CD4⁺ T helper cells stimulated in response to placental ischemia mediate hypertension during pregnancy

Abstract

We have shown that hypertension in response to chronic placental ischemia is associated with elevated inflammatory cytokines and CD4⁺ T cells. However, it is unknown if these cells play an important role in mediating hypertension in response to placental ischemia. Therefore, we hypothesize that, reduced uterine perfusion pressure, (RUPP) induced CD4⁺T cells increase blood pressure during pregnancy. To answer this question, CD4⁺T cells were isolated from spleens at day 19 of gestation from control normal pregnant and pregnant RUPP rats, cultured, adjusted to 10⁶ cells/100µl saline for intraperitoneal injection into normal pregnant rats at day 13 of gestation. On day 18, in the experimental groups of rats, arterial catheters were inserted, and on day 19 mean arterial pressure (MAP) was analyzed. Inflammatory cytokines and anti-angiogenic factor, soluble fms-like tyrosine kinase, sFlt-1, were determined via enzyme linked immunosorbant assay. MAP increased from 104±2 mmHg in normal pregnant to 124±2 mmHg in RUPP rats (P<0.001) and to 118±1mmHg in rats receiving RUPP CD4⁺T cells (P<0.001). Circulating tumor necrosis factor-alpha and sFlt-1 were elevated in recipients of RUPP CD4⁺T cells to levels similar to control RUPP rats. In contrast, virgin rats injected with normal pregnant or RUPP CD4⁺T cells exhibited no blood pressure changes compared to control virgin rats. Importantly, MAP did not change in recipients of normal pregnant CD4⁺T cells (109 ± 3 mmHg). These data support the hypothesis that reduced uterine perfusion pressure induced CD4⁺ T cells play an important role in the pathophysiology of hypertension in response to placental ischemia.

INTRODUCTION

Preeclampsia is an immune based disease that affects 5–7% of pregnancies in the United States¹, which has been increased by 40% within the last decade². Preeclampsia is characterized by new onset hypertension with proteinuria, immune activation and imbalances between pro-angiogenic (vascular endothelial growth factor/platelet derived growth factor (VEGF/PLGF)) and anti-angiogenic factor, soluble fms-like tyrosine kinase-1(sFlt-1), during pregnancy. The pathophysiologic mechanism(s) underlying this disease are yet to be elucidated^3–5. Previous studies have demonstrated that women with preeclampsia have increased placental and plasma levels of inflammatory cytokines compared to women with normal pregnancies^{6, 7}. Further evidence implicates an imbalance between regulatory T cells (Treg) and effector T cells, subclasses of CD4⁺ T lymphocytes, in preeclamptic women^{8, 9}.

There is considerable evidence for the importance of a subclass of CD4⁺ T lymphocytes known as T helper (Th) 17 cells in the development and progression of chronic inflammatory and autoimmune diseases^8–12. Th17 cells secrete interleukin-17 (IL-17) which is a pro-inflammatory cytokine, strongly associated with allergic phenomena^{11, 12}. IL-17 producing CD4⁺ T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases such as lupus, allograft rejection, autoimmune arthritis, psoriasis and multiple sclerosis and are associated with airway remodeling and potential progression to asthma^{11, 12}. Furthermore, IL-17 induces the synthesis of other cytokines such as interleukin-6 (IL-6), granulocyte colony stimulating factor, tumor necrosis factor-alpha (TNF-α), interleukin-8 and macrophage colony stimulating factor, all of which are associated with preeclampsia and we have previously shown to be associated with hypertension in response to reductions in uterine placental perfusion (RUPP) in pregnant rats^{1–9, 13–17}.

