Genetic and therapeutic targeting of properdin in mice prevents complement-mediated tissue injury
Yuko Kimura, Lin Zhou, Takashi Miwa, Wen-Chao Song
Yuko Kimura, Lin Zhou, Takashi Miwa, Wen-Chao Song
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Research Article Inflammation

Genetic and therapeutic targeting of properdin in mice prevents complement-mediated tissue injury

Abstract

The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface–bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement–mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement–mediated injury and support its therapeutic targeting in complement-dependent human diseases.

Authors

Yuko Kimura, Lin Zhou, Takashi Miwa, Wen-Chao Song

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Figure 4

Purification of mouse properdin and generation of a function-blocking anti-mouse properdin Ab.

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Purification of mouse properdin and generation of a function-blocking an...
(A) Elution of properdin from the Mono S column of FPLC with a salt gradient (20–300 mM in 20 minutes). Protein concentration in the FPLC fractions was monitored by OD280, and its activity was determined by LPS-induced AP complement activation assay using Cfp–/– mouse serum. Data are presented as percent recovery of AP complement activity in Cfp–/– mouse serum (normalized to WT mouse serum). Fractions 5–12 (10 μl each) were analyzed for purity by separation on a 10% SDS-PAGE gel under reducing condition and staining with Coomassie blue. The most active fractions from the last FPLC step (fractions 8–10; red font), confirmed to contain a protein of near homogeneity with the expected molecular weight of mouse properdin, were pooled. (B) Western blot assay showing reactivity of a rabbit polyclonal anti-mouse properdin Ab with plasma properdin in WT mice. No signal was detected in Cfp–/– mouse plasma. Plasma samples were either analyzed directly (2 μl) or precipitated with 5% PEG (from 12.5 μl). (C and D) Ab neutralization of plasma properdin in vitro. Rabbit anti-mouse properdin IgG F(ab′)2 (C), but not nonimmune rabbit IgG F(ab′)2 (D), blocked LPS-induced AP complement activation in WT mouse serum. EDTA sera were used as negative controls. (E and F) Ab neutralization of plasma properdin in vivo. WT mice were injected with rabbit anti-mouse properdin IgG F(ab′)2 (E) or nonimmune rabbit IgG F(ab′)2 (F) (2 mg/mouse i.p.). Serum samples were collected at the indicated times and assayed for LPS-induced AP complement activity.