Abstract: The immunochemical depletion from serum of C3b inactivator (KAF) has been performed using purified F(ab′)2 antibody. In vitro KAF depletion leads to spontaneous activation of the `alternate pathway of complement fixation' as evidenced by conversion of glycine-rich β-glycoprotein, depletion of cobra venom factor—C3 proactivator and conversion of C3. The status of the complement system following in vitro KAF depletion accurately mimics that found in vivo in the unique KAF-deficient patient. The activation of the alternative pathway whether by KAF depletion or by conventional alternative pathway activators is prevented by the immunochemical depletion of C3. It therefore appears that the alternative pathway is essentially a C3b-feedback pathway which is normally controlled by the activity of KAF.

Abstract: The contributions of the classical and alternate pathways of complement activation to the biological effects of endotoxin have been examined in the guinea pig, with particular reference to thrombocytopenia, leukopenia, and the development of the hypercoagulable state. Injection of endotoxin into normal guinea pigs led to a 95% fall in the level of circulating platelets within 15 min as well as a fall in circulating granulocytes. C4-deficient guinea pigs, known to have a complete block in the activity of the classical complement pathway, but with the alternate pathway intact, sustained no fall in platelets. The development of granulocytopenia proceeded normally. Endotoxin did activate the alternate complement pathway in C4D guinea pigs, as evidenced by the fall in C3-9 titers. With restoration of serum C4 levels, endotoxin-induced thrombocytopenia was observed in C4D animals. Thus, function of the classical complement pathway was an absolute requirement for the development of thrombocytopenia. Experiments performed in cobra venom factor (CVF)-treated normal guinea pigs, with normal levels of C1, C4, and C2, but with less than 1% of serum C3-9 demonstrated the importance of the late components in the development of thrombocytopenia but not leukopenia.C4-deficient guinea pigs had normal clotting times demonstrating that C4 was not required for normal clotting. In addition, development of the hypercoagulable state, evidenced by a marked shortening of the clotting time, was not observed on injection of endotoxin into C4D animals. Therefore, development of the hypercoagulable state paralleled the development of thrombocytopenia and required function of the classical complement pathway. Again, the importance of the late components of complement was emphasized by the failure of CVF-treated normal animals to develop hypercoagulability. These results demonstrate that endotoxin is capable of activating both the classical and alternate complement pathways in guinea pigs but that function of the classical pathway is an absolute requirement for the development of thrombocytopenia and the hypercoagulable state.

Abstract: SummaryDuration of survival was comparable in normal and C4D guinea pigs infected iv with cryptococci. Survival was shortened in animals depleted of late complement components by treatment with CVF. Some animals received a single treatment with CVF to allow recovery of complement components after challenge with cryptococci. This resulted in lower counts of fungi in peripheral blood, lung, and liver, but no improvement in levels of fungi in brain, or in survival when compared with animals continuously depleted of C3–9. Late complement components were therefore important in clearance of cryptococci from extraneural sites. However, once infection was established in brain, complement levels in the central nervous system may have been too low to aid in destruction or organisms.The authors wish to thank Miss Margret Huber for expert technical assistance and Dr. David Alling for help with statistical evaluation.

Abstract: The complement system is a mediator of a number of biologically important reactions which range from cytotoxicity of antibody-sensitized cells, bacteria, and viruses to mediation of inflammatory processes. The components of the system are a number of normal serum proteins which are present in the circulation as functionally inactive precursor molecules. Activation is the term applied to the process which enables these proteins to participate in a complement reaction. Activation is not a single event, rather it is a dynamic process, as each component must be activated under appropriate conditions in a certain sequence in order to sustain an ongoing complement reaction.

Abstract: A new cytolytic pathway is described whereby cells sensitized with cytotoxic antibody can be specifically lysed in the complete absence of intact, classical complement pathway function. Evaluation of this model with sera deficient in the 4th (C4), 2nd (C2), and 6th (C6) components of complement has revealed that this new lytic mechanism, termed the C1-bypass activation pathway, is initiated by the antibody-mediated activation of C1. Utilizing components of the previously described alternate complement pathway, this pathway bypasses C4 and C2 to activate the third to ninth components of complement (C3-9) with induction of membrane damage.

Abstract: A B s T R A C T The contributions of the classical and alternate pathways of complement activation to the biological effects of endotoxin have been examined in the guinea pig, with particular reference to thrombocytopenia, leukopenia, and the development of the hypercoagulable state. Injection of endotoxin into normal guinea pigs led to a 95% fall in the level of circulating platelets within 15 min as well as a fall in circulating granulocytes. C4-deficient guinea pigs, known to have a complete block in the activity of the classical complement pathway, but with the alternate pathway intact, sustained no fall in platelets. The development of granulocytopenia proceeded normally. Endotoxin did activate the alternate complement pathway in C4D guinea pigs, as evidenced by the fall in C3-9 titers. With restoration of serum C4 levels, endotoxin-induced thrombocytopenia was observed in C4D animals. Thus, function of the classical complement pathway was an absolute requirement for the development of thrombocytopenia. Experiments performed in cobra venom factor (CVF)-treated normal guinea pigs, with normal levels of Cl, C4, and C2, but with less than 1% of serum C3-9 demonstrated the importance of the late components in the development of thrombocytopenia but not leukopenia. C4-deficient guinea pigs had normal clotting times demonstrating that C4 was not required for normal clotting. In addition, development of the hypercoagulable state, evidenced by a marked shortening of the clotting time, was not observed on injection of endotoxin into C4D animals. Therefore, development of the hypercoagulable state paralleled the development of thrombocvtopenia and required function of the classical complement

