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Mixed lymphocyte reaction in healthy women with rheumatoid factor. lack of association with hla-dw4

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690
MIXED LYMPHOCYTE REACTION IN
HEALTHY WOMEN WITH
RHEUMATOID FACTOR
LACK OF ASSOCIATION WITH HLA-Dw4
EDGAR G . ENGLEMAN, ERNEST E. SPONZILLI, MARION E. BATEY, SAVITRI RAMCHARAN,
and H U G H 0. McDEVITT
In independent studies, 51% and 36% of patients
with rheumatoid arthritis have been shown to possess
HLA-Dw4 compared to 7%and 13% of controls. In one
study Dw4 positive patients more frequently had rheumatoid factor in their sera than did Dw4 negative patients. In
order to determine if the Dw4 specificity is associated with
the disease or with the presence of rheumatoid factor, the
frequency of this HLA antigen has been determined in 24
healthy women known to have rheumatoid factor (median
titer 1 :160). Only 3 were found to have the Dw4 specificity, suggesting that this specificity is not associated with
rheumatoid factor in the absence of rheumatoid arthritis.
From the Division of Immunology, Department of Medicine,
Stanford University School of Medicine, Stanford, California and the
Kaiser-Permanente Medical Center, Walnut Creek, Cahforhia.
Supported by a grant from the National Institutes of Health
(All 1313), a grant from the Kroc Foundation (Santa Ynez, California), and a contract with the Center for Population Research, National Institute of Child Health and Human Development.
Edgar G. Engleman, M.D.: Postdoctoral Fellow, Division of
Immunology, Stanford University School of Medicine, supported by a
senior fellowship of the American Cancer Society, California Division;
Ernest E. Sponzilli, M.D.: Attending Physician, Kaiser-Permanente
Medical Center: Marion E. Batey: Senior Technologist, MLC Typing
Laboratory, Divison of Immunology, Stanford University School of
Medicine; Savitri Ramcharan, M.D., Ph.D.: Research Director, Walnut Creek Contraceptive Drug Study; Hugh 0. McDevitt, Professor of
Medicine, Stanford University School of Medicine.
Address reprint requests to Edgar G. Engleman, M.D., Division of Immunology M-21 I , Stanford University School of Medicine,
Stanford, California 94305.
Submitted for publication December 27, 1977; accepted February 2, 1978.
Arthritis and Rheumatism, Vol. 21, No. 6 (July-August 1978)
The number of documented associations between
HLA and disease has risen steadily over the past several
years. One of the most intriguing of these associations
for rheumatologists is the association between rheumatoid arthritis and HLA-Dw4. As initially described by
Stastny (1) and confirmed by McMichael et al. (2),
either 5 1% or 36% of patients with rheumatoid arthritis
possessed the Dw4 specificity, compared to 7% or 13%
of controls, respectively. The explanation for this association is unknown, as is the explanation for every other
HLA and disease association. Nonetheless, because it is
anticipated that immune response genes will be found in
the HLA-D region (3) and because such genes have been
shown to quantitatively control antibody responses to
specific proteins in several species (4). the possibility
that such genes or their products play a pathogenetic
role in rheumatoid arthritis must be considered.
I n this regard, when rheumatoid arthritis patients
who were Dw4 positive were compared with those who
were Dw4 negative, a higher percentage of patients with
Dw4 had rheumatoid factor (2). This correlation between rheumatoid factor positivity and the presence of
HLA-Dw4, although tentative, suggests a possible explanation for the association between rheumatoid arthritis and HLA-Dw4: the presence of an immune response gene, in linkage disequilibrium with HLA-Dw4,
could code for an antibody response to immunoglobulin. By definition, such a gene would be found more
frequently in persons with HLA-Dw4 than in a control
population.
69 1
R F IN HEALTHY WOMEN
To test this hypothesis, the frequency of HLADw4 was determined in a group of women who had
significant titers of rheumatoid factor but who, according to a review of their medical records, lacked clinical
disease. Twenty-four such individuals with “false-positive” tests for rheumatoid factor were typed for Dw4
using the same homozygous typing cell that was used to
demonstrate an association between the Dw4 specificity
and rheumatoid arthritis (2). The results indicate that
the Dw4 specificity is found no more frequently in the
rheumatoid factor positive group than in controls.
Therefore, the hypothesis has not been supported.
MATERIALS AND METHODS
Patient Selection. The subjects were among 14,856
women who underwent testing in the Automated Multitest
Laboratory a t the Kaiser-Permanente Medical Center in Walnut Creek, California, from December 1968 through September 1972. The results of a survey for rheumatoid factor positivity and the effects of oral contraceptive use on its prevalence in
this population have been reported previously ( 5 ) . In this
survey a latex agglutination slide test (Hyland Laboratories,
Costa Mesa, California) was performed and, when reactive, a
tube dilution procedure followed. For the current study, letters
soliciting participation were sent to all women who had rheumatoid factor titers of 1 : 160 or greater, who lacked a diagnosis of rheumatoid arthritis, and who were still Kaiser Health
Plan participants. Of 21 persons contacted, 17 participated in
the study. Seven additional “false-positive’’ subjects with rheumatoid factor titers of 1 :80 were selected at random to bring
to 24 the total number tested. All 24 of these women were
Caucasian. Twelve had rheumatoid factor testing repeated a t
an average interval of 16 months, and of these, 10 remained
positive at a titer of I :80 or greater.
