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. 1999 Sep;98(1):104–110. doi: 10.1046/j.1365-2567.1999.00862.x

Regulation of major histocompatibility complex class II antigens on human alveolar macrophages by granulocyte–macrophage colony-stimulating factor in the presence of glucocorticoids

J J Caulfield 1, M H Fernandez 1, A R Sousa 1, S J Lane 1, T H Lee 1, C M Hawrylowicz 1
PMCID: PMC2326900  PMID: 10469240

Abstract

Alveolar macrophages (AM) present antigen poorly to CD4+ T cells and respond weakly to interferon-γ (IFN-γ) for up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecule expression. In atopic asthma, however, AM exhibit enhanced antigen-presenting cell (APC) activity. Since granulocyte–macrophage colony-stimulating factor (GM-CSF) is increased in the airways of asthmatic patients, we have investigated its role in modulating the APC function of AM. The effects of glucocorticoids were also studied since earlier studies showed optimal induction of MHC antigens on monocytes by GM-CSF in their presence. GM-CSF in the presence, but not the absence, of dexamethasone enhanced the expression of HLA-DR, -DP and -DQ antigens by AM. However AM and monocytes differed in the optimal concentration of steroid required to mediate this effect (10−10 m and 10−7 m, respectively). Induction of MHC antigens was glucocorticoid specific and independent of IFN-γ. These studies suggest the existence of an IFN-γ-independent pathway of macrophage activation, which may be important in regulating APC function within the lung.

INTRODUCTION

The lung represents a unique immunological environment where, in normal healthy individuals, there is little or no immune activation despite constant exposure to foreign material in inhaled air. This is reflected in the functional phenotype of alveolar macrophages (AM) which are highly phagocytic but present antigen to T lymphocytes poorly in comparison to blood monocytes.1–6 AM differ from monocytes in the regulation of their antigen-presenting function. For example, AM are negative for, or express low levels of, the costimulatory molecules CD80 (B7-1) and CD86 (B7-2), and these molecules are not up-regulated in vitro in response to interferon-γ (IFN-γ).7 Furthermore, early studies show a failure of AM to up-regulate expression of major histocompatibility complex (MHC) class II antigens4 or to show improved accessory function following exposure to IFN-γ.8

Increased immune activation is observed in the lung in chronic inflammatory conditions such as asthma. It seems probable that an independent pathway acts on resident lung antigen-presenting cell (APC) populations to activate infiltrating memory T cells, although this has not yet been clearly defined. Although expression of IFN-γ is not up-regulated in the lung in asthma,9 patients show increased expression of granulocyte–macrophage colony-stimulating factor (GM- CSF) in airway epithelial cells10 and monocytes and AM produce two- to threefold higher concentrations of GM-CSF as compared with healthy individuals.11,12 GM-CSF is reported to activate human blood monocytes for increased APC-associated function13–15 and appears a likely candidate for regulation of the observed increase in human lung macrophage antigen-presenting function in asthma.

Our previous work has demonstrated that maximal induction of MHC class II antigens by GM-CSF on human blood-derived monocytes16 and eosinophils17 is only observed when glucocorticoid hormones or their synthetic analogues are also present in culture. We have proposed that stimulation with GM-CSF in the presence of endogenous steroid hormones represents an important pathway for the activation of lung macrophages in asthma. Since clear differences occur in regulation of monocyte and AM APC function, the present study was designed to test this hypothesis directly. GM-CSF alone had little effect, but stimulation with GM-CSF plus glucocorticoids increased MHC class II expression by AM. These studies highlight further differences in the regulation of AM and monocyte APC function, since that latter require two to three logs higher concentrations of steroid to mediate this effect. They support a role for GM-CSF in promoting increased APC-associated functions in the human lung under conditions of chronic inflammation such as asthma.

MATERIALS AND METHODS

Reagents and monoclonal antibodies

Purified recombinant human GM-CSF was purchased from R & D Systems (Abingdon, UK), IFN-γ from Boehringer-Ingelheim (Ingelheim, Germany), dexamethasone from Sigma (Poole, UK) and mifepristone (RU 486) was a kind gift from Dr Sitruk-Ware, Exelgyn, Paris, France. Fluorescein isothiocyanate (FITC) -conjugated isotype controls and mouse anti-human monoclonal antibodies (CD3, CD14, CD16) used to demonstrate monocyte purity were purchased from Becton Dickinson-Pharmingen (Oxford, UK), anti-CD80 and anti-CD86 from The Binding Site (Birmingham, UK), rabbit anti-mouse phycoerthyrin (PE) -conjugated secondary antibodies from Dako (Glostrup, Denmark). Murine hybridomas secreting antibody against human HLA-DP [clone B7/21, immunoglobulin G1 (IgG1)], HLA-DQ (clone SPV-L3, IgG2a), HLA-DR (clone L243, IgG2a) and anti-IFN-γ antibody (clone 4SB3, IgG1) were grown in a Miniperm system (Hereaus, Osterode, Germany) and antibody isolated from the culture supernatants using MabTrap protein G columns (Pharmacia, St Albans, UK). FITC conjugation of anti-HLA-D antibodies was performed using standard techniques. All antibodies were used at a final concentration of 1 μg/ml.

