Oxidative burst in lipopolysaccharide-activated human alveolar macrophages is inhibited by interleukin-9

Interleukin (IL)-9 is known to regulate many cell types involved in T-helper type 2 responses classically associated with asthma, including B- and T-lymphocytes, mast cells, eosinophils and epithelial cells. In contrast, target cells mediating the effects of IL-9 in the lower respiratory tract remain to be identified. Therefore, the authors evaluated the activity of IL-9 on human alveolar macrophages (AM) from healthy volunteers. AM preincubated with IL-9 before lipopolysaccharide (LPS) stimulation exhibited a decreased oxidative burst, as previously shown with IL-4. The inhibitory effect of IL-9 was abolished by anti-hIL-9Rα monoclonal antibody, and presence of IL-9 receptors on AM was demonstrated by immunofluorescence. Both IL-4 and IL-9 failed to modulate tumour necrosis factor-α, IL-8 and IL-10 release by LPS-stimulated AM. However, several observations suggested that IL-9 and IL-4 act through different mechanisms: 1) interferon-γ antagonised the IL-4- but not the IL-9-mediated inhibition of AM oxidative burst; 2) expression of CD14 was downregulated by IL-4 but not by IL-9 and 3) production of tumour growth factor-β by activated AM was potentiated by IL-9 and not by IL-4, and was required for the IL-9-mediated inhibition of AM oxidative burst. These observations provide additional information concerning the activity of interleukin-9 in the lung, related to inflammatory or fibrosing lung processes.

Recently, interleukin (IL)-9 has been shown to play an important role in T-helper (Th) type 2 responses, especially in asthma [1]. More specifically, it has been shown that IL-9 potentiates IL-4-induced immunoglobulin (Ig)E production [2], stimulates mast cell proliferation and differentiation [3], activates eosinophil maturation [4] and stimulates mucus production by bronchial epithelial cells [5]. In contrast with IL-4, which has been implicated in cell regulation both in the airways and alveolar spaces, the role of IL-9 in the lower respiratory tract is poorly characterised with regards to potential target cells. Thus, IL-9 can modulate inflammatory and fibrogenic processes occurring in the lower respiratory tract, as shown in mice exposed to silica particles in which IL-9 exerts a protective anti-fibrotic activity [6], but the lung target cells mediating these effects have not been identified.
The current authors recently showed [7] that IL-9 is capable of inhibiting in vitro human blood monocytes activated by lipopolysaccharide (LPS) for the release of oxygen metabolites and tumour necrosis factor (TNF)-a, similarly to IL-4. This monocyte deactivation by IL-9 probably accounts for the beneficial activity of IL-9 observed in the model of lethal endotoxaemia [8]. In contrast with other macrophage inhibitory cytokines of the Th2 family, such as IL-4, no effect of IL-9 has so far been reported on lung macrophages. Moreover, it has been reported that differentiated macrophages such as synovial [9] or alveolar macrophages (AM) [10] can exhibit different responses to the same stimulation when compared to blood monocytes. Therefore, in this study the effects of IL-9 on human AM, which represent the resident phagocytes involved in the first-line defence of the lung [11], were evaluated. More specifically, the oxidative burst and cytokine release, including the release of the profibrotic cytokine tumour growth factor (TGF)-b, by AM activated by LPS, was studied. These effects were compared with IL-4 and interferon (IFN)-c, the prototypic Th2 and Th1 cytokines, respectively, known to modulate the activation state of AM especially with regards to the respiratory burst.

Materials and methods
Positive recombinant human (rh)IFN-c and TGF-b were purchased from Genzyme (Cambridge, UK). RhIL-9 and IL-4 as well as anti-human IL-9R (a chain) monoclonal antibodies (mAbs) were produced at the Ludwig Institute, Brussels branch, Belgium.

