Reactive Lysis: The Complement-Mediated Lysis of Unsensitized Cells

R e a c t i v e lysls is the consequence of the interaction of two reagents. One, previously called indicator, is a normal s e r u m protein and has been identified as C7 (1); the other is generated b y c o m p l e m e n t activation from certain sera (referred to as " r e a c t o r " sera) and has been called a c t i v a t e d reactor. I n this paper the n a t u r e of the activated reactor is explored and the course of the reaction analyzed. Materials and MethodsThese are described in the preceding paper (1). Diisopropyl fluorophosphate (DFP) was obtained from Boots Pure Drug Co., Nottingham, England.1:10 phenanthroline hydrate was obtained from Hopkins & Williams, Ltd., Chadwell Heath, Essex, England. A 0.5 ~ solution was made in dimethylformamide and this was diluted 1 : 50 in the E/EDTA/agarose mixture. 1Quantitation of Activated Reactor.--An estimate of activated reactor was obtained by filling a standard size well in an E/EDTA/agarose plate with activated reactor and allowing diffusion to occur for 18 hr at room temperature. The plate was then immersed in 1: 50 guinea pig EDTA complement and incubated at 37°C for 6 hr. The diameter of the zone of lysis was measured. RESULTS Fractionation of Activated ReactorAbsorption with Zymosan (4 mg/ml) and Precipitation as Euglobulin.--These were performed in the usual way. Reactor activity in the euglobulin fraction, when redissolved in 0.3 N NaCI, pH 7.2, was stable on storage both at 4°C and at -20°C. Addition of glycerol to a final concentration of 25% allows the activated reactor euglobulin to be stored, certainly for some months, at --20°C without loss of activity.

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“…and inhibited by a reaction between antibody and indicator. This pattern was shown only by antibody to indicator (C7) (1).…”

Section: Molecular Weight Of Activatedmentioning

confidence: 99%

“…The activated reactor euglobulin was fractionated on DEAE-cellulose in 40% glycerol containing buffers essentially as described for the fractionation of C7 in (1).…”

Section: Fractionation On Dielhylaminoethyl ( Deae)-cellulose--mentioning

confidence: 99%

Reactive Lysis: The Complement-Mediated Lysis of Unsensitized Cells

Lachmann

Thompson

1970

The Journal of Experimental Medicine

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228

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“…Discussion We have adapted a system for reactive lysis (18,19), using purified human C5b6, C7, C8, and C9, that produces significant lysis of normal human E . With this system, we have previously shown that PNH-III E lyse 3-5-fold more than normal E at a given C input (5), and that this enhanced lysis is not accompanied by increased C7 binding (6) .…”

Section: Resultsmentioning

confidence: 99%

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes. Studies on C9 binding and incorporation into high molecular weight complexes.

Rosenfeld¹,

Jenkins²,

Leddy³

1986

The Journal of Experimental Medicine

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The human disease paroxysmal nocturnal hemoglobinuria (PNH)' may be regarded as an experiment of nature that has already provided, and should continue to provide, unique insights on the cytolytic function of complement (C) and its regulation (1-4). The studies of our laboratory have been focused particularly on elucidating the process by which the most sensitive type of PNH erythrocyte (E) (designated type III) are abnormally sensitive to in vitro reactive lysis, i.e., to membrane attack initiated by purified C5b6, C7, C8, and C9 in the absence of the earlier activation and amplification steps (5).In an earlier report (6), we presented evidence that for a given input of purified C5b6, type III PNH E (PNH-III E) did not bind '21 I-C7 to a greater extent than normal human E. Rather, the data indicated that PNH E underwent two-fourfold greater lysis for a given number of C7 molecules bound. Compatible results were obtained in less extensive, unpublished studies with radiolabeled C5b6. Compelling independent evidence from other laboratories (7) indicates that the basic unit of the membrane attack complex involves one molecule each of C5b, C6, C7, and C8 in the formation of the C5b-8 complex, to which a variable number of C9 molecules then bind to produce the fully effective membrane attack complexes. Therefore, although C8 uptake has not been directly measured in our system, it seems justified to infer that in reactive lysis the assembly of C5b-8 complexes on PNH-III E from the fluid phase is quantitatively normal . The excessive lysis of the PNH E by the full C5b-9 sequence could result from a quantitative abnormality affecting C9 binding and/or polymerization within the C5b-9 complex, or from a qualitative difference in the disposition or function of the terminal C components after they have become bound to the PNH membrane. The previously reported deficiency of a membrane complement regula-

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“…The phenomenon of 'bystander activation' was described more than 30 years ago, but the biological significance of bystander activation has remained unclear. To demonstrate bystander activation in this work, the original description of 'reactive lysis' (Thompson and Lachmann, 1970), was adapted to detect macrophage inflammasome activation. In the original reactive lysis experiments, the complement C5 convertase was formed on the surface of a complement-activating particle, such as inulin, by incubating inulin with complete serum in 5 mM Mg 2+ and EGTA at 17°C.…”

Section: Bystander Deposition Of the Mac Complex Causes Il-1β Releasementioning

confidence: 99%

Complement-mediated ‘bystander’ damage initiates host NLRP3 inflammasome activation

Suresh

Chandrasekaran

Sutterwala

et al. 2016

Journal of Cell Science

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Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1β and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (T H 17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity.

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Reactive Lysis: The Complement-Mediated Lysis of Unsensitized Cells

Cited by 165 publications

References 10 publications

Reactive Lysis: The Complement-Mediated Lysis of Unsensitized Cells

Reactive Lysis: The Complement-Mediated Lysis of Unsensitized Cells

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes. Studies on C9 binding and incorporation into high molecular weight complexes.

Complement-mediated ‘bystander’ damage initiates host NLRP3 inflammasome activation

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