/ . Embryol. exp. Morph. Vol. 39, pp. 195-202, 1977
Printed in Great Britain
195
Reactivity of rat placental cells with alloantisera
By FREDERICK G. FERGUSON 1 AND JOY PALM (deceased)
From the Pennsylvania State University and the
Wistar Institute of Anatomy and Biology, Philadelphia
SUMMARY
The nature of the protection afforded the placental structure against the immunologic
consequences that could result from maternal exposure to paternal alloantigens remains
obscure. One problem has been difficulty in antigenically defining the individual cellular
components of the intact placenta. These studies were designed to examine serologically,
with specific antisera, five cell types isolated from the rat placenta. The results indicate that
non-Ag-B antigens are present on the cytotrophoblast cells, while Ag-B antigens are absent
on all the cells except an undefined population of fibroblasts. Of particular significance is the
obvious absence of detectable surface antigens on the cells directly in contact with the
maternal tissues, the trophoblast mononuclear giant cells.
INTRODUCTION
Attempts to define the alloantigenicity of placental tissue, which is potentially an allograft in an outbred population, have led to diverse conclusions.
This diversity may be due to the types of cells which comprise the developing
placenta at different stages of pregnancy. As well as having distinct biological
potentials, each of these cell types may have distinct alloantigenic characteristics.
In rats, a recent study of placental cells in culture had as its purpose the
definition of individual cell types at a specific time during pregnancy for a
subsequent serological analysis (Ferguson & Palm, 1976). Five cell types were
observed in the cultures from 12-day placentas. These included two cell types of
apparent fetal origin (mononuclear giant cells, cytotrophoblast cells) and two
cell types of apparent maternal origin (small round cells, multinucleated giant
cells). The fifth was a fibroblastic cell which occurred in small number initially,
but gradually overgrew the cultures. The reactivity of each cell type with
various alloantisera was examined in these studies with indirect immunofluorescence.
Serologically detectable rat cellular antigens include those determined by
the major histocompatibility locus, Ag-B (Palm, 1971; Palm & Black, 1971),
the Ag-C blood group antigens (Palm & Black, 1971), a male specific antigen,
presumably the counterpart of the Y-factor of mice (Hausman & Palm, 1973),
and various non-Ag-B antigens (DeWitt & McCullough, 1975). Tests of
1
Author's address: The Pennsylvania State University Centralized Biological Laboratory,
University Park, Pennsylvania 16802, U.S.A.
196
F. G. FERGUSON AND J. PALM
Table 1. Immunofluorescent reactivity of alloantisera
with DA placental cells
Antiserum
BN anti-DA
BNanti-BN.B4
BN anti-Buffalo
BN anti-Buffalo
BN anti-Buffalo
DA anti-BN
Absorbed
with
—
—
—
BN.B4Rbc's
DA Rbc's
—
Alloantigens
detectable
Ag-B4, non-Ag-B
Ag-B4
Ag-B4, non-Ag-B
Non-Ag-B
None
None
placental cells from DA strain rats with sera containing antibodies of broad
specificity were negative, except for immunofluorescent activity associated with
a small undefined population of fibroblasts. However, antisera containing
antibodies to one or more non-Ag-B antigens were found to react selectively
with cytotrophoblast cells.
MATERIALS AND EXPERIMENTAL PROCEDURES
Placental tissues derived from female DA rats at 12-13 days of pregnancy
were the source of the cells examined in this study. The DA animals originated
from a strain obtained from the Wistar Institute in 1972, which since has been
maintained at Penn State University.
Alloantisera, DA anti-BN, BN anti-BN.B4, BN anti-Buffalo, and BN
anti-DA, were produced in female rats by skin grafting and/or not less than
three intraperitoneal or subcutaneous injections, at appropriate intervals, of
30-40 x 106 lymphocytes/ml from female rats. The sera were evaluated by
cytotoxicity (Sanderson, 1965) and hemagglutination (Stimpfling, 1964). Portions of the BN anti-Buffalo alloantiserum were absorbed in two ways:
(1) with red blood cells from a congenic BN.B4 rat strain to remove antibodies
to antigenic specificities which are shared by the Ag-B4 and Ag-B6 loci and to
leave non-Ag-B antibodies (Palm & Black, 1971); and (2) as a control, with
red blood cells from the DA rat strain to remove shared Ag-B and non-Ag-B
antibodies. The resulting alloantisera were known to contain antibodies specific
for alloantigens determined both by the Ag-B major histocompatibility locus
and non-Ag-B loci (Table 1).
The placentas were carefully shelled out from the uterus, separated from the
fetal membranes and the labyrinth, minced with scissors, and thoroughly
washed with calcium and magnesium-free Hanks' balanced salt solution.
