Clint. exp. Immunol. (1978), 31, 397-407.
Changes in lymphocyte accumulation and proliferation in the lymph nodes draining the pregnant uterus J. D. ANSELL, CATHERINE M. McDOUGALL, GILLIAN SPEEDY* & C. J. INCHLEY Immunobiology Unit, Department of Zoology, University ofEdinburgh, Edinburgh
(Received 2 August 1977) SUMMARY
Changes in weight, lymphocyte accumulation and cellular proliferation have been measured in the lymph nodes draining the uterus during inter- and intra-strain pregnancies and compared with similar effects after other antigenic stimuli. From the data obtained it was concluded that 'paternal' antigenic stimulation from the conceptus initiated an immune response in these nodes. The mechanisms of the subsequent suppression ofthis response are discussed. INTRODUCTION Exposure of mice to the transplantation antigens of allogeneic tissue is normally followed by wellcharacterized changes in the draining lymphoid tissues. In the case of skin allografts, for instance, the regional lymph nodes show a pronounced enlargement, associated with the proliferation of lymphocytes in the paracortical region, and increased accumulation of circulating cells (Scothorne & McGregor, 1955; Parrott & de Sousa, 1970; Zatz & Lance, 1971). Similar, but more rapid, changes also take place in the draining lymph nodes following the subcutaneous administration of xenogeneic erythrocytes or other antigens (Davies et al., 1969; Zatz & Lance, 1971). The accumulation of lymphocytes from the circulating pool reflects their importance in the initiation of immune responses (Ford & Gowans, 1967; Sprent, Miller & Mitchell, 1971; Rowley et al., 1972), and thus both lymphocyte trapping and lymphocyte proliferation are indicative ofthe early stages ofa developing immune reaction. A similar increase in size and weight to that observed after skin allografting has been found in lymph nodes draining the uterus during allogeneic (inter-strain) pregnancies (Beer & Billingham, 1971; Maroni & de Sousa, 1973; Beer, Scott & Billingham, 1975). In one instance, an increase in DNA synthesis within these nodes on the 16th day of pregnancy has also been reported (Maroni & de Sousa, 1973). These observations are consistent with the view that immune responses may be initiated in the mother towards paternally-derived antigens of the foetus, presumably as a consequence of the accumulation of foetal antigens within the draining nodes. On the other hand, the protection afforded to the foetus, despite its established antigenicity (Simmons & Russell, 1966; Olds, 1968; Searle et al., 1976; Carter, 1976a), may be due in part to some interference in the normal sequence of immunological events-localization of antigen, lymphocyte trapping, blast transformation and proliferation and the generation of effector and memory cells. To establish that changes in the lymph node weight are indicative of an immune response initiated against foetal antigens, we have investigated the localization and accumulation of 51Cr-labelled lymph * Present address: MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road South, Edinburgh. Correspondence: Dr J. D. Ansell, Immunobiology Unit, Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT. 0099-9104/78/0300-0397 $02.00 (D 1978 Blackwell Scientific Publications
