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Previous Article | Next Article 
Infection and Immunity, November 2004, p. 6463-6470, Vol. 72, No. 11
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.11.6463-6470.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
School of Pharmacy,1 Institute of Infection, Immunity and Immunology, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom2
Received 6 January 2004/ Returned for modification 18 February 2004/ Accepted 24 June 2004
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ABSTRACT |
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INTRODUCTION |
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P. aeruginosa produces a number of quorum-sensing signal molecules (QSSM) which have been chemically characterized as N-acylhomoserine lactones (AHL) and a quinolone (10, 29, 50). The two major AHL [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] and two minor AHL [N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL)], together with 2-heptyl-3-hydroxy-4 (1H)-quinolone (PQS), coregulate the expression of multiple exogenous virulence determinants (11, 27, 49).
Apart from regulating the expression of virulence factors, AHL themselves may function as virulence determinants per se, in that some molecules apparently modulate immunological responses and other modulate eukaryotic functions (reviewed by Pritchard et al. [31]). Consequently, there is increasing evidence that QSSM impact directly on the host. AHL have been detected at nanomolar to micromolar concentrations in lung tissues and sputum samples from CF patients experiencing P. aeruginosa infections (13, 23, 37). Wu et al. (52) provided additional compelling evidence for the in situ presence of AHL when mice were coinfected with P. aeruginosa and an Escherichia coli AHL biosensor; AHL were detected in lungs when mice were challenged intratracheally with P. aeruginosa. Additional evidence in support of AHL production in vivo was reported by Stickler et al. (42) during analyses of sections of indwelling urethral catheters that had become colonized by bacterial biofilms while in use. PQS could also be detected in the sputum of CF patients (16) and during early colonization of CF patient airways (7). Furthermore, in a number of animal models of infection, mutation of the las and/or rhl quorum-sensing system attenuated the ability of P. aeruginosa to colonize and subsequently kill the host (26, 36, 39, 44, 51). However, in contrast, a LasR-negative mutant was as virulent as the wild type in a murine model of corneal infection (30), a finding which probably reflects the LasR/3-oxo-C12-HSL-independent activation of the rhl system (28).
Given the detection of QSSM ex vivo and their involvement in driving the disease process associated with infection, it became of interest to assess the inherent immune-modulatory capacity of these signal molecules. In early immunological experiments, 3-oxo-C12-HSL was shown to suppress interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-
) secretion by lipopolysaccharide (LPS)-stimulated macrophages and to suppress T-cell proliferation (45). In contrast, T-helper 2 (Th2)-dependent antibody secretion was enhanced by 3-oxo-C12-HSL at low micromolar concentrations. This led to the hypothesis that this particular QSSM could in fact steer T-cell responses away from a host-protective T-helper 1 (Th1) phenotype, possibly promoting pathogen survival (45). Bioactivity has subsequently been shown to be dependent on an intact molecular structure and a side chain length of C8 or longer. As a result, C4-HSL is inactive in the immunological assays used to date (4).
The bioactivity of 3-oxo-C12-HSL in immunological systems was confirmed by data which showed Cox-2 expression and cytokine and chemokine mRNA induction by this compound in addition to gamma interferon secretion by T cells and NF-
ß activation in keratinocytes (39). These observations led the authors to postulate that 3-oxo-C12-HSL was in fact promoting a Th1 environment, in contrast to the findings by Telford et al. (46). These published papers, however, are not totally contradictory, since the effects seen by Smith et al. (39) tended to be at high dose whereas those seen by Telford et al. (46) were at low dose. Furthermore, recent in vivo data support the immunomodulatory activity of 3-oxo-C12-HSL (35).
It was also established that 3-oxo-C12-HSL stimulated the release of IL-8 from human epithelial cells (12, 38), and a LasR-expressing strain of P. aeruginosa was able to elicit the production of IL-8 when human epithelial cells were exposed to the bacteria (44). IL-8 is the major chemokine associated with neutrophil migration across the airway epithelium (19), and it is found in increased amounts in the lungs of CF patients (9). Chronic influx of activated neutrophils into infected airways is responsible for damage to the airway and for progressive respiratory insufficiency in CF patients (47, 53). As well as IL-8, other chemokines and cytokines were induced by 3-oxo-C12-HSL in other experimental systems (39). Paradoxically, 3-oxo-C12-HSL can cause neutrophil apoptosis (45), illustrating the true complexity of the host-pathogen relationship.
