Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy

Abstract

Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.

Keywords: amphotericin B; blood cell count; blood pressure; cobra venom factor; complement activation; infusion reactions; thromboxane; zymosan.

Figure 1

Effects of direct complement activators, amphotericin B-containing and empty liposomes, on the complement system in anesthetized rats. Complement activation was assessed as complement C3 consumption expressed as percentage decreases in C3 concentration relative to the baseline (time 0). (A) Effects of direct complement activators, zymosan and cobra venom factor (CVF); (B) Effects of the amphotericin B-containing liposome AmBisome applied at 2 different doses. (C) Effects of empty liposomes, AmBisombo and PEG-2000-chol. The data are mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.

Figure 2

Blood pressure changes following in vivo administration of direct complement activators, amphotericin B-containing liposomes and empty vesicles. The doses and number of animals are specified in the keys; MABP, mean arterial blood pressure. (A) Effects of zymosan and cobra venom factor (CVF); (B) AmBisome at two doses; and (C) empty liposomes (AmBisombo at 2 doses and PEG-2000-chol). (D) Correlation between the lowest MABP and C3 consumption at the same time, including the data with CVF, zymosan, and AmBisome at both doses. Except for saline, CVF, and AmBisombo, the data in [17] were replotted with permission of the publisher. The data show percentages of change relative to baseline (t = 0 min), mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.

Figure 2

Blood pressure changes following in vivo administration of direct complement activators, amphotericin B-containing liposomes and empty vesicles. The doses and number of animals are specified in the keys; MABP, mean arterial blood pressure. (A) Effects of zymosan and cobra venom factor (CVF); (B) AmBisome at two doses; and (C) empty liposomes (AmBisombo at 2 doses and PEG-2000-chol). (D) Correlation between the lowest MABP and C3 consumption at the same time, including the data with CVF, zymosan, and AmBisome at both doses. Except for saline, CVF, and AmBisombo, the data in [17] were replotted with permission of the publisher. The data show percentages of change relative to baseline (t = 0 min), mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.

Figure 3

Effects of direct complement activators (A), amphotericin B-containing (B), and empty liposomes (C) on plasma platelet (PLT) counts in anesthetized rats. The doses and number of animals are specified in the keys. The data show percentages of change relative to baseline (t = 0 min), mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.

Figure 4

Effects of direct complement activators (A), amphotericin B-containing (B), and empty liposomes (C) on plasma white blood cell (WBC) counts in anesthetized rats. The doses and number of animals are specified in the keys. The data show percentages of change relative to baseline (t = 0 min), mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.

Figure 5

Effects of direct complement activators (A), amphotericin B-containing (B), and empty liposomes (C) on plasma white blood cell (WBC) counts in anesthetized rats. The doses and number of animals are specified in the keys. The data show % change relative to baseline (t = 0 min), mean ± SEM. Statistical analysis was performed using two-way repeated measurements ANOVA followed by Dunnett’s multiple comparisons post-hoc test. Significant differences (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) are shown relative to the group treated with saline.