A pattern of protein expression in peripheral blood mononuclear cells distinguishes rheumatoid arthritis patients from healthy individuals

We compared the expression levels of proteins in peripheral blood mononuclear cells (PBMCs) of healthy control individuals to those of patients diagnosed with rheumatoid arthritis (RA) using a proteomics approach. Using two-dimensional electrophoresis we identified 18 proteins that were 2-fold or more highly expressed in patients than in controls, and 11 proteins that were 2-fold or more highly expressed in controls than in patients. Some of these differentially expressed proteins, identified by MALDI-TOF spectrometry, have previously been shown to play a potential role in the pathogenesis of RA. Hierarchical cluster analyses of the data segregated the samples into two groups, one which contained only controls and the other which contained only patients, and was used to compare the expression pattern of these 29 proteins in individual samples with the median expression pattern determined in the healthy control and in the RA patient groups. This analyses was able to predict whether a sample was derived from a rheumatoid arthritis patient or from a healthy individual, suggesting that a comparison of such protein expression patterns may be of diagnostic value.     

Transcellular distribution heterogeneity of Annexin A5 represents a protective response to lupus-related thrombophilia: a pilot Proteomics-based study

Lupus-related vascular events are becoming a formidable obstacle to the improvement of long-term prognosis of systemic lupus erythematosus (SLE) and the existent findings lack for systematization. Proteomics is a strategic approach but its applications in this regard are rare and primarily involve proteome acquisition or biomarker screening, rather than functional identification. To provide further insight, we investigated the proteomic diversity of peripheral blood mononuclear cells (PBMCs) in SLE and the possible role of the identified Annexin A5 (AnxA5) in pathogenesis. The study involved 214 SLE and 183 healthy women. The two-dimensional electrophoresis gel images showed 649 ± 25 and 676 ± 19 protein spots from the PBMCs of the patients and controls, respectively. From these protein spots, 30 differentially expressed proteins were chosen, and 16 of these proteins were identified by mass spectrometer. Western blotting confirmed the over-expressed candidate, AnxA5, from the PBMCs of the patients (SLE:control=1.607:1, P=0.0004), but ELISAs indicated decreased levels of sera AnxA5 in the patients compared to healthy donors (SLE vs. control=26.8 ± 3.0 vs. 49.0 ± 3.3 ng/mL, P<0.0001). A positive correlation was demonstrated between the manifestation of thrombosis and AnxA5 (Mann-Whitney Z=-2.084, P=0.037), not anti-AnxA5, while searching for correlations between clinical parameters and the two molecular levels of patient sera. The coagulation assays using plasma from SLE patients revealed that elevated AnxA5 could shorten prothrombin time, activated partial thromboplastin time and prolonged thrombin time (P<0.001). Our data demonstrated the proteomic differences in the PBMCs between SLE patients and healthy persons. Moreover, the heterogeneous transcellular distribution, increased intracellular concentrations and decreased serum levels of AnxA5 represent a protective response to lupus-related thrombophilia; AnxA5 mostly participate in the common coagulation pathway in the thrombogenesis of SLE.     

Cyclosporin A inhibits the production of IL-17 by memory Th17 cells from healthy individuals and patients with rheumatoid arthritis

Recent evidence from several studies indicated that IL-17-producing Th17 cells can represent the key effector cells in the induction and development of autoimmune disorders. Cyclosporine A (CsA) is a commonly used immunosuppressant to treat lots of autoimmune diseases including rheumatoid arthritis (RA). Here, we demonstrated that PBMCs and purified CD4⁺ T cells from healthy individuals and patients with RA could be induced to produce large amounts of IL-17 after stimulation with anti-CD3 plus anti-CD28 mAbs. Phenotypic analysis indicated that the majority of IL-17-producing cells were Th17 cells with memory phenotype. The addition of CsA into cell cultures significantly inhibited the IL-17 production by Th17 cells at protein and at mRNA levels. Compared to the PBMCs from normal individuals, PBMCs from the patients with RA produced higher levels of IL-17 that was also significantly inhibited by CsA both at protein and at mRNA levels. The mechanism might be the effect of CsA on the T cells activation because the expression of CD69 and CD25 molecules on T cells was markedly reduced in the presence of CsA. Taken together, these results demonstrated that CsA suppressed the IL-17 production and inhibited the Th17 cells differentiation from both healthy individuals and patients with RA.     

