J Appl Genet 46(2), 2005, pp. 171-177
Immunorelevant gene expression in LPS-challenged bovine
mammary epithelial cells
Ravi Pareek1, Olga Wellnitz2, Renate Van Dorp3, Jeanne Burton3, David Kerr1
1
Department of Animal Science, University of Vermont, Burlington, USA
2
Department of Physiology, Technical University of Munich, Germany
3
Department of Animal Science, Immunogenetics Laboratory and Center for Animal Functional Genomics, Michigan State
University, East Lansing, Michigan, USA
Abstract. Infection of the bovine mammary gland, in addition to causing animal distress, is a major economic
burden of the dairy industry. Greater understanding of the initial host response to infection may lead to more accu-
rate selection of resistant animals or to novel prophylactic or therapeutic intervention strategies. The epithelial
cell plays a role in the host response by alerting the immune system to the infection and providing a signal as to
where the infection is located. To understand this process better, a cDNA microarray approach was used to search
for potential signals produced by mammary epithelial cells in response to exposure to Escherichia coli
lipopolysaccharide (LPS). Total RNA from separate cultures of epithelial cells from 4 Holstein cows was har-
vested 6 h after LPS challenge or control conditions. For each cow, RNA from control or LPS-exposed cells was
transcribed to cDNA and labeled with Cy3 or Cy5, then pooled and applied to a bovine total leukocyte (BOTL)
microarray slide containing 1278 unique transcripts. Dye reversal was used so that RNA from two of the control
cultures was labeled with Cy3 while RNA from the other two control cultures was labeled with Cy5. From the re-
sulting microarray data we selected 4 of the 9 genes significantly (P < 0.02) induced (>1.25-fold) in response to
LPS exposure for more detailed analysis. The array signal intensity for 3 of these genes, RANTES/CCL5, IL-6 and
T-PA, was relatively low, but quantitative real-time RT-PCR (Q-RT-PCR) analysis revealed that they were in-
duced 208-fold, 10-fold and 3-fold, respectively. The gene that showed the greatest fold induction by microarray
analysis (2.5-fold) was CXCL5. This gene had a relatively strong signal intensity on the array and was easily de-
tected by northern blot analysis, which indicated a 10-fold induction. This cell culture model system provides evi-
dence for an important role of the mammary epithelial cell in initiating the innate response to infection.
Key words: bovine total leukocyte microarray, BOTL, lipopolysaccharide, mammary gland.
position. Depending on the duration and severity
Introduction
of disease, the productive performance of infected
dairy cattle may be diminished for the remainder
Bovine mastitis is the most important infectious
of the lactation cycle (Wilson et al. 2004). On av-
disease of dairy cattle, affecting both the quality
erage, losses associated with mastitis cost Ameri-
and quantity of milk produced in the world. Masti-
can dairy producers about 2 billion dollars
tis occurs with the highest frequency around partu-
annually (Sordillo and Streicher 2002), and world-
rition, when protective inflammatory responses at
wide losses are estimated at 25 billion dollars an-
the blood-mammary barrier are delayed or hin-
nually.
dered (Burton and Erskine 2003; Diez-Fraile et al.
The innate immune system represents the first
2003; Paape et al. 2002). Mastitis can cause de-
line of defense in the host response to infection
struction of milk-synthesizing tissues, resulting in
and is poised to recognize and respond immedi-
decreased milk production and altered milk com-
Received: September 12, 2004. Revised: February 26, 2005. Accepted: March 03, 2005.
Correspondence: D.E. Kerr, Department of Animal Science, Terrill Hall, University of Vermont, 570 Main Street, Burlington,
