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Hemolysis by the complement of tanned erythrocytes coated with cobra venom factor: A sensitive method to detect the alternative complement pathway activity of serum
Journal of Immunological Methods, 46 (1981) 85--95
85
Elsevier/North-Holland Biomedical Press
HEMOLYSIS BY THE COMPLEMENT OF T A N N E D ERYTHROCYTES COATED WITH COBRA VENOM FACTOR: A SENSITIVE METHOD TO DETECT THE ALTERNATIVE COMPLEMENT PATHWAY ACTIVITY OF SERUM
HIDECHIKA O K A D A and HIROKO T A N A K A
National Cancer Center Research Institute, Tsukiji, Tokyo 104, Japan (Received 19 February 1981, accepted 6 May 1981)
Sheep (ESh), human (EhU), rabbit (E tab) or guinea pig (E gp) erythrocytes were treated with tannic acid and coated with cobra venom factor (CoVF), which activates the alternative complement pathway (ACP). Tanned erythrocytes (TE) coated with CoVF (TECoVF) were efficiently hemolyzed by guinea pig serum (GPS) and/or rabbit serum (RabS) in Mg2+-EGTA-GVB (gelatin veronal-buffered saline containing 2 mM MgC12 and 10 mM ethyleneglycol-bis(~3-aminoethyl ether)N,Nt-tetraacetate). The reactivity of TE shCoVF, TEhU-CoVF and TErab-CoVF to the ACP of guinea pig and/or rabbit increased with the increased amount of CoVF fixed on TE until it was sensitive enough to be hemolyzed by serum diluted over 80 times in Mg2+-EGTA-GVB. The hemolysis of TECoVF by GPS was confirmed to be the result of ACP activation by the findings that the reaction was inhibited in EDTA-GVB, heating of GPS at 50°C diminished its hemolytic potency, and fractions of factor B and factor D were essential to the sensitization of TECoVF for hemolysis by GPS in EDTA-GVB. On the other hand, none of the TE coated with CoVF were hemolyzed by human serum (HuS) diluted over 1 : 40. Although the low efficiency o f HuS in TE-CoVF hemolysis remains to be explained, TE-CoVF will be useful for the detection o f ACP activity of guinea pig and rabbit sera. INTRODUCTION
Cobra venom has been demonstrated to have anticomplementary activity (Flexner and Noguchi, 1903) and cobra venom factor (CoVF) has been separated from cobra toxin as an anticomplementary factor (Nelson, 1966). CoVF is now known to activate the alternative pathway of the complement (Mfiller-Eberhard and FjellstrSm, 1971), which causes the hemolysis of adjacent erythrocytes (E) and this phenomenon has been utilized as an assay of ACP activity (Brai and Osler, 1972). In this communication, we have fixed CoVF on tanned E (TE) according to Boyden's method (1951) with a modification (Okada et al., 1970) that should promote more efficient hemolysis in the reaction with serum complement. We found that CoVFcoated TE (TE-CoVF) are so highly sensitive to guinea pig and/or rabbit sera that they are hemolyzed at a dilution of over 1 : 80 in Mg2*-EGTA-GVB (gelatin veronal-buffered saline containing 2 mM MgC12 and 10 mM ethyleneglycol-bis-(/~-aminoethylether)-N,N'-tetraacetate, pH 7.5). 0022-1759/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
86 MATERIALS AND METHODS
Diluents EDTA-GVB is a veronal-buffered saline containing 40 mM ethylenediaminetetraacetate (EDTA) and 0.1% gelatin (pH 7.5). Mg2÷-EGTA-GVB is a veronal-buffered saline containing 0.1% gelatin, 2 mM MgC12 and 10 mM ethyleneglycol-bis-(~-aminoethyl ether)N,N'-tetraacetate (pH 7.5). Veronalbuffered saline containing 0.15 mM CaC12, 0.5 mM MgC12 and 0.1% gelatin (GVB 2+) was also used. They were used as diluents in ttie complement reaction to control the presence or absence of Mg2+ and/or Ca 2+ in the reaction solution. For tannic acid treatment of E and fixation of CoVF to the TE, 0.11 M phosphate buffer (0.11 M PB, pH 7.2) was used. Veronal-buffered saline containing 0.4% gelatin (0.4% GVB, pH 7.5) was also prepared for washing away any CoVF that had not been fixed to TE.
Cobra venom factor (CoVF) CoVF was purified from venom of the snake Naja naja oxiana (Sigma, St. Louis, MO) according to the method of Ballow and Cochrane (1969). To test its activity, a dilution of the CoVF was incubated at 37°C for 1 h with an equal volume of guinea pig serum (GPS) diluted 1 : 10 in GVB 2+ and the residual complement activity determined by hemolytic activity on antibodysensitized sheep E. The amount of CoVF required to deplete 50% of the hemolytic complement activity in 1.0 ml of 1 : 1 0 GPS in GVB 2÷ was defined as 1 unit. The CoVF preparation used for the experiments reported here showed an anticomplementary activity of 2400 units/ml and an optical density of 0.54 at 280 nm. Radiolabelling of CoVF with 12sI was performed with the chloramine T method (McConahey and Dixon, 1966) and the 12sIlabeled CoVF was used to monitor the amount of CoVF fixed on tanned erythrocytes.
Phospholipase A2 (PLA2) PLA2 from bee venom (DL100 mice: i.v. 7.5 mg/kg) was purchased from Boehringer Mannheim (F.R.G.).
Erythrocytes (E ) Sheep blood in Alsever's solution was purchased from Nippon Bio-Test Lab., Tokyo. Human blood and rabbit blood were obtained by venipuncture with a syringe containing 2 ml of 0.1 M EDTA (pH 7.5) to mix immediately with the 8 ml of blood withdrawn. Strain 2 guinea pig blood was obtained by heart puncture with a syringe containing 0.1 M EDTA (0.25 volume of blood to be obtained). From the blood sample suspensions of sheep (ESh), human (Ehu), guinea pig (E g°) and rabbit E (E tab ) w e r e prepared by washing each 3 times with EDTA-GVB and 3 times with 0.11 M PB. Erythrocytes were suspended at 1 × 109/ml in 0.11 M PB.
