Transcriptome and Proteome Factors Regulating Neutrophil Function in Periparturient Dairy Cows

NON-TECHNICAL SUMMARY: Mastitis is an inflammatory disease of the mammary gland caused by invading bacteria and is highly prevalent around calving in dairy cows. Mastitis costs the dairy industry approximately 2 billion dollars annually in the US and 25 billion dollars worldwide. Current mastitis treatments based on antibiotic therapy are not effective against this disease and raise important food safety and public health concerns regarding the potential introduction of bacterial antibiotic resistance. Therefore, there is an urgent need to develop alternative strategies to prevent and treat coliform mastitis especially in the periparturient cow. The white blood cell called the neutrophil is the primary cellular defense against bacteria invaders. The primary functions of neutrophils are to locate, engulf (eat) and kill invading bacteria. At calving these neutrophil functions are impaired. Therefore, the goal of this research project is to gain a clear understanding of the basic mechanisms that mediate changes in the neutrophil functionality at calving. To achieve this goal functional genomic and proteomic tools will be used to identify genes and proteins that regulate important neutrophil functions. In the future these identified genes and proteins will be used to develop novel treatments and/or prevention strategies for mastitis.
<P>

APPROACH: This project will use state of art functional genomics and proteomic technologies to answer our fundamental question. Neutrophil gene and protein expression will be monitored throughout the peripartum period using bovine microarrays for gene expression profiling and two-dimensional gel electrophoresis and high-throughput mass spectrometry for protein profiling. Bioinformatics will be used to identify significant gene and protein expression changes in periparturient dairy cows. These identified genes and proteins will then be evaluated by knockdown and over expression techniques to determine they ability to effect neutrophil phagocytosis and/or killing of mastitis causing bacteria. We believe that by identifying neutrophil genes and proteins that modulate phagocytosis and/or bacterial killing we will be able to develop effective mastitis treatments that will enhance the cow's own defense mechanism against invading pathogens.

<P>
PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: This project was disseminated through the presentation of two invited seminars. The title of the first presentation was "Microarray Analysis of the Bovine Neutrophil Transcriptome During Stress: Making Sense Out of Chaos" and was presented January 19, 2007 on the campus of Virginia Polytechnic Institute and State University for the Molecular Cell Biology & Biotechnology Program seminar series by Dr. Jeanne Burton, a major collaborator on this project. The second presentation was entitled "Neutrophil Transcriptome Changes During Stress: Friend or Foe?" presented February 4-7, 2007 at the University of Minnesota for the Comparative Biology Program seminar series. This seminar also was prepared and presented by the late Dr. Jeanne Burton. <BR>PARTICIPANTS: The key individual participants to this project are Drs. Patty Weber and the late Jeanne Burton who served as co-principal investigators/project directors and were responsible for project coordination and oversight. The key collaborators of the project are Drs Paul Coussens, Robert Halgren, Juan Pedro Steibel, Robert Tempelman, Ronald Erskine, and Lorraine Sordillo. Drs. Coussens, and Halgren facilitated the development of the new BLO-Plus array. Drs. Steibel and Tempelman serve as statistical consultants to this project and Drs. Erskine and Sordillo provide their expertise in the area of bovine mastitis, especially as it relates to periparturient dairy cows. <BR>TARGET AUDIENCES: A target audience for this project is basic researchers in the fields of animal genomics, statistics and bioinformatics for functional genomic research, as well as research scientists interested in the development of well-annotated gene expression databases for the agricultural and aquacultural animal species. Dairy producers are another target audience for this research. The project results are disseminated to these producers through the Michigan Dairy Review, an online newsletter.
