Effects of pepsin and pepstatin on reflux tonsil hypertrophy in vitro

Abstract

There is evidence that pepsin can aggravate tonsil hypertrophy. Pepstatin is a potent inhibitor of pepsin activity and could protect patients against reflux tonsil hypertrophy by inhibiting pepsin. We examined the effects of pepstatin on the development of tonsil hypertrophy to investigate pepsin's role in the pathogenesis of tonsil lesions. We investigated whether pepstatin suppresses pepsin-mediated lymphocyte proliferation in tonsil hypertrophy. Forty-nine children with tonsil hypertrophy and twenty-two adults with tonsillitis were recruited to the study prior to surgery. Tonsil tissue from each patient was harvested and assessed for changes in the number of lymphocytes and macrophages in the presence of pepsin and pepstatin. We found that the proportions of CD4- and CD14-positive cells were significantly lower (p < 0.05), but that the proportions of CD19- and CD68-positive cells were significantly higher (p < 0.05), in children than in adults. There were significantly more CD4-positive cells after pepsin treatment, but these numbers were reduced by pepstatin. The levels of both interleukin-2 (IL-2) and interferon gamma (IFN-γ) increased significantly in response to pepsin, but were reduced when pepsin was inhibited by pepstatin. The level of IL-10 is reduced in pepsin-treated CD4 cells and the level is restored by pepstatin. IL-2 blocking reduced the increased CD4 cell number by pepsin. But, an additive or a synergic effect is not founded in combined with IL-2 blocking and pepstatin. Pepsin-positive cells did not co-localize with CD20 and CD45 cells, but they were found surrounding CD20- and CD45-positive hypertrophic tonsil cells. Pepsin-positive cells co-localized with CD68-positive cells. It is probable that pepsin from extraesophageal reflux aggravates tonsil hypertrophy and pepstatin exerts a protective effect by inhibiting pepsin activity.

Fig 1. Immune cell population from hypertrophic tonsils and patients with tonsillitis.

Tissues were homogenized and washed with cold phosphate-buffered saline, and tonsil cells were stained with anti-CD4, CD14, CD68, and CD19 antibodies to identify T lymphocytes, monocytes, macrophages, and B cells, respectively. Values represent mean ± standard error of the mean (SEM). *p < 0.05 (patients <16 years, n = 46; patients >16 years, n = 20).

Fig 2. Effect of pepsin on proliferation of cells from patients with tonsil hypertrophy and tonsillitis.

Tonsil cells from pediatric patients with hypertrophic tonsils (<16 years. A) and from patients with tonsillitis (>16 years. B) were isolated using magnetic-activated cell sorting (MACS), treated with pepsin and pepstatin for 7 d, and counted. Fold changes were calculated relative to final values in the absence of pepsin and pepstatin (set as “1”). Values represent mean ± SEM. *p < 0.05 (n = 35). 1P, 1.0 μg/ml pepsin; 0.5 PS, 0.5 μg/ml pepstatin; 1 PS, 1.0 μg/ml pepstatin; CTL, no pepsin or pepstatin.

Fig 3. Effect of pepsin on secretion of interleukin-2 (IL-2) and interferon gamma (IFN-γ), and interleukin 10 (IL-10) by CD4-positive cells and anti-pepsin effect of IL-2 blocking.

CD4-positive cells were isolated using MACS and treated with pepsin and pepstatin for 7 d. The IL-2, IFN-γ, and IL-10 levels in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA) (A). IL-2 is blocked by IL-2 blocking antibody (anti-IL-2) on day 3 and day 7. The cell number is counted in each treated groups (B). Values represent mean ± SEM. *p < 0.05 (n = 17). 1P, 1.0 μg/ml pepsin; 0.5 PS, 0.5 μg/ml pepstatin; 1 PS, 1.0 μg/ml pepstatin; CTL, no pepsin or pepstatin.

Fig 4. Co-localization of pepsin and lymphocytes in hypertrophic tonsil sections.

No co-localization of pepsin with CD4- (A) and CD19 (B)-positive cells was detected. Co-localization of pepsin and CD68-positive cells was detected in the surrounding germinal centers (C, upper) and crypt epithelium (C, lower). Scale bar equals 50 μm.