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RT-PCR & real-time RT-PCR
applications in
Mammary Gland & Mammary
Gland Immunology
Short-term
changes of mRNA expression of various inflammatory factors and milk
proteins in mammary tissue during LPS-induced mastitis
S. Schmitz, M.W. Pfaffl, H.H.D. Meyer, R.M. Bruckmaier (2004)
Domestic Animal Endocrinology 26(4) 111-126
During mammary gland infection,
non-specific responses are the
predominant ones. The goal of this studywas to investigate
themRNAexpression of various soluble immune components and of the major
milk proteins during the acute phase of mammary inflammation. Five
healthy lactating cows were intramammary infused in one quarter with
100µg Escherichia coli-endotoxin (lipopolysac- charide, LPS) and
the contralateral quarter with saline (9 g/l) serving as control.
Mammary biopsy samples of both quarters were taken immediately before
and at 3, 6, 9 and 12 h after infusion and mRNA expression of various
factors was quantified via real-time RT–PCR. Blood samples for
determination of leukocyte number were taken simultaneously with the
biopsy samples
and rectal temperature was measured at 1-h intervals. Rectal
temperature
increased until 5 h (P < 0.05) after LPS administration and remained
elevated until 9 h after LPS inoculation. Blood leukocyte number
decreased
(P < 0.05) from 0 to 3 h from 7.7±1.1×109 l−1 to
5.7±1.0×109 l−1 and thereafter recovered to pre-treatment
levels until 12 h after LPS challenge. In LPS-treated quarters, tumor
necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P
< 0.05) to highest values at 3 h after LPS
challenge. Lactoferrin, lysozyme, inducible nitric oxide synthase
increased
(P < 0.05) and peaked at 6 h after challenge, and
platelet-activating
factor acetylhydrolase-mRNA expression tended to increase (P = 0.07).
mRNA expression of insulin-like growth factor-I and of alphaS1-casein
(CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change
significantly,
whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin
decreased
(P < 0.05) in both quarters and that of kappa-CN only in the LPS
quarter.
mRNAexpression of some investigated factors (tumor necrosis
factor-alpha,
lysozyme, 5-lipoxygenase,beta-lactalbumin) changed in control quarters,
however in all respective factors less than in the LPS quarters (P <
0.05).
In conclusion, mRNA expression of most inflammatory factors increased
within
hours, whereas that of most milk proteins remained unchanged.
mRNA
expression of immune factors and milk proteins in mammary tissue
and milk cells and their concentration in milk during subclinical
mastitis
S. SCHMITZ, M.W. PFAFFL, M. MILLER, J. BUCHBERGER, T. MEYER, H.
SAUERWEIN,
R.M. BRUCKMAIER
Milk Science International 59 (7/8) 2004
5-Lipoxygenase,
Cyclo-oxygenase-2 and Tumor necrosis factor
alpha
(TNF-alpha) gene expression in somatic milk
cells
Wittmann SL,
Pfaffl M., Meyer HHD & Bruckmaier RM (2002)
Milk Science
International, 57(2): 63-66.
Summary
The goal of this
work was to investigate whether different fractions of somatic cells in
cow milk do produce prostaglandins, leukotrienes and the cytokine tumor
necrosis factor alpha (TNFalpha). mRNA expressions of TNFalpha
and of key enzymes of prostaglandin and leukotriene biosynthesis,
cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), respectively, were
quantitatively
determined. Eleven clinically healthy Brown Swiss cows were either
defined
as control (C) group (n=5; all quarters < 150 000 cells/ml)
or as group with partially elevated quarter somatic cell counts (SCC;
n=6) with at least one quarter > 150 000 cells/ml (H) and one
quarter < 150 000 cells/ml (L). Total quarter milk from one quarter
of control animals and from two quarters of cows with partially
elevated
SCC (one of H and one of L) was collected. Cells were fractionized into
macrophages and lymphocytes (MAC+LYM) and polymorphonuclear leukocytes
(PMN) using a density gradient. RNA was isolated, reversely transcribed
and quantitative PCR was carried out. mRNA expression was similar for
5-LO,
COX-2 and TNFalpha: no differences between C and L quarters were
detected.
