Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human NeutrophilsNeutrophil Methods and Protocols

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, rely on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, role, and physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis.