thogens that infect cattle. These normally short-lived white blood

elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the

www.elsevier.com/locate/vetimm

Veterinary Immunology and Immunopathology 105 (2005) 197219* Corresponding author. Tel.: +1 517 353 9702/9815 (Laboratory); fax: +1 517 353 1699.

E-mail address: burtonj@msu.edu (J.L. Burton).

0165-2427/$ see front matter # 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.vetimm.2005.02.012circulation 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 tocells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into theNeutrophils are the first line of immunity against most paGene expression signatures in neutrophils exposed

to glucocorticoids: A new paradigm to help explain

neutrophil dysfunction in parturient dairy cows

Jeanne L. Burton a,b,*, Sally A. Madsen a, Ling-Chu Chang a,Patty S.D. Weber a, Kelly R. Buckham a, Renate van Dorp b,

Mary-Clare Hickey c, Bernadette Earley c

a Immunogenetics Laboratory, Department of Animal Science, Michigan State University,

1205E Anthony Hall, East Lansing, MI 48824, USAb Center for Animal Functional Genomics, Department of Animal Science, East Lansing, MI 48824, USA

c Animal Health and Welfare Department, Teagasc, Grange Research Center, Co Meath, Ireland

Abstract

rient dairy cows may experience heightened incidence and severity

rtisol; Dexamethasone

from bone marrow is clearly detectable in cattle24 hfollowing glucocorticoid challenge and may contri-

of glucocorticoid receptors (GR). In most mammalian

cells, including bovine neutrophils (Chang et al.,

2004), GR exists in two main isoforms, GRa and GRb(Hollenberg et al., 1985). However, only GRa hasglucocorticoid binding capacity (Bamberger et al.,

1996; Kino et al., 2001). We have shown that bovine

blood neutrophils have 8-fold higher GRa mRNAabundance compared with the cells more immature

counterparts developing in bone marrow (Fig. 1).

Thus, bovine blood neutrophils are predicted to be

y and Immunopathology 105 (2005) 197219

samples assayed in duplicate. The RNA samples were obtained from

bone marrow neutrophil-lineage cells and blood neutrophils of

healthy steers (n = 12) using Percoll density gradients (Webersteroid response (Burton and Kehrli, 1995; Weber

et al., 2004). Thus, when studying in vivo effects of

glucocorticoids on bovine neutrophils, it is relevant to

do so in both blood and bone marrow pools of the cells.

In cattle, circulating neutrophils are sensitive to

myeloblasts and promyelocytes, F2 cells 55% late-immaturemyelocytes and metamyelocytes, and F3 cells 70% band andsegmented neutrophils. The P-value indicates the level of signifi-

cance for the main effect of granulocyte maturation fraction,

obtained by general linear models analysis of the data, and a and

b above individual bars indicate significant (P 0.05) differences inbute to additional waves of neutrophilia later in the et al., 2004). Bone marrow F1 cells contained70% early-immatureinduction of parturition and partly explaining why some partu

of inflammatory diseases like mastitis.

# 2005 Elsevier B.V. All rights reserved.

Keywords: cDNA microarray; Neutrophils; Bone marrow cells; Co

1. Background

Elevated blood levels of glucocorticoid hormone

during stress and exogenous administration of the

steroid have been associated with increased suscept-

ibility to infectious diseases in cattle that affect

production, including mastitis in parturient dairy cows

and shipping fever in transportation stressed feedlot

animals (Roth and Kaeberle, 1982; Burton and

Erskine, 2003). Our groups are interested in the

effects of glucocorticoids on bovine neutrophils

because of the critical role these leukocytes play in

innate immune defense against such diseases. We have

studied bovine neutrophils following administration of

the potent glucocorticoid, dexamethasone (Burton

et al., 1995; Burton and Kehrli, 1995; Weber et al.,

2001, 2004; Chang et al., 2004) and during parturition

when there is a surge in blood concentrations of the

endogenous glucocorticoid, cortisol (Preisler et al.,

2000; Weber et al., 2001; Madsen et al., 2002, 2004;

Chang et al., 2004). Pronounced neutrophilia occurs in

both cases, showing that glucocorticoids profoundly

alter neutrophil homeostasis. The vast majority of

circulating cells in the early phase of glucocorticoid-

induced neutrophilia are segmented neutrophils that

have de-marginated from blood vessels due to down-

regulated expression of surface adhesion molecules

(Burton et al., 1995; Weber et al., 2004) and, possibly,

up regulation of anti-adhesion molecules (Goulding

et al., 1998; La et al., 2001). However, significant

release of mature (segmented and band) neutrophils

J.L. Burton et al. / Veterinary Immunolog198glucocorticoids because they possess high expressionFig. 1. Bovine blood neutrophils express8-fold higher abundanceof mRNA for the hormone-binding alpha isoform of the glucocor-

ticoid receptor (GRa) than immature neutrophil-lineage cells devel-

oping in bone marrow. Data were generated by quantitative slot blot

analysis with b-actin as the control gene (Madsen et al., 2002), GRa

and b-actin cDNA probes described in Weber et al. (2001), and RNAmean (S.E.M.) GRa mRNA abundance by maturation fraction.

highly sensitive to changes in circulating glucocorti-

coid concentrations.

In cells not exposed to stress or therapeutic levels of

J.L. Burton et al. / Veterinary Immunology and Immunopathology 105 (2005) 197219 199

Fig. 2. The surge in blood cortisol during bovine parturition (a) is

correlated (r = 0.71; P = 0.06) with rapid down regulation ofglucocorticoid receptor-alpha (GRa) mRNA abundance in blood

neutrophils (b), possibly due to the ability of glucocorticoid to

reduce the rate of transcription of its own receptor gene (c). Blood

for serum cortisol assays and neutrophil GRa mRNA abundance

assessment (a and b) was collected from three primiparous Holstein

cows on days 7, 0, 0.25, and 1 relative to parturition (on day 0).Serum cortisol was measured in duplicate by EIA (Assay Designs,

Inc., Ann Arbor, MI) and GRa mRNA by quantitative real time RT-

PCR (Madsen et al., 2004). GRa mRNA abundance on days 0, 0.25,

and 1 were relative to expression on day7 (b) and calculated usingthe 2DDCt method (Livak and Schmittgen, 2001). Nuclear run onassay was used to generate data in panel (c). Briefly, Percoll-isolated

blood neutrophils of four healthy Holstein steers (34 months of

age) were left untreated (Control) or treated with 107 M ofdexamethasone (Dex) for 4 h prior to isolation of the cells nuclei,

which were snap frozen before being used to transcribe new radi-

olabeled mRNA in vitro. GRa and b-actin (control) cDNA probes

(Weber et al., 2001) were spotted in excess on nylon membrane

strips and the blots probed with the newly synthesized, radiolabeled

mRNA. Densitometry was used to quantify abundance of tran-

scribed GRa and b-actin mRNA, and the GRa transcription rateglucocorticoids, most GRa are located in thecytoplasm as complexes with accessory proteins that

maintain the receptors in a high affinity hormone-

binding state (Bamberger et al., 1996). When

glucocorticoid concentrations rise inside the cell

due to elevated extracellular concentrations, hormone

binding to GRa activates the receptor causing it todissociate from its protein complex. This allows GRato translocate into the cells nucleus (Eicher and

Burton, 2004). In the nucleus, hormone-bound GRaemploys multiple mechanisms to change expression of

hormone sensitive genes, including binding to DNA

and other transcription factors to influence rates of

gene transcription and to mRNAs already existing in

the cytoplasm to affect their stability (Bamberger

et al., 1996; Newton, 2000; Beato and Klug, 2000;

Almawi and Melemedjian, 2002).

In previous studies, we demonstrated that blood

neutrophils of dairy cows express functional GRaproteins, staining brightly with fluorescently labeled

hormone when collected during mid and late lactation

and exiting the cells cytosol in response to the cortisol

surge at parturition (Preisler et al., 2000; Fig. 2a). We

also showed that glucocorticoid-induced loss of GRafrom the cytosol is time- and dose-dependent when

cultured neutrophils are treated with the steroid in

vitro (Chang et al., 2004 and our unpublished data). In

both scenarios of endogenous and exogenous gluco-

corticoid challenge, GRa loss from the neutrophilscytosol is associated with acute and pronounced

changes in expression of two genes that regulate

neutrophil behavior, including margination (CD62L;

Weber et al., 2001, 2004) and programmed cell death

(Fas; Chang et al., 2004). Thus, GRa activation byglucocorticoids in circulating neutrophils causes

distinctive phenotypic changes in the cells via altered

expression of hormone-sensitive genes. However,

these changes are relatively short-lived (48 h),possibly due to the fact that GRa activation rapidlydecreases abundance of its own mRNA (Fig. 2b) by

was expressed as the mRNA abundance ratio of GRa:b-actin. P-

values associated with each graph show the level of significance for

the main effect of parturition (a and b) or treatment (c), and ac

above individual bars indicate significant (P 0.05) differences in

means (S.E.M.) shown in each plot.

Erskine, 2003; Burvenich et al., 2003). Also, though

y andneutrophil-lineage cells developing in bone marrow

express low levels of GRa (Fig. 1), we sought tounderstand if glucocorticoid challenge could never-

theless impact gene expression in this only renewable

source of blood neutrophils. To begin to address these

questions we collected blood neutrophils from dairy

cows around the parturient cortisol surge, and bone

marrow cells from dairy steers treated with a high dose

of dexamethasone. Total RNA isolated from these

cells was used to obtain preliminary gene expression

profiles by cDNA microarray and quantitative real

time RT-PCR (Q-RT-PCR) analyses.

2. Gene expression profiling of blood neutrophils

during the surge in cortisol at parturition

In our first cDNA microarray experiment, blood

from four primiparous Holstein cows was collected

close to the surge in parturient cortisol. These animals

were healthy and did not experience retained placenta,

metritis, or mastitis at or following parturition.

Neutrophils were enriched from the blood samples

to 9498% purity using Percoll density gradients (as in

Weber et al., 2001) and their gene expression patterns

profiled using our groups third generation BOTL (for

bovine total leukocyte) cDNA microarrays (clones

searchable at http://www.nbfgc.msu.edu). As we

reported in Madsen et al. (2004), expression changes

(P < 0.05) for 302 genes were detected over the timerepressing the transcription rate of its gene (Fig. 2c).

Thus, choices of blood sampling times relative to

glucocorticoid challenges in vivo and in vitro are

critical in experiments designed to study neutrophil

gene expression changes in response to the hormone.

Results of our studies cited above led us to ask

several questions about possible additional impacts of

glucocorticoids on the bovine neutrophil system. In

particular, we wanted to know what other genes might

be affected in blood neutrophils during the cortisol

surge at parturition. This is important because such

knowledge may elucidate why blood neutrophils have

depressed bactericidal activities that correspond with

increased susceptibility to opportunistic infectious

diseases, such as coliform mastitis, during parturition

(reviewed in Kehrli and Harp, 2001; Burton and

J.L. Burton et al. / Veterinary Immunolog200the cows transitioned from the dry period (day 7relative to parturition) through parturition (day 0) and

into the first day of lactation (days 0.25 and 1).

Pronounced changes occurred for most affected genes

at parturition and 6 h postpartum (day 0.25), when

blood cortisol concentrations and neutrophilia were

highest (Fig. 2a and Madsen et al., 2004).

2.1. Apoptosis gene expression profiles during the

cortisol surge at parturition

The largest ontological cluster of affected known

genes (42 genes) in our microarray study encoded

apoptosis regulatory proteins. Quantitative real time

RT-PCR was used to substantiate expression changes

for some of the best known of these apoptosis genes

(Fig. 3). Percoll enriched blood neutrophils from three

additional primiparous parturient cows were used for

RNA isolations and cDNA synthesis in this work, as per

our described methods (Madsen et al., 2004; Weber

et al., 2004). Beta-actin was selected as the control gene

for Q-RT-PCR because its amplification efficiency was

the same as that for all test genes and abundance of its

mRNA in bovine blood neutrophils does not change

through the peripartum period (Weber et al., 2001;

Madsen et al., 2002). Using this approach, we verified

that expression of genes encoding 4 death inducing

signaling complex (DISC) proteins (i.e., FADD, Daxx,

FLASH, and RIP), which are associated with Fas

activation by its ligand (FasL; Fig. 3), were profoundly

down regulated during parturition (Madsen et al.,

2004). In addition, parturition down-regulated gene

expression for the pro-apoptotic Bcl-2 family member,

Bak, while simultaneously up regulating expression of

genes encoding the anti-apoptotic Bcl-2 homologue,

A1, and the potent pro-survival chemokine, IL-8

(Fig. 3; Madsen et al., 2004). These preliminary gene

expression data supported a hypothesis that parturition

reprograms neutrophils for extended survival in blood.

In addition, IL-8 is known to enhance cell survival by

signaling the down regulation of Bak gene expression

(Grutkoski et al., 2002). Therefore, parturition may

also induce an autocrine short-loop in neutrophils

whereby induction of IL-8 suppresses Bak expression

to support continued survival of the cells. In turn,

survival induction in neutrophils could partly explain

the neutrophilia that occurs in conjunction with the

cortisol surge at parturition (Fig. 2a; Preisler et al.,

Immunopathology 105 (2005) 1972192000; Weber et al., 2001).

y andJ.L. Burton et al. / Veterinary ImmunologSince publishing those original data we have Q-RT-

PCR profiled expression patterns for an additional six

apoptosis regulatory genes, including pro-apoptotic

Fas, FasL, and DAP5, and anti-apoptotic TRAF6,

TANK, and BAFF (Fig. 3). The first five genes listed

were significantly down regulated between parturition

and the first day of lactation, while BAFF was up

regulated at parturition (Fig. 4). BAFF is a relatively

newly identified member of the growing family of

TNF ligands and is best known for its critical role in

development of long-lived mature peripheral B cells

(Chin et al., 1999; Mak and Yeh, 2002; Mackay and

Ambrose, 2003). Its pronounced up regulation in

blood neutrophils at parturition argues in favor of cell

survival. Down regulation of the TRAF-6 and TANK

genes is not necessarily congruent with a survival

Fig. 3. Blood neutrophils are normally programmed to undergo rapid apopt

plasma membrane and spontaneous death initiated through mitochondrial s

(FasL) recruits a variety of adaptor proteins [the death initiating signaling

cytoplasmic tails, which then recruit and sequentially activate caspases 8

oxygen species (ROS) generated during oxidative metabolism (not shown) a

of cytochrome c (cyt c) and Smac/DIABLO (not shown), which activate casp

spontaneous form of apoptosis is normally uninhibited in circulating neutro

of key anti-apoptotic molecules like A1 (Bcl-2 homologue) and IL-8 (chem

that the cells continue translating death effector proteins even as they unde

caspase-induced cleavage of proteins that are critical for DNA repair, cyt

neutrophils are distinguished from viable cells by their reduced size, flippin

of the plasma membrane, and irreversible fragmentation of genomic DNA (

induce expression of genes encoding the potent anti-apoptotic proteins A1,

events and extend neutrophil life span by quenching mitochondrial mem

signaling pathways derived from death receptors such as Fas and TNF-aImmunopathology 105 (2005) 197219 201gene expression signature during parturition because

protein products of these genes act to desensitize cells

to pro-apoptotic signals from activated TNF-areceptors (Fig. 3; Bradley and Pober, 2001; Wu and

Arron, 2003). However, TRAF-6 and TANK act

downstream of other key death inducing molecules,

including Fas and FasL, which are highly potent in

their ability to induce apoptosis in bovine neutrophils

and were dramatically down regulated in the cells

during parturition (Fig. 4). Presence of DAP5 proteins

in cells ensures that continued translation of death

effector proteins occurs even as cells undergo apoptosis

(Fig. 3; Henis-Korenblit et al., 2000, 2002). Thus, the

down regulation of DAP5 gene expression that we

observed during parturition (Fig. 4) would be expected

to have a positive impact on neutrophil survival. So,

osis through a combination of death receptor signaling initiated at the

ignaling. For example, ligation of membrane bound Fas by its ligand

complex (DISC) molecules FADD, FLASH, RIP, and Daxx] to Fas

and 3 to effect cell death. At the mitochondrial membrane, reactive

nd pro-apoptotic Bcl-2 family members Bak and Bax induce release

ase 9 to directly and indirectly activate caspase 3 and cell death. This

phils because electron transport chain activity is high and expression

okine) is low or absent. Furthermore, expression of DAP-5 ensures

rgo apoptotic cell death. In both pathways, cell death is mediated by

oskeletal stability, and plasma membrane integrity. Thus, apoptotic

g of inner leaflet phosphatidylserine (PS) residues to the outer leaflet

hypodiploidy). Factors that reduce mitochondrial ROS production or

IL-8, BAFF, TRAF-6, and TANK temporarily prevent these cellular

brane destabilizing activities of ROS, Bak, and Bax and blocking

(TNF-R).

y andJ.L. Burton et al. / Veterinary Immunolog202while the down regulation we observed in TRAF6 and

TANK gene expression may lead to neutrophil

apoptosis in some circumstances, it is possible that

parturient physiology overrides such effects by

simultaneously down regulating gene expression for

pro-apoptotic Fas, FasL, DAP5, FADD, Daxx, FLASH,

RIP, and Bak and up regulating gene expression for

anti-apoptotic A1, BAFF, and IL-8.

