IMMUNOLOGY AND MOLECULAR BIOLOGY In Situ Detection and Quantification of Bursa of Fabricius Cellular Proliferation or Apoptosis in Normal or Steroid-Treated Neonatal Chicks S. E. Higgins,* ,1 L. R. Berghman,* R. W. Moore,* ,2 D. J. Caldwell,* D. Y. Caldwell,* I. Tizard,* and B. M. Hargis* ,1,3 *Departments of Poultry Science and Veterinary Pathobiology, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843-2472 ABSTRACT Apoptosis, or programmed cell death, is believed to be the mechanism for depletion of lympho- cytes recognizing self-antigens following clonal expan- sion in the bursa of Fabricius. Although bursal apoptosis has previously been shown to increase following in vivo exposure to glucocorticoids, the microanatomical site of induced or normal apoptosis has not been unequivocally established. Presently, we adapted the existing terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling (TUNEL) assay for use with neonatal bursae. Similar to previous reports, TUNEL revealed that normal apoptosis is preferentially, but not exclusively, ongoing in bursal follicular cortical cells. Administration of a sin- gle dose of a synthetic glucocorticoid (dexamethasone) or androgen (19-nortestosterone) did not significantly (P < 0.05) alter follicular lymphocyte numbers or apoptosis (Key words: bursa of Fabricius, apoptosis, proliferation, lymphocytes, steroid) 2002 Poultry Science 81:1136–1141 INTRODUCTION The bursa of Fabricius in chickens is the sole site of B- lymphocyte maturation and differentiation (Glick, 1988). External antigens may be introduced into the developing bursa of Fabricius through a process known as cloacal drinking, with subsequent presentation to specialized phagocytic epithelial cells that can ingest and present antigens to developing lymphocytes in the follicular me- dulla (Glick, 1977; Ekino et al., 1985). Within the bursal follicle, lymphocytes are undergoing the process of gene conversion allowing for antigenic diversity (Ratcliffe, 1989). Mature B-lymphocytes from the medulla and cor- tex can emigrate to the blood and peripheral lymphoid organs, where they await activation by contact with the 2002 Poultry Science Association, Inc. Received for publication August 9, 2001. Accepted for publication March 1, 2002. 1 Present Address: Department of Poultry Science, University of Ar- kansas, Fayetteville, Arkansas 72701. 2 Present Address: USDA-ARS-SPARC, 2881 F&B Road, College Sta- tion, TX 77845. 3 To whom correspondence should be addressed: bhargis@uark.edu. 1136 per unit of area at the time points evaluated post-adminis- tration (6 or 24 h). However, administration of 19-Nortes- tosterone increased the interfollicular epithelial thickness, a change usually associated with edema, within 6 h fol- lowing treatment. Additionally, administration of the an- drogen 19-nortestosterone significantly decreased the number of proliferating cells as detected using mouse anti-proliferating cell nuclear antigen (PCNA) as a pri- mary immunohistochemical antibody. In normal (control) bursal sections, occasional follicles consisting of predomi- nantly apoptotic cells were observed (0.26% of follicles). Such follicles were consistently one-tenth the area of nor- mal follicles. This incidental finding may suggest occa- sional occurrence of a common signal for deletion, such as a common integral or clonal mistake, viral infection, or an aberrant paracrine signal. specific antigen that they are individually capable of rec- ognizing (Paramithiotis and Ratcliffe, 1994). Thus, this emigration to, and colonization of, peripheral lymphoid organs and tissues provides a means for continued ability to respond to new antigens and maintainance of humoral immunity once the bursa of Fabricius regresses at puberty (Paramithiotis and Ratcliffe, 1994). Despite the fact the future B-lymphocyte repertoire of the bird is dependent upon these emigrated cells that escape the bursal microenvironment, only 5% of all B lymphocytes ever emigrate to the periphery; the rest of the cells die in situ (Glick, 1988; Paramithiotis and Rat- cliffe, 1994). The process by which B-lymphocytes die in situ without inflammation is called apoptosis (Motyka and Reynolds, 1991; Paramithiotis et al., 1995; Jacobsen et al., 1996). Recently, a transgenic chicken model express- ing a mutant IgM lacking a light chain has been used to further explore the process of B-lymphocyte follicular colonization and selection processes (Sayegh et al., 1999a). Abbreviation Key: DAB= diaminobenzidine; PCNA= proliferating cell nuclear antigen; TBS=Tris-buffered saline; TUNEL= terminal deoxy- nucleotidal transferase-mediated dUTP nick-end labeling. Downloaded from https://academic.oup.com/ps/article-abstract/81/8/1136/1551242 by guest on 28 July 2018