Open Access

Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus

  • Nils T Veith1,
  • Thomas Tschernig1Email author,
  • Birgitt Gutbier2,
  • Martin Witzenrath2,
  • Carola Meier1,
  • Michael Menger3 and
  • Markus Bischoff4
Respiratory Research201415:85

https://doi.org/10.1186/s12931-014-0085-2

Received: 20 February 2014

Accepted: 20 July 2014

Published: 5 August 2014

Abstract

Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.

Findings

Collectins are a part of surfactant proteins and have significant functions in the opsonization and clearance of bacteria in the pulmonary microenvironment. Absence of surfactant protein A (SP-A) leads to increased susceptibility for bacterial infections [1]–[3]. Interestingly, SP-A binds to the S. aureus extracellular adherence protein, Eap, thereby enhancing phagocytosis and killing of S. aureus by alveolar macrophages [4]. In this study the role of SP-A in the phagocytosis of S. aureus in the peripheral alveoli was investigated in vivo by intravital microscopy. Fluorescence labelling and intratracheal inoculation of S. aureus as well as intravital microscopy were performed essentially as described before [5]. Briefly, exponential growth phase cells of S. aureus strain Newman were labelled using 5-[6]-carboxyfluorescein diacetate succinimidyl ester. The suspension (100 μl containing 2 × 108 colony forming units) was injected into the inspiration limb. Mice (7 wild-type and 10 SP-A−/− on C57BL/6 background, 20 to 25 g body weight; [1]) were anesthetized and ventilated after tracheotomy. Fluorescence-labelled viable bacteria were administered and a thoracotomy was performed. Imaging commenced thirty minutes after the application of bacteria. Three 5 min intervals were recorded and then the experiment was concluded. The complete anterior part of the right thorax was removed surgically. A micromanipulator was used to position a cover-glass horizontally over the surface of the lung. By using a drop of warm saline the lung surface was attached by adhesion forces to the lower surface of the cover-glass. Ventilation was discontinued occasionally for up to 15 seconds to avoid ventilation-induced movements and to obtain a comparable inflation of the lung (PEEP 4 cm H2O). The upper right lung lobe was imaged by means of a fluorescence microscope and a filter set for blue light (450–490 nm) epi-illumination. Microscopic images were recorded by means of a charge-coupled device video camera and digitally recorded for subsequent off-line analyses. For each time interval (0–5 min, 5–10 min, 10–15 min) randomly selected fields (3 per interval and animal) were chosen to determine the number of active cells of phagocytosis. Data are given as mean values ± standard deviation (SD). At the end of the experiment all lungs revealed normal morphology. No edema was found after this short viewing period (Figure 1A-B). The fluorescent staphylococci reached the alveoli and were ingested within minutes. Phagocytes which had internalized labelled bacteria shone brightly in the outer alveoli (Figure 1C-D). As known from previous studies, most of the phagocytes detected early after inoculation are alveolar macrophages [5]. In all intervals a significantly lower number of phagocytes filled with staphylococci were found in SP-A deficient mice than in WT (Figure 1E-F), confirming recent findings indicating SP-A to mediate pulmonary clearance of Staphylococcus aureus[4]. To proove that interaction of SP-A with the bacterial cell wall-associated Eap contributes to phagocytosis, four experiments were performed using C57BL/6 mice and cells of the Eap-deficient S. aureus Newman derivative mAH12 (Newman eap::ermB; [6]) under identical conditions. Here, results similar to those with the SP-A deficient mice and S. aureus Newman wild type cells were obtained (Figure 1E-H). These findings further support the above-mentioned study on the roles of SP-A and Eap in promoting phagocytosis of staphylococci by alveolar macrophages [4].
Figure 1

Ingestion of fluorescence-labelled S. aureus Newman and Newman eap cells by alveolar macrophages over time in wild-type and SP-A −/− mice (n = 4–10). (A, B) Normal lung histology was observed after the experiment in the SP-A deficient animals (A) and in the wild-type animals (B). (C, D) A representative image of intravital microscopy shows bright, shining phagocytes in the alveoli which have ingested fluorescent bacteria. Clearly less of those phagocytes could be seen in SP-A deficient animals (C) compared to wild-type animals (D). (E- H) The count of such cells is presented in three subsequent intervals (E , F , G). A slight increase was observed over time (H).

Declarations

Acknowledgements

The authors thank Ann Soether for editing the language. This study was supported by a grant of the German Ministry for Education and Research (BMBF) #01 KI 070103 and by a grant of the Deutsche Forschungsgemeinschaft (DFG) SFB-TR 84, TP C06.

Authors’ Affiliations

(1)
Institute of Anatomy and Cell Biology
(2)
Department for Infectious and Pulmonary Disease, Charité Universitätsmedizin Berlin
(3)
Institute for Clinical and Experimental Surgery, University of Saarland
(4)
Institute for Microbiology and Hygiene, Saarland University, Medical Faculty

References

  1. LeVine AM, Bruno MD, Huelsman KM, Ross GF, Whitsett JA, Korfhagen TR: Surfactant protein A-deficient mice are susceptible to group B streptococcal infection. J Immunol. 1997, 158: 4336-4340.PubMedGoogle Scholar
  2. Kudo K, Sano H, Takahashi H, Kuronuma K, Yokota S, Fujii N, Shimada K, Yano I, Kumazawa Y, Voelker DR, Abe S, Kuroki Y: Pulmonary collectins enhance phagocytosis ofMycobacterium aviumthrough increased activity of mannose receptor.J Immunol 2004, 172:7592–7602.,PubMedView ArticleGoogle Scholar
  3. Kuronuma K, Sano H, Kato K, Kudo K, Hyakushima N, Yokota S, Takahashi H, Fujii N, Suzuki H, Kodama T, Abe S, Kuroki Y: Pulmonary surfactant protein A augments the phagocytosis ofStreptococcus pneumoniaeby alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A.J Biol Chem 2004, 279:21421–21430.,View ArticleGoogle Scholar
  4. Sever-Chroneos Z, Krupa A, Davis J, Hasan M, Yang CH, Szeliga J, Herrmann M, Hussain M, Geisbrecht BV, Kobzik L, Chroneos ZC: Surfactant Protein A (SP-A)-mediated clearance ofStaphylococcus aureusinvolves binding of SP-A to the staphylococcal adhesion Eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A.J Biol Chem 2011, 286:4854–4870.,View ArticleGoogle Scholar
  5. Tschernig T, Veith NT, Schramm R, Laschke MW, Roller J, Rosenbruch M, Theegarten D, Bischoff M, Meier C, Menger MD: Direct visualisation of microparticles in the living lung. Exp Toxicol Pathol. 2013, 65: 883-886. 10.1016/j.etp.2012.12.007.PubMedView ArticleGoogle Scholar
  6. Hussain M, Haggar A, Heilmann C, Peters G, Flock JI, Herrmann M: Insertional inactivation of Eap inStaphylococcus aureusstrain Newman confers reduced staphylococcal binding to fibroblasts.Infect Immun 2002, 70:2933–2940.,PubMedPubMed CentralView ArticleGoogle Scholar

Copyright

© Veith et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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