In contrast, Tregs, a subset of CD4⁺ T cells expressing the transcription factor forkhead box P3 (FoxP3), have anti-inflammatory properties and can cause quiescence of autoimmune diseases. T regs have recently been shown to be decreased in preeclamptic women compared to normal pregnant women. In a recent study the frequencies of circulating T regs (FoxP3⁺ CD4⁺Tcells), interferon gamma (IFNγ), interleukin-10 (IL-10), and IL-17 were compared at the end of the third trimester of healthy and preeclamptic pregnancies⁸. Th17 cells and Treg cells were detected by flow cytometry while IL-17 was measured by enzyme linked immunosorbant assay (ELISA). Preeclamptic women showed abnormal ratios of FoxP3⁺ Treg to IL-17-expressing CD4⁺ T cells. The percentage of CD4⁺ IL-17-producing T cells were decreased significantly in normal pregnant women compared with preeclamptic pregnancies⁸. In addition, IL-17 and IFNγ were increased while IL-10 was decreased among preeclamptics compared to normal pregnant women. Although preeclampsia is associated with increased IL-17 and decreased Tregs it is unknown if placental ischemia is a stimulus for the imbalance between Th17 vs. T regs or if there are other maternal or paternal factors leading to the dysregulation of these immune factors. Furthermore it remains undetermined if this dysregulation among CD4⁺ Thelper subtypes lends to the development of hypertension in response to placental ischemia. Therefore this study was designed to test the hypothesis that placental ischemia stimulates a CD4⁺ T helper cell imbalance which contributes to excess pro-inflammatory cytokine production, a shift in angiogenic factors and leads to the development of hypertension during pregnancy.

METHODS

All studies were performed in 250g timed-pregnant Sprague Dawley rats (Harlan, Indianapolis, IN). Animals were housed in a temperature controlled room with a 12:12 light:dark cycle. All experimental procedures in this study were in accordance with the National Institutes of Health guidelines for use and care of animals and were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.

Protocol 1. Effect of placental ischemia on blood pressure and CD4⁺T cell dysregulation

The RUPP procedure reduces the uterine perfusion pressure with the application of a constrictive silver clip (0.203 mm) to the aorta superior to the iliac bifurcation performed while ovarian collateral circulation to the uterus is reduced with restrictive clips (0.100 mm) to the bilateral uterine arcades at the ovarian end^{14, 18}. Under isoflurane anesthesia restrictive clips are implanted on day 14 of gestation, while on day 18 of gestation carotid arterial catheters were inserted for blood pressure measurements. The catheters inserted are V3 tubing (SCI) which is tunneled to the back of the neck and exteriorized. On day 19 of gestation arterial blood pressure was analyzed after placing the rats in individual restraining cages. Arterial pressure was monitored with a pressure transducer (Cobe III Transducer CDX Sema) and recorded continuously after a one hour stabilization period^{14, 18}.

Determination of circulating T lymphocytes

Circulating CD4⁺ T cell populations were measured from peripheral blood leukocytes (PBL) collected at day 19 of gestation from normal pregnant (NP) rats and from pregnant RUPP rats. We utilized flow cytometry analysis to detect specific CD4⁺ T cell populations; CD4⁺FoxP3⁺ and CD4⁺RORγ⁺ (retinoic acid receptor-related organ receptor gamma) isolated from RUPP and NP rats PBLs. Intracellular staining of FoxP3 and RORγ transcription factors were also performed to determine the percent positive cells belonging to the specific subtypes of either regulatory T cells (FoxP3⁺) or Th17 cells (RORγ⁺).

At the time of tissue harvest, plasma was collected and PBLs were isolated from plasma by centrifugation on a cushion of Ficoll-Hypaque (Lymphoprep, Accurate Chemical Corp) according to the manufacturer’s directions. For flow cytometric analysis equal numbers of leukocytes (1 × 10⁶) were incubated for 30 minutes at 4°C with antibodies against mouse CD4 (BD Biosciences, San Jose, CA). After washing, cells were labeled with the secondary fluorescein isothiocyanate (FITC) antibody (Southern Biotech, Birmingham, AL) for 30 minutes at 4°C. Cells were washed and permeabilized according to manufacturer’s directions using the FoxP3 staining kit (eBioscience, San Diego, CA) followed by intracellular staining with anti-mouse/rat FoxP3 conjugated to phycoerythrin (PE) (BD Pharmingen) or anti-rat RORγ conjugated to PE (BD Pharmingen) for 30 min at 4°C. As a negative control, for each individual rat, cells were treated exactly as described above except they were incubated with anti-FITC and anti-PE secondary antibodies alone. Subsequently, cells were washed and resuspended in 500µl of Roswell Park Memorial Institute medium (RPMI) and analyzed for single and double staining on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The percent of positive staining cells above the negative control was collected for each individual rat and mean values for each experimental group (NP and RUPP) was calculated.

