Immunology 1979 36 671

Studies of human IgM anti-IgG cryoglobulins I.

PATTERNS OF REACTIVITY WITH AUTOLOGOUS AND ISOLOGOUS HUMAN IgG AND ITS SUBUNITS

SARAH LARRIAN JOHNSTON & G. N. ABRAHAM Departments of Medicine andMicrobiology, University of Rochester School of Medicine and Dentistry, Rochester, New York, U.S.A.

Received 27 April 1978; acceptedfor publication 28 June 1978

Summary. Monoclonal human anti-IgG preparations purified from mixed IgM-IgG cryoglobulins were tested for their antigenic specificity by haemagglutination-inhibition assay. A panel of fourteen IgG preparations of the four gamma chain subclasses were prepared from myeloma sera and used as inhibitors of haemagglutination. Each of six IgM anti-globulins demonstrated different reactivity profiles with these IgG preparations. In addition, the fraction of the serum IgG which had bound to and cryoprecipitated with the IgM preparations, termed 'antigen-IgG', was purified and assayed for subclass content. The y chain subclasses found in the 'antigen-IgG' fractions showed that each IgM cryoprecipitated an IgG from serum which had different quantities of the subclasses present. These 'autologous' reactivity patterns were in instances different from the specificities expected from the results obtained with the myeloma proteins. When all antigen-IgG pools were tested with each IgM, some antiglobulins showed stronger reactivity with isologous than with their own, antigen-IgG pools. The IgM anti-IgG preparations were also compared

in reactivity with IgG and its subunits in order to localize the antigenic determinant(s) with which these autoantibodies react. Heavy chains showed far greater reactivity than Fc fragment for 5/6 IgM preparations. Light chains, F(ab')2, pFc' and Fab were non-reactive. A relationship between the length of papain digestion and Fc reactivity was demonstrated. Based on the data, possible locations for the antigenic determinant(s) were considered.

INTRODUCTION Anti-IgG autoantibodies or rheumatoid factors found in a variety of diseases are predominantly IgMs which have numerous and diverse patterns of reactivity for antigenic determinants on IgG (Heimer, Schwartz & Freyberg, 1962; Milgrom, Witebsky, Goldstein & Loza, 1962; Williams & Kunkel, 1963). Numerous factors which influence the binding of the antigen IgG have been defined for non-cryoprecipitable IgM rheumatoid factors. Some data indicate that differences exist between and reactivity may depend upon, the species of the IgG antigens (Williams & Kunkel, 1963; Butler & Vaughan, 1965). Further, the interactions between IgM rheumatoid factors and IgG has also been

Correspondence: Dr George N. Abraham, Box 695, University of Rochester Medical Center, Rochester, N.Y. 14642, U.S.A. 00 1 9-2805/79/0400-0671$02.00 © 1979 Blackwell Scientific Publications

671

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Sarah Larrian Johnston & G. N. Abraham

shown to depend upon the locus of the antigenic determinant(s) on the heavy chain or Fc fragment. Many reports have shown that rheumatoid factors react with IgG-Fc fragment (Dissanayake, Hay & Roitt, 1977), heavy chains (Allen & Kunkel, 1966) and Fc region or heavy chain peptides (Henney, Jefferis & Stanworth, 1968; Taylor & Abraham, 1973). Most of theIgM rheumatoid factor specificities have been shown to be present in the pFc' segment (the third y chain constant region domain) of IgG (Gaarder & Natvig, 1970; Steward, Turner, Natvig & Gaarder, 1973). Further, it has been demonstrated that particular antigenic configurations of IgG favour its interaction with rheumatoid factors (Henney, 1969; Dissanayake et al., 1977). The diverse spectrum of the antigenic specificities has been repeatedly stressed. As examples, Allen & Kunkel (1966) demonstrated that IgM rheumatoid factors frequently reacted with a determinant termed 'Ga' on the Fc region of IgGl, 2 and 4 myeloma proteins. Gaarder & Natvig (1970) extended these observations and showed that fourteen out of fourteen rheumatoid factors contained anti-Ga specificity, ten anti-Gm(a) (a specificity on IgGI), one anti-Gm(g) (IgG3), and six a new specificity which was termed anti- 'non-a' present on Gm(a-) IgGl and all IgG2 and IgG3. The Ga antigen was broadly localized to the Fc region and 'non-a' more specifically to the pFc region of IgG. These are thus important and frequent specificities of non-cryoprecipitable rheumatoid factors. Numerous other specificities have also been described. These have been recently summarized by Johnson & Faulk (1976) and include anti-Gm(x), (b), (g), (b'), 'non-b', '4 non-a' and anti-IgG4, all of which are less frequent specificities. Cumulatively, these data seem to indicate that non-cryoprecipitable rheumatoid factors are not limited in the variety of their antigenic specificities to the Fc region and/or H chain of human IgG. Similar types of data which describe the reactivity of cryoprecipitable IgM anti-IgG preparations with IgG are not as complete. MacKenzie, Goldberg, Barnet & Fundenberg (1968) defined differences in broad patterns of serological specificity for seven monoclonal cryo-IgM preparations in their reactivity with native and aggregated, human and rabbit IgG preparations. Broad reactivity with human and various primate IgG preparations was demonstrated for one IgM cryoglobulin by Stone & Metzger (1969). Stone & Metzger (1968) and Chavin &

