Evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin-8 in the cow

Abstract

BackgroundAfter ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL.Methods and ResultsPolymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs.ConclusionPMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.

Upregulation of Interferon-stimulated Genes and Interleukin-10 in Peripheral Blood Immune Cells During Early Pregnancy in Dairy Cows

“…Additionally, IFNT mRNA is first expressed in 16-cell stage in vitro fertilized embryos on day 4 [11], and IFNT protein was detected in the trophectoderm of nonhatched blastocysts on day 7 using confocal microscopy [12]. These findings suggest that IFNT signals are transmitted before the MRP (around days [15][16][17][18][19] in cows. On the other hand, general alterations occur in the maternal inflammatory and immune system during pregnancy.…”

“…The suspension was layered onto Ficoll-Paque solution (Lymphoprep, Axis-Shield, Oslo, Norway) and then centrifuged at 1000 × g at 10 C for 30 min as described previous study [16][17][18]. The plasma and buffy coat (population of mononuclear cells) were separated as PBMCs layer.…”

“…To check the purity of PBMCs and PMNs before use in the experiment, the PBMCs and PMNs were subjected to a flow cytometric evaluation (Beckman Coulter, Brea, CA, UAS), and they were found to be >98% and >95% pure, respectively (Fig. 2) [16][17][18]. To analyze mRNA expression, PMNs and PBMCs were placed into 1.5-ml microcentrifuge tubes with 400 µl of TRIzol reagent and stored at −80 C until analysis.…”

“…PBMCs and PMNs were isolated from whole blood collected via jugular venipuncture on days 4-5, 7-8 and 10-12 of the estrous cycle (day of ovulation = day 1; non-AI cows, n = 4) as described previously [17,18]. Isolated PBMCs and PMNs were resuspended at a concentration of 5 × 10 6 cell/ml in RPMI 1640 medium containing 0.1% FBS, gentamicin (50 mg/l) and amphotericin B (2.5 mg/l) purchased from Sigma (St. Louis, MO, USA).…”

“…2) [16][17][18]. To analyze mRNA expression, PMNs and PBMCs were placed into 1.5-ml microcentrifuge tubes with 400 µl of TRIzol reagent and stored at −80 C until analysis. …”