Scand, J. Immunol, 7, 503-509, 1978

Binding Affinity of Human Autoantibodies: Studies of Cryoglobulin IgM Rheumatoid Factors and IgG Autoantibodies to Albumin O. WAGER & A.-M. TEPPO Municipal Bacteriological Laboratory, Aurora Hospital, Helsinki, Finland

Wager, O. & Teppo, A.-M. Binding AfBnity of Human Autoantibodies: Studies of Cryoglobulin IgM Rheumatoid Factors and IgG Autoantibodies to Albumin. Scand. J. Immunol. 7, 503-509, 1978. The binding affinity of cryoglobulin IgM rheumatoid factors (RF) for human IgG and of human IgG anti-albumin autoantibodies for HSA was measured by the molecular sieving technique. The binding affinities ofthe two autoantibodies were consistently low (lO^-lO^ 1/M) as compared to the affinities of corresponding hyperimmune animal antibodies (lO'-lO* 1/M). The findings were discussed in relation to theories on human autoimmunity. The existence of strict autotolerance at the T cell level and of autoreactivity at the low affinity B cell level was considered to be best compatible with the findings of this study and with the major known facts of autoimmunity. O, Wager, Municipal Bacteriological Laboratory, Aurora Hospital, SF-00250 Helsinki 25, Finland,

Burnet [5] predicted the existence of a phase in the development of the lymphocyte's immunological competence, during which contact of self-antigens with cell receptors leads to deletion of the corresponding clones and thus to natural tolerance against self-antigens. Recent experimental evidence for and extension of this concept come from studies by Nossal [15] and Klinman [8, 13] with co-workers. They showed that murine B cells mature through an early and short tolerance-sensitive phase, during which binding of antigen to cell receptors leads to elimination of that clone (clonal abortion). Clonal abortion was achieved with low epitope concentrations and without the help of T cells. It was inferred [13] that the affinity requirements for B cell tolerogenesis and immunogenesis were the same, and that binding of antigen to cell receptors is not sufficient for stimulation, if the affinity does not exceed a minimal threshold. Extrapolating the above

affinity postulates to human autoimmunity, the autoantigen-binding B cells present in normal subjects and in greater numbers in subjects with corresponding autoimmune responses [cf. 4] appear to represent low-affinity B cell clones that have escaped clonal abortion. The modern theories on the initiation of autoimmune responses to an essential part rely on assumption that autoreactive resting B clones are inducible to response by native autoantigens provided that T cell helper effect can be recruited by various putative bypass or circumvention mechanisms [1, 2, 4, 9, 19, 28]. Affinity parameters of the products of the autoreactive B clones therefore acquire considerable theoretical interest. The binding affinity of circulating antibodies are reportedly representative of the affinity of the B cell surface Ig [3]. We have studied two human autoantibodies reactive with native ubiquitous autoantigens, the. IgM rheumatoid factors (RF) [27, 28, 31]

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and more recently also the IgG anti-human serum albumin (HSA) autoantihodies [11, 26]. Speculations on the possible mechanisms of elaboration of these two autoantibodies led us to appreciate the potential significance of the affinity parameters in relation to establishment and breakdown of autotolerance. By reviewing the literature we could find surprisingly few observations on binding affinity of human autoantibodies for their native autoantigens [7, 10, 12,25,32]. In this paper we report the results of binding affinity measurements on polyelonal and monoclonal IgM RFs and on IgG anti-HSA autoantibodies and discuss the observed low affinities in relation to the theories of human autoimmunity.

MATERIALS AND METHODS IgM rheumatoid factor (RF) preparations, RF-active cryoglobulin IgM components (Cryo-IgM) were isolated from sera of patients with IgM-IgG cryoglobulinaemia. Five of the Cryo-IgM components (AS, HK, IV, KH, RH) have been studied previously [29, 30]. Carefully separated serum from 50-100 ml of clotted blood, that had been preincubated for 30 min in a water-bath at 37°C, was kept at +4°C until maximal cryoprecipitation had taken place, usually for 2-3 days. The cryoprecipitate was spun down and washed with cold 0.04 M phosphate-buffered saline (PBS) (pH 6.5) until the washings were protein-free. Special efforts were made to recover a population of RF molecules representative of the afiinity of the RF-active cryo-IgM components. For this end, the optimal conditions for dissociation ofthe lgG-IgM cryoglobulins were studied in preliminary experiments (Table I). Aliquots of each ofthe eight cryoprecipitates were first suspended in 5 ml of PBS, and 1 vol ofthe suspensions incubated at 37°C for 1 h with 4 vol of the various buffers, and then centrifuged at the same temperature. The relative concentrations of IgG and IgM in the supernatants were determined by radial immunodiffusion at 37°C. According to the maximal mean IgM concentrations obtained in these preliminary experiments, the cryoprecipitates were dissolved at 37''C in PBS (pH 7.8) containing I M NaCl. The IgM and IgG components were separated at 37°C on Sephadex G-200 columns (35x2.5 cm) equilibrated with the above buffer. The platelet aggregation test (Pl.A.) [16, 17] was applied for assay of aggregated IgG in the IgG preparations. Less than 1 (xg/ml of heat-aggregated IgG is detectable by the Pl.A.-test. Control IgM preparations. IgM was isolated from individual (AMT) and pooled (V) normal human sera and from IgM M-component sera (AR, RA, AR6) by precipitation with 2.2 M (NH4)2SO4 followed by filtration on Sephadex G-200 (PBS, pH 7.2). The monoclonal IgM of the M-component sera was freed from con-