However, expression was markedly elevated in H quarters. mRNA
expression
of all parameters tested was higher in MAC+LYM than in the PMN
fraction.
In conclusion, this investigation underlines that somatic milk cells
are
able to synthesize 5-LO, COX-2 and TNFalpha.
Gene Expression of
Immunologically Important Factors
in Blood Cells, Milk Cells and Mammary Tissue
of Cows
M. W. Pfaffl, S.
L. Wittmann, H. H. D. Meyer & R. M. Bruckmaier (2003)
Jornal
of Dairy Science 86: 538–545
Summary
Cytokines,
eicosanoids and lactoferrin are involved in the mammary gland´s
immune response to invading microorganisms. The goal of this work
was to investigate the synthesis of these immunologically important
factors in somatic milk cells, blood cells and mammary tissue of cows
with different somatic cell count levels, i.e. different immunological
activity. On the level of mRNA expression, the cytokine tumor necrosis
factor a (TNFa), lactoferrin (Lf) and specific key enzymes of
leukotriene
and prostaglandin biosynthesis, 5-lipoxygenase (5-LO) and
cyclooxygenase-1
(COX-1) and –2 (COX-2), respectively, were determined. All fifteen
experimental
cows were clinically healthy with no visible mammary disease. Eight
cows
were defined as control group with all quarters < 150 000 cells/ml
(C)
while seven cows had partially elevated quarter somatic cell counts,
with
at least one quarter > 150 000 cells/ml (H) and one quarter
<
150 000 cells/ml (L). Total quarter milk from one quarter of control
group
and from two quarters of cows with partially elevated cell counts (one
of H and one of L) was collected at one milking and a blood sample was
taken simultaneously. In addition, mammary tissue samples were taken
from
the respective quarters on the following day during slaughter. Total
RNA
from milk, blood and tissue cells was isolated and reverse
transcription
and quantitative polymerase chain reaction (RT-PCR) was carried out.
All
factors investigated were not significantly different between groups in
blood cells and between C and L quarters in milk cells and mammary
tissue. TNFa and COX-2 mRNA expression was higher in milk cells and
mammary tissue of H than in L quarters, except for COX-2 in mammary
tissue. Generally TNFa and COX-2 showed their highest expression in
milk cells, 5-LO in blood cells, whereas lactoferrin was mainly
expressed by the mammary tissue. COX-1
was similarly expressed in all tested samples.
Acute
Phase Inflamatory Reaction In The Bovine Mammary Gland
R. M. Bruckmaier, S. Schmitz and M. W. Pfaffl (2002)
Mammary
Gland Health and Disease
Immune cells and bioactive substances in function of
susceptibility
and spreading of infections in human and animals
Ghent University, Belgium
Multidisciplinary
joint meeting
EU COST NETWORK, COST ACTION B20
MAMMARY GLAND BIOLOGY, WG 1 & 2 MEETING
AND COST ACTION 844
Immunological
and inflammatory factors such as cytokines, lipid mediators and
bacteriostatic
enzymes are elevated during mastitis in dairy cows. A study
was conducted to determine changes of
mRNA expression of various of these factors in the mammary tissue of
cows during 12 h after
induction of mastitis via intramammary administration of
lipopolysaccharide (LPS). Five healthy
lactating cows were injected in one quarter with 100 µg E.
