Giorgio Senaldi

Activation of the complement system in primary sclerosing cholangitis

Recent evidence, including the presence of circulating immune complexes, suggests that abnormalities of humoral immunity may be important in the pathogenesis of primary sclerosing cholangitis. The aim of the present study was to determine whether activation of the complement system is present in patients with this disease, as this would be supportive evidence of a role for circulating immune complexes in primary sclerosing cholangitis. Plasma complement fragments C3d and C4d, and serum C3 and C4, their respective parent molecules, have been assayed. Both C3d and C4d were elevated in patients with primary sclerosing cholangitis compared with patients with extrahepatic obstructive cholestasis and normal controls (p less than 0.01 in all instances). C3 was elevated in both patient groups, in whom it was similar, compared with normal controls (p less than 0.001 in both cases), whereas C4 was similar in all groups. Elevated levels of circulating immune complexes were identified in 21 of 24 (88%) patients with primary sclerosing cholangitis, but in none of the normal controls. These findings support the hypothesis that in primary sclerosing cholangitis circulating immune complexes are associated with activation of complement via the classical pathway.

Methods for assessing complement activation in the clinical immunology laboratory.

Complement activation is a key component of the pathogenesis of immune-mediated tissue damage in many diseases. Assessment of complement activation in current practice is largely based on the measurement of intact C3 and C4 or the determination of complement haemolytic function. These parameters reflect activation only indirectly, are insensitive and open to influence by factors other than complement conversion. New approaches to evaluate complement activation directly using sensitive techniques have been developed, and several could be adopted easily in most laboratories. These concentrate on the detection of activation fragments, neoantigens or complexes that only arise as a direct result of complement activation. The wide application of these techniques in research and clinical practice would enhance our understanding of the pathogenesis of a range of inflammatory and infectious diseases.

Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.