lmmunocheraistry, 1976.VoL 13, pp. 793-800. PergamonPress. Printedin GreatBritain
STUDY OF THE INTERACTION BETWEEN MONOCLONAL IgM PROTEINS AND THE COMPLEMENT SYSTEM GEORGE F/~IST, M,ARIA CSECSI-NAGY, GEORGE A. MEDGYESI, JUDIT KULICS and J,~NOS GERGELY Department of Immunochemistry, National Institute of Haematology and Blood Transfusion, H-1502, Budapest; and Department of Immunology of Lor~ind Ertvrs University, H-2131 GSd, Hungary (Received 6 May 1976)
Al~traet--The capacity to activate the complement system and to fix isolated C1 of different monoclonal IgM proteins and various fragments was studied. All the 8 different IgM proteins tested fixed isolated C1, but the dose of the IgM preparations necessary for the fixation of the 50~o of the available C1 activity strongly differed from each other. The complement activating (anti-complementary) effect of the IgM preparations was weak. (Fc#)5 fragments prepared by tryptic digestion from 4 different IgM proteins and separated by gel filtration at pH 8.0 could fix C1 and their anti-complementary effect significantly increased with respect to the original lgM preparations. It was demonstrated that the Cl-fixing ability of the (Fc#)5 fragment was lost after gel filtration at pH 3.0 and a Cl-fixing low tool. wt peptide fraction was released from the (Fc/t)5. This peptide recombined with the non-Cl-fixing (Fc#)5 and restored its ability for the Cl-fixation. It was shown, furthermore, that the (Fc#)5 preparation which could not fix isolated C1, activated the complement system via the alternative pathway.
INTRODUCTION The activation of the complement system is one of the most important effector functions of immunoglobulins. It is known that some immunoglobulin classes (IgG, IgM) can, and others (IgA, IgE) can not activate complement through the classical pathway, and that the complexing to soluble or particulate antigens significantly enhance the ability of IgM and IgG antibodies for the complement activation. Ishizaka et al. (1967) reported firstly on the anti-complementary effect of monomer IgM preparations. Normal IgM preparations and Waldenstr/Sm proteins induced a weak effect, aggregation strongly increased their activity. Glovsky and Fudenberg (1971) demonstrated a C1 or C! subcomponent contamination in IgM preparations. They suggested the anticomplementary effect of these preparations to be associated with the presence of activated C1. MacKenzie et al. (1971) using a Clq-fixation assay concluded that IgM proteins are heterogeneous in respect to binding of these subcomponents. In contrast, Augener et al. (1971) did not found significant differences in the C1 fixation ability of 12 monoclonal IgM proteins. Plaut et aL (1972) studied firstly the anti-complementary effect of fragments of IgM proteins cleaved by trypsin and demonstrated (Fcl05 and Fc~t fragments to be 4-7 times more effective on molar basis than the whole IgM molecule. Sledge and Bing (1973) found that IgM and (Fc#)5 fixed isolated radiolabelled C l q approximately to the same extent. Recently Hurst et al. (1974, 1975) reported on the interaction of the complement system and the various fragments of a monoclonal IgM protein (DAU). According to their study, tryptic (Fc#)5 fragments can not fix isolated human C1 but after reduction to monomer Fc/~ a tM~ 13/10--A
significant Cl-fixation was observed. They separated from the Fc part of the DAU protein two Cl-fixing fragments and they suggested that a part of the CH4 domain including 24 amino acids is responsible for the complement activating ability of the IgM molecule. The data summarized above include some contradictory findings concerning the interaction between IgM and complement system. Our recent experiments were initiated to clarify the bases of these controversial results and to collect further data on the intramolecular localization of the site (or sites) responsible for the ability of IgM molecule to fix isolated C1 and to activate the complement system. As each method used for the study of the immunoglobulin-complement interaction has some limitations and disadvantages, three methods (Cl-fixation assay, anti-complementary reaction and C4-consumption in normal human serum) were used in parallel. In the present study we tested 8 different monoclonal IgM samples and all were found to fix isolated C1. Marked differences in the C1 binding capacity were observed. (Fc/~)5 fragments were also found to fix C1 and this activity was associated with a small mol. wt peptide fraction.
MATERIALS
AND
METHODS
1. Preparation of IgM
Human Waldenstrrm IgM was prepared according to Tomasi (1973), separated on Sephadex G-200 in 0.14M NaC1. 2. Trypsin digestion
Trypsin digestion was performed according to Piaut and Tomasi (1970) and Zik~in and Bennett (1973). The IgM 793
794
GEORGE FLIST et al.
was dissolved at a concentration of 20 mg/ml in 0.05 M Tris-HC1 buffer pH 8.0, containing 0.005 M CaC12. Trypsin (SERVA, DCC-treated, 1 x crystlyophil, salt-free) was dissolved in 0.001 M HC1 at 1~o concentration and added to a pre-heated solution of IgM (20 min, 60°C) to give a substrate-to-enzyme ratio of 50:1. Digestion was stopped by cooling and immediately gel filtrated on Sephadex G-200 in 1~o NH4HCO3 (pH 8.0) or in 1 M acetic acid (pH 3.0).
or with 0.1 ml VBS for control and incubated at 37°C for 60min. Thereafter haemolytic complement activity was determined by standard method (Mayer, 1962) and expressed in CHso units. The anti-complementary activity of IgM preparations was given as the difference between CHso units measured in the control and IgM preparation containing samples and was measured by the dose of IgM consuming 1 CH~o units out of 5 units available.
4. Immunodiffusion Radial double immunodiffusion was performed in 1.5~ agar; anti-# sera were obtained from SEVAC (Prague), as well as SPCI (Paris), anti-~c and anti-2 from HYLAND (U.S.A.).
10. Measurement of the activation of the alternativ~ pathway in human serum Activation of the alternative pathway by IgM preparations was measured by the method of Fine et al. (1972) slightly modified by us. IgM preparation (0.1 ml) was incubated with 0.1 ml of human serum chelated with EGTA (ethylene glycol tetraacetic acid, final concentration 10 mM) and supplemented with MgCI2 (final concentration 8 mM) at 37°C for 60 min. Then the serum was recalcified and the residual haemolytic activity was measured. The results were evaluated as by the measurement of anti-complementary activity.
5. Diluents Veronal-buffered saline (VBS) containing 0.001 M Ca + +, 0.0005 M Mg+* and 0.1~o of gelatine was made according to Mayer (1962). Veronal-mannit buffer was prepared as outlined by Rapp and Borsos (1963). Buffer of low ionic strength (# = 0.065, G-VBS-M) was made by mixing 4 vol of VBS with 6 vol of veronal-mannit buffer. 0.1 M solution of EDTA pH 7.4 was prepared according to the formula given by Frank et al. (1964). Lower concentrations of EDTA were obtained by diluting this isotonic solution with appropriate amount of VBS without Ca ÷+ and Mg + ÷.
11. Measurement of the Cl-fixing activity of IgM preparations C 1-fixation by the IgM preparations was measured according to the method of Augener et al. (1971), using a dilution of human C1 sufficient to yield approx 63~ haemolysis. The C1 activity measured in the IgM containing and control samples was expressed in z values (average number of C1 molecules per cell). The extent of the Cl-fixation of the IgM preparations was given as the dose required for the fixation of 50~o of C1 activity (z value) measured in the control sample (Ct + buffer) and was determined graphically as outlined by Hurst et al. (1974).
3. Polyacrylamide disc electrophoresis Mol. wt was estimated according to Weber and Osborn (1969). Polymers of ribonuclease (REANAL) treated by diethyl pyroearbonate according to Wolf et al. (1970) were used as standards.