87
Tannic acid treatment Tannic acid treatment of E to fix CoVF o n t o the cell membrane was performed according to Boyden's m e t h o d (1951) with a slight modification (Okada et al., 1970). One volume o f 1 X 109/ml E in 0.11 M PB was mixed with an equal volume o f tannic acid (50 pg/ml) solution in 0.11 M PB and incubated at 37°C for 1 h. The tannic acid-treated E (TE) were washed 3 times with 0.11 M PB and suspended at 1 X 109/ml in the same medium. The TE suspension was mixed with an e q u a l volume o f C o V F diluted in 0.11 M PB and incubated at 4°C overnight and then at 37°C for 20 min. The TE treated with C o V F ( T E ~ o V F ) were washed with 0.4% GVB 3 times and suspended in Mg2*-EGTA-GVB. The a m o u n t of C o V F fixed on TE ranged b e t w e e n 3 and 15% o f the input C o V F when monitored b y ~2SI-labeled CoVF. In an experiment, C o V F was replaced b y PLA2. Complement sou rce Freshly prepared human serum (HuS), strain 2 guinea pig serum (GPS), C4 deficient guinea pig serum (C4DGPS) and rabbit serum (RabS) were stored at --70°C until use. Determination o f extent o f hemolysis After incubation of E with serum diluted in Mg2÷-EGTA-GVB or in EDTA~3VB, the reaction mixtures were diluted with E D T A ~ V B (5 times or more), centrifuged at 800 × g for 5 min and the OD414 o f the supernatants determined spectrophotometrically on a double beam Hitachi spectrop h o t o m e t e r model 200. E hemolyzed with distilled water was prepared as a 100% hemolysis control. Serum color controls were prepared by diluting serum with EDTA-GVB up to the same volume of the experiment mixture. The hemolysis rate (y) of the experimental reaction was calculated from the optical densities of experimental reaction, 100% hemolysis and serum color as follows: hemolysis rate (y) = experimental -- serum color 100% hemolysis
50% hemolysis (HLso) unit by complement One HLs0 unit/ml was taken as the serum activity which caused 50% hemolysis of an equal volume of 1 X 10S/ml E in Mg2*-EGTA-GVB. The HLs0 value was determined in a manner similar to the determination of CHs0. (Mayer, 1961). Briefly, the function y ( x ) - - y ( o ) / 1 - - y ( x ) was plotted against the reciprocal dilution (x) on logarithmic paper: where y(o) and y(x) stand for the hemolysis rate w i t h o u t serum and that at a serum dilution o f x respectively. From the dose
88 RESULTS
Hemolysis of TE'~-CoVF by GPS in Mg2÷-EGTA-GVB E sh were treated with tannic acid and fixed with varying amounts of CoVF as described in Materials and Methods. 100 pl of the 1 × 108/ml TE shCoVF were mixed with an equal volume of GPS diluted in Mg2÷-EGTA-GVB and incubated at 37°C for 60 min with shaking. Following addition of 800 pl of EDTA~:~VB to the reaction mixtures, the tubes were centrifuged at 800 X g for 5 rain, the OD414 o f the supernatant determined and the percentage hemolysis calculated. Table 1 lists the percentage hemolysis of TE ~a treated with varying amounts of CoVF by varying concentrations of GPS in Mg2+-EGTA-GVB. Although the background hemolysis (without GPS) was relatively high (up to 15%) due to the frangibility of TE sh, the sensitivity to hemolysis by GPS was increased remarkably, depending on the amount of CoVF coated. The dilution of GPS which caused 50% hemolysis of each TESh~oVF was calculated from the percent hemolysis. TESh~oVF prepared with 1200 units/ml of CoVF was so sensitive to GPS as to be 50% hemolyzed by 1/152 GPS in Mg2÷-EGTA-GVB. TE ~h not treated with CoVF had a tendency to be hemolyzed by GPS although a net hemolysis by 1/10 GPS was only 17.2%. In the control experiment where EDTA~:~VB was used instead of Mg2÷-EGTA-GVB as a diluent, no specific hemolysis of TE ~hCoVF by GPS was observed.
Hemolysis by HuS of TE'h-CoVF The same lots of TESh~oVF suspensions used for the above experiment with GPS shown in Table 1 were also incubated with an equal volume of dilutions of HuS in Mg2+-EGTA-GVB at 37°C for 60 min. Although TE ~h TABLE 1 Hemolysis by GPS of TE sh coated with CoVF. CoVF a
% hemolysis in Mg2+-EGTA-GVB b by GPS diluted to
HLs0 c
(U/ml) 0 4 12 40 120 400 1 200
(/ml) 1/10
1/20
1/40
1/80
1/160
1/320
0
29.6 44.9 72.8 105.0 104.0 106.0 102.6
25.9 36.5 38.6 84.3 93.6 103.8 105.6
23.8 24.6 35.1 60.9 79.7 100.0 102.2
22.4 19.7 25.4 35.9 51.4 80.4 87.9
20.2 17.4 18.4 23.1 27.3 41.5 49.6
18.7 14.5 16.1 17.4 16.7 22.3 27.6
15.0 13.9 12.3 13.5 7.4 12.8 15.1
<5 4.4 14 45 79 125 152
a Concentration of CoVF reacted with 5 × 10S/ml TE sh to prepare TEsh-CoVF. b Replacement of Mg:+-EGTA-GVB with EDTA-GVB completely suppressed the hemolysis by GPS over the hemolysis of GPS-absent controls. c Reciprocal dilution of GPS for 50% hemolysis.
89
was hemolyzed by HuS in Mg2+-EGTA-GVB (HLs0 was 7.2/ml), no clear dose
Hemolysis sensitivity of erythrocytes from different species E h", E gp and E ~ab were treated with tannic acid (50 #g/ml) and coated with varying amounts of CoVF (0, 4, 12, 4 0 , 1 2 0 , 4 0 0 or 1200 units/ml) to prepare TEhU-CoVF, TEgP~oVF and TE~aD~oVF, respectively. They were suspended at 1 X 10S/ml in Mg2*-EGTA-GVB and incubated with an equal volume of serially diluted GPS, HuS or RabS in Mg2+-EGTA-GVB (1/10, 1/20, 1/40, 1/80, 1/160, 1/320 or 0). Sera diluted in EDTA~3VB were used as controls. From the hemolytic rates, the HLs0 titer of each serum on each TE-CoVF was calculated. As shown in Table 3, TEhU-CoVF became sensitive to GPS and RabS depending on the increase of CoVF used. TEgP-CoVF was efficiently hemolyzed by RabS and TErab-CoVF was efficiently hemolyzed by GPS. On the other hand, none of the erythrocytes coated with CoVF became significantly sensitive to HuS.
Hemolysis by C4DGPS in the presence of unfixed CoVF To examine the hemolytic sensitivity of E, TE or TE-CoVF (prepared by the reaction of 5 × l 0 s TE in 400 units/ml CoVF) to C4DGPS in the presence of CoVF in the fluid phase, 50/A of 2 X 108/ml of each type of E, 50 pl of CoVF (2400, 240, 24, 2.4 or 0 units/ml) and 100 pl of C4DGPS (1/4, 1/8, 1/16, 1/32, 1/64, 1/128 or 0) were mixed and incubated at 37°C for 1 h. After the reaction the extent of hemolysis was determined spectroTABLE 2 Hemolysis by HuS of TE sh coated with CoVF.
CoVF a (U/ml)
0 4 12 40 120 400 1 200
% hemolysis in Mg2+-EGTA-GVB b by HuS diluted to
HLs0 (/ml)
1i10
1/20
1/40
1/80
1/160
0
46.5 45.8 44.9 63.7 51.1 48.7 55.3
29.2 29.9 26.9 37.5 28.6 24.8 28.8
18.6 19.8 15.7 15.0 20.1 14.6 16.5
17.9 18.3 17.3 14.4 18.3 13.2 15.6
18.8 19.3 16.3 15.6 18.5 12.2 14.9
12.6 12.6 12.1 11.8 14.9 10.3 13.5
7.2 6.9 7.2 12.2 8.6 8.7 9.7
a Concentration o f CoVF reacted with 5 x 10S/ml TE Ih to prepare TEsh-CoVF. b Replacement o f Mg2+-EGTA-GVB with EDTA-GVB completely suppressed the hemolysis by HuS over the hemolysis of HuS-absent controls. c Reciprocal dilution o f HuS for 50% hemolysis.
