Differential expression analysis of proteins from neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows

Neutrophils play an important role in the host immune system's defense against pathogens. It has been established that neutrophil functionality is suppressed in dairy cows at parturition. The periparturient immunosuppression seen in dairy cattle is associated with an increase in the incidence of mastitis. Using amine-reactive isobaric tagging reagents we have measured relative protein expression from normal prepartum neutrophils and neutrophils isolated during immunosuppression at parturition. We found over 40 proteins that are differentially expressed at parturition compared to prepartum. In addition, we measured relative protein expression from normal neutrophils and neutrophils obtained from cows treated with an immunosuppressive dose of dexamethasone. We found over 70 proteins are differentially expressed during dexamethasone treatment. We then compared protein expression changes in dexamethasone-induced immunosuppression to periparturient immunosuppression. A number of proteins underwent similar expression changes in both dexamethasone and periparturient immunosuppressed neutrophils. Most significantly, we found a significant number of proteins whose relative expression was not the same for these two different conditions that cause neutrophil dysfunction. The data demonstrates that there are both similarities and differences in neutrophil protein expression in the naturally occurring immunosuppression observed at parturition compared to dexamethasone-induced immunosuppression in the bovine neutrophil.     

An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cows

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 > or = 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.     

Glucocorticoid receptor down-regulation in neutrophils of periparturient cows

OBJECTIVE: To determine effects of parturition on glucocorticoid receptor (GR) expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts in periparturient dairy cows. ANIMALS: 23 Holstein cows. PROCEDURE: Blood samples were collected from 8 multiparous and 5 primiparous periparturient cows at various times from 28 days before parturition until 14 days after parturition. Glucocorticoid receptor expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts were determined. Results were compared with results from control samples obtained from 5 multiparous and 5 primiparous Holstein cows in midpregnancy. RESULTS: Neutrophils from periparturient cows had 49% reduction in GR expression at calving, compared with GR expression 2 to 4 weeks before calving, and 39% reduction, compared with neutrophils from cows in midpregnancy. Reduction in neutrophil GR expression began 1 week before calving and was most severe at calving and 24 hours after calving; a significant difference in GR expression was detected between primiparous and multiparous cows. Serum cortisol concentrations and total leukocyte and neutrophil counts were significantly increased at calving and returned to baseline values by 24 hours after calving. Significant negative correlations were detected between neutrophil GR expression and serum cortisol concentration, total leukocyte count, and neutrophil count. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced GR expression in blood neutrophils of periparturient dairy cows was associated with increased serum cortisol concentrations, leukocytosis, and neutrophilia. Thus, GR down-regulation in neutrophils may be involved in periparturient neutrophil dysregulation and may cause increased susceptibility to mastitis.     

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.     

Proteomic survey of bovine neutrophils

Mastitis is a major economic concern for the dairy industry. Conditions such as parturition cause a transient immunosuppression that leads to increased incidence of mastitis. One facet of periparturient immunosuppression is a functional impairment of the blood and milk neutrophils in dairy cows. To better understand the biology of the bovine neutrophil we report the first proteomic analysis of the bovine neutrophil. We have identified over 250 proteins using one-dimensional electrophoresis followed by reverse-phase chromatography in line with electrospray tandem mass spectrometry. A large number of metabolic proteins were identified, including most of the enzymes required for generation of NADPH and ATP. In addition, many proteins were identified that participate in cell mobility and phagocytosis. All the bovine members of the cathelicidin family were identified, as well as other proteins with immunological functions. Proteins important for cell signaling, vesicular transport, control of apoptosis and other functions were identified giving an overview of the bovine neutrophil proteome.     

Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.     

Alterations in bovine neutrophil function during the periparturient period

Neutrophils from 8 Holstein heifers were evaluated for function during the periparturient period. Random migration, ingestion of bacteria, superoxide anion production, native (nonenhanced) chemiluminescence, iodination, and antibody-dependent, cell-mediated cytotoxicity by neutrophils were determined. Foremilk samples were evaluated for bacteria. Significant (P less than 0.05) increases in random migration of neutrophils, iodination, and chemiluminescence were evident 2 weeks before parturition and then decreased dramatically by the first week after parturition. These impairments of neutrophil function after parturition may be manifested as a severe cumulative deficit in the native defenses afforded by the neutrophil.     

Influence of sex steroids on the viability and CD11b, CD18 and CD47 expression of blood neutrophils from dairy cows in the last month of gestation

In the period around parturition, cows experience an increased susceptibility for the development of Escherichia coli mastitis. This increased susceptibility has been correlated with a decreased functionality of neutrophils. In the current study, it is suggested that the decreased neutrophil functionality may be induced by the extensive alterations in sex steroid levels occurring around parturition. It was first hypothesized that 17beta-estradiol and progesterone influence the viability, apoptosis and necrosis of blood neutrophils from cows in their last month of gestation. Subsequently, it was hypothesized that 17beta-estradiol modulates the expression of CD11b, CD18 or CD47 thereby explaining its influence on the migration of bovine neutrophils. Neither 17beta-estradiol nor progesterone significantly influenced viability, apoptosis or necrosis in spontaneous apoptosis conditions. However, when apoptosis was induced with TNF-alpha and gliotoxin, progesterone exerted a survival effect (P<0.05). In addition, 17beta-estradiol treatment of bovine blood neutrophils significantly decreased the expression of CD47 (P<0.05) but not of CD11b or CD18. It can be concluded that 17beta-estradiol and progesterone do not affect spontaneous apoptosis of bovine blood neutrophils while a survival effect was observed for progesterone on induced neutrophils apoptosis. Moreover, our results concerning the influence of 17beta-estradiol on the CD11b, CD18 and CD47 expression extend previous demonstrations of the suppressive effect of 17beta-estradiol on neutrophils migration and indicate that the altered expression of CD47 may contribute to this phenomenon.     

Expression of bovine parathyroid hormone/parathyroid hormone- related protein (PTH/PTHrP) receptor mRNA in the mammary gland of cows

To examine the PTH/PTHrP receptor in the mammary gland, molecular cloning of bovine PTH/PTHrP receptor and measurement of its mRNA expression were carried out in cows during the periparturient period. The PTH/PTHrP receptor gene was partially cloned, and expression of bovine PTH/PTHrP receptor mRNA was detected in various tissues of the cow. In the mammary gland, PTH/PTHrP receptor mRNA expression was constantly low during the periparturient period, whereas PTHrP mRNA expression dramatically increased after parturition. This suggested that expression of PTH/PTHrP receptor mRNA in the mammary gland is not affected by lactation in cows.     

Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination, and anti-bacterial function

The transportation of beef cattle results in a stress response that is associated with increased susceptibility and severity of respiratory diseases, presumably due to an alteration in immune function. Neutrophils are phagocytic immune cells important in lung defense and are also targets of the stress response. The objective of this study was to determine if a 9h transportation of young bulls by road induced changes in the expression of candidate genes known to be important in neutrophil-mediated defense and inflammation in the lung. These neutrophil genes encompassed functions of apoptosis (A1 and Fas), tissue remodeling (MMP-9), vascular margination (L-selectin), bacterial killing (BPI), and wound healing (betaglycan), as well as responsiveness of the cells to stress-induced increases in glucocorticoid hormones (GRalpha). To explore gene expression changes, blood was collected, plasma harvested, and neutrophils isolated from six Belgian Blue x Friesian bulls (231+/-7.0 kg in weight; 282+/-4 days of age) at -24, 0, 4.5, 9.75, 14.25, 24, and 48h relative to commencement of a 9h road transportation by truck. Plasma cortisol concentrations were elevated at 4.5 and 9.75h, peaking at 50.64+/-4.46 ng/mL (P<0.0001) and confirming that the animals experienced stress. Blood neutrophil count was elevated between 4.5 and 14.25h (P<0.0001), reaching a peak that was over 3-fold higher than the -24h concentration. Neutrophil Fas gene expression was acutely down-regulated (P=0.02) by transportation stress, while expressions of MMP-9, l-selectin, and BPI were profoundly up-regulated (P=0.003, 0.002, and <0.001 respectively). However, no changes in neutrophil expressions of betaglycan, GRalpha, and A1 were detected. It is concluded that a 9h transportation of young bulls induces a gene expression signature in blood neutrophils that increases their circulating numbers and may enhance their pro-inflammatory and anti-bacterial potential.     

