Inhibition of terminal complement complex formation alleviates murine antibody-mediated TRALI

Abstract

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-triggered mortality. Recently, we demonstrated the critical role of Fc-dependent complement activation in anti-CD36-mediated murine TRALI. In this study, we found that C5-/- mice were protected, and administration of anti-C5 rescued wild-type mice from anti-CD36-mediated TRALI. However, C5aR1-/- mice were not protected against anti-CD36-mediated TRALI, implying a possible role of C5b-9 (membrane attack complex [MAC]). Accordingly, elevated levels of MAC were detected in bronchoalveolar lavage fluid and lung tissue of mice with anti-CD36-induced TRALI. Inhibition of MAC formation by administration of anti-C7-blocking monoclonal antibody alleviated TRALI in mice, suggesting the critical role of the MAC in the pathology of anti-CD36-mediated TRALI. Furthermore, anti-C7 treatment also led to favorable outcome in murine TRALI induced by anti-major histocompatibility complex class 1, indicating the potential broader applicability of MAC inhibitors in the treatment of antibody-mediated TRALI. Therefore, this approach may be promising to further explore the treatment of patients with TRALI.

Graphical abstract

Figure 1.

Analysis of anti-CD36–mediated TRALI in wild-type, C5^−/−, C5aR1^−/−, and wild-type mice receiving C5aR1 or C5aR2 agonist. Wild-type, C5^−/−, and C5aR1^−/− C57BL/6J male mice were treated with different reagents as indicated. The signs of TRALI including rectal temperatures and lung W/D weight ratios changes in wild-type mice (A-B), C5^−/− mice (C-D), C5aR1^−/− mice (E-F), and wild-type mice receiving C5aR1 agonist (PMX53) or C5aR2 agonist (P32) (G-H) are presented. In the control experiment (A-B), untreated wild-type mice (naïve), or treated with LPS, with LPS alone, or LPS and afterward isotype control or mAb GZ1 (0.4 mg/kg) were shown (n = 5 in each cohort). Similar experiments were performed with C5^−/− mice (C-D). In addition, rectal temperatures and lung W/D weight ratios of C5^−/− mice supplemented with C5a (100 ng) and C5a together with mAb GZ1 (0.4 mg/kg) are shown (n = 5 in each cohort). Similar experiments as wild-type mice (see earlier) were performed with C5aR1^−/− mice (E-F). (G-H) Rectal temperatures and lung W/D weight ratios of LPS-treated wild-type mice receiving either mAb GZ1 (n = 10), GZ1 together with PMX53 (10 mg/kg; n = 5), or (P32: 3 mg/kg; n = 5) or isotype control (n = 5). Rectal temperatures could be measured in only 9 of 10 LPS + GZ1–treated mice (1 mouse died within 30 minutes) and in 3 of 5 LPS + GZ1 + PMX53–treated mice (2 mice died within 30 minutes). Statistical analysis was performed with 1-way analysis of variance with Bonferroni correction for multiple comparisons. Each dot represents 1 mouse and error bars represent the standard deviations. ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05. ns, nonsignificant.

Figure 2.

TRALI induced by anti-CD36 led to upregulation of C5b-9 in the lungs and could be inhibited by administration of anti-C7. (A) Immunohistochemical detection of C5b-9 (MAC) in mice lung tissue from untreated wild-type mice (naïve) compared with LPS pretreated and received afterward mAb GZ1 (0.4 mg/kg) or isotype control, and pretreated with anti-C7 before GZ1 (n = 5 in each group). Lung tissue sections were stained with rabbit anti-C5b-9 (top panel) or normal rabbit IgG (normal IgG control, bottom panel), and images were taken at ×80 original magnification. Representative images from each indicated group are shown. Scale bars, 25 μm. (B) C5b-9 concentration in BALF of the indicated mouse groups were measured by enzyme-linked immunosorbent assay. In the prophylactic approach, anti-C7 (dose: 1 mg/200 μL in PBS; n = 5), or anti-C7 F(ab′)₂ (dose: 1 mg/200 μL in PBS; n = 5) was administered before TRALI induction with mAb GZ1 (0.4 mg/kg). In the therapeutic approach, TRALI was first induced with mAb GZ1 (0.4 mg/kg), then mice were treated with anti-C7 (dose: 1 mg/200 μL in PBS; n = 10), or anti-C7 F(ab′)₂ (dose: 1 mg/200 μL in PBS; n = 10) after TRALI. PBS was treated as a control. Rectal temperatures (C) and lung W/D weight ratios (D) were measured as described earlier. Statistical analysis was performed with 1-way analysis of variance with Bonferroni correction for multiple comparisons (B-D). Each dot represents 1 mouse and error bars represent the standard deviations. ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05. ns, nonsignificant.

Figure 3.

Anti-C7 inhibited anti-MHC 1–induced murine TRALI. Wild-type BALB/c male mice were treated with mAbs against MHC 1 (anti-MHC 1: mAb 34-1-2S) for TRALI induction as indicated. In the prophylactic approach, anti-C7 (dose: 1 mg/200 μL in PBS) was injected before TRALI induction with mAb 34-1-2S (4.5 mg/kg). In the therapeutic approach, TRALI was first induced with mAb 34-1-2S (4.5 mg/kg), then treated with anti-C7 (dose: 1 mg/200 μL in PBS) after TRALI. PBS was treated as a control. Rectal temperatures (A) and lung W/D weight ratios (B) were measured as described earlier (n = 5 in each cohort). Statistical analysis was performed with 1-way analysis of variance with Bonferroni correction for multiple comparisons. Each dot represents 1 mouse and error bars represent the standard deviations. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01.