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The serum capacity to solubilize immune complexes (ICSC) measured by an enzyme-anti-enzyme complex probe
Journal of Immunological Methods, 77 (1985) 119-130 Elsevier
119
JIM03391
The Serum Capacity to Solubilize Immune Complexes (ICSC) Measured by an Enzyme-Anti-Enzyme Complex Probe Paola Migliorini l, Giorgio Chieregatti 2, Giorgio Trovatello 3, Sebastiana Cantarella 3, Daniela Fenoglio 3 and Franco Celada 3.. J Istituto di Patologia Medica, Universith di Pisa, Pisa, 2 Farmitalia Carlo Erba, Divisione Diagnostici, Rodano (Milan), and 3 Cattedra di Immunologia, Universith di Genova, Viale Benedetto XV, 10, Genoa, Italy
(Received 11 September 1984, accepted 31 October 1984)
The capacity to solubilize immune complexes can be readily measured by incubating the test serum with a suspension of an immune precipitate formed by/3-galactosidase and anti-fl-galactosidase antibody, and then reading the enzyme units (EU) liberated in the clear supernatant. Our method is rapid and inexpensive; it can be performed in plates and read in scanning colorimeters. Although on large numbers of observations the ICSC is significantly correlated with the CH50, a few discordant cases suggest that solubilization and haemolysis are functions of the alternative and classical pathways of complement respectively. Key words: immune complexes - solubilization - fl- galactosidase - complement
Introduction
Antigen-antibody precipitates can be solubilized by fresh serum (Miller and Nussenzweig, 1975). The first description of this simple phenomenon 15 years ago caused some surprise, and thereafter a number of laboratories have investigated the mechanism of solubilization involved and clarified its sequential steps (Takahashi et al., 1977, 1978). The key event in this process appears to be the assembly of C3 convertase, by alternative pathway of complement activation, on the immune precipitate. As a result of the subsequent cleavage of the C3 component, the nascent C3b binds to the Fc region of antibody moieties. Since hydrophobic bonds established between adjacent Fc regions during lattice formation constitute an important factor in the immune precipitation, their disruption by the intercalating C3b causes
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
120 release of soluble immune complexes in the liquid phase. Thus, the liberated complexes bear complement components tightly linked on their surface and are unable at this stage to further activate complement or to react with membrane-bound Fc receptors (Takahashi et al., 1980). The above mechanism is probably operational in vivo as one of the physiological mechanisms of handling immune complexes by preventing the formation or accelerating the dispersal of harmful insoluble complexes. For these reasons measuring the capacity of individual sera to solubilize immune complexes is considered to be a valuable tool in the study of immune complex related diseases, immune deficiencies and immune disturbances in general (Aguado et al., 1981; Schifferli et al., 1981). For this task in vitro assays have been constructed essentially based on the addition to the patient's serum of artificial antigen-antibody precipitates where the antigen or the antibody was radioactive, and the measurement of radioactivity released in the supernatant after incubation and centrifugation (Aguado et al., 1981; Schifferli et al., 1981; Baatrup et al., 1983). The results obtained showed that ICSC was often impaired in Ic-mediated diseases and that in some of them (e.g., SLE), the degree of impairment correlated closely with the disease activity (Aguado et al., 1981; Schifferli et al., 1981; Sakurai et al., 1982; Celada et al., 1984). In order to avoid the necessity of handling radioactive materials in the serological laboratory and to circumvent the decay problems posed by the use of a radioactive probe, we have developed a new test that employs a 'probe complex' where the antigen is enzymatically active E. coli fl-galactosidase. In this paper we describe the characteristics of the probe and the results obtained in normal and pathological sera.
Materials and Methods
Enzyme E. coli fl-galactosidase from E. coli was used as described earlier (Migliorini et al., 1983). The enzyme was purified from E. coli strain 3300 by DEAE-Sephadex (Celada et al., 1971). It was stored in 40% ammonium sulphate at 4°C. The preparation used had a specific activity of 144,000 E U / m g . A nti-fl-gal antisera The following antisera were used to prepare the probe complex: (1) donkey, primary response; (2) donkey, secondary response; (3) rabbit, secondary response. All antisera were prepared in the laboratories of Farmitalia Carlo Erba, as described in earlier reports (Gnemmi et al., 1981; Migliorini et al., 1983). Donkeys were immunized by injection in 2 sites of 1 mg fl-gal in 1 ml complete Marcol adjuvant and boosted after 3 months injecting in 2 sites 0.5 mg in incomplete Marcol adjuvant (1 : 1), 0.5 ml/site. NZW rabbits were immunized by injecting in the footpads 100 ~g fl-gal in complete Freund's adjuvant and boosted after 2 weeks by injecting 10/~g of soluble antigen intradermally in several sites of the back.
121
Precipitation curve The precipitation tests were performed by mixing 2200 EU of fl-gal in 0.2 ml with the same amount of dilutions of donkey antiserum or rabbit antiserum. After 3 h at RT, the tubes were centrifuged for 30 min at 3000 x g and supernatants measured for residual enzyme activity.
Precipitation of the probe complex Each probe complex (PC) was numbered after the antiserum used (1, 2, 3). The PC 1 was prepared by mixing I vol. of fl-gal (2200 EU/ml), with 1 vol. of antiserum at the chosen dilution in veronal-buffered saline (VBS). After 1 h at 37°C and 22 h at 4°C, the PC was centrifuged at 4°C, 10,000 x g for 10 min. The precipitate was washed twice in 2 vols. of cold VBS and recentrifuged in the same conditions. The precipitate was then carefully resuspended in 8 vols. of cold phosphate-buffered saline containing 0.15 mM CaC12, 0.5 mM MgC12, 0.1% gelatine (PBS-G) and stored at 4°C. Its activity, checked at 1 month intervals, was stable for at least 3 months. In order to disperse evenly the PC, it was vortexed vigorously before delivering each series of 8 samples. After washing, 73% of initial enzyme activity was recovered in the precipitate, corresponding to 3.6/~g/ml of fl-gal at the final dilution of the PC. PC 2 and PC 3 were prepared and used at the same concentration as the PC 1.
