Proinflammatory cytokine gene polymorphisms in Behçet's disease

Beh?et's disease (BD) is a chronic, systemic disease, characterized by oral and genital lesions, and ocular inflammation. There is evidence indicating altered levels of proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in patients with BD. This study involved 150 patients with BD and 140 healthy controls, and investigated the role of proinflammatory cytokine gene polymorphisms in the disease. The frequency of the TNF-α (-238) G/G genotype was significantly higher in the patient group, compared to the controls (p < 0.001), whilst the G/A genotype was significantly lower in the patients with BD (p < 0.001). Patients with BD showed a significant increase in the TNF-α (- 308, - 238) GG haplotype (p < 0.001), whilst there was a significant decrease in the GA haplotype (p < 0.001). The heterozygous, IL-6 (- 174) C/G genotype (p = 0.005), and the IL-6 (- 174, nt565) haplotype CG (p < 0.001), were significantly decreased in the patient group. The increased production of proinflammatory cytokines in BD could be a consequence of specific, cytokine gene polymorphisms. Particular genotypes and haplotypes in TNF-α were over-represented in BD, which may, in turn, predispose individuals to this disease.     

Behçet's disease

Behçet's disease is characterized by recurrent aphthous stomatitis, uveitis, genital ulcers, and skin lesions. The role of the HLA-B*51 gene has been confirmed in recent years, although its contribution to the overall genetic susceptibility to Behçet's disease was estimated to be only 19%. The production of a variety of cytokines by T cells activated with multiple antigens has been shown to play a pivotal role in the activation of neutrophils. As regards the treatment, anti-tumor necrosis factor alpha therapy has been shown to be effective for mucocutaneous symptoms as well as for sight-threatening panuveitis, although a randomized, controlled trial is required.     

Natural killer cells control a T-helper 1 response in patients with Behçet's disease

Introduction

Behçet's disease (BD) is a multisystem inflammatory disorder, in which a T-helper 1 (Th1)-polarized immune response plays a major role in the pathogenic process. We evaluated the regulatory role of natural killer (NK) cells in Th1-biased immune responses in patients with BD.

Methods

We studied 47 patients with BD, including 10 with active disease (aBD) and 37 with inactive disease (iBD), and 29 healthy controls. The activation status and cytotoxic activity of NK cells were examined by flow cytometry. The levels of mRNAs for immune modulatory and cytotoxic molecules in NK cells were determined by quantitative PCR. The IL-12 signal strength in NK cells was determined by assessing the phosphorylation state of its downstream component, signal transducer and activator of transduction 4, by immunoblotting. Finally, NK cells' ability to modulate the Th1 response was evaluated by co-culturing NK cells and T cells without cell contact.

Results

CD69⁺-activated NK cells were significantly increased in aBD compared with iBD or control samples, although their cytotoxic activities were similar. The iBD NK cells showed downregulated IL-12 receptor β₂ mRNA levels compared with aBD or control NK cells. The increased IL-13 expression was detected in a subset of BD patients: most of them had iBD. The IL-13 expression level in iBD patients was significantly higher than the level in controls, but was not statistically different compared with the level in aBD patients. The gene expression profile in iBD patients was consistent with the NK type 2 phenotype, and the shift to NK type 2 was associated with disease remission. NK cells from iBD patients showed impaired IL-12-induced signal transducer and activator of transduction 4 phosphorylation. Finally, iBD, but not control, NK cells suppressed IFNγ expression by aBD-derived CD4⁺ T cells in vitro.

Conclusions

NK cells may control disease flare/remission in BD patients via NK type 2-mediated modulation of the Th1 response.

