During or soon after RSV infection, superinfections of the respiratory tract with bacteria such as NTHI occur frequently. In the present study, the effect of RSV infection on NTHI attachment to A549, a human Type II-like alveolar epithelial cell line, was analyzed by quantititative culture or by flow cytometry using fluorescence-labeled NTHI. Additionally, the effect of NTHI and RSV on production of IL-8, a potent chemoattractant for neutrophils, was investigated using an ELISA kit. A549 cells were grown as a confluent monolayer in tissue culture inserts and were inoculated with NTHI (A549:NTHI = 1:1,5,10,100) and/or RSV (multiplicity of infection = 1 and 5). The attachment of NTHI was evaluated at 24 and 48 h of RSV infection, whereas, IL-8 production was evaluated at 12, 24, 48 and 56 of NTHI/RSV infection.

Compared to non-RSV control cells, NTHI attachment to A549 cells infected with RSV was significantly increased (>×2; P<0.001). This increase in NTHI attachment was dependent on time as well as dose of RSV infection. Furthermore, NTHI induced a dose- and time-dependent increase in IL-8 production which was greater in A549 cells simultaneously incubated with NTHI and RSV than those incubated with RSV alone, although the differences were most pronounced at 12 and 24 h {eg. mean IL-8 (ng/ml) at 24 h: RSV=0.97 vs. RSV+NTHI=1.85; P<0.05}. The effect of dead and live NTHI on IL-8 production was comparable.

In summary, RSV infection enhances the attachment of NTHI to respiratory epithelial cells. NTHI further enhances the production of IL-8 by RSV-infected epithelial cells. In clinical settings, increased attachment of NTHI to RSV infected cells may lead to the development of otitis media, sinusitis and lower airway complications. Furthermore, enhanced production of IL-8 during combined RSV/NTHI infection may increase the disease severity related to the toxic products released by activated neutrophils.