Performance of the tests A micro- (A) and a macro-method (B) were devised. Because of the similarities, they are described together. Fifty microlitres of the tested serum, 50/~1 of PBS and 25 /~1 of PC suspension were placed in a polystyrene tube (B) or in a well of a 96-well microtitre plate (A). Tubes and plates were incubated for 2 h at 37°C, then the tubes were centrifuged at 3000 x g and 4°C for 20 min (B) and the plates at 2000 rpm at room temperature for 40 min (A). Fifty microlitres of the supernatants (A) and (B) were transferred to another tube (B) and another plate (A) and enzyme activity measured by hydrolysis of ortho-nitrophenyl-fl-D-galactopyranoside (Manca et al., 1980) and expressed as enzyme units (EU), 1 EU being the amount of enzyme that hydrolyzes 10 -9 moles of substrate in 1 rain. Plates were read in a Multiskan photometer (Flow Laboratories) equipped with a 420 mm filter. Samples were tested in duplicate and the reference serum in quadruplicate. ICSC was calculated as per cent ratio of EU solubilized by the sample to EU solubilized by the reference serum. In tubes containing buffer only, ICSC (spontaneous solubilization of the PC) never exceeded 5%.
Purification of rheumatoid factor (RF) RF was isolated from the serum of a patient with WaldenstriSm macroglobulinaemia and high RF titre. It was precipitated by 33% saturated ammonium sulphate, dialyzed against acetate buffer 0.01 M, p H 5 and passed through a Sephadex G-200 column equilibrated with the same buffer. The excluded protein was concentrated and dialyzed against PBS, and adjusted to a protein concentration
122 of 2 mg/ml. Radial immunodiffusion showed the presence of IgM and traces of IgG.
Conglutinin-coated beads Conglutinin (K) was isolated from bovine serum according to Lachmann et al. (1973). Polystyrene beads were incubated in a solution of K containing 10/~g/ml in VBS at pH 7.4. The volume of the solution was 0.25 m l / b e a d and the incubation was 24 h at room temperature. Subsequently the beads were saturated with bovine albumin 3%. Determination of anti-B-gal antibody affinity Among anti-/3-gal antibodies found in the serum of immunized animals, one restricted family has been characterized by its ability to activate the defective enzyme produced by certain mutant E. coli strains (Rotman and Celada, 1968). These activating antibodies demonstrate one-hit kinetics; therefore it is possible to measure their affinity even when a complexed antigen is used (Celada et al., 1973). Fifty microlitres of donkey (1 : 100) and rabbit (1 : 100) antiserum were dispensed in flat-bottom microtitre plates (Sterilin M 29A) with 20/~1 of increasing concentrat i o n s of m u t a n t 6101 in b u f f e r B (BB), c o n t a i n i n g 10 m M (hydroxymethyl)aminomethane (Tris), 10 mM MgC1 z, 0.1 M NaC1 and 0.05 M 2-mercaptoethanol; its pH was adjusted to 7.2 with acetic acid. After 3 h at 37°C, 200 t~l of 2-nitrophenyl-/3-D-galactopyranoside (ONPG), 3 x 10 3 M were added and EU present in each well were calculated from the OD at 420 nm. On the assumption that every 6101 molecule bound by activating antibody was active, the affinity was calculated by double reciprocal plotting of bound vs. free antigen. It was expressed as 1 / k D a (in M - l ) corresponding to the concentration of free antigen at which the binding reached 50% of the maximum. Normal and pathological sera Normal sera were obtained from healthy laboratory staff members. Pathological sera were collected at the Rheumatic Disease Unit, University of Pisa. The diagnosis of systemic lupus erythematosus and rheumatoid arthritis were made according to well accepted criteria (Ropes et al., 1958; Tan et al., 1982). The diagnosis of essential mixed cryoglobulinaemia was made on the basis of the typical findings (purpura, weakness, arthralgias/arthritis, presence of serum cryoglobulins), after having ruled out other known causes of cryoglobulinaemia by careful clinical and laboratory investigation. Measurement of CH50 The determination of CH50, defined according to Kabat and Mayer (1961) as the quantity of complement required for 50% lysis of sensitized erythrocytes, was performed by a microtitre plate method (see Celada et al., 1984). Normal and pathological sera in 7 progressive dilutions ranging from 1 : 400 to 1 : 100 were tested for their ability to lyse sensitized sheep RBC after incubation for 30 min at 37°C. The total ~eaction volume was 250/~1, and the final RBC concentration 0.2%. After
123
centrifugation for 15 min at 2000 rpm, the haemoglobin concentration in the supernatant was measured in a Multiskan photometer. The 50% lysis point was determined by a computerized procedure that also calculated the number of 50% haemolytic U / m l original serum (HU). Under these conditions the CH50 level ranged in normal sera between 720 and 1180 U / m l .
Results
Choice of the antigen-antibody ratio in the probe complexes Fig. 1 shows the precipitation curves obtained by adding antisera 1, 2 3 at doubling dilutions to a fixed amount of fl-gal. Precipitable complexes formed at dilutions ranging from 1 : 8 to 1 : 64. The latter, at which few soluble complexes were
%EU 100"
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1:256 DILUTIONS
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Fig. 1. Precipitation curves of the 3 anti-fl-gal antisera: - - , Ab 1 (donkey, primary response); . . . . . . , Ab 2 (donkey, secondary response); . . . . . , Ab 3 (Rabbit, secondary response). Conditions aa in Materials and Methods. Ordinate: fl-gal EU found in supernatant, in percent of input (2200 EU)." Abscissa: dilution of antisera.
124 ICSC
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60
80
100
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Fig. 2. Compound illustration of 2 time-dependent events studied on PC 1. The continuous line shows the kinetics of solubilization (as per cent of the ICSC of a normal reference serum (ordinate). The dotted line shows the kinetics of appearance of C3b-containing soluble complexes in the supernatant. This parameter was studied by measuring (ordinate) the binding of PC (fl-gal activity) to plastic beads coated with bovine conglutinin.
detectable, was chosen as the a g : a b ratio for PC 2 a n d 3. A c c o r d i n g to the same criterion, an a n t i s e r u m dilution of 1 : 32 was chosen for PC 1.
