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Rheumatoid Factor Autoantibodies
in Health and Diseasea
RIZGAR A. MAGEED,b MARIE B@RRETZEN,cSASHA P. MOYES,b KEITH M. THOMPSON,C
AND JACOB B. NATVIGC
bKennedy Institute of RheumatologyHammersmith, London W6 7c x United Kingdom
=Instituteof Immunology and RheumatologyThe National Hospital0172 Oslo, Norway
INTRODUCTION
Since their discovery, a vast amount of work has been done on the incidence,
specificity, and genetics of human rheumatoid factors (RF) in a great many laborato-
ries. In the main, these studies were inspired by the association of RFwith RA and the
hope that understanding mechanisms of their production may help define disease
pathogenesis. However, studies of RF production and incidence have shown that RF
are not restricted to patients with RA and that genes encoding RF are inherited in the
normal human population. Furthermore, the docum entation that RF specificity can bepart of the natural antibody reperto ire during early fetal life and that norm al adults can
produce RF following imm unization are important indications for their physiological
role in normals and in understanding mechanisms of B cell regulation in health and
disease.
Th e initial attempts to define RF structure and genetics benefited from serological
analysis with anti-idiotypic reagents and amino acid sequence analysis of RFparapro-
teins, or “M” components, from patients with mixed cryoglobulinemia, Walden-
strom’s macroglobulinemia, and chronic lymphocytic leukemia. More recently,
nucleotide sequence analysis of RF genes isolated from immortalized human B cell
hybridomas from normals and RA patients in our laboratories, and others, provided
significant insight into how RF are produced and likely to be r e g ~ l a t e d . ~ ~ere, we
show that RF produced in normal individuals are different from those found in the
synovium of RApatients. RF in normals re frequently encoded by a limited set of IgV
genes, do not appear to switch isotype, and have few replacement mutations in the
CDRs. These observations support the concept that RF are under strict control to
preven t the emergence of high-affinity RF in normals.
asupport for this work was provided by the Arthritis and Rheumatism Council of GreatBritain (to R. A. Mageed and S. P. Moyes) an d by the Norwegian Women’s Health Organization
(NKS) and the Norwegian Research Council (to M. Berretzen, K. M. Thompson, and J. B.
Natvig).
296
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298 ANNALS NEW YORK ACADEMY OF SCIENCES
bind com plement, perpetuating the inflammatory processes and con tributing to tissue
damage.I6 The production of IgG RF is particularly important due to the ability of
IgG RF to self-associate and form large aggregates with high complement-fixing
potential.
The involvement of T cells in the synovial inflamm ation and the interaction with B
cells have been the focus of numerous studies.T cell involvement in RA is suggested
by the presence ofT cell infiltrates in the synovium and by the genetic association with
HLA -DRl and DR4 haplotypes.17J8 Synovial T cell infiltrates seem to be activated,
but lack specificity for any particular a~ to a n ti g en .' ~nly specificity for m ycobacterial
antigens has clearly been demonstrated.20 Furthermore, investigation of possible
clonal dominance w ithin T cell populations in rheumatoid synov ium has shown little
evidence for interclonal similarity or dom inance of T cell populations, suggesting that
the synovialT cell population may have been selected by their activation state rather
than by specificity.21
STRUCTURE-FUNCTIONALASPECTS OF HUMAN RF
The term RF is common for antibodies with specificities for determinan ts within
the Fc region of IgG. Antibod ies with these specificities are de tectable in a variety of
autoimmune and infectious diseases at varying levels and freq~encies?~-~~any
different specificities have been described for different RF. In general, IgG determi-nants recogn ized by RF can be categorized into the following groups: (1) subclass-
specific antigens, or isotypic antigens, found on all or som e of the four subclasses of
human IgG; (2) genetically defined alloantigens of the Gm type; (3) cross-reactive
antigens shared by human and animal IgG; (4) species-specific antigens found in
human IgG, but not animal IgG; 5 ) neoantigens expressed on aggregated, denatured,
or enzymatically digested IgG; and (6) determinants expressed on the Fab region of
IgG (antibodies with specificity for these determinants are often referred to as
antiglobulins, not RF). Extensive studies have shown that reactivity with an isotypic
antigen expressed on I gG l, 2, and 4, but not IgG3 (the Ga determinant) is the mainspecificity known for RF.25-28Despite some minor differences, the overall conclusion
has been that most FW,ncluding RF established from normal individuals, bind sites
related to the Ga deter1ninant.2~This site appears to be related to or overlaps with the
site of staphylococcal protein A (SPA) binding.30 Furthermore, pH titration and
chemical modification studies implicate histidine and tyrosine residues on the IgG Fc
in the binding of RF to the Ga dete~ min ant.~ 'he epitope is situated in the C y2 -C y3
interface region of IgG and involves three polypeptide loops, two from the Cy2 and
one from the C y3 domain, which come together in close proximity in the C y2 -C y3
interface region. The amino acids involved in the binding are likely to include
histidine at positions 310, 433, and 435 and tyrosine at 436. Despite the differing
causes of RF production, the specificity to this region appears to be generally
preserved.