Subjects

Volunteers for bronchoalveolar lavage were healthy non-smokers between the ages of 20 and 40 years. Asthma was defined as reversible obstruction of the airways and all asthmatic subjects fulfilled the American Thoracic Society criteria for asthma. All asthmatic subjects (three male; one female) had mild asthma, were not on inhaled steroids for 3 months prior to the study and were well at the time of lavage. Mean 1-second forced expiratory volume (FEV1) and provocation concentration-20 (PC20) for histamine challenge for asthmatic volunteers were 90% (83, 94, 93 and 89, respectively) and 0·37 mg/ml (0·3, 0·47, 0·49 and 0·21, respectively) compared with 96% and 3·57 mg/ml for non-asthmatic subjects (all male), respectively. Atopy was defined as the presence of a positive skin test reactions (3 mm greater than the diluent control reaction) to at least one of a panel of aeroallergens (Dermatophagoides pteronyssinus, cat dander and grass pollen).

Bronchoalveolar lavage

Fibreoptic bronchoscopy was performed, as previously described,11 on non-asthmatic or atopic, asthmatic volunteers with full, informed consent and adherence to guidelines approved by the UMDS Ethical Committee. A single 100 ml aliquot of normal buffered saline was instilled into a subsegmental bronchus of the left side middle lobe and gentle suction was applied. The lavage fluid was centrifuged at 200 g for 10 min and the cell pellet was washed three times in Hanks’ balanced salt solution. A differential cell count was performed. Cells were adhered to plastic for 60 min, washed to remove any non-adherent cells, and the adhered cells were resuspended in RPMI-1640+5% fetal calf serum (FCS) supplemented with 2·5 μg/ml amphotericin B (Gibco, Paisley, UK).

Monocyte purification

Peripheral blood mononuclear cells (PBMC) were separated by centrifugation at 800 g for 20 min over Lymphoprep (Gibco) and then spun through a discontinuous Percoll (Sigma) gradient comprising layers of 1·057, 1·070, 1·085 and 1·122 g/ml at 800 g for 20 min. Monocytes were harvested at the 1·057 and 1·070 g/ml interface and the enriched fraction was depleted of residual CD2+ T cells by incubation overnight at 4° with sheep red blood cells (Difco, East Molesey, UK) conjugated to 2-aminoethylisothio-uronium bromide (Sigma) as previously described.16 Monocyte purity was assessed using a Becton Dickinson FACScan flow cytometer.

Cell stimulation cultures and immunofluorescence labelling

Monocytes and AM were cultured in RPMI-1640+5% FCS supplemented with 2 mm l-glutamine (Gibco) and 2 μg/ml ciprofloxacin (Bayer, Newberry, UK) at 1×106 cells/ml in 24-well plates (Nunc, Roskilde, Denmark) at 37°. They were stimulated with previously determined optimal concentrations of IFN-γ (200 ng/ml) or GM-CSF (5 ng/ml) and the indicated concentrations of dexamethasone. Following titration experiments, anti-IFN-γ neutralizing antibody was used at 20 μg/ml. Cells were harvested from the plates by incubation with 3·3 mm ethylenediamine tetraacetic acid (EDTA) on ice for 30 min, washed and incubated with the relevant antibody for 30 min on ice. Where necessary, a second layer of PE-conjugated goat anti-mouse antibody was added for a further 30 min prior to fixation in 1% paraformaldehyde and analysis by flow cytometry. Monocytes and AM were gated using forward (size) versus side (granularity) scatter dot plots to exclude any debris or lymphocytes from further analysis. Results are expressed as geometric means of fluorescence intensity (MFI) for the test antibody minus the MFI values of matched isotype controls.

Statistical analysis

A Student’s t-test (paired, two-tailed) was used to determine statistically significant differences between groups. A value of P < 0·05 was used to determine significant differences between means.

RESULTS

Characteristics of monocyte and AM populations

Isolated monocytes were >99% viable as assessed by trypan blue exclusion and over 90% pure as determined by flow cytometry. From 12 bronchoalveolar lavage preparations, mean yields were 5·4×106 AM and the percentage of AM was 83% and 17% mononuclear cells. These were then adhered to plastic to enrich for AM, of which >95% were viable.

