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Increased Frequency of HLA-Cw3 and HLA-Dw4 in Rheumatoid Arthritis.

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ARTHRITIS
1037
&
RHEUMATISM
OFFICIAL JOURNAL OF T H E AMERICAN RHEUMXTISM ASSOCIATION
SECTION OF T H E *ARTHRITIS FOUNDATION
INCREASED FREQUENCY OF HLA-Cw3
AND HLA-Dw4 IN RHEUMATOID
ARTHRITIS
ANDREW J. McMICHAEL, TAKEHIKO SASAZUKI, HUGH 0. McDEVITT, and ROSE 0. PAYNE
Thirty-nine patients with rheumatoid arthritis were
HLA typed for the full range of A and B locus specificities
and for Cw3 and Dw4. HLA-Dw4 was significantly more
frequent in patients (36%) than in controls (13%). The
frequency of Cw3 was also increased, pr.obably because
this allele is in linkage disequilibrium with Dw4.
HLA antigens are polymorphic cell surface glycoproteins whose function is now exciting considerable
interest (1). They are genetically controlled by at least
four loci on chromosome 6, HLA-A, B, C, and D. Until
recently, serologic methods have been available only to
identify A and B series antigens. At the VIth International Histocompatibility Testing Workshop, the C
series antigens were defined and can now be identified
serologically. Also D locus antigens, responsible for the
stimulation in mixed lymphocyte reaction (MLR) and
identified by this test, were characterized.
From the Divisions of Immunology and Hematology, Department of Medicine, Stanford University School of Medicine, Stanford. California.
Supported in part by NIH grants AI I1313 and H L D 3365,
and by a grant from the Kroc Foundation. A. J. McMichael was
supported by the Medical Research Council (U. K . ) .
Andrew J. McMichael, M.B., Ph.D., M.R.C.P.: Research
Fellow: Takehiko Sasazuki, M.D.: Research Fellow; Hugh 0.
McDevitt, M.D.:
Professor of Medicine and Head of the Immunology
Division: Rose 0. Payne. Ph.D.: Professor of Medicine.
Address reprint requests t o Andrew J. McMichael, M.B.,
Nuffield Department of Surgery, RadclifT Infirmary, Oxford, England.
Submitted for publication September 10, 1976; accepted December 3. 1976.
Arthritis and Rheumatism, Vol. 20, No. 5 (June 1977)
A possible function of these antigens is to regulate immune responses. In mice the equivalent of the
HLA-A and B antigens (H-2D and K ) are involved in Tlymphocyte response to viruses and other agents that
attach to or alter cell surfaces (2). The mouse equivalent
of the D locus, the I region, is closely associated with
specific immune response (Ir) genes which quantitatively
control antibody responses to specific protein or peptide
antigens (3). The association of HLA antigens with disease may therefore indicate that these kinds of immune
processes play pathogenetic roles in these diseases.
Rheumatoid arthritis is a disorder in which immunologic abnormalities occur; the most notable abnormality is the frequent presence of rheumatoid factor, an
antiimmunoglobulin (4). There has been some controversy about whether there are genetic etiologic factors in this disease. Some reports have suggested that
there is a slightly higher than expected frequency of
disease in monozygotic twins of patients as well as in
first-degree relatives of patients (5,6). Previous studies
have failed to demonstrate an association of an HLA-A
or B locus antigen with rheumatoid arthritis (7,8). However Stastny found a patient whose lymphocytes failed
to stimulate those of several other patients in the mixed
lymphocyte reaction (MLR) (9). I t was thought that this
individual was homozygous for a D locus specificity, R.
which was thus significantly increased in frequency in
this disease.
This report extends these findings. An HLA-D
homozygous typing cell from a healthy donor, shown
previously to be representative of the HLA-Dw4 speci-
1038
McMICHAEL ET AL
ficity, was tested in 39 patients and found t o occur more
frequently than normal. There was also a significantly
increased frequency of a C locus antigen, Cw3. Clinical
analysis showed that patients with Dw4 h a d a slightly
more severe form of the disease.
