Mesenchymal stem cell-conditioned medium reduces disease severity and immune responses in inflammatory arthritis

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis

Neutralization of IL-17 ameliorates uveitis but damages photoreceptors in a murine model of spondyloarthritis

INTRODUCTION: Uveitis, or intraocular inflammatory disease, is a frequent extra-articular manifestation of several forms of arthritis. Despite the frequent co-occurrence of uveitis and arthritis, little is understood of the eye's predisposition to this disease. We recently described a previously unreported uveitis in a murine model of spondyloarthropathy triggered by autoimmunity to aggrecan, a prominent proteoglycan (PG) macromolecule in cartilage. In contrast to the joint and spine, wherein interferon-gamma (IFNγ) deficiency reduced disease, IFNγ deficiency worsened uveitis. Given the regulatory role of IFNγ on the Th17 response and the current focus of anti-interleukin-17 therapeutics in patients with uveitis and spondyloarthritis, we sought to determine the extent to which interleukin (IL)-17 mediates uveitis in the absence of IFNγ. METHODS: Antigen specific T cell cytokine production was measured in splenocyte cultures using multiplex-ELISA. Transgenic (Tg) mice expressing the T cell receptor (TCR) recognizing the dominant arthritogenic epitope in the G1 domain of PG (TCR-Tg), also lacking IFNγ, were immunized with PG. Mice were then systemically administered an anti-IL-17 neutralizing antibody. The onset and severity of peripheral arthritis was evaluated by clinical scoring criteria and histology. Uveitis was assessed using intravital videomicroscopy, which visualizes leukocyte trafficking within the vasculature and tissue of the iris, and by histology. RESULTS: TCR-Tg splenocytes stimulated in vitro with recombinant G1 peptide demonstrated exacerbated production of cytokines, such as macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-1β, and most notably IL-17A as a consequence of IFNγ deficiency. In vivo, IL-17 inhibition prevented the component of PG-induced arthritis that occurs independently of IFNγ. Blockade of IL-17 ameliorated the ongoing leukocyte trafficking responses within the iris vasculature and tissue, which coincided with reduced infiltration of leukocytes within the anterior and posterior eye segments. However, the anti-IL-17 treatment resulted in unanticipated photoreceptor toxicity. CONCLUSIONS: These data support a protective, regulatory role for IFNγ in suppression of IL-17-mediated intraocular disease and to a lesser extent, joint disease. The unanticipated photoreceptor toxicity raises some caution regarding the use of anti-IL-17 therapeutics until the mechanism of this potential effect is determined

Molecular manipulation with the arthritogenic epitopes of the G1 domain of human cartilage proteoglycan aggrecan

Systemic immunization of BALB/c mice with human cartilage proteoglycan (PG) aggrecan induces progressive polyarthritis. The G1 domain of the PG aggrecan molecule contains most of the T cell epitopes, including three immunodominant (‘arthritogenic’) and at least six subdominant T cell epitopes. The three dominant T cell epitopes (P49, P70 and P155) were deleted individually or in combination by site directed mutagenesis, and the recombinant human G1 (rhG1) domain (wild type and mutated) proteins were used for immunization. Close to 100% of BALB/c mice immunized with the wild-type (nonmutated) rhG1 domain developed severe arthritis, which was 75% in the absence of P70 (5/4E 8) epitope, and very low (< 10% incidence) when all three dominant T cell epitopes were deleted. The onset was delayed and the severity of arthritis reduced in animals when dominant T cell epitopes were missing from the immunizing rhG1 domain. The lack of T cell response to the deleted epitope(s) was specific, but the overall immune response against the wild-type rhG1 domain of human PG was not significantly affected. This study helped us to understand the dynamics and immune-regulatory mechanisms of arthritis, and supported the hypothesis that the development of autoimmune arthritis requires a concerted T cell response to multiple epitopes, rather than the immune response to a single arthritogenic structure

Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes

Tumor necrosis factor stimulated gene-6 (TSG-6) has been previously shown to be induced in vitro in several cell types by proinflammatory cytokines, and in vivo in pathological conditions such as rheumatoid arthritis. In this study, we report the complete coding sequence for the mouse TSG-6 protein, and the exon-intron structure and the chromosomal localization of the gene. We have identified a 1605 nt cDNA. sequence from mouse cumulus cell-oocyte complexes (COCs) induced to expand in vivo. The sequence contains an open reading frame of 825 nt that codes for the 275 amino acid TSG-6 protein. The gene contains six exons separated by 1.1-5.8 kb introns and has been localized to the murine chromosome 2 by linkage analysis. Comparative reverse transcription-polymerase chain reaction studies have revealed that TSG-6 mRNA is specifically expressed after COC expansion induced in vivo, identifying the first non-pathological process in which TSG-6 may play an important role. Since TSG-6 binds to hyaluronan and interacts with inter-alpha-trypsin inhibitor (I alpha I), molecules that are essential for matrix formation by COCs, this protein may have a structural role in the matrix or may enhance the antiproteolytic effect of I alpha I to protect the matrix from degradation. (C) 1997 Elsevier Science B.V

Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes

Tumor necrosis factor stimulated gene-6 (TSG-6) has been previously shown to be induced in vitro in several cell types by proinflammatory cytokines, and in vivo in pathological conditions such as rheumatoid arthritis. In this study, we report the complete coding sequence for the mouse TSG-6 protein, and the exon-intron structure and the chromosomal localization of the gene. We have identified a 1605 nt cDNA. sequence from mouse cumulus cell-oocyte complexes (COCs) induced to expand in vivo. The sequence contains an open reading frame of 825 nt that codes for the 275 amino acid TSG-6 protein. The gene contains six exons separated by 1.1-5.8 kb introns and has been localized to the murine chromosome 2 by linkage analysis. Comparative reverse transcription-polymerase chain reaction studies have revealed that TSG-6 mRNA is specifically expressed after COC expansion induced in vivo, identifying the first non-pathological process in which TSG-6 may play an important role. Since TSG-6 binds to hyaluronan and interacts with inter-alpha-trypsin inhibitor (I alpha I), molecules that are essential for matrix formation by COCs, this protein may have a structural role in the matrix or may enhance the antiproteolytic effect of I alpha I to protect the matrix from degradation. (C) 1997 Elsevier Science B.V