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Chronic otitis media with effusion (OME) is a frequent disease during childhood, and its complications and sequelae often persist into the adult years (7). Two important factors in the development of OME are obstruction or dysfunction of the eustachian tube and bacterial infection. In chronic OME, the majority of cases are caused by gram-negative bacteria (GNB), whereas in acute OME gram-positive bacteria are also frequently isolated (1). Lipopolysaccharide (LPS) is a component of GNB. LPS alone has been shown to induce mucosal inflammation with accumulation of effusion in the middle ears of chinchillas (2) and guinea pigs (14). Furthermore, LPS has been detected in human middle ear effusions (3), and the level of it was found to be significantly higher in children with chronic OME than in children with acute OME (13). Finally, LPS is also thought to be cytotoxic to ciliated epithelial cells (10).

We recently developed an animal model of chronic OME using a combination of eustachian tube obstruction (ETO) and LPS injection (12). This procedure induces an increase in secretory cells of the epithelium and degeneration of cilia, which results in a disturbance of the mucociliary clearance system (MCS) of the middle ear. Comparable mucosal changes have been observed in humans with chronic OME (9, 15). The MCS is considered to be an important defense system of the middle ear cavity, and disturbance of this system is suspected to be an important factor in the development of chronic OME.

Bactericidal/permeability-increasing protein (BPI), a 55-kDa cationic protein present in the granules of polymorphonuclear neutrophils (PMNs), is an antimicrobial protein that has been implicated in the host defensive response to GNB infection (5). In addition to being bactericidal against GNB, BPI binds to the highly conserved lipid A portion of LPS with high affinity and can inhibit its actions (11). Previous investigations have shown that a 21-kDa recombinant amino-terminal fragment of BPI (rBPI₂₁) protects animals against the effects of GNB and LPS (6). Furthermore, in humans, rBPI₂₁ appears to be safe and non-immunogenic and is in phase II/III clinical trials with apparent therapeutic benefit (4, 6, 8). In the present study, we aimed to assess the in vivo capacity of rBPI₂₁ to prevent mucosal damage in chronic OME.

Induction of chronic OME. During anesthesia with nitrous oxide, the eustachian tube was reached by a ventral approach, medially to the posterior belly of the digastric muscle, and obstructed by plugging a small piece of Gelfoam (Upjohn Co.) into the tube. Moreover, a drop of tissue glue (Historesin; Braun, Melsungen, Germany) was used to keep the Gelfoam in the tube. In addition, 50 µl of LPS solution (2 µg/ml) in phosphate-buffered saline (PBS) prepared from Salmonella typhimurium (L-6511; Sigma, Zwijndrecht, The Netherlands) was injected through the tympanic membrane. As a control, the other ear was injected with 50 µl of PBS.

Histopathological findings. By LM and SEM, control middle ears remained apparently normal during the whole period. The hypotympanum of the middle ear consisted of thin, one-layered squamous epithelium, containing very few ciliated cells (Fig. 1A). In the tympanic orifice of the eustachian tube, a more pseudostratified, cuboidal, or cylindrical epithelium was observed, which contained an abundant number of ciliated cells and few secretory cells (Fig. 2A). This part represents the mucociliary clearance system of the middle ear.

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Light micrographs of the hypotympanum at 12 weeks after PBS injection (A), ETO and LPS injection (B), and ETO plus LPS with rBPI21 injection after 2 days (C). OME induction caused thickening of the mucosa (between arrows) and infiltration of inflammatory cells (arrowheads). This was not observed after rBPI21 injection. Magnification, ×200; bar, 10 µm.

View larger version (106K): [in this window] [in a new window]   FIG. 2.   SEM of the tympanic orifice at 12 weeks after (A) PBS injection, (B) ETO and LPS injection, and (C) ETO plus LPS with rBPI21 injection after 2 days. Abundant goblet cells (arrows) and ciliated cells are present. OME induction caused degeneration of cilia and swollen epithelial cells (asterisks). This was not observed after rBPI21 injection, and the cilia had a normal appearance. Magnification, ×1,250; bar, 10 µm.

View larger version (76K): [in this window] [in a new window]   FIG. 3.   Numbers of ciliated cells ± standard deviations (n = 4) counted in the tympanic orifice after PBS injection, ETO with LPS injection (ETO+LPS), and ETO plus LPS with rBPI21 injection after 2 days (ETO+LPS+BPI). Statistical comparisons were made by the Tukey HSD test. *, significantly different from PBS; **, significantly different from ETO+LPS of the same week. Injection of rBPI21 after 2 days prevented the decrease in the numbers of ciliated cells caused by OME induction.

Treatment with BPI. Two days after induction of chronic OME, 50 µl of 2-mg/ml rBPI₂₁, a recombinant 21-kDa amino-terminal fragment of BPI (Xoma LLC., Berkeley, Calif.), was injected into the middle ear cavity. To ensure complete neutralization of LPS, an amount of rBPI₂₁ equal to 1,000 times the LPS concentration was used (11). It was decided to inject the BPI after 2 days to investigate the effect of BPI during an acute inflammation reaction for prevention of chronic OME. Injection of BPI prevented the thickening of the middle ear mucosa in the hypotympanum seen after ETO in combination with LPS injection. No infiltration of inflammatory cells in the subepithelial layer was observed (Fig. 1C). In the tympanic orifice, an abundance of ciliated cells were present and no significant increase in secretory cells was seen (Fig. 3 and 4). BPI prevented the induction of mucosal changes in the rat middle ear (Fig. 2C) seen after ETO and LPS injection.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   Numbers of goblet cells ± standard deviations (n = 4) counted in the tympanic orifice after PBS injection, ETO with LPS injection (ETO+LPS), and ETO plus LPS with rBPI21 injection after 2 days (ETO+LPS+BPI). Statistical comparisons were made by the Tukey HSD test. *, significantly different from PBS; **, significantly different from ETO+LPS of the same week. Application of rBPI21 after OME induction significantly inhibited the increase in the numbers of goblet cells.