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Inhibition of inflammatory arthritis using fullerene nanomaterials.

Dellinger AL, Cunin P, Lee D, Kung AL, Brooks DB, Zhou Z, Nigrovic PA, Kepley CL - PLoS ONE (2015)

Bottom Line: It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts.Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls.In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina Greensboro, Joint School of Nanosceince and Nanoengineering, Greensboro, North Carolina, United States of America.

ABSTRACT
Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

No MeSH data available.


Related in: MedlinePlus

Fullerene derivatives reduce degranulation and cytokine production from synovial fibroblast from RA patients, mouse BMMC, and human MC (hMC), and osteoclast formation from human PBMC.Fig 1A shows FcγRII- BMMC incubated with fullerene derivatives overnight (10 μg/ml). The next day anti-FcγRII/III antibody 2.4G2 or isotype control was added followed by cross-linking donkey anti-rat (DAR) F(ab)2. Cells were centrifuged and β-hexosaminidase release or IL-1 production determined in supernatants or lysates, respectively. Data shown are means ± SE of triplicate samples that is representative of three experiments. All data was statistically significant with P values < 0.05. In Fig 1B tissue MC were incubated with fullerene derivatives (10 μg/ml) overnight, washed and preformed IgG anti-NP–NP-BSA immune complexes [8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA [35]], were incubated with MC for 30 minutes or four hours. Supernatants and cell lysates were prepared for mediator release analysis as described. Data is expressed as mean ± SE from three individual experiments. P values < 0.05 by ANOVA when experimental values are compared with the Ab-only control (not shown). Fig 1C shows fullerene derivatives can inhibit cytokine production from rheumatoid arthritis-derived synovial fibroblasts. Synovial fibroblasts from RA patients were preincubated with or without various fullerene derivatives (10 μg/ml) overnight, washed, and incubated with TNF-α (10 ng/ml for 12 hours). Supernatants were saved and cytokines measured in the supernatants. The percent inhibition of the treated cells was calculated based on the release of cytokines from non- fullerene derivative treated cells. Fig 1D shows the ability for fullerene derivatives to inhibit osteoclast formation. Human PBMC were incubated without (negative) or with RANK ligand (30 ng/ml) and GMCSF (25 ng/ml). After one hour fullerene derivatives were added (10 μg/ml) and remained throughout. In order to verify the differentiation of mononuclear cells to osteoclasts, after eight days of culture, cells were analyzed for tartrate resistant acid phosphatase (TRAP) activity by cytochemistry. The cells with the reddish color represent osteoclast formation and are quantified in the graph (bottom). Results are representative of two separate experiments. Magnification 40X.
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pone.0126290.g001: Fullerene derivatives reduce degranulation and cytokine production from synovial fibroblast from RA patients, mouse BMMC, and human MC (hMC), and osteoclast formation from human PBMC.Fig 1A shows FcγRII- BMMC incubated with fullerene derivatives overnight (10 μg/ml). The next day anti-FcγRII/III antibody 2.4G2 or isotype control was added followed by cross-linking donkey anti-rat (DAR) F(ab)2. Cells were centrifuged and β-hexosaminidase release or IL-1 production determined in supernatants or lysates, respectively. Data shown are means ± SE of triplicate samples that is representative of three experiments. All data was statistically significant with P values < 0.05. In Fig 1B tissue MC were incubated with fullerene derivatives (10 μg/ml) overnight, washed and preformed IgG anti-NP–NP-BSA immune complexes [8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA [35]], were incubated with MC for 30 minutes or four hours. Supernatants and cell lysates were prepared for mediator release analysis as described. Data is expressed as mean ± SE from three individual experiments. P values < 0.05 by ANOVA when experimental values are compared with the Ab-only control (not shown). Fig 1C shows fullerene derivatives can inhibit cytokine production from rheumatoid arthritis-derived synovial fibroblasts. Synovial fibroblasts from RA patients were preincubated with or without various fullerene derivatives (10 μg/ml) overnight, washed, and incubated with TNF-α (10 ng/ml for 12 hours). Supernatants were saved and cytokines measured in the supernatants. The percent inhibition of the treated cells was calculated based on the release of cytokines from non- fullerene derivative treated cells. Fig 1D shows the ability for fullerene derivatives to inhibit osteoclast formation. Human PBMC were incubated without (negative) or with RANK ligand (30 ng/ml) and GMCSF (25 ng/ml). After one hour fullerene derivatives were added (10 μg/ml) and remained throughout. In order to verify the differentiation of mononuclear cells to osteoclasts, after eight days of culture, cells were analyzed for tartrate resistant acid phosphatase (TRAP) activity by cytochemistry. The cells with the reddish color represent osteoclast formation and are quantified in the graph (bottom). Results are representative of two separate experiments. Magnification 40X.

