ORIGINAL ARTICLE
Maternal Circulating Dendritic Cell Subtypes at Delivery and During the 1-Year Postpartum Period Hye Ryun Lee1, Byoung Jae Kim2, Sue Shin3,4,5, Hye Won Jeon2, Eun Youn Roh3,4,5, Jong Hyun Yoon3,4,5, Eun Young Song3 1
Department of Laboratory Medicine, Gyeongsang National University Hospital, Jinju, Korea; Department of Obstetrics and Gynecology, Boramae Hospital, Seoul, Korea; 3 Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea; 4 Seoul Metropolitan Government Public Cord Blood Bank (Allcord), Seoul, Korea; 5 Department of Laboratory Medicine, Boramae Hospital, Seoul, Korea 2
Keywords Dendritic cells, mean fluorescence intensity, postpartum Correspondence Sue Shin, Department of Laboratory Medicine, Seoul National University Boramae Hospital, Boramaro 5 gil 20, Dongjak-gu, 156-707, Seoul, Korea. E-mail: [email protected] and Hye Won Jeon, Department of Obstetrics and Gynecology, Seoul National University Boramae Hospital, Boramaro 5 gil 20, Dongjak-gu, 156-707, Seoul, Korea. E-mail: [email protected] Lee HR and Kim BJ contributed equally to this study as co-first authors. Shin S and Jeon HW contributed equally to this study as co-corresponding authors. Submission September 7, 2013; accepted November 18, 2013. Citation Lee HR, Kim BJ, Shin S, Jeon HW, Roh EY, Yoon JH, Song EY. Maternal circulating dendritic cell subtypes at delivery and during the 1-year postpartum period. Am J Reprod Immunol 2014; 71: 210–216
Problem Dendritic cells (DCs) play an important role in maintaining pregnancy by inducing tolerance toward the fetus. Such an immunologic change in the mother should be restored to normal after delivery, but few studies have reported postpartum maternal immune recovery, in terms of the types circulating DCs. Method of study The level of each DC subtype and HLA-DR-positive immunoreactivity of the blood from 29 pregnant women with uncomplicated labor was serially analyzed by flowcytometry at delivery and at 1.5, 6, and 12 months after delivery. DC subtypes were characterized as myeloid, lymphoid, and less differentiated (ldDC). Mean fluorescence intensity (MFI) was evaluated for HLA-DR expression for each DC subtype. Results The total number and the percentage of DCs at delivery were lower than those at 12 months postpartum. The ldDC fractions were significantly higher at delivery and at 1.5 months than at 12 months postpartum. The MFI of HLA-DR expression on ldDCs at delivery was lower than that at 12 months postpartum. The myeloid-to-lymphoid DC ratio did not differ over the 1-year postpartum period. Conclusion The maternal alteration in DCs rapidly normalized within 1.5 months, except for the ldDC fraction, which persisted between 1.5 and 6 months after delivery.
