American Journal of Respiratory and Critical Care Medicine

We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1 α (MIP-1 α ), interferon- γ (IFN- γ ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1 β were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1 α , IFN- γ , and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1 α , IFN- γ , and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1 β stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.

Macrophages are widely recognized as cells that play a central role in the regulation of immune and inflammatory activities as well as in tissue remodeling. The fulfillment of these activities is mediated by complex and multifactorial processes involving products derived from macrophages (1). Macrophages usually elaborate powerful suppressive signals to limit the proliferative potential of T cells, thus maintaining local immunologic homeostasis (2). In asthma, macrophages may be stimulated by specific allergen to augment T-cell proliferation (3), which may result from a different profile of cytokines released from these macrophages. For example, increased release of granulocyte-macrophage colony-stimulating factor (GM-CSF) may inhibit the immunosuppressive effect of macrophages (4). Indeed, macrophages from asthmatic subjects release increased amounts of several pro-inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-6, and GM-CSF (5-7).

Inhaled steroids used for the treatment of asthma reduce the number of infiltrating eosinophils, T cells, macrophages, and mast cells in the airways submucosa (8). Suppression of pro-inflammatory cytokine release such as GM-CSF, IL-4, IL-5, and RANTES from many inflammatory and resident airway cells is a likely mechanism of steroid action (9-11). Pro-inflammatory cytokine expression by macrophages may also be inhibited such as the release of GM-CSF, IL-6, and MIP-1α, as has been demonstrated in vitro (12, 13). However, it is not clear how anti-inflammatory cytokines such as IL-10 are modulated by corticosteroids in vivo. IL-10, a product of alveolar macrophages and T cells (14, 15), inhibits the release of several pro-inflammatory cytokines such as TNFα, IL-1β, IL-6, IL-8, macrophage inflammatory protein-1α (MIP-1α), and GM-CSF from monocytes and macrophages (14, 16-18) and T-cell proliferation (19, 20). While topical corticosteroids can inhibit pro-inflammatory cytokine expression, it is not known whether they can modulate the production and expression of the anti-inflammatory cytokine IL-10 in asthma. We have therefore investigated the effect of inhaled steroid therapy on the release of IL-10 and the pro-inflammatory cytokines GM-CSF, IFN-γ, and MIP-1α from alveolar macrophages of mild asthmatic patients ex vivo.


Twelve subjects with mild stable asthma (Table 1) who were receiving treatment with only the inhaled β2-adrenergic agonist aerosol, albuterol, for intermittent relief of wheeze were recruited. All patients demonstrated > 15% improvement in FEV1 following 200 μg of albuterol and in airway hyperresponsiveness to methacholine with a provocative concentration of methacholine producing a 20% fall in FEV1 (PC20) of < 4 mg/ml. All patients were atopic as defined by two or more positive skin prick tests to common allergens. None of the subjects studied had received oral or inhaled corticosteroids for the preceding 12 mo or any other treatment apart from inhaled β2 agonists. Current smokers or ex-smokers of > 5 pack-years and patients with FEV1 < 80% predicted were excluded.


nAge*(yr )Sex (M:F )FEV1 *(% pred )Log PC20 *(mg/ml )
Normal 724.0 ± 1.53:4101 ± 3.4> 1.78
Asthmatic1228.8 ± 1.36:696.7 ± 3.6−0.30 ± 0.17

*Mean ± SEM.

In order to compare the responses of monocytes and alveolar macrophages obtained from asthmatic subjects during placebo and budesonide treatment, we studied seven normal nonatopic nonsmoking volunteers (Table ), who gave no history of asthma or of any respiratory disease and who had normal lung function and airway responsiveness to methacholine (PC20 > 16 mg/ml).