<BR><BR>
IMPACT: 2007/01 TO 2007/12<BR>
The goal of this research project is to gain a clear understanding of the molecular and cellular mechanisms that mediate changes in the neutrophil function in the periparturient dairy cows. This is accomplished through the use of transcriptome and proteome profiling as well as phenotypic evaluation of neutrophil functions relative to parturition. During this reporting year we have developed an alternative bovine blood neutrophil isolation procedure that is more efficient and economical than our previous density gradient centrifugation protocol. The new protocol reduces the time required to purify neutrophils in half without a lost in neutrophil purity. Also we participated in the development of the newest generation of the Bovine Long Oligo (BLO-Plus) microarray (http://cafg.msu.edu/BLO Update/blo update.html). The BLO-Plus array consists of a total of 9600 unique, that include all 70-mer oligos from the first generation BLO, plus 70-mers representing all sequences from BOTL5, key developmental neutrophil mRNAs, and sequences from selected KEGG pathways. All sequences represent predicted bovine mRNAs, and are spotted in duplicate on the array. BLO-Plus also includes long oligo set for ten bovine control genes and ten Stratagene Alien Genes spotted multiple times across the array. This new BLO-Plus array has been spotted and is being validated presently. The BLO-Plus array will be useful tool in the forth-coming gene expression profiling experiments. Finally, we have continued our quest to identify specific antibodies that cross react with key bovine neutrophil proteins for use in immunoprecipitation and Western Blot Analysis. This year we have tested 8 antibodies from Santa Cruz Biotechnology for cross reactivity in bovine neutrophil whole cell lysates, as well as in cytosolic and nuclear fractions. Human polyclonal antibodies to Bactericidal/permeability-increasing protein, glucocorticoid receptor, c-Jun and NFkappa-B have been shown to cross react with these proteins in bovine neutrophil samples. These antibodies will enhance our research effort towards understand protein expression changes in neutrophils across the peripartum period in dairy cows.
<BR><BR>

PROGRESS: 2006/01/01 TO 2006/12/31<BR>
Blood neutrophils from stressed cattle have several functional changes relative to cells collected before stress occurs. Parturition is one such stressful event and is the primary research model under study in our program. Previous research from our group and others has shown that neutrophils from parturient cows take up inert particles as well or better than neutrophils from prepartum animals, but that the capacity of the cells for bacterial uptake and killing is reduced. This has been blamed for the increased susceptibility to opportunistic bacterial infections and diseases in cows shortly after calving. However, little is known about the causative factors involved or the molecular bases for parturition stress-induced neutrophil changes and disease susceptibility. Over the past two years we have documented that neutrophils from unstressed cattle express abundant cytosolic receptors for the main stress hormone, glucocorticoid, and that these receptors respond to glucocorticoid challenge with rapid (minutes) translocation into the cell's nucleus. We also have shown that the nuclear translocation of glucocorticoid receptors in neutrophils is associated with transcriptional regulation of more than 200 genes encoding a variety of proteins involved in inflammation regulatory pathways. Most recently, we documented several of the main functional classes of genes that are affected when bovine neutrophils are directly exposed to glucocorticoids in vitro, including those that mediate the cell's protective stress response (e.g., Symplekin, MT-II, HSPs), adhesion to and migration through blood vessels (e.g., CD62L, CD18), tissue extracellular matrix degradation and remodeling (e.g., MMPs, TIMPs, TGF-beta, betaglycan), apoptosis (e.g., Fas, FasL, caspase 8, A1, Bak, Blimp-1, TOMM70A, TFAR-19), mitochondrial electron transport chain activity (e.g., cyto b, PSST), wound healing (e.g., PPAR-gamma), and antibacterial activities (e.g., BPI, LC3). Together, the neutrophil gene expression signature we have documented suggests that glucocorticoid genetically reprograms circulating neutrophils into a state of final maturation that involves their altered tissue trafficking and extended life span with increased potential for facilitating tissue remodeling and wound healing but with changed capacity for fighting bacteria. The biological reason for this occurring during the surge in endogenous glucocorticoid at parturition is still unknown but we speculate that it may be connected to need of the reproductive tract for long lived neutrophils that facilitate processes related to labor onset, delivery, and subsequent organ involution.
<BR><BR>
IMPACT: 2006/01/01 TO 2006/12/31<BR>
In dairy cows, immune defense against diseases caused by opportunistic bacteria absolutely depends upon aggressive antibacterial activities of blood-derived neutrophils, but these are depressed around calving time when disease susceptibility is high. Thus, prevention of ever-prevalent parturient diseases, such as mastitis and metritis, has been elusive. This has led to societal concerns over the welfare of dairy cows and contamination of the environment and dairy foods with antibiotics and other drugs, and continues to cost the US dairy industry multibillions of dollars in lost annual revenues. While parturition stress-induced tissue remodeling and wound healing activities of blood neutrophils documented in this project might to be pivotal to the processes of calving itself, it appears from results of this work that these may come at the cost of effetive bacteria fighting capacity of the cells. Confirming this through results of future research could lead to development of novel preventatives and therapeutics that target and exploit the affected neutrophil gene pathways we have identified, leading to better disease control, safer dairy food products, and economic prosperity for the UAS dairy industry.
