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Studies in murine models have suggested the involvement of the complement anaphylatoxins (C3a and C5a) in the development of allergic asthma. We investigated the effects of inhibiting complement activation after sensitization but before allergen challenge on the development of allergic airway inflammation and airway hyperresponsiveness. To prevent complement activation, we used a recombinant soluble form of the mouse membrane complement inhibitor complement receptor-related gene y (Crry) fused to the IgG1 hinge, CH2 and CH3 domains (Crry-Ig), which has decay-accelerating activity for both the classic and alternative pathways of complement as well as cofactor activity for factor I-mediated cleavage of C3b and C4b. C57BL/6 mice were sensitized (Days 1 and 14) and challenged (Days 24-26) with ovalbumin. Crry-Ig was administered after allergen sensitization either as an intraperitoneal injection or by nebulization before allergen challenge. Crry-Ig significantly prevented the development of airway hyperresponsiveness, decreased airway and lung eosinophilia as well as the numbers of lung lymphocytes, decreased levels of interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and decreased serum ovalbumin-specific IgE and IgG1. These results suggest that prevention of complement activation may have a therapeutic role in the treatment of allergic airway inflammation and asthma in sensitized individuals.

This unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes C1qrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. Two CH(50) assay procedures are presented that test total classical pathway function; one is carried out in test tubes, whereas the alternate protocol describes a variation that is carried out in 96-well microtiter plates. Three support protocols describe preparing serum and erythrocyte-antibody complexes (EA) for use in CH(50) assays,and titrating of hemolysin for use in EA preparation. As an alternative to functional assays, radial immunodiffusion (RID) methods are also presented. These can be used to measure the concentrations of most circulating complement proteins and to evaluate the functional status of C1-esterase inhibitor. A fourth support protocol provides a method to determine antibody concentrations for RID assays.

In an earlier study, we presented evidence that plicatic acid (PA) activated C in normal human serum (NHS) by an immunoglobulin-independent mechanism that did not involve factor B of the alternative pathway and that required Ca++ for initiation. The present paper further verifies that PA acts through the classical pathway. In PA-treated NHS, titers of C1, C4, C3, and C5 decreased after incubation at 37 degrees C, but in C2-deficient serum, only the titers of C1 and C4 were decreased after identical PA treatment. Activation of purified precursor C1 did not occur when it was incubated with PA at concentrations that would have produced C1 consumption in serum. Thus, PA added to serum initiates activation of the classical pathway, but is incapable of activating C1 directly. PA added to a mixture of native C4 and activated C1s did not alter the kinetics or the extent of C4 inactivation by the C1s. However, when mixtures of C4, C1s, and C1-In were incubated with PA, the ability of the inhibitor to inactivate the C1s was markedly reduced. These data indicate that the mechanism by which PA activates C in serum may involve interference with the normal regulatory mechanism that controls C1 activity.

10 mg of monosodium urate crystals reduced the CH50 of 1 ml of human serum by 57% after 30 min at 37 degrees C. C1, C4, and C3 depletion of 52, 68, and 46% were typical of classical pathway activation. C1 binding and activation occurred when urate crystals were incubated with isolated precursor C1, and required the intact macromolecule, C1qrs. Activation of isolated C1 by urate crystals was not diminished by F(ab')2 anti-Fc under conditions in which C1 activation by aggregated immunoglobulin (G) was blocked by the F(ab')2 antibody.