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MAGEED et aL: RF 299
IDIOTYPIC AND GENETIC BASIS OF HUMAN RF PRODUCTION
Idiotypic Analysis
The main questions addressed in studying the RF idiotype have been whethernatural RF found in patients with lymphoproliferative disorders and pathogenic RF n
RA patients are related and whether they are direct copies of germline genes or
products of somatic mutations. The first set of human RF to be extensively studied
were IgM paraproteins (or “M ” components) isolated from the serum of patients w ith
lymph oproliferative disorders (Waldenstrom’s macrog lobulinemia and cryoglobuline-
mia). Anti-idiotypic antisera to these monoclonal antibodies were obtained following
absorption of rabbit antisera with paraproteins from different individuals and with
polyclonal IgG. This approach allowed the d efinition of three cross-reactive idiotypic
families, or groups, designa ted Wa, Po, and Bla.’*32 decade later, practically similarprotocols were followed in the selection of mouse monoclonal antibodies identifying
cross-reactive idiotopes expressed by RF para protein^ ^^ A rapidly growing library of
antibody sequences (derived from mRNA or DNA) from hybridomas whose protein
products reacted with the antibodies has enab led the determ ination of the contribution
of germline genes to RF production and has established the range of genes whose
protein products were recognized by the monoclonal anti-idiotypic a n t i b o d i e ~ . ~ ~
We investigated the expression of V gene markers and cross-reactive idiotopes
(CRI) using various murine monoclonal anti-idiotypic antibodies. These included G 6
and G 8 , which recognize VH1-associated CRI;33+35 6 and D12, which recognizeVH3-associatedCRI;36and Lc 1 , which recognizes protein products of the vH4 family
of gene s3’ V, gene family products were determined by C 7 (specific for the VK 3
family) and C6 (specific for the V 3b ~ u b f a m i l y ) . ~ ~7.109 was used to identify a C RI
that was encoded by the VK3b-subfamily ariable region gene, K ~ 3 2 5 . ~ ~he m ajority
of the RF paraproteins and RF from imm unized donors expressed at least one of these
markers (TABLE ). Within individual donors, particularly the normal immunized
donor, MR , groups of RF were found that shared identical patterns of V gene markers,
suggesting a restricted r e ~ p o n s e .~
Analyses of the monoclonal RF paraproteins showed a high degree of CRIe x p r e s s i ~ n . ~ ~he m ost-striking feature was the predominant use of the VK 3-associated
light-chain subgroup and CRI, with more than 90% expressing the V,3 light-chain
s ~ b g r o u p . ~ . ~ ~urthermore, it transpired that at least 60% of all IgMK RF express light
chains encoded by two V,3 genes whose protein products are recognized by the
monoclonal an ti-idiotypic antibodies 17.109 and 6B6.6.40Similarly, albeit to a lesser
extent, the RF paraproteins were also shown to widely react with the G6 and G8
monoclonal anti-idiotypic antibodies. When the VL and VH genes were considered
together, three different combinations of VH-VL could be iden tified. The largest grou p
of these RF were those that expressed kappa light chains reactive with the m onoclonal
antibody 17.109 in association with VH genes w hose produc ts were reactive with the
monoclonal antibody G6, which was subsequently shown to be a serological marker
for the DP- 10 and h vl 26 3 genes.40A second group of these RF expressed a V K 3 ight
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MAGEED et al : RF 301
V,3 in the R F from normals, together with a distribution of VH gene segments of the
v"1, vH3, and vH4 families, closely resembled the distribution seen among the RF
paraproteins. In contrast, RF from the synovial membrane of RA patients predomi-
nantly used heavy-chain segments encoded by genes from the vH3 family. These data
imply that RF paraproteins may be representative of R F used in normal physiological
responses, but that have undergone neoplastic transformation. Th e overall conclusion
