Monocyte function in pregnant and non-pregnant dairy cows
R.W. Sweere (2010), Cornell University, Utrecht University Veterinary Medicine.
Abstract
The main goal of this project was to investigate the differences in monocyte population and
function between pregnant and non-pregnant dairy cows. Evaluating the differences in
monocyte function may help to understand the immunosuppression of periparturient dairy
cows. Blood was collected from three pregnant cows and four non-pregnant cows. The
peripheral blood mononuclear cells (PBMCs) were incubated in six well plates for 3 hours,
after which the monocytes were collected. Each cow was sampled twice, with 2-5 days in
between. There was no difference in the total blood monocyte population as a percentage of
the circulating PMBCs between pregnant and non-pregnant dairy cows, but pregnant cows
showed a slightly increase in CD14+ cells as a percentage of the whole blood monocyte
population. Dairy cows in late gestation (270 days) showed a decrease in MHCII expression
on the CD14+ subpopulation compared with non-pregnant cows and cows in early gestation
(120 days). This project showed that monocytes function of non-pregnant, early pregnant and
late pregnant dairy cows are different. More research is needed to determine the magnitude of
the differences and the consequences for cow health.
Keywords: Dairy cows, monocytes, pregnancy, MHC function.
Introduction
During the periparturient period, the dairy cow is more susceptible to infectious disease due to
immunosuppression. This can be explained by certain changes in the regulation of the
immune system that take place during pregnancy (Luppi et al 2002, Raghupathy 1997,
Szekeres-Bartho et al 2001, Wegman et al 1993). These changes are required for the survival
and growth of the fetus, but at the same time the immune system needs to maintain a normal,
robust response to pathogens (Luppi et al 2002, Wegman et al 1993).
The immune system is divided into a more primitive innate immune response, and an adaptive
immune response, both contain humoral and cellular components. The humoral immune
response is characterized by complement binding and the secretion of antibodies against
specific pathogens (Luppi et al 2002, Wegman et al 1993). The cellular component involves
the activation of macrophages, natural killer cells, t-lymphocytes and the release of various
cytokines in response to an antigen.
Normal pregnancy is characterized by a dominant humoral immune response (SzekeresBartho et al 2001, Wegman et al 1993). Wegmann et all showed that, during pregnancy in
human there is a T helper (Th2) dominant situation with an increased level of interleukins 4,
IL-5, IL-6 and IL-10. Those cytokines are involved in various phases of B-cell development
for the humoral immune response. This Th2 dominant situation is important because the Th1
cells, helper cells for cell-mediated immunity, secrete cytokines IL-2, IFN-γ and TNF that are
generally harmful to the maintenance of pregnancy. There is evidence that the high IL-10
level (Th2) influences the monocyte population and monocyte activation in humans
(Woszczek et al 2008). The influence on the monocyte population in dairy cows in unknown,
but it is assumed to act the same way.
The shift towards Th2 results in increased susceptibility of the mother to certain infections;
immunoglobulin synthesis is increased, cell-mediated responses are decreased (Wegman et al
1993). Cell mediated responses restrain the activation or macrophages, natural killer cells and
cytotoxic T cells, and that part of the immune response is partly lost during pregnancy to help
the fetus survive.
The former
Monocytes play an important role in the immune system. They have the ability to differentiate
into different subsets of tissue macrophages and dendritical cells with specific functions.
Monocytes and their macrophage and dendritic cell progeny serve three main functions in the
immune system; phagocytosis, antigen presentation and cytokine production (Woszczek et al
2008). Those different functions make monocytes a very interesting cell population, with a
big influence in the first attack of pathogens as well in the antigen presentation, T-cell
activation and therefore the specific immune response.
Evaluating the difference in monocyte function between pregnant (late dry period) and nonpregnant cows, may help in the understanding of immunosuppression of dairy cows in the
periparturient period.