Kinetics of solubilization and nature of solubilized complexes This p a r a m e t e r was s t u d i e d with PC 1. T h e a m o u n t of solubilized P C increased steadily in the first 10 m i n of i n c u b a t i o n a n d a p p r o a c h e d the m a x i m u m after 1 h (Fig. 2). I n tubes c o n t a i n i n g heat d e c o m p l e m e n t e d serum, no e n z y m e activity was detectable in the s u p e r n a t a n t , i n d i c a t i n g that the r e a c t i o n is c o m p l e m e n t d e p e n d e n t a n d that no free e n z y m e is released s p o n t a n e o u s l y u n d e r the c o n d i t i o n s of the test. O n the o t h e r h a n d , fresh a n d M g - E G T A chelated serum solubilize the same a m o u n t of PC in 2 h. It is k n o w n that solubilization of i m m u n e p r e c i p i t a t e s d e p e n d s u p o n the intercal a t i o n of C3b in the lattice a n d that solubilized c o m p l e x e s still retain C3b on their surface. Since b o v i n e conglutinin b i n d s I C through their C3b i moiety, we investigated the structure of solubilized PC evaluating its b i n d i n g ability to c o n g l u t i n i n - c o a t e d beads.
125 ICSC" 100n,,
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Fig. 3. Effect of serum dilution on the ICSC, using different PC preparations. Ordinate: ICSC, as per cent of normal reference serum. Abscissa: serum dilution. The means and standard deviations of 5 sera are given. - - , PC 1; . . . . . . , PC 2; . . . . . , PC 3.
The supernatants obtained at the different times in the above experiment were incubated with K-coated beads for 24 h at 4°C. After 2 washings in VBS-Tween 80 0.05%, enzyme activity on the beads was measured. The binding curve, closely parallel to the kinetics of solubilization (Fig. 2), indicates the presence of C3b on solubilized PC.
Immune complex solubilizing capacity in normal sera tested with different probe complexes Five normal sera were titrated against PC 1, 2, 3. As can be seen in Fig. 3 the solubilization capacity decreases with increasing dilutions at a rate strikingly different for the different PC preparations. The rate of I C S C decrease was f o u n d to be related to the affinity of the antibodies used to form the complex. The association constant of serum 3 was 2 × 108 M - ] , of serum 2 about 2 × 107 M -1, while that of serum 1 very m u c h lower although, because of the heterogeneity of the primary response, an association constant was not calculated.
Interfering substances The interference of rheumatoid factor (RF) on the measurement of I C S C has been suggested in some papers (Tincani et al., 1983). We investigated the influence of R F on the solubilization of PC by adding a fixed a m o u n t (20 t~g) of a purified I g M monoclonal R F to normal sera (Fig. 4). With PC 1, the solubilization capacity of 1 : 2 diluted sera was scarcely affected by the addition of R F except when the complement content was lowered by dilution, R F has an inhibitory effect on ICSC, that becomes striking when ICSC is 50-60%. O n the contrary, with PC 2 and 3 the inhibition exerted by R F was evident even on sera diluted 1 : 2.
126 ICSC 100"
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Fig. 4. Interference of rheumatoid factor on ICSC. Ordinate: ICSC, as % of normal reference serum. Abscissa: dilution of sera. For each PC, the ICSC is shown as a function of serum dilution, in the absence (O) or in the presence (e) of a constant amount of IgM monoclonal RF. The means and standard deviations of 4 sera are given.
I C S C in pathological sera Fig. 5 shows ICSC levels measured by PC 1 in n o r m a l a n d pathological sera. In 65 n o r m a l sera, the m e a n ICSC level was 97 _+ 8%. M e a n m i n u s 2 SD of n o r m a l sera (80%) was considered the lower limit of normality. ICSC was low in 2 0 / 5 3 SLE sera; 1 3 / 4 1 E M C sera; 1 / 1 5 r h e u m a t o i d arthritis sera a n d 1 1 / 1 4 synovial fluids from r h e u m a t o i d arthritis patients. I n 88 unselected specimens, collected for CHS0 m e a s u r e m e n t , a parallel evaluation of ICSC was o b t a i n e d (Fig. 6). The 2 parameters are strongly correlated ( r = 0.43, P < 0.001), but some sera showed n o r m a l C H 5 0 in spite of low ICSC a n d one had high ICSC with low CHS0 level. PC 1 a n d 3, c o m p a r e d in 38 unselected pathological sera, were highly correlated ( r = 0.59, P < 0.0001); however, sera had lower ICSC levels when measured with PC 3. This higher n u m b e r of 'positive' results suggests a higher sensitivity for the PC 3 assay.
127 ICSC 120 -
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Fig. 5. ICSC in normal sera vs. SLE, EMC, RA patients and (SF), synovial fluid of rheumatoid arthritis patients. The determinations were performed with PC 1.
Discussion
The method described in this paper measures a body fluid's capacity to solubilize immune complexes by probing it with an enzyme-antibody complex whose liberation in the soluble phase can be followed with ease and precision by a simple determination of /3-galactosidase activity. The results of this test have been shown to be significantly in agreement with those of methods employing a radiolabelled complex (125I-BSA-anti-BSA); e.g., a comparative study performed by Corvetta et al. (1984) on 52 sera yielded a high correlation index (r = 0.73, P < 0.001). Nevertheless, the probe complex system has several advantages, all facilitating the preparation of
128 ICSC 12o•
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~ 40-
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5 0' 0
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1o'o 0
1500' CH50
Fig. 6. Correlation of ICSC and CH50 in the same sera. Ordinate: ICSC (measured on PC 3). The value 80% represents the lower limit set for normal values. Abscissa: haemolytic capacity expressed as CH50 units (HU). The value of 720 HU represents the lower limit set for normal sera. industrial kits. It avoids the risks and administrative problems of using radioactive material. The PC reagents are stable over months and can be prepared in large batches lasting for a number of tests. The manipulations are simple and the assay can be performed in microplates with considerable economy of materials and time. After this positive presentation, we would like to raise discussion about the following points: (a) by which mechanism is the PC solubilized; (b) what 'section' of the complement system is the ICSC representing and is it possible to extrapolate the results on the entire system; (c) what is the influence of the quality of the antibody used in the PC on the ICSC. The present, rather mechanistic view of the solubilizing event is based on the accepted concept that any macromolecule is kept in solution if and until its molecular weight is counterbalanced by an adequate number of surface hydrophilic groups, that keep it, so to speak 'floating' in the fluid. Deposition of C3b splits the large IC aggregates into fragments which become hydrophilic presumably by the introduction of new hydrophilic groups. Suggestive evidence, if not proof, that solubilization works through C3b deposition is our finding that the proportion of probe complex solubilized at any time is closely paralleled by the amount of activity that can be bound by beads coated with conglutinin - - a protein endowed with specific affinity for C3b. Our data also show some evidence that most of the C3 activation in question is alternative pathway dependent since approximately the same amount of PC is solubilized if the serum is treated with E G T A - M g which blocks the classical pathway. Malasit et al. (1983) found that under similar conditions the solubilization process was slower than in fresh serum but the final amount of complex solubilized was the same and so was the profile of the sucrose density gradient analysis. In our case the incubation time (2 h) was probably too long to permit differences in kinetics to become manifest. A further point concerns the variables that can affect the sensitivity of the ICSC test, and which can also be used by the investigator as handles to adjust the characteristics of the assay to the clinical needs.
