Polyglycolic acid-induced inflammation: role of hydrolysis and resulting complement activation.

Tissue and organ replacement have quickly outpaced available supply. Tissue bioengineering holds the promise for additional tissue availability. Various scaffolds are currently used, whereas polyglycolic acid (PGA), which is currently used in absorbable sutures and orthopedic pins, provides an excellent support for tissue development. Unfortunately, PGA can induce a local inflammatory response following implantation. Therefore, we investigated the molecular mechanism of inflammation in vitro and in vivo. Degraded PGA induced an acute peritonitis, characterized by neutrophil (PMN) infiltration following intraperitoneal injection in mice. Similar observations were observed using the metabolite of PGA, glycolide. Dissolved PGA or glycolide, but not native PGA, activated the classical complement pathway in human sera, as determined by classical complement pathway hemolytic assays, C3a and C5a production, and C3 and immunoglobulin deposition. To investigate whether these in vitro observations translated to in vivo findings, we used genetically engineered mice. Intraperitoneal administration of glycolide or dissolved PGA in mice deficient in C1q, factor D, C1q and factor D, or C2 and factor B demonstrated significantly reduced PMN infiltration compared to congenic controls (WT). Mice deficient in C6 also demonstrated acute peritonitis. However, treatment of WT or C6 deficient mice with a monoclonal antibody against C5 prevented the inflammatory response. These data suggest that the hydrolysis of PGA to glycolide activates the classical complement pathway. Furthermore, complement is amplified via the alternative pathway and inflammation is induced by C5a generation. Inhibition of C5a may provide a potential therapeutic approach to limit the inflammation associated with PGA-derived materials following implantation.

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q.

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

Inhibition of C5 or absence of C6 protects from sepsis mortality.

Inhibiting complement anaphlytoxin C5a during sepsis may prevent sepsis mortality. Although human anti-C5 antibodies exist, their therapeutic use in microbial sepsis has been avoided because of the hypothesis that inhibiting C5b will prevent formation of the bactericidal membrane attack complex (MAC) and worsen clinical outcome. We wished to test the hypothesis that inhibition of C5 would improve outcomes in sepsis. Sepsis was induced in rats by laparotomy and cecal ligation and puncture (CLP) by an IACUC-approved protocol. Sham animals underwent laparotomy without CLP. Following CLP rats were randomized to receive a single IV dose of purified IgG ant-C5 antibody (Ab) or control IgG Ab. Anti-C5 Ab treated rats (n = 20) had significantly lower mortality vs. controls (n = 21), 20% vs. 52% (P = 0.019, log-rank). Analysis of bacterial load by culture of spleen and liver homogenates showed a reduction in colony forming units in anti-C5 Ab treated rats vs. control IgG (P = 0.003 and 0.009, respectively). Anti-C5 treatment reduced lung injury as measured by total MPO content of lung tissue (P = 0.024). Finally, rats genetically deficient in C6 production, unable to form MAC but capable of producing C5a and C5b, were protected from CLP-induced sepsis mortality. Our results show that in anti-C5 antibody therapy prevents CLP sepsis-induced mortality and improves lung injury. Inhibition of the complement MAC does not increase bacterial load or mortality, therefore, the use of anti-C5 therapy may be beneficial rather than detrimental in sepsis.