90 TABLE 3 R e c i p r o c a l d i l u t i o n o f sera w h i c h caused 50% h e m o l y s i s ( H L s 0 / m l ) o f n a t i v e E o r T E coated with varying amounts of CoVF. Erythrocytes
CoVF a
HLs0 (/ml) of
(U/ml) GPS
HuS
RabS
E hu T E hu
0 0 4 12 40 120 400 1200
<10 <10 12 18 25 44 74 110
<10 <10 <10 <10 <10 <10 12 15
<10 14 21 53 66 99 113 123
EgP TEgP
0 0 4 12 40 120 400 1 200
<10 13 15 13 13 10 14 18
<10 10 <10 <10 <10 13 14 18
<10 14 13 <10 15 24 63 88
S rab T E tab
0 0 4 12 40 120 400 1 200
<10 <10 <10 17 33 47 60 83
~'10 13 13 14 14 14 14 15
~"10 <10 <10 <10 <10 <10 14 25
a C o n c e n t r a t i o n o f C o V F used for c o n j u g a t i o n .
photometrically. As shown in Table 4, even with the optimum concentrations of CoVF, the HLs0 of C 4 D G P S remained at most 11.6/mi and 40/ml on native E hu and E sh respectively,and at most 52/mi and 34/mi on T E hu and T E sh respectively.O n the other hand, the presence of C o V F in the fluid phase was inhibitory on the hemolytic reaction of T E h U ~ o V F and T E shC o V F by C4DGPS. This indicated that fluid-phase activation of the complement caused it to react with the C o V F fixed on the cell membrane. As shown in Table 4, tannic acid treatment of E hu and E sh made them sensitive to hemolytic reaction by C 4 D G P S in the absence of C o V F in the fluid phase. Although the mechanism underlying the increased reactivity with G PS remains to be studied, tannic acid treatment might have induced the ACPactivating ability,as in the case of neuraminidase treatment (Fearon, 1978; Paungburn and MiiUer-Eberhard, 1978).
91
TABLE 4 HLs0/ml of C4DGPS on E, TE and TE-CoVF in the presence of fluid phase CoVF. E species
Ehu TE hu TEhU-CoVF Esh TE sh TE sh.CoVF
Concentration (U/ml) of CoVF in fluid phase 0
0.6
6
60
600
2.2 13.3 100.0 <1.0 4.5 140.0
2.5 17.0 87.0 3.7 7.6 140.0
5.3 32.0 63.0 25.5 22.5 92.0
11.0 52.0 48.5 40.0 34.0 48.5
11.6 27.0 26.5 27.0 21.5 22.7
Effect of phospholipase A2 on hemolysis by GPS of TE In the C o V F preparation, there might have been a contamination b y phospholipase as indicated b y Lachmann et al. (1976). 0.4 ml samples o f 1 × 109/ml TE hu in 0.11 M PB were mixed with an equal volume of a serial dilution o f PLA2 in 0.11 M PB (100, 10, 1, 0.1, 0.01 or 0.001 pg/ml or buffer alone). After incubation overnight at 4°C, followed b y incubation at 37°C for 20 min, the TEhU-PLA2 were washed and suspended at 1 × 108/ml in Mg2+-EGTA~:~VB. 100 pl of the TEhU-PLA2 were incubated with an equal volume of diluted GPS at 370C for 1 h and t~he hemolytic rate was determined. PLA2 treatment did n o t increase the sensitivity of erythrocytes to hemolytic reaction b y GPS, indicating that PLA2 was n o t involved in the increased hemolytic sensitivity o f T E ~ o V F to GPS.
Factors essential for TE-Co VF hemolysis Partial purification was performed to confirm the factors in GPS essential to T E ~ o V F hemolysis. 95 ml of GPS were mixed with 285 ml o f 5 mM phosphate buffer containing 1 mM EDTA (pH 7.5) and settled in an ice bath for 1 h and centrifuged at 16 000 X g for 30 rain to remove the precipitated euglobulin fraction. The supernatant was applied on a DE-52 column (5.5 cm × 40 cm), equilibrated with 5 mM phosphate buffer containing 50 mM NaC1 and 1 mM EDTA. (pH 7.5), and the effluent collected in 15 ml samples. The hemolytic activity was assayed b y incubating the fractions in Mg2+-EGTA-GVB with T E r a b ~ o V F at 37°C for 20 min, followed b y addition o f 1/10 GPS in EDTA-GVB and further incubation at 37°C for 1 h. A hemolytically active fraction was obtained soon after the main protein peak o f effluent. The active fractions, from tube numbers 56 to 64, w e r e p o o l e d and concentrated to 4.4 ml under negative pressure in a visking tube. The concentrated sample was filtered through a Sephadex G-100 column (2.5 cm × 40 cm) in 0.3 M NaC1 containing 1 mM EDTA (pH 7.5) and effluent was collected as 5-ml fraction. On the gel filtration t w o peaks with absor-
92
bances at 280 nm (OD2s0) were observed. The peaks were at Fr 14 and Fr 22 with OD280 values of 1.55 and 0.33. Although neither Fr 14 or Fr 22 from the column had any activity to sensitize T E r a e ~ o v F to hemolysis b y the addition of 1/10 GPS in EDTA-GVB, the presence of a 1/5 dilution of either fraction enabled any of the other fractions to cause hemolysis of TECoVF, after a further incubation with GPS in E D T A ~ ] V B at 37°C. The synergistic ability of Fr 14 and Fr 22 is demonstrated in Fig. 1. Fr 14 and Fr 22 were diluted 1 : 15 in Mg2*-EGTA-GVB and mixtures prepared of the following combinations: Fr 14 and Fr 22, Fr 14 and buffer, and Fr 22 and buffer. Two volumes of the mixtures were mixed with 1 vol of 1 × 108/ml TErab-CoVF (prepared with 400 units/ml CoVF) for the period indicated at 30°C and then 300 #1 of the reaction mixtures were added with 200 ~1 of EDTA-GVB and 200 /A o f 1/10 GPS in EDTA-GVB. After further incubation at 37°C for 1 h, the extent of hemolysis was determined spectrophotometrically. To be hemolyzed, TErab-CoVF was sensitized by incubation with a mixture of Fr 14 and Fr 22 b u t n o t with Fr 14 or Fr 22 alone (Fig. 1). Since Fr 14 and Fr 22 correspond with fractions B and D in a comparison with the results reported b y Brade et al. (1972), the hemolysis of TE-CoVF b y serum is suggested to be induced b y the reaction of ACP.
Effect o f heating on hemolytic potency o f GPS on TE-CoVF GPS was incubated at 50°C for 16 and 32 min. After the heating, GPS were chilled immediately in an ice bath and serially diluted in Mg2*-EGTA GVB. To 100 pl o f the diluted GPS, 100 pl of 1 X 108/ml T E S h ~ o V F (prepared coating TE am with 400 units/ml CoVF) was added and the mixture incubated at 37°C for 60 min. As shown in Fig. 2, hemolytic activity of GPS
40 •
r22
3O
"6
20
"r
10
20
30
Time i n r n ; n u t e s
Fig. 1. The ability of Fr 14 and/or F r 22, from the Sephadex G-100 gel filtration, to sensitize TErab-CoVF for hemolysis by GPS in EDTA-GVB. TErab-CoVF was mixed with Fr 14 and/or Fr 22 in Mg2+-EGTA-GVB and incubated at 30°C for the period indicated on the abscissa. Both Fr 14 and Fr 22 were required for the sensitization of TErab-CoVF.