Franklin (1969) have clearly established that the antigenic determinant(s) recognized by the IgM cryo anti-IgG preparations are located on the Fc region of IgG. Balazs & Frohlich (1966) have shown that a 19S cryo-IgM may react with aggregated but not non-aggregated human IgG heavy chains. Cryoprecipitable IgM preparations may also exhibit diverse patterns of reactivity with human IgG in vivo. The antigen IgGs present in thirteen mixed cryoglobulins were studied by Cream, Howard & Virella (1972). The distribution of the IgG subclasses found in a few IgM-IgG cryoglobulins was different from that of the IgG in the same sera. The heterogeneity detected in the cryoglobulin gamma chain subclasses reflected a diversity of specificity for the IgM antiglobulins. In two of these cryoglobulins, the IgG consisted entirely of IgG3 and in two others, there was a predominance of IgG3. In the present paper, data are presented which show the reactivity spectra for a select population of IgM anti-IgG preparations isolated from sera of patients with mixed IgM-IgG cryoglobulinemia. The antigenic specificities of the IgM preparations for a panel of IgG preparations of the four heavy chain subclasses were compared with the subclasses found in the IgG which cryoprecipitated bound to the IgM preparations as their antigens. The same IgM preparations were also compared in their ability to react with intact IgG and various aggregated and non-aggregated, enzymatically and chemically produced IgG fragments in order to determine if the autoreactive determinant(s) for this group of IgM preparations was similar and on the same region of the IgG molecule. The specificity patterns obtained for these IgM preparations were not like those previously described for non-cryoprecipitable IgM rheumatoid factors and were each individually discrete from the other. MATERIALS AND METHODS

IgM antiglobulins The IgM anti-IgG cryoglobulins were isolated from serum or plasma by cryoprecipitation, washing of the cryoglobulin in chilled buffer, and DEAE cellulose chromatography at 370 as previously described (Johnston, Abraham & Welch, 1975).

IgG subclass bank In order to establish the specificities of the IgM

Human IgM anti-IgG cryoglobulins I

cryo-antiglobulins, a bank of fourteen monoclonal IgG myeloma proteins, whose gamma chain subclass had been previously determined by J. P. Leddy * were purified from the serum of patients with multiple myeloma. Proteins were precipitated with pH 7 0 ammonium sulphate at 50% saturation, solubilized in and dialysed against 0-01 M sodium phosphate (starting buffer) pH 7 6, and applied to a DEAE cellulose column equilibrated in the same buffer. All IgG preparations eluted in the starting buffer except IgG4 Str which eluted at a slightly higher phosphate concentration. The peak fractions were pooled and analysed by immunodiffusion (ID) and immunoelectrophoresis (IEP) in agar gel with rabbit antisera specific for human y, a, p, K and A chains, and with a polyvalent horse antiserum to whole human serum. Some IgG preparations contaminated with electrophoretically heterogeneous IgG after chromatography were subjected to zone electrophoresis in starch with 0-06 M sodium barbital pH 8-6. Only fractions which were immunoelectrophoretically restricted and without contaminant were used in this study. IgA and IgM were precipitated from myeloma plasma at 50% saturation with neutral ammonium sulphate and purified by DEAE-cellulose and Sephadex G-200 column chromatography. Both were shown to be free of contaminants by ID and IEP with appropriate antisera. Pooled human IgG initially obtained as human fraction II (Pentex), was dialysed against 0-02 M sodium phosphate pH 7-6 and purified by DEAE cellulose column chromatography. The peak column fractions which eluted with the 002 M starting buffer were utilized for papain and pepsin digestion. Antigen IgG and residual serum IgG The population of IgG which had bound to and precipitated with the IgM anti-IgG, termed 'antigenIgG', was separated from the IgM mixed cryoglobulin by 370 DEAE-cellulose column chromatography as previously described (Johnston et al., 1975). Cryoglobulins were washed three times and cryoprecipitated twice before separating the antigenIgG. For comparison, the remaining 'residual' serum-IgG was precipitated from cryoglobulindepleted serum by 16% sodium sulphate saturation, solubilized in and dialysed against 0015 M phosphate pH 7 3 and applied to a DEAE-cellulose column. * Department of Medicine, University of Rochester School of Medicine, Rochester, New York, U.S.A.

673

The fall-through peaks were concentrated, dialysed against BBS and applied to Sephadex G-200 columns. The chromatogram for each sample consisted of only one peak. The fractions of high optical density were pooled and shown to be free of contaminants by ID analysis. These IgG preparations were termed 'residual-IgG'.

