B2 cells contribute to hypertension and natural killer cell activation possibly via AT1-AA in response to placental ischemia

Abstract

Preeclampsia, new onset hypertension during pregnancy, is associated with activated T helper cells (Th) and B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA). The reduced uterine perfusion pressure (RUPP) model of placental ischemia recapitulates these characteristics. We have shown that Th-B cell communication contributes to AT1-AA and symptoms of preeclampsia in the RUPP rat. B2 cells are classical B cells that communicate with Th cells and are then transformed into memory B cells. We hypothesize that B2 cells cause hypertension, natural killer (NK) cell activation, and complement activation during pregnancy through the production of AT1-AA. To test this hypothesis, total splenic B cells and B2 cells were isolated from normal pregnant (NP) or RUPP rats on gestational day (GD)19 and adoptively transferred into GD12 NP rats. A group of recipient rats was treated with a specific inhibitor peptide of AT1-AA. On GD19, mean arterial pressure was measured, tissues were collected, activated NK cells were measured by flow cytometry, and AT1-AA was measured by cardiomyocyte assay. NP recipients of RUPP B cells or RUPP B2 cells had increased mean arterial pressure, AT1-AA, and circulating activated NK cells compared with recipients of NP B cells. Hypertension in NP recipients of RUPP B cells or RUPP B2 was attenuated with AT1-AA blockade. This study demonstrates that B cells and B2 cells from RUPP rats cause hypertension and increased AT1-AA and NK cell activation in response to placental ischemia during pregnancy.NEW & NOTEWORTHY This study demonstrates that placental ischemia-stimulated B2 cells induce hypertension and circulating natural killer cell activation and angiotensin II type 1 receptor production in normal pregnant rats.

Keywords: B cells; autoantibodies; preeclampsia.

Graphical abstract

Figure 1.

In this study, pregnant Sprague–Dawley rats underwent reduced uterine perfusion pressure (RUPP) surgery on gestational day (GD)14 and carotid catheterization on GD18. On GD19, normal pregnant (NP) and RUPP blood pressure was measured, tissues were collected, and spleens were harvested for B cells and B2 cells. The same day as tissue harvest, isolated B cells were adoptively transferred into GD12 pregnant Sprague–Dawley rats. Some of the rats that received RUPP B cells or RUPP B2 cells underwent mini-osmotic pump surgery to administer “n7aac” peptide to inhibit agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA). All animals underwent carotid catheterization on GD18, and blood pressure was measured and tissues were collected on GD19.

Figure 2.

The gating strategy used for B cells and B2 cells. The purity of B cells was above 80% for total B cell adoptive transfer, and the purity of B cells was ∼98% for adoptive transfer. Lymphocytes were gated in a forward (FSC) and side scatter (SSC) plot, and then doublets were excluded. Singlet lymphocytes were then gated using fluorescence minus one (FMO) controls. Cells were gated using CD3 (VioBlue), CD68 (PE-Vio770), CD45R (APC-Vio770), and CD43 (FITC). B cells were considered CD3⁻CD68⁻CD45R⁺ cells; B2 lymphocytes were considered CD3⁻CD68⁻CD45R⁺CD43⁻ cells.

Figure 3.

Mean arterial pressure (MAP) was increased in response to reduced uterine perfusion pressure (RUPP; n = 15) compared with the normal pregnant (NP) group (n = 11, P < 0.0001), as previously described. Adoptive transfer of NP B cells (n = 6) did not significantly increase MAP, but adoptive transfer of RUPP B cells (n = 10) significantly increased MAP (P < 0.001) compared with recipients of NP B cells. Infusion of agonistic autoantibodies against the angiotensin II type 1 receptor-inhibiting “n7aac” peptide (7AA; n = 6) significantly reduced MAP in recipients of RUPP B cells (P < 0.01). Adoptive transfer of RUPP B2 cells (n = 12) significantly increased MAP compared with recipients of NP B cells (P < 0.05), and infusion of n7aac peptide (n = 6) attenuated the increase in MAP associated with adoptive transfer of B2 cells (P < 0.01). Data were compared using one-way ANOVA and are reported as means ± SE. *Statistically significant (P < 0.05) change compared with the referenced group.

Figure 4.

