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Outer surface lipoproteins of Borrelia burgdorferi vary in their ability to induce experimental joint injury.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 7, July 2004, pp 2360–2369
DOI 10.1002/art.20337
© 2004, American College of Rheumatology
Outer Surface Lipoproteins of Borrelia burgdorferi Vary in
Their Ability to Induce Experimental Joint Injury
Stephen Batsford,1 John Dunn,2 and Michael Mihatsch3
Objective. To examine the ability of bacterial
lipoproteins from the spirochete Borrelia burgdorferi to
cause in vivo tissue injury (arthritis).
Methods. Outer surface proteins (OSPs) from B
burgdorferi were used in a rat model of antigen-induced
allergic arthritis. Intraarticular challenge with recombinant OspA, OspB, and OspC in nonlipidated (peptide) and lipidated forms was performed in the left knee
joint; the contralateral joint received buffer as control.
Inflammation was monitored by technetium scintigraphy and histology.
Results. Nonlipidated (peptide) OspA, OspB, and
OspC did not induce arthritis; the only exception was
polymerized OspA, which was tested in preimmunized
rats. Lipidated OspA from 2 different strains and
lipidated OspC induced severe arthritis, whereas lipidated OspB failed to induce injury. A synthetic analog
of the OSP lipid modification, lipopeptide Pam3Cys-SerLys4-OH, either alone or coupled to bovine serum
albumin, also failed to induce injury. Injury did not
develop in control groups that were given the appropriate buffers or lipopolysaccharide. This showed that
lipidated borrelial OSPs can be potent arthritogens but
vary greatly with respect to their injury-inducing potential. The possession of a lipid modification is essential
but is not sufficient to render an OSP arthritogenic.
Conclusion. This is the first study to demonstrate
that individual lipoproteins from B burgdorferi can
induce experimental joint injury in vivo. These results
may help elucidate the pathogenesis of Lyme arthritis
and, above all, underline the importance of bacterial
lipoproteins as major virulence factors.
Lipoproteins are now being recognized as prime
virulence factors of bacteria. For many decades this
group of molecules played the role of poor relation to
the lipopolysaccharides (LPS), but particularly the discovery that lipoproteins can act as ligands for the
Toll-like receptor (TLR) complex has rekindled interest
(1). In earlier studies, a number of naturally occurring
bacterial lipoproteins and their synthetic lipopeptide
analogs were shown to possess impressive biologic activities. The Braun protein, a prototype lipoprotein, can
stimulate lymphocytes (2), and the corresponding synthetic lipopeptide is in fact now used as an adjuvant (3).
Membrane lipoproteins and the corresponding lipopeptides from mycoplasma could induce mononuclear
phagocytes to secrete cytokines; a particular lipopeptide
from Mycoplasma fermentans, macrophage activating
lipopeptide 2, was effective even at concentrations in the
picogram range (4). The outer surface lipoproteins
(OSPs) of Borrelia burgdorferi have been shown to
exhibit similar stimulatory effects (5–8).
Results of recent in vitro studies indicated that
the lipid moiety plays a key role, and that the CD14/
TLR-2 receptors can be involved (9–11). Lipoproteins
have been shown to be capable of inducing tissue injury
in animals, mimicking certain common clinical manifestations of the corresponding infection in man. First,
synthetic lipopeptide analogs of treponemal and borrelial lipoproteins caused acute dermal lesions in mice
(12). Second, an OSP complex extracted from B burgdorferi induced severe, chronic arthritis in rats (13). In
the current study, 3 major OSPs (OspA, OspB, and
OspC) derived from B burgdorferi were studied. Their
ability to induce arthritis in a rat model of antigeninduced allergic arthritis (14) was analyzed in detail,
using a battery of purified recombinant antigens. The
injury-inducing potential of the lipid modification and
1
Stephen Batsford, PhD: Albert Ludwigs University,
Freiburg, Germany; 2John Dunn, PhD: Brookhaven National Laboratory, Upton, New York; 3Michael Mihatsch, MD: Institute of Pathology, Kantonspital Basel, Basel, Switzerland.
Address correspondence and reprint requests to Stephen
Batsford, PhD, Department of Immunology, Institute of Medical
Microbiology, University of Freiburg, Hermann-Herder-Strasse 11,
D-79104, Freiburg, Germany. E-mail: bats@ukl.uni-freiburg.de.
Submitted for publication July 21, 2003; accepted in revised
form March 5, 2004.
2360
INDUCTION OF EXPERIMENTAL JOINT INJURY IN VIVO BY OSPs FROM B BURGDORFERI
the peptide regions was evaluated, and the role of the
immune response to the OSPs in inducing inflammation
in vivo was studied. Because the pathogenesis of organ
manifestations of B burgdorferi infection is still quite
unclear, this information may provide valuable insights
into the mechanisms involved.
