Mannan-specific immunoglobulin G antibodies in normal human serum accelerate binding of C3 to Candida albicans via the alternative complement pathway

Abstract

Candida albicans activates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allows C. albicans to initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca2+ with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding to C. albicans incubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab')2 fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system and C. albicans.

FIG. 1

Effect of antimannan IgG on the kinetics for C3 deposition on C. albicans yeast cells via the alternative pathway. Yeast cells were incubated in a C3 binding medium containing 40% NHS, 40% EGTA-chelated NHS, 40% mannan-absorbed and EGTA-chelated NHS, or 40% mannan-absorbed and EGTA-chelated NHS supplemented with antimannan IgG at 88 μg per ml of reaction mixture, which was 40% of antimannan IgG concentration found in the pooled serum used for this experiment.

FIG. 2

Effect of antimannan IgG on alternative pathway activity reconstituted from six purified alternative pathway proteins. Yeast cells were incubated in a C3 binding medium containing the isolated alternative pathway proteins alone or with purified antimannan IgG at 30 μg per ml of reaction mixture.

FIG. 3

Dose effect of exogenous antimannan IgG on binding of C3 to C. albicans via the classical or alternative pathway. C3 binding reactions were supplemented with various amounts of antimannan IgG per milliliter of binding mixture that contained either 40% mannan-absorbed serum to assess the ability of yeast cells to activate the classical pathway following a 6-min incubation or 40% mannan-absorbed and EGTA-chelated serum to assess the ability of yeast cells to activate the alternative pathway following a 12 min incubation.

FIG. 4

Effect of Fab or F(ab′)₂ fragments of antimannan IgG on alternative pathway mediated-C3 deposition to yeast cells. Yeast cells were incubated in a C3 binding medium containing 40% mannan-absorbed and EGTA-chelated NHS that was supplemented with various amounts of intact antimannan IgG or its Fab or F(ab′)₂ fragments.

FIG. 5

Effect of exogenous antimannan IgG on formation of alternative pathway initiation sites. Yeast cells were incubated in a complement binding medium that contained 40% mannan-absorbed NHS and 26 μg of antimannan IgG per ml of reaction mixture (classical pathway intact), 40% mannan-absorbed and EGTA-chelated NHS (antibody-independent activation of the alternative pathway), or 40% mannan-absorbed and EGTA-chelated NHS and 26 μg antimannan IgG per ml reaction mixture (antibody-dependent activation of the alternative pathway). Yeast cells were stained with FITC-labeled goat anti-human C3 antibodies.