Korean J Physiol Pharmacol.  2012 Dec;16(6):393-398. 10.4196/kjpp.2012.16.6.393.

Dexamethasone Induces FcgammaRIIb Expression in RBL-2H3 Cells

• Silwal P
• Lee MN
• Lee CJ
• Hong JH
• Namgung U
• Lee ZW
• Kim J
• Lim K
• Kweon GR
• Park JI
• Park SK

• KMID: 2285447
• DOI: http://doi.org/10.4196/kjpp.2012.16.6.393

Abstract

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress FcepsilonRI-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE (FcepsilonR) I and increased the mRNA levels of the inhibitory Fc receptor for IgG FcgammaRIIb. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG (FcgammaR) I and FcgammaRIII. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced FcgammaRI and FcgammaRIII mRNA levels potently, while FcepsilonRI and FcgammaRIIb were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only FcgammaRIIb protein expression was significantly enhanced by Dex treatment, while FcgammaRI, FcgammaRIII and FcepsilonRI expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor FcgammaRIIb.

Keyword

Degranulation; Fc receptor; Glucocorticoid; Mast cells; Transcription

MeSH Terms

Animals
Autoimmune Diseases
Cycloheximide
Dactinomycin
Dexamethasone
Glucocorticoids
Immunoglobulin E
Immunoglobulin G
Mast Cells
Rats
Real-Time Polymerase Chain Reaction
Receptors, Fc
RNA, Messenger
Cycloheximide
Dactinomycin
Dexamethasone
Glucocorticoids
Immunoglobulin E
Immunoglobulin G
RNA, Messenger
Receptors, Fc

Figure

• 

  Fig. 1 Inhibition of degranulation by Dex in RBL-2H3 cells. RBL-2H3 cells were cultured for 18 h with IgE (at 100 ng/ml) and Dex (100 nM) was treated for indicated times. After sensitization with IgE specific to DNP overnight, cells were washed and then stimulated with antigens (DNP-HSA) for 15 min. Data are expressed as mean±SD, n=3. **p<0.01 versus no antigen treatment.

• 

  Fig. 2 mRNA levels of FcεRI and FcγRs by Dex. Transcript levels of FcεRI (A), FcγRI (B), FcγRIII (C) and FcγRIIb (D) were measured following treatment with 100 nM Dex to RBL-2H3 cells for the indicated time. Real time PCRs were performed with cDNAs made from reverse transcriptase reaction from RNA extracts. Results are expressed as fold change relative to control. Filled squares are Dex treatment and filled circles are control. Data are expressed as mean±SD, n=3. **p<0.01 versus no Dex treatment.

• 

  Fig. 3 Effects of actinomycin D and cycloheximide on Fc receptor transcript levels. Actinomycin D (AcD, 200 ng/ml) or cycloheximide (ChX, 1 µg/ml) was added 20 min before treatment of 100 nM Dex and transcript levels were determined 6 h thereafter. Expression analysis was performed by real-time PCR. Data are expressed as mean±SD, n=3. **p<0.01 versus no treatment.

• 

  Fig. 4 Organization of FcγRIIb genes. (A) Exons and introns are indicated by the numbered boxes and solid lines, respectively. Dashed lines indicate non-transcribed regions. (B) Genes surrounding the FcγRIIb gene.

• 

  Fig. 5 Effects of Dex treatment on cell surface expressions of Fcε RI, FcγRI, FcγRIIb and FcγRIII. Cultured RBL-2H3 cells were treated with Dex (100 nM) for 18 h, and cells were stained with antibodies specific to FcεR (A), FcγRI (B), FcγRIIb (C) and Fcγ RIII (D). The surface expressions of Fc receptors were verified by cell fluorescence analysis. Dex treatment or control is represented with filled or empty area, respectively. Data is a representative from three separate experiments.

• 

  Fig. 6 Effects of downregulation of FcγRIIb by transfection of anti-FcγRIIb siRNA. The effects of transfection with siRNA against FcγRIIb on its transcript level (A) and degranulation efficiency by the cross-linking of FcεRI (B). Cells were incubated 24 h after transfection with the siRNA. After cells were incubated for 18 h in the presence or absence of 100 nM Dex, degranulation efficiency was determined by β-hexosaminidase assay, and cell surface expression was evaluated by flow cytometric analysis. Data are expressed as mean±SD, n=3. Flow cytometric data is a representative from three separate experiments.

Reference

1. Secor VH, Secor WE, Gutekunst CA, Brown MA. Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med. 2000. 191:813–822.

2. Lee DM, Friend DS, Gurish MF, Benoist C, Mathis D, Brenner MB. Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science. 2002. 297:1689–1692.

3. Sun J, Sukhova GK, Wolters PJ, Yang M, Kitamoto S, Libby P, MacFarlane LA, Mallen-St Clair J, Shi GP. Mast cells promote atherosclerosis by releasing proinflammatory cytokines. Nat Med. 2007. 13:719–724.

4. Liu J, Divoux A, Sun J, Zhang J, Clément K, Glickman JN, Sukhova GK, Wolters PJ, Du J, Gorgun CZ, Doria A, Libby P, Blumberg RS, Kahn BB, Hotamisligil GS, Shi GP. Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice. Nat Med. 2009. 15:940–945.