2.2. Apoptosis gene expression profiles correlate

with blood cortisol profiles during parturition

Because blood neutrophils express abundant

GRa mRNA (Fig. 1) and protein (Chang et al.,

Fig. 4. Expression changes in six neutrophil genes (Fas, FasL, TRAF6, TA

Data were generated using quantitative real time RT-PCR (Madsen et al., 20

gene (Weber et al., 2001; Madsen et al., 2002). Test RNA for these ass

primiparous Holstein cows on days 7, 0, 0.25, and 1 relative to parturitiorelative to expression on day7 and calculated using the 2DDCt method (Lof significance for the main effect of day relative to parturition obtained thro

bars indicate significant (P 0.05) differences in mean (S.E.M.) geneconditions used to generate these data are listed under the Links icon

www.cafg.msu.edu/).Immunopathology 105 (2005) 1972192004), we postulated that cortisol may have been

one blood factor responsible for inducing apoptosis

gene expression changes in circulating neutrophils

of the parturient cows that, by in large, appeared to

favor cell survival. To test this possibility, we

performed correlation analyses on these data sets

using the PROC CORR procedure of SAS. Results

in Table 1 show that blood cortisol profiles were

significantly (P 0.05) associated with geneexpression profiles for A1, BAFF, Bak, DAP5,

Fas, FLASH, IL-8, and TRAF6, and tended

(P = 0.06) to be associated with expression profiles

for FasL and Daxx. However, other unidentified

factors also must have participated in the regulation

NK, DAP5, and BAFF) whose protein products regulate apoptosis.

04), with all samples run in duplicate and b-actin used as the control

ays was obtained from Percoll-isolated blood neutrophils of three

n (on day 0). Gene expression changes for days 0, 0.25, and 1 were

ivak and Schmittgen, 2001). P-values above each graph are the level

ugh repeated measures analysis of the data, and ac above individual

expression by day relative to parturition. The PCR primers and

on our Center for Animal Functional Genomics web site (http://

y andof specific components of the apoptosis program in

bovine neutrophils at parturition because correla-

tions between cortisol and gene expression profiles

for FADD, RIP, and TANK were not detected

(Table 1). It is unlikely that estradiol was one of

J.L. Burton et al. / Veterinary Immunolog

Table 1

Pearson product moment correlations (r-values) between serum

cortisol profiles and expression profiles for thirteen apoptosis reg-

ulatory genes in Percoll-isolated blood neutrophils from parturient

primiparous Holstein cows (n = 3)

Gene name Effect on apoptosis r-value P-value

A1 Anti-apoptotic 0.90 0.002

BAFF Anti-apoptotic 0.75 0.04

Bak Pro-apoptotic 0.82 0.01Daxx Pro-apoptotic 0.72 0.06DAP5 Pro-apoptotic 0.73 0.05FADD Pro-apoptotic 0.33 0.32Fas Pro-apoptotic 0.75 0.04FasL Pro-apoptotic 0.71 0.06FLASH Pro-apoptotic 0.80 0.02IL-8 Anti-apoptotic 0.73 0.01

RIP Pro-apoptotic 0.55 0.24TRAF-6 Anti-apoptotic 0.75 0.04TANK Anti-apoptotic 0.65 0.11Gene expression changes were first detected by cDNA microarray

analysis (Madsen et al., 2004) and further characterized using

quantitative real time RT-PCR (Fig. 4).these factors because we (unpublished data) and

others (Winters et al., 2003) could not detect

estrogen receptor proteins in bovine blood neutro-

phils. Also, correlations between the cows serum

estradiol profiles and neutrophil apoptosis gene

expression profiles were not significant (P > 0.10;not shown). While bovine neutrophils also do not

express progesterone receptor mRNA or protein (our

unpublished data; Winters et al., 2003), it is possible

that fluctuations in serum progesterone around

parturition contributed to the apoptosis gene

expression profiles we observed because progester-

one can act as a GRa antagonist (Preisler et al.,2000). Thus, it is possible that the parturient drop in

blood progesterone as cortisol levels soar may have

facilitated glucocorticoid activity on the expression

of some apoptosis genes. In support of this

possibility, we observed significant (P < 0.05)correlations between serum progesterone profiles

and neutrophil gene expression profiles for Fas

(r = 0.703), RIP (r = 0.796), BAFF (r = 0.793), IL-8 (r = 0.724), and TANK (r = 0.785).2.3. Cortisol in parturient serum is partly

responsible for inducing survival in blood neutrophils

To substantiate that the relationships between

serum cortisol and apoptosis gene expression profiles

we observed translate into changed apoptosis status of

the cells, we attempted to phenotype apoptosis of

neutrophils in fresh whole blood samples collected

from a group of primiparous cows prepartum and at

parturition. However, effects of parturition on apop-

tosis status were not clear because extremely high

animal variation existed in the staining of neutrophils

with Annexin-V-FITC, a reagent that binds with

phosphatidylserine residues that have flipped from the

inner to the outer leaflet of the plasma membrane

during early stages of apoptosis (Fig. 3; Vermes et al.,

1995). For example, the mean percentage of Annexin-

V-FITC+ neutrophils in blood from 16 cows collected

10 days prepartum was 48.9% (16.20; S.E.M.),with a range of 19.183.2%. This wide animal

variation in Annexin-V-FITC staining in fresh cells

is likely related to normal variation in proportions of

variously aged neutrophil populations in whole blood

(Shidham and Swami, 2000). In turn, this is a function

of variation in the rates of bone marrow release, cell

exit from blood into tissues, and clearance of

circulating apoptotic neutrophils by the bodys

phagocytic network (Homburg and Roos, 1996; Savill,

1997). Thus, it was difficult to conclude whether or not

neutrophils in fresh blood samples were less apoptotic

overall at parturition versus prepartum.

When we instead enriched neutrophils from

prepartum blood samples on Percoll density gradients,

4% of the freshly isolated cells stained with Annexin-V-FITC. It thus appears that our neutrophil isolation

protocol is relatively selective in its enrichment of blood

neutrophils that have no overt signs of this early stage of

apoptosis. In fact, the recovery of neutrophils in this

procedure averages 75% when neutrophilia is notpresent (and higher when it is). The majority of

neutrophil loss (76%) occurs during removal of the

buffy coat and top one-third of the red cell pack prior to

addition of the remaining red cell pack to the Percoll

gradient for neutrophil isolation. A more minor loss

(24%) results from neutrophils that remain on or in the

Percoll layer because the cells are not dense enough to

penetrate through it. The dense neutrophils that do

Immunopathology 105 (2005) 197219 203penetrate the Percoll gradient to form the cell pellet at

parturient serum for 12 or 24 h, followed by apoptosis

phenotyping. Results in Fig. 6a show that RU486 (a

GRa/progesterone receptor antagonist), but nottamoxifen (an estrogen receptor antagonist), inhibited

the ability of the parturient serum to support

neutrophil survival, and did so to the same extent as

the steroid extracted serum. Thus, cortisol and (or)

