T and NK cell subset changes with microbial extracts and human HSP60-derived peptides in Behçet's disease.

OBJECTIVE: Microorganisms such as streptococcus and autoantigens such as 60 kD heat-shock protein (HSP60) are implicated in the etiopathogenesis of Behçet's disease (BD). METHODS: Peripheral blood mononuclear cells from patients with BD (n = 16) and healthy controls (HC) (n = 11) were cultured for 5 days with extracts of S. sanguis-KTH-1 (SS), E. coli (EC) and a mixed peptide combination from human HSP60 (aa 136-50, 179-97, 224-58 and 336-51) reported to be associated with BD. T and NK cell subset changes were determined by flow cytometry. RESULTS: In unstimulated 5-day cultures gammadelta+ (both CD4+gammadelta+ and CD8+gammadelta+), CD8+alphabeta+, CD4+CD56+ and CD8+CD11b+ cells were increased in BD compared to HC. In antigen-stimulated cultures of BD patients CD3+ and alphabeta+ T cells responded to HSP60 peptides whereas EC stimulated only CD16/ CD56+ NK cells. In the control group, similar to BD, alphabeta+ and CD4+ T cells responded to HSP60 peptides, however SS and EC mainly activated cytotoxic T cell subsets (CD8+CD11b and CD4+CD56+ T cells). CONCLUSION: Significant increases in unstimulated T cell subsets suggest the presence of an in vivo T cell activation in BD. In both patients and controls similar patterns of responses were observed against different microorganisms, however the role of human HSP60 peptides as immunodominant, crossreactive antigens could not be demonstrated.

The effects of nitric oxide donors and inhibitors on neutrophil functions in Behçet's disease.

OBJECTIVE: The effects of nitric oxide donor SNAP and nitric oxide inhibitors L-NMMA and AG on the functions of neutrophils in patients with Behçet's Disease (BD) were investigated in vitro. METHODS: Oxidative burst and phagocytosis of neutrophils were evaluated by flow cytometry in patients with Behçet's disease (n = 32), inflammatory (n = 17) and healthy controls (n = 14), in the presence of L-NMMA, AG and SNAP. RESULTS: The stimulation index of oxidative burst was found to be significantly decreased following PMA stimulation in patients with active BD compared to inflammatory and healthy controls. Oxidative function of neutrophils were inhibited in all 3 groups in the presence of L-NMMA, AG and SNAP L-NMMA inhibited the oxidative burst of neutrophils obtained from healthy controls more than inflammatory controls and BD (80% vs 52% and 53% respectively, p = 0.001). No significant difference of phagocytosis inhibition was found with L-NMMA, AG and SNAP and there were also no differences between the groups (% 9-39). CONCLUSION: Nitric oxide donors and inhibitors may have a therapeutic role in Behçet's disease by suppresing neutrophil activity.

Effect of interferon-alpha(2a) on neutrophil adhesion and phagocytosis in chronic myeloid leukemia and Behçet's disease.

Alpha-interferon (alpha-IFN) is implicated in a Behçet's disease (BD)-like syndrome observed in a small number of chronic myeloid leukemia (CML) patients. The effect of alpha-IFN on neutrophil adhesion and phagocytosis in CML patients, BD patients and healthy volunteers was investigated to clarify the reason for this observation. Ten subjects were studied for each group by incubating neutrophils with various doses of alpha-IFN. Basal neutrophil adhesions for CML patients, BD patients and healthy volunteers were similar. However, BD patients had greater basal phagocytosis than CML patients, and both groups had greater basal phagocytosis than healthy volunteers. Neutrophil adhesion and phagocytosis of CML patients increased following incubation with higher doses of alpha-IFN, and phagocytosis approached the high levels observed with BD neutrophils. This study provides evidence that alpha-IFN activates neutrophils in CML patients in a dose-dependent manner, and leads to a neutrophil function profile that resembles BD.

HSP 60 expression in mucocutaneous lesions of Behçet's disease.

