Low molecular weight rheumatoid factors in rheumatoid arthritis sera

lmmunochermstry, 1974, Vol i1 pp 41%421 Pergamon Press

Printed in Great Britain

LOW MOLECULAR WEIGHT R H E U M A T O I D FACTORS IN R H E U M A T O I D ARTHRITIS SERA J P A U L L Y E T and D E N O R M A N S E L L Department of Mlcrobmlogy, University of Virginia School of Medicine, Charlottesville, VA 22903, U S A (Ftrst recewed 27 October 1973, m rewsed form 20 December 1973) Abstract--Low mol wt (7S IgG) ant1 IgG rheumatoid factors (RF) were isolated from the serum of two pahents w~th rheumatoid arthritis The interaction of these RF preparations with the Fc fragment of human IgG was studied in the ultracentrifuge and compared with 19S IgM RF isolated from the same sera The measured binding constant for IgG RF was ldenUcal to that of the IgM RF m one patient, whereas m the other patient, the IgG RF exlubited a lower binding constant than did the IgM RF

INTRODUCTION Antl-IgG rheumatoid factors (RF) occurring m the serum of rheumatoid arthrttls pauents are predominantly of the 19S IgM lmmunoglobuhn class (Kunkel and Tan, 1964) and react wRh normal IgG to form a 22S complex (Franklin, 1961, Normansell and Stanworth, 1968, Normansell, 1970) The presence of low mol wt RF m certain rheumatoid sera and joint flmds has been referred from the presence of lntermedmte complexes (10-17S) m these flmds (Villa et al, 1960, Chodlrker and Tomasl, 1963, Schrohenloher, 1966, Hannestad, 1967, Jasm et al, 1970, Winchester et al, 1968, 1970, 1971) These complexes consist mainly of IgG (Schrohenloher, 1966, Hannestad, 1967, Winchester et al, 1971) and appear to revolve F a b - F c mteractmns (Schrohenloher, 1966), although complexes due to F c - F c interactions have been described (Helmer et at, 1970) In a dafferent approach, RF actwlty assocmted with IgG and IgA globuhns has been demonstrated by adsorption to, and elutlon from, IgG lmmunoadsorbents (Helmer and Levm, 1966, T o m g l a m and Roltt, 1967) In wew of the generally accepted concept of maturation of the immune response (Elsen and Slskmd, 1964, Werblln and Slskmd, 1972), it was antmpated that the binding constant of IgG RF for IgG might be ingher than that of the 19S IgM RF We report here studies of two sera winch contained large amounts of both intermediate and 22S complexes, indicating the presence of both 7S and 19S RF m the serum at the same ttme Both species of RF were isolated from each of the sera and their reachvlty wRh IgG measured It was found that the bmchng constant of the 7S RF did not exceed that of the 19S RF m eRher case MATERIALS AND METHODS

Isolatzon of low mol wt RF Prehminary experiments established that the intermediate complexes reversibly dissociated to 7S material at pH 4 0 Accordingly, serum samples at pH 4 0 (0-05 M phosphatecitrate buffer containing 0 15 M NaC1) were chromategraphed on Sephadex G200(110 x 3 1 cm) m tlus buffer The 7S fraction was concentrated by ultrafiltratlon to 5mg/ml and re-chromatographed on the same column to ehminate any traces of higher and lower reel wt material After concentration to 5 mg/ml, the 7S fraction was digested wRh pepsm (Sigma, 1 I00 w/w, in 0 1 M sodium acetate pH 4 0) for 18 hr at 37°C Digestion was terminated by adjustment to pH 8 0 followed by dialysis against 0 01 M potassium phosphate buffer, pH 7 3 contalmng 0 15 M NaC1 (PBS) Rheumatoid factors were isolated from the 5S digest by specific precipitation with soluble aggregates of human IgG, prepared as described below The 5S digest and the soluble IgG aggregates were mixed gently at eqmvalence (determined by prior titration) and allowed to stand at 25°C for 1 hr The precipitate was collected by centrffugatlon (6800 g, 10 mm), washed with cold PBS and carefully resnspended in a small volume of PBS at 25°C The pH of the suspension was slowly lowered to 4 0 with 0 1 M cRric acid, resulting in solublhzatlon of the precipitate, and the mixture was then separated on Sephadex G200, pH 4 0 at 25°C The RF peak was adjusted to pH 7, concentrated and dialyzed against PBS The product sedlmented as a symmetrical 5S peak m the ultracentrifuge lsolatmn of 19S RF High reel wt RF was Isolated from the sera by chromatography of the euglobuhn fraction on Sephadex G200 at pH 4 5, as previously described (Norrnansell, 1970) Specific Isolation of rheumatoid factors was accomplished by specific preclpRation wRh soluble aggregates of human IgG m the same was as for the low reel wt RF, except that a 5% Agarose column, at pH 4 5, was used to separate soluble aggregates and RF The purified RF exhibited a sharp 19S peak m the ultracentrifuge with no detectable aggregates or fragments Preparatmn of soluble aggregates of human lgG Commercml Cohn FnII (Mann Research Labs, Lederle, Inc ) at 20 mgm/ml in PBS was heat aggregated at 63°C for 10 rain, cooled and any Insoluble material removed by cen-