Study Design

The study was a 12-wk, double-blind, randomized, cross-over study comparing the effects of inhaled corticosteroid, budesonide (800 μg twice daily), to that of placebo. Each treatment was administered for 4 wk, separated by a 4-wk wash-out phase. All patients were reviewed at Day 14 of each treatment limb, and at Day 26, spirometry and airway responsiveness to methacholine were measured. At Day 28, venous blood samples were obtained and fiberoptic bronchoscopy was performed. The study was approved by the Royal Brompton Hospital Ethics Committee, and all patients gave their informed consent.

Fiberoptic Bronchoscopy

Subjects attended our bronchoscopy suite at 8:30 a.m. after having fasted from midnight and were pretreated with atropine (0.6 mg intravenously) and midazolam (5 to 10 mg intravenously). Using local anesthesia with lidocaine (4%) to the upper airways and larynx, a fiberoptic bronchoscope (Olympus BF10; Southend-on-Sea, Essex, UK) was passed through the nasal passages into the trachea. Bronchoalveolar lavage (BAL) was performed from the right middle lobe using four successive aliquots of 60 ml of 0.9% NaCl. BAL cells were spun (500 × g; 10 min) and washed twice with Hanks' buffered salt solution (HBSS). Cytospins were prepared and stained with May-Grünwald stain for differential cell counts. Cell viability was assessed using the trypan blue exclusion method. Cells (5 × 105) were placed in guanidinium thiocyanate and left at −70° C for later RNA extraction.


IL-1β was purchased from R&D Laboratories (Oxford, UK). Human recombinant MIP-1α and anti-MIP-1α antibody was a gift from T. J. Schall (DNAX, Palo Alto, CA). Complete culture medium contained RPMI-1640 (ICN-Flow, High Wycombe, UK), 10% heat-inactivated fetal calf serum (Sera Lab, Crawley, UK) 2 mM l-glutamine, and penicillin (100 U/ml)-streptomycin (100 μg/ml) (both from ICN-Flow). Culture plates were from Falcon (London, UK). Lipopolysaccharide (Escherichia coli), avidin-peroxidase, and ABTS were from Sigma (Poole, UK). [32P]dCTP, [125I]NA, and hybond N-filters were from Amersham International (Amersham, UK). Ficoll-Hypaque was from Pharmacia Biotech (Hertfordshire, UK). Round-bottom, 96-well plates were from Greiner Labortechnik Ltd (London, UK).

Isolation of Peripheral Blood Monocytes

Peripheral venous blood was mixed with acid-citrate dextrose (1:6, vol/vol) and sedimented on dextran (6% in 0.9% NaCl) for 40 min. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation. Cells were washed twice with HBSS and plated at a concentration of 5 × 106 monocytes/2 ml in 6-well plates for Northern analysis or 1 × 106/ml in 24-well plates for reverse-transcription polymerase chain reaction (RT-PCR) and collection of supernatants for cytokine assays. After adherence at 37° C for 90 min, cells were washed twice with HBSS prior to stimulation. Cell viability was consistently > 96%, as assessed by trypan blue exclusion; after adherence, > 95% of these cells were monocytes, as assessed by nonspecific esterase staining.

Isolation of Alveolar Macrophages

BAL fluid was filtered through sterile gauze and centrifuged at 500 × g for 10 min. Cells were washed twice in HBSS, counted, and plated in complete media at a concentration of 1 × 106/ml in a 24-well plate. In normal subjects, the total cell yield was 12 ± 7 × 106 cells with a differential cell count of 93 ± 2% macrophages, 6.6 ± 2% lymphocytes, and 0.4 ± 1% neutrophils. In asthmatic subjects, the yield was 11 ± 4.4 × 106 cells with 88.6 ± 3.1% macrophages, 8.6 ± 2.6% lymphocytes, 1.1 ± 0.4% neutrophils, and 1.6 ± 1.0% eosinophils. Cells were > 85% viable, as assessed by trypan blue exclusion.