Freshly isolated AM showed considerable heterogeneity in basal MHC class II expression. Mean MFI expression (±SEM) of HLA-DR, -DP and -DQ was 35 (±9), 26 (±7) and 14 (±7), respectively, for 12 subjects. Basal levels of costimulation molecules were low: mean MFI values for CD40, CD80 and CD86 were 18 (±10), 2 (±1) and 19 (±10), respectively.

GM-CSF, in combination with glucocorticoids, up-regulates MHC class II molecule expression on AM at lower concentrations compared to monocytes

The glucocorticoid dexamethasone was tested for its capacity, in conjunction with 5 ng/ml GM-CSF, to enhance MHC class II molecule expression on human AM. GM-CSF alone did not increase MHC class II antigen expression. The combination of GM-CSF and dexamethasone up-regulated expression of HLA-DR, HLA-DP and HLA-DQ antigens on AM (Figs 1 and 2). The optimal concentrations of dexamethasone varied between donors. 1×10−10 m (range 1×10−11–10−9 m) was the ideal concentration in most subjects (Fig. 2). High concentrations of dexamethasone (10−8– 10−6 m) on occasion reduced MHC class II expression when compared to cells cultured with GM-CSF alone. Expression of HLA-DQ was low compared with the matched isotype immunoglobulin control and all further data therefore are for HLA-DP and HLA-DR only.

Figure 1.

Figure 1

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Immunofluorescent staining profiles of AM (a–c) and monocytes (d–f) labelled with anti-HLA-DR antibody. Cells were cultured in medium alone (a, d), or stimulated with 5 ng/ml GM-CSF (b, e) or GM-CSF plus optimal concentration of dexamethasone (c, f). All HLA-DR profiles (filled histogram) are shown compared with isotype-matched control antibody profiles (open histogram). Cells were obtained from a non-asthmatic, healthy donor.

Figure 2.

Figure 2

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GM-CSF and low concentrations of dexamethasone up-regulate MHC class II antigen expression on AM. Expression of HLA-DR (a), HLA-DP (b) and HLA-DQ (c) was measured on AM stimulated for 4 days with GM-CSF alone (open symbols) or GM-CSF plus a range of dexamethasone concentrations (closed symbols). Data for medium alone were equivalent or lower to values for GM-CSF only. Data shown are from two representative, non-asthmatic, healthy donors.

Table 1 shows mean±SEM MFI of expression of HLA-DP (n = 9; four asthmatic and five non-asthmatic donors) and HLA-DR (n = 8; four asthmatic and four non-asthmatic donors) antigens on AM stimulated with GM-CSF and a range of dexamethasone concentrations. There was a significant increase in MHC class II expression following culture with GM-CSF plus an optimal concentration of dexamethasone compared with GM-CSF alone for HLA-DP (P = 0·01) and HLA-DR (P = 0·03). However in the small sample size tested, no significant difference in MHC class II expression between atopic, asthmatic and non-asthmatic subjects was observed. Mean (±SEM) MFI expression of HLA-DR expression by AM cultured with GM-CSF plus an optimal concentration of dexamethasone by four non-asthmatic subjects was 98 (±44), compared with 85 (±35) for four asthmatic volunteers. For HLA-DP, mean (±SEM) MFI by five non-asthmatic volunteers was 88 (±3), compared with 52 (±12) for four asthmatic subjects. All patients were mildly asthmatic and well at the time of study.

Titration of dexamethasone for GM-CSF-mediated induction of MHC class II antigens. Mean MFI expression±SEM of HLA-DP (n = 9; four asthmatic and five non-asthmatic donors) and HLA-DR (n = 8; four asthmatic and four non-asthmatic donors) antigens by AM

graphic file with name imm0098-0104-t1.jpg

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GM-CSF plus dexamethasone significantly up-regulated HLA-DP (P = 0·01) and HLA-DR (P = 0·004) antigen expression on peripheral blood monocytes. However, as previously reported,16 monocytes optimally responded to two log higher concentrations of dexamethasone (optimally 10−7m; Table 2) than AM. Dexamethasone alone did not increase MHC class II expression on either monocytes or AM.

