Cytokine production of neutrophils is limited to interleukin-8

Since granulocytes are one of the first cell types at sites of inflammation, investigation of their capacity to produce cytokines has concentrated on interleukin-1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha). However, the results are subject to controversy. Incapability to produce cytokines as well as a broad panel of cytokines induced by isolation procedures are reported. The purpose of this study was to investigate the capacity of non-prestimulated neutrophils to produce the above-mentioned cytokines in response to stimulation with lipopolysaccharide or zymosan. In reverse transcription-polymerase chain reactions, we found IL-8 mRNA directly after isolation in unstimulated cells, whereas mRNA for IL-1 beta, IL-6 and TNF-alpha only appeared after stimulation. By means of flow cytometry we ruled out the possibility of prestimulation of the neutrophils during isolation, proving that IL-8 mRNA is produced constitutively by neutrophils. In enzyme-linked immunosorbent assays we found that, compared with controls, only IL-8 was released at significantly higher levels after 24 hr of stimulation, giving a further indication that neutrophils have an immunoregulatory influence driven by IL-8. We can confirm neither a constitutive nor a post-stimulatory release of IL-1 beta, IL-6, or TNF-alpha by neutrophils, as had been reported by others. These observations may be due to prestimulation, handling and culturing of the granulocytes, or to monocyte contamination. Collectively, our results show that granulocytes have a preformed capacity to produce the cytokine IL-8 and that the production of proinflammatory cytokines by neutrophils is limited to IL-8.