T(H)2-like chemokine patterns correlate with disease severity in patients with recurrent respiratory papillomatosis

Abstract

Recurrent respiratory papillomatosis (RRP), characterized by the recurrent growth of benign tumors of the respiratory tract, is caused by infection with human papillomavirus (HPV), predominantly types 6 and 11. Surgical removal of these lesions can be required as frequently as every 3 to 4 wks to maintain a patent airway. There is no approved medical treatment for this disease. In this study, we have characterized the T(H)2-like chemokine profile (CCL17, CCL18, CCL20, CCL22) in patients with RRP and asked whether it was modulated in patients who had achieved significant clinical improvement. CCL17, CCL18 and CCL22 messenger RNAs (mRNAs) were increased in papillomas compared with clinically normal laryngeal epithelium of the RRP patients. Overall, CCL20 mRNA expression was not increased, but there was intense, selective CCL20 protein expression in the basal layer of the papillomas. Patients with RRP expressed more CCL17 (p = 0.003), CCL18 (p = 0.0003), and CCL22 (p = 0.007) in their plasma than controls. Plasma CCL18 decreased over time in three patients enrolled in a pilot clinical trial of celecoxib, and the decrease occurred in conjunction with clinical improvement. There was a significant correlation between sustained clinical remission in additional patients with RRP and reduced levels of CCL17 (p = 0.01), CCL22 (p = 0.002) and CCL18 (p = 0.05). Thus, the change in expression of these three plasma T(H)2-like chemokines may, with future studies, prove to serve as a useful biomarker for predicting disease prognosis.

Figure 1

Chemokine levels are different in papilloma tissues, uninfected laryngeal tissues of RRP patients, and control laryngeal tissues. The T_H2-like chemokines CCL17, CCL18, CCL20 and CCL22, and the T_H1-like chemokine CCL19 were quantified by reverse transcription real-time PCR in papilloma tissues (black box; n = 9–16) and adjacent tissues from RRP patients (gray box; n = 12–19) and depicted as fold change compared with true normal laryngeal tissue from controls (white box; n = 6–7).

Figure 2

CCL18 localizes to scattered cells in the spinous layer of papilloma tissues and CCL20 localizes to the basal layer. CCL18 and CCL20 were detected by immunohistochemistry in papillomas and clinically normal adjacent epithelium of patients with severe RRP. (A) CCL18 stained scattered cells in the papillomas (thin arrow), but (B) showed only faint diffuse staining in normal adjacent tissues. (C) CCL20 was localized predominately to the basal layer of papillomas, adjacent to the basement membrane, while (D) normal adjacent tissue had uniform staining throughout the epithelium. (E,F) In situ hybridization, reprinted by permission from Steinberg, et al., 1988 (1), shows HPV-6/11 viral expression in the suprabasal layer. A–D scale bar = 20 μm; E and F scale bar = 100 μm; large arrows point to the basement membrane.

Figure 3

T_H2-like chemokines are overexpressed in the plasma of patients with RRP and correlate with disease severity. (A) CCL17, (B) CCL18 and (C) CCL22 were measured in plasma of patients with RRP and controls. The concentration (mean ± SEM) of each chemokine is shown. Concentrations of all three chemokines are higher in patients with RRP: CCL17 pg/mL (severe 317.9 ± 84.0, mild/mod 169.7 ± 22.2, control 98.0 ± 14.3, p = 0.003); CCL18 ng/mL (severe 81.9 ± 12.8, mild/mod 72.7 ± 6.7, control 36.2 ± 8.8, p = 0.0003); and CCL22 pg/mL (severe 686.5 ± 105.1, mild/mod 512.8 ± 31.7, control 411.4 ± 34.0, p = 0.007). Levels are significantly different among patients with severe RRP (n = 10), patients with mild/moderate RRP (n = 16) and controls (n = 14), as determined by ANOVA, Kruskal-Wallis test.

Figure 4

Plasma CCL18 levels decrease in concert with improved clinical status in patients treated with celecoxib, a COX-2 inhibitor. Three patients (A,B,C) in the pilot clinical trial had CCL18 levels (solid line) measured by a single-plex ELISA in plasma samples collected at varying times during the study. Clinical improvement, denoted by reduction in severity score (gray bars), correlated with decreasing CCL18 concentrations.

Figure 5

Plasma CCL17 and CCL22 levels decrease over time in patients who achieve remission of disease. Plasma samples collected every 3 months from seven patients (separate colors) enrolled in an ongoing celecoxib clinical trial who became disease-free for at least 6 months were analyzed for (A) CCL17, (B) CCL22, and (C) CCL18 concentrations via multiplex ELISA. Data is shown relative to time (months) after complete clinical remission. Linear regression was performed for each patient, and a test of repeated measures using severity group as a covariant was employed. CCL17 (p = 0.01) and CCL22 (p = 0.002) strongly correlated with the number of months since remission, CCL18 did not.