Immune Netw.  2017 Jun;17(3):171-178. 10.4110/in.2017.17.3.171.

Effects of Cellular 11β-hydroxysteroid Dehydrogenase 1 on LPS-induced Inflammatory Responses in Synovial Cell Line, SW982

• Cho YS
• Kim KN
• Shim JH

• KMID: 2383355
• DOI: http://doi.org/10.4110/in.2017.17.3.171

Abstract

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive cortisone into active cortisol, which has pleiotropic roles in various biological conditions, such as immunological and metabolic homeostasis. Cortisol is mainly produced in the adrenal gland, but can be locally regenerated in the liver, fat, and muscle. Its diverse actions are primarily mediated by binding to the glucocorticoid receptor. SW982, a human synovial cell line, expresses 11β-HSD type 1, but not type 2, that catalyzes the conversion of cortisone to cortisol. In this study, therefore, we investigated the control of lipopolysaccharide (LPS)-induced inflammatory responses by prereceptor regulation-mediated maintenance of cortisol levels. Preliminarily, cell seeding density and incubation period were optimized for analyzing the catalytic activity of SW982. Additionally, cellular 11β-HSD1 still remained active irrespective of monolayer or spheroid culture conditions. Inflammatory stimulants, such as interleukin (IL)-1β, tumor necrosis factor (TNF)α, and LPS, did not affect the catalytic activity of 11β-HSD1, although a high dose of LPS significantly decreased its activity. Additionally, autocrine effects of cortisol on inflammatory responses were investigated in LPS-stimulated SW982 cells. LPS upregulated pro-inflammatory cytokines, including IL-6 and IL-1β, in SW982 cells, while cortisol production, catalyzed by cellular 11β-HSD1, downregulated LPS-stimulated cytokines. Furthermore, suppression of NFκB activation-mediated pro-inflammatory responses by cortisol was revealed. In conclusion, the activity of cellular 11β-HSD1 was closely correlated with suppression of LPS-induced inflammation. Therefore, these results partly support the notion that prereceptor regulation of locally regenerated cortisol could be taken into consideration for treatment of inflammation-associated diseases, including arthritis.

Keyword

11β-hydroxysteroid dehydrogenase; Cortisone; Cortisol; NFκB; IL-6; IL-1β

MeSH Terms

Adrenal Glands
Arthritis
Cell Line*
Cortisone
Cytokines
Homeostasis
Humans
Hydrocortisone
Inflammation
Interleukin-6
Interleukins
Liver
Oxidoreductases*
Receptors, Glucocorticoid
Tumor Necrosis Factor-alpha
Cortisone
Cytokines
Hydrocortisone
Interleukin-6
Interleukins
Oxidoreductases
Receptors, Glucocorticoid
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Determination of cortisol production by 11β-HSD 1 activity in SW982 cells. Cortisol was detected by (A) TLC, and (B) HTRF assay using a cortisol-specific antibody. Cells were plated at various densities of 5×104, 1×105, 5×105, 1×106 and incubated with medium containing radioactive cortisone for 48 h. Culture media were subjected to extraction with organic solvent and then TLC development as described in the Materials and Methods section. The ability of cells to convert cortisone/cortisol was further optimized in the HTRF assay by differing cell-seeding density (top of 1B) and incubation times (bottom of 1B). Cells (1×105) were incubated in 160 nM cortisone-containing medium in the presence or absence of 1 µM CBX or GA for 6 h, and then the medium was aliquoted to measure cortisol levels. E and F indicate cortisone and cortisol, respectively.

• 

  Figure 2 Microscopic evaluation and 11β-HSD 1 activity in cells grown under monolayer or spheroid culture conditions. (A) Cells were grown in monolayer or spheroid conditions and then observed microscopically using 10× ocular lens and 2× objective lens. (B) Cortisol levels in culture medium from SW982 cells grown under different conditions were measured. Cells were incubated in DMEM containing 160 nM cortisone as a substrate of 11β-HSD1.

• 

  Figure 3 Effects of pro-inflammatory cytokines or LPS on 11β-HSD1 activity. Cells at a density of 106 were stimulated with IL-1β, LPS, or TNFα at the concentrations of 10 and 100 ng/mL for 24 h. Cortisone (160 nM) was added right before stimulation, and then the culture medium was aliquoted to measure conversion of cortisone to cortisol in the HTRF assay. Each bar represents the mean±S.D. of 2 separate experiments. *p<0.05 versus untreated control.

• 

  Figure 4 Effect of cortisone/cortisol on LPS-induced inflammatory cytokines in SW982 cells. SW982 cells were stimulated with 10 ng/mL LPS and then the profiles of pro-inflammatory cytokines were primarily investigated. To check the anti-inflammatory effect of cortisol, cells were incubated in DMEM containing cortisone. Pro-inflammatory cytokines, including IL-1β and IL-6, were monitored in cells stimulated with LPS in the presence or absence of cortisone.

• 

  Figure 5 Suppression of LPS-mediated NFκB activation by cortisone. To examine the suppression of LPS-stimulated inflammatory signal by cortisone, phosphorylation of IB, an indicator of NFκB activation, was investigated by western blotting.

• 

  Figure 6 The schematic summary of cortisone-mediated suppression of inflammatory responses to LPS. LPS mediates inflammatory responses via NFκB signaling pathway. SW982 cells effectively converted cortisone to active cortisol, releasing it into the culture medium to act back on themselves in an autocrine manner.

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