Terminal Complement Components Research Articles

Failure of Terminal Complement Components to Induce Lysis of Moloney Virus Transformed Lymphocytes

Summary Antibody-dependent complement-mediated lysis of a viral-induced lymphoma cell was confined to the G1 phase of the cell cycle. During the portion of the cell cycle in which the cells were resistant to complement-mediated lysis (S, G2, and M), C5 and C8 became bound to the plasma membrane and ultrastructural lesions characteristic of complement membrane attack developed after incubation of the cells with antibody and complement. Quantitative studies revealed that the plasma membrane of cells bound equivalent numbers of C8 molecules in all phases of the cell cycle. These findings emphasize the importance of events occurring subsequent to activation and attachment of the cytolytic effector system for destruction of this tumor cell.

Alternate Complement Pathway: Elevation of Cobra Venom-Induced Hemolytic Activity in Sera of Tumor-Bearing Mice

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.

Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells.

The effect of five different reactions which activate complement (antibody activation, reduction in ionic strength, acidification, cobra venom factor (CoF) activation, and inulin activation) upon normal and PNH cells was investigated, using normal serum and serum devoid of the fourth component of complement (C4) activity from patients with hereditary angioneurotic edema (HANE) as a source of complement. Both normal and HANE serum lysed paroxysmal nocturnal hemoglobinuria (PNH) cells when complement was activated by acidification, CoF and inulin, indicating that the terminal steps of complement were activated in the absence of C4, presumably by the alternate or properdin pathway. Normal but not HANE serum lysed cells coated with anti-I, indicating that complement was activated by the C1-dependent classic pathway. HANE serum partially supported lysis by serum at reduced ionic strength, indicating that the activation of terminal complement components had occurred through both of the pathways of activation. The amount of the third component of human complement (C3) which was bound to the membrane of lysed and unlysed cells by these procedures was determined by anti-C3 absorption and was found to differ for each method of complement activation. In general, more C3 was bound to lysed cells than to unlysed cells. For given conditions, more was bound to PNH cells than to normal cells. However, very much less bound C3 was required for lysis of the PNH cells than for lysis of normal cells. These two phenomena, especially the latter, account for the marked lysis of PNH cells when complement is activated. Normal cells treated with AET (aminoethylisothiouronium bromide) did not bind more C3 than untreated cells and the lysed cells had less bound C3 than lysed PNH cells.

The lysis of paroxysmal nocturnal haemoglobinuria red cells by serum and cobra factor.

Summary. The complement‐sensitive red cells of patients with paroxysmal nocturnal haemoglobinuria (PNH) are haemolysed in the presence of serum and a protein factor present in cobra venom (CoF). The reaction of CoF and a serum factor, C3 proactivator, requires Mg2+. Once this interaction has taken place, the activation of C3 and the terminal components of complement can occur in the presence of EDTA and result in the lysis of PNH red cells.The concentration of reactants may be adjusted so that all of the complementsensitive red cells of PNH patients and the red cells of no other patients are lysed. A simple but specific and precise test for the presence of the complement‐sensitive red cells of PNH was devised. Normal human red cells treated with trypsin and neuraminidase are not lysed, whereas these cells treated with glutathione or 2‐aminoethylisothiouronium bromide (AET) are lysed.

Activation of Complement by Endotoxin: A Role for γ2 Globulin, C1, C4 and C2 in the Consumption of Terminal Complement Components by Endotoxin-Coated Erythrocytes