<BR><BR>
PROGRESS: 2005/01/01 TO 2005/12/31<BR>
Blood-derived neutrophils are the first line of immunity against most pathogens that infect cattle. These white blood cells develop in bone marrow and are released into the blood circulation as short-lived, terminally differentiated cells. In the circulation, neutrophils marginate on blood vessel endothelial cells in search of infection and migrate through them into the infection focus under the influence of pro-inflammatory cytokines. Once migrated, neutrophils perform their bactericidal functions in the tissue and die there by apoptosis without causing inflammatory damage. However, factors such as heightened cytokine levels that prolong neutrophil life span also contribute to exaggerated inflammatory responses by the cells leading to severe tissue damage. In dairy cows, parturition dramatically increases susceptibility to severe acute mammary gland inflammation, called mastitis. Mastitis is caused by bacterial invasion of the teat canal but neutrophils migrating to the site normally contain the infection and die rapidly to prevent further tissue damage. The same is not true at parturition, though the reasons for this are unknown. Bovine parturition appears to be extremely pro-inflammatory for circulating neutrophils. For example, we have observed significantly higher concentrations of serum TNF-a, G-CSF, and MMP-9 at parturition than before or after parturition. These factors are well known to stimulate pro-inflammatory activities of neutrophils, including margination, migration, apoptosis delay, and tissue extracellular matrix degradation. We have also documented a dramatic increase in serum glucocorticoid and a drop in progesterone at bovine parturition, which correlated with extended longevity of neutrophils and increased blood neutrophil counts, both pro-inflammatory conditions. Gene expression analyses of neutrophils from parturient cows further supported that parturition induces a pro-inflammatory scenario for neutrophils. Currently, in vitro experiments are underway to determine if the neutrophil gene expression signature we have documented at parturition leads to altered inflammatory behaviors of the cells, and if so, which blood factors might have induced these behaviors. Results to date suggest that bovine parturition comes with complex changes in circulating factors that influence the pro-inflammatory program of blood neutrophils, possibly altering the cells' life span and tissue degrading activities and tipping the delicate balance between health and disease.
<BR><BR>
IMPACT: 2005/01/01 TO 2005/12/31<BR>
Blood neutrophils are critical to host defense against bacterial infections, and regulate inflammation and tissue remodeling/repair in sick or injured animals. In dairy cows, defense against mastitis causing bacteria absolutely depends upon aggressive bactericidal activities of blood-derived neutrophils as well as rapid apoptotic death of spent cells in the infection focus. Prevention of this ever-prevalent disease has been elusive, leading to poor welfare of affected animals, lost productivity in affected herds, billions of dollars of lost annual revenues for the US dairy industry, and antibiotic contamination of the environment and dairy foods. Because neutrophil life span and tissue degrading activity and the cytokines and hormones that regulate these activities are pivotal control points in the resolution of inflammation, results of this project may soon elucidate novel neutrophil and serum factors as possible targets for development of non-antibiotic preventatives and therapies for better control mastitis in postpartum cows and leading to improved animal welfare, farm economies, and safer dairy food products.