RT-PCR for IL-10

RNA was extracted using a modification of the method of Chomczynski and Sacchi (21) and its purity assessed by spectrophotometry. Reverse transcription of 1 μg of total RNA was performed using AMV-reverse transcriptase (15 U), 10 mM of dATP, dCTP, dGTP, and dTTP, oligo dT15 primer (0.2 μg), RNase inhibitor (30 U), and 5× AMV buffer in a total volume of 8.5 μl (all from Promega, Southampton, UK). RNA was denatured at 65° C for 10 min. The remaining ingredients were then added as a master mix, and samples were incubated at 42° C for 60 min followed by 4 min at 90° C. The cDNA was subsequently diluted to a final volume of 100 μl in nuclease-free water. For PCR, 5 μl of the cDNA solutions were used. PCR was performed using 0.5 μg/μl of forward and reverse primers, dATP, dGTP, dTTP, and dCTP, at a concentration of 10 mM each, Taq polymerase (0.5 U), and buffer A in a final volume of 25 μl (all from Promega) set for 29 cycles for IL-10 and 28 cycles for GAPDH at a denaturing temperature of 94° C for 30 s, specific annealing temperature of 65° C for IL-10 and 58° C for GAPDH, and extension temperature of 72° C for 30 s. Primers for IL-10 were 5′ ATGCCCCAAGCTGAGAACCAAGACCCA and 3′ TCTCAAGGGGCTGGGTCAGCTATCCCA, giving a product of 351 base pairs. Primers for GAPDH were 5′ TCTAGACGGCAGGCTAGGTCCACC and 3′ CCACCCATGGCAAATTCCATGGCA, giving a product of 598 base pairs. PCR was carried out in a Techne multiwell thermocycler (Techne, Cambridge, UK). The number of cycles was chosen after determination of the linear phase of the product amplification curve from serial sampling with increasing cycles of amplification. Products were distinguished by electrophoresis on a 1.5% agarose, ethidium bromide–stained gel and then visualized using ultraviolet luminescence.

Southern blot analysis was performed. The 1.5% agarose gels were denatured, neutralized, and blotted onto nylon membranes. DNA was cross-linked to the membrane by ultraviolet light. Membranes were incubated for 4 h at 65° C in buffer D (6× SSC, 5× Denhardt's solution, 20 mM NaH2PO4, 0.1% wt/vol SDS, 250 μg/ml sonicated salmon sperm DNA) and hybridized at 65° C overnight in the same buffer with 32P-radiolabeled gene-specific internal oligonucleotide probes directed against a sequence internal to the specific PCR product. Filters were washed twice in 3× SSC/0.1% SDS for 15 min at 65° C, once with 1× SSC/0.1% SDS for 30 min at 65° C, and finally twice with 0.1× SSC/0.1% SDS for 30 min at 65° C. Each filter was then exposed for 6 to 24 h to Kodak (Rochester, NY) X-OMAT film and developed. In addition, 5 μl of each PCR product was dot-blotted and hybridized according to the above-described method to allow quantitation by Cerenkov counting of the filters. Data are expressed as the ratio of IL-10 to GAPDH cDNA.

Radioimmunoassay for MIP-1 α

Recombinant MIP-1α was iodinated (2.5 μg) using iodogen as previously described (22). The competitive binding assay was as previously described for the complement activation product C5a (23). Briefly, cell culture supernatant fluid (100 μl) was mixed with 11% polyethylene glycol 6,000 containing 0.5% protamine sulfate (100 μl), [125I]ligand (6 fmol in 50 μl), and antibody (50 μl of rabbit anti-MIP-1α antibody diluted 1:50). After 24 h of incubation at room temperature, radioactivity was precipitated using 25 μl of goat anti-rabbit IgG (Nordik, UK). The lower limit of adequate measurement (the concentration required to inhibit [125I]ligand binding by 20%) was 12 pM. This assay was specific for MIP-1α and did not cross-react with other chemokines, including MIP-1β, RANTES, and monocyte chemoattractant protein-1 (MCP-1).