TABLE I. The effect of buffers on the relative concentration of IgM dissociated from eight cryoprecipitates at 37°C. Mean diameter in mm of IgM precipitate zone by radial immunodifTusion. For details cf. Methods.

Bufrers 0.1 M Na-acet., 0 1 M Na-acet.,

2 M NaCl, pH 3.0 pH3.0

PBS, pH 7.4 PBS, 1 M NaCI,pH 7.8 0.1 M glys.-HCI , pH 2.3 0.1 M Tris-HCI, 6 M urea, pH 8.6

Mean diameter of IgM zone (mm) 6.9 7.3 8.0 8.3 6.2 4.7

taminating polyclonal IgM (that usually contained some RF-activity) by preparative agarose gel electrophoresis (2% agarose gel in barbiturate buffer, pH 8.6). Other hutnan serum protein preparations. Isolation of IgG from 'tailing' albumin (TA) sera and from normal human sera, and papain digestion of IgG were carried out as described elsewhere [26]. IgG monomers were freed from aggregated IgG by Sephadex G-200 column chromatography and tested by the Pl.A.-test. Preparation of the (Fab')2 fragment by pepsin digestion was performed in 0.1 M Na-acetate buffer, pH 4.5, at 37°C for 4 h using 1 mg pepsin per 10 mg IgG [22]. The pH was then brought to 8.0 with saturated TRIS-solution and the (FabOj fragments separated by Sephadex G-50 filtration in PBS. HSA (human albumin grade III, Sigma Chemical Co.) was further purified as described elsewhere [26]. Antisera, Antisera against human IgG were raised in rabbits (Table II). Porcine anti-HSA serum was a gift from Orion Diagnostica, Espoo. Rabbit and swine serum protein preparations. Isolation of IgG and IgM fractions from rabbit and swine antisera was carried out by precipitation with 2.2 M (NH4)2SO4 and by Sephadex G-200 gel filtration. The IgM fractions were used without further purification; the IgG fractions were concentrated by Amicon B 15 concentrator (Amicon Corporation, Lexington, USA) to give a protein concentration of 3 mg/ml, and further purified by DEAE filtration (DEAE-Sephadex A-50, Pharmacia, Uppsala) in Tris-phosphate buffer (pH 8.2). Radiolabelling of IgG and HSA, The IgG and HSA preparations were iodinated with ''''I (Radiochemical Centre, Amersham, England) by the lactoperoxidase method [14]. Iodinated proteins were separated from unbound iodine by Sephadex G-25 filtration. 96-99% of the counts were precipitable by 10% trichloroacetic acid. By the Pl.A.-test aggregated IgG was not detected in the '"I-IgG preparations. Binding affinity measurements. The equilibrium molecular sieving technique [23-25] was applied to measure the binding of the '^^I-IgG (Ag) by the IgM RF preparations (Ab) using three different Ag levels (in the range of 5-20 (xg/tube). At these levels 30-70% of the Ag was bound by the Ab. 200 (xl aliquots of Ag (0.01 M phosphate buffer, pH 6.0, 0.15 M NaCl) were

Bittding Affinity of Human Autoantibodies

505

TABLE II. Maturation of antibody affinity for human IgG in rabbits immunized with human IgG

Binding constants {K,J a t -H22°C Rabbit 929 Schedule Prebleeding Day 0, stim. with IgG* Day 6, bleeding Day 9, stim. with IgGf Day 21, bleeding Day 35, bleeding Day 49, bleeding Day 66, bleeding Day 98, bleeding

Rabbit

Rabbit

930

932

Serum

IgG

IgM

Serum

Serum

3x10'

NT

NT

2x10'

9x 10=

NT

l x 10"

1 X 10"

5x 10"

7x 10°

8x10" 4x10" lxlO'

8xlO« 1 XlO'

1 X 10" 2x106

5x 10" 5xlO«

6xlO«

6x10' 1x10*

1 XlO' 7x]0«

3x10" 2x10'' 3x10"

1 XlO' 1 XlO' 2x10'

NT

2x10" 3xlO« 2x10' 1 X 10'

