close

Вход

Забыли?

вход по аккаунту

?

Lymphoproliferation in primary sjgren's syndrome. evidence of selective expansion of a b cell subset characterized by the expression of cross-reactive idiotypes

код для вставкиСкачать
1128
LYMPHOPROLIFERATION IN
PRIMARY SJOGREN’S SYNDROME
Evidence of Selective Expansion of a B Cell Subset Characterized by the
Expression of Cross-Reactive Idiotypes
FAZEL SHOKRI, RIZGAR A. MAGEED, B. ROBERT MAZIAK, NORMAN TALAL, NICHOLAS AMOS,
BRYAN D. WILLIAMS, and ROY JEFFERIS
Objective. To evaluate the possibility that lymphoproliferation in primary Sjogren’s syndrome (SS)
arises within a subset of B cells.
Methods. A panel of monoclonal antibodies
(MAb) specific for rheumatoid factor (RF)-associated
cross-reactive idiotypes (CRI) and anti-V, and anti-V,
subgroup antibodies were used to define the clonality of
B lymphocytes undergoing neoplastic transformation in
5 patients with primary SS. Anti-CRI antibodies were
also used to study longitudinal variations in serum
paraprotein levels and in vitro regulation of IgM and
IgM-RF production by peripheral blood lymphocytes.
The levels of CRI, IgM, and IgM-RF were quantitated
Presented in part at the First Breton Workshop on Autoimmunity, Brest, France, March 1990.
From the Department of Immunology, University of Birmingham, Birmingham, United Kingdom; the Department of Immunology, School of Public Health, University of Medical Sciences of
Tehran, Tehran, Iran; the Kennedy Institute of Rheumatology,
London, and the Department of Medicine, University of Wales,
Cardiff, United Kingdom; and the Department of Medicine, University of Texas Health Science Center, San Antonio.
Supported by a grant from the Arthritis and Rheumatism
Council of the United Kingdom.
Fazel Shokri, PhD: Assistant Professor of Immunology,
Department of Immunology, School of Public Health, University of
Medical Sciences of Tehran; Rizgar A. Mageed, PhD: Kennedy
Institute of Rheumatology; B. Robert Maziak, BSc: Research Assistant, Department of Immunology, University of Birmingham;
Norman Talal, MD: Professor of Medicine and Microbiology, Department of Medicine, University of Texas Health Science Center;
Nicholas Amos, MD: Department of Medicine, University of Wales;
Bryan D. Williams, MD: Department of Medicine, University of
Wales; Roy Jefferis, DSc: Professor of Immunology, Department of
Immunology, University of Birmingham.
Address reprint requests to Fazel Shokri, PhD, Department
of Immunology, School of Public Health, University of Medical
Sciences of Tehran, Tehran, P.O. Box 6446-14155, I.R. Iran.
Submitted for publication October 6, 1992; accepted in
revised form January 28, 1993.
Arthritis and Rheumatism, Vol. 36, No. 8 (August 1993)
in serum and culture supernatants by enzyme-linked
immunosorbent assay. Heavy and light chain isotypes
and V, subgroups of the paraproteins were determined
by immunoelectrophoresis, immunofixation, and Western blotting.
Results. Paraproteins from all patients expressed
an epitope associated with VJIIb sub-subgroup of light
chains. Three of the paraproteins were cryoglobulins
with RF activity, all of which expressed the VJIIbassociated CRI (detected by MAb 17-109) and the
V,I-associated CRI (detected by MAb G6 and GS).
None of the paraproteins expressed the V,III-associated
CRI (detected by MAb B6 and D12). The CRI were
consistently expressed over a period of 5-6 years. The
anti-CRI and anti-subgroup antibodies substantially inhibited spontaneous production of IgM-RF and IgM by
peripheral blood B lymphocytes from 3 of the SS
patients.
Conclusion. These results suggest that lymphoproliferation in primary SS is highly selective, and that
the anti-CRI antibodies can be used as an aid to early
diagnosis as well as for monitoring and modulating the
lymphoproliferative process in primary SS.
Primary Sjogren’s syndrome (SS), a chronic
autoimmune disease of unknown cause, is characterized clinically by dryness of the eyes and mouth. The
keratoconjuctivitis sicca and xerostomia are due to
lymphocytic infiltration and destruction of the salivary
and lacrimal glands, which occur in the absence of
coexistent chronic inflammatory connective tissue diseases, such as rheumatoid arthritis (RA) and systemic
lupus erythematosus (1). These patients have an increased incidence of monoclonal gammopathies (2,3)
and non-Hodgkin’s lymphoma (4). There have also
SELECTIVE EXPRESSION OF CRI IN PRIMARY SS
been reports of a high incidence of monoclonal free
light chains, immunoglobulins, and mixed monoclonal
cryoglobulins without evidence of lymphoid malignancy ( 3 3 , as well as clonal rearrangement of immunoglobulin genes in salivary gland lymphocytes derived from benign lymphoepithelial lesions (6).
Transition from autoimmune proliferation to
lymphoproliferation and malignant transformation
may be a multistage process in which a particular
subpopulation of B cell clones is selected (7). In
patients with primary SS, monoclonal B cell proliferation has been reported to occur predominantly among
rheumatoid factor (RF)-producing clones (8). Thus,
such selection may be operative on a subset of B cells
expressing a particular V, and/or V, gene(s) encoding
for a defined specificity.
