Lipid mediator-induced expression of bactericidal/ permeability-increasing protein (BPI) in human mucosal epithelia

Abstract

Epithelial cells which line mucosal surfaces are the first line of defense against bacterial invasion and infection. Recent studies have also indicated that epithelial cells contribute significantly to the orchestration of ongoing inflammatory processes. Here, we demonstrate that human epithelial cells express bactericidal/permeability-increasing protein (BPI), an antibacterial and endotoxin-neutralizing molecule previously associated with neutrophils. Moreover, we demonstrate that such BPI expression is transcriptionally regulated by analogs of endogenously occurring anti-inflammatory eicosanoids (aspirin-triggered lipoxins, ATLa). Initial studies to verify microarray analysis revealed that epithelial cells of wide origin (oral, pulmonary, and gastrointestinal mucosa) express BPI and each is similarly regulated by aspirin-triggered lipoxins. Studies aimed at localization of BPI revealed that such expression occurs on the cell surface of cultured epithelial cell lines and dominantly localizes to epithelia in human mucosal tissue. Functional studies employing a BPI-neutralizing anti-serum revealed that surface BPI blocks endotoxin-mediated signaling in epithelia and kills Salmonella typhimurium. These studies identify a previously unappreciated "molecular shield" for protection of mucosal surfaces against Gram-negative bacteria and their endotoxin.

Figure 1

BPI induction by ATLa. Confluent epithelial monolayers were exposed to indicated concentrations or time periods of ATLa (1 μM). (A) Quantitative microarray data for BPI in epithelial cells exposed to indicated conditions. (B) Total RNA was isolated from ATLa-exposed KB cells (1 μM, 8 h) and examined for BPI transcript by semiquantitative RT-PCR (increasing numbers of PCR cycles). β actin transcript was examined under similar conditions as an internal standard. (C) KB cells were exposed to indicated concentrations of ATLa for 8 h and examined for BPI transcript by using 28 cycles of PCR. (D–F) Indicated epithelial cell lines were exposed to indicated periods of ATLa, and BPI transcript was determined by 26 cycles of RT-PCR. β actin transcript was used as an internal standard.

Figure 2

Localization of BPI to the cell surface. (A) BPI was localized by confocal microscopy in nonpermeabilized Caco2 cells exposed to vehicle (Top) or ATLa (1 μM, 24 h, Middle and Bottom). BPI adsorbed antisera was used as a control (Bottom). Shown here are confocal sections through the mid-zone, subjunctional portion of epithelial monolayers. Representative experiment from n = 2. (B) T84 cells were preexposed to ATLa (1 μM) for indicated periods of time. Cell surface proteins were nonspecifically labeled with biotin, BPI was immunoprecipitated from cell lysates, resolved by SDS/PAGE, and Western blots were probed with avidin peroxidase. Also shown is the immunoprecipitation control (omission of primary Ab) as well as a biotinylated BPI standard. Representative experiment from n = 3.

Figure 3

BPI functionally regulates epithelial endotoxin responses. (A) Transcriptional induction of ICAM-1 was examined in KB cells in response to indicated concentrations of endotoxin (LPS) in the presence of NHS (5% vol/vol) or IL-1 (10 ng/ml) for 8 h. Total RNA was isolated and used to assess ICAM-1 transcripts by RT-PCR. β actin transcript was used as an internal standard. (B) Endotoxin-induced ICAM-1 expression was examined in the presence polyclonal anti-BPI (+) or in the presence of NGS (−). β actin transcript was used as an internal standard. (C) KB cells were preexposed to ATLa (1 μM, 8 h), and cell surface ELISA was used to screen ICAM-1 protein induction by endotoxin/5% NHS (additional 24 h) in the presence of anti-BPI or control NGS. (*, significantly different from NGS; P < 0.025)

Figure 4

ATLa enhances BPI-dependent bacterial killing. Adherent Caco2 cells were incubated with vehicle or indicated concentrations of ATLa (24 h) and the role of BPI in Caco2 killing of S. typhimurium over a 60-min period was assessed. (A) Concentration-dependent increases in bacterial killing with previous exposure to ATLa. (Inset) A killing curve in the absence of ATLa. (B) Relative contribution of BPI was examined by incubation anti-BPI or control antisera preadsorbed with rBPI. Shown here are pooled results from three experiments. (**, indicates significantly different from control condition; P < 0.01)

Figure 5

Localization of epithelial BPI in human mucosal tissues. Normal human esophageal (A and B) or colon (C and D) specimens were obtained, fixed in 10% buffered formalin, paraffin embedded, and sectioned. After antigen retrieval, sections were stained with rabbit polyclonal BPI antisera (A and C) or control sera (BPI preadsorbed Ab, B and D) followed by peroxidase-coupled secondary Ab and visualized by peroxidase method. Sections were visualized at ×200 magnification. Arrows indicate areas of dominant BPI localization.