Literature on monocyte function in dairy cattle is scarce. Most of the publications investigated
the leukocyte population or bovine cytokines and their role in the pathophysiology of a
mastitis infection.
Human monocyte function has been studied but the main goal of the majority of these studies
was getting a better understanding of dendritical cell function.
The main objective of this study was to investigate whether the changes during pregnancy,
affect the immune status of the animal and whether pregnant dairy cows are therefore more
likely to get infected by pathogens.
To fully understand the immune status of pregnant dairy cows it is important to look at
monocyte function. We hypothesized that there is a difference in monocyte function and
population in vitro, between pregnant and non-pregnant dairy cows. Also we expect a
difference in MHCII expression. A lower expression of MHCII is hypothetically associated
with a decreased response of monocyte function.
In order to investigate monocyte function, PMBCs were collected from bovine blood samples
and the monocyte population was selected by using a CD14 marker; a specific marker for
monocytes. Monocyte function was scored by looked at the expression of MHC class II on the
monocyte surface using a MHC II marker.
Materials and Methods
The experimental design and all handling of the animals for this study were approved by the
Institutional Animal Care and Use Committee (Cornell University) and the American
Association for Laboratory Animal Science.
The animals used in this study were adult Holstein-Frisian cows, pregnant (n=3; cow numbers
8709, 8415, 8556) or non-pregnant (n=4; cow numbers 8144, 8152, 8132, 8193). Within the
pregnant group 8709 is in early gestation, 122 days, and 8415/8556 are in late gestation, 270
days. Blood samples were obtained from each cow via coccygeal venipuncture, in 10 ml
tubes with heparin.
Isolation of PMBC
PBMCs were isolated from the buffycoat by Ficoll-Hypaque density gradient centrifugation.
The PBMCs were washed twice with 20ml Phosphate Buffered Saline (PBS)and centrifuged
for 20 minutes at 1200 rpm..
To get a clear detection of white blood cells with Flow Cytometry analysis, it is important to
remove the majority of the red blood cells with osmotic shock. The cell pallet was resuspended in 20 ml 0,2% NaCl solution for 30 seconds. Then 20 ml 1,6 % NaCl was added,
and the solution was centrifuged at 1200 rpm for 10 minutes. This time, the cell pallet was resuspended in 30 ml pre-warmed RPMI 1640.
Isolation of monocytes from PBMCs
Monocytes were collected by incubation of the PBMCs in RPMI 1640 in a 6 well culture
plate, at 370C and 5%CO2. Monocytes were allowed to adhere for 3h, supernatants and non
adherent cells were discarded. In order to isolate the monocytes, the plastic surface of the 6
well culture plate was washed with pre-warmed PBS. After the washing there was a 30 ml
solution of PBS with the monocytes in it.
Preparing for Flow Cytometry
After centrifuging for 15 minutes at 1500 rpm, the cell pallet was washed twice with 2 ml of
FACS buffer (PBS+0.5%BSA+0.03%NaN3).
The cell pallet was re-suspended with 2 ml of fixating buffer (4 ml FACS, 1 ml 10%
formaldehyde, 20 µl buffered-formaldehyde-ace- tone (BFA)/ml fixation buffer) followed by
incubation at 40C for 20 minutes in the dark. After two more washings of the suspension with
2 ml of FACS buffer, the samples were centrifuged for 5 minutes at 1500 rpm.
For the Flow Cytometric analysis 3 control samples were used, one without antibodies
(unlabeled), one labeled with antibodies for IL-10 and one with antibodies for IFN-g. With
those control samples you can set your cell population, in which you can look for monocytes,
and also you can see if there is a reaction of the cells with any antibody in general.
A volume of 100 µl of the suspension was incubated with 10 µl of the antibody at 40C in the
dark for 30 minutes.