coli-LPS (O26:B6) and the contralateral
quarter with saline (9 g/l) serving as control. mRNA expression in
mammary biopsy samples of the
various inflammatory factors and milk proteins at 0, 3, 6, 9 and 12 h
after LPS administration was
quantified by real-time RT-PCR. Blood samples were taken following the
same time course and
rectal temperature was measured at 1-h intervals. Temperature increased
until 5 h (P<0.05) after
LPS administration and decreased to pretreatment levels within 24 h
after LPS-challenge. Blood
leukocyte number decreased (P<0.05) from 0 to 3 h from
7.7±1.1 x 109/l to 5.7±1.0 x 109/l and
thereafter recovered to pretreatment levels until 12 h after
LPS-challenge. In LPS-challenged
quarters tumor necrosis factor α and cyclooxygenase-2 mRNA expression
increased to highest
values (P<0.05) at 3 h after LPS-challenge. Lactoferrin,
lysozyme, inducible nitric oxide synthase
mRNA expression increased (P<0.05) and peaked at 6 h after
challenge, while platelet-activating
factor acethylhydrolase mRNA increased only numerically. mRNA
expression of the investigated
factors did not change in control quarters. mRNA expression
of insulin-like growth factor-1, 5-
lipoxygenase and of αS1-casein (CN), αS2-CN, β-CN and β-lactoglobulin
did not change
significantly, whereas mRNA expression of α-lactalbumin decreased
(P<0.05) in LPS-treated and
control quarters and that of κ-CN only in the LPS-treated quarters. In
conclusion, mRNA expression
of most inflammatory factors changed within hours, whereas that of most
milk proteins remained unchanged.
Detection and
quantification of mRNA-expression
of alpha- and beta-adrenergic
receptor subtypes in
the
mammary gland of dairy cows
T. Inderwies, M. W. Pfaffl, H. H. D. Meyer, J. W. Blum &
R. M. Bruckmaier
Domestic Animal Endocrinology 2003
24(2): 123-135
Summary
Adrenergic receptors
are pharmacologically classified into the receptor types alpha-1,
alpha-2, beta-1, beta-2, and beta-3. Structural differences and varying
affinities in radioligand binding studies lead to a further
classification of alpha-1- and alpha-2-receptors into subtypes which
are termed alpha-1A(formerly C), alpha-1B, and alpha-1D-(formerly AD),
and alpha-2AD, alpha-2B, and alpha-2C, respectively. mRNA-expression
of all but one alpha-adrenergic receptor subtypes and of all
beta-adrenergic receptor types was measured quantitatively in total RNA
extracted from
mammary tissue of ten lactating dairy cows by real-time reverse
transcriptase
(RT) polymerase chain reaction (PCR). mRNA expression of
alpha1-adrenergic
receptors was highest for the alpha-1A-subtype followed by alpha-1B,
whereas
the alpha-1D-subtype could not be detected. The highest mRNA expression
of alpha-2-adrenergic receptors was found for the alpha-2AD-subtype,
followed by alpha-2B and alpha-2C. Within the beta-adrenergic
receptors, the
beta-2-receptor type was most highly expressed, followed by beta-1 and beta-3. In conclusion, eight of nine
adrenergic receptors classified to date were detected
and relatively quantified in the mammary gland of dairy cows.
Milking characteristics and
their relation to adrenergic receptor mRNA expression
and ligand binding in the mammary gland of dairy cows
T. Inderwies, M. W. Pfaffl & R. M.
Bruckmaier (2003)
Domestic Animal Endocrinology 2003 (25):
275-286
Stimulation
of  - and  -adrenergic receptors in the
bovine mammary gland affects milking characteristics such as milk yield
and peak flow rate. The aim of this study was to detect possible
correlations between milkability, adrenergic receptor binding capacity
and receptor expression at the mRNA level. In addition, dose–response
relationships of - and -adrenergic receptor stimulation
were evaluated after application of an - and -adrenergic receptor agonist,
respectively in different dosages. Density and distribution of
adrenergic receptor binding sites in the region around the large
mammary ducts were investigated as well as adrenergic receptor mRNA
expression. Milk flow of one-quarter was recorded in 10 cows without or
with additional - and -adrenergic receptor stimulation
in three dosages each. After slaughter, mammary tissue was taken from
the region around the large mammary ducts in the previously
investigated quarters. Protein and RNA were extracted for measuring 1-, 2-, and 2-adrenergic receptor
binding sites and mRNA expression levels by real-time polymerase chain
reaction (RT-PCR). Peak flow rate without additional adrenergic
receptor stimulation was negatively correlated with 2-adrenergic receptor
binding (maximal binding capacity, Bmax) and
positively correlated with 2-adrenergic receptor
expression at the mRNA level (crossing point (CP) of the real-time
PCR). During -adrenergic receptor stimulation,
there was a positive correlation between milkability and 2-adrenergic receptor
mRNA expression, whereas during -adrenergic receptor stimulation
no correlations were detected. Dose–response relationships were
existing during -adrenergic receptor
stimulations, but not during -adrenergic receptor stimulations
at four dosages each including control milking. Significant changes in
milk yield and peak flow rate mainly occurred after application of an -adrenergic receptor agonist. In
conclusion, high mRNA expression levels or binding capacities of
adrenergic receptors do not necessarily lead to according reactions in
vivo, concerning milk yield and peak flow rate. To influence milking
characteristics, individual reactions of the cow on adrenergic
stimulation have to be considered.