Enzyme-linked Immunosorbent Assays for IL-10, IFN- γ , and GM-CSF

These cytokines were assayed using a quantitative sandwich enzyme immunoassay technique. For IL-10 and IFN-γ, commercially available kits were used (Quantikine; R&D Systems, Abingdon, Oxon, UK). Briefly, monoclonal anti-IL-10 or anti-IFN-γ antibody was coated onto a microtiter plate, to which standards and samples were added. An enzyme-linked polyclonal antibody specific for IL-10 or IFN-γ was added to the wells to sandwich-immobilized IL-10 or IFN-γ. Addition of a stabilized chromogen and hydrogen peroxide allowed a color development in proportion to the amount of IL-10 or IFN-γ assayed by measurement of optical density using a spectrophotometer set to 450 nm. The lower limit of detection was 1.5 and 2 pg/ml for IL-10 and IFN-γ, respectively.

For GM-CSF assay, round-bottom plates were coated overnight at 4° C with a rat anti-human GM-CSF monoclonal antibody of (50 μl of 2 μg/ml). After washing with phosphate-buffered saline (PBS)/Tween, the antibody was blocked with PBS/10% fetal calf serum (200 μl; 2 h). GM-CSF standard and samples were added to the plate overnight at 4° C and washed with PBS/Tween (four times). A biotinylated secondary anti-GM-CSF antibody (100 μl of 2 μg/ml in PBS/10% fetal calf serum) was added for 45 min followed by 1:400 avidin-peroxidase solution (100 μl). After washing, GM-CSF was measured colometrically at 405 nm and quantified by interpolation from a standard curve. The lower limit of detection was 16 pg/ml.

Data Analysis

Data are reported as mean ± SEM. Data obtained within the asthmatic group during the placebo and active periods were compared using the nonparametric Wilcoxon's test. To compare the data between asthmatics and normal volunteers, the nonparametric Mann-Whitney test was used. A p value of < 0.05 was considered to be significant.

Effect of Budesonide on FEV1, Bronchial Responsiveness, and BAL Cells

There was no significant difference in FEV1 and PC20 between the start and the end of the placebo period. However, mean FEV1 improved from 3.60 ± 0.15 L to 3.80 ± 0.22 L (p < 0.05) and log PC20 from −0.40 ± 0.16 to 0.30 ± 0.21 (p < 0.005) after 1 mo of steroid treatment. There was no significant effect of steroid treatment on total BAL cell counts, but eosinophil counts were significantly reduced from 1.6 ± 1.0% to 0.6 ± 0.3% following budesonide (p < 0.05).

Time Course of IL-10 mRNA Expression and Protein Release

To determine the time course of IL-10 mRNA expression and protein production, peripheral blood monocytes and alveolar macrophages from normal subjects were plated and stimulated with LPS (1 μg/ml). There was a time-dependent increase of IL-10 protein and mRNA peaking at 24 h in both monocytes and macrophages. Expression of IL-10 mRNA and protein release was higher in monocytes than in macrophages (Figure 1).

Effects of Budesonide on IL-10 Expression and Release

There was no significant difference in IL-10 mRNA expression and protein release between the steroid-treated and placebo-treated periods and between the blood monocytes from normal subjects compared with asthmatics (Figure 2). However, in alveolar macrophages, IL-10 mRNA was significantly lower during placebo treatment compared with budesonide treatment at baseline and 24 h after stimulation with LPS and IL-1β (Figure 3). Significantly lower IL-10 protein was released during the placebo treatment compared with the budesonide treatment at baseline and 24 h after stimulation with LPS and IL-1β. Compared with the control group of normal volunteers, placebo-treated asthmatics showed a significantly lower IL-10 protein release at baseline and after IL-1β stimulation, although the difference after LPS stimulation did not reach statistical significance (Figure 3).