from these studies is that RF produced in the synovial tissue of RA patients are
encoded by a wider range of IgV genes than the RF paraproteins and R F in normal
TABLE 2. Frequency of VHGen e Expression by RF Paraproteins and Monoclonal
IgM RF Produced by Hybridomas Established from Peripheral Blood of Immunized
Norm als and Synovial Membrane of RA Patients"
No. of No. of RF No. of RF
Family Individual Expressing Expressing ExpressingV H Paraproteins from Normals from RA SM
Used VHGene Used the Gene the Gene theG ene Total
DP-10DP-75HG31-13
total
3 DP-54DP-49DP-46b18DP-35DP-47DP-42v3-53DP-3 1
total
4 4.18DP-7 1
VH4.35DP-65DP-66
total
7 DP-2
Overall total
1
1
I
0
00
3
10
310
1
1
0
8
17
1 11
0 21 1
1
3 15
0 43 42 30 1
1 21 20
0
18 19
1 1
0 10 1
0
1 1
2 5
1 2
14 41
"Summary of the data on VH ene use in RF-producing B cell hybridomas generated in our
laboratories. For the sake of simplicity, data generated in other laboratories are not included.Only IgM RF paraproteins and IgM RF-producing lineshybridomas that have been well
characterized are included. IgG and I gA RF-producing lineshybridomas are not given. Normal
PBL and RA SM indicate the original sources of the RF-producing cells used to establish the
lineshybridomas.
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302 ANNALS NEW YORK ACADEMY OF SCIENCES
human immunized donors (HID). Furthermore, there is an increase in the representa-
tion of vH3 genes in the synovial RF hybridomas.
Interestingly, when the values of RF expressing the CRI detected by the monoclo-
nal antibodies G6, G8, B6, D12, and LC1 were assessed in monozygotic twins,
concordant or discordant for RA, it appeared that the absolute levels of the CRImarkers were higher than in normals and that the values were very similar within
twins, irrespective of disease status, but different between unrelated twins.42 These
findings were taken to imply that there may be additional regulatory mechanisms,
possibly genetic or idiotypic, that control the level of natural R F and that some of
these regulatory mechanisms may be inherently abnormal in individuals with a
genetic predisposition to develop
NATURE OF SOMATIC MUTATION IN FtF
The increase in the aftinity of antibodies to antigen and isotype switching is often
referred to as maturation of the immune response. This maturation requires that the
Ag-binding s ite of the antibody molecule be structurally different from antibodies in
the primary response. This is typically achieved by som atic mutation and repertoire
shift. Sequence analysis of IgV gene mRNA from a large collection of B cell
hybridomas with specificity for a variety of different antigens has shown that such
structural changes are often achieved through mutations leading to amino acid
replacements in the CDRS ~ In such responses, hypermutation of IgV genes is
followed by bo th positive and negative selection pressures through w hich B cells withhigh-affinity receptors are selected for expansion. B cells with no mutations or with
limited mutated receptors that have low affinity for antigen die, presumably by
apoptosis. Besides an ncrease in affinity, positively selected B cells show a character-
istic elevation of the ratio of R:S in the C D R S . ~urprisingly, R F produced in the
healthy immunized normals also show evidence in their IgV genes of extensive
somatic mutations (9-16 mutations per VHgene). There was, however, evidence for
selection against replacement mutations in the CDRs, suggesting that high-affinity
variants were negatively selected. Without any selective pressures, random mutations
in CDRl+2 of the DP-10 gene segment would generate R:S ratios of 4.4 s Theoverall R:S ratio of the C D R l + 2 in the VH region of the RF rom the immunized
donor MR, who had 10R F encoded by the D P- 10 gene, was significantly lower at 0.4
p < 0.0027, Fisher’s exact test, two-sided) (TABLE ). In contrast, RF from RA
synovia, bone marrow, and peripheral blood did not show a similar counterselection of
replacement mutations in the CDRs as seen in RF from the normal immunized donors.
