Lia Pulsatelli

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.

Tocilizumab: a novel therapy for patients with large-vessel vasculitis

OBJECTIVE Treatment of large-vessel vasculitis (LVV) remains challenging. Patients usually respond to glucocorticoid (GC) therapy, but often relapse on tapering of the GC dose or after GC withdrawal. In addition, GCs are fraught with numerous adverse events. The aim of this study was to assess the efficacy and safety of the anti-IL-6 receptor (IL-6R) antibody tocilizumab (TCZ) in patients with LVV. METHODS Four patients with active LVV (two with GCA and two with Takayasu arteritis) received monthly TCZ infusions (8 mg/kg bodyweight) for 6 consecutive months. Two patients were treatment naïve, while two had relapsing disease. Disease activity and drug tolerability were assessed clinically and by laboratory tests at study entry and subsequently every month for 6 months of TCZ treatment, while an [(18)F]fluorodeoxyglucose PET (PET/CT) scan was performed before and after treatment. In addition, a semi-quantitative clinical evaluation was performed at baseline and at 3 and 6 months using the Indian Takayasu activity score and the Kerr indices. After TCZ, MTX was used as maintenance therapy. RESULTS All patients treated with TCZ therapy had a satisfactory clinical and laboratory response, while PET/CT findings significantly improved in all cases. No serious adverse events were noted. Only one patient had a transient increase in liver enzymes. CONCLUSIONS In this small group of patients with LVV, treatment with TCZ was effective and well tolerated. Further, larger studies are required to confirm our findings.

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.

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.

Different IL-8 production by T and NK lymphocytes in elderly subjects.

A gradual decline in the functional activity of the immune system is described with advancing age. The adaptive immune system seems the most severely affected, but some age-associated modifications also occurs in NK cells. Several studies investigated the age related changes of cytokine production, while little is known about chemokines, whose importance in regulating immune-response becomes even more evident. In this study we investigated whether the ability of T lymphocytes and NK cells to produce IL-8, either spontaneously or after activation, respectively with anti-CD3 monoclonal antibody or interleukin 2 (IL-2) was affected by age. We demonstrated that: (a) T lymphocytes and NK cells spontaneously produced detectable amounts of IL-8; (b) anti-CD3 stimulation of T lymphocytes significantly increased IL-8 production and the increment was more evident in the nonagenarian subjects; (c) similarly, IL-2 stimulation of NK cells rose the production of IL-8 but the amount produced by the old was lower than the one produced by the young group. Because of the co-stimulatory role of chemokines on NK responses and given the demonstrated importance of NK cells in defence against viral infections, the decreased production of IL-8 can be involved in the defective functional activity of NK cells from old subjects.

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.

Signaling Pathways in Cartilage Repair

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair.

Does Platelet-Rich Plasma Freeze-Thawing Influence Growth Factor Release and Their Effects on Chondrocytes and Synoviocytes?

PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1β and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

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.