RESUMO
BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. RESULTS: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFß1 (p = 0.038). CONCLUSION: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.
Assuntos
Linfócitos T CD4-Positivos/citologia , Lúpus Eritematoso Sistêmico/imunologia , Lavagem Peritoneal , Baço/citologia , Linfócitos T Reguladores/citologia , Animais , Antígenos CD/análise , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos Ly/análise , Antígenos Ly/imunologia , Antígenos CD28/análise , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/imunologia , Imunossupressores , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/análise , Lectinas Tipo C/imunologia , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/imunologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/induzido quimicamente , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , TerpenosRESUMO
Because collagen type V (Col V) can be exposed in tissue injury, we hypothesized that oral administration of this collagen species modulates the inflammation and remodeling of experimental synovitis, avoiding joint destruction, and that the modulation may differ according to the temporal administration. Arthritis (IA, n = 20) was induced in Lewis rats by intraarticular (ia) injection of 500 µg of methylated bovine serum albumin (mBSA) emulsified in complete Freund's adjuvant (CFA) (10 µl) followed by an intraarticular booster of mBSA (50 µg) in saline (50 µl) administered at 7 and 14 days. The control group received saline (50 µl, ia). After the first intraarticular injection, ten IA animals were supplemented via gavage with Col V (500 µg/300 µl) daily for 30 days (IA/Suppl). The control group received saline (50 µL) and Col V supplement in the same way (Suppl). Col V oral administration in IA/Suppl led to 1) inhibited edema and severe inflammatory cell infiltration, 2) decreased collagen fiber content, 3) decreased collagen type I, 4) inhibited lymphocyte subpopulations and macrophages, 5) inhibited IL-1ß, IL-10, IL-17 and TNF-α production and 6) increased expression of caspase-9 in the synovial tissue. In conclusion, Col V supplementation decreased synovial inflammation and the fibrotic response, possibly by increased the apoptosis of inflammatory cells.
Assuntos
Artrite Experimental/tratamento farmacológico , Colágeno Tipo V/farmacologia , Membrana Sinovial/efeitos dos fármacos , Administração Oral , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Caspase 9/metabolismo , Citocinas/metabolismo , Edema/tratamento farmacológico , Edema/imunologia , Edema/patologia , Adjuvante de Freund , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Ratos Endogâmicos Lew , Soroalbumina Bovina , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
OBJECTIVE: To evaluate the effect of low dose methotrexate alone or in combination with glucocorticoid treatment on titanium implant osseointegration. METHODS: Groups of 6-8 adult New Zealand White rabbits were treated for 18 weeks with saline (control), methotrexate, glucocorticoid, or methotrexate plus glucocorticoid. The animals received a titanium implant in the tibia at week 6. Lumbar spine and tibia bone mineral densities were analyzed before and after treatment. Histomorphometric analysis of bone cortical thickness, total bone area around the implant, and percent of bone to implant contact was performed. RESULTS: After 18 weeks, the change in the bone mineral density in the lumbar spines and tibias in the methotrexate group was comparable to the control group (0.035 vs. 0.055 g/cm² and 0.021 vs. 0.041 g/cm², respectively). In contrast, both the glucocorticoid group and glucocorticoid plus methotrexate group had significant reductions at both sites. Histomorphometric analysis of the tibia in the control and methotrexate groups revealed no significant changes in cortical thickness (133 vs. 126 μm), total bone area around the implant (33 vs. 30 percent), or bone to implant contact (40 vs. 38 percent). In contrast, glucocorticoid group had significant reductions compared to controls in tibia cortical thickness (99 vs. 133 μm), total bone area around the implant (24 vs. 33 percent), and bone to implant contact (27 vs. 40 percent). Similar reductions were observed in the glucocorticoid plus methotrexate group. CONCLUSIONS: Our results demonstrate that low dose methotrexate treatment does not affect titanium implant osseointegration, suggesting that this therapy is safe for surgical procedures requiring a titanium implant.
Assuntos
Animais , Masculino , Coelhos , Antirreumáticos/administração & dosagem , Metotrexato/administração & dosagem , Osseointegração/efeitos dos fármacos , Tíbia , Titânio , Absorciometria de Fóton , Densidade Óssea , Glucocorticoides/administração & dosagem , Teste de Materiais , Modelos Animais , Fatores de Tempo , Resultado do TratamentoRESUMO
The present study evaluates the effect of isolated fractions of Harpagophytum procumbens (devil's claw) on cyclooxygenase (COX-1 and COX-2) activities and NO production using a whole blood assay. The activity of COX-1 was quantified as platelet thromboxane B(2) production in blood clotting and COX-2 as prostaglandin E(2) production in LPS-stimulated whole blood. Total NO(2) (-)/NO(3) (-) concentration was determined by Griess reaction in LPS stimulated blood. Assays were performed by incubation of isolated fractions obtained by flash chromatography monitored with HPLC, TLC and identified by (1)HNMR, containing different amounts of harpagoside with blood from healthy donors. Indomethacin and etoricoxib were the positive controls of COX-1 and COX-2 Inhibition. Data shows that fraction containing the highest concentration of harpagoside inhibited indistinctively COX-1 and COX-2 (37.2 and 29.5% respectively) activity and greatly inhibited NO production (66%). In contrast the fraction including iridoid pool increased COX-2 and did not alter NO and COX-1 activities. The fraction containing cinnamic acid was able to reduce only NO production (67%). Our results demonstrated that the harpagoside fraction is the main responsible for the effect of devils claw on these enzyme activities. However, other components from devil's claw crude extract could antagonize or increase the synthesis of inflammatory mediators.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Glicosídeos/farmacologia , Harpagophytum/química , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Piranos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Análise Química do Sangue , Coagulação Sanguínea , Plaquetas/metabolismo , Cinamatos/farmacologia , Dinoprostona/biossíntese , Etoricoxib , Feminino , Humanos , Indometacina/farmacologia , Mediadores da Inflamação/metabolismo , Iridoides/farmacologia , Lipopolissacarídeos , Piridinas/farmacologia , Sulfonas/farmacologia , Tromboxano B2/biossínteseRESUMO