Protocol 2: Effect of Adoptive transfer of RUPP CD4⁺ T helper cells on blood pressure in normal pregnant rats

This protocol was designed following that of previous investigators demonstrating an important role for T cells to mediate hypertension in pregnant mice¹⁹ and was therefore performed to determine the importance of the imbalance of CD4⁺ T helper cells stimulated in response to RUPP to mediate hypertension during pregnancy. Spleens from NP and RUPP rats were isolated at the time of sacrifice and immediately placed in ice-cold phosphate buffered saline (PBS), pH 7.0. Spleens were homogenized in culture dishes with RPMI medium containing 10% fetal bovine serum and filtered through a 100µm cell strainer to obtain single cell suspensions. CD4⁺ T lymphocytes were isolated from the splenocytes via magnetic separation using CD4⁺ Dynabeads according to the manufacturer’s recommended protocol (Invitrogen, Carlsbad, CA). Once released from the Dynabeads, CD4⁺ Th1 cells were washed in PBS and cultured in RPMI containing HEPES (25mM), Glutamine (2 mM), Pen/Strep (100U/ml), 1.022 ng/ml interleukin-2 and 4 ng/ml interleukin-12 for 24 hours at 5% CO₂ at 37° C in a humidified atmosphere. Following centrifugation, cell pellets were washed with saline and adjusted to 1 × 10⁶ cells/100 µl saline for injection into recipient normal pregnant rats. Cell culture media was retained for cytokine analysis via ELISA.

Adoptive Transfer

At gestational day 13, 1×10⁶ CD4⁺ T cells/100µl saline (obtained as described above) were administered to normal pregnant rats or virgin rats via intraperitoneal injection. Blood pressure and circulating inflammatory cytokines were compared between the groups from plasma collected on day 19. The groups examined were as follows: NP rats; NP rats injected with NP CD4⁺ T cells (NP + NP CD4⁺ T cells); RUPP rats; NP rats injected with RUPP CD4⁺ T cells (NP + RUPP CD4⁺ T cells); virgin (V); virgin rats receiving NP CD4⁺ T cells (V + NP CD4⁺ T cells) and virgin rats receiving RUPP CD4⁺ T cells (V + RUPP CD4⁺ T cells).

Measurement of arterial pressure in chronically instrumented conscious rats

Arterial pressure was determined in all groups of pregnant rats at day 19 of gestation or six days following injection as previously described in protocol 1 and as we have previously published^{14, 18}.

Determination of cytokine production

Supernatants collected from CD4⁺ T lymphocytes cultured for 24h and plasma from all pregnant rats injected with CD4⁺ T cells were measured for sFlt-1, IL-6 and TNF-α concentrations using commercial ELISA kits available from R&D Systems (Quantikine) according to the manufacturer’s protocol. IL-17 concentrations were measured using commercial ELISA kits available from eBioscience. Sensitivity of ELISAs is reported in the instruction manual provided by the manufacturer. The minimal detectable dose for the rat quantikine IL-6 ELISA was 21pg/ml and maximal detectable dose being 4000 pg/ml with an intra-assay/inter-assay precision of 8.8 and 10% CV respectively. The minimal detectable dose for the TNF-α quantikine ELISA was less than 5pg/ml and maximal being 900 pg/ml with an intra-assay/inter-assayprecision of 5 and 9.7 % CV, respectively. The minimal detectable dose for the sFlt-1 ELISA was 3.8 pg/ml and maximal was 800 pg/ml with an intra-assay/inter-assay precision of 7.2 and 8.2 % CV, respectively. The minimal detectable dose for IL-17 ELISA was 30 pg/ml and maximal was 4000 pg/ml with an intra-assay/inter-assay precision of <10 and <10 % CV, respectively.

Statistical Analysis

All data are expressed as mean ± S.D. Differences between control and experimental groups were analyzed using the Student’s t-test. Differences between multiple groups were analyzed via one-way analysis of variance (ANOVA) and post-hoc analyses were obtained through Bonferroni post hoc test. Values of P < 0.05 were considered significant.

RESULTS

Protocol 1. Effect of placental ischemia on blood pressure and CD4⁺T cell dysregulation

As seen with previous studies¹⁴ animals in the RUPP (n=10) group had significant increases in mean arterial pressure compared to NP rats (n=10) (125±2 RUPP vs. 106±2 mmHg NP; P<0.001; Figure 1A). Figures 1B and C demonstrate that CD4⁺ T lymphocytes are elevated in the circulation of our preeclamptic RUPP pregnant rat model compared to NP rats (46±4% RUPP vs 32±3% NP). In addition, we demonstrate that FoxP3⁺ T regulatory cells have a tendency to decrease and were 47% less in RUPP rats compared to NP rats (.085±1% RUPP vs. 0.178±.2% NP) however this difference did not reach statistical significance with a P value of 0.06, which may be due to difficulty in detection of these cell within the circulation (Figure 2). Furthermore, in Figure 2 we demonstrate that circulating CD4⁺ Th17 cells, identified by expression of the RORγ transcription factor, are significantly elevated in response to placental ischemia during pregnancy compared to NP rats (22±8% RUPP vs. 7±2% NP, P<0.05).

