Michael K. Pangburn

Initiation of the alternative complement pathway due to spontaneous hydrolysis of the thioester of C3.

Initiation of the alternative pathway of complement involves the random attachment of C3b on all biological particles in contact with plasma. This process requires continuous proteolytic generation of metastable C3b. The responsible protease arises as a result of conformational changes in C3 that occur upon thioester hydrolysis. The product, C3(H2O), is functionally C3b-like and forms a C3 convertase with Factors B and D. The resulting biomolecular enzyme, C3(H2O),Bb, is a serine protease that cleaves C3 generating metastable C3b. The rate of spontaneous generation of C3(H2O) under physiological conditions is between 0.2 and 0.4%/hr. C3(H2O),Bb produces only three to five metastable C3b molecules per enzyme before being inactivated by regulatory proteins. The thioester bond in metastable C3b has been estimated to be 10(10) times more reactive than that in native C3 and mediates attachment of C3b to biological particles. Once bound, C3b is subject to two competing processes: (1) inactivation and (2) a chain reaction-like amplification process that can deposit large numbers of C3b molecules on the particles within a very short period of time after the initial C3b binds. On activators of the alternative pathway, inactivation of C3b is restricted and amplification of C3b results in activation of the cytolytic pathway of complement.

The role of C3 fragments in endocytosis and extracellular cytotoxic reactions by polymorphonuclear leukocytes

Abstract Human polymorphonuclear leukocytes (PMNL) display the ability to bind particles coated with C3b or C3bi in the absence of immunoglobulin. Using highly purified, defined fragments of C3, we have extended the eivdence which indicates that these cells possess two distinct C3 receptors which are specific for their respective ligands but not for C3d. PMNL rosettes with erythrocytes (E) bearing only C3b (EC3b) were inhibited by soluble C3b but not native C3, C3bi, C3c, or C3d. EC3bi rosettes were inhibited by soluble C3bi but not native C3, C3b, C3c, or C3d. Engagement of either C3 receptor elicited comparable biological responses from PMNL. Particle bound C3b or C3bi stimulated the production of active oxygen species by PMNL as detected using a chemiluminescence (CL) assay. CL responses occurred in the total absence of immunoglobulin and were inhibited by sodium azide or superoxide dismutase but not by catalase. Particle bound C4b also elicitied a CL response from PMNL. Engagement of multiple C3 receptors was necessary to stimulate the PMNL. Although soluble, monomeric C3b had no effect on PMNL, polymeric forms of C3b induced a CL response. Using a 51 Cr release assay, the antibody-dependent, PMNL-mediated killing of Raji cells was enhanced three- to fourfold when targets carried C3 fragments on their surface. Antibody was not required to effect phagocytosis of C3 fragment coated Escherichia coli . Fluoresceinlabeled E. coli 04 were preopsonized by incubation with a mixture of the six purified alternative pathway proteins at their respective physiological concentrations which were totally devoid of immunoglobulin. Incubation of the treated E. coli with purified PMNL led to ingestion of the bacteria as assessed by a combination of phase contrast and fluorescent light microscopy and scanning and transmission electron microscopy. Using radiolabeled E. coli 075, either C3b or C3bi was found to be sufficient to induce antibody-independent ingestion of the bacteria by PMNL. These studies thus comprise the first description of PMNL reactions which depend upon or are enhanced by C3bi. By utilizing well-defined and purified C3 fragments, these studies also clearly illustrate the participation of cell bound C3 fragments in promoting both antibody-dependent as well as antibody-independent cytotoxic reactions mediated by PMNL.

Raji cell injury and subsequent lysis by the purified cytolytic alternative pathway of human complement.

Abstract Lysis of Raji cells could be effected by the 11 proteins of the purified cytolytic alternative pathway of complement (PCAP) not including immunoglobulins. Lysis was dose dependent and slow, reaching completion only after 10–20 hr. Cellular uptake of complement proteins occurred early during incubation as evidenced by the binding of radiolabeled properdin and C9. 86 Rb release paralleled C9 uptake and was complete in 2 hr. After inhibition of cell metabolism by puromycin the kinetics of cell lysis paralleled C9 uptake and 86 Rb release. Raji cells appear to be weak activators of the alternative pathway since properdin and C9 deposition was approximately 10 times slower than their deposition on strong pathway activators. Control of cell-bound C3b by β1H was reduced and intermediate to strong activators and nonactivators. The results indicate that activation of the alternative pathway by Raji cells leads to formation and binding of the membrane attack complex which in turn produces functional membrane lesions. The initial membrane injury does not result in immediate cell death. The time course of cell lysis was 7 times slower than that of formation of the initial lesion.

C3 modified at the thiolester site: Acquisition of reactivity with cellular C3b receptors

Isolated human C3 protein has been reported to have variable affinity for cellular C3b receptors. The question was investigated therefore as to whether native C3 or a derivative form of the protein exhibits receptor affinity. It was found that native C3 lacks the ability to react with C3b receptors. However, C3 modified at the thiolester site, either by treatment with chaotropic agents or methylamine or by freezing and thawing, expressed inhibitory activity in the C3b-mediated rosetting assay or immune adherence. The extent of inhibition was comparable to that caused by C3b. The ability of modified C3 to react with cellular C3b receptors constitutes an additional functional property of C3b-like C3.