A. Facchini

Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis

OBJECTIVE To evaluate the sites of expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and inducible nitric oxide synthase (iNOS) in patients with inflammatory and degenerative joint diseases. METHODS Cytokines and iNOS were detected by immunohistochemistry analysis of synovial and cartilage biopsy specimens obtained at knee arthroscopy in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), osteoarthritis (OA), and traumatic knee arthritis. Cytokine and iNOS expression was quantified using computerized image analysis. RESULTS IL-1beta, TNFalpha, and iNOS were highly expressed by synovial cells (lining layer cells, infiltrating leukocytes, endothelial cells) from patients with inflammatory arthritides and significantly less by synovial cells from patients with OA and traumatic arthritis. In contrast, the latter patients showed high chondrocyte expression of cytokines and iNOS while RA and PsA patients had only minor chondrocyte positivity. In both joint compartments, IL-1beta expression, TNFalpha expression, and iNOS expression were strongly correlated. CONCLUSION The enhanced and coordinated expression of IL-1beta, TNFalpha, and iNOS by chondrocytes strongly supports the hypothesis that chondrocytes are the major site of production of mediators of inflammation in human OA, thus playing a primary role in the pathogenesis of this disease.

Human chondrocytes express functional chemokine receptors and release matrix-degrading enzymes in response to C-X-C and C-C chemokines

OBJECTIVE Human chondrocytes produce different C-X-C and C-C chemokines under basal conditions and upon activation with proinflammatory cytokines. We investigated whether human chondrocytes also have chemokine receptors and examined the effects of chemokines on chondrocyte activity. METHODS The expression of chemokine receptors was determined by immunochemical analysis of frozen sections from normal and osteoarthritic cartilage and by flow cytometry of isolated cells. The messenger RNA expression for chemokine receptors was studied by reverse transcriptase-polymerase chain reaction. Isolated chondrocytes were stimulated with different chemokines, and the responses were evaluated by assaying the release of matrix metalloprotease 3 (MMP-3) and of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase in the supernatants. RESULTS A wide variety of chemokine receptors (CCR-1, CCR-2, CCR-3, CCR-5, CXCR-1, and CXCR-2) was detected on human chondrocytes. Interaction of these receptors with the corresponding ligands induced the release of MMP-3. This response was abrogated by pretreatment of the cells with Bordetella pertussis toxin, demonstrating involvement of G proteins of the Gi type. The response decreased in the presence of cycloheximide, indicating dependence on protein synthesis. Chemokines also induced the exocytosis of N-acetyl-beta-D-glucosaminidase, which was prevented by receptor blockage with anti-CCR-3 and by treatment with B pertussis toxin. Chondrocytes obtained from osteoarthritic tissue showed an increased expression of CCR-3 and possibly of CXCR-1, and an augmented release of matrix-degrading enzymes compared with chondrocytes from normal donors. CONCLUSION Our findings suggest the existence in human chondrocytes of a novel catabolic pathway, primed by chemokines and their receptors, that leads to the breakdown of cartilage matrix components.

Endothelin-1 in idiopathic pulmonary fibrosis.

AIMS--To evaluate whether endothelin-1 is involved in the pathology of idiopathic pulmonary fibrosis (IPF). METHODS--Plasma endothelin-1 concentrations were evaluated in 37 patients with IPF and 27 normal controls by radioimmunoassay. In addition, expression of endothelin-1 in lung tissue was evaluated in biopsy specimens obtained from four patients with IPF. Three biopsy specimens of normal lung were used as controls. Endothelin-1 immunoreactivity was detected using immunohistochemistry. RESULTS--Elevated endothelin-1 plasma concentrations were found in patients with IPF compared with controls and a positive correlation was found with duration of disease. No significant difference was observed between treated and untreated patients with IPF. Increased endothelin-1 immunoreactivity was found in lungs of three of four patients with IPF. Endothelin-1 positive consisted mainly of small vessel endothelial cells. Some scattered macrophages were also positive. CONCLUSIONS--Elevated plasma concentrations and expression of endothelin-1 in lung tissue are suggestive of increased production of endothelin-1 in at least a proportion of patients with IPF. Consequently, endothelin-1 activity could play a role in the fibrogenic process of the disease.

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone‐forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody‐dependent cell‐mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector‐to‐target cell ratios. We conclude that the impaired functional activity (T or NK‐dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

Natural killer function in flow cytometry: I. Evaluation of NK lytic activity on K562 cell line

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Distribution and lytic activity of NK cell subsets in the elderly

Old subjects present an increased number of NK cells associated with a decreased lytic activity of isolated and cloned CD16 cells. Recently, two new surface molecules of 58 kDa, identified by the monoclonal antibodies GL183 and EB6, have been described. The presence of these molecules, which can be coexpressed on CD16+ cells allows the recognition of the NK cell subsets whose cytolytic activity is restricted to different allospecificities. This study investigated a group of old subjects to determine whether a particular distribution or a different lytic activity of NK subsets, defined by MoAbs GL183 and EB6, is involved in the altered cytolytic activity found during ageing. Further, we investigated whether the ageing process might be responsible for a restriction of the NK cell repertoire involved in the recognition of allogenic cells. We found that old and young subjects have a similar proportion of double positive and double negative GL183/EB6 cells, while in the old group single positive subsets were increased. The lytic activity of sorted NK subsets isolated from old and young subjects was similar, although double positive and double negative cells from the old presented a lower cytotoxic activity. The addition of IFN-beta or rIL-2 to the culture medium restored the lytic activity to the level found in young subjects. These data show that the decreased NK lytic activity found in the old subjects is shared out among the different NK subsets and normal aged subjects do not lose the NK repertoire found in the young.