We have recently demonstrated a substantial
increase in serum levels of V,I-associated crossreactive idiotypes (CRI) in primary SS patients compared with patients with RA or with normal individuals (9,lO). We have also reported a case of primary
SS with lymphoma, in which serum paraprotein with
RF activity expressing V,I-associated CRI w a s identified (1 1).
The present study was designed t o investigate
structural similarities of serum paraproteins from 5
patients with primary SS and monoclonal gammopathies, using a panel of anti-CRI antibodies defining
RF-associated CRI and anti-V, and anti-V, subgroup
antibodies. Anti-CRI antibodies were also used to
study longitudinal variations in t he level of serum
paraproteins, and in vitro regulation of IgM and
IgM-RF production b y peripheral blood lymphocytes
collected from some of the patients. Our results suggest that lymphoproliferation in primary SS is a highly
selective process, and that anti-CRI antibodies can b e
used to aid early diagnosis and modulation of lymphoproliferation in primary SS.
MATERIALS AND METHODS
Protein purification. IgM-RF paraproteins KO, He,
and Fr were isolated from the plasma of patients with
essential mixed cryoglobulinemia by affinity chromatography, using a heat-aggregated IgG-Sepharose 4B column.
Polyclonal IgG was purified from a normal human immunoglobulin preparation (Lister Institute, London, UK) as the
“breakthrough” fraction from a DE-52 column (Whatman,
Maidstone, Kent, UK) in 0.01M phosphate buffer, pH 7.0.
IgGl Fc protein (Per) from a patient with heavy chain
disease was purified by affinity chromatography as described
elsewhere (12).
1129
Anti-CRI and anti-subgroup antibodies. The production and characterization of monoclonal antibodies (MAb)
G6 and G8 (VJ-associated CRI), B6 and D12 (VJIIassociated CRI), and 17-109 (VJIIb-associated CRI) have
been described in detail elsewhere (13-16). MAb C6 has
specificity for an epitope expressed on all V,IIIb light chains
tested. It was produced to the IgM-RF KO, which is known
to express V,IIIb light chain (Mageed RA: unpublished
observations). Heavy chain variable region subgroups I, 11,
and I11 were determined by immunoblotting using rabbit
antisera directed against synthetic peptides corresponding to
consensus first-framework sequences (17).
MAb specific for human IgM (AF6 and BUl) and IgG
(8a4) were produced in the Department of Immunology,
University of Birmingham (commercially available from
Unipath-Oxoid, Bedford, UK). Antibodies were purified
from ascitic fluid by ion-exchange chromatography on
DEAE-cellulose or by affinity chromatography on protein
A-Sepharose 4B as previously described (18). All MAb are
of the IgGl isotype, except BUI and 17-109, which are
IgG2a and IgG2b, respectively. F(ab’), fragments of MAb
were prepared by pepsin digestion in 0.1M sodium acetate
buffer, pH 4.0, with an enzyme-to-antibody ratio of 1:40
(weight/weight) at 37°C for 8 hours. Undigested IgG was
removed on a protein A-Sepharose 4B column. The purity
of the F(ab’), preparations was confirmed by sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis on a
10% gel.
Mononuclear cell preparation and stimulation. Fresh
heparinized peripheral blood collected from the patients was
diluted 1:2 with phosphate buffered saline (PBS) and overlayed on an equal volume of Ficoll-Paque (Pharmacia,
Uppsala, Sweden). Following centrifugation at 350g for 20
minutes at room temperature, mononuclear cells were collected from the interface, washed twice with RPMI 1640
medium (Gibco, Paisley, Scotland), and resuspended in
culture medium, consisting of RPMI 1640 containing Lglutamine, 10% heat-inactivated fetal calf serum (Gibco), 200
IU/ml penicillin, 100 pg/ml streptomycin, and 2-mercaptoethanol (5 x lO-’M). The cells were incubated in the
presence or absence of either F(ab‘), or intact preparations
of anti-CRI antibody G6 or intact IgG of MAb C6, BU1, and
G4 (a mouse IgGl antibody of irrelevant specificity used as
a control). The cells were incubated at 37°C in 5% CO, in a
humidified chamber.
Mononuclear cells from patients DJ and ES were first
incubated with G6 or G4 for 8 hours, washed twice with
medium, and resuspended in fresh complete culture medium. Culture supernatants were collected for IgM and
IgM-RF quantitation after 7 days of incubation.
Quantitation of LgM and IgM-RF in sera and culture
supernatants. The quantitation method is described in detail
elsewhere (19). Briefly, microtiter enzyme-linked immunosorbent assay (ELISA) plates (Linbro; Flow, High Wycombe, Buckinghamshire, UK) presensitized with 10 pg/ml
of MAb AF6 or with 20 pg/ml Fc-Per were incubated with
serial dilutions of the sera or culture supernates for 2 hours
at 37°C. Titrations of IgM-RF KO and Fr were used to
construct standard curves for IgM and IgM-RF, respectively. Bound IgM was revealed using horseradish peroxidaseconjugated sheep anti-p chain-specific antisera (The Binding
SHOKRI ET AL
1130
Table 1. Clinical and serologic features of 5 patients with primary Sjogren’s syndrome
Patient/
ageisex
Disease
duration
(years)
Clinical
findings*
Serologic findings?
Therapy$
ESI76lF
DJ/46/F
SS41/53/F
EWl481F
SSKl7llM
23
10
13
20
?