129
A first variable is the amount of precipitating PC used, since the complement concentration needed for complete solubilization will be relative to that amount (Baatrup et al., 1983). A second variable is the intrinsic affinity of the antibody used to prepare the PC. From all the experiments with different antibodies shown in the present article we can conclude that regardless of the animal species in which the antiserum was raised, hyperimmune, high affinity antibodies produce immune precipitates that are more difficult to solubilize and will therefore require higher complement concentrations than those formed by low affinity, primary response antibodies. As a consequence, it is possible to grade the degree of stringency and the sensitivity of the test by selecting an antibody of the desired affinity - all other conditions including the total amount of PC remaining equal. The highly significant correlation between CH50 and ICSC is explicable by the very fact that C3 is a key component of both complement activation pathways. Any severe C3 deficiency will affect the classical pathway since it will limit in the formation of the C5 convertase C1423, and will affect ICSC for want of C3b deposition. On the other hand, intermediate deficiencies of C3 will be detected by the ICSC test but may not affect the haemolytic test, since C3 is not a limiting factor in the classical cascade. This situation leads to the discrepancy between low ICSC and normal CH50, which is found rather infrequently but which can also be caused by the presence of nephritic factor (C3NeF), an autoantibody that stabilizes the C3 convertase of the alternative pathway. The opposite situation, (i.e., normal ICSC, low CH50) would be expected to arise in genetic deficiencies of C2, C4 or C5-C9. We can conclude that ICSC values reflect closely the condition of the alternative pathway. This means that the ICSC test would be best used in parallel with an assay specific for the classical pathway. Such a combination would permit a comprehensive assessment of the entire complement system. On the other hand, considering the ease and rapidity of performing a determination by our method and the high correlation with CH50, ICSC could also be used as a first screening, instead of the more delicate haemolytic assay.
Acknowledgements This work was in part supported by Grant No. 83/00527.57 Tecnologie Biomediche e Sanitarie from the Italian Research Council (C.N.R.). Ms. Luisa Di Rosa is gratefully acknowledged for her secretarial help.
References Aguado, M.T., L.H. Perrin, P.A. Miesher and P.H. Lambert, 1981, Arthritis Rheum. 24, 1225. Baatrup, G., I. Peterson, S.E. Svehag and I. Brandslund, 1983, J. Immunol. Methods 59, 369. Celada, F., J. Ellis, K. Bodlund and B. Rotman, 1971, J. Exp. Med. 134, 751. Celada, F., A.J.L Macario and E. Conway De Macario, 1973, Immunochemistry 10, 797. Celada, F., P. Migliorini, F. Manca, G. Trovatello, S. Cantarella, G. Rossi, S. Bombardieri, S. Barra, S. Canesi, D. Giordano, A. Parodi, A. Rebora, M.D. Sabbadini and P. Saluscev, 1984, in: Recent Advances in Lupus Erythematosus, eds. P.H. Lambert, L. Perrin and S. Izui (Academic Press, London) pp. 179-183.
130 Corvetta, A., G. Marchigiani, P. Migliorini and G. Danieli, 1984, Proc. II Int. Symposium on Plasma Exchange in Nephrology, Viareggio, May 18, 1984 (under the patronage of the Italian Societies of Nephrology, Immunohematology and Rheumatology) (abstract), p. 13. Gnemmi, E., P. Migliorini, G. Chieregatti, F. Manca, S. Cantarella and F. Celada, 1981, Lab. J. Res. Lab. Med. 8, 5. Kabat, E. and M. Mayer, 1961, Experimental Immunochemistry, II edition (C.C. Thomas, Springfield, IL) p. 136. Lachmann, P.J., M.J. Hobart, P.W. Aston, 1973, Handbook of Experimental Immunology, Vol. 1, ed. D.M. Weir (Blackwell, Oxford) Ch. 5. Malasit, P., S.R. Bartolotti and J.H. Humphrey, 1983, Immunology 48, 779. Manca, F., P. Migliorini, S. Bombardieri and F. Celada, 1980, Clin. Immunol. Immunopathol. 16, 131. Migliorini, P., G. Trovatello, S. Cantarella, F. Manca, S. Bombardieri and F. Celada, 1983, J. Immunol. Methods 59, 245. Miller, G.W. and V. Nussenzweig, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 418. Ropes, M.W., G.A. Bennet, S. Cobb, S. Jacox and R.A. Jessar, 1958, Bull. Rheum. Dis. 9, 175. Rotman, B. and F. Celada, 1968, Proc. Natl. Acad. Sci. U.S.A. 60, 660. Sakurai, T., T. Fujita, J. Kono, T. Kabashima, K. Yamane, N. Tamura and H. Kashiwagi, 1982, Clin. Exp. Immunol. 48, 37. Schifferli, J.A., S.M. Morris, A. Dash and D.K. Peters, 1981, Clin. Exp. Immunol. 46, 557. Takahashi, M., B.F. Tack and V. Nussenzweig, 1977, J. Exp. Med. 145, 86. Takahashi, M., S. Takahashi, V. Brade and V. Nussenzweig, 1978, J. Clin. Invest. 62, 349. Takahashi, M., S. Takahashi and S. Hirose, 1980, Prog. Allergy 27, 131. Tan, E.M., A.S. Cohen, J.F. Fries, A.T. Masi, D.F. McGhane, N.F. Rothfield, J. Green Schaller, N. Talal and R.J. Winchester, 1982, Arthritis Rheum. 25, 1271. Tincani, A., P. Migliorini, C. Ferri, G. Balestrieri and S. Bombardieri, 1983, Folia Allergol. Immunol. Clin. 30, 22.
119
JIM03391
The Serum Capacity to Solubilize Immune Complexes (ICSC) Measured by an Enzyme-Anti-Enzyme Complex Probe Paola Migliorini l, Giorgio Chieregatti 2, Giorgio Trovatello 3, Sebastiana Cantarella 3, Daniela Fenoglio 3 and Franco Celada 3.. J Istituto di Patologia Medica, Universith di Pisa, Pisa, 2 Farmitalia Carlo Erba, Divisione Diagnostici, Rodano (Milan), and 3 Cattedra di Immunologia, Universith di Genova, Viale Benedetto XV, 10, Genoa, Italy
(Received 11 September 1984, accepted 31 October 1984)
The capacity to solubilize immune complexes can be readily measured by incubating the test serum with a suspension of an immune precipitate formed by/3-galactosidase and anti-fl-galactosidase antibody, and then reading the enzyme units (EU) liberated in the clear supernatant. Our method is rapid and inexpensive; it can be performed in plates and read in scanning colorimeters. Although on large numbers of observations the ICSC is significantly correlated with the CH50, a few discordant cases suggest that solubilization and haemolysis are functions of the alternative and classical pathways of complement respectively. Key words: immune complexes - solubilization - fl- galactosidase - complement
Introduction
Antigen-antibody precipitates can be solubilized by fresh serum (Miller and Nussenzweig, 1975). The first description of this simple phenomenon 15 years ago caused some surprise, and thereafter a number of laboratories have investigated the mechanism of solubilization involved and clarified its sequential steps (Takahashi et al., 1977, 1978). The key event in this process appears to be the assembly of C3 convertase, by alternative pathway of complement activation, on the immune precipitate. As a result of the subsequent cleavage of the C3 component, the nascent C3b binds to the Fc region of antibody moieties. Since hydrophobic bonds established between adjacent Fc regions during lattice formation constitute an important factor in the immune precipitation, their disruption by the intercalating C3b causes
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
120 release of soluble immune complexes in the liquid phase. Thus, the liberated complexes bear complement components tightly linked on their surface and are unable at this stage to further activate complement or to react with membrane-bound Fc receptors (Takahashi et al., 1980). The above mechanism is probably operational in vivo as one of the physiological mechanisms of handling immune complexes by preventing the formation or accelerating the dispersal of harmful insoluble complexes. For these reasons measuring the capacity of individual sera to solubilize immune complexes is considered to be a valuable tool in the study of immune complex related diseases, immune deficiencies and immune disturbances in general (Aguado et al., 1981; Schifferli et al., 1981). For this task in vitro assays have been constructed essentially based on the addition to the patient's serum of artificial antigen-antibody precipitates where the antigen or the antibody was radioactive, and the measurement of radioactivity released in the supernatant after incubation and centrifugation (Aguado et al., 1981; Schifferli et al., 1981; Baatrup et al., 1983). The results obtained showed that ICSC was often impaired in Ic-mediated diseases and that in some of them (e.g., SLE), the degree of impairment correlated closely with the disease activity (Aguado et al., 1981; Schifferli et al., 1981; Sakurai et al., 1982; Celada et al., 1984). In order to avoid the necessity of handling radioactive materials in the serological laboratory and to circumvent the decay problems posed by the use of a radioactive probe, we have developed a new test that employs a 'probe complex' where the antigen is enzymatically active E. coli fl-galactosidase. In this paper we describe the characteristics of the probe and the results obtained in normal and pathological sera.
Materials and Methods
Enzyme E. coli fl-galactosidase from E. coli was used as described earlier (Migliorini et al., 1983). The enzyme was purified from E. coli strain 3300 by DEAE-Sephadex (Celada et al., 1971). It was stored in 40% ammonium sulphate at 4°C. The preparation used had a specific activity of 144,000 E U / m g . A nti-fl-gal antisera The following antisera were used to prepare the probe complex: (1) donkey, primary response; (2) donkey, secondary response; (3) rabbit, secondary response. All antisera were prepared in the laboratories of Farmitalia Carlo Erba, as described in earlier reports (Gnemmi et al., 1981; Migliorini et al., 1983). Donkeys were immunized by injection in 2 sites of 1 mg fl-gal in 1 ml complete Marcol adjuvant and boosted after 3 months injecting in 2 sites 0.5 mg in incomplete Marcol adjuvant (1 : 1), 0.5 ml/site. NZW rabbits were immunized by injecting in the footpads 100 ~g fl-gal in complete Freund's adjuvant and boosted after 2 weeks by injecting 10/~g of soluble antigen intradermally in several sites of the back.
121
Precipitation curve The precipitation tests were performed by mixing 2200 EU of fl-gal in 0.2 ml with the same amount of dilutions of donkey antiserum or rabbit antiserum. After 3 h at RT, the tubes were centrifuged for 30 min at 3000 x g and supernatants measured for residual enzyme activity.
Precipitation of the probe complex Each probe complex (PC) was numbered after the antiserum used (1, 2, 3). The PC 1 was prepared by mixing I vol. of fl-gal (2200 EU/ml), with 1 vol. of antiserum at the chosen dilution in veronal-buffered saline (VBS). After 1 h at 37°C and 22 h at 4°C, the PC was centrifuged at 4°C, 10,000 x g for 10 min. The precipitate was washed twice in 2 vols. of cold VBS and recentrifuged in the same conditions. The precipitate was then carefully resuspended in 8 vols. of cold phosphate-buffered saline containing 0.15 mM CaC12, 0.5 mM MgC12, 0.1% gelatine (PBS-G) and stored at 4°C. Its activity, checked at 1 month intervals, was stable for at least 3 months. In order to disperse evenly the PC, it was vortexed vigorously before delivering each series of 8 samples. After washing, 73% of initial enzyme activity was recovered in the precipitate, corresponding to 3.6/~g/ml of fl-gal at the final dilution of the PC. PC 2 and PC 3 were prepared and used at the same concentration as the PC 1.