Abstract: The present study demonstrates the ability of human autologous RBC stroma to activate the alternate complement pathway (C3-Activator system, properdin system), as evidenced by the generation of C3-Activator (C3A) from C3-Proactivator (C3PA) when RBC ghosts and sonicated ghosts are incubated with autologous serum. Intact RBC's, hemoglobin, and concentrated platelet stroma, on the other hand, are inactive in this regard. We postulate that this in vitro activity of RBC stroma may occur intravascularly when erythrocytes are damaged by immune or nonimmune mechanisms. The ensuing interaction of activated complement components with platelets leading to release of platelet factor three (PF-3) may constitute a mechanism for activation of the coagulation system during acute hemolytic episodes.

Abstract: Three independent lines of investigation suggested the requirement of C1 for lysis in the deficient sera: 1) C4D serum depleted of C1 by low ionic strength precipitation sustained a marked loss of lytic activity which was overcome by the use of the complement-cell intermediate EAC1. 2) C2D serum depleted of C1 by use of a monospecific anti-C1q immunoabsorbent sustained marked loss of hemolytic activity. Addition of properdin to such sera did not restore hemolytic activity. These absorbed sera were able to lyse the cellular intermediate EAC1. 3) Mg-EGTA completely inhibited lysis in C2D and C4D serum by this pathway while allowing normal function of the alternate complement pathway. Function of C4 and C2 in this lytic mechanism was further excluded by the absence of C2 consumption in C4D serum incubated with heavily sensitized RBC, the failure of anti-C4 to inhibit the lytic reaction, and the inability to lyse heavily sensitized RBC by the sequential addition of purified C1, C2, and C EDTA. Participation of components of the alternate complement pathway was suggested by: 1) conversion of the C3 proactivator by heavily sensitized RBC, 2) abolition or marked reduction of the cytotoxic capacity of C4D and C2D serum heated at 50°C for 30 min, conditions which did not deplete C1, and 3) abolition of lysis in C4D or C2D treated with 0.03 M hydrazine. This lytic mechanism utilizes the later acting complement components as shown by complete absence of lysis in rabbit serum lacking C6.

Abstract: Cultured human lymphoid cells sensitized with human histocompatibility (HL-A) antibodies were able to activate the human complement system in vitro. Some HL-A alloantisera selectively activated the alternate complement pathway while other antisera activated only the classical pathway. A third group of alloantisera was equally able to initiate complement action by way of either pathway. The mechanism of complement activation did not correlate with the HL-A antigen present on the cells or the HL-A specificity of the alloantisera, indicating that the antigenic determinants or distribution on the cell surface play on direct role in selecting the pathway of activation. In this completely homologous system the alternate pathway was found to have the same cytolytic potential as the classical pathway. Thus, an altered or damaged membrane is not a prerequisite for the production of cytolytic damage by the alternate pathway. A complete understanding of the mechanism of interaction of membrane bound antigens and antibodies with the complement system may provide a versatile tool for the investigation of membrane antigen expression.

Abstract: Allergic histamine release from rabbit platelets can be initiated through the alternate pathway of C activation. The release reaction proceeds in the absence of the Fc portion of the antibody molecule. The time course of release due to this pathway is slower than that after activation of the classical C sequence. Platelet injury due to immune complexes through both pathways requires the sixth C component.

Abstract: Summary The site of C3 deposition in the direct Arthus reaction induced in a strain of guinea pigs with a nonoperative classical complement pathway but a functional alternate complement pathway (C4 deficient animals) was examined. The area of hemorrhage, induration, and erythema was identical in these animals and controls. The lesions in homozygous C4 deficient animals showed normal infiltration of neutrophils and normal deposition of C3 by immuno-fluorescent visualization; thus the alternate complement pathway could successfully mediate this inflammatory event.

Abstract: We have recently reported that sera of cancer patients possess greater lytic capacity for unsensitized erythrocytes after interaction with a cobra venom protein, CVF, (1). This antibody independent lytic process results from utilization of the terminal complement components (C3 to C9) through an alternate pathway that can be initiated by an enzymatically active complex formed by CVF, C3 proactivator (properdin factor B) and other serum constituents (2–4). A hemolytic assay for this reaction sequence has been described in (5). For minimizing the number of variables inherent in studies of this type with sera of hospitalized patients, an experiment was designed to evaluate levels of CVF-mediated lytic activity in the sera of tumor-bearing mice. Toward this end, mice of both sexes (25 ± 2g), were inoculated subcutaneously with 0.5 mg of 3-methylcholanthrene (3MC) dissolved in sterile corn oil. The largest experimental group contained mice of the Swiss-Webster strain maintained as an outbred colony in this Institute.

Abstract: Relative to pooled adult serum, most cord sera have normal or slightly diminished opsonic activity. Fifteen percent of the cord sera had markedly impaired opsonic power. This activity did not correlate with immunoglobulin or C3 levels but was associated with significantly subnormal concentrations of GBG, a component of the properdin system. The deficient opsonic activity of some cord serum is thus due to hypofunction of the alternate complement pathway.