Haemagglutination-inhibition assay Ripley procedure. The specificity patterns of the IgM anti-IgG cryoglobulins for IgG were determined by a haemagglutination-inhibition assay which utilized human 0-positive red blood cells coated with Ripley anti-IgG, anti-CD antibody (Waller & Vaughan, 1956). The ability of monoclonal IgG preparations of various heavy chain subclasses to inhibit the haemagglutination of coated cells by an IgM cryoglobulin defined its reactivity pattern. Thrice-washed human group 0 Rh-positive cells were sensitized for one hour at 370 with Ripley anti-Rh serum. Doubling dilutions of 0025 ml of inhibitor IgG (initial concentration 0-7 mg/ml) in HEPES buffer pH 6-5 (Gibco) were made in microtitre haemagglutination plates and 0-025 ml of IgM antiglobulin was added to each well. IgM anti-IgG was used at the lowest concentration which produced a 4 + haemagglutination end point which was determined just prior to each test. The microtitre plates were mixed, incubated at room temperature for 2 h and 0025 ml of a 05% suspension of sensitized red cells was added to each well. The plates were mixed and the haemagglutination patterns read after a 2 h incubation period at 40. Negative cell and positive antibody controls were run with each test. The negative haemagglutination effect of inhibitor alone was established in separate tests. Chromic chloride method. Proteins were also coupled to 0-positive red cells with chromic chloride (Poston, 1974). For most proteins, red cells sensitized at a concentration of 1 mg/ml gave the best haemagglutination titres. In some instances the antisubclass antisera produced optimal haemagglutination only when higher protein concentrations (3-6 mg/ml) were utilized for sensitization. The assay utilized doubling dilutions of inhibitor proteins, a constant amount of the antibody or IgM anti-IgG and antigen-coated red cells in the same quantities as above.

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Sarah Larrian Johnston & G. N. Abraham

Antisera to IgG Subclasses Rabbit and monkey antisera with antibody specific for human IgG subclass determinants (supplied mainly by J. P. Leddy), were purified on solid phase adsorbant columns of Sepharose 4B (Pharmacia) conjugated with purified IgG of the appropriate subclass. The IgG was coupled as described by March, Parikh & Cuatrecasas (1974) except that pulverized cyanogen bromide was quickly mixed into an alkaline solution (0 5 ml in 1 N NaOH + 14-5 ml H20) immediately before use. The quantity of protein bound ranged from 3 3 to 5 4 mg/ml of packed Sepharose. Each protein was coupled separately and the various antigen-Sepharose conjugates were mixed to obtain the specific adsorbant needed. Antisera were initially adsorbed on a conjugated Sepharose mixture of human K and A light chains plus IgAA and IgMK, and reacted only with IgG by ID analysis after this adsorption. These antisera were then passed through a Sepharose-IgG mixture which lacked the specific y chain subclass utilized for immunization. For example, an antiserum specific for IgG1 was passed through a column that contained various preparations of Sepharose conjugated with IgG2K and A, IgG3 K and A, and IgG4 A, termed a 'lack-i' column. In order to determine the specificity of these antisera, K and A proteins comprising the four IgG heavy chain subclasses were individually coupled to human group 0 red cells and tested by microtitre haemagglutination assay. In order to avoid idiotypic reactions, the original immunizing protein was not used for sensitization. Only antisera which reacted equally well with K and A species of the desired IgG subclass were used for study. If an antiserum was produced by immunization with IgG4A, then IgG4K was coated onto red cells to avoid the possibility of slight preferences for a similar light chain type. If only IgG4 proteins inhibited such a system, the antiserum was selected as a suitable reagent. At least two highly specific antisera for each subclass met the above requirements. Pepsin digestion of Human IgG A 1% solution of purified pooled human IgG was dialysed against 0-1 M sodium acetate buffer pH 4-5 and digested at a 1:100 pepsin (Schwarz-Mann) to protein ratio for 18 h at 370 as described by Turner, Bennich & Natvig (1970). The digest was dialysed against 0-1 M borate buffered 0-15 M NaCl pH 7-8 (BBS) and applied to a calibrated Sephadex

G-100 column. The F(ab')2 and pFc' fragments were identified by column elution position, ID and IEP. Only individual column fractions from the elution peaks which contained purified preparations of either fragment were utilized.

