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Yersinia Enterocolitica In the synovial membrane of patients with Yersinia-induced Arthritis.

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Using a monospecific rabbit antibody against
Yersiniu enterocolitica outer membrane protein 1, we
examined synovial biopsy specimens from 7 patients
with Yersiniu-induced arthritis. Yersinia were demonstrated in the synovial membrane by indirect immunofluorescence in 4 patients with Yersiniu-induced arthritis, but not in 6 control patients with Salmonella-induced
arthritis or with rheumatoid arthritis. These findings
suggest the persistence of Yersinia in the joints of
patients with Yersinia-induced arthritis.
In the past 20 years, enteropathogenic Yersiniu
have been identified with increasing frequency as the
causative agent of acute bacterial gastroenteritis and
extraintestinal complications such as reactive arthritis
and erythema nodosum (1-6). Until recently, Yersiniainduced reactive arthritis was diagnosed on the basis
of a clinical picture of arthritis following gastroenteritis, and elevated agglutination antibody titers (Widal's
reaction) for Yersiniu (1,3,4,6,7). The recent use of
immunoblot analysis of class-specific serum antibodies
against plasmid-encoded Yersiniu proteins has made
From the Division of Rheurnatology, Medizinische Hochschule Hannover and the Institute for Hygienics and Microbiology,
University of Wiirzburg, Federal Republic of Germany.
Michael Hammer, MD: Senior Physician, Division of Rheumatology, Medizinische Hochschule Hannover; Henning Zeidler,
MD: Professor of Internal Medicine and Rheumatology and Head,
Division of Rheumatology, Medizinische Hochschule Hannover;
Silvia Klirnsa, Medical Technical Assistant, Division of Rheumatology, Medizinische Hochschule Hannover; Jurgen Heesemann, MD:
Professor of Microbiology and Head, Institute for Hygienics and
Microbiology, University of Wurzburg.
Address reprint requests to Michael Hammer, MD, Division of Rheumatology, Medizinische Hochschule Hannover,
Konstanty-Gutschow-Str. 8, 3000 Hannover 61, FRG.
Submitted for publication April 19, 1990; accepted in revised form June 22, 1990.
Arthritis and Rheumatism, Vol. 33, No. I2 (December 1990)
serologic testing for Yersiniu more sensitive and more
specific, in comparison with Widal's reaction (8,9).
Yersiniu were detected in intestinal biopsy material of
patients with seronegative spondylarthropathy by
means of indirect immunofluorescence, and this
finding showed a strong correlation with the presence
of specific IgA antibodies (10). The persistence of
Yersiniu in lymph nodes and intestinal tissues for
several years was also demonstrated using the same
method (9).
Recently, 2 studies concerning the detection of
Yersiniu-derived antigenic material in synovial fluid
and synovial fluid cell preparations from patients with
Yersinia-induced arthritis have been reported (1 1,12).
However, the synovial membrane has not been the
subject of extensive investigations in Yersinia-induced
arthritis. There is only 1 published report dealing with
the possible persistence of bacterial antigens in the
synovial membrane of a patient with reactive arthritis
following a Yersiniu pseudotuberculosis infection (13).
However, this report gave no detailed specifications of
the method used to detect antigen nor of the appearance of immunofluorescence staining patterns. Therefore, we studied the synovial membrane of patients
with Yersiniu-induced arthritis to determine whether
Yersiniu could be detected at this site, analogous to
recent findings of bacteria or bacterial antigens present
at intraarticular sites in patients with Chlamydiuinduced arthritis (14,15) and Lyme arthritis (16,17).
Patients. Seven patients (2 women and 5 men, age
range 2 7 4 5 years) from our outpatient clinic, with a diagnosis of Yersinia-induced arthritis based on serologic find-
Table 1.
Clinical characteristics of the patients studied*
Duration of
Yersinia arthritis
Yersinia arthritis
Yersinia arthritis
Yersinia arthritis
Yersinia arthritis
Yersinia arthritis
Yersinia arthritis
Salmonella arthritis
Salmonella arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
1 :320
I :20
antibodies to
Y enterocolitica
I :20
* RF = rheumatoid factor, determined by the Rose-Waaler test (titer >1:40).
t Detected by immunoblotting.
