Complement activation directly induced by Helicobacter pylori Audun E. Berstad, Kolbjørn Høgåsen, Geir Bukholm, Anthony P. Moran, Per Brandtzaeg  Gastroenterology  Volume 120, Issue 5, Pages 1108-1116 (April 2001) DOI: 10.1053/gast.2001.23248 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Two cagA+ and 2 cagA− strains of H. pylori, Zymosan A, or saline (negative control) were incubated with nonimmune human serum for different time intervals at 37°C before complement activation was blocked by the addition of EDTA. The complement activation products (A) C3bc and (B) TCC were subsequently measured. Mean ± SD of 3 representative experiments are indicated. Concentrations of complement products are given as AU/mL. After 15 minutes, H. pylori generated significantly more C3bc (P = 0.01), but less TCC (P = 0.004), than Zymosan. Gastroenterology 2001 120, 1108-1116DOI: (10.1053/gast.2001.23248) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Levels of (A) C3bc and (B) TCC generated after incubation of H. pylori with preabsorbed nonimmune human serum. Classic pathway activation, or both classic and alternative pathway activation, were blocked by adding EGTA-Cl2 or EDTA, respectively, before incubations as indicated. Zymosan A (OD 0.8 and 10 mg/mL) was included as control for alternative pathway activation, and HAIGG (1 mg/mL) as control for the classic pathway. Saline served as negative control. Each column represents mean ± SD of 4 separate experiments. *P < 0.01 for reduction of activation by EGTA-Cl2. Gastroenterology 2001 120, 1108-1116DOI: (10.1053/gast.2001.23248) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Complement activation by smooth LPS in nonimmune human serum. (A) C3bc and (B) TCC were generated in a dose-dependent manner. Mean ± SD of 5 separate experiments is plotted. *P < 0.01 for reduction of activation by EGTA-Cl2. Gastroenterology 2001 120, 1108-1116DOI: (10.1053/gast.2001.23248) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Serum sensitivity of H. pylori (strain L01) after 15-minute contact with nonimmune (■) or heat-inactivated (○; 56°C, 30 minutes) nonimmune human serum at various concentrations as indicated. Serum sensitivity of type strain NCTC 11637 (▾) after contact with nonimmune human serum is shown for comparison. The number of bacterial colonies is given in percentage of that obtained after incubation with medium alone (100% reference). Mean ± SD of 5 separate experiments is plotted. *P < 0.001 for increase in number of colonies of strain L01 after heat inactivation of serum. Gastroenterology 2001 120, 1108-1116DOI: (10.1053/gast.2001.23248) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Two-color immunofluorescence staining (double exposures) of complement deposits on H. pylori after incubation with preabsorbed nonimmune human serum for 5 minutes. (A) Paired staining for C3bc (Texas red) and H. pylori (FITC, green) shows colocalization (yellow), signifying the presence of C3bc on several of the bacteria. (B) Paired staining for TCC (Texas red) and H. pylori (FITC) likewise shows colocalization (yellow), signifying terminal complement activation with the formation of the membrane attack complex (or TCC). Bacteria associated with complement activation appear swollen, suggesting lytic activity. (C) The bacteria appear rod shaped and do not stain for C3bc after blocking of complement activation with EDTA before bacterial incubation (staining protocol as in A; original magnification, 1000×). Gastroenterology 2001 120, 1108-1116DOI: (10.1053/gast.2001.23248) Copyright © 2001 American Gastroenterological Association Terms and Conditions