METHODS

Experimental procedures

Antigen arthritis was induced in ovalbumin preimmunised rabbits.^2,6 Animals were divided into three experimental groups: non-treated rabbits (n=16), meloxicam (MXC) treated (Boehringer Ingelheim) (4 mg/kg/day, n=14) and diclofenac (DCF) treated (Sigma) (5 mg/kg/8 h, n=14). Drugs were intramuscularly injected from 24 hours before the intra-articular injection until the animals were killed, 48 hours after the intra-articular injection. Eight healthy age matched animals were employed as controls.

Determination of PGE₂ in synovial fluid

PGE₂ concentration in the synovial fluid (SF) was determined by PGE₂ enzyme immunoassay (Assay Designs, Inc).

Identification of recruited cells and chemokines in the synovial membrane

Paraffin embedded synovial membrane (SM) was stained with haematoxylin and eosin. A 0–3 points scoring scale estimated the density of leucocyte populations.

Immunoreactivity to IL8 and MCP-1 was assessed by peroxidase techniques.² The density of IL8 and MCP-1 staining was scored 0–4.

RT-PCR studies

Total RNA from the SM was isolated, reverse transcribed and amplified with Access RT-PCR System (Promega),² employing specific primers of rabbit IL8 and MCP-1.²

Statistical analysis

Data were expressed as mean (SEM). Descriptive statistics, analysis of variance test, and comparison of means by independent t test were carried out. Differences were considered significant for p<0.05.

RESULTS

Infiltration of leucocytes and production of PGE₂

Untreated rabbits showed a large synovial effusion that was reduced by MXC treatment, but not by DCF (table 1). A high cell concentration was found in the SF in the untreated group (80% of the total cells were polymorphonuclear cells (PMN)). Only MXC administration diminished total cell count and PMN concentration (table 1). None of the drugs diminished the SF MN influx compared with the untreated group (table 1).

Tissue damage was partially prevented by both NSAIDs (web extra fig W1). MXC treated rabbits showed a diminution in the PMN infiltration. DCF did not have a significant effect. Neither MXC nor DCF modified SM MN cell infiltration (table 1).

Increased levels of PGE₂ were found in the SF of untreated rabbits (16 (4) ng/ml). Both NSAIDs reduced PGE₂ concentration. DCF induced a greater fall in PGE₂ levels than MXC.

IL8 and MCP-1 immunolocalisation

IL8 and MCP-1 followed a similar pattern of distribution. Mononuclear cells and the lining layer were strongly positive (fig 1). Extracellular IL8 was largely detected in neutrophilic collections and in the endothelium. Both NSAIDs down regulated IL8 immunoreactivity (figs 1A–D, I) mainly by a reduction in the areas corresponding to neutrophilic collections. For the MCP-1 signal, there were no differences between the groups (figs 1E–H, J).

IL8 and MCP-1 gene expression in the SM

The high immunoreactivity to IL8 and MCP-1 in the injured tissues was accompanied by an enhancement in the expression of both genes. IL8 expression was reduced with both treatments, although it was more evident in MXC treated animals. None of the drugs diminished the MCP-1 message (fig 2).

DISCUSSION

We have studied the effect of two NSAIDs on cell infiltration, local PGE₂, and IL8 and MCP-1 synovial expression in a model of experimental arthritis. The clinical benefit of both drugs was related to a diminution of joint swelling, reduction of the effusion and infiltration by PMN. Only MXC treatment showed significant effects on SF volume and PMN infiltration. The MXC group showed a smaller diminution of PGE₂ levels in SF than did DCF. Our results showed a lack of correlation between PGE₂ inhibition and clinical improvement. Previous evidence has shown that PGE₂ depletion may lead to an up regulation of several proinflammatory mediators, such as tumour necrosis factor α and IL1β in the inflamed SM.⁶ COX derived eicosanoids exert some regulating functions important for the resolution of inflammation.⁷

We found a differential regulation of PMN and MN migration by the NSAIDs. Cell count and density scoring in each group paralleled the respective induction of the chemokines studied. NSAIDs action appeared selective for PMN influx, and related to a down regulation of IL8 local expression. MCP-1 expression and synthesis remained largely activated in the SM of treated groups. This may explain why both treatments increase MN density in the SM and SF. This is in agreement with previous data describing a lack of effect of NSAIDs at the chronic stage of this experimental disease, when inflammatory events are dominated by macrophages and lymphocytes. Recent unpublished data from our laboratory show that MXC and DCF further increase MCP-1 expression in IL1β stimulated synoviocytes. These and other data suggest that PGE₂ may act as an MCP-1 repressor agent.⁸ This effect seems to be related to the PGE₂ synthesis inhibition afforded by NSAIDs.

Our results indicate that a total PGE₂ depletion may not be desirable. We have shown that NSAIDs down regulate IL8 production in antigen arthritis, but may favour the recruitment of MN. A full understanding of prostaglandin activities as stimulating or repressor factors of inducible genes is desirable to design a better therapeutic approach with NSAIDs in joint diseases.