progesterone were likely factors responsible for

neutrophil survival induction by parturient serum. In

a final experiment, we showed that cortisol was the

predominant steroid responsible for neutrophil survi-

val-induction by parturient serum because only

y andthe bottom of the tube may thus represent the youngest

population of neutrophils in blood, presumably those

that were recently released from the maturation pool in

bone marrow. We suspect that use of Percoll isolated

cells standardized the starting population of neutrophils

in our apoptosis phenotyping assay because, when

allowed to age in culture over 48 h, these cells had

significantly extended viability when collected from

cows at parturition versus prepartum (Madsen et al.,

2003a). This observation did support those from our

microarray and Q-RT-PCR experiments, which sug-

gested that the cells gain a predominantly pro-survival

gene expression signature around parturition. Addi-

tionally, we have shown that sera collected from the

cows at parturition (day 0) supported viability in

significantly more Percoll-isolated steer neutrophils

over 48 h in culture than sera collected prepartum (day

7) or postpartum (days 0.25 and 1; Madsen et al.,2004). This suggested, as our correlation analysis did,

that parturient blood does indeed contain factors that

promote extended neutrophil life span. However, given

the differences in Annexin-V-FITC staining character-

istics between neutrophils in fresh whole blood samples

versus Percoll-isolated cells, it would seem important

for investigators to carefully consider which neutrophil

isolation method to use for such experiments and to

characterize the apoptosis status of neutrophils

employed in various assays. We selected the Percoll

density gradient method to isolate neutrophils because

it was the quickest way to obtain high numbers of very

pure neutrophils with enough quantity of high quality

RNA needed for our for our microarray and Q-RT-PCR

work. We felt that this was critical to our ability to

generate gene expression data from which we could

draw conclusions about effects of parturition on gene

expression changes in neutrophils. However, we

acknowledge that our conclusions about the apoptosis

gene expression signatures and phenotypes observed

around parturition may apply only to younger popula-

tions of the cells and not to all circulating neutrophils.

With this caveat in mind, we continued with several

additional experiments using Percoll-isolated neutro-

phils to study potential relationships between gluco-

corticoids in serum and neutrophil apoptosis

phenotypes.

Given our in vitro apoptosis results and the

correlations we detected between blood cortisol

J.L. Burton et al. / Veterinary Immunolog204profiles and neutrophil apoptosis gene expressionaround parturition (Table 1), we sought to determine if

the cortisol component of parturient blood is one

factor capable of inducing survival in cultured bovine

neutrophils. We pooled the parturient (day 0) serum

samples, split the pool into two aliquots, and used

activated charcoal to remove cortisol (Szafarczyk

et al., 1995; McCarty and Schwartz, 1999) from one

aliquot (Table 2). Charcoal treatment also removed

progesterone and estradiol from this aliquot (Table 2)

and, perhaps, other unidentified factors. We added

these sera at 20% of the volume of culture medium to

Percoll-isolated steer neutrophils as the cells aged in

vitro for 12 h or 24 h prior to apoptosis phenotyping.

Representative data from one steer in Fig. 5 show that

compared to the steroid extracted serum (panel b),

intact parturient serum (panel a) significantly

increased the proportion of viable neutrophils in

culture at 12 h (top panels of Fig. 5) and reduced DNA

fragmentation in the cells at 24 h (bottom panels of

Fig. 5). To clarify which extracted steroid may have

been responsible for this effect, neutrophils from

additional steers were pretreated for 10 min with

steroid receptor antagonists prior to addition of intact

Immunopathology 105 (2005) 197219

Table 2

Concentrations (pg/ml) of cortisol, estradiol, and progesterone in

pooled parturient serum and charcoal-treated parturient serum with

steroids extracted

Hormone Parturient

serum

Steroid extracted

parturient serum

Fetal bovine

serum

Cortisol 29,000 a 150 c 985 b

Estradiol 1,500 a 75 c 869 b

Progesterone 900 a 110 c 370 b

Concentrations in commercial fetal bovine serum are included for

comparison. P < 0.0001 for within hormone differences (ac) in

serum sources.cortisol (not estradiol or progesterone) added back

y andJ.L. Burton et al. / Veterinary Immunologto the steroid extracted serum reconstituted its

neutrophil survival-inducing capacity back to that of

intact parturient serum (Fig. 6b). These results are in

accordance with the well-documented ability of

glucocorticoids to induce survival of human and

rodent neutrophils (reviewed in Fanning et al., 1999;

Maianski et al., 2004). Thus, while we acknowledge

that interactions between cortisol and progesterone

may modify neutrophil survival characteristics around

parturition in vivo, and that non-steroid survival

enhancing factors (e.g., endotoxin, cytokines) may

have been removed from our serum during charcoal

treatment, it is clear from these experiments that

glucocorticoids alone are sufficient to enhance the

survival of neutrophils cultured in steroid extracted

parturient serum. Combined with the numerous

Fig. 5. Bovine neutrophils cultured in parturient serum (a) retain viability

panels) than neutrophils cultured in parturient serum from which steroids ha

healthy Holstein steer (3 months old) were used in this assay. Top panels sh

culture for 12 h, and bottom panels show PI staining of genomic DNA afte

cells aged in culture for 24 h. Data from 10,000 neutrophils are represent

generate these plots are described in Madsen et al. (2003a, 2003b, 2004)Immunopathology 105 (2005) 197219 205significant correlations shown in Table 1, we propose

that survival induction during the surge in cortisol

contributes an important new piece to the story about

how bovine parturition affects some circulating

neutrophils. We are currently performing in vitro

experiments to determine if effects of glucocorticoids

on neutrophil apoptosis gene expression are direct,

mediated via GRa, and translate into alteredabundance of corresponding proteins. So far this

seems to be the case for glucocorticoid-induced down

regulation of Fas gene expression, which associates

with inhibited spontaneous and sFasL-induced cas-

pase 8 activity (Fig. 7) and may partly explain the

extended longevity of isolated blood neutrophils

treated with glucocorticoids in vitro (Chang et al.,

2004).

(top panels) and show less fragmentation of genomic DNA (bottom

ve been extracted by charcoal (b). Percoll-isolated neutrophils from a

ow Annexin V-FITC/propidium iodide (PI) staining of cells aged in

r plasma membrane permeabilization and RNAseA treatment of the

ed in each plot. The staining and flow cytometric methods used to

.

y andJ.L. Burton et al. / Veterinary Immunolog206Delayed apoptosis at parturition is difficult to

reconcile in light of the well-documented migration

and bactericidal dysfunctions of blood neutrophils

from parturient cows (Cai et al., 1994; Shuster et al.,

1996; Kimura et al., 1999; Kehrli and Harp, 2001;

Mehrzad et al., 2001, 2002). These dysfunctions are

normally characteristic of aged neutrophils that are

apoptotic and no longer useful to host immune defense

(Whyte et al., 1993; Narayanan et al., 1997; Tanji-

Matsuba et al., 1998; Whyte et al., 1999; Van

Oostveldt et al., 2001, 2002). In humans, normal

apoptosis of aging neutrophils is a process that

associates with considerable disabling of potentially

injurious effector functions of the cells, limiting

transendothelial migration of blood neutrophils and

phagocytosis-induced degranulation of tissue neutro-

phils to prevent significant inflammatory damage

Fig. 6. Cortisol concentration differences largely explain differences in s

fragmentation (bottom panels; % hypodiploid cells after 24 h in culture) in n

serum. Percoll-isolated neutrophils were from blood of two healthy Holstein

(a), cortisol/progesterone receptors were antagonized with RU486 (104 Mto culturing cells in 20% PS or ES. In (b), cortisol (Cort), estradiol (Estr), o

in PS (Table 2) prior to use in neutrophil cultures. PS alone, ES alone, and E

staining and flow cytometric methods used to generate these data are shown

above individual bars within plot indicate significant (P 0.05) differencImmunopathology 105 (2005) 197219(Heasman et al., 2003). Given that our survival gene

expression signatures and phenotypes were observed

in Percoll-isolated neutrophils that, on average, may

have been younger than the total population present in

blood, it is possible that neutrophils naturally aged in

the circulation are not rescued from apoptosis during

parturition and that these were the dysfunctional cells

noted by others who did not isolate the cells on Percoll

gradients for use in bactericidal assays (e.g., Cai et al.,

1994; Mehrzad et al., 2001, 2002). Older neutrophils

that have de-marginated under the influence of

increasing cortisol at parturition (Preisler et al.,

2000; Weber et al., 2001) may be the source of such

dysfunctional cells because oxidative burst activity is

observed to be lowest around the time when

neutrophilia peaks (Mateus et al., 2002; Mehrzad

et al., 2002). If so, the parturient cortisol surge (and

urvival (top panels; % viable cells after 12 h in culture) and DNA

eutrophils exposed to intact (PS) or steroid extracted (ES) parturient

steers (3 months old) and cultured in duplicate for each treatment. In

), and estrogen receptors with tamoxifen (104 M), for 10 min priorr progesterone (Prog) were added back to ES at concentrations found

