Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

“…The fact that the native trout C5a, cleavage product of C5 [23], was recognized by using the anti-trout C5a pAb, suggests the existence of C5a anaphylatoxin in the PUEF-8 as previously suggested by Li et al [26]. The functional receptor for trout C5a (C5aR) was reported previously, which is present on B lymphocytes, granulocytes as well as macrophage-like cells [24,25].…”

“…Whereas, the complement-activated trout serum, as the positive control, displayed strong chemoattractive ability (Fig. 1E), for that it contains multiple complement elements, including C5a, C3a, C4a et al [26]. All these results indicate that the C5a portion of recombinant G-C5a fusion protein was correctly folded and thus retained its natural chemoattractive ability to leukocytes.…”

“…Furthermore, recombinant trout C5a showed functions similar to mammalian C5a, such as respiratory burst and chemotaxis [24,25]. In addition, a trout serum fraction (8-kDa particle-uptake enhancing fraction, PUEF-8) composed of trout anaphylatoxins, potentially including C5a, was found to enhance the phagocytic capacity of trout leukocytes [26]. All of these findings lead us to hypothesize that trout C5a may act as a molecular adjuvant.…”

“…As previously described [26], to generate PUEF-8, 0.5 ml trout serum was incubated with 50 ml of rabbit erythrocytes (Cocalico Biologicals) for 30 min at room temperature. The reaction mixture was then diluted with 0.5 ml of PBS (containing 20 mM EDTA, pH 7.2), loaded onto a Superdex-200 FPLC column (GE Healthcare) and eluted (0.5 ml per fraction) at room temperature with PBS, pH 7.2.…”

“…Western blotting was carried out as previously described [26]. The native C5a, recombinant C5a, or the C5a portion in G-C5a fusion protein were detected using rabbit anti-trout C5a pAbs (0.5 mg/ml), followed by HRP-conjugated anti-rabbit IgG (GE Healthcare) (1: 4000).…”