     

MDR1 gene polymorphisms may be associated with Behçet's disease and its colchicum treatment response

Behçet's disease (BD) is a chronic multisystem disorder. Infectious agents, immune system mechanisms, and genetic factors are implicated in the etiopathogenesis of BD, which remains to be explained. The human MDR1 (ABCB1) gene encoder P-glycoprotein (P-gp) plays a key role in drug disposition, serves as a protective mechanism against xenobiotics, and provides additional protection for the brain, testis, and fetus. We investigated the genotype and haplotype distributions of three MDR1 gene polymorphisms (C1236T, G2677T/A, and C3435T) in 104 BD patients and 130 control subjects. The genotyping analysis was performed by using PCR–RFLP methods.     

Selenium and Behçet's disease

Behçet's disease is an inflammatory disorder of unknown etiology, characterized by recurrent oral and genital aphthous ulcers, ocular inflammation, and skin lesions of erythema nodusum and acneiformeruptions. Selenium (Se) affects all components of the immune system, i.e., the development and expression of nonspecific, humoral, and cell-mediated responses. In general, a deficiency in Se appears to result in immunosuppression, whereas supplementation with low doses of Se appears to result in augmentation and/or restoration of immunoglogic functions. In this study, the distribution of Se and IgG, IgM in serum were compared in samples from healthy adult control and Behçet's disease patients. The serum Se levels were measured by AA-30-40 Varian Spectra, and immunoglobulins were measured by immunodiffusion technique. The mean (SD) serum Se level of 54.24 ± 8.06 ng/mL among Behçet's disease subjects was significantly different (P<0.01) from that in the control subjects (90.01 ± 9.94 ng/mL). We also measured IgG and IgM as 10.01 ± 2.74 mg/mL and 1.26 ± 0.29 mg/mL, respectively for patients, and 15.08 ± 4.73 mg/mL and 1.58 ± 0.43 mg/mL for controls. The mean values of IgG and IgM for patients were significantly (P<0.05) different from the values of controls. It seems, therefore, that a deficiency in selenium impedes the humoral immune response.     

Expression of Bcl-2 in inflammatory sites from patients with active Behçet's disease

Behçet''s disease (BD) is a current systemic vasculitis of unknown aetiology. Eyes, skin, joints, the oral cavity, genital system, blood vessels, central nervous system and lung are usually involved. Defective regulation of programmed cell death (apoptosis) may play a role in the development of (BD), and the proto-oncogene Bcl-2 is involved in the control of apoptosis in immunocompetent cells. We therefore wished to investigate the expression of Bcl-2 in the peripheral lymphocytes and in two inflammatory sites of patients with active BD: bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) lymphocytes. Levels of Bcl-2 expression in the lymphocytes of patients with BD and, for comparison, in the lymphocytes of healthy controls and non-inflammatory neurological diseases (NIND), were studied by two-colour cytofluorography and RNA analysis. In BD patients, a significant proportion of T cells expressed increased amounts of Bcl-2 protein, both in peripheral blood and in inflammatory sites. Mononuclear cells of patients with BD showed increased amount of Bcl-2 messenger RNA. The in vitro incubation of T lymphocytes with IL-10, significantly increased the Bcl-2 expression, specifically in T lymphocytes from inflammatory sites. In active BD, stimulation of HSV-1 T lymphocytes slightly increased Bcl-2 expression, not significantly different from unstimulated HSV-1 T cells. The occurrence of circulating T lymphocytes with abnormally high Bcl-2 expression in peripheral circulation and in inflammatory sites may be explained in part by the increased in vivo activation levels, and by aetiopathological agent(s): our findings seem to indicate an important role in the chronic inflammation in BD.     

