Tania Silvestri

Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis

OBJECTIVE To evaluate the sites of expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and inducible nitric oxide synthase (iNOS) in patients with inflammatory and degenerative joint diseases. METHODS Cytokines and iNOS were detected by immunohistochemistry analysis of synovial and cartilage biopsy specimens obtained at knee arthroscopy in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), osteoarthritis (OA), and traumatic knee arthritis. Cytokine and iNOS expression was quantified using computerized image analysis. RESULTS IL-1beta, TNFalpha, and iNOS were highly expressed by synovial cells (lining layer cells, infiltrating leukocytes, endothelial cells) from patients with inflammatory arthritides and significantly less by synovial cells from patients with OA and traumatic arthritis. In contrast, the latter patients showed high chondrocyte expression of cytokines and iNOS while RA and PsA patients had only minor chondrocyte positivity. In both joint compartments, IL-1beta expression, TNFalpha expression, and iNOS expression were strongly correlated. CONCLUSION The enhanced and coordinated expression of IL-1beta, TNFalpha, and iNOS by chondrocytes strongly supports the hypothesis that chondrocytes are the major site of production of mediators of inflammation in human OA, thus playing a primary role in the pathogenesis of this disease.

RANTES and MIP-1α production by T lymphocytes, monocytes and NK cells from nonagenarian subjects

While numerous previous studies have investigated age-related changes of cytokine production, little is known about chemokines, the importance of which in regulating immune response is becoming increasingly evident. In this study, a group of healthy subjects over 90 years old is compared to a group of young subjects, we evaluated the ability of monocytes, T lymphocytes and NK cells: (1) to produce RANTES and MIP-1alpha, either in basal conditions or after stimulation with, respectively, LPS, anti-CD3 MoAb and IL-2; (2) to express the corresponding chemokine receptors (CCR1, CCR3, CCR5). We demonstrate that: (a) monocytes, T lymphocytes and NK cells spontaneously produced detectable amounts of chemokines, both in young and old subjects; (b) monocyte-dependent RANTES and MIP-1alpha production induced by LPS was up-regulated in nonagenarian subjects as anti-CD3-induced secretion from T cells; (c) RANTES and MIP-1alpha production by IL-2 stimulated NK cells was reduced in elderly subjects; (d) CCR1, CCR3 and CCR5 were widely expressed on monocytes, but less expressed on T lymphocytes and NK cells. The diversity within PBMC might reflect their different states of activation and/or responsiveness, influencing the ability to develop rapid innate and long-lasting adaptive immune responses.

Soluble receptor activator of nuclear factor-κB ligand (sRANKL)/ osteoprotegerin balance in ageing and age-associated diseases

Recently, novel members of the TNF/TNF receptor superfamily, receptor activator of nuclear factor-κB ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin (OPG), have been identified as paracrine mediators of both the immune system and bone functions. The balance of RANK/RANK-L and OPG is critical for osteoclastogenesis modulation and physiological bone remodeling. In order to evaluate whether RANKL/OPG balance is modified by ageing, we analyzed, by imunoassay, systemic levels of OPG and sRANKL in healthy elderly subjects (age range from 70 to over 90 years) and in patients affected by two age-related diseases, osteoarthritis (OA) and polymyalgia rheumatica (PMR), characterized by bone metabolism alteration and involvement of the immune system. We demonstrated that (a) plasma concentrations of OPG increased significantly with age; (b) conversely, sRANKL significantly declined in the group of subjects aged between 81 and 90 years, being similar to the young controls in the other age groups; (c) in OA and PMR, circulating OPG did not differ from plasma levels found in age-matched control groups, while sRANKL concentration was significantly increased compared to controls. Hence, in ageing, the sRANKL/OPG system appears to be modified, with prominent changes in circulating OPG levels; in OA and PMR, the sRANKL/OPG balance alteration was shown to be mainly due to the increase of plasma sRANKL concentration.

VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION IN POLYMYALGIA RHEUMATICA

OBJECTIVE To evaluate peripheral production and synovial expression of vascular endothelial growth factor (VEGF) in polymyalgia rheumatica (PMR). METHODS Circulating levels of VEGF in PMR (serum concentration and in vitro release by peripheral blood mononuclear cells [PBMC]) were investigated by enzyme-linked immunosorbent assay. Local expression of VEGF in shoulder synovial tissue was investigated by immunohistochemical analysis. Investigations were performed in patients with active, untreated disease and in patients treated with corticosteroids. RESULTS VEGF serum concentrations were significantly higher in untreated PMR patients than in normal control subjects. During steroid treatment, VEGF serum concentrations reached their lowest level after the sixth month of treatment. PBMC isolated from untreated PMR patients spontaneously secreted a higher amount of VEGF compared with PBMC from control subjects. Corticosteroid therapy did not affect the ability of PBMC to produce VEGF. Immunohistochemical staining performed on shoulder synovial tissue showed VEGF expression in both the lining layer and the sublining area. In 3 of 4 treated patients, no VEGF staining was found in synovial tissue during corticosteroid therapy. VEGF expression correlated with vessel density, but was not associated with alphavbeta3 and alphavbeta5 integrin expression. CONCLUSION Peripheral and local VEGF releases have different responses to steroid treatment in PMR. The lack of response to corticosteroids by peripheral VEGF production supports the hypothesis that systemic involvement is dominant in PMR. At the synovial level, VEGF production is linked to vascular proliferation and is thus directly involved in the pathogenesis of synovitis.