Alveolar macrophage isolation
Human AM were obtained from seven nonsmoking healthy volunteers by bronchoalveolar lavage (BAL) following a standardised technique [12], after written consent from all volunteers and approval of the BAL procedure by the local ethical committee. BAL cells, represented by w90% of macrophages, as assessed on Giemsa-stained cytospins, were allowed to adhere to plastic in complete Roswell Park Memoral Institute medium (cRPMI) (Bio-Whittaker, Walkersville, MD, USA) for 1 h at 37uC to remove nonadherent cells (mainly lymphocytes) by washings with cRPMI. Finally, AM represented w95% of total adherent cells at microscopical examination and flow cytometry. Cell viability assessed by the trypan blue exclusion test was at least 90%.
Cytokine release assay AM (0.5610 6 ?well -1 ) were distributed in 24-well plates (Falcon), preincubated for 24 h with cytokine and further stimulated by LPS as for the oxidative burst assessment. Supernatants were harvested and frozen at -20uC until cytokine titration. Release of TNF-a was quantified by a cytotoxicity bioassay using WEHI 64 cells clone 13, as previously described [14], and rhTNF-a (Boehringer Mannheim Gmbh, Mannheim, Germany) as a standard. IL-8, IL-10 and TGF-b1 concentrations were determined by enzymelinked immunosorbent assay. A kit from CLB (Amsterdam, the Netherlands) was used for IL-10 quantitation, following the manufacturer9s protocol. A kit from Biosource International (Camarillo, CA, USA) allowed the determination of TGF-b1 after the release from its latent complexes by acid treatment of supernatants; TGF-b1 was also assessed in crude supernatants. For IL-8, 96-well plates were coated overnight at 4uC with 4 mg?mL -1 anti-hIL-8 mAb (clone 6217.11, Sigma) in 100 mM sodium carbonate buffer, pH 9.6. After washings in PBS containing 0.1% v/v Tween 20 and blocking for 1 h at 37uC with 1% w/v bovine serum albumin in the same buffer, rhIL-8 standards (Biosource International) and supernatants were incubated for 2 h at 37uC. Plates were then incubated with 20 ng?mL -1 biotinylated polyclonal anti-hIL-8 Ab (R&D Systems, Minneapolis, MN, USA) in blocking buffer and, after washings, with horseradish peroidase-conjugated streptavidin (Sigma). The reaction was then developed in 0.03% v/v H 2 O 2 substrate and 0.42 mM 3,39,5,59tetramethylbenzidine as chromogen in 100 mM sodium acetate/citric acid buffer, pH 4.9, stopped with 2 M dihydrogen sulphate, and read in a plate spectrometer at 450 nm. The sensitivity of the TNF-a bioassay was 0.2 pg?mL -1 , and that of IL-8, IL-10 and TGF-b1 immunoassays 10 pg?mL -1 , 2 pg?mL -1 and 2 pg?mL -1 , respectively. All supernatants were assayed in duplicate.

Immunofluorescence staining
Surface expression of the IL-9 receptor (a chain and IL-2Rc subunit) on AM was assayed by indirect immunofluorescence. AM (0.2610 6 ?well -1 ) were incubated at 4uC for 1 h with anti-hIL-9Ra mAb (AH9R2 or AH9R7), or with anti-hIL-2Rc mAb (clone 38024.11, mIgG1; R&D Systems), diluted at 10 mg?mL in RPMI containing 3% foetal bovine serum (FBS). After three washings with RPMI-3% FBS, AM were incubated at 4uC for 1 h with 10 mg?mL -1 fluoresceine isothiocyanate (FITC)-conjugated F(ab9) 2 fragments of sheep anti-mouse IgG (SAM-FITC; Sigma) in the same medium. AM incubated with isotypic Ig and thereafter with SAM-FITC represented negative controls. After three washings, AM were fixed in 2% v/v formaldehyde in PBS-3% FBS for 15 min at room temperature, gently scraped with a rubber policeman and kept in the dark at 4uC until flow cytometric analysis performed on a FACscan (Becton Dickinson, Mountainview, CA, USA). An additional staining for CD14 was performed on AM preincubated for 24 h with cytokine by direct immunofluorescence using FITC-conjugated anti-CD14 mAb (clone MøP9, mIgG2b; Becton Dickinson).
For confocal microscopy, AM (0.2610 6 ?coverslip -1 ) were cultured for 2 h in 24-well plates, washed with cRPMI, and immunostained for IL-9R as for flow cytometric analysis with AH9R2 mAb. After washings with PBS-3% FBS and fixation by 2% formaldehyde in the same buffer, cells were mounted on slides with 2.5%,4-diacylbicyclo 2,2,2-octane (Sigma) in Mowiol (Calbiochem-Novabiochem, Darmstadt, Germany), and analysed by a MRC-1024 confocal microscope (Bio Rad Laboratories, Richmond, CA, USA) using a 636 objective under oil immersion. Images were digitally recorded and reproduced with a photoprinter. Both for flow cytometry and confocal microscopy, IL-9R negative and positive control cells consisted respectively in wild-type and hIL-9Rtransfected Baf-3 cells.