Poster
mRNA expression of
5-Lipoxygenase, Cyclooxygenase-2 and
Tumor necrosis factor alpha in milk somatic
cells
S.L.
Wittmann, M.W. Pfaffl, H.H.D.Meyer & R.M. Bruckmaier, (2000)
Symposium of
Immunology of Ruminant Mammary Gland ; 11. - 14. Juni in Stresa, Italien
Cytokines
and inflammatory mediators are involved in immune response in various
tissues. Within the mammary gland of dairy cows a substantial amount of
immune cells needs to be transported from blood circulation into milk
to provide cellular immune defence despite of frequent removal of milk,
including cells. Cytokines and inflammatory mediators, such as
prostaglandins
and leukotrienes, exhibit potent chemokinetic and chemotactic activity
for leukocytes and enhance the bactericidal activity of phagocytes.
Moreover, they cause increasing vascular permeability and hyperalgesia
during inflammatory disorders [1,2,3]. To assess the origin of proteins
in milk, the detection of the protein-encoding mRNAs by reverse
transcription (RT) polymerase
chain reaction (PCR) is a suitable method. Whereas the mRNA of
cytokines
can be determined with RT-PCR, prostaglandins and leukotrienes, which
are
arachidonic acid (C20:4 fatty acid) derivatives (eicosanoids), cannot
be
measured on the mRNA level. Alternatively, it is possible to measure
the
mRNA expression of specific key enzymes of their synthetic pathways.
The
goal of this work was to investigate whether leukotrienes and
prostaglandins
are produced by different fractions of somatic cells in cow milk.
Therefore,
we have developed a quantitative RT-PCR method to determine the mRNA
expression
of the key enzymes in leukotriene and prostaglandin biosynthesis, i.e.
5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2), respectively. The
expression of tumor necrosis factor alpha (TNFa ), a cytokine known to
be crucial during early inflammatory stages in the mammary gland [3],
has been studied concomitantly.
Material
and Methods
Animals.
Eleven lactating Brown Swiss dairy cows with no clinical signs of
mammary disease were used. Somatic cell counts (SCC) of total quarter
milk was measured with a fluoro-opto-electronic method using a
Fossomatic
cell counter (Foss Electric, Denmark). Cows were defined as control
group, if all quarters were < 150 000 cells/ml (C) and as group with
partially elevated quarter SCC, if minimum one quarter was > 150 000
cells/ml (H) and one quarter < 150 000 cells/ml (L).
Cell
isolation
Total quarter
milk from one quarter of control group and from two quarters of cows
with partially elevated SCC (one of H and one of L) was collected at
one morning milking. The milk was centrifuged and the cell pellet was
washed three times in phosphate buffered saline (PBS), pH 7.4. The cell
fractions were separated using a density gradient (Lymphocyte
Separation Medium, ICN, Aurora, OH, USA). Macrophages and lymphocytes
(Mac+Lym) were distributed within the interphase, polymorphonuclear
leukocytes (PMN) were located in the pellet.