6. Preparation of complement components Functionally purified human C1 was prepared as described by Tamura and Nelson (1968). Guinea pig C2 was obtained in functionally purified form according to the method of Nelson et al. (1966). 7. Preparation of sheep erythrocyte-antibody--complement complexes EACI~p was prepared according to Tamura and Nelson (1968), EAC4,u was made as described by Borsos and Rapp (1967). 8. Measurement of C4 titre in human serum The haemolytic activity of C4 in human serum was measured by effective molecule titration as outlined by Nelson et al. (1966). 9. Measurement of anti-complementary activity of immunoglobulin preparations Fresh pooled human serum (0.1 ml) was mixed with various doses of IgM or IgM fragments in 0.1 ml of VBS
12. Estimation of the Cl-fixin# activity of lgM preparations contaminated with CI IgM preparations were serially diluted parallel in two sets of tubes (volume of dilutions 0.2 ml, dilution factor 1:3). C1 dilution (0.2 ml, z = 1-1.5) was added to the first set of tubes, 0.2 mi diluent was added to the second ones. After incubation at 30°C for 1 0 imn , EAC4 cells were added and C1 haemolytic activity (z values) was determined. C1 activity fixed by the different doses of IgM was calculated as follows: z value measured in control sample (C1 + buffer) was added to the z values measured in samples containing the corresponding dose of IgM without C1 (second set of tubes). This sum represented the C1 activity available in the tubes containing both IgM and C1 (first set of tubes). Finally, the z values measured in the samples in the first set of tubes (IgM + C1) were subtracted from the corresponding sums. This difference was taken as the extent of C1 fixation of the different doses of IgM preparations. A protocol of calculation is given in Table 1.
Table 1. Estimation of Cl-fixing activity of IgM preparations contaminated with C1 by calculation N u m b e r of tube I g M preparation, 0.2 ml//~g C l dilution, ml Diluent, ml z Value C1 activity available (B + C) C I activity fixed (B + C) - A C1 activity fixed ° in ~ of control (C)
IA 300 0.2 -1.11 . . 1.03 96
2A
3A
100 0.2 -0.97 . 0.85 79
33
4A 11
0.2
0.2
0.73
0.78
. 0.65 61
0.43 40
IB 300
2B
3B
100
. . 0.2 1.07 2.14
4B
33
. 0.2 0.75 1.82
Il
. 0.2 0.31 1.38
0.2 0.14 1.21
C (control) -0.2 0.2 1.07
-.
.
.
.
.
aDose required for the fixation of the 50~o of the C1 activity measured in control tube: 19/~g (determined graphically as outlined by Hurst et al., 1974).
Interactions of IgM and Complement 13. Analytical ultracentrifu#ation Sedimentation velocity experiments were performed in a MOM Model Ultracentrifuge, type 3170/b (Hungarian Optical Works, Budapest) using Sehlieren optics. A 12 mm 3° single-sector cell in an A 60-2 rotor was used. All sedimentation measurements were performed at 20° + 0.1°C at various rotor speeds, the rotor speed was regulated + 50 rev/min during the run.
RESULTS In the first part of experiments the Cl-fixation ability and the anti-complementary effect of monoclonal IgM proteins were compared. Preliminary experiments indicated C1 contamination in each preparation, 1 mg of the preparations contained 0.7-7.2 CH6a units of C1. The C1 contamination rendered difficult the estimation of the Cl-fixation effect of IgM preparations: the C1 activity measured in samples containing mixture of IgM and isolated CI originated both from the activity of the CI contamination and that of the isolated C1 added to the mixture. Consequently in some IgM doses this activity exceeded the control values measured in control samples without IgM. There are different possibilities to overcome this difficulty: C1 contamination either has to be eliminated or alternatively, it can be quantitated and taken into account by a correction when calculating the results. Both ways were followed in these studies. CI contamination was eliminated by heat treatment at 56°C for 6 min. (According to our preliminary experiments, this treatment destroyed 95~ of the haemolytic activity of a diluted C1 preparation.) When the subtractive method was used, different doses of IgM preparations were incubated together with a dilution of C1 (z --- 1.0-1.5) or without C1, z values were determined in each sample and the C1 consumption of the IgM preparation was calculated as described in Materials and Methods.
Study of the Cl-fixing and anti-complementary effects of I#M preparations Table 2 shows the Cl-fixation and the anti-complementary effect of 8 different monoclonal IgM preparations. The figures for results of Cl-fixation obtained by the 2 methods were at least roughly parallel. Great differences were observed, however,
795
between the Cl-consumption activity of the different IgM preparations: the dose necessary for the consumption of the 50% of the available C1 activity varied between 4-80 #g. The anti-complementary activity of 6 preparations was determined and was found to be weak: 100-> 500 #g was necessary for consumption of 1 CHso unit out of 5 units. According to Augener et al. (1971) the presence of aggregates can strongly enhance the Cl-fixing ability of IgM preparations. To control the aggregate content 3 different IgM preparations (Fig. 1)--2 strongly and 1 weakly active--were studied by analytical ultracentrifugation. Only one preparation (MAU) contained a considerable quantity of larger aggregates. The ultracentrifugation patterns of the other strongly Cl-fixing protein (VIR), as well as that of the weakly active preparation (CAN) showed one predominant peak, the sedimentation coefficient of which was 18.2 and 18.7 S, respectively. These findings suggest that the differences observed in the extent of the Cl-fixation of the different IgM proteins can not be explained by the presence of large aggregates in the preparations.
Study of the Cl-fixi~ and anti-complementary effects of (Fc#)5 preparations separated at pH 8.0 Table 3 compares the Cl-fixing and anti-complementary effects of 4 different (Fc#)5 fragments obtained by gel filtration at pH 8.0. The immunochemical purity of these (Fc#)5 preparations was controlled by radial double irnmunodiffusion using anti-#, anti-x and anti-2 sera. Each (Fc#)5 fragment fixed isolated C1, and on the weight basis the Cl-fixing activity of the (Fc#)5 and the corresponding IgM preparation was about the same. The anti-complementary effect of each (Fc#)5 preparation, however, was 6 times higher on the average as compared to the original IgM proteins. Figure 2 shows the polyacrylamide disc electrophoresis in SDS (SDS-PAGE) patterns of the (Fc#)5 preparations. Besides the bands corresponding to the products of (Fc#)5, a low mol. wt component can be seen in 3 gels out of 4. The staining of these bands was roughly parallel with the Cl-consumption activity of the (Fc#)5 preparations. The greatest intensity was observed in the case of VIR protein (Cl-fixation 20 #g). In the case of CAN preparation (Cl-fixation t20 #g) no low mol. wt component was observed.
Table 2. Cl-fixing and anti-complementary effects of monoclonal IgM preparations
Preparation FAR MAU VIR BOL O'BE CAN CZU SCI-IA
Cl-fixation (dose fixing the 50% of the CI activity available, #g) A Heat-treatment of calculation lgM for 6 min at method ° 56°C 30 4 6 53 25 80 5 10
°See Materials and Methods. N.T. not tested.
48 10 17 N.T. 30 120 N.T. N.T.
Anti-complementary effect (dose consuming 1.0 CHso units from the 5.0 units available, #g) B Heat-treated untreated (56°C, 6 min) preparation preparation 100 450 300 > 500 240 360 N.T. N.T.
300 1000 450 > 500 840 1050 N.T. N.T.
A/B 0.30 0.01 0.02 < 0.10 0.10 0.22 ---
GEORGE FOSTet al.
796
Fig. I. Sedimentation velocity pattern of 3 purified monoclonal IgM preparations. (a) VIR, (b) CAN, (c) MAU in saline, 3 mg/ml. Conditions were: speed, 44,000rev/min; temperature, 20°C; photograph, (a) and (b) 14rain, (c) 24rain after reaching full speed. S2o,w was found to be (a) 18.2S, (b) 18.7S; 21.9 S, (c) 19.0 S; 25.2 S; 32.1 S; 36.8 S; 44.8 S. Sedimentation was from left to right.
