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HLA В Э-DR4 is not a requisite for autoimmunity to collagen in rheumatoid arthritis.

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Immunogenetic studies in rheumatoid arthritis
have demonstrated an increased frequency of the HLADR4 alloantigen in individuals with both the sporadic
and familial forms of the disease. To investigate the
significance of this association, we ascertained whether
the possession of DR4 is necessary for the expression of
cellular and humoral immunity to native human type I1
collagen. Our results indicate that the presence of DR4
is not required for the development of autoimmunity to
collagen in rheumatoid arthritis.
lmmunogenetic studies in rheumatoid arthritis
(RA) have demonstrated that there is an increased
prevalence of the alloantigen HLA-DR4 in individuals
with the sporadic form of the disease (1,2). Preliminary
findings in studies of families having multiple probands
with RA indicate increased frequcncies of HLA-DR4
From the Division of Rheumatic Diseass, Department of
Medicine, Case Western Reserve Cniversity School of Medicine.
Cleveland. Ohio; the Department of Medicine, Harvard Medical
School; and the Department of Rheumatology and Immunology.
Hrigham and Women’, Hospital. Boston. ,Massxhusetts.
Supportcd in part by research grants RR-05410. AM-21490,
AM-20850. and RR-05669 from the L.S. Puhlic Health Service. and
by grants Irom the Northeast Ohio Arthritis Foundation. the Ohio
Department of Health (No. 525-01). the New England Peabody
Home Foundation. and the Massachusetts Chapter of the American
Red Cross.
Gary M. Kammer, MD: Assistant Professor of Medicine.
Case Western Reserve Cniversity School of Medicine and recipient
of the Daland Fellowship from the American Philosophical Association: David E. Trentham, MD: Asbociate Professor of Medicine,
Harvard Medical School and recipient of an Arthritis Senior Invcstigator Award from the Arthritis Foundation.
Address reprint requests toGary M. Kammer, M D , Department of Medicine, Case Western Reserve University School of
Medicine. 2074 Ahinyton Road. Cleveland. OH 44106.
Submitted for publication September I . 1983: accepted in
revised form November 28. 1983.
Arthritis and
Rheumatism, Vol.
No. 5 (May 1984)
in unaffected first-degree relatives and homozygosity
for DR4 in probands (3). Moreover. HLA-DR4 is
sometimes present on the extended haplotype HLAB15 (W62), DR4, BFS, C2C, C4A3. C4B3; the C4B3
allele is significantly increased in RA and is in linkage
disequilibrium with HLA-DR4 (4). These associations
suggest that susceptibility to RA may be determined in
part by the inheritance of genes in linkage disequilibrium with HLA-DR4 (5).
Since human immune response (Ir) genes appear to be localized near the D locus on chromosome 6
(6,7), it i s conceivable that the gene coding for HLADR4, or a linked gene. prcdisposes the proband to
defective immunoregulation of a specific immunogen(s), the eventual result being the clinical expression
of RA. The observation that sensitization to the cartilage and ocular type of collagen (native type 11) can
induce inflammatory arthritis in rodents (8,9), and the
presence of immunity to human collagen in RA (10-13)
and psoriatic arthritis (13). are consistent with collagen
functioning as a tissue-restricted autoantigen involved
in thc pathogenesis of chronic inflammatory arthritis.
Moreover, responsiveness to type I 1 collagen in rats
(14) and mice (15) is also under immunogenetic control. For these reasons, we investigated whether the
possession of the HLA-DR4 locus, which may regulate the expression of a cell surface la molecule
necessary for antigen presentation by accessory cells
(16), is required for the development of immunity to
native homologous type I1 collagen in humans.
Study subjects. Thirty-one consecutive cthnically
mixed white individuals (17 female, 14 male) with the
diagnosis of active. classic or definite RA (17) werc studied.
Table 1. Distribution of HLA-DR alloantigens in 31 rheumatoid
arthritis (RA) patients and 21 control subjects
% individuals bearing the alloantiaen
* The high proportion of HLA-DR5,6 in controls reflects the
selection of 4 asymptomatic first-degree relatives from a multiple
disease RA family.
