Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway

Abstract

The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a(-/-) mice, which lack AT(1A) receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT(1)) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT(1) receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT(1) signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT(1) receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.

Figure 1

Angiotensin binding in mouse spleen. (a) Total [¹²⁵I]angiotensin II binding in a section of normal mouse spleen. (b) [¹²⁵I]Angiotensin II binding in the presence of excess concentrations of the AT₁ receptor antagonist losartan.

Figure 2

[¹²⁵I]Angiotensin II radioligand binding in mouse splenocytes. [¹²⁵I]Angiotensin II radioligand binding in cell suspensions prepared from whole spleen from Agtr1a^+/+ (open bars) and Agtr1a^–/– (filled bars) mice. Total binding is shown in the left, and binding in the presence of the AT₁ receptor antagonist losartan is shown on the right.

Figure 3

AT_1A receptor mRNA expression in cell populations isolated from spleen. AT_1A receptor mRNA expression in spleen assessed by RT-PCR. Total RNA was isolated, and RT-PCR was performed using primers specific for the AT_1A receptor (7). The PCR products were size fractionated on an agarose gel that was stained with ethidium bromide. The 457-bp PCR product is easily seen in samples from isolated splenic T cells (lane 2), B cells (lane 3), and macrophages (lane 4), or unseparated splenocytes (lane 5) from a wild-type mouse. No product was detected in RNA isolated from splenocytes of an AT_1A receptor–deficient mouse (lane 6).

Figure 4

Angiotensin II stimulates splenocyte proliferation. Concentrations of angiotensin II in the range of 0.01–3 μM were added to single-cell suspensions of splenocytes from wild-type (open bars) and Agtr1a^–/– mice (filled bars). Proliferation was assessed 48 hours later as [³H]thymidine incorporation. *P < 0.001 versus 0 μM angiotensin II; **P < 0.0001 versus 0 μM angiotensin II; ^¶P < 0.03 versus Agtr1a^+/+; ^†P < 0.008 versus Agtr1a^+/+; ^‡P < 0.001 versus Agtr1a^+/+.

Figure 5

MLRs and responses to mitogens by splenocytes from Agtr1a^+/+ and Agtr1a^–/– mice. (a) MLR using splenocytes from C57BL/6 (H-2^b) Agtr1a^+/+ and Agtr1a^–/– mice as responders and irradiated splenocytes from (BALB/c × DBA/2)F₁ (H-2^d) mice as stimulators. [³H]Thymidine incorporation is significantly reduced in splenocytes from Agtr1a^–/– mice (filled bars) compared with Agtr1a^+/+ controls (open bars) across a wide range of stimulator concentrations. *P < 0.001 versus Agtr1a^–/–. (b) Proliferative responses in MLR were measured after 3, 4, and 5 days in culture comparing C57BL/6 wild-type (open bars) and Agtr1a^–/– (filled bars) responder cells. *P < 0.04 versus Agtr1a^+/+; ^†P < 0.0001 versus Agtr1a^+/+. (c) The effects of concanavalin A (left) and anti-CD3 antibodies (right) on proliferation of splenocytes isolated from Agtr1a^+/+ (open bars) and Agtr1a^–/– mice (filled bars). Proliferation is once again measured as [³H]thymidine incorporation. *P < 0.01 versus Agtr1a^+/+; ^†P < 0.002 versus Agtr1a^+/+.

Figure 6

The effects of RAS inhibitors on lymphocyte proliferation. A range of concentrations of the ACE inhibitor enalapril or the AT₁ receptor antagonist losartan were added to a 1-way MLR. Proliferation was measured as specific incorporation of [³H]thymidine. Both losartan (open bars) and enalapril (filled bars) caused significant, dose-dependent reductions in proliferation of wild-type splenocytes in MLR. *P < 0.005 versus no drug.

Figure 7

The effects of calcineurin inhibition on angiotensin II–stimulated lymphocyte proliferation. Splenocyte suspensions from C57BL/6 wild-type mice were cultured with 1 μM angiotensin II along with 0.3 or 3 ng/mL cyclosporine, or its vehicle alone; after 24 hours [³H]thymidine incorporation was determined. Baseline proliferation, in the absence of angiotensin II, was similar between the groups: 2,094 ± 125 cpm for vehicle; 1,791 ± 168 cpm for 0.3 ng/mL cyclosporine; and 1,789 ± 245 cpm for 3 ng/mL cyclosporine. *P < 0.02 versus vehicle.

Figure 8

Enhanced effect of cyclosporine to prolong allograft survival in AT_1A receptor-deficient recipients. Heterotropic cardiac allografts were placed in Agtr1a^+/+ (filled symbols) and Agtr1a^–/– (open symbols) mice. Recipient animals received cyclosporine for 7 days at 2 doses, 10 mg/kg per day (diamonds) and 20 mg/kg per day (triangles). These subtherapeutic doses of cyclosporine had no significant effect on graft survival in the Agtr1a^+/+ mice. Cyclosporine caused a modest but significant prolongation of survival in the Agtr1a^–/– animals (P < 0.014 versus wild-type).