Lilia Marinova-Mutafchieva

Dynamics of proinflammatory cytokine expression in the joints of mice with collagen‐induced arthritis (CIA)

This report contains a description of the cellular localization and kinetics of proinflammatory cytokine expression in murine CIA, a model for rheumatoid arthritis. Tissue cryostat sections of undecalcified paws from type II collagen‐immunized DBA/1 mice, taken 1–10 days after the onset of clinical arthritis, were examined for the presence of tumour necrosis factor‐alpha (TNF‐α), IL‐1β and IL‐6 using an indirect immunoperoxidase technique. In parallel, interferon‐gamma (IFN‐γ) production by lymph node cells, stimulated in vitro with type II collagen, was assessed as a marker of T cell activity. The main areas of TNF‐α, IL‐1β and IL‐6 expression were in the synovial lining layer and in tissue contiguous with cartilage and bone (the marginal zone), in particular at sites of pannus formation and joint erosion. There was a progressive increase in the number of TNF‐α‐, IL‐1β‐ and IL‐6‐positive cells from day 1 to day 10 of arthritis, during which time IFN‐γ production by CD4+ T cells from draining lymph nodes declined sharply. A further finding of potential significance was that TNF‐α was consistently detected at day 1 of arthritis, whereas IL‐1β‐positive cells were not found until day 3, suggesting that the expression of TNF‐α precedes that of IL‐1β.

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6‐hydroxydopamine model of Parkinson’s disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson’s disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6‐hydroxydopamine (6‐OHDA) model of Parkinson’s disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6‐OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen‐cluster of differentiation 11b – anti‐complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage‐myeloid associated antigen immunoreactivity was observed at day 3 post‐lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage‐myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.

Evaluation of TNF-alpha and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-alpha/anti-CD4 therapy.

We have evaluated the effects of anti-TNF-α, anti-IL-1, and combined anti-TNF-α/anti-CD4 therapy in collagen-induced arthritis. Blockade of TNF-α or IL-1 before disease onset delayed, but did not prevent, the induction of arthritis. When treatment was initiated after onset of arthritis, anti-TNF-α, anti-IL-1β, and anti-IL-1R (which blocks IL-1α and IL-1β) were all found to be effective in reducing the severity of arthritis, with anti-IL-1R and anti-IL-1β showing greater efficacy than anti-TNF-α. Anti-IL-1β was equally as effective as anti-IL-1R, indicating that IL-1β plays a more prominent role than IL-1α in collagen-induced arthritis. An additive effect was observed between anti-TNF-α and anti-IL-1R in the prevention of joint erosion and in normalization of the levels of serum amyloid P. Combined anti-TNF-α/anti-CD4 therapy also caused normalization of serum amyloid P levels. The therapeutic effect of anti-TNF-α plus anti-CD4 was comparable to that of anti-TNF-α plus anti-IL-1R, suggesting that combined anti-TNF-α/anti-CD4 therapy prevents both TNF-α- and IL-1-mediated pathology. Anti-TNF-α treatment reduced IL-1β expression in the joint and, conversely, anti-IL-1β treatment reduced TNF-α expression. Combined anti-TNF-α/anti-CD4 treatment almost completely blocked the expression of IL-1β, thereby confirming the ability of this form of combination therapy to prevent IL-1β-mediated pathology.

Mesenchymal cells expressing bone morphogenetic protein receptors are present in the rheumatoid arthritis joint.