BACKGROUND: Heat shock protein (60 kd HSP) has been implicated in the etiology of Behçet's disease, but its expression at sites of inflammation is unknown. OBJECTIVE: Our aim was to investigate local HSP 60 expression and to quantify T-cell receptor (TCR) gamma delta-positive cells, which are known to respond to HSP peptides. METHODS: Patients with active Behçet's disease (n = 21) and controls (n = 18) were included. Flow cytometric analysis was performed on peripheral blood to investigate TCR gamma delta-positive cell counts. Biopsies were performed on active skin lesions, and immunohistochemical analysis was performed by a streptavidin-biotin method using the monoclonal ML-30 antibody; HSP staining intensity and distribution were evaluated in a blinded fashion. Immunohistochemical studies were performed to quantify TCR gamma delta-positive cells at lesional sites. RESULTS: Mucocutaneous lesions of patients with Behçet's disease had statistically significantly increased expression of HSP 60/65. Peripheral blood TCR gamma delta-positive cell counts were similar in both groups. However, lesional skin of patients with Behçet's disease had significantly increased gamma delta-positive T-cell counts. CONCLUSION: Up-regulation of HSP expression was found at lesional skin sites in Behçet's disease. The increased number of TCR gamma delta-positive cells, which are known to respond to HSP peptides, may support the function of HSPs in the etiology of Behçet's disease. However, these findings may also be an epiphenomenon that needs to be further investigated.

Anticardiolipin antibodies in Behçet's disease: a reassessment.

OBJECTIVE: To assess the frequency and clinical relevance of anticardiolipin antibodies (aCL) in Behçet's disease (BD). METHODS: IgG, IgM and IgA aCL isotypes were investigated by ELISA in 128 patients with BD, 143 healthy controls and 20 systemic lupus erythematosus (SLE) patients. RESULTS: The IgA binding index (BI) was slightly elevated in BD compared with healthy controls (120+/-53 vs 107+/-46, P=0.02), whereas IgG and IgM aCL levels were not significantly different (IgG, BD 2.5+/-2.4 G phospholipid (GPL), healthy controls 2.8+/-3.6 GPL, P=0.6; IgM, BD 0.7+/-0.9 M phospholipid (MPL), healthy controls 0.9+/-1.3 MPL, P=0.6). The frequency of aCL positivity was 7% in BD (IgG 0.8%, IgM 1.6%, IgA 4.6%), 50% in SLE and 5.6% in healthy controls. IgA BI was elevated in the HLA-B5-negative group compared with HLA-B5-positive patients (P<0.005). In a literature review, the frequency of aCL was found to be 9.5% in studies from Turkey compared with 25.5% in other series (P<0.0001). CONCLUSION: These results do not suggest a primary role for aCL in BD. A significantly lower frequency of aCL in Turkish BD patients than in other series indicate that regional determinants, whether environmental or genetic, might also play a role in controlling aCL production in BD.

Neutrophil activation in Behçet's disease.

OBJECTIVE: Neutrophils are implicated in the pathogenesis of Behçet's disease (BD). Various functions of neutrophils are studied to clarify this role. METHODS: The oxidative burst and phagocytic functions of neutrophils and surface molecules associated with neutrophil activation (CD10, CD14 and CD16) were investigated in BD patients by flow cytometric methods. Patients with inflammatory arthropathies, sepsis and healthy controls were also studied. RESULTS: In the oxidative burst experiments, after fMLP and PMA stimulation, stimulation index was found to be significantly decreased in patients both with BD and sepsis compared to healthy controls and inflammatory arthropathies (p < 0.001 and p < 0.01, respectively). The phagocytosis of labelled E. coli particles in patients with BD was not different from that of the healthy controls, while it was decreased in diseased controls (p < 0.001). The surface density of neutral endopeptidase (CD10) and the mean percentage of LPS receptor (CD14) was found to be significantly higher in both BD patients and diseased controls (p < 0.001). The mean percentage of CD16 expression was only low in patients with sepsis (p < 0.001), whereas CD16 intensity on cells was found to be lower in patients with BD as well as in sepsis (p < 0.01). CONCLUSION: These findings indicate the presence of in vivo pre-activated neutrophils in BD. A similar activation was also a feature of severe inflammatory disorders.

