Journal List > Immune Netw > v.14(6) > 1033445

Im, Gerelchuluun, and Lee: Evidence for Direct Inhibition of MHC-Restricted Antigen Processing by Dexamethasone

Abstract

Dexamethasone (Dex) was shown to inhibit the differentiation, maturation, and antigen-presenting function of dendritic cells (DC) when added during DC generation or maturation stages. Here, we examined the direct effects of Dex on MHC-restricted antigen processing. Macrophages were incubated with microencapsulated ovalbumin (OVA) in the presence of different concentrations of Dex for 2 h, and the efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Dex inhibited both class I- and class II-restricted presentation of OVA to T cells; this inhibitory effect on antigen presentation was much more potent in immature macrophages than in mature macrophages. The presentation of the exogenously added OVA peptide SIINFEKL was not blocked by Dex. In addition, short-term treatment of macrophages with Dex had no discernible effects on the phagocytic activity, total expression levels of MHC molecules or co-stimulatory molecules. These results demonstrate that Dex inhibits intracellular processing events of phagocytosed antigens in macrophages.

Abbreviations

Dex

dexamethasone

DC

dendritic cell

M-CSF

macrophage-colony stimulating factor

rhM-CSF

recombinant human macrophage colony stimulating factor

INTRODUCTION

Glucocorticoids have been effectively used for several decades as potent immunosuppressive agents in the treatment of inflammatory and autoimmune diseases. The immunosuppressive activities of glucocorticoids are primarily attributed to their influence on T cells and monocytes/macrophages (1,2,3). Glucocorticoids inhibit the secretion of cytokines from T cells and their proliferation induced by various stimuli (4,5,6,7). Glucocorticoids also inhibit several immunologically relevant activities of monocytes and macrophages. They block the production of cytokines, the expression of surface receptors for complement and immunoglobulins, phagocytosis and pinocytosis, and bactericidal and fungicidal activities of monocyte/macrophages (8,9,10,11).
Dexamethasone (Dex) is a synthetic glucocorticoid exerting 25 times more potent immunosuppressive activity than cortisol. Dex is particularly interesting because it promotes tolerance in vivo by enriching tolerogenic macrophages, while inducing apoptosis of effector T cells (12,13,14). Dex was also shown to severely impair the differentiation, maturation, and function of dendritic cells (DCs) and macrophages (15,16,17). The effects of Dex on DCs and macrophages, however, were investigated in cells cultured in vitro in the presence of Dex for two to several days.
In the present study, we examined the direct effects of Dex on the MHC-restricted presentation of exogenous antigens. Macrophages were generated from mouse bone marrow cells and allowed to phagocytose microencapsulated ovalbumin (OVA) in the presence of Dex for 2 h. The efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Our results show that Dex inhibits the intracellular processing events of phagocytosed antigens in macrophages. We also discovered that immature macrophages are much more sensitive to the Dex-induced inhibition of MHC-restricted antigen processing than mature macrophages.

MATERIALS AND METHODS

Cell lines and reagents

The T-cell hybridoma cell lines B3Z86/90.14 (B3Z) and DOBW were kindly provided by Dr. Nilabh Shastri (University of California, Berkeley, CA, USA) and Dr. Clifford V. Harding (Case Western Reserve University, Cleveland, OH, USA), respectively (18,19). Recombinant human M-CSF was purchased from PeproTech (Rocky Hill, NJ, USA). Dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Generation of macrophages from bone marrow cells

Macrophages were generated from mouse bone marrow using recombinant human macrophage colony stimulating factor (rhM-CSF). Briefly, bone marrow cells obtained from femurs of C57BL/6 or Balb/c mice were cultured in a 6-well plate (5×106/well) in culture media supplemented with 20 U/ml rhM-CSF. At days 3 and 4 after the initiation of the culture, non-adherent cells were discarded by gentle shaking and replacement of the culture medium with fresh medium containing rhM-CSF. Immature macrophages were harvested on day 6 using cell stripper solution. Lipopolysaccharide (100 ng/ml) was added to immature macrophage cultures for maturation. Cells were cultured for 2 additional days and then harvested using cell stripper solution.

