Biomedical subjects

Jeanne L Burton

Publications and source records attributed to Jeanne L Burton.

8 recordsLinked to original sources

PubMed2006

Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils.

Neutrophils are critical for innate immune defense against microbial invasion but can also cause inflammatory tissue damage if their life span is not tightly regulated. Antiinflammatory glucocorticoids delay spontaneous apoptosis in human, rodent, and bovine neutrophils, but mechanisms involved are unknown. We hypothesized here that glucocorticoids delay neutrophil apoptosis by altering expression of key Bcl-2 apoptosis regulatory proteins, A1 and Bak, via activation of the cell's glucocorticoid receptors. To test this hypothesis, isolated bovine blood neutrophils were exposed to dexamethasone with and without glucocorticoid receptor antagonism (RU486) and aged ex vivo over 0-24 h for assessment of various spontaneous apoptosis pathway indicators and A1 and Bak abundance. Results show that dexamethasone preserved neutrophil mitochondrial membrane integrity, delayed caspase-9 activation, and reduced the rate of spontaneous apoptosis. Also, dexamethasone increased A1 and decreased Bak mRNA abundance. RU486 pretreatment of the cells abrogated each of these dexamethasone effects. Dexamethasone-induced increases in A1 mRNA were reflected in A1 protein increases, which also were observed in circulating neutrophils of dexamethasone-treated animals. Bak protein decreases were observed in neutrophils of the dexamethasone-treated animals but not in isolated neutrophils, suggesting that stimuli additional to (and perhaps regulated by) glucocorticoid are required to affect Bak protein expression changes in neutrophils. Collectively, our results are unique in demonstrating a mechanism behind glucocorticoid regulation of spontaneous apoptosis and implicate steroid receptor activation and subsequent regulation of A1 and Bak as contributors to mitochondrial membrane stability, reduced caspase-9 activity, and delayed apoptosis in bovine neutrophils exposed to glucocorticoids.

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PubMed2005

Gene expression signatures in neutrophils exposed to glucocorticoids: a new paradigm to help explain "neutrophil dysfunction" in parturient dairy cows.

Neutrophils are the first line of immunity against most pathogens that infect cattle. These normally short-lived white blood cells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into the circulation where they marginate on inflamed blood vessel endothelial cells and migrate through them into the area of infection. Once migrated, neutrophils do not reenter the circulation, but rather, perform their bactericidal functions and die by apoptosis in the tissue. The cytokine and hormonal milieu of the blood and extracellular tissue fluid can influence neutrophil development and immunity-related activities, but the molecular basis of these phenotypic changes and physiological benefits or drawbacks of them are poorly understood. In the current paper, we review new gene expression information that resulted from two of our functional genomics studies designed to evaluate effects of glucocorticoid hormones on bovine neutrophils. This work provides one model to describe complex changes that occur in neutrophils as the cells respond to glucocorticoids, which might act to alter the cells' functional priorities and tip the delicate balance between health and disease during stress, including at parturition. A bovine immunobiology microarray and real time RT-PCR were used to study blood neutrophils collected during the natural surge of endogenous glucocorticoid (cortisol) in parturient dairy cows and bone marrow neutrophils collected from glucocorticoid (dexamethasone)-treated dairy steers. The gene expression signatures we observed led us to perform additional phenotyping of the neutrophils and correlation analyses, which together painted a picture suggesting that glucocorticoids have key roles in modulating neutrophil development, life span, and tissue defense functions during parturition and hormone therapy. Based on these observations, we postulate that glucocorticoids orchestrate adaptive changes in the entire neutrophil system that support increased cell numbers and longevity in blood and heightened remodeling activity in tissues, while at the same time decreasing some important antimicrobial defense activities of the cells. Thus, our functional genomics studies have enabled us to elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the induction of parturition and partly explaining why some parturient dairy cows may experience heightened incidence and severity of inflammatory diseases like mastitis.

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PubMed2004

Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils.

One anti-inflammatory action of glucocorticoids is down-regulation of surface L-selectin on circulating neutrophils. However, it is unclear if this is a result of release of affected bone marrow neutrophils or if the steroid has direct effects on L-selectin expression in existing blood neutrophils. We recently demonstrated that circulating neutrophils from cattle with high blood concentrations of endogenous glucocorticoid had reduced L-selectin mRNA, suggesting that the steroid interrupted L-selectin gene expression. In the current study, dexamethasone (DEX) was administered to cattle in vivo, and blood and bone marrow neutrophils were studied simultaneously within the animal to determine which pool of cells responds to glucocorticoids with inhibited L-selectin expression. Purified blood neutrophils were also treated with DEX +/- RU486 in vitro, and glucocorticoid effects on L-selectin expression were determined. Our results indicate that glucocorticoid-induced suppression of L-selectin, which accompanies neutrophilia, is likely mediated by direct effects of glucocorticoid receptor activation on intracellular reservoirs of L-selectin mRNA and protein in cattle, predominantly in blood neutrophils.