Papain digestion Papain digestion of human pooled IgG and an IgGI myeloma protein were performed according to Nisonoff (1964) with two exceptions; digest mixtures were incubated for 9 h at 370 and protein concentrations were 10 mg/mi. The digests were dialysed against 0-005 M sodium phosphate pH 7-6 and applied to DEAE-cellulose columns. Fab fragment which eluted with the 0 005 M starting buffer was contaminated with a small amount of Fc fragment as shown by ID and IEP. A stepwise gradient to 0-4 M NaCl in 0-02 M sodium phosphate pH 7-6 eluted first, a small peak of Fc fragment and then a larger peak which contained Fc fragment and a small amount of Fc' fragment. In order to obtain a preparation of Fab fragment free of contaminating Fc fragment, the fractions containing Fab fragment were dialysed against 0 005 M sodium acetate pH 5-6 and applied to a carboxymethyl cellulose (CMC) column in the same buffer. No protein eluted with the 0 005 M acetate. A stepwise gradient to 0-01 M sodium phosphate pH 5 6 followed by a continuous gradient to 0 5 M NaCl (in 0-01 M phosphate) eluted fractions which contained pure Fc and Fab fragments, respectively. Fc' fragment was prepared by a 21 h papain digestion of human IgG and purified by CMC chromatography. Fc' fragment was eluted in the starting buffer (0-01 M sodium acetate pH 5 6) and produced precipitin arcs of characteristic anodal mobility by IEP (Turner & Bennich, 1968). Timed papain digestions Aliquots from papain digests of IgG and IgGl performed as described above, were extracted at 0-25, 1, 4, and 11 h after addition of papain and applied directly to Sephadex G-100 columns equilibrated with BBS. The first elution peak contained undigested IgG which diminished in size as the 11 h digestion period was approached. The second peak, which was quite assymetrical, contained Fc fragment in its ascending and Fab fragment in its descending portions. This peak became more symmetrical as the 11 h digestion period was approached. Individual column fractions when

Human IgM anti-IgG cryoglobulins I

675

contained heavy chains and the light chain peak concentrated and reapplied to acidic Sephadex G-100 columns. Only one peak was produced for either sample. The fractions of high optical density were pooled, dialysed against 0-01 M acetic acid and shown to be pure heavy or light chain by SDS polyacrylamide gel electrophoresis. Both preparations remained crystal clear.

analysed by immunodiffusion and immunoelectrophoresis were shown to contain pure Fab and Fc fragments. These fractions were dialysed against 0-15 M NaCI and utilized in haemagglutinationinhibition experiments. A third peak which was minor after 1 h, but more prominent after 11 h was felt to contain small peptides because of its delayed elution position.

were

Reduction and alkylation Reduction and alkylation of a 1 % solution of human IgG was performed by standard techniques (Fleischman, Pain & Porter, 1962) with minor reagent modifications (Waterhouse, Abraham & Vaughan, 1973). Sephadex G-100 chromatography in 1 M propionic acid produced a chromatogram with a bifid first peak and a typical light chain second peak. By SDS polyacrylamide gel electrophoresis (Steiner & Blumberg, 1971) the first portion of the double peak showed heavy and light chains in approximately equal amounts as judged by the intensity of the stain, and a small amount of material which only penetrated the very top of the gel. In order to obtain unequivocally pure and non-aggregated proteins the second portion of the first peak which

RESULTS Specificity of IgM anti-IgG preparations for IgG preparations of various subclasses Fourteen monoclonal K and IgG preparations of the four y chain subclasses, were tested for their ability to inhibit the reaction between each cryo-IgM and RBC sensitized with IgG (anti-Rh), termed 'isologous' reactions. Human monoclonal IgA and IgM were used as controls for negative inhibition. Each of the six IgM preparations showed preferential reactivity for particular y chain subclasses as well as individual IgG preparations within a subclass (Table 1). Additionally, each IgM was sufficiently unique in its specificity profile such that it could not

Table 1. Inhibition of the haemagglutination between anti-Rh coated human 0-positive erythrocytes and the various IgM anti-IgG cryoglobulins by IgG preparations of the four y-chain subclasses. Inhibitor IgG preparations were doubly diluted from an initial maximum concentration of 0 7 mg/ml to a final minimum of 0 006 mg/ml. The concentration of inhibitor at which an initial 3-4+ haemagglutination endpoint was restored, i.e. where no inhibition was noted, is indicated Lowest concentration of IgG (mg/ml) producing haemagglutination-inhibition of the indicated IgM anti-IgG cryoglobulin

Inhibitor Ric Woo Ham Mit Thi Pig Kus Upd Can Coo Dic Tos Dun Str Pot Hoe

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NI NI NI

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IgM-Luc

IgM-Jos

IgM-Cra

NI NI NI NI NI NI 0 09 0 09

0 09 0 045 0-012 0 18 NI 0 35 07 NI

NI

NI

NI NI NI NI NI NI NI

0 35 NI NI NI NI NI NI

0-18 0 045 0-09 0 35 0 35 0-18 0 09 0 045 0-012 0 006 0 006 0 006 0 045 0-7 NI NI

0 045 0-023 0 006 0 09 07 0 045 0 09 0 045 0 09 0 045 0 09 0 09 0 09

NI, non-inhibitory at an inhibitor concentration of 0-7 mg/ml.