doi:10.1111/aji.12188
Introduction Dendritic cells (DCs) play an important role in the adaptive immune responses to foreign antigens and self-tolerance.1 In addition, DCs play an important 210
role in maintaining pregnancy through immunologic tolerance.2 After delivery, rapid recovery of normal maternal immune function is desirable. In normal pregnancy, the maternal immune system is tolerant to the partially histocompatible fetus. American Journal of Reproductive Immunology 71 (2014) 210–216 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
MATERNAL DENDRITIC CELLS DURING 1-YEAR POSTPARTUM
A shift from T-helper cell type 1 (Th1) to T-helper cell type 2 (Th2) activity is important in immune tolerance during pregnancy.3–5 Different DC subtypes are involved in this shift. The two main DC subtypes include myeloid DCs (mDCs) and lymphoid DCs (lDCs). MDCs express the integrin CD11c, produce interleukin-12, and induce the differentiation of na€ıve T lymphocytes into Th1 cells, whereas lDCs express CD123, produce type 1 interferon (IFN) and IFN-a, and may influence Th1/Th2 differentiation.6,7 Apart from mDCs and lDCs, a third DC subtype that weakly expresses CD123, termed as less differentiated DCs (ldDCs), has been recognized.8 In this study, we investigated the DC subtypes circulating in the maternal blood at the time of delivery and at 1.5, 6, and 12 months after delivery to determine the duration required for immunologic recovery to the prepartum status. During the postpartum period, complete immunologic recovery requires approximately 1 year9; therefore, in this study, we used the values at 12 months after delivery as the baseline reference values for comparison. Materials and methods Study Subjects and Sample Collection In this study, we enrolled 29 pregnant women (median age, 31 years; range, 22–43 years) with uncomplicated full-term labor (gestational week: median, 39 weeks; range, 36–41 weeks) from October 2009 to March 2010. The study was reviewed and approved by the Institutional Review Board of Seoul National University Boramae Hospital (062009-98). Cord blood was collected at delivery, and maternal peripheral blood was collected at delivery and at 1.5, 6, and 12 months postpartum. Cord blood was aseptically collected in utero from the umbilical vein by gravity into a collection bag containing citrate phosphate dextrose adenine-1 as an anticoagulant. Maternal blood was collected in tubes containing acid citrate dextrose-1 at delivery and in tubes containing ethylenediaminetetraacetic acid at 1.5, 6, and 12 months after delivery, within 1 week of the scheduled visit. At each blood draw, all participants were asked whether they experienced any febrile symptoms and their body temperature was assessed. American Journal of Reproductive Immunology 71 (2014) 210–216 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunophenotyping of Circulating DC Subtypes Immunophenotyping for DC subtypes from whole blood samples was analyzed as previously described.10,11 Briefly, 50 lL of whole blood was incubated with mouse anti-human CD123-PE, CD11c-APC, HLA-DR-PerCP, and a lineage cocktail 1-fluorescein isothiocyanate (Lin1-FITC) (containing a mixture of FITC-conjugated anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 antibodies) (BD Biosciences, San Jose, CA, USA) for 25 min at room temperature in the dark, according to manufacture instructions. Isotypematched mouse anti-human IgG isotype controls were used for each monoclonal antibody, to assess non-specific antibody binding and determine background fluorescence levels. After antibody incubation, 2 mL of BD FACSTM lysing solution (BD Biosciences) diluted 1:10 in distilled water was added and samples were fixed for another 15 min in the dark. Cells were washed twice and resuspended in 0.5 mL phosphate-buffered saline (PBS), and labeled cells were analyzed with a FACSAriaTM flowcytometer (Becton Dickinson) using FACSDivaTM Software (Becton Dickinson). From each sample, 100,000 events were acquired and analyzed. Among the gated mononuclear cells, DCs were negative for lineage cocktail markers but expressed HLA-DR. Among the DC population, cells expressing CD11c and variable/low CD123 were characterized as mDCs: CD11c+/CD123, whereas CD11cnegative cells expressing CD123 with high intensity were characterized as lDCs: CD11c /CD123++. Cells that were negative for CD11c that expressed low or no CD123 levels were characterized as ldDCs: CD11c /CD123. The mDCs, lDCs, and ldDCs were expressed as percentages of total DCs. The intensity of HLA-DR expression by each DC subtype was estimated from the mean fluorescence intensity (MFI). Measurement of Absolute Leukocyte and Calculation of DC Numbers Total leukocyte counts and relative percentages of each type of leukocytes were determined from the collected cord blood samples and maternal blood samples. The number of leukocytes was measured by automated cell counter, XE-2100 (Sysmex, Kobe, Japan). Absolute DC counts (cells/lL) were calculated
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LEE ET AL.
