Increased cellular proliferation and inflammatory cytokines in tonsils derived from children with obstructive sleep apnea

Abstract

Adenotonsillar hypertrophy is the major pathophysiological mechanism underlying obstructive sleep apnea (OSA) and recurrent tonsillitis (RI) in children. The increased expression of various mediators of the inflammatory response in tonsils of patients with OSA prompted our hypothesis that the enhanced local and systemic inflammation in children with OSA would promote tonsillar proliferation. Mixed cell cultures from tonsils recovered during adenotonsillectomy in children with OSA and RI were established, and proliferative rates were assessed. Cells were also cultured to determine the levels of proinflammatory cytokines and antioxidant protein levels and mRNA expression. Global cell proliferative rates from OSA tonsils were significantly higher than RI (p < 0.01), with CD3, CD4, and CD8 cell proliferation being higher in OSA (p < 0.05). Moreover, proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1alpha, were highly expressed in OSA-derived tonsils. Furthermore, thioredoxin (TRX), an antioxidant protein, was also highly expressed in OSA tonsils at the mRNA and protein levels (p < 0.01). Thus, T cells are in a highly proliferative state in the tonsils of children with OSA and are associated with increased production of proinflammatory cytokines and TRX, when compared with children with RI.

Figure 1

Tonsillar cellular proliferation in children with OSA (n=25) and RI (n=20) in a mixed cell culture system. Cell proliferation was assessed using a colorimetric method and is expressed in relative 490 nm absorbance units. **P<0.01

Figure 2

Proliferation of CD3+ and CD19+ cells in tonsils from children with OSA and RI in a mixed cell culture system. A. Illustrative example of the flow cytometry-based strategy for detection of CD3+ and CD19+ cell proliferation. B. CD3+ (■) and CD19+ (□) cell proliferation in tonsillar cultures in a mixed cell culture system in children with OSA (n=12) and RI (n=11). Data are expressed as the percentage of BrdU+ cells out of total CD3+ and CD19+cell populations. *P<0.05

Figure 3

CD4+ and CD8+ expression in intact tonsillar tissues and in a mixed cell culture system in children with OSA and RI. A. Representative immunohistochemical assessment for CD4+ and CD8+ in tonsils from children with OSA and RI. A higher abundance of CD4+ (Green) and CD8+ (Red) is apparent in the OSA children. Scaling bars indicate 200 µm for upper panels and 50 µm for lower panels. B. CD4 (■) and CD8 (□) mRNA expression in a mixed tonsillar cell culture system in children with OSA (n=12) and RI (n=11). Data are expressed as relative fold increases relative to RI. *P<0.05

Figure 4

TRX expression at both protein and mRNA levels in children with OSA (n=12) and RI (n=11) in a mixed tonsillar cell culture system. A. TRX mRNA expression in OSA children compared to RI children, Data are expressed as fold increase relative to RI. B. Representative immunoblots are shown of TRX (detected at 12 kD) and β-actin. Data are summarized graphically in relative intensity units. **P<0.01

Figure 5

Pro-inflammatory cytokine expression at both protein and mRNA levels in children with OSA and RI in a mixed tonsillar cell culture system. A. TNF-α (■), IL-6 (□), and IL-1α () pro-inflammatory cytokine concentrations in cell-free supernatants in OSA children (n=23) compared to RI children (n=19). B. TNF-α (■), IL-6 (□), and IL-1α () mRNA expression in OSA children (n=12) compared to RI children (n=11). Data are expressed as fold increase. *P<0.05, **P<0.01