Figure 1. Hypertension in response to placental ischemia is associated with elevated circulating CD4⁺T helper cells.

Panel (A) shows the blood pressure (mmHg) in normal pregnant (NP, n=10) compared to placental ischemic (RUPP, n=10) rats. Panel (B) shows the percent CD4⁺ T cells in freshly isolated peripheral blood leukocytes (PBL) collected from NP and RUPP rats on day 19 of gestation. (*P<0.05 compared to NP). Percent of CD4⁺ staining cells above the negative control was collected for each individual rat and mean values for each experimental group (NP and RUPP) was calculated. Representative NP and RUPP PBL scatter plots and staining profiles are shown in (C). PBL aliquots from NP or RUPP were stained with anti-CD4 followed by a FITC conjugated secondary antibody.

Figure 2. Placental ischemia is a stimulus for CD4⁺ T helper cell imbalance.

Circulating peripheral blood leukocytes (PBLs) isolated from normal pregnant (NP, n=10) and placental ischemic (RUPP, n=10) rats were collected, double stained for CD4, permeabilized and stained intracellularly for FoxP3 or RORγ and analyzed by flow cytometry. Top panel shows the graphed results indicating that regulatory T cells (FoxP3⁺CD4⁺) (A) are decreased and Th17 cells (RORγ⁺CD4⁺) (B) are increased in RUPP compared to NP rats. (*P<0.05 compared to NP). Percent double-positive, CD4⁺FoxP3⁺ and CD4⁺RORγ⁺ staining cells above the negative control was collected for each individual rat respectively and mean values for each experimental group (NP and RUPP) was calculated. Panels (C–D) illustrate representative scatter plots and staining profiles of PBL double stained with either anti-CD4 and anti-FoxP3 or anti-CD4 and anti-RORγ.

CD4⁺ T cell Secretion of TNF-α, IL-6, IL-17 and sFlt-1

CD4⁺ T cells isolated from NP and RUPP spleens were cultured overnight and TNF-α, IL-6, IL-17 and sFlt-1 concentrations were determined from cell culture media. After 24h of incubation, TNF-α secretion from RUPP CD4⁺ T cells was significantly increased compared to NP CD4⁺ T cells (250±50 RUPP vs. 133±23 pg/ml NP) (P<0.05, Figure 3A). Likewise, IL-6 secretion from RUPP CD4⁺ T cells was 778±29 compared to 287±12 pg/ml (P<0.05, Figure 3B) from NP CD4⁺ T cells. IL-17 secretion from RUPP CD4⁺ T cells was significantly increased compared to NP CD4⁺ T cells (714 ± 30 RUPP vs. 245 ±45 pg/ml NP) (P<0.05, Figure 3C), as was sFlt-1 secretion (2500 ± 650 RUPP vs. 1046 ±280 pg/ml NP) (P<0.05, Figure 3D).

Figure 3. Inflammatory and anti-angiogenic factors are secreted from CD4⁺T cells.

ELISA results from supernatant collected from normal pregnant (NP, n=10) and placental ischemic (RUPP, n=10) derived CD4⁺ T cell culture. TNF-α (A), IL-17 (B), and IL-6 (C), and sFlt-1(D). (*P<0.05 compared to NP).

Protocol 2: Effect of Adoptive transfer of RUPP CD4⁺ T helper cells on blood pressure and cytokines in normal pregnant rats

In order to determine a role for the CD4⁺ T cells stimulated in response to placental ischemia to mediate an increase in blood pressure, CD4⁺ T cells were isolated from splenocytes obtained from new groups of NP and RUPP rats utilized in Protocol 2. Mean arterial pressure in RUPP rats (n=15) was elevated; 124±2 mmHg compared to 104±2 mmHg in NP controls (n=13); P<0.001; Figure 4. Adoptive transfer of CD4⁺ T cells from RUPP rats into NP rats (n=18) resulted in significant increases in arterial pressure compared to NP controls (118±1 mmHg; (P<0.001) and compared to 109±3 mmHg in NP recipients of NP CD4⁺ T cells (n=8). Furthermore, neither adoptive transfer of NP CD4⁺ T cells or RUPP CD4⁺ T cells into virgin rats increased blood pressure compared to virgin controls. Blood pressure (MAP) in control virgins was 135±4 mmHg (n=12) compared to 131±5 mmHg in virgin + RUPP CD4⁺ T cells (n=8) and 139±2 mmHg in virgin + NP CD4⁺ Tcells (n=3).