Study of Cl-fixin9 and anti-complementary effects of a (Fc#)5 preparation separated by gel filtration at pH 3.0 Based on this observation one can suppose that the Cl-fixing capacity of (Fc#)5 is due to low mol. wt components adsorbed or non-covalently bound to the (Fcp)5. Therefore, fragments obtained from two IgM proteins (VIR, CAN) by tryptic hydrolysis were separated on Sephadex G-200 column equilibrated with 1 M acetic acid. SDS-PAGE patterns of eluted fractions of VIR protein following reduction are shown in Fig. 3. The Cl-fixation and the anti-complementary effect of the 3 fractions, corresponding to the (Fc#)5, Fab/z and peptide fragments are shown in Table 4. Two (Fc#)5 fragments (VIR and CAN) fixed C1 only at a very high concentration. No significant Cl-fixation by the Fab fragment of these proteins was observed. The peptide component of VIR protein, however, could fix isolated C! efficiently. Table 4 shows the activation of the alternative pathway induced by the (Fc/~)5 (pH 3.0) fragments, too. Both (Fc#)5 fragments could activate the alternative pathway and approximately the same doses of (Fcp)5 (VIR) consumed 1.0 CHso out of 5 both in
normal human serum and in Mg ÷ +-EGTA serum. On the basis of these experiments, therefore, it seems that the Cl-fixation of (Fc/~)5 fragments isolated at slightly alkaline pH can be due to an adsorbed or non-covalently linked low mol. wt peptide which was separated from the (Fc#)5 fragment, if the gel filtration of tryptic digest of IgM was performed at acidic pH. To give a further support to this hypothesis, a 2-step experiment was done. IgM (VIR) was digested by trypsin, the (Fc#)5 fragment was isolated on Sephadex G-200 column at pH 8.0, the (Fc#)5 containing fraction freeze-dried and filtered on Sephadex G-200 column at pH 3.0. Two fractions were obtained, the first corresponded to the (Fc#)5 fragment, the other contained the low mol. wt peptides. Table 5 shows the CI consumption and the anticomplementary effect of the (Fc#)5 fraction obtained at the first as well as (Fc/~)5 and peptide fraction isolated at the second gel filtration step. (Fc#)5 fragments obtained from the tryptic digest by gel filtration at pH 8.0 strongly fixed isolated C1, but after gel filtration at pH 3.0 they have lost almost completely their Cl-fixation ability. On the other hand, during the second gel filtration step, a fraction
Table 3. Cl-fixing and anti-complementary effects of (Fc#)5 fragments prepared by tryptic digestion from IgM proteins and separated by gel filtration at pH 8,0
IgM preparation FAR VIR O'BE CAN
A Cl-fixation (dose fixing the 50~ of the CI activity available, ,ug)
B Anti-complementary effect (dose consuming 1.0 CHs0 units from the 5.0 units available, ,ug)
A/B
50 20 30 120
30 25 50 40
1.67 0.80 0.60 3.00
Interactions of IgM and Complement
797
studied either immediately, or after dialysis overnight against saline for the elimination of the cysteine, or was alkylated with iodoacetic acid and dialysed. No significant Cl-fixation was observed in either case.
Study of the C4-consumption in normal human serum induced by IgM and IgM fragments The ability of IgM (VIR) and their fragments to fix isolated C1 and to activate the classical pathway were compared. Classical pathway activation was studied by C4-consumption assay in normal human serum. A heat-aggregated human IgG preparation was included as a control (Table 5). The proportion of doses necessary for the Cl-fixation and complement activation was about 0.01 with aggregated IgG. In the case of original IgM preparation this proportion was less than 0.003. The relatively high complement activating effect (proportion 0.05) of the (Fc/05 (pH 8.0) preparation is remarkable.
DISCUSSION
(a)
(b)
(c)
(d)
Experiments described in this paper were performed to study the following and not fully clarified problems concerning interaction of IgM and complement system. (1) Is the IgM class homogeneous or heterogeneous in respect to the Cl-fixation?
Fig. 2. SDS-PAGE patterns of 4 different tryptic (Fc#)5 fragments isolated by gel filtration at pH 8.0. (a) FAR, (b) CAN, (c) VIR and (d) O'BE. Low mol. wt peptide components is indicated by an arrow.
of low mol. wt peptides was released from (Fc/z)5 (obtained by gel filtration at pH 8.0) and this peptide strongly fixed C1.
Recombination experiment with the (Fcb05 and peptide fraction isolated by gel filtration at pH 3.0 (Fc#)5 and peptide fraction obtained from the tryptic digest by gel filtration at pH 3.0 were mixed at 4°C and dialyzed against buffer of pH 8.0. The mixture was concentrated by pressure dialysis and finally gel filtrated on Sephadex G-200, at pH 8.0, to remove unbound small mol. wt peptides. The Cl-fixation activity of the fraction containing (Fc/05 fragment was compared with that of the original materials..The original (Fc/~)5 fragment did not fix C1 (Cl-fixing dose > 1000/~g), the peptide fraction showed a marked Cl-fixing activity (Cl-fixing dose 21 #g). The (Fc#)5 fragment recombined with the peptide fixed C1, too (Cl-fixing dose 70.4~tg). Therefore it seems that the non-Cl-fixing (Fc/~)5 fragment had at least partly regained its ability to fix isolated C1 after recombination with the Cl-fixing peptide.
Study of the Cl-fixing effect of the Fc# fragment In the subsequent experiment the (Fc#)5 obtained by acidic gel filtration was reduced by 0.05 M cysteine. The generated monomer Fc~ fragment was
(a)
(b)
(c)
(d)
Fig. 3. SDS-PAGE patterns of (a) (Fc#)5, (b) Fab# and (c) low mol. wt peptide containing fraction, obtained from tryptic digest of VIR IgM protein, by gel filtration at pH 3.0, (d) polymerized ribonuclease. All samples were reduced in 1~ SDS.
798
GEORGE FkIST et al.
Table 4. Cl-fixing and anti-complementary effects of different fragments of monoclonal IgM proteins obtained by tryptic digestion and separated by gel filtration on Sephadex G-200 column at pH 3.0
IgM preparation
Fragment
A CI-fixation (dose fixing the 50% of the CI activity available, #g)
B Anti-complementary effect (dose consuming 1.0 CH~o units from the 5.0 units available, ~g)
A/B
C Activation of the alternative pathway (dose consuming 1.0 CHso units out of 5.0 units in Mg ++ - EGTA)
VIR
(Fc#)5 Fab peptide
600 226 20
19 140 60
31 1.6 0.33
20 N.T. N.T.
CAN
(Fc#)5 Fa b peptide
1003 1000 500
113 120 143
8 8 3.5
190 N.T. N.T.
N.T. not tested. (2) Cl-fixation by IgM is followed or not by the activation of the classical complement pathway? (3) (Fcp)5 fragments obtained by tryptic hydrolysis of the IgM molecule can or cannot fix isolated C1 and activate the complement system? (4) To what extent can Cl-fixing capacity be attributed to a relatively small submolecular region?
plementary effect in undiluted human serum. The weak complement activating ability of each IgM preparation could be due to C1 contamination, since this activity decreased after heat-treatment of IgM at 56°C for 6 min. On the other hand it is possible that a part of the anti-complementary effect is mediated through the alternative pathway.
(1) All the 8 IgM preparations studied were contaminated with C1. The extent of this contamination varied between large limits. These findings are in agreement with those of Glovsky and Fudenberg (1971). MacKenzie et al. (1971) found marked differences in the C 1-fixing capacity of the different IgM proteins. In contrast, Augener et al. (1971) did not observe such differences at the study of 12 monoclonal IgM preparations. The results of our experiments agree with those of MacKenzie et al. (1971), and it was demonstrated that the differences between Cl-fixation activity were not necessarily associated with the presence of large aggregates in the preparations. The discrepancies between these findings may be explained by the fact that Augener et al. (1971) isolated their IgM preparations from heat-treated serum in contrast to MacKenzie et al. (1971) and to our recent studies.