RNA
Isolation and RT-PCR
Total RNA was
isolated using TriPure (Roche, Basle, Switzerland) according to the
manufacturers instructions. Synthesis of first strand cDNA was
performed with MMLV-RT (Promega, Madison, WI, USA) and random hexamer
primers. Quantitative analysis of PCR products was carried out in the
LightCycler (Roche, Basle, Switzerland) using external DNA standard
dilutions. DNA standards were generated by cloning the RT-PCR products
into pCR 4.0 vector (Invitrogen, Groningen, NL).
Statistical
evaluations
Differences
between C and L quarters and differences between Mac+Lym and PMN cell
fractions were tested for significance (p<0.05) using
Wilcoxon´s rank sum test. Differences between L and H quarters
(within animal) were tested for significance (p<0.05) by
Wilcoxon´s signed rank
test.
Results
Mean SCC were
27±5 and 34±20 x103/ml in C and L quarters,
respectively, and significantly higher in H quarters (741±289
x103/ml). As shown in table 1, mRNA expressions behaved similarly for
5-LO, COX-2 and TNFa. There were no differences between C and L
quarters,
but expression levels were markedly elevated in H quarters. mRNA
expression
of protein tested was higher in Mac+Lym than in PMN cell fractions.
Discussion
and Conclusions
Our results
indicate that 5-LO, COX-2 and TNFa are, at least to a certain extent,
produced by somatic milk cells, i.e. represent milk borne factors.
Expression of each compound studied was locally elevated in quarters
with more immunological activity, i.e. higher SCC, indicating that the
somatic milk cells themselves are involved in the maintenance of immune
response in milk. However, most likely these factors are also
synthesized by
mammary epithelial cells. Further investigations are in progress.
References
1 Rose,
D.M., Giri, S.N., Wood, S.J. & Cullor, J.S. Role of leukotriene B4
in the
pathogenesis
of Klebsiella pneumoniae-induced bovine mastitis. American Journal
of Veterinary Research 50: 915-918 (1989)
2
Persson, K., Larsson, I. & Hallén Sandgren, C. Effects of
certain inflammatory
mediators on
bovine neutrophil migration in vivo and in vitro. Veterinary Immunology
and Immunopathology 37: 99-112 (1993)
3
Sordillo, L.M., Shafer-Weaver, K. & DeRosa, D. Immunobiology of the
mammary
gland. Journal
of Dairy Science 80: 1851-1865 (1997)
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Inflammatory mediators in mammary gland
immunology:
Quantification
of key factors by LightCycler real-time PCR
S. L.
Wittmann, M. W Pfaffl., H.H.D. Meyer & R. M. Bruckmaier (2000)
2nd.
LightCycler Symposium und Anwender Workshop, Roche Diagnostics
GmbH,
19.-20.
September in Heidelberg
Introduction
Inflammatory
mediators, such as cytokines, prostaglandins and leukotrienes, exhibit
potent chemokinetic and chemotactic activity for leukocytes and enhance
the bactericidal activity of phagocytes. Moreover, they cause
increasing vascular permeability and hyperalgesia during inflammatory
disorders [1,2,3]. The goal of this work was to investigate whether
leukotrienes and prostaglandins are produced by different fractions of
somatic cells in cow milk. Therefore, we have developed a quantitative
RT-PCR method to determine the mRNA expression of the key enzymes
in leukotriene and prostaglandin biosynthesis, i.e. 5-lipoxygenase
(5-LO) and cyclooxygenase2 (COX-2), respectively. The
expression of tumor necrosis factor
alpha (TNFa), a cytokine known to be crucial during early inflammatory
stages in
the
mammary gland [3], has
been
studied concomitantly.
Material
and Methods
Animals
Brown Swiss
dairy cows (n=11) with no clinical signs of mammary disease were used.
Somatic cell counts (SCC) of total quarter milk was measured with a
fluoro-opto-electronic method using a Fossomatic® cell counter
(Foss Electric, Denmark). Based on the SCC results animals were devided
in two groups.