VT 05405, USA; e-mail: david.kerr@uvm.edu
172 R. Pareek et al.
ately to the earliest stages of infection (Hoffmann DMEM/F12, 10% FBS, ITS supplement
et al. 1999). The inherent capability of the innate (5 µg mL 1 insulin, 5 µg mL 1 transferrin and
immune system to respond to a vast number of 0.005 µg mL 1 sodium selenite; Invitrogen,
pathogens is mediated by its ability to recognize Carlsbed, CA), penicillin G (100 u mL 1) and
highly conserved motifs shared by diverse patho- streptomycin (100 µg mL 1). They were then split
gens. These motifs, commonly referred to as into two 75-mL flasks/cow (15 × 106 cells/flask)
pathogen-associated molecular patterns (PAMPs), in challenge media (3% FBS medium). When
include some bacterial cell wall components, like ~80% confluence was obtained, the medium was
lipopolysaccharide (LPS), peptidoglycan and replaced with fresh challenge media, with or with-
lipoteichoic acid (Aderem and Ulevitch 2000). out 1µgmL 1 LPS (0111:B4; Sigma). Total RNA
Although milk macrophages may play a role in was harvested after 6 hrs by TRIzol (Invitrogen,
the triggering of the inflammatory response, stud- Carlsbed, CA) and mRNA was isolated by
ies at various epithelial sites strongly suggest that oligo(dT)-latex beads with the NucleoTrap Kit
epithelial cells are capable of responding to bacte- (BD Biosciences, San Jose, CA).
rial intrusion and play a major part in the initiation
of inflammation. A better knowledge of the medi- Bovine total leukocyte (BOTL) microarray
ators involved in the initial recognition and signal-
In the present study we used 4 BOTL microarray
ing of bacterial invasion could help in devising
slides (one for each cow), in which samples de-
strategies to modulate the defense of the udder
rived from control and LPS-treated cultures com-
(Rainard and Riollet 2003).
peted for hybridization to the arrayed cDNA spots.
We, and others, have used a bovine mammary
The spots on the BOTL slides represent
epithelial cell culture system to investigate factors
1278 genes, spotted in triplicate, drawn from a bo-
produced by these cells in response to infection by
vine leukocyte library and additionally supple-
Staphylococcus aureus or exposure to Escherichia
mented with candidate genes involved in
coli LPS (Wellnitz and Kerr 2004; Boudjellab et
apoptosis and inflammation. A listing of the genes
al. 1998; Talhouk et al. 1990; Okada et al. 1997).
is available on the Center for Animal Functional
These studies demonstrated that epithelial cells re-
Genomics (CAFG) website (www.nbfgc.msu.edu).
spond with enhanced production of IL-8, TNFá,
The general cDNA spotting design used on
and lactoferrin. The role of IL-8 in attracting neu-
the BOTL microarray is described in detail on
trophils to the site of infection is well known, and
the website of the Center for Biotechnology Infor-
may be a key feature of the epithelial cell re-
mation Gene Expression Omnibus (NCBI GEO;
sponse. In an effort to uncover additional epithe-
http://www.ncbi.nlm.nih.gov/geo/, platform ac-
lial cell signals of infection, we employed a
cession number GPL 363). Control genes spotted
microarray-based screening approach. We were
within and across the 48-patch microarrays (9 × 9
particularly interested in the early responses and
series of spots per patch) included 96 spots of syn-
thus examined the RNA from our bovine epithelial
thetic Q cDNA (external controls), 144 spots of
cell model following a 6-hour exposure to E. coli
GAPDH, 75 spots of ß-actin and 75 spots of
LPS.
RPL-19 (internal controls).
For probe synthesis, 1 µg of polyA+-enriched
RNA was converted to cDNA (42°C for 65 min,
Material and methods
then 70° for 5 min) using the Atlas PowerScript
fluorescent labeling kit (BD Biosciences, San
LPS treatment of mammary epithelial cells
Jose, CA). Dye couplings and labeled cDNA
purifications were performed according to
The procedures for cell culture and cryopreserva-
the manufacturer s instructions (Amersham Bio-
tion have been previously described (Wellnitz and
sciences, Piscataway, NJ). For two cows
Kerr 2004). In brief, mammary tissue was col-
the LPS-challenged cultures were labeled with
lected at slaughter from 4 lactating Holstein cows.
