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It is well documented that blood neutrophils from parturient dairy cows do not perform as well as neutrophils from nonparturient cows in laboratory assays of adhesion, migration, or phagocytosis-induced respiratory burst. However, little is known about the possible molecular basis for parturition-induced changes in neutrophils. cDNA microarray analysis was used in the current study to explore parturition-induced changes in gene expression profiles in bovine blood neutrophils. Total RNA from isolated blood neutrophils of four parturient Holstein cows was obtained before, during, and after parturition, reverse transcribed into cDNA, and sequentially labeled with Cy3 or Cy5 dyes prior to paired hybridizations to 1,056 member bovine total leukocyte (BOTL-3) microarrays in a loop design. Resulting gene expression data were LOWESS normalized by array and analyzed using a mixed model approach. Results showed that expression profiles for 302 BOTL-3 genes were influenced by parturition. BLASTn analysis and preliminary clustering of affected genes by biological function indicated that the largest proportion (14%) of changed genes encode proteins critical to regulation of apoptosis. Independent confirmation of altered expression for 16 of these genes was achieved using quantitative real-time RT-PCR (Q-RT-PCR). A predominantly survival phenotype inferred from the microarray and Q-RT-PCR results was substantiated by monitoring apoptosis status of blood neutrophils from castrated male cattle cultured in the presence of sera from parturient cows. Thus our combined gene expression and apoptosis phenotyping results suggest that bovine parturition may induce prolonged survival in normally short-lived blood neutrophils.

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with >700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with ≥1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

Blood neutrophils are extremely short-lived cells that are programmed for rapid apoptosis after differentiation in bone marrow. Recently, glucocorticoids have been shown to prolong survival of human and rodent neutrophils, but the mechanisms and implications for leukocyte homeostasis and health are unclear. In this study, we investigated the effects of endogenous and exogenous glucocorticoids on Fas expression in bovine neutrophils because Fas is a major death receptor that stimulates apoptosis in circulating cells. Our study subjects were four periparturient dairy cows whose blood concentrations of cortisol peaked at calving, 15 dexamethasone-treated steers and three untreated steers whose neutrophils were exposed to dexamethasone in vitro. Fas mRNA abundance changes in collected neutrophils were monitored numerous times relative to the in vivo glucocorticoid challenges, and the relationships between these data and circulating neutrophil counts were estimated by correlation analyses. Fas mRNA and protein abundance, caspase 8 activity, and survival of neutrophils in vitro were also monitored in the presence and absence of dexamethasone. In the periparturient cows, Fas mRNA abundance in circulating neutrophils showed a sharp decrease between calving and 12 h postpartum. Based on PROC CORR analysis (SAS), this correlated negatively with blood neutrophil count (r=-0.634; P=0.0009) and serum cortisol concentration (r=-0.659; P<0.0001), but showed no relationship with serum progesterone or estradiol concentrations (P > or =0.09). Administration of dexamethasone to steers also caused a pronounced reduction in neutrophil Fas mRNA abundance that persisted for 12 h and correlated negatively with blood neutrophil count (r=-0.748; P=0.0021). In vitro, dexamethasone caused dose-dependent loss of GR proteins from the cytosol of neutrophils concurrently with Fas mRNA downregulation, which was inhibited by the glucocorticoid receptor (GR) antagonist, RU486. Dexamethasone treatment of cultured neutrophils also reduced surface Fas expression, spontaneous and sFasL-induced caspase 8 activity, and rate of apoptosis in the cells. Taken together, these in vivo and in vitro results suggest that glucocorticoids inhibit Fas expression in bovine blood neutrophils via GR activation, possibly contributing to the cells' increased longevity in culture and the pronounced neutrophilia observed in parturient cows and hormone-treated steers. We thus conclude that glucocorticoid-activated GR may change the homeostasis of circulating neutrophils, in part through its negative effects on Fas gene expression and downstream apoptosis signaling pathways.