The functional class was determined according to the criteria
of the American Rheumatism Association. The mean age
was 59 years (range 30-87). and the mean duration of disease
was 14 years (range 2-35). Of these patients, 74% were
judged to be in functional class I11 or IV. and 91% manifested radiographic cvidence of bone erosions. IgM rheumatoid
factor was present in 77% at the time of study (titer range
1 :%I:10,240). Twenty-two of the 3 I patients were receiving
nonsteroidal antiinflammatory drugs: 14 were receiving gold
salts; 8 methotrexate; 7 penicillamine; 7 salicylates; 4 hydroxychloroquine; and 2 prednisone (510 rndday).
Twenty-one sex- and race-matched healthy subjects
(hospital personnel, blood bank donors, or KA family members, mean age 49 years, range 29-85) were also selected for
study. Eleven were unaffected members of multiple disease
families (3), and 10 were unrelated individuals who possessed HLA-DR4. None of the latter 10 donors possessing
DR4 had a history of arthritis or polyarthralgia, had family
members with known RA, or were seropositive for IgM
rheumatoid factor.
Histocompatibility typing. HLA typing was performed for alleles of the A, B, C, and DR loci as previously
described (3,181; the antisera used for determination of the
DR alleles were those designated by the 8th Histocompatibility Workshop (19).
Collagen. Previously prepared (20) pepsin-solubilized
native human type I1 collagen was used.
Preparation of cells and serum. Approximately 100 ml
of blood was obtained by venipuncture from each subject,
and mononuclear cells were isolated from heparinized aliquots by FicolllMetrizoate (Accurate Chemical and Scientific Corp., Westbury, NY) density gradient centrifugation.
The cells were washed in Hanks' balanced salt solution
(HBSS; Gibco, Grand Island, NY), supplemented with 10
mM HEPES (Sigma Chemical Co., St. Louis, MO), and 2%
heat-inactivated fetal calf serum (FCS, Reheis Co., Phoenix,
AZ) was added. The cells were then divided into 2 fractions.
Cells from 1 fraction were resuspended in RPMI 1640
(Gibco), 10 rnM HEPES, 2 mM L-glutamine. 10 pg/ml
gentamicin, 10% heat-inactivated, pooled human AB serum
(courtesy of Dr. L. Keating, Red Cross, Cleveland, OH),
and 7.5 Fg/ml meclofenamate, and used without preservation in a thymidine incorporation assay. Cells in the other
fraction were cryopreserved for later use in a leukocyte
inhibitory factor (LIF) assay. This was accomplished by
resuspending the cells in KPMI 1640 containing HEPES, Lglutamine, gentamicin. and 5% FCS, diluted 1:l in media
containing 60% (volume/volume) HBSS,20% DMSO (Fisher
Scientific. Fair Lawn, NJ), and 20% FCS. Cryopreservation
was carried out iltilizing controlled rate freezing with a
Union Carbide Freezer, and the cells wcre stored in liquid
nitrogen .
Serum was collected at venipuncture and was stored
at -70°C. Aliquots of serum and cryopreserved cells were
shipped on dry ice by express mail for the antibody and LIF
assays, respectively. Assays for collagen immunity were
performed on coded specimens; identities were not revealed
until the results of all assays in the study had been obtained.
Cellular assays. Cells from all subjects were tested
for sensitivity to collagen by thymidine incorporation and
LIF assays. Fresh cells were used in a 6-day thymidine
incorporation assay using techniques and optimal concentrations of native particulate collagen as previously described
(1 I ) . In most experiments, immunocornpetence of the donor,
as well as culture conditions in the thymidine incorporation
assay, were assessed by alloantigenic stimulation in a concurrent mixed lymphocyte culture assay (IMLC). In the
MLC, cryopreserved allogeneic blood mononuclear cells
irradiated with 3,000 rad were co-cultured for 6 days at I x
lo5 cells/well in a ratio of I :1 with the responding cells from
the test subject. Results are expressed, in part, as mean
counts per minute from quadruplicate cultures performed in
the presence of a stimulus after subtraction of the cpm from
cultures conducted in the absence of stimulation (Acpm). To
determine the frequency of responses in this assay (111, a
stimulation index (SI) was calculated as follows:
s1 =
cpm with collagen or alloantigen
cpm without antiuen
An SI 2 2 represents a significant response to human type I1
collagen (1 1 ) or in the MLC assay.
