Myeloperoxidase negatively regulates the expression of proinflammatory cytokines and chemokines by zymosan-induced mouse neutrophils

Daiki Endo, Takayuki Saito, Yu Umeki, Kazuo Suzuki, Yasuaki Aratani

We have previously reported that myeloperoxidase-deficient (MPO(-/-)) neutrophils produce greater amounts of macrophage inflammatory protein-2 (MIP-2) upon in vitro stimulation with zymosan than wild-type neutrophils. This study aimed to examine the effect of MPO deficiency on the expression of other cytokines and chemokines. Wild-type and MPO(-/-) neutrophils isolated from peritoneal cavity were stimulated with zymosan in vitro. Secretion of MIP-1α, MIP-1β, interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α by neutrophils was quantified by ELISA. mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) in neutrophils was analyzed by western blot. For in vivo studies, mice were inoculated with zymosan intranasally, and the levels of these cytokines and chemokines were measured in the lungs. The MPO(-/-) neutrophils stimulated by zymosan expressed and secreted significantly higher levels of MIP-1α, MIP-1β, IL-1α, IL-1β, and TNF-α than the stimulated wild-type cells. Expression of all of these inflammatory mediators was blocked by pre-treatment with BAY11-7082, U0126, and SB203580, which are inhibitors of nuclear factor (NF)-κB, ERK1/2, and p38 MAPK, respectively. Enhanced expression of these inflammatory mediators is associated with elevated activation of ERK1/2 in stimulated MPO(-/-) neutrophils. In vivo, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-1α, MIP-1β, IL-1α, IL-1β, and TNF-α relative to the responses seen in wild-type mice within 24 h of zymosan administration. MPO deficiency upregulates the expression of several proinflammatory cytokines and chemokines in mouse neutrophils.