Serum samples had been stored frozen for up to 3 yr Ahquots were thawed, heat mactwated at 56°C for 30 mm and clarified at 80,000 g for 30 mm 417

418

J PAUL LYET and D E NORMANSELL

trffugatmn High mol wt soluble aggregates (20-30S) were preclpstated with sodmm sulfate (0 62 M) (Chrlstmn, 1958), collected by centrffugatlon (6800 g, 10 mm) and redlssolved in PBS The precipitation step was repeated and the aggregates separated on Sephadex G200 at pH4 0 The fractmn eluted at the void volume was adjusted to pH 7 Rechromatography of thss fraction on Sephadex G200 at pH 4 0 indicated that it was.stable, no breakdown to lower mol wt material could be detected For the specific preclpltatmn of 19S RF, the aggregates were separated on an Agarose 5F column at pH 4 5 rather than on Sephadex G200 Only the void volume fraction was used for the prec~pstat~on F~7 Jragments Fc7 fragments were prepared from an IgG~ myeloma protein (To Oand from pooled normal human FnlI by papam dlgestmn followed by separatmn of Fab 7 and Fcy fragments on CM and DEAE 52 cellulose (Whatman) (Frankhn, 1960) Measurement of bmdmg constants The general technique used for measuring bmdmg constants m the ultracentrifuge has been described (Normansell, 1971) Briefly, a series of solutions containing RF and Fc), in molar ratios ranging from 3 1 to 1 25 were prepared using mlcroburettes (Gflmont) Control solutmns were prepared for each reactant alone, at three appropriate dilutions, to determine their concentration m the ultracentrifuge Reaction mixtures, and controls, were incubated for at least 1 hr at 25°C prior to ultracentrffugatmn For both 19S and 5S RF, reactmn with 3 5S Fcy resulted in the formatmn of a soluble complex sed~mentlng faster than either reactant alone The interaction between 19S RF and Fc7 was momtored by the photoelectric scanner, the concentration being obtained &rectly from the pen deflections For the lnteractmn between 5S RF and Fc 7, the components could not be resolved sufficiently by the scanner, so concentrations were obtained from the areas of Schheren peaks using a plammeter Replicate determinaUons of concentration from Schheren peak areas and from photoelecmc scanner pen deflections agreed to wsthln +5 per cent and to wsthm +2 per cent respectively The amount of Fc7 bound to RF was obtained from the &fference between the amount of Fc7 initially added and the amount which did not brad to RF The binding constant was calculated from a conventional scatchard plot of the binding data RESULTS

