Prevalence of anti-endothelial cell antibodies in idiopathic pulmonary arterial hypertension

To the Editors:

Pulmonary arterial hypertension (PAH) is a rare disease often resulting in right-sided heart failure and premature death 1. PAH is idiopathic (IPAH), heritable, or related to conditions such as connective tissue diseases (CTD) 2. IPAH prognosis remains poor despite improved patient survival with current treatment options. Therefore, elucidating the pathophysiology of IPAH is important for the discovery of novel therapeutic approaches. Inflammation and immune reactivity have been implicated in the pathophysiology of IPAH. In 2005, Tamby et al. 3 described the presence of anti-endothelial cell antibodies (AECA) in IPAH, pointing at the involvement of humoral immunity. AECA are a heterogeneous family of auto-antibodies capable of reacting with different endothelial cell (EC) structures 4. In PAH, pulmonary EC dysfunction is considered a key player in the initiation and progression of the disease 5. Systemic sclerosis (SSc) is a paradigm of autoimmune PAH, as 10–15% of these patients develop PAH 6. Interestingly, in SSc, immunoglobuliln (Ig)G AECA targeting cell-surface antigens have indeed been shown to induce EC dysfunction 7, 8. Thus, IgG AECA-induced endothelial dysfunction may be the initiating event in IPAH. Whereas IgG auto-antibodies are considered pathogenic in various autoimmune diseases, IgM auto-antibodies have been proposed to be protective in these diseases 9.

Regarding a pathophysiological role of AECA, it is important to identify whether AECA are reactive with EC-surface antigens. To corroborate and further investigate the role of the humoral immune system in IPAH, we aimed to study the prevalence of IgG, as well as IgM AECA targeting cell-surface EC antigens using a cell-based ELISA with viable human umbilical vein EC (HUVEC).

We screened five study cohorts for the presence of AECA. 1) 29 IPAH patients; mean pulmonary artery pressure (P̄_pa) 45.5 mmHg as measured by right heart catheterisation. 2) 18 CTD-associated PAH patients (14 SSc, two systemic lupus erythematosus and two mixed connective tissue disease); P̄_pa 42.5 mmHg as measured by right heart catheterisation; total lung capacity (TLC) >70% of predicted. 3) 51 SSc patients without PAH (nine diffuse SSc and 42 limited SSc); right systolic ventricular pressure <35 mmHg, as estimated by echocardiography; TLC >70% pred. 4) 40 type II diabetes mellitus (DM II) patients with vascular involvement. 5) 40 healthy individuals. The CTD-associated PAH patients and SSc patients without PAH were included as a comparison cohort with known immunopathological involvement. DM II patients served as controls with known vascular dysfunction. The healthy individuals were included to determine optimal cut-off values for AECA positivity. All subjects gave their informed consent prior to participation. Age was similar in all five study cohorts (p = 0.18). Previous use of immunosuppressive drugs occurred in four out of 18 CTD-associated PAH patients as compared with 14 out of 51 in SSc and zero out of 29 in IPAH patients.

HUVEC isolation and culture was carried out as previously described 10. Experiments were executed with confluent HUVEC monolayers between passages 3 and 5. HUVEC monolayers displayed the typical cobblestone morphology of ECs. HUVEC were phenotypically characterised as ECs by flow cytometry: CD31+ (96.5%), CD146+ (98.6%), Ulex europaeus lectin positive (99.3%), von Willebrand factor positive (98.9%) and CD90- (0.3%).

A cyto-ELISA with unfixed HUVEC was performed to detect AECA specifically targeting EC-surface antigens 10. Experiments were performed three times in triplicate. Cut-off points based on the optical density (OD) values from the IPAH cohort compared to the healthy individuals were calculated using receiver operating characteristic curve analysis. Sera were considered positive for IgG and IgM AECA if the OD value was >0.322 and 0.137, respectively. The intra- and inter-assay coefficients of variation for the HUVEC ELISA were 5.15% and 19.89%, respectively. The mean kappa concordance value of the HUVEC ELISA was 0.87 (p<0.0001).

IgG AECA prevalence in the IPAH cohort was 62.1% (18 out of 29) compared to 77.8% (14 out of 18) in the CTD-associated PAH and 43.1% (23 out of 51) in the SSc without PAH cohorts (table 1⇓). IgG AECA prevalence in the IPAH cohort did not differ significantly from that of the CTD-associated PAH and SSc without PAH cohorts (p = 0.261 and p = 0.104, respectively). Of the 18 patients with CTD-associated PAH, 14 were diagnosed with SSc. The prevalence of IgG AECA in these 14 SSc associated PAH patients was 71.4% (10 out of 14). IgG AECA prevalence in the IPAH and CTD-associated PAH cohorts was significantly higher compared to the DM II cohort (p<0.0001 and p<0.0001, respectively) and the healthy individuals (p = 0.015 and p = 0.001, respectively).