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach

In order to identify target antigens of anti-endothelial cell (anti-EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2-DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 microg/mL. Targeted spots were identified by MS. The HUVEC proteome was composed of 884 protein spots. Among these, 61 +/- 25.8 (mean +/- SD) spots were recognized by serum IgG from healthy individuals, with marked differences from one individual to another. Among these spots, 11 were recognized by serum IgG from all healthy individuals tested. These spots corresponded to six different proteins with several spots corresponding to different isoforms of the same protein. Target antigens were: cytoskeletal proteins (beta-actin, alpha-tubulin, and vimentin); glycolytic enzymes (glucose-3-phosphate-deshydrogenase and alpha-enolase); and prolyl-4-hydroxylase beta subunit, a member of the disulfide isomerase family. This study shows that the repertoire of IgG AECA is heterogeneous among healthy individuals. IgG from all of the healthy individuals tested recognized a restricted set of highly conserved ubiquitous proteins playing key roles in cell biology and maintenance of homeostasis.     

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.     

Serum protein profile of rheumatoid arthritis treated with anti-TNF therapy (infliximab)

We analyzed the changes in the serum protein profile by infliximab using two-dimensional gel electrophoresis and mass spectrometry. More than 50 gel spots were seen to increase or decrease in correlation with clinical improvements of RA. The spots corresponding to CRP, C3, and Apo J showed reduced staining intensity, while the spots corresponding to Apo A-I, RBP, and transthyretin were enhanced. The protein profile of RA patients treated with infliximab was mostly similar to that of normal healthy controls except for several protein spots. This suggested that infliximab normalized the serum protein profile of RA patients, leading to modification in the serum lipid profile and antioxidant status in RA.     

Expression of VSTM1-v2 Is Increased in Peripheral Blood Mononuclear Cells from Patients with Rheumatoid Arthritis and Is Correlated with Disease Activity

Rheumatoid arthritis (RA) is a chronic, systematic autoimmune disease that mainly affects joints and bones. Although the precise etiology is still unknown, Th17 cell is being recognized as an important mediator in pathogenesis of RA. VSTM1-v2 is a novel cytokine which has recently been reported to promote the differentiation of Th17 cells. This study is performed to study whether VSTM1-v2 can be recognized as a biomarker of RA, and is correlated to IL-17 expression. We obtained peripheral blood mononuclear cells (PBMCs) from 40 patients with RA and 40 age- and sex- matched healthy controls by standard Ficoll-Paque Plus density centrifugation. The mRNA expression levels of VSTM1-v2 and IL-17A in PBMCs were detected by real time-PCR. Disease activity parameters of RA were measured by routine methods. Our results showed that VSTM1-v2 mRNA expression in PBMCs from RA patients was significantly increased in comparison of that in healthy individuals. The VSTM1-v2 mRNA expression level was positively correlated with IL-17A mRNA expression level, DAS28, CRP and ESR, but was not correlated to RF, Anti-CCP or ANA. VSTM1-v2 might be a biomarker of RA and a novel factor in the pathogenesis of RA.     

Interleukin-28A promotes IFN-γ production by peripheral blood mononuclear cells from patients with Behçet’s disease

Behçet’s disease (BD) is an autoimmune disease of unknown etiology. Interleukin-28A (IL-28A) promotes immune responses and may participate in the pathogenesis of autoimmune diseases. To examine the role of IL-28A in the pathogenesis of BD, we measured the expression of IFN-γ and IL-17 by IL-28A-stimulated peripheral blood mononuclear cells (PBMCs) from 19 patients with BD and 16 healthy individuals. We found that IFN-γ and IL-17 were undetectable in the sera from BD patients and control subjects. The mRNA expression and protein production of IFN-γ by IL-28A-stimulated PBMCs from BD patients were significantly increased compared to healthy individuals. No significant difference was observed in the mRNA expression and protein production of IL-17 by IL-28A-stimulated PBMCs between BD patients and normal individuals.     

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW ∼32 kDa and ∼62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the ∼62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.     

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.     

Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis

Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4⁺ and CD4^- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24⁺Vβ11⁺CD3⁺ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA.     

Structural studies on IgG oligosaccharides of patients with primary Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease, and some patients have been found to have SS complicated with rheumatoid arthritis (RA), in which IgG is known to carry abnormal N-linked oligosaccharides. In order to investigate the relationship between SS and RA, the structures of N-linked oligosaccharides of IgG from 12 primary SS patients without RA, 9 RA patients, and 8 healthy individuals were analyzed using reversed-phase high-performance liquid chromatography, in combination with sequential exoglycosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. All of the IgG samples obtained from primary SS patients, RA patients, and healthy individuals contained the same series of biantennary complex-type oligosaccharides, but the ratio of each oligosaccharide differed among these 3 groups. The incidence of galactose-lacking N-linked oligosaccharides obtained from the IgG of RA patients was significantly higher than that from healthy individuals, but that from the serum IgG of primary SS patients varied among individuals. The patients with primary SS were classified into two groups based on the galactosylation levels of IgG oligosaccharides; one group exhibits galactosylation levels as low as those of RA patients and another exhibits levels similar to those of healthy individuals. Measurement of levels of rheumatoid factor (RF) revealed that primary SS patients with a high incidence of RF belonged to the low galactosylation group, as did RA patients. These results suggest that appearance of IgG carrying abnormal N-linked oligosaccharides in primary SS may be related to future complication with RA.     