Abstract Sheep erythrocytes (E) coated with endotoxin (LPS) from Salmonella typhosa or Veillonella alcalescens (E-LPS) have been employed to define more precisely the nature of a pathway by which LPS activates the terminal components of complement (C). E-LPS consumed C in normal guinea pig serum in a manner identical to free LPS, that is, with marked consumption of C3 through C9 and little utilization of C1, C4 and C2. A γ2-migrating globulin, isolated from normal guinea pig serum by DEAE-cellulose chromatography, was required for C-mediated lysis of E-LPS in diluted guinea pig serum. Absorption experiments with E-LPS indicated that the heat stable γ2 globulin showed specificity for the LPS employed. Cobra venom factor, known to activate C by the alternate pathway (C3 proactivator system), was equally able to deplete C activity in normal serum or serum absorbed with E-LPS. E-LPS reacted with γ2 globulin, C1, C4, C2, followed by C-EDTA resulted in lysis of E-LPS. Deletion of γ2 globulin or any of the earlier acting C components from the reaction prevented lysis. The small amounts of the early acting C components employed, particularly C4 and C2, indicated that it would be difficult to measure their loss in whole serum after activation with LPS by conventional hemolytic assay. This could explain the finding of a relative sparing of C1, C4 and C2 during consumption of C3 through C9 by LPS in normal guinea pig serum. Thus, a classical C pathway involving a “natural” γ2 globulin and early components of C has been defined as a mechanism by which endotoxin can efficiently activate the terminal components of C.

PREVENTION OF THE LOCALIZED AND GENERALIZED SHWARTZMAN REACTIONS BY AN ANTICOMPLEMENTARY AGENT, COBRA VENOM FACTOR

Both localized and generalized Shwartzman reactions were induced in the same rabbits by simultaneous administration of preparatory intravenous and intradermal doses of endotoxin followed in 24 hr by the provocative dose. Control rabbits with more than 80% positive responses showed corresponding changes of platelet, white blood cell, fibrinogen, and hemolytic complement levels. Circulating fibrinogen and fibrin degradation products were detected shortly after the preparatory dose and persisted for at least 3 days. Rabbits given cobra venom anticomplementary factor showed hypocomplementemia (less than 10% of normal), leukocytosis, and elevated fibrinogen levels. After the administration of endotoxin, only one of 15 CVF-treated animals developed a Shwartzman reaction and that was mild. These rabbits showed only minor changes of platelet and fibrinogen levels throughout the experiment although their white blood cell responses were similar to those of the control group. No detectable fibrinogen and fibrin degradation products appeared in circulation, and the hemolytic complement activity increased gradually beginning with the preparatory dose of endotoxin. Thus depletion of terminal complement components (mainly C3) in rabbits is protective against the development of both localized and generalized Shwartzman reactions; its mechanism of action is probably through the sparing of platelets by inhibiting platelet-endotoxin interaction. The essential role of the complement system in Shwartzman reaction indicates that this coagulopathy probably represents a manifestation of immunologic injury.

Kinetics of Bovine Complement

Abstract Although bovine serum is non-hemolytic in standard hemolytic complement assays, it reacts with sensitized sheep erythrocytes to form an intermediate which can be lysed with EDTA-treated guinea pig serum. The optimum condition for the formation of EACBo consists of incubating sensitized sheep erythrocytes and bovine serum at 2°C for 10 min in a low ionic strength buffer (glucose-gelatin-barbital-buffered saline r/2 = 0.075) containing calcium 0.00015 M and magnesium 0.0001 M. In constrast to the corresponding human, guinea pig or equine intermediates, the maximum formation of the bovine lytic intermediate is inversely associated with incubation temperature and is enhanced to a greater extent by magnesium ions. The formation of a bovine lytic intermediate with sheep cells sensitized by rabbit antibody suggests that there is no incompatibility of interaction between early-acting bovine complement components and guinea pig terminal complement components or sensitized sheep cells. Thus the inability of bovine serum to lyse sheep erythrocytes probably is a functional difference of the bovine terminal complement components.