MATERIALS AND METHODS
Thirty-nine patients were arbitrarily selected from the
Immunology Clinics at Stanford University Medical Center
and the Veterans Administration Hospital, Palo Alto, California. All had either definite or classic rheumatoid arthritis by
the ARA criteria (10). Thirty-six were Caucasian, 2 were
black, and 1 was Mexican-American; 28 were female and 11
male. All were receiving aspirin, and 14 were on gold, 9 on
plaquenil, 6 on penicillarnine, 2 on cytotoxic agents, and 10 on
low-dose (< 10 mg/day) prednisone. All patients attending the
Stanford Medical Center were examined by one of the investigators, and the clinical data were recorded on an ARA
Uniform Database for Rheumatic Disease ( 1 I). The clinical
data were analyzed directly from this chart.
Fifteen milliliters of venous blood were taken from
each patient into heparinized tubes for the HLA study. HLAA, 0, and C antigen typing was carried out by the fluorochromasia method (12). Because of the scarcity of well-defined C
antigen antisera, we tested only for the C locus product, Cw3,
by using four antisera-5404A Zick, 3429A N844, 7162A
UPS, and 7163B D4603.
HLA-D antigen was typed by using fresh peripheral
blood lymphocytes in the mixed lymphocyte culture (MLC)
method described in detail by Sasazuki e? al (13). Patient
lymphocytes (5 X 10') were mixed with an equal number of
irradiated (6OOO rad) stimulator lymphocytes either from other
patients or from D locus homozygous cell donors. The latter
stimulator cells were stored frozen in liquid nitrogen and
thawed immediately before the culture was set up. The cultures
were incubated at 37°C for 6 days, then tritiated thymidine
was added. After further overnight incubation, the cells were
harvested and the incorporation of thymidine into DNA was
measured by liquid scintillation counting. Failure to stimulate
DNA synthesis in a responder cell in this test indicates that it
shares D locus antigens with the stimulator cell (14).
Patients were tested in groups of 5 , and each individual
was tested as a responder a'gainst irradiated stimulators comprising the other patients in the group and one or more homozygous typing cells. For each responder-stimulator combination, the relative response (RR) was calculated by the
formula: RR = (test cpm - low control cpm)/(high control
cpm - low control cpm) X 100. The low control was an
autologous cell mixture, and the high control was taken as the
median value of the five highest stimulations achieved.
Control data for HLA-A, B, C, and D typing were
derived from findings in 51 healthy volunteer blood donors
who had been typed with these sera and cells as part of the
continuing International Histocompatibility Testing Workshop studies.
RESULTS
Table 1 shows the results of HLA-A a n d B antigen typing. As in previous studies, neither A nor B
Table 1. H L A Phenotypes of 39 Rheumatoid Arthritis Patients
Patient
I
2
3
4
5
6
1
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
23
24
25
26
21
28
29
30
31
32
33
34
35
36
31
38
39
A
2, W28
1,2
2, W32
2.9
2,9
192
W28, W29
3, W28
1,9
2
ND
ND
ND
ND
1, I I
11, W31
1, W31
W30
9, I I
9, W29
W29
W30
2, W25
1 , W19
192
2, W29
1.3
1,9
2
2, W28
1,11
I
9, W30
9, I 1
ND
I , 28
ND
ND
ND
B
Cw3
Dw4
W15
W40. TY
w40,12
13, W22
w 3 5 , w 18
8,12
1,21
w35,12
W35,,1
w21, w 2 2
ND
ND
ND
ND
8, W15
W16,W22
W40,Wll
w35
W35, W40
12, W40
12
12, W42
8, W15
8, W 14
1, w 3 5
12, w 1 7
8, W18
8, w 3 5
1, 12
1. w 2 2
8, w 3 5
W l l , W40
w35
21, w 3 5
ND
8, W14
ND
ND
ND
antigen frequencies deviated significantly from normal.
HLA-C typing was carried out with antisera specific only for HLA-Cw3. This antigen was tested because
it is in linkage disequilibrium with the B locus specificity
HLA-BW40, which has been previously described to be
slightly more frequent in patients with rheumatoid arthritis (7,8). Cw3 is also in linkage disequilibrium with
HLA-Dw4, which we found to be significantly more
frequent in rheumatoid arthritis patients (see below).
