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T cell proliferation induced by Borrelia burgdorferi in patients with lyme borreliosis. Autologous serum required for optimum stimulation

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393
T CELL PROLIFERATION INDUCED BY
BORRELIA B URGDORFERI IN PATIENTS WITH
LYME BORRELIOSIS
Autologous Serum Required for Optimum Stimulation
ANDREAS KRAUSE, VOLKER BRADE, CHRISTOPH SCHOERNER, WERNER SOLBACH,
JOACHIM R. KALDEN, and GERD R. BURMESTER
The cellular immune response to Borrelia burgdorferi was studied in 24 patients with seropositive and
seronegative Lyme borreliosis, 30 patients with arthritides of different origin (non-Lyme arthritides), and 20
normal blood donors. By far, the strongest T cell
stimulation was induced by incubation with autologous
serum; there was a significantly lower response or no
response after incubation with allogeneic or heterologous sera. In patients with Lyme borreliosis, including
seronegative patients, there was a strikingly elevated
proliferation in response to whole B burgdorferi bacteria
(mean 64,750 dpm) compared with that of normal
donors (mean 19,700 dpm; P < 0.0001) and especially
that of non-Lyme arthritis patients (mean 11,600 dpm;
P < 0.0001). Levels of proliferation declined significantly in patients with Lyme borreliosis after successful
antibiotic treatment. Parallel cultures using B burgdorf e n and Treponema phagedenis as antigens showed that
cells from patients with Lyme borreliosis responded
significantly more to B burgdorferi than to Tphagedenis,
~-
From the Institute of Clinical Immunology and Rheumatology, Department of Medicine 111, and the Institute of Clinical Microbiology, University of Erlangen-Nuemberg, Erlangen, Germany.
Andreas Krause is a postdoctoral fellow of the Deutsche
.
Forschungsgemeinschaft, grant Kr 99711.1.
Andreas Krause, MD: Postdoctoral Fellow, Department of
Medicine 111; Volker Brade, MD: Professor of Microbiology, Institute of Clinical Microbiology; Christoph Schoerner, MD, MSc:
Institute of Clinical Microbiology; Werner Solbach, MD: Professor
of Microbiology, Institute of Clinical Microbiology; Joachim R.
Kalden, MD: Professor of Medicine, Department of Medicine 111;
Gerd R. Burmester, MD: Professor of Rheumatology and Clinical
Immunology, Department of Medicine 111.
Address reprint requests to Gerd R. Burmester, MD, Institute of Clinical Immunology and Rheumatology, Department of
Medicine 111, Krankenhausstrasse 12, D-8520 Erlangen, Germany.
Submitted for publication May IS, 1990; accepted in revised
form October 8, 1990.
Arthritis and Rheumatism, Vol. 34, No. 4 (April 1991)
but this did not occur with cells from individuals with
non-Lyme arthritides. There was no correlation between disease stages and proliferation values. These
data indicate that lymphocyte proliferation assays may
provide an important tool for the diagnosis of Lyme
borreliosis, most notably in patients with arthritides and
in those who are seronegative. Conversely, the lack of
reactivity appears to be a strong indicator of the absence
of active Lyme disease. It seems to be crucial, however,
to use autologous sera in these assays.
Lyme borreliosis is a multisystem disorder
caused by the spirochete Borrelia burgdorferi (1-3).
Because of the vector required by the spirochete,
lxodes ticks, the disorder occurs primarily in persons
who have been in or who live near wooded areas of
countries that have a moderate climate. Thus, many
cases have been reported in North America and Europe, although different ticks were found to have
transmitted the bacterium (2,3). The course of the
disease can be separated into 3 stages, which demonstrate different clinical symptoms (2,3). Only a few
patients experience all 3 stages, and some even
present with disease manifestations of the late stage,
but no history of previous symptoms (4-8). Therefore,
in contrast to the typical skin lesions or neurologic
signs of early disease (4-7,9), the manifestations of the
chronic stages impose severe diagnostic problems.
Many clinical manifestations are nonspecific (4-lo),
and results of serologic tests, though mostly positive
during these later disease stages (11-14), are often
difficult to interpret, since the detection of antibodies
to B burgdorferi frequently only signifies a previous
infection, and not necessarily active disease (11-15).
KRAUSE ET AL
394
Table 1. Clinical characteristics of patients with Lyme borreliosis, patients with arthritis not caused by Lyme borreliosis (non-Lyme arthritis),
and normal control subjects*
No. with positive
n
Patients with Lyme borreliosis
ECM (convalescence)
Stage I1
Arthritis
Patients with non-Lyme arthritis
Rheumatoid arthritis
Oligoarthritis
Psoriatic arthritis
Other
Normal controls
24
3
5
16
30
13
11
3
3
20
Males/
females
12/12
211
1I4
917
14/16
419
714
1I2
112
1317
No. with
Mean age
(range)
IFA titer
(range of titers)
No. with
No. with
tick bite
ECM
43 (8-69)
20 (1:32-1:4,096)
0 (-1
4 (1:32-1:512)
16 (1:32-1:4,096)
3 (1:32-1:128)
14
3
3
8
8
3
I
4
-
57 (49-64)
29 (8-49)
45 (26-69)
45 (18-77)
54 (24-68)
39 (18-77)
41 (3249)
32 (21-48)
28 (21-39)
1(1:64)
2 (1:32-1:128)
0 (-)
0 (-)
I (1:321
-
-
neurologic
manifestations
-
-
4
2
2
-
* Oligoarthritis patients had reactive arthritides and HLA-B27-associated spondylarthropathies.Neurologic manifestations were of stage I1
disease (disseminated early infection), especially meningopolyneuritis. IFA = immunofluorescence assay; ECM = erythema chronicum
migrans.