Statistical analysis
Data were obtained from experiments performed in triplicate and repeated at least three times, and results are expressed as means¡SEM. As indicated, some results were obtained from experiments performed in duplicate or repeated only twice, and then expressed as means¡SD. The differences observed between the different groups were analysed by a t-test and Bonferroni9s correction was applied when multiple comparisons were performed with a same control condition. A pv0.05 was considered to be significant.

IL-9 and IL-4 inhibit the oxidative burst in LPS-stimulated AM
Stimulation by LPS for 20 h increased DCFH oxidation approximately two-fold as compared with unstimulated AM ( fig. 1). Although IL-9 did not significantly modulate the oxidative burst in unstimulated AM (data not shown), preincubation for 24 h with IL-9 downregulated the intracellular oxidative burst in LPS-stimulated AM (6.5¡0.5 versus 10.4¡ 0.3 nmol DCF?mg protein -1 , pv0.001) ( fig.1) to its baseline level. A similar inhibitory effect was observed with IL-4 (6.1¡0.3 versus 10.4¡0.3 nmol DCF?mg protein -1 , pv0.001) ( fig.1). In contrast, preincubation of AM with IFN-c slightly increased the oxidative burst in response to LPS although this effect was not statistically significant ( fig. 1).

IL-9-mediated inhibition of the oxidative burst in AM is dose-and time-dependent
AM were preincubated with various concentrations of IL-9 and were evaluated after LPS stimulation for the intracellular oxidative burst. A dose/response curve was observed considering the inhibition by IL-9 of the respiratory burst in LPS-stimulated AM ( fig. 2). In addition, a preincubation period of 24 h before LPS stimulation was required to observe the inhibitory effect of IL-9 on AM oxidative burst. Thus, no significant inhibition occurred when AM were preincubated with IL-9 for 4 or 8 h, and no additional inhibition was observed for preincubation periods w24 h (data not shown).

IFN-c abrogates the IL-4, but not the IL-9, inhibitory effect on the oxidative burst in LPS-stimulated AM
The influence of IFN-c on the inhibition mediated by IL-9 and IL-4 on the oxidative burst in LPSstimulated AM was evaluated by co-incubating cells with IL-9 or IL-4 and IFN-c. Inhibition of the respiratory burst by IL-9 in LPS-stimulated AM was maintained in the presence of IFN-c (6.6¡0.1 versus 6.5¡0.5 nmol DCF?mg protein -1 , NS) ( fig. 3). In contrast, IFN-c completely abrogated the inhibitory  IL-9 inhibitory effect on the oxidative burst in LPS-stimulated AM is specifically blocked by neutralising anti-IL-9Ra mAb In order to confirm the specificity of the observed effect of IL-9 on the AM respiratory burst, AM were pretreated with neutralising anti-hIL-9Ra mAb (AH9R7) before IL-9 incubation. Pretreatment of AM with AH9R7 (10 mg?mL -1 ) 1 h before addition of IL-9 abolished 90¡5% (mean¡SEM) of the IL-9 effect on LPS-stimulated DCFH oxidation. In contrast, no significant change was observed on the IL-9-mediated inhibitory effect with mIgG2b used as isotypic control (table 1), nor on the IL-4-mediated inhibition with anti-hIL-9Ra mAb (data not shown).   cytokines. In LPS-stimulated AM, the preincubation with IL-9 or IL-4 slightly reduced the TNF-a production, but without reaching statistical significance (74.8¡10. 2 . 4). Similar to TNF-a, no significant modulation of the IL-8 release was observed in AM pretreated with IL-9, nor with IL-4 ( fig. 4). The LPS-induced IL-10 production was clearly inhibited by IFN-c (22.5¡4 versus 45.3¡9.2 pg?mL -1 , pv0.001), and also slightly reduced by IL-9 and IL-4 but without reaching statistical significance (38.4¡5.7 and 35.1¡9.8 pg?mL -1 , respectively, versus 45.3¡9.2 pg?mL -1 , NS) ( fig. 4).