Cell
isolation
Total quarter
milk from one quarter of control group and from two quarters of cows
with partially elevated SCC (one of H and one of L) was collected at
one morning milking. The milk was
centrifuged and the cell pellet was
washed three times in PBS (phosphate buffered saline). The cell
fractions were separated using a density gradient (LSM®, ICN,
Aurora, USA). Macrophages and lymphocytes (Mac+Lym) were distributed
within the inter-phase, polymorphonuclear leukocytes (PMN) were located
in the pellet.
Statistical
evaluations
Differences
between C and L quarters were tested for significance (p<0.05) using
Wilcoxon´s rank sum test. Differences between L and H quarters
and differences between Mac+Lym and PMN cell fractions (within animal)
were tested for significance (p<0.05) by Wilcoxon´s signed
rank test.
Results
and Discussion
In
one control animal gene expression
of all factors determined was
higher than in the
other four animals despite
similarly low SCC. The reasons are unclear. Our
results indicate that 5-LO, COX-2 and TNFa are, at least to a certain
extent, produced by somatic milk cells, i.e. represent milk borne
factors. Expression of each compound studied was locally elevated in
quarters
with more immunological activity, i.e. higher SCC, indicating that the
somatic milk cells themselves are involved in the maintenance of immune
response in milk. However, most likely these factors are also
synthesized
by mammary epithelial cells. Further investigations are in progress.
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mRNA expression of immunologically important factors
and milk proteins
in
mammary tissue of dairy cows during LPS induced mastitis
S. Schmitz,
M.W. Pfaffl, H.H.D. Meyer, R.M. Bruckmaier
ASAS meeting
21.-25. July 2002 in Quebec
Journal of
Dairy Science 85, supplement 1, p 8
Inflammatory
factors
are known to change during mastitis. This study was conducted to
determine
changes of mRNA expression of various immunologically important factors
in
mammary tissue during the first 12 h of lipopolysaccharid (LPS)
stimulated
mastitis. Five healthy lactating cows were intramammary injected in one
quarter
with 100 µg E.coli-LPS (O26:B6) and the contralateral quarter
with
saline (9 g/l) serving as control. mRNA expression in mammary biopsy
samples
of various factors at 0, 3, 6, 9 and 12 h after LPS administration was
quantified
by real-time RT-PCR. Blood samples were taken following the same time
course
and rectal temperature was measured at 1-h intervals. Temperature
increased
until 5 h (P<0.05) after LPS administration and decreased to
preinfusion
level within 24 h after LPS-challenge. Blood leukocyte (WBC) number
decreased
(P<0.05) from 0 to 3 h from 7.7±1.1 x 109/l to 5.7±1.0
x
109/l and thereafter recovered to pretreatment levels until 12 h after
LPS-challenge.
In LPS-challenged quarters tumor necrosis factor a and cyclooxygenase-2
mRNA
expression increased to highest values (P<0.05) at 3h after
LPS-challenge.
Lactoferrin, lysozyme, inducible nitric oxide synthase increased
(P<0.05)
and peaked at 6 h after challenge, while platelet-activating factor
acetylhydrolase
mRNA increased only numerically. mRNA expression of the investigated
factors
did not change in control quarters. mRNA expression of insulin-like
growth
factor-1, 5-lipoxygenase and of aS1-Casein (CN), aS2-CN, b-CN and
b-lactoglobulin
did not change significantly, whereas mRNA expression of a-lactalbumin
decreased
(P<0.05) in both quarters and that of k-CN only in the LPS quarter.
In
conclusion, mRNA expression of most inflammatory factors changed within
hours,
whereas that of most milk proteins remained unchanged.
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 (German) 
http://www.tec.agrar.tu-muenchen.de/landtech/deutsch/projekte/eurotier2002/literatur.html
mRNA
Nachweis immunrelevanter Faktoren im Eutergewebe von
Milchkühen
während einer E. Coli Endotoxin induzierten Mastitis
Michael W.
Pfaffl, Susanne Schmitz & Rupert M. Bruckmaier
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