Figure 1 demonstrates the amounts of complexes in the two sera at pH 7 3 and 4 0 The 7S component of each serum, isolated at pH 4 0 on Sephadex G200, contamed no detectable aggregates or fragments at pH 4 0, but when adjusted to pH 7 0, about half of the 7S fraction reformed mterme&ate complexes (10-15S), in&catmg that approx 20 per cent of the 7S fractmn possessed RF activity Pepsin &gestlon of this fractmn abohshed the formation of complexes at pH 7 - F i g 2(a) and (b) However, such pepsm &gested materml reacted with normal IgG to form c o m p l e x e s - Fig 2(c) These results clearly establish the rheumatoid factor nature of the reactant molecules, complex formation bemg medtated through F a b - F c interaction RF was specifically isolated from thss pepsin &gest

by precipitation with soluble aggregated IgG Maximum precipitation occurred at a two-fold excess, by weight, of soluble aggregated IgG over RF, the final yield of RF representing 80 per cent of the 7S RF present in the serum The 5S RF so obtained ~as fully reactive with added normal human IgG or Fc), fragments to form complexes, with no unreacted 5S material [Fig 2(d)] RF Isolated from the 7S fraction of the sera, prior to pepsin digestion, sedlmented as a single 7S peak at pH 4 0, but as Intermediate complexes (10-14S) when adjusted to pH 7 3 with no remaining 7S material Double diffusion precipltln analysl~ using monospecific anti human antisera indicated that only h, .~ and 7 antigens could be detected in the specifically isolated 5S and 7S RF preparations There was no trace of ct or/~ determmants The IgM RF isolated from each of these sera se&mented at 19S at pH 7 0 Addmon of excess 7S IgG to the 19S RF resulted m conversion of all 19S materml to the 22S complex The IgM RF contained only h, / and/~ antigens except for a trace of 7 antigens (less than 5 per cent) The Scatchard plots obtained for the interaction between the RF preparation and Fc?, fragments are shown m Fig 3 and the binding constants obtained from these plots are given m Table 1 For ES 5S F(ab')2 RF reactlv5ty with FCTTol and pooled normal human Fc, a straight hne was fitted by least squares analysis The other three hnes were hyperbolae and were fitted by computer analysis of the data The equations used were (1) ES 19S RF, v = x/(0 9 5 x - 0 375), t = 0 85, (55) MB 19S RF, v = 1/(0 9 8 x - 0 078) t = 0 99 (m) MB 5S RF, ~ =(2 9 5 / ' Q - 1 09, 1=0 99, where z=mdex of determination for each equatxon The binding constants were determined from the free antigen concn at half saturation of the binding sites, i e the ordinate value at r = 1 The binding constants for 19S IgM RF and 5S F(ab')z IgG RF isolated from patient MB were essentsally identical, whereas the binding constant for the 5S F(ab')2 IgG RF isolated from patient ES was significantly lower than that for the 19S RF from that serum Moreover, an identical low result was obtained for ES 5S RF when it reacted with Fc 7 from pooled normal human IgG instead of Fc from the IgGl myeloma DISCUSSION

The two sera studied here offered the opportunity of comparing the reactlvtty of high and low mol wt RF for the same antigen The identity of the 7S IgG RF rests on the pepsin digestion studies The intermediate complexes were dissociated by acid pH but reformed on neutralization to pH 7 The 7S fraction of the sera, isolated at acid pH, also reformed complexes when neutralized to pH 7 Pepsin digestion of the 7S fraction however, eliminated complex formation, but did not eliminate the capacity to react w~th added intact normal IgG, or Fc 7, at pH 7 0 These data show that the reactivity is due to Fab regions directed against Fc7 an-

IgG Rheumatoid Factors

32'

A

32'

B

419

80'

80'

Fig 1 Effectof pH on the complexes m MB and ES serum (a) MB serum at pH 7 3 (upper) and pH 4 0 (lower) (b) ES serum at pH 7 3 (upper) and pH 4 0 (lower) Photographs taken at indicated times at 60,000 rev/mm and 20°C Phase plate angle 65° Centrffugatlon to the right tlgens, that is, RF activity Fc-Fc interactions (Helmer et al, 1970) and non-specific Fc-Fab interactions are ruled out Moreover, these 7S RF preparations possessed the same IgG subclass specafic~tyas the 19S IgM RF isolated from these sera complex formation did not occur with IgG of the ~,3 subclass (Normansell, 1973, Lyet and Normansell, unpubhshed data) Ant~gemc analysis of the punfied 7S RF from each serum indicated the presence of ~ heavy chain antigens and x and 2 hght chain antigens only This result would suggest a polyclonal response The absence of # heavy chain determinants and the absence of detectable 19S materml m the 7S RF preparations indicate that the lntermedmte complexes were due to IgG rheumatoid factors and not to IgM or subumts of IgM rheumatoid factors (Stage and Manmk, 1971)