93 50
I00
150
(HLso)
unheated
50°C16min ~
50°= 32 min
Fig. 2. HLso/ml on T E S h ~ o V F of GPS before or after heating at 50°C for 16 min or 32 rain. The hemolytic activity-was determined in the absence (solid b a r s ) o r presence (open bars) of the factor B supplemented fraction.
was reduced from 132 HLs0/ml to 83 HLs0/ml (37% reduction) and 26 HLs0/ ml (80% reduction) by heating at 50°C for 16 rain and for 32 rain respectively. To 100/~l of the GPS dilution, 100 #l of 1 × 10 s T E s n ~ o v F supplemented with a 1/4 dilution of Fr 14 used as in Fig. 1 (factor B fraction) were added to examine the extent of hemolysis on TESh~oVF after the reconstitution of the heated serum with the factor B fraction. The hemolytic activity of 50°C heated GPS after this reconstitution, recovered to 140 HLs0/ ml from 83 HLs0/ml (16-rain heated GPS) and to 92 HLs0/ml from 26 HLs0/ml (32-rain heated GPS). The values of 140 HLs0/ml and 92 HLs0/ml were 81% and 53% of the 173 HLs0/ml of unheated GPS in the presence of the supplemented factor B fraction. The findings suggest that factor B of ACP, which is labile at 50°C heating, will be essential in the hemolytic reaction of TE-CoVF by serum in Mg2*-EGTA-GVB. DISCUSSION
The interaction mechanism of CoVF has been well analyzed in human ACP (Miiller-Eberhard and Fjellstrom, 1971; Cooper, 1973). CoVF binds factor B, and in the presence of factor D forms a CoVF • Bb complex which has C3 convertase activity. ACP activation of serum induced by CoVF results in the hemolysis of incidental E, and this has been successfully applied as a hemolytic assay of ACP by Brai and Osier (1972). To increase the hemolytic efficiency of CoVF-induced hemolysis by ACP activation of the serum complement, we fixed by CoVF onto E which had been treated with tannic acid according to Boyden (1951), with slight modification (Okada et al., 1970). The fixation of CoVF on the E membrane results in the activation of serum ACP only on the cell surface and not in the fluid phase. The results in this
94 report demonstrate that this was the case. The addition of CoVF in the fluid phase inhibited the hemolysis of T E ~ o V F by GPS, indicating that the fluidphase activation of ACP by CoVF reduced the supply of complement factors to react on the surface-bound CoVF. In other words, activation of complement by. membrane-bound CoVF effectively results in the hemolysis of the E. TEhu-CoVF, T E ' h ~ o V F and TErab-CoVF were so sensitive that they were hemolyzed by GPS diluted over I : 80 when E were coated with a large amount of CoVF (Tables 1 and 3). The hemolysis of T E ~ o V F by GPS was the result of ACP activation since the hemolysis occurred in Mg2*-EGTA GVB but not in EDTA-GVB. Fractions of factors B and D were essential for the sensitization of TE-CoVF to hemolysis b y a subsequent reaction with GPS in EDTA-GVB (Fig. 1). Heating GPS at 50°C reduced its hemolytic potency, which was recovered by supplementation with the factor B fraction (Fig. 2). Although the CoVF preparation might have been contaminated with PLA2 (Lachmann et al., 1976), the enzyme would not have sensitized erythrocytes for hemolysis by serum since PLA2 coated TE hu (TE-PLA2) was not sensitive to GPS. Although GPS or RabS efficiently hemolyzed TEhu~oVF, TE shCoVF, TEgP~:~oVF and/or TErab~:~oVF, HuS did not effectively take advantage of the presence of CoVF fixed on the cell membrane (Tables 2 and 3). This might be due to the restricted efficiency of later components of the complement, since the addition of GPS in EDTA-GVB following a 20 min incubation of T E ~ o V F with HuS in Mg2+-EGTA~:~VB enhanced the hemolysis of HuS reacted-TE~oVF (unpublished data). Although little appreciable enhancement of hemolysis was noted by the addition of 1/10 homologous serum in EDTA-GVB, following a 20-rain incubation of T E ~ o V F with a dilution of GPS or RabS in Mg2*-EGTA-GVB, the .addition of serum to EDTA-GVB may be used to examine the ACP potency of serum. This procedure will eliminate a possible restriction of hemolysis by a shortage of lateacting components of the complement, especially in those cases such as C6deficient rabbit serum and serum in which C7 has been exhausted. The optimum incubation condition prior to the addition of EDTA-serum is under investigation. Although T E ~ o V F is not sufficiently sensitive to HuS, it is highly sensitive, and useful, in the detection of the ACP activity of GPS and RabS. However, the sensitivity of T E ~ o V F depends on the amount of CoVF fixed and so the ACP titer will need to be standardized with a standard serum sample to compare the activity of different experiments. Another disadvantage of TE-CoVF was the frangibility of TE, which caused 5--15% spontaneous hemolysis without the reaction of complement, although this was not a serious problem. It is noteworthy that tannic acid treatment made E hu and E sh sensitive to hemolytic reaction by GPS. Tannic acid treatment may induce or enhance the ACP-activating capacity of E by a mechanism similar to that observed on neuraminidase-treated E ' h (Fearon, 1978; Pangburn and Miiller-Eberhard, 1978). Recently, ACP of mouse and rat sera have been shown to form a CoVF-Bb complex in the presence of EDTA (Goldman et al., 1979). In preliminary
95 experiments, T E ~ o V F was also sensitive to rat and mouse c o m p l e m e n t in E D T A in accordance with their results (data n o t shown). The o p t i m u m react i o n c o n d i t i o n o f T E - C oV F f or t he d e t e c t i o n o f m a r i n e ACP activity is now u n d e r investigation. ACKNOWLEDGEMENTS We t h a n k Mr. Masahiro Y o k o t a f or his excellent assistance and Miss Chiaki Iida f o r ty p in g the manuscript. This w o r k was support ed by Grants-in-Aid f r o m th e Ministry o f E d u c a t i o n , Science and Culture, Japan and f r o m t he Adult Disease Clinic Memorial F o u n d a t i o n . REFERENCES Ballow, M. and C.G. Cochrane, 1969, J. Immunol. 103,944. Boyden, S.V., 1951, J. Exp. Med. 93, 107. Brade, V., C.T. Cook, H.S. Shin and M.M. Mayer, 1972, J. Immunol. 109, 1174. Brai, M. and A.G. Osier, 1972, Proc. Soc. Exp. Biol. Med. 140, 1 116. Cooper, N.R., 1973, J. Exp. Med. 137, 451. Fearon, D.T., 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 1 971. Flexner, S. and H. Noguchi, 1903, J. Exp. Med. 6,277. Goldmari, J.H., S. Bangalore and M.B. Goldman, 1979, J. Immunol. 123, 2 421. Lachmann, P.J., L. Halbwachs, A. Gewurz and H. Gewurz, 1978, Immunology 31,961. Mayer, M.M., 1961, in: Experimental Immunochemistry, 2nd edn., eds. E.A. Kabat and M.M. Mayer (Charles C. Thomas, Springfield, IL) p. 133. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29,185. Miiller-Eberhard, H.J. and K. FjellstrSm, 1971, J. Immunol. 107, 1 666. Nelson, Jr., R.A., 1966, Survey Ophthal. 11,498. Okada, H., S. Kawachi and K. Nishioka, 1970, Biochim. Biophys. Acta 208, 541. Pangburn, M.K. and H.J. Miiller-Eberhard, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 2 416.