In vivo expression of inflammatory cytokine receptors in the joint compartments of patients with arthritis

To test a hypothesis of compartmentalized pathogenesis of different types of arthritis, namely inflammatory arthritis (IA) and osteoarthritis (OA), synovial and cartilage biopsies were examined for the expression of TNF and IL-1 receptors. In cartilage, we found constitutive expression of all receptors in normal tissues, and decreased expression of signal-transducing receptors in pathological chondrocytes. In synovium, there was a lower expression of signal-transducing receptors in cases of OA compared to those of IA. In OA, the three signal-transducing receptors were more abundantly expressed in cartilage, while in IA they were mainly present in synovial tissue (TNFRp75 being expressed more than p55). IL-1 decoy receptor type II was low or absent in synovial tissues, but present in cartilage. The increased expression of TNFRp75 and IL-1RI in OA cartilage, compared to IA, in addition to the abundant local cytokine production, strengthens the hypothesis of autocrine/paracrine action by inflammatory cytokines in the pathogenesis of cartilage damage.

Serum Interleukin-6 Receptor in Polymyalgia Rheumatica : A Potential Marker of Relapse/Recurrence Risk

OBJECTIVE To investigate the modulation of systemic levels of soluble interleukin-6 receptor (sIL-6R) and soluble gp130 (sgp130) in untreated and treated polymyalgia rheumatica (PMR) patients during a followup period of at least 24 months in order to evaluate the relationship of these molecules with clinical outcome and their feasibility to provide a prognostic tool in clinical practice. METHODS We analyzed sIL-6R and sgp130 serum levels in 93 PMR patients, and 46 age-matched normal controls, at disease onset and at 1, 3, 6, 12, and 24 months of followup during corticosteroid therapy by enzyme-linked immunosorbent assay. RESULTS No difference in sIL-6R and sgp130 levels was observed between PMR patients and normal controls at disease onset or during followup. A significant correlation was found between the number of relapses and sIL-6R concentrations at baseline and after 1, 3, and 12 months of therapy. No correlation was found between sgp130 levels and the number of relapses. Cox multivariate analysis indicated that the best model for predicting relapses was identified by sIL-6R levels and the hemoglobin value at baseline. We found that high sIL-6R levels combined with low hemoglobin values resulted in a 10.1-fold increased risk of relapse. CONCLUSION Our data support the identification of a potential prognostic marker of PMR outcome that might have important implications in clinical practice. Because targeting sIL-6R with blocking antibodies has proven useful in other rheumatic disorders, our results could suggest the opportunity to evaluate sIL-6R-blocking treatment in patients with PMR and elevated levels of sIL-6R at disease onset.

358 ASSOCIATION OF THE INTERLEUKIN-4/INTERLEUKIN-4 RECEPTOR GENETIC VARIANTS WITH HAND OSTEOARTHRITIS

total variation consisted of Il-2, Il-6, G-CSF, Il-10 and Il-7, the second component explaining 15% of total variation consisted of MCP-1, MIP1b and Il-8, the third component, which explained 12% of total variation consisted of Il-7, Il-5 and CRP. CRP was represented negative in the third component, indicating an inverse relation with the cytokines in this component. Genetic analysis revealed that three SNPs in the SELS gene formed 4 common haplotypes, one of which, GAG (frequency 3.5%) showed significant association to the first component (P = 0.019) and to the third component (P = 0.036). Furthermore, OA subtype analysis showed that the second component (mainly representing chemokine variation) was significantly associated to hand ROA and discus degeneration (P=0.029 and P=0.010 respectively) as well as the physical component score (PCS) derived from the SF36 questionnaire (P=0.042). Further analysis of these associations showed that the association of hand ROA and the PCS are not independent. The CRP related component also showed a strong association to the PCS (P=0.007). The SELS haplotypes showed no association to OA subtypes in the GARP study. The QL analyzed cytokines showed no associations to either OA subtypes or the SELS genetic variation. Conclusions: Genetic variation in the SELS gene associates to two components, which were revealed by a principal component analysis of 17 cytokines and chemokines and CRP. The CRP containing component also showed association to the physical component score derived from the SF36 questionnaire. A third component was identified which mainly represents chemokine variation. This chemokine representing component showed association to hand OA and discus degeneration as well as the physical component score, indicating chemokines are involved in the etiology of OA.