Effects of Budesonide on MIP-1 α , GM-CSF, and IFN- γ Release

Blood monocytes. Inhaled budesonide had no effect on MIP-1α mRNA expression as assessed by Northern analysis or on MIP-1α protein release. There was no significant difference in mRNA expression and protein release between the placebo and budesonide treatments and between the asthmatic and normal control groups at baseline and after 24 h of stimulation with LPS and IL-1β. However, there was a significantly higher amount of GM-CSF release from blood monocytes of the placebo period after stimulation with LPS and IL-1β compared with the budesonide treatment period and with the normal group. IFN-γ release was not detectable from supernatants of monocytes from asthmatic or normal subjects.

Alveolar macrophages. MIP-1α protein release was significantly higher in the placebo period compared with the budesonide period and also compared with the normal control group, at baseline and after 24 h of stimulation with LPS and IL-1β (Figure 4). Similar results were obtained with the release of GM-CSF and IFN-γ. IFN-γ release was not detectable in supernatants from alveolar macrophages of normal subjects. MIP-1α and GM-CSF release was higher from macrophages compared with monocytes.

Effect of Dexamethasone on Stimulated IL-10 Release from Macrophages In Vitro

To determine the effect of corticosteroids on IL-10 release from human alveolar macrophages, we stimulated macrophages (1 × 106) obtained from six normal volunteers with LPS (1 μg/ml), with and without prior addition of dexamethasone (10−6 M) for 2 h in 24-well plates. Supernatants were harvested 24 h after addition of LPS. LPS alone induced release of IL-10 of 486 ± 76 pg/ml, whereas after dexamethasone incubation the release was significantly inhibited to 85.8 ± 24.1 pg/ml (p < 0.01).

We have shown that alveolar macrophages of patients with mild asthma release significantly less IL-10 than those of normal nonasthmatic subjects at baseline and following stimulation by exogenous stimuli such as LPS and IL-1β. In addition, they express a lesser amount of IL-10 mRNA, indicating that this difference in IL-10 release is due to a reduction in IL-10 transcription. These results are generally in line with recent data indicating that IL-10 levels obtained from BAL fluid of asthmatics are lower compared with those of normal subjects, associated with reduced IL-10 mRNA levels in BAL cells (24). On the other hand, the capacity for alveolar macrophages to produce pro-inflammatory cytokines MIP-1α, IFN-γ, and GM-CSF was increased in asthma, further indicating the overall pro- inflammatory contribution of alveolar macrophages in asthma.

Chronic inhaled corticosteroid therapy led to an increased capacity for alveolar macrophages to express IL-10 mRNA and to release IL-10 from alveolar macrophages to levels observed from those obtained from normal subjects. By contrast, the increased release of the pro-inflammatory cytokines MIP-1α, IFN-γ, and GM-CSF from alveolar macrophages of asthmatic subjects was inhibited. Thus, overall, there was an anti-inflammatory effect of inhaled steroid therapy. IL-10 possesses inhibitory properties on macrophage function such as reducing the release of pro-inflammatory cytokines, and the proliferation of T cells by preventing the production of IL-2. Our data therefore indicate that one of the mechanisms by which inhaled steroids inhibit asthmatic inflammation is to enhance IL-10 expression and release from alveolar macrophages. Conversely, airway inflammation may be persistent in asthma through a reduction of IL-10 expression in alveolar macrophages.