Mentions: A panel of 40 fullerene derivatives was tested for the ability to inhibit Fcγ receptor-dependent degranulation and cytokine production from human and mouse MC. Previous studies demonstrated an overnight incubation with 10 μg/ml was optimal for MC stabilization to FcεRI-dependent [25] and-independent [46] stimulation and was thus used for these studies. Approximately 15% of the fullerene derivatives tested significantly (p<0.05) inhibited both degranulation and IL-1β (Fig 1A–1C). As demonstrated previously examining FcεRI-dependent mediator release [25], several fullerene derivatives exhibited inhibitory capabilities on both degranulation and cytokine production in Fcγ-stimulated BMMC (Fig 1A) and IC-stimulated human tissue-derived MC (Fig 1B) which was dependent on the side chain moieties added to the carbon cage. Cytokine release from TNF-α-challenged synovial fibroblasts was significantly inhibited by 25% for all fullerene derivatives tested (Fig 1C). The two most efficacious cytokine blockers (ALM and TGA) also inhibited the formation of bone resorbing osteoclasts (Fig 1D). Thus, fullerene derivatives inhibit critical parameters important for the pathologies associated with inflammatory arthritis as assessed by in vitro models.


Inhibition of inflammatory arthritis using fullerene nanomaterials.

Dellinger AL, Cunin P, Lee D, Kung AL, Brooks DB, Zhou Z, Nigrovic PA, Kepley CL - PLoS ONE (2015)

Fullerene derivatives reduce degranulation and cytokine production from synovial fibroblast from RA patients, mouse BMMC, and human MC (hMC), and osteoclast formation from human PBMC.Fig 1A shows FcγRII- BMMC incubated with fullerene derivatives overnight (10 μg/ml). The next day anti-FcγRII/III antibody 2.4G2 or isotype control was added followed by cross-linking donkey anti-rat (DAR) F(ab)2. Cells were centrifuged and β-hexosaminidase release or IL-1 production determined in supernatants or lysates, respectively. Data shown are means ± SE of triplicate samples that is representative of three experiments. All data was statistically significant with P values < 0.05. In Fig 1B tissue MC were incubated with fullerene derivatives (10 μg/ml) overnight, washed and preformed IgG anti-NP–NP-BSA immune complexes [8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA [35]], were incubated with MC for 30 minutes or four hours. Supernatants and cell lysates were prepared for mediator release analysis as described. Data is expressed as mean ± SE from three individual experiments. P values < 0.05 by ANOVA when experimental values are compared with the Ab-only control (not shown). Fig 1C shows fullerene derivatives can inhibit cytokine production from rheumatoid arthritis-derived synovial fibroblasts. Synovial fibroblasts from RA patients were preincubated with or without various fullerene derivatives (10 μg/ml) overnight, washed, and incubated with TNF-α (10 ng/ml for 12 hours). Supernatants were saved and cytokines measured in the supernatants. The percent inhibition of the treated cells was calculated based on the release of cytokines from non- fullerene derivative treated cells. Fig 1D shows the ability for fullerene derivatives to inhibit osteoclast formation. Human PBMC were incubated without (negative) or with RANK ligand (30 ng/ml) and GMCSF (25 ng/ml). After one hour fullerene derivatives were added (10 μg/ml) and remained throughout. In order to verify the differentiation of mononuclear cells to osteoclasts, after eight days of culture, cells were analyzed for tartrate resistant acid phosphatase (TRAP) activity by cytochemistry. The cells with the reddish color represent osteoclast formation and are quantified in the graph (bottom). Results are representative of two separate experiments. Magnification 40X.
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Related In: Results  -  Collection