Nephritis, vasculitis, RP
Vasculitis, hepatospl.
Lymphoma, vasculitis, RP
Lymphaden., hepatospl., WM
Lymphoma, lymphaden.
Cryos., anti-Ro, anti-La, RF, low C3 and C4
Cryos., anti-Ro, anti-La, RF, low C3
Cryos., anti-Ro, R F
Paraprotein, low IgG, low C3
Paraprotein
Plasma., chlor., pred.
Plasma., CYC
Not defined
Plasma., chlor.
Radia., chlor., pred.
* RP = Raynaud’s phenomenon; hepatospl. = hepatosplenomegaly; lymphaden. = lymphadenopathy; WM = Waldenstrom’s macroglobuhnemia.
t Cryos. = cryoglobulins; R F = rheumatoid factor.
$- Plasma. = plasmapheresis; chlor. = chlorambucil; pred. = prednisolone; CYC = cyclophosphamide; radia. = radiation.
Site, Birmingham, UK). The plates were developed with
o-phenylenediamine substrate, and optical densities were
measured at 492 nm using a multiscan ELISA reader (Flow).
The concentration of the IgM or IgM-RF was determined by
reading from the standard curves.
Quantitation of CRI+ IgM and VJIIb+ IgM. A
capture ELISA was employed using microtiter plates sensitized with 10 pg/ml F(ab’), fragments of the monoclonal
anti-CRI or anti-subgroup antibodies as previously described
(19). Due to an inability to prepare F(ab’), fragments from
the mouse IgG2b isotype (20), intact IgG of anti-CRI antibody 17-109 was used. F(ab’), fragments of G4 antibody and
an intact preparation of OKMl (IgGZb), which have irrelevant specificities, were used as negative controls. Quantitation was achieved after subtracting any nonspecific binding
and by extrapolation from standard curves constructed for
IgM-RF KO (G6, G8, C6, and 17-109) and He (B6 and D12).
Inhibition of RF binding to IgGl Fc. The ability of G6,
G8, and 17-109 antibodies to inhibit the binding of serum
IgM-RF from patients DJ, ES, and SS41 to IgGl Fc-(Per)
was tested in an ELISA as described previously (10,ll).
Electrophoresis, immunoelectrophoresis (IEP), and
immunofixation (IF). High-resolution gel electrophoresis was
performed using Universal agarose film (Corning, Corning,
NY). The samples were run in barbital buffer, pH 8.0
(Gelman, Ann Arbor, MI), for 40 minutes at constant current. For IEP, 20 pl of antisera specific for IgM, IgG, and
IgA, K or A heavy and light chains (The Binding Site) was
applied to appropriate troughs and incubated in a humidified
chamber overnight at room temperature. The agarose film
was then pressed and washed twice in saline, dried, and
stained for 10 minutes with 0.6% (weight/volume) naphthol
blue-black (Sigma, St. Louis, MO).
For IF, 80 pl of each antiserum was applied to the
surface of the gel, which had been covered by a template for
antiserum application. A strip of the gel was cut off as a
reference and fixed with 80 pl of the fixative containing 5%
(w/w) 5-sulfosalicylic acid in 5% (w/w) acetic acid. The gel
was then washed and stained as above.
Western blotting. V, subgroups I, 11, and I11 were
determined by immunoblotting using polyclonal anti-V,
reagents. Immunoblotting was performed essentially as described previously (21). Briefly, approximately 10 pg of the
proteins in disruption buffer containing 20% SDS, 1% bromphenol, and 3% sucrose in 0.2 mM Tris HCI, pH 7.0, was
loaded onto a 10% polyacrylamide slab gel containing 0.1%
SDS. Proteins were subjected to electrophoresis at 150V for
3.5 hours and then transferred onto nitrocellulose membrane
(Schleicher & Schuell, Hamburg, FRG) at 600 mA for 2
hours. The sheets were quenched by incubation with 2.5%
dried skim milk, and then incubated at 4°C with a 1500
dilution of the anti-V, antisera or a 1: 1,000 dilution of MAb
specific for p (AF6) or y (8a4) chains, in PBS containing
0.05% Tween 20 (v/v). Following extensive washes with
PBS, bound proteins were revealed with horseradish
peroxidase-conjugated sheep anti-rabbit or anti-mouse Ig
(The Binding Site) and developed in 0.05% diaminobenzidine
tetrahydrochloride substrate (Sigma).
Clinical samples. Serum and/or peripheral blood were
collected from 19 patients with primary SS without apparent
monoclonal gammopathies, 5 primary SS patients with detectable serum monoclonal paraprotein, and from l l normal
individuals. The diagnostic criteria for primary SS and the
major clinical and serologic characteristics of the primary SS
patients with monoclonal gammopathy are described in the
Results section.
RESULTS
Clinical manifestations in the primary SS patients studied. Serum samples from 5 patients with
primary SS and monoclonal gammopathy were studied. Patients ES, DJ, and EW were living in Wales,
patient SSK in the United States, and patient SS41 in
Greece. All were diagnosed as having primary SS
based on the presence of at least 2 of the following 3
criteria: dryness of the eyes (positive Schirmer’s and
rose-bengal staining results), dryness of the mouth,
with decreased parotid flow rate, and parotid gland
enlargement (1). Major clinical and serologic findings
are listed in Table 1.
