Polarized Cytokine Secretion Figure 2. Cytokine and chemokine secretion by 16HBE14o- cells stimulated with poly-L-arginine. 16HBE14o- cells were stimulated with vehicle control (A) or 50 mM poly-L-arginine (B) for 3 hours, and cell supernatants were analyzed using a cytokine antibody array. (C) Summarized data showing the average optical intensity relative to positive control spots. Results represent the mean 6 S.E. for 6-7 sets of each antibody array membrane. (c) Internal positive control, (1) IL-8, (2) IL-1a, (3) IL-6, (4) IL-6sR, (5) IL-12p40, (6) RANTES, (7) TNFa. (*, p,0.05, Student’s t-test compared with untreated control). doi:10.1371/journal.pone.0012091.g002 IL-6 release into both apical and basolateral compartments when compared to the time-matched control. Similar results were obtained when we measured the polarized secretion of IL-8 into apical (Fig. 3C) or basolateral (Fig. 3D) compartments. Fig. 4A shows the concentration effect of poly-L-arginine on IL-6 release in apical and basolateral compartments. Exposure of the cultured epithelia to 1 to 50 mM poly-L-arginine for 3 hours stimulated a concentration-dependent increase from 1 to 10 mM in IL-6 release into both apical and basolateral compartments, which gradually decreased. Exposure to various concentrations of poly-L- arginine for 3 hours induced a significantly higher concentration of IL-6 release into the apical compartment than into the basolateral compartment for all concentrations tested. The data clearly demonstrated polarized secretion of IL-6 into the apical compartment. Similar results were obtained for IL-8 (Fig. 4B), showing that there is a concentration-dependent and predomi- nately apical secretion by the epithelia. To assess if there is significant diffusion of IL-6 or IL-8 from the apical to the basolateral compartment, exogenous recombinant IL-6 or IL-8 (4000 pg/ml) were added to the apical compartment, and their concentrations in the basolateral compartment were measured. After 6 hours, the IL-6 and IL-8 levels were undetectable (n = 3), indicating that cytokine secretion into the apical compartment does not significantly contribute to the amount detected on the other side of the compartment. Experiments were also performed in which poly-L-arginine (50 mM) was added to the basolateral compartment for 3 hours and the IL-6 released into the apical and basolateral compart- ments was measured. The epithelia secreted a much higher level of IL-6 into the apical (32506263 pg/ml) than the basolateral (232646 pg/ml, n = 3, p,0.05) compartment, suggesting that there is polarized secretion of IL-6, regardless of whether the stimulus was applied from the apical or basolateral side of the membrane. To demonstrate whether poly-L-arginine treatment affected mRNA expression of IL-6 and IL-8, cells were stimulated for 3 or 6 hours with 10 mM poly-L-arginine, and mRNA expression was measured with qRT-PCR. Increased IL-6 and IL-8 mRNA PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12091