<BR><BR>
PROGRESS: 2004/01/01 TO 2004/12/31<BR>
Blood neutrophils from stressed cattle, including parturient cows, do not perform as well as neutrophils from non-stressed animals in some laboratory assays of bactericidal function. While little is known about the molecular bases for these stress-induced neutrophil changes, nor the biological reason why they occur, we have recently shown that the cells express abundant cytosolic receptors for stress glucocorticoids and that these receptors respond to glucocorticoid challenge in vitro with nuclear translocation and transcriptional down regulation of two important neutrophil inflammatory genes, CD62L and Fas. These same gene expression changes were observed in blood neutrophils during experimental challenge of cattle with glucocorticoids, and at calving when there is a normal surge in endogenous glucocorticoid release into blood. In this project we used cDNA microarray and other more quantitative gene expression techniques to explore additional genes influenced by glucocorticoids. Neutrophils were collected from the mature circulating pool in blood of parturient cows, and from the pool of developing cells in bone marrow of hormone-treated steers. Total RNA isolated from these cells was reverse transcribed into cDNA and sequentially labeled with Cy3 and Cy5 prior to paired hybridizations on series of BOTL microarrays (http://www.cafg.msy.edu) using loop designs. Resulting gene expression data were LOESS normalized by array and analyzed using a mixed model approach. Significant (P < 0.05) parturition-induced expression changes were observed in 302 genes, 53 of which when pursued in further gene and protein expression and phenotype validation experiments told a story of dramatically extended cell survival with a newly acquired capacity for massive tissue extracellular matrix degradation but partially inhibited bactericidal activity. Many of these gene expression changes were correlated with blood glucocorticoid concentrations, and could be replicated by treating neutrophils from unstressed donor steers with parturient serum or purified steroid in vitro. Preliminary analysis of the bone marrow RNA revealed that glucocorticoid also has pronounced positive effects on the development of neutrophils from myeloid-lineage cells. This, in combination with the gene expression signature we observed in mature blood neutrophils of parturient cows could explain why parturition and glucocorticoid treatment are associated with pronounced increases in blood neutrophil counts. While we still do not know the precise biological reason for such glucocorticoid-induced neutrophilia, we are currently testing a hypothesis that it relates to a requirement for massive neutrophil recruitment and tissue remodeling activity in the reproductive tract and placenta in parturient cows. Thus, our combined observations to date suggest that glucocorticoid is a key hormone of parturition that orchestrates adaptive changes in the entire neutrophil system in support of reproductive track remodeling but which may also leave peripheral tissues devoid of innate immune defense against opportunistic bacteria, such as the coliforms that cause severe mastitis in many newly calved cows.
<BR><BR>
IMPACT: 2004/01/01 TO 2004/12/31<BR>
Blood neutrophils are critical to host defense against bacterial infections, and regulate inflammation and tissue remodeling/repair in sick or injured animals. In dairy cows, defense against mastitis causing bacteria absolutely depends upon aggressive bactrericidal activities of blood-derived neutrophils. Prevention of this ever-prevalent disease has been elusive, leading to poor welfare of affected animals, lost productivity in affected herds, billions of dollars of lost annual revenues for the US dairy industry, and antibiotic contamination of the environment and dairy foods. Because extended longevity and enhanced tissue degrading activities of blood neutrophils appears to be pivotal to reproductive tract/placental remodeling that allows parturition to proceed but may also contribute to the severity of mastitis around calving, results of our research could soon lead to development of new management tools and strategies that exploit these neutrophil gene expression pathways for better mastitis control and safer dairy food products.

<BR><BR>

PROGRESS: 2003/01/01 TO 2003/12/31<BR>
It is well documented that blood neutrophils from parturient dairy cows do not perform as well as neutrophils from non-parturient 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 4 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 1056 member bovine total leukocyte (BOTL-3) microarrays in a loop design. Resulting gene expression data were LOESS 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 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. This experiment showed that neutrophils from the donor steers survived significantly longer in parturient serum than in pre- or post-partum serum. Because glucocorticoids have potent effects on expression of inflammatory genes and fluctuate dramatically in blood at parturition, we hypothesized that this class of steroids was responsible for the neutrophil survival induction ex vivo. To test this we divided a pool of serum from 4 cows (collected at parturition) into two aliquots, left one aliquot untreated (P; with 29,000 pg/ml cortisol, 1500 pg/ml estradiol, 900 pg/ml progesterone), and treated the other aliquot with activated charcoal to extract steroids (E; with 150 pg/ml cortisol, 75 pg/ml estradiol, 110 pg/ml progesterone). Neutrophils survived significantly (P < 0.001) longer in P than E when apoptosis was assessed flow cytometrically by annexin-V-FITC binding and propidium iodide (PI) staining, as well as PI staining in conjunction with RNaseA treatment (to examine nuclear fragmentation). Reconstitution of E with cortisol and dexamethasone, but not with progesterone or estradiol, resulted in survival induction near levels achieved with P. The glucocorticoid receptor antagonist, RU486, inhibited the survival-inducing capacity of P, E + cortisol, and E + dexamethasone. Thus, our combined gene expression and apoptosis phenotyping results suggest that the glucocorticoid surge of bovine parturition induces prolonged survival in normally short-lived blood neutrophils by reprogramming the cell's apoptosis gene systems. Implications of extended neutrophil survival for disease susceptibility in parturient cows awaits further investigation.