RF from RA atients had between 2 and 18 nucleotide mutations compared with 9-1 6
in immunized normals (TABLE and FIGURE).
Nucleotide sequencing of the light chains of 5 of the RF established from the
normal immunized donor, MR, confirmed the clonal relatedness and showed that all
used the Kv325 light-chain germline gene rearranged to the JK1gene segment with
identical VK-J, junctions (RF-MR2, R F-MR13, RF-MR16, RF-MR28, and RF-MR30). Hypermutation was also evident in the light chains, but to a lesser degree than
in the heavy chains. Four of the 5 related clones had 1 shared replacement mutation in
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MAGEED et al.: RF 303
the light chain. These data confirmed that RF from immunized donors clearly undergo
somatic hypermutation.
ISOTYPE SWITCH IN RF-PRODUCING B CELLS
Following the initial interaction between virgin B cells and Ag, gennline gene-
encoded an tibodies of the IgM isotype are produced. The ability of the immune system
to adapt to combat antigenic challenge is manifested by further differentiation of
Ag-specific B cells to produce antibodies with high affinity and of different i ~ o t y p e s . ~ ~
Individual B cells with specificity for Ag can switch isotype while retaining the same
recombined IgV genes and an tibody specificity. These features of the immune system
have been widely used to assess the nature and mechanisms of induction of immune
responses to self-antigens in murine models of autoim mune disease^:^.^' Specifically,
TABLE 3. Total Number of Nucleotide Changes Observed in the VHGenes of RF
Derived from Peripheral Blood of Imm unized Normals, Synovial Membrane of RA
Patients, and IgM Anti-Red Blood Cell Rh(D) Antigen from Immunized Normals
Average Total No . of Total No. ofNo. Of Nucleotide Nucleotide
No. of Nucleotide Changes ChangesAntibodies Changes Seen in Seen in
CDR and 2ncluded from the 1.2 , and 3Source of in the Germline R:S R:SAntibody Analysis Gene R S Ratio R S Ratio
immunizednormals 10 12.0 17 16 1.1 7 16 0.4
RA SM 7 16.1 30 34 0.9 33 14 2.4
anti-Rh(D) 7 4.3 8 12 0.7 8 8.0
RF from
the ability to detect autoan tibodies of the IgG, and to a lesser extent IgA, isotypes withincreased affinity and similar specificity to autoantigens has been used as an indicator
of specific autoantigen-driven imm une responses in autoan tibody production.
One of the most-characteristic serological features of RA patients with severe
active disease and extra-articular symptom s is high levels of IgG W 8 High levels of
IgG RF are often associated with circulating immune complexes and complement
activation. The finding of large-size immune complexes is particularly important in
RA synovial inflammation because such complexes can contribute to pathogenesis
through com plement activation, cell recruitment, and cartilage and bone damage.I6 In
addition to the finding of high levels of IgG RF in the serum of a high proportion of
RA patients, IgG RF-producing B cell hybridomas were also isolated from three RA
patients in our stud ies.49 n con trast, none of the imm unized norm al donors produced
IgG RF or IgA RF, mplying that the switch of isotype to IgG and IgA is a more
common feature of R F-produc ing B cells in RA, but not norm al, individuals.