CY5 and control cultures with CY3. Dye labeling
Epithelial cells were obtained after collagen-
was reversed for the other two cows.
ase-based digestion of the tissue. Cell numbers
Cow-specific pairs of labeled cDNA were in-
were multiplied by culture on a plastic substratum,
cubated at 70°C for 5 min just prior to application
passaged several times, and then cryopreserved.
to the array under a cover slip. Hybridization was
Aliquots of cells (passage 3) from all 4 cows were
allowed to proceed at 50°C overnight in a humidi-
thawed for culture, and grown separately to con-
fied chamber (HYBAID; Labnet, Woodbridge,
fluence in a growth medium consisting of
Gene expression in LPS-challenged cells 173
NJ). The cover slips were then removed and the ar- and 85-bp regions, respectively. For each PCR
rays washed twice for 15 min at me- product the melting curve was determined.
dium-stringency (45°C, 1 × SSC, 0.05% SDS) on
The comparative threshold cycle number (2 Ct)
an orbital shaker. An additional wash (0.1 × SSC)
method was used after a validation experiment
was applied for 5 min. Finally, arrays were re-
demonstrated that efficiencies of target and refer-
moved, rinsed with H2O, and centrifuged in open
ence (ß-actin) were approximately equal. Ct val-
50-mL conical tubes (500 g for 3 min at room tem-
ues define the threshold cycle of PCR, at which
perature) to dry. Array scanning was done using
amplified products were detected. Our results are
a GeneTAC LS IV scanner (Genomic Solutions,
represented as Ct values, where Ct is the differ-
Ann Arbor, MI) and accompanying software (ver-
ence in threshold cycles for target and ß-actin as an
sion 3.01). Spot analyses were performed using
internal control. Fold changes in expression for
GeneTAC Integrator 3.3.0 microarray analysis
LPS relative to control ( Ct) were calculated
software (Genomic Solutions, Ann Arbor, MI).
from the arithmetic formula 2 Ct as described in
detail by Livak and Schmittgen (2001).
Northern blot and Q-RT-PCR analysis
Verification of altered expression of CXCL5 was
Statistical analysis
pursued through the use of northern blot. Total
After the BOTL microarray experiment, total in-
RNA (8 g/lane) was separated on agarose gel
tensity values for each dye channel were loaded
containing 1.1% formaldehyde and blotted to
into SAS for data normalization and analysis. Data
Gene Screen hybridization membranes
normalization was performed considering a robust
(NEN/Perkin Elmer) in 1 M ammonium acetate.
local regression technique (Yang et al. 2002) using
Membranes were sequentially probed with
32
the LOWESS (Locally-weighted Regression) pro-
P-labeled CXCL5, and bovine ß-actin PCR prod-
ucts. Differences in gene expression were ana- cedure of SAS 8 (SAS/STAT Software, Cary,
NC). The normalized data were analyzed with
lyzed with a phosphoimager (STORM, Molecular
Dynamics). Expression of CXCL5 was normalized a 2-step mixed model advocated by Wolfinger et
to ß-actin expression levels. al. (2001). Significance of values of expressed
Microarray-indicated changes in RANTES, genes was measured by Student s t-test in LPS
IL-6, and T-PA expression were verified with versus control spots.
quantitative real-time RT-PCR (Q-RT-PCR). To- Differences between control and LPS chal-
tal RNA (1.5 g) was treated with DNase lenge in the 4 genes selected for further validation
(DNAfree; Ambion) according to the manufac- by Q-RT-PCR or northern blot analysis were
turer s protocol, then reverse-transcribed to cDNA tested for significance by paired t-test. A P value
with random hexamer primers by using the Super- of <0.01 was considered significant. One sample
script First-Strand Synthesis System (Invitrogen, for the T-PA gene expression was lost due to
Carlsbed, CA) according to the manufacturer s a technical error.
protocols. Real-time PCR was performed with
the SYBR Green JumpStart Taq Ready Mix
Results
(Sigma) on an ABI Prism 7700 Sequence Detector
at the Vermont Cancer Center core facility. Bo-
In general, the results from the microarray analysis
vine specific primers for T-PA were 5 -CAA TGA
lacked sensitivity. However, cells from the indi-
CAT CGC GCT GCT-3 for forward; and 5 -TGG
vidual cows appeared to yield consistent results,
GTA CAA CCT GAC GTG AGC-3 for reverse;
which can be seen by a representative northern
primers for RANTES were 5 -GCC AAC CCA
blot analysis of CXCL5 (Figure 1). In total, the sta-
GAG AAG AAGTG-3 for forward; and 5 -CTG
tistical analysis revealed that 54 genes were signif-
CTT AGG ACA AGA GCG AGA-3 for reverse;
icantly induced by LPS, but the overall fold
primers for IL-6 were 5 -TGAGGGAAA
changes were quite low. Considering the robust-
TCAGGAAAATGT-3 for forward; and
ness of the system, we concentrated our efforts on
5 -CAGTGTTTGTGGCTGGAGTG-3 for re-
the 9 genes that had the greatest fold changes
verse; primers for ß-actin were 5 -GCA AAT
(Table 1). Of these genes, BLASTn analysis and
GCT TCT AGG CGG ACT-3 for forward; and
gene ontology information as well as Genecards"!