AIM OF THE STUDY: This study assessed the involvement of endogenous glucocorticoids (GCs) in the anti-arthritic properties of bee venom (BV) on antigen-induced arthritis (AIA) in rabbits. MATERIALS AND METHODS: BV (1.5-6 microg/kg/day) was injected for 7 days before AIA induction, whereas the control group received sterile saline. The total and differential leukocyte count, PGE(2) levels in synovial fluid and synovial membrane cell infiltrate were evaluated. The contribution of GCs to BV action was assessed in rabbits treated with BV plus metyrapone, an inhibitor of GC synthesis, or RU-38 486, a steroid antagonist. RESULTS: Treatment with BV (1.5 microg/kg/day) reduced the leukocyte count and PGE(2) level (18571+/-1909 cells/mm(3) and 0.49+/-0.05 ng/mL, respectively) as well as the cellular infiltrate compared with the control group (40968+/-5248 cells/mm(3) and 2.92+/-0.68 ng/mL, p<0.05). The addition of metyrapone to BV treatment completely reversed the inhibition of AIA, whereas RU-38 486 was ineffective. CONCLUSION: Our data show that bee venom treatment prevents the development of antigen-induced arthritis in rabbits through the action of GCs.
Assuntos
Anti-Inflamatórios/farmacologia , Antígenos/administração & dosagem , Artrite Experimental/tratamento farmacológico , Venenos de Abelha/farmacologia , Glucocorticoides/fisiologia , Animais , Anti-Inflamatórios/uso terapêutico , Venenos de Abelha/uso terapêutico , Dinoprostona/metabolismo , Contagem de Leucócitos , Metirapona/administração & dosagem , Mifepristona/administração & dosagem , CoelhosRESUMO
Increased neutrophil chemotaxis and hyperresponsiveness to streptococci have been considered to play a role in Behçet's disease (BD) pathogenesis. Our aim was to correlate TLR2 expression and chemotactic responses stimulated by bacterial lipoteichoic acid (LTA) in BD neutrophils. Thus, we assessed expressions of TLR2 and the correlate receptors CD14, CD114 (G-CSF receptor), CD116 (GM-CSF receptor) and also TLR4 on circulating neutrophils and monocytes of patients with active BD. Serum concentration of soluble CD14 (sCD14) was also measured. Neutrophil chemotactic responses from BD patients and healthy controls under LTA stimulation were assessed. Disease activity was evaluated by Behçet's Disease Current Activity Form (BDCAF). Receptor expressions were measured by flow cytometry, neutrophil chemotaxis was assessed in a Boyden chamber and sCD14 was measured by enzyme-linked immunosorbent assay. TLR2 expression was higher only on BD monocytes compared with healthy controls (39.9 +/- 13.1 vs. 33.6 +/- 5.3, p = 0.019). Expressions of all other receptors were similar in BD and control group. Of particular interest, TLR2 expression on neutrophils was similar in both groups and, when stimulated with LTA, BD neutrophils showed chemotactic responses similar to the controls. Neutrophils exhibited increased chemotaxis only when incubated with BD plasma. Serum sCD14 concentration was higher in BD patients than in controls (1,920.8 +/- 563.6 vs. 1,623.2 +/- 391.3 ng/ml, p = 0.008), and it was positively correlated with both CD14 expression on circulating monocytes membrane (p = 0.035) and BDCAF scores (p = 0.025). In conclusion, isolated BD neutrophils do not overexpress TLR2 neither overreact to LTA. However, because TLR2 expression was higher on BD monocytes, sCD14 from monocyte origin correlated with disease activity and neutrophil hyperchemotaxis occurred on strict dependence of plasmatic soluble factors, we suggest a possible role for bacterial stimulation of monocytes via TLR2 producing neutrophil-stimulating pro-inflammatory factors in BD.
Assuntos
Síndrome de Behçet/imunologia , Quimiotaxia de Leucócito/imunologia , Imunidade Inata/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Adolescente , Adulto , Antígenos CD/sangue , Síndrome de Behçet/sangue , Síndrome de Behçet/fisiopatologia , Biomarcadores/sangue , Células Cultivadas , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Índice de Gravidade de Doença , Ácidos Teicoicos/farmacologia , Receptores Toll-Like/sangue , Adulto JovemRESUMO
Studies have demonstrated the beneficial effects of fangotherapy on relieve of pain improving function of rheumatic patients. Herein, we investigated the effect of Brazilian black mud in protect articular damage in chronic arthritis induced in rats. Mud was daily applied (40 degrees C/30 min) during the course of arthritis and was compared with warm water and no treated groups. At 21th day after arthritis induction synovial fluid and membrane were analyzed regarding cellular influx, hyperplasia and vascular proliferation. Cartilage structure, cell count, proteoglycan and collagen amount were also analyzed by three pathologists blinded to the treatment. Mud treatment diminished leukocyte migration into the synovial membrane and articular cavity when compared with both control groups. Regarding cartilage, an increase in collagen, number of chondrocytes and more conserved tissue structure was observed in mud-treated animals. These results demonstrate a protective effect of Brazilian mud on this model of arthritis, suggesting that this therapy may be useful as a complementary approach to treat articular diseases.