Proteomic analysis of gingival crevicular fluid for discovery of novel periodontal disease markers

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.     

The significant increase of FcγRIIIA (CD16), a sensitive marker, in patients with coronary heart disease

Our previous studies suggest that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is closely correlated to coronary heart disease (CHD). However, whether or not deregulated FcγRIIIA expression is involved in the development of CHD remains largely unclear. Herein, we investigated the FcγRIIIA mRNA expression in the leukocytes, the serum protein level of soluble CD16 (sCD16) and membrane CD16 on monocytes from 100 diagnosed CHD patients and 40 healthy individuals. Our results demonstrated that there was a significant increase of FcγRIIIA at the mRNA level in leukocytes, and at the protein level for both sCD16 in sera and membrane CD16 on monocytes from CHD patients compared to the healthy control. Similarly to the soluble CD14 (sCD14), the level of macrophage colony stimulating factor (M-CSF) in sera was also higher in CHD patients than that in the control individuals. Furthermore, the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), in sera and the mean fluorescent intensity of intercellular adhesion molecule 1 (ICAM-1, CD54) on CD14(+) CD16(+) monocytes were increased in CHD patients. Overall, these data demonstrated that FcγRIIIA (CD16) is involved in the pathogenesis of CHD by activating monocytes and stimulating inflammation. The significant increase of CD14(+) CD16(+) monocytes in CHD patients therefore suggested that the increase of the FcγRIIIA level might be a sensitive marker for the CHD diagnosis.     

肺鳞癌患者与健康人血清的差异蛋白质组学研究

为筛选肺鳞癌的血清标志物,采用二维凝胶电泳(2-DE)技术分离I期肺鳞癌患者和健康人的血清蛋白质,PDquest图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白,然后应用蛋白质印迹和免疫组化方法分别检测差异蛋白——结合珠蛋白-2(haptoglobin-2,HP-2)在肺鳞癌患者血清和健康人血清以及肺鳞癌组织和癌旁正常支气管上皮组织中的表达.建立了肺鳞癌患者和健康人血清的2-DE图谱,图像分析软件识别了1O个差异蛋白质点,质谱鉴定了4种差异蛋白;蛋白质印迹分析显示,HP-2在肺鳞癌血清中的表达水平显著高于健康人(P<0.05),但其表达水平与肺鳞癌的临床分期无明显相关性;免疫组化结果显示,HP-2在肺鳞癌组织中的表达水平高于癌旁正常支气管上皮组织(P<0.05).研究结果提示:HP-2是候选的肺鳞癌血清分子标志物,血清中HP-2水平对肺鳞癌诊断可能具有一定的参考价值;肺鳞癌组织中HP-2表达上调可能是患者血清中HP-2表达升高的原因之一.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

A Potential Role of Myeloid DAP12-Associating Lectin (MDL)-1 in the Regulation of Inflammation in Rheumatoid Arthritis Patients

The pathogenic roles of myeloid DAP12-associating lectin-1(MDL-1) and DAP12 in human rheumatoid arthritis (RA) remain unknown. Frequencies of MDL-1-expressing monocytes in 22 active RA patients, 16 inactive RA patients, 12 osteoarthritis (OA) patients and 10 healthy controls (HC) were determined by flow-cytometry analysis. The mRNA expression levels of MDL-1 and DAP12 on PBMCs were evaluated by quantitative PCR, and their protein expression levels in the synovium were examined by immunohistochemistry. Significantly higher median percentages of circulating MDL-1-expressing monocytes were observed in active RA patients (53.6%) compared to inactive RA patients (34.1%), OA patients (27.9%), and HC (21.2%). Levels of MDL-1 and DAP12 gene expression in PBMCs and their protein expression in the synovium were significantly higher in active RA patients than in inactive RA or OA patients. MDL-1 levels were positively correlated with parameters of disease activity, articular damage, and levels of proinflammatory cytokines. MDL-1 activator (Dengue virus type 2 antigen) stimulation on PBMCs resulted in significantly enhanced levels of proinflammatory cytokines in RA patients compared to those in OA patients or HC, indicating that MDL-1 activation is functional. Frequencies of MDL-1-expressing monocytes and levels of MDL-1 and DAP12 gene expression significantly decreased after effective therapy. Concordant overexpression of MDL-1 and DAP12 were correlated with increased production of proinflammatory cytokines in RA patients, suggesting their roles in regulating articular inflammation.