Detection and characterization of the paraproteins. High-resolution gel electrophoresis, immunoelectrophoresis, and immunofixation were performed
on the patients’ sera and cryoprecipitates to demonstrate their monoclonality and isotypy. All the para-
SELECTIVE EXPRESSION OF CRI IN PRIMARY SS
Figure 1. Immunofixation of serum and cryoprecipitates (cryo.)
from 2 patients (ES and DJ) with primary Sjogren’s syndrome and
monoclonal gammopathy.
proteins were found to be IgMK. Results of IF of sera
and cryoprecipitates from patients DJ and ES are
illustrated in Figure 1. The cryoglobulins were char-
1131
acterized as type 11, composed of a monoclonal
IgM-RF and a polyclonal IgG.
Quantitation of IgM, IgM-RF, and CRI+ IgM in
serum. A capture ELISA was used to measure serum
levels of IgM, IgM-RF, and IgM-bearing CRI. The
paraprotein present in each sample was found to
express the V,IIIb-associated sub-subgroup epitope
recognized by C6 antibody (Table 2). Sera from patients ES, DJ, and SS41 contained 3 monoclonal
IgM-RF components with cryoglobulin activity, which
expressed the VHI-associated CRI, detected by MAb
G6 and G8, and the VJIIb-associated CRI, recognized
by MAb 17-109.
The paraproteins present in the other 2 patients’
samples were not cryoglobulins, did not display RF
activity, and were found to express only the V,IIIbassociated light chain epitope (recognized by C6 antibody). None of the CRI were expressed on these
paraproteins. The serum sample from patient EW
contained >lo0 mg/ml of IgM paraprotein which expressed the C6 epitope. The VHIII-associated CRI
detected by MAb B6 and D12 were not expressed in
any of the paraproteins.
Levels of CRI, IgM, and IgM-RF were also
quantitated in serum samples from 19 patients with
primary SS without apparent monoclonal gammopathies and in sera from 11 normal subjects. The
results of these assays are given for comparison in
Table 2.
Western blot analysis of the V, subgroups of
CRI+ and CRI- paraproteins. The V, heavy chain
subgroup of 2 V,I-associated CRI+ (ES and DJ) and
I CRI- paraprotein (EW) was analyzed by Western
blotting, using polyclonal anti-V, subgroup antisera.
The IgM from patients ES and DJ expressed only V,I
subgroup, whereas that from patient EW did not react
Table 2. Quantitation of IgM, IgM-RF, and CRI+ IgM in serum from patients with primary SS and monoclonal gammopathy*
CRI detected by MAb
IgM
SS patients
ES
DJ
SS41
EW
SSK
SS controls
(n = 19)
Normal controls
(n = 11)
2,160
1,890
7,100
>loo
1,390
1,300
(300)
1,270
(237)
IgM-RF
1,240
740
6,400
90
24
133
(48)
1.2
(0.4)
G6
930
960
3,070
110
1.2
16.5
(5.7)
0.83
(0.3)
G8
1,400
1,080
5,020
420
9.1
31.8
(9.8)
5.0
(1.1)
B6
160
200
59
125
17.5
44.6
(11.1)
88.3
(24.2)
* Values are in pdml, except for Sjogren’s syndrome (SS) patient E W s IgM and C6 values,
D12
I65
160
156
115
36
118
(19.4)
198
(46)
C6
17-109
2,400
1,460
5,400
> 100
1,380
NT
660
340
2,350
50
8.5
NT
NT
6.8
(2.1)
which are expressed in mgiml. Values for the 2
control groups are the mean (SEM). The SS patient control group did not have evidence of a monoclonal gammopathy. IgM-RF = IgM
rheumatoid factor; CRI = cross-reactive idiotype; MAb = monoclonal antibody; NT = not tested.
SHOKRI ET AL
1132
with any of the anti-VH subgroup reagents tested
(Figure 2). The y heavy chain present in the cryoprecipitates from patients ES and DJ reacted with all
anti-subgroup antisera, indicating polyclonality of the
IgG present.
Inhibition of binding of RF paraproteins to IgG
by anti-CRI antibodies. F(ab’), fragments of G6 and G8
antibodies almost completely inhibited the binding of
RF from patients ES and SS41, and to a lesser extent,
the binding of RF from patient DJ, to IgGl Fc (Figure
3). An intact preparation of antibody 17-109 also gave
substantial inhibition of the binding of RF from both
patients SS41 and ES to IgG (results not shown).
X Inhibition
100 I
0.6
0.2
- ES
-x--
1
ES
+
3.2
Inhibitor (uglrnl)
DJ
--+-DJ
--(i
SSII
.-e-.
SSJ
I
50
12.5
-
555
Figure 3. Inhibition of binding of rheumatoid factor (RF) cryoglobulins to IgG by anti-cross-reactive idiotype antibodies. Various
concentrations of monoclonal antibodies G6 (solid lines) and G8
(broken lines) were used to inhibit the binding of serum RF from 4
Sjogren’s syndrome patients to immobilized human IgC.
Figure 2. Western blot analysis of cryoprecipitates (patients ES
and DJ) and serum (patient EW) for V, subgroup expression
(analyzed as described in Materials and Methods).
No significant inhibition was observed with
serum from 2 patients with primary SS but no detectable monoclonal gammopathy. There was a large
amount of polyclonal IgM-RF (75 and 600 pg/ml) and
considerable levels of G6 (27 and 44 pg/ml) and G8 (17
and 69 pglml) CRI. The results obtained for I of these
patients (SS5) are illustrated in Figure 3.