Tc1/Tc2 ratio in the inflammatory process in patients with Behçet's disease

BACKGROUND: Peripheral blood CD8+ T cells expressing interferon gamma and interleukin-4 (IL-4), and lacking CD28 molecules, were responsible for the dynamic interplay between peripheral blood and inflammatory sites. INTRODUCTION: The aim of the current study was to define in Behçet''s disease (BD), CD8+ T-cell subsets using CD28 and CD11b monoclonal antibodies, and the characterization of the Tc1/Tc2 ratio and perforin expression. METHODS: Flow cytometry was used for intracytoplasmic cytokines and perforin expression. Effector cells were investigated by adhesion of CD8+ T cells to human microvascular endothelial cells and by chemotaxis using beta-chemokine. RESULTS: Interferon-gamma-producing CD8+ T cells in active and remission BD patients were increased, which induce a significant increase of the Tc1:Tc2 ratio in BD. CD8(+)CD28(-)CD11b+ T cells were found to be more expanded in BD patients than in age-matched healthy controls. The expression of CD11b molecules in active BD allowed to CD8(+)CD28+/CD8(+)CD28- subsets to adhere to human microvascular endothelial cells, with more efficiency in BD. Using MIP-1alpha, we observed that the migratory process of CD28(-)CD11b(+) is more important in BD. CD28(-)CD11b+ exhibited an increased perforin expression in BD patients. CONCLUSION: Taken together these results suggest the presence of immune activation, probably in response to a profound inflammation affecting BD patients. The physiopathological significance of these results were toward autoimmune diseases and/or infectious process.     

Serum prolidase enzyme activity and oxidative status in patients with Behçet's disease

Abstract

Objectives

To assess serum prolidase enzyme activity and oxidative stress in patients with Behçet's disease (BD).

Methods

The study population consisted of BD patients (n = 42) and healthy participants (n = 29). BD patients were classified as active (n = 18) or inactive (n = 24) according to disease activity. Serum prolidase enzyme activity, total antioxidant status (TAS), total oxidative status (TOS), oxidative stress index (OSI), and malondialdehyde (MDA) levels were measured.

Results

In BD patients with active disease, serum prolidase activity was significantly higher compared with the inactive and control participants. Serum prolidase activity was also significantly higher in all BD patients in comparison with controls. Serum prolidase activity was also positively correlated with OSI, C-reactive protein, and active BD. MDA, TOS, and OSI levels were all significantly higher in the BD group when compared with the healthy control participants. Serum TAS levels were significantly lower in BD patients in comparison with healthy controls.

Conclusion

High prolidase activity may indicate critical biological activities relevant to pathological events in BD, and this activity may be a biological indicator of disease. Further studies are needed to verify these findings.     

Arachidonic acid metabolites and colchicine in Beh?et's disease (BD)

Vasculitis is accepted to be the basis of Beh?et's disease (BD) which is a multisystem disease, and the arachidonic acid(AA) metabolites acting as balancing mediators in the organism are accepted to be responsible for the vasculitis. In this study, we examined the prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) levels of the patients with BD before and after colchicine therapy. We found a statistical decrease in the PGE2 and LTC4 levels after colchicine therapy compared to the previous levels, concluding that colchicine inhibits the inflammation and the polymorphonuclear leukocyte (PML) chemotaxis by inhibiting the cyclooxygenase and lipoxygenase pathways.     

Oxidases and oxygenases in regulation of neutrophil redox pathways in Behçet's disease patients

This study aimed to determine plasma and neutrophil oxidase activities that may contribute to vascular inflammation in Beh?et's disease (BD) patients. Cyclooxygenase (COX), NADPH oxidase and myeloperoxidase (MPO) activity was determined in neutrophils isolated from BD patients and healthy controls. Functional assay of NADPH oxidase was significantly increased in BD patients, both at basal conditions and in response to fMLP stimulation. There was a significant increase in plasma MPO activity in the disease group as compared to controls. Total COX activity was significantly increased in BD neutrophils. The increase in total COX activity was accompanied with enhanced activity of COX-2, differentiated by using the COX-1 isoform-specific inhibitor SC-560. Neutrophil nitrate/nitrite levels showed no significant difference in BD; however, plasma nitrate/nitrite contents in BD patients were significantly greater compared to controls. In conclusion, increased plasma MPO, neutrophil NADPH and COX activities may contribute to intravascular inflammation documented in BD patients.     

