METHODS

Three 60 ml samples of bronchoalveolar lavage (BAL) fluid were taken from nine stable non-smoking lung transplant recipients (table 1) as previously described.⁶ The selection and procurement of the pulmonary donor,⁷ immunosuppression,⁷ bronchoscopy and BAL, and preparation of cell free BAL supernatants⁶ were carried out as in previous studies.

Detection of N-acylhomoserine lactones (AHLs) in BAL fluid

BAL fluid supernatants (900 μl) were extracted twice with equal volumes of HPLC grade dichloromethane and evaporated to dryness overnight as described by Middleton et al.³ The extracts were reconstituted in 200 μl acetonitrile and 5 μl was analysed by thin layer chromatography. To detect the presence of long chain (C10–14) AHLs, a bioluminescence based E coli biosensor strain (E coli JM109) harbouring the AHL reporter plasmid pSB1075 was used.³ In all cases light emission resulting from the detection of AHLs was captured using a Berthold LB980 photon video camera (E G and G Berthold UK Ltd, Milton Keynes, UK). Synthetic AHLs were used as positive controls. The investigator performing the AHL assay was blind to the patient clinical data.

RESULTS

The clinical details of the study population at the time of the bronchoscopy are shown in table 1, together with the results of clinical microbiological assessment.

BAL was successfully performed in all the lung transplant recipients (median return 90 ml, range 66–120 ml). Neutrophil counts were variable and moderately raised, confirming our previous results (median 3%, range 0–82%).^6,8,9

From the nine extracted BAL fluid supernatants analysed, five exhibited AHL activity with the E coli pSB1075 biosensor, suggesting the presence of long chain AHLs (fig 1, table 1). Interestingly, there was no correlation between the level of AHLs detected (+, ++, +++) or their absence and pretransplant diagnosis, positive BAL microbiological culture (table 1), or lung function.

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Figure 1

Representative bioluminescence capture resulting from the activation of the N-acylhomoserine lactone (AHL) biosensor pSB1075. SN1, 2 and 6=positive patient samples denoted by light emission (white arrows); C12=N-dodecanoyl-L-homoserine lactone standard (10 μg/ml); 3 oxo C12=N-(3-oxododecanoyl)-L-homoserine lactone standard (1 μg/ml).

DISCUSSION

In this study we provide the first evidence of quorum signals in a series of clinically stable human lung allograft recipients, with AHL activity detectable even in subjects with no evidence of rejection, BOS, or infection (clinically or following BAL microbiology).

Many bacteria coordinate gene expression and hence phenotype in a cell density dependent manner employing quorum sensing signal molecules. Of these, the AHLs measured in this study have been intensely investigated in P aeruginosa and B cepacia infection of patients with CF.^3,5

This study supports previous findings that pathophysiological changes are present even in clinically stable lung allograft recipients.^6,8,9 Our reports of increased airway neutrophil and interleukin (IL)-8 levels in clinically stable allograft recipients are potentially complementary to the present study, and we have also shown increased levels of soluble CD14 in BAL fluid.^6,8,9 CD14 is a surface protein, shed following activation, which is found on macrophages and activated neutrophils. It serves as the cellular receptor for lipopolysaccharide, a major component of Gram negative bacteria such as P aeruginosa and B cepacia which produce AHLs.

AHL activity may be particularly strategic, exhibiting pharmacological properties and modulating T cell and macrophage function, as well as being involved in control of virulence factors and biofilm formation and eliciting host IL-8 production.^2,10 This is the first description of quorum signals in lung transplantation, and our findings are limited by the preliminary cross sectional nature of this study. Longitudinal studies are therefore required to track AHL levels and to elucidate potential relationships with infection, BOS, and the pathophysiological deterioration of lung allografts.

The inclusion of rapid DNA based identification methods for P aeruginosa and other bacteria in allografts may be useful in future studies. There is also a need to include appropriate patient controls. This type of study based on the detection of AHL signal molecules may lead to the establishment of a link between the presence of AHLs in allografts and BOS. This may aid the search for novel treatments which interfere with the production and mode of action of these quorum sensing signal molecules.