The binding constants determined for the IgG RF from both rheumatoid sera are clearly of a lesser magnitude than would be expected from a companson of pubhshed values for 7S IgG antibodies from chromeally sttmulated experimental animals (Werblln and Slsklnd, 1972) The Ko for MB IgG RF is identical to that for the 19S IgM RF in the same patient, whereas that for ES IgG RF is lower than that for the 19S IgM RF The low binding constants cannot be due to the use of a umque myeloma Fc since an identical result was obtained for Fc~ prepared from pooled normal human IgG Comparison between different stzed RF molecules, 19S IgM RF and 5S F(ab'7)2 used m this study, is based on the observaUons that 19S IgM, 7S IgM subunlts and Fab# fragments have smaltar binding constants for haptens (Onoue et al, 1965, 1968, Stone

420

J PAUL LYET and D E N O R M A N S E L L

A

8

¢

D

Fig 2 Ultracentnfugal analysxs of the 7S fractions of MB and ES serum, isolated on Sephadex G200 at pH 4 0 (a) 7S fraction from ES serum at pH 4 0 (upper) and at pH 7 3 (lower) (b) 7S fraction from MB serum at pH 7 3 after (upper) and before (lower) pepsin digestion (c) Pepsin &gested 7S fraction from ES serum alone (upper) and mixed with an equal weight of normal human IgG (lower) (d) Specifically precipitated 5S [F(ab')2] R F from serum MB alone (upper) and mixed with a three-fold molar excess of human FCyrot (lower) Photographs taken after 64 (a), 40 (b), 64 (c) and 80 (d) mm at 60 000 rev/mm and 20°C Phase plate angle 65' Centnfugatlon to the right

Table 1 Bmdmg constants for the reaction of 7S lgG and 19S IgM RF with human Fc7 Binding constant for reaction with

Patient MB ES * Not done

RF 7S 19S 7S 19S

IgG IgM IgG IgM

IgGTol L/M --* 2 x l0 s 2 x l0 s

FCyro~ L/M 2x 1x 4x 2×

l0 s l0 s 104 l0 s

Pooled normal human Fc7 L/M --4 x 104

IgG Rheumatoid Factors

421

patients w~th severe rheumatoid arthritis is predormnantly a 19S IgM response The conventional maturation of the response resulting m high affimty 7S IgG antibodies falls to occur, or else, the high affinity antibodies are preferentially cleared However, since both IgG and IgA rheumatoid factors have been demonstrated (Tornglanl and Roltt, 1967) m many patients, it Is clear that the response Is not highly restricted The poss~bdlty that the response represents a prolonged primary response Is one explanation but the exact nature of the antigen involved remains obscure

40

30' _J x

Acknowledgements--This work was supported by USPHS Grant AM 15248 from NIAMDD and by USPHS Training Grant AI 00102 (JPL)

20

REFERENCES

IO

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2

Fig 3 Scatchard plots for the reactlwty of IgG and IgM RF with human Fcy The data for IgM RF reactlwty has been normahzed to two binding sites per moleculefor ease of comparison /X A ES 5S RF reaction with pooled norreal human F ~ © O ES 5S RF reaetaon with Fcyro~ [] • [] MB 5S RF reaction vath FcYrol 0------0 ES 19S RF reacUon with Fcyr.~ ~ MB 19S RF reaction with Fcyrol

and Metzger, 1968) and that 19S IgM RF and 7S subunits of IgM RF have stmllar binding constants for IgG (Normansell, 1970) Whether both IgG RF and IgM RF have specificity for the same antigenic site on the Fc fragment remains to be determined There appears to be just one funcUonal antlgemc site for RF per IgG Fc fragment since both the IgG RF and IgM RF form soluble complexes with monomeric 7S 'antigen' IgG This is a somewhat curious phenomenon when the mtact, purified 7S IgG RF is considered Although this molecule reversibly self-associates under physiological condmons, it does not form a precipitate even on standmg at 25°C for 24 hr or 4°C for 1 week The complexes formed are dimers and tnmers with some tetramers and presumably represent a mlmmum energy structure It would appear that the antl-IgG response m these

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