Performance of the tests A micro- (A) and a macro-method (B) were devised. Because of the similarities, they are described together. Fifty microlitres of the tested serum, 50/~1 of PBS and 25 /~1 of PC suspension were placed in a polystyrene tube (B) or in a well of a 96-well microtitre plate (A). Tubes and plates were incubated for 2 h at 37°C, then the tubes were centrifuged at 3000 x g and 4°C for 20 min (B) and the plates at 2000 rpm at room temperature for 40 min (A). Fifty microlitres of the supernatants (A) and (B) were transferred to another tube (B) and another plate (A) and enzyme activity measured by hydrolysis of ortho-nitrophenyl-fl-D-galactopyranoside (Manca et al., 1980) and expressed as enzyme units (EU), 1 EU being the amount of enzyme that hydrolyzes 10 -9 moles of substrate in 1 rain. Plates were read in a Multiskan photometer (Flow Laboratories) equipped with a 420 mm filter. Samples were tested in duplicate and the reference serum in quadruplicate. ICSC was calculated as per cent ratio of EU solubilized by the sample to EU solubilized by the reference serum. In tubes containing buffer only, ICSC (spontaneous solubilization of the PC) never exceeded 5%.
Purification of rheumatoid factor (RF) RF was isolated from the serum of a patient with WaldenstriSm macroglobulinaemia and high RF titre. It was precipitated by 33% saturated ammonium sulphate, dialyzed against acetate buffer 0.01 M, p H 5 and passed through a Sephadex G-200 column equilibrated with the same buffer. The excluded protein was concentrated and dialyzed against PBS, and adjusted to a protein concentration
122 of 2 mg/ml. Radial immunodiffusion showed the presence of IgM and traces of IgG.
Conglutinin-coated beads Conglutinin (K) was isolated from bovine serum according to Lachmann et al. (1973). Polystyrene beads were incubated in a solution of K containing 10/~g/ml in VBS at pH 7.4. The volume of the solution was 0.25 m l / b e a d and the incubation was 24 h at room temperature. Subsequently the beads were saturated with bovine albumin 3%. Determination of anti-B-gal antibody affinity Among anti-/3-gal antibodies found in the serum of immunized animals, one restricted family has been characterized by its ability to activate the defective enzyme produced by certain mutant E. coli strains (Rotman and Celada, 1968). These activating antibodies demonstrate one-hit kinetics; therefore it is possible to measure their affinity even when a complexed antigen is used (Celada et al., 1973). Fifty microlitres of donkey (1 : 100) and rabbit (1 : 100) antiserum were dispensed in flat-bottom microtitre plates (Sterilin M 29A) with 20/~1 of increasing concentrat i o n s of m u t a n t 6101 in b u f f e r B (BB), c o n t a i n i n g 10 m M (hydroxymethyl)aminomethane (Tris), 10 mM MgC1 z, 0.1 M NaC1 and 0.05 M 2-mercaptoethanol; its pH was adjusted to 7.2 with acetic acid. After 3 h at 37°C, 200 t~l of 2-nitrophenyl-/3-D-galactopyranoside (ONPG), 3 x 10 3 M were added and EU present in each well were calculated from the OD at 420 nm. On the assumption that every 6101 molecule bound by activating antibody was active, the affinity was calculated by double reciprocal plotting of bound vs. free antigen. It was expressed as 1 / k D a (in M - l ) corresponding to the concentration of free antigen at which the binding reached 50% of the maximum. Normal and pathological sera Normal sera were obtained from healthy laboratory staff members. Pathological sera were collected at the Rheumatic Disease Unit, University of Pisa. The diagnosis of systemic lupus erythematosus and rheumatoid arthritis were made according to well accepted criteria (Ropes et al., 1958; Tan et al., 1982). The diagnosis of essential mixed cryoglobulinaemia was made on the basis of the typical findings (purpura, weakness, arthralgias/arthritis, presence of serum cryoglobulins), after having ruled out other known causes of cryoglobulinaemia by careful clinical and laboratory investigation. Measurement of CH50 The determination of CH50, defined according to Kabat and Mayer (1961) as the quantity of complement required for 50% lysis of sensitized erythrocytes, was performed by a microtitre plate method (see Celada et al., 1984). Normal and pathological sera in 7 progressive dilutions ranging from 1 : 400 to 1 : 100 were tested for their ability to lyse sensitized sheep RBC after incubation for 30 min at 37°C. The total ~eaction volume was 250/~1, and the final RBC concentration 0.2%. After
123
centrifugation for 15 min at 2000 rpm, the haemoglobin concentration in the supernatant was measured in a Multiskan photometer. The 50% lysis point was determined by a computerized procedure that also calculated the number of 50% haemolytic U / m l original serum (HU). Under these conditions the CH50 level ranged in normal sera between 720 and 1180 U / m l .
Results
Choice of the antigen-antibody ratio in the probe complexes Fig. 1 shows the precipitation curves obtained by adding antisera 1, 2 3 at doubling dilutions to a fixed amount of fl-gal. Precipitable complexes formed at dilutions ranging from 1 : 8 to 1 : 64. The latter, at which few soluble complexes were
%EU 100"
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l I
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Fig. 1. Precipitation curves of the 3 anti-fl-gal antisera: - - , Ab 1 (donkey, primary response); . . . . . . , Ab 2 (donkey, secondary response); . . . . . , Ab 3 (Rabbit, secondary response). Conditions aa in Materials and Methods. Ordinate: fl-gal EU found in supernatant, in percent of input (2200 EU)." Abscissa: dilution of antisera.
124 ICSC
EUb
100
10
80
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. . . . . . . . . . . . . . . . .
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60
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4
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[
20
40
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I
60
80
100
120
TIME (mm)
Fig. 2. Compound illustration of 2 time-dependent events studied on PC 1. The continuous line shows the kinetics of solubilization (as per cent of the ICSC of a normal reference serum (ordinate). The dotted line shows the kinetics of appearance of C3b-containing soluble complexes in the supernatant. This parameter was studied by measuring (ordinate) the binding of PC (fl-gal activity) to plastic beads coated with bovine conglutinin.
detectable, was chosen as the a g : a b ratio for PC 2 a n d 3. A c c o r d i n g to the same criterion, an a n t i s e r u m dilution of 1 : 32 was chosen for PC 1.