Longitudinal measurement of IgM, IgM-RF, and
CRI+ IgM in serum. Total IgM, IgM-RF, and CRI+
IgM levels were measured in serum samples collected
from patients ES and DJ over a period of 5 years and
6 years, respectively (stored at -20°C). While IgM-RF
and the CRI constituted a large proportion of the total
IgM in patient ES, they constituted only a minor
proportion of the IgM in patient DJ (Figures 4A and
B). Longitudinal variations in the level of CRI reflected those of total IgM and IgM-RF in both patients.
A parallel and gradual increase in levels of IgM-RF
and CRI over a period of 5 years was observed in
patient DJ. These levels dropped sharply in December
1989. Patient ES had high levels of IgM-RF and CRI at
the time the first serum sample was collected. Apart
from a sharp increase in 1987, there was a gradual
decrease in the CRI and IgM-RF levels in this patient.
No significant variations were found in the levels of
V,III-associated CRI, as detected by the MAb B6, in
either patient.
In vitro down-regulation of IgM and IgM-RF
production by anti-CRI and anti-sub-subgroup antibodies. Peripheral blood mononuclear cells (PBMC) from
patients ES, DJ, and EW were cultured. High levels of
SELECTIVE EXPRESSION OF CRI IN PRIMARY SS
mglml
“1
Patient ES
1988
*
1987
1988
1989(MAY)
- IgM
+
IgMRF
*
G6
* 06
*
17-109
-G
BE
1989(DEC)
A
7
A
Patient D J
/ \
6t
I
1133
lesser extent in patient DJ. The intact preparation of a
mouse IgGl MAb (G4) with irrelevant specificity inhibited neither IgM nor IgM-RF production by either
patient’s PBMC. When PBMC from patient EW were
cultured with intact molecules of MAb BU1 or C6,
there was a substantial inhibition of IgM production
only in those treated with C6 antibody (Figure 6),
though a modest decrease in IgM was also observed
with BU1 antibody.
Essential controls were included to assess the
possibilities that the presence of anti-p, anti-CRI, or
anti-subgroup antibodies in the culture supernatants
interfere with IgM quantitation. Using an inhibition
ELISA, neither G6, C6, nor BU1 inhibited the binding
of the capture anti-IgM antibody AF6 to the standard
protein monoclonal IgM-RF Kok (data not shown).
Cultures treated with G6 antibody and G4 control
antibody were washed and incubated in fresh culture
medium as described in Materials and Methods.
DISCUSSION
584
1985
1988
1987
1988
1989(MAY) 1989(DEC)
Our previous studies have demonstrated a significant elevation in the level of V,I-associated CRI in
serum from patients with primary SS compared with
that from patients with other systemic autoimmune
complications and with that from normal individuals
(9,lO). The serum level of CRI in primary SS patients
B
Figure 4. Longitudinal quantitation of IgM, IgM rheumatoid factor
(IgM RF), and cross-reactive idiotype-positive IgM in serum from
patients ES (A) and DJ (B).
IgM were produced spontaneously by the PBMC of all
3 patients, and patient EW in particular. A spontaneous production of IgM-RF was also observed in PBMC
cultures from patients ES and DJ (Figure 5), but not
patient EW. In vitro stimulation of the PBMC with
Staphylococcus aureus Cowan I or phorbol myristate
acetate did not result in increased production of IgM
or IgM-RF (data not presented).
To study the effect of the anti-CRI antibodies
on spontaneous production of IgM and IgM-RF,
mononuclear cells from patients DJ and ES were
cultured with the intact IgG and F(ab’), fragments of
G6 antibody. Both preparations of the G6 antibody,
particularly the intact IgG molecules, gave almost
complete inhibition of IgM-RF production in both
patients (Figure 5). A substantial decrease in IgM
production was also observed in patient ES and to a
I
Cell
mp.ii.nt
DJ
m~.ii.nt
0 4 - l n l . 08-FI.b’12
08-lnl.
ES
Figure 5. In vitro down-regulation of spontaneous production of
IgM and IgM rheumatoid factor (IgMRF) by anti-cross-reactive
idiotype antibody G6. Peripheral blood mononuclear cells from
patients DJ and ES were cultured alone or in the presence of
monoclonal antibodies G6 or G4 (control), and the total levels of
IgM and IgMRF were measured in culture supernatants, as described in Materials and Methods. int. = intact.
SHOKRI ET AL
1134
was closely associated with the presence of autoantibodies to SS-A, SS-B, and type I1 cryoglobulins. The
cryoglobulins from 1 patient with lymphoma were
found to display equivalent quantities of IgM-RF and
the V,I-associated CRI, which suggests monoclonality (1 1).
In the present study, we have extended our
previous observations by analyzing the paraproteins
collected from 5 patients with primary SS and monoclonal gammopathies. The presence of serum monoclonal Ig was confirmed by electrophoresis, IEP, and
IF techniques. All 5 paraproteins expressed K light
chain and IgM heavy chain isotype. Quantitation of
the CRI and K light chain subgroups in the patients’
sera indicated that all may be of the V,IIIb subsubgroup, as identified by C6 antibody. Three of the
paraproteins expressed the V,IIIb-associated CRI (detected by MAb 17-109) and the VHI-associated CRI
(detected by MAb G6 and G8). These 3 paraproteins
were found to be cryoglobulins having RF activity.