T h e results in Table 1 show that Cw3 was found in 10 of
the 29 patients tested. This frequency is higher than the
antigen frequency in t h e normal population, 17% a n d is
significant (x* = 6.3, P < 0.01, relative risk 2.6).
H L A - D typing was carried out only with a ho-
1039
HLA-Cw3 A N D HLA-Dw4 IN RA
mozygous typing cell, BM, specific for HLA-Dw4. This
cell donor had been shown to be very similar to the cell
defining R in a colloborative experiment with Stastny
(13). In that study R did not stimulate BM, and both
typing cells belonged to the cluster HLA-Dw4. Although cells typing for the well-defined D locus specificities Dw2 and Dw3 were available for study, they were
not tested because Dw2 and 3 are in strong linkage
disequilibrium with HLA-B7 and B8, respectively, so
that an increase in the frequency of these specificities
would be accompanied by an increased frequency of B7
or B8. This increased frequency was not observed (Table
1).
The cell donor, BM, who is healthy and does not
have rheumatoid arthritis or a family history of the
disease, is clearly homozygous for the D locus antigen
Dw4. Figure 1 shows the MLR of the family. The results
are given as counts per minute (cpm), and a cpm value
greater than lo' is taken to indicate stimulation. BM
cells failed to stimulate either parent and all but one of
the siblings. This finding, plus the fact that siblings 112,
113, and I15 were mutually nonstimulatory with the
HLA identical father 11, indicates that BM is hornozygous for HLA-D. The only anomolous reaction,
I12/BM', can probably be attributed to back stimulation, in which blastogenic factors are released by the
irradiated cells recognizing the foreign determinants of
the responding cell (15).
The cell BM was used as a stimulator with all the
rheumatoid arthritis patients. A lack of response was
taken to indicate that the patient possessed this antigen.
Because relative responsiveness was used in the calculations (Figure 2), nonspecific low responsiveness of a
patient's lymphocytes to all stimulating cells was eliminated as a cause of false typing reactions. Figure 2 shows
the relative response of all 39 rheumatoid arthritis patients to this cell compared to 42 normal controls. The
response to cell BM is bimodal, and with a cutoff point
of less than 35% to define nonresponsiveness, 14 of 39
patients were typed by BM compared to 5 of 42 controls. I f the 3 non-Caucasian patients were excluded, 13
of 36 were typed by BM. Since BM has been shown to
type for the specificity HLA-Dw4 (13), these results
show that 36% of patients with rheumatoid arthritis
have Dw4, compared to 12% of controls. At the VIth
Histocompatibility Testing Workshop, an antigen frequency of 15.6%was found for Dw4 in over 3000 Caucasians. When this control value is used, the association of
Dw4 with rheumatoid arthritis is highly significant: xz =
12.2, P < 0.005, relative risk 3.0.
MLC reactions were also carried out between
patients. One cell was found (Patient 1, Table 1) which
I
1 [EM]
2,12
2.12
2
3
4
5
2.12
W19,W14
2.12
W19,W14
2.12
2,12
2,12
W19,W14
MLR*
14
2
2
6
(1)
2
70
56
51
63
24
(4)
63
92
48
I1 2
4
55
22
(2)
1
3
I1 3
1
37
4
1
(1)
3
11 5
3
32
4
4
5
(5)
I 1
(2)
13
I 2
41
I1 1
Fig 1. Family tree and MLC reactions in the family of the HLA-Dw4
typing cell B M . HLA-A and B types are also shown for each member.
*Results expressed as cpm X lo-'. tlrradiated (stimulating) cells.
was typed by BM and failed to stimulate other patients
typed as Dw4 positive. Because this patient's HLA-A, B
phenotype showed only one B antigen-Bw15-it
is
likely that it is homozygous for this HLA-B antigen
and also for Dw4. It was not possible to carry out a
family study with this patient.
