Changes in lymphocyte subsets during normal pregnancy

Changes in lymphocyte subsets during normal pregnancy

European Journal of Obstetrics & Gynecology and Reproductive Biology 76 (1998) 147–151 Changes in lymphocyte subsets during normal pregnancy *, R. St...

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European Journal of Obstetrics & Gynecology and Reproductive Biology 76 (1998) 147–151

Changes in lymphocyte subsets during normal pregnancy *, R. Strohmeier, M. Stegmuller, ¨ ¨ M. Kuhnert E. Halberstadt Department of Gynecology and Obstetrics, University Hospital Frankfurt /Main, Frankfurt, Germany Received 17 February 1997; received in revised form 18 July 1997; accepted 25 August 1997

Abstract Peripheral blood lymphocytes from healthy women were studied during pregnancy and postnatally, and were compared with lymphocytes from an age-matched non-pregnant control group. Compared with non-pregnant women, the total white cell count was significantly increased at all pregnancies and also post-partum. In pregnancy the absolute number and percentage of T lymphocytes was slightly elevated while almost no changes in B cells were found. No significant changes were found in the percentage of suppressor / cytotoxic (CD8 1 ), of helper / inducer (CD4 1 ) T lymphocytes, nor of CD4 1 / CD8 1 ratio at any stage of pregnancy and puerperium. The most remarkable changes of the immune system occurred in the group of HLA-DR1 and CD56 1 activated T cells. The cell numbers showed a significant increase in the first trimester (,14 weeks) and decreased slightly from stage to stage. Lower values in NK (natural killer) cells and higher levels of IL-2 receptor positive T lymphocytes did not reach significant levels of change.  1998 Elsevier Science Ireland Ltd. Keywords: Pregnancy; Lymphocyte subsets; Flow cytometry; Immune system

1. Introduction To achieve a successful pregnancy, a competent immune system protects the maternal organism from foreign material yet is immunologically tolerant of the semiallograft fetus. Maternal immunologic adaptations are required to downregulate antifetal activities in order to maintain the semiallogenic conceptus. Several theories exist on how the immune system accomplishes these tasks. Downregulation of the cell-mediated immunity in conjunction with an increase in humoral immunity is widely discussed [1,2]. Local immune responses to pregnancy in the uterus or the fetoplacental unit synchronous with changes in the systemic immune system as seen in mice [3], may also occur in man. It is still not completely understood to what extent changes in the cellular immune system contribute to the regulation of the immunophysiology of pregnancy. The circulating number of total white cells, lymphocytes as well as lymphocyte subpopulations were investigated in

*Corresponding author. Tel.: 149 69 63015190.

several studies during normal pregnancy [4–6] and regarding pre-eclampsia [7,8]. Contradictory results concerning changes in number and proportion of lymphocyte subtypes in normal pregnancy make it difficult to include immunological data in the management of abnormal pregnancy. In the present study we are examining peripheral blood lymphocytes (PBL) for expression of phenotypic and activation markers in order to gain more knowledge of the immune profile of normal pregnant women throughout pregnancy and post-partum.

2. Materials and methods

2.1. Patients Twenty-three healthy women (aged 24–44 years; mean 34.7) without any risk factors were studied during pregnancy. The respective numbers of blood samples were taken in different stages of pregnancy as follows: 16 in the first trimester (,14 weeks), 28 in the second trimester (week 14–23), 27 in the third trimester (week 24–35), 21

0301-2115 / 98 / $19.00  1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0301-2115( 97 )00180-2

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in late pregnancy (week 36–termination of pregnancy), and nine within 1 week post-partum. The control group consisted of 23 age-matched non-pregnant healthy women. All blood samples were drawn in the morning, stored at room temperature, and stained within 6 h and measured within 24 h.

2.2. Antibody panels To define and evaluate the light-scatter gate that distinguishes lymphocytes from granulocytes, monocytes, unlysed or nucleated red blood cells and debris, the panel CD45 / CD14 was used. It contained FITC-labeled CD45 for the identification of leucocytes and PE-labeled CD14 for the identification of monocytes. An isotype control reagent was used to set the fluorescence-1 and fluorescence-2 quatrand markers around the unstained (negative) lymphocyte population to establish a boundary between negative and positive cell populations and to estimate non-antigen-specific antibody binding. It contained FITC-labeled IgG 1 and PE-labeled IgG 2 a , murine monoclonal antibodies that react specifically with keyhole limpet hemocyanin, an antigen not present on human leucocytes. FITC-labeled CD3 was used for the identification of T lymphocytes and PE-labeled CD19 for the identification of B lymphocytes. The paired monoclonal antibody conjugates CD4 / CD8 were used to simultaneously characterize helper / inducer and suppressor / cytotoxic lymphocytes. The panel CD3 / HLA-DR was used to enumerate T lymphocytes, DR1 non-T lymphocytes (primarily B lymphocytes), and activated T lymphocytes. It contained FITC-labeled CD3 for the identification of T lymphocytes and PE-labeled Anti-HLA-DR for the identification of DR1 non-T lymphocytes and activated T lymphocytes. The panel CD3 / CD161CD56 was used to identify T and NK lymphocytes. It contained FITC-labeled CD3 to identify T lymphocytes. It also contained PElabeled CD16 and PE-labeled CD56 to identify NK lymphocyte populations as well as T-lymphocyte subsets. FITC-labeled CD25 in combination with PE-labeled CD3 was used for the identification of IL-2 receptor positive T lymphocytes. A brief summery of the antibodies is given in Table 1.