None of the paraproteins tested expressed the VHIIIassociated CRI, which is normally expressed at severalfold higher frequency than the V,I-associated CRI
(10,22). Serum levels of the VHI CRI and IgM-RF were
also highly elevated in primary S S patients without
monoclonal gammopathy compared with levels in normal subjects (Table 2) (12). These results suggest a
progression from a polyclonal, benign, lymphocyte
infiltration to a monoclonal lymphoid neoplasm in
primary SS. This is supported by the demonstration of
a polyclonal distribution of K and A light chains in the
affected tissues prior to the onset of lymphoid malignancy (23).
The differences in serum levels of each CRI,
particularly the CRI detected by MAb 17-109 and
VJIIb light chains in one cryoglobulin and between
the 3 cryoglobulins tested, may reflect differences in
the binding affinity of each antibody for the corresponding idiotope or epitope (Table 2). In contrast to
the other CRI, the level of 17-109 was measured using
a paraprotein (Kok) other than the immunogen (Sie),
which may partly account for such differences. Two
strategies were employed to determine whether the
paraproteins in the serum were clonally related to the
CRI in the same serum: Western blot analysis using
different anti-V, subgroup antisera, and inhibition of
binding of the RF cryoglobulins to IgG by anti-CRI
antibodies. These assays were performed on the cryoglobulins and serum samples from 3 patients. The 2
cryoglobulins tested reacted only with anti-VHI antisera, while the paraprotein from patient EW, which
nglml
1000
Patient EW
7 50
500
250
0
cell
CB
EUl
Figure 6. In vitro down-regulation of the spontaneous production
of IgM by anti-sub-subgroup antibody C6 and anti-human IgM
antibody BUl . Peripheral blood mononuclear cells from patient EW
were cultured alone or in the presence of monoclonal antibodies C6
or BUI, and the total IgM was measured in culture supernatants, as
described in Materials and Methods.
did not express any of the CRI, was not typable with
the anti-V, reagents (Figure 2). Both anti-V,Jassociated CRI antibodies, G6 and G8, almost completely inhibited the binding of RF cryoglobulins to
IgG. Antibody 17-109 also inhibited the binding of 2 of
the RF cryoglobulins tested (data not presented).
Together, these results suggest that the cryoglobulins
from patients ES, DJ, and SS41 are encoded by closely
related germline genes or minimally mutated germline
genes from the VHI and VJIIb families. Considering
that these patients were from different countries, this
degree of similarity between the paraproteins suggests
the involvement of a highly selective mechanism in the
lymphoproliferation of primary SS.
The presence and variations in the levels of
cryoglobulins in 2 patients were studied longitudinally,
over a period of 5-6 years. Parallel variations in levels
of RF and CRI were observed in both patients (Figures
4A and B). These results suggest that quantitation of
CRI may have several important implications. First,
they may be used to aid early diagnosis of lymphoproliferation in primary SS. Low levels of CRI were seen
in the serum from patient DJ taken at early stages of
SELECTIVE EXPRESSION OF CRI IN PRIMARY SS
the disease, when a monoclonal band may not be
detectable by routine paraprotein assays. Second,
since longitudinal variations in the level of a paraprotein can be monitored, they may prove useful as an
index of clinical status. Although at this stage we have
no data establishing a correlation between longitudinal
variations in CRI levels and clinical status, monitoring
of CRI expression in such patients may prove to be
clinically valuable. Third, since all the CRI were
present for a long period of time, somatic mutation
may not have a major influence on this subpopulation
of B cells, though minor mutations not detectable by
the MAb we used may contribute to diversification of
the antibodies. Idiotypic stability and lack of somatic
mutation have also been reported in some other lymphoproliferative disorders (24-28), and treatment with
anti-CRI antibodies may be an effective and selective
therapeutic strategy for such neoplasms.
The G6 anti-CRI antibody completely inhibited
spontaneous production of IgM-RF by PBMC from 2
patients (Figure 5). These results are consistent with
our recent findings of idiotypic suppression in patients
with chronic lymphocytic leukemia (CLL) (29) and
with those by other investigators, showing idiotypic
suppression of RF production in other clinical subsets
(24,30,3 1). In vivo studies of experimental animals
have also shown the potential implication of antiidiotype antibodies in the prevention of cryoglobulinemia
and associated pathologic complications (32).
In summary, we have demonstrated that the
paraproteins produced by neoplastic B cells from
patients with primary SS may largely be products of
limited numbers of germline genes or minimally mutated germline genes, identified by the expression of
V,I-associated and VJIIb-associated CRI, detected
by MAb G6 and G8 and MAb 17-109, respectively
(25,26). The same families of V, and V, genes are
frequently used in a proportion of patients with essential mixed cryoglobulinemia, Waldenstrom’s macroglobulinemia, and CLL (26,33-37). The leukemic cells
in CLL express the CD5 molecule and produce autoantibodies; the same subpopulation of B cells is found
in increased numbers in peripheral blood and salivary
gland samples from patients with primary SS (38-40).
Interestingly, the percentage of these cells has been
shown to be higher in primary SS patients with detectable monoclonal paraproteins than in those without
(38), and remission of malignant lymphoma in some
primary SS patients has been shown to be accompanied by restoration of CD5+ B cell levels to normal
(39). Perhaps the lymphoproliferation in primary SS is
1135
highly selective and a distinct subpopulation of B cells,
possibly those expressing the CD5 molecule, is selected and may be the same subpopulation of B cells
involved in such other B cell neoplasms as Waldenstrom’s macroglobulinemia, essential mixed cryoglobulinemia, and chronic lymphocytic leukemia.