* 1.2 mg with FCA subcutaneously. t 2.2 mg intravenously. incubated with 100 \J.\ of the Ab for 1 h at 5, 15, 22 and 37°C in siliconized glass tubes. Four parallel analyses were performed at each Ag and temperature level. After preincubation 500 [AI of a thick slurry of pre-swollen and unaerated Sephadex G-200 was added to each tube. Control tubes contained Ag, Sephadex slurry and PBS. Equilibration was allowed to take place for 4 h at 37°C, for 7 h at 25''C, for 10 h at 15°C and overnight at 5°C. The tubes were shaken occasionally. After centrifugation, radioactivity of 50 jxl of the supernatants were determined by gamma counter (Wallac, Sweden). The amount of Ag bound by Ab as well as the total amount of Ab were determined as described by Soothill et at. [23]. The binding constants (ATA l/mol) were determined from Karush' equation and expressed as mean value of twelve determinations (four parallel analyses, three Ag levels). The binding of the I " I . H S A (Ag) by the anti-HSA preparations (Ab) also was measured by the sieving technique with the following modifications. Sephadex G-50 was used instead of Sephadex G-200, the test volumes of Ag and Ab were 80 (xl; the dilutions of the Ab used bound more than 30% of available Ag (50 [j.g/ tube).

RESULTS Maturation of immtme response against htiman IgG Before binding affinity assays were performed on the human rheumatoid factors, maturation of the anti-human IgG response was studied experimentally in rabbits immunized with polyclonal human IgG. Some of the binding con-

stants observed are shown in Table 11 and all o them summarized in the lower section of Table III. A pattern of gradual increase ofthe binding affinities was observed more or less clearly in all of the six rabbits followed-up to 3 months. Binding affinity of IgM rheumatoid factors The binding constants (Table 111) of the monoclonal and polyclonal cryo-IgM RFs for human monomeric IgG were lower (10''-]0'' l/mol) than the affinities of the IgM fractions of rabbit anti-human IgG sera (10»-10' l/mol) for human IgG and Fc but they were higher than the binding constants of monoclonal and polyclonal human IgM devoid of R F activity (10^ l/mol). The binding constants of the cryo-IgM RFs measured at diflFerent temperatures were of the same order of magnitude; a weak trend towards lower values at higher temperatures, however, could be seen. The affinities of the IgM RFs contained in the IgM fraction of the supernatants left after removal of the serum cryoprecipitates were approximately equal to the affinities of the corresponding cryoglobulin TgM RFs. Binding affinity of IgG anti-HSA autoatttibodies As can be seen in Table IV, the binding constants of the IgG isolated from TA sera as well

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O. Wager & A.-M. Teppo

TABLE III. Affinity of IgM rheumatoid factor (RF) and control preparations for human IgG Binding eonstani'CI^A) Measured at: Specimen tested Polyclonal Cryo-IgM RF L.P. Polyclonal Cryo-IgM RF E.L. Polyclonal Cryo-IgM RF H.M. Polyclonal Cryo-IgM RF H.K. Monoclonal Cryo-IgM RF K.H. Polyclonal Cryo-IgM RF LV. Polyclonal Cryo-IgM RF R.H. Cryosupernate IgM'"' of R.H. Cryosupernate IgM'"' of R.H. Monoclonal Cryo-IgM RF A.S. Cryosupernate IgM'"' of A.S. Cryosupernate IgM'"' of A.S. Monoclonal IgM A.R. Monoclonal IgM E.A. Monoclonal IgM A.R6 Polyclonal IgM AM.T Polyclonal IgM V.

For

Cryo-IgG'" Cryo IgG'2' Cryo IgG'=' Cryo IgG'2' Cryo IgG"" Cryo IgG'2' Cryo I g G ' " Cryo IgG'2) Polycl. IgG Cryo-IgG'" Cryo IgG"" Polycl. IgG Polycl. IgG Polycl. IgG Polycl. IgG Polycl. IgG Polycl. IgG

Anti-lgG

5°C

I5°C

22°C

37°C

5x10' 5x10'

3x10' 4x 10' 2x10' l x 10' 2x10' 1x10' 8x10" 2x10' NT NT 6x10" NT NT NT NT 3X10' 7x10^

3X10' 3x10' 2x10'

3x10' 2x 10' 1 X 10' 1 X 10' 1 X 10' IxlO' 4x 10" 2x 10' NT 3x 10" 4x 10" NT NT NT NT 4x 10' 8x10^

9(3)

2x10'

2x10' lx 10' 2X10' 2x10' NT 5x10" 1 xlO» NT NT NT NT 3x10^ 1x10'

V(3)

lx 10' 1x10' 4x 10" 1x10' 5x10' 5x10" 5x10" IxlO' 5x10' 9x10'^ 1 XlO'' 3xlO» 6x10^

titrc (Latex) ^128 ^128 >I28 ^128 > 128 5:128 2:128 > 128 >128 S= 128 5 128

>I28 5 128