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Elsevier/North-Holland Biomedical Press
HEMOLYSIS BY THE COMPLEMENT OF T A N N E D ERYTHROCYTES COATED WITH COBRA VENOM FACTOR: A SENSITIVE METHOD TO DETECT THE ALTERNATIVE COMPLEMENT PATHWAY ACTIVITY OF SERUM
HIDECHIKA O K A D A and HIROKO T A N A K A
National Cancer Center Research Institute, Tsukiji, Tokyo 104, Japan (Received 19 February 1981, accepted 6 May 1981)
Sheep (ESh), human (EhU), rabbit (E tab) or guinea pig (E gp) erythrocytes were treated with tannic acid and coated with cobra venom factor (CoVF), which activates the alternative complement pathway (ACP). Tanned erythrocytes (TE) coated with CoVF (TECoVF) were efficiently hemolyzed by guinea pig serum (GPS) and/or rabbit serum (RabS) in Mg2+-EGTA-GVB (gelatin veronal-buffered saline containing 2 mM MgC12 and 10 mM ethyleneglycol-bis(~3-aminoethyl ether)N,Nt-tetraacetate). The reactivity of TE shCoVF, TEhU-CoVF and TErab-CoVF to the ACP of guinea pig and/or rabbit increased with the increased amount of CoVF fixed on TE until it was sensitive enough to be hemolyzed by serum diluted over 80 times in Mg2+-EGTA-GVB. The hemolysis of TECoVF by GPS was confirmed to be the result of ACP activation by the findings that the reaction was inhibited in EDTA-GVB, heating of GPS at 50°C diminished its hemolytic potency, and fractions of factor B and factor D were essential to the sensitization of TECoVF for hemolysis by GPS in EDTA-GVB. On the other hand, none of the TE coated with CoVF were hemolyzed by human serum (HuS) diluted over 1 : 40. Although the low efficiency o f HuS in TE-CoVF hemolysis remains to be explained, TE-CoVF will be useful for the detection o f ACP activity of guinea pig and rabbit sera. INTRODUCTION
Cobra venom has been demonstrated to have anticomplementary activity (Flexner and Noguchi, 1903) and cobra venom factor (CoVF) has been separated from cobra toxin as an anticomplementary factor (Nelson, 1966). CoVF is now known to activate the alternative pathway of the complement (Mfiller-Eberhard and FjellstrSm, 1971), which causes the hemolysis of adjacent erythrocytes (E) and this phenomenon has been utilized as an assay of ACP activity (Brai and Osler, 1972). In this communication, we have fixed CoVF on tanned E (TE) according to Boyden's method (1951) with a modification (Okada et al., 1970) that should promote more efficient hemolysis in the reaction with serum complement. We found that CoVFcoated TE (TE-CoVF) are so highly sensitive to guinea pig and/or rabbit sera that they are hemolyzed at a dilution of over 1 : 80 in Mg2*-EGTA-GVB (gelatin veronal-buffered saline containing 2 mM MgC12 and 10 mM ethyleneglycol-bis-(/~-aminoethylether)-N,N'-tetraacetate, pH 7.5). 0022-1759/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
86 MATERIALS AND METHODS
Diluents EDTA-GVB is a veronal-buffered saline containing 40 mM ethylenediaminetetraacetate (EDTA) and 0.1% gelatin (pH 7.5). Mg2÷-EGTA-GVB is a veronal-buffered saline containing 0.1% gelatin, 2 mM MgC12 and 10 mM ethyleneglycol-bis-(~-aminoethyl ether)N,N'-tetraacetate (pH 7.5). Veronalbuffered saline containing 0.15 mM CaC12, 0.5 mM MgC12 and 0.1% gelatin (GVB 2+) was also used. They were used as diluents in ttie complement reaction to control the presence or absence of Mg2+ and/or Ca 2+ in the reaction solution. For tannic acid treatment of E and fixation of CoVF to the TE, 0.11 M phosphate buffer (0.11 M PB, pH 7.2) was used. Veronal-buffered saline containing 0.4% gelatin (0.4% GVB, pH 7.5) was also prepared for washing away any CoVF that had not been fixed to TE.
Cobra venom factor (CoVF) CoVF was purified from venom of the snake Naja naja oxiana (Sigma, St. Louis, MO) according to the method of Ballow and Cochrane (1969). To test its activity, a dilution of the CoVF was incubated at 37°C for 1 h with an equal volume of guinea pig serum (GPS) diluted 1 : 10 in GVB 2+ and the residual complement activity determined by hemolytic activity on antibodysensitized sheep E. The amount of CoVF required to deplete 50% of the hemolytic complement activity in 1.0 ml of 1 : 1 0 GPS in GVB 2÷ was defined as 1 unit. The CoVF preparation used for the experiments reported here showed an anticomplementary activity of 2400 units/ml and an optical density of 0.54 at 280 nm. Radiolabelling of CoVF with 12sI was performed with the chloramine T method (McConahey and Dixon, 1966) and the 12sIlabeled CoVF was used to monitor the amount of CoVF fixed on tanned erythrocytes.
Phospholipase A2 (PLA2) PLA2 from bee venom (DL100 mice: i.v. 7.5 mg/kg) was purchased from Boehringer Mannheim (F.R.G.).
Erythrocytes (E ) Sheep blood in Alsever's solution was purchased from Nippon Bio-Test Lab., Tokyo. Human blood and rabbit blood were obtained by venipuncture with a syringe containing 2 ml of 0.1 M EDTA (pH 7.5) to mix immediately with the 8 ml of blood withdrawn. Strain 2 guinea pig blood was obtained by heart puncture with a syringe containing 0.1 M EDTA (0.25 volume of blood to be obtained). From the blood sample suspensions of sheep (ESh), human (Ehu), guinea pig (E g°) and rabbit E (E tab ) w e r e prepared by washing each 3 times with EDTA-GVB and 3 times with 0.11 M PB. Erythrocytes were suspended at 1 × 109/ml in 0.11 M PB.