2.3. Flow cytometric analysis Staining and flow cytometric analysis was performed according to the instructions given in the Becton and Dickinson Simultest TM IMK Plus, Catalogue No. 92-0017. Briefly, 100 m l of each sample of anticoagulated (EDTA) whole blood was incubated for 15 min at room temperature with monoclonal antibodies to each surface marker. Two ml of Lysing Solution (Becton and Dickinson) were added and incubated for a further 10 min. The Lysing Solution contained a fixative (15% of formaldehyde). The cells were then washed twice in phosphate-buffered saline (PBS). Two-colour flow cytometry was performed on a FACScan flow cytometer (Becton and Dickinson) with the appropriate filter combinations for the excitation and measurement of PE (phycoerythrin) and FITC (isothiocyanate). A total of 10 4 acquired events were measured for each sample. The results were reported as a percentage of antibody positive cells of total lymphocytes in suspension corrected for non-specific binding by antibody controls. An independent differential white cell count for each sample was performed in the automatic system Multicoulter T-450 (Coulter Electronics, Krefeld, FRG). The resulting data were used to calculate the absolute number of cells for each parameter.

2.4. Statistics The significance of differences between groups was computed with the Kruskal-Wallis analysis of variance with multiple comparison according to Dunn [8]. All statistical analysis was done with the BIAS SoftwareProgram [9].

3. Results Compared with the non-pregnant control group, the total white cell count was significantly increased at all pregnancies and also in the postnatal period (see Table 2). In the first trimester (,14 weeks) of pregnancy the absolute number of white cells was elevated by 40% and

Table 1 Monoclonal antibodies used in the study Antibodies

Main cellular reactivity

IgG 1 -FITC1IgG 2 a -PE Leucogate Anti-CD45-FITC1anti-CD14-PE Anti-CD3-FITC1anti-CD19-PE Anti-CD4-FITC1anti-CD8-PE

Control Lymphocytes, monocytes, granulocytes T and B lymphocytes T helper / inducer cells T cytotoxic / suppressor cells Activated T lymphocytes NK lymphocytes and MHC-independent cytotoxic T lymphocytes IL-2 receptor positive T lymphocytes

Anti-CD3-FITC1anti-HLA-DR-PE Anti-CD3-FITC1anti-CD161CD56-PE Anti-CD25-FITC1anti-CD3-PE

¨ M. Kuhnert et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 76 (1998) 147 – 151

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Table 2 The changes in white cell count (WCC) and in percentage of lymphocytes, monocytes and granulocytes in non-pregnant women, at varying times throughout pregnancy and post-partum

Non-pregnant (n523) I trimester ,14 weeks (n516) II trimester weeks 14–23 (n528) III trimester weeks 24–35 (n527) Late pregnancy week 36–termination (n521) Postnatal within 1 week (n59)

WCC

Lymphocytes

Monocytes

Granulocytes

6.761.2 9.563** 10.562.5**** 10.863.2**** 10.162.8**** 9.762.6*

27.269.1 22.064.5 17.363.9*** 15.464.2**** 18.368.1*** 20.466.3*

4.961.9 4.661.8 4.961.4 4.861.8 5.661.5 5.462.5

68.769.1 73.564.9 77.764.2**** 79.965.1**** 76.068.7** 74.966.2*

Data with * indicate statistically significant differences relative to non-pregnant control values. *P,0.05; **P,0.005; ***P,0.0005; ****P,0.00005.

