37 I

HLA-D LOCUS TYPING IN ANKYLOSING SPONDYLITIS AND REITER’S SYNDROME

KIP KEMPLE, RICHARD A. GATTI, WOLFGANG LEIBOLD, JAMES KLINENBERG, and RODNEY BLUESTONE

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 re- sponses within each experiment. Contrary to reports of diminished lymphocyte responses in AS patients, hyper- responsiveness in both A S and R S patients was found.

From the Division of Hematology, Oncology, and Irnmunol- ogy, Department of Pediatrics, and the Department of Medicine, Ce- dars-Sinai Medical Center and University of California at Los Ange- les 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 Cen- ter; 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.

A number of studies in the past several years have attempted to confirm and extend the initial obser- vations that ankylosing spondylitis and Reiter’s syn- drome 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 clari- fied the actual mechanisms through which the histo- compatibility system is influencing disease pathogene- sis. 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 im- mune 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 histocompa- tibility 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 ankylos- ing spondylitis and Reiter’s syndrome in order to deter-

Arthritis and Rheumatism. Vol. 22, No. 4 (April 1979)

372 KEMPLE ET AL

Table 1. HLA-D typing responses

Dw7 Dw8 Nottyped Dwl* DW2 Dw3 Dw4 Dw5 Dw6

Normal controls (45)t 29 35 -- 7 7 29 24 28 15 18 18 Ankylosing spondylitis (44) 34 24 22 35 I9 14 22 19 36

Reiter’s syndrome ( 3 I ) 16 29 23 15 21 2x 26 23 32 P value NS$ N S NS NS NS N S NS NS

P value N S N S NS NS NS NS N S NS

* 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.

MATERIALS AND METHODS Typing was performed on 44 patients with ankylosing

spondylitis and 3 1 patients with Reiter’s syndrome. The modi- fied Rome (New York) criteria were used to define patients as having either definite (36 patients) or probable (8 patients) an- kylosing spondylitis (5). Patients were considered to have defi- nite Reiter’s syndrome if they had all of the major triad, i.e. arthritis, conjunctivitis, and nonbacterial urethritis (24 pa- tients). They were considered to have probable Reiter’s syn- drome if they manifested arthritis plus one of the major fea- tures (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 experi- ments, 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 lympho- cytes as stimulator cells (6,7). This method has been used be- fore by the authors (6) and by Netzel et a1 (7.8) and is de- scribed in detail elsewhere (6.9.10). Briefly, the stimulator typing cells were obtained initially as peripheral blood lym- phocytes 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 cul- ture. In these experiments, typing cell lines (LCL-HTCs) rep- resenting 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 LCL- HTCs for each.

Typing cells 5 X lo4 were frozen in 5% dimethyl sul- foxide (DMSO) in round bottom typing plates following treat- ment with mitomycin C (80 pg/ml). Responder blood was collected in sterile heparinized tubes and lymphocytes were separated on a Ficoll-Hypaque gradient. Responder lympho- cytes ( 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 re- sponsiveness between different individuals, a computer pro- gram was developed that adjusted for this variation and as- signed 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 In- dices (11) were used to assign Dw specificities by arbitrarily se- lecting an I1 of 50 as the cutoff value: I1 below 50 were consid- ered 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 I 1 was carried out using a f test to compare the fre- quencies of typing responses to various HLA-D alleles be- tween patient and control groups.

RESULTS 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 be- tween either patient group and the normal control pop- ulation. The last column on the right includes the per- centages 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 pa- tients is due in part to the fact that patients were gener- ally typed in single experiments only, while most con- trols were studied several times. Homozygosity for a particular allele would also increase this figure since our methods do not distinguish “blanks” from homo- zygotes. 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 speci- ficities. While this would not influence percent-positive

HLA-D LOCUS TYPING 373

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, sug- gesting 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 leuko- cyte culture responsiveness. Table 2 shows that the mean stimulation score for patients with AS was signifi- cantly greater than the mean score for the normal con- trols. A similar but less impressive increase was docu- mented for Reiter’s syndrome patients.

DISCUSSION 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 con- sidered to explain these associations. The hypothesis mentioned in the introduction involves abnormal im-

Table 2. MLC responsiveness*

Mean raw stimulator

score (cpm) P value

Normal controls (4S)t 21.100 Ankylosing spondylitis (44) 38.700 < 0.00 I Reiter’s syndrome (31) 26.700 < u.0os

* MLC responsiveness scores represent the mean of responses for each donor tested to the entire panel o f LCL-l1TCs. This score in- cludes 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 dis- equilibrium with the B27 antigen. Alternative mecha- nisms suggest that the B27 antigen itself is involved in disease pathogenesis, either acting as a surface receptor for infectious agents or possibly cross-reacting immuno- logically 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 ini- tial associations were noted to involve serologically de- fined 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 dis- equilibrium between these antigens and the HLA-D an- tigens Dw2 and Dw3, respectively. Also, rheumatoid ar- thritis now appears to be associated with the HLA-D antigen Dw4, without any association with the sero- logically defined HLA-A, -B or -C antigens (23). With some reservations, this kind of data linking specific dis- eases with HLA-D locus alleles can be taken as evi- dence in favor of disease susceptibility genes which are themselves located close to the D locus.

Evidence is presented here that there is no asso- ciation between ankylosing spondylitis or Reiter’s syn- drome and any individual HLA-D allele. These results are in agreement with those of other groups who have evaluated ankylosing spondylitis patients by conven- tional 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). Al- though 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 link- age between Dw7 and DRw7.

There are important negative results that set an- kylosing spondylitis and Reiter’s syndrome apart from the other diseases mentioned above. They are the only adequately studied diseases to date which have histo- compatibility 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 B27- negative 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 elu- sive to date.

The reason for the increased level of MLC re- sponsiveness 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 selec- tive responsiveness could, in fact, have pathogenetic im- plications and is a topic for further study. However, all experiments to date indicate that EBV- and EBV+ per- sons do not differ in the level of responsiveness to EBV- transformed LCL cells (9,3 I ) .

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