For the LIF assay, cryopreserved cells were thawed
and washed with HBSS; viability at this stage consistently
exceeded %% as determined by ethidium bromide-acridine
staining. The design of the code enabled virtually all LIF
assays to test cells from both RA patients and healthy
subjects. The LIF assay was performed using native particulate collagen and streptokinase-streptodornase (SK-SD) at
optimal concentrations of 50 pg/ml and 50 units/ml, respectively, as test antigens (1 1.13,20,21). Planimetry was performed by the same investigator without knowledge of the
identity of the supernatant medium being tested
(lI,l3,20,21). LIF activity was expressed as 5% migration
inhibition, where:
Area of migration in the supernatant
medium from cultures with antigen
96 inhibition = 1.0 x 100
Area of rnigralion in the supernatant
medium from cultures without antigen
Migration inhibition exceeding approximately 25% has been
previously calculated to indicate significant activity induced
by human type I1 collagen, whereas 220% represents a
positive response to SK-SD ( I I).
49 1
I - ,
0 Negative
- a\o
Figure 1. Pattern of the cellular immune response to native human type 11 collagen in rheumatoid arthritis. T w o assays were performed to
identify cellular sensitivity to collagen; the results depicted indicate whether the patient was a responder o r a nonresponder to collagen in each
assay. Of 31 patients, 22 (71%) responded significantly by collagen-induced thymidine incorporation (A) and 13 (42%) by the production of
leukocyte inhibitory factor (LIF) (B).The presence of HLA-DR4 did not correlate with collagen responses in either assay. When sufficient cells
were available. patients were also tested with alloantigenic (mixed lymphocyte culture, M I X ) and streptokinase-streptodornase (SK-SD)
stimulation in the thymidine incorporation and LIF assays. respectively. ['HI-IdR = 'H thymidine: NS = not significant.
Antibody assay. Serum samples were examined for
antibodies to collagen by passive microhemagglutination
(22); titers of 2 2 - logz dilution are considered significant
(1 l,l3,21).
Statistical analysis. Statistical significance was assessed by the chi-square test with Yates' correction, by
least-squares linear regression, or by the Student's t-test. A
P value of 50.05 was considered statistically significant.
Distribution of HLA-DR alloantigens. The distribution of HLA-DR alloantigens observed in this
study is given in Table 1 . The proportion of patients
with RA bearing HLA-DR4 was 71%. Because we
purposely selected as healthy study subjects either
first-degree family members from cohorts having multiple individuals with RA (3,181 or normal blood donors possessing HLA-DR4, the prevalence of the DR4
alloantigen in this population was 69%. For this reason, the distribution of other HLA-DK alloantigens
differed from that observed in other healthy control
populations (23).
Cellular sensitivity to collagen. When cellular
sensitivity to type 11 collagen was measured by the
thymidine incorporation assay, '22 (71%) of the 31
patients with RA exhibited a significant response
(Figure I). The background cpm, used to derive the
stimulation indices, were similar in the collagen responders and nonresponders (mean cpm ? SEM of
874 2 112 cpm for the responders versus 902 2 76 cpm
for the nonresponders, P not significant). Of the
responders, 17 (77%) possessed HLA-DR4. There
was also no significant difference in the magnitude of
thymidine incorporation to collagen when the DR4positive responders were compared with the DR4negative responders (mean SI of 5.3 2 1.1 for DR4positive responders versus 4.8 _t 0.6 for DR4-negative
responders, P not significant).
Using the LIF assay, 13 (42%) of the 31 RA
Table 2. Distribution of specific HLA-DR alloantigens in
rheumatoid arthritis patients possessing autoantibodies to type II
Patient no.
Antibody titer
( - log,)
HI,A-DK genotype
I I6
I 1-
16 healthy subjects who were tested possessed such
antibodies (- logz titers of 2 , 2, and 4, respectively).
These 3 subjects were each HLA-DR5,6 asymptomatic siblings from a family with 2 probands having the
patients responded significantly to collagen. Five
(38%) of the responders in this assay were positive for
HLA-DR4. Similar quantities of LIF activity induced
by collagen were found when the DK4-positive responders were compared with the DR4-negative responders (mean % LIF of 37 ? 2 in the DR4-positive
responders versus 35 2 5 in the DR4-negative responders, P not significant). In contrast, of the 21
healthy subjects, only 1 responded to collagen by
thymidine incorporation, and a different subject reacted in the L,IF assay. All of the study subjects tested
responded significantly in the MLC assay, and cells
from 77% produced LIF in response to SK-SD.
