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. 2017 Jul;140(1):89-100.e2.
doi: 10.1016/j.jaci.2016.11.015. Epub 2016 Dec 12.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

Griet A Van Roey  1 , Christopher C Vanison  1 , Jeffanie Wu  1 , Julia H Huang  1 , Lydia A Suh  2 , Roderick G Carter  2 , James E Norton  2 , Stephanie Shintani-Smith  1 , David B Conley  1 , Kevin C Welch  1 , Anju T Peters  2 , Leslie C Grammer  2 , Kathleen E Harris  2 , Kathryn E Hulse  2 , Atsushi Kato  3 , Whitney W Stevens  2 , Robert C Kern  3 , Robert P Schleimer  3 , Bruce K Tan  4
Affiliations

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

Griet A Van Roey et al. J Allergy Clin Immunol. 2017 Jul.

Abstract

Background: Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known.

Objectives: After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS.

Methods: Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA.

Results: C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P < .05) and correlated with levels of C5a (P < .01), local immunoglobulins (especially IgM, P < .0001), and anti-double-stranded DNA IgG (P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P < .0001).

Conclusion: Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.

Keywords: Chronic rhinosinusitis; antibodies; basement membrane; classical complement pathway; complement.

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Conflict of interest statement

Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

Figures

Figure 1
Figure 1
The complement activation pathway. The three major pathways of complement activation; classical, lectin and alternative, all lead to the formation of C3 convertases which leads to the generation of the anaphylatoxins C3a and C5a and the formation of the lytic membrane attack complex (C5b-9). We evaluated the complement activation neoepitopes highlighted by the boxes. The anaphylatoxin C5a was also evaluated. Dotted arrows indicate enzymatic processes. MBL, mannose-binding lectin; MASP, MBL-associated serine protease.(68)
Figure 2
Figure 2
Complement activation products C5b-9, C4d and C1rs-C1inh were elevated in NP compared to UT. Tissue homogenates were tested by ELISA and levels were normalized to total protein for A) C5b-9, B) C4d and C) C1rs-C1inh. Data represents means ± SEMs. *P<0.05, **P<0.01, *** P<0.001, and ****P<0.0001, Kruskal-Wallis test.
Figure 3
Figure 3
Complement components C5b-9 and C4d are deposited linearly along the basement membrane of the nasal epithelium. OCT embedded tissue sections were stained for C5b-9 in red (A, C, E and F), C4d in green (B, D and E) and Laminin in green (F and G). Representative image of NP tissue.
Figure 4
Figure 4
Percentage of nasal epithelium lined with complement. Stitched whole tissue section images were analyzed for positive subepithelial C5b-9 staining using Image J and represented as a percentage of the length of epithelium visualized. Data represents means ± SEMs. *P<0.05, and *** P<0.001, Kruskal-Wallis test.
Figure 5
Figure 5
Correlations between complement fixing antibody isotypes and C1rs-C1inh- activation product of the classical pathway in NP tissue. A, Correlation between IgM and C1rs-C1inh. B, Correlation between IgG3 and C1rs-C1inh. C, Correlation between IgG1 and C1rs-C1inh. D, Correlation between IgG2 and C1rs-C1inh. E, Correlation between IgG4 and C1rs-C1inh. F, Correlation between total immunoglobulin and C1rs-C1inh. (Spearman’s coefficient)
Figure 6
Figure 6
C5a levels in sinonasal tissue and correlations with complement activation products. A. Levels of C5a B. Correlation between C1rs-C1inh and C5a. C, Correlation between C4d and C5a. D, Correlation between C5b-9 and C5a. E, Correlations between activated C1, C4d, c5b-9 and C5a with ECP and anti-dsDNA IgG (Spearman’s coefficient).
Figure 7
Figure 7
Elevated levels of anti-basement membrane antibodies in NP tissue. Antibodies were purified from NP tissue homogenates and compared with antibodies purified from serum from the same CRSwNP and control patients. 5mg/µl of total extracted antibody were tested on an anti-Matrigel ELISA. Data represents means ± SEMs. ****P<0.0001, Kruskal-Wallis test.
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