Remitting seronegative symmetrical synovitis with pitting oedema associated with chronic lymphocytic leukaemia.

We describe a case of remitting seronegative symmetrical synovitis with pitting oedema (RS3PE syndrome) in a 67-year-old man. Immunophenotyping studies and histology of biopsy specimens revealed chronic lymphocytic leukaemia (CLL). Polyarthritis and oedema were revealed by small doses of corticosteroids. The association of RS3PE syndrome with rheumatological and malign disorders are discussed.

IL-8 producing cells in patients with Behçet's disease.

OBJECTIVE: IL-8 is thought to be the principal chemokine responsible for neutrophil activation and tissue infiltration in patients with Behçet's disease (BD). In various studies serum levels of IL-8 were reported to be increased. METHODS: IL-8 mRNA was purified from both peripheral whole blood samples and separated lymphocytes, granulocytes and monocytes of patients with BD and compared to that from healthy (HC) and disease controls. IL-8 sequences were revealed by PCR amplification using appropriate sequence-specific primers. mRNA levels were determined semi-quantitatively using an image analyser in comparison with beta-actin. RESULTS: Although the differences did not reach statistical significance, BD patients tended to have higher IL-8 mRNA levels compared to HC in whole blood samples (2.0 +/- 1.4 vs 1.5 +/- 1.2) as well as in their lymphocytes (2.7 +/- 1.6 vs 1.5 +/- 0.9). No differences were observed between BD and HC in monocyte or granulocyte IL-8 mRNA levels. CONCLUSION: Our results suggest that the cellular source of IL-8 is diverse in BD with a possible major contribution by lymphocytes.

Role of hepatocyte growth factor in the development of dendritic cells from CD34+ bone marrow cells.

BACKGROUND AND OBJECTIVE: Hepatocyte growth factor (HGF) is known to augment the effects of stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoetin, and granulocyte colony-stimulating factor, all of which are involved in hematopoiesis. HGF is also known to have a role in immune responses. The aim of this study was to investigate whether HGF is involved in the development of dendritic cells (DC) from CD34+ bone marrow cells. DESIGN AND METHODS: CD34+ cells obtained from three healthy donors were incubated in various combinations of HGF, GM-CSF, and tumor necrosis factor (TNF) for 12 days. Developing cell populations were analyzed for surface markers, morphology and functional capacities by flow cytometry, light microscopy and mixed lymphocyte reaction, respectively. RESULTS: Incubation with HGF alone generated greater number of dendritic cells from CD34+ bone marrow cells than incubation with GM-CSF, or a combination of GM-CSF with TNF. HGF was also found to potentiate the effect of GM-CSF on DC and monocyte development. The effects of HGF were inhibited by the concurrent use of TNF. INTERPRETATION AND CONCLUSIONS: HGF appears to be a significant factor in the development of dendritic cells from CD34+ bone marrow cells.

Anti-MHC autoimmunity in Behçet's disease: T cell responses to an HLA-B-derived peptide cross-reactive with retinal-S antigen in patients with uveitis.

