Department of Clinical Microbiology, 9301 Rigshospitalet, Juliane Maries Vej 22, DK-2100, Copenhagen Ø, Denmark1
Institute of Microbiology, Technical University of Denmark, DK-2800, Lyngby, Denmark2
Hygiene Institute, University of Tübingen,D-72074, Tübingen, Germany3
Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, USA4
Bartholin Institute, Kommunehospitalet, Copenhagen, Denmark5
Author for correspondence: Hong Wu. Tel: +45 35456427. Fax: +45 35456412. e-mail: wuhong{at}get2net.dk
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ABSTRACT |
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Keywords: lasI and rhlI, N-acylhomoserine lactones, chronic lung infection, cystic fibrosis, rat model
Abbreviations: AHL, N-acylhomoserine lactone; CF, cystic fibrosis; IFN-, interferon
; IL, interleukin; LIMP, lung index of macroscopic pathology; MN, mononuclear leukocyte; p.i., post-infection; PMN, polymorphonuclear leukocyte; St-Ag, P. aeruginosa standard antigen
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INTRODUCTION |
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P. aeruginosa can produce a number of cell-associated and extracellular virulence factors which contribute to its pathogenesis (Doring et al., 1984 , 1985
, 1987
; Jaeger, 1994
; Van Delden & Iglewski, 1998
). The extracellular virulence factors include proteases (elastase, Staphylolytic protease and alkaline protease), pigments (pyocyanin, pyoverdine), haemolysins, exoenzyme S and exotoxin A (Pollack, 1984
; Van Delden & Iglewski, 1998
). In P. aeruginosa, it has been demonstrated that the production of most of the extracellular virulence factors is controlled by quorum sensing systems in vitro (Pesci et al., 1997
; Pesci & Iglewski, 1997
; Van Delden & Iglewski, 1998
). Quorum sensing systems exert their action by small diffusible signal molecules called N-acylhomoserine lactones (AHLs) (Salmond et al., 1995
; Fuqua et al., 1996
; Fuqua & Greenberg, 1998
). The signal molecules are synthesized from precursors by a synthetase (a LuxI homologue) and they interact with a transcriptional activator (a LuxR homologue) to induce the expression of target genes (Fuqua et al., 1996
). In P. aeruginosa there are at least two different quorum sensing systems: las and rhl (Gambello & Iglewski, 1991
; Seed et al., 1995
; Ochsner & Reiser, 1995
), which code for synthetases (LasI/RhlI) and cognate transcriptional regulators (LasR/RhlR). The lasIlasR system has been shown to modulate expression of lasI itself (Seed et al., 1995
), lasB (elastase) (Passador et al., 1993
; Pearson et al., 1997
), lasA (Staphylolytic protease) (Gambello et al., 1993
), apr (alkaline protease) (Gambello et al., 1993
), the xcp secretion pathway (Chapon-Herve et al., 1997
), twitching motility (Glessner et al., 1999
) and rhlR (Latifi et al., 1996
; Pesci et al., 1997
). The rhlIrhlR system modulates expression of rhlI itself (Latifi et al., 1996
), rhlAB (rhamnolipid biosynthesis) (Ochsner & Reiser, 1995
; Pearson et al., 1997
), lasB (elastase) (Brint & Ohman, 1995
; Pearson et al., 1995
, 1997
), twitching motility (Glessner et al., 1999
) and rpoS (Latifi et al., 1996
). The lasI and rhlI products are N-oxododecanoyl homoserine lactone (OdDHL, 3OC12-HSL or PAI-1) (Pearson et al., 1994
) and N-butyryl homoserine lactone (BHL, C4-HSL or PAI-2) (Pearson et al., 1995
; Winson et al., 1995
), respectively. Knowledge of how quorum sensing systems of P. aeruginosa operate during infection may help us to find a new approach to the treatment of chronic P. aeruginosa lung infections (Finch et al., 1998
; Hartman & Wise, 1998
). A few in vivo studies have demonstrated that the virulence of P. aeruginosa is associated with quorum sensing. For instance, the importance of a functional lasR gene has been shown in a neonatal mouse model of pneumonia (Tang et al., 1996
). The virulence of lasI and rhlI mutants has been reported in a burned-mouse model (Rumbaugh et al., 1999
), and we have previously demonstrated that the production of AHLs from P. aeruginosa can be directly detected in the lung tissues of mice with P. aeruginosa infection (Wu et al., 2000
). OdDHL has been shown to induce an imbalance of the Th1/Th2 (T helper cells) response in vitro, i.e. suppressing interleukin (IL)-12 synthesis, enhancing antibody and IgG1 responses, and promoting IgE production from blood cells stimulated by IL-4 (Telford et al., 1998
). All these data indicate that the quorum sensing systems of P. aeruginosa play an important role during the infectious process.