0-18 NI NI

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Sarah Larrian Johnston & G. N. Abraham

be considered exactly like any other. IgM-Luc reacted preferentially with IgGl, IgM-Gly with IgG2, and IgM-Jos with IgG3. IgM preparations Teh and Pla, reacted with some examples of IgG1, IgG2 and IgG4 but with sufficient variations within subclasses such that the patterns of reactivity may signify specificity not related to subclass determinants. Finally, only two IgM preparations (Cra and Jos) reacted with IgG of all subclasses. Reactivity of IgM anti-IgGs with autologous IgG An inferential method for determining the reactivity of an IgM anti-IgG in vivo is to assay the portion of the serum IgG which cryoprecipitates bound to the IgM, i.e. the 'antigen-IgG'. The 'antigen-IgG' was thus isolated from each cryoglobulin and the unbound or 'residual-IgG' from the cryoglobulin depleted sera of five patients. The IgG subclasses in these pools were identified by comparing their ability to inhibit the haemagglutination between antisera specific for an IgG subclass and red cells chromic chloride coated with IgG of the same subclass. When the 'antigen' had a greater inhibitory ability than 'residual' IgG, this was termed 'subclass enrichment'. The data obtained by use of one antiserum specific for each IgG subclass are shown in Table 2 but at least one other antiserum for each subclass was tested and the results were substantiated. A normal human IgG pool was included in these studies as a reference. As previously noted, four of the five IgM preparations shown had reacted with isologous IgGl, but the data in Table 2 show IgGl in each antigen pool. That of Gly, Jos and Pla did not have the potent inhibitory capability of the Teh and Luc IgG pools. Thus, the quantities of IgGI are most likely equivalently reduced in both the antigen and residual IgG pools of these three cryoprecipitates. Further, no 'antigen' IgG showed a significant IgGI enrichment when compared to its residual IgG. Since this subclass comprises 60-75 % of all serum IgG, any enrichment in the antigen pool may easily be masked in an haemagglutination-inhibition assay which used doubling dilutions and has an assumed two-well

experimental error. Similar experiments were performed with antisera specific for IgG2, IgG3 and IgG4. IgM preparations, Teh, Pla and Jos, were shown above to react with isologous IgG2 (Table 1). The antigen IgG pools, however, contained less IgG2 than the residual IgG

pools. Further, when compared to an equivalent concentration of pooled human IgG, their residual IgG preparations contained the same and their antigen IgG preparations, reduced amounts of IgG2. IgM-Gly preferentially reacted with autologous IgG2 and none was detected in the residual IgG which indicated that IgM-Gly had bound all serum IgG2. As shown in Table 2, four IgM preparations cryoprecipitated autologous IgG3 and the antigen IgG preparations of three (Jos, Pla and Luc) demonstrated IgG3 enrichment. These were unexpected findings since three of these cryo-IgM preparations (Pla, Luc and Teh) previously showed no reactivity with isologous IgG3 myeloma proteins (Table 1). Use of two other specific antisera confirmed that very little of this subclass was present in the Gly-IgG pools. The strong reactivity of IgM-Jos with autologous IgG3 and the y-3 enrichment in the antigen pool correlated well with the isologous specificity reactions. Finally, IgM preparations, Pla and Jos reacted with isologous and autologous IgG4. IgM-Luc which failed to react with two isologous IgG4 preparations, however, cryoprecipitated autologous IgG4 as evidenced by its presence in the antigen-IgG pool. The IgG pools of Gly and Teh showed a lack and deficiency of IgG4, respectively. The antigen and residual IgG pools were also tested with several antisera specific for human K and A light chains in order to determine if there was selectivity for a particular light chain type by a heavy chain subclass enriched in either pool. Pooled human IgG K and A was attached to red cells with chromic chloride, the antigen and residual IgG preparations adjusted to equivalent concentrations and compared in their ability to inhibit the haemagglutination of cells by various antisera. There were no differences in the inhibitory capabilities (i.e. the K: A ratios) for the two IgG populations of the five patients (not shown).

Reactivity of each IgM for the other antigen and residual IgG preparations The same five IgM preparations were tested for reactivity with the other antigen and residual IgG pools as another measure of their specificities and to determine if any of the antigen or residual IgG preparations were exceptionally potent in their reactivity with the other cryo-IgM preparations. In

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this set of experiments, the five antigen and residual IgG pools were compared in ability to inhibit the haemagglutination between each IgM and anti-Rh coated red cells. As noted in Table 3, the residual or antigen IgG preparation isolated from some other patient's cryoglobulin or serum, was equal to or greater in reactivity with each IgM than the patient's own antigen or residual IgG, and in every instance. As examples, the potent reactivity of the Luc antigenIgG with IgM-Teh, and the Teh residual IgG preparation with IgM-Gly should be noted. Finally, these assays substantiated and provided other evidence for subtle differences in the specificities between IgMs Pla and Teh originally noted above. As seen from Table 3 the Luc-IgG preparations were clearly more reactive with IgM-Teh than IgM-Pla. Thus, the minor difference in the specificity pattern previously shown in Table 1 was highlighted.

