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1.  Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen induced arthritis: influence on macrophage activation 
Annals of the Rheumatic Diseases  2004;63(11):1470-1477.
Objective: To investigate the indirect effects of anti-CD4 treatment on the functions of macrophages (CD4– in mice) in the acute and early chronic phase of mouse antigen induced arthritis (AIA).
Methods: C57BL/6 mice with AIA were treated intraperitoneally with the anti-CD4 mAb GK1.5 or control rat IgG on days –1, 0, 1, 3, 5, and 7. Proinflammatory cytokines (IL1ß, IL6, and TNFα) were quantified by sandwich ELISA in joint extracts, serum, and supernatants of ex vivo stimulated spleen/lymph node cells or peritoneal macrophages (+LPS/IFNγ). Nitric oxide (NO) levels in supernatants of ex vivo stimulated peritoneal macrophages were measured by the Griess reaction. Proteolytic activity in joint homogenates was analysed by gelatin, casein, and elastin zymography, and substrate assays.
Results: Anti-CD4 treatment significantly reduced joint swelling in acute (days 3, 5) and early chronic AIA (day 7) and diminished inflammation and destruction scores in late chronic AIA (day 21). On day 3, anti-CD4 treatment significantly reduced IL6 levels in all compartments. IL1ß was reduced in joint extracts, unaffected in serum or cells from lymphoid organs, and increased in stimulated peritoneal macrophages. TNFα was significantly increased in the joints, decreased in serum, and otherwise unchanged. NO production by stimulated peritoneal macrophages was significantly reduced by anti-CD4 treatment. Lower activity of matrix metalloproteinases and neutrophil elastase was seen in joint extracts of anti-CD4 treated animals than in IgG treated AIA controls.
Conclusion: CD4+ T cell directed treatment had strong local and systemic effects on macrophages. These indirect effects may contribute to the reduction of destructive mediators/joint destruction in AIA.
doi:10.1136/ard.2003.013060
PMCID: PMC1754787  PMID: 15479897
2.  Identification of the advanced glycation end products N -carboxymethyllysine in the synovial tissue of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2002;61(6):488-492.
Background: Generation of advanced glycation end products (AGEs) is an inevitable process in vivo and can be accelerated under pathological conditions such as oxidative stress. In serum and synovial fluid of patients with rheumatoid arthritis (RA) raised AGE levels have been found.
Objective: To determine the presence of N -carboxymethyllysine (CML; marker of oxidative stress) in RA synovial tissue by immunohistology.
Methods: Frozen synovial tissue samples from 10 patients with RA and eight controls (four patients without joint disease and four patients with osteoarthritis (OA)) were treated with rabbit-anti-CML-IgG and goat-antirabbit-IgG. Immunostaining was visualised by streptavidine-alkaline phosphatase (chromogen fuchsin). Cell differentiation was performed with antibodies against CD68, CD45RO, and CD20.
Results: CML was detected in the synovial lining, sublining, and endothelium in 10/10 RA and 4/4 OA synovial specimens. In RA some macrophages (CD68+) and T cells (CD45RO+) showed positive immunostaining for CML, whereas B cells were negative. Staining in OA synovial sublining was weak compared with RA.
Conclusions: CML was detected for the first time in RA and OA synovial tissue. Different patterns of immunostaining in RA and OA and the presence of CML on macrophages and T cells, suggest a role for CML in the pathogenesis of RA. This might be due to presentation of new epitopes which can maintain or even trigger an autoimmune response.
doi:10.1136/ard.61.6.488
PMCID: PMC1754129  PMID: 12006318
3.  Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2002;61(5):391-397.
Objective: To study the pattern and cell type-specificity of collagenase 3, membrane-type 1 matrix metalloproteinase (MT1-MMP), and gelatinase A mRNA expression in the synovial membrane in rheumatoid arthritis (RA).
Methods: The mRNA expression of collagenase 3, MT1-MMP, and gelatinase A was characterised by northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridisation. In situ hybridisation was performed in combination with the immunohistochemical detection of cell type-specific antigens.
Results: Synovial membrane specimens from 19 of 21 patients with RA expressing collagenase 3 mRNA were positive for MT1-MMP and gelatinase A mRNA. In control samples from patients without destructive inflammatory joint diseases collagenase 3 mRNA was not expressed and only in two of seven cases was a coexpression of MT1-MMP and gelatinase A mRNA detected. Fibroblast-like cells of the synovial membrane were found to be the predominant source of collagenase 3, MT1-MMP, and gelatinase A mRNA expression in lining and sublining layers as well as at the synovial membrane-cartilage interface. Additionally, the expression of MT1-MMP mRNA was detected in endothelial cells. Collagenase 3 mRNA expression was found in about 5% of CD68 positive macrophages.
Conclusions: Collagenase 3 mRNA is expressed simultaneously with MT1-MMP and gelatinase A mRNA in fibroblast-like cells of the synovial membrane in RA. These results suggest (a) a broad extracellular proteolytic potential of fibroblast-like cells and (b) an important role of cell surface associated procollagenase 3 activation by MT1-MMP and gelatinase A for cartilage degradation by invading fibroblast-like cells.
doi:10.1136/ard.61.5.391
PMCID: PMC1754087  PMID: 11959761
4.  Endotoxin-Induced Lung Inflammation Is Independent of the Complement Membrane Attack Complex 
Infection and Immunity  2000;68(3):1626-1632.
Several products of the activated complement system are known to modulate endothelial cell function in vitro. It has been shown that the membrane attack complex (MAC) (C5b-C9) can enhance tumor necrosis factor alpha (TNF-α)-induced expression of P- and E-selectin and intercellular adhesion molecule type 1 in cell cultures of human umbilical vein endothelial cells. In the present study the potential role of this synegism for lung injury during endotoxin-mediated septic shock in vivo was examined using a model of C6-deficient PVG (C−) (RT1C) rats and the congenic PVG (C+) (RT1C) strain. Following administration of a high (5 mg/kg) or low (0.5 mg/kg) dose of lipopolysaccharide (LPS) (Escherichia coli O55:B5), we determined the expression of cytokines, chemokines, and adhesion molecules as well as the recruitment of leukocytes in the lung. Challenge with intraperitoneal i.p. injections of LPS resulted in a strong induction of TNF-α, interleukin-1α/β, cytokine-induced neutrophil chemoattractant, interferon-inducible protein 10, macrophage inflammatory proteins 1α and 2, macrophage chemotactic protein 1, and P-selectin. However, there were no significant differences between PVG (C−) and PVG (C+) rats. Immunoperoxidase staining showed a similar increase of lung infiltration by CD11b/c+ leukocytes in both rat strains. We therefore conclude that the described synergism between TNF-α and the MAC of the complement system on the induction of endothelial adhesion molecules is dispensable for inflammatory processes during endotoxin-mediated septic shock in vivo.
PMCID: PMC97323  PMID: 10678982
5.  Requirement and role of C5a in acute lung inflammatory injury in rats. 
Journal of Clinical Investigation  1996;98(2):503-512.
The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.
PMCID: PMC507456  PMID: 8755663

Results 1-6 (6)