Riccardo Meliconi

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.

Endothelin-1 in idiopathic pulmonary fibrosis.

AIMS--To evaluate whether endothelin-1 is involved in the pathology of idiopathic pulmonary fibrosis (IPF). METHODS--Plasma endothelin-1 concentrations were evaluated in 37 patients with IPF and 27 normal controls by radioimmunoassay. In addition, expression of endothelin-1 in lung tissue was evaluated in biopsy specimens obtained from four patients with IPF. Three biopsy specimens of normal lung were used as controls. Endothelin-1 immunoreactivity was detected using immunohistochemistry. RESULTS--Elevated endothelin-1 plasma concentrations were found in patients with IPF compared with controls and a positive correlation was found with duration of disease. No significant difference was observed between treated and untreated patients with IPF. Increased endothelin-1 immunoreactivity was found in lungs of three of four patients with IPF. Endothelin-1 positive consisted mainly of small vessel endothelial cells. Some scattered macrophages were also positive. CONCLUSIONS--Elevated plasma concentrations and expression of endothelin-1 in lung tissue are suggestive of increased production of endothelin-1 in at least a proportion of patients with IPF. Consequently, endothelin-1 activity could play a role in the fibrogenic process of the disease.

New findings in osteoarthritis pathogenesis: therapeutic implications

This review focuses on the new perspectives which can provide insight into the crucial pathways that drive cartilage-bone physiopathology. In particular, we discuss the critical signaling and effector molecules that can activate cellular and molecular processes in both cartilage and bone cells and which may be relevant in cross talk among joint compartments: growth factors (bone morphogenetic proteins and transforming growth factor), hypoxia-related factors, cell–matrix interactions [discoidin domain receptor 2 (DDR2) and syndecan 4], signaling molecules [WNT, Hedgehog (Hh)]. With the continuous progression of our knowledge on the molecular pathways involved in cartilage and bone changes in osteoarthritis (OA), an increasing number of potentially effective candidates for OA therapy are already under scrutiny in clinical trials to ascertain their possible safe use in an attempt to identify molecules active in slowing or halting OA progression and reducing joint pain. We then review the principal molecules currently under clinical investigation.

Clinical associations in patients with hand osteoarthritis

Objectives: To investigate the clinical associations of hand osteoarthritis (HOA) and their relationships with radiographic features. Methods: A total of 446 patients with hand osteoarthritis (HOA; 233 with erosive HOA (EHOA) and 213 with non-EHOA) and 307 controls were evaluated. Demographic and clinical data from patients and controls were recorded based on medical records/clinical reports and an anamnesis of drug consumption. Posteroanterior radiographs of both hands were obtained from all HOA patients and were assessed using the Kellgren and Lawrence (K&L) and Kallman scoring systems. Results: After adjustment for age, gender, and body mass index (BMI), HOA patients showed a significantly increased odds ratio (OR) for hypercholesterolaemia [OR 2.10, 95% confidence interval (CI) 1.39–3.16, p < 0.0005] and autoimmune thyroiditis (OR 4.85, 95% CI 1.77–13.29, p = 0.002), as well as for knee (OR 1.63, 95% CI 1.09–2.44, p = 0.018) and hip OA (OR 1.87, 95% CI 1.07–3.27, p = 0.029). No significant increase for systemic hypertension, ischaemic heart disease, and diabetes mellitus was found. Patients with EHOA and non-EHOA showed similar risks for the above-mentioned co-morbidities. A similar occurrence of clinical associations was also observed in patients with HOA alone and in those with generalized OA. No association between radiographic scores and clinical associations was observed. Conclusions: Patients with HOA present a direct association with hypercholesterolaemia (and autoimmune thyroiditis) but do not show increased ischaemic cardiovascular manifestations compared to controls. No significant association between radiographic scores and co-morbidities was found.

Mapping of topoisomerase II α epitopes recognized by autoantibodies in idiopathic pulmonary fibrosis

Autoantibodies against DNA topoisomerase II α have been identified in the sera of patients with idiopathic pulmonary fibrosis (IPF). To map topoisomerase II autoepitopes, we tested by ELISA and immunoblotting the IPF anti‐topoisomerase II‐positive sera against a series of recombinant proteins which covered the full length of topoisomerase II α. Specific patterns of reactivity were observed, indicating the existence of multiple epitopes on topoisomerase II, either highly complex or conformational/discontiguous or conformational/contiguous ones. The latter resided in amino acid residues 854–1147 and 1370–1447. A detailed analysis of these regions was undertaken, but we were not able to pinpoint a sequential peptide‐sized epitope, or any significant homology with foreign pathogens. Further, we observed a significant correlation between the progression from a contiguous to a quaternary/tertiary structure‐dependent autoepitope and the disease duration but not with the disease severity. Therefore, this result supports the hypothesis that anti‐topoisomerase II autoreactivity evolves following an antigen‐driven process.

Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1β-stimulated production of chemokines and matrix-degrading enzymes in vitro.

Background In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. Methodology/Principal Findings The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. Conclusions/Significance Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

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.

Clinical and radiographic distribution of structural damage in erosive and nonerosive hand osteoarthritis.

To characterize the clinical and radiographic joint phenotype in erosive hand osteoarthritis (EHOA) and non‐EHOA.

Serum markers of immune activation and liver allograft rejection

We monitored the immune response after liver transplantation in 20 patients by measuring the serum levels of soluble interleukin-2 receptor (sIL-2R), soluble CD8 (sCD8), serum amyloid-A protein (SAA), and tumor necrosis factor-α (TNF-α). In six patients data were available to extend the follow-up period to one year. In all patients mean sIL-2R levels increased in the first month after liver transplantation, and subsequently decreased to values similar to pre-OLT ones, while SAA mean levels rose in the first week after OLT only in patients with rejection. sCD8 levels did not significantly rise after OLT, and TNF-α was undetectable in most cases. During episodes of rejection, rejector patients had significantly higher levels of sIL-2R, sCD8, and SAA than stable (without complications) patients. Conversely, no significant difference between rejectors and patients with other complications existed for any of the markers studied. These results diminish the importance of these serum markers of immune activation as laboratory tools in the differential diagnosis of acute rejection from other complications. However, sIL-2R, SAA, and sCD8 levels correlated with the histological grade of rejection and therefore can be utilized to monitor patients with an established diagnosis of acute rejection.