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Dexamethasone palmitate ameliorates macrophages-rich graft-versus-host disease by inhibiting macrophage functions.

Nishiwaki S, Nakayama T, Murata M, Nishida T, Terakura S, Saito S, Kato T, Mizuno H, Imahashi N, Seto A, Ozawa Y, Miyamura K, Ito M, Takeshita K, Kato H, Toyokuni S, Nagao K, Ueda R, Naoe T - PLoS ONE (2014)

Bottom Line: We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD.DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro.This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; Japan Society for the Promotion of Science, Japanese Red Cross Nagoya First Hospital, Nagoya, Aichi, Japan; Department of Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya, Aichi, Japan.

ABSTRACT
Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

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Effect of dexamethasone palmitate on macrophages in vitro.A: The viability of mouse macrophage-like RAW264.7 cells after dexamethasone sodium phosphate (DSP) or dexamethasone palmitate (DP) treatment (48 hours) was evaluated by using a colorimetric assay (left panel). The percentage viability was calculated as follows: (O.D value in the presence of each concentration of steroid/O.D value without steroid) ×100. The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). The viability of splenic T lymphocytes after exposure to DSP or DP (25 nM each, 48 hours) was assessed by trypan blue exclusion (right panel). Viable cells were determined as Trypan blue- negative cells. The percent viability was calculated as follows: (viability in DSP or DP group/viability in control group) ×100 (%). The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). B: CCR2 expression on the surface of mouse primary peritoneal macrophages after DSP or DP treatment was evaluated by FACS. The results are representative of three independent experiments (left panel). C: The migration of peritoneal macrophages towards CCL2 after DSP or DP treatment was analyzed using transwell assays. For quantitative analysis, four fields were randomly selected, and migrated cells were counted under a light microscope (×200). The results reflect the mean ± SD of four independent determinations. Representative results of three independent experiments are shown (right panel). Statistical significance: *P<0.05 and **P<0.01.
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pone-0096252-g002: Effect of dexamethasone palmitate on macrophages in vitro.A: The viability of mouse macrophage-like RAW264.7 cells after dexamethasone sodium phosphate (DSP) or dexamethasone palmitate (DP) treatment (48 hours) was evaluated by using a colorimetric assay (left panel). The percentage viability was calculated as follows: (O.D value in the presence of each concentration of steroid/O.D value without steroid) ×100. The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). The viability of splenic T lymphocytes after exposure to DSP or DP (25 nM each, 48 hours) was assessed by trypan blue exclusion (right panel). Viable cells were determined as Trypan blue- negative cells. The percent viability was calculated as follows: (viability in DSP or DP group/viability in control group) ×100 (%). The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). B: CCR2 expression on the surface of mouse primary peritoneal macrophages after DSP or DP treatment was evaluated by FACS. The results are representative of three independent experiments (left panel). C: The migration of peritoneal macrophages towards CCL2 after DSP or DP treatment was analyzed using transwell assays. For quantitative analysis, four fields were randomly selected, and migrated cells were counted under a light microscope (×200). The results reflect the mean ± SD of four independent determinations. Representative results of three independent experiments are shown (right panel). Statistical significance: *P<0.05 and **P<0.01.

Mentions: Based on these results, we hypothesized that GVHD with many macrophages would be ameliorated by inhibiting macrophage functions. We therefore compared DP with conventional DSP on macrophage functions. Both DSP and DP inhibited proliferation of RAW 264.7 cells in a dose dependent manner. However, DP possessed a significantly higher ability than DSP (Figure 2A left panel). DP decreased the viability of RAW 264.7 cells by 75% at a concentration of 10 µM, which is 25-fold lower than the concentration at which DSP similarly worked (by 71% at 250 µM) (Figure 2A left panel). Interestingly, the toxic effect of DP on splenic T lymphocytes is rather weak toxic than DSP, when tested at 25 µM as dexamethasone (Figure 2A right panel). DP also significantly decreased CCR2 expression on the surface of primary peritoneal macrophages (Figure 2B) and RAW 264.7 cells (data not shown), and subsequently decreased migration of primary macrophages towards CCL2 (Figure 2C) compared to DSP. This decreased number of macrophage migration could not be attributed to decreased number of the input cells, as 3 hour-treatment with DSP or DP at 25 µM minimally affected on macrophage viabilities (data not shown). These results clearly suggest that DP attenuates macrophage functions more efficiently than DSP.


