STUDIES ON IMMUNE CELLULAR INJURY : I. CYTOTOXIC EFFECTS OF ANTIBODY AND COMPLEMENT

A model system for the investigation of immune cellular injury in primary isolates of a fixed-tissue cell of human origin has been described, using metabolism-inhibition and uptake of trypan blue as independent criteria of cytoxicity. Cytotoxic effects on human amnion cells were produced by the combination of specific rabbit antibody and factors in normal human serum which were indistinguishable from the components of hemolytic complement and calcium and magnesium ions. The data have been discussed in relation to the apparent multiplicity of serologic systems which can effect cellular injury. The nature of the cell and the source of antibody or normal serum constituents have been emphasized as factors which may influence the experimental selection of a given humoral mechanism

STUDIES ON IMMUNE CELLULAR INJURY : II. FUNCTIONAL ROLE OF C'1 ESTERASE IN IMMUNE CYTOTOXICITY

An initial separation of reaction stages in immune cytoxicity is described. Primary isolates of normal human amnion cells were reacted first with specific rabbit γ2-globulin antibody, then with human C'1 esterase in the presence of Ca++, and finally with human C'2, C'3, and C'4 in the presence of Mg++. Cytotoxicity, as measured by uptake of trypan blue, occurred only when these reactions were performed in the order given and did not occur when any of the constituents was omitted. Inhibition of immune cytotoxicity was achieved with partially purified preparations of human serum inhibitor of C'1 esterase under certain experimental conditions. The data are discussed in relation to the role of C'1 esterase in initiating complement action and the partial biochemical definition of an early event in immune cytotoxicity

COMPLEMENT AS A MEDIATOR OF INFLAMMATION : ENHANCEMENT OF VASCULAR PERMEABILITY BY PURIFIED HUMAN C'1 ESTERASE

Purified preparations of the esterase derived from the first component of complement (C'1 esterase) increased vascular permeability in guinea pig skin, an effect inhibited by triprolidine, an antihistaminic agent, but not by soy bean trypsin inhibitor. The permeability-increasing and esterolytic properties of C'1 esterase were inhibited in parallel by the serum inhibitor of C'1 esterase, diisopropylphosphofluoridate and extremes of temperature and pH. Moreover, the permeability-increasing and esterolytic properties evolved in parallel when C'1 esterase was generated from its subcomponents. How C'1 esterase induces changes in vascular permeability remains unexplained, although the possibility that its action is mediated through a histamine-like agent is attractive

C'1 ESTERASE EFFECT ON ACTIVITY AND PHYSICOCHEMICAL PROPERTIES OF THE FOURTH COMPONENT OF COMPLEMENT

Highly purified C'1 esterase of human serum is capable of inactivating isolated fourth component of human complement (β1E-globulin). Inactivation is accompanied by changes in electrophoretic and ultracentrifugal properties of β1E-globulin. If non-sensitized sheep erythrocytes are present during the action of C'1 esterase on β1E-globulin, a complex is formed consisting of cells and cytolytically active fourth component (EC'4). Thus, inactivation of β1E-globulin by C'1 esterase appears to be preceded by a state of activation enabling β1E-molecules to combine with cell membrane receptors. Acceptor groups appear to be present also in 7S γ-globulin and in β1E-globulin itself, since C'1 esterase can induce the formation of β-β and of β1E-7S γ-globulin complexes

COMPLEMENT AS A MEDIATOR OF INFLAMMATION : II. BIOLOGICAL PROPERTIES OF ANAPHYLATOXIN PREPARED WITH PURIFIED COMPONENTS OF HUMAN COMPLEMENT

Interaction in free solution of highly purified preparations of human C'1 esterase, C'4, C'2, and C'3, in the presence of Mg2+, resulted in rapid generation of an activity indistinguishable by biological criteria from anaphylatoxin. The formation of anaphylatoxin was associated with immunoelectrophoretic conversion of C'3 to anodically faster migrating proteins and was unaffected by the presence or absence of added C'5. The biological properties of human anaphylatoxin prepared in this manner include: contraction and desensitization of isolated guinea pig ileum, failure to contract isolated rat uterus, enhancement of vascular permeability in guinea pig skin, degranulation of mast cells in guinea pig mesentery preparations, and liberation of histamine from suspensions of rat peritoneal mast cells. The smooth muscle-contracting and permeability enhancing properties were fully blocked by an antihistaminic drug, triprolidine. No cross-desensitizing activity on guinea pig ileum was demonstrable between rat and human or guinea pig and human anaphylatoxins but a closer biological relationship between rat and guinea pig anaphylatoxins was observed. It is concluded that anaphylatoxin is a product of the complement system. Its possible relationship to apparently similar activities currently being obtained in other laboratories has been discussed

