydrate (Kanto Chemical, Japan) per ml Tris-Hydrochloride buffer (0.05 M, pH 7.6) containing 0.03% H₂O₂, and slightly counter-stained with hematoxylin. Control sections were stained as described above, but omitting the mAbs.

The frequency of occurrence of immino-stained cells was observed in 40 fields (each field was 0.158 mm²) of light microscope at × 40 magnification.

Results and discussion

The major lymphoid organs of chicken (spleen, thymus and cecal tonsil) were very small at hatching. Their size increased gradually with the advancement of age, and at about 15 weeks of age the size of lymphoid organs attained a peak (data not presented). The mAbs, immunoreactive with T-cell subsets, were stained in these organs in all groups. The development of T-cell subsets also peaked at 15 weeks of age (data not presented).

In the spleen, the CD3⁺ cells were located in periarterial lymphatic tissues (PALT) of the white pulp and the venous sinusoids (VS) of the red pulp. The CD4⁺ cells were stained both in the PALT and VS (Fig. 1). In contrast, CD8⁺ cells were preferentially located only in the VS from birth to 5 weeks (Fig. 2a). The CD8⁺ cells started to infiltrate the PALT from 9 weeks of age and peaked at 15 weeks of age (Fig. 2b). gd-TCR⁺ cells were present almost exclusively in the VS with strong peroxidase reaction. From 9 weeks these cells were also specially found in the PALT, and their peak in the PALT was noticed from 15 to 19 weeks (Fig. 3). In contrast to the gd-TCR⁺ cells, the ab-TCR⁺ cells were preferentially located in the PALT although some of the sinusoidal CD8⁺ cells were ab-Vb1-TCR⁺. The frequencies of occurrence of ab1-Vb2-TCR⁺ cells were very low and randomly stained in the two splenic compartments. In the thymus, CD3, gd-TCR, ab-Vb1-TCR, and ab-Vb2-TCR⁺ cells were distributed both in the cortex and medulla throughout the postnatal staged of development, although gd-TCR⁺ cells were sparse in the cortex. The CD4⁺ cells stained uniformly both in the cortex and medulla from one day- to 5 weeks old. From 9 to 78 weeks, CD4⁺ cells were relatively depopulated from the medulla, and predominant in the cortex (Fig. 4a-b). The CD8⁺ cells were predominant only in the cortex throughout the postnatal period of development.

Fig. 1. Section of the spleen obtained from 4-week old chicken. CD4⁺ cells are stained both in the venous sinus (VS) and around the periarterial lymphatic tissues (PALT) (A) (arrowheads). Immunohistochemical staining (ABC method).
80 ×; bar = 32 µm.

Fig. 2. Section of the spleen taken from chickens at different postnatal stages of development. CD8⁺ cells are located only in the venous sinus (VS) at 5 weeks of age (2a), and at 15 weeks (2b) these cells are located both in the venous sinus (VS) and around the PALT (A) (arrowheads). Immunohistochemical staining (ABC method).
80 ×; bar = 32 µm.

Fig. 3. Section obtained from the spleen of 19-week-old chicken. Numerous gd-TCR⁺ cells are located around the PALT (arrowheads); VS, venous sinus. Immunohistochemical staining (ABC method). 80 ×; bar = 32 µm.

Fig. 4. Section of thymus obtained from chickens at different postnatal stages of development. CD4⁺ cells are distributed in the cortex (C) and medulla (M) in one-day-old chicken (4a), and at 19 weeks of age (4b) these cells are depopulating from the medulla (asterisks). Immunohistochemical staining (ABC method).
80 ×; bar = 64 µm.

In the cecal tonsil, CD3, CD4, CD8, gd-TCR, ab-Vb1-TCR or ab-Vb2- TCR⁺ cells were observed from one-day to 78 weeks of age and were found both in the lamina propria and the epithelium including CD8⁺ cells (Fig. 5). CD4⁺ and ab-Vb2-TCR⁺ cells were occasionally observed in the epithelium throughout the postnatal period of development.

Fig. 5. Section of cecal tonsil taken from 5-week-old chicken. CD8⁺ cells are located both in the epithelium (E) and the lamina propria (LP). Immunohistochemical staining (ABC method). 80 ×; bar = 32 µm.

In the present study development of T-cell sub-populations in the lymphoid organs of postnatal chickens peaked at about 15 weeks of age, indicating age-related development of T-cells in these organs. However, the gd-TCR⁺ cells were gathered in the PALT from 9 weeks of age and attained a peak from 15 to 19 weeks. These results were not in agreement with the data of BUCY et al. (1988), who reported that gd-TCR⁺ cells are preferentially located only in the venous sinusoids of chicken spleen. Although BUCY et al. (1988) did not mention the age of the chicken, this discrepancy may be due to the use of chickens less than 9 weeks of age. Similarly, CD4⁺ cells were depopulated from the medulla of the thymus from 9 weeks of age, and CD8⁺ cells were abundantly stained both in the lamina propria and the epithelium of the cecal tonsil. KON-OGURA et al. (1993) reported that CD4⁺ cells were stained both in the cortex and the medulla of the thymus, and CD8⁺ cells were found restrictedly in the sub-epithelial spaces of the chicken cecal tonsil. These inconsistent results could be also due to the fact that KON-OGURA et al. (1993) used chickens of from 5 to 7 weeks of age.

In conclusion, we suggest that the dynamic changes of T-cell sub-population in the lymphoid tissue of the postnatal chickens are age dependent.

Acknowledgement
The authors wish to thank Prof. Dr. Toshihiko Iwanaga, Laboratory of Anatomy, Postgraduate School of Veterinary Medicine, Hokkaido University, Japan for his valuable suggestions made during this collaborative research.

References

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Received: 26 January 1998
Accepted: 29 June 1998

KHAN, M. Z. I., Y. HASHIMOTO, M. ASADUZZMAN: Postnatalni razvoj suppopulacija T-stanica u limfoidnim organima pilica. Vet. arhiv 68, 183-189, 1998.

SAZETAK

Imunohistokemijskim metodama je istrazivan postnatalni razvoj suppopulacija T-stanica u limfoidnim organima bijelih Leghorn pilica. Pri valjenju su limfoidni organi pilica bili vrlo maleni, a njihov dalji rast stavljan je u korelaciju s dobi. CD3, CD4, CD8, gd-TCR, ab-Vb1-TCR i ab-Vb2-TCR pozitivne stanice bojane su u svih dobnih skupina pilica, a najveca ucestalost je utvrdena u dobi od 15 tjedana. Znacajni pad je utvrden u dobi od 78 tjedana. Rezultati pokazuju da je postanatalni razvoj suppopulacija T-stanica u pilica ovisan o dobi.

Kljucne rijeci: T-stanice, limfoidni organi, imunohistokemija, pilici, bijeli Leghorn/Dekalb pilici