MATERIALS AND METHODS
Animals. Male Wistar rats (Harlan, Borchen, Germany) were used throughout. Body weight ranged from 100 to
120 gm at the beginning of the experiments. Animal experiments were approved by the local government agency (license
no. G-00/43).
Antigens. The following borrelial OSPs were prepared
as both truncated nonlipidated (peptide) and full-length lipidated forms (the strain[s] of origin of the sequence is shown in
parentheses): OspA (B31 and K48, both B burgdorferi sensu
stricto), OspB (B31), and OspC (B31-lipidated using the
leader sequence from OspB of B31, K48-truncated). The
details of plasmid construction, expression systems, and purification of recombinant products have been published elsewhere (15). Expression of OspC with the original leader
sequence could not be achieved due to the occurrence of
severe toxicity problems in the expression system, which were
overcome by replacing the OspC leader with OspB (Dunn J,
Lade B: unpublished observations).
The 83-kd structure protein (synonyms, 94- and 100-kd
protein) of B burgdorferi sensu stricto (strain GeHo) was
prepared in recombinant form as previously described (16). All
of the truncated OSP preparations (OspA, OspB, OspC) and
the 83-kd protein were water soluble; all lipidated OSP preparations were solubilized in phosphate buffered saline (PBS)
containing 0.1% Triton X-100. All preparations were screened
for contamination with LPS by the Limulus coagulation test
(Acila, Frankfurt, Germany); concentrations found were between 0.1% and 1.0%.
The water-soluble lipopeptide Pam3Cys-Ser-Lys4OH (molecular weight 1,620 daltons (Boehringer, Mannheim,
Germany) was used. When required, it was covalently coupled
to bovine serum albumin (BSA), as described below. LPS,
prepared from Escherichia coli EH100, was used as an aqueous
solution at 200 ␮g/ml.
Protein polymerization. Truncated recombinant OspA
and OspB (both derived from strain B31) were polymerized
with dimethyl suberimidate, a bifunctional iminoester that
crosslinks lysine side chains without altering the net charge
(17,18). The OSP preparation was brought to a concentration
of 10 mg/ml in 0.1M NaHPO4 (pH 10.0), and then 2 mg of
dimethyl suberimidate per milliliter of protein solution was
added in portions over a 30-minute period, with stirring, at
20°C. The reaction mixture was dialyzed against PBS at 4°C,
centrifuged at 1,000g for 10 minutes, and the supernatant was
stored at ⫺20°C.
The synthetic lipopeptide Pam3Cys-Ser-Lys4-OH was
coupled to BSA to create an analog lipoprotein containing a
nonarthritogenic peptide moiety. For this purpose, BSA at
concentrations of 10 mg/ml and 20 mg/ml was coupled with
Pam3Cys-Ser-Lys4-OH at concentrations of 0.5 mg/ml and 1
2361
mg/ml, respectively, as described above. Before use, the conjugate was diluted to a BSA concentration of 1 mg/ml.
Size-exclusion chromatography. Polymerized OspA
and OspB were separated on a HiLoad 26/60 Superdex 75 prep
grade column run on an FPLC system (Amersham Biosciences,
Freiburg, Germany). The Superdex column was equilibrated in
PBS, samples containing 10 mg of protein were loaded and run
in PBS at a flow rate of 3 ml/minute, 5-ml fractions were
collected, and the effluent was monitored at 280 nm. Selected
fractions were pooled and concentrated in an ultrafiltration
cell using YM10 membranes (Millipore, Bedford, MA). Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (see below) showed that both polymerized
OspA and polymerized OspB could be separated into monomeric, dimeric, and oligomeric (trimer and larger) fractions.
SDS-PAGE. Protein preparations were analyzed in
12% acrylamide gels and stained with Coomassie blue R250,
using molecular mass standards in the range of 14–93 kd
(Amersham Biosciences) (19).
Arthritis induction. Test substances (usually 50 ␮g of
test protein in 50 ␮l of buffer) were injected directly into the
left knee joint; the contralateral right knee joint received 50 ␮l
of buffer alone as a control. In the case of LPS, the dose was
reduced to 10 ␮g. In some experiments, rats were preimmunized by injecting 100 ␮g of antigen, emulsified in Freund’s
complete adjuvant, subcutaneously into the dorsal region to
avoid joint interference. Two booster injections with the same
dose were given at 2-week intervals, and experiments were
initiated 1–2 weeks later.