A retrospective analysis of 31 patients attending
the Stanford Clinic was made to determine whether any
clinical differences could be distinguished in the subgroup of patients positive for Dw4 compared to those
who were negative. Subjects were compared for the severity of the arthritis, presence of nodules, and evidence
of extraarticular disease. The laboratory data compared
were the latex titer, sedimentation rate, PCV, and evidence of proteinuria. Also the treatments were compared, again to estimate disease severity. The data suggested that the Dw4 positive patients had slightly more
severe disease. Although the physical signs compared
showed no real difference, they required more aggressive
therapy to control their disease. Only 1 of 12 Dw4
positive patients was being managed on aspirin alone,
compared t o 6 of 19 Dw4 negative patients. In addition,
the Dw4 positive patients had a latex titer (geometric
that was slightly higher than the Dw4
mean 10 X 2T.0*1.3)
negative group (geometric mean 10 x 26.2*0.8).
McMICHAEL ET AL
m'm
m 8 m
mm
8
8
8
m
8
8
x
8
888
8
o
8
m
8
8
*.
:
8
**
8
mm
mm
:
8
8
*i*
8
8
01
PAT IENT S
C 0 N T R0 L S
Fig 2. Relative responses for 39 rheumatoid arthritis patients and 42
normal controls typed with the HLA-Dw4 typing cell Dw4.
DISCUSSION
These results show a significant association between HLA-Dw4 and rheumatoid arthritis (P < 0.005).
The relative risk of getting rheumatoid arthritis for Dw4
positive persons is 3.0. The frequency of Dw4 found in
this group of patients is comparable to that found by
Stastny for the HLA-D specificity R. In a collaborative
experiment with Stastny we found that R and Dw4 were
very similar in their reactions with a normal population
group (13). It should be noted that, because only one
Dw4 typing cell was used in these studies, the frequency
of an antigen of the Dw4 cluster (1) in this group of
patients might be a little higher. Individual typing cells
are rarely exactly alike in the reactions they give, and
there is now some evidence that Dw4 may include two
or more antigenic subgroups (16). It is also possible that
other D locus antigens may be more frequent in rheumatoid arthritis. However, because most of those identified
SO far are in strong linkage disequilibrium with B locus
antigens which are not themselves increased in frequency in the disease, these D locus antigens are probably a new type. With this possibility in mind, MLR
tests were carried out between patients because this
group might be expected to show an increased frequency
of homozygotes for any antigen that is increased in
frequency in the disease; one probable Dw4 homozygote
was the only cell found. Stastny (9) has similarly found a
Dw4 homozygous cell and also one that may identify a
second specificity. As more typing cells become available it will be valuable to perform complete D antigen
profiles of rheumatoid arthritis patients.
There was also an increased frequency of the C
locus antigen Cw3. Disease association with C locus
antigens are rare, but in this instance it is probably
secondary to the Dw4 association, because Dw4 and
Cw3 are in linkage disequilibrium with a delta value of
0.03 (13). Hence the Cw3-Dw4 haplotype actually occurs with a frequency of 0.039 compared to the expected
frequency of 0.009. The association of Cw3 with rheumatoid arthritis was less significant than the association
of Dw4, as judged by lower x z and relative risk values.
The association of HLA-Dw4 with rheumatoid arthritis
is therefore probably primary, with the putative disease
susceptibility gene more closely linked to the D locus
than the C locus. It is interesting that the C locus association was apparent when an association with the B locus
which maps between the C and the D locus on the sixth
chromosome was not found. This may be because the
polymorphism of the C locus ( 5 antigens defined) is less
than of that of the B locus (22 antigens defined).
These findings apply to a group of patients attending the Stanford Immunology Clinic. This group
may not be representative of the whole spectrum of
rheumatoid arthritis because they are more likely to
include severe cases. In support of this likelihood, in a
clinic where conservative approaches to therapy are favored, only 7 patients were receiving aspirin alone.
Therefore it is important to determine whether Dw4
positive patients have more severe disease. In a similar
study Jersild et a1 found that multiple sclerosis patients
with HLA-Dw2 (LD7a) had more active disease (17). A
retrospective analysis of the clinical data showed that
Dw4 positive patients appeared to require more aggressive drug therapy and had slightly more severe disease
on clinical assessment. These differences could have oc-
1041
HLA-Cw3 A N D HLA-Dw4 IN R A
curred by chance, however, and this point will not be
resolved until several other series of patients are reported. It is therefore not clear at this point whether
HLA-Dw4 defines a disease subgroup.