THE INFLUENCE OF CALCIUM IONS ON THE INACTIVATION OF HUMAN COMPLEMENT AND ITS COMPONENTS BY PLASMIN

Ca++ potentiates the inactivation of human complement by streptokinase-activated plasmin and chloroform-activated bovine plasmin. The optimum concentration of Ca++ varies between 10–2 M and 10–3 M, decreasing with increasing concentrations of streptokinase. The susceptibility of the components of complement to inactivation by plasmin is different in the presence and absence of Ca++. C'2 and C'4 are most readily inactivated by plasmin in the presence of Ca++, while C'1 disappears first in the absence of Ca++. A large part of this C'1 disappearance is due to spontaneous inactivation. Similarities are pointed out between the influence of Ca++ on the inactivation of the components of complement by antigen-antibody systems and by plasmin

THE MACROMOLECULAR NATURE OF THE FIRST COMPONENT OF HUMAN COMPLEMENT

Kinetic and ultracentrifugal experiments demonstrated that the previously described subcomponents of human C'1, designated C'1q, C'1r, and C'1s, interacted with each other in liquid phase to form a macromolecule which was then capable of converting sensitized erythrocytes (EA) to the state EAC'1. The apparent sedimentation constants of C'1q, C'1r, and C'1s and of the macromolecular product of their interaction were approximately 11S, 7S, 4S, and 18S respectively. Association of C'1 subcomponents was prevented and dissociation of macromolecular C'1 was effected by Na3HEDTA and Na2MgEDTA but not by Na2CaEDTA. The rate of formation of macromolecular C'1 was a function of concentration of subcomponents and temperature of interaction, with an apparent energy of activation of 21,000 calories per mol. Ultracentrifugal studies further indicated the macromolecular nature of C'1 in normal human serum. In the absence of EDTA, C'1 sedimented with the serum macroglobulins and C'1 subcomponents were not detected. Conversely, in the presence of EDTA, macromolecular C'1 was not demonstrable and individual C'1 subcomponents could be measured in lighter fractions. The significance of these observations in relation to previous studies on C'1 subcomponents, the role of Ca++ in C'1 function, and the subunit structure of Enzymes has been discussed

THE RELATIONSHIP OF GLYCINE-RICH β-GLYCOPROTEIN TO FACTOR B IN THE PROPERDIN SYSTEM AND TO THE COBRA FACTOR-BINDING PROTEIN OF HUMAN SERUM

Factor B activity of the properdin system was found to be identical with purified glycine-rich β-glycoprotein (GBG) but was distinct from the normal human serum protein capable of forming a C3-inactivating complex with a protein from cobra venom (CoF). Factor B activity coincided with electrophoretically separated GBG genetic variants, whereas the CoF-binding protein did not. GBGase destroyed factor B as it cleaved GBG but did not destroy the C3-inactivating activity of the CoF-binding protein. During incubation of serum with CoF, GBG did not change in molecular size, nor was there any coincidence in the immunoelectrophoretic mobilities of CoF and GBG. It was not possible to precipitate labeled CoF incubated with serum by anti-GBG, nor labeled GBG from serum incubated with CoF by anti-CoF. The CoF-binding capacity of serum was 2 mg/100 ml or less or under 6.5% of the serum concentration of GBG. When labeled CoF was added to serum below the binding capacity, complete complexation of the CoF was demonstrated, whereas CoF was largely uncomplexed when CoF was added in amounts equimolar to GBG

STUDIES ON IMMUNE HUMAN HEMOLYSIS : I. THE KINETICS OF THE DONATH-LANDSTEINER REACTION AND THE REQUIREMENT FOR COMPLEMENT IN THE REACTION

The Donath-Landsteiner reaction was studied using low and high titer antisera and purified antibody, normal and PNH erythrocytes, and human serum complement. The requirement for complement in both the cold and warm phases of the reaction depended upon the level of antibody used and the sensitivity of the cells to hemolytic antibodies. Complement was not necessary in the cold phase using PNH cells and a potent source of antibody, but complement was required to be present at some stage if hemolysis were to occur. Optimal conditions for the cold phase were at 1°C. for 30 minutes at pH 7.4. Ca++ ions were required. Hemolysis in the warm phase occurred within one minute, was optimal at 32°C., and required Mg++. The relation of these observations to previous reports is discussed with respect to discrepant observations on the nature of the Donath-Landsteiner reaction

STUDIES ON THE ACTIVATION OF A PROESTERASE ASSOCIATED WITH PARTIALLY PURIFIED FIRST COMPONENT OF HUMAN COMPLEMENT

It has been found that under a wide range of physico-chemical conditions a positive correlation exists between the rate of disappearance of hemolytically active, partially purified first component of human complement and the rate of activation of an esterase hydrolyzing N-acetyl-L-tyrosine ethyl ester. Both reactions follow the kinetic equation for second order autocatalysis, with an apparent energy of activation of 31,000 calories per mol. They occur optimally at pH 7.3–7.7 and are inhibited by ionic strengths greater than 0.15, by 5 x 105 M ethylenediaminetetraacetic acid, and by a heat-labile serum inhibitor which appears unrelated to any component of complement. The activation of first component to esterase resembles closely the activation of trypsinogen to trypsin. Partially purified first component, containing plasminogen, may also be activated to esterase by addition of streptokinase. The significance of these data with respect to the postulated existence of first component as a proesterase and its possible role in complement-"fixation" is discussed