From the above, it is clear that QSSM possess inherent bioactivity in eukaryotic systems. In the present study, we sought to determine for the first time whether PQS, a chemically distinct QSSM, was capable of modulating immune responses in a manner similar to that described for 3-oxo-C12-HSL, by using a parallel panel of assays to that in which 3-oxo-C12-HSL was first shown to have immune-modulatory activity (46). In human peripheral blood mononuclear cells (hPBMC) activated with a panactivating lectin (concanavalin A [ConA]), both 3-oxo-C12-HSL and PQS displayed immune suppression in terms of cell proliferation and cytokine (IL-2) release. When a more specific T-cell stimulant (anti-CD3/anti-CD28 antibodies) was used, both QSSM suppressed cell proliferation but only 3-oxo-C12-HSL down-regulated the release of IL-2. Following LPS activation of monocytes in hPBMC, the release of TNF- was unaffected by the treatment with PQS; in fact, the release of TNF- was induced at concentrations above 10 µM PQS. In comparison, 3-oxo-C12-HSL inhibited TNF- release, as reported previously (46).
Taken in their entirety, the data presented in this paper highlight the inherent immune-modulatory potential of PQS and the fact that it acts differently from another immune-modulatory QSSM, 3-oxo-C12-HSL. Although the full impact of the presence of two chemically distinct immune-active QSSM on this complex host-pathogen interface remain to be determined, PQS and 3-oxo-C12-HSL as immune modulants certainly warrant inclusion in any future biological equation addressing this relationship. Furthermore, the possibility of developing QSSM as novel immune modulants, as therapeutics, or to dissect immunological signaling pathways remains an attractive proposition.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assay of cell viability. Cell viability was determined using a colorimetric method, utilizing solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) (Promega) and an electron-coupling reagent (phenozine methosulfate). MTS was bioreduced by viable cells into a formazan product that was soluble in tissue culture medium (1, 8, 34). After an incubation of 4 h at 37°C in 5% CO2-air, the production of formazan was determined by measuring the absorbance of the compound at 450 nm with a spectrophotometric 96-well plate reader (Dynex).
Proliferation of hPBMC stimulated with ConA and anti-CD/anti-CD28 antibodies. ConA (Sigma, Poole, United Kingdom) and anti-CD3 with anti-CD28 antibodies (BD Pharmingen) were used to assess the effect of QSSM on T-cell proliferation and IL-2 secretion. Proliferation was assessed by the incorporation of [3H]thymidine into the DNA of PBMC, and the release of cytokines in the culture supernatants was determined by sandwich enzyme-linked immunosorbent assay (ELISA). Blood specimens were obtained with consent from healthy human volunteers (aged between 20 and 50 years). hPBMC were isolated from heparinized whole blood by buoyant density centrifugation over Histopaque 1077 (Sigma) at 600 x g for 20 min. PBMC harvested from the buffy layers were washed twice with RPMI 1640 medium and resuspended in complete cell culture medium (CTCM) consisting of RPMI 1640 medium with 5% fetal calf serum, 2 mM L-glutamine, and 5 x 10–5 M 2-mercaptoethanol. QSSM, of which stock solutions of 0.1 M were soluble in dimethyl sulfoxide (DMSO), were initially diluted into CTCM and then tested in triplicate wells at concentrations ranging from 0.78125 to 100 µM in 200 µl of CTCM containing 100,000 hPBMC which were separately challenged with 1 µg of ConA per ml and with 100 ng of mouse anti-human CD3 monoclonal antibody (clone UCHT1; BD Pharmingen) per ml plus 5 µg of mouse anti-human CD28 monoclonal antibody (clone CD28.2; BD Pharmingen) per ml. Cell stimulations of hPBMC were incubated for 24 h at 37°C in 5% CO2-air, and 50-µl portions of culture supernatants were removed for the determination of IL-2 (see below). The cells were returned for a further 24-h culture; this was followed by pulsing with 0.25 µCi of [3H]thymidine (Amersham) in a 10-µl volume made up in RPMI 1640 medium. After a further incubation of 24 h, cells were harvested onto 96-well filter plates (Unifilter Filtermate Harvester; Packard Bioscience Ltd.). Following the addition of scintillant (MicroScint-O; Packard Bioscience Ltd.) to each well in the filter plates, the radioactivity in the filters was measured with a ß-scintillation counter (TopCount; Packard Bioscience Ltd.) as specified by the manufacturer.