Immune response to retinal autoantigens plays a central role in the pathogenesis of uveitis. A synthetic peptide (B27PD) from a common sequence of various HLA-B molecules associated with uveitis, such as HLA-B27 and 51, which shares amino acid homologies with a retinal-S antigen (S-Ag)-derived peptide (PDSAg), was shown to be immunogenic in human and experimental uveitis in the rat. In this study we investigated T cell responses to B27PD and PDSAg in patients with Behçet's disease and posterior uveitis (BD-posterior uveitis; n = 33) in comparison with non-Behçet anterior uveitis (AU, n = 14), Behçet's patients without uveitis (BD, n = 15) and healthy controls (HC, n = 32) in a 6-day proliferation assay. Patients with BD and posterior uveitis had significantly higher responses (stimulation index (SI) 2.8 +/- 1.3) than those with AU (SI 1.5 +/- 0.4), BD without uveitis (SI 1.1 +/- 0.4) and HC (SI 1.1 +/- 0.6) for B27PD (P < 0.0001). Responses to PDSAg were also higher in BD with posterior uveitis patients (SI 3.3 +/- 1.6) than AU (SI 1.5 +/- 0.4), BD without uveitis (SI 1.2 +/- 0.3) and HC (SI 1.1 +/- 0.6) (P < 0. 0001). A significant correlation between the responses to PDSAg and B27PD (r = 0.56, P < 0.001) was observed. Elevated levels of IL-2 and tumour necrosis factor-alpha were also observed in culture supernatants obtained from peripheral blood mononuclear cells after stimulation with the peptides, but no correlation was found between the proliferative responses and cytokine levels. These results suggest that cellular immunity to cross-reactive HLA-B and S-Ag-derived peptides might play a role in the pathogenesis of posterior uveitis in BD.

T cell responses to 60/65 kDa heat shock protein derived peptides in Turkish patients with Behçet's disease.

OBJECTIVE: Sequence homology and cross reactivity between microbial and human heat shock proteins (HSP) led to the concept that HSP might be involved in the etiopathogenesis of Behçet's disease (BD). We investigated T cell responses to 8 synthetic peptides derived from the mycobacterial 65 kDa and homologous human 60 kDa HSP in patients with BD. METHODS: T cell proliferative responses to synthetic peptides were studied in 49 patients with BD and 46 disease (DC) and 34 healthy controls (HC) with 3H-thymidine uptake test. RESULTS: Positive T cell responses to one or more of the mycobacterial peptides were observed in 52% (12/23) of patients with BD compared with 17% (3/18) of DC (p = 0.02) and to homologous human peptides in 57% (13/23) of BD and 11% (2/18) of DC (p < 0.01). Responses to the mixtures of 4 mycobacterial peptides were also significantly higher in BD compared with controls (stimulation index in BD 4.7 +/- 3.5 vs DC 2.0 +/- 1.2, HC 1.6 +/- 0.4; BD vs DC and HC, p < 0.001). Similar elevated responses to the mixture of 4 human peptides was also observed in patients with BD (BD 3.4 +/- 2.3; DC 1.9 +/- 0.8; HC 1.4 +/- 0.6; BD vs DC, p < 0.01; BD vs HC, p < 0.001). CONCLUSION: These results suggest that cellular immunity against the 65 kDa mycobacterial and 60 kDa human HSP derived peptides is significantly increased in Turkish patients with BD compared to controls, as observed in the UK and Japan.

Morphological and functional characteristics of short-term and long-term bone marrow cultures in chronic myelogenous leukemia.

Clonogenic capacity of bone marrow progenitors and stromal layers established from bone marrow of 12 patients with CML and 13 healthy controls were evaluated. The initial BFU-E and CFU-GM contents were slightly higher in the CML patients (p > 0.05) in contrast to CFU-GEMM. CFU-GEMM was lower in the patients compared to healthy controls (p < 0.001). In long-term cultures, the number of non-adherent cell population and total clonogenic progenitor cell content decreased gradually in both groups. Weekly evaluation of stromal confluency of adherent cells revealed that establishment of adherent stromal layer was slower in CML patients than in control samples (p < 0.05). At the end of fourth week, the number of samples presenting confluency was 41.7% in the CML group compared with 92.3% in the controls. The initial CD34 positive cell content of the bone marrow samples was similar in both groups. Although CD34 positive cell number in the adherent stromal layer was well preserved in the control group at the end of 4 weeks, this figure decreased significantly in the CML group. The numbers of total adherent cells as well as the total clonogenic progenitor content of adherent layer were also lower in the CML group (3.03% vs 98.2%). When normal CD34+ cells were cultured on IFN-alpha-treated stromal layer followed by the assessment of the long-term culture initiating cells, a reduced capacity to support hemopoietic growth was observed with IFN-alpha-treated normal stroma. This reduction was even higher when CML stroma was treated with IFN-alpha followed by the seeding of the normal CD34+ cells on this stromal layer (26.9% vs 42.8%). These findings show that stromal cells are abnormal in CML patients as well as the progenitor cells, and IFN-alpha treatment causes further defects of the stromal cells.