Preparation of OVA-nanospheres

Nanospheres containing OVA were prepared using a homogenization/solvent evaporation method with 400µl of OVA-containing water (50 mg/ml OVA) and 2 ml of ethyl acetate containing poly(lactic-co-glycolic acid) (100 mg/ml, Sigma-Aldrich) as described previously (Lee et al., 2010). Fluorescein isothiocyanate (FITC)-containing PLGA-nanospheres were prepared by adding FITC to the ethyl acetate phase together with PLGA. The OVA content was determined using a micro-bicinchoninic acid assay kit (Pierce, Rockford, IL, USA) after lysis of the nanospheres with a lysis buffer containing 0.1% SDS and 0.1 N NaOH.

MHC class I-restricted presentation assay

Class I MHC-complexed OVA peptide quantities on macrophages were assessed using B3Z cells (20). Briefly, macrophages (1×105/well) generated from bone marrow cells of C57BL/6 mice (H-2b) were incubated with the indicated amounts of Dex for 2 h, and then OVA-nanospheres were added (50µg as OVA). After 2 h incubation at 37℃, the plate was washed twice with pre-warmed PBS (300µl/well) and then fixed with ice-cold 1.0% paraformaldehyde (100µl/well) for 5 min at room temperature, followed by washing of the plate three times with PBS (300µl/well). Class I MHC-complexed OVA peptide quantities were assessed by IL-2 secretion assays after culturing the paraformaldehyde-fixed macrophages with CD8.OVA cells (2×104/well) for 18 h as described previously (20).

MHC class II-restricted presentation assay

Class II MHC-complexed OVA peptide quantities on macrophages were assessed using DOBW cells (20). Briefly, macrophages (1×105/well) generated from bone marrow cells of BALB/C mice (H-2d) were incubated with the indicated amounts of Dex for 2 h, and then OVA-nanospheres were added (50µg as OVA). After 2 h incubation at 37℃, unphagocytosed nanospheres were removed by suction and then fixed with ice-cold 1.0% paraformaldehyde (100µl/well) for 5 min at room temperature. Class II MHC-complexed OVA peptide quantities were assessed by IL-2 secretion assays after culturing the paraformaldehyde-fixed macrophages with DOBW cells (2×104/well) for 18 h as described previously (20).

Phagocytosis activity

Twenty minutes after nanospheres containing both OVA and FITC (1 mg/well as OVA) were added to macrophages, unphagocytosed nanospheres were removed by washing with pre-warmed PBS. The cells were harvested, fixed in 1% paraformaldehyde in PBS, and a flow cytometric analysis was performed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA).

Phenotype analysis

Cells were stained with monoclonal antibodies recognizing murine cell surface markers as described previously (21) and flow cytometry was performed. The monoclonal antibodies (anti-H2-Kb, anti-I-Ab, anti-CD80, anti-CD86, anti-CD40, and anti-CD54) and an isotype-matched control antibody were purchased from BD Biosciences.