Kinetics of solubilization and nature of solubilized complexes This p a r a m e t e r was s t u d i e d with PC 1. T h e a m o u n t of solubilized P C increased steadily in the first 10 m i n of i n c u b a t i o n a n d a p p r o a c h e d the m a x i m u m after 1 h (Fig. 2). I n tubes c o n t a i n i n g heat d e c o m p l e m e n t e d serum, no e n z y m e activity was detectable in the s u p e r n a t a n t , i n d i c a t i n g that the r e a c t i o n is c o m p l e m e n t d e p e n d e n t a n d that no free e n z y m e is released s p o n t a n e o u s l y u n d e r the c o n d i t i o n s of the test. O n the o t h e r h a n d , fresh a n d M g - E G T A chelated serum solubilize the same a m o u n t of PC in 2 h. It is k n o w n that solubilization of i m m u n e p r e c i p i t a t e s d e p e n d s u p o n the intercal a t i o n of C3b in the lattice a n d that solubilized c o m p l e x e s still retain C3b on their surface. Since b o v i n e conglutinin b i n d s I C through their C3b i moiety, we investigated the structure of solubilized PC evaluating its b i n d i n g ability to c o n g l u t i n i n - c o a t e d beads.
125 ICSC" 100n,,
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i DILUTIONS
1:11~
Fig. 3. Effect of serum dilution on the ICSC, using different PC preparations. Ordinate: ICSC, as per cent of normal reference serum. Abscissa: serum dilution. The means and standard deviations of 5 sera are given. - - , PC 1; . . . . . . , PC 2; . . . . . , PC 3.
The supernatants obtained at the different times in the above experiment were incubated with K-coated beads for 24 h at 4°C. After 2 washings in VBS-Tween 80 0.05%, enzyme activity on the beads was measured. The binding curve, closely parallel to the kinetics of solubilization (Fig. 2), indicates the presence of C3b on solubilized PC.
Immune complex solubilizing capacity in normal sera tested with different probe complexes Five normal sera were titrated against PC 1, 2, 3. As can be seen in Fig. 3 the solubilization capacity decreases with increasing dilutions at a rate strikingly different for the different PC preparations. The rate of I C S C decrease was f o u n d to be related to the affinity of the antibodies used to form the complex. The association constant of serum 3 was 2 × 108 M - ] , of serum 2 about 2 × 107 M -1, while that of serum 1 very m u c h lower although, because of the heterogeneity of the primary response, an association constant was not calculated.
Interfering substances The interference of rheumatoid factor (RF) on the measurement of I C S C has been suggested in some papers (Tincani et al., 1983). We investigated the influence of R F on the solubilization of PC by adding a fixed a m o u n t (20 t~g) of a purified I g M monoclonal R F to normal sera (Fig. 4). With PC 1, the solubilization capacity of 1 : 2 diluted sera was scarcely affected by the addition of R F except when the complement content was lowered by dilution, R F has an inhibitory effect on ICSC, that becomes striking when ICSC is 50-60%. O n the contrary, with PC 2 and 3 the inhibition exerted by R F was evident even on sera diluted 1 : 2.
126 ICSC 100"
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Fig. 4. Interference of rheumatoid factor on ICSC. Ordinate: ICSC, as % of normal reference serum. Abscissa: dilution of sera. For each PC, the ICSC is shown as a function of serum dilution, in the absence (O) or in the presence (e) of a constant amount of IgM monoclonal RF. The means and standard deviations of 4 sera are given.
I C S C in pathological sera Fig. 5 shows ICSC levels measured by PC 1 in n o r m a l a n d pathological sera. In 65 n o r m a l sera, the m e a n ICSC level was 97 _+ 8%. M e a n m i n u s 2 SD of n o r m a l sera (80%) was considered the lower limit of normality. ICSC was low in 2 0 / 5 3 SLE sera; 1 3 / 4 1 E M C sera; 1 / 1 5 r h e u m a t o i d arthritis sera a n d 1 1 / 1 4 synovial fluids from r h e u m a t o i d arthritis patients. I n 88 unselected specimens, collected for CHS0 m e a s u r e m e n t , a parallel evaluation of ICSC was o b t a i n e d (Fig. 6). The 2 parameters are strongly correlated ( r = 0.43, P < 0.001), but some sera showed n o r m a l C H 5 0 in spite of low ICSC a n d one had high ICSC with low CHS0 level. PC 1 a n d 3, c o m p a r e d in 38 unselected pathological sera, were highly correlated ( r = 0.59, P < 0.0001); however, sera had lower ICSC levels when measured with PC 3. This higher n u m b e r of 'positive' results suggests a higher sensitivity for the PC 3 assay.
127 ICSC 120 -
i. 100 .lllt °eeoc °o° • o•• J•
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7
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RA
SF
Fig. 5. ICSC in normal sera vs. SLE, EMC, RA patients and (SF), synovial fluid of rheumatoid arthritis patients. The determinations were performed with PC 1.
Discussion
The method described in this paper measures a body fluid's capacity to solubilize immune complexes by probing it with an enzyme-antibody complex whose liberation in the soluble phase can be followed with ease and precision by a simple determination of /3-galactosidase activity. The results of this test have been shown to be significantly in agreement with those of methods employing a radiolabelled complex (125I-BSA-anti-BSA); e.g., a comparative study performed by Corvetta et al. (1984) on 52 sera yielded a high correlation index (r = 0.73, P < 0.001). Nevertheless, the probe complex system has several advantages, all facilitating the preparation of
128 ICSC 12o•
.f'
.
:
•
i,. • ""~ t
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• .:.
" "i,
uJ 8(3 u Z Lu Lu u.