(3) (Fc#)5 preparations isolated from different IgM proteins were studied and the Cl-fixation activity of each preparation was nearly identical with that of original IgM molecules. This finding corresponds to the results of Sledge & Bing (1973), who demonstrated that IgM and (Fc#)5 fixed C1 nearly to the same extent. More recently, however, Hurst et al (1974) did not find any Cl-fixation by a tryptic (Fc#)5 fragment of a Waldenstrrm protein isolated from the digest by means of gel filtration at slightly alkaline pH purified further by Bio-Gel P-150. In SDS-PAGE disc electrophoresis of our (Fc#)5 fragments a low mol. wt component was found in all but one preparations and the intensity of its zone showed a rough correlation with the Cl-fixation activity of the (Fc#)5 preparations. It was supposed, therefore that the Cl-fixation of (Fc/z)5 preparations may be due to the presence of a peptide non-covalently bound or adsorbed to the fragments. This hypothesis was supported by the results of the subsequent experiments: (Fc#)5 isolated from the tryptic digest by gel filtration at acidic pH lost its Cl-binding capacity. The same results were
(2) It was found in good accordance with the results of other authors (Ishizaka et al., 1967; Glovsky & Fudenberg, 1971 ; Plaut et al., 1972) that IgM preparations have only a weak capacity to activate the complement system as measured by their anti-com-
Table 5. Comparison of Cl-fixation, anti-complementary effect and classical pathway activation (consumption of C4 in normal human serum) induced by aggregated IgG, IgM and different tryptic fragments of IgM
No. 1.
Preparation Aggregated human IgG Monoclonal lgM
II.
A Cl-fixation (dose fixing the 50% of the CI activity available, #g)
B Anti-complementary effect (dose consuming 1.0 CH~ o units from the 5.0 units available, #g)
C Dose consuming the 50~ of the C4 activity available in normal human serum, ,ag 18.9
A/B --
A/C
0.17
N.T.
5.5
300
>1600
0.02
640
13.00
--
> 150
> 150
--
--
(VIR) IU.
IV. V.
Tryptic (Fc#)5 from If separated by gel filtration at pH 8.0 (Fop)5 obtained from III by gel filtration at pH 3.0 Peptide fraction from III by gel filtration at pH 3.0
N.T. not tested.
400 22.1
799
Interactions of IgM and Complement Table 6. Comparison of the efficiency of Cl-fixation of various IgM proteins and IgM fragments on weight basis and on molar basis Efi$eiency of CIfixation on molar basis (compared to the original lgM)
MoL wt
Cl-fixation (dose fixing the 50% of the CI activity available, #g)
Part 1° IgM (VIR) (Fc#)5b pcptidc c
840,000 340,000 10,000
5.5 7.8 2.1
1.0 0.29 0.003
Part 2a IgM (DAU) Fc# C.4 fragment (CNBr5)2 fragment
840,000 34,000 6800 40,000
1.5 40 20 46
1.0 0.002 0.001 0.002
Preparation
*Data from the present work. ~III preparation in Table 5. clV preparation in Table 5. dData obtained from the paper of Hurst et al. (1974).
obtained, when the Cl-fixing (Fc#)5 preparation had been rechromatographed in an acidic medium. Nor did the Fc# fragment obtained by reduction of the (Fc#)5 preparation fix isolated C1. During the gel filtration of the (Fc#)5 at pH 3.0 a fraction containing a peptide (or peptides) of low mol. wt was eluted which fixed C1. After incubation and gel filtration of this fraction with the non-Cl-fixing (Fc#)5 fragment at slightly alkaline pH, the (Fc#)5 obtained could fix again isolated C1. These results suggest that the peptide recombined with the (Fcg)5 fragment and the Cl-fixing ability of the latter was at least partly recovered. Since the activity of the recombinate was weaker than that of the (Fc/~)5 fragment isolated by gel filtration at pH 8.0 immediately after the tryptic digestion of IgM, it can not be excluded that the decreased Cl-fixing capacity of (Fc#)5 following the gel filtration at acidic pH may be the consequence of a structural change induced by the acidic pH. (Fc#)5 fragments isolated by gel filtration both at slightly alkaline or acidic pH independently of their Cl-fixing capacity, were able to activate the whole complement system approximately to the same extent. The anti-complementary effect of the (Fc#)5 fragment was at least 6 times stronger on weight basis than that of the original IgM molecule. Similarly, marked C4-consumption in normal human serum was observed with a Cl-fixing (Fc#)5 preparation. Similar findings were reported by Plaut et al. (1972). On the other hand, if the anti-complementary effect of an (Fc#)5 preparation was measured in normal human serum chelated with EGTA and supplemented with Mg ++ ions, in which the complement system can be activated only via the alternative pathway (Fine et al., 1972), significant complement consumption was observed. A non-Cl-fixing (Fc#)5 preparation was demonstrated to activate the complement system almost exclusively through the alternative pathway. These experiments suggest that 2 additional activities were generated or at least strongly increased on the (Fc#)5 fragment as compared to the original IgM molecule: the ability to activate the classical pathway (as revealed by C4-consumption in human normal serum) and the ability to activate the alternative pathway. It was reported that immune complexes contain-
ing IgM antibodies strongly activate the classical pathway (Hyslop et al., 1970) and some immune complexes are capable to the activation of the alternative pathway, too (Zikfin & Miller, 1975). These activities are localized on the (Fc#)5 part of the IgM molecules. Therefore, it seems that tryptic cleavage induces structural changes on the (Fc#)5 part of molecules, which are similar to the changes occurring during the interaction of IgM antibodies and antigens. It can not be decided at the present time that the appearance or increase of the classical pathway activating capacity of (Fc#)5 fragment is due to the change of CIfixing site present already on the whole IgM molecules or to the expression of a new complement activating site. (4) A Cl-fixing low mol. wt peptide fraction was isolated. On molar basis the Cl-fixing activity of these peptides was very weak. In Table 6 ability of IgM, (Fc#)5 and peptide preparations to fix isolated C1 were compared on weight and molar basis with the data from the paper of Hurst et al. (1974). The efficiency of Cl-fixation by (Fc#)5 preparation was comparable on molar basis with that of the whole IgM molecule, but neither our other fragments nor the fragments isolated by Hurst et al. (1974) from DAU protein fixed C1 efficiently: 50-1000 times more molecules were necessary of these fragments for the fixation of the 50% of the available C1 ability, than from the original IgM preparation. Therefore, if we suppose that the isolated peptides represent the Cl-fixing site of the IgM molecule, it seems that the structure of the regions of molecule surrounding this site after complexing of IgM with antigen, strongly influence and enhance the efficiency of the Cl-fixation. Alternatively, it is possible that not the whole Cl-fixing site, but only its parts were isolated and efficient Cl-fixation could occur only if these parts worked together. The results of recombination experiments support the first possibility rather than the second one.
REFERENCES
Augener W., Grey H. M., Cooper N. R. & Mi~iler-Eberhard H. J. (1971) lmmunochemistry 8, 1011. Borsos T. & Rapp H. J. (1967) J. lmmun. 99, 263.
800
GEORGE FUST et al.