Figure 4. Adoptive transfer of RUPP CD4⁺T cells increases blood pressure in normal pregnant rats.

Blood pressure results (mmHg) in normal pregnant (NP, n=13), placental ischemic (RUPP,n=15), NP+NP CD4⁺T cells (n=8) and NP+RUPP CD4⁺ T cells (n=18) on day 19 of gestation. (*P<0.05 compared to NP).

Effect of Adoptive Transfer on Circulating TNF-α, IL-6, IL-17 and sFlt-1

Circulating cytokines, TNF-α, IL-6, IL-17 and sFlt-1, were measured in plasma collected from control and experimental pregnant rats. We have previously shown that circulating levels of TNF-α, IL-6, IL-17 and sFlt-1 are all increased in RUPP rats compared to NP rats^{17, 19–20}. TNF-α increased significantly in RUPP rats compared to NP rats and increased significantly with adoptive transfer of RUPP CD4⁺ T cells to NP rats (Figure 5A). IL-17 increased in RUPP rats compared to NP rats, but was not elevated to the same degree in NP + RUPP CD4⁺ T cells (Figure 5B). In addition IL-6 increased in RUPP compared to NP, but was not significantly elevated in NP + RUPP CD4⁺ T cell rats (Figure 5C). Circulating sFlt-1 increased significantly in RUPP rats compared to NP rats and increased significantly with adoptive transfer of RUPP CD4⁺ T cells to NP rats. Importantly there was no significant increase with adoptive transfer of NP + NP CD4⁺ T cells (P<0.01) compared to NP rats (Figure 5D). Although IL-6 and IL-17 were elevated in NP + RUPP CD4⁺ Tcell recipient rats compared to NP rats, these cytokines did not reach the level of those seen in control RUPP rats. This may be indicative of the lack of endogenous immune stimulus, such as the original antigen presentation stimulating these cells in response to placental ischemia that is not present in the NP rats that received RUPP T cells. Nonetheless these data may be indicators that although placental ischemic T cells play a role in the hypertension in RUPP rats they are not the sole factor influencing the phenotype seen during preeclampsia. Because adoptive transfer did not increase blood pressure in virgin rats, cytokine analysis was not performed in this study group.

Figure 5. Adoptive transfer of RUPP CD4⁺T cells increases circulating inflammatory cytokines and anti-angiogenic factors in normal pregnant rats.

ELISA results from plasma collected from normal pregnant (NP, n=13), placental ischemic (RUPP,n=15), NP+NP CD4⁺T cells (n=8) and NP+RUPP CD4⁺ T cells (n=18) on day 19 of gestation to measure circulating TNF alpha(A), IL-17 (B), IL-6 (C) and sFlt-1 (D) (*P<0.05 compared to NP).

DISCUSSION

Preeclampsia is associated with immune activation and an imbalance among T lymphocyte regulatory mechanisms with a tendency toward the Th1/Th2 paradigm to play a role in mediating the pathophysiology associated with the disease. Previous studies demonstrated that adoptive transfer of activated Th1-like splenocytes into pregnant mice elicited high blood pressure, proteinuria and an inflammatory response similar to that observed in preeclamptic women⁹. However, the stimulus for T lymphocytes during preeclampsia remains unknown. With this study we demonstrate that placental ischemia is a stimulus for elevated circulating CD4⁺ T lymphocytes during pregnancy (Figure 1). Furthermore, we demonstrated that placental ischemic rats have lower levels, of regulatory CD4⁺FoxP3⁺ T cells and significantly increased autoimmune associated CD4⁺ T helper 17 cells (Figure 2)^8–10. We report that CD4⁺ T cells isolated from placental ischemic rats, as expected secrete elevated levels of inflammatory cytokines, but surprisingly also secrete excess anti-angiogenic factor, sFlt-1 in vitro (Figure 3). To determine if this imbalance among CD4⁺ T cells in response to placental ischemia mediates hypertension, angiogenic imbalance and/or inflammation during pregnancy, we adoptively transferred RUPP CD4⁺ T helper cells into NP and virgin rats. We demonstrate that adoptive transfer of RUPP CD4⁺ T helper cells caused a significant increase in mean arterial pressure as well as circulating inflammatory cytokines, and stimulated the release of sFlt-1 (Figures 4 and 5). However this imbalance in immune cells when injected into virgin rats had no effect on blood pressure.

