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

PURPOSE. Previous studies have shown that ovariectomy and hypophysectomy cause regression of the lacrimal gland and have implicated androgens as trophic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apoptosis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-induced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS. Groups of sexually mature female New Zealand White rabbits were ovariectomized and killed at various time periods up to 9 days. Additional groups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At each time period, sham-operated rabbits were used as controls. Lacrimal glands were removed and: processed for analysis of apoptosis as assessed by DNA fragmentation and fur morphologic examination, DNA fragmentation was determined 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), rabbit CD18, and La antigen. Morphology was evaluated by bath light and electron microscopy. RESULTS, The time course of apoptosis exhibited two phases, a rapid and transient phase and a second prolonged phase. A transient phase peaked at approximately 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 time periods were 1.77% +/- 0.08% and 0.82%, +/- 0.21%, respectively. The percentage of degraded DNA at 24 hours after ovariectomy was not different from controls examined at the same interval after sham operation. The percentage 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. Dihydrotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections indicated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ovariectomy 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 observed in acinar regions 6 days after ovariectomy, Dihydrotestosterone prevented this necrosis. Increased numbers of RTLA(+), CD18(+), and La+ interstitial cells were also evident 6 days after ovariectomy. In addition, ovariectomy increased La expression in ductal cells. Dihydrotestosterone treatment prevented the increase in numbers of lymphoid cells and La expression. Dihydrotestosterone also promoted the appearance of mitotic figures in acinar cells and increased the sizes of acini by 43% (P< 0.05). CONCLUSIONS. Glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosis, These processes are accompanied by increased lymphocytic infiltration. These results suggest that a critical level of androgen is necessary to maintain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necrosis, and an autoimmune response. Because apoptotic and necrotic cell fragments may be sources of autoantigens that can be processed and presented to initiate an autoimmune reaction, we surmise that cell death triggered by androgen withdrawal may trigger an autoimmune response such as that encountered in Sjogren's syndrome. Therefore, replacement of androgens in states of low androgen levels, such as after menopause, might help to cure primary lacrimal deficiency and prevent Sjogren's autoimmunity. The possibility of the role of an autocrine factor, a paracrine factor, or both, promoted by androgens, and the action of other hormones, such as gonadotropin releasing factor, on lacrimal gland cells needs to be investigated.