Reactivity of IgM preparations with subunits of IgG Studies performed to determine if the cryo-IgM preparations had similar patterns of reactivity with enzymatic fragments and heavy chains of IgG are shown in Table 4. The IgM preparations are arranged in order of increasing reactivity with Fc fragment. As noted, papain digestion of human IgG for 9 h abolished reactivity with IgM preparations Teh and Gly and markedly diminished that with the remaining IgM preparations. Purified Fc fragment showed no reactivity with IgM preparations Teh and Gly, was weakly reactive with IgM preparations Jos and Pla, more so with IgM-Cra, and strongest with IgM-Luc. The separated papain fragments and the unfractionated digest were strong inhibitors of the anti-IgG antiserum R-174. The same preparations of Fab and Fc fragments were heated at 56° for 1 h and retested. No differences or changes in the degree of inhibition were noted for any of the IgM preparations, i.e. Fc fragment untreated and heattreated were equivalent in reactivity or non-reactivity. Thus the process did not convert non-inhibitory fragments into inhibitory ones. Fab fragment was always non-reactive. Pepsin digestion abolished reactivity with intact IgG since neither the unfractionated IgG pepsin digest, F(ab')2 nor pFc' fragments were able to inhibit reactivity of the six IgM preparations tested. Both the pepsin and papain fragments retained

ability to inhibit the rabbit antiserum, R-174 included as a control. Heavy and light chains were also tested. Light chains were non-inhibitory. Heavy chains were approximately equivalent in reactivity to intact IgG with IgM preparations Teh, Gly and Cra, more reactive with IgM preparations Pla and Jos, and less reactive with IgM-Luc. Further, heavy chains were more reactive than Fc fragment with all IgM preparations except Luc. Of interest is the fact that unmodified, intact IgG was a better inhibitor of IgM-Luc reactivity than was reduced and alkylated IgG or heavy chains. Curiously, heat aggregation of human IgG for one hour at 560 slightly increased its reactivity with all antiglobulins and particularly with IgM-Cra. Previous experiments from this laboratory (Abraham, Clark & Vaughan, 1972) had shown that Fc fragments from an IgGl myeloma protein (All) and the heavy chain disease protein Cr (kindly donated by Dr Edward C. Franklin, New York University Medical Center, New York, N.Y.) were strongly reactive with an IgA anti-IgG in direct and competitive binding assays. Since papain digests of IgG (9 h) were either minimally or non-reactive with four of five IgM preparations, attempts were made to demonstrate the reactivity of Fc fragment with IgM preparations Teh, Pla, Jos and Gly. In these experiments, Fc fragments were isolated after a much shorter period of papain digestion of pooled IgG and IgGI (All). In order to increase the sensitivity of the assay, an alternative method of the haemagglutinationinhibition test was used which employed a constant inhibitor concentration and serially diluted antibody. Data obtained are shown in Table 5a for IgM preparations, Pla, Gly and Jos. Intact All was as strong a reactor as IgG. Fc fragment from All isolated after 15 min of digestion reacted with each IgM to various degrees. The inhibition by All-Fc was best for IgM-Teh (not shown) and Pla, less for Gly and nearly non-existent for Jos. This lack of reactivity between All and IgM-Jos may be due to its previously shown preferential binding of IgG3. The IgG 1 Fc piece Cr, was a potent reactor with all IgM preparations. It was felt that when reactivity was initially minimal as for IgM preparations, Teh and Pla (Table 4), this may have been caused by further degradation of Fc fragment with papain as the digestion period increased. In order to test this

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+1+1 +1+1 00o 00o

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+l Cl -

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Sarah Larrian Johnston & G. N. Abraham

680

Table 4. Inhibition of haemagglutination (HA) between the IgM anti-IgG preparations and IgG (anti-Rh) coated erythrocytes. The penultimate dilution of each IgM which produced a 4+ HA endpoint was inhibited by IgG, its subunits and polypeptide chains. The lowest molar concentration x 10-3 of each inhibitor at which the 3+ or 4+ HA endpoint was restored is indicated. The initial molarity and the equivalent in mg/ml shown in the Table for each protein, is the maximal concentration utilized of each inhibitor

Initial concentration inhibitor Inhibitor

Papain digest IgG Fab fragment Fc fragment Pepsin digest IgG F(ab')2 fragment pFc' fragment Reduced and alkylated IgG H chain (recycled) L chain (recycled) IgG IgG heated

(mg/ml)

MX 10-5

1.0 3-7 3-7 70 4-7 17

0-67 7-4 7-4 6-7 4-7 6-7

1.0 10

0-67 2-0 4-7 0-67 0-67

1V2 1.0 1-0

Lowest molar concentration (x 10-5) of inhibitor producing haemagglutinationinhibition of the indicated IgM anti-IgG cryoglobulin IgM-Gly

IgM-Teh

IgM-Jos

IgM-Pla

IgM-Cra

IgM-Luc

R-174

NI NI

NI NI NI NI NI NI

0 34 NI 09 NI NI NI

0 34 NI 1-8 NI NI NI

0 34 NI 0 45 NI NI NI

0 34 NI 0-12 NI NI NI

0-0013 0-014 0-014 0 007 0-014 0-23

0 04 0-25 NI 0 16 0-08

0-02 0 004 NI 0005 0-0013

0 0025 0 004 NI 0 04 0-01

0 0025 0 004 NI 0-01 0 0025

0-0013 0 004 NI 0005 0-0013

0-08 05 NI 0-02 0-01

0-0013 0-015

NI NI NI

NI

NI 0-0013 0 0013

NI, Non-inhibitory at the maximal concentration of protein. Other conditions of the assay are the same as those in Table 1 and given in Materials and Methods. Table 5a. Haemagglutination-inhibition of serially diluted IgM by a constant concentration of inhibitor. IgG is DEAE processed human fraction II, IgG, (All) a myeloma protein. Fc fragment was isolated after 15 min and 1 h digestion periods of IgG1 (All) and IgG (F-lI) respectively

