Dynamics of macrophages and inflammatory cytokine concentrations in the cervix of late pregnant cows

Abstract

Cervical ripening is associated with M1 macrophages and inflammatory cytokines in mice and rats, but its mechanism in cattle is unclear. In this study, to elucidate some of the mechanisms of normal cervical maturation in cows, we examined the expressions of M1 macrophages and mRNA of interleukin (IL)-1α, 1β, 6, 8, and 10, as well as TNFα in cervical tissue and the mRNA expressions and protein concentrations of these cytokines in cervical mucus from late pregnancy to parturition. Forty-one Japanese Black cows were sampled at 200, 230, and 260 d of gestation and then at 7 d intervals until parturition to obtain cervical mucus and tissue samples. The collected tissues were fluorescently immunostained with anti-Iba-1 antibodies, and the macrophage infiltration was checked. Cervical mucus was mixed with PBS, the concentrations of the above cytokines in the supernatant after centrifugation were measured, and the concentration per protein weight was used as the measurement. Total RNA was extracted from the cervical tissue and mucus, and the mRNA expression of various cytokines was analyzed using real-time PCR. In cervical tissue, macrophages stained with anti-Iba-1 antibody were observed from five to six weeks before calving until the week of calving. There was also a trend toward a concurrent increase in the mRNA expression of inflammatory cytokines, especially IL-6, three weeks before calving (P < 0.05). In cervical mucus, the concentrations of IL-1α, IL-1β, IL-8, and TNFα increased (P < 0.05) zero to three weeks before calving compared to 12–14 weeks before calving. In addition, IL-1α mRNA increased three weeks before and during the week of parturition, and IL-8 mRNA increased three weeks before and two weeks after (P < 0.05). These results suggest that cervical ripening in cows begins five to six weeks before calving when macrophages infiltrating cervical tissue may produce large amounts of IL-6, inflammatory cells infiltrate cervical mucus, and IL-1α, IL-1β, IL-8, and TNFα levels in the mucus increase toward calving.