as follows: leukocyte count (lL) 9 the percentages of lymphocytes and monocytes 9 the percentage of DCs among the mononuclear cells (from flowcytometry analyses). Statistical Analysis Student’s t-test was used to compare groups, and differences were considered to be statistically significant when the P-value was
Maternal Circulating Dendritic Cell Subtypes at Delivery and During the 1-Year Postpartum Period Hye Ryun Lee1, Byoung Jae Kim2, Sue Shin3,4,5, Hye Won Jeon2, Eun Youn Roh3,4,5, Jong Hyun Yoon3,4,5, Eun Young Song3 1
Department of Laboratory Medicine, Gyeongsang National University Hospital, Jinju, Korea; Department of Obstetrics and Gynecology, Boramae Hospital, Seoul, Korea; 3 Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea; 4 Seoul Metropolitan Government Public Cord Blood Bank (Allcord), Seoul, Korea; 5 Department of Laboratory Medicine, Boramae Hospital, Seoul, Korea 2
Keywords Dendritic cells, mean fluorescence intensity, postpartum Correspondence Sue Shin, Department of Laboratory Medicine, Seoul National University Boramae Hospital, Boramaro 5 gil 20, Dongjak-gu, 156-707, Seoul, Korea. E-mail: [email protected] and Hye Won Jeon, Department of Obstetrics and Gynecology, Seoul National University Boramae Hospital, Boramaro 5 gil 20, Dongjak-gu, 156-707, Seoul, Korea. E-mail: [email protected] Lee HR and Kim BJ contributed equally to this study as co-first authors. Shin S and Jeon HW contributed equally to this study as co-corresponding authors. Submission September 7, 2013; accepted November 18, 2013. Citation Lee HR, Kim BJ, Shin S, Jeon HW, Roh EY, Yoon JH, Song EY. Maternal circulating dendritic cell subtypes at delivery and during the 1-year postpartum period. Am J Reprod Immunol 2014; 71: 210–216
Problem Dendritic cells (DCs) play an important role in maintaining pregnancy by inducing tolerance toward the fetus. Such an immunologic change in the mother should be restored to normal after delivery, but few studies have reported postpartum maternal immune recovery, in terms of the types circulating DCs. Method of study The level of each DC subtype and HLA-DR-positive immunoreactivity of the blood from 29 pregnant women with uncomplicated labor was serially analyzed by flowcytometry at delivery and at 1.5, 6, and 12 months after delivery. DC subtypes were characterized as myeloid, lymphoid, and less differentiated (ldDC). Mean fluorescence intensity (MFI) was evaluated for HLA-DR expression for each DC subtype. Results The total number and the percentage of DCs at delivery were lower than those at 12 months postpartum. The ldDC fractions were significantly higher at delivery and at 1.5 months than at 12 months postpartum. The MFI of HLA-DR expression on ldDCs at delivery was lower than that at 12 months postpartum. The myeloid-to-lymphoid DC ratio did not differ over the 1-year postpartum period. Conclusion The maternal alteration in DCs rapidly normalized within 1.5 months, except for the ldDC fraction, which persisted between 1.5 and 6 months after delivery.
