Cytokines and soluble receptors of the interleukin-1 family in Schnitzler syndrome.

OBJECTIVES: Schnitzler syndrome (SchS) is an autoinflammatory disorder characterized by chronic urticaria, fever, and monoclonal gammopathy. The success of interleukin-1 (IL-1) blocking therapies suggests a crucial role for IL-1 in disease induction. The aim of this study is to perform a comprehensive analysis of IL-1 family cytokines and soluble receptors in a group of SchS patients. METHOD: Three patients fulfilling the criteria for the diagnosis of SchS were recruited; 80 blood donors formed the control group. IL-1 family cytokines (IL-1α, IL-1ß, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, sIL-1R4), and antagonists [IL-1Ra, IL-18 binding protein (IL-18BP)] were measured by a multiarray enzyme-linked immunosorbent assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Cytokine levels were compared by the Mann-Whitney test. RESULTS: IL-18 and free IL-18 were increased in patients compared with controls (p = 0.005 and p = 0.0082, respectively), while IL-18BP levels were not different. IL-1α, IL-1ß, and IL-33 were undetectable in both patients and controls. The soluble receptors sIL-1R1, sIL-1R2, and ST2/sIL-1R4, and the IL-1 antagonist IL-1Ra were all within normal ranges; sIL-1R3 was significantly lower in patients than in controls (p = 0.039). CONCLUSIONS: The data indicate that SchS is characterized by increased circulating levels of free IL-18, possibly leading to a higher activation of innate/inflammatory effector cells. At variance with other inflammatory diseases, the lack of increase in sIL-1R1 and sIL-1R2 and the decreased levels of sIL-R3 imply a failure in the counterbalancing mechanism aimed at inhibiting excessive IL-1ß in tissues.

Association between anti-citrullinated alpha enolase antibodies and clinical features in a cohort of patients with rheumatoid arthritis: a pilot study.

In recent years several antibodies against citrullinated peptides (ACPAs) have been identified in patients with rheumatoid arthritis (RA) and their pathogenic, diagnostic and prognostic significance is under intense investigation. Among ACPAs, those targeting citrullinated alpha enolase (anti-CEP1) have been identified in RA but data about their ability to predict the development of erosive disease are conflicting. Furthermore, no data are currently available concerning their possible association with extra-articular manifestations (EAMs) in RA. The aim of this study was to investigate the prevalence and significance of anti-CEP1 from a prognostic point of view. In this pilot study we confirmed that anti-CEP1 Abs are associated with higher prevalence of bone erosions, but we also provided the first evidence of an association between anti-CEP1 Abs and RA interstitial lung disease (ILD). These results provide the basis to investigate the association between anti-CEP1 Abs and EAMs in larger cohorts of RA patients to possibly confirm its role as biomarker for RA-ILD.

Fingerprinting of anti-citrullinated protein antibodies (ACPA): specificity, isotypes and subclasses.

Anti-citrullinated protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific autoantibodies that recognize the amino acid citrulline, resulting from the post-translational modification of arginine. Peptidyl arginine deiminase, the enzyme responsible for citrullination, is present in humans in different isoforms with different tissue distribution, enzymatic activity and target specificity; nonetheless, the number of proteins citrullinated in physiological or pathological conditions is wide, but not every citrullinated protein is a target for antibodies. In pre-RA patients the immune response to citrullinated antigens is initially restricted, expands with time and, after the onset of the disease, is relatively stable. ACPA are heterogeneous in terms of not only fine specificity but also isotype and IgG subclasses usage. This heterogeneity may be relevant for the immunopathogenesis of RA, conditioning the interaction of antibodies with complement and Fc receptors.

Two Takayasu arteritis patients successfully treated with rituximab.

Takayasu arteritis (TA) is a rare form of chronic large vessel vasculitis of unknown origin involving the aorta and its major branches. Recently, the involvement of B lymphocytes in TA has been suggested, and active refractory TA patients were successfully treated with B cell depletion therapy (BCDT). We report two cases of patients with TA successfully treated with anti-CD20 monoclonal antibody (rituximab). The favorable outcome of rituximab treatment in our patients also support the view that BCDT can be a useful option for refractory TA, and its potential should be evaluated in controlled trials.

Free IL-18 and IL-33 cytokines in chronic spontaneous urticaria.