Immunotluorescence staining for the Y enterolifica outer membrane protein I (YOPI) was performed with rabbit monospecific anti-YOP1
ings and on a recent history of oligoarthritis, agreed to
undergo synovial biopsy. In 6 patients, there was an episode
of diarrhea 2-8 weeks before the onset of arthritis, and in 1
patient, an appendectomy was performed 4 weeks prior to
the onset of arthritis because of abdominal pain and fever,
before the Yersinia infection (with pseudoappendicular syndrome) was diagnosed.
The control group consisted of 2 patients with reactive arthritis following Salmonella fyphi infection and 4
patients with rheumatoid arthritis according to the American
Rheumatism Association 1987 revised criteria (18). The age
range of these control patients (3 women and 3 men) was
22-52 years. None of the patients had received antibiotic
therapy before serum samples were taken and synovial
membrane biopsies were performed. The duration of arthritis before biopsy and other clinical characteristics of the
patients are shown in Table 1.
Specific serum agglutinins against Yersinia enterocolitica serotypes 0 : 3 and 0 : 9 and against Y pseudotuberculosis serotypes 1-111 were determined by standard methods
(I). Specific IgG and IgA antibodies against plasmid-encoded
proteins of Y enterocolitica were demonstrated in sera by
immunoblotting techniques, as previously described (8,lO).
Biopsy procedure. Synovial membrane biopsy specimens were obtained, under local anesthesia, from 4 different
areas of the suprapatellar 'pouch of an affected knee joint.
Samples were obtained either with a Parker-Pearson needle
or with biopsy forceps with a length of 25 cm and with oval,
spoon-type yaws, like those used in intestinal endoscopy.
Biopsy material was quick frozen in liquid nitrogen, followed
by preparation of tissue sections (5p) with a cryostat. The
sections were placed on glass slides coated with 0.01%
poly-L-lysine and were fixed in acetone for 10 minutes. The
slides were stored at -70°C until further staining.
Monospecific rabbit antibodies. Monospecific rabbit
antiserum against purified Yersinia outer membrane protein
1 (YOP1) was obtained as described previously (2,10,19).
The crude antiserum was affinity purified using YOPl antigen blotted onto a nitrocellulose sheet as the affinity matrix.
YOP1-specific antibodies were eluted with 0.1M glycine HCI
buffer, pH 2.0. The specificity of the antibodies was defined
by indirect immunofluorescence and by immunoblotting
(Figure 1). Pellets of plasmid-containing Y enterocolitica 0 : 3
and plasmid-free isogenic derivatives served as positive and
negative controls, respectively, and were compared with the
findings in the synovial tissue. Additional positive controls
were obtained by using sections of spleen from Yersiniainfected BALBIc mice. Additional negative control sections
were made at each staining procedure, where instead of the
monospecific antibodies, rabbit preimmune serum or phosphate buffered saline (PBS) was used.
Staining procedures. Four biopsy sections from different areas of the suprapatellar pouch of each patient were
examined. Indirect immunofluorescence was performed according to standard methods. Before tissues were stained,
slides containing pellets of Y enterocolitica 0:3 were stained
with different dilutions of the antibodies in order to evaluate
the optimal dilutions for bright and clearly fluorescent staining of the bacterial cell wall. Briefly, the slides were overlaid
with a 1: 100 dilution of the primary antibodies, followed by
fluoresceinated anti-rabbit IgG (1:20). Identical dilutions
were used in patients and controls. Samples were incubated
with the primary antibody for 30 minutes at room temperature, washed with PBS, then incubated with secondary
Figure 1. A, Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of outer membrane proteins of Yersinia enterocolitica
serotype 0:3. + = plasmid positive; - = plasmid negative. Molecular weight standards are shown on the left. B, lmmunoblot
incubated with affinity-purified rabbit antibodies to the 220-kd
Yersinia outer membrane protein 1 (YOP 1) and developed with
alkaline phosphatase-conjugated swine anti-rabbit IgG. YOP 1 =
55-kd subunit of YOP 1.
antibody for 30 minutes at room temperature and washed
again with PBS. All slides were read, without knowledge of
the clinical diagnosis, on a Leitz (Wetzlar, FRG) lightfluorescence microscope. At least 10 slides of each biopsy
area and a total of more than 50 slides per patient were
reviewed for 15-20 minutes per slide.