<BR><BR>
IMPACT: 2003/01/01 TO 2003/12/31<BR>
Blood neutrophils are critical to host defense against bacterial infections, and regulate inflammation and tissue remodeling/repair in sick or injured animals. In dairy cows, defense against mastitis, retained placenta and metritis at calving absolutely depends upon functional longevity of blood neutrophils. Prevention of these ever-prevalent diseases has been elusive, leading to poor welfare of affected animals, lost productivity in affected herds, billions of dollars of lost annual revenues for the US dairy industry, and antibiotic contamination of the environment and dairy foods. Because the life-death balance of blood neutrophils is pivotal to mammary gland and reproductive tract health at calving, results of our research could soon lead to development of targeted preventatives and useful management strategies that exploit neutrophil apoptosis pathways for better control of mastitis, retained placenta, and metritis.
<BR><BR>
PROGRESS: 2002/01/01 TO 2002/12/31<BR>
Past work has shown that parturition has pronounced negative effects on bovine immune cell functions, culminating in increased susceptibility to production diseases in high producing dairy cows. However, the causes and genetic mechanisms leading to immunosuppression and disease susceptibility in periparturient dairy cows are complex and largely unknown. In other studies we have demonstrated that bovine leukocytes express receptor mRNA for steroid hormones of reproduction, including glucocorticoids, which are known to influence expression of many genes in other cell systems. The overall hypothesis of the current study was that bovine leukocyte sensitivity to glucocorticoid hormones exhibits genetic variation. If true, this could help explain genetic variation in susceptibility to production diseases such as mastitis and metritis. Test animals included 60 pedigreed Holstein bulls treated on three consecutive days with a synthetic glucocorticoid (dexamethasone) and 5 untreated control bulls. Five indicator traits of neutrophil glucocorticoid sensitivity (circulating neutrophil counts and two measures on each of CD62L and CD18 expression) and eight traits of mononuclear leukocyte sensitivity to glucocorticoid challenge (percentages and mean expressions of MHC I and MHC II, and percentages of CD4 T cells, CD8 T cells, gamma delta T cells, and B cells) were monitored before, during, and after the glucocorticoid challenge. Random regression models with treatment-specific serial correlation were used to estimate genetic and non-genetic sources of variation at these times. Results from the neutrophil traits are complete and the mononuclear leukocyte traits are currently under analysis. Significant genetic variation was observed for neutrophil CD18 expression, with longitudinal heritability estimates ranging from 0.10 to 0.54 and influenced by glucocorticoid administration. Significant genetic variation was also observed for blood neutrophil counts (heritability estimates ranging from 0.11 to 0.24) but was not influenced by glucocorticoid administration. Estimated genetic correlations between circulating neutrophil counts and various indicators of CD62L and CD18 expression were large and negative (-0.44 to -0.78). These results imply significant genetic variability and pleiotropic effects for neutrophil traits that are important for stress-induced disease susceptibility in dairy cattle and which might be exploited by genetic selection. Results from the mononuclear leukocyte traits should be complete in 2003.
<BR><BR>
IMPACT: 2002/01/01 TO 2002/12/31<BR>
Successful genetic improvement of immunocompetence and disease resistance in dairy cattle depends on the availability of biologically relevant traits and (or) molecular genetic markers on which to base genetic selection. We have identified several neutrophil traits, previously shown to be related to mastitis susceptibility in glucocorticoid-stressed cattle, that exhibit significant genetic variation in a small group of pedigreed Holstein bulls. These traits warrant further investigation for potential usefulness as indicators of health status of the daughters of these bulls.

