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. 2016 May 3;18(11):1570–1582. doi: 10.1111/cmi.12597

Figure 2.

Figure 2

Granulocytic CEACAM3 is essential for oxidative burst, degranulation and chemokine release in response to Moraxella‐UspA1. A. Pull down: BBH18 wild‐type bacteria and the UspA1‐deficient mutant BBH18.1 were incubated with CEACAM proteins as described in the Materials and Methods section. 10% of inputs, 10% of supernatants (unbound proteins) and 50% of eluates (bound proteins) were analysed by Western blot using a pan‐human CEACAM cross‐reactive anti‐CEA antibody (DAKO). Note the binding of CEACAM1, CEACAM3 and CEACAM6 to the wild‐type bacteria only and the absence of any binding of CEACAM8 and the CEACAM1‐ΔN mutant lacking the N‐terminal Ig‐domain responsible for the CEACAM1‐UspA1 interaction. B. CEACAM3‐specific degranulation in response to Moraxella infection. Cells were incubated with Cytochalasin B prior to infection with Moraxella catarrhalis BBH18 or the UspA1 deletion strain (1 h, MOI 50) alone or after pre‐incubation (1 h, 30 µg/ml) with the CEACAM3‐blocking antibody [Col‐1] as well as CEACAM1‐ or CEACAM6‐blocking antibodies. Supernatants were collected and analysed for myeloperoxidase (MPO) release, as described in Material and Methods. The untreated control has been set to 100%. C. Oxidative burst determined by Dichlorofluorescein (DCF)‐dependent fluorescence of granulocytes stimulated for 1 h with M. catarrhalis wild‐type strain BBH18 (MOI 100) or the corresponding, UspA1 deletion strain (MOI 100) alone or after preincubation (1 h) with the CEACAM3‐blocking antibody [Col‐1] or a CEACAM1‐blocking antibody. PMA served as positive control, unstimulated cells served as negative control (unfilled). D. CXCL8 or (E) CCL3 were determined by ELISA in supernatants of human granulocytes stimulated for 16 h with Pam3Cys (1 µg/ml), M. catarrhalis strain BBH18 (MOI 50) alone or after pre‐incubation (1 h, 30 µg/ml) with the CEACAM3‐blocking antibody [Col‐1], CEACAM1‐, CEACAM6‐blocking antibodies or IgG2a control antibody. Data are representative from one out of three experiments (A, C) or represent mean and SD of at least three (B, D, E) experiments done in duplicates (p < 0.05).