Overproduction of IL-18 has been described in chronic urticaria. To evaluate free IL-18 and IL-33 in chronic spontaneous urticaria (CSU). IL-18, its inhibitor IL-18BP, IL-33 and its soluble receptor ST2 (sST2) were measured (ELISA) in the sera of 73 CSU patients. Free IL-18 was calculated (law of mass action). Autologous serum skin test (ASST) was performed in all patients. Total IL-18, IL-18BP and free IL-18 serum levels were significantly higher in CSU than in controls. IL-18 and IL-18BP increased significantly in both ASST-positive and negative subgroups. Free IL-18 resulted significantly higher in the ASST-negative, but not in the ASST-positive subgroup. No differences in IL-33/sST2 levels were detected between CSU and controls. Increased levels of free IL-18 and IL-18BP, but not IL-33, was detected in CSU. Whether IL-18 up-regulation is a consequence of inflammation or one of the causes of the pathology needs to be addressed.

Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases.

OBJECTIVES: A defined role for natural killer (NK) cells and their activating receptors in autoimmunity has not been clearly established. The aim of this study was to evaluate the levels of the CD3-CD56+ NK cells and their expression of receptors and co-receptors in the peripheral blood of patients with systemic autoimmune disorders. METHODS: Thirty-four subjects with systemic sclerosis (SSc), 14 with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), 14 with systemic lupus erythematosus (SLE), and 14 healthy donors were studied. The activating receptors NKp46, NKp44, NKp30, NKG2D, and DNAM-1 and the co-receptors NTB-A and 2B4 were analysed by flow cytometry on peripheral blood NK cells. RESULTS: In SSc, AAV, and SLE we detected a significant decrease in the percentage of CD3-CD56+ NK cells compared to healthy controls. No differences in the expression of NKp46, NKp44, and NKp30 were identified. On the contrary, NKG2D and DNAM-1 expression was decreased in SLE, but not in SSc and AAV, NTB-A was decreased in SLE, and 2B4 in both SLE and SSc. No differences were detected between active and inactive SLE patients. In SSc, only patients affected by pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) had a low expression of DNAM-1, 2B4, and NKp30. CONCLUSIONS: These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3-CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti-cyclic citrullinated peptide (CCP) and anti-VCP1 antibodies.

Anti-citrullinated protein/peptide antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and can be measured using different citrullinated substrates. In this paper we describe a new viral citrullinated peptide - VCP2 - derived from the Epstein-Barr virus-encoded protein EBNA-2 and analyse its potential as substrate for ACPA detection. Analysing sera from 100 RA patients and 306 controls, anti-VCP2 immunoglobulin (Ig)G were found in 66% of RA sera, IgM in 46% and IgA in 39%, compared with less than 3% of control sera. Anti-VCP2 IgG was associated with erosive arthritis, the presence of rheumatoid factor and anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Anti-VCP2 antibodies were detected in 1% and anti-VCP1 antibodies in 4% of CCP-negative RA sera; conversely, 3% of the VCP-negative sera were CCP-positive. Taken together, these data suggest that VCP2 could offer a valuable tool for ACPA detection. Inhibition assays showed that two non-overlapping epitopes - a citrulline-glycine stretch shared between VCP1 and VCP2 and the N-terminal portion of the VCP2 sequence - were targeted by anti-VCP2 antibodies. Moreover, in some RA sera that tested positive in CCP and VCP2 assays, preincubation with VCP2 inhibited binding to CCP, whereas in other sera the binding was unaffected. Thus, the reactivity with more than one ACPA substrate might be due in some RA patients to antibody populations with different specificities, and in others to cross-reactive antibody populations. Finally, affinity-purified anti-VCP2 antibodies immunoprecipitated deiminated Epstein-Barr virus nuclear antigen (EBNA-2) from an EBNA-2-transfected cell line, suggesting that viral sequences may be involved in the generation of the ACPA response.

Osteopontin is expressed and functional in human eosinophils.

BACKGROUND: Eosinophils are critically involved in allergic inflammation and tissue remodeling. Osteopontin (OPN) is a glycoprotein molecule which exhibits pro-fibrogenic and pro-angiogenic properties and has recently also been implicated in allergic diseases. In this study, we investigated the expression and function of OPN in human eosinophils. METHODS: Osteopontin mRNA (RT-PCR) and protein (immunofluorescence) expression in peripheral blood eosinophils from atopic human subjects were evaluated. Soluble OPN release was determined in resting and activated eosinophils. The contribution of OPN to eosinophil-induced angiogenesis was determined using the chick embryo chorio- allantoic membrane (CAM) assay and OPN-induced eosinophil chemotaxis was determined (ChemoTx System microplate wells). Finally, OPN expression in bronchoalveolar lavage (BAL) fluids from mild asthmatic and normal control subjects was determined. RESULTS: Osteopontin is expressed in human eosinophils and is increased following GM-CSF and IL-5 activation. Eosinophil-derived OPN contributes to eosinophil-induced angiogenesis. Recombinant OPN promotes eosinophil chemotaxis in vitro and this effect is mediated by alpha(4)beta(1) integrin binding. Soluble OPN is increased in the bronchoalveolar lavage fluid from mild asthmatic subjects and correlates with eosinophil counts. CONCLUSIONS: We therefore conclude that OPN is likely to contribute to the process of angiogenesis observed in the airways in asthma.