S with 107 M of added dexamethasone (Dex) were the controls. Thein Fig. 5 and described in Madsen et al. (2003a, 2003b, 2004). a and b

es in mean (S.E.M.) cell phenotype across culture scenarios.

y andJ.L. Burton et al. / Veterinary Immunolog

Fig. 7. Bovine blood neutrophils are extremely sensitive to con-

stitutive and FasL-induced caspase 8 activation, and dexamethasone

(Dex) reduces these sensitivities. Neutrophils for these experiments

were isolated from blood of three healthy Holstein steers (34

months old) using Percoll density gradients. Duplicate aliquots of

neutrophils were either pretreated for 4 h with Dex or left untreated

and then cultured for an additional 3 h in the presence or absence of

soluble Fas ligand (sFasL) before measuring caspase 8 activity

(ApoAlert Caspase Colorimetric Assay Kit; ClonTech). ad indicate

significant (P 0.05) differences in mean (S.E.M.) caspase 8activity across treatments.possibly other factors) may act on the one hand to shut

down potentially harmful inflammatory processes in

already aged neutrophils while, on the other hand,

temporarily rescuing younger neutrophils from apop-

totic cell death by reprogramming expression of key

apoptosis regulatory genes. If this is true and our data

are representative, one is led to question the

physiological reasons that youthful blood neutrophils

would be induced to live longer than normal around

parturition.

2.4. Parturient cortisol profiles also correlate with

expression profiles in some key immune response

and tissue remodeling genes

In an attempt to gain a handle on this question, we

opted to study expression profiles for additional

neutrophil genes revealed by our microarray analysis

to be altered around parturition. We used Q-RT-PCR to

profile expression changes for 12 genes, 6 of which

encode proteins that regulate traditional immune

defense functions of the cells and the other 6 that are

less well studied but have key roles in extracellular

matrix degrading activities of the cells. The immunityrelated genes included CD62L and CD18 (vascular

adhesion and migration), IL-8 receptor beta (chemo-

kinesis), IkK alpha (NF-kB-mediated transcription ofpro-inflammatory genes), FcRN (opsonized phagocy-

tosis), and PSST [regulation of reactive oxygen species

(ROS) generation and redox status of the cells].

Though numerically down regulated on days 0.25

and 1 postpartum, gene expression for IkK-alpha andFcRN was not significantly (P > 0.20) affected byparturition in the Q-RT-PCR analysis (Fig. 8) as it was

in the microarray analysis. The reason for this

discrepancy between assays is not known. However,

this suggested as others studies have, that the synthesis

of pro-inflammatory mediators and phagocytic activ-

ity of neutrophils may remain intact around parturition

(Guidry et al., 1976; Hoedemaker et al., 1992b;

Shuster et al., 1996; Goto et al., 1997; Zerbe et al.,

2000). Additional Q-RT-PCR data in Fig. 8 confirmed

our microarray results by showing pronounced

(P < 0.01) down regulation of CD62L, CD18, IL-8receptor beta, and PSST gene expression between

parturition and the first day of lactation. Some of these

gene expression profiles were correlated (P = 0.05)

with serum cortisol (Table 3), suggesting three things:

(i) that the ability of blood neutrophils to get into

infected tissue is hampered at parturition, even in

young cells; (ii) that redox homeostasis in neutrophils

is out of regular balance during parturition; and (iii)

that the increase in blood cortisol at parturition may be

involved in both.

For decades, investigators have suggested that

slowed neutrophil migration from blood into infected

mammary glands is a key factor in the heightened

susceptibility of parturient cows to coliform mastitis

(e.g., Hill et al., 1979; Shuster et al., 1996). This is

congruent with our neutrophil gene expression results

for CD62L, CD18, and IL-8 receptor beta, and the

documented role of glucocorticoids in the regulation

of at least two of these genes (Burton et al., 1995;

Burton and Kehrli, 1995; Weber et al., 2001, 2004;

Tempelman et al., 2002). However, without supporting

functional data, the putative imbalance in redox status

via down-regulated PSST is more difficult to interpret.

PSST contains the redox group used by NADH:ubi-

quinone oxidoreductase (Complex I of the electron

transport chain) to couple the transfer of two electrons

from NADH! ubiquinone to the translocation of

Immunopathology 105 (2005) 197219 207four protons across the mitochondrial inner membrane

J.L. Burton et al. / Veterinary Immunology and Immunopathology 105 (2005) 197219208

Fig. 8. Expression changes in six neutrophil genes (IkK-alpha, CD62L, FcRN, IL-8 receptor beta, CD18, and PSST) whose protein products

regulate key inflammatory and immune functions of the cells. Data were generated using quantitative real time RT-PCR (Madsen et al., 2004),

with all assays run in duplicate and b-actin serving as the control gene (Weber et al., 2001; Madsen et al., 2002). Test RNAs were obtained from

Percoll-isolated blood neutrophils of three primiparous Holstein cows on days 7, 0, 0.25, and 1 relative to parturition (on day 0). Geneexpression changes for days 0, 0.25, and 1 were relative to expression on day7 and calculated using the 2DDCt method (Livak and Schmittgen,2001). P-values above each graph are the level of significance for the main effect of day relative to parturition obtained through repeated

measures analysis of the data, and ac above individual bars indicate significant (P 0.05) differences in mean (S.E.M.) gene expression byday relative to parturition. The PCR primers and conditions used to generate these data are listed under the Links icon on our Center for Animal

Functional Genomics web site (http://www.cafg.msu.edu).

Table 3

Pearson product moment correlations (r-values) between serum cortisol profiles and expression profiles for six immune function regulatory

genes in Percoll-isolated blood neutrophils from parturient Holstein cows (n = 3)

Gene name Immune function r-value P-value

IkK-alpha Regulates NF-kB activity 0.49 0.34CD62L Vascular margination 0.66 0.09FcRN Antibody-mediated phagocytosis 0.38 0.24IL-8 receptor b Chemokinesis and cell activation 0.60 0.17CD18 Transendothelial migration 0.70 0.06PSST Mitochondrial electron transport chain; redox homeostasis 0.73 0.05Gene expression changes were first detected by cDNA microarray analysis (Madsen et al., 2004) and further characterized using quantitative real

time RT-PCR (Fig. 8).

y and(Brandt et al., 2003). This reaction is critical for

respiratory metabolism, but it also produces abundant

ROS that are toxic to mitochondria (Genova et al.,

2003), destabilizing the organelles membranes and

promoting release of cytochrome c that activates

apoptosis-inducing caspase cascades (Nunez et al.,

1998; see Fig. 3). If future cell phenotyping

experiments validate that redox potential is indeed

reduced in neutrophils at parturition, possibly under

the influence of cortisol (Table 3), down-regulated

PSST could help explain the increased survival we

have observed in these neutrophils. In light of this

possibility, it is interesting to note that using

differential display RT-PCR, we previously detected

pronounced down regulation of mitochondrial cyto-

chrome b (a component of Complex III of the electron

transport chain) in Percoll-isolated neutrophils of

parturient cows (Madsen et al., 2002). Thus, down

regulation of mitochondrial respiratory metabolism

during parturition may be an additional mechanism

that prolongs the life of circulating neutrophils via

reduced levels of intracellular ROS, with cortisol

potentially acting as an initiating signal. However, this

may also contribute to the reduced bactericidal

activity of neutrophils that presumably underlies

mastitis susceptibility at parturition (Kehrli and Harp,

2001; Mehrzad et al., 2001).

The next question with which we struggled is why

parturition with its associated surge in cortisol would

potentially restrict key bacteria-fighting properties of

neutrophils while at the same time extending cell

longevity. The answer to this question still eludes us.