397
398
J. D. Ansell et al.
node cells, the changes in DNA synthesis in nodes draining the uteri of pregnant mice during the first and third pregnancies, and in a pregnancy after pre-immunization with paternal strain skin. The extent to which these observations reflect normal immune responses to antigens is assessed by comparison with results obtained after immunization with allogeneic skin, allogeneic erythrocytes and xenogeneic erythrocytes. MATERIALS AND METHODS Animals. Female mice of the inbred strain BALB/c (H2d) bred in this department and maintained under conventional conditions were used in these experiments. They were mated at 8-12 weeks of age with males of the same strain, or with CBA/H males (H2) obtained from the Edinburgh University Centre for Laboratory Animals. Pregnant females were timematched relative to the point at which vaginal plugs were found (day 1=24 hr after observation of the plug). For experiments on third pregnancies, the first and second litters were removed within 7 days of birth and the males removed during the second pregnancy. Synchrony of the third mating was achieved by returning each male to its original female on the same day. To maximize the size of the groups assayed at each point, determinations were made at 8-10 days and 14-16 days of pregnancy, and 1-2 days post-partum. The assay of lymph nodes draining the uterus was confined to the lumbar nodes, while the brachial lymph nodes were also removed as non-draining controls, together with the livers and spleens. 51Cr-labelled lymph node cells. Syngeneic (BALC/c female) lymph node cells were labelled with [51Cr]sodium chromate by the method previously described (Bainbridge & Gowland, 1966; Inchley et al., 1976). 5 x 106 labelled cells were injected intravenously on the day of assay and 24 hr later the mice were killed. Lymph nodes, livers and spleens were removed, and the radioactivity within them was determined. In control experiments, pregnant mice were injected with cells which had been frozen and thawed, or with a volume of the first supernatant from the washing of 51Cr-labelled cells (mostly free sodium chromate). Both these suspensions localized mainly in the liver, with some radioactivity also appearing in the spleen. There was no increased localization in the lumbar lymph nodes in either case, although the injection of supernatant resulted in some radioactivity appearing in the placenta. Conversely, no radioactivity was found in the placenta after the injection of whole labelled lymph node cells. [1 25I]5-iodo-2'-deoxyuridine ([1 25I]UdR). The amount of DNA synthesis in paired lumbar or brachial lymph nodes was assessed by the incorporation of [125I]UdR (Radiochemical Centre, Amersham) according to the technique of Pritchard & Micklem (1972). Antigens. Sheep red blood cells (SRBC; Tissue Culture Services Ltd, Slough, England) were washed four times and made up for injection in 0-85% saline. Mouse red blood cells (MRBC) were obtained fresh as required, immediately diluted in a large volume of saline and prepared as for SRBC. Whole-thickness allogeneic or syngeneic skin grafts were applied to beds prepared on the shaved right flank of virgin BALB/c mice. Bandages were removed after 10 days and the grafts inspected daily for macroscopic signs ofrejection. Statistics. Lymphocyte accumulation is expressed as a percentage increase of 51Cr label over control levels, these being given a value of 100%. [125I]UdR incorporation is expressed as the mean log counts per 10 min. Estimates of significance were made using the Student's t-test or paired t-tests where appropriate.
RESULTS
Changes in lymph node weights during pregnancy. The previous observation that inter-strain mating causes hypertrophy of the lymph nodes draining the uterus was confirmed for the strain combination used in these experiments (Fig. 1). In BALB/c females pregnant by CBA males, an increase in the weight of the lumbar nodes was found in both the first and third pregnancies, with peak hypertrophy on days 14-16 in both cases. The increase was more pronounced during a third pregnancy. On the other hand, little or no change in weight could be ascribed to the effects of pregnancy alone, as measured in syngeneically mated BALB/c females. Distribution of,5' Cr-labelled lymph node cells in pregnant mice Syngeneically or allogeneically mated BALB/c females were injected with 51Cr during and after the first and third pregnancies, and the distribution of radioactivity after 24 hr was compared with that in virgin controls (Figs 2 and 3). Following inter-strain mating, increasing localization of labelled cells was seen in the paired lumbar nodes. This was significantly higher than with the syngeneically mated (P< 005) and virgin control levels (P< 0.001) by days 14-16 days of pregnancy. A similar level of localization was seen at 14-16
399
Lymph nodes in pregnancy
.I..
I co
;0
'am.0 0' 2.