Inhibitor Diluent IgGl-Fc (All) 15 min IgG-Fc 1 h Cr-Fc IgG (Fii & All)

Conc. (OD-280) 04 13 0-5 04

Gly-lgM

Pla-IgM 4 2 3 0 0

3 0 1 0 0

2 0 0 0 0

2 0 0 0 0

1 0 0 0 0

notion, human IgG and IgGl All were digested for varying times, the Fc fragments isolated and tested for reactivity with IgM-Teh. The results are shown in Table 5b. The reactivity of the Fc fragments was clearly related to the length of digestion. Fc fragment isolated after 15 min of digestion was the most reactive while that isolated after 21 h of digestion was only marginally reactive. Fc, F(ab')2 and pFc' fragments obtained from the pooled IgG showed no reactivity when tested at the same molar concentrations as IgGl. The heavy chain disease protein-Cr showed potent reactivity.

0 0 0 0 0

0 0 0 0 0

4 3 2 1 + 0 0 3 2 1 0 0 0 0 3 2 1 0 0 0 0 0 000000 0 0 0 0 0 0 0

Jos-lgM 4 4 4 0 1

4 4 3 0 0

3 3 2 0 0

3 2 1 0 0

3 1 0 0 0

2 1 0 0 0

1 0 0 0 0

DISCUSSION

Previous reports have demonstrated that rheumatoid factors including the IgM cryo-anti-IgG preparations react with a variety of antigenic determinants on IgG (Heimer et al., 1962; MacKenzie et al., 1968; Cream et al., 1972). In the present study, six IgM cryoglobulins with anti-IgG activity were compared in reactivity with the various heavy chain subclasses of IgG and five with autologous IgG preparations, and IgG fragments and subunits. Three methods were utilized to compare the

681

Human IgM anti-IgM cryoglobulins I Table 5b. Haemagglutination-inhibition of serially diluted Teh-IgM by a constant concentration of Fc fragment isolated from the IgGi myeloma protein, All, after the indicated period of papain digestion. Inhibitor concentrations are given in optical density at 280 nm

Conc. (OD-280)

Inhibitor

Diluent IgGI-All All-Fc All-Fc All-Fc All-Fc

All-Fc' Cr-Fc

15 min 55 min 4h 21 h 21 h

Teh-IgM 4 0 2 3 3 3 4 0

03 0-3 03 0-16 03 0-14 0-2

3 0 1 2 2 2 3 0

3 0 + 1 1 2 3 0

2 2 1 + 0 0 0 0 000 1 + 0 0 1 + + 0 1 1 + 0 2 1 1 + 0 0 0 0

R-174

4 4 0 0 1 + 1 1 1 1 2 1 ND ND

3 2 0 0 + 0 +0 +0 + +

2 0 0 0 0 0

1 0 0 0 0 0

+ 0 0 0 0 0

ND, Not determined.

antigenic specificities. First, the reactivity patterns with isologous myeloma IgG preparations of the four y chain subclasses were established. Second, the IgG subclasses present in the antigen-IgG which cryoprecipitated with five of six IgM preparations were compared to those of the residual or unbound IgG isolated from the cryoglobulin depleted serum. The data obtained provided information on the ability of each IgM to select a particular IgG subclass from its own serum milieu, termed 'subclass enrichment'. Finally, the IgM preparations were compared in reactivity with each of the autologous and isologous antigen and residual IgG pools. Numerous conclusions can be derived from these data. First, no IgM anti-IgG had a pattern of reactivity with isologous IgG preparations which was identical to another. Further, for four of six, the specificity patterns precluded reactivity with any of the previously mentioned genetic determinants. None possessed the anti-'non-a' specificity, an antigen on the pFc' of Gm(a-) IgG I, and all IgG2 and IgG3. Two IgM preparations, however, reacted in this assay with IgG 1, 2 and 4 with enough similarity to suggest that they may be reactive with a closely related antigen or a different portion of the same determinant, possibly the Ga antigen (Allen & Kunkel, 1966). Second, the specificity profiles obtained by this commonly utilized assay did not allow one to predict with certainty the subclasses of IgG with which the cryo-IgM preparations would react, and precipitate from their serum. Next, in certain instances and as previously noted by Cream et al. (1972), the IgM preparations precipitated an IgG

