RESUMO
Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)(2) (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-ß-induced collagen mRNA expression and interleukin (IL)-1ß-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1ß/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.
Assuntos
Proliferação de Células , Colágeno/biossíntese , Citocinas/biossíntese , Mucosa Nasal/metabolismo , Pólipos Nasais/metabolismo , Inibidores de Proteassoma/farmacologia , Adulto , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/antagonistas & inibidores , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Pessoa de Meia-Idade , Mucosa Nasal/efeitos dos fármacos , Pólipos Nasais/tratamento farmacológico , Inibidores de Proteassoma/uso terapêuticoRESUMO
BACKGROUND: In vitro culture of nasal polyp cells is frequently used in the investigation of inflammatory mechanisms and effect of treatments in nasal polyposis. Research outcomes may, however, be influenced by the culture methodology used. METHODS: Nasal polyp and nasal mucosa in vitro fibroblast cultures were pre-treated with foetal bovine serum (FBS)-free culture medium or medium supplemented with either FBS or charcoal-stripped (cs) FBS. Cells were then stimulated with FBS or csFBS, with or without different doses of dexamethasone for 4 and 24h. IL-6, IL-8, GM-CSF and VEGF release and cell viability were measured. RESULTS: The highest cytokine levels were found in growth-arrested cells stimulated with 10% FBS. csFBS poorly stimulated cytokine release. Nasal polyp released larger IL-8 amounts than nasal mucosa fibroblasts. Dexamethasone decreased cytokine production dose- and time-dependently in both nasal mucosa and nasal polyp fibroblasts. The IC25 of IL-8 inhibition by dexamethasone was higher in nasal polyp than in nasal mucosa fibroblasts. Cell viability did not differ among treatments. CONCLUSIONS: Cytokine production by in vitro cultured nasal fibroblasts is affected by the culture conditions used and is inhibited by dexamethasone in both fibroblast types. Our results highlight the importance of culture methodology on nasal polyp research outcomes.
Assuntos
Técnicas de Cultura de Células , Mediadores da Inflamação/fisiologia , Mucosa Nasal/fisiopatologia , Pólipos Nasais/fisiopatologia , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/fisiologia , Citocinas , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoAssuntos
Asma Induzida por Aspirina/tratamento farmacológico , Asma Induzida por Aspirina/genética , Fibroblastos/metabolismo , Glucocorticoides/uso terapêutico , Inflamação/genética , Pólipos Nasais/tratamento farmacológico , Pólipos Nasais/genética , Aspirina/imunologia , Asma Induzida por Aspirina/complicações , Resistência a Medicamentos/genética , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/imunologia , Pólipos Nasais/complicaçõesRESUMO
BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment. OBJECTIVE: We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma. METHODS: NP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRß, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRß localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing. RESULTS: Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/ß MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone's capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRß expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone's capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone's capacity to suppress FBS-induced CXCL8 release. CONCLUSION: The bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.
Assuntos
Asma/complicações , Fibroblastos/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasais/complicações , Receptores de Glucocorticoides/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Doença Crônica , Citocinas/biossíntese , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Expressão Gênica , Humanos , Lipopolissacarídeos/imunologia , Mucosa Nasal/imunologia , Transporte Proteico , Receptores de Glucocorticoides/genética , Rinite/complicações , Rinite/genética , Rinite/imunologia , Sinusite/complicações , Sinusite/genética , Sinusite/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Nasal polyposis (NP) is treated with topical glucocorticoids (GC). Some patients require endoscopic nasal surgery because GC treatment is ineffective. To exert its function, the GC needs to bind with the GC receptor (GR) and the GC-GR complex moves to the cell nucleus. Our aim was to establish whether the poor response to GC is due to an alteration in the translocation of the GR to the nucleus. METHODS: We performed nasal fibroblast cell cultures from seven healthy controls and 12 patients with NP and asthma. Fibroblasts were incubated with budesonide or dexamethasone (10(-7) M) for different times (30 min to 4 h) and GR translocation was analyzed by immunocytochemistry. RESULTS: Both GC induced GR translocation in every group, doubling its concentration in the cell nucleus (30 min) compared to baseline. There were no differences in GR translocation between controls and patients, nor differences related to the severity of asthma or intolerance to non-steroidal anti-inflammatory drugs (NSAIDs). Atopic subjects showed a decrease in GR translocation with budesonide (1 h, 3 h and 4 h, P<0.05) and dexamethasone (30 min and 2 h, P<.05). CONCLUSIONS: The insensitivity to GC treatment does not appear to be due to an alteration in GR translocation to the nucleus. Neither does the severity of asthma or intolerance to NSAIDs appear to alter GR translocation. The association between atopy and the alteration in GR function deserves further investigation.
