Table of Contents

MATERIALS AND METHODS

RESULTS

ACKNOWLEDGMENT

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Paper 7

YOSHITAKATANI, HARMALE TIWANA, SINSUKE HUKUDA, JUNICHI NISHIOKA, MARK FIELDER, CLYDE WILSON, SUKHVINDER BANSAL, and ALAN EBRINGER

ABSTRACT.

Objective. To determine whether patients with ankylosing Spondylitis (AS) and patients with rheumatoid arthritis (RA) from Japan have antibodies to Klebsiella pneumoniae and Proteus mirabilis and to assess whether such antibodies are activated against peptides sharing sequences with HLA-B27.

Methods. Serum samples from 152 Japanese patients, 52 with AS, 50 with RA, and 50 healthy controls, were tested against 3 bacteria (K. pneumoniae, P. mirabilis, and Escherichia coll) and 3 synthetic peptides (HLA-B27, pullulanase-D, and scrambled pullulanase-D control peptide) by ELISA under coded conditions. Samples were tested for elevations in IgG, IgA, and IgM antibody classes in patients with active AS or RA, in patients with RA with probable disease, and in patients with inactive AS. Disease activity was determined by an elevated serum C-reactive protein (> 10 mg/1) level and elevated erythrocyte sedimentation rate (> 20 mm/h).
Results. Patients with active AS showed specific elevations in serum IgA antibody levels against K. pneumoniae compared to patients with RA and controls ( p < 0.001). No such elevation was seen in the IgG and IgM antibody classes. Patients with inactive AS showed no elevation in any class of antibody against K. pneumoniae compared to controls or patients with RA. Patients with active or probably active RA showed significant elevations in IgG antibody levels against P. mirabilis compared to AS and controls (p < 0.001). Patients with AS (active or inactive), RA (active or probably active), and controls showed no elevations in any antibody class to E. coli. Both active and inactive AS patients had specific autoantibodies against HLA-B27 peptide compared to patients with RA and controls (active AS: IgG, IgA, IgM, p < 0.001; inactive AS: IgG and IgA, p < 0.001). Patients with active AS had IgG and IgA antibodies against pullulanase-D peptide, which contains a sequence that cross reacts with HLA-B27 compared to controls (p < 0.001).
Conclusion. These results provide the first evidence of AS and RA patients in Japan having specific elevations of antibody to K. pneumoniae and P. mirabilis, respectively. This suggests that K. pneumoniae in AS and P. mirabilis in RA may play a role in triggering and/or exacerbating these diseases. (JRheumatol 7997;24:109-14)

Key Indexing Terms: ANKYLOSING SPONDYLITIS KLEBSIELLA
ANTIBODY HLA-B27
RHEUMATOID ARTHRITIS PROTEUS

Ankylosing Spondylitis (AS) and rheumatoid arthritis (RA) are HLA linked inflammatory disorders. Over 90% of patients with AS possess HLA-B27 and a similar percentage of patients with RA possess HLA-DR1 or DR4'-³. In Japan, the frequency of HLA-B27 is only 0.41% in the general population⁴, and the prevalence of AS is reported to be 0.03-0.04%⁵-⁶. In contrast, the frequency of HLA-DR4 (mainly DW15 in Japanese) is about 40%, but the prevalence of RA is 0.3-0.5% in Japanese⁶-⁷, lower than the

From Department of Orthopaedic Surgery, Shiga University of Medical Science, Otxu. Japan; Life Science Centre. King i College. London, UK. K Tani, MO; S. Hukuda. MO: J. Nishioka, MD. Shiga University of Medical Science: H. Ttwana. PhD; M. Fielder. PhD; C. Wilson. PhD; S. Bansal, PhD; A. Ebringer. MD. King's College. Address reprint requests to Dr Y. Tani, Department of Orthopaedic Surgery. Shiga University of Medical Science. Tsukinowa-cho, Seta. Otsu. Shiga 520-21, Japan.
Submitted May 17. 1996 revision accepted July 3, 1996.

Caucasian rate⁸-⁹. These data suggest that the pathogenesis of AS and RA are mediated by factors, such as the immuno-genetic status of the patient and the involvement of environmental agents, similar to those observed in other countries.

An increase in anti-Klebsiella antibodies in patients with AS during the active phase has been reported in different geographical locations¹⁰'". Moreover, crossreactivity between Klebsiella and HLA-B27 has been reported by several groups¹²"¹⁴. In addition to the relationship between AS and Klebsiella, elevated levels of antibodies to Proteus have been identified in patients with active RA in Europe and America¹⁵'¹⁸. An amino acid homology between an outer membrane hemolysin protein of P. mirabilis and the susceptibility sequence in HLA-DRI and DR4 subtypes (DW14, DW15) has been reported¹⁹.

These reports suggest the possibility of bacteria being the causative agents of AS and RA. To explore these possibilities, we measured IgG, IgA, and IgM class specific antibodies to K. pneumoniae, P. mirabilis, and synthetic peptides containing HLA-B27 sequences by ELISA in Japanese patients with AS and RA.