Dry period management and optimization of post-partum reproductive management in dairy cattle

Dry period and early post-partum management are decisive factors for fertility in lactating dairy cows. Previous studies have shown that decreased dry matter intake (DMI) and increased non-esterified fatty acids (NEFA) negatively affect fertility and subsequent milk production. The traditional dry period decreases DMI prior to parturition, resulting in a decrease in energy intake. A negative energy balance increases NEFA concentration, and increased NEFA may impair the immune system, especially by decreasing neutrophil function prior to parturition. Earlier studies have shown that post-partum health disorders, including retained placenta and metritis, were correlated with periparturient neutrophil function. In addition, decreased DMI is also linked to a reduced body condition score (BCS) in dairy cows. These events in the periparturient period negatively affect fertility. Some manipulation, such as shortening the dry period, may be a solution to increased DMI in the periparturient period, preventing post-partum disorders and subsequent fertility issues. This article aims to explain the effects of shortening the dry period on reproduction and early post-partum treatments to improve fertility. In addition, timed artificial insemination protocols will be discussed for use during the post-partum period to improve fertility in dairy cows.     

Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle.

L-selectin (CD62L) gene expression in neutrophils is commonly referred to as "constitutive" because circulating neutrophils require a constant supply of this adhesion molecule for continuous trafficking into peripheral tissues. Under normal circumstances, marginating blood neutrophils and neutrophils that become activated for migration into infected tissues rapidly shed surface CD62L that is ligated to the vascular endothelium. However, this does not shut down CD62L gene expression because these cells continue to express surface CD62L. In contrast, glucocorticoid challenges resulting from stress and hormone injections result in gradual and chronic down-regulation of CD62L on the surface of blood neutrophils. Rather than being associated with migration, this type of CD62L down-regulation associates with pronounced neutrophilia and increased susceptibility to infections. Nothing is currently known about glucocorticoid regulation of CD62L expression in neutrophils. In other cell systems, however, this steroid hormone binds to cytoplasmic glucocorticoid receptors (GR) that influence expression of glucocorticoid-responsive genes at multiple pre-translational levels. Thus, the hypothesis of the present study was that glucocorticoid challenge suppresses CD62L mRNA expression in blood neutrophils. Suppressed CD62L gene expression might help explain the chronic down-regulation of surface CD62L in neutrophils and accompanying neutrophilia. The main objectives of the study were to monitor neutrophil CD62L mRNA abundance before and during subtle and severe glucocorticoid challenges and to determine if CD62L mRNA expression correlates with degree of glucocorticoid challenge. Parturient dairy cows and dexamethasone-treated steers were used as models of subtle and severe (respectively) glucocorticoid challenges. Data presented from both models support the hypothesis and show for the first time that glucocorticoids regulate neutrophil CD62L at a pre-translational level. Results also showed that inhibited CD62L mRNA expression correlated precisely with down-regulated surface expression of CD62L on neutrophils and peak neutrophilia during severe glucocorticoid challenge. Therefore, results of this study indicate that bovine neutrophils are highly sensitive to the blood environment, displaying full capacity to alter CD62L gene expression and trafficking patterns in response to changing glucocorticoid levels. This may serve animals well when heightened inflammatory responses begin to lead to tissue damage, but may be detrimental to overall health if animals are exposed to opportunistic pathogens while stressed or undergoing glucocorticoid therapy. Although this study did not elucidate how glucocorticoids inhibit neutrophil CD62L mRNA expression, presented data implicate GR as possibly being involved because neutrophils from cattle in both models expressed GR mRNA. Further in vitro studies using purified populations of neutrophils will be required to determine if GR is directly involved in glucocorticoid regulation of CD62L gene expression and, if so, at what level.     

The immune status of the bovine uterus during the peripartum period

The post-partum period in cattle is characterised by an increased risk of infection of the uterus, as the anatomical barriers are broached during parturition and remain open for several days. Infection of the uterus is largely influenced by the balance between bacterial contamination and the local and systemic immune status during pregnancy and around parturition. Infectious diseases are more prevalent during this period, because of an impaired immune status before and immediately after parturition. Neutrophils play a primary role in the defence of the uterus against infection. Influx of neutrophils into the uterus is thought to be mediated by chemoattractants, chemokines and adhesion molecules, such as beta2-integrin (complement receptor 3) and L-selectin (CD62L). Other cellular components activated in the uterus during this period include lymphocytes, eosinophils, mast cells and macrophages. The major classes of immunoglobulins (IgM, IgA and IgG), either by passive diffusion or local production, play an important protective role in the uterus by acting as opsonins to enhance phagocytosis, stimulating the complement pathways or blocking pathogens from adhering to mucosal surfaces. Endometrial cells express toll-like receptor 4 (TLR4), which recognises lipopolysaccharides of Escherichia coli and other Gram negative bacteria, the most common causes of bovine endometritis. Activation of TLR4 triggers the production of tumour necrosis factor alpha and other pro-inflammatory cytokines. The periparturient period is also characterised by an increased secretion of prostaglandin F(2alpha), which enhances uterine immune defences.     