enriched for a particular subclass which indicated a highly preferential reactivity. It is possible that the antigen-IgG possesses a unique antibody activity of its own. In contrast, other data indicated that selection of IgG in serum by some IgM preparations was a random and non-specific process, since there were no serological differences between the antigen and residual IgG pools. Perhaps in these instances the IgG recovered is not the original antigen, i.e. the actual IgG antigen may no longer be present or IgG may be cross-reactive with the actual antigen. This hypothesis is supported by the finding that in two instances, the residual or antigen-IgG from some other patient was actually more reactive with an IgM than either of the patient's own IgG pools. These studies also confirmed the clinical impression that patients with primary idiopathic mixedcryoglobulinemia may be humorally immunodeficient. Assays of the residual and antigen IgG pools did not permit precise quantitation, but it was nevertheless quite apparent that there were marked and selective IgG subclass deficiencies in at least two instances. The reasons for this are unknown. The remaining experiments were concerned with localization of the antigenic region on the IgG molecule. This was determined by assaying the reactivity of various IgG subunits with these IgM preparations. A finding not previously noted was that the IgM anti-IgG preparations have marked variability in their reactivity with IgG-Fc fragment. Two of six did not react, two of six reacted weakly and two of six well with Fc fragment prepared from pooled human IgG. Further, the

682

Sarah Larrian Johnston & G. N. Abraham

molar concentration of Fc fragment required to demonstrate this reactivity was greater and reactivity much less than for intact IgG or partially reduced and non-aggregated y chains in every instance but one. IgM-Luc demonstrated preferential reactivity with intact IgG and Fc piece. This reactivity was diminished by partial reduction and alkylation of IgG, i.e. cleaving the interchain disulphide bonds without separation of heavy and light chains. Thus, in one instance the reactivity depended upon particular conformations expressed only on intact IgG, while in the others the reactive determinants were more readily expressed on heavy chains. These latter may possibly represent unique peptide antigens with a stable conformation. In order to localize the reactive domain of the Fc region, pFc' fragment was utilized as an inhibitor. This fragment represents the entire third heavy chain constant region domain as well as a small portion of the carboxy terminal portion of the Cy2 domain (Turner & Bennich, 1968; Turner et al., 1970). Remarkably it was non-reactive in all instances. In order to substantiate this finding, another preparation of pFc' fragment provided by Dr Robert Painter (University of Toronto, Toronto, Canada) was also assayed with IgM preparations Teh and Gly, and shown to be non-reactive. The reactivity with pFc' fragment of the various y chain subclasses was one type of major antigenic specificities of noncryoprecipitating rheumatoid factors (Gaarder & Natvig, 1970; Steward et al., 1973). Despite these results, the IgGI heavy chain disease protein Cr demonstrated potent reactivity with all of these IgM preparations. Thus, Fc fragment was isolated after short term papain digestion of IgG. Fifteen minutes of papain digestion of the IgGI myeloma protein (All) produced Fc fragment which reacted with each IgM, but the reactivity was less than that for either intact TgG or the Cr-protein. Further, as shown with IgM-Teh, as the papain digestion time increased, a progressive loss in reactivity of Fc fragment was noted. The major antigenic determinant with which at least four of six of these IgM cryoglobulins reacted must have been altered or degraded during papain digestion. These data are compatible with and explained by results obtained by Wang & Wang (1977) who noted that papain was capable of enzymatically digesting IgGl myeloma proteins in a stepwise fashion. The first cleavage site was located in the hinge region, at the amino terminal side of the inter-heavy chain

disulphide bridges; the second, nine amino acid residues away at the carboxy-terminal side of these bridges. Further, papain cleavage lead to degradation of the Fc fragment into small peptides. This portion of the Fc fragment is preserved in the Crprotein (Dr Bias Fragione, personal communication). Based on the data obtained in the present study, there are only a few possible locations for the antigenic determinant(s) with which these IgM preparations react. Such a determinant could be located in or near a region of the heavy chain such that enzymatic digestion near the site destroys its integrity and/or conformation. This may be located on or near the hinge region or at the amino terminal end of the second constant region domain of gamma chain. This notion is supported by data which show that no IgM reacted with the pFc' fragment, i.e. the Cy3 domain. The data, however, do not preclude the possibility that the actual location of the reactive area differs for each antiglobulin. Additional information which concerns the nature of the changes in the reactivity between IgM and the Fc fragments will be obtained by primary amino acid sequence analysis of each Fc fragment obtained from the timed papain digestions. When the actual antigenic sites are determined, it may be that the IgM cryoglobulins will be seen to comprise a unique group of anti-IgG autoantibodies separate from the more common non-cryoprecipitable rheumatoid factors.

ACKNOWLEDGMENTS

This study was supported by USPHS Training Grant AI-00028, USPHS Research Grant Al-lI 550, a New York State Health Research Council Research Grant, The Monroe County Chapter of the Arthritis Foundation, and the David Welk Memorial Fund. The generosity of Dr Robert Painter in donating the IgGI Nel constant region domains in order to confirm our results is greatly appreciated. Portions of this manuscript were written during an academic leave in the Irvington House Institute of the New York University Medical Center, New York, N.Y. G.N.A. is the recipient of USPHS Allergic Diseases Academic Award AI-70834. We thank Mrs Sally Ann Hart for preparation of a portion of this manuscript. Dr John P. Leddy provided helpful comments and advice during the research and preparation of the manuscript.

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