VOLUME
54 (4) 1999
CHANGES IN THE CELLULAR SUBPOPULATIONS OF PERIPHERAL BLOOD
LEUKOCYTES DURING THE REPRODUCTIVE CYCLE OF DAIRY COWS
- PRELIMINARY OBSERVATIONS
R. Meirom, I. Samina and J. Brenner
Division of Virology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel
Summary
By means of flow cytometry and peripheral blood mononuclear cell proliferation
studies, we investigated the cellular profiles of CD4+, CD8+ and B-lymphocytes at
three selected time points representing the first, second and third trimesters of five
gravid dairy cows. B-cells that constitute approximately 20% of the lymphocyte
population tended to decrease during the 1st trimester (early pregnancy) with a steep
increase at mid- pregnancy. CD4+ cells circulate with a relative percentage of 18-20%
during the entire reproductive cycle but a small peak was noted during the first
trimester. CD8+ cells varied in number only during the second trimester where they
reached about 30% of the lymphocyte population. Thereafter they decreased to 1518%, at which level they are normally present. The stimulatory indices of cells
obtained from gravid cows were lower than those obtained from control (empty)
cows.
Introduction
Previous studies with dairy cattle have shown that some in vitro lymphocyte and
neutrophil functions are impaired during the periparturient period (1,2,3). This
impairment corresponded to an increased susceptibility to infections, notably mastitis
in the weeks before and immediately after calving (4,5). Harp et al., (6) have observed
a lower percentage of T helper (CD4+ bearing) cells in cows during the prepartum
than in the postpartum periods.
Other studies (7,8) have indicated the existence of an intricate but precise
balance between the bovine immune system and gonadal hormone levels. This
balance is also reflected by numerical changes in the sub-populations of
circulating white blood cells (6,9,10). For instance, estrogens were shown to
be either immuno- suppressive or immunostimulatory depending on the
species and humoral status of the animal (11). Grossman suggested that
immunosuppression of T cell activity occurs at sites of estrogenic activity,
(e.g. the female reproductive tract (12).
Previously we focused our studies on cellular changes occurring during the
estral and the luteal phases of the non-gravid cow (9), while in a second study
we measured humoral (IgG) fluctuations during the period around parturition
(13). We also measured cytokine (IL-6 and TNF-a) activities at these time
points, the dry period and the day after parturition (9).
As dairy cows have a continuous productive/reproductive cycle, it was of
interest to investigate the peripheral blood (PB) cellular profiles at additional
time points in the reproductive cycle. This report presents the cellular profiles
of CD4+, CD8+ and B-lymphocytes at three selected time points representing
the first, second and third trimesters of the gravid dairy cow, and compares
them with ten empty cows.
Materials and Methods
Animals
Five pregnant Holstein cows were studied. Jugular blood was collected in
EDTA as anti-coagulant.
Enumeration of blood cells was carried out by standard methods (Contraves,
Digicell, Zurich, Switzerland). The five cows were tested twice in each
trimester (early pregnancy, less than the third months of pregnancy; mid - 4-5
months; and late; 7-8 months). The mean of the two tests was taken as one.
Ten non-gravid cows served as controls. Only data pertaining to the period
between 7 days post partum and the first confirmed insemination were
included in the study. Each bleed from the experimental cows was matched by
two blood samples from empty cows (a total of 10 cows).
Flow cytometry
Peripheral mononuclear cells (PBMC) were separated on a Ficoll-Hypaque
gradient. Cell populations were determined using fluorescence-activated cell
analysis (FACS) (FACscan Becton-Dickinson, San Jose, CA). The antibodies
used for analysis were as follows: CACT138A for CD4, CACT80C for CD8
and BAQ44A for B cells (VRMD, Pullman, WA) (14).
Cell proliferation
PBMCs were also taken for stimulatory cell proliferation assays to assess the
degree of activation.
For activation of PBMNC, they were co-cultivated with ConA. The degree of
activation was measured by incorporation of labeled thymidine. 105 PBMC
suspended in RPMI were placed in each well of a 96-well plate together with
100 µl RPMI, 10% FCS and antibiotics. Then 5µg/ml of ConA was added in
100 µl volume. Incubation was for 72h at 370C in 5% CO2. Six h. before the
end of incubation, 1µci of radioactive thymidine (Nuclear Research
Statistics
Student's t test for paired samples was used to evaluate the relative changes
(expressed as the percentage total lymphocyte count) of circulating B, CD4+
and CD8+ lymphocytes during the reproductive cycle and to compare the
mean percentage values of the sub-populations in the 1st, 2nd and. 3rd
trimesters (Tables 1 and 2)
Student's independent t test was used to compare the mean percentages of
circulating B, CD4+ and CD8+ lymphocytes at the 1st and 3rd trimesters
respectively, with the mean percentages of the sub-populations of 10 empty
(control) cows.
Results
Table1 shows the percentage of circulating B cells, CD4+ and CD8+
lymphocyte sub-populations of 5 gravid cows and 10 control-empty cows. In
addition, Figures 1-3 depict the individual as well as the generalized group
trend (± standard deviation) and the concordance of both. As shown in these
figures, almost all the cows under observation exhibited the same trends
regarding the relative changes in all the cellular sub-populations analyzed.
Cell proliferation
In Table 2 the sequel of cellular activation are expressed as the stimulatory index (SI).
A certain degree of variation can be noted but no statistical significance was found
between the indexes of the three trimesters or between them and empty cows.
Generally, the SIs of cells obtained from gravid cows were lower than those obtained
from the control (empty) cows.
Fig 1: Proportions of CD4, CD8 and Ig-Bearing (B) cells in the peripheral bloos of a cow during the
reproductive cycle.
Discussion
In comparing our previous results (8,13) with the current data, we calculate
that the following cellular interactions exist among the three major
lymphocyte sub-populations of B, CD4+ and CD8+ cells (see Figure 4).
B-Cells - These cells are responsible for immunoglobulin production. During
the empty period, they are present in the PB where they constitute
approximately 20% of the lymphocyte population with the first nadir
occurring on the day of estrus. Their percentages decrease during the 1st
trimester (early pregnancy) with a steep increase at mid- pregnancy
(approximately fifth month of pregnancy). Brenner et al., have shown changes
in the serum IgG levels that could be completely or partially associated with
hormonal fluctuations (13). Thus, some of these fluctuations may be related to
shifts in B-cell counts during the reproductive cycle.
Harp et al., (6) who observed a lower percent of T helper (CD4+ bearing cells)
in cows during prepartum. Whether this indicates immunosuppression can be
deduced from previous studies with dairy cattle that had impaired lymphocyte
functions during the periparturient period (1,2,3).
CD8+ cells - this group of cells varied in number only during the second
trimester where they reached about 30% of the lymphocyte populati.
Thereafter they tended to decrease to 15-18%, at which level they are
normally present (14). When activated these cells include cells with activity
against intracellular (viruses, bacteria and protozoa) pathogens as well as
regulatory (immunosuppression) activity. To relate these activities with the
reproductive cycle and changes in the health status of the animal is of
paramount in veterinary importance.
The dry period is known (5,13) to be influenced by hormonal changes and
must be investigated further. Also the post-parturient period is also of great
interest to veterinarian and immunologists (1-3,6,13).
Normally, ruminant dams are not exposed to fetal antigens and PBMC should
remain at the same degree of activation, in this state of "no antigen contact".
How then can we explain the marginal slight decrease in activation potential
that was noted in our study? In such circumstances this unresponsiveness
could be one of many signs, which characterize the immunological tolerance
designed to avoid (fetal) rejection.
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