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Infection and Immunity, March 2008, p. 994-1002, Vol. 76, No. 3
0019-9567/08/$08.00+0     doi:10.1128/IAI.01185-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response to Blastomyces dermatitidis

California Institute for Medical Research and Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, California,¹ Veterans Administration Medical Center, Cincinnati, Ohio,² Department of Medicine, Stanford University School of Medicine, Stanford, California³

Received 28 August 2007/ Returned for modification 23 September 2007/ Accepted 19 November 2007

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-) production. Since macrophage production of TNF- is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF- production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF- production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF- production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF- production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF- production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-β-Glucan stimulated TNF- production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-β-glucan antibody inhibited TNF- production by PM. With anti-1,3-β-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-β-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-β-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-β-glucan by anti-1,3-β-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-β-glucan sites or sterically masking 1,3-β-glucan sites, thus preventing 1,3-β-glucan stimulation of PM for TNF- production.

Published ahead of print on 10 December 2007.

Editor: A. Casadevall