RESULTS

Dex inhibits MHC-restricted processing of exogenous antigens

In order to examine the effects of Dex on the class I MHC-restricted processing of exogenous antigens, macrophages were allowed to phagocytose OVA-nanospheres in the presence or absence of Dex for 2 h and then fixed with paraformaldehyde. The efficacy of class Kb-restricted-OVA peptide presentation was evaluated using OVA-specific CD8 T cell hybridoma cells (B3Z), which express β-galactosidase when activated by OVA[257-264]-H-2Kb complexes. Dex dose-dependently inhibited class I MHC-restricted presentation of exogenous OVA (Fig. 1A). The inhibitory effect of Dex on class I MHC-restricted exogenous antigen presentation was much more potent in immature macrophages than in mature macrophages; the IC50 of Dex was approximately 1µM for immature macrophages and 10µM for mature macrophages.
In order to examine the effects of Dex on the class II MHC-restricted processing of exogenous antigens, macrophages were allowed to phagocytose OVA-nanospheres in the presence or absence of Dex for 2 h. The macrophages were then fixed with paraformaldehyde, and the amount of OVA peptide-class II MHC complexes was measured using OVA-specific CD4 T cell hybridoma DOBW cells. Dex dosedependently inhibited class II MHC-restricted presentation of exogenous OVA (Fig. 1B). This effect was also much more potent in immature macrophages than in mature macrophages; the IC50 of Dex was approximately 0.4µM for immature macrophages and 20µM for mature macrophages.
The exogenously added OVA peptide SIINFEKL can bind to cell surface Kb molecules, and thus does not require intracellular processing. In order to prove that Dex inhibits intracellular events of antigen processing pathways, macrophage cells were incubated with the peptide (1µM) for 2 h in the presence of different Dex concentrations. The cells were then washed, fixed, and examined for their stimulatory activities on T cell hybridoma B3Z cells. Dex did not inhibit presentation of the exogenously added synthetic peptide in immature or mature macrophages (Fig. 1C).

Short-term treatment of macrophages with Dex does not affect their phagocytic activity or the expression of cell surface molecules

In order to test whether the antigen presentation-inhibitory activity of Dex (Fig. 1) was due to prevention of phagocytic activity, nanospheres containing both OVA and FITC were added to immature and mature macrophages pre-treated with Dex for 4 h. Cells were washed, cooled on ice, and then harvested by gentle pipetting. Flow cytometric analysis of the harvested cells demonstrated that Dex did not inhibit phagocytic activity of either immature or mature macrophages (Fig. 2A).
The effects of Dex on the expression of class I (K-2Kb) and II MHC molecules (I-Ab) and co-stimulatory molecules such as B7-1 and B7-2 were also examined with 4 h Dex-treated macrophages. Short-term treatment with Dex did not result in discernible effects on the expression levels of MHC molecules (Fig. 2B) or co-stimulatory molecules (Fig. 2C).

DISCUSSION

The major question addressed in the present study was whether Dex can directly inhibit MHC-restricted antigen processing pathways. Several studies have shown that Dex severely impairs the antigen-presenting function of macrophages and DCs (15,16,17). Nonetheless, these studies used macrophages or DCs that were differentiated or matured in the presence of Dex. Thus, the direct effect of Dex on MHC-restricted antigen processing has not yet been clarified. In the present study, we show evidence that Dex directly inhibits intracellular processing events of phagocytosed antigens in macrophages. Short-term exposure (4 h) of macrophages to Dex sufficed to inhibit both class I and class II MHC-restricted antigen processing pathways. We also discovered that immature macrophages are more sensitive to the Dex-mediated suppression of MHC-restricted antigen processing.
Exposure of macrophages or DCs to Dex during differentiation or maturation suppresses the phagocytic activity and the expression of co-stimulatory molecules such as CD80, CD86, and CD40 receptors (22,23,24,25). In our experimental conditions, where Dex inhibits MHC-restricted antigen processing, Dex did not exert inhibitory effects on the phagocytic activity or the total MHC expression levels or co-stimulatory molecules. It is noteworthy that we pre-treated macrophages with Dex for 2 h, and then allowed 2 h for phagocytosis and processing of exogenous OVA in the presence of Dex. Furthermore, Dex did not inhibit the presentation of the exogenously added OVA peptide SIINFEKL under our experimental conditions. These results demonstrate that Dex directly inhibits intracellular processing events of phagocytosed antigens in macrophages.