IL-2 production from ConA and anti-CD3/anti-CD28 antibody-stimulated hPBMC. When T cells in hPBMC are challenged with the above stimuli of ConA and anti-CD3/anti-CD28 antibodies, the cytokine IL-2 is released into the culture supernatants. The levels of cytokine produced in the culture supernatants after 24 h were determined in a sandwich ELISA. Briefly, 96-well Nunc MaxiSorp (Life Technologies, Paisley, United Kingdom) plates were coated overnight at 4°C with "capture" mouse anti-human IL-2 monoclonal antibody (BD Pharmingen). After the plates were blocked with 1% (wt/vol) bovine serum albumin (Sigma), 50-µl volumes of cell culture supernatants were added and incubated overnight at 4°C; standard human IL-2 concentrations (BD Pharmingen) ranging from 7.8125 to 500 pg/ml were included for each plate. The "captured" IL-2 was sandwiched with a biotinylated mouse anti-human IL-2 monoclonal antibody. The presence of biotinylated antibodies was detected by the addition of streptavidin-peroxidase (BD Pharmingen). After thorough washes, the assay mixture was developed using tetramethyl benzidine subtrate (Sigma). The enzyme reaction was stopped with 2.5 M H2SO4, and the colorimetric development was read at 450 nm with a spectrophotometric 96-well plate reader. The concentrations of IL-2 in the culture supernatants were determined by extrapolation from the reference standard IL-2 curve.
TNF- production from LPS-stimulated hPBMC.
Bacterial LPS stimulates the production of a variety of cytokines, including TNF-, from hPBMC; these cytokines in turn influence the development of T cells, supporting a Th1-conducive milieu. hPBMC prepared from whole blood by buoyant density centrifugation were resuspended in CTCM. QSSM were again tested at similar dilutions to those used earlier in 200 µl of CTCM, containing 10–5 µg of E. coli O55:B5 LPS per ml (Sigma) and 100,000 hPBMC. Following incubation for 24 h at 37°C in 5% CO2-air, the cell culture supernatants were collected and tested for TNF-. Levels of TNF- are determined by sandwich ELISA, as described above for the IL-2 detection assay, using a capture mouse anti-human TNF- monoclonal antibody (BD Pharmingen). Cell culture supernatants were added and incubated overnight at 4°C; standard human TNF- concentrations (BD Pharmingen) ranging from 31.25 to 2,000 pg/ml were included for each plate. Biotinylated mouse anti-human TNF- monoclonal antibody was added, and bound biotinylated antibody was detected with streptavidin-peroxidase. The assay mixture was developed as described above for the IL-2 detection assay. The concentrations of TNF- in the culture supernatants were determined by extrapolation from the reference standard TNF- curve.
Data analysis. Blood samples were collected from four individual donors by a qualified phlebotomist with informed subject consent (ethical committee approval is not required for this procedure at the University of Nottingham), and hPBMC responses were assayed in triplicate. Triplicate data for all donors were calculated for mean and standard error values. One-way analysis of variance (ANOVA) followed by Dunnett's test was used for all analyses, comparing dilutions of QSSM with the control stimulus. Values for 50% inhibitory concentration (IC50) were calculated from nonlinear regression analysis by using GraphPad Prism software.
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RESULTS |
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PBMC proliferation and IL-2 secretion following stimulation with anti-CD3/anti-CD28 antibodies. The specific stimulation of T cells by the engagement of the T-cell receptor CD3 complex with specific antibodies requires a further antibody coligation of CD28, a coreceptor of T-cell activation (19, 47). The CD28 pathway provides intracellular coactivation signals which are required for the production of cytokines, such as IL-2 and gamma interferon (2, 19, 47), to drive T-cell proliferation. This method of T-cell activation was used to further investigate the immunological effects of QSSM of P. aeruginosa. Both 3-oxo-C12-HSL and PQS consistently inhibited T-cell proliferation when the cells were cross-linked with anti-CD3 and anti-CD28 antibodies (Fig. 3A). The IC50 of PQS and 3-oxo-C12-HSL were 1.73 µM (95% confidence interval, 1.54 to 1.93 µM) and 44.47 µM (95% confidence interval, 37.22 to 53.13 µM), respectively.
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LPS-stimulated TNF- secretion from hPBMC.
In an LPS-driven TNF- secretion assay, 3-oxo-C12-HSL at 50 µM and above suppressed secretion, supporting the earlier data of Telford et al. (46) (Fig. 4). In contrast, PQS significantly promoted secretion above 25 µM. Given the failure of PQS to affect hPBMC cell viability, the stimulatory effect of this compound on TNF- secretion above 25 µM warrants further attention, since this would appear to be a stimulatory event. In contrast, the effects of 3-oxo-C12-HSL in suppressing TNF- secretion at doses which are beginning to affect cell viability (Fig. 2A) are suggestive of a causal link between the two events.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Notwithstanding the latter, the present study clearly advances our knowledge of potential molecular interactions at the P. aeruginosa host/pathogen interface by demonstrating that two structurally diverse QSSM, 3-oxo-C12-HSL and PQS, which are in a hierarchical cascade involved in the regulation of numerous essential P. aeruginosa virulence determinants (11, 25, 27), can exert differential modulatory effects on mammalian immune responses in vitro. PQS and 3-oxo-C12-HSL significantly reduced the ability of lymphocytes to respond to ConA (IC50, 0.9 and 18.24 µM, respectively). The antiproliferative activity of PQS occurred without any effect on cell viability, while 3-oxo-C12-HSL suppressed proliferation before cell viability was affected, exhibiting what we term its immune-suppressive window, with an IC50 of 18.24 µM.