However, biomedical literature suggests that homeo-

static functions of neutrophils likely exist that are

outside of the cells traditional bactericidal roles

(Smith, 1994). For example, the granular nature of

neutrophils make them outstanding vectors for

targeted delivery of proteolytic factors to tissues in

need of remodeling, such as the reproductive tract

during parturition. In fact, neutrophils have a central

role in human parturition (Kelly et al., 1994; Kelly,

1996; Thomson et al., 1999; Winkler et al., 1999a;

Bowen et al., 2002; Osman et al., 2003). When the

progesterone block on parturition wanes at term, blood

vessels in the human cervix and lower uterine tract are

enabled to express adhesion molecules and secrete

high levels of IL-8 into the extracellular tissue matrix

J.L. Burton et al. / Veterinary Immunolog(Winkler et al., 1999a, 1999b). These act to recruitlarge numbers of blood neutrophils to precisely the

sites where extracellular matrix degradation is

required for cervical softening, separation of maternal

and fetal membranes, and rupture of the fetal

membrane (Bowen et al., 2002; Fortunato and Menon,

2002). Neutrophils become activated at these sites,

probably under the influence of IL-8 (Luo et al., 2000),

which causes them to increase plasma membrane

expression the elastase, MMP-8 (Maymon et al., 2000;

Owen et al., 2004) and release their granule stores of

the gelatinase, MMP-9 (Junqueira et al., 1980; Osmers

et al., 1992). These matrix metalloproteinases

(MMPs) degrade structural proteins in fetal mem-

branes and the cervix, such that their activity

correlates with collagenolysis, cervical dilation, and

duration of labor in women at term (Winkler et al.,

2000). Cattle studies have also suggested that IL-8,

recruited blood neutrophils, and activity of their

MMPs figure predominantly in bovine parturition

(Klucinski et al., 1990; Eiler and Hopkins, 1992;

Hoedemaker et al., 1992b; Hussain and Daniel, 1991,

1992; Maj and Kankofer, 1997; Chassagne et al.,

1999; Kimura et al., 2002; Mateus et al., 2002). In fact,

these studies extended information available in the

biomedical literature by linking inadequate levels/

activity of these pro-inflammatory mediators at

parturition to retained placentas (Maj and Kankofer,

1997; Kimura et al., 2002) and showing that the bovine

placenta secretes factor(s) that shut down oxidative

burst activity in the recruited neutrophils (Hoede-

maker et al., 1992a, 1992b; Kimura et al., 2002). Thus,

the process of parturition appears to be a massive

inflammatory process, with the fetus, placenta, and

reproductive tract playing active roles in the recruit-

ment, activation, and altered functional priority of

blood neutrophils. Is it possible, then, that the

reproductive tract has a more urgent demand for long

lived neutrophils with elevated tissue degrading

capacity during parturition than peripheral tissues

have of traditional short-lived neutrophils endowed

with potent antimicrobial activity? We think this is an

important question that warrants further consideration

because of its possibilities for improving our under-

standing about disease susceptibility, especially to

retained placenta, metritis, and mastitis in parturient

dairy cows.

In this light, the other cluster of six neutrophil

Immunopathology 105 (2005) 197219 209function genes we selected to profile by Q-RT-PCR for

y andJ.L. Burton et al. / Veterinary Immunolog210expression changes during parturition relate to tissue

remodeling activity of the cells. The genes included

MMP-8 and MMP-9, their tissue inhibitors (TIMP-2

and TIMP-3), TGF-b (a pleiotropic cytokine withprofibrotic properties that can inhibit neutrophil

chemotaxis and possibly MMP-8; Moilanen et al.,

2002; Chakir et al., 2003; Ghio et al., 2003), and

granzyme B (a potent apoptosis-inducing protein

secreted by neutrophils and other immune cells to kill

infected and otherwise altered host cells; Talanian

et al., 1997; Trapani and Sutton, 2003; Andrade et al.,

2004; Wagner et al., 2004). Results in Fig. 9 suggest

Fig. 9. Expression changes in six neutrophil genes (MMP-8, TIMP-2, G

regulate tissue remodeling functions of the cells. Data were generated using

run in duplicate and b-actin used as the control gene (Weber et al., 2001; M

blood neutrophils of three primiparous Holstein cows on days 7, 0, 0.25,days 0, 0.25, and 1 were relative to expression on day7 and calculated usieach graph are the level of significance for the main effect of day relative to

and ac above individual bars indicate significant (P 0.05) differences inPCR primers and conditions used to generate these data are listed under the

(http://www.cafg.msu.edu).Immunopathology 105 (2005) 197219that neutrophils do indeed become programmed at

parturition for increased tissue remodeling capacity,

with a gene expression signature that clearly favors

extracellular matrix degradation. Correlation analysis

showed strong associations between these gene

expression profiles and serum cortisol profiles

(Table 4), some additional relationships (P < 0.04)with serum progesterone (r = 0.815 for TGF-b;r = 0.744 for TIMP-2; r = 0.741 for TIMP-3), but no

associations (P > 0.10) with serum estradiol (notshown). In support of these correlation data, gluco-

corticoids have been shown to block synthesis and

ranzyme B, MMP-9, TIMP-3, and TGF-b) whose protein products

quantitative real time RT-PCR (Madsen et al., 2004), with all assays

adsen et al., 2002). Test RNAs were obtained from Percoll-isolated

and 1 relative to parturition (on day 0). Gene expression changes for

ng the 2DDCt method (Livak and Schmittgen, 2001). P-values aboveparturition obtained through repeated measures analysis of the data,

mean (S.E.M.) gene expression by day relative to parturition. TheLinks icon on our Center for Animal Functional Genomics web site

y and

tisol p

cows

sin 1

s

atory

lysis (

time RT-PCR (Fig. 9).activity of granzyme B and TGF-b in other cell systems(Li et al., 2003; Sasson and Amsterdam, 2003) and

increase secretion of MMP-9 by monocytes and

neutrophils while inhibiting their expression of

TIMP-2 (Valencia et al., 2003; Mirowska et al.,

2004). Interestingly, elevated MMP-9 and decreased

TIMP-2 expression have been found to be associated

with premature rupture of fetal membranes in women

(Fortunato and Menon, 2001). While we have yet to

demonstrate that our gene expression profiles equate

with heightened tissue degrading activity of blood

neutrophils at parturition, it would make sense that this

was the case because of the critical role of these cells in

remodeling the extracellular matrix of the reproductive

tract and placental at term. In turn, this could provide an

explanation for the relationships between cortisol (and

perhaps progesterone) and the neutrophil gene expres-

sion profiles we have documented (Figs. 4, 8 and 9).

Parturition appears to occur at the temporary expense of

innate immune defense, possibly as a way of protecting

reproductive tract cells and the fetus from damagingJ.L. Burton et al. / Veterinary Immunolog

Table 4

Pearson product moment correlations (r-values) between serum cor

genes in Percoll isolated blood neutrophils from parturient Holstein

Gene name Immune function

MMP-8 Neutrophil elastase; collagenase 2 activity

MMP-9 Gelatinase B activity

TIMP-2 Tissue inhibitor of gelatinases A and B

TIMP-3 Tissue inhibitor of collagenase 1, stromely

Granzyme B Apoptosis induction in damaged host cell

TGF-b MMP and TIMP regulation; anti-inflamm

Gene expression changes were first detected by cDNA microarray anaeffects of neutrophil-generated ROS.

3. Contribution of bone marrow to

glucocorticoid-induced changes in blood

neutrophils

In addition to direct effects of parturient cortisol

(and perhaps progesterone) on existing blood neu-

trophils, it is possible that young neutrophils released

from bone marrow under the influence of these

steroids contributed to the neutrophilia and accom-

panying gene expression signatures we observed in

Percoll-isolated blood neutrophils of our parturientcows. If true, bone marrow could participate in two

main ways, through increased production of neutro-

phils (granulopoiesis) and increased release of mature

neutrophils into blood. In rat bone marrow for

example, stress levels of exogenous glucocorticoid

target immature neutrophil-lineage cells, increasing

their proliferation and survival and enhancing avail-

ability of the mature neutrophil pool for release into

blood (Laakko and Fraker, 2002). In addition,

proportions of neutrophils in bone marrow and blood

appear to be positively correlated with blood cortisol

concentration in neonatal horses (Chavatte et al.,

1991). Interestingly, glucocorticoid-induced granulo-

poiesis in humans may be related to the ability of this

steroid to dose-dependently increase blood concentra-

tions of a key neutrophil development and activating

cytokine, granulocyte colony stimulating factor (G-

CSF; Jilma et al., 1998). Regardless of mechanism of

action, it seemed relevant to study gene expression in

bone marrow cells of glucocorticoid challenged cattle

to try and learn more about the blood neutrophil

Immunopathology 105 (2005) 197219 211

rofiles and expression profiles for six tissue remodeling regulatory

(n = 3)

r-value P-value

0.70 0.02

0.70 0.02

0.75 0.04, and gelatinases A and B 0.75 0.04

0.75 0.040.73 0.05

Madsen et al., 2004) and further characterized using quantitative realpicture that we observed during parturition in dairy

cows.