Measurement of delayed-type hypersensitivity. Delayedtype hypersensitivity was measured in the experimental groups
by injecting 5 ␮g of antigen in 5 ␮l of buffer intracutaneously
into the pinna of the ear; the contralateral ear was injected
with PBS as a control. Increases in ear thickness were measured with an engineer’s micrometer after 48 hours, and results
were expressed as the mean ⫾ SEM increase (mm ⫻ 10⫺2).
99m
Tc scintigraphic uptake measurements. Joint inflammation was assessed by comparing the uptake of 99mTcpertechnetate between knee joints (20). Each rat received 2
MBq of 99mTc in 0.2 ml of PBS, subcutaneously into the neck
region. Gamma radiation emanating from both knee joints of
the hind legs was measured using a collimated photoscintillation crystal. The rats’ hind legs were kept in a defined position,
and their bodies were shielded with a lead screen. Radioactivity was measured 3 times over each joint in an alternating
manner, for a duration of 0.5 minutes, and the mean values
were used to calculate the left:right (L:R) ratio.
Joint retention of 131I-labeled OSP preparations. Onemilligram portions of nonlipidated OspA, lipidated OspA, and
lipidated OspB were labeled with 131I (Amersham Biosciences) by the chloramine-T–method (21). Free 131I was
removed by passage through a Sephadex PD-10 column (Amersham Biosciences). Before use, 131I-labeled samples were
mixed with unlabeled, chloramine-T–treated protein, and each
rat received 25 ␮g of the 131I-labeled sample (2 ⫻ 104 Bq)
directly into the left knee joint; the right knee joints received
PBS at 1, 10, and 60 minutes and at 2, 6, and 24 hours.
Radioactivity was measured over both knee joints with a
collimated counter, in a manner similar to that used to
measure 99mTc uptake (see above).
2362
BATSFORD ET AL
Table 1. Effect of intraarticular challenge with truncated, nonlipidated outer surface proteins (OSPs)
Group
1
2
3
4
5
6
(n
(n
(n
(n
(n
(n
⫽
⫽
⫽
⫽
⫽
⫽
5)
5)
5)
5)
4)
4)
Technetium scintigraphy‡
Challenge preparation
(sequence)*
Immunized
(DTH test)*
Histology†
Result (no./total)
Value, L:R ratio
Monomeric OspA (B31)
Monomeric OspB (B31)
Monomeric OspC (K48)
Polymerized OspA (B31)
Polymerized OspA (B31)
Polymerized OspB (B31)
Yes
Yes
No
No
Yes (42 ⫾ 5.3)
Yes (22 ⫾ 7.5)
Normal
Normal
Normal
Normal
Injury
Normal
Normal (5/5)
Normal (5/5)
Normal (4/5)
Normal (5/5)
Raised (4/4)
Normal (3/4)
1.10 ⫾ 0.03
1.09 ⫾ 0.02
1.16 ⫾ 0.02
1.03 ⫾ 0.02
1.37 ⫾ 0.03§
1.07 ⫾ 0.06
* Values for delayed-type hypersensitivity (DTH) testing are the mean ⫾ SEM increase in ear thickness (mm ⫻ 10⫺2) measured at 48 hours (for
control ears, the increase was 2.9 ⫾ 1.3 mm ⫻ 10⫺2). Intraarticular challenge and immunization were with the same antigen for all B31 sequences.
† In each group, 2 additional rats were killed for histologic examination on days 2 and 7, respectively; classification is for both rats.
‡ The L:R ratio represents 99mTc uptake in left:right knee joints on day 2 (number of rats with a ratio of ⬍1.2 [cutoff level] for normal and ⬎1.2
for raised values/number in group; in groups 3 and 6, one rat had borderline levels (1.2–1.25). Values are the mean ⫾ SEM.
§ P ⫽ 0.05 versus control groups 4 and 7 in Table 3.
Histology. In each group studied (see Tables 1, 2, and
3), animals were killed on days 2 and 7 after intraarticular
challenge, for histologic examination. Knee joints were detached and fixed in 4% buffered formalin, decalcified, and
embedded in paraffin. Standard frontal sections of the joints
were stained with hematoxylin and eosin.
Statistical analysis. Data from scintigraphic measurements revealed varying inhomogeneity and were subjected to a
log-log transformation followed by analysis of variance. Differences between groups were tested by Tukey’s studentized
range test, and within-group differences between days 2 and 7
were tested by paired t-tests. P values less than 0.05 were
considered significant. Statistical analysis was performed using
the general linear model procedure plus t-test functions in the
SAS program, release 6.12 (SAS Institute, Cary, NC). Data on
retention of OSPs in knee joints were compared using Fisher’s
t-test for unpaired data.
