Unexpected Off-Target Activities for Recombinant C5a in Human Macrophages

Area of Science:

• Immunology
• Complement System Biology

Background:

• The anaphylatoxin C5a is a key mediator of complement activation, influencing immune cell functions via C5a receptors (C5aR1, C5aR2).
• Native C5a possesses N-linked glycosylation at Asn64, a feature absent in commonly used Escherichia coli-derived recombinant C5a.

Purpose of the Study:

• To compare the functional effects of recombinant human C5a versus plasma-purified native human C5a on primary human macrophages.
• To investigate potential differences in signaling pathways and cytokine production induced by these C5a variants.

Main Methods:

• Human primary monocyte-derived macrophages were stimulated with both recombinant and plasma-purified C5a.
• Cell signaling pathways (Syk, NF-κB) and cytokine production (IL-6, IL-10) were analyzed.
• Experiments were conducted to rule out endotoxin contamination as a confounding factor.

Main Results:

• Both recombinant and plasma-purified C5a activated C5aR1 and C5aR2 signaling similarly.
• However, recombinant C5a, but not purified or synthetic C5a, induced IL-6 and IL-10 production independently of C5a receptors.
• This cytokine production was mediated by Syk and NF-κB signaling pathways.

Conclusions:

• The glycosylation status of C5a significantly influences its functional activity in primary human immune cells.
• Caution is advised when using only recombinant C5a in functional assays, especially concerning cytokine production.
• Future studies using recombinant C5a should be validated with purified/synthetic C5a or C5aR1 inhibitors to ensure relevance.