The reduced uterine perfusion pressure (RUPP; n = 15) procedure significantly reduced fetal weight (P < 0.01; A) and placental efficiency (P < 0.05; C) compared with the normal pregnant (NP) group (n = 11), as previously described. Adoptive transfer of NP B cells (n = 7) significantly increased fetal weight compared with the NP group (P < 0.05) without affecting placental efficiency. Adoptive transfer of neither RUPP B cells (n = 10) nor RUPP B2 cells (n = 10) changed pup weight or placental efficiency. There were no changes in placental weight (B) associated with the RUPP procedure or with B cell adoptive transfer. The RUPP (n = 15) procedure significantly reduced placental efficiency compared with the NP group (n = 11, P < 0.05). There were no changes in placental efficiency seen in response to B cell adoptive transfer. Data were compared using one-way ANOVA and are reported as means ± SE. 7AA, n7aac peptide. *Statistically significant (P < 0.05) change compared with the referenced group.

Figure 5.

There were no changes in total circulating natural killer (NK) cells (A) or total placental NK cells (B) in any of the adoptive transfer groups (n = 5–11). Adoptive transfer of normal pregnant (NP) B cells into NP rats did not change NK cell activity in the circulation (C) or in the placenta (n = 4) (D). Adoptive transfer of reduced uterine perfusion pressure (RUPP) B cells into NP rats significantly increased cytolytic NK cells in the circulation (P < 0.01) and placentas (P < 0.01) compared with either NP or NP + NP B cell rats (n = 6 or 7). Adoptive transfer of RUPP B2 cells into NP rats increased cytolytic NK cells in the circulation (P < 0.05) compared with NP rats (n = 7-8). Data were compared using one-way ANOVA and are reported as means ± SE. *Statistically significant (P < 0.05) change compared with the referenced group.

Figure 6.

The reduced uterine perfusion pressure (RUPP; n = 4) procedure significantly increased circulating agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA; P < 0.001) compared with the normal pregnant (NP) group (n = 4), as previously described. Adoptive transfer of NP B cells into NP rats (n = 4) did not increase circulating AT1-AA compared with NP rats. Adoptive transfer of either RUPP B cells (n = 6, P < 0.001) or RUPP B2 cells (n = 4, P < 0.001) into NP rats significantly increased serum AT1-AA levels compared with NP + NP B cell rats. Data were compared using one-way ANOVA and are reported as means ± SE. *Statistically significant (P < 0.05) change compared with the referenced group.

Figure 7.

A: the reduced uterine perfusion pressure (RUPP; n = 5) procedure significantly increased circulating complement component C3 (P < 0.05) compared with the normal pregnant (NP) group (n = 5). Adoptive transfer of NP B cells (n = 5), RUPP B cells (n = 6), or RUPP B2 cells (n = 5) did not change circulating complement component C3. B: the RUPP (n = 5) procedure significantly increased circulating complement component C3a (P < 0.05) compared with the NP group (n = 7). Adoptive transfer of NP B cells (n = 5), RUPP B cells (n = 6), or RUPP B2 cells (n = 5) did not change circulating complement component C3a. Data were compared using one-way ANOVA and are reported as means ± SE. 7AA, n7aac peptide. *Statistically significant (P < 0.05) change compared with the referenced group.

Figure 8.

A: the reduced uterine perfusion pressure (RUPP; n = 5) had no change in renal C1q deposition compared with the normal pregnant (NP) group (n = 5). NP + NP B cells (n = 5), NP + RUPP B cells (n = 6), and NP + RUPP B2 cells (n = 6) had decreased renal C1q compared with the NP group (P < 0.05). There was no change in renal C3a between NP (n = 5) and RUPP (n = 5) groups. B: there was no change in renal C3a between NP (n = 5) and RUPP (n = 5) groups. There was no change associated with adoptive transfer of RUPP B cells (n = 6), but adoptive transfer of RUPP B2 cells (n = 6) significantly increased renal C3a (P < 0.05) compared with recipients of NP B cells (n = 5). C: there was no change in renal perforin in any of the groups (n = 4-6). D: the RUPP group (n = 4) had significantly increased granzyme A (P < 0.05) compared with the NP group (n = 4). Adoptive transfer of RUPP B cells (n = 6) or RUPP B2 cells (n = 6) significantly increased renal granzyme A (P < 0.05) compared with recipients of NP B cells (n = 5). E: the RUPP (n = 4) procedure significantly increased granzyme B (P < 0.05) compared with the NP group (n = 4). Adoptive transfer of RUPP B cells (n = 6) or RUPP B2 cells (n = 6) significantly increased renal granzyme B (P < 0.05) compared with recipients of NP B cells (n = 5). F: the RUPP (n = 4) procedure significantly increased renal granzyme K (P < 0.05) compared with the NP group (n = 5). There was no change associated with adoptive transfer of NP B cells (n = 5), RUPP B cells (n = 6), or RUPP B2 cells (n = 6). Data were compared using one-way ANOVA and are reported as means ± SE. 7AA, n7aac peptide. *Statistically significant (P < 0.05) change compared with the referenced group.