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304 ANNALS NEW YORK ACADEMY OF SCIENCES
?Rl ..**..*'E~~'****.* .*ttt*ttt~~~.* * **..
4 13 24 26 27 28 29 3 0 3 1 32 33 34 35 31 ( 3 53 54 56 57 59 63
AGC T A T DCT A W AGC GTU ACA CCT ATC DOT ACA M C Au:P-10 CTD AAG CCT GG OOC ACC TK AGC
hsM ERp m1 4 __.._ _._ _._- - G
Flu69 7 74 I1 79 80 82 83 84 00 89 93 94 95
ACG ATC GAA GC GCC TAC GAQ CTD ACC TCT GAG 0 TAT TACP-I0
l L 4 s a FRp'ISI .._ .._ .._ __.a .__
~p m14 A -T- __._ A a --t
Rp-- A -0- __.-- - - _ _ - - - - - --- - - - --t
~ p - M R 3 3 _.__.A ... _._.. .a .. t
~p-w _._C A ...__. __.a . -t
Rp rnl - - - --- c --c
FIGURE 1 Nucleotide sequences of the rearranged DP-I0 VH genes of IgM RF-producinghybridomas established from the synovial membrane (SM) of an RA patient, from bone marrow(BM) and peripheral blood lymphocytes (PBL ) of other RA patients, and from PBL of a normalhuman immunized donor (HID), MR, in comparison with the DP-10 ge m lin e gene sequence.Nucleotide sequences of the reamanged genes from RF-producing hybridomas are comparedwith the sequence of the DP-10 germline gene to show nucleotide mutations. B ases similar tothe germline gene are given as dashes, mutations that result in amino acid replacements aregiven in uppercase letters, and silent mutations are given in lowercase letters. Only co dons that
are different from the germline in the HID RF are given in the figure. RF established from thebone marrow (BM) B cells and peripheral blood (PBL ) of RA patients are included in the figureto show that RF rom R A patients, irrespective of their original anatomical compartment, havedifferent patterns of mutations compared with HID RF. The data are based o n references 4543.
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MAGEED ef al.: RF 305
CLONA L EXPANSION AND AFFINITY M ATURATION
It is now recognized that generation of mutations in the rearranged IgV genes
alone would not be adequate to account for the phenom enon of affinity maturation in
antibodies produced during the secondary phase of imm une responses.43A process (or
processes) capable of selectively activating and expanding B cells producing high-
affinity antibodies is also essential. In T cell-dependent immune responses, the
process of selective activation and expansion of higher affinity antibodies occurs
initially in the T cell areas of the spleen and lymph nodes.50 Subsequently, selected B
cells migrate to B cell follicles where they expand and initiate a germinal center
reaction, leading to clonal expansion of the higher affinity antibody-producing B
cells. This feature of the immune response has been used to study the kinetics of
immune responses to exogenous antigen^.^' Such studies have been achievable with
small hapten antigens, such as oxazolone and nitrophenyl, but are thought to bedifficult with complex antigens, which have numerous immunogenic epitopes. In our
studies, however, two IgM RF (RF-SJ1 and RF-SJ2) isolated from the same RA
patients were found to be clonally related.52The VHof RF-SJ2 had only 2 nucleotide
differences from its germline counterpart (GL-SJ), whereas RF-SJ1 had accumulated
18 nucleotide mutations. The light chain of RF-SJ1 also had acquired 18 nucleotide
mutations compared with the RF-SJ2. Functional affinity studies showed that the
extensively mutated antibody (RF-SJI) had a 100-fold higher affinity fo r human IgG
than the RF-SJ2. These observations provide further evidence that RF in RA do
undergo m aturation including clonal expansion.The derivation of the clonally related RF from the normal immunized donor MR
allowed a detailed examination of the relationship between somatic hypermutation
and affinity (TABLE).Among this group of RF, there is very little increase in affinity
with the accumulation of mutations in the FRs. On average, the RF had Kd values
of 2 X M). When compared with the affmity of RF from
RA patients, using the same gene segments, the mean K d was approximately 10-fold
lower, that is, 3 X M to 5 X lo-’ M) (TABLE).RF in RA
thus do not appear to be subject to regulatory mechanisms that control their affinity.