8-10
14-16 Aost-partum 8-I0
14-16 Post-partum Days of pregnancy First pregnancy Third pregnancy Do
I
. .,, _+ ,
FIG. 1. The mean weight (i s.e.) of paired lumbar lymph nodes during the first and third pregnancies. (E) Node weights in syngeneic pregnancies; (5) node weights in allogeneic pregnancies; (- -) node weights in virgin control mice. 'Syngeneic' levels in all groups do not differ from controls. Differences of allogeneic levels from virgin controls: *P< 0 001; t P< 0 01; t P< 0 05. Five to six animals per group. - -
2I0r-
(a)
aa 0
0
8-10 First pregnancy
14-16 14-16 Second pregnancy Third pregnancy Days of pregnancy FIG. 2. Mean lymphocyte localization (± s.e.) in paired (a) lumbar and (b) brachial nodes during first and third pregnancies. (ri) Localization in syngeneic pregnancy; (5) localization in allogeneic pregnancy; (--- -) localization in virgin control mice. The data is expressed as a percentage of the mean virgin control level of localization, this being given a value of 100"%. Differences from virgin controls: * P
Changes in lymphocyte accumulation and proliferation in the lymph nodes draining the pregnant uterus J. D. ANSELL, CATHERINE M. McDOUGALL, GILLIAN SPEEDY* & C. J. INCHLEY Immunobiology Unit, Department of Zoology, University ofEdinburgh, Edinburgh
(Received 2 August 1977) SUMMARY
Changes in weight, lymphocyte accumulation and cellular proliferation have been measured in the lymph nodes draining the uterus during inter- and intra-strain pregnancies and compared with similar effects after other antigenic stimuli. From the data obtained it was concluded that 'paternal' antigenic stimulation from the conceptus initiated an immune response in these nodes. The mechanisms of the subsequent suppression ofthis response are discussed. INTRODUCTION Exposure of mice to the transplantation antigens of allogeneic tissue is normally followed by wellcharacterized changes in the draining lymphoid tissues. In the case of skin allografts, for instance, the regional lymph nodes show a pronounced enlargement, associated with the proliferation of lymphocytes in the paracortical region, and increased accumulation of circulating cells (Scothorne & McGregor, 1955; Parrott & de Sousa, 1970; Zatz & Lance, 1971). Similar, but more rapid, changes also take place in the draining lymph nodes following the subcutaneous administration of xenogeneic erythrocytes or other antigens (Davies et al., 1969; Zatz & Lance, 1971). The accumulation of lymphocytes from the circulating pool reflects their importance in the initiation of immune responses (Ford & Gowans, 1967; Sprent, Miller & Mitchell, 1971; Rowley et al., 1972), and thus both lymphocyte trapping and lymphocyte proliferation are indicative ofthe early stages ofa developing immune reaction. A similar increase in size and weight to that observed after skin allografting has been found in lymph nodes draining the uterus during allogeneic (inter-strain) pregnancies (Beer & Billingham, 1971; Maroni & de Sousa, 1973; Beer, Scott & Billingham, 1975). In one instance, an increase in DNA synthesis within these nodes on the 16th day of pregnancy has also been reported (Maroni & de Sousa, 1973). These observations are consistent with the view that immune responses may be initiated in the mother towards paternally-derived antigens of the foetus, presumably as a consequence of the accumulation of foetal antigens within the draining nodes. On the other hand, the protection afforded to the foetus, despite its established antigenicity (Simmons & Russell, 1966; Olds, 1968; Searle et al., 1976; Carter, 1976a), may be due in part to some interference in the normal sequence of immunological events-localization of antigen, lymphocyte trapping, blast transformation and proliferation and the generation of effector and memory cells. To establish that changes in the lymph node weight are indicative of an immune response initiated against foetal antigens, we have investigated the localization and accumulation of 51Cr-labelled lymph * Present address: MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road South, Edinburgh. Correspondence: Dr J. D. Ansell, Immunobiology Unit, Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT. 0099-9104/78/0300-0397 $02.00 (D 1978 Blackwell Scientific Publications