Figures and Tables

Figure 1
Dex inhibits MHC-restricted processing of exogenous antigens. Immature or mature macrophages were incubated with the indicated amounts of Dex for 2 h, followed by addition of OVA-nanospheres (50µg as OVA). After another 2 h incubation at 37℃, the class I and class II MHC-complexed OVA peptide quantities were assessed using OVA-specific CD8 T cell hybridoma B3Z cells (A) and OVA-specific CD4 T cell hybridoma DOBW cells (B), respectively. The indicated amounts of Dex were added to macrophages cultures together with the antigenic OVA-peptide SIINFEKL. The cells were harvested, washed, and the amount of SIINFEKL-H-2Kb complex measured using B3Z cells. Each data point represents the mean±SD of values obtained from three individual experiments.
in-14-328-g001
Figure 2
Short-term treatment of macrophages with Dex does not affect their phagocytic activity or the expression of cell surface molecules. Immature and mature macrophages were incubated with Dex (10µM) for 2 h, followed by addition of nanospheres containing both OVA and FITC. After an additional 2 h incubation, the cells were washed, harvested, and flow cytometric analysis was performed (A). The shaded histograms represent the phagocytic activity in the presence of Dex, and the thick line histograms in the absence of Dex. The effects of Dex on the expression of class II MHC molecules (I-Ab) and co-stimulatory molecules such as B7-1 were also examined in the macrophages treated with Dex (10 µM) for 4 h (B, C). The shaded histograms represent the expression levels of the cell surface molecules in the presence of Dex, and the thick line histograms in the absence of Dex.
in-14-328-g002

Abbreviations

Dex

dexamethasone

DC

dendritic cell

M-CSF

macrophage-colony stimulating factor

rhM-CSF

recombinant human macrophage colony stimulating factor

ACKNOWLEDGEMENTS

This study was supported by the research grant of the Chungbuk National University in 2012.

Notes

The authors have no financial conflict of interest.