IL-4 but not IL-9 inhibits surface expression of CD14 LPS receptor on AM
CD14 was detected at the surface of AM, although to a much less extent than on blood monocytes [7]. CD14 expression at the surface of AM was downregulated by preincubation with IL-4 (mean fluorescence intensity (MFI) 42¡4 versus 84¡6, pv0.001). Conversely, IFN-c increased CD14 on AM, although this effect was below the level of statistical significance (MFI 111¡5 versus 84¡6, NS) (table 2). In contrast, IL-9 did not change the expression level of surface CD14 on AM (table 2).

Inhibition by IL-9 of the oxidative burst in LPS-activated AM depends on TGF-b1
Considering that TGF-b has been previously identified as a major macrophage deactivating factor and that its secretion was stimulated by IL-9, the possibility that TGF-b might mediate the inhibitory effect of IL-9 on the oxidative burst in AM was addressed. Interestingly, similarly to blood monocytes [7], the IL-9-induced inhibition of the respiratory   burst in LPS-stimulated AM was abrogated by the blockade of TGF-b1 activity by a specific mAb, and not by control mIgG1 (fig. 6). This abrogation by anti-TGF-b1 mAb was not observed on the inhibition induced by IL-4 (data not shown).

AM express specific receptors for IL-9
Specific surface receptors for IL-9 were identified on AM. The presence of IL-9Ra chain was thus demonstrated on AM by flow cytometry and confocal microscopy, after immunostaining using different specific mAbs (AH9R2 and AH9R7) ( fig. 7). Positive control cells (hIL-9Ra-transfected Baf-3 cells) exhibited a similar pattern of surface fluorescence, whereas no shift of the fluorescence histogram was observed in wild-type Baf-3 control cells (data not shown). The level of expression of IL-9Ra was not significantly changed by IL-9, IL-4 or IFN-c on AM cultured for 24 h with these cytokines (data not shown). The other subunit of the IL-9R complex, IL-2Rc (cc), was also identified on AM ( fig. 7), although the expression level of this latter receptor appeared quite variable among the different volunteers (data not shown).