Placental cell suspensions were prepared by two 10-min trypsinizations (0-1 %)
with constant stirring at 37 °C. The resulting cells were pelleted by centrifugation at 1000g for lOmin at 5 °C. The supernatant was decanted and the top
layer of the pellet, consisting of cells other than the red blood cells, was removed
Reactivity of rat placental cells
197
and resuspended in cold Hanks' balanced salt solution (Beer, Billingham &
Yang, 1972). The centrifugation was repeated and the top layer of this pellet
was finally resuspended in cold phosphate-buffered saline.
Three cell types, as determined by size, were present in the cell suspension
including: (1) giant cells, (2) intermediate-sized cells, and (3) small round cells.
With phase contrast microscopy it was determined that the giant cells included
both multinucleated and mononucleated cells. The intermediate-sized cells
consisted of both cytotrophoblasts and fibroblasts which usually could be distinguished from one another by their size, nuclear and cytoplasmic characteristics. The small round cells were easily distinguishable. In addition, these morphologic determinations were confirmed by cell culture studies (Ferguson &
Palm, 1976). For each test placentas from at least three different pregnancies
were evaluated.
For preparation of the fluorescein-labeled rabbit anti-rat IgG, the rat IgG
was separated by ammonium sulfate precipitation and DEAE-cellulose column
chromatography with 0-0175 M phosphate buffer, pH 7-6. The eluate from the
ascending slope of the first peak was collected, concentrated by ultrafiltration
and resuspended in phosphate-buffered saline. Protein determinations were
done by the method of Lowry (Lowry, Rosebrough, Farr & Randall, 1951).
This protein was characterized with immunoelectrophoresis and found to consist only of IgG. Rabbits were immunized with the rat IgG. Rabbit IgG was
separated and characterized in the same manner as the rat IgG and then was
conjugated with fluorescein according to the method of Clark and Shephard
(Clark & Shephard, 1963). The fluorescein:protein ratio of the conjugate was
5:1.
Indirect immunofluorescence tests were performed according to the procedure described by Moller (Moller, 1961). Approximately 5 x 105 cells were
mixed with 0-05 ml of each of the alloantisera diluted 1:10 and incubated for
30 min at 37 °C. After incubation the cells were washed three times with
phosphate buffered saline and were resuspended in 0-05 ml of the fluorescein
conjugate. The cells and the conjugate were incubated for 30 min more at the
same temperature, again washed with PBS, and finally resuspended in one drop
of 50% PBS and glycerol. The cell suspension was placed on an alcohol-cleaned
slide, coverslipped, and examined under a fluorescent microscope.
Specificity controls for the immunofluorescent test are indicated in Table 2.
This combination of controls excluded the possibility of non-specific fluorescence. With these controls there was no evidence of maternal IgG fixed to
the various cells (Voisin & Chaouat, 1974). Some non-specific uptake or pinocytosis did occur when the slides were not examined immediately after preparation. This pinocytotic activity was most pronounced in the small round cells
and mononucleated trophoblast giant cells, the former even taking up some
fluorescent material under the most controlled conditions. To reduce the effect
of this, the slides were examined immediately after preparation.
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F. G. FERGUSON AND J. PALM
Table 2. Controls for specificity of immunofluorescent test
1. Cells + conjugate without intermediate IgG
2. Cells + positive antiserum+ unlabeled rabbit
anti-mouse IgG + conjugate (Blocking Test)
3. Cells + absorbed positive antiserum + conjugate
4. Cells + normal serum + conjugate
5. Cells + unrelated antiserum + conjugate
Fig. 1 (A) DA placental fibroblast treated with BN anti-DA antiserum to show the
characteristic positive petichial membrane immunofluorescence; (B) DA placental
cytotrophoblast cell treated with BN anti-Buffalo antiserum demonstrating the
delicate membrane immunofluorescence over a portion of its surface, x 400.
RESULTS
With the BN anti-DA serum only the smaller sized intermediate cells, which
were determined by phase microscopy and tissue culture studies to be fibroblasts, were positive (Fig. 1 A). All of the other cells, including the cytotrophoblast cells, lacked discernible surface antigens. The same results were obtained
with the BN anti-BN.B4 antiserum to the Ag-B4 alloantigens.
In contrast, immunofluorescent examination of similar cells with BN antiBuffalo antiserum indicated the presence of petichial surface alloantigens on
both types of intermediate sized cells, cytotrophoblast cells as well as fibroblasts (Fig. IB). Absorption of the BN anti-Buffalo antiserum with BN.B4
red blood cells, which removed any serologically detectable antigenic specificities which the Ag-B6 and Ag-B4 major loci share, failed to remove the
Reactivity of rat placental cells
199
Fig. 2. DA giant trophoblast cell treated with alloantiserum to show the characteristic negative surface immunofluorescent reaction. The diffuse internal fluorescence
and presence of an occasional fluorescent globule can be seen in this cell, x 400.
immunofluorescent reactivity of the DA cytotrophoblast cells and the fibroblasts. In this case the surface antigen being detected was a non-Ag-B antigen.