Titration of dexamethasone for GM-CSF-mediated induction of MHC class II antigens. Mean±SEM of HLA-DP and HLA-DR (n = 7) antigens by peripheral blood monocytes

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Differential kinetics of MHC class II antigen up-regulation by IFN-γ compared with GM-CSF plus glucocorticoids

Stimulation of monocytes with 200 ng/ml IFN-γ led to a significant (P < 0·01) increase in HLA-DR and HLA-DP on peripheral blood monocytes (Table 2). This up-regulation occurred within 24 hr of culture, and decreased within 96 hr to levels of unstimulated cells. In contrast, GM-CSF plus dexamethasone did not up-regulate HLA-DP or HLA-DR at 24 hr, but a two-to threefold increase in expression was observed at 96 hr or later (Fig. 3a). Similar kinetics of MHC class II up-regulation was observed on AM (Fig. 3b). Stimulation of AM (Table 1) with 200 ng/ml IFN-γ increased HLA-DR and HLA-DP expression, although this was not statistically significant for either HLA-DR or HLA-DP. Although one donor demonstrated a large increase of HLA-DP and HLA-DR expression in response to IFN-γ, three others showed little or no up-regulation (Fig. 3b and data not shown).

Figure 3.

Figure 3

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Kinetics of up-regulation of HLA-DR by IFN-γ versus GM-CSF plus dexamethasone. Monocytes (a) or AM (b) were cultured in medium or stimulated with IFN-γ, GM-CSF, or GM-CSF plus an optimal concentration of dexamethasone, for 1–7 days as indicated. Mean±SEM of seven and four non-asthmatic, healthy donors, respectively. Similar data were observed for HLA-DP.

GM-CSF plus glucocorticoid-mediated HLA-DR up-regulation is independent of IFN-γ

To determine whether the up-regulation of MHC class II molecules by GM-CSF plus glucocorticoids was IFN-γ independent, a neutralizing anti-IFN-γ antibody (clone 4SB3) was used. On monocytes, HLA-DR antigen up-regulation by GM-CSF plus 1×10−7 m dexamethasone was unaffected by the presence of neutralizing antibody (Fig. 4a). IFN-γ-mediated up-regulation of HLA-DR antigen at the optimal time of 24 hr was abolished by 4SB3, but was unaffected by an isotype-matched IgG1 control antibody. Comparable data was observed following 96 hr of culture (data not shown). Similarly, stimulation of AM with GM-CSF plus dexamethasone was unaffected by the presence of the neutralizing anti-IFN-γ or the control antibody (Fig. 4b). These results suggest that there are two separate pathways for MHC class II molecule up-regulation in human AM and monocytes, and that GM-CSF and glucocorticoids act independently of IFN-γ.

Figure 4.

Figure 4

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Induction of HLA-DR expression on monocytes and AM by GM-CSF plus dexamethasone is independent of IFN-γ. Monocytes (a) or AM (b) were cultured with IFN-γ, GM-CSF and/or dexamethasone as indicated in the presence or absence of 20 μg/ml neutralizing anti-IFN-γ antibody or control isotype-matched antibody and the effects on HLA-DR expression assessed at 24 hr or 96 hr. Monocyte data is expressed as mean±SEM of four non-asthmatic, healthy donors. AM data is from one non-asthmatic donor. Comparable data was obtained from a second donor.

A glucocorticoid receptor antagonist, mifepristone (RU 486), was used to demonstrate that GM-CSF plus glucocorticoid-mediated HLA-D up-regulation is mediated via the glucocorticoid receptor. Mifepristone at 1×10−6 m abolished the up-regulation of HLA-DR antigens observed with the culture of monocytes from four separate donors with GM-CSF and 1×10−7 m dexamethasone (data not shown). This effect was dose dependent and no inhibition was observed at 1×10−8 m mifepristone. Co-culture of AM with GM-CSF plus dexamethasone with 1×10−6 m mifepristone inhibited up-regulation of HLA-DR (data not shown). This concentration of mifepristone had no effect on HLA-DR expression by AM or monocytes cultured with GM-CSF alone (data not shown). Comparable results were observed with two separate donors.

Expression of costimulation molecules by AM

The effects of GM-CSF plus dexamethasone stimulation on AM expression of additional surface molecules involved with APC function were studied, specifically CD40, CD80 and CD86. After 72 hr in culture, CD40, CD80 and CD86 levels were not significantly increased in the presence of IFN-γ (mean±SEM; 15±6, 4±2, 19±2, respectively) or GM-CSF plus low concentrations of dexamethasone (13±5, 6±4, 10±2, respectively, at 1×10−10 m dexamethasone), as compared with medium controls (16±9, 2±1, 9±3, respectively).