We wished to further elucidate the involvement of quorum sensing in chronic P. aeruginosa lung infection. In this study, we used a P. aeruginosa PAO1 lasI rhlI double mutant (Pearson et al., 1997 ) to infect rats intratracheally and compared the severity of the resulting lung infection with that caused by its wild-type counterpart PAO1. The bacteria were embedded in alginate beads and the resultant infection mimics the P. aeruginosa infection found in the CF lung (Pedersen et al., 1990
). Immune parameters, lung bacteriology and lung pathology were evaluated at four different time points on days 3, 7, 14 and 28 post-inoculation.
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METHODS |
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Challenge procedures and blood sample collection.
Before challenge, all rats were anaesthetized by subcutaneous injection of a 1:1 mixture of etomidate (Janssen) and midazolam (Roche) at a dose of 1·5 ml (kg body weight)-1 and tracheotomized (Johansen et al., 1993 ). Intratracheal challenge with alginate beads was performed as described by Johansen et al. (1993)
. Each rat received 0·1 ml alginate beads containing 5x107 c.f.u. PAO1 or PAO1 lasI rhlI. The incision was sutured with silk and healed without any complications. The animals were killed by using 20% (w/v) pentobarbital (DAK) at a dosage of 2 ml (kg body weight)-1 and blood samples were obtained by cardiac puncture.
Macroscopic pathology of the lungs.
Eighteen to nineteen lungs from each time point in each group were macroscopically described in situ and after removal from the thoracic cavities. The macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP) as described by Song et al. (1998) according to the modified formula: LIMP=lung area with pathological changes divided by the area of the whole lung. The macroscopic lung pathology included lung abscess, consolidation, atelectasis and haemorrhage.
Lung histopathology.
Lung histopathology was carried out with eight lungs selected randomly from each group of rats at each time point. The following parameters were evaluated.
Size of lung abscesses.
This was expressed as diameter (mm). Lung sections were made at the middle of a lung abscess to get its maximal diameter, which was measured with a micro-ruler under the microscope.
Classification of acute or chronic inflammation.
This was assigned by a scoring system based on the proportion of polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (MNs) in the inflammatory foci. Acute inflammation was defined as an inflammatory infiltration dominated by PMNs (PMNs 90%, MNs<10%), whereas chronic inflammation was defined as a predominance of MNs (MNs>90%, PMNs<10%), which included lymphocytes, plasma cells and the presence of granulomas (Johansen et al., 1994 ).
Mast cell count.
Toluidine blue staining was performed to detect mast cells in the lung tissues (Kiernan, 1981 ). Ten representative fields (viewed at 500x magnification) were selected along the inflammatory foci to count the number of mast cell as described previously (Song et al., 1997
).
Preparation of PMNs.
PMNs were isolated from 9 to 12 citrated peripheral blood specimens in each group of rats by dextran sedimentation and sodium metrizoate/Ficoll (lymphoprep; Nyegaard) separation (Kharazmi et al., 1984a ). The remaining erythrocytes were removed by hypotonic lysis. PMNs were then counted and the concentration was adjusted to 107 cells ml-1 in Krebs Ringers solution with 5 mM glucose. The purity and cell viability were both >97%.
Blood PMN chemiluminescence.
A luminol-enhanced assay was performed with a luminometer (model 1251; LKB-Wallac), which was placed in an air-conditioned thermostat-controlled environment at 21±1 °C. Zymosan and luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) were purchased from Sigma. A total volume of 1 ml of a mixture containing 0·1 ml PMN suspension, 0·2 ml serum-opsonized zymosan at 10 mg ml-1 and 0·7 ml luminol at 10-4 mol l-1 was used. The peak chemiluminescence (in mV) and the time taken to peak were measured.
Lung bacteriology.
Ten to eleven random lung samples from each group of rats at each time point were prepared for quantitative bacteriological examination as described by Johansen et al. (1993) . Each lung was mixed with 3 ml cold sterile PBS and the mixture was homogenized in a blender. Appropriately diluted samples were plated on Blue agar plates (a modified ConradiDrigalsky medium selective for Gram-negative rods and containing lactose, pH 7·0; State Serum Institute, Copenhagen) to determine the number of bacterial c.f.u. after 2024 h incubation at 37 °C.
ELISA. Serum antibody responses.