397
398
J. D. Ansell et al.
node cells, the changes in DNA synthesis in nodes draining the uteri of pregnant mice during the first and third pregnancies, and in a pregnancy after pre-immunization with paternal strain skin. The extent to which these observations reflect normal immune responses to antigens is assessed by comparison with results obtained after immunization with allogeneic skin, allogeneic erythrocytes and xenogeneic erythrocytes. MATERIALS AND METHODS Animals. Female mice of the inbred strain BALB/c (H2d) bred in this department and maintained under conventional conditions were used in these experiments. They were mated at 8-12 weeks of age with males of the same strain, or with CBA/H males (H2) obtained from the Edinburgh University Centre for Laboratory Animals. Pregnant females were timematched relative to the point at which vaginal plugs were found (day 1=24 hr after observation of the plug). For experiments on third pregnancies, the first and second litters were removed within 7 days of birth and the males removed during the second pregnancy. Synchrony of the third mating was achieved by returning each male to its original female on the same day. To maximize the size of the groups assayed at each point, determinations were made at 8-10 days and 14-16 days of pregnancy, and 1-2 days post-partum. The assay of lymph nodes draining the uterus was confined to the lumbar nodes, while the brachial lymph nodes were also removed as non-draining controls, together with the livers and spleens. 51Cr-labelled lymph node cells. Syngeneic (BALC/c female) lymph node cells were labelled with [51Cr]sodium chromate by the method previously described (Bainbridge & Gowland, 1966; Inchley et al., 1976). 5 x 106 labelled cells were injected intravenously on the day of assay and 24 hr later the mice were killed. Lymph nodes, livers and spleens were removed, and the radioactivity within them was determined. In control experiments, pregnant mice were injected with cells which had been frozen and thawed, or with a volume of the first supernatant from the washing of 51Cr-labelled cells (mostly free sodium chromate). Both these suspensions localized mainly in the liver, with some radioactivity also appearing in the spleen. There was no increased localization in the lumbar lymph nodes in either case, although the injection of supernatant resulted in some radioactivity appearing in the placenta. Conversely, no radioactivity was found in the placenta after the injection of whole labelled lymph node cells. [1 25I]5-iodo-2'-deoxyuridine ([1 25I]UdR). The amount of DNA synthesis in paired lumbar or brachial lymph nodes was assessed by the incorporation of [125I]UdR (Radiochemical Centre, Amersham) according to the technique of Pritchard & Micklem (1972). Antigens. Sheep red blood cells (SRBC; Tissue Culture Services Ltd, Slough, England) were washed four times and made up for injection in 0-85% saline. Mouse red blood cells (MRBC) were obtained fresh as required, immediately diluted in a large volume of saline and prepared as for SRBC. Whole-thickness allogeneic or syngeneic skin grafts were applied to beds prepared on the shaved right flank of virgin BALB/c mice. Bandages were removed after 10 days and the grafts inspected daily for macroscopic signs ofrejection. Statistics. Lymphocyte accumulation is expressed as a percentage increase of 51Cr label over control levels, these being given a value of 100%. [125I]UdR incorporation is expressed as the mean log counts per 10 min. Estimates of significance were made using the Student's t-test or paired t-tests where appropriate.
RESULTS
Changes in lymph node weights during pregnancy. The previous observation that inter-strain mating causes hypertrophy of the lymph nodes draining the uterus was confirmed for the strain combination used in these experiments (Fig. 1). In BALB/c females pregnant by CBA males, an increase in the weight of the lumbar nodes was found in both the first and third pregnancies, with peak hypertrophy on days 14-16 in both cases. The increase was more pronounced during a third pregnancy. On the other hand, little or no change in weight could be ascribed to the effects of pregnancy alone, as measured in syngeneically mated BALB/c females. Distribution of,5' Cr-labelled lymph node cells in pregnant mice Syngeneically or allogeneically mated BALB/c females were injected with 51Cr during and after the first and third pregnancies, and the distribution of radioactivity after 24 hr was compared with that in virgin controls (Figs 2 and 3). Following inter-strain mating, increasing localization of labelled cells was seen in the paired lumbar nodes. This was significantly higher than with the syngeneically mated (P< 005) and virgin control levels (P< 0.001) by days 14-16 days of pregnancy. A similar level of localization was seen at 14-16
399
Lymph nodes in pregnancy
.I..
I co
;0
'am.0 0' 2.
8-10
14-16 Aost-partum 8-I0
14-16 Post-partum Days of pregnancy First pregnancy Third pregnancy Do
I
. .,, _+ ,
FIG. 1. The mean weight (i s.e.) of paired lumbar lymph nodes during the first and third pregnancies. (E) Node weights in syngeneic pregnancies; (5) node weights in allogeneic pregnancies; (- -) node weights in virgin control mice. 'Syngeneic' levels in all groups do not differ from controls. Differences of allogeneic levels from virgin controls: *P< 0 001; t P< 0 01; t P< 0 05. Five to six animals per group. - -
2I0r-
(a)
aa 0
0
8-10 First pregnancy
14-16 14-16 Second pregnancy Third pregnancy Days of pregnancy FIG. 2. Mean lymphocyte localization (± s.e.) in paired (a) lumbar and (b) brachial nodes during first and third pregnancies. (ri) Localization in syngeneic pregnancy; (5) localization in allogeneic pregnancy; (--- -) localization in virgin control mice. The data is expressed as a percentage of the mean virgin control level of localization, this being given a value of 100"%. Differences from virgin controls: * P
