Figure 4.

Rats with pulmonary hypertension (PH) circulate autoantibodies. (A) Control (CTL) lung sections incubated with plasma from normal (no PH) rats is not bound by sequentially applied Alexa 488–conjugated antirat IgG. In contrast, control lung sections incubated with plasma from rats with monocrotaline (MCT) induced PH can be bound by antirat IgG (B, green signal indicated by yellow arrows) indicating that the PH plasma contains IgG that bind self-antigens (autoantibodies). The inset in B shows resistance sized vessels (50–500 μm internal diameter) are also labeled with autoantibodies in PH plasma. (C and D) Lung sections from rats with hypoxic PH or MCT-PH incubated with control plasmas lack autoantibody binding. MCT-PH lung sections incubated with fluor-labeled phosphatidyl inositol 3 kinase (Pi3K) or vimentin (E, red signal, yellow arrows) indicate the presence of autoantibodies in situ. (F) Lung sections from MCT rats, but not control rats, demonstrate the presence of rat IgGs (green) localized to regions with CD45RA⁺ B cells (red). (G) Plasma IgG titers increase in MCT-PH rats compared with control rats, peaking at Week 4 and correlating with increasing mean pulmonary artery (PA) pressure. (H) Immunoblots using rat plasma as the primary antibody reveal autoimmune epitope spreading. Compared with the lack of autoantibodies in control rat plasma, MCT-PH plasma contains autoantibodies that increasingly recognize a wider array of tissue lysates, peaking at 4 weeks post-MCT. Representative images taken from lung sections from n = 6 animals per group, with two to three sections per rat from three independent experiments. Bars = 20 μm. ELISAs were performed in three separate experiments in triplicate on rat plasmas from n = 3 rats per group. Immunoblots shown are representative of tissue lysates run in three separate experiments. Blotting used pools (n = 3) of indicated rat plasmas, performed independently using three separate pools.