OBJECTIVE To evaluate the presence of cells of an early mesenchymal lineage, as judged by the expression of bone morphogenetic protein receptors (BMPRs), in the joints of normal individuals and patients with rheumatoid arthritis (RA). METHODS Synovial fluids, single cell suspensions of cultured fibroblast-like synoviocytes (FLS), and synovial tissues were examined by immunohistology with antibodies to BMPR type IA (BMPRIA), BMPRIB, and BMPRII and then quantified using computerized image analysis. Other antibodies were evaluated by cytofluorography. RESULTS In primary cultures of joint effusions from patients with RA and other forms of inflammatory arthritis, there were large adherent cells with the appearance of either fibroblasts or stromal cells that stained with antibodies to mesenchymal elements-CD44, type I collagen, alpha-actin, and vimentin-but not with antibodies to hematopoietic markers. These cells proliferated rapidly, expressed BMPRIA and BMPRII, and soon became the predominant cells in culture. They were retained through multiple passages and persistently displayed surface vascular cell adhesion molecule 1. Immunohistochemical analysis of cultured RA FLS (passages 3, 4, and 6; n = 6) revealed that 11.6% were BMPR-positive, while only 2.0% of osteoarthritis FLS (passage 4; n = 3) were BMPR-positive, and 1 normal synovial culture had no BMPR-positive cells. In all RA synovial membranes examined (n = 9), BMPRI- and BMPRII-expressing cells were identified in the intimal lining and were also scattered in the subintima. These cells constituted approximately 25% and approximately 7% of the cells in each area, respectively. Double immunostaining showed no coexpression of BMPR-positive cells with CD68, CD34, or CD3. Cells expressing BMPR were not seen in any normal synovial samples (n = 4). Strong staining for BMPR was identified on cells at the invasive front of the pannus and at sites of cartilage erosion. CONCLUSION The inflamed RA joint contains BMPR-positive mesenchymal cells. Their origin is still speculative, but since their counterparts in the bone marrow are essential for osteoclastogenesis, support lymphocyte development and maturation, and protect T cells and B cells from programmed cell death, the BMPR-positive cells may be essential elements in the pathogenesis of RA and other inflammatory forms of chronic synovitis.

Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease.

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.

Therapeutic actions of cyclosporine and anti-tumor necrosis factor alpha in collagen-induced arthritis and the effect of combination therapy

OBJECTIVE To define the mechanisms of action of 2 novel drugs, cyclosporine and anti-tumor necrosis factor alpha (TNFalpha), in collagen-induced arthritis and to determine the effect of combination therapy. METHODS Type II collagen-immunized DBA/1 mice with established arthritis were treated with cyclosporine alone, anti-TNFalpha alone, cyclosporine plus anti-TNFalpha, or saline. RESULTS Cyclosporine was found to ameliorate arthritis, suppress interferon-gamma (IFNgamma) production by CD4+ T cells, and reduce TNFalpha expression in arthritic joints. However, cyclosporine did not directly inhibit TNFalpha production by macrophages, indicating that the decrease in TNFalpha expression observed in vivo was probably an indirect consequence of the reduction in type 1 T helper cell activity. Anti-TNFalpha also reduced IFNgamma production by T cells, indicating that TNFalpha is involved in the cellular immune response to collagen. Combined treatment with cyclosporine plus anti-TNFalpha had an additive therapeutic effect. CONCLUSION Although cyclosporine and anti-TNFalpha target different points in the inflammatory pathway, there is an overlap in the consequences of their actions in vivo.

A comparative study into the mechanisms of action of anti-tumor necrosis factor alpha, anti-CD4, and combined anti-tumor necrosis factor alpha/anti-CD4 treatment in early collagen-induced arthritis.