3.1. Microarray analysis of bone marrow cells in

dexamethasone treated steers

We did not have access to cows bone marrow in

our parturition experiments and turned instead to our

steer model of glucocorticoid challenge (Weber et al.,

2004) to perform a second microarray experiment.

Briefly, four Holstein steers served as untreated

controls (no glucocorticoid challenge) and another

four steers were treated with two doses of dexa-

methasone (0.1 mg/kg of body weight) at times 0 and

y and6 h. Bone marrow was harvested from exposed

sternebrea of all animals 12 h after the first

dexamethasone dose was administered into the treated

steers (procedure described in Weber et al., 2004). The

samples were divided into two aliquots, one for

microscopic slide preparation and flow cytometric

analysis of neutrophil-lineage cells (protocols

described in Weber et al., 2004) and the other for

RNA isolation and microarray analysis of gene

expression differences.

The microarray work was performed using our

fourth generation BOTL cDNA microarrays, which

were upgraded to include triplicate spots for an

additional 200 leukocyte genes relative to the thirdgeneration BOTL arrays used in our parturition study

(Madsen et al., 2004). All conditions for cDNA

synthesis, dye labeling, and array hybridizations and

readings were as described previously (Madsen et al.,

2004). Each sample RNA was labeled with both Cy3

and Cy5 dyes so that there was assay replication at the

level of dye. Samples from control and dexamethasone

treated steers were randomly paired with each other

for array hybridizations. Resulting total fluorescence

intensity values for Cy3 and Cy5 from each spotted

cDNA were imported into SAS and median values for

triplicate spots of each gene log2-transformed for

LOWESS normalization, which was done within array

and patch to account for potential dye biases (Yang

et al., 2002). Because the literature reveals lack of

consensus about the best way to statistically analyze

cDNA microarray data, we opted to use two

commonly advocated approaches for our bone marrow

data set. One approach is general linear model based

(Kerr et al., 2000; Kerr and Churchill, 2001) and the

other involves a two-step linear mixed model

(Wolfinger et al., 2001). Our preliminary list of bone

marrow expressed genes affected by dexamethasone

administration was developed based upon the criterion

that P < 0.05 in both analyses. Least squares estimatesresulting from the general linear model analysis were

used to indicate putative direction of change in

expression of these affected genes.

3.2. Dexamethasone-induced expression changes in

key hematopoiesis regulatory genes

Based on our two-pronged data analysis, we have

J.L. Burton et al. / Veterinary Immunolog212tentatively identified 10 genes for which dexametha-sone caused expression changes 12 h post-adminis-

tration in vivo (Table 5). Of these, three genes

(FANCA, IkB Kinase alpha, and JUN-D) with knownroles in promoting proliferation of immature myeloid-

lineage cells, were up regulated in bone marrow of the

dexamethasone treated animals relative to controls.

The remaining seven genes were down regulated in

bone marrow cells of the treated steers. Protein

products of two of these genes negatively regulate

proliferation of hematopoietic cells (CD164; GRO-

beta), another is potently pro-apoptotic in neutrophils

(Bax-alpha; Fig. 3), and two more help drive

differentiation and maturation of hematopoietic cells

(endothelin receptor; IL-7 receptor alpha). Another

gene (receptor type tyrosine kinase RSE) encodes a

protein known for its role in promoting hematopoiesis

of monocyte- and platelet-lineage cells. The last

identified gene encodes VE cadherin, which when

down regulated enables transendothelial migration of

circulating CD34+ hematopoietic cell precursors into

bone marrow by loosening endothelial cellcell

adhesions in bone marrow blood vessels.

While we still need to confirm and characterize

these gene expression profiles in bone marrow cells

using Q-RT-PCR, and show that they are reflected in

phenotypic changes in the cells, the signature shown in

Table 5 suggests that dexamethasone may have acted

to promote proliferation and survival of immature

neutrophil-lineage cells in cattle. Substantiating this

gene expression signature, microscopic examination

and differential counting of granulocyte maturation

pools in our bone marrow samples revealed an

increased proportion of early immature myeloblasts

and promyelocytes in dexamethasone treated steers

relative to control animals (Fig. 10a). In addition, bone

marrow from the treated animals showed modest

reductions in proportions of mature band and

segmented neutrophils (Fig. 10a), also observed as

a small decrease in G1+ immunostained cells

(Fig. 10b), compared with bone marrow from control

steers. This presumably reflected an increase in the

release of newly matured neutrophils into blood.

Nonetheless, production and release of neutrophils

appeared to be well balanced during the glucocorticoid

challenge because there was no gross effect of

dexamethasone administration on overall cellularity

of CD45+ leukocyte-lineage cells in bone marrow

Immunopathology 105 (2005) 197219(Fig. 10c).

y and

lated

crease

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script

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C1918J.L. Burton et al. / Veterinary Immunolog

Table 5

Dexamethasone up-regulated expression of three genes and down-regu

12 h post hormone administration

Clone name or actual gene name of spotted BOTL cDNA

Up-regulated genes

Clone name: BOTL0100013_E10. FANCA; a nuclear protein, in

related with cell proliferation in bone marrow in acute myelogen

(E = 1050, TC190561, BM251970)a

Gene name: IKB KINASE ALPHA. Activates NF-kB pathway o

transcription; up-regulated expression in myeloid blasts

Gene name: JUN-D PROTO-ONCOGENE. Part of the AP-1 tran

complex; highly expressed in bone marrow cells of multiple mye

Down-regulated genes

Clone name: BOTL0100005XH03R. CD164; CD34+b cell adhesi

acts as potent negative regulator of proliferation (E = 1020.69, TBM 251379)aIt thus appears likely that glucocorticoids influence

neutrophil production and release from bone marrow

in cattle, which may have elevated the number of

youthful neutrophils in blood of the parturient dairy

cows in our previous study. However, additional

reprogramming in circulating neutrophils under the

influence of parturient cortisol (and possibly proges-

terone) must also have occurred in the cows because

the complex survival/tissue remodeling gene expres-

sion signature in their blood neutrophils was not

detected in bone marrow cells of our dexamethasone

treated steers. Differences in gene expression profiles

between bone marrow and blood neutrophils may have

been due in part to differences in GRa expression(Fig. 1) and related transcription factors between these

two neutrophil pools. On the other hand, we did not

Gene name: BAX-ALPHA. Pro-apoptotic Bcl-2 family member (see F

Gene name: ENDOTHELIN RECEPTOR. Critical role in tissue devel

and differentiation, cell proliferation, bone remodeling, and apoptosis;

expression known to be inhibited by dexamethasone

Gene name: IL-7 RECEPTOR ALPHA. Activation via IL-7 binding le

survival, proliferation, differentiation, and maturation of hematopoietic

Gene name: GRO-Beta. Growth-regulated oncogene beta; a chemokine

dose-dependently suppresses colony formation of myeloid progenitors

Gene name: RECEPTORTYPE TYROSINE KINASE RSE. Stromal ce

expression; involved in hematopoiesis regulation especially of

megakaryocytes and myelomonocytic precursors

Gene name: VE CADHERIN. Vascularendothelial cadherin; mediates

cellcell adhesion in bone marrow, down regulation associated with C

transendothelial migration into bone marrow

a E = expectation value, TC = TIGR cluster number, and BM = GenBan

microarrays (also see http://www.cafg.msu.edu).b CD34+ cells are leukocyte-lineage precursor cells that can be found in

differentiation during hematopoiesis.Immunopathology 105 (2005) 197219 213

expression of seven genes in bone marrow cells collected from steers

P-value

from GLM

P-value from two-step

mixed model

d expression

ukemia

0.003 0.005

0.014 0.005

ion factor

patients

0.003 0.024

ceptor,

57,

0.042 0.041purify neutrophil-lineage cells from the bone marrow

samples as we did for the blood cells, and this would

certainly cause differences in gene expression profiles

between the two sample types. Furthermore, we

examined gene expression in bone marrow of the

dexamethasone treated animals at only one time point,

which was not necessarily representative of our

sampling times in the parturient cows. Also, the blood

environment at parturition in cows is more complex

than a simple surge in cortisol, and other factors such as

peptide hormones, cytokines, and prostaglandins also

may have influenced gene expression in both popula-

tions of neutrophils. Regardless of these problems

comparing the results from our two microarray

experiments, preliminary bone marrow gene expres-

sion data derived from our dexamethasone treated

ig. 3) 0.007 0.061

opment 0.037 0.022

ads to cell

cells

0.036 0.042

that 0.042 0.020

ll 0.010 0.050

endothelial

D34+ cell

0.049 0.052

k number for BOTL sequences spotted on fourth generation BOTL

the blood circulation but home to bone marrow for proliferation and

y andJ.L. Burton et al. / Veterinary Immunolog214steers are interesting enough to warrant additional

functional genomic analysis of bone marrow neutro-

phils collected from periparturient cows. Such studies

would benefit from continued expansion and annotation

of our BOTL microarray, including its significant

enrichment with genes collected from a bone marrow

cDNA library.