Discussion
The authors have previously shown that IL-9, a Th2 cytokine [15], inhibits the production of reactive oxygen intermediates (ROI) and TNF-a by human blood monocytes [7]. It is shown here that IL-9 can also regulate some important functions of human AM. Thus, pretreatment of AM with IL-9 inhibited their subsequent production of ROI after LPS stimulation. In contrast, the LPS-stimulated cytokine release by AM was not significantly affected by IL-9 nor by IL-4. Interestingly, in contrast with IL-4, which exhibited a similar deactivating activity on AM respiratory burst, IL-9 stimulated the production of TGF-b1 by AM. Moreover, TGF-b1 was required for the inhibitory effect of IL-9 on AM oxidative burst to be seen.
In contrast with IFN-c, which represents a major priming factor for AM respiratory burst [16] and cytokine release [17], Th2 cytokines (such as IL-4, IL-10 and IL-13) and TGF-b have generally been described as macrophage-deactivating factors. In particular, BHASKARAN et al. [18] showed that IL-4 deactivates AM for the oxidative burst response to LPS. Correspondingly, in this study it has been shown that IL-9 inhibits the production of ROI in AM stimulated by LPS. While ROI are probably required to clear the lung from intracellular pathogens [19], the role of these toxic metabolites has also been demonstrated in lung tissue injury in vivo [20]. Thus, the inhibition of H 2 O 2 generation in activated AM by IL-4 and IL-9 might have an important role in the prevention of tissue injury during some inflammatory processes occurring in the alveoli. In this regard, it is of interest that the inhibitory effects mediated by IL-4 and IL-9, which could appear as redundant, are differently modulated by IFN-c, probably produced concomitantly during immune responses in the lung. Thus, the inhibition of the oxidative burst in LPSactivated AM by IL-4 is antagonised by IFN-c as previously shown [21], in contrast to that of IL-9.
In contrast with the regulation of AM oxidative burst, IL-4 and IL-9 did not change the release of several cytokines by AM. Thus, both IL-4 and IL-9 failed to significantly modulate the release of TNF-a, IL-8 and IL-10 by LPS-stimulated AM, although there was a tendency to inhibition especially for IL-10 production. Several studies have reported that IL-4 inhibits the release of inflammatory mediators by activated AM, namely TNF-a and IL-1b [22][23][24], IL-6 [25], monocyte chemotactic peptide-1 [10], IL-12 [26] or prostaglandin E 2 [27,28]. However, other studies failed to demonstrate a significant effect of IL-4 on cytokine production in AM [29], as well as in synovial macrophages or in monocyte-derived macrophages five experiments. c) IL-9Ra staining of AM with AH9R2 mAb was also evaluated by confocal microscopy (inset, scale=mm). [9]. One possible explanation for the relative loss of IL-9 and IL-4 activity on tissue macrophages, as compared to blood monocytes, was a reduced expression of the IL-2Rc chain, the common subunit notably shared by receptors for IL-4 and IL-9 [30]. However, the present data show that both IL-4 and IL-9 can regulate AM, notably for the oxidative metabolism, and that IL-2Rc is expressed by these tissue macrophages.
The production of TGF-b by activated AM is strongly potentiated by IL-9, but not by IL-4. Interestingly, using antibodies to neutralise TGF-b1, it was shown that the upregulation by IL-9 of the TGF-b pathway is required for the IL-9-mediated inhibition of AM oxidative burst to be seen. This is in agreement with previous studies indicating that TGF-b is a major macrophage deactivating factor, notably for the production of ROI [31]. In addition, this observation could explain, at least in part, the need for a preincubation period for IL-9 to inhibit the oxidative burst in LPS-activated AM. Conversely, TGF-b, which is produced as a latent procytokine complex, has been identified as a major fibrosing agent [32,33]. Although a Th2 inflammatory profile was associated with fibrotic lung diseases such as idiopathic pulmonary fibrosis [34] or bleomycininduced lung fibrosis [35], this relationship between Th2 inflammation and tissue fibrosis remains poorly understood. LEE et al. [36] recently showed that IL-13 induces lung fibrosis through the activation of TGF-b, suggesting that TGF-b might represent a link between Th2 inflammation and lung fibrosis. The present data, showing that TGF-b upregulation can be induced by IL-9 in activated AM, supports this concept. Conversely, ARRAS et al. [6] reported that IL-9 inhibits rather than potentiates lung fibrosis in a mouse model of silicosis, and this effect was associated with an inhibition of the silica-induced Th2 polarisation. Thus, other mechanisms could occur in vivo with regard to the cytokine network or to alternative regulatory pathways. More specifically, it has been shown that H 2 O 2 can induce connective tissue growth factor (CTGF) [37] which mediates the effect of TGF-b on fibroblast collagen synthesis [38]. Therefore, IL-9 might induce a profibrotic activity in the lung through increased TGF-b secretion by activated AM, while the downregulation of the oxidative burst might prevent tissue fibrosis by limiting the induction of CTGF.
The modulation of surface expression of CD14 on AM represents a potential regulatory mechanism of cell activation, since this receptor is required to induce the signalling events leading to the synthesis of inflammatory cytokines elicited by LPS [39]. The current authors have confirmed that CD14 expression on AM is downregulated by IL-4, as previously shown [27,40]. This is in contrast with IL-9, which had no effect on CD14 expression in AM. In addition, the authors observed that IFN-c can neutralise the reduction of CD14 on AM induced by IL-4 (data not shown). These effects might represent mechanisms of deactivation or priming of AM by IL-4 and IFN-c, respectively, with regard to further LPS stimulation.
In conclusion, the authors report that interleukin-9 inhibits the production of reactive oxygen species in activated alveolar macrophages, similarly to interleukin-4. In contrast with the release of tumour necrosis factor-a, interleukin-8 and -10, which is not significantly affected by interleukin-9 or -4, the production of tumour growth factor-b1 by activated alveolar macrophages is strongly enhanced by interleukin-9 and this tumour growth factor-b1 upregulation mediates the inhibitory effect of interleukin-9 on the oxidative burst. Finally, the modulation of CD14 expression which appears as a potential mechanism of alveolar macrophage deactivation by interleukin-4, as well as of priming by interferon-c, is not observed with interleukin-9. These observations highlight regulatory pathways of alveolar macrophage activation by T-helper 2 cytokines and provide new information on interleukin-9 relating to alveolar macrophage biology.