OBJECTIVE Anti-tumor necrosis factor alpha (anti-TNFalpha) therapy is very effective in rheumatoid arthritis (RA), whereas depleting anti-CD4 therapy is relatively ineffective. To explain the differences in efficacy between these 2 therapies, we used an animal model of RA to compare their effects on different aspects of the disease process. METHODS Mice with collagen-induced arthritis were treated with depleting anti-CD4 monoclonal antibodies (mAb), anti-TNFalpha mAb, or phosphate buffered saline. Another group was given a combination of anti-TNFalpha plus anti-CD4. The treatments were compared for their ability to down-regulate the expression of proinflammatory cytokines and adhesion molecules, reduce the cellularity of the joint, and inhibit Th1 activity. RESULTS Anti-TNFalpha significantly reduced the numbers of cells expressing TNFalpha, interleukin-1beta (IL-1beta), very late activation antigen 4 (VLA-4), vascular cell adhesion molecule 1 (VCAM-1), and numbers of CD4+ T cells and macrophages in the joint. Anti-CD4 treatment led to a small reduction in the expression of TNFalpha, IL-1beta, VLA-4, and VCAM-1, but this did not reach statistical significance. Depleting anti-CD4 was also surprisingly ineffective in eliminating CD4+ T cells from the joint. Anti-TNFalpha therapy was also more effective than anti-CD4 in reducing Thl activity, as assessed by the production of interferon-gamma in lymph node cell cultures. There was a synergistic relationship between anti-TNFalpha and anti-CD4 in the reduction of histologic score and inhibition of TNFalpha/IL-1beta expression in the joints. CONCLUSION The efficacy of the 3 treatments correlated with their ability to modulate the expression of inflammatory cytokines and adhesion molecules in the joint, reduce the cellularity of the joint, and inhibit Th1 activity. This kind of analysis may prove useful in the testing of novel therapies for RA.

Full and partial peroxisome proliferation-activated receptor-gamma agonists, but not delta agonist, rescue of dopaminergic neurons in the 6-OHDA Parkinsonian model is associated with inhibition of microglial activation and MMP expression

BACKGROUND Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that has been shown to have anti-inflammatory and matrix metalloproteinase (MMP) inhibitor properties. PPARγ agonists have been shown to have neuroprotective effects in various neurodegeneration models where inflammation is implicated, including models of Parkinsons disease. However, no studies have looked at the effects of partial PPARγ agonists. EXPERIMENTAL APPROACH The neuroprotective effects of the PPARγ full agonist, pioglitazone (20 mg/kg), partial PPARγ agonist GW855266X (15 mg/kg) and PPAR-δ full agonist GW610742X (10 mg/kg) were investigated in the 6-hydroxydopamine (6-OHDA) model of Parkinsons disease when administered prior to or post 6-OHDA lesioning. The integrity of the nigrostriatal system was assessed by assessing the numbers dopaminergic neurons in the substantia nigra (SN) and by assessing striatal dopamine content. The degree of microglia activation in the SN was also immunohistochemistry assessed utilizing the marker OX-6 for activated microglia and CD-68 a marker for phagocytic microglia. Additionally we performed immunocytochemistry for MMP3 in the SN. Finally, we investigated whether a period of drug withdrawal for a further 7 days affected the neuroprotection produced by the PPARγ agonists. KEY RESULTS Both pioglitazone and GW855266X protected against 6-OHDA induced loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine when administered orally twice daily for either 1) 7 day prior to and 7 days post lesioning or 2) for 7 days starting 2 days post lesioning when neurons will be severely traumatized. 6-OHDA lesioning was associated with an increase in microglia activation and in numbers of MMP-3 immunoreactive cells which was attenuated by pioglitazone and GW855266X. Neuroprotective effects were not replicated using the PPARδ agonist GW610742X. Subsequent withdrawal of both pioglitazone and GW855266X, for a further 7 days negated any neuroprotective effect suggesting that long-term administration may be required to attenuate the inflammatory response. CONCLUSIONS AND IMPLICATIONS For the first time a partial PPAR-γ agonist has been shown to be neuroprotectory when administered post lesioning in a parkinsonian model. Effects may be via the inhibition of microglial and MMP activation and support further research.

Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-β expression in the joint

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self‐limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro‐ and anti‐inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and IL‐6] and anti‐inflammatory cytokines [IL‐10, IL‐1ra, transforming growth factor (TGF)‐β1, TGF‐β2 and TGF‐β3] in joints was carried out over the course of the disease. Both pro‐ and anti‐inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti‐inflammatory cytokines, particularly TGF‐β1 and TGF‐β2, increased. Surprisingly, TNF‐α continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF‐α during the period of disease remission had no effect on TGF‐β expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro‐ and anti‐inflammatory cytokine expression in the joints. Of the anti‐inflammatory cytokines studied, TGF‐β1 and TGF‐β2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.