The finding that more of the Dw4 positive patients were receiving drugs such as gold, penicillamine,
and prednisone raises the possibility that these medications might lower responsiveness in the MLC. However,
because relative responses were used in the data analysis, the frequency of typing responses would not be
affected; the response relative to that given by a particular patient’s cells to several stimulating cells was calculated. Similarly, the possibility that rheumatoid arthritis
itself suppresses the MLR would not result in false
typing reactions.
The increased frequency of particular HLA antigens in certain diseases probably indicates a role for that
antigen or a closely linked gene product in the pathogenesis of the disease. The major histocompatibility complexes of the mouse and guinea pig have clearly been
shown to be intimately involved in regulating immune
responses (18). Products (Ir genes) of the mouse I region
probably analogous to the human D locus region, control antibody responses to protein antigens. Thus mice
with particular H-2 (equivalent to HLA) types are
higher responders or lower responders to selected antigens. Many human diseases are more strongly associated with D locus antigens than with A, B, or C
products (17-20). This fact is now strikingly demonstrated in rheumatoid arthritis where no A or B association could be clearly demonstrated.
The implication of this finding is that it is a gene
closely linked to the D locus that is directly involved in
the pathogenesis of this disease. The most likely candidate, according to the present understanding of the
HLA complex, would be an Ir gene. It is also possible,
however, that linked genes controlling complement or
cell surface interactions with pathogens could be involved. Because the mean titer of rheumatoid factor was
higher ih Dw4 positive patients, it is possible that the
disease susceptibility gene for rheumatoid arthritis
linked to the HLA system might be an I r gene involved
in the immune response to immunoglobulin. Further
studies on the presence of rheumatoid factor in patients’
families and in healthy HLA-Dw4 positive individuals
might clarify this point.
That Dw4 is present in 36% of rheumatoid arthritis patients may indicate that the HLA-linked disease
susceptibility gene is not essential. Or it may merely
reflect a relatively weak linkage disequilibrium between
an essential gene and Dw4. This gene may thus occur
most frequently in association with Dw4, but may also
occur with other haplotypes. The phenomenon of linkage disequilibrium, in which two linked alleles occur
together more often than expected, is often seen in the
HLA system. It may mean that certain HLA haplotypes
had selective advantage in recent evolutionary history,
e.g., by conferring immunologic resistance to particular
infectious diseases. Alternatively, it is possible that an
allele has arisen by mutation on a particular haplotype
and has not yet reached equilibrium in the population.
This possibility is attractive in this instance because
rheumatoid arthritis seems to have appeared only recently (21).
There is now no direct way to identify disease
susceptibility genes. However methods of distinguishing
human t r genes may become available (22), and if an Ir
gene is pathogenetically involved in rheumatoid arthritis, a higher degree of association may be found with an
t r gene marker. Serologic dissection of the human D
locus region is also becoming possible. Van Rood et a1
(23) and Mann et a1 (24) have found serologic specificities-present only on B lymphocytes and thus analogous to D region antigens-which are highly specific for
gluten-sensitive enteropathy patients. Winchester et a1
(25) have found a B lymphocyte antigen highly specific
for multiple sclerosis. Other groups have initiated
screening programs by using pregnancy antisera and
have found several B-cell specific alloantigens that
partly correlated with known D region specificities
(26,27). Such serologic methods, by analogy with the
mouse, will probably result in splitting of the D region
into two or more loci. Thus a part of Dw4, present also
in other Dw specificities, may be found with a very high
association with rheumatoid arthritis. Or a cell surface
marker may be found that is distinct from but in linkage
disequilibrium with Dw4, and which is closer t o the
disease susceptibility gene for rheumatoid arthritis.
Finally, only a small percentage of persons with
HLA-Dw4 develop rheumatoid arthritis, probably because the disease is multifactorial. It may be polygenic,
which would be consistent with the previously conflicting genetic data. There might also be interaction of gene
products with environmental agents. A simple Ir
gene-infectious agent hypothesis seems unlikely because
of the lack of familial clustering. However it would be
plausible if one or more other genes also played a part in
the disease pathogenesis.
ACKNOWLEDGMENTS
The authors thank Carolyn Hedrick and Wendy
Leong for expert technical assistance and Dennis McShane,
M.D., for preliminary computer analysis of the clinical data.
McMICHAEL ET AL
1042
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