<BR><BR>

PROGRESS: 2001/01/01 TO 2001/12/31<BR>
Past work has shown that parturition has pronounced negative effects on bovine immune cell functions, culminating in increased susceptibility to production diseases in high producing dairy cows. However, the causes and genetic mechanisms leading to immunosuppression and disease susceptibility in periparturient dairy cows are complex and largely unknown. In other studies we have demonstrated that bovine leukocytes express receptor mRNA for the steroid hormones of reproduction, which are known to influence expression of many genes in other cell systems. Therefore, our overall hypothesis of the current study was that bovine leukocytes become dysfunctional around parturition due to negative influences of steroid hormones on expression of key cellular genes. To begin testing this hypothesis, we collaborated with the Center for Animal Functional Genomics (Animal Science, Michigan State University) to develop a size selected normalized bovine total leukocyte (BOTL) cDNA library (http://gowhite@ans.msu.edu) for cDNA microarray analysis of gene expression changes in bovine leukocytes. From this library, 1000 cloned inserts were sequenced and 724 of these representing unique expressed sequence tags (ESTs) spotted in triplicate onto first generation cDNA microarrays. Leukocyte RNA samples from one parturient Holstein cow collected 14 days before and 6 hours after parturition were reverse transcribed into cDNA, labeled with Cy5 (14 days before) or Cy3 (6 hours after) fluorescent dyes, the labeled samples mixed together, and the mixture used to interrogate the cDNA microarray. Results demonstrated 2-fold or greater expression 14 days prepartum compared to 6 hours postpartum (P values less than 0.05) for 18 of the spotted ESTs. This finding begins to support our hypothesis by showing that parturition negatively influenced gene expression in bovine leukocytes. DNA sequences for these 18 ESTs were subjected to BLASTn analysis, which revealed that the genes represented by them are involved in normal cellular functions such as signal transduction, transcription, energy metabolism, fatty acid synthesis, and enzymatic degradation in lysosomes. One classic immune response gene encoding the beta-chain of the MHC II complex used for antigen presentation in macrophages and B lymphocytes was also dramatically down regulated in expression following parturition. Future experiments related to this study will utilize RNA from isolated bovine leukocyte populations (neutrophils, T and B lymphphocytes, monocytes) of at least 12 cows, and second generation microarrays containing approximately 250 additional ESTs (from genes known to be involved in innate, humoral, and cell-mediated immune responses), to determine which gene expression changes are influenced by parturition in which cell types. We also plan to test what factors of parturition might be inducing the gene expression changes we observe (e.g., steroid hormones).
<BR><BR>
IMPACT: 2001/01/01 TO 2001/12/31<BR>
White blood cells called neutrophils must be able to make contact with blood vessel walls to protect underlying tissues from bacterial infections. This process depends on expression of L-selectin molecules by the neutrophils. Our work shows that stress hormones inhibit the expression of L-selectin in bovine neutrophils and that this causes increased susceptibility to infectious diseases, such as mastitis of the mammary gland. We are currently studying the mechanisms by which stress hormones reduce L-selectin expression in bovine neutrophils. It is anticipated that increased knowledge in this area will open doors to possibilities for discovery of practical strategies that will allow dairy producers to better manage infectious diseases in cows during unavoidable periods of husbandry stress.