Dexamethasone palmitate ameliorates macrophages-rich graft-versus-host disease by inhibiting macrophage functions.

Nishiwaki S, Nakayama T, Murata M, Nishida T, Terakura S, Saito S, Kato T, Mizuno H, Imahashi N, Seto A, Ozawa Y, Miyamura K, Ito M, Takeshita K, Kato H, Toyokuni S, Nagao K, Ueda R, Naoe T - PLoS ONE (2014)

Effect of dexamethasone palmitate on macrophages in vitro.A: The viability of mouse macrophage-like RAW264.7 cells after dexamethasone sodium phosphate (DSP) or dexamethasone palmitate (DP) treatment (48 hours) was evaluated by using a colorimetric assay (left panel). The percentage viability was calculated as follows: (O.D value in the presence of each concentration of steroid/O.D value without steroid) ×100. The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). The viability of splenic T lymphocytes after exposure to DSP or DP (25 nM each, 48 hours) was assessed by trypan blue exclusion (right panel). Viable cells were determined as Trypan blue- negative cells. The percent viability was calculated as follows: (viability in DSP or DP group/viability in control group) ×100 (%). The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). B: CCR2 expression on the surface of mouse primary peritoneal macrophages after DSP or DP treatment was evaluated by FACS. The results are representative of three independent experiments (left panel). C: The migration of peritoneal macrophages towards CCL2 after DSP or DP treatment was analyzed using transwell assays. For quantitative analysis, four fields were randomly selected, and migrated cells were counted under a light microscope (×200). The results reflect the mean ± SD of four independent determinations. Representative results of three independent experiments are shown (right panel). Statistical significance: *P<0.05 and **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4012982&req=5

pone-0096252-g002: Effect of dexamethasone palmitate on macrophages in vitro.A: The viability of mouse macrophage-like RAW264.7 cells after dexamethasone sodium phosphate (DSP) or dexamethasone palmitate (DP) treatment (48 hours) was evaluated by using a colorimetric assay (left panel). The percentage viability was calculated as follows: (O.D value in the presence of each concentration of steroid/O.D value without steroid) ×100. The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). The viability of splenic T lymphocytes after exposure to DSP or DP (25 nM each, 48 hours) was assessed by trypan blue exclusion (right panel). Viable cells were determined as Trypan blue- negative cells. The percent viability was calculated as follows: (viability in DSP or DP group/viability in control group) ×100 (%). The results reflect the mean ± SD of three independent determinations (representative experiment of three performed). B: CCR2 expression on the surface of mouse primary peritoneal macrophages after DSP or DP treatment was evaluated by FACS. The results are representative of three independent experiments (left panel). C: The migration of peritoneal macrophages towards CCL2 after DSP or DP treatment was analyzed using transwell assays. For quantitative analysis, four fields were randomly selected, and migrated cells were counted under a light microscope (×200). The results reflect the mean ± SD of four independent determinations. Representative results of three independent experiments are shown (right panel). Statistical significance: *P<0.05 and **P<0.01.
Mentions: Based on these results, we hypothesized that GVHD with many macrophages would be ameliorated by inhibiting macrophage functions. We therefore compared DP with conventional DSP on macrophage functions. Both DSP and DP inhibited proliferation of RAW 264.7 cells in a dose dependent manner. However, DP possessed a significantly higher ability than DSP (Figure 2A left panel). DP decreased the viability of RAW 264.7 cells by 75% at a concentration of 10 µM, which is 25-fold lower than the concentration at which DSP similarly worked (by 71% at 250 µM) (Figure 2A left panel). Interestingly, the toxic effect of DP on splenic T lymphocytes is rather weak toxic than DSP, when tested at 25 µM as dexamethasone (Figure 2A right panel). DP also significantly decreased CCR2 expression on the surface of primary peritoneal macrophages (Figure 2B) and RAW 264.7 cells (data not shown), and subsequently decreased migration of primary macrophages towards CCL2 (Figure 2C) compared to DSP. This decreased number of macrophage migration could not be attributed to decreased number of the input cells, as 3 hour-treatment with DSP or DP at 25 µM minimally affected on macrophage viabilities (data not shown). These results clearly suggest that DP attenuates macrophage functions more efficiently than DSP.

Bottom Line: We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD.DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro.This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; Japan Society for the Promotion of Science, Japanese Red Cross Nagoya First Hospital, Nagoya, Aichi, Japan; Department of Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya, Aichi, Japan.

ABSTRACT
Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Show MeSH
Related in: MedlinePlus