References

1. Boumpas DT, Paliogianni F, Anastassiou ED, Balow JE. Glucocorticosteroid action on the immune system: molecular and cellular aspects. Clin Exp Rheumatol. 1991; 9:413–423.
2. Marx J. How the glucocorticoids suppress immunity. Science. 1995; 270:232–233.
crossref
3. Kitajima T, Ariizumi K, Bergstresser PR, Takashima A. A novel mechanism of glucocorticoid-induced immune suppression: the inhibiton of T cell-mediated terminal maturation of a murine dendritic cell line. J Clin Invest. 1996; 98:142–147.
crossref
4. Arya SK, Wong-Staal F, Gallo RC. Dexamethasone-mediated inhibition of human T cell growth factor and gamma-interferon messenger RNA. J Immunol. 1984; 133:273–276.
5. Culpepper JA, Lee F. Regulation of IL 3 expression by glucocorticoids in cloned murine T lymphocytes. J Immunol. 1985; 135:3191–3197.
6. Wu CY, Fargeas C, Nakajima T, Delespesse G. Glucocorticoids suppress the production of interleukin 4 by human lymphocytes. Eur J Immunol. 1991; 21:2645–2647.
crossref
7. Furue M, Ishibashi Y. Differential regulation by dexamethasone and cyclosporine of human T cells activated by various stimuli. Transplantation. 1991; 52:522–526.
crossref
8. Waage A, Slupphaug G, Shalaby R. Glucocorticoids inhibit the production of IL6 from monocytes, endothelial cells and fibroblasts. Eur J Immunol. 1990; 20:2439–2443.
crossref
9. Heidenreich S, Kubis T, Schmidt M, Fegeler W. Glucocorticoid-induced alterations of monocyte defense mechanisms against Candida albicans. Cell Immunol. 1994; 157:320–327.
crossref
10. Almawi WY, Beyhum HN, Rahme AA, Rieder MJ. Regulation of cytokine and cytokine receptor expression by glucocorticoids. J Leukoc Biol. 1996; 60:563–572.
crossref
11. Fushimi T, Okayama H, Seki T, Shimura S, Shirato K. Dexamethasone suppressed gene expression and production of interleukin-10 by human peripheral blood mononuclear cells and monocytes. Int Arch Allergy Immunol. 1997; 112:13–18.
crossref
12. Chen X, Oppenheim JJ, Winkler-Pickett RT, Ortaldo JR, Howard OM. Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3(+)CD4(+) CD25(+) T regulatory cells in vivo and enhances their capacity to suppress EAE. Eur J Immunol. 2006; 36:2139–2149.
crossref
13. Chen X, Murakami T, Oppenheim JJ, Howard OM. Differential response of murine CD4+CD25- and CD4+CD25- T cells to dexamethasone-induced cell death. Eur J Immunol. 2004; 34:859–869.
crossref
14. Zheng G, Zhong S, Geng Y, Munirathinam G, Cha I, Reardon C, Getz GS, van RN, Kang Y, Wang B, Chen A. Dexamethasone promotes tolerance in vivo by enriching CD11clo CD40lo tolerogenic macrophages. Eur J Immunol. 2013; 43:219–227.
crossref
15. Rozkova D, Horvath R, Bartunkova J, Spisek R. Glucocorticoids severely impair differentiation and antigen presenting function of dendritic cells despite upregulation of Toll-like receptors. Clin Immunol. 2006; 120:260–271.
crossref
16. Piemonti L, Monti P, Allavena P, Sironi M, Soldini L, Leone BE, Socci C, Di CV. Glucocorticoids affect human dendritic cell differentiation and maturation. J Immunol. 1999; 162:6473–6481.
17. Pan J, Ju D, Wang Q, Zhang M, Xia D, Zhang L, Yu H, Cao X. Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway. Immunol Lett. 2001; 76:153–161.
crossref
18. Karttunen J, Sanderson S, Shastri N. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci U S A. 1992; 89:6020–6024.
crossref
19. Harding CV, Song R. Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules. J Immunol. 1994; 153:4925–4933.
20. Lee YR, Lee YH, Im SA, Yang IH, Ahn GW, Kim K, Lee CK. Biodegradable nanoparticles containing TLR3 or TLR9 agonists together with antigen enhance MHC-restricted presentation of the antigen. Arch Pharm Res. 2010; 33:1859–1866.
crossref
21. Lee YR, Yang IH, Lee YH, Im SA, Song S, Li H, Han K, Kim K, Eo SK, Lee CK. Cyclosporin A and tacrolimus, but not rapamycin, inhibit MHC-restricted antigen presentation pathways in dendritic cells. Blood. 2005; 105:3951–3955.
crossref
22. Matyszak MK, Citterio S, Rescigno M, Ricciardi-Castagnoli P. Differential effects of corticosteroids during different stages of dendritic cell maturation. Eur J Immunol. 2000; 30:1233–1242.
crossref
23. Woltman AM, de Fijter JW, Kamerling SW, Paul LC, Daha MR, van KC. The effect of calcineurin inhibitors and corticosteroids on the differentiation of human dendritic cells. Eur J Immunol. 2000; 30:1807–1812.
crossref
24. Orlikowsky TW, Dannecker GE, Spring B, Eichner M, Hoffmann MK, Poets CF. Effect of dexamethasone on B7 regulation and T cell activation in neonates and adults. Pediatr Res. 2005; 57:656–661.
crossref
25. Salgado CG, Nakamura K, Sugaya M, Tada Y, Asahina A, Fukuda S, Koyama Y, Irie S, Tamaki K. Differential effects of cytokines and immunosuppressive drugs on CD40, B7-1, and B7-2 expression on purified epidermal Langerhans cells1. J Invest Dermatol. 1999; 113:1021–1027.
crossref
TOOLS
ORCID iDs

Sun-A Im
https://orcid.org/http://orcid.org/0000-0003-2179-0278

Chong-Kil Lee
https://orcid.org/http://orcid.org/0000-0001-9070-341X

Similar articles