Identification of kinectin as a novel Behçet's disease autoantigen

There has been some evidence that Beh?et's disease (BD) has a significant autoimmune component but the molecular identity of putative autoantigens has not been well characterized. In the initial analysis of the autoantibody profile in 39 Chinese BD patients, autoantibodies to cellular proteins were uncovered in 23% as determined by immunoblotting. We have now identified one of the major autoantibody specificities using expression cloning. Serum from a BD patient was used as a probe to immunoscreen a lambdaZAP expression cDNA library. Candidate autoantigen cDNAs were characterized by direct nucleotide sequencing and their expressed products were examined for reactivity to the entire panel of BD sera using immunoprecipitation. Reactivity was also examined with normal control sera and disease control sera from patients with lupus and Sj?gren's syndrome. Six independent candidate clones were isolated from the cDNA library screen and were identified as overlapping partial human kinectin cDNAs. The finding that kinectin was an autoantigen was verified in 9 out of 39 (23%) BD patient sera by immunoprecipitation of the in vitro translation products. Sera from controls showed no reactivity. The significance of kinectin as a participant in autoimmune pathogenesis in BD and the potential use of autoantibody to kinectin in serodiagnostics are discussed.     

Serum levels of soluble P-selectin are increased and associated with disease activity in patients with Behçet's syndrome

Beh?et's syndrome (BS) is a relapsing, chronic, inflammatory disease characterized by endothelial dysfunction, atherothromboembogenesis, and leukocytoclastic vasculitis with complex immunologic molecular interactions. Generalized derangements of the lymphocyte and neutrophil populations, activated monocytes, and increased PMNLs motility with upregulated cell surface molecules such as ICAM-1, VCAM-1, and E-selectin, which are found on the endothelial cells, leukocytes, and platelets, have all been demonstrated during the course of BS. Our aim is to investigate the association of serum concentrations of soluble P-selectin in patients with BS, and to evaluate whether disease activity has an effect on their blood levels. This multicenter study included 31 patients with BS (15 men and 16 women) and 20 age- and sex-matched healthy control volunteers (11 men and nine women). Neutrophil count, erythrocyte sedimentation rate, and acute-phase reactants as well as soluble P-selectin levels were determined. The mean age and sex distributions were similar (P > .05) between BS patients (35 years) and control volunteers (36 years). Serum levels of soluble P-selectin in patients with BS (399 +/- 72 ng/mL) were significantly (P < .001) higher when compared with control subjects (164 +/- 40 ng/mL). In addition, active BS patients (453 +/- 37 ng/mL) had significantly (P < .001) elevated levels of soluble P-selectin than those in inactive period (341 +/- 52 ng/mL). This study clearly demonstrated that serum soluble P-selectin levels are increased in BS patients when compared with control subjects, suggesting a modulator role for soluble P-selectin during the course of platelet activation and therefore, atherothrombogenesis formation in BS, especially in active disease.     

Interleukin-6 in peripheral blood and inflammatory sites in Behçet's disease

Interleukin-6, a potent pro-inflammatory cytokine, might be involved in Beh?et's disease (BD) pathological pathways. We investigated IL-6 levels in sera and synovial fluids collected from BD patients. The IL-6 production was also studied in vivo, by measuring its activity in culture supernatants of PBMC and alveolar macrophages, stimulated or not with LPS. The patients with BD were compared to RA patients and healthy controls. High IL-6 levels were observed in sera, synovial fluid and LPS stimulated PBMC supernatants, from active BD patients, similar to those of RA patients. Alveolar macrophages production of IL-6 was significantly elevated in two active BD patients with an interstitial pneumonia, when compared to controls. These elevated levels of IL-6 suggest its involvement in the inflammatory sites of BD, which may be related to the progression of the acute lesions, at least in the joints and in the lungs.     