Serologic findings. Four of 7 patients who were
diagnosed as having Yersinia-induced arthritis had a
significant agglutination titer (>1:80) against Y enterocolitica 0:3 or 0:9 (Table 1). Immunoblotting showed
that specific IgG antibodies against' Yersinia plasmidencoded proteins were found in all 7 patients, and
specific IgA antibodies were found in 5 of 7 patients.
Only 1 of the control patients had IgG antibody, and
none had a significant titer in the agglutination reaction
(Table 1).
Immunofluorescence results. Oval-to-roundshaped particles with membranous fluorescence in the
synovial membrane of 4 patients were identified as
Yersinia. Typical findings in 2 patients and in a positive control (Yersinia-infected spleen of a BALBk
mouse) are shown in Figure 2. Careful evaluation of
the slides was necessary to avoid missing the characteristic cell wall staining pattern of the bacteria. Nonspecific staining or artifacts with irregularly formed
particles and spotty or granular fluorescence patterns
occurred occasionally both in positively stained sections and in negative controls, and were clearly distinguishable from Yersinia (Figure 2A).
Generally, the bacteria were thinly scattered
and, in most sections, occurred in small numbers or in
clumps, often in densely infiltrated areas of the synovial lining cell layer or the subsynovium, and only
sporadically in deeper interstitial tissue. In patients 1,
3, and 4, adjacent sections stained with hematoxylin
and eosin showed moderate to high infiltration with
mononuclear cells and granulocytes, as well as fibrin
exudation and partial destruction and regeneration of
the lining cell layer, which consisted mostly of 2-4
layers (Figure 2D). In patients 2 , 5 , 6 , and 7, there was
only scattered lymphocytic infiltration without fibrin
exudation, and most of the lining cell layer appeared
normal. The total number of precisely identifiable
Yersinia bacilli ranged from 12 to more than 50 per
patient. Positive results occurred in only 2 or 3 of 10
slides, and did not occur in each of the 4 biopsied areas
of the suprapatellar pouch. Based on the immunofluorescence method used, with no or very weak background staining of the synovial cells, it was not possible
to differentiate between intracellular and extracellular
localization of the bacilli.
Yersinia could not be demonstrated in biopsy
specimens of patients with Salmonella-induced arthritis or rheumatoid arthritis, even though the same
number of slides was examined. Additional control
sections, using rabbit preimmune serum or PBS instead of the rabbit antibodies, showed no evidence of
oval- or round-shaped particles with membranous fluorescence.
In this study, we found bacillus-like particles in
the synovial membranes of patients with Yersiniainduced arthritis. Since the persistence of Yersinia in
the intestinal tissues of patients with seronegative
spondylarthropathy has been demonstrated by indirect
immunofluorescence (lo), we decided to apply this
procedure in the detection of Yersinia in affected joints
of patients with Yersinia-induced arthritis.
One difficulty in studying patients with a short
history of undifferentiated or reactive arthritis is how
to obtain synovial membrane samples for investigation, in addition to synovial effusions. We solved this
problem in the present study by obtaining biopsy
specimens from the suprapatellar pouch of the affected
knee joints, using a Parker-Pearson needle or biopsy
Figure 2. Immunofluorescence staining of Yersiniu in the synovial membrane of patient I (A) and patient 2 (B), and in the spleen of a
Yersiniu-infected BALB/c mouse (C), using monospecific rabbit antibodies against Yersiniu outer membrane protein 1 . (Original magnification
X 1,OOO.) D, Hematoxylin and eosin-stained adjacent section from patient 2 (original magnification x 250).
forceps. Another problem is that patients with reactive
arthritis are frequently referred to a rheumatologist
only when the disease has not remitted after several
months of symptoms. At this point in the course,
inflammation is, in most cases, not as active as at the
onset of symptoms, and patients are reluctant to
undergo invasive diagnostic procedures. These circumstances make the recruitment of additional patients and the obtaining of joint material for investigation more difficult.