ACKNOWLEDGMENTS
We thank Dr. M. Goodall for supplying t h e monoclonal antibodies, Prof. H. M. Moutsopoulos for the generous supply of serum samples and clinical records of some of
the patients, and Dr. G. Silverman for the kind gift of
anti-V, antisera.
REFERENCES
1. Manthorpe R. Oxholm P, Prause JH, Schiodt M: The Copenhagen criteria for Sjogren’s syndrome. Scand J Rheumatol
Suppl61:19-21, 1986
2. Sugai S,Shimizu S, Kunda S: Lymphoproliferative disorders in
patients with Sjogren’s syndrome. Scand J Rheumatol Suppl
611118-122,1986
3. Tzioufas AG, Manousakis MN, Costello R, Silk M, Papadopoulos NM, Moutsopoulos HM: Cryoglobulinemia in autoimmune rheumatic diseases: evidence for circulating monoclonal
cryoglobulins in patients with primary Sjogren’s syndrome.
Arthritis Rheum 29:1098-1104, 1986
4. Kassan SS, Thomas TL, Moutsopoulos HM, Hoover R, Kimberly RP, Budman DR: Increased risk of lymphoma in sicca
syndrome. Ann Intern Med 89:888-892, 1978
5. Walters MT, Stevenson FK, Herbert A, Cawley MID, Smith
JL: Urinary monoclonal free light chains in primary Sjogren’s
syndrome: an aid to the diagnosis of malignant lymphoma. Ann
Rheum Dis 45:210-219,1986
6. Fishleder A, Tubbs R, Hesse B , Levine H: Uniform detection of
Ig gene rearrmgement in benign lymphoepithelial lesions. N
Engl J Med 3l6:1118-1121,1987
7. Tala1 N: Maintenance of autoimmunity. J Autoimmun 1:703709, 1988
8. Sugai S, Shimizu S, Tachibana J, Kunda S: The high incidence
of rheumatoid factor idiotypes in monoclonal Igs associated
with Sjogren’s syndrome, The Second International Conference
on Sjogren’s syndrome. Texas, 1988
9. Shokri F , Mageed RA, Kitas GD, Moutsopoulos HM, Jefferis
R: The level of rheumatoid factor associated cross-reactive
idiotypes in Sjogren’s syndrome, rheumatoid arthritis, SLE and
healthy individuals (abstract). Br J Rheumatol 28 (suppl 1):40,
1989
10. Shokri F, Mageed RA, Kitas GD, Katsikis P, Moutsopoulos
HM, Jefferis R: Quantitation of cross-reactive idiotype positive
rheumatoid factor produced in autoimmune rheumatic diseases:
an indicator of clonality and €3-cell proliferative mechanisms.
Clin Exp Immunol 85:20-27,1991
11. Shokri F, Mageed RA, Kitas GD, Katsikis P, Moutsopoulos
HM, Jefferis R: Monoclonal anti-cross reactive idiotype antibodies as possible probes for lymphoproliferation in primary
Sjogren’s syndrome. Br J Rheumatol 28:458459, 1989
12. Nik-Jaafar MI, Lowe JA, Ling NR, Jefferis R: Immunogenic
and antigenic epitopes of immunoglobulins. VII. The topographical distribution of Fc-gamma epitopes and the relationship of an
1136
13.
14.
15.
16.
17.
18.
19.
20
21.
22.
23.
24.
25.
26.
isoallotypic specificity to the presence of histidine 435. Mol
Immunol 21:137-143, 1984
Mageed RA, Dearlove M, Goodall DM, Jefferis R: Immunogenic and antigenic epitopes of immunoglobulins. XVII. Monoclonal anti-idiotypes reactive with common and restricted idiotopes to the heavy chain of human rheumatoid factors.
Rheumatol Int 6:179-183, 1986
Mageed RA, Goodall DM, Jefferis R: A highly conserved
conformational idiotope on human IgM rheumatoid factor paraproteins of the Wa cross-reactive idiotype family defined by a
monoclonal antibody. Rheumatol Int 1057-63, 1990
Crowley JJ, Mageed RA, Silverman GJ, Chen PP, Kozin F,
Jefferis R, Carson DA: The incidence of new human crossreactive idiotype linked to the subgroup V,III heavy chains.