87
Tannic acid treatment Tannic acid treatment of E to fix CoVF o n t o the cell membrane was performed according to Boyden's m e t h o d (1951) with a slight modification (Okada et al., 1970). One volume o f 1 X 109/ml E in 0.11 M PB was mixed with an equal volume o f tannic acid (50 pg/ml) solution in 0.11 M PB and incubated at 37°C for 1 h. The tannic acid-treated E (TE) were washed 3 times with 0.11 M PB and suspended at 1 X 109/ml in the same medium. The TE suspension was mixed with an e q u a l volume o f C o V F diluted in 0.11 M PB and incubated at 4°C overnight and then at 37°C for 20 min. The TE treated with C o V F ( T E ~ o V F ) were washed with 0.4% GVB 3 times and suspended in Mg2*-EGTA-GVB. The a m o u n t of C o V F fixed on TE ranged b e t w e e n 3 and 15% o f the input C o V F when monitored b y ~2SI-labeled CoVF. In an experiment, C o V F was replaced b y PLA2. Complement sou rce Freshly prepared human serum (HuS), strain 2 guinea pig serum (GPS), C4 deficient guinea pig serum (C4DGPS) and rabbit serum (RabS) were stored at --70°C until use. Determination o f extent o f hemolysis After incubation of E with serum diluted in Mg2÷-EGTA-GVB or in EDTA~3VB, the reaction mixtures were diluted with E D T A ~ V B (5 times or more), centrifuged at 800 × g for 5 min and the OD414 o f the supernatants determined spectrophotometrically on a double beam Hitachi spectrop h o t o m e t e r model 200. E hemolyzed with distilled water was prepared as a 100% hemolysis control. Serum color controls were prepared by diluting serum with EDTA-GVB up to the same volume of the experiment mixture. The hemolysis rate (y) of the experimental reaction was calculated from the optical densities of experimental reaction, 100% hemolysis and serum color as follows: hemolysis rate (y) = experimental -- serum color 100% hemolysis
50% hemolysis (HLso) unit by complement One HLs0 unit/ml was taken as the serum activity which caused 50% hemolysis of an equal volume of 1 X 10S/ml E in Mg2*-EGTA-GVB. The HLs0 value was determined in a manner similar to the determination of CHs0. (Mayer, 1961). Briefly, the function y ( x ) - - y ( o ) / 1 - - y ( x ) was plotted against the reciprocal dilution (x) on logarithmic paper: where y(o) and y(x) stand for the hemolysis rate w i t h o u t serum and that at a serum dilution o f x respectively. From the dose
88 RESULTS
Hemolysis of TE'~-CoVF by GPS in Mg2÷-EGTA-GVB E sh were treated with tannic acid and fixed with varying amounts of CoVF as described in Materials and Methods. 100 pl of the 1 × 108/ml TE shCoVF were mixed with an equal volume of GPS diluted in Mg2÷-EGTA-GVB and incubated at 37°C for 60 min with shaking. Following addition of 800 pl of EDTA~:~VB to the reaction mixtures, the tubes were centrifuged at 800 X g for 5 rain, the OD414 o f the supernatant determined and the percentage hemolysis calculated. Table 1 lists the percentage hemolysis of TE ~a treated with varying amounts of CoVF by varying concentrations of GPS in Mg2+-EGTA-GVB. Although the background hemolysis (without GPS) was relatively high (up to 15%) due to the frangibility of TE sh, the sensitivity to hemolysis by GPS was increased remarkably, depending on the amount of CoVF coated. The dilution of GPS which caused 50% hemolysis of each TESh~oVF was calculated from the percent hemolysis. TESh~oVF prepared with 1200 units/ml of CoVF was so sensitive to GPS as to be 50% hemolyzed by 1/152 GPS in Mg2÷-EGTA-GVB. TE ~h not treated with CoVF had a tendency to be hemolyzed by GPS although a net hemolysis by 1/10 GPS was only 17.2%. In the control experiment where EDTA~:~VB was used instead of Mg2÷-EGTA-GVB as a diluent, no specific hemolysis of TE ~hCoVF by GPS was observed.
Hemolysis by HuS of TE'h-CoVF The same lots of TESh~oVF suspensions used for the above experiment with GPS shown in Table 1 were also incubated with an equal volume of dilutions of HuS in Mg2+-EGTA-GVB at 37°C for 60 min. Although TE ~h TABLE 1 Hemolysis by GPS of TE sh coated with CoVF. CoVF a
% hemolysis in Mg2+-EGTA-GVB b by GPS diluted to
HLs0 c
(U/ml) 0 4 12 40 120 400 1 200
(/ml) 1/10
1/20
1/40
1/80
1/160
1/320
0
29.6 44.9 72.8 105.0 104.0 106.0 102.6
25.9 36.5 38.6 84.3 93.6 103.8 105.6
23.8 24.6 35.1 60.9 79.7 100.0 102.2
22.4 19.7 25.4 35.9 51.4 80.4 87.9
20.2 17.4 18.4 23.1 27.3 41.5 49.6
18.7 14.5 16.1 17.4 16.7 22.3 27.6
15.0 13.9 12.3 13.5 7.4 12.8 15.1
<5 4.4 14 45 79 125 152
a Concentration of CoVF reacted with 5 × 10S/ml TE sh to prepare TEsh-CoVF. b Replacement of Mg:+-EGTA-GVB with EDTA-GVB completely suppressed the hemolysis by GPS over the hemolysis of GPS-absent controls. c Reciprocal dilution of GPS for 50% hemolysis.
89
was hemolyzed by HuS in Mg2+-EGTA-GVB (HLs0 was 7.2/ml), no clear dose
Hemolysis sensitivity of erythrocytes from different species E h", E gp and E ~ab were treated with tannic acid (50 #g/ml) and coated with varying amounts of CoVF (0, 4, 12, 4 0 , 1 2 0 , 4 0 0 or 1200 units/ml) to prepare TEhU-CoVF, TEgP~oVF and TE~aD~oVF, respectively. They were suspended at 1 X 10S/ml in Mg2*-EGTA-GVB and incubated with an equal volume of serially diluted GPS, HuS or RabS in Mg2+-EGTA-GVB (1/10, 1/20, 1/40, 1/80, 1/160, 1/320 or 0). Sera diluted in EDTA~3VB were used as controls. From the hemolytic rates, the HLs0 titer of each serum on each TE-CoVF was calculated. As shown in Table 3, TEhU-CoVF became sensitive to GPS and RabS depending on the increase of CoVF used. TEgP-CoVF was efficiently hemolyzed by RabS and TErab-CoVF was efficiently hemolyzed by GPS. On the other hand, none of the erythrocytes coated with CoVF became significantly sensitive to HuS.
Hemolysis by C4DGPS in the presence of unfixed CoVF To examine the hemolytic sensitivity of E, TE or TE-CoVF (prepared by the reaction of 5 × l 0 s TE in 400 units/ml CoVF) to C4DGPS in the presence of CoVF in the fluid phase, 50/A of 2 X 108/ml of each type of E, 50 pl of CoVF (2400, 240, 24, 2.4 or 0 units/ml) and 100 pl of C4DGPS (1/4, 1/8, 1/16, 1/32, 1/64, 1/128 or 0) were mixed and incubated at 37°C for 1 h. After the reaction the extent of hemolysis was determined spectroTABLE 2 Hemolysis by HuS of TE sh coated with CoVF.
CoVF a (U/ml)
0 4 12 40 120 400 1 200
% hemolysis in Mg2+-EGTA-GVB b by HuS diluted to
HLs0 (/ml)
1i10
1/20
1/40
1/80
1/160
0
46.5 45.8 44.9 63.7 51.1 48.7 55.3
29.2 29.9 26.9 37.5 28.6 24.8 28.8
18.6 19.8 15.7 15.0 20.1 14.6 16.5
17.9 18.3 17.3 14.4 18.3 13.2 15.6
18.8 19.3 16.3 15.6 18.5 12.2 14.9
12.6 12.6 12.1 11.8 14.9 10.3 13.5
7.2 6.9 7.2 12.2 8.6 8.7 9.7
a Concentration o f CoVF reacted with 5 x 10S/ml TE Ih to prepare TEsh-CoVF. b Replacement o f Mg2+-EGTA-GVB with EDTA-GVB completely suppressed the hemolysis by HuS over the hemolysis of HuS-absent controls. c Reciprocal dilution o f HuS for 50% hemolysis.
