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

<p>Abstract</p> <p>Background</p> <p>After 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.</p> <p>Methods and Results</p> <p>Polymorphonuclear 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.</p> <p>Conclusion</p> <p>PMNs 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.</p

Rapid Accumulation of Polymorphonuclear Neutrophils in the Corpus luteum during Prostaglandin F2α-Induced Luteolysis in the Cow

Prostaglandin F2α (PGF2α) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF2α administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF2α injection. The number of PMNs was increased at 5 min after PGF2α administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF2α directly stimulated P-selectin protein expression at 5–30 min in luteal endothelial cells (LECs). Moreover, PGF2α enhanced PMN adhesion to LECs, and this enhancement by PGF2α was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF2α is crucial in PMN migration. In conclusion, PGF2α rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF2α may involve an acute inflammatory-like response due to rapidly infiltrated PMNs

Expression of FPr in PMNs, PBMCs, and the mid CL and transmigration assay of PMNs by PGF_2α and IL-8.

<p>The mRNA and protein levels of FPr (A and B) in PMNs, PBMCs and the mid CL tissue (n = 4 in each group). All values are the means ± SEM (relative to β-actin mRNA levels). Representative photograph of a western blot is shown for FPr and β-actin (B). The number of PMNs migration by PGF_2α (10⁻⁶ M) and IL-8 (50 ng/ml) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g007" target="_blank">Fig 7C</a> (n = 4 in each group). The number of migrating PMNs after PGF_2α administration is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g007" target="_blank">Fig 7D</a>. White bar indicates control (PMNs before PGF_2α administration, IL-8 un-stimulate) and black bars indicate IL-8 (10 ng/ml)-stimulated PMNs (at 0 min, 5 min, 30 min, and 2 h after PGF_2α administration, n = 4 in each time point). Values are shown as the means ± SEM. Different superscript indicate significant differences (P<0.05) as determined by ANOVA followed by the Bonferroni's multiple comparison test.</p

Effect of PGF_2α on P-selectin in LECs.

<p>The levels of P-selectin expression after PGF_2α (10⁻⁶ M) treatment in LECs. Fig. 5A shows P-selectin expression in LECs in control and Fig. 5B shows P-selectin expression in the PGF_2α treatment group at 5 min (n = 4 in each group). Scale bars indicate 5 µm. Fig. 5C shows the intensity of P-selectin expression at 5, 15 and 30 after treatment, respectively (% of control). All values are the means ± SEM. Different superscript indicate significant differences (P<0.05) as determined by Student's <i>t</i>-test.</p

PMN numbers in the bovine CL during PGF_2α-induced luteolysis.

<p>The typical images of PMNs within the CL at 0 min (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1A</a>), 5 min (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1C</a>), 30 min (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1D</a>), and 12 h (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1E</a>) during PGF_2α-induced luteolysis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1B</a> indicates extended figure of PMN within the CL and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g001" target="_blank">Fig. 1F</a> shows number of PMNs during PGF_2α-induced luteolysis (n = 4−5 in each time), respectively. Black arrows show PMNs in the CL. Values are shown as the means ± SEM. Different superscripts indicate significant differences (P<0.05) as determined by ANOVA followed by Bonferroni's multiple comparison test.</p

Expression of IL-8 and P-selectin mRNA in LCs and LECs.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029054#pone-0029054-g003" target="_blank">Fig. 3</a> indicates mRNA expression of IL-8 (A) and P-selectin (B) in LCs and LECs (n = 5 in each group). All values are the means ± SEM. Different superscript indicate significant differences (P<0.05) as determined by Student's <i>t</i>-test.</p