Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration

PURPOSE. Previous studies have shown that ovariectomy and hypophysectomy ca use regression of the lacrimal gland and have implicated androgens as troph ic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apopt osis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-indu ced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS. Groups of sexually mature female New Zealand White rabbits were ov ariectomized and killed at various time periods up to 9 days. Additional gr oups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At ea ch time period, sham-operated rabbits were used as controls. Lacrimal gland s were removed and: processed for analysis of apoptosis as assessed by DNA fragmentation and fur morphologic examination, DNA fragmentation was determ ined using the TdT-dUTP terminal nick-end labeling assay and by agarose gel electrophoresis, Labeled nuclei were quantified by automated densitometry. Sections were also stained for RTLA (rabbit thymic lymphocyte antigen), ra bbit CD18, and La antigen. Morphology was evaluated by bath light and elect ron microscopy. RESULTS, The time course of apoptosis exhibited two phases, a rapid and tra nsient phase and a second prolonged phase. A transient phase peaked at appr oximately 4 to 6 hears after ovariectomy. The values for degraded DNA as a percentage of total nuclear area were 4.28% +/- 0.79% and 4.26% +/- 0.54%, respectively. The values for sham-operated controls examined at the same ti me periods were 1.77% +/- 0.08% and 0.82%, +/- 0.21%, respectively. The per centage of degraded DNA at 24 hours after ovariectomy was not different fro m controls examined at the same interval after sham operation. The percenta ge of degraded DNA 6 days after ovariectomy was significantly increased (8. 5% +/- 2.4%), compared with sham-operated animals at the same time period ( 0.68% +/- 0.03%). DNA laddering was more pronounced after ovariectomy. Dihy drotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections ind icated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ov ariectomy showed nuclear chromatin condensation principally in plasma cells . Increased numbers of macrophages were also evident, Significant levels of cell degeneration and cell debris, characteristic of necrosis, were observ ed in acinar regions 6 days after ovariectomy, Dihydrotestosterone prevente d this necrosis. Increased numbers of RTLA(+), CD18(+), and La+ interstitia l cells were also evident 6 days after ovariectomy. In addition, ovariectom y increased La expression in ductal cells. Dihydrotestosterone treatment pr evented the increase in numbers of lymphoid cells and La expression. Dihydr otestosterone also promoted the appearance of mitotic figures in acinar cel ls and increased the sizes of acini by 43% (P< 0.05). CONCLUSIONS. Glandular atrophy observed after ovariectomy is likely to proc eed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosi s, These processes are accompanied by increased lymphocytic infiltration. T hese results suggest that a critical level of androgen is necessary to main tain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necro sis, and an autoimmune response. Because apoptotic and necrotic cell fragme nts may be sources of autoantigens that can be processed and presented to i nitiate an autoimmune reaction, we surmise that cell death triggered by and rogen withdrawal may trigger an autoimmune response such as that encountere d in Sjogren's syndrome. Therefore, replacement of androgens in states of l ow androgen levels, such as after menopause, might help to cure primary lac rimal deficiency and prevent Sjogren's autoimmunity. The possibility of the role of an autocrine factor, a paracrine factor, or both, promoted by andr ogens, and the action of other hormones, such as gonadotropin releasing fac tor, on lacrimal gland cells needs to be investigated.