For the detection of the monocytes we used the antibody: CD14 marker (specific monocyte
marker), with M-M7 (cell line BAQ151A, isotope IgG1). For the detection of the MHC
function we used: Bola-DR marker, with MHCII, HLA-DRα (cell line TH14B, isotope
IgG2a)
After incubation, the cells were washed twice with 2ml of FACS buffer and centrifuged for 5
minute at 1500 rpm. Supernatant was discarded and re-suspended in 1 ml FACS
Analyze by flow cytometry
Data was analyzed using a BD FACSCalibur Flow Cytometry. 50,000 events per samples
were collected. Unlabeled, IL-10 and IFN-γ controls were used instead of monoclonal
antibodies for detection of non-specific staining in order to set the criteria for positive cell
population.
Marker positive cells were expressed as a percentage of mononuclear cells. The results were
exported to Flowjo (Flow Cytometry Analysis Software, Tree Start Inc. 1997-2010) for
further data processing.
The statistical analysis was conducted using software (SPSS). In order to investigate the
significant difference between pregnant and non-pregnant cows, an Independent- Samples T
Test was used. P values less than 0.05 were considered significant.
Results
CD14-MHCII positive cells in circulating PMBC population
All monocytes were found positive for both CD14 and MHCII markers (Kim et al 2010).
Figure 1 shows the percentage of the monocyte population within the PBMCs for the pregnant
and non-pregnant cows. There is variation between the individual cows, but the experimental
results do not show a significant difference between the pregnant and non-pregnant group
(p=0,855) (Figure 1B).
Monocyte population CD14++/CD14+
Figure 2 demonstrates two examples of cell analysis by Flow Cytometry with expression of
the markers for monocytes (CD14) and MHCII. In Figure 2, Q2 represents the cells from the
PBMC population that are positive for both CD14 and MHCII, the monocyte population.
Within this population two subpopulations can be defined by CD14-expression; there is a
population of monocytes with high CD14 expression (CD14++) and one with low CD14
expression (CD14+).
Figure 3 displays the ratio of CD14+ monocytes versus the whole blood monocyte population.
In the non-pregnant group the ratio differs from 0,14 to up to 3,40 and in the pregnant group
the ratio ranges from 0,31 to 0,39. The two groups were not significantly different (result not
shown).
Because we were interested in the trend of the ratio of CD14+ monocytes versus the
monocyte population from non-pregnant to late gestation, Figure 4 shows the different cows
with their different stages of gestation together in one graph. Figure 4 visualizes a small
increase of the ratio from non-pregnant to pregnant and a big decrease at the end of gestation.
MHCII expression on subpopulations CD14++ and CD14+ monocytes
Non-pregnant:
The histograms in Figure 5 show two separate subpopulations of monocytes and their
individual MHCII expression. Histogram A and B represent two non-pregnant cows (8152
and 8144) and show a clear difference in MHCII expression between CD14+CD16+ and
CD14++CD16- monocytes. The CD14+CD16+ cells have a higher expression of MHCII in
comparison with the CD14++ CD16- cells. This corresponds with the findings of Kim et al
(2010), Ziegler-Heitbrock (2007) and Passlick et al (1989) in human.
The two other non-pregnant cows (8132, 8193) had contrasting results for the MHCII
expression on the CD14+CD16+ monocytes; they have less expression of MHCII. The health
status of the non-pregnant cows was unknown, but at least one of them had an abscess on the
left hind leg and some of them were lame on one leg.
Pregnant:
Results from the pregnant cow in early gestation (8709) correspond with results as mentioned
in the literature and are not significantly different from 2 of the non-pregnant cows (8144,
8152) used in our experiment; MHCII expression is higher on the CD14+CD16+ monocytes
(Fingerle et al 1993, Kim et al 2002, Passlick et al 1989, Zieglr-Heitbrock 2007). This
suggests that there isn’t any difference for MHCII expression between non-pregnant cows and
cows in early gestation.