The opposing relationships between IL-10 release on the one hand and MIP-1α, IFN-γ, and GM-CSF release on the other hand suggest that these cytokines may have effects on each other. One possibility is that endogenously released IL-10 can influence the release of pro-inflammatory cytokines. An antibody to IL-10 has been shown to enhance the release of MIP-1α, TNFα, IL-1β, and IL-6 from monocytes (14, 16, 18). Thus, IL-10 may act as an auto-regulatory brake on macrophages and have a modulating effect on the release of other pro-inflammatory cytokines. Conversely, IFN-γ and GM-CSF can also inhibit IL-10 release in blood monocytes (25-27). Furthermore, the inhibition of IL-10 by IFN-γ was associated with induction of TNFα and IL-1β synthesis (26, 27). It is more plausible that the pro-inflammatory cytokines may be influencing the levels of IL-10, since the time course of expression of IL-10 is, as we have shown, delayed in relation to the other pro-inflammatory cytokines such as MIP-1α and GM-CSF, which are expressed quickly with peak responses within a few hours after cell stimulation (6, 12). Therefore, corticosteroids may primarily lead to suppression of pro-inflammatory cytokine release such as IFN-γ and GM-CSF, in turn causing a secondary rise in IL-10 release. This possibility is also more likely because corticosteroids directly inhibit IL-10 release from monocytes (24) and alveolar macrophages (present study) in vitro, indicating that up-regulation of IL-10 mRNA in vivo is likely to be an indirect effect of corticosteroids. The changes in IL-10 release secondary to corticosteroid therapy were mirrored by similar changes in IL-10 mRNA in both normal and asthmatic alveolar macrophages, indicating that the corticosteroid effects occurred at the transcriptional level. Binding of activated corticosteroid receptors to positive consensus glucocorticoid responsive elements present on the IL-10 promoter may lead to increased IL-10 expression, but, on the other hand, binding of activated corticosteroid receptors with consensus sequences for transcription factors such as AP-1 present in the IL-10 promoter (28) may underlie the in vitro inhibitory effects of corticosteroids on IL-10 expression. However, it is unclear why chronic steroid therapy should lead to one mechanism while acute in vitro exposure leads to another mechanism.

We observed several differences between monocytes and macrophages. Monocytes produced more IL-10 and also more of the pro-inflammatory cytokines MIP-1α and GM-CSF than did macrophages. However, the differences between normal and asthmatic subjects was only observed in the alveolar macrophage in terms of release of pro- and anti-inflammatory cytokines. The underlying mechanism for this increased release in pro-inflammatory cytokines together with a decrease in IL-10 release is not known. It is possible that, as discussed above, there is an overexpression of IFN-γ and/or other pro-inflammatory cytokines that inhibit IL-10 expression and synthesis. In addition, the effect of corticosteroids in increasing IL-10 release was not observed in monocytes but in macrophages. This may not be surprising because the effect of inhaled corticosteroids would be mostly on airway macrophages rather than on blood monocytes. However, we found that monocytes also demonstrated decreased release of GM-CSF (but not of MIP-1α and IFN-γ) as observed in alveolar macrophages after corticosteroid therapy, which may reflect the actions of systemically absorbed budesonide.

IL-10 has numerous anti-inflammatory effects that would be beneficial in suppressing inflammatory mechanisms associated with asthma. In addition to inhibiting the production of IFN-γ and IL-2 by Th-1 lymphocytes (15), IL-10 also inhibits pro-inflammatory cytokine production (such as IL-1β, IL-6, and IL-8) by mononuclear phagocytes (14, 17, 18, 29) at the level of cytokine gene transcription (30) and IL-4 and IL-5 production by Th-2 T cells (31). Corticosteroids convert the cytokine response of the alveolar macrophage of the asthmatic subject into the range found in normal subjects, and this may underlie the improved functional effects observed, such as the attenuation of bronchial hyperresponsiveness.

In summary, we have shown that chronic inhaled corticosteroid therapy alters the balance of pro- and anti-inflammatory cytokine expression and release from alveolar macrophages in asthma by increasing the production of the anti-inflammatory cytokine, IL-10, while reducing the production of the pro-inflammatory cytokines MIP-1α, GM-CSF, and IFN-γ.

Supported by a Program Grant from the British Medical Research Council (to K.F.C. and P.J.B.), a grant from Astra Draco, and fellowships from the Overseas German Academic Exchange Service (to M.J.) and Deutsche Forschungsgemeinschaft (to J.S.).

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Correspondence and requests for reprints should be addressed to Dr. K. F. Chung, National Heart & Lung Institute, Dovehouse St., London SW3 6LY, UK.


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