<BR><BR>

PROGRESS: 2000/01/01 TO 2000/12/31<BR>
Past work has shown that glucocorticoid hormones negatively affect bovine neutrophils causing increased disease susceptibility, such as mastitis in parturient dairy cows. We have demonstrated that bovine neutrophils express abundant cytosolic glucocorticoid receptors, thus explaining the sensitivity of these cells to glucocorticoids. Bovine neutrophils exposed to glucocorticoids at parturition or following hormone injections lose ability to contact blood vessels and to migrate into infected tissues. A critical molecule involved in endothelial capture of blood neutrophils is L-selectin. Surface expression of L-selectin is dramatically down regulated on bovine neutrophils in response to glucocorticoids, leading to pronounced neutrophilia and increased susceptibility to mastitis. In the current study we were curious to know if glucocorticoids mediate L-selectin down-regulation at a gene expression level. We used two models of glucocorticoid challenge to test this possibility, parturient dairy cows and hormone-treated dairy steers. A 711 bp bovine L-selectin cDNA probe that hybridized with a single 3kb transcript during Northern blot analysis of RNA from bovine neutrophils and other immune cells was developed and used in this study. Blood was sampled from 4 primiparous test cows before, at, and after parturition and RNA isolated from enriched neutrophils (greater than 95 percent pure) and subjected to Northern blot analysis. Results showed clear reductions in L-selectin mRNA levels at parturition, so quantitative slot blot analysis was performed to allow statistical analysis of the parturition effect. Blots were probed with the L-selectin cDNA, stripped, and re-probed with a beta-actin cDNA, and L-selectin abundance expressed as the L-selectin-to-beta-actin mRNA ratio. When analyzed statistically, L-selectin mRNA levels were shown to be significantly reduced from calving through d 7 of lactation, in strong negative correlation with blood glucocorticoid concentrations and circulating neutrophil counts. These results suggested that the glucocorticoids of parturition acted on L-selection expression at a pre-translational level. To substantiate this, a synthetic glucocorticoid (dexamethasone) was administered into one dairy steer and L-selectin mRNA abundance monitored 24 h post hormone administration. One untreated steer was used as a control. Results again showed a dramatic reduction in L-selectin mRNA abundance following glucocorticoid challenge, which was highly correlated with down regulated surface expression and pronounced neutrophilia. Together with the parturient cow data, we concluded from this work that L-selectin down-regulation in response to glucocorticoids occurs at a pre-translational level in bovine neutrophils. Our future work will determine if reduced L-selectin mRNA is due to reduced rates of L-selectin gene transcription in neutrophils of glucocorticoid-challenged dairy cattle, and if hormone-activated glucocorticoid receptors mediate this effect. If so, we may have identified a key molecular pathway linking husbandry stress with disease susceptibility in dairy cattle.
<BR><BR>
IMPACT: 2000/01/01 TO 2000/12/31<BR>
White blood cells called neutrophils must be able to make contact with blood vessel walls to protect underlying tissues from bacterial infections. This process depends on expression of L-selectin molecules by the neutrophils. Our work shows that stress hormones inhibit the expression of L-selectin in bovine neutrophils and that this causes increased susceptibility to infectious diseases, such as mastitis of the mammary gland. We are currently studying the mechanisms by which stress hormones reduce L-selectin expression in bovine neutrophils. It is anticipated that increased knowledge in this area will open doors to possibilities for discovery of practical strategies that will allow dairy producers to better manage infectious diseases in cows during unavoidable periods of husbandry stress.
<BR><BR>

PROGRESS: 1999/01/01 TO 1999/12/31<BR>
Glucocorticoid hormones negatively influence the phenotype of immune cells during stress, which culminate in increased susceptibility to infectious diseases. We hypothesized that elevated glucocorticoids at calving contribute to immunosuppression and susceptibility to bacterial infections of the mammary gland, reproductive tract, and respiratory system in dairy cows. Glucocorticoids mediate their actions on cells via cytoplasmic receptors (GR), so one objective of our project was to determine a possible link between leukocyte GR and immunity at parturition. We developed a GR fluorescent labeling technique and flow cytometric analysis for this work. Blood was sampled multiple times before, at, and after parturition in 13 cows and in 10 mid lactation control cows. GR expression was recorded as the mean fluorescence intensity (MFI) of labeled GR in neutrophils, monocytes, and lymphocytes. The MFI values were reduced in all leukocyte populations from 7 days before to 7 days after calving, with extreme nadirs occurring for 24 h following calving. Nadir values correlated with peak glucocorticoid levels at calving and with dramatic increases in circulating neutrophil counts. Results implied that leukocyte GR become occupied by endogenous glucocorticoids around parturition, which is consistent with known negative effects of ligand-activated GR on leukocyte phenotypes that render the host susceptible to acute bacterial infections. Therefore, we concluded that GR may be one link between parturition, immunosuppression, and disease susceptibility in dairy cows. The second and ongoing objective of this work was to determine if a genetic basis for glucocorticoid-altered leukocyte phenotypes exists in cattle. Blood neutrophil expression of the adhesion molecules CD62L and CD18 were the first traits studied; both molecules are required for timely inflammatory responses that contain acute infections. Dexamethasone, a synthetic glucocorticoid, was administered 3 times into 60 pedigreed Holstein bulls and blood sampled multiple times before, during, and after the injections. Neutrophil CD62L and CD18 were monitored by immunostaining and flow cytometry. Data were expressed as MFI. Bulls were identified by sire and maternal grandsire and had known BoLA-DRB3 genotypes. A mixed effects smoothing spline model in the context of ASREML was used to assess genetic and environmental variance; fixed effects were treatment, group, BoLA-DRB3 alleles; covariates were day, body weight; random effects were bull, spline, lack of fit for the spline, and errors. Appropriate two-way interactions were also included. Daily heritability estimates considered both random parametric and non-parametric effects. Dexamethasone profoundly down-regulated CD62L but had more subtle negative effects on CD18 MFI. The hormone exposed additive genetic variation in CD62L MFI (peak h2 of 0.20 when CD62L down-regulation was maximum) but did little to influence genetic variance in CD18 MFI, which always was moderately heritable (h2 greater than 0.13). Preliminary conclusions are that neutrophil CD62L sensitivity to glucocorticoids and basal CD18 expression could be altered by genetic selection.