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PubMed2004

Microarray analysis of gene expression in blood neutrophils of parturient cows.

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.

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PubMed2004

Effects of glucocorticoids on Fas gene expression in bovine blood neutrophils.

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.

Animals↗

PubMed2003

Development and testing of a high-density cDNA microarray resource for cattle.

A cDNA microarray resource has been developed with the goal of providing integrated functional genomics resources for cattle. The National Bovine Functional Genomics Consortium's (NBFGC) expressed sequence tag (EST) collection was established in 2001 to develop resources for functional genomics research. The NBFGC EST collection and microarray contains 18,263 unique transcripts, derived from many different tissue types and various physiologically important states within these tissues. The NBFGC microarray has been tested for false-positive rates using self-self hybridizations and was shown to yield robust results in test microarray experiments. A web-accessible database has been established to provide pertinent data related to NBFGC clones, including sequence data, BLAST results, and ontology information. The NBFGC microarray represents the largest cDNA microarray for a livestock species prepared to date and should prove to be a valuable tool in studying genome-wide gene expression in cattle.

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PubMed2003

Immunity and mastitis. Some new ideas for an old disease.

The biggest challenge for host immune defense against mastitis-causing bacteria in dairy cows is to quickly recruit large enough numbers of opsonizing molecules and mature neutrophils into milk such that intramammary pathogens are cleared before they multiply significantly and the inflammatory response gets out of control. Currently, this challenge is best facilitated when established mastitis control procedures are practiced on the farm, including proper hygiene, milking procedures, and regular administration of approved mastitis vaccines. However, mastitis is still a significant problem. New animal functional genomics research is beginning to allow scientists to solve the puzzle of mastitis susceptibility. Results of this type of research offer the hope of giant leaps toward a clear identification of molecular genetic variation and potential gene targets for therapies and immune manipulations that could significantly reduce the risk of clinical mastitis in traditionally susceptible cows.

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PubMed2002

Altered expression of cellular genes in neutrophils of periparturient dairy cows.

The periparturient dairy cow undergoes a plethora of physiological changes, including changes in the immune system that lead to profound effects on animal health. Of the immune cells affected at parturition, the neutrophil has been of particular interest due to its primary role in innate immune defense against mastitis. Immune functions of bovine neutrophils are known to be depressed around parturition, but it has not been discerned at what level these alterations occur, including the possibility that parturition induces changes in expression of key genes. The hypothesis of the present study was that blood neutrophils respond to the physiology of parturition by altering the expression of genes needed for normal cellular functions. The main objectives of the study were to detect and characterize parturition induced changes in neutrophil gene expression, to determine if altered gene expression was significantly associated with the main steroid hormones of bovine parturition, and to obtain putative identities of differentially expressed neutrophil genes. Differential gene expression was detected and characterized through mRNA abundance changes in neutrophils, and steroid hormone concentrations by RIA assay of periparturient serum samples. Preliminary assessment of differential gene expression was done using differential display reverse transcription polymerase chain reaction (DDRT-PCR) followed by secondary screening using high throughput cDNA dot blot hybridization. Altered gene expression was confirmed using Northern blot hybridization and detailed expression patterns characterized using quantitative slot blot analysis. The identities of two fully characterized transcripts with clear parturition induced repression (P< or =0.02) in neutrophils had high DNA sequence homology with genes that encode bovine mitochondrial cytochrome b (cytb) and rig/ribosomal protein S15 (rig/RPS15). These proteins are critical for normal respiratory metabolism and translation in cells, respectively. The gene expression profiles for cytb were significantly related to serum progesterone concentration (r=0.44) and for rig/RPS15 to progesterone and estradiol concentrations (r=0.35, 0.36, respectively). Eleven additional transcripts showed evidence of parturition induced repression in neutrophils and were putatively identified as representing genes of the citric acid cycle and various DNA binding proteins. Results of this study show for the first time that genes regulating basic life functions of bovine neutrophils may be repressed by parturition, possibly due to influences of steroid hormones.

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