Serum soluble intercellular adhesion molecule 1 and interleukin 8 levels in familial Mediterranean fever.

OBJECTIVE: Familial Mediterranean fever (FMF) is a disease of unknown etiology characterized by recurrent attacks of polyserositis (peritonitis, pleuritis, and arthritis) and fever. We measured levels of soluble intercellular adhesion molecule 1 (sICAM-1) and interleukin 8 (IL-8), which are important mediators in leukocyte-endothelial adhesion and leukocyte accumulation in tissues. METHODS: sICAM-1 and IL-8 levels were studied in 30 patients with FMF during attacks and remission, along with 23 healthy and 26 disease controls. sICAM-1 and IL-8 levels were measured with commercial ELISA systems. RESULTS: Median levels of sICAM-1 were significantly elevated in patients with FMF during attacks (FMF-a) and remission periods (FMF-r) compared to healthy controls (HC) (FMF-a: 600 ng/ml, FMF-r: 520 ng/ml, HC: 353 ng/ml; FMF-a vs. HC: p<0.0001, FMF-r vs. HC: p = 0.002). IL-8 levels were also significantly elevated in FMF-a compared to HC (37 vs. 25 pg/ml; p = 0.009), but not during remission (26 pg/ml; p = 0.7). A significant correlation was observed between sICAM-1 and IL-8 levels (r = 0.33, p = 0.01). sICAM-1 levels also correlated significantly with erythrocyte sedimentation rate, C-reactive protein, and fibrinogen levels of patients with FMF. CONCLUSION: Increased levels of sICAM-1 and IL-8 in FMF suggest that neutrophils are active with increased adhesion in FMF. Since increased levels of sICAM-1 are also observed during remission, subclinical disease activity and inflammation seem to be present in some patients.

Evaluation of the Turkish translation of a disease activity form for Behçet's syndrome.

OBJECTIVE: This study examined the interobserver and intra-observer reliability of the Turkish version of the Behçet's Disease Current Activity Form (BDCAF), which was obtained by a translation and back-translation process. METHODS: Fifty Behçet's syndrome (BS) patients were assessed by four rheumatologists in separate morning and afternoon sessions. RESULTS: The results showed good intra- and interobserver agreement for the oro-genital ulcers and eye involvement of BS, but there was poor agreement between (kappa score = 0.14) and within observers (range for kappa scores 0.09-0.25) for their overall impression of disease activity. Individual low kappa scores were also noted for erythema nodosum, vascular involvement, central nervous system involvement and gastrointestinal involvement. CONCLUSION: These results suggest that the Turkish version of BDCAF may be useful for assessing the classic triad of BS (oro-genital ulceration and eye involvement), but more experience is needed for its other parts.

Oligoclonal T cell expansions in patients with Behçet's disease.

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vbeta gene products in CD4+ and CD8+ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4+ T cell compartment, oligoclonal TCR Vbeta expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8+ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vbeta5.1 subset was observed in five BD patients among CD8+ T cells. Other elevations of TCR Vbeta subsets were heterogeneously distributed with one to three different Vbeta subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vbeta group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments.

Double G0/G1 peak in the DNA histogram of aberrant marker positive acute leukemia patients is associated with a poor clinical outcome.

