We have defined how monosaccharide composition and linkage specificity of β-glucans (β1→3 and β1→6 linked) together influence activation of the alternative pathway (AP). Importantly, the role played by properdin in linkage-specific β-glucan-mediated AP activation has also been defined. β-Glucans are found naturally in fungi, algae, plants, and some bacteria; both β1→3 and β1→6 glucans are major constituents of the cell walls of fungi. A small percentage of the fungal cell wall is composed of chitin (β1→4 d
-acetylglucosamine). As shown previously (25
), we also demonstrated that all glycan particles (GP) with either β1→3 and/or β1→6 glucan linkage activated C3 when the AP was intact. In contrast, chitin or chitosan (β1→4 linkages) activated the AP (measured by C3a and C5a generation) only minimally. In the absence of functional properdin, only glucan particles that express surface-exposed β1→3 linkages (GP, scleroglucan, and curdlan) generated C3a and C5a, albeit at concentrations about 10-fold lower than when properdin was functional. Pustulan (contains only β1→6 glucan linkages) was a potent AP activator in the presence of properdin but generated minimal C3a and C5a when properdin function was blocked. These data suggest that glucan particles that express surface-exposed β1→3 glucan linkages can activate C3 in the absence of properdin, but particles with only β1→6 glucan linkages require properdin to activate C3.
Following C3 activation, the metastable C3b molecule must quickly bind to an electron-donating –OH group through the thioester bond; failure to do so results in hydrolysis of the thioester, and the C3b molecule remains in solution. Free C3b can form alternative pathway C3 and C5 convertases (C3b,Bb and C3bC3b,Bb) in solution that can further activate C3 and C5 and generate more C3a and C5a, respectively. However, the amount of C3b deposited on the glycan particles depends on the availability of hydroxyl (–OH) groups on ringed monosaccharides, principally hexoses, and serves to explain the apparent discordance between C3a and C5a generation () and C3 fragment deposition (). The particulate β1→3 glucans used in this study exist primarily in a triple helix conformation (37
). Based on the results of glucan fiber diffraction studies (37
), the hydroxyl groups at positions 2 and 4 are buried within the polysaccharide chain participating in intra- and interchain hydrogen bonds. The hydroxyls at position 6 are exposed to solvent and are the likely electron donors for reaction with C3. Removal of primary –OH groups from position 6 of monosaccharides decreases the efficiency of C3 binding (38
). Consistent with our observations (), the β1→3 particulate glucans with exposed free –OH groups at position 6 (curdlan, GP, and scleroglucan) bound C3 more effectively than β1→6 glucans, whose position 6 is occupied by the linkage to the adjacent monosaccharide (pustulan). Also consistent with our observations, the β1→3 particulate glucans with free –OH groups at position 6 masked by mannans (zymosan and glucan-mannan particles [GMP]) bound C3 less effectively. Higher efficiency of β1→3 glucan binding to nascent C3 likely facilitates C3b binding to –OH at position 6, even when the AP lacks properdin ().
Release of anaphylatoxins C3a and C5a during activation of complement has also been shown to promote neutrophil influx to sites of infection. Human neutrophils release properdin upon stimulation (12
), which likely serves to augment AP activation (39
). β1→6 glucans, in particular, stimulate human neutrophils and mediate engulfment, production of reactive oxygen species, and expression of heat shock proteins more efficiently than β1→3 glucans (40
). In one study, human neutrophils rapidly ingested beads coated with β1→6 glucan, but not beads coated with β1→3 glucan (40
), suggesting that stimulation of neutrophils may be directed by the specific location of C3b binding on glycan surfaces. The effects may not be limited to neutrophils, as recently, it was shown that C3a and C5a generation can regulate interleukin 17A (IL-17A) responses (41
), a critical component of host defense against many fungal infections. Selective reduction of properdin function may be of therapeutic benefit. For example, in a model of cardiopulmonary bypass, treatment with antiproperdin antibodies resulted in significantly reduced neutrophil and platelet activation (14
). However, our data suggest that neutralizing properdin could increase the risk of fungal infections by decreasing AP activation.
In addition to affecting innate immune responses, generation of C3a and C5a can affect adaptive immune responses (41
). For example, recently, Lajoie et al. demonstrated the abilities of C3a and C5a to reciprocally regulate TH
1 and TH
17 responses in an airway hyperresponsiveness mouse model (41
). As TH
1 and TH
17 responses are critical components of host defense against many fungal infections (43
), the contribution of complement activation by fungal cell walls to TH
skewing deserves further study.
We have shown that purified properdin in its native forms (dimers, trimers, or tetramers) binds directly to zymosan (27
), but data from the present study suggest that properdin does not bind directly to either GP or zymosan in the context of serum but instead binds to deposited C3 (). Thus, it seems unlikely that properdin itself initiates AP activation in the presence of serum, confirming that properdin serves to stabilize AP C3 convertases (26
). Serum amyloid P component has been reported to interfere with properdin’s ability to bind to complement activator surfaces (30
). Although zymosan and GMP contain both β1→3 and β1→6 glucan linkages and activated the AP in the presence of properdin, C3 was minimally activated by these particles in the absence of properdin. We speculate that mannans present on the surfaces of zymosan and GMP may limit covalent linkage formation between nascent C3b and the underlying β1→3 glucans. The finding that properdin is required for AP activation by mannan-coated particles may be clinically relevant, considering the fact that most pathogenic fungi have an outer mannan layer.
Because hollow and porous β-glucan particles allow for high antigen loading (20
), they can serve as an effective antigen-presenting cell receptor-targeted vaccine delivery system. Recently, orally administered β-glucan particles have also been shown to function as effective adjuvants for tumor immunotherapy (45
). Although the relative contributions of complement receptors and β-glucan receptors remain to be defined, efficient coating of β1→3 linkage-containing particles such as GP with C3 fragments likely contributes to their excellent ability to act as an adjuvant. Goodridge et al. (46
) recently demonstrated that Dectin-1, a pattern recognition receptor, was activated only by particulate β-glucans and not by soluble β-glucans (46
). Particulate β-glucans clustered around the receptor to form synapse-like structures activating phagocytosis (46
). Binding of C3 fragments to antigens facilitates their uptake by subcapsular sinus macrophages in lymph nodes (47
). Transfer of the antigen-C3 fragment complexes to mature B cells involves binding of the C3d-coated antigen to the B cell coreceptor (CD21/CD19/CD81), which lowers the threshold for B cell activation (48
) and results in B cell expansion and migration to the T cell-B cell boundary in lymph nodes. Following interaction with follicular T helper cells, B cells undergo somatic cell hypermutation and class switch recombination and develop into plasma cells or memory B cells (reviewed in reference 47). Our observations regarding how properdin facilitates AP activation by glucans in fungal cell walls contributes to our understanding of the complexity of complement activation by fungi and has implications for the use of glucan particles in biomedical applications.