Gene expression by inflammatory neutrophils: stimulation of interleukin-1 beta production by rheumatoid synovial fluid.

Abstract

The neutrophl has been thought of as a terminally differentiated cell with little or no capacity or requirement for de novo protein synthesis. It has recently been demonstrated that neutrophils are biosynthetically active and that the biosynthesis of some cellular components is essential to maintain the neutrophil in an active state [I]. It has also been shown that neutrophls can synthesise and secrete a range of cytokines in v i m including Interleukin (1L)-I p, IL-6, IL-8 and tumour necrosis factor-a (TNFa), all of which are present in the synovial fluid of rheumatoid joints [2,3]. L I P has been implicated as a mediator of tissueand bone-damage in rheumatoid arthritis and among its many effects it has been shown to stimulate osteoclasts and chondrocytes to resorb bone 131. As neutrophils can comprise 80% of the cell population in a rheumatoid joint, their production of IL-IP in the joint could be an important contributing factor in the perpetuation of the disease. This production in the joint could have many effects including the stimulation of production of further IL1 P by forming a positive feedback loop. IL-1p has also been shown to enhance adhesion molecule expression on vascular endothelium thereby promoting the accumulation of further neutrophils and other leukocytes [4]. Here we show the synthesis of IL-lp by neutrophils in response to rheumatoid synovial fluid. Neutrophils were isolated from fresh buffy coats as described previously [5] and suspended in RPMI 1640 medium. Suspensions (2x10’ celldml) were re-incubated at 37°C with gentle agitation in the presence of [’ S]-methionine for 15 min. Following this, either granulocyte macrophage-colony stimulating factor (GM-CSF) (14 ng/ml), lipopolysaccharide (LPS) (5 pg/ml), TNFa (25 nglml), synthetic soluble or insoluble immune complexes (10 %, v/v) or cell free synovial fluid (5 %, v/v) were added. Incubation was continued at 37°C with gentle agitation for time periods of up to 24 h. After incubation, cells were pelleted and any IL-Ip present in the cells or culture medium was immunoprecipitated as described previously [6]. The immuno-precipitated samples were then analysed by SDS-PAGE and fluorography. P