With these sera, the giant cells lacked definite surface fluorescence except for
an occasional cell which showed non-specific internal fluorescence or a large
patchy globule on or near its surface (Fig. 2).
The surface fluorescence of the fibroblasts was more intense and more
petichial in nature than that seen on the cytotrophoblast cells. The cytotrophoblast fluorescence was evenly distributed and was less intense. Often with cytotrophoblast only a part of the cell surface would show a positive fluorescent
reaction. This could be the result of the cell preparation techniques or it could
be due to the nature of the antigen distribution. Specific fluorescence on the
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F. G. FERGUSON AND J. PALM
Table 3. Antigen detection on individual DA rat placental cell types
Cell type
Antiserum
BN anti-DA
BNanti-BN.B4
BN anti-Buffalo
BN anti-Buffalo (absorbed
withBN.B4Rbc's)
DA anti-BN
Giant
trophoblast
Cytotrophoblast
-
-
-
-
—
+
+
—
—
_
MultiMononucleated nucleated
_
_
_
Fibroblast
+
+
+
+
_
small round cells was difficult to confirm due to the extreme pinocytotic activity
of these cells.
It appears, therefore, that the fibroblast-type cells are the only placentarelated cell population which consistently demonstrates both non-Ag-B and
Ag-B surface antigens at the 12-13 day of pregnancy (Table 3). In addition,
with appropriately prepared antisera there is undoubtedly some type of nonAg-B antigen(s) expressed on cytotrophoblast cells. The antigen(s) is absent or
is not demonstrable under the conditions used here on the trophoblast giant
cells.
DISCUSSION
These studies provide further serologic support of the apparent lack of
detectable surface alloantigens on either the rodent giant mononuclear trophoblast cells or human syncytiotrophoblast cells (Seigler & Metzgar, 1970; Edidin,
1972), those cells known to be directly in contact with maternal tissues. Whether
this is actually an absence (Simmons, Cruse & McKay, 1967), in contrast to
a masking (Kirby, Billington, Bradbury & Goldstein, 1964) of the antigens has
not been resolved. To date the only serologically detectable antigens on these
outermost cells have been species or tissue specific (Koren, Behrman & Paine,
1969). In the mouse there is good evidence this antigen absence is an antigen
loss which occurs with implantation (Hakansson & Surdquist, 1975). It has
been suggested that the loss is due to the physiologic and membrane changes
which occur as embryonic cells mature (Muggleton-Harris & Johnson, 1976).
The presence of alloantigens on the rat embryonic fibroblasts is not unexpected. Using immunofluorescent techniques, antigens have been found on
fibroblasts derived from rat embryos from as early as nine days of gestation
(Ferguson, 1972). In addition, there is good evidence to indicate that early
embryonic tissues in the mouse possess non-H-2 antigens (Palm, Heyner &
Brinster, 1971; Muggleton-Harris & Johnson, 1976), and perhaps some H-2
antigens (Heyner, 1973; Patthey & Edidin, 1973; Jenkinson & Billington,
1974;Searlee*fl/. 1974).
Reactivity of rat placental cells
201
The pinocytotic activity of the small round cells is interesting. As suggested
previously, these cells have many similarities to monocytes, cells known to
develop into epithelioid and multinucleate cells. Our tissue culture studies
suggest that they are derived from maternal placenta-associated tissue and are
phagocytic (Ferguson & Palm, 1976).
These results which demonstrate non-Ag-B antigen(s) on cytotrophoblast
cells are the first direct serologic indication of a surface antigen on rat placentaderived cells at this stage of gestation. The exact nature of the non-Ag-B
antigen(s) being detected is undetermined. However, because of the strain and
the sex of rats utilized, it is not the Ag-C antigen or the Y antigen. Apparently,
based on the immunization procedure and on the removal of reactivity by
absorption with DA red blood cells, the DA cells share the antigen with Buffalo
cells. The positive results with the BN anti-Buffalo serum, in contrast to the
negative results with the BN anti-DA serum, suggest a difference between the
Buffalo and DA cells in the immunogenicity of the antigen(s) when used to
immunize BN animals.
The presence of antigens on 12-13-day rodent cytotrophoblast cells from
hemochorial placental tissue, which is in intimate contact with maternal tissue,
suggests the possibility of occasional exposure of the mother to alloantigens
during pregnancy. The significance of the presence of such antigens and of the
possible immune interactions has not been determined at this time, however,
antigenic disparity could have a role in such things as the increase in placental
size which is found in allogenic matings (Billington, 1964) and the extent of the
decidual reaction (Hetherington & Humber, 1975). In addition, the antigens
could have a role in offspring selection which is related to genetically determined histocompatibility antigen differences (Michie & Anderson, 1966; Hull,
1969; Palm, 1969).
This communication is authorized for publication as paper No. 4940 in the journal
series of the Pennsylvania Agricultural Experiment Station. Joy Palm (deceased) was
supported by United States Public Health Service Grants CA-100097 and CA-10815
from NCI.
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