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Hla-d locus typing in ankylosing spondylitis and reiter's syndrome.

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37 I
HLA-D typing of 44 patients with ankylosing
spondylitis (AS) and 31 patients with Reiter’s syndrome
(RS) did not show increased frequency of any particular
Dw allele in either population of patients as compared
to controls. Such studies also allowed each patient’s
general response to be compared with other general responses within each experiment. Contrary to reports of
diminished lymphocyte responses in AS patients, hyperresponsiveness in both A S and R S patients was found.
From the Division of Hematology, Oncology, and Irnmunology, Department of Pediatrics, and the Department of Medicine, Cedars-Sinai Medical Center and University of California at Los Angeles School of Medicine, Los Angeles, California.
Supported in part by NCI Grant CA-18892-01, The Amie
Karen Cancer Fund, and The Deutsche Forschungsgemeinschaft
(SFB 54).
Kip Kemple, MD: UCLA School of Medicine; Richard A.
Gatti, MD: Cedars-Sinai Medical Center; Wolfgang Leibold, DVM:
lnstitut fur Pathologie der Tierarztlichen Hochschule, Hannover,
West Germany; James Klinenberg, MD: Cedars-Sinai Medical Center; Rodney Bluestone, MB, MRCP: Chief, Rheumatology Section,
VA Wadsworth Hospital.
Address reprint requests to Dr. Rodney Bluestone, Chief,
Rheumatology Section. VA Wadsworth Hospital, Wilshire & Sawtelle
Boulevards, Los Angeles, California 90073.
Submitted for publication November 13, 1978; accepted in
revised form December 26, 1978.
Arthritis and Rheumatism. Vol. 22, No. 4 (April 1979)
A number of studies in the past several years
have attempted to confirm and extend the initial observations that ankylosing spondylitis and Reiter’s syndrome are highly associated with the HLA antigen B27
(1,2). While family and population studies in this area
have improved understanding of the genetic basis of
disease susceptibility, they have not substantially clarified the actual mechanisms through which the histocompatibility system is influencing disease pathogenesis. One of the favored hypotheses to explain the
association of diseases with histocompatibility antigens
involves the existence of disease susceptibility genes.
These genes are presumed to be analogous to the immune response (Ir) genes which have been relatively
well characterized in several different animal models.
Immune response genes in the mouse have been
mapped and are located within the major histocompatibility complex H2 (3). The actual Ir loci are intimately
related to the major loci coding for mixed lymphocyte
culture (MLC) determinants in the mouse (4). Because
of this chromosomal association of Ir and MLC genes
there has been anticipation that certain human disease
susceptibility genes might be associated with MLC
(HLA-D) determinants. The authors performed a series
of HLA-D typing experiments in patients with ankylosing spondylitis and Reiter’s syndrome in order to deter-
Table 1. HLA-D typing responses
Normal controls (45)t
Ankylosing spondylitis (44)
P value
Reiter’s syndrome ( 3 I )
P value
* Numbers indicate percent o f positive reaponses
t Number in each group
f NS = not significant
mine whether any abnormality exists in the distribution
of HLA-D alleles in these patient groups.
Typing was performed on 44 patients with ankylosing
spondylitis and 3 1 patients with Reiter’s syndrome. The modified Rome (New York) criteria were used to define patients as
having either definite (36 patients) or probable (8 patients) ankylosing spondylitis (5). Patients were considered to have definite Reiter’s syndrome if they had all of the major triad, i.e.
arthritis, conjunctivitis, and nonbacterial urethritis (24 patients). They were considered to have probable Reiter’s syndrome if they manifested arthritis plus one of the major features (7 patients). Most of the patients with spondylitis were
typed for the B27 antigen with 30 positive and 7 negative.
Twenty-three patients with Reiter’s syndrome were positive
and 4 were negative for the B27 antigen. In the same experiments, a group of 45 normal donors was also HLA-D typed.