M (range, 0 63 X
M (range, 8 X
REPERTOIRE SHIFT IN THE IgV GENES ENCO DING RF
In addition to diversifying the antibody repertoire of an immune response by
somatic mutation, maturation of the antibody response is frequently accompanied by a
shift in the repertoire of the IgV genes used.53Studies of primary immune responses to
defined hapten antigens, such as oxazolone, in mice revealed that, although the
primary response was entirely dominated by a particular pair of VHand VL germline
genes, only 20 of the antibodies utilized the same com bination in the secondary and
tertiary responses.a This modification in the repertoire is called “repertoire shift.”
In humans, studies of immune responses to rabies vaccine have provided evidence
for repertoire shift.54For RF, there are limited data to suggest that repertoire shift may
contribute to the diversity of the RF repertoire in R A synovia (TABLE ).
Repertoire shift signifies that IgV genes utilized by low-affinity antibodies in the
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3 6 ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE . Functional ffi ni ti es of IgM RF rom Different Sourcesa
Nucleotide NucleotideMutations
in FR1+ 2+3ource
and CloneExamined R S
Mutationsin C D R l + 2
R S
HID PBLRF-MR 1RF-MR30RF-MR2RF-MR 16RF-MR37RF-MR33RF-MR25RF-MR14
RF-MR28
RA SMRF-SJlRF-SJ2RF-SJ3RF-SJ4RF-TS 1RF-TS2RF-TS5RF-KL1
Ki (MI
2 x 10-65 x 10-6
1 x 10-66 x2 x 10-69 x 10-72 x 10-6
2 x 10-6
1 x 10-6
2.4 x2.7 x4.6 x7.9 x 10-87.7 x 10-87.1 X 10-8
6.8 X lo-’2.1 x 10-7
EMCKAS ND ND ND ND 2.5 x 10-7
“The data are based on references 45 and 52. Only IgM R F paraprotein KAS and IgM
RF-producing lineshybridomas that have been characterized for specificity and gene use are
given. HID PEL and RA SM indicate the original RF-producing cells used to establish the
lineshybridomas. EM C indicates that the RF paraproteinKAS was from a patient with essential
mixed cryoglobulinem ia. All RF-producing hybridomas were sequenced at the mR NA level,
whereas paraprotein KAS was sequenced at the am ino acid level. ND = not determined. The
dissociation constant, Kd as deduced from the Scatchard plot using an ELISA assay for
binding to native human IgG.