<BR><BR>
IMPACT: 1999/01/01 TO 1999/12/31<BR>
Preliminary results from this project implicate the stress hormones, glucocorticoids, in suppression of immunity at calving dairy cows. Furthermore, there appears to be a genetic basis for immune cell sensitivity to glucocorticoids, at least for traits related to acute inflammation. Therefore, it may be feasible to alter immunosuppression phenotypes of parturient cows via genetic selection. If so, this could be one way to permanently improve health of parturient cows.
<BR><BR>

PROGRESS: 1998/01/01 TO 1998/12/31<BR>
Mastitis is a bacterial invasion of the bovine mammary gland that requires neutrophils for its defense. Antibodies can also be important in as much as they facilitate neutrophil killing of mastitis-causing bacteria. Neutrophil functions and antibody responses are suppressed at parturition, a time blood cortisol and mastitis susceptibility are high. Physiological and genetic linkages between periparturient immunosuppression and mastitis susceptibility have been casually implied but expense of the in vitro assays and disease challenges to monitor these traits has precluded rigorous statistical and genetic analyses. Also, the biological significance of these assays is unknown. The current MAES project attempts to overcome these limitations and to answer some of the basic mechanistic questions on periparturient immunosuppression and mastitis susceptibility. Hypotheses of this project are: (1) immunosuppression and mastitis susceptibility are linked in a cause-and-effect relationship in periparturient dairy cows; (2) glucocorticoids and the glucocorticoid receptor (GR) are intimately involved in this relationship; and (3) polymorphism in GR and related genes underlie population variation in this relationship. Neutrophils of cows stressed by glucocorticoid injections or parturition lose their ability to migrate from blood. One mechanism of glucocorticoid-induced inhibition of neutrophil migration has been elucidated by the PI and her colleagues as acute down-regulation of the neutrophil adhesion molecule, CD62L. Under the current project, the PI discovered that bovine neutrophils contain large numbers of the receptor for glucocorticoids, called GR, and that GR is down-regulated at calving when blood cortisol is high and CD62L is repressed. These data are the first that circumstantially link GR with neutrophil migration dysfunction in periparturient cows. Current research under this project will determine whether down-regulation of neutrophil GR and CD62L are linked at the gene transcription level in neutrophils via glucocorticoid-activated GR. To do this work, the bovine GR cDNA has be isolated, cloned, and is currently being sequenced in the PI's laboratory. Research under this project has also revealed significant additive genetic variation in glucocorticoid-induced down-regulation of neutrophil CD62L, with heritability estimates as high as .55. To elucidate the molecular basis of this genetic variation, hypothesis testing has been taken to the level of DNA polymorphism identification in a first of several candidate genes, the bovine GR gene. To date, two polymorphisms have been identified in a protein-coding region of the gene that encodes a functional domain in the protein receptor that is required for transactivation. These polymorphisms seem to segregate in 5 dairy and beef breeds that have variable susceptibility to mastitis. Segregation and association with milk SCC of these RFLPs in families of two major dairy breeds are currently being pursued. Large groups of highly pedigreed and American and Danish cattle are being used for this purpose.