RESULTS
Effect of intraarticular challenge with nonlipidated (truncated) OSPs. The preparations tested, the
numbers of rats tested and their status (naive or preimmunized), and the ability of the preparation to induce
joint inflammation are shown in Table 1 (the 99mTc
scintigraphic L:R ratios shown are for day 2 only).
As can be seen in Table 1, monomeric, truncated
(nonlipidated), water-soluble OspA, OspB, and OspC did
not induce injury (groups 1, 2, and 3). Polymerized samples
were also tested, because, in the model used, the size of the
test molecule is known to be an important parameter
influencing induction of arthritis (20,22). Polymerized
Table 2. Effect of intraarticular challenge with full-length, lipidated outer surface proteins (OSPs)
Technetium scintigraphy‡
Group
1
2
3
4
5
6
(n
(n
(n
(n
(n
(n
⫽
⫽
⫽
⫽
⫽
⫽
6)
6)
4)
6)
5)
3)
7 (n ⫽ 6)
Histology†
Result
(no./total)
Value, L:R ratio
Yes (56 ⫾ 7.9)
No
BSA
No
Yes (48 ⫾ 4.6)
No
Injury
Injury
Injury
Injury
Borderline injury
Borderline injury
Raised (6/6)
Raised (6/6)
Raised (4/4)
Raised (4/6)
Normal (5/5)
Normal (3/3)
1.59 ⫾ 0.11§
1.61 ⫾ 0.07§
1.45 ⫾ 0.12§
1.30 ⫾ 0.07§
1.12 ⫾ 0.02
1.10 ⫾ 0.08
No
Injury
Raised (4/6)
1.34 ⫾ 0.12§
Challenge preparation (sequence)*
Immunized (DTH test)*
Lipidated OspA (B31)
Lipidated OspA (B31)
Lipidated OspA (B31)
Lipidated OspA (K48)
Lipidated OspB (B31)
Lipidated OspB (B31),
150 ␮g (triple dose)
Lipidated OspC (B31)
⫺2
* Values for delayed-type hypersensitivity (DTH) testing are the mean ⫾ SEM increase in ear thickness (mm ⫻ 10 ) measured at 48 hours (for
control ears, the increase was 2.9 ⫾ 1.3 mm ⫻ 10⫺2). Intraarticular challenge and immunization were with the same antigen, except where indicated
otherwise. BSA ⫽ bovine serum albumin.
† In groups 3, 4, and 6, two additional rats were killed (on days 2 and 7, respectively) for histologic examination; classification is for both rats. In
groups 1, 2, and 5, four additional rats were killed (two on day 2, and two on day 7), and in group 6, three additional rats were killed (two on day
2, and one on day 7). See text for detailed description.
‡ The L:R ratio represents 99mTc uptake in left:right knee joints on day 2 (number of rats with L:R ratios of ⬍1.2 [cutoff level] for normal and ⬎1.2
for raised values/number in group). In groups 4 and 7, two rats had an L:R ratio of ⬍1.2. Values are the mean ⫾ SEM.
§ P ⫽ 0.05 versus control groups 4 and 7 in Table 3.
INDUCTION OF EXPERIMENTAL JOINT INJURY IN VIVO BY OSPs FROM B BURGDORFERI
2363
Table 3. Effect of intraarticular challenge with synthetic lipopeptides, conjugates, and control preparations
Technetium scintigraphy‡
Group
1
2
3
4
5
6
7
(n
(n
(n
(n
(n
(n
(n
⫽
⫽
⫽
⫽
⫽
⫽
⫽
4)
4)
5)
4)
4)
4)
8)
Challenge preparation
(sequence)*
Immunized
(DTH test)*
Histology†
Lipopeptide Pam3CSK4
Pam3CSK4 BSA conjugate
83-kd protein (GeHo)
Triton X-100, 0.1%
Triton X-100, 1.0%
Lipopolysaccharide
Phosphate buffered saline
No
No
Yes (36 ⫾ 5.4)
No
No
No
No
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Result
(no./total)
Normal
Normal
Normal
Normal
Normal
Normal
Normal
(4/4)
(4/4)
(5/5)
(4/4)
(3/4)
(4/4)
(8/8)
Value, L:R
ratio
1.12 ⫾ 0.02
1.05 ⫾ 0.02
1.04 ⫾ 0.01
1.04 ⫾ 0.03
1.11 ⫾ 0.06
1.14 ⫾ 0.02
1.01 ⫾ 0.02
* Values for delayed-type hypersensitivity (DTH) testing are the mean ⫾ SEM of the increase in ear thickness (mm ⫻ 10⫺2) measured at 48 hours
(for control ears, an increase of 2.9 ⫾ 1.3 mm ⫻ 10⫺2). Intraarticular challenge and immunization were with the same antigen (all B31 sequences),
except where indicated otherwise. Lipopeptide Pam3CSK4 ⫽ Pam3Cys-Ser-Lys4-OH; BSA ⫽ bovine serum albumin.