90 TABLE 3 R e c i p r o c a l d i l u t i o n o f sera w h i c h caused 50% h e m o l y s i s ( H L s 0 / m l ) o f n a t i v e E o r T E coated with varying amounts of CoVF. Erythrocytes
CoVF a
HLs0 (/ml) of
(U/ml) GPS
HuS
RabS
E hu T E hu
0 0 4 12 40 120 400 1200
<10 <10 12 18 25 44 74 110
<10 <10 <10 <10 <10 <10 12 15
<10 14 21 53 66 99 113 123
EgP TEgP
0 0 4 12 40 120 400 1 200
<10 13 15 13 13 10 14 18
<10 10 <10 <10 <10 13 14 18
<10 14 13 <10 15 24 63 88
S rab T E tab
0 0 4 12 40 120 400 1 200
<10 <10 <10 17 33 47 60 83
~'10 13 13 14 14 14 14 15
~"10 <10 <10 <10 <10 <10 14 25
a C o n c e n t r a t i o n o f C o V F used for c o n j u g a t i o n .
photometrically. As shown in Table 4, even with the optimum concentrations of CoVF, the HLs0 of C 4 D G P S remained at most 11.6/mi and 40/ml on native E hu and E sh respectively,and at most 52/mi and 34/mi on T E hu and T E sh respectively.O n the other hand, the presence of C o V F in the fluid phase was inhibitory on the hemolytic reaction of T E h U ~ o V F and T E shC o V F by C4DGPS. This indicated that fluid-phase activation of the complement caused it to react with the C o V F fixed on the cell membrane. As shown in Table 4, tannic acid treatment of E hu and E sh made them sensitive to hemolytic reaction by C 4 D G P S in the absence of C o V F in the fluid phase. Although the mechanism underlying the increased reactivity with G PS remains to be studied, tannic acid treatment might have induced the ACPactivating ability,as in the case of neuraminidase treatment (Fearon, 1978; Paungburn and MiiUer-Eberhard, 1978).
91
TABLE 4 HLs0/ml of C4DGPS on E, TE and TE-CoVF in the presence of fluid phase CoVF. E species
Ehu TE hu TEhU-CoVF Esh TE sh TE sh.CoVF
Concentration (U/ml) of CoVF in fluid phase 0
0.6
6
60
600
2.2 13.3 100.0 <1.0 4.5 140.0
2.5 17.0 87.0 3.7 7.6 140.0
5.3 32.0 63.0 25.5 22.5 92.0
11.0 52.0 48.5 40.0 34.0 48.5
11.6 27.0 26.5 27.0 21.5 22.7
Effect of phospholipase A2 on hemolysis by GPS of TE In the C o V F preparation, there might have been a contamination b y phospholipase as indicated b y Lachmann et al. (1976). 0.4 ml samples o f 1 × 109/ml TE hu in 0.11 M PB were mixed with an equal volume of a serial dilution o f PLA2 in 0.11 M PB (100, 10, 1, 0.1, 0.01 or 0.001 pg/ml or buffer alone). After incubation overnight at 4°C, followed b y incubation at 37°C for 20 min, the TEhU-PLA2 were washed and suspended at 1 × 108/ml in Mg2+-EGTA~:~VB. 100 pl of the TEhU-PLA2 were incubated with an equal volume of diluted GPS at 370C for 1 h and t~he hemolytic rate was determined. PLA2 treatment did n o t increase the sensitivity of erythrocytes to hemolytic reaction b y GPS, indicating that PLA2 was n o t involved in the increased hemolytic sensitivity o f T E ~ o V F to GPS.
Factors essential for TE-Co VF hemolysis Partial purification was performed to confirm the factors in GPS essential to T E ~ o V F hemolysis. 95 ml of GPS were mixed with 285 ml o f 5 mM phosphate buffer containing 1 mM EDTA (pH 7.5) and settled in an ice bath for 1 h and centrifuged at 16 000 X g for 30 rain to remove the precipitated euglobulin fraction. The supernatant was applied on a DE-52 column (5.5 cm × 40 cm), equilibrated with 5 mM phosphate buffer containing 50 mM NaC1 and 1 mM EDTA. (pH 7.5), and the effluent collected in 15 ml samples. The hemolytic activity was assayed b y incubating the fractions in Mg2+-EGTA-GVB with T E r a b ~ o V F at 37°C for 20 min, followed b y addition o f 1/10 GPS in EDTA-GVB and further incubation at 37°C for 1 h. A hemolytically active fraction was obtained soon after the main protein peak o f effluent. The active fractions, from tube numbers 56 to 64, w e r e p o o l e d and concentrated to 4.4 ml under negative pressure in a visking tube. The concentrated sample was filtered through a Sephadex G-100 column (2.5 cm × 40 cm) in 0.3 M NaC1 containing 1 mM EDTA (pH 7.5) and effluent was collected as 5-ml fraction. On the gel filtration t w o peaks with absor-
92
bances at 280 nm (OD2s0) were observed. The peaks were at Fr 14 and Fr 22 with OD280 values of 1.55 and 0.33. Although neither Fr 14 or Fr 22 from the column had any activity to sensitize T E r a e ~ o v F to hemolysis b y the addition of 1/10 GPS in EDTA-GVB, the presence of a 1/5 dilution of either fraction enabled any of the other fractions to cause hemolysis of TECoVF, after a further incubation with GPS in E D T A ~ ] V B at 37°C. The synergistic ability of Fr 14 and Fr 22 is demonstrated in Fig. 1. Fr 14 and Fr 22 were diluted 1 : 15 in Mg2*-EGTA-GVB and mixtures prepared of the following combinations: Fr 14 and Fr 22, Fr 14 and buffer, and Fr 22 and buffer. Two volumes of the mixtures were mixed with 1 vol of 1 × 108/ml TErab-CoVF (prepared with 400 units/ml CoVF) for the period indicated at 30°C and then 300 #1 of the reaction mixtures were added with 200 ~1 of EDTA-GVB and 200 /A o f 1/10 GPS in EDTA-GVB. After further incubation at 37°C for 1 h, the extent of hemolysis was determined spectrophotometrically. To be hemolyzed, TErab-CoVF was sensitized by incubation with a mixture of Fr 14 and Fr 22 b u t n o t with Fr 14 or Fr 22 alone (Fig. 1). Since Fr 14 and Fr 22 correspond with fractions B and D in a comparison with the results reported b y Brade et al. (1972), the hemolysis of TE-CoVF b y serum is suggested to be induced b y the reaction of ACP.
Effect o f heating on hemolytic potency o f GPS on TE-CoVF GPS was incubated at 50°C for 16 and 32 min. After the heating, GPS were chilled immediately in an ice bath and serially diluted in Mg2*-EGTA GVB. To 100 pl o f the diluted GPS, 100 pl of 1 X 108/ml T E S h ~ o V F (prepared coating TE am with 400 units/ml CoVF) was added and the mixture incubated at 37°C for 60 min. As shown in Fig. 2, hemolytic activity of GPS
40 •
r22
3O
"6
20
"r
10
20
30
Time i n r n ; n u t e s
Fig. 1. The ability of Fr 14 and/or F r 22, from the Sephadex G-100 gel filtration, to sensitize TErab-CoVF for hemolysis by GPS in EDTA-GVB. TErab-CoVF was mixed with Fr 14 and/or Fr 22 in Mg2+-EGTA-GVB and incubated at 30°C for the period indicated on the abscissa. Both Fr 14 and Fr 22 were required for the sensitization of TErab-CoVF.