HLA-D typing was performed by a modified one-way
mixed leukocyte culture technique which substitutes cultured
lymphoblastoid cell lines (LCL) for peripheral blood lymphocytes as stimulator cells (6,7). This method has been used before by the authors (6) and by Netzel et a1 (7.8) and is described in detail elsewhere (6.9.10). Briefly, the stimulator
typing cells were obtained initially as peripheral blood lymphocytes from individuals known to be homozygous at the
HLA-D locus. The cells were infected with Epstein-Barr virus
(EBV) resulting in their transformation into lymphoblastoid
cell lines which could then be maintained indefinitely in culture. In these experiments, typing cell lines (LCL-HTCs) representing HLA-D alleles w I-w8 were included. Alleles Dwl,
2, and 3 were represented by as many as six. four, and six
LCL-HTCs, respectively, in some experiments. Typing for
other alleles depended upon responses to one or two LCLHTCs for each.
Typing cells 5 X lo4 were frozen in 5% dimethyl sulfoxide (DMSO) in round bottom typing plates following treatment with mitomycin C (80 pg/ml). Responder blood was
collected in sterile heparinized tubes and lymphocytes were
separated on a Ficoll-Hypaque gradient. Responder lymphocytes ( 1 x 105/well) were added and the plates were incubated
for 5 days at 37°C with 5% CO, and 70-90% humidity, each
stimulator-responder pair being cultured in triplicate. The
cultures were exposed to I pCi/well of ’H-thymidine for 16
hours, then harvested and counted in a liquid scintillation
counter. Results were expressed initially in counts per minute.
Because there was substantial variation in MLC responsiveness between different individuals, a computer program was developed that adjusted for this variation and assigned an lnteraction Index to each responder-stimulator
combination (10). The program also corrected for differences
in stimulating potential between different LCL-HTCs and for
the effects of autologous-type stimulation (9). Interaction Indices (11) were used to assign Dw specificities by arbitrarily selecting an I1 of 50 as the cutoff value: I1 below 50 were considered typing responses. This computer program identified
HLA-D alleles in a manner which appeared very comparable
to other methods (9-12). Further statistical analysis of the
computed I1 was carried out using a f test to compare the frequencies of typing responses to various HLA-D alleles between patient and control groups.
Table I lists the percentage of typing responses
for each Dw allele in the three groups tested. It can be
seen that each of the Dw specificities is well represented
in each patient group and that there are no statistically
significant differences in typing pattern distributions between either patient group and the normal control population. The last column on the right includes the percentages of responders who could not be typed for one
or both Dw alleles assuming two alleles per donor. The
apparent increase in non-typing responses among patients is due in part to the fact that patients were generally typed in single experiments only, while most controls were studied several times. Homozygosity for a
particular allele would also increase this figure since our
methods d o not distinguish “blanks” from homozygotes. In addition, the patient typing experiments
were less comprehensive with regard to the number of
stimulator LCL-HTCs used: for technical reasons, some
patients were not tested against all eight HLA-D specificities. While this would not influence percent-positive
for a given allele, it would result in a greater number of
incompletely characterized phenotypes.
The overall percentage of non-typing responses
by this method was comparable to that seen by other
groups performing HLA-D typing by conventional
methods (13). It appears that there was no substantial
excess of non-typing responses among the patients, suggesting that it is unlikely that there are any unique Dw
determinants among patients with AS or RS. When the
small number of B27 negative individuals among the
AS and RS patients was analyzed separately, there was
again no discernible difference from controls in their
typing patterns.
While there was no difference between patients
and controls in typing patterns, there was, on the other
hand, a difference in the overall level of mixed leukocyte culture responsiveness. Table 2 shows that the
mean stimulation score for patients with AS was significantly greater than the mean score for the normal controls. A similar but less impressive increase was documented for Reiter’s syndrome patients.