† In addition to the rats used for scintigraphy, 1 rat was killed for histologic examination on day 2 and day 7, respectively; classification is for both
rats.
‡ The L:R ratio represents 99mTc uptake in left:right knee joints on day 2 (number of rats with an L:R ratio of ⬍1.2 [cutoff level]/number in group).
In group 5, one rat had borderline levels (1.2–1.25). Values are the mean ⫾ SEM. No significant differences between groups were observed.
OspB (group 6) did not induce arthritis, even in immunized
rats, whereas oligomeric OspA induced a transient arthritis
(that persisted for a maximum of 7 days) in previously
immunized rats (group 5). Examination of joint sections
from group 5 revealed moderate swelling of the synovial
membrane and limited polymorphonuclear neutrophil infiltration, without pronounced cartilage destruction. Due
to the limited quantities available, OspC was not tested in
the polymerized form.
Effect of intraarticular challenge with fulllength, lipidated OSPs. The preparations tested, the
numbers of rats tested and their status (naive or preimmunized), and the ability of the preparations to induce
joint inflammation are shown in Table 2 (the 99mTc
scintigraphic L:R ratios shown are for day 2 only).
Lipidated OspA derived from the strain B31
sequence induced severe arthritis in both naive and
preimmunized rats (Table 2 and Figure 1). The severity
of injury induced was similar in naive rats (group 2), in
rats preimmunized with lipidated OspA (group 1), and
in rats preimmunized with BSA (group 3) (Table 2).
Histologic examination revealed massive swelling and
cellular infiltration of the synovial membrane, together
with focal, complete destruction of the cartilage surface
in all of the rats examined (Table 2). Some rats in these
groups had persisting arthritis and were followed up. An
example of the severe changes seen on day 29 is shown
in Figures 2A and B. A second lipidated OspA derived
from the strain K48 sequence, and lipidated OspC
derived from the strain B31 sequence also induced joint
inflammation in naive rats (groups 4 and 7, respectively;
Table 2); preimmunized rats were not tested. In groups
4 and 7, the 99mTc scintigraphic values were lower than
those in rats given lipidated OspA derived from the
strain B31 sequence (groups 1–3); the differences did
not reach significance, however, and the histologic
changes were qualitatively similar but milder.
Figure 1. Ratio of the uptake of 99mTc in the right and left knee joints
(L:R ratio) of groups of rats injected with different lipidated (Lip.)
outer surface protein (OSP) preparations on days 2 and 7. On day 2,
values in rats receiving lipidated OspA (sequences B31 and K48) and
lipidated OspC (B31) were significantly raised, and values in rats
receiving lipidated OspB (B31) were not. By day 7, only values in
groups receiving lipidated OspA (B31) and lipidated OspC (B31) were
significantly raised. Values are the mean ⫾ SEM simple ratios.
2364
BATSFORD ET AL
Figure 2. Histologic appearance of knee joints from rats challenged with lipidated
outer surface protein (OSP) preparations. A, Example of chronic joint injury induced
by lipidated OspA (B31) in a preimmunized rat 29 days after challenge, showing
severe chronic inflammation of the synovial membrane with focal complete destruction of the cartilage. B, Higher magnification of area in A. C, Example of minimal
damage induced by lipidated OspB (B31) in some rat knee joints 2 days after
challenge. Low power magnification shows no significant abnormalities. D, Higher
magnification of area in C shows discrete area of slight chronic inflammation of the
synovial membrane. (Hematoxylin and eosin stained; original magnification ⫻ 16 in
A and C; ⫻ 80 in B and D.)
Intraarticular injection of lipidated OspB derived
from B31 sequence induced only borderline injury in
preimmunized rats (group 5). There was no increased
uptake of 99mTc in the joint challenged at any time point
examined (Figure 1), the histologic picture was normal
in 2 of the 4 rats examined, and changes were minimal in
the other 2 rats (Figures 2C and D). In an additional
experiment in naive rats, increasing the dose of lipidated
OspB injected from 50 ␮g to 150 ␮g (group 6) did not
increase the uptake of 99mTc (Table 2), and histologic
examination still revealed only minimal changes in 2 of
the 3 rats examined (the other rat was normal).