Fine P. P., Marney S. R., Jr., Colley D. G., Sergent J. S. & Des Prez R. M. (1972) J. lmmun. 109, 807. Frank M. M., Rapp H. J. & Borsos T. (1964) J. Immun. 93, 409. Glovsky M. M. & Fudenberg H. (1971) Immunochemistry 8, 129. Hurst M. M., Volanakis J. E., Hester R. B., Stroud R. M. & Bennett J. C. (1974) J. exp. Med. 140. 1117. Hurst M. M., Volanakis J. E., Stroud R. M. & Bennett J. C. (1975) J. exp. Med. 142, 1322. Hyslop N. E., Dourmashkin R. R., Green N. M. & Porter R. R. (1970) J. exp. Med. 131, 783. Ishizaka T., Ishizaka I., Salmon S. & Fudenberg H. (1967) J. Immun. 99, 82. MacKenzie M. R., Creevy N. E. & Heh M. (1971) J. lmmun. 106, 65. Mayer M. M. (1962) Experimental Immunochemistry
(Edited by Kabat E. A. & Mayer M. M.), p. 111. Thomas, Springfield, IL. Nelson R. A., Jr., Jensen J., Gigli I. & Tamura N. (1966) Immunochemistry 3, 111. Plaut A. G. & Tomasi T. B. (1970) Proc. hath. Acad. Sci. U.S.A. 65, 318. Plaut A. G., Cohen S. & Tomasi T. B., Jr. (1972) Science 176, 55. Rapp H. J. & Borsos T. (1963) J. Immun. 91, 826. Sledge C. R. & Bing D. H. (1973) J. biol. Chem. 248, 2818. Tamura N. & Nelson R. A., Jr. (1968) J. Immun. 101, 1333. Tomasi T. B. (1973) Proc. natn. Acad. Sci. U.S.A. 70, 3410. Weber K. & Osborn M. (1969) J. biol. Chem. 244, 4406. Wolf B., Michelin Lausarot P., Lesnaw J. A. & Reichmann M. E. (1970) Biochim. biophys. Acta 200, 180. Zik~n J. & Bennett J. C. (1973) Eur. J. lmmun. 3, 415. Zik~in J. & Miler 1. (1975) Immunochemistry 12, 813.
STUDY OF THE INTERACTION BETWEEN MONOCLONAL IgM PROTEINS AND THE COMPLEMENT SYSTEM GEORGE F/~IST, M,ARIA CSECSI-NAGY, GEORGE A. MEDGYESI, JUDIT KULICS and J,~NOS GERGELY Department of Immunochemistry, National Institute of Haematology and Blood Transfusion, H-1502, Budapest; and Department of Immunology of Lor~ind Ertvrs University, H-2131 GSd, Hungary (Received 6 May 1976)
Al~traet--The capacity to activate the complement system and to fix isolated C1 of different monoclonal IgM proteins and various fragments was studied. All the 8 different IgM proteins tested fixed isolated C1, but the dose of the IgM preparations necessary for the fixation of the 50~o of the available C1 activity strongly differed from each other. The complement activating (anti-complementary) effect of the IgM preparations was weak. (Fc#)5 fragments prepared by tryptic digestion from 4 different IgM proteins and separated by gel filtration at pH 8.0 could fix C1 and their anti-complementary effect significantly increased with respect to the original lgM preparations. It was demonstrated that the Cl-fixing ability of the (Fc#)5 fragment was lost after gel filtration at pH 3.0 and a Cl-fixing low tool. wt peptide fraction was released from the (Fc/t)5. This peptide recombined with the non-Cl-fixing (Fc#)5 and restored its ability for the Cl-fixation. It was shown, furthermore, that the (Fc#)5 preparation which could not fix isolated C1, activated the complement system via the alternative pathway.
INTRODUCTION The activation of the complement system is one of the most important effector functions of immunoglobulins. It is known that some immunoglobulin classes (IgG, IgM) can, and others (IgA, IgE) can not activate complement through the classical pathway, and that the complexing to soluble or particulate antigens significantly enhance the ability of IgM and IgG antibodies for the complement activation. Ishizaka et al. (1967) reported firstly on the anti-complementary effect of monomer IgM preparations. Normal IgM preparations and Waldenstr/Sm proteins induced a weak effect, aggregation strongly increased their activity. Glovsky and Fudenberg (1971) demonstrated a C1 or C! subcomponent contamination in IgM preparations. They suggested the anticomplementary effect of these preparations to be associated with the presence of activated C1. MacKenzie et al. (1971) using a Clq-fixation assay concluded that IgM proteins are heterogeneous in respect to binding of these subcomponents. In contrast, Augener et al. (1971) did not found significant differences in the C1 fixation ability of 12 monoclonal IgM proteins. Plaut et aL (1972) studied firstly the anti-complementary effect of fragments of IgM proteins cleaved by trypsin and demonstrated (Fcl05 and Fc~t fragments to be 4-7 times more effective on molar basis than the whole IgM molecule. Sledge and Bing (1973) found that IgM and (Fc#)5 fixed isolated radiolabelled C l q approximately to the same extent. Recently Hurst et al. (1974, 1975) reported on the interaction of the complement system and the various fragments of a monoclonal IgM protein (DAU). According to their study, tryptic (Fc#)5 fragments can not fix isolated human C1 but after reduction to monomer Fc/~ a tM~ 13/10--A
significant Cl-fixation was observed. They separated from the Fc part of the DAU protein two Cl-fixing fragments and they suggested that a part of the CH4 domain including 24 amino acids is responsible for the complement activating ability of the IgM molecule. The data summarized above include some contradictory findings concerning the interaction between IgM and complement system. Our recent experiments were initiated to clarify the bases of these controversial results and to collect further data on the intramolecular localization of the site (or sites) responsible for the ability of IgM molecule to fix isolated C1 and to activate the complement system. As each method used for the study of the immunoglobulin-complement interaction has some limitations and disadvantages, three methods (Cl-fixation assay, anti-complementary reaction and C4-consumption in normal human serum) were used in parallel. In the present study we tested 8 different monoclonal IgM samples and all were found to fix isolated C1. Marked differences in the C1 binding capacity were observed. (Fc/~)5 fragments were also found to fix C1 and this activity was associated with a small mol. wt peptide fraction.
MATERIALS
AND
METHODS
1. Preparation of IgM
Human Waldenstrrm IgM was prepared according to Tomasi (1973), separated on Sephadex G-200 in 0.14M NaC1. 2. Trypsin digestion
Trypsin digestion was performed according to Piaut and Tomasi (1970) and Zik~in and Bennett (1973). The IgM 793
794
GEORGE FLIST et al.
was dissolved at a concentration of 20 mg/ml in 0.05 M Tris-HC1 buffer pH 8.0, containing 0.005 M CaC12. Trypsin (SERVA, DCC-treated, 1 x crystlyophil, salt-free) was dissolved in 0.001 M HC1 at 1~o concentration and added to a pre-heated solution of IgM (20 min, 60°C) to give a substrate-to-enzyme ratio of 50:1. Digestion was stopped by cooling and immediately gel filtrated on Sephadex G-200 in 1~o NH4HCO3 (pH 8.0) or in 1 M acetic acid (pH 3.0).
or with 0.1 ml VBS for control and incubated at 37°C for 60min. Thereafter haemolytic complement activity was determined by standard method (Mayer, 1962) and expressed in CHso units. The anti-complementary activity of IgM preparations was given as the difference between CHso units measured in the control and IgM preparation containing samples and was measured by the dose of IgM consuming 1 CH~o units out of 5 units available.
4. Immunodiffusion Radial double immunodiffusion was performed in 1.5~ agar; anti-# sera were obtained from SEVAC (Prague), as well as SPCI (Paris), anti-~c and anti-2 from HYLAND (U.S.A.).
10. Measurement of the activation of the alternativ~ pathway in human serum Activation of the alternative pathway by IgM preparations was measured by the method of Fine et al. (1972) slightly modified by us. IgM preparation (0.1 ml) was incubated with 0.1 ml of human serum chelated with EGTA (ethylene glycol tetraacetic acid, final concentration 10 mM) and supplemented with MgCI2 (final concentration 8 mM) at 37°C for 60 min. Then the serum was recalcified and the residual haemolytic activity was measured. The results were evaluated as by the measurement of anti-complementary activity.
5. Diluents Veronal-buffered saline (VBS) containing 0.001 M Ca + +, 0.0005 M Mg+* and 0.1~o of gelatine was made according to Mayer (1962). Veronal-mannit buffer was prepared as outlined by Rapp and Borsos (1963). Buffer of low ionic strength (# = 0.065, G-VBS-M) was made by mixing 4 vol of VBS with 6 vol of veronal-mannit buffer. 0.1 M solution of EDTA pH 7.4 was prepared according to the formula given by Frank et al. (1964). Lower concentrations of EDTA were obtained by diluting this isotonic solution with appropriate amount of VBS without Ca ÷+ and Mg + ÷.