Joint Examination. According to their medical records,
none of the subjects had a history of rheumatic disease. Nonetheless, at the time of venipuncture for HLA-D typing, all
subjects were asked about possible symptoms and/or family
history of rheumatic disease, and their peripheral joints were
examined by one of the investigators. Individuals who complained of joint pains were examined by a second physician.
Mixed Lymphocyte Reactions. Mixed lymphocyte reactions were performed using fresh peripheral blood lymphocytes according t o the method of Sasazuki et al. (6). Donor
lymphocytes (5 X lo’), enriched over Ficoll-Hypaque gradients, were cocultured with an equal number of irradiated
(6,000 rads) stimulator lymphocytes obtained either from
other donors or from a donor homozygous for HLA-Dw4.
The cultures were incubated at 37OC for 6 days and aHthymidine was then added. After further overnight incubation,
the cells were harvested and the incorporation of thymidine
into D N A was measured by liquid scintillation counting. Failure t o stimulate D N A synthesis in a responder cell in this test
indicates that it shares D locus antigens with the stimulator
cell (7).
Women positive for rheumatoid factor were tested in
groups of six, and each individual was tested as a responder
against irradiated stimulators consisting of the other subjects
in the group, at least 4 normal volunteers, and one or more
homozygous typing cells. For each responder-stimulator combination the relative response ( R R ) was calculated according
to the formula:
test cpm - low control cpm
x 100
RR =
high control cpm - low control cpm
The low control was an autologous cell culture and the high
control was the median value of the 5 highest allogeneic responses.
HLA-Dw4 typing was carried out with a Dw4 homozygous stimulator cell, BM. This was the cell used in a recent
study (2) to demonstrate an association between rheumatoid
arthritis and HLA-Dw4. Evidence that this cell is homozygous
for the Dw4 specificity was provided in the report of that
study. A lack of response to BM in the mixed lymphocyte
reaction indicates that a subject possesses the Dw4 antigen.
RESULTS
Figure 1 shows the relative responses of 24 rheumatoid factor positive subjects and 30 normal donors to
the BM cell. The responses of 39 patients with classic
rheumatoid arthritis to BM, as obtained previously (2),
are included for comparison. With a cutoff point of less
than 35% to define nonresponsiveness, 3 of 24 (12.5%)
I
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4
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WOMEN
WITH
RHEUMATOID
FACTOR
CONTROLS
PATIENTS
WITH
RHEUMATOID
ARTHRITIS
Figure 1. Relative responses of 24 healthy subjects with
factor, 30 normal controls. and 39 patients with classic
arthritis to the Dw4 typing cell, BM. The responses of
arthritis patients to BM were obtained in a previous study
provided for comparison.
rheumatoid
rheumatoid
rheumatoid
( 2 ) and are
692
ENGLEMAN ET AL
women with rheumatoid factor were typed as Dw4 compared to 5 of 30 (16.7%) controls and 13 of 39 (36%)
patients with rheumatoid arthritis. In the Sixth International Histocompatibility Testing Workshop, an antigen frequency of 15.6% was found for HLA-Dw4 in
more than 3,000 Caucasians (8). The differences between the rheumatoid factor positive group, the control
group, and the established frequency of the Dw4 specificity in Caucasians are not significant ( P greater than
0.1 by two-tailed t tests).
Table 1 provides additional information about
the 24 rheumatoid factor positive women studied. Rheumatoid factor titers varied from 1 : 80 to 1 : 1,280 with a
median of 1 : 160 and a geometric mean of 1 :320. There
was no association in this group between rheumatoid
factor titer and patient age or between rheumatoid factor titer and oral contraceptive use. Subjects with titers
of 1:320 or greater had higher levels of serum gammaglobulin than subjects with titers of 1 : 160 or lower ( P
less than 0.01 by a two-tailed t test). Three subjects
complained of pain in metacarpophalangeal joints and/
or proximal phalangeal joints, but physical examination
confirmed joint swelling (metacarpophalangeal) in
only 1 subject. Three additional subjects claimed that a
first-degree relative had rheumatoid arthritis. The Dw4
specificity was found in none of the 3 persons with joint
pains and in only 1 of 3 persons with a positive family
history for rheumatoid arthritis.
