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The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface - PubMed

  • ️Thu Jan 01 1998

The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface

R Bals et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The airway surface is an important host defense against pulmonary infection. Secretion of proteins with antimicrobial activity from epithelial cells onto the airway surface represents an important component of this innate immune system. Defensins are the best characterized epithelial-derived peptide antibiotics. A member of another family of peptide antibiotics called cathelicidins recently was identified from human bone marrow. We show in this paper that this human peptide named LL-37/hCAP-18 also may play a role in innate immunity of the human lung. In situ hybridization localized high levels of LL-37/hCAP-18 RNA to surface epithelial cells of the conducting airway as well as serous and mucous cells of the submucosal glands. LL-37/hCAP-18 peptide with antimicrobial activity was partially purified from airway surface fluid from human lung and a human bronchial xenograft model. The synthetic peptide LL-37 demonstrated antibiotic activity against a number of Gram-negative and Gram-positive organisms including Pseudomonas aeruginosa; bacterial killing of LL-37 was sensitive to NaCl and was synergistic with lactoferrin and lysozyme. In summary, we show that LL-37/hCAP-18 is a peptide with broad antimicrobial activity that is secreted onto the airway surface from epithelial cells of the human lung.

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Figures

Figure 1
Figure 1

Tissue distribution of LL-37/hCAP-18 expression in human tissues. A filter dotted with mRNA from a number of human tissues was hybridized to a LL-37/hCAP-18 probe. Signals were quantified by using a PhosphorImager system and normalized to the expression of the housekeeping gene ubiquitin. Data are expressed as relative hybridization signals and are plotted on a log scale. Only those tissues that demonstrated a significant signal over background are presented, except for thyroid gland, which is an example of a negative organ. The experiment was repeated on three occasions with virtually identical results.

Figure 2
Figure 2

In situ hybridization of LL-37/hCAP-18 in epithelia of nonpulmonary tissues. A subset of tissues positive by dot blot analysis was analyzed by in situ hybridization using a probe specific for LL-37/hCAP-18. In each case, tissue sections were hybridized to both an antisense and a sense probe. The following tissues were analyzed: stomach (antisense, A and sense, B), small bowel (antisense, C and sense, D), colon (antisense, E and sense, F), and pancreas (antisense, G and sense, H). In each case the magnification is ×100.

Figure 3
Figure 3

Detection of LL-37/hCAP-18 RNA in airway tissue and cultivated respiratory epithelial cells by RT-PCR. Poly(A)+ RNA was isolated from human lung (lanes 1 and 2) and cultured airway epithelial cells (lanes 3 and 4) and subjected to RT-PCR by using two different sets of primers specific for LL-37/hCAP-18. The PCR mixtures were fractionated on an agarose gel that was stained with ethidium bromide (Left). DNA from the gel was transferred to a filter, which was hybridized to a LL-37/hCAP-18 probe (Right). An autoradiograph of this experiment is shown. Lanes 1 and 3 represent reactions using the primer combination fall 1 and fall 2, whereas lanes 2 and 4 are reactions using combination fall 1 and fall 8. Molecular weight markers measured as base pairs are shown along the left border.

Figure 4
Figure 4

Fluorescent in situ hybridization of LL-37/hCAP-18 in proximal human lung. Sections of human lung were hybridized to a LL-37/hCAP-18 RNA probe and immunolabeled by using an antibody to lysozyme as described in Materials and Methods. (A–F) The bronchial surface epithelium, hybridized to the LL-37/hCAP-18 antisense (A) or sense probe (D), stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (B and E) and visualized by Nomarski optics (C and F). (G–L) Specific evaluation of the submucosal glands, showing the hybridization to the LL-37/hCAP-18 antisense (G) or sense probe (J), the colocalization with lysozyme by immunohistochemistry (H and K), and the visualization by Nomarski optics (I and L). Arrows indicate serous cells and arrowheads indicate mucous cells. In each case the magnification is ×100.

Figure 5
Figure 5

Western blot analysis of HPLC purified airway surface fluid. Airway surface fluid from human lung and a human bronchial xenograft model was extracted, lyophylized, resuspended in water, and fractionated over an HPLC column. Fractions that showed antimicrobial activity and positive immunoreactivity with the anti-LL-37/hCAP-18 antibody in dot blot analysis were analyzed by Western blot. Shown are the results of the Western blot for fractions 35 and 36 from the xenograft (XG) and 57 to 60 from human lung (Lung). The control is 40 ng of synthetic LL-37 (lane LL); 20 μg of a low molecular weight fraction from total mouse lung was loaded into lane ML. Bars along the left border indicate molecular mass markers of 2.3, 3.4, 6.5, 14.3, 21.5, 30.0, and 46.0 kDa.

Figure 6
Figure 6

Synergism of LL-37 with other antimicrobial proteins in killing of various bacteria. The MIC for LL-37 was determined against a number of bacteria (two strains of E. coli, P. aeruginosa, E. faecalis, and S. aureus). The MICs were determined with or without the addition of medium E or the presence of lysozyme (500 μg/ml) and lactoferrin (1 mg/ml for all except S. aureus, which was 250 μg/ml). Pancreatic phospholipase A2 (1.25 mg/ml) was used as negative control. This experiment was repeated on three occasions with essentially identical results.

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References

    1. Sherman M P, Ganz T. Annu Rev Physiol. 1992;54:331–350. - PubMed
    1. Toews G B. Semin Respir Infect. 1993;8:160–167. - PubMed
    1. Ganz T, Lehrer R-I. Pharmacol Ther. 1995;66:191–205. - PubMed
    1. Ganz T, Lehrer R-I. Immunology. 1994;6:584–589. - PubMed
    1. Diamond G, Zasloff M, Eck H, Brasseur M, Maloy W L, Bevins C L. Proc Natl Acad Sci USA. 1994;88:3952–3956. - PMC - PubMed

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