Anti‐topoisomerase II α autoantibodies in systemic sclerosis—association with pulmonary hypertension and HLA‐B35

We have previously detected autoantibodies against topoisomerase II α (anti‐topo II α) in sera from patients with idiopathic pulmonary fibrosis. To determine whether anti‐topo II α is also present in systemic sclerosis (SSc) patients with pulmonary involvement, we screened sera from 92 patients and 34 healthy controls. Presence of anti‐topo II α was investigated with respect to clinical and serological features, including the frequencies of HLA class I and II alleles. Anti‐topo II α was detected in 20/92 (21.7%) patients. No association was found with either anti‐topoisomerase I (Scl‐70 or anti‐topo I) or anti‐centromere antibodies. However, anti‐topo II α was associated with the presence of pulmonary hypertension (PHT) (as opposed to pulmonary fibrosis), and with a decrease of carbon monoxide diffusing capacity. Anti‐topo II α was strongly associated with the presence of the class I antigen HLA‐B35. No significant association was found with HLA class II antigens. HLA‐B35 also turned out to be associated with the presence of PHT. These results indicate that in SSc patients, the presence of anti‐topo II α is associated with PHT, and that the simultaneous presence of HLA‐B35 seems to add to the risk of developing PHT.

Serum copper/zinc superoxide dismutase levels in patients with rheumatoid arthritis.

Rheumatoid arthritis is characterized by a chronic hypertrophic synovitis leading to destruction of connective tissue and functional damage of cartilage and bone structures. Reactive oxygen species play an important role in tissue injury in this disease. To clarify the role of the cellular antioxidant system in the protection against oxygen free radicals, we examined the levels of copper/zinc superoxide dismutase in the sera of patients with rheumatoid arthritis. We used an enzyme-linked immunosorbent assaywhich determines the concentration of copper/zinc superoxide dismutase indepedently from its enzymatic activity. We found that patients with rheumatoid arthritis have higher serum copper/zinc superoxide dismutase levels than control subjects. Copper/zinc superoxide dismutase also correlated positively with serum levels of both neopterin and rheumatoid factor, sensitive markers for disease activity in rheumatoid arthritis. These results support the hypothesis that the increased amount of copper/zinc superoxide dismutase is probably inadequate to exert an effective antioxidant protection but can result in a pro-inflammatory, pathogenic effect enhancing tissue damage. Furthermore, copper/zinc superoxide dismutase might be used as a marker of inflammatory activity in rheumatoid arthritis.

Mapping of topoisomerase II α epitopes recognized by autoantibodies in idiopathic pulmonary fibrosis

Autoantibodies against DNA topoisomerase II α have been identified in the sera of patients with idiopathic pulmonary fibrosis (IPF). To map topoisomerase II autoepitopes, we tested by ELISA and immunoblotting the IPF anti‐topoisomerase II‐positive sera against a series of recombinant proteins which covered the full length of topoisomerase II α. Specific patterns of reactivity were observed, indicating the existence of multiple epitopes on topoisomerase II, either highly complex or conformational/discontiguous or conformational/contiguous ones. The latter resided in amino acid residues 854–1147 and 1370–1447. A detailed analysis of these regions was undertaken, but we were not able to pinpoint a sequential peptide‐sized epitope, or any significant homology with foreign pathogens. Further, we observed a significant correlation between the progression from a contiguous to a quaternary/tertiary structure‐dependent autoepitope and the disease duration but not with the disease severity. Therefore, this result supports the hypothesis that anti‐topoisomerase II autoreactivity evolves following an antigen‐driven process.

VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION IN POLYMYALGIA RHEUMATICA

OBJECTIVE To evaluate peripheral production and synovial expression of vascular endothelial growth factor (VEGF) in polymyalgia rheumatica (PMR). METHODS Circulating levels of VEGF in PMR (serum concentration and in vitro release by peripheral blood mononuclear cells [PBMC]) were investigated by enzyme-linked immunosorbent assay. Local expression of VEGF in shoulder synovial tissue was investigated by immunohistochemical analysis. Investigations were performed in patients with active, untreated disease and in patients treated with corticosteroids. RESULTS VEGF serum concentrations were significantly higher in untreated PMR patients than in normal control subjects. During steroid treatment, VEGF serum concentrations reached their lowest level after the sixth month of treatment. PBMC isolated from untreated PMR patients spontaneously secreted a higher amount of VEGF compared with PBMC from control subjects. Corticosteroid therapy did not affect the ability of PBMC to produce VEGF. Immunohistochemical staining performed on shoulder synovial tissue showed VEGF expression in both the lining layer and the sublining area. In 3 of 4 treated patients, no VEGF staining was found in synovial tissue during corticosteroid therapy. VEGF expression correlated with vessel density, but was not associated with alphavbeta3 and alphavbeta5 integrin expression. CONCLUSION Peripheral and local VEGF releases have different responses to steroid treatment in PMR. The lack of response to corticosteroids by peripheral VEGF production supports the hypothesis that systemic involvement is dominant in PMR. At the synovial level, VEGF production is linked to vascular proliferation and is thus directly involved in the pathogenesis of synovitis.