Effects of synthetic lipopeptides and LPS. The
water-soluble lipopeptide Pam3Cys-Ser-Lys4-OH (see
Materials and Methods) did not produce joint inflammation following intraarticular injection (group 1; Table
3). In a further experiment, lipopeptide Pam3Cys-SerLys4-OH was covalently coupled to BSA to produce an
analog of a larger lipoprotein; this preparation also
failed to induce joint injury (group 2; Table 3). No joint
INDUCTION OF EXPERIMENTAL JOINT INJURY IN VIVO BY OSPs FROM B BURGDORFERI
2365
antigens used for immunization (see Tables 1, 2, and 3),
without subsequent antigen challenge, could cause arthritis.
Retention of lipidated OspA and OspB in joints.
The kinetics of elimination of truncated nonlipidated
and full-length lipidated OspA, as well as full-length
lipidated OspB, are shown in Figure 3. No significant
differences were observed within the 24-hour observation period.
DISCUSSION
Figure 3. External radioactivity measurements at various times after
intraarticular injection of 25 ␮g of 131I-labeled outer surface protein
(OSP) preparations. Values are the mean ⫾ SEM of groups of 4 rats.
There were no significant differences at any time point studied.
injury was seen after intraarticular challenge with 10 ␮g
of LPS (group 6; Table 3). This quantity of LPS far
exceeds that present as a contaminant in the OSP
preparations (0.05–0.5 ␮g of LPS per 50 ␮g of OSP; see
Materials and Methods).
Control experiments. For comparison, a group of
rats were immunized and challenged with recombinant
83-kd protein derived from strain GeHo of B burgdorferi
sensu stricto (cytoplasmic structure protein [synonyms,
94-kd and 100-kd protein]), and no injury was induced
(group 3; Table 3). Because the full-length lipidated
OSP preparations were solubilized in 0.1% Triton
X-100, it was important to exclude an effect of this
detergent on the joint. A group of 4 rats were given
intraarticular injections of 50 ␮l of 0.1% Triton X-100,
and no injury was produced (group 4; Table 3 and Figure
1). Increasing the concentration of Triton X-100 to 1%
resulted in only a borderline increase in 99mTc uptake
and minimal histologic changes in 1 of 4 rats (group 5;
Table 3). Rats given only PBS (group 7) showed no
evidence of injury. The major control in the experimental system used was the contralateral knee joint, which
received only buffer (PBS or 0.1% Triton X-100), and no
signs of inflammation were seen in any of these joints
when they were examined histologically. This latter
finding also effectively excludes the possibility that induction of a strong immune response to the OSP
Only limited information on the potential of
bacterial lipoproteins to induce tissue injury in vivo is
available. Synthetic lipohexapeptide analogs of treponemal and borrelial lipoproteins have been shown to cause
acute skin lesions in mice (12). We previously demonstrated that a model of chronic arthritis could be induced
in rats with a butanol-extracted, water-soluble OSP
complex from B burgdorferi (13). That observation
prompted the current set of experiments, which represent the first study in which the pathogenicity of individual recombinant versions of OSPs was directly studied in vivo. Differences in the arthritogenic potential of
the 3 OSPs studied, OspA, OspB, and OspC, could be
shown.
The concept that borrelial lipoproteins may be
key virulence factors in Lyme disease is also supported
by in vitro data demonstrating that lipidated OspA
induced proliferation and apoptosis of astrocytes (astrogliosis) (23), and might thus contribute to the pathogenesis of neuroborreliosis. The authors of that study obtained a similar result using a synthetic analog of the
lipid modification Pam3Cys, showing that this moiety
also played a central role in this context.
The model used here, known as antigen-induced
allergic arthritis, allows individual testing of selected
substances. Cationic molecules are particularly effective
in this experimental setting (24), which has, in part, been
attributed to their prolonged persistence following attachment to anionic structures in the joint, as originally
shown for chemically cationized antigens (20,22), and
subsequently extended to a cationic antigen from the
bacillus Yersinia enterocolitica (25). In the case of B
burgdorferi, both individual OSPs and the OSP complex
originally prepared by butanol extraction are cationic (26).
The results presented here allow some conclusions to be drawn concerning properties of borrelial
OSPs that may confer pathogenicity. Clearly, the lipid
modification plays a central role, because its absence
drastically reduced the severity of arthritis induced in
2366
rats. The observation that synthetic lipopeptide analogs
of treponemal and borrelial lipoprotein induced dermal
inflammation in vivo (12) provides direct experimental
support for this viewpoint. In addition, as discussed
above (23), a synthetic analog of the lipid moiety was
shown to be effective in an in vitro test system (23), in
which it could induce astrogliosis. However, in the
current study, a synthetic analog of the lipid moiety
alone failed to induce joint injury, which could indicate
that the mechanisms involved are organ/structure specific. The latter observation cannot be simply explained
by the difference in the size of the molecules (31 kd for
monomeric lipidated OspA, and 1.6 kd for lipopeptide
Pam3Cys-Ser-Lys4-OH). First, coupling the synthetic
lipopeptide to a larger protein (BSA) (product in monomeric form ⬃69 kd) failed to produce an arthritogen.