Association of reduced heme oxygenase-1 with excessive Toll-like receptor 4 expression in peripheral blood mononuclear cells in Behçet's disease

Introduction

Toll-like receptors (TLRs) mediate signaling that triggers activation of the innate immune system, whereas heme oxygenase (HO)-1 (an inducible heme-degrading enzyme that is induced by various stresses) suppresses inflammatory responses. We investigated the interaction between TLR and HO-1 in an inflammatory disorder, namely Behçet's disease.

Methods

Thirty-three patients with Behçet's disease and 30 healthy control individuals were included in the study. Expression levels of HO-1, TLR2 and TLR4 mRNA were semiquantitatively analyzed using a real-time PCR technique, and HO-1 protein level was determined by immunoblotting in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes. In some experiments, cells were stimulated with lipopolysaccharide or heat shock protein-60; these proteins are known to be ligands for TLR2 and 4.

Results

Levels of expression of HO-1 mRNA were significantly reduced in PBMCs from patients with active Behçet's disease, whereas those of TLR4, but not TLR2, were increased in PBMCs, regardless of disease activity. Moreover, HO-1 expression in PBMCs from patients with Behçet's disease was repressed in the presence of either lipopolysaccharide or heat shock protein-60.

Conclusion

The results suggest that upregulated TLR4 is associated with HO-1 reduction in PBMCs from patients with Behçet's disease, leading to augmented inflammatory responses.     

Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease

Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10^-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.     

Circulating intercellular adhesion molecules in blood and bronchoalveolar lavage in Behçet's disease

The aim of this study was to evaluate circulating intercellular adhesion molecule-1 (cICAM-1) in serum and in bronchoalveolar lavage (BAL), as a marker for the inflammatory process in patients with active Behçet''s disease (BD). Circulating ICAM-1 was tested by an enzyme linked immuno-sorbent assay in serum and in BAL of patients with BD. These values were compared to those of patients with tuberculosis and to healthy controls. Increased levels of circulating ICAM-1 were found in serum from patients with active BD compared to healthy controls (p < 0.01). Similar levels of serum cICAM-1 were found in BD and tuberculosis. Additionally, both BD and tuberculosis patients exhibited high levels of cICAM-1 in BAL fluid, suggesting that this increase may be a result of the immune system activation in inflammatory sites. Circulating ICAM-1 seemed to have a good discriminative power in identifying active BD, being elevated in all active stages (p < 0.01) compared to remission BD stage. No differences were found in active BD patients depending upon the clinical manifestations. These results suggest that cICAM-1 may be involved in leucocyte adhesion and migration into the vessel wall of the lung. Circulating forms are derived from molecules expressed on the surface of activated cells, as a result of an inflammatory process.     

Expressions of vascular endothelial growth factor and CD34 in oral aphthous lesions of Behçet's disease

OBJECTIVE: Beh?et's disease (BD) is an immunoin-flammatory vasculitis with an unknown etiopathogenesis. Vascular endothelial growth factor (VEGF) is a cytokine-stimulating angiogenesis. It has been suggested to play a role in inflammation and pathogenesis of vasculitic processes. STUDY DESIGN: VEGF and CD34 expressions were assessed in samples taken from oral aphthous lesions. The patients were evaluated for disease activity, duration of lesions, serum C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR). RESULTS: Twenty-six patients were included. Fourteen (53.9%) had increased CRP levels and 12 (46.1%) had increased ESR levels. Positive VEGF and CD34 staining were detected in 46.2% and 69.2% of biopsy samples, respectively. There was good positive correlation between them. The frequency of positive VEGF and CD34 staining was statistically higher in lesions with a duration of more than 6 days. No correlation was found between positive VEGF staining and serum CRP level, ESR and disease activity. CONCLUSION: Increased VEGF expression in correlation with CD34 positivity in oral aphthous lesions may show the role of VEGF in pathogenesis of these vasculitic lesions. We have concluded that VEGF may play a role during the course of oral aphthous lesions in BD.