The two late pregnant cows (8415, 8556) show a lower expression of MHCII on the
CD14+CD16+ cells on both sample time points, in comparison to the MHCII expression of
the CD14++CD16- cells (Figure 6). Additionally there is a decrease in the percentage
CD14+CD16+ monocytes within the two sample time points. Because both cows that are in
late gestation show the same changes in MHCII expression and have a CD14low population,
it is reasonable to believe that these changes are due to the immunological changes around
parturition/late gestation.
Figure 7 displays the timeline for MHCII expression. It demonstrates a big decrease of
MHCII expression from early gestation to late gestation.
Discussion
The main objective of this research project was to investigate whether there is a difference
between the monocyte population of pregnant and non-pregnant dairy cows. Overall, the
design of the study was too small and incomplete to draw reliable conclusions.
This study investigated 3 pregnant cows (with variation in gestation length) and 4 nonpregnant cows. The health status of the non pregnant cows at time of sampling was unknown.
Some of them were lame at one leg and two cows had an abscess on one of the legs. The
influence of these abnormalities/local infections on the blood monocyte population is ignored
in this study. Ziegler-Heitbrock (2007) and Fingerle et al (1993) showed that only diseases
with active involvement of monocytes will result in changes in the monocyte population.
There is no evidence that a local infection will change the CD14+/CD14++ subpopulations or
the MCHII expression. But because of the role of CD14+ cells in tissue macrophage and
dendritic cell differentiation it could be possible that even during a local infection the CD14+
monocyte population will change.
Between the individual cows there was some variation in the percentage of CD14+ from the
whole monocyte population. It is possible that there is a biological variance between
individual cows, but by sampling the cow several times with a few days in between sample
time points, variance caused by technique and cow variation can be decreased. When
comparing two groups, pregnant and non-pregnant cows, minimal variation within those
groups is desired.
However, despite of the restrictions of the study, this project gives a good first impression on
the monocyte population in dairy cows. Up till now, little literature about the bovine
monocyte population and no publications about dairy cows have mentioned the presence two
different monocyte populations, CD14+ and CD14++.
Different studies investigated the CD14++/CD14+ population in human. Table 1 shows the
differences between the CD14+ and CD14++ monocytes described in the literature. The
difference in myeloid markers depends mostly on the CD16 and CD14 molecules (Kim et al
2010).
Kim et al (2010) showed that amongst mediators involved in the down-regulation of HLA-DR
on monocytes, glucocorticoids act on both CD14++ and CD14+ subsets, whereas IL-10 is
only active on CD14++CD16- monocytes, a subset that expresses higher levels of IL-10
receptor.
Ziegler-Heitbrock (2007) point out that CD14+CD16+ monocytes can be developed from
CD14++CD16- cells and this indicates that the CD14+CD16+ subpopulation is the more
mature population of monocytes. Another point that has been made is that the HLA-DR
expression is higher on the CD14+CD16+ monocytes, and together with the higher expression
of CD32, CD40, CD80 and CD86 (all markers for antigen presenting and co-stimulating) this
would predict a higher antigen presenting cell activity in these cells. T cell activation was
much higher in the CD14+CD16+ population.
Both Kim et al (2010) and Ziegler-Heitbrock (2007) declare that after stimulation with LPS,
CD14+CD16+ monocytes are a major source of TNF, whereas the IL-10 transcript is absent
or at low level. This led us to coin the CD14+CD16+ cells pro-inflammatory monocytes.
Fingerle et al (1993) showed that CD14+CD16+ monocytes have higher expression of HLADR, as mentioned before and lower expression of CD11b and CD33. This indicates that the
CD14+CD16+ cells develop towards tissue macrophages.
This project corresponds with the findings in human that there is no difference in the
percentage of monocytes in circulating PBMC between pregnant and non-pregnant
individuals.
We showed that there are two different subpopulations of monocytes, CD14++ and CD14+.