4. Glucocorticoid reprogramming of the bovine

neutrophil system: a new paradigm to help explainneutrophil dysfunction in parturient dairy cows

Given our collective observations, we propose that

the sudden surge in blood cortisol at parturition

Fig. 10. Dexamethasone administration appeared to promote balanced gran

segmented cells) in treated dairy steers (a) such that gross changes in overall

of the tissue were not altered. Bone marrow was collected from the steers (

were obtained from microscopic enumeration of MayGrunwald stained (S

cells that were isolated by maturation stage on Percoll density gradients

immunostaining with the bovine granulocyte marker G1 (b) and the pan leu

the protocol described in Weber et al. (2004). a and b in panel (a) indicate sig

treated steers within granulocyte-lineage pool.Immunopathology 105 (2005) 197219induces adaptive changes in the neutrophil system

that supports expansion and release of bone marrow

cells and extended life span of blood cells that have

re-prioritized functional activities favoring tissue

remodeling over antibacterial defense. Because the

surge in parturient cortisol is fetus-derived and

necessary for rapid shut down of progesterone

secretion by the placenta and corpus luteum to

enable parturition, the gene expression profiles we

observed in the dams blood neutrophils may have

been driven by the fetus need to be born. Such

changes in neutrophil gene expression may enable

recruitment of large numbers of the cells into the

reproductive tract and placenta to facilitate separa-

tion of maternalfetal membranes, rupture of the

ulopoiesis and bone marrow release of mature neutrophils (band and

cellularity of the granulocytic (b) and total leukocyte (c) populations

n = 8) as described in the text and in Weber et al. (2004). Data in (a)

igma Chemical Co., St. Louis, MO) neutrophil-lineage bone marrow

(see legend of Fig. 1). The percentage of total bone marrow cells

kocyte marker CD45 (c) were determined flow cytometrically using

nificant (P < 0.05) differences between control and dexamethasone-

However, as long as parturition occurs rapidly and

y andfetal membrane, and softening of the cervix for

delivery of the calf. We propose that such

reprogramming of bone marrow and blood neutro-

phils is normal and appropriate because the seven

cows used in our studies proceeded through par-

turition without complication and remained healthy

well into lactation. We are not the first to put forward

such ideas. Sendo et al. (1996) speculated that

neutrophil survival induction by glucocorticoids acts

in some way to benefit innate functions of the cells

by augmenting the supply of youthful neutrophils

available for defense of body tissues. In light of our

observations, we suggest that the dysfunction

label so commonly used to describe neutrophils

from parturient cows be changed to altered

function.

That said, our gene expression results did suggest

that parturient changes in neutrophil life span and

tissue remodeling capacity is accompanied with

temporary disabling of some antibacterial functions

of the cells. In particular, the ability of the cells to

marginate on and migrate through inflamed endothe-

lium in infected peripheral tissues, and to mount

effective respiratory burst needed to kill pathogens

they phagocytose in these tissues, may be reduced

for a short period of time around parturition. In some

animals, this could translate into increased suscept-

ibility to disease caused by opportunistic pathogens

and, combined with the cells new longevity and

tissue degrading status, increased severity of tissue

inflammation mediated by neutrophils that do make

their way into infected tissue as the blood cortisol/

progesterone profile changes. However, if the

margination/migration capacity of blood neutrophils

is disabled at parturition, one is led to question how

the cells manage to traffic to the reproductive tract

and placenta. The answer to this question may lie in

the endothelial cells that line blood vessels of the

uterus, cervix and placenta, which may up regulate

their own set of adhesion molecules in response to

cortisol/progesterone imbalance, effectively pluck-

ing neutrophils out of blood. This possibility would

be interesting to address in future studies. We also

recognize that blood and tissue factors other than

cortisol and progesterone might be involved in the

reprogramming of neutrophils at parturition, and

these will be important to identify in the future. In

J.L. Burton et al. / Veterinary Immunologlight of our presented observations and the questionswithout interruption, these risky consequences of the

glucocorticoid hit on neutrophils are expected to be

quite short-lived because the steroid also down

regulates expression of its own receptor in neutrophils

and the population of blood neutrophils turns over

rapidly. Thus, apoptosis status and traditional bacter-these raise, our next steps will be to try and unravel

the biological consequences of the neutrophil gene

expression signatures we have documented to the

processes of parturition and defense of the mammary

gland in parturient cows.

5. Summary comments

Studies using sophisticated molecular probes and

global gene expression analyses show that neutrophils

are not simply short-lived terminally differentiated

cells, but rather, have mRNA synthetic capacity that

contributes to their elasticity (Goulding et al., 1998;

Newburger et al., 2000; Subrahmanyam et al., 2001;

Maianski et al., 2002; Madsen et al., 2002, 2004).

Indeed, these leukocytes probably participate in

previously unanticipated ways to a whole host of

immune, inflammatory, and tissue remodeling

responses that keep animals alive during stress (Smith,

1994; Cassatella, 1999; Brinkmann et al., 2004).

Through our functional genomics studies we have

shown that intriguing responses of blood neutrophils

and bone marrow cells occur during exposure to

glucocorticoids in vivo. While it has long been stated

that glucocorticoids induce generalized immune

suppression, our results offer an additional possibility.

Glucocorticoids may be a critical component of front

line host defense during stress, enhancing bone

marrow production of neutrophils, prolonging survival

of these new cells in blood, and directing their

functional priorities to deal with extracellular matrix

remodeling in the event that tissues become damaged

during fight-or-flight. One event in nature when this

system appears to be absolutely necessary is parturi-

tion. During parturition, the reproductive tract requires

large numbers of neutrophils that are committed to the

massive tissue remodeling job that ensures the fetus is

born alive, even if this occurs at the expense of

reduced antibacterial defense in peripheral tissues.

Immunopathology 105 (2005) 197219 215icidal functions of the cells may return to normal

for their thoughtful input and assistance during

Bamberger, C.M., Schulte, H.M., Chrousos, G.P., 1996. Molecular

determinants of glucocorticoid receptor function and tissue

J.L. Burton et al. / Veterinary Immunology and Immunopathology 105 (2005) 197219216sensitivity to glucocorticoids. Endocr. Rev. 17, 245259.

Beato, M., Klug, J., 2000. Steroid hormone receptors: an update.

Hum. Reprod. Update 6, 225236.

Bradley, J.R., Pober, J.S., 2001. Tumor necrosis factor receptor-

associated factors (TRAFs). Oncogene 20, 64826491.

Bowen, J.M., Chamley, L., Keelan, J.A., Mitchell, M.D., 2002.

Cytokines of the placenta and extra-placental membranes: roles

and regulation during human pregnancy and parturition. Pla-

centa 23, 257273.

Brandt, U., Kerscher, S., Drose, S., Zwicker, K., Zickermann, V.,microarray experimentation and analyses of the

presented data sets. Thanks are also extended to Dr.

Jennifer Jacob, Dr. Trine Toelboell, and Dr. Anders

Toelboell, Ms. Rebecca Darch, and Mr. Bob Kreft for

their help during blood and bone marrow collections.

The work presented in this paper was partly funded

through support from the Michigan Agricultural

Experiment Station (project numbers MICL01691

and MICL01836), the USDA-NRI program (grant

number 2001-35204-10798), and the USDA-IFAFS

program (grant number 2001-52100-11211).

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