We have recently demonstrated that TNF-α serves as a stimulus for the anti-angiogenic factor sFlt-1 in pregnant rats²¹. However, the role of immune cells stimulated in response to RUPP to secrete these cytokines, anti-angiogenic factors or mediate blood pressure effects during pregnancy were not examined. In this study we clearly demonstrate a role for CD4⁺ T cells, both in vitro and in vivo, to secrete and/or stimulate sFlt-1 in response to placental ischemia. To our knowledge this is one of the first studies to demonstrate that T lymphocytes stimulated in response to placental ischemia play an important role in mediating the release of this potent anti-angiogenic factor.

Although preeclampsia is associated with altered CD4⁺ T cell ratios, it is unknown if placental ischemia is a stimulus for the imbalance^{8, 10–12, 21}. The percentage of CD4⁺IL-17-producing T cells (Th17 cells) were increased significantly in preeclamptic pregnancies while Tregs (FoxP3⁺ Tcells) were decreased in women exhibiting preeclampsia compared to those with normal pregnancies^8–10. In addition, IL-17 was increased among preeclamptics compared to normal pregnant women¹⁰, IL-17 is a pro-inflammatory cytokine having many immune regulatory functions critical for Th17 cells and is strongly associated with autoimmune diseases such as asthma, lupus and allograft rejection. IL-6 is the principal stimulus switching pro-regulatory signals into a Th17 mediator, thus making IL-6 the most important regulator of self versus non-self immune discrimination^{11, 12}. Figure 2 demonstrates that the autoimmune associated Th17 cells (indicated by intracellular staining of the ROR γ⁺ transcription factor) are elevated in RUPP placental ischemic rats. Although not reaching statistical significance, we demonstrate Treg cells (indicated by intracellular staining of the FoxP3⁺ transcription factor) are lower in RUPP rats compared to NP rats. We also show increased TNF-α, IL-6 and IL-17 in RUPP vs. NP rats. Collectively, these data support the theory that placental ischemia is a stimulus for the loss of self versus non-self regulatory T cell responses during pregnancy. This may be simply due to early rises in IL-6 thus leading to altered Treg/Th17 or alternatively it may be due to upregulation of specific antigen stimulated by placental ischemia. However, neither of these questions was addressed in this study. Nevertheless, we demonstrate a role for placental ischemic stimulated CD4⁺ T helper cells to increase blood pressure, anti-angiogenic factors and TNF-α during pregnancy. Future studies identifying a role for IL-17 and Th17 cells in mediating the pathophysiology of hypertension in response to placental ischemia are planned in our laboratory. These studies will be important to clarify a role of T helper subsets in stimulating autoantibodies and other cytokines that mediate the pathophysiology of preeclampsia.

PERSPECTIVES

Although the data presented in this study demonstrate that CD4⁺ T cells are important in mediating hypertension during pregnancy, there are still a number of unanswered questions in this field of investigation. Despite the significant increase in mean arterial pressure and circulating levels of inflammatory cytokines due to adoptive transfer of CD4⁺ T cells, the role of activated T helper cells and specific subsets in mediating impaired renal hemodynamics, proteinuria or agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and endothelin-1 (ET-1) during pregnancy is unclear. Measurement of circulating AT1-AA and tissue ET-1 from rats in the adoptive transfer model as well as experiments measuring renal hemodynamics and proteinuria in this model will contribute to further defining the pathophysiological role of activated T helper cells in mediating hypertension during pregnancy. In addition studies examining a role for CD4⁺ T cells to mediate endothelial cell activation and dysfunction will be important to understand the affects of T cell activation on the maternal vasculature during pregnancy²². Future studies are designed to further examine the role of placental ischemic induced IL-17 producing Th17 cells to mediate hypertension, ET-1 and AT1-AA during pregnancy. Furthermore, experiments blocking T cell activation or suppressing CD4⁺ T cells in response to placental ischemia are underway in our laboratory. Knowledge gained from these types of studies could lead to better treatment and early diagnostic strategies for women developing preeclampsia.