Kinetics of Bovine Complement

Abstract With the use of conglutination assay and different anticomplementary agents, the lytic intermediate formed during interaction of sensitized sheep erythrocytes and diluted bovine serum was shown indirectly to be SHEACBo142. Lysis of this cellular intermediate by guinea pig C3-9 was completed in about 6 min of incubation at 37°C at ionic strengths between 0.075 and 0.125. In contrast to the lability of other corresponding intermediates, SHEACBo142 was extremely stable in low ionic strength buffer (GGBS++) with divalent cations: up to 18 hr at 37°C and 6 days at 2°C. Change of the ionic strength of the buffer to iso-ionicity resulted in gradual decay of the lytic intermediate, whereas presence of EDTA induced a precipitous decay of SHEACBo-142, whose reactivity could be restored with bovine euglobulin or functionally pure human C1. The apparent paradox, that is, that bovine complement was non-hemolytic to sensitized sheep erythrocytes and inhibitory to the lytic activity of guinea pig terminal complement components on SHEACBo142 but hemolytic to sensitized rabbit erythrocytes, further emphasized that in vitvo hemolytic complement assays do not necessarily correlate with in vivo functional activity of complement-dependent or -mediated processes.

Isolation of the Precursor and the Active Form of the C3 Activator from Human Serum

Abstract It had previously been shown that a protein isolated from the venom of Naja naja, the cobra factor, is capable of forming a complex with a β globulin present in human serum. This complex is able to cleave C3 in free solution and apparently activates the terminal complement components beginning with C3. The C3 proactivator (C3PA) which participates in these reactions by forming the enzymatically active complex with cobra factor was isolated from human serum. It was found to be homogeneous on disc- and immunoelectrophoresis. Cleavage of the C3PA into at least two fragments was observed by treatment of serum with a variety of naturally occurring lipopolysaccharides and polysaccharides such as endotoxin, zymosan, inulin or agar. This reaction was accompanied in all instances by the conversion of C3 to its electrophoretically faster form. The larger of the two C3PA fragments (m.w. approximately 60,000) has γ globulin mobility and is antigenically deficient.

Effect of C4 depletion on the utilization of the terminal components of guinea-pig complement by endotoxin.

ENDOTOXIN and antigen-antibody complexes (Ag-Ab) are potent inflammatory agents which induce marked pathophysiological effects in a number of animal species. Both these agents lead to increased vascular permeability, local accumulations of polymorphonuclear leucocytes and alterations in the coagulability of blood1. There is mounting evidence that these similarities derive from endotoxins and Ag-Ab being activators of the complement (C) system, an important mediator of inflammation1'2. When endotoxins or preformed Ag-Ab are reacted with normal guinea-pig (GP) serum so as to induce equivalent consumption of whole haemolytic C, there are, however, striking differences in the consumption profiles of the individual C components3. Although interaction of Ag-Ab with GP serum results in the consumption of CI, C4, and C2 as well as C3 to C9, the interaction of endotoxin with GP serum leads to a marked depletion of C3 to C9 with little or no detectable loss of C1,C4 and C2 (ref. 3).

Glomerular deposition of properdin in acute and chronic glomerulonephritis with hypocomplementemia.

Kidney tissue from 97 patients was studied by immunofluorescent techniques using antiserum to purified properdin. All patients with acute poststreptococcal glomerulonephritis showed deposition of properdin and the third component of complement (C3), either as "humps" on the basement membrane, or in the mesangium. In all cases of chronic membranoproliferative glomerulonephritis, properdin and C3 were localized in the glomeruli, most commonly in a lobular pattern on the basement membrane. Activation of C3 by the properdin system may explain the depressed serum levels of C3 and terminal complement components even though levels of earlier components are normal, and the deposition of C3, often without immunoglobulins, in the kidneys of patients with acute glomerulonephritis or chronic membranoproliferative glomerulonephritis.

Formation and function of a complement-activating enzyme generated from factors of guinea pig serum and cobra venom.

AbstractAn enzymatic complex can be formed by factors from guinea pig serum and cobra venom, which is able to activate C3 bypassing C1, C4 and C2. Formation and action of the enzyme are described. The action on C3 results in an activation of the terminal complement components and in membrane destruction provided suitable membrane receptors are available.