Second, lipidated OspB (34 kd) did not produce injury,
even in preimmunized rats, in spite of possession of the
same lipid modification. Even challenge with the 3-fold
dose of lipidated OspB (raised from 50 ␮g to 150 ␮g)
failed to cause significant injury. The leader sequence of
OspB was, for technical reasons (see Materials and
Methods), also used to produce recombinant, lipidated
OspC. This latter molecule also induced arthritis, which
indicates that the lipid modifications of OspA and OspB
differ little in respect to injury-inducing potential.
In contrast, the peptide portions of the OSP
alone are not very effective in inducing injury. When
nonlipidated, pure-peptide OspA and OspB were tested,
only polymerized peptide OspA derived from strain B31
induced mild, transient injury and then only in preimmunized rats. All of these data indicate that the fatty
acid modification is essential, but not sufficient, for
inducing injury; the peptide region appears to contribute. The evidence suggests that the OspB peptide sequence may differ in a critical manner. On comparing
some relevant properties of OspA and OspB, the kinetics of joint retention of both lipidated OspA and lipidated OspB were found to be similar and did not differ
from the kinetics of retention of nonlipidated OspA.
This finding excludes more rapid elimination of OspB as
an explanation for its failure to induce injury. It is also
unlikely that differences in NF-␬B activation are involved, because all lipidated OSPs (OspA, OspB, and
OspC) were able to activate this major inflammatory
pathway in selected target cell populations, whereas the
nonlipidated versions were not effective (6,27) (R. Lohrmann, J. Dunn, S. Batsford. Abstract P304, VIII International Conference on Lyme Borreliosis, Munich, Germany, 1999).
Genes coding for lipoproteins are proportionally
BATSFORD ET AL
more abundant in B burgdorferi compared with other
bacterial pathogens (28). The B burgdorferi genome
contains ⬎150 genes coding for putative lipoproteins,
compared with a known 22 genes in another spirochete,
Treponema pallidum (29). In B burgdorferi these genes
account for ⬎5% of the chromosomal open reading
frames and 14.5–17% of functionally complete open
reading frames on plasmids. In contrast, chromosomal
lipoprotein genes account for 2.1% and 1.3% of the
open reading frames in the spirochete T pallidum and
the bacillus Helicobacter pylori, respectively (28).
Expression of OSP gene products in the tick
vector and the mammalian host has been studied previously (30). In unfed ticks, B burgdorferi usually expresses
OspA and more rarely expresses OspC (31–33). Following attachment to the host and feeding, the spirochetes
multiply, shed or down-regulate OspA, and up-regulate
OspC within the tick (32,34). This pattern of OSP gene
expression is probably maintained both during transmission to and in the early phase of infection in the
mammalian host (29,32,33). In persisting infections it is
thought that a reversal eventually occurs, with OspA
being up-regulated and OspC down-regulated. The observation that antibody to OspC is a marker of acute
infection in humans (35,36), whereas antibody to OspA
appears at later stages (37), is consistent with this
presumed pattern of gene expression in humans. A
detailed analysis of 137 lipoprotein genes from B burgdorferi in a murine model revealed that 116 genes were
transcribed within 10 days of transmission; thereafter,
many genes were down-regulated, until finally only 40 of
137 were expressed (29). In contrast to the fluctuations
seen with the OspA and OspC genes, the OspB gene
appeared to be continuously expressed, which provides
an interesting parallel to the relative abilities of these
particular gene expression products to induce injury in
the model described.
B burgdorferi does not produce true LPS, and
genomic analysis has shown that LPS biosynthetic genes
are absent (28). The recombinant proteins used in the
current study contained LPS in quantities comprising
0.1–1.0% of total protein, presumably arising as a contaminant during production and purification. Based on
the standard 50-␮g intraarticular challenge protocol,
rats would then have received 50–500 ng of LPS in
addition to that in OSP sample. Many of these recombinant samples induced no injury despite the LPS contamination, and lipidated OspA (B31) had the lowest
level of LPS (50 ng/50 ␮g OspA), although it induced the
most severe injury. Direct challenge with as much as 10
␮g of LPS also produced no detectable joint injury.