primary response may not be capable of, or adequate for, encoding high-affinity
antibodies that dominate the secondary response. The reasons for this may lie in
intrinsic properties of the sequence or may reflect regulatory influences of B cell
selection in germinal centers.55 n responses to exogenous an tigens, the limiting factor
in determining repertoire is Ag concentration. Levels of exogenous antigens decline
gradually, reaching a minimum at the end of the acute response. This leads to
competition for the limited concentration of Ag, thus allowing only B cells with the
highest affinity to survive. In responses to self-antigens in normal individuals,however, the process of B cell recruitment in germinal centers is regulated by a
number of mechanisms, including high levels of soluble self-antigens that block
self-reactive B cells from reaching germinal centers and prevent maturation.’6
Some of these regulatory processes may be abnormal in the ectopic germinal
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MAGEED ef uL: RF 307
TABLE . IgV Gene Use in RF Paraproteins and Monoclonal FW-producing Clones
Established by Hybridoma Technology from Peripheral Blood of Normal Immunized
Donors and the Synovial Membrane of RA Patients0
Closest ClosestVH Germline GermlineRF Family VHGene VLFamiIy VLGene Source
RF-lT9RF-MR514RF-TT5
RF-MRCRF-MRlRF-TrM7BOR
KASHULRF-TS 1RF-TS2RF-TS3RF-TS4RF-TS5RF-SJ1RF-SJ2RF-SJ3RF-SJ4RF-KLIB42c93D53H4H6HAFIOC6G4D5G9A2A2
333111
31
1
131
3333343334311
31
343
DP-54DP-54DP-54DP-10DP-10DP-10DP-54DP- I0
DP-10DP-I0DP-I0DP-494.16DP-49DP-35DP-46DP-46DP-49DP-66DP-47DP-478-1B4.41DP-47DP-75hvlflONDDP-46DP-46DP-79DP-49
K3aK3aK3a~ 3 b~ 3 b~ 3 bK3a~ 3 b
~ 3 b~ 3 b~ 3 bK3a
ND
A1
A l
~ 3 b~ 3 b
A1
h3A3A3
A
~ 3 b~ 3 bx3
K2
K1
K 1
K4
K 1
K1
Kv328Kv328Kv328Kv325Kv325Kv325Kv328Kv325
Kv325Kv325Kv325Kv328A23NDllL102lv117lv117Kv325Kv305Vdlv1042
15011150
11150
k18lv80 1HK102
v gv glv318OlUOZ
HID PBLHID PBLHID PBLHID PBLHID PBLHID PBLEMCEMC
EMCEMCRA SMRA SMRASMRA SMRA SMRA SMRA SMRA SMRA SMJRA SMRA SMRA SMRA SMRA SMRA SMRA SMRA SMRA SMRA SMRA SMRA SM
The data are based on references 4, 5 45, and 57-63. Only IgM R F paraproteins or IgM
RF-producing lineshybridomas that have been well characterized are given. HID PBL and RA
SM indicate the original RF-producing cells used to establish the linesfhybridomas. JRA refers
to juvenile rheumatoid arthritis. M7, BOR, KAS, and HUL are RF paraproteins from patients
with essential mixed cryoglobulinemia EMC)orWaldenstrom’s macroglobulinemia.These RF
were sequenced at the protein level. ND = not determined.
centers in synovial membranes of patients with RA, leading to immune response
maturation.
SUMMARY
Recent advances in molecular biological and human cell hybridization technology
have significantly advanced the knowledge of mechanisms that underlie human
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308 ANNALS NEW YORK ACADEMY OF SCIENCES
rheumatoid factor (RF) production. These advances have provided insight into the
etiopathogenesis of synovial inflammation and lymphocyte recruitment in rheumatoid
arthritis (RA)joints.
We have examined the mechanisms that lead to RF production in RA patients and
those that regulate RF production in normals. The studies revealed structural featuresthat distinguish RF produced in normals from those produced in RA synovial tissue.
There are significant differences in the use of VL and V H genes between the two RF
populations. Furthermore, IgV genes encoding synovial RF in RA have extensive
evidence for nucleotide changes, leading to amino acid replacement in the complemen-
tarity determining regions (CDRs). In addition, RF produced in RA synovia show
evidence for affinity maturation, isotype switch to IgG RF, and repertoire shift
indicative of a continued recruitment of B cells. Together with computer modeling and
crystallographic studies, our data suggest that the mechanisms that operate on RF
selection in RA synovia are similar to immune responses to exogenous antigens. Incontrast, RF established from human immunized donors (HID) are characterized by a
very low ratio of replacement to silent R:S) ucleotide changes in the CDRl+2. In
addition, there is little increase in affinity with increasing num bers of mutations. There
is thus evidence for regulatory mechanisms that limit affinity maturation of RF n
normals.
ACKNOWLEDGMENTS
We would like to thank D. G. W illiams, of the Kennedy Institute of Rheumatology,for useful discussions and for the literature survey used in TA BLE .
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