was even higher later in pregnancy (about 47%). While the percentage of monocytes did not differ significantly from the non-pregnant, the percentage of lymphocytes decreased in the first trimester (,14 weeks) by 19% and further in the course of pregnancy with the lowest value in the third trimester (weeks 24–35). Women in this stage of pregnancy had only 56.6% of lymphocytes of non-pregnant women (see Table 2). The percentage of granulocytes increased during pregnancy with the highest values in the third trimester (weeks 24–35) followed by a slight decrease in late pregnancy. The WCC was still significantly elevated in the post-partum period (P50.02) while the elevated percentage of lymphocytes, monocytes and granulocytes did not reach a significant level in this period. In pregnancy the absolute number as well as the percentage of T lymphocytes (CD3 1 ) was slightly elevated in comparison with non-pregnant subjects (see Table 4), but only the increase in the percentage of T cells in late pregnancy was significant (see Table 3). In this period the percentage of B lymphocytes (CD19 1 ) decreased. But the decrease as well as the elevation in the first trimester (,14 weeks) was not significant. There was no significant change in the percentage of suppressor / cytotoxic (CD8 1 ), of helper / inducer (CD4 1 ) lymphocytes, nor in CD4 1 / CD8 1 ratio, in any stage of pregnancy and puerperium. There was also no difference found in comparing pregnant with non-pregnant women (see Table 3). An increase was seen in absolute numbers of

CD4 1 and CD8 1 lymphocytes in the first trimester (,14 weeks) of pregnancy but the increases were not significant. Subsequently, the CD4 1 and CD8 1 levels were similar in pregnant and non-pregnant subjects (see Table 4). The most remarkable changes of the immune system occurred in the group of HLA-DR activated T lymphocytes. The cell numbers showed an almost three-fold increase in the first trimester (,14 weeks) of pregnancy (P50.001) and decreased slightly from stage to stage remaining significantly higher compared with controls. The level of controls was restored in the post-partum period. Similarly, the number of CD56 1 activated T lymphocytes rose in the first trimester (,14 weeks) and fell slightly in the subsequent stages (see Table 4). Significantly higher values were measured in the percentage in the second trimester (weeks 14–23) (P50.05) and in the third trimester (weeks 24–35) of pregnancy (P50.004) (see Table 3). The increase in cell numbers was significant in the first and second trimester (weeks 14–23) (P50.02) and even more in the third trimester (weeks 24–35) (P50.004) although extremely high differences between individual women were seen. In the course of pregnancy the NK cell (CD16 1 ) level was 20–30% lower in number and slightly lower in percentage than in non-pregnant women. The values stayed lower during pregnancy and recovered during the postpartum period. But the changes were not significant. Higher values in comparison with non-pregnant women

Table 3 Percentages of T and B lymphocytes and their subsets in non-pregnant women, at varying gestational ages and post-partum

T lymphocytes B lymphocytes Suppressor / cytotoxic lymphocytes Helper / inducer lymphocytes Helper / suppressor ratio NK lymphocytes HLA-DR1 activated T lymphocytes CD56 1 and CD3 1 lymphocytes IL-2 receptor1T lymphocytes

Non pregnant (n523)

I trimester ,14 weeks (n516)

II trimester weeks 14–23 (n528)

III trimester weeks 24–35 (n527)

Late pregnancy week 36– termination (n521)

Postnatal within 1 week (n59)

73.066.3 11.064.0 35.765.6 48.065.2 1.4160.4 14.066.3 6.363.8 5.363.3 9.664.8

77.765.0 12.462.9 37.469.9 48.666.9 1.3960.5 10.364.7 12.369.0* 9.967.8 8.965.1

76.864.9 12.064.4 33.768.2 49.968.8 1.660.6 10.464.0 12.168.6* 12.5611.4* 11.063.5

76.166.7 12.163.5 39.0610.2 50.069.1 1.4760.6 12.265.4 12.867.7* 12.969.6* 10.465.0

79.266.7 10.163.5 36.869.0 51.868.4 1.560.5 11.065.4 10.766.1 9.366.7 10.364.5

73.766.4 10.262.7 33.767.5 50.7867.8 1.660.6 15.167.1 6.263.7 5.763.6 11.864.2

Results are given as mean percentages of lymphocytes6S.D. The lymphocyte subsets are calculated as a percentage of total lymphocytes in the lymphocyte acquisition gate corrected for non-lymphocytes. Data with * indicate statistically significant differences relative to non-pregnant control values. *P,0.05.

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Table 4 Absolute cell counts of T and B lymphocytes and their subsets in 1 nl whole blood in non-pregnant women, at varying gestational ages and postnatal

T lymphocytes B lymphocytes Suppressor / cytotoxic lymphocytes Helper / inducer lymphocytes NK lymphocytes HLA-DR1 activated T lymphocytes CD56 1 / CD3 1 lymphocytes IL-2 receptor1T lymphocytes

Non-pregnant (n523)

I trimester ,14 weeks (n516)

II trimester weeks 14–23 (n528)

III trimester weeks 24–35 (n527)

Late pregnancy week 36– termination (n521)

Postnatal within 1 week (n59)