INDUCTION OF EXPERIMENTAL JOINT INJURY IN VIVO BY OSPs FROM B BURGDORFERI
Taken together, these results indicate that LPS did not
contribute to injury in these studies.
Local inflammation initiated by OSPs, particularly OspA, is a potential major mechanism in the acute
phase of Lyme arthritis. In addition to the experimental
evidence presented here and previously (13), development of arthritis in humans during B burgdorferi infection is associated with humoral and cellular immune
responses to borrelial antigens, including OspA (37–39).
A temporal relationship between the appearance of
antibody to OspA and the onset of joint inflammation
was previously reported (40). The available data do not
support the idea that a vigorous immune response to
OSP antigens alone may precipitate joint inflammation.
As reported here, immunization of rodents with OspA
and OspB did not induce arthritis unless a direct antigen
challenge was made subsequently. This is consistent with
a further study that showed that hamsters previously
immunized with recombinant OspA developed severe
arthritis only after challenge with whole infectious B
burgdorferi organisms (41). Active immunization of humans with lipidated OspA (42) (see below) or dogs with
a bacterin-type vaccine (43) has not yet produced evidence of arthritis. It should be stressed that, in the
model described, acute articular inflammation was seen
without a specific immune response. This is consistent
with the observation that transgenic mice, tolerant to
OspA and OspB, still developed arthritis and other
organ manifestations during subsequent experimental
infection in the absence of specific immunity (44).
In Lyme arthritis the organism usually cannot be
cultured from joint fluid specimens, yet the strong local
inflammatory response suggests that antigen is present.
Genetic material specific for B burgdorferi can be detected in samples of synovial fluid from almost all
untreated patients. According to one report, OspA
sequences were present in 96% of untreated patients
(45). Interestingly, Persing et al (46) have shown that
there is a molecular imbalance in Lyme arthritis, because plasmid-coded genes such as OspA are much
more abundant than genomic genes.
The following scenario may help explain the
occurrence and perpetuation of Lyme arthritis: after
infection, small quantities of spirochetes enter the joint
space and shed OSPs, particularly OspA. Molecular
imbalance might also help lead to increased local antigen production. These OSPs then attach to joint structures and initiate inflammation, probably via TLRs.
Studies on the role of this receptor system in the
induction of arthritis following intraarticular application
of lipidated OspA in genetically manipulated mice are
2367
currently under way in our laboratory. The subsequent
onset of a vigorous immune response (humoral and
cellular) may lead to further aggravation and chronic
inflammation. It has been suggested that autoimmunity
could also develop at this stage (e.g., via molecular
mimicry between an immunodominant T cell epitope of
OspA and human lymphocyte function–associated antigen 1 [hLFA-1], an adhesion molecule expressed on T
cells in the synovium) (47). This notion is controversial,
and recently Th1 cell reactivity to hLFA peptides has
been attributed to the promiscuous nature of the T cell
receptor (T cell degeneracy) (48,49).
The current studies are also relevant for vaccine
safety considerations, because a vaccine based on lipidated OspA is licensed in North America (50,51), and
lipidated OspC is a serious candidate antigen for further
vaccine development (42,52). To date, use of the OspA
vaccine has not been associated with significant numbers
of untoward reactions in joints or other organs (50,51).
However, because vaccine efficacy is ⬃70–80%, concern
must be raised concerning individuals who are sensitized
to OspA but subsequently still become infected. At
present, three 30-␮g intramuscular injections of lipidated OspA are recommended for protection. Using this
latter regimen, it is not very likely that lipidated OspA
could enter any joint in quantities sufficient to trigger
local inflammation, although great caution should be
used if the dose of antigen is significantly increased or if
DNA vaccines are introduced.
Experiments with lipoproteins derived from the
bacterium B burgdorferi, the causative agent of Lyme
disease, demonstrate that these molecules can be capable of inducing severe tissue injury in vivo. This class of
protein should certainly be regarded as a major virulence factor. Particular lipoproteins derived from B
burgdorferi, the surface-expressed OSPs A and C, could
play an important role in the induction of arthritis, a
frequent clinical complication of Lyme disease.
ACKNOWLEDGMENTS
We thank Jürgen Schulte Mönting (Institute of Medical Biometry and Informatics, Freiburg, Germany) for performing the statistical analysis. Recombinant 83-kd protein
from B burgdorferi was provided by Sebastian Rauer (Department of Neurology, University Hospital, Freiburg, Germany).
The LPS we used was a kind gift from Marina Freudenberg
(Max-Planck Institute of Immunobiology, Freiburg, Germany).
The skilled technical assistance of Oliver Schweier and Barbara Lade is acknowledged.
2368
BATSFORD ET AL
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