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pone.0126290.g001: Fullerene derivatives reduce degranulation and cytokine production from synovial fibroblast from RA patients, mouse BMMC, and human MC (hMC), and osteoclast formation from human PBMC.Fig 1A shows FcγRII- BMMC incubated with fullerene derivatives overnight (10 μg/ml). The next day anti-FcγRII/III antibody 2.4G2 or isotype control was added followed by cross-linking donkey anti-rat (DAR) F(ab)2. Cells were centrifuged and β-hexosaminidase release or IL-1 production determined in supernatants or lysates, respectively. Data shown are means ± SE of triplicate samples that is representative of three experiments. All data was statistically significant with P values < 0.05. In Fig 1B tissue MC were incubated with fullerene derivatives (10 μg/ml) overnight, washed and preformed IgG anti-NP–NP-BSA immune complexes [8.8 μg/ml anti-NP Ab with 0.13 μg/ml NP-BSA [35]], were incubated with MC for 30 minutes or four hours. Supernatants and cell lysates were prepared for mediator release analysis as described. Data is expressed as mean ± SE from three individual experiments. P values < 0.05 by ANOVA when experimental values are compared with the Ab-only control (not shown). Fig 1C shows fullerene derivatives can inhibit cytokine production from rheumatoid arthritis-derived synovial fibroblasts. Synovial fibroblasts from RA patients were preincubated with or without various fullerene derivatives (10 μg/ml) overnight, washed, and incubated with TNF-α (10 ng/ml for 12 hours). Supernatants were saved and cytokines measured in the supernatants. The percent inhibition of the treated cells was calculated based on the release of cytokines from non- fullerene derivative treated cells. Fig 1D shows the ability for fullerene derivatives to inhibit osteoclast formation. Human PBMC were incubated without (negative) or with RANK ligand (30 ng/ml) and GMCSF (25 ng/ml). After one hour fullerene derivatives were added (10 μg/ml) and remained throughout. In order to verify the differentiation of mononuclear cells to osteoclasts, after eight days of culture, cells were analyzed for tartrate resistant acid phosphatase (TRAP) activity by cytochemistry. The cells with the reddish color represent osteoclast formation and are quantified in the graph (bottom). Results are representative of two separate experiments. Magnification 40X.
Mentions: A panel of 40 fullerene derivatives was tested for the ability to inhibit Fcγ receptor-dependent degranulation and cytokine production from human and mouse MC. Previous studies demonstrated an overnight incubation with 10 μg/ml was optimal for MC stabilization to FcεRI-dependent [25] and-independent [46] stimulation and was thus used for these studies. Approximately 15% of the fullerene derivatives tested significantly (p<0.05) inhibited both degranulation and IL-1β (Fig 1A–1C). As demonstrated previously examining FcεRI-dependent mediator release [25], several fullerene derivatives exhibited inhibitory capabilities on both degranulation and cytokine production in Fcγ-stimulated BMMC (Fig 1A) and IC-stimulated human tissue-derived MC (Fig 1B) which was dependent on the side chain moieties added to the carbon cage. Cytokine release from TNF-α-challenged synovial fibroblasts was significantly inhibited by 25% for all fullerene derivatives tested (Fig 1C). The two most efficacious cytokine blockers (ALM and TGA) also inhibited the formation of bone resorbing osteoclasts (Fig 1D). Thus, fullerene derivatives inhibit critical parameters important for the pathologies associated with inflammatory arthritis as assessed by in vitro models.

Bottom Line: It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts.Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls.In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina Greensboro, Joint School of Nanosceince and Nanoengineering, Greensboro, North Carolina, United States of America.

ABSTRACT
Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

No MeSH data available.


Related in: MedlinePlus