The concentrations of serum IgM, IgA, IgG, IgG1 and IgG2a against P. aeruginosa standard antigen (St-Ag) in 15 serum samples from each group of rats at each time point were determined by ELISA as reported previously (Johansen & Høiby, 1992 ; Johansen et al., 1993
). The serum antibody titres expressed as ELISA units were obtained by dividing the mean absorbance of the samples by the mean absorbance of an internal standard expressing between 0·30 and 0·40 absorbance units.
Cytokine production
The concentrations of IL-4 and IFN- in 1011 supernatants of the lung homogenate (from the detection of lung bacteriology) from each group of rats at each time point were determined by ELISA kits (Nordic BioSite AB). Standard curves for IL-4, ranging from 8 to 500 pg ml-1 (lower detection limit 2 pg ml-1), and IFN-
, ranging from 10 to 2000 pg ml-1 (lower detection limit 10 pg ml-1), were constructed.
Statistical analyses.
The categorical data were analysed by the chi-squared test. The MannWhitney U test was used to compare the data between two groups.
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RESULTS |
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Macroscopic lung pathology
To calculate the LIMP, which was used as an indicator of the severity of the lung pathology, we measured the area of the lungs exhibiting pathological changes. The major pathological changes observed were lung consolidation, abscesses, adhesion, haemorrhage and atelectasis. On days 3 and 7 p.i., lung adhesion was rarely found, while lung consolidation with haemorrhage and abscesses were the main pathological changes. However, from day 14 p.i., lung adhesion and single huge or multiple lung abscesses became the important characteristics, particularly in the PAO1 group. A few lung samples with atelectasis were found on day 28 p.i. in both groups of rats. The LIMP in the lasI rhlI group was higher (P<0·007) on day 3 p.i. but declined significantly on days 14 and 28 p.i. (P<0·003 and P<0·008, respectively) compared to the PAO1 group. On day 7 p.i., no difference was observed between the two groups (Fig. 1). In the lasI rhlI group, each of the differences in the LIMP between days 3 and 7 p.i. (P<0·04), days 7 and 14 p.i. (P<0·001), and days 14 and 28 p.i. (P<0·02) was significant. In contrast, each of the differences in the LIMP between days 3 and 7 p.i. and days 7 and 14 p.i. in the PAO1 group was not significant; only the difference between days 14 and 28 p.i. was significant (P<0·005). These results indicated that PAO1 caused more persistent and severe lung pathological changes than PAO1 lasI rhlI.
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Anti-P. aeruginosa St-Ag antibody responses. During the early immune response, serum IgM and IgG levels in the lasI rhlI group were significantly higher than those in the PAO1 group (P<0·007). However, from day 14 p.i., the situation changed. The IgG titre found in the lasI rhlI group was much lower than that in the PAO1 group (P<0·05), and the IgM response in both groups dropped markedly (Fig. 4). The production of IgA in the two groups of rats did not differ significantly (data not shown).
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DISCUSSION |
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A stronger oxidative burst response in peripheral blood PMNs is indirect evidence of PMN activation in the lung. During the initial infection process host phagocytes commonly play a major role in the defence against invading micro-organisms. Among the phagocytes, blood PMNs are the most active and important. Previous studies have shown that some P. aeruginosa virulence factors, such as alkaline protease and elastase, could interfere with phagocytosis of neutrophils (Kharazmi et al., 1984a , b
, 1986
), especially inhibiting the oxidative burst response of neutrophils to opsonized zymosan. Our results suggest that PAO1 lasI rhlI produces less virulence factors and lessens the suppression of the PMN oxidative burst in early stages of lung infection, which would be helpful for the lung phagocytes to kill the bacteria. On the other hand, wild-type P. aeruginosa PAO1 can be induced to produce extracellular products which inhibit PMN chemiluminescence (Table 3
), one of the major antimicrobial systems of PMNs. The increased chemiluminescence is required for the bactericidal activity of PMNs but it might also, in some circumstances, worsen the tissue damage (Kharazmi et al., 1989
) (Fig. 1
). The release of elastase and other lysosomal enzymes from neutrophils are other likely candidates participating in the process of tissue damage (Doring & Dauner, 1988
; Doring, 1994
). IFN-
is known to enhance the oxidative burst of PMNs in response to lipopolysaccharide, to increase neutrophil-mediated antibody-dependent cytotoxicity, and to facilitate phagocytosis and bactericidal activity by increasing the expression of high affinity IgG receptors and C3b receptors on PMNs (Hokland & Berg, 1981
; Petroni et al., 1988
; Roilides et al., 1992
). The results of lung bacteriology showed that the lung bacterial clearance in the lasI rhlI group was significantly faster than that in the PAO1 group, suggesting that it may be associated with an early stronger PMN chemiluminescence response in the lasI rhlI group. On day 7 p.i., PMN chemiluminescence in both groups increased markedly compared with that found on day 3 p.i., suggesting the suppression of PMN activity on day 3 p.i., which might be associated with the septicaemia found at the same time (Solberg et al., 1982
). Spleen culture showed that P. aeruginosa could be detected in 6070% of the rats in both groups on day 3 p.i., indicating septicaemia. On day 7 p.i., the pathogen was no longer found in the spleen (data not shown). However, the difference of PMN chemiluminescence between the two groups was not significant on days 7 and 14 p.i. This might be due to the formation of serum IgM directed against virulence factors in the PAO1 group, thereby removing the inhibition of PMN chemiluminescence by virulence factors.