Ankylosing spondylitis and Reiter’s syndrome
have been related to each other on the basis of common
clinical features including sacroiliitis or spondylitis, a
similar peripheral arthritis in association with anterior
uveitis and, finally, a noteworthy coexistence of both
diseases in some families ( 14,15). The immunogenetic
basis of this relationship has been strengthened and to
some extent clarified by the finding of a dramatically
high frequency of the HLA antigen B27 in both of these
diseases, i.e. greater than 90% in AS and 80-90% in RS
(1,2). A number of possible mechanisms have been considered to explain these associations. The hypothesis
mentioned in the introduction involves abnormal im-
Table 2. MLC responsiveness*
Mean raw
score (cpm)
Normal controls (4S)t
Ankylosing spondylitis (44)
Reiter’s syndrome (31)
P value
< 0.00 I
< u.0os
* MLC responsiveness scores represent the mean of responses for
each donor tested to the entire panel o f LCL-l1TCs. This score includes responses 10 approximately 7-40 stimulator cells.
t Number in each group.
mune responses (disease susceptibility) genes. These
genes are presumed to occur in marked linkage disequilibrium with the B27 antigen. Alternative mechanisms suggest that the B27 antigen itself is involved in
disease pathogenesis, either acting as a surface receptor
for infectious agents or possibly cross-reacting immunologically with other foreign antigens.
While there is no conclusive evidence allowing
one to select among these mechanisms at present, there
is an increasing amount of data relevant to the question.
A number of other diseases have now been associated
with various histocompatibility antigens. While the initial associations were noted to involve serologically defined antigens such as HLA-B7 in multiple sclerosis (16)
and HLA-B8 in celiac disease and Sjogren’s syndrome
( 17, 18), more recent studies have implicated a stronger
association of these diseases with HLA-D antigens or B
cell alloantigens ( 19-22). Thus, the weaker associations
with B7 and B8 appear to reflect a linkage disequilibrium between these antigens and the HLA-D antigens Dw2 and Dw3, respectively. Also, rheumatoid arthritis now appears to be associated with the HLA-D
antigen Dw4, without any association with the serologically defined HLA-A, -B or -C antigens (23). With
some reservations, this kind of data linking specific diseases with HLA-D locus alleles can be taken as evidence in favor of disease susceptibility genes which are
themselves located close to the D locus.
Evidence is presented here that there is no association between ankylosing spondylitis or Reiter’s syndrome and any individual HLA-D allele. These results
are in agreement with those of other groups who have
evaluated ankylosing spondylitis patients by conventional HLA-D typing with limited typing cell panels,
patient-versus-patient MLC testing (24-26), or serologic
typing of the closely related HLA-DR alleles (27). Although the latter study included only 27 patients, these
workers noted a complete absence of DRw7 which the
present data fail to support if one assumes a strong linkage between Dw7 and DRw7.
There are important negative results that set ankylosing spondylitis and Reiter’s syndrome apart from
the other diseases mentioned above. They are the only
adequately studied diseases to date which have histocompatibility associations restricted to the B locus. On
the other hand, Duquesnoy et a1 have recently noted a
strong association of ankylosing spondylitis with HLA
Cwl and Cw2 in both B27-positive and B27-negative
patients (28). If this observation is borne out, it will sup-
3 74
port the hypothesis that a disease susceptibility gene for
ankylosing spondylitis is located near the B and C loci
since these two loci themselves lie very close together
within the major histocompatibility complex on the
short arm of chromosome 6. Complicating the picture
further is the observation by Arnett et a1 that 5 of 6 B27negative patients with Reiter’s syndrome carried one of
the HLA-B antigens known to cross-react serologically
with B27, i.e. B7 or Bw22 (29). This suggests some direct
pathogenetic involvement of the antigen molecules
themselves. The actual genetic mechanism remains elusive to date.
The reason for the increased level of MLC responsiveness among AS and RS patients in the present
study is unclear, especially in that this result conflicts
with the findings of Nibkin et a1 who found low levels of
MLC response to pooled allogeneic lymphocytes among
ankylosing spondylitis patients, family members, and
healthy B27-positive individuals (30). This discrepancy
raises the possibilities that ankylosing spondylitis and
Reiter’s syndrome patients are more responsive to
unique antigens on our lymphoblastoid cell lines (LCL),
possibly Epstein-Barr virus-related antigens. Such selective responsiveness could, in fact, have pathogenetic implications and is a topic for further study. However, all
experiments to date indicate that EBV- and EBV+ persons do not differ in the level of responsiveness to EBVtransformed LCL cells (9,3 I ) .
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locus, reiter, hla, syndrome, ankylosis, spondylitis, typing
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