Sphingosine-1-phosphate attenuates proteoglycan aggrecan expression via production of prostaglandin E(2 )from human articular chondrocytes

BACKGROUND: Sphingosine-1-phosphate (S1P), a downstream metabolite of ceramide, induces various bioactivities via two distinct pathways: as an intracellular second messenger or through receptor activation. The receptor for S1P (S1PR) is the family of Endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG). We have here attempted to reveal the expression of EDG/S1PR in human articular chondrocytes (HAC), exploring the implications of S1P in cartilage degradation. METHODS: Articular cartilage specimens were obtained from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or traumatic fracture (representing normal chondrocytes) who underwent joint surgery. Isolated HAC were cultured in vitro by monolayer and stimulated with S1P in the presence or absence of inhibitors of signaling molecules. Stimulated cells and culture supernatants were collected and subjected to analyses using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). RESULTS: All of the tested HAC samples showed positive results in terms of EDG/S1PR expression in basal condition. When HAC was stimulated with S1P, a significant increase in prostaglandin (PG) E(2 )production was observed together with enhanced expression of cyclooxygenase (COX)-2. S1P stimulated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in HAC, and the PGE(2 )induction was abrogated by PD98059 and SB203580. Pertussis toxin inhibited the PGE(2 )induction from HAC by S1P, suggesting an essential role for Gi protein. S1P also attenuated the expression of proteoglycan aggrecan, a component of cartilage matrix, in HAC at transcriptional level. CONCLUSION: It was suggested that the S1P-induced PGE(2 )was at least in part involved in the aggrecan-suppressing effect of S1P, seeing as COX inhibitors attenuated the effect. Accordingly, S1P might play an important role in cartilage degradation in arthritides

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

OBJECTIVE: To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions. METHODS: Mature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4. RESULTS: cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2) in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent. CONCLUSIONS: Regulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis

Effect of IL15 on T cell clonality in vitro and in the synovial fluid of patients with rheumatoid arthritis

OBJECTIVE—Recent studies have suggested that interleukin (IL) 15 induces T cell accumulation in synovial lesions of rheumatoid arthritis (RA). This study aimed at determining whether this cytokine could explain in vivo T cell clonality in RA.
METHODS—Peripheral blood mononuclear cells (PBMC) from patients with RA were stimulated in vitro with IL15 or IL2. After isolation of mRNA from stimulated cells and synovial T cells, genes coding the V-D(N)-J (CDR3) region of T cell receptor β chains were amplified by a reverse transcriptase polymerase chain reaction. A single strand conformation polymorphism analysis was used to detect the clonotype(s) of accumulating T cells. Nucleotide and amino acid sequencing was also performed.
RESULTS—Stimulation of PBMC with IL15 resulted in oligoclonal expansion of T cells. However, IL15 induced clones from PBMC were mostly different from the dominantly expanding T cell clones in synovial fluid. Furthermore, IL15 and IL2 responding clones were only partially identical.
CONCLUSIONS—Although IL15 results in clonal accumulation of T cells, T cell clonality in rheumatoid joints could not be explained by the effect of IL15 alone. The results indicated the requirement of other factor(s), in addition to IL15, in the pathological process affecting RA joints. The results also suggested different responses by each T cell clone to IL15 or IL2.


Anti-Scl-70 Antibodies in Autoimmune Hypothyroidism

The relationship between autoimmune thyroiditis and systemic sclerosis is controversial. Data exist on the presence of thyroid autoantibodies in patients with systemic sclerosis but, as far as we could ascertain, anti-Scl-70 antibodies, which are highly specific for systemic sclerosis, have not been investigated in autoimmune hypothyroidism. This study compares the presence of anti-Scl-70 in females with autoimmune hypothyroidism (n = 24) and in healthy age-matched female controls (n = 26). Free thyroxine levels were similar in both groups. Thyroid stimulating hormone (TSH), antithyroid peroxidase (anti-TPO), antithyroglobulin (anti-Tg) and index values for anti-Scl-70 levels were significantly higher in patients with autoimmune hypothyroidism compared with controls, although the anti-Scl-70 test was negative in both groups. Anti-TPO, anti-Tg and TSH significantly correlated with anti-Scl-70. In conclusion, autoimmune hypothyroidism seems to be associated with a higher index level of anti-Scl-70, yet a negative anti-Scl-70 antibody test. This suggests that autoimmune hypothyroidism might have common aetiological factors with systemic sclerosis

Characterisation of cartilage intermediate layer protein (CILP)-induced arthropathy in mice

Objectives: To characterise cartilage intermediate layer protein (CILP)-induced arthropathy in mice. Methods: The first and second halves of the nucleotide triphosphate pyrophosphohydrolase (NTPPHase) non-homologous region of human CILP were prepared as recombinant proteins (C1 and C2, respectively), including three overlapping fragments of C2 (C2F1, C2F2, and C2F3). C57BL/6 mice were immunised with these proteins to induce arthritis. In addition, a separate group of mice were immunised repeatedly with the mixture of C1 and C2 to see the effect of chronic immunisation. Arthritis developed in the mice, and cellular and humoral immune responses against CILP were analysed. Results: Immunisation with C2 and with the mixture C2F1/C2F2/C2F3 caused the severest arthritis to develop in mice. Immunisation with one of C1, C2F1, C2F2, or C2F3 caused milder arthritis, even though each of the fragments carried T cell epitopes. Immunisation either with C1 or C2 alone evoked cellular and humoral immune responses to both the C1 and C2 proteins. Further, the repeated immunisation with the C1/C2 mixture caused tendon calcification and bone irregularity, together with decreased NTPPH activity. Conclusions: The results show that multiple T cell epitopes are needed for the development of CILP-induced arthritis, and present the characteristic new model of mild arthropathy accompanied by extra-articular calcifications. An immune response to putative murine CILP/NTPPH may be involved in the ectopic calcifications in the arthritic mice