Maternal background strain influences fetal–maternal trafficking more than maternal immune competence in mice
Introduction
Fetal cell microchimerism (FCM) is a term that describes the presence of fetal cells in maternal blood and organs during pregnancy and post-partum. It has been studied in both humans and animal models (Johnson and Bianchi, 2004, Lissauer et al., 2007, Dutta and Burlingham, 2009). In humans, most studies on FCM have used the Y chromosome as a marker of fetal cells, thereby limiting the study subject population to women who have had sons (Johnson et al., 2001). Because of this and other inherent limitations of human studies, our laboratory began to use a murine model of FCM in which males that are transgenic for the green fluorescent protein (GFP) are used for breeding. The model we initially used incorporated hemizygous males with the transgene present on an autosome, so its inheritance was independent of fetal gender. Fifty percent of the offspring were GFP-positive (Khosrotehrani et al., 2004, Khosrotehrani et al., 2005). With this model, we showed that the frequency of FCM was highest in the maternal lung, peaked just prior to parturition, and declined rapidly post-partum (Khosrotehrani et al., 2005, Fujiki et al., 2008a). Following this work, we incorporated a strain that was homozygous for the Gfp transgene, resulting in 100% fetal cell detection. This strain was used earlier by other investigators to study fetal cell microchimerism in the maternal mouse brain (Tan et al., 2005). In a prior study from our group using this homozygous transgenic model, we demonstrated that fetal cells in maternal organs expressed a variety of cell surface antigens, including CD34, CXCR4 (CD184), SLAMF1 (CD150), CD45, integrin alpha M (CD11b), integrin beta 1 (CD29), CD44, and endoglin (CD105) (Fujiki et al., 2009).
The role of immune competence and background strain in FCM was first studied in the mouse by Bonney and Matzinger (1997). Using PCR amplification of a Y chromosome sequence, they showed that fetal cell trafficking occurs in only 20% of wild-type pregnancies, and that early in pregnancy immune compromised (SCID) and normal females have equivalent levels of trafficking. FCM in SCID mice increased relative to normal females as pregnancy progressed. This suggested that SCID mice were not able to clear fetal cells as efficiently as wild-type mice, which implied involvement of the maternal immune system. In the same study, Bonney and Matzinger (1997) demonstrated higher levels of FCM in pregnant females following allogenic matings. In another study, our laboratory showed that there was a non-statistically significant trend toward higher levels of cell-free fetal DNA in maternal plasma from allogenic compared with congenic females (Khosrotehrani et al., 2004). We suggested that this was due to a more robust maternal immune response to fetal allo-antigens, which resulted in increased placental apoptosis and levels of fetal DNA in the maternal circulation. In a follow-up investigation, we also demonstrated that there was a statistically significantly higher level of FCM in maternal solid tissues from congenic than from allogenic matings, particularly in the spleen and liver, although only five allogenic pregnancies were analyzed (Khosrotehrani et al., 2005). In an investigation that specifically examined the effect of histocompatibility between mother and fetus on cell migration through the placenta, FCM was higher in allogenic than syngenic gestations (Vernochet et al., 2007).
Taken together, these studies suggest that genetic differences between the mother and fetus, as well as the maternal immune system, can influence FCM. A consistent limitation of these previous studies, however, was that PCR amplification was used to measure fetal DNA sequences. PCR has a sensitivity of detection of 1 fetal in 105 maternal cells. In our experience, flow cytometry is a more robust method for the detection of fetal cells, as it has significantly higher sensitivity (5 in 107), and allows for the simultaneous assessment of cell surface phenotype (Fujiki et al., 2008b, Fujiki et al., 2009).
The purpose of the current study was to determine if FCM is affected by maternal background strain and/or immune competence using the most sensitive technique available, flow cytometry. We examined maternal organs that we have previously shown to contain the highest frequency of fetal cells (lung, liver, spleen, bone marrow, and blood) from immunocompetent and immunodeficient females that were allogenic and congenic with their breeding partners. We further sought to characterize the fetal cells through analysis of specific cell surface markers, including antigens expressed by hematopoietic cells (CD34), myeloid cells (CD11b), and mesenchymal cells (CD29, CD44, and CD105) (Fujiki et al., 2009).
Section snippets
Animals
All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Tufts University School of Medicine Division of Laboratory Animal Medicine (DLAM). All institutional guidelines regarding the ethical use of experimental animals were followed.