On day 7 p.i., higher lung IFN- and IL-4 production as well as lower serum IgM, IgG and IgG2a responses were detected in the PAO1 group than the lasI rhlI group. This may reflect a delayed immune response due to the cleaving effects on immunoglobulins by the virulence factors produced by wild-type PAO1 (Doring et al., 1981
, 1984
; Bainbridge & Fick, 1989
) (Figs 3
, 4
and 5
). The lower lung IFN-
on day 3 p.i. may be partly due to the inactivation of IFN-
by the alkaline protease and elastase of PAO1 (Horvat et al., 1989
). The higher production of lung IFN-
on day 7 p.i. in the PAO1 group could be explained by the formation of neutralizing antibody (IgM) in the serum. Cytokines are released from macrophages, lymphocytes and appropriately stimulated cells during the infection. In lung foci produced by P. aeruginosa infection, the accumulation of different cytokines would lead to local induction of the Th cell response towards the Th1/Th2 type. High-level production of IFN-
activates macrophages and facilitates the production of the IgG2a subclass, which correlates with the Th1 response. The Th1 response favours cell-mediated immunity (Mosmann & Coffman, 1989
; Mosmann & Sad, 1996
). High-level production of IL-4 would stimulate the humoral immunity and promote the production of the IgG1 subclass, which associates with the Th2 response (Haczku et al., 1996
; Mosmann & Sad, 1996
). The Th1 response would benefit the host fighting against chronic P. aeruginosa lung infection and the Th2 response correlates with a poor prognosis (Johansen et al., 1996
; Moser et al., 1997
; Song et al., 1997
). Telford et al. (1998)
suggested on the basis of an in vitro study that N-oxododecanoyl homoserine lactone, a major signal molecule produced by LasI, inhibits the production of IL-12, a Th1-supportive cytokine, and induces a Th2-like response (increases IgG1 level and stimulates the production of IgE with IL-4). Our results provide in vivo evidence regarding production of cytokines during P. aeruginosa lung infection and supports the view that AHLs originating from the quorum sensing systems of P. aeruginosa are important in the pathogenesis of chronic pulmonary infection.
From days 14 to 28 p.i., milder pathology, lower mast cell count, higher IFN- production in lungs, and lower serum IgG and IgG1 responses were found in the PAO1 lasI rhlI infected group when compared with the PAO1 infected group, indicating a Th1-like immune response. Mast cells and serum IgG and IgG1 are involved in Th2 responses (Johansen et al., 1996
; Krishnan et al., 1996
; Mosmann & Sad, 1996
; Moser et al., 1997
). Chronic P. aeruginosa lung infection in CF patients is characterized by the persistent and significant antibody response in serum and the remarkable infiltration of PMNs in the lung (Høiby et al., 1990
). In the present study, a higher serum IgG level was found in the PAO1 infected rats than the PAO1 lasI rhlI infected rats during the chronic lung infection. The increased level of serum antibodies could lead to the formation of a larger quantity of immune complexes in the lung foci and this is thought to play an important role in the immunopathology of CF. In addition, the larger number of mast cells in the lung foci leading to the release of PMN chemoattractants, together with the activation of complement by the immune complexes would result in significant infiltration of PMNs into the lung foci and damage of the lung tissues (Abraham & Malaviya, 1996
). Moreover, a high antibody titre in CF patients with chronic P. aeruginosa lung infection has been correlated with a poor prognosis (Høiby et al., 1986
, 1990
). The results of antibody responses revealed that P. aeruginosa PAO1 with the ability of producing AHLs inhibited the immune response during the early phase of infection but stimulated the humoral immune reaction during the chronic infection.
Our results suggest that functional quorum sensing systems significantly affect the severity of P. aeruginosa lung infection in both acute and chronic phases. AHL signal molecules as well as receptors (LasR and RhlR) are therefore promising new targets in the quest for a new type of therapy of CF patients with chronic P. aeruginosa lung infection.
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ACKNOWLEDGEMENTS |
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Received 13 September 2000;
revised 11 December 2000;
accepted 11 January 2001.