The lymphoid organs during pregnancy in the mouse. A comparison between a syngeneic and an allogeneic mating

In the present experiments the changes that occur in the central and peripheral lymphoid organs during a first pregnancy in the mouse have been followed up in BALB/c females (H-2d) mated either to males of the same inbred strain or to C3H/Bi males, a strain differing at the major histocompatibility locus (H-2k). Three parameters were examined: weight, morphology and DNA synthesis. The results to be presented strengthen existing evidence that sensitization of the female to foetal transplantation antigens of paternal origin may occur during a first pregnancy and suggest that this process develops at least in part via the lymph nodes draining the uterus.     

Induction of "allogeneic effect"-like reaction by syngeneic TNP-modified lymphoid cells

TNP-substituted SRBC-immune spleen cells, when injected into cyclophosphamide-treated recipients, are recognized by T lymphocytes and produce, in the presence of specific antigen (SRBC), significantly more PFC than nonsubstituted cells. Labeling of immune B cells is more important in producing the augmented responses than is the labeling of immune T cells. TNP determinant has to be bound directly to the transferred immune cells to produce enhanced antibody responses, as when recipients were injected with non-substituted immune cells and TNP-substituted non-immune cells simultaneously, no increase in PFC number was noted (lack of a bystander effect). When recipients were rendered tolerant to TNP, two separate effects were observed, dependent on the mode of inducing unresponsiveness. In mice which were treated with TNP over an extended period of time, lack of recognition of TNP was demonstrated, such that TNP-substituted cells failed, when transferred, to produce an augmented response. When a short-term tolerogenic regime was used, the adoptively transferred TNP-labeled cells gave a very poor response (greater than 95% inhibition) due to in vivo suppression and/or killing. These results, together with the lack of influence of tolerance induced to unrelated hapten (DNP or OX), confirm the antigen specificity of the phenomenon. The reaction observed by us shows a striking resemblance with, but not identical to, the "allogeneic effect" produced by MHC encoded alloantigens. Our results extend the list of analogous immune reactions induced by MHC encoded alloantigens and TNP-derivatized self.     

Distribution of MEL-14+ cells in various lymphoid tissues

The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.     

Acute phase serum proteins in syngeneic and allogeneic mouse pregnancy.

The levels of two murine acute phase proteins, serum amyloid P component (SAP) and haptoglobin, have been measured in the serum of C57BL/10 female mice during syngeneic and allogeneic pregnancy. Both syngeneic and allogeneic pregnancy resulted in alterations in the levels of these proteins as compared to those observed in virgin females. Syngeneic mating resulted in an increase in concentration of both proteins during the final 3 days of pregnancy. During allogeneic pregnancy, SAP levels, after a transient increase on day 4, rose from days 6-8 and, after remaining relatively stable, increased from day 12 to reach maximum levels on day 18 of pregnancy. Levels fell dramatically during the immediate post-partum period. In contrast, although levels of haptoglobin also increased from days 6-8, for the remainder of pregnancy these increased levels remained stable. The implications of these findings are discussed in relation to the mechanisms of regulation of acute phase reactants and the immunological relationship between the mother and fetus.     

Maternal regulator cells during murine pregnancy

Helper and suppressor cells have been identified in lymph nodes of multiparous pregnant mice using one-way mixed lymphocyte reactions. Primiparous C57Bl and C57Bl/10ScSn females during pregnancy and immediately post-partum gave unaltered MLR reactivity compared to virgin controls when stimulated with paternal or third-party alloantigens. In contrast, multiparity produced strain-dependent alterations in responsiveness: C57Bl females being hyporesponsive while C57Bl/10ScSn mice were hyper-responsive to these antigens. To evaluate regulator activity, the cells were treated with mitomycin C and mixed with normal syngeneic MLR responder populations. Results showed that mixtures of para-aortic and inguinal lymph node cells from pregnant C57Bl females suppressed while those from pregnant C57Bl/10ScSn increased normal virgin MLR responses. Treatment of the regulator cells with anti-Thy-1.2 plus complement showed that suppressor activity was predominantly non-T cell in nature while C57Bl/10ScSn helper activity was wholly T cell-dependent. The regulator cells appeared to be non-specific since they were found in syngeneically as well as allogeneically mated females but covert specificity could not be totally excluded. Embryo resorption in the C57Bl strain was associated with helper rather than suppressor activity. These results suggest that suppressor cells are not essential for the maintenance of pregnancy and that the presence of regulator cells probably reflects the normal homeostatic mechanisms controlling the maternal immune response to the conceptus.     