11. Measurement of the Cl-fixing activity of IgM preparations C 1-fixation by the IgM preparations was measured according to the method of Augener et al. (1971), using a dilution of human C1 sufficient to yield approx 63~ haemolysis. The C1 activity measured in the IgM containing and control samples was expressed in z values (average number of C1 molecules per cell). The extent of the Cl-fixation of the IgM preparations was given as the dose required for the fixation of 50~o of C1 activity (z value) measured in the control sample (Ct + buffer) and was determined graphically as outlined by Hurst et al. (1974).
3. Polyacrylamide disc electrophoresis Mol. wt was estimated according to Weber and Osborn (1969). Polymers of ribonuclease (REANAL) treated by diethyl pyroearbonate according to Wolf et al. (1970) were used as standards.
6. Preparation of complement components Functionally purified human C1 was prepared as described by Tamura and Nelson (1968). Guinea pig C2 was obtained in functionally purified form according to the method of Nelson et al. (1966). 7. Preparation of sheep erythrocyte-antibody--complement complexes EACI~p was prepared according to Tamura and Nelson (1968), EAC4,u was made as described by Borsos and Rapp (1967). 8. Measurement of C4 titre in human serum The haemolytic activity of C4 in human serum was measured by effective molecule titration as outlined by Nelson et al. (1966). 9. Measurement of anti-complementary activity of immunoglobulin preparations Fresh pooled human serum (0.1 ml) was mixed with various doses of IgM or IgM fragments in 0.1 ml of VBS
12. Estimation of the Cl-fixin# activity of lgM preparations contaminated with CI IgM preparations were serially diluted parallel in two sets of tubes (volume of dilutions 0.2 ml, dilution factor 1:3). C1 dilution (0.2 ml, z = 1-1.5) was added to the first set of tubes, 0.2 mi diluent was added to the second ones. After incubation at 30°C for 1 0 imn , EAC4 cells were added and C1 haemolytic activity (z values) was determined. C1 activity fixed by the different doses of IgM was calculated as follows: z value measured in control sample (C1 + buffer) was added to the z values measured in samples containing the corresponding dose of IgM without C1 (second set of tubes). This sum represented the C1 activity available in the tubes containing both IgM and C1 (first set of tubes). Finally, the z values measured in the samples in the first set of tubes (IgM + C1) were subtracted from the corresponding sums. This difference was taken as the extent of C1 fixation of the different doses of IgM preparations. A protocol of calculation is given in Table 1.
Table 1. Estimation of Cl-fixing activity of IgM preparations contaminated with C1 by calculation N u m b e r of tube I g M preparation, 0.2 ml//~g C l dilution, ml Diluent, ml z Value C1 activity available (B + C) C I activity fixed (B + C) - A C1 activity fixed ° in ~ of control (C)
IA 300 0.2 -1.11 . . 1.03 96
2A
3A
100 0.2 -0.97 . 0.85 79
33
4A 11
0.2
0.2
0.73
0.78
. 0.65 61
0.43 40
IB 300
2B
3B
100
. . 0.2 1.07 2.14
4B
33
. 0.2 0.75 1.82
Il
. 0.2 0.31 1.38
0.2 0.14 1.21
C (control) -0.2 0.2 1.07
-.
.
.
.
.
aDose required for the fixation of the 50~o of the C1 activity measured in control tube: 19/~g (determined graphically as outlined by Hurst et al., 1974).
Interactions of IgM and Complement 13. Analytical ultracentrifu#ation Sedimentation velocity experiments were performed in a MOM Model Ultracentrifuge, type 3170/b (Hungarian Optical Works, Budapest) using Sehlieren optics. A 12 mm 3° single-sector cell in an A 60-2 rotor was used. All sedimentation measurements were performed at 20° + 0.1°C at various rotor speeds, the rotor speed was regulated + 50 rev/min during the run.
RESULTS In the first part of experiments the Cl-fixation ability and the anti-complementary effect of monoclonal IgM proteins were compared. Preliminary experiments indicated C1 contamination in each preparation, 1 mg of the preparations contained 0.7-7.2 CH6a units of C1. The C1 contamination rendered difficult the estimation of the Cl-fixation effect of IgM preparations: the C1 activity measured in samples containing mixture of IgM and isolated CI originated both from the activity of the CI contamination and that of the isolated C1 added to the mixture. Consequently in some IgM doses this activity exceeded the control values measured in control samples without IgM. There are different possibilities to overcome this difficulty: C1 contamination either has to be eliminated or alternatively, it can be quantitated and taken into account by a correction when calculating the results. Both ways were followed in these studies. CI contamination was eliminated by heat treatment at 56°C for 6 min. (According to our preliminary experiments, this treatment destroyed 95~ of the haemolytic activity of a diluted C1 preparation.) When the subtractive method was used, different doses of IgM preparations were incubated together with a dilution of C1 (z --- 1.0-1.5) or without C1, z values were determined in each sample and the C1 consumption of the IgM preparation was calculated as described in Materials and Methods.
Study of the Cl-fixing and anti-complementary effects of I#M preparations Table 2 shows the Cl-fixation and the anti-complementary effect of 8 different monoclonal IgM preparations. The figures for results of Cl-fixation obtained by the 2 methods were at least roughly parallel. Great differences were observed, however,
795
between the Cl-consumption activity of the different IgM preparations: the dose necessary for the consumption of the 50% of the available C1 activity varied between 4-80 #g. The anti-complementary activity of 6 preparations was determined and was found to be weak: 100-> 500 #g was necessary for consumption of 1 CHso unit out of 5 units. According to Augener et al. (1971) the presence of aggregates can strongly enhance the Cl-fixing ability of IgM preparations. To control the aggregate content 3 different IgM preparations (Fig. 1)--2 strongly and 1 weakly active--were studied by analytical ultracentrifugation. Only one preparation (MAU) contained a considerable quantity of larger aggregates. The ultracentrifugation patterns of the other strongly Cl-fixing protein (VIR), as well as that of the weakly active preparation (CAN) showed one predominant peak, the sedimentation coefficient of which was 18.2 and 18.7 S, respectively. These findings suggest that the differences observed in the extent of the Cl-fixation of the different IgM proteins can not be explained by the presence of large aggregates in the preparations.
Study of the Cl-fixi~ and anti-complementary effects of (Fc#)5 preparations separated at pH 8.0 Table 3 compares the Cl-fixing and anti-complementary effects of 4 different (Fc#)5 fragments obtained by gel filtration at pH 8.0. The immunochemical purity of these (Fc#)5 preparations was controlled by radial double irnmunodiffusion using anti-#, anti-x and anti-2 sera. Each (Fc#)5 fragment fixed isolated C1, and on the weight basis the Cl-fixing activity of the (Fc#)5 and the corresponding IgM preparation was about the same. The anti-complementary effect of each (Fc#)5 preparation, however, was 6 times higher on the average as compared to the original IgM proteins. Figure 2 shows the polyacrylamide disc electrophoresis in SDS (SDS-PAGE) patterns of the (Fc#)5 preparations. Besides the bands corresponding to the products of (Fc#)5, a low mol. wt component can be seen in 3 gels out of 4. The staining of these bands was roughly parallel with the Cl-consumption activity of the (Fc#)5 preparations. The greatest intensity was observed in the case of VIR protein (Cl-fixation 20 #g). In the case of CAN preparation (Cl-fixation t20 #g) no low mol. wt component was observed.
Table 2. Cl-fixing and anti-complementary effects of monoclonal IgM preparations
Preparation FAR MAU VIR BOL O'BE CAN CZU SCI-IA
Cl-fixation (dose fixing the 50% of the CI activity available, #g) A Heat-treatment of calculation lgM for 6 min at method ° 56°C 30 4 6 53 25 80 5 10
°See Materials and Methods. N.T. not tested.