Are the fluorescent particles present in the
synovial membrane really Yersiniu? There are several
important indications in support of this assumption.
First, the size and form of the fluorescent particles
correspond closely with those of gram-negative bacteria.
The positive control sections of a pellet of plasmidbearing Yersiniu and the spleen sections of Yersiniuinfected BALB/c mice showed identical-lookingfluorescent rods (see Figure 2).
Second, the fluorescent particles found in intestinal tissues by Hoogkamp-Korstanjeet al, which were
described as Yersinia, feature an identical fluorescence morphology (9). Also, when these bacilli were
detected at the initial site of infection, they were thinly
scattered in the tunica propria of colonic biopsy material or were localized in small groups in the mesenteric lymph nodes (2,lO). Nevertheless, the long-term
persistence of Yersiniu in the gut or in lymphoid tissue
has been demonstrated (9).
Third, based on previous findings and immuno-
blotting (Figure I), it could be assumed that the
antibodies used react only with YOPl epitopes of
Yersinia species and that this protein is not shared by
other gram-negative bacteria (10,19). Furthermore,
possible false-positive results caused by nonspecific
and irregularly formed fluorescent particles or background staining could be excluded by evaluating only
cell wall staining of rod-shaped bacilli.
The presence of serum IgA antibodies against
plasmid-encoded “released” proteins of Yersinia appears to indicate the persistence of the bacteria or
bacterial antigen (9,lO). Three of our patients who had
Yersinia in the synovial membrane had IgA antibodies
specific for this organism. In 2 patients with serum IgA
antibodies, bacteria were not found in the synovial
membrane. Disease duration and the degree of the
local inflammation in the biopsied area may influence
the detectability of the causative agent. Therefore,
early arthroscopic biopsy of maximally inflamed areas
of the synovial membrane may be more informative in
future studies.
Although Yersinia were clearly identified in the
synovial membrane, the number of detectable bacilli
seen was very small. This observation is consistent
with the positive fluorescence pattern seen in only
I-10% of synovial fluid cells in 9 patients with
Yersinia-induced arthritis who tested positive for Yersinia antigens (12). In Chlamydia-induced arthritis also,
the microorganisms are described as being scattered in
the synovial membrane (14). Similar observations
have also been made in Lyme disease, in which it was
very difficult to find spirochetes or spirochetal antigen
in tissue (16,17).
The morphology observed by immunofluorescence seems to indicate the presence of intact Yersinia
bacilli in the synovial membrane. A recent study
demonstrated the collagen binding capacity of Yersinia
and showed that this interaction is closely related to
the presence of YOPl (19). In the present study, we
made no attempt to recover bacilli from synovial
biopsy specimens by bacterial culture methods. In
future studies, the identification of Yersinia should be
confirmed by specific bacteriologic techniques or, alternatively, by the use of species-specific DNA probes
for hybridization studies, or DNA primers for the
polymerase chain reaction. The adaptation of these
techniques for synovial biopsy material is in progress.
The presence and possible persistence of lipopolysaccharide (LPSkontaining particles, which may
be resistant to degradation by the host, or of intact
gram-negative bacilli in the immunologically active
synovial tissue, is known to be a strong inducer of
inflammation (20,21). In addition, elevated serum LPS
levels (endotoxin levels) were found in patients with
ankylosing spondylitis during periods of increased
disease activity (22). Therefore, insufficient clearing of
the causative agents in genetically predisposed patients with reactive arthritis may be the essential factor
in the chronicity of the disease.
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