D-lactic acid interferes with the effects of platelet activating factor on bovine neutrophils

D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.     

Effect of proinflammatory mediators and glucocorticoids on L-selectin expression in peripheral blood neutrophils from dairy cows in various stages of lactation

OBJECTIVE: To determine whether proinflammatory mediators and glucocorticoids affect CD62L(L-selectin) expression on peripheral blood neutrophils from cows in various stages of lactation. ANIMALS: 100 healthy dairy cows during early (13.1 +/- 0.79 days after parturition; n = 31), peak (58.7 +/- 1.64 days after parturition; 31), and mid (137.2 +/- 2.59 days after parturition; 38) lactation. PROCEDURE: In vitro effects of relevant proinflammatory mediators that are released in response to mastitis caused by gram-negative bacteria such as lipopolysaccharide (endotoxin), tumor necrosis factor-alpha, and platelet-activating factor (PAF) on CD62L expression on bovine neutrophils were assessed by flow cytometry. Influences of cortisol and dexamethasone on CD62L expression on bovine neutrophils were also investigated. RESULTS: Basal CD62L expression on neutrophils from cows during early, peak, and mid lactation were similar. Lipopolysaccharide and tumor necrosis factor-alpha had no effect on CD62L expression on neutrophils from cows at any stage of lactation. Conversely, PAF elicited a time- and dose-dependent, down regulatory effect on CD62L expression. However, no differential shedding of CD62L from neutrophils of cows at any stage of lactation were detected. In addition, no effects on CD62L expression on bovine neutrophils after whole blood incubation with cortisol or dexamethasone were observed. Incubation with glucocorticoids did not prevent the down regulatory effect of PAF on CD62L expression. CONCLUSIONS AND CLINICAL RELEVANCE: Comparable basal CD62L expression on bovine neutrophils and equal amounts of CD62L shedding from bovine neutrophils during all stages of lactation suggest that variations in CD62L density are not a likely cause of susceptibility of cows to coliform-induced mastitis during early lactation.     

Effects of protein supply and reproductive status on local and systemic immune responses to Teladorsagia circumcincta in sheep

The effects of protein supply and reproductive status on circulating antibody responses and local inflammatory cell counts were investigated in parasitized sheep, with local immune responses assessed through a recently refined abomasal cannulation methodology. We hypothesized that if breakdown of immunity has a nutritional basis, then protein scarcity would result in a breakdown of immunity to Teladorsagia circumcincta in both periparturient and non-reproducing (barren) ewes. Twin-bearing and barren, abomasally cannulated ewes were fed at either 0.8 or 1.3 times protein requirements from 3 weeks before until 6 weeks after parturition (n = 6). All sheep were trickle infected at a rate of 10,000 infective larvae (L3) per day, for 3 days per week throughout the experiment. Faecal egg counts remained virtually zero in all barren ewes, whilst protein supplementation reduced faecal egg counts in the periparturient ewes during most of the periparturient period. Final worm burdens, taken at 6 weeks into lactation, were lower for the barren ewes than for the lactating ewes, whilst protein supplementation reduced worm burdens in the latter. Protein supply did not affect mucosal mast cell counts, which were consistently higher for the barren ewes than the periparturient ewes, but were temporarily decreased around parturition. Barren ewes and protein supplemented lactating ewes had higher globule leukocyte counts than the unsupplemented lactating ewes. Protein supplementation increased eosinophil counts in the lactating ewes though only during the later part of the lactation period. Plasma IgA anti-L3 antibody was similar for all ewes, but IgE anti-L3 antibody was higher for the protein supplemented periparturient ewes compared to the unsupplemented periparturient ewes and all barren ewes. It is likely that the combination of low protein requirements and large body protein reserves did not result in breakdown of immunity to T. circumcincta for the barren ewes. These results suggest that changes in mucosal mast cell and eosinophil counts are not necessarily associated with changes in host resistance to T. circumcincta. However, the data support the view that increased globule leukocyte counts and plasma IgE anti-L3 antibody may be associated with nutritionally improved expression of immunity in periparturient ewes.