There have been several attempts to use cell
assays as a way to enhance the diagnostic repertoire
and to learn more about the host’s immune response to
this infectious agent. However, the results reported so
far have shown conflicting data. Sigal and coworkers
(16) and Weyand and Goronzy (17) described rather
moderate immune responses of peripheral blood lymphocytes, especially those from patients with early
stages of infection, whereas Dattwyler et a1 (18-20)
found vigorous T cell responses, even in patients who
were seronegative.
The objective of the present investigation was
to study the cellular immune response against B burgdorferi, focusing particularly on the establishment of
optimum conditions for T cell proliferation. The patient population consisted of large groups of subjects
with an ethnically homogeneous (Bavarian) background, which may differ from the American counterpart. The data demonstrate that autologous serum is
crucial for an optimum cellular response. This enhancing effect was not restricted to B burgdorferi antigens.
It could also be demonstrated with tetanus toxoid. The
effect was observed in cells from patients with Lyme
borreliosis as well as cells from most of the normal
donors.
The serum factors leading to this enhanced T
cell proliferation to B burgdorferi have not yet been
identified; isolated immunoglobulins did not show enhancing effects. Moreover, enhanced stimulation by
serum factors was also evident in seronegative patients. However, the possibility of a nonspecific effect
of Lyme borreliosis patient sera leading to enhanced T
cell proliferation was ruled out by crossover experi-
ments, which did not show increased proliferation of T
cells from normal donors when the cells were incubated in the presence of sera from Lyme borreliosis
patients. In the autologous system, proliferation after
stimulation with B burgdorferi was significantly elevated in cells from patients with Lyme borreliosis
compared with those from normal donors. Of particular interest, the lowest values were obtained with cells
from patients with arthritides not caused by Lyme
borreliosis (non-Lyme arthritides). Parallel cultures
revealed a significantly higher response to B burgdorferi than to Treponema phagedenis only by cells from
patients with Lyme borreliosis.
PATIENTS AND METHODS
Patients and control subjects. The clinical characteristics of the patients and control subjects studied are summarized in Table 1. Twelve of the 24 patients with Lyme
borreliosis were male, and 12 were female. Their age ranged
from 8 years to 69 years (mean 43). Fourteen patients
reported a tick bite. Twenty patients had antibodies to B
burgdorferi, as determined by immunofluorescence assay
and Western blot analysis; 4 patients (3 with erythema
chronicum migrans [ECM] and 1 with stage I1 Lyme disease)
had no detectable antibodies.
At the time of the first examination, 3 patients were
in the convalescence phase from typical ECM, and all of
them had been treated with antibiotics. Five patients presented with symptoms of a disseminated early infection
(stage 11) (4), including chronic fatigue, pain in the joints,
muscles, or tendons, and lymphadenopathy. Two of these
patients had a meningopolyneuritis. Two other individuals
had ophthalmologic complications, with choroiditis and retinal vasculitis, respectively. These 2 patients had been
treated with doxycycline prior to the present investigation.
The remaining 16 patients with Lyme borreliosis had
INDUCTION OF T CELL PROLIFERATION BY B BURGDORFERl
late infection (stage 111), with oligoarthritis, which included
the knee joint in 13 patients, and constitutional symptoms.
Two of these patients had a history of neurologic manifestations, 4 individuals remembered having had an ECM, and
1 of them had acrodermatitis chronica atrophicans. Three
patients experienced mild neurologic symptoms, including
subtle ataxia and difficulties in concentrating. Eight of these
16 patients had already received antibiotic therapy.
An additional group of patients were studied in
parallel. These 30 individuals, 16 females and 14 males, had
arthritides of different origin (referred to as “non-Lyme”
arthritis). The patients’ ages ranged from 18 years to 77 years
(mean 45). Thirteen patients had rheumatoid arthritis (RA),
as diagnosed according to the American Rheumatism Association criteria (21). Eleven patients presented with reactive
oligoarthritides, including Reiter’s syndrome or HLA-B27associated seronegative spondylarthropathies. In 3 patients,
psoriatic arthritis had been diagnosed. The remaining 3
patients had pauciarticular arthritides caused by BehCet’s
disease, systemic sclerosis, and ankylosing spondylitis, respectively.
Three of these patients with non-Lyme arthritis (1
with RA, 2 with seronegative spondylarthropathies) had IgG
antibodies to B burgdorferi (titers 1:32, 1:64, 1:128, respectively). None of these patients had a history of skin lesions
or neurologic symptoms. All 3 patients had been treated
according to established regimens with antibiotics, but there
had been no clinical improvement. These patients were
therefore considered not to have active Lyme disease.
The control group consisted of 20 healthy normal
donors (8 women and 12 men) ages 21-39 (mean 28). One
individual had a low titer of IgG antibody against B burgdorferi (1:32), but no history or symptoms compatible with
Lyme borreliosis.
Preparation of mononuclear cells and T cells. Mononuclear cells (PBMC) were isolated from heparinized peripheral venous blood by Ficoll-Hypaque (Nycomed, Oslo,
Norway) density-gradient centrifugation. After