In order to investigate the relationship between aberrant marker expression and DNA ploidy, 61 adult patients with acute leukemia (39 AML and 22 ALL) were studied. Aberrant marker expression was observed in 20 patients (16/39 of AML and 4/22 of ALL patients). In flow cytometric DNA analysis aneuploidy was observed in 18 patients (9/39 of AML and 9/22 of ALL patients). The incidence of aneuploidy in patients with aberrant marker expression was 35% whereas this was 26.8% in patients without aberrant marker expression. Furthermore, 7 patients with aberrant marker expression showed an aneuploid, double G0/G1 peaks appearance whereas the remaining 11 patients with aberrant marker expression had euploid DNA content. Double G0/G1 appearance was not observed in patients without aberrant marker expression. Further analyses revealed that this did not correlate with apoptosis. All 7 patients, who had both aberrant marker expression and double G0/G1 peak had a poor clinical outcome with a short survival and all died within three months whereas three-months survival was 67% for AML, 69% for ALL patients and 81% for patients with aberrant marker expression respectively (p<0.01). Our data indicate that the evaluation of the DNA ploidy in patients with aberrant marker expression may be of prognostic importance.

Phenotypic characteristics of B cells in Behçet's disease: increased activity in B cell subsets.

OBJECTIVE: Increased numbers of spontaneous Ig secreting B cells and elevated immunoglobulin levels have been described in Behçet's disease (BD), in addition to changes in numbers and activities of T cells, natural killer cells, and monocyte-macrophages. We investigated other characteristics of B cells in BD. METHODS: B lymphocyte subsets (CD19+CD5+, CD19+CD13+, CD19+CD28+, CD19+CD33+, CD19+CD80+, CD5+CD19+CD45RA+, CD5+CD19+CD45RO+) were phenotypically evaluated in 50 patients with BD, 80 healthy subjects, and 20 other patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and sepsis. RESULTS: Although the B cell number (CD19+) was normal, CD13 and CD33 positive B cells were more numerous in BD and sepsis compared to healthy controls and patients with RA and SLE. The percentage of CD45RO positive B cells was higher in both BD and sepsis, while the percentage of CD80 positive B cells was high only in BD. There was no increase in the CD5+CD19+ B cell subset, previously shown to be increased in several autoimmune diseases. Naive (CD45RA) and memory (CD45RO) status of CD5+CD19+ and CD5-CD19+ B cells showed that CD45RA expression was higher in CD5+CD19+ B cells, whereas expression of both CD45RA and CD45RO was higher in the CD5-CD19+ B cell group compared with healthy controls. CONCLUSION: Although the total B cell number was normal, increased levels of activated and memory B cell subsets suggest a modified B cell function in BD, which may be related to a weak stimulus by an unknown external antigen.

Increased CD4+CD16+ and CD4+CD56+ T cell subsets in Behçet's disease.

Behçet's disease is a systemic vasculitis of unknown etiology. Various immune abnormalities have previously been shown in Behçet's disease. We investigated T lymphocyte subsets associated with cytotoxic activity and natural killer (NK) cells by flow cytometry in 37 patients with Behçet's disease, 38 healthy controls, and 17 diseased control patients. Compared to the healthy controls, CD4+CD16+ and CD4+CD56+ subsets were found to be higher in the Behçet's disease group as well as in the disease control group (CD4+CD16+: BD = 5 +/- 3, DC = 14 +/- 14, HC = 3 +/- 2, P = 0.001; CD4+CD56+: BD = 11 +/- 5, DC = 18 +/- 17, HC = 8 +/- 6, P = 0.01). CD8+CD16+ and CD8+CD56+ T cell subsets were at normal levels in Behçet's disease but found to be elevated in disease controls. Similarly, NK cells (CD16+CD56+) were high only in the disease control group. Significant increases in CD4+CD16+ and CD4+CD56+ cell subsets in Behçet's patients and disease controls suggest that T cell activation patterns of these subsets in Behçet's disease are similar to those in other inflammatory disorders.