48 10 17 N.T. 30 120 N.T. N.T.
Anti-complementary effect (dose consuming 1.0 CHso units from the 5.0 units available, #g) B Heat-treated untreated (56°C, 6 min) preparation preparation 100 450 300 > 500 240 360 N.T. N.T.
300 1000 450 > 500 840 1050 N.T. N.T.
A/B 0.30 0.01 0.02 < 0.10 0.10 0.22 ---
GEORGE FOSTet al.
796
Fig. I. Sedimentation velocity pattern of 3 purified monoclonal IgM preparations. (a) VIR, (b) CAN, (c) MAU in saline, 3 mg/ml. Conditions were: speed, 44,000rev/min; temperature, 20°C; photograph, (a) and (b) 14rain, (c) 24rain after reaching full speed. S2o,w was found to be (a) 18.2S, (b) 18.7S; 21.9 S, (c) 19.0 S; 25.2 S; 32.1 S; 36.8 S; 44.8 S. Sedimentation was from left to right.
Study of Cl-fixin9 and anti-complementary effects of a (Fc#)5 preparation separated by gel filtration at pH 3.0 Based on this observation one can suppose that the Cl-fixing capacity of (Fc#)5 is due to low mol. wt components adsorbed or non-covalently bound to the (Fcp)5. Therefore, fragments obtained from two IgM proteins (VIR, CAN) by tryptic hydrolysis were separated on Sephadex G-200 column equilibrated with 1 M acetic acid. SDS-PAGE patterns of eluted fractions of VIR protein following reduction are shown in Fig. 3. The Cl-fixation and the anti-complementary effect of the 3 fractions, corresponding to the (Fc#)5, Fab/z and peptide fragments are shown in Table 4. Two (Fc#)5 fragments (VIR and CAN) fixed C1 only at a very high concentration. No significant Cl-fixation by the Fab fragment of these proteins was observed. The peptide component of VIR protein, however, could fix isolated C! efficiently. Table 4 shows the activation of the alternative pathway induced by the (Fc/~)5 (pH 3.0) fragments, too. Both (Fc#)5 fragments could activate the alternative pathway and approximately the same doses of (Fcp)5 (VIR) consumed 1.0 CHso out of 5 both in
normal human serum and in Mg ÷ +-EGTA serum. On the basis of these experiments, therefore, it seems that the Cl-fixation of (Fc/~)5 fragments isolated at slightly alkaline pH can be due to an adsorbed or non-covalently linked low mol. wt peptide which was separated from the (Fc#)5 fragment, if the gel filtration of tryptic digest of IgM was performed at acidic pH. To give a further support to this hypothesis, a 2-step experiment was done. IgM (VIR) was digested by trypsin, the (Fc#)5 fragment was isolated on Sephadex G-200 column at pH 8.0, the (Fc#)5 containing fraction freeze-dried and filtered on Sephadex G-200 column at pH 3.0. Two fractions were obtained, the first corresponded to the (Fc#)5 fragment, the other contained the low mol. wt peptides. Table 5 shows the CI consumption and the anticomplementary effect of the (Fc#)5 fraction obtained at the first as well as (Fc/~)5 and peptide fraction isolated at the second gel filtration step. (Fc#)5 fragments obtained from the tryptic digest by gel filtration at pH 8.0 strongly fixed isolated C1, but after gel filtration at pH 3.0 they have lost almost completely their Cl-fixation ability. On the other hand, during the second gel filtration step, a fraction
Table 3. Cl-fixing and anti-complementary effects of (Fc#)5 fragments prepared by tryptic digestion from IgM proteins and separated by gel filtration at pH 8,0
IgM preparation FAR VIR O'BE CAN
A Cl-fixation (dose fixing the 50~ of the CI activity available, ,ug)
B Anti-complementary effect (dose consuming 1.0 CHs0 units from the 5.0 units available, ,ug)
A/B
50 20 30 120
30 25 50 40
1.67 0.80 0.60 3.00
Interactions of IgM and Complement
797
studied either immediately, or after dialysis overnight against saline for the elimination of the cysteine, or was alkylated with iodoacetic acid and dialysed. No significant Cl-fixation was observed in either case.
Study of the C4-consumption in normal human serum induced by IgM and IgM fragments The ability of IgM (VIR) and their fragments to fix isolated C1 and to activate the classical pathway were compared. Classical pathway activation was studied by C4-consumption assay in normal human serum. A heat-aggregated human IgG preparation was included as a control (Table 5). The proportion of doses necessary for the Cl-fixation and complement activation was about 0.01 with aggregated IgG. In the case of original IgM preparation this proportion was less than 0.003. The relatively high complement activating effect (proportion 0.05) of the (Fc/05 (pH 8.0) preparation is remarkable.
DISCUSSION
(a)
(b)
(c)
(d)
Experiments described in this paper were performed to study the following and not fully clarified problems concerning interaction of IgM and complement system. (1) Is the IgM class homogeneous or heterogeneous in respect to the Cl-fixation?
Fig. 2. SDS-PAGE patterns of 4 different tryptic (Fc#)5 fragments isolated by gel filtration at pH 8.0. (a) FAR, (b) CAN, (c) VIR and (d) O'BE. Low mol. wt peptide components is indicated by an arrow.
of low mol. wt peptides was released from (Fc/z)5 (obtained by gel filtration at pH 8.0) and this peptide strongly fixed C1.
Recombination experiment with the (Fcb05 and peptide fraction isolated by gel filtration at pH 3.0 (Fc#)5 and peptide fraction obtained from the tryptic digest by gel filtration at pH 3.0 were mixed at 4°C and dialyzed against buffer of pH 8.0. The mixture was concentrated by pressure dialysis and finally gel filtrated on Sephadex G-200, at pH 8.0, to remove unbound small mol. wt peptides. The Cl-fixation activity of the fraction containing (Fc/05 fragment was compared with that of the original materials..The original (Fc/~)5 fragment did not fix C1 (Cl-fixing dose > 1000/~g), the peptide fraction showed a marked Cl-fixing activity (Cl-fixing dose 21 #g). The (Fc#)5 fragment recombined with the peptide fixed C1, too (Cl-fixing dose 70.4~tg). Therefore it seems that the non-Cl-fixing (Fc/~)5 fragment had at least partly regained its ability to fix isolated C1 after recombination with the Cl-fixing peptide.
Study of the Cl-fixing effect of the Fc# fragment In the subsequent experiment the (Fc#)5 obtained by acidic gel filtration was reduced by 0.05 M cysteine. The generated monomer Fc~ fragment was
(a)
(b)
(c)
(d)
Fig. 3. SDS-PAGE patterns of (a) (Fc#)5, (b) Fab# and (c) low mol. wt peptide containing fraction, obtained from tryptic digest of VIR IgM protein, by gel filtration at pH 3.0, (d) polymerized ribonuclease. All samples were reduced in 1~ SDS.
798
GEORGE FkIST et al.
Table 4. Cl-fixing and anti-complementary effects of different fragments of monoclonal IgM proteins obtained by tryptic digestion and separated by gel filtration on Sephadex G-200 column at pH 3.0
IgM preparation
Fragment
A CI-fixation (dose fixing the 50% of the CI activity available, #g)
B Anti-complementary effect (dose consuming 1.0 CH~o units from the 5.0 units available, ~g)
A/B
C Activation of the alternative pathway (dose consuming 1.0 CHso units out of 5.0 units in Mg ++ - EGTA)
VIR
(Fc#)5 Fab peptide
600 226 20
19 140 60
31 1.6 0.33
20 N.T. N.T.
CAN
(Fc#)5 Fa b peptide
1003 1000 500
113 120 143
8 8 3.5
190 N.T. N.T.
N.T. not tested. (2) Cl-fixation by IgM is followed or not by the activation of the classical complement pathway? (3) (Fcp)5 fragments obtained by tryptic hydrolysis of the IgM molecule can or cannot fix isolated C1 and activate the complement system? (4) To what extent can Cl-fixing capacity be attributed to a relatively small submolecular region?
plementary effect in undiluted human serum. The weak complement activating ability of each IgM preparation could be due to C1 contamination, since this activity decreased after heat-treatment of IgM at 56°C for 6 min. On the other hand it is possible that a part of the anti-complementary effect is mediated through the alternative pathway.
