I. von Zabern

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H2O2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH. is the active oxygen species that converts C5. The conversion product of C5, termed C5(H2O2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H2O2) is much more stable than C5b obtained by complement convertases. Although C5(H2O2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H2O2) is not chemotactic. C5(H2O2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.

Mechanisms of complement activation by crystalline cholesterol

The mechanism by which cholesterol crystals activate complement in human serum has been studied. Crystals treated with serum and washed with buffer contain a fixed C3/C5 convertase. Its generation is dependent on the presence of divalent cations (and of factor B). The cholesterol-fixed convertase is subject to decay and can be regenerated by factors B and D. C2 in combination with C1 is not essential but enhances the convertase formation. These findings indicate that it is predominantly the alternative C3/C5 convertase C3bBb(P) that assembles on cholesterol during exposure to human serum. By the use of different antisera and immunofluorescence a C3 fragment, probably C3b, was demonstrated on serum-treated crystals. Its fixation is resistant to washing with urea, and with buffers of differing pH: by hydroxylaminolysis the C3 fragment dissociates from the crystals. This indicates a covalent ester bond linking the labile binding site of activated C3 to the hydroxyl group of cholesterol. Cholesterol acetate does not fix C3 nor acquire a C3-cleaving activity upon contact with serum. In addition, cholesterol crystals bind factor I (C3b inactivator) and in this way may facilitate fixation and amplification of the alternative C3/C5 convertase.

Effect of radiographic contrast media on complement components C3 and C4: generation of C3b-like C3 and C4b- like C4

Various water soluble iodinated radiographic contrast media (RCM) have been studied for their effect on complement components C3 and C4, purified and in serum. Hepatotropic RCM, and at higher concentration also some nephrotropic RCM, were found to exert a direct effect on purified C3 and C4. RCM treated human C3 and C4 are characterized by (a) loss of haemolytic function, (b) retention of activity in the formation of fluid phase C3 convertases and (c) an antigenic relationship to activated C3 and C4 (C3b and C4b, respectively). This direct alteration of C3 and C4 can probably also occur in serum since loss of haemolytic function is observed at similar RCM concentrations after incubation of serum and of purified components. It is concluded that RCM treated C3 and C4 represent altered forms of these components that resemble C4b and C3b in activity and conformation (C3b-like C3 and C4b-like C4). The alteration is probably caused by binding of RCM, exerting a mild denaturing effect. C3b-like C3 is a potential activator of the alternative pathway, and both C3b-like C3 and C4b-like C4 are known to be cleaved by serum inactivators. A possible pathological significance of the generation of C3b-like C3, C4b-like C4 and their split products remains to be evaluated.

Effect of Different Penicillin Derivatives on Complement Components in Human Serum

The effect of different penicillin derivatives on the human complement system has been studied in vitro. Four penicillins tested (benzylpenicillin, oxacillin, methicillin, azlocillin) were found to inactivate total hemolytic complement dose dependently. This effect is caused by direct inactivation of C2, C5 and one or more of the components C6-9 as well as by activation of the alternative pathway. At relatively high concentrations, penicillins induce a conformational change in C3 and C4, leading to the C3b-like or C4b-like forms of these molecules, respectively. Prominent effects on the complement system occur at penicillin concentrations exceeding those achieved in plasma under regular therapeutic use. However, a synergism of penicillins with drugs acting similarly on the complement system, such as iodinated radiographic contrast media, may be expected.

Effect of metrizamide, a nonionic radiographic contrast agent, on human serum complement: comparison with ionic contrast media

The nonionic radiographic contrast material (RCM) metrizamide causes consumption of total complement activity in normal human serum (NHS) in vitro in the absence and to a lesser extent also in the presence of EDTA. The depression of titers of total complement is related to an inactivating effect of metrizamide on component C2. Furthermore, metrizamide induces activation of the alternative pathway as evidenced by the appearance of C3 and factor B cleavage products in NHS, dependent on the presence of divalent cations. Alternative pathway activation is probably mediated by an antagonizing effect of metrizamide on the inactivation of C3b. Unlike ionic RCM, the nonionic substance metrizamide does not lead to cleavage of the internal thiolester bond present in native C3 and C4, at concentrations that produce potent consumption of C3 activity in NHS.

Function of the activated fourth component of complement (C4b) in activation of C2

In course of pathway activation fourth component complement is by acCT into (8800 M,) C4b (197 M,). It well known C4b forms reversible complex the second of compleC2, with essential participation Mg2+ [ 1,2]. Furthermore, C4b serves as an acceptor for CZ, activated by cleavage by Ci; the complex formed, C4b2a,b is independent of Mg2+ and has proteolytic activity for C3 and C5. The enzymic site resides in the C2a part of the labile complex 12-41, expressed only when C2a is bound, not after its irreversible dissociation or when generated by CT in the absence of C4b. facilitates C2 cleavage by acting on the Ci complex, an explanation proposed in [5]. Facilitation of C2 cleavage by interaction of C4b with C2 [6] or by action of C4b as product acceptor [8] seem now to be excluded.

Activation of the Alternative Pathway of Human Complement by Sulfhydryl Compounds of Analytic and Therapeutic Use

Thiol-containing drugs (dimercaprol, dimercaptopropanesulfonate, captopril, penicillamine, N-acetylcysteine) and the standard reducing agent beta-mercaptoethanol, activate the alternative pathway of complement as shown by in vitro experiments. Depending on the substance tested, at concentrations of 0.5-5 mM, cleavage products of C3 and factor B were demonstrable in serum by immunoelectrophoresis. The regulatory protein factor I proved to be very sensitive to thiols; this observation offers an explanation for the alternative pathway activating effect of these substances. At concentrations of thiols that initiate the alternative pathway, the classical pathway was not or only to a minor extent activated; however, the activity of C2, C5 and one or several of the components C6-9 was directly affected. Alkylation of the thiol group of the compounds tested, abrogated their effects on the complement system.

Effect of different sulfonamides on the human serum complement system

Incubation of normal human serum with different sulfonamides led to a dose-dependent inactivation of total hemolytic complement activity. A decrease of the activities of C1, C2, C3, C5 and one or several of the components C6-9 was observed after treatment of normal human serum with the sulfonamide sulfisomidine. Inactivation of complement components by sulfonamides seems to result from direct interaction with the drug as well as, to a minor extent, from activation of the alternative pathway. At relatively high concentrations, sulfonamides caused a conformational change in C3 and C4. The observed structural change is equivalent to that induced by cleavage of the internal thiolester bond in these molecules. The generation of such structurally altered C3 (C3b-like C3) as well as an antagonizing effect of sulfonamides towards the action of the regulatory protein factor I might be responsible for alternative pathway activation. The physiological relevance in vivo of the observed effects of sulfonamides remains to be assessed.

Function of the activated fourth component of complement (C4b) in activation of C2 (1982) FEBS Letters 144, 195-198.

page 230, column 1, lines 14 to 9 from below should read: before elutriation. The amount of sinusoidal cells recovered from a liver of 5 g varied from 160200x lo6 cells, of which -20% were Kupffer cells, 27% lymphocytes and 53% endothelial cells. After elutriation > 90% of the ceils could be covered. The viability of the elutriated cells was ~90%. The average instead of: before elution. The amount of sinusoidal cells recovered from a liver of 5 g varied from 160200x IO6 cells, of which -20% were Kupffer cells, 27% lymphocytes and 53% endothelial cells. After elution, >90% of the cells could be recovered. The viability of the eluted cells was > 90%. The average