Mol Immunol 27537-94, 1990
Carson DA, Fong S: A common idiotype on human RF identified by a hybridoma antibody. Mol Immunol20:1081-1087, 1983
Silverman GJ, Goldfein RD, Chen PP, Mageed RA, Jefferis R,
Goni F, Frangione B, Fong S, Carson DA: Idiotypic and
subgroup analysis of human rheumatoid factors: implications
for structural and genetic basis of autoantibodies in humans. J
Clin Invest 82:469-475, 1987
Ey PL, Prowse SJ, Jenkin CR: Isolation of pure IgGI, IgG2a
and IgG2b immunoglobulins from mouse serum using protein
A-Sepharose. Immunochemistry 15:429-436, 1978
Shokri F, Mageed RA, Tunn E, Bacon P, Jefferis R: Quantitative and qualitative expression of V,I-associated cross-reactive
idiotypes within IgMRF from patients with early synovitis. Ann
Rheum Dis 49:156154, 1990
Parham P: On the fragmentation of monoclonal IgGl, IgG2a and
IgG2b from Balbk mice. J Immunol 131:2895-2902. 1983
Towbin H, Stahelin T, Gordon J: Electrophoretic transfer of
proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A
76:4350-4354, 1979
Shokri F, Mageed RA, Maziak BR, Jefferis R: Expression of
V,III-associated cross-reactive idiotypes on human B-lymphocytes: association with staphylococcal protein A (SPA) binding
and Staphylococcus aureus Cowan I (SAC) stimulation. J Immunol 146:93&940, 1991
Zulman J, JaEe R, Talal N: Evidence that the malignant
lymphoma of Sjogren’s syndrome is a monoclonal 9-cell neoplasm. N Engl J Med 299:1215-1220, 1978
Pasquali JL, Martin T, Knapp AM, Levallois H, Farrdji A:
Monoclonal rheumatoid factor secreting cells in a patient with
mixed cryoglobulinemia: homogeneity and stability of the idiotypic production and in vitro idiotypic suppression. J Immunol
143:18261831, 1989
Kipps TJ, Tomhave E, Chen PP, Carson DA: Autoantibodyassociated kappa light chain variable region gene expressed in
chronic lymphocytic leukemia with little or no somatic mutation. J Exp Med 1672340452, 1988
Kipps TJ, Tomhave E, Pratt LF, Duffy S, Chen PP, Carson DA:
Developmentally restricted immunoglobulin heavy chain variable region gene expressed at high frequency in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 86:5913-5917,
1989
SHOKRI ET AL
27. Meeker TC, Grimaldi CJ, O’Rouke R, Loeb J, Juliusson G ,
Einhorn S: Lack of detectable somatic hypermutation in the
V-region of the IgH chain gene of a human chronic B-lymphocytic leukemia. J Immunol 141:399&3998, 1988
28. Levy R, Levy S, Cleary M, Carroll W, Kon S , Bird J, Sklar J:
Somatic mutations in human B-cell tumors. Immunol Rev
96~43-58, 1987
29. Shokri F , Mageed RA, Richardson P, Jefferis R: Modulation
and high frequency expression of autoantibody associated
cross-reactive idiotypes linked to the V,I subgroup in CD5
expressing B-lymphocytes from patients with chronic lymphocytic leukemia (B-CLL). Scand J Immunol (in press)
30. Koopman W, Schrohenloher RE, Barton J, Greenleaf E: Suppression of in vitro monoclonal human rheumatoid factor synthesis by anti-idiotype antibodies: target cells and molecular
requirements. J Clin Invest 72:1410-1419, 1983
31. Takeuchi T, Hosono 0, Koide J, Homma M, Abe T: Suppression of rheumatoid factor synthesis by antiidiotypic antibody in
rheumatoid arthritis patients with cross-reactive idiotypes. Arthritis Rheum 28:873-881, 1985
32. Spertin F, Donati Y , Welle I, Izui S, Lambert PH: Prevention of
murine cryoglobulinemia and associated pathology by monoclonal anti-idiotype antibodies. J Immunol 143:2508-2513, 1989
33. Kunkel HG, Winchester RJ, J o s h FJ, Capra JD: Similarities in
the light chains of antigammaglobulins showing cross-idiotypic
specificities. J Exp Med 139:128-136, 1974
34. Newkirk MM, Mageed RA, Jefferis R, Chen PP, Capra JD:
Complete amino acid sequences of the variable regions of two
human IgMRF, BOR and KAS of the Wa idiotypic family
revealed restricted use of heavy and light chain variable and
joining region gene segments. J Exp Med 166556564, 1987
35. Kipps TJ, Fong S, Tomhave E, Chen PP, Goldfein RD, Carson
DA: High frequency expression of a conserved kappa light
chain variable region gene in CLL. Proc Natl Acad Sci U S A
84:29 16-2920, 1987
36. Sthoeger ZM, Wakai M, Tse DB, Vinciguerra VP, Allen SL,
Budman DR, Litchman SM, Schulman P, Weiselberg LR,
Chiorazzi N: Production of autoantibodies by CD5-expressing
B-lymphocytes from patients with chronic lymphocytic leukemia. J Exp Med 169:255-268, 1989
37. Borche L, Annick L, Binet J-L, Dighiero G: Evidence that
chronic lymphocytic leukemia B-lymphocytes are frequently
committed to production of natural autoantibodies. Blood 76:
562-569, 1990
38. Youinou P, MacKenzie L, Masson GL, Papadopoulos NM,
Jouquan J, Pennec YL, Angelidis P, Katsikis P, Moutsopoulos
HM, Lydyard PM: CD5-expressing B-lymphocytes in the blood
and salivary glands of patients with primary Sjogren’s syndrome. J Autoimmun 1:185-194, 1988
39. DauphinCe M, Tovar Z, Talal N: B cells expressing CD5 are
increased in Sjogren’s syndrome. Arthritis Rheum 31:642-647,
1988
40. Deacon EM, Mathews JB, Potts JC, Hamburger J, Mageed RA,
Jefferis R: Expression of RF-associated cross-reactive idiotypes
by glandular 9-cells in Sjogren’s syndrome. Clin Exp Immunol
83:286285, 1991
Документ
Категория
Без категории
Просмотров
3
Размер файла
937 Кб
Теги
expressions, subsets, idiotype, syndrome, selective, sjgren, primary, cross, reactive, cells, expansion, lymphoproliferative, characterized, evidence
1/--страниц
Пожаловаться на содержимое документа