~ 40-
O 0
5 0' 0
720 HU
1o'o 0
1500' CH50
Fig. 6. Correlation of ICSC and CH50 in the same sera. Ordinate: ICSC (measured on PC 3). The value 80% represents the lower limit set for normal values. Abscissa: haemolytic capacity expressed as CH50 units (HU). The value of 720 HU represents the lower limit set for normal sera. industrial kits. It avoids the risks and administrative problems of using radioactive material. The PC reagents are stable over months and can be prepared in large batches lasting for a number of tests. The manipulations are simple and the assay can be performed in microplates with considerable economy of materials and time. After this positive presentation, we would like to raise discussion about the following points: (a) by which mechanism is the PC solubilized; (b) what 'section' of the complement system is the ICSC representing and is it possible to extrapolate the results on the entire system; (c) what is the influence of the quality of the antibody used in the PC on the ICSC. The present, rather mechanistic view of the solubilizing event is based on the accepted concept that any macromolecule is kept in solution if and until its molecular weight is counterbalanced by an adequate number of surface hydrophilic groups, that keep it, so to speak 'floating' in the fluid. Deposition of C3b splits the large IC aggregates into fragments which become hydrophilic presumably by the introduction of new hydrophilic groups. Suggestive evidence, if not proof, that solubilization works through C3b deposition is our finding that the proportion of probe complex solubilized at any time is closely paralleled by the amount of activity that can be bound by beads coated with conglutinin - - a protein endowed with specific affinity for C3b. Our data also show some evidence that most of the C3 activation in question is alternative pathway dependent since approximately the same amount of PC is solubilized if the serum is treated with E G T A - M g which blocks the classical pathway. Malasit et al. (1983) found that under similar conditions the solubilization process was slower than in fresh serum but the final amount of complex solubilized was the same and so was the profile of the sucrose density gradient analysis. In our case the incubation time (2 h) was probably too long to permit differences in kinetics to become manifest. A further point concerns the variables that can affect the sensitivity of the ICSC test, and which can also be used by the investigator as handles to adjust the characteristics of the assay to the clinical needs.
129
A first variable is the amount of precipitating PC used, since the complement concentration needed for complete solubilization will be relative to that amount (Baatrup et al., 1983). A second variable is the intrinsic affinity of the antibody used to prepare the PC. From all the experiments with different antibodies shown in the present article we can conclude that regardless of the animal species in which the antiserum was raised, hyperimmune, high affinity antibodies produce immune precipitates that are more difficult to solubilize and will therefore require higher complement concentrations than those formed by low affinity, primary response antibodies. As a consequence, it is possible to grade the degree of stringency and the sensitivity of the test by selecting an antibody of the desired affinity - all other conditions including the total amount of PC remaining equal. The highly significant correlation between CH50 and ICSC is explicable by the very fact that C3 is a key component of both complement activation pathways. Any severe C3 deficiency will affect the classical pathway since it will limit in the formation of the C5 convertase C1423, and will affect ICSC for want of C3b deposition. On the other hand, intermediate deficiencies of C3 will be detected by the ICSC test but may not affect the haemolytic test, since C3 is not a limiting factor in the classical cascade. This situation leads to the discrepancy between low ICSC and normal CH50, which is found rather infrequently but which can also be caused by the presence of nephritic factor (C3NeF), an autoantibody that stabilizes the C3 convertase of the alternative pathway. The opposite situation, (i.e., normal ICSC, low CH50) would be expected to arise in genetic deficiencies of C2, C4 or C5-C9. We can conclude that ICSC values reflect closely the condition of the alternative pathway. This means that the ICSC test would be best used in parallel with an assay specific for the classical pathway. Such a combination would permit a comprehensive assessment of the entire complement system. On the other hand, considering the ease and rapidity of performing a determination by our method and the high correlation with CH50, ICSC could also be used as a first screening, instead of the more delicate haemolytic assay.
Acknowledgements This work was in part supported by Grant No. 83/00527.57 Tecnologie Biomediche e Sanitarie from the Italian Research Council (C.N.R.). Ms. Luisa Di Rosa is gratefully acknowledged for her secretarial help.
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130 Corvetta, A., G. Marchigiani, P. Migliorini and G. Danieli, 1984, Proc. II Int. Symposium on Plasma Exchange in Nephrology, Viareggio, May 18, 1984 (under the patronage of the Italian Societies of Nephrology, Immunohematology and Rheumatology) (abstract), p. 13. Gnemmi, E., P. Migliorini, G. Chieregatti, F. Manca, S. Cantarella and F. Celada, 1981, Lab. J. Res. Lab. Med. 8, 5. Kabat, E. and M. Mayer, 1961, Experimental Immunochemistry, II edition (C.C. Thomas, Springfield, IL) p. 136. Lachmann, P.J., M.J. Hobart, P.W. Aston, 1973, Handbook of Experimental Immunology, Vol. 1, ed. D.M. Weir (Blackwell, Oxford) Ch. 5. Malasit, P., S.R. Bartolotti and J.H. Humphrey, 1983, Immunology 48, 779. Manca, F., P. Migliorini, S. Bombardieri and F. Celada, 1980, Clin. Immunol. Immunopathol. 16, 131. Migliorini, P., G. Trovatello, S. Cantarella, F. Manca, S. Bombardieri and F. Celada, 1983, J. Immunol. Methods 59, 245. Miller, G.W. and V. Nussenzweig, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 418. Ropes, M.W., G.A. Bennet, S. Cobb, S. Jacox and R.A. Jessar, 1958, Bull. Rheum. Dis. 9, 175. Rotman, B. and F. Celada, 1968, Proc. Natl. Acad. Sci. U.S.A. 60, 660. Sakurai, T., T. Fujita, J. Kono, T. Kabashima, K. Yamane, N. Tamura and H. Kashiwagi, 1982, Clin. Exp. Immunol. 48, 37. Schifferli, J.A., S.M. Morris, A. Dash and D.K. Peters, 1981, Clin. Exp. Immunol. 46, 557. Takahashi, M., B.F. Tack and V. Nussenzweig, 1977, J. Exp. Med. 145, 86. Takahashi, M., S. Takahashi, V. Brade and V. Nussenzweig, 1978, J. Clin. Invest. 62, 349. Takahashi, M., S. Takahashi and S. Hirose, 1980, Prog. Allergy 27, 131. Tan, E.M., A.S. Cohen, J.F. Fries, A.T. Masi, D.F. McGhane, N.F. Rothfield, J. Green Schaller, N. Talal and R.J. Winchester, 1982, Arthritis Rheum. 25, 1271. Tincani, A., P. Migliorini, C. Ferri, G. Balestrieri and S. Bombardieri, 1983, Folia Allergol. Immunol. Clin. 30, 22.