The effect of ultraviolet irradiation on the localization of syngeneic and allogeneic lymphoid cells and on alloimmunogenicity.

Ultraviolet (u.v.) irradiation was found to have only a modest inhibitory effect on the alloimmunogenecity of murine lymphoid cells in vivo when the response was assessed by the primary cytotoxic antibody response. Ultraviolet irradiation had no effect on the initial (1 hr) organ localization of chromium-labelled lymphoid cells in syngeneic or allogeneic recipients using either CBA or DBA/2 cells. However the 24 hr localization in peripheral lymph nodes was considerably and similarly depressed in syngeneic and allogeneic recipients. This effect was shown, by alloantibody treatment of recipients of allogeneic cells, to be due mainly to lack of entry into lymph nodes after 1 hr. In agreement with much of the published data, control data for these experiments indicated little difference in lymph node localization of lymph node cells in allogeneic and syngeneic recipients using both CBA and DBA/2 cells in both CBA and DBA/2 recipients. The localization of spleen cells in lymph nodes was found to be rather more sensitive to the allogeneic environment though not as much as has been previously described for spleen cells in contrast to the long established data on lymph node cells. A marked difference in the degree of lymph node localization of CBA and CBA/2 cells was found regardless of the recipient strain.     

Cytokine profile in collagen-induced arthritis: Differences between syngeneic and allogeneic pregnancy

OBJECTIVE: To identify the differences in cytokine profile between allogeneic and syngeneic pregnancy in mice with collagen-induced arthritis (CIA). METHODS: Mice (strain B10.RIII) were injected with bovine collagen. Females were mated with males of the same strain (syngeneic pregnancy) or with males of strain B10. Q (allogeneic pregnancy). Concentrations of cytokines were measured during pregnancy and after delivery, and the onset and evolution of arthritis was followed in all female animals throughout the study period. RESULTS: In female mice that developed CIA, cytokine concentrations were lower in allogeneic pregnancies than syngeneic pregnancies. When paired cytokine concentrations were compared in each animal during and after pregnancy, MCP-1 was lower during gestation than after delivery in both groups of pregnant mice, IL-6 was lower during gestation than after delivery only in allogeneic pregnancies, and IL-10 was lower during gestation than after delivery in allogeneic pregnancies, whereas in syngeneic pregnancies IL-10 was higher during gestation than after delivery. CONCLUSIONS: Allogeneic pregnancy was associated with less arthritis because of lower concentrations of proinflammatory cytokines (IL-6 and others), not because of an increase in the concentration of antiinflammatory cytokines (IL-10).     

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7^low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7^high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7^low, while few cytokine‐expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN‐γ.     

Inhibition of the growth of murine tumour cells in vitro by serum from non-immune syngeneic and allogeneic mice.

Sera from DBA/2 and Quackenbush mice (which are non-immune for mastocytoma and Sarcoma 180 respectively) contain a heat-labile (56 degrees for 30 min) component(s) that inhibits the in vitro growth of DBA Mastocytoma P-815 X-2 and Sarcoma 180. Adsorption of the sera with tumour cells at 4 degrees did not eliminate the factor(s), suggesting that it is not an antibody. In liquid suspension cultures inhibitory activity was observed at concentrations of mouse serum of 10--20% and in semisolid agar clonogenic cell assays at concentrations as low as 1%. The influences of the inhibitor(s) for both tumours and in both culture systems were parallel. However, there was a quantitative difference in susceptibility to other environmental factors (FCS concentration, bicarbonate concentration, and O2 tension) between the two tumours. These results parallel the in vivo findings where intravenously injected mastocytoma cells produced more tumours than did Sarcoma 180.     

