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Histochemistry and Cell Biology

, Volume 147, Issue 6, pp 707–719 | Cite as

Localization and pneumococcal alteration of junction proteins in the human alveolar–capillary compartment

  • Andrea Peter
  • Diana Fatykhova
  • Olivia Kershaw
  • Achim D. Gruber
  • Jens Rueckert
  • Jens Neudecker
  • Mario Toennies
  • Torsten T. Bauer
  • Paul Schneider
  • Maria Schimek
  • Stephan Eggeling
  • Norbert Suttorp
  • Andreas C. Hocke
  • Stefan HippenstielEmail author
Original Paper

Abstract

Loss of alveolar barrier function with subsequent respiratory failure is a hallmark of severe pneumonia. Although junctions between endo- and epithelial cells regulate paracellular fluid flux, little is known about their composition and regulation in the human alveolar compartment. High autofluorescence of human lung tissue in particular complicates the determination of subcellular protein localization. By comparing conventional channel mode confocal imaging with spectral imaging and linear unmixing, we demonstrate that background fluorescent spectra and fluorophore signals could be rigorously separated resulting in complete recovery of the specific signal at a high signal–to-noise ratio. Using this technique and Western blotting, we show the expression patterns of tight junction proteins occludin, ZO-1 as well as claudin-3, -4, -5 and -18 and adherence junction protein VE-cadherin in naive or Streptococcus pneumoniae-infected human lung tissue. In uninfected tissues, occludin and ZO-1 formed band-like structures in alveolar epithelial cells type I (AEC I), alveolar epithelial cells type II (AEC II) and lung capillaries, whereas claudin-3, -4 and -18 were visualised in AEC II. Claudin-5 was detected in the endothelium only. Claudin-3, -5, -18 displayed continuous band-like structures, while claudin-4 showed a dot-like expression. Pneumococcal infection reduced alveolar occludin, ZO-1, claudin-5 and VE-cadherin but did not change the presence of claudin-3, -4 and -18. Spectral confocal microscopy allows for the subcellular structural analysis of proteins in highly autofluorescent human lung tissue. The thereby observed deterioration of lung alveolar junctional organisation gives a structural explanation for alveolar barrier disruption in severe pneumococcal pneumonia.

Keywords

Tight junctions Streptococcus pneumoniae Pneumonia Occludin Claudin Spectral confocal microscopy 

Notes

Acknowledgements

Parts of this work are included in the doctoral thesis of Andrea Peter.

Compliance with ethical standards

Support statement

This study was supported by the Transregional Collaborative Research Center SFB-TR84 of the Deutsche Forschungsgemeinschaft (Grants B6 to A.C. Hocke and S. Hippenstiel, Z1a to A.C. Hocke as well as Z1b to A.D. Gruber and B1 to N. Suttorp) and the German Federal Ministry of Education and Research (Grant C1 to S. Hippenstiel and C8 to A.C. Hocke; PROGRESS—Pneumonia Research Network on Genetic Resistance and Susceptibility for the Evolution of Severe Sepsis).

Conflict of interest

The authors have no financial conflict of interest.

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Andrea Peter
    • 1
    • 2
  • Diana Fatykhova
    • 1
  • Olivia Kershaw
    • 3
  • Achim D. Gruber
    • 3
  • Jens Rueckert
    • 5
  • Jens Neudecker
    • 5
  • Mario Toennies
    • 4
  • Torsten T. Bauer
    • 4
  • Paul Schneider
    • 6
  • Maria Schimek
    • 7
  • Stephan Eggeling
    • 7
  • Norbert Suttorp
    • 1
  • Andreas C. Hocke
    • 1
  • Stefan Hippenstiel
    • 1
    • 8
    Email author
  1. 1.Department of Internal Medicine/Infectious Diseases and Pulmonary MedicineCharité-Universitätsmedizin BerlinBerlinGermany
  2. 2.Department for Bioanalytics, Institute of BiotechnologyTechnische Universität BerlinBerlinGermany
  3. 3.Department of Veterinary PathologyFreie Universität BerlinBerlinGermany
  4. 4.Lungenklinik HeckeshornHELIOS Klinikum Emil von BehringBerlinGermany
  5. 5.Department of General, Visceral, Vascular and Thoracic SurgeryCharité-Universitätsmedizin BerlinBerlinGermany
  6. 6.Department for General and Thoracic SurgeryDRK ClinicsBerlinGermany
  7. 7.Vivantes Netzwerk für Gesundheit, Klinikum NeuköllnKlinik für Thoraxchirurgie, BerlinBerlinGermany
  8. 8.Department of Internal Medicine/Infectious Diseases and Pulmonary MedicineCharité-Universitätsmedizin BerlinBerlinGermany

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