Wild-type C57BL/6J (congenic, immunocompetent; n = 18), FVB/NJ (allogenic, immunocompetent; n = 15), or B6129S7-Rag1tm1Mom/J (with a C57BL/6J background; from here on in referred to as “RAG”; congenic, immunodeficient; n = 16) females,
Results
The raw numbers of GFP-positive cells relative to 107 maternal cells and the proportion of fetal cells that were antibody-positive from all three female strains are shown in Table 1. In C57BL/6J and FVB/NJ females, the median number of fetal cells in solid organs was consistently highest in the lung, followed by the liver and spleen. In RAG females, the median numbers of GFP+ fetal cells were roughly equivalent in the lung and liver. The number of fetal cells was lowest in bone marrow and blood
Discussion
In this study, maternal background strain was a more important determinant of FCM than maternal immune competence. The results showed higher proportions of fetal cells in lung, liver and spleen from allogenic females compared with both congenic immunocompetent and immunodeficient females. However, while allogenic females had the highest proportion of GFP-positive fetal cells, the relative proportions of fetal cells that expressed the markers analyzed here (CD 11b, 29, 34, 44 and 105) were lower
Acknowledgements
The authors wish to thank Stephen Kwok and Allen Parmelee for flow cytometry expertise and Stephanie Pritchard for manuscript preparation. This study was supported by NIH grant R01 HD049469-05 to DWB. Homozygous transgenic mice were provided by Dr. Masaru Okabe, Research Institute for Microbial Disease, Osaka University, Japan through Riken BioResource Center, Ibaraki, Japan.
References (16)
- et al.
Natural history of fetal microchimerism during and following murine pregnancy
J. Reprod. Immunol.
(2005) - et al.
FISH analysis of 142 EGFP transgene integration sites into the mouse genome
Genomics
(2002) - et al.
Bi-directional cell trafficking between mother and fetus in mouse placenta
Placenta
(2007) - et al.
Comparison of allergic lung disease in three mouse strains after systemic or mucosal sensitization with ovalbumin antigen
Immunogenetics
(2009) - et al.
The maternal immune system's interaction with circulating fetal cells
J. Immunol.
(1997) - et al.
Tolerance to non-inherited maternal antigens in mice and humans
Curr. Opin. Organ Transplant.
(2009) - et al.
Fetomaternal trafficking in the mouse increases as delivery approaches and is highest in the maternal lung
Biol. Reprod.
(2008) - et al.
Quantification of green fluorescence protein by in vivo imaging, PCR and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism
Cytometry
(2008)
Cited by (13)
Engraftment potential of maternal adipose-derived stem cells for fetal transplantation
2020, HeliyonCitation Excerpt :These results suggest that maternal PDGFRα+Sca-1+ ADSC grafts can be used as the donor cells for in utero transplantation to suppress the recipient's immune system, including maternal immunity more than PDGFRα+Sca-1+ ADSC grafts from green congenic mice. The extent of congenicity between wild-type C57BL/6J and GFP transgenic mice on an inter-substrain hybrid betweenC57/BL6J and C57BL/6CrSlc background are likely to be different, as they were derived and are maintained in different ways including derivation directly from an embryonic stem cell line, backcrossing [45]. The rationale behind in utero transplantation with maternal ADSCs is that maternal stem cells will eventually colonize the fetus's neural stem cell pool of the central nervous system and delay neural metabolic disease progression through tropic support, anti-inflammatory, or intrinsic immune modulatory properties.
Potential role of exosomes in reproductive medicine and pregnancy
2019, Exosomes: A Clinical CompendiumMicrochimerism in Mouse Pregnancy
2014, The Guide to Investigation of Mouse PregnancyPregnancy-acquired fetal progenitor cells
2013, Journal of Reproductive ImmunologyCitation Excerpt :The maternal immune system appears to influence the level of fetal microchimerism, although this is considered contentious. Mouse studies provide some evidence of histocompatibility being associated with greater levels of microchimerism (Bonney and Matzinger, 1997; Khosrotehrani and Bianchi, 2005), although, recent studies imply that maternal background strain may be more important (Fujiki et al., 2008b; Kallenbach et al., 2011). Human data reporting that fetal microchimerism occurs even in the absence of complete matching at different MHC loci provide some support for this (Lambert et al., 2004; Nelson et al., 1993).
Immunological Tolerance During Fetal Development. From Mouse to Man.
2012, Advances in ImmunologyCitation Excerpt :In this case, it is necessary to use GFP+/− female mice mated to GFP−/− males, followed by analysis of GFP−/− offspring for evidence of maternal microchimerism. Although the ability to detect maternal microchimerism was dependent upon the strain of mouse and whether matings were performed with syngeneic, allogeneic, or outbred mating pairs (Kallenbach et al., 2011), small numbers of maternal cells have been observed in the fetus, with the highest levels in the fetal blood at mid-gestation and lower levels in the fetal liver and thymus (Nijagal et al., 2011). Analysis of GFP+ cells at the fetomaternal interface shows that fetal cells associate with maternal blood sinuses whereas maternal cells were found scattered in the placental labyrinth (Vernochet et al., 2007).