5. Takai T. Roles of Fc receptors in autoimmunity. Nat Rev Immunol. 2002. 2:580–592.

6. Smith KG, Clatworthy MR. FcgammaRIIB in autoimmunity and infection: evolutionary and therapeutic implications. Nat Rev Immunol. 2010. 10:328–343.

7. Scharenberg AM, Lin S, Cuenod B, Yamamura H, Kinet JP. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering. EMBO J. 1995. 14:3385–3394.

8. Rosen J, Miner JN. The search for safer glucocorticoid receptor ligands. Endocr Rev. 2005. 26:452–464.

9. Clark AR. Anti-inflammatory functions of glucocorticoid-induced genes. Mol Cell Endocrinol. 2007. 275:79–97.

10. Ito K, Chung KF, Adcock IM. Update on glucocorticoid action and resistance. J Allergy Clin Immunol. 2006. 117:522–543.

11. Smoak K, Cidlowski JA. Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling. Mol Cell Biol. 2006. 26:9126–9135.

12. Ing NH. Steroid hormones regulate gene expression posttranscriptionally by altering the stabilities of messenger RNAs. Biol Reprod. 2005. 72:1290–1296.

13. Stahn C, Löwenberg M, Hommes DW, Buttgereit F. Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists. Mol Cell Endocrinol. 2007. 275:71–78.

14. Stellato C. Post-transcriptional and nongenomic effects of glucocorticoids. Proc Am Thorac Soc. 2004. 1:255–263.

15. Hiragun T, Peng Z, Beaven MA. Dexamethasone up-regulates the inhibitory adaptor protein Dok-1 and suppresses downstream activation of the mitogen-activated protein kinase pathway in antigen-stimulated RBL-2H3 mast cells. Mol Pharmacol. 2005. 67:598–603.

16. Kassel O, Sancono A, Krätzschmar J, Kreft B, Stassen M, Cato AC. Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J. 2001. 20:7108–7116.

17. Park SK, Beaven MA. Mechanism of upregulation of the inhibitory regulator, src-like adaptor protein (SLAP), by glucocorticoids in mast cells. Mol Immunol. 2009. 46:492–497.

18. Hiragun T, Peng Z, Beaven MA. Cutting edge: dexamethasone negatively regulates Syk in mast cells by up-regulating SRC-like adaptor protein. J Immunol. 2006. 177:2047–2050.

19. Ozawa K, Szallasi Z, Kazanietz MG, Blumberg PM, Mischak H, Mushinski JF, Beaven MA. Ca²⁺-dependent and Ca²⁺-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca²⁺ and purified isozymes in washed permeabilized cells. J Biol Chem. 1993. 268:1749–1749.

20. Benhamou M, Ninio E, Salem P, Hieblot C, Bessou G, Pitton C, Liu FT, Mencia-Huerta JM. Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone. J Immunol. 1986. 136:1385–1392.

21. Ovcharenko I, Nobrega MA, Loots GG, Stubbs L. ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res. 2004. 32:W280–W286.

22. Yamaguchi M, Hirai K, Komiya A, Miyamasu M, Furumoto Y, Teshima R, Ohta K, Morita Y, Galli SJ, Ra C, Yamamoto K. Regulation of mouse mast cell surface Fc epsilon RI expression by dexamethasone. Int Immunol. 2001. 13:843–851.

23. Ishmael FT, Fang X, Galdiero MR, Atasoy U, Rigby WF, Gorospe M, Cheadle C, Stellato C. Role of the RNA-binding protein tristetraprolin in glucocorticoid-mediated gene regulation. J Immunol. 2008. 180:8342–8353.

24. Petroni KC, Shen L, Guyre PM. Modulation of human polymorphonuclear leukocyte IgG Fc receptors and Fc receptor-mediated functions by IFN-gamma and glucocorticoids. J Immunol. 1988. 140:3467–3472.

25. Pfirsch-Maisonnas S, Aloulou M, Xu T, Claver J, Kanamaru Y, Tiwari M, Launay P, Monteiro RC, Blank U. Inhibitory ITAM signaling traps activating receptors with the phosphatase SHP-1 to form polarized "inhibisome" clusters. Sci Signal. 2011. 4:ra24.

26. Kanamaru Y, Pfirsch S, Aloulou M, Vrtovsnik F, Essig M, Loirat C, Deschênes G, Guérin-Marchand C, Blank U, Monteiro RC. Inhibitory ITAM signaling by Fc alpha RI-FcR gamma chain controls multiple activating responses and prevents renal inflammation. J Immunol. 2008. 180:2669–2678.

27. Ujike A, Ishikawa Y, Ono M, Yuasa T, Yoshino T, Fukumoto M, Ravetch JV, Takai T. Modulation of immunoglobulin (Ig)E-mediated systemic anaphylaxis by low-affinity Fc receptors for IgG. J Exp Med. 1999. 189:1573–1579.

28. Falanga YT, Chaimowitz NS, Charles N, Finkelman FD, Pullen NA, Barbour S, Dholaria K, Faber T, Kolawole M, Huang B, Odom S, Rivera J, Carlyon J, Conrad DH, Spiegel S, Oskeritzian CA, Ryan JJ. Lyn but not Fyn kinase controls IgG-mediated systemic anaphylaxis. J Immunol. 2012. 188:4360–4368.

29. Sylvestre DL, Ravetch JV. A dominant role for mast cell Fc receptors in the Arthus reaction. Immunity. 1996. 5:387–390.