The potassium channel KCa3.1 as new therapeutic target for the prevention of obliterative airway disease

Abstract

Background: The calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD.

Methods: Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28.

Results: Subepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes.

Conclusions: Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.

Figure 1. Graft infiltrating cells and systemic cellular immune response

Graft infiltration of F4/80+ macrophages and CD3+ lymphocytes within the subepithelial area on POD5 is shown by immunohistochemistry (A; magnification 400×). Mean numbers of F4/80+ macrophages (N=5 for no medication, N=6 for syngeneic, N=6 for KCa3.1−/−, N=5 for TRAM-34) and CD3+ T cells (N=4 for all groups) are expressed as cells/mm² (B; †p<0.001 vs. no medication). On POD5, Elispot assays revealed attenuated systemic responses of IFN-γ producing Th1 cells (C) and IL-4 producing Th2 cells (D) in the KCa3.1 −/− (N=4) and TRAM-34 (N=5) groups (*p<0.05 vs. no medication; †p<0.001 vs. no medication (N=3), N=3 for syngeneic).

Figure 2. Histopathology, luminal obliteration, and donor-reactive antibodies

Representative cross sections of tracheal grafts on POD 28 stained with Masson-Goldner Trichrome at a magnification 75× are depicted (A). The average percent luminal obliteration is shown (B; *p<0.05 vs. no medication; †p<0.001 vs. no medication; N=7 for no medication and TRAM-34, N=5 for syngeneic, N=8 for KCa3.1 −/−). Mean fluorescence of IgG demonstrates a significant reduction in DSAs in the syngeneic (N=5) and KCa3.1 −/− (N=9) groups (C; p=0.001 and p=0.018, respectively, vs. no medication (n=7); N=7 for TRAM-34).

Figure 3. Airway epithelium

Representative sections of the tracheal epithelia on POD 28, stained with Masson-Goldner trichrome, are shown at a magnification of 400× (A). The respiratory epithelium of the syngeneic group (N=5) is widely preserved (B). The KCa3.1−/− (N=10) and TRAM-34 (N=8) groups show cuboidal or flattened epithelia, whereas the epithelium in the no medication group (N=8) is totally destroyed.

Figure 4. KCa3.1 expression and in vitro proliferation assay

KCa3.1 mRNA-expression in tracheal grafts was analyzed by semi-quantitative RT-PCR (A; *p<0.008; †p<0.001 vs. no medication (N=6), N=4 for syngeneic, N=8 for KCa3.1−/−, N=5 for TRAM-34). Representative stainings for the KCa3.1 channel in tracheal graft are shown at a magnification of 400× (B). The results of the in vitro proliferation assay for WT or KCa3.1−/− splenocytes are shown as [³H]-TdR incorporation normalized to the ConA-stimulated controls (C; *p<0.05 vs.controls).