Kinetics of regulatory T cells during murine pregnancy

PROBLEM: The semi-allogeneic fetus is usually tolerated by the maternal immune system. This was proposed to be modulated by CD4+CD25+foxp3+ regulatory T cells (Treg). We aimed to determine the kinetics of Treg during murine gestation and investigate whether changes in Treg levels respond to hormonal variations during pregnancy or generated changes in the local indolamine dioxygenase (IDO) expression. METHOD OF STUDY: We included in our studies the well-known CBA/JxDBA/2J abortion-prone combination using CBA/JxBALB/c as controls. CBA/JxC57/BL6 and BALB/cxC57/BL6 were included as further controls. Animals were killed on days 0, 2, 5, 8, 10, and 12 of pregnancy to measure the levels of Treg, pregnancy-related hormones and IDO expression. RESULTS: A Treg augmentation in normal pregnancy combinations could be observed on day 2 in several organs contrary to the observations made in abortion-prone mice. No differences in hormonal levels could be seen among all groups. IDO was expressed exclusively in placenta starting from day eight, showing no variations among the groups. CONCLUSION: Differences in Treg levels and pregnancy outcome do not correlate with changes in hormonal levels. In addition, as Treg augmentation takes place early and it is observed mainly in the decidual component of the fetal-maternal interface, IDO does not seem to be the pathway underlying Treg protective activity as proposed for humans.     

Thymic and body weight during first syngeneic and allogeneic pregnancy in the rat, and the effects of strain difference

Two hundred female rats, 91-101 days of age, were used in this investigation which provided data on thymic weight, body weight and where appropriate the number of progeny. The observations began in oestrus phase virgin females from the two highly inbred strains AO (RT1u/AgB2) and DA (RT1a/AgB4), continued at five day intervals throughout syngeneic and allogeneic pregnancy in both strains and were completed in the immediate post-partum period. This experimental protocol permitted sequential observations throughout gestation and allowed syngeneic:allogeneic and interstrain comparisons to be made. The syngeneic:allogeneic comparisons demonstrated significant differences in the body weight response and the number of progeny while the AO:DA comparisons demonstrated significant differences in the thymic weight and body weight responses.     

Immunoglobulin-secreting stomach cells in peptic ulcer

The mucous membrane of the fundal and pyloric parts of 26 stomachs resected for peptic ulcer was examined. The edges and bottoms of the ulcers were studied. The cells secreting immunoglobulin (Ig) of A, M, and G classes were detected by indirect immunoperoxidase method. The proportions (in per cent) of lymphocytes, plasma cells and Ig-containing cells were determined stereometrically. The intact mucous membrane of the fundal part contains 2.17% lymphocytes and 7.5% plasma cells. The portions of IgA-, IgM- and IgG-secreting cells were 0.99%, 0.39%, and 0.13%, respectively. In atrophic antral gastritis, plasma cell infiltration of the mucosa increases 2 1/2-fold, the number of lymphocytes remaining unchanged. The number of immunocytes secreting IgA and IgM increases almost 4-fold and that of secreting IgG over 13-fold. In the ulcer edges, plasma cell infiltration of the stroma reached 10.83% whereas the number of Ig-secreting cells was the same as in the pyloric part in the same patients. The activation of the secretory immune system in peptic ulcer is due to significant antigenic stimulation. The most predominant are the IgA-containing cells. A marked increase of IgG synthesis indicates the defect of autoregulation of the local immunity and may be of pathogenetic importance for the development of autoaggression which would facilitate progression and chronicity of the ulcer.     