Thus, there was no evidence of impaired cellular responsiveness in the RA patients and control
subjects who did not react to collagen. Among the
individual RA patients. there was no correlation between positive responses to collagen in the 2 assays,
and responses to collagen and SK-SD were unrelated.
Likewise, the presence or absence of collagen sensitivity did not correlate with any other HLA-DK alloantigen, RA disease parameter, or type of drug
Antibodies to collagen. Hemagglutinating antibodies to type I1 collagen were found in the serum
specimens of 13 (42%) of the 31 patients with RA
(mean - log2 titer of 3.1 ? 0.4, range 2-6) (Table 2).
There was no association between the presence of
collagen antibodies and clinical aspects. Although 6
(46%)of the 13 RA patients with antibodies were DRIpositive, this finding did not attain statistical significance (x’ = 1.9, P not significant). Three (19%) of the
The present study was undertaken to determine
whether there is any association in RA between the
expression of the DR4 alloantigen and autoimmunity
to native type I1 collagen. It is pertinent to examine
this relationship, since the DK4 locus is situated in the
region of the putative Ir genes and because patients
with R A frequently exhibit immunity to this host
protein (10-13,20,24,25). Our results indicate that
there is no correlation between DK4 and either cellular
or humoral immunity to human type I1 collagen. Thus,
the significance of the relationship between DR4 and
RA remains obscure.
The absence of an association between DR4
and autoimmunity to collagen, as reported herein,
supports the observations of some researchers, but is
in opposition to those of others. Utilizing denatured
human type I collagen as a test antigen in a thymidine
incorporation assay, one study identified no relationship in RA between DR4 and cellular autoimmunity to
collagen (26). In contrast, another report concluded
that the immune response to collagen is regulated by Ir
genes encoding the DR4 locus. With the use of an LIF
assay, all RA patients possessing DR4 exhibited a
positive response to denatured bovine collagen, and
the responders who were tested also reacted to chick
types 1 and I1 collagen (25,27). Moreover, DR4-positive normal subjects also produced LIF activity to
bovine collagen, further suggesting the existence of
specific Ir genes for collagen linked to genes encoding
DR4, which permit the development of immunity to
collagen (25).
In the present study, however, cells from only I
of 10 healthy subjects possessing DR4 exhibited cellular reactivity to collagen by LIF production, and there
were no positive responses to collagen by the thymidine incorporation or passive hemagglutination assays
in the DR4-positive healthy group. Although the precise factors leading to these disparate results remain to
be established, potential variables may be epitope or
solubility differences of the collagens utilized as test
antigens, or the methodology and interpretation of the
assays used. I n this study, positive responses to
collagen occurred more frequently using the thymidine
incorporation, as opposed t o the LIF. assay. This
result is opposite to the frequency observed with the 2
assays in a previous study, where the LIF assay
proved to b e more sensitive (11). This difference
illustrates the inherent variability of currently used in
vitro assays of cellular sensitivity.
There was no significant relationship between a
DR alloantigen and humoral immunity to collagen in
this study. This finding is in contrast to a recent report
demonstrating a significant association in RA between
antibodies t o native type I1 collagen, purified from an
unidentified species, and the DR3 and DR7 alloantigens (28). Nonetheless, the observation in that report
(28) of an increased frequency of DRI in t h e patients
with antibodies to collagen (39%) underscores the
potential significance of t h e antibody distribution in
the present study.
In addition, the identification of autoantibodies
to collagen in 3 siblings within a family with 2 probands having such antibodies is consistent with the
possibility that immunity to collagen in humans is
influenced by an as yet unidentified immunogenetic
mechanism. Further studies to attempt to clarify these
issues are warranted.
The authors wish to thank Ms Judith Smith, Roselynn Dynesius-Trentham, and Donna Rowland for technical
assistance and Ms Dee Condon for manuscript preparation.
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dr4, hla, requisite, arthritis, autoimmunity, collagen, rheumatoid
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