DISCUSSION
The data do not support the hypothesis that a
gene linked to the HLA-Dw4 locus is responsible for
production of rheumatoid factor in the individuals studied. This does not completely rule out the possibility
that immune response genes linked to the Dw4 locus are
responsible for the synthesis of rheumatoid factor in
patients with rheumatoid arthritis. It is possible, for
example, that the rheumatoid factors in our “false-positive” subjects differ from the rheumatoid factors present
Table 1. Characteristics of 24 Women with Rheumatoid Factor Tested for HLA-Dw4
Patient
I
2
3
4
5
6
1
8
9
10
11
12
13
14
IS
16
17
18
19
20
21
22
23
24
* RF
t OC
$ RA
Ageat
Initial
RFTest*
Initial
RF
Titer
Repeat
RF
Testing
Serum
Globulin
(gm%)
45
41
44
34
24
51
38
21
42
33
39
48
27
44
54
53
49
33
49
44
21
22
41
41
80
80
160
80
80
160
1280
160
320
320
160
80
80
320
++
+++
+
+
+++
+++
++
+
++
++
+
+
++
+
+
+
+
+
++
++
++
++
I .28
I .47
1.23
1.21
0.11
0.91
2.00
I .61
2.18
2.30
1.02
I .03
I .69
2.51
0.96
0.98
I .27
0.76
1.29
I .30
I .80
2.01
1.17
I .52
160
640
160
80
1280
160
160
320
320
160
OC
Use?
Signs
ofRA$
P
-
P
N
N
P
N
N
C
N
P
N
C
C
P
N
P
P
C
N
P
C
C
N
C
=
current; P = past; N
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
-
-
-
-
-
+
-
-
-
+
-
-
=
HLA-Dw4
+
= Rheumatoid factor.
Use = Oral contraceptive use: C
= Rheumatoid arthritis.
Family
History
ofRA
never.
-
-
-
-
693
RF IN HEALTHY WOMEN
in patients with rheumatoid arthritis and that the genes
responsible for their synthesis would differ correspondingly. More likely, genes linked to HLA-Dw4 mediate
susceptibility to rheumatoid arthritis via a mechanism
other than the control of rheumatoid factor synthesis.
Such a contention is supported not only by the current
study, which demonstrates a low frequency of genes
common to the clinical disease, in women with rheumatoid factor, but also by the frequent occurrence of rheumatoid arthritis in the absence of rheumatoid factor (9).
Thus, patients with rheumatoid arthritis who are HLADw4 positive may have a higher frequency of rheumatoid factor simply because their disease is more severe.
A low rate of “conversion” from positivity for
rheumatoid factor to rheumatoid arthritis was suggested
in the present study. Only 1 of the 24 rheumatoid factor
positive subjects fulfilled the American Rheumatism Association criteria for probable rheumatoid arthritis, and
this individual’s symptoms antedated testing for rheumatoid factor. Thus, despite the fact that the individuals
included in our study had rheumatoid factor at a titer
equal to or greater than 1 :80, none developed clinically
significant rheumatoid arthritis during a mean 7 year
period. It must be noted that .only persons who remained participants in the Walnut Creek Kaiser Health
Plan in 1977 were available for inclusion in the present
study and that, therefore, the tested group does not
necessarily represent a random sampling of rheumatoid
factor positive individuals. With this precaution, however, the data would suggest that a positive test for
rheumatoid factor in the absence of clinical manifestations of rheumatoid disease has a benign prognosis.
REFERENCES
1. Stastny P: Mixed lymphocyte culture typing cells from patients with rheumatoid arthritis. Tissue Antigens 4 5 7 l579, 1974
2. McMichael AJ, Sasazuki T, McDevitt HO, Payne RO:
Increased frequency of HLA-Cw3 and HLA-Dw4 in rheumatoid arthritis. Arthritis Rheum 2 0 1037-1042, 1977
3. McDevitt HO, Engleman EG: Association between genes
in the major histocompatibility complex and disease susceptibility. Arthritis Rheum (Supplement) 20:S9-S20, 1977
4. Benacerraf B, McDevitt HO: The histocompatibility linked
immune response genes. Science 175:273-279, 1972
5. Sponzilli EE, Ramcharan S, Wingerd J: Rheumatoid factor
(anti-gammaglobulin) in women: effects of oral contraceptive use on its prevalence. Arthritis Rheum 19:602606, 1976
6. Sasazuki T, McMichael AJ, Payne R, et al: New and previously recognized MLC alleles. Histocompatibility Testing
1975. Edited by F Kissmeyer-Nielsen. Copenhagen, M unksgaard, 1976, pp 464-469
7
I.
Mempel W, Gross-Wilde H, Bauman P, et al: Population
genetics of MLC responses: typing for MLC determinants
using homozygous and heterozygous reference cells.
Transpl Proc 41529, 1973
8. Thorsby E, Piazza A: Joint report from the Sixth International Histocompatibility Workshop Conference 11. Typing for HLA-D (LD-I or MLC) determinants. Histocompatibility Testing 1975. Edited by F Kissmeyer-Nielsen.
Copenhagen, Munksgaard, 1975, p 451
9. Plotz CM. Singer JM: The latex fixation test. 11. Results in
rheumatoid arthritis. Am J Med 212393496, 1956
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