Using flow cytometry it is possible to get a clear view on those populations. We showed that
there is a change in the ratio of CD14+/total monocyte population in the trend from nonpregnant towards pregnant cows. It is likely that the CD14+ population has the same function
in dairy cows as in human, which is antigen presenting and differentiating into mature tissue
macrophages and dendritic cells. Our late pregnant cows show a big decrease in that
CD14+population, we can’t explain why there is a downfall in antigen-presenting cells at that
point of pregnancy.
The CD14+ population does produce cytokines similar to those produced in Th1 response,
TNF and IFNγ. Wegmann et al (1993) showed that those cytokines are harmful to the fetus, a
possible explanation for a decrease of cytokine secretion.
But when we assume that this decrease in CD14+ is caused by the Th2 shift and
immunosuppression (IL-10) during gestation, it should be there during the whole gestation
and not just only at the end of pregnancy.
Comparing the MHCII expression on the CD14+ population there is no difference between
non-pregnant and early pregnant cows in this project. Those cows showed, just like Fingerle
et al (1993), Kim et al (2010), Passlick et al (1989) and Ziegler-Heitbrock (2007) did in
human, that the expression of MHCII in healthy individuals is higher on the CD14+
population. We don’t have an explanation for the decrease in CD14+population in late
pregnant cows.
An intramammair E.Coli challenge study during the late dry period showed that the immune
response increases after parturition, and just a little pre parturition post challenge. This could
be explained by the low expression of MHCII on monocytes at the end of gestation. The
reason why this happens remains unclear.
Conclusion
There is still a lot unknown about the immunosuppression period in dairy cows. Because this
period has a big influence on the milk production and health status of those animals it is
important to understand these changes in immune response. With a complete understanding of
the immunological changes during pregnancy in dairy cows we can possibly implement a
variety of production practices that thereby improve the milk production yield and animal
health.
With this project we showed that during pregnancy there are changes in the monocyte
population, which result in a difference in expression of MHCII and probably a difference in
immune response. Because a large part of monocyte function, the subpopulations and their
changes during pregnancy in dairy cows remains unknown it could be of great value to study
this carefully and thereby help to get a better understanding of the immunosuppression during
pregnancy.
Attachments
B
Figure 1. CD14-MHCII positive cells in circulating PBMCs, non-pregnant vs pregnant dairy cows. The box plot
shows the mean for the two separate groups, pregnant and non-pregnant, and there standard deviation.
A
B
Figure 2. CD14 and MHCII expression in PBMC population. Q2 shows the cells positive for CD14 and MHCII, the monocyte
population.
Figure3. Ratio CD14+/CD14total monocyte population, non-pregnant versus pregnant
Figure 4. Ratio CD14+/CD14total population in timeline non pregnant – early gestation – late
gestation.
A
B
D
C
E
Figure 5. MHCII expression on CD14++CD16- and CD14+CD16+ monocytes. Blue line represents the
CD14+CD16- subpopulation, the black line the CD14++CD16- subpopulation.
A
Figure 6. MHCII expression on CD14++CD16- and CD14+CD16+ monocytes. It shows the two sample
time points, 4 and 7 days pre calving, of the two late pregnant cows
Figure 7. MHCII expression on CD14+ monocytes in timeline non pregnant – early gestation – late
gestation.
Table 1. Different markers and molecules expressed by CD14++ and CD14+ monocytes (2,5,8,12).
Summary from different publications about CD14+ and CD14++ monocytes in human.
Meyloid marker
CD16
CD14
CD33
Fc RI, CD64
Molecules AP/costim
HLA-DR
CD40
CD80
CD86
CD32
CD11a
CD11b
CD11c
Mannose Receptor
CD49d
CD14low
Monoctytes
CD14high
monocytes
High
Low
Low
Low
Low
High
High
High
High
High
High
High
High
Low
Low
Low
Low
Low
High
Low
Equal
Low
High
Low
High
Equal
High
Low
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