Interferon-gamma and growth factor production by murine T cells derived from three different lymphoid tissues

Various antigen-presenting cells and the environment in different lymphoid tissues have been suggested to influence the type of lymphokine produced by T cells. We have investigated the mitogen-induced proliferation, interferon-gamma (IFN-gamma) and growth factor production by cells isolated from spleen, mesenteric and peripheral (axillary, brachial and inguinal) lymph nodes (LN). We found that stimulation with concanavalin A or staphylococcus enterotoxin B induced IFN-gamma synthesis in spleen cells but not in LN cells. Proliferation and growth factor production were comparable in the three cell populations. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), which is commonly used as a substitute for accessory cells, did not influence the IFN-gamma synthesis by LN cells. The growth factor production was, on the other hand, elevated by the addition of PMA. A high number of IFN-gamma-producing peripheral LN cells were obtained if they were stimulated in the presence of splenic adherent cells. The growth factor synthesis was marginally affected by the presence of these cells. Thus, splenic adherent cells provide a co-stimulatory signal to the T cell necessary for IFN-gamma synthesis.     

Protection against syngeneic tumor grafts induced by inoculation with normal allogeneic tissues.

Subcutaneous injection of normal allogeneic kidney and liver tissues extended survival of DBA/2Cr mice challenged with a lethal dose of syngeneic L5178Y lymphoma cells. Immunization with a combination of tissues from five strains provided far more protection than immunization with tissue from any single strain. It is suggested that the basis of this protective effect is a cross-reactivity or identity between tumor-associated transplantation antigens (TATA) on the L5178Y tumor and non-H-2 alloantigens normally expressed by some allogeneic tissues.     

In vitro preactivated human T cells engraft in SCID mice and migrate to murine lymphoid tissues.

Mice with severe combined immunodeficiency (SCID) accept grafts of human T and B lymphocytes derived from resting peripheral blood mononuclear cells (PBMC). We wished to determine whether activated human T cells engraft and migrate into lymphoid tissues in SCID mice. PBMC (50 x 10(6)) activated in vitro in a 4-day mixed lymphocyte culture (MLC) were injected into the peritoneum of 12 SCID mice. In 11 of 12 animals killed at 3 or 4 weeks after injection, human cells were detected in cells pooled from lymphoid organs by flow cytometry and by immunohistochemical staining of frozen tissue sections. The percentage of CD45+ cells in the 11 mice ranged from 2% to 45% and the absolute numbers of CD45+ cells recovered from lymphoid organs ranged from 4 x 10(6) to 90 x 10(6). Up to 93% of the human cells expressed the CD3 antigen together with either CD4 or CD8. Human T cells were localized in periarteriolar areas in murine spleens, whereas in the lymph nodes and gut mucosa, the T cells did not show the pattern for T-dependent areas found in human lymphoid tissue. Numerous human plasma cells were detected in the spleen and gut mucosal crypts of engrafted SCID mice. Human IgG was detected in the serum of all 11 engrafted SCID mice. The functional activity of human T cells recovered from murine splenic tissue was very low 3-4 weeks after engraftment.     

Molecular interactions during pregnancy: Distribution of interleukin-6 in maternal and embryonic tissues during the first trimester

Interleukin-6 (IL-6) distribution was investigated in coelomicfluid, amniotic fluid, maternal serum, decidua and placentalvillous tissue collected from 16 normal pregnancies between7 and 12 weeks of gestation. IL-6 levels in fluids and tissueswere measured by an immunoenzymatic assay and mouse monoclonalantibodies specific for IL-6 were used to localize immunohistochemicallyIL-6 in decidual and placental tissue. IL-6 was detected inall samples of coelomic and amniotic fluids and in most extractsof placental and decidual tissues. IL-6 concentration was significantly(P < 0.005) higher in the coelomic fluid than in amnioticfluid and was positively correlated with gestational age. Immunostainingfor IL-6 was present in both syncytiotrophoblast and extra-villoustrophoblast. IL-6 was in significantly (P < 0.001) higherconcentration in decidual than in placental tissues. These dataindicate that IL-6 is normally present in coelomic and amnioticfluids of early pregnancy and that IL-6 concentrations mainlyresult from the accumulation of trophoblastic-derived IL-6.During the first trimester IL-6 could play a role in tissueremodelling associated with placentation but also in the haematopoiesisfunction of the secondary yolk sac and in the generation ofnew vessels in placental villous tissue. coelomic fluid/early placenta/first trimester/interleukin-6/pregnancy