DISCUSSION

The present study demonstrates that GM-CSF, in the presence of low ‘physiological’ concentrations of dexamethasone, up-regulates MHC class II antigen expression on human AM in all individuals tested. Strikingly, blood monocytes and AM exhibit a two to three log difference in their sensitivity to steroid. Treatment of AM with GM-CSF and dexamethasone had no significant effect upon costimulatory molecule expression. This pathway of MHC class II induction is independent of IFN-γ and the kinetics of expression differs from that observed with IFN-γ stimulation.

AM are generally believed to be poor APC, although they have been shown to present recall antigens to CD4+ T cells,5,6,18,19 albeit with less efficiency than autologous monocytes. However AM from patients with allergic asthma show an increased capacity to activate T cells.20 Expression of MHC class II antigens is essential for antigen-specific activation of CD4+ T cells. Induction of increased and sustained levels of MHC class II antigens, and therefore of effective antigen dose, is predicted to increase T-cell activation. However, treatment of AM with GM-CSF and dexamethasone, or IFN-γ, had no effect on expression by AM of costimulation molecules. Two previous reports confirm this low level of costimulatory molecule expression on AM.7,20 Although CD86 expression is not elevated, the observed increased allergen presentation by AM in atopic asthmatic patients appears to be partially dependent on CD86.20 Essentially all T cells present are of the CD45RO+ memory T-cell phenotype in the asthmatic lung,21,22 and their activation requirements are less stringent than for CD45RA+ naive T cells.23 These studies imply that the low levels of costimulatory signals provided by AM are sufficient to activate T cells within the lung.

A number of additional studies using rodent models suggest that GM-CSF is a likely candidate for regulating lung APC function. Experiments in rodents have shown that AM are immunosuppressive, inhibiting the function of dendritic cells and T-cell proliferation6,24,25 and this can be reversed in vitro by GM-CSF.26 Recently, it has been demonstrated that local expression of GM-CSF within the airways induces an APC-dependent, allergic sensitization.27 Mice infected intranasally with an adenovirus construct expressing GM-CSF and repeatedly exposed to ovalbumin (OVA) developed an antigen-specific airway eosinophilia, tissue pathology and OVA-specific T-cell memory. This response did not occur in mice lacking either MHC class II antigens or interleukin-5 (IL-5). The authors concluded that GM-CSF expression within the airways increased local antigen presentation and facilitated the development of an antigen-specific, eosinophilic, inflammatory response to an otherwise innocuous antigen.

Previous studies demonstrate that GM-CSF and IL-3, or GM-CSF, IL-3 and IL-5, act in synergy with glucocorticoids such as dexamethasone or hydrocortisone to increase MHC class II antigen expression on human blood monocytes and eosinophils, respectively.16,17 The fundamental difference between the current and these earlier studies is the optimal concentration of steroid required to induce these effects: typically 10−7 m for monocytes and eosinophils vs. 10−10 m for AM. Whilst the reasons for the differential sensitivity of monocytes and AM to corticosteroid are unclear, possible explanations include: differences in glucocorticoid receptor density or availability of associated molecules, the isoform of the glucocorticoid receptor present or even differences in metabolism of steroid by the two cell types.

At least two other examples exist of comparably low concentrations of steroid up-regulating immunological parameters. Calandra et al28 report glucocorticoid up-regulation of macrophage migration inhibitory factor (MIF) by macrophages and T cells where the optimal response was induced by 10−14–10−12 m dexamethasone. The authors proposed a role for MIF in counter-regulation of the inhibitory effects of glucocorticoids. Long-term administration of low doses of glucocorticoid results in enhanced nitric oxide and IL-1β secretion by lipopolysaccharide pulsed rat AM.29 These and other studies highlight an important role for physiological concentrations of glucocorticoid hormones in regulating critical immune parameters and the complexity of glucocorticoid action upon immune responses.

In summary, we have described a novel pathway where glucocorticoids, at ‘physiological’ concentrations enhance GM-CSF-induced functions of AM associated with an increased capacity to activate T lymphocytes. Since GM-CSF levels, but not the well-defined regulator of APC function IFN-γ, are increased in the lung during chronic inflammation associated with asthmatic disease, we propose that this represents an important pathway of immune activation contributing to pathology associated with the disease.

Acknowledgments

This work was funded by The National Asthma Campaign & Glaxo Wellcome. The authors thank Dr Boris Lams and Dr Cecilia Trigg of the Department of Respiratory Medicine and Allergy, Guy’s Hospital, and Dr Chris Corrigan of Imperial College of Science, Technology and Medicine for provision of the BAL samples and Dr Malcolm Johnson from Glaxo Wellcome and Dave Richards from the Department of Respiratory Medicine and Allergy, Guy’s Hospital for constructive comments on the work. They also thank Mrs Sheila Lusher and Ms Monique Plieger for secretarial support.

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