93 50
I00
150
(HLso)
unheated
50°C16min ~
50°= 32 min
Fig. 2. HLso/ml on T E S h ~ o V F of GPS before or after heating at 50°C for 16 min or 32 rain. The hemolytic activity-was determined in the absence (solid b a r s ) o r presence (open bars) of the factor B supplemented fraction.
was reduced from 132 HLs0/ml to 83 HLs0/ml (37% reduction) and 26 HLs0/ ml (80% reduction) by heating at 50°C for 16 rain and for 32 rain respectively. To 100/~l of the GPS dilution, 100 #l of 1 × 10 s T E s n ~ o v F supplemented with a 1/4 dilution of Fr 14 used as in Fig. 1 (factor B fraction) were added to examine the extent of hemolysis on TESh~oVF after the reconstitution of the heated serum with the factor B fraction. The hemolytic activity of 50°C heated GPS after this reconstitution, recovered to 140 HLs0/ ml from 83 HLs0/ml (16-rain heated GPS) and to 92 HLs0/ml from 26 HLs0/ml (32-rain heated GPS). The values of 140 HLs0/ml and 92 HLs0/ml were 81% and 53% of the 173 HLs0/ml of unheated GPS in the presence of the supplemented factor B fraction. The findings suggest that factor B of ACP, which is labile at 50°C heating, will be essential in the hemolytic reaction of TE-CoVF by serum in Mg2*-EGTA-GVB. DISCUSSION
The interaction mechanism of CoVF has been well analyzed in human ACP (Miiller-Eberhard and Fjellstrom, 1971; Cooper, 1973). CoVF binds factor B, and in the presence of factor D forms a CoVF • Bb complex which has C3 convertase activity. ACP activation of serum induced by CoVF results in the hemolysis of incidental E, and this has been successfully applied as a hemolytic assay of ACP by Brai and Osier (1972). To increase the hemolytic efficiency of CoVF-induced hemolysis by ACP activation of the serum complement, we fixed by CoVF onto E which had been treated with tannic acid according to Boyden (1951), with slight modification (Okada et al., 1970). The fixation of CoVF on the E membrane results in the activation of serum ACP only on the cell surface and not in the fluid phase. The results in this
94 report demonstrate that this was the case. The addition of CoVF in the fluid phase inhibited the hemolysis of T E ~ o V F by GPS, indicating that the fluidphase activation of ACP by CoVF reduced the supply of complement factors to react on the surface-bound CoVF. In other words, activation of complement by. membrane-bound CoVF effectively results in the hemolysis of the E. TEhu-CoVF, T E ' h ~ o V F and TErab-CoVF were so sensitive that they were hemolyzed by GPS diluted over I : 80 when E were coated with a large amount of CoVF (Tables 1 and 3). The hemolysis of T E ~ o V F by GPS was the result of ACP activation since the hemolysis occurred in Mg2*-EGTA GVB but not in EDTA-GVB. Fractions of factors B and D were essential for the sensitization of TE-CoVF to hemolysis b y a subsequent reaction with GPS in EDTA-GVB (Fig. 1). Heating GPS at 50°C reduced its hemolytic potency, which was recovered by supplementation with the factor B fraction (Fig. 2). Although the CoVF preparation might have been contaminated with PLA2 (Lachmann et al., 1976), the enzyme would not have sensitized erythrocytes for hemolysis by serum since PLA2 coated TE hu (TE-PLA2) was not sensitive to GPS. Although GPS or RabS efficiently hemolyzed TEhu~oVF, TE shCoVF, TEgP~:~oVF and/or TErab~:~oVF, HuS did not effectively take advantage of the presence of CoVF fixed on the cell membrane (Tables 2 and 3). This might be due to the restricted efficiency of later components of the complement, since the addition of GPS in EDTA-GVB following a 20 min incubation of T E ~ o V F with HuS in Mg2+-EGTA~:~VB enhanced the hemolysis of HuS reacted-TE~oVF (unpublished data). Although little appreciable enhancement of hemolysis was noted by the addition of 1/10 homologous serum in EDTA-GVB, following a 20-rain incubation of T E ~ o V F with a dilution of GPS or RabS in Mg2*-EGTA-GVB, the .addition of serum to EDTA-GVB may be used to examine the ACP potency of serum. This procedure will eliminate a possible restriction of hemolysis by a shortage of lateacting components of the complement, especially in those cases such as C6deficient rabbit serum and serum in which C7 has been exhausted. The optimum incubation condition prior to the addition of EDTA-serum is under investigation. Although T E ~ o V F is not sufficiently sensitive to HuS, it is highly sensitive, and useful, in the detection of the ACP activity of GPS and RabS. However, the sensitivity of T E ~ o V F depends on the amount of CoVF fixed and so the ACP titer will need to be standardized with a standard serum sample to compare the activity of different experiments. Another disadvantage of TE-CoVF was the frangibility of TE, which caused 5--15% spontaneous hemolysis without the reaction of complement, although this was not a serious problem. It is noteworthy that tannic acid treatment made E hu and E sh sensitive to hemolytic reaction by GPS. Tannic acid treatment may induce or enhance the ACP-activating capacity of E by a mechanism similar to that observed on neuraminidase-treated E ' h (Fearon, 1978; Pangburn and Miiller-Eberhard, 1978). Recently, ACP of mouse and rat sera have been shown to form a CoVF-Bb complex in the presence of EDTA (Goldman et al., 1979). In preliminary
95 experiments, T E ~ o V F was also sensitive to rat and mouse c o m p l e m e n t in E D T A in accordance with their results (data n o t shown). The o p t i m u m react i o n c o n d i t i o n o f T E - C oV F f or t he d e t e c t i o n o f m a r i n e ACP activity is now u n d e r investigation. ACKNOWLEDGEMENTS We t h a n k Mr. Masahiro Y o k o t a f or his excellent assistance and Miss Chiaki Iida f o r ty p in g the manuscript. This w o r k was support ed by Grants-in-Aid f r o m th e Ministry o f E d u c a t i o n , Science and Culture, Japan and f r o m t he Adult Disease Clinic Memorial F o u n d a t i o n . REFERENCES Ballow, M. and C.G. Cochrane, 1969, J. Immunol. 103,944. Boyden, S.V., 1951, J. Exp. Med. 93, 107. Brade, V., C.T. Cook, H.S. Shin and M.M. Mayer, 1972, J. Immunol. 109, 1174. Brai, M. and A.G. Osier, 1972, Proc. Soc. Exp. Biol. Med. 140, 1 116. Cooper, N.R., 1973, J. Exp. Med. 137, 451. Fearon, D.T., 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 1 971. Flexner, S. and H. Noguchi, 1903, J. Exp. Med. 6,277. Goldmari, J.H., S. Bangalore and M.B. Goldman, 1979, J. Immunol. 123, 2 421. Lachmann, P.J., L. Halbwachs, A. Gewurz and H. Gewurz, 1978, Immunology 31,961. Mayer, M.M., 1961, in: Experimental Immunochemistry, 2nd edn., eds. E.A. Kabat and M.M. Mayer (Charles C. Thomas, Springfield, IL) p. 133. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29,185. Miiller-Eberhard, H.J. and K. FjellstrSm, 1971, J. Immunol. 107, 1 666. Nelson, Jr., R.A., 1966, Survey Ophthal. 11,498. Okada, H., S. Kawachi and K. Nishioka, 1970, Biochim. Biophys. Acta 208, 541. Pangburn, M.K. and H.J. Miiller-Eberhard, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 2 416.