Although the results of assays involving mitogens do not directly correlate with physiological activities of cells, they do provide a first indication of the pharmacological activity of test compounds. ConA stimulates T cells independently of accessory cells and activates via the T-cell receptor and other cell surface receptors. To investigate the effects of 3-oxo-C12-HSL and PQS in a more specific T-cell stimulation assay, anti-CD3 antibody was used in combination with anti-CD28 antibody to drive T-cell proliferation. As was observed in lectin stimulation assays, PQS was more potent than 3-oxo-C12-HSL in suppressing T-cell proliferation (IC50, 1.73 and 44.47 µM, respectively). With respect to IL-2 production in response to T-cell activation with lectin and anti-CD3/anti-CD28 (20, 48) antibodies, PQS exhibited a different activity from 3-oxo-C12-HSL in that only 3-oxo-C12-HSL inhibited the release of this cytokine when T cells were stimulated with anti-CD3/anti-CD28. This would suggest that 3-oxo-C12-HSL is acting upstream of IL-2 secretion while PQS is preventing proliferation by acting downstream of IL-2 secretion, activities reminiscent of cyclosporin A and rapamycin, respectively. Rapamycin acts at a later stage of the cell cycle, namely, at the transition from the G1 to the S phase, thus inhibiting the proliferation of cells in response to growth factors such as IL-2 (18, 32), while cyclosporin A selectively prevents the transcription of early T-cell activation genes (22). The bioactivity of the IL-2 released and the integrity of the IL-2 receptor apparatus are in the process of being determined.
Further differential immune-modulatory activity was observed when TNF- secretion was assessed in assays where LPS was used to drive TNF- secretion from hPBMC, with 3-oxo-C12-HSL (suppressive) and PQS (stimulatory) exhibiting contrasting effects. The biological significance of these findings could be significant. PQS and 3-oxo-C12-HSL are both present endobronchially at low micromolar levels in CF patients; this could be considered to be immunologically active (7, 13, 14, 37, 52). Therefore, it is conceivable that Pseudomonas bacteria in the lungs of CF patients, while communicating through the release of QSSM, stimulate the bronchial epithelium to release IL-8 (12, 38) while suppressing lymphocyte function, although it has yet to be determined whether QSSM can access bronchial airway lymphoid tissue. However, their documented effects on bronchial epithelial cells suggest that uptake into tissues is possible, and the innate hydrophobicity of the QSSM tested could further facilitate uptake into cells and tissues, although recent evidence suggests that human airway epithelia can inactivate 3-oxo-C12-HSL (6).
While it is inappropriate to speculate about the full clinical significance of our findings until bioavailability has been determined, the innate bioactivity of these chemically defined QSSM is clear; these activities may yet be harnessed to treat immunological disease, in the same way that cyclosporin A and rapamycin have been exploited (18). Furthermore, a full molecular understanding of the role of these QSSM at the host/pathogen interface and their interplay with each other and with molecules produced by host tissue will surely advance our knowledge in a way which could support better treatment of Pseudomonas infections and colonizations in the lungs of CF patients and elsewhere.
In summary, the data presented in the present paper (i) confirm the immune-modulatory potential of 3-oxo-C12-HSL, (ii) describe for the first time the immune-modulatory activity of PQS, and, furthermore, (iii) clearly demonstrate the different immunological activities of these compounds. The immune response in immunologically and physiologically competent persons is vigorous and effective in dealing with P. aeruginosa. The production of a dual wave of immune modulants in compromised patients, in combination with other immunologically confounding virulence factors such as exotoxin A and pyocyanin (reviewed in reference 31), may confer a survival advantage on the infecting microorganism and hence may enable P. aeruginosa to reach a population density sufficient to trigger the expression of diverse proinflammatory tissue-damaging and -degrading virulence determinants. However, the true impact of immune modulatory QSSM on patient status awaits a full scientific investigation, using relevant disease models and patient material.
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ACKNOWLEDGMENTS |
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Gary Telford is acknowledged for his continued support and for sound advice on the statistical analysis of data.
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FOOTNOTES |
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----, 3-oxo-C12-HSL in cell viability; CT P, ConA-stimulated cell proliferation control; CT V, Cell viability medium control. (B) -
-, 3-oxo-C6-HSL; ----
