Activation of the alternative complement pathway by fungal melanins

Abstract

Melanins are complex biological pigments formed by the oxidative polymerization of phenolic and/or indolic compounds. These pigments have been implicated in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases. Recent studies have demonstrated that melanins have antigenic and anti-inflammatory properties. These findings led us to further explore the interaction of melanins with the immune system. Melanin particles ("ghosts") were isolated from in vitro-melanized Cryptococcus neoformans cells and Aspergillus niger conidia and then incubated in normal human serum containing (125)I-labeled complement C3. The results demonstrated deposition of C3 fragments onto the melanin ghosts as early as 1 min after incubation, with maximum deposition occurring after 12 min for C. neoformans-derived melanin ghosts and after 25 min for A. niger-derived melanin ghosts. The blocking of classical pathway activation did not affect the kinetics or total deposition of C3 onto the melanin ghosts, indicating that melanins activate complement through the alternative pathway. Immunofluorescence analysis of lungs from BALB/c mice injected intratracheally with C. neoformans-derived melanin ghosts demonstrated deposition of C3 fragments onto the ghosts. Small granulomas were also observed surrounding the ghosts. However, melanization of the C. neoformans cell wall did not alter the kinetics or total deposition of C3 fragments onto the fungal cells. The finding that melanin surfaces can activate the complement system suggests a potential mechanism for the pathogenesis of some degenerative and/or autoimmune processes that involve melanized cells as well as another potential role for melanin in the virulence of melanin-producing microorganisms.

FIG. 1.

Complement activation by C. neoformans- and A. niger-derived melanin ghosts after incubation in 40% NHS containing ¹²⁵I-labeled C3. Numbers of C3 molecules should be multiplied by 10⁶.

FIG. 2.

IF analysis of complement activation by C. neoformans-derived melanin ghosts after incubation in 40% NHS (magnification, ×250).

FIG. 3.

Light (A) and fluorescent (B) analyses of complement activation by C. neoformans-derived melanin ghosts in lung tissues of BALB/c mice (magnification, ×250). (C) Hematoxylin and eosin staining of lung tissues of BALB/c mice, demonstrating the formation of small granulomas surrounding C. neoformans-derived melanin ghosts (magnification, ×100). Arrows indicate melanin ghosts.

FIG. 4.

Complement activation by melanized and nonmelanized C. neoformans 3501 cells after incubation in 40% NHS containing ¹²⁵I-labeled C3. Numbers of C3 molecules should be multiplied by 10⁶.