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
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).

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