AECA levels of the SSc-associated PAH cohort were significantly higher in comparison to SSc patients without PAH (p = 0.011). The use of immunosuppressive drugs had no significant impact on IgG AECA levels.

IgM AECA prevalence in the IPAH cohort was 44.8% (13 out of 29) compared to 61.1% (11 out of 18) in the CTD-associated PAH cohort and 41.2% (21 out of 51) in the SSc without PAH cohort (table 1⇑). IgM AECA prevalence in the IPAH cohort did not differ significantly from that of the CTD-associated PAH and SSc without PAH cohorts (p = 0.278 and p = 0.751, respectively). IgM AECA prevalence in the IPAH and CTD-associated PAH cohorts was significantly higher in comparison to the DM II cohort (p = 0.014 and p = 0.001, respectively) and the healthy individuals (p = 0.027 and p = 0.002, respectively).

Again, the use of immunosuppressive drugs had no significant impact on IgM AECA levels.

In the IPAH cohort the IgG/IgM ratio correlated with pulmonary vascular resistance (R = 0.433, p = 0.039) and correlated inversely with cardiac index (R =  -0.420, p = 0.046). Such a correlation could not be demonstrated in the CTD-associated PAH cohort.

AECA are present in patients with established PAH. Our data corroborate the findings described by Tamby et al. 3. Moreover, by using a cell-based ELISA with viable ECs, we detected functionally relevant AECA able to target cell-surface antigens. Dysfunction of pulmonary ECs is considered pivotal in IPAH pathogenesis. The initiating stimulus or injury resulting in EC dysfunction, however, is still a matter of speculation. We hypothesise that an autoimmune attack by IgG AECA may be the initial trigger leading to EC dysfunction. However, IgG AECA was not found in all PAH patients. There may be several explanations for this. First, the phenotype expressed by the EC is of crucial importance. Ideally, patients’ own pulmonary ECs should be used to detect AECA. These ECs may harbour the unique endothelial epitope phenotype influenced by individual factors, such as shear stress, viral infections and pulmonary inflammatory characteristics (e.g. cytokine profile). To better mimic the pulmonary environment we also determined the prevalence of IgG AECA on normal human pulmonary artery ECs. No difference in AECA prevalence was found compared to the results obtained by the HUVEC ELISA (data not shown). Secondly, some PAH patients were treated with immunosuppressants that may have suppressed AECA levels. However, no statistically significant difference was observed between AECA levels of treated and untreated patients.

It may be questioned whether AECA in IPAH play a primary pathogenic role or whether they arise as a consequence of EC damage. The observation of low prevalence of IgG AECA in our diabetic control group with established vascular damage supports a primary role for IgG AECA in IPAH. The higher IgG AECA prevalence in SSc patients with PAH compared to SSc patients without PAH also supports their pathogenic role. The role of IgM AECA is also unclear. IgM AECA prevalence in IPAH, CTD-associated PAH, and SSc without PAH did not differ. However, the finding that the IgG/IgM AECA ratio correlates with severity of disease in the IPAH cohort suggests that IgM AECA may be protective, for instance by binding to relevant endothelial epitopes, thus hindering IgG binding.

The mechanisms by which IgG AECA might lead to EC dysfunction are also incompletely understood. In SSc, which is considered a paradigm of autoimmune PAH, AECA targeting EC-surface antigens have been shown to upregulate adhesion molecule expression and to induce EC apoptosis 7, 8. Moreover, it is possible that downstream signalling may have an impact on adjacent pulmonary fibroblasts. However, our study does not address the functional aspects of IgG AECA. Therefore, further studies to define putative pathogenic potentials of IgG AECA in IPAH are required.

In conclusion, we report the presence of IgM and IgG AECA specifically targeting EC-surface antigens in IPAH, further corroborating the involvement of autoimmune processes in the pathophysiology of this disease.

Special thanks go to H. Bilo (Dept of Internal Medicine/Diabetes Centre, Isala Clinic, Zwolle, the Netherlands) who provided the serum samples from the diabetes mellitus type II patients.

Statement of interest

Statements of interest for J. Damoiseaux and for the study itself can be found at www.erj.ersjournals.com/misc/statements.dtl