The role of eosinophil major basic protein in angiogenesis.

BACKGROUND: Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. METHODS: Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. RESULTS: Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. CONCLUSIONS: These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

Angiogenesis in asthma.

Asthma is a chronic inflammatory disease of the airways characterized by infiltration and activation of inflammatory cells and by structural changes, including subepithelial fibrosis, smooth muscle cells hypertrophy/hyperplasia, epithelial cell metaplasia and angiogenesis. These structural changes are thought to correlate with asthma severity and to account for the development of progressive lung function deterioration. The mechanism underlying airway angiogenesis in asthma and its precise clinical relevance have not yet been completely elucidated. This review provides recent data showing the contribution of allergic inflammation in increased airway vascularity and potential therapeutical approaches in asthma treatment by acting on bronchial microvascular changes.

Enhanced osteopontin expression in a murine model of allergen-induced airway remodelling.

BACKGROUND: Airway remodelling is a central pathophysiological feature of chronic asthma. A wide variety of cytokines and growth factors are likely to be involved in the development of airway remodelling. Osteopontin (OPN) is a cytokine with pro-fibrotic properties; however, its role in airway remodelling in asthma has not been explored. OBJECTIVE: To determine the expression and cellular sources of OPN in a murine model of chronic allergen-induced airway remodelling. METHODS: BALB/c mice were sensitized and exposed to ovalbumin (OVA) or saline inhalations for 5 weeks and killed 24 h after the last inhalation. The following parameters of inflammation and remodelling were assessed: differential cell counts in bronchoalveolar lavage (BAL) fluid lung collagen content (colorimetric biochemical assay) and peribronchial smooth muscle content (immunohistochemistry, followed by image analysis). OPN expression in BAL and lung tissue was determined by PCR and ELISA. The cellular source and distribution of OPN were evaluated by immunohistochemistry and immunofluorescence. RESULTS: OPN expression is up-regulated in lung tissue and in BAL fluid of OVA-treated mice and correlates with collagen content and peribronchial smooth muscle area. In addition, OPN significantly increases collagen deposition in vitro in a murine lung cell line. Cells producing OPN include the airway epithelium and cells of the submucosal inflammatory infiltrate (T cells, eosinophils, and macrophages). Positive staining for OPN was also observed in bronchial tissue from human asthmatic subjects. CONCLUSION: OPN expression in the lungs is increased in a murine model of allergen-induced chronic airway remodelling, suggesting a role for this cytokine in airway remodelling in asthma.

The pro-fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor-beta.

BACKGROUND: Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring. OBJECTIVE: The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts. METHODS: Expression of NGF, trkA(NGFR) and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (alpha-SMA) (cell surface ELISA) and contraction of 3D collagen gels. RESULTS: NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkA(NGF) and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing alpha-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-beta1 (TGF-beta1) production by fibroblasts, and following specific TGF-beta neutralization, all the NGF-induced effects were completely abrogated. CONCLUSION: Our findings indicate that NGF, via TGF-beta induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.

Mast cells in allergy and beyond.

Mast cells (MC) are highly granulated tissue dwelling cells, widely distributed throughout the body in connective tissues and on mucosal surfaces. They are derived from bone marrow progenitors that migrate into the blood and subsequently into the tissues, where they undergo final maturation. Mast cell proliferation, differentiation, survival and activation are regulated by stem cell factor, the ligand for the c-kit tyrosine kinase receptor, expressed on the mast cell surface. They release a large number of pro-inflammatory and immunoregulatory mediators after activation induced by either immunoglobulin E-dependent or immunoglobulin E-independent mechanisms. Mast cells have been most widely studied in the context of allergic reactions and parasite infections, but there is now compelling evidences that they are important players in innate and acquired immunity, wound healing, fibrosis, tumors and autoimmune diseases. This review will discuss current advances in these fields.