12606338 194674 6206217 8336238 2536170 94645 92670 145658

15956531 247692 7766299 10346378 194695 2606191** 2366215* 1836123

13326271 211694 5836177 8586249 174667 207678* 1866133* 186657

12166305 193666 6146188 8066267 192687 1956113* 1786130 159688

13586453 168695 6456265 8776320 1926117 1906145 1126125 169689

14076469 191669 6226167 9686365 266687 115679 108677 2376132

Results are given as mean number of cells6S.D. Data with * indicate statistically significant differences relative to non-pregnant control values. *P,0.05; **P,0.005.

occurred in the number and percentage of IL-2 receptor positive T lymphocytes (CD25 1 ) in pregnancy and postpartum. Because of the wide range of individual data no statistical significance was reached (see Table 3 and Table 4).

4. Discussion In the present study we have successfully demonstrated an increase in total white cell counts mostly due to an elevated number of granulocytes at all pregnancies and a significant decrease in lymphocytes from the first trimester (,14 weeks) to late pregnancy. No significant changes were found in T and B lymphocytes, suppressor / cytotoxic, nor in helper / inducer T cells. The NK lymphocytes also did not change significantly in number nor in percentage. The main changes in the T cell subsets appear to be a significantly elevated number and percentage of HLA-DR1 activated cells and CD56 1 T cells. The rise in leukocytes during early pregnancy is well documented [4,10], while all changes among lymphocyte subpopulations are still being discussed. MacLean et al. [4] found no change in total lymphocyte count, while Johnstone et al. [5] found a significant reduction in lymphocytes of about 6% in early pregnancy, which is consistent with our findings. In positive agreement with our results Scridama et al. [11] and Sumiyoshi et al. [12] demonstrated unchanged numbers of T lymphocytes but, contradictory results were obtained by Vanderbeeken et al. and Moore et al. [13,14], who found a reduction of T cells in pregnancy. Significantly lower numbers of CD3 1 and CD4 1 T lymphocytes in comparison to non-pregnant subjects were reported [6] as well as singular decrease in CD4 1 T cells [5,11]. However, Sumiyoshi et al. [12] found a reduction in helper / inducer T cells (CD4 1 ) and an increase in suppressor / cytotoxic (CD8 1 ) T cells. The expression and release of IL-2-R is the consequence of the interaction of

the responding cells with an antigen stimulus. As the amount of IL-2-R positive T cells is found to be unchanged in our study as in others [4], we conclude there is neither activation nor suppression regarding the T H 1 cytokine regulation [1] in normal pregnancy. However, Bettin et al. [7] found elevated numbers of IL-2 receptor positive cells in patients with pre-eclampsia. Similarly in our investigations no changes in the circulating number and percentage of NK cells was found by Gregory et al. [15] but they did demonstrate that the cytolytic activity of these cells decreased. Others have reported the presence of a steroid-inducible immune-suppressive protein during pregnancy, which is capable of inhibiting NK cell activity as well as proliferation in a mixed lymphocyte reaction test system [2]. B cell increase as well as unchanged number and activity [16] and a negative regulation of B lymphopoiesis were found in earlier studies [17–19]. A significant increase occurred in our study in HLADR1 and major histocompatibility complex (MHC)-independent cytotoxic T lymphocytes (CD3 1 / CD56 1 ). HLA-DR1 T lymphocytes are known to be elevated in states of immune activation [20], which may be caused by infection [21], or impending rejection in the case of transplant monitoring [22]. Trophoblast antigens come into contact with maternal cells and tissues and are capable of stimulating maternal immune responses to paternal HLA [23], a situation comparable to transplantation, but normally no rejection of the embryo occurs. It remains still unexplained why even higher levels of HLA-DR1 T lymphocytes are not deleterious to the fetus. Very little information is available on the CD3 1 / CD56 1 type of activated T lymphocytes. This phenotype is rare in peripheral blood (|1–5%) but expands enormously in cell culture in the presence of IFN-g, IL-2, monoclonal antibodies against CD3, and IL-1-a. In vitro studies showed that the cells with the highest cytotoxicity against cellular targets expressed both the T cell marker CD3 and the NK cell marker CD56. These cells were derived from

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CD3 1 CD56 2 T cells and had also potent antitumor activity in mice with severe combined immunodeficiency [24]. Except for a decrease in lymphocytes we found no evidence that the healthy pregnant woman is significantly immunocompromised regarding cellular immunity. To overcome problems with contradictory results and wide ranges of individual data, a correlation to the course of individual pregnancy may be helpful. Regarding the major changes in activated T cells we suggest that further investigations should focus on functional activation and / or suppression of the cellular immune system rather than on number and percentage of lymphocyte subpopulations.

Acknowledgements We thank Monika Reißmann for her excellent technical assistance and Angie Green for language advice.

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