Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor

Abstract

Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. We investigated 25 preeclamptic patients and compared them with 12 normotensive pregnant women and 10 pregnant patients with essential hypertension. Antibodies were detected by the chronotropic responses to AT1 receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Immunoglobulin from all preeclamptic patients stimulated the AT1 receptor, whereas immunoglobulin from controls had no effect. The increased autoimmune activity decreased after delivery. Affinity-column purification and anti-human IgG and IgM antibody exposure implicated an IgG antibody directed at the AT1 receptor. Peptides corresponding to sites on the AT1 receptor's second extracellular loop abolished the stimulatory effect. Western blotting with purified patient IgG and a commercially obtained AT1 receptor antibody produced bands of identical molecular weight. Furthermore, confocal microscopy of vascular smooth muscle cells showed colocalization of purified patient IgG and AT1 receptor antibody. The protein kinase C (PKC) inhibitor calphostin C prevented the stimulatory effect. Our results suggest that preeclamptic patients develop stimulatory autoantibodies against the second extracellular AT1 receptor loop. The effect appears to be PKC-mediated. These novel autoantibodies may participate in the angiotensin II-induced vascular lesions in these patients.

Figures

Figure 1

Increase in beat number of spontaneously beating neonatal rat cardiomyocytes when exposed to immunoglobulin from 25 patients with preeclampsia, 7 patients after delivery, and 12 patients with normal pregnancy. Nulliparous patients are shown with filled circles, while multiparous patients are designated with an x. Ten pregnant patients with essential hypertension were studied; five (open circles) had immunoglobulins that increased the beating rate. This increase was abolished by prazosin, implying involvement of the α1-adrenoceptor in these five patients. **Different from preceding group (P < 0.01).

Figure 2

(a) Dose–response relationship between Ang II concentrations and spontaneous beating rate. A plateau was observed after 0.1 μM. (b) Dose–response relationship of immunoglobulin fractions from two preeclamptic women (each measured 10 times) on the spontaneous beating rate. Dilutions from 1:20 to 1:400 were prepared. A linear response from 1:400 to 1:40 was observed, after which a maximum effect was observed. Ang II, angiotensin II.

Figure 3

Increase in beat number induced by immunoglobulin from patients with preeclampsia, complete inhibition of the response by losartan (five patients; open bars), and additional inhibition by prazosin (five patients; hatched bars). The responses indicate primary involvement of the AT1 receptor and an additional response involving the α1 adrenoceptor. The specificity of the AT1 response is shown, in that the AT2 receptor blocker PD 123319 had no effect on any of the responses.

Figure 4

Increase in beat number induced by immunoglobulin from patients with preeclampsia, the response after preexposure of the immunoglobulin to a peptide consisting of the second extracellular loop of the AT1 receptor, and additional inhibition by prazosin. The response indicates inhibition of antibody directed against the AT1 receptor by the peptide, thereby verifying the specificity of the response. We included a control peptide consisting of the second extracellular loop of the β1-adrenergic receptor. This peptide had no effect on the spontaneous beating rate.

Figure 5

Short, overlapping peptides corresponding to the second extracellular loop of the AT1 receptor were prepared. These peptides were exposed to affinity-purified antibody preparations. We tested the effect on the spontaneous beating rate of neonatal rat cardiomyocytes. The sequence AFHYESQ abolished the response.

Figure 6

Western blot (representative of 4 experiments) of vascular smooth muscle cell (VSMC) extract. Column (a) shows bands (arrows) produced by a commercial antibody directed against the AT1 receptor. Column (b) shows bands produced by column-purified IgG from preeclamptic patients. Column (c) shows identically handled column-purified IgG from nonpreeclamptic pregnant patients. Two bands are probably the result of glycosylated and nonglycosylated forms of the receptor.

Figure 7

Confocal photomicrograph (representative of four experiments) of VSMCs exposed to affinity column–purified IgG from preeclamptic patient serum and commercially available AT1 receptor antibody. (a) A Cy3-labeled anti–human IgG antibody produced the red staining (left). The same cells were simultaneously exposed to commercially available AT1 receptor antibody (center) with a Cy2-labeled secondary anti–human IgG antibody. Superimposition (right) revealed that these antibodies colocalize (yellow). (b) The same procedure with IgG fraction from a pregnant nonpreeclamptic patient. Only staining from the commercially available AT1 receptor antibody is seen.

Figure 8

Increase in beat number induced by immunoglobulin from four patients with preeclampsia followed by protein kinase C inhibition with calphostin C. Calphostin C resulted in elimination of the response in 30 min.