The Predominant Infiltration of Neutrophils at the Early Stage of Development into the Cyclic Bovine Corpus Luteum.

Abstract

In the ovary, ovulation induces a kind of inflammation with infiltration of a large number of macrophages and granulocytes and these leukocytes secrete inflammatory cytokines within the ovulatory/ovulated follicle. After ovulation, the corpus luteum (CL) rapidly develops within one week together with active angiogenesis and progesterone synthesis in the cow. In the life span of CL, the immune cell population presents throughout the estrous cycle. Especially in the regressing CL, T-lymphocytes and macrophages significantly increase, and the luteolytic function of these leukocytes is well illustrated. In the early CL, several types of immune cells including macrophages and eosinophils infiltrate and the primary function of these immune cells is considered to be phagocytosis, but they may also have other critical functions for the CL development. Therefore, we first characterized the distribution of mRNA expression in leukocytes especially for angiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO) and tumor necrosis factor alpha (TNF). Blood samples were collected from cyclic cows and lymphocytes and granulocytes were purified using Ficoll solution. The mRNA expressions of chemokine receptor CXCR1 and CXCR2 were higher in granulocytes than lymphocytes. The mRNAs of VEGF, VEGF receptor (VEGFR) 1, interleukin-8 (IL8) and TNF were expressed in both lymphocytes and granulocytes, but VEGFR2 and inducible NO synthase mRNAs were expressed only in granulocytes. These results suggest that bovine leukocytes have a potential to act on angiogenesis. In the next, we focused on granulocytes in the early developing CL and investigated the distribution of neutrophils and eosinophils by periodic acid-schiff staining and luna staining, respectively, with related mRNA expressions. CLs were collected at a local slaughterhouse and divided into 4 groups: Days 1-2, 3-4, 5-6 and 7-8 of the estrous cycle (n=5-8 for each luteal phase). The number of neutrophils and eosinophils in the CL were significantly higher on Days 1-2 than Days 5-6 and 7-8. The mRNA expression of IL8, chemoattractant of neutrophils, was highest on Days 1-2 as well as the number of neutrophils in the CL, although mRNA expression of CCL5, major chemoattractant of eosinophils, was not high on Days 1-2. To investigate the function of IL8 as the possible luteal chemotactic mediator, we tested the migration of neutrophils using transmigration assay with the bovine IL8 and supernatants of the CL culture. In transmigration assay, the CL supernatants as well as IL8 (10-100 ng/ml) stimulated neutrophil migration. Although we tested the capacity of migration in neutrophils collected from circulating blood from Day 2, 5 and 10 of the estrous cycle (n=5 for each group), the levels of neutrophil migration were constant in all groups, suggesting that the migration of neutrophils rather depends on the level of IL8 in the CL but not on the stage of estrous cycle or the capacity of neutropils. In conclusion, the bovine granulocytes and lymphocytes express mRNAs for angiogenic factors, and neutrophils and eosinophils exist at the largest number in the early stage of development CL, besides those in regressing CL. Also, the bovine CL is able to active a migration of neutrophils possibly by chemoattractant such as IL8. Further study should be addressed on the possible interactions between the CL and immune cells to enhance luteal development in addition to phagocytosis in the cow. (poster)