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.

SUMMARYThis 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-a), IL-1b and IL-6 using an indirect immunoperoxidase technique. In parallel, interferon-gamma (IFN-g) 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-a, IL-1b 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-a-, IL-1b-and IL-6-positive cells from day 1 to day 10 of arthritis, during which time IFN-g production by CD4+ T cells from draining lymph nodes declined sharply. A further finding of potential significance was that TNF-a was consistently detected at day 1 of arthritis, whereas IL-1b-positive cells were not found until day 3, suggesting that the expression of TNF-a precedes that of IL-1b.

Objective. To determine the involvement of mesenchymal progenitor cells in the induction of collageninduced arthritis (CIA).Methods. DBA/1 mice were immunized with type II collagen in adjuvant or adjuvant alone, and the presence of mesenchymal cells in the joints of prearthritic mice was studied by immunohistochemistry.Results. An analysis of the joints on day 10 postimmunization (at least 10 days before the onset of arthritis) revealed synovial hyperplasia without leukocytic infiltration. Large, round cells expressing bone morphogenetic protein receptors (BMPRs), which serve as markers for primitive mesenchymal cells, were present in increased numbers in the bone marrow adjacent to the joint, in the synovium itself, and within enlarged bone canals that connect the bone marrow to the synovium. Similar changes were observed in mice given adjuvant without collagen. Adjuvant-induced infiltration of BMPR ؉ cells and enlargement of bone canals were abrogated by anti-tumor necrosis factor (anti-TNF) treatment and were absent in TNFR p55/ p75 ؊/؊ mice. Increased numbers of bone marrow cells and enlarged bone canals were observed in nonimmunized TNF transgenic mice (which spontaneously develop arthritis).Conclusion. These findings suggest that in CIA, there is an antigen-independent (innate) prearthritic phase that prepares the joint for the subsequent immune-mediated arthritis. The induction phase involves marrow-derived mesenchymal cells and requires the presence of TNF.

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 syno-vial 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,-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 25% and 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.

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.