doi:10.1111/aji.12188
Introduction Dendritic cells (DCs) play an important role in the adaptive immune responses to foreign antigens and self-tolerance.1 In addition, DCs play an important 210
role in maintaining pregnancy through immunologic tolerance.2 After delivery, rapid recovery of normal maternal immune function is desirable. In normal pregnancy, the maternal immune system is tolerant to the partially histocompatible fetus. American Journal of Reproductive Immunology 71 (2014) 210–216 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
MATERNAL DENDRITIC CELLS DURING 1-YEAR POSTPARTUM
A shift from T-helper cell type 1 (Th1) to T-helper cell type 2 (Th2) activity is important in immune tolerance during pregnancy.3–5 Different DC subtypes are involved in this shift. The two main DC subtypes include myeloid DCs (mDCs) and lymphoid DCs (lDCs). MDCs express the integrin CD11c, produce interleukin-12, and induce the differentiation of na€ıve T lymphocytes into Th1 cells, whereas lDCs express CD123, produce type 1 interferon (IFN) and IFN-a, and may influence Th1/Th2 differentiation.6,7 Apart from mDCs and lDCs, a third DC subtype that weakly expresses CD123, termed as less differentiated DCs (ldDCs), has been recognized.8 In this study, we investigated the DC subtypes circulating in the maternal blood at the time of delivery and at 1.5, 6, and 12 months after delivery to determine the duration required for immunologic recovery to the prepartum status. During the postpartum period, complete immunologic recovery requires approximately 1 year9; therefore, in this study, we used the values at 12 months after delivery as the baseline reference values for comparison. Materials and methods Study Subjects and Sample Collection In this study, we enrolled 29 pregnant women (median age, 31 years; range, 22–43 years) with uncomplicated full-term labor (gestational week: median, 39 weeks; range, 36–41 weeks) from October 2009 to March 2010. The study was reviewed and approved by the Institutional Review Board of Seoul National University Boramae Hospital (062009-98). Cord blood was collected at delivery, and maternal peripheral blood was collected at delivery and at 1.5, 6, and 12 months postpartum. Cord blood was aseptically collected in utero from the umbilical vein by gravity into a collection bag containing citrate phosphate dextrose adenine-1 as an anticoagulant. Maternal blood was collected in tubes containing acid citrate dextrose-1 at delivery and in tubes containing ethylenediaminetetraacetic acid at 1.5, 6, and 12 months after delivery, within 1 week of the scheduled visit. At each blood draw, all participants were asked whether they experienced any febrile symptoms and their body temperature was assessed. American Journal of Reproductive Immunology 71 (2014) 210–216 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Immunophenotyping of Circulating DC Subtypes Immunophenotyping for DC subtypes from whole blood samples was analyzed as previously described.10,11 Briefly, 50 lL of whole blood was incubated with mouse anti-human CD123-PE, CD11c-APC, HLA-DR-PerCP, and a lineage cocktail 1-fluorescein isothiocyanate (Lin1-FITC) (containing a mixture of FITC-conjugated anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 antibodies) (BD Biosciences, San Jose, CA, USA) for 25 min at room temperature in the dark, according to manufacture instructions. Isotypematched mouse anti-human IgG isotype controls were used for each monoclonal antibody, to assess non-specific antibody binding and determine background fluorescence levels. After antibody incubation, 2 mL of BD FACSTM lysing solution (BD Biosciences) diluted 1:10 in distilled water was added and samples were fixed for another 15 min in the dark. Cells were washed twice and resuspended in 0.5 mL phosphate-buffered saline (PBS), and labeled cells were analyzed with a FACSAriaTM flowcytometer (Becton Dickinson) using FACSDivaTM Software (Becton Dickinson). From each sample, 100,000 events were acquired and analyzed. Among the gated mononuclear cells, DCs were negative for lineage cocktail markers but expressed HLA-DR. Among the DC population, cells expressing CD11c and variable/low CD123 were characterized as mDCs: CD11c+/CD123, whereas CD11cnegative cells expressing CD123 with high intensity were characterized as lDCs: CD11c /CD123++. Cells that were negative for CD11c that expressed low or no CD123 levels were characterized as ldDCs: CD11c /CD123. The mDCs, lDCs, and ldDCs were expressed as percentages of total DCs. The intensity of HLA-DR expression by each DC subtype was estimated from the mean fluorescence intensity (MFI). Measurement of Absolute Leukocyte and Calculation of DC Numbers Total leukocyte counts and relative percentages of each type of leukocytes were determined from the collected cord blood samples and maternal blood samples. The number of leukocytes was measured by automated cell counter, XE-2100 (Sysmex, Kobe, Japan). Absolute DC counts (cells/lL) were calculated
211
LEE ET AL.
as follows: leukocyte count (lL) 9 the percentages of lymphocytes and monocytes 9 the percentage of DCs among the mononuclear cells (from flowcytometry analyses). Statistical Analysis Student’s t-test was used to compare groups, and differences were considered to be statistically significant when the P-value was