Assuntos
Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Asma/patologia , Budesonida/farmacologia , Dexametasona/farmacologia , Resistência a Medicamentos/fisiologia , Fibroblastos/metabolismo , Glucocorticoides/farmacologia , Pólipos Nasais/patologia , Receptores de Glucocorticoides/metabolismo , Transporte Ativo do Núcleo Celular , Antiasmáticos/farmacocinética , Antiasmáticos/uso terapêutico , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios não Esteroides/efeitos adversos , Asma/complicações , Asma/tratamento farmacológico , Asma Induzida por Aspirina/tratamento farmacológico , Asma Induzida por Aspirina/patologia , Budesonida/farmacocinética , Núcleo Celular/metabolismo , Células Cultivadas/efeitos dos fármacos , Dexametasona/farmacocinética , Fibroblastos/efeitos dos fármacos , Glucocorticoides/farmacocinética , Glucocorticoides/uso terapêutico , Humanos , Mucosa Nasal/patologia , Pólipos Nasais/tratamento farmacológico , Pólipos Nasais/cirurgia , Receptores de Glucocorticoides/agonistas , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Treatment with glucocorticoids (GCs) is the cornerstone of nasal polyp (NP) therapy, but some patients respond poorly to them. Fibroblasts are involved in both inflammation and remodelling of NP. We aimed to evaluate whether NP fibroblasts are less sensitive to GCs' anti-proliferative and anti-inflammatory effects, compared to nasal mucosa (NM) fibroblasts. METHODS: Fibroblasts were obtained from NP (n = 8) from asthmatic patients undergoing endoscopic surgery and NM (n = 8) from patients undergoing nasal corrective surgery. Fibroblasts were stimulated with DMEM at 0.5% or 5% FBS, or TGF-ß (5 ng/ml), with or without dexamethasone (10(-11) to 10(-5)M) for different times. Cell proliferation, collagen mRNA expression and IL-6 and IL-8 release were measured. RESULTS: After 3-days, dexamethasone dose-dependently inhibited proliferation of NM (p < 0.001) but not that of NP fibroblasts. Dexamethasone (10(-6)M) reduced by 25% the proliferation of NM fibroblasts. Dexamethasone also inhibited proliferation of NM (p < 0.01) but not that of NP fibroblasts at 5-days. TGF-ß induced collagen-1α1, -1α2, and -3α1 mRNA levels in both NM and NP fibroblasts (p < 0.05), and dexamethasone did not alter TGF-ß-induced collagen mRNA levels in either fibroblast type at 24 h. Dexamethasone dose-dependently decreased (p < 0.05) FBS-induced IL-6 and IL-8 release in both NM and NP fibroblasts at 4 h, although at 10(-8)M, dexamethasone inhibited cytokine production in NM (p < 0.05) but not in NP fibroblasts. CONCLUSIONS: This impaired sensitivity of nasal polyp fibroblasts to in vitro glucocorticoid effects concurs in part with the poor clinical response that these nasal polyp patients show to glucocorticoid treatment.