(1) All the 8 IgM preparations studied were contaminated with C1. The extent of this contamination varied between large limits. These findings are in agreement with those of Glovsky and Fudenberg (1971). MacKenzie et al. (1971) found marked differences in the C 1-fixing capacity of the different IgM proteins. In contrast, Augener et al. (1971) did not observe such differences at the study of 12 monoclonal IgM preparations. The results of our experiments agree with those of MacKenzie et al. (1971), and it was demonstrated that the differences between Cl-fixation activity were not necessarily associated with the presence of large aggregates in the preparations. The discrepancies between these findings may be explained by the fact that Augener et al. (1971) isolated their IgM preparations from heat-treated serum in contrast to MacKenzie et al. (1971) and to our recent studies.
(3) (Fc#)5 preparations isolated from different IgM proteins were studied and the Cl-fixation activity of each preparation was nearly identical with that of original IgM molecules. This finding corresponds to the results of Sledge & Bing (1973), who demonstrated that IgM and (Fc#)5 fixed C1 nearly to the same extent. More recently, however, Hurst et al (1974) did not find any Cl-fixation by a tryptic (Fc#)5 fragment of a Waldenstrrm protein isolated from the digest by means of gel filtration at slightly alkaline pH purified further by Bio-Gel P-150. In SDS-PAGE disc electrophoresis of our (Fc#)5 fragments a low mol. wt component was found in all but one preparations and the intensity of its zone showed a rough correlation with the Cl-fixation activity of the (Fc#)5 preparations. It was supposed, therefore that the Cl-fixation of (Fc/z)5 preparations may be due to the presence of a peptide non-covalently bound or adsorbed to the fragments. This hypothesis was supported by the results of the subsequent experiments: (Fc#)5 isolated from the tryptic digest by gel filtration at acidic pH lost its Cl-binding capacity. The same results were
(2) It was found in good accordance with the results of other authors (Ishizaka et al., 1967; Glovsky & Fudenberg, 1971 ; Plaut et al., 1972) that IgM preparations have only a weak capacity to activate the complement system as measured by their anti-com-
Table 5. Comparison of Cl-fixation, anti-complementary effect and classical pathway activation (consumption of C4 in normal human serum) induced by aggregated IgG, IgM and different tryptic fragments of IgM
No. 1.
Preparation Aggregated human IgG Monoclonal lgM
II.
A Cl-fixation (dose fixing the 50% of the CI activity available, #g)
B Anti-complementary effect (dose consuming 1.0 CH~ o units from the 5.0 units available, #g)
C Dose consuming the 50~ of the C4 activity available in normal human serum, ,ag 18.9
A/B --
A/C
0.17
N.T.
5.5
300
>1600
0.02
640
13.00
--
> 150
> 150
--
--
(VIR) IU.
IV. V.
Tryptic (Fc#)5 from If separated by gel filtration at pH 8.0 (Fop)5 obtained from III by gel filtration at pH 3.0 Peptide fraction from III by gel filtration at pH 3.0
N.T. not tested.
400 22.1
799
Interactions of IgM and Complement Table 6. Comparison of the efficiency of Cl-fixation of various IgM proteins and IgM fragments on weight basis and on molar basis Efi$eiency of CIfixation on molar basis (compared to the original lgM)
MoL wt
Cl-fixation (dose fixing the 50% of the CI activity available, #g)
Part 1° IgM (VIR) (Fc#)5b pcptidc c
840,000 340,000 10,000
5.5 7.8 2.1
1.0 0.29 0.003
Part 2a IgM (DAU) Fc# C.4 fragment (CNBr5)2 fragment
840,000 34,000 6800 40,000
1.5 40 20 46
1.0 0.002 0.001 0.002
Preparation
*Data from the present work. ~III preparation in Table 5. clV preparation in Table 5. dData obtained from the paper of Hurst et al. (1974).
obtained, when the Cl-fixing (Fc#)5 preparation had been rechromatographed in an acidic medium. Nor did the Fc# fragment obtained by reduction of the (Fc#)5 preparation fix isolated C1. During the gel filtration of the (Fc#)5 at pH 3.0 a fraction containing a peptide (or peptides) of low mol. wt was eluted which fixed C1. After incubation and gel filtration of this fraction with the non-Cl-fixing (Fc#)5 fragment at slightly alkaline pH, the (Fc#)5 obtained could fix again isolated C1. These results suggest that the peptide recombined with the (Fcg)5 fragment and the Cl-fixing ability of the latter was at least partly recovered. Since the activity of the recombinate was weaker than that of the (Fc/~)5 fragment isolated by gel filtration at pH 8.0 immediately after the tryptic digestion of IgM, it can not be excluded that the decreased Cl-fixing capacity of (Fc#)5 following the gel filtration at acidic pH may be the consequence of a structural change induced by the acidic pH. (Fc#)5 fragments isolated by gel filtration both at slightly alkaline or acidic pH independently of their Cl-fixing capacity, were able to activate the whole complement system approximately to the same extent. The anti-complementary effect of the (Fc#)5 fragment was at least 6 times stronger on weight basis than that of the original IgM molecule. Similarly, marked C4-consumption in normal human serum was observed with a Cl-fixing (Fc#)5 preparation. Similar findings were reported by Plaut et al. (1972). On the other hand, if the anti-complementary effect of an (Fc#)5 preparation was measured in normal human serum chelated with EGTA and supplemented with Mg ++ ions, in which the complement system can be activated only via the alternative pathway (Fine et al., 1972), significant complement consumption was observed. A non-Cl-fixing (Fc#)5 preparation was demonstrated to activate the complement system almost exclusively through the alternative pathway. These experiments suggest that 2 additional activities were generated or at least strongly increased on the (Fc#)5 fragment as compared to the original IgM molecule: the ability to activate the classical pathway (as revealed by C4-consumption in human normal serum) and the ability to activate the alternative pathway. It was reported that immune complexes contain-
ing IgM antibodies strongly activate the classical pathway (Hyslop et al., 1970) and some immune complexes are capable to the activation of the alternative pathway, too (Zikfin & Miller, 1975). These activities are localized on the (Fc#)5 part of the IgM molecules. Therefore, it seems that tryptic cleavage induces structural changes on the (Fc#)5 part of molecules, which are similar to the changes occurring during the interaction of IgM antibodies and antigens. It can not be decided at the present time that the appearance or increase of the classical pathway activating capacity of (Fc#)5 fragment is due to the change of CIfixing site present already on the whole IgM molecules or to the expression of a new complement activating site. (4) A Cl-fixing low mol. wt peptide fraction was isolated. On molar basis the Cl-fixing activity of these peptides was very weak. In Table 6 ability of IgM, (Fc#)5 and peptide preparations to fix isolated C1 were compared on weight and molar basis with the data from the paper of Hurst et al. (1974). The efficiency of Cl-fixation by (Fc#)5 preparation was comparable on molar basis with that of the whole IgM molecule, but neither our other fragments nor the fragments isolated by Hurst et al. (1974) from DAU protein fixed C1 efficiently: 50-1000 times more molecules were necessary of these fragments for the fixation of the 50% of the available C1 ability, than from the original IgM preparation. Therefore, if we suppose that the isolated peptides represent the Cl-fixing site of the IgM molecule, it seems that the structure of the regions of molecule surrounding this site after complexing of IgM with antigen, strongly influence and enhance the efficiency of the Cl-fixation. Alternatively, it is possible that not the whole Cl-fixing site, but only its parts were isolated and efficient Cl-fixation could occur only if these parts worked together. The results of recombination experiments support the first possibility rather than the second one.
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