5 -CAA TCT CAT CTC GTT TTC TGC G-3 for
studies for functional genomics in humans indi-
reverse. The above primers flanked 205, 119, 110
174 R. Pareek et al.
Figure 2. Quantification of northern blot analysis of
CXCL5 gene expression in control and LPS-treated
Figure 1. Northern blot analysis of CXCL5 gene
mammary epithelial cells derived from 4 cows and treated
expression in RNA (8µg/lane) from control (CTL) and
for 6 hours with LPS (1µg mL 1). *Treatment means are
LPS-treated mammary epithelial cells, derived from
significantly different (P<0.01).
4 cows, and treated for 6 hours with LPS (1µg mL 1).
ß-actin expression was used as a lane loading control.
CXCL5, which encodes an epithelial-derived
Bands were visualized with a phosphoimager.
chemokine (Figure 2).
Three other genes, which had a relatively low
cated that 5 genes were likely components of
signal intensity on the microarray slides, were se-
the innate immune system, and 4 genes were re-
lected for further examination by the very sensi-
lated to apoptosis regulation.
tive Q-RT-PCR technique. The expression of
In accordance with our goal of screening for
RANTES, which encodes a CC cytokine, was dra-
signals produced by epithelial cells to indicate
matically increased (P < 0.01) by LPS exposure.
LPS exposure, we selected 4 genes from
The mean Ct for RANTES declined from
the microarray data for further analysis. Based on
11.1 Ä… 1.0 to 3.4 Ä… 0.3 in response to LPS.
the relatively strong signal intensity from
This corresponds to a 208-fold, LPS-mediated, in-
the microarray chips, the expression of CXCL5
duction in RANTES gene expression (Figure 3a).
was examined by traditional northern blot analy-
With a similar analysis, the Ct of IL-6, whose
sis. This technique revealed that CXCL5 was con-
product is known for its cytokine activity in
sistently induced in LPS-treated cells of all 4 cows
the acute-phase response, declined (P < 0.01) from
(Figure 1). Subsequent quantification of band in-
10.0 Ä… 0.3 to 6.7 Ä… 0.3, which corresponds to
tensity indicated that LPS challenge caused
a 10.5-fold induction (Figure 3b). The expression
a 10.1-fold increase (P < 0.01) in the expression of
of T-PA, whose product is involved in
Table 1. List of top ten up-regulated genes in mammary epithelial cells in response to LPS treatment for 6 hours, as
detected by microarray analysis (P < 0.02)
Fold
Gene name Gene product Ontology and/or molecular function of gene product
change
CXCL5 epithelial-derived neutrophil-activating neutrophil activator 2.46
protein ENA-78
IL-1ß converting en- apoptosis-related cysteine protease caspase activator 1.44
zyme/Caspase-13B
IL-6 B-cell stimulatory factor 2 cytokine activity for acute-phase response; final dif- 1.39
ferentiation of B-cells into Ig-secreting cells
T-PA plasminogen activator (tissue type) binds to fibrin with high affinity, activates plasmin 1.36
IAP-1 NSD inhibitor of apoptosis protein 1 apoptotic suppressor 1.36
Caspase-13A apoptosis-related cysteine protease caspase activator 1.33
KIAA1554 double cortin domain-containing pro- intracellular signaling cascade 1.27
tein 2 (RU2S protein).
RANTES/CCL5 member 5 of small inducible cytokine chemoattractant for blood monocytes 1.26
subfamily A (Cys-Cys)
Beta 2-microglobulin beta 2-microglobulin (beta2M) beta-chain of major histocompatibility complex 1.25
class I molecules, acting as MHC class I receptor
PIAP putative inhibitor of apoptosis unknown 1.25
Gene expression in LPS-challenged cells 175
Figure 3. RANTES (a), IL-6 (b), and T-PA (c) gene expression in control (CTL) and LPS-treated mammary epithelial
cells, as measured by quantitative RT-PCR. ÄCt values represent Ct values of RANTES, IL-6 and T-PA after subtraction
of Ct values for ß-actin (internal control). *P < 0.01 between control and treated cells.
plasminogen activity, was also induced by LPS fection in this epithelial cell culture system
treatment, although to a much lesser extent. Nor- (Wellnitz and Kerr 2004).
malized cycle threshold values of T-PA declined Our microarray gene list indicated that the pro-
(P < 0.01) from 9.65 Ä… 0.7 to 8.2 Ä… 0.8 due to LPS duction of two novel chemokines, RANTES and
exposure, corresponding to a 3-fold induction of CXCL5, was also induced by LPS.
T-PA gene expression (Figure 3c). The chemokines are grouped into 4 families ac-
cording to the number and arrangement of con-
served N-terminal cysteine motifs: C, CC, CXC,
Discussion
and CX3C. The CXC chemokines are subdivided
into two classes based on the presence or absence
of a tripeptide motif, Glu-Leu-Arg (ELR), which
The use of microarrays in conjunction with other
is N-terminal to the conserved CXC region. Mem-
genomic and proteomic methods should provide
bers that contain the motif (ELR+) are potent
complementary tools for selection of livestock
chemoattractants for neutrophils and promoters of
with superior health and performance attributes.
angiogenesis, whereas those that do not contain
In the present study, we further exploit our model
the motif (ELR ) are potent chemoattractants for
system (Wellnitz and Kerr 2004) to examine
the direct effect of LPS on bovine mammary epi- mononuclear cells. Representatives of the ELR+
CXC chemokines are structurally similar, includ-
thelial cells by gene expression profiling as well as
ing IL-8/CXCL8, and ENA-78/CXCL5 (Cole et
independent confirmation of expression changes
al. 2001). RANTES is a member of the CC family
by quantitative techniques. A similar approach
of chemokines.
was used to profile parturition-induced changes in
The remarkable induction of chemokine gene
expression of genes of bovine blood neutrophils
expression by the epithelial cell lends strong sup-
(Madsen et al. 2004).
A key component of the host innate immune re- port to its role in stimulating migration of
sponse to mastitis is a rapid increase in concentra- polymorphonuclear neutrophil leukocytes
(PMNs) into the mammary gland to provide de-
tions of cytokines in milk (Bannerman et al.
2004a; 2004b). Bovine lymph also contains in- fense against invading mastitis pathogens (Paape
et al. 2003). These neutrophil-attracting che-
creased levels of cytokines, such as IL-8 and
mokines, known to be produced by epithelial cells,
TNF-á (Persson Waller et al. 2003) in response to
could lead to cell-specific markers for immune re-
LPS exposure. However, the contribution of
the mammary epithelial cell to this response is un- sponse in future in vitro studies. A recent study
known. Our microarray and Q-RT-PCR results in- (Ruddy et al. 2004) has demonstrated that IL-17
and TNFá cooperatively induce the production of
dicated that there was a marked induction of IL-6
production by bovine epithelial cells after stimula- the LPS-inducible chemokine CXCL5 in
the preosteoblast murine cell line, MC3T3. Sig-
tion with LPS. This cytokine, along with IL-1ß
and TNFá, are known to play major roles in the in- naling deficiency of IL-17 in mice causes dramatic
reductions in CXCL5 and subsequent neutrophil
flammatory and febrile responses to infection
chemotaxis, resulting in an increased susceptibil-
(Virta et al. 2002; Dinarello 1996). Furthermore,
we have previously demonstrated a rapid induc- ity to bacterial infection. The structurally similar
tion of TNFá in response to LPS and S. aureus in- IL-8 is also known to be produced by mammary
176 R. Pareek et al.
sponses following intramammary infection. Clin
epithelial cells in response to LPS (Wellnitz and
Diagn Lab Immunol 11:463 472.
Kerr 2004, Boudjellab et al. 1998).
Bannerman DD, Paape MJ, Hare WR, Hope JC, 2004b.
Further evidence of epithelial signaling of in-
Characterization of the bovine innate immune re-
fection is indicated by the >200-fold induction of
sponse to intramammary infection with Klebsiella
RANTES gene expression. RANTES/CCL5 is one
pneumoniae. J Dairy Sci 87: 2420 2432.
of several CC cytokine genes involved in
Boudjellab N, Chan-Tang HS, Li X, Zhao X, 1998.
immunoregulatory and inflammatory processes.
Interleukin 8 response by bovine mammary epithe-
lial cells to lipopolysaccharide stimulation. Am J
The CC cytokines are proteins characterized by
Vet Res 59: 1563 1567.
two adjacent cysteines. The cytokine encoded by
Burton JL, Erskine RJ, 2003. Mastitis and immunity:
this gene functions as a chemoattractant for blood
some new ideas for an old disease. Veterinary
monocytes, memory T helper cells and
Clinics of North America: Food Animal Practice
eosinophils. Several molecules along with
19: 1 45.
RANTES, which mediate leukocyte trafficking in
Cole AM, Ganz T, Liese AM, Burdick MD, Liu L,
the immune system, are expressed in the mam-
Strieter RM, 2001. Cutting edge: IFN-inducible
mary gland (Nishimura 2003) and found in milk ELR- CXC chemokines display defensin-like
antimicrobial activity. J Immuno 167: 623 627.
(Bannerman et al. 2004a; 2004b). The present
Diez-Fraile A, Meyer E, Burvenich C, 2003.
study implicates the epithelial source of this
Sympathoadrenal and immune system activation
chemokine.
during the periparturient period and their associa-
Our results indicate that LPS-challenged mam-
tion with bovine coliform mastitis. A review. Vet Q
mary cells may contribute indirectly to reduced
25: 31 44.
milk quality and tissue damage through
Dinarello CA, 1996. Cytokines as mediators in
up-regulation of T-PA gene expression. Conver- the pathogenesis of septic shock. Curr Top
Microbiol Immunol 216: 133 165.
sion of plasminogen to plasmin generates a source
Hoffmann JA, Kafatos FC, Janeway CA, Ezeko-
of proteolytic activity in the bovine mammary
witz RA, 1999. Phylogenetic perspectives in innate
gland (Zavizion et al.1996). Confirmation of T-PA
immunity. Science 284: 1313 1318.
gene expression by Q-RT-PCR supports previous
Livak KJ, Schmittgen TD, 2001. Analysis of relative
studies (Zachos et al. 1992) on the effects of masti-
gene expression data using real-time quantitative
tis and stage of lactation on plasminogen activator
PCR and the 2(-Delta Delta C(T)) method. Methods
(PA) activity in milk, where PA activity is in-
25: 402 408.
creased during severe mastitis.
Madsen SA, Chang LC, Hicky MC, Rosa GJ,
In conclusion, the present study has used Coussens PM, Burton JL, 2004. Microarray analy-
sis of gene expression in blood neutrophils of
microarray technology to uncover a remarkable
parturient cows. Physiol Genomics 16: 212 221.
induction of CXCL5 and RANTES gene expression
Nishimura T, 2003. Expression of potential lympho-
in bovine mammary epithelial cells in response to
cyte trafficking mediator molecules in the mam-
LPS. Without the array data, it is unlikely that we
mary gland. Vet Res 34: 3 10.
would have chosen these two candidate genes for
Okada H, Ito T, Ohtsuka H, Kirisawa R, Iwai H,
further investigations. Marked induction of IL-6
Yamashita K, Yoshino T, Rosol TJ, 1997. Detection
and T-PA expression by bovine epithelial cells af- of interleukin-1 and interleukin-6 on cryopreserved
ter stimulation with LPS confirms that the prod- bovine mammary epithelial cells in vitro. J Vet Med
Sci 59: 503 507.
ucts of those genes act as a pro-inflammatory
Paape M, Bannerman D, Zhao X, Lee J, 2003. The bo-
cytokine and as an agent stimulating tis-
vine neutrophil: Structure and function in blood and
sue-degradation, respectively.
milk. Vet Res 34: 597 627.
Paape M, Mehrzad J, Zhaom X, Detilleuxm J,
Acknowledgements. This study was supported by
Burvenich C, 2002. Defense of the bovine mam-
the grant VT-AS-034CG (D.E.K.) from the Vermont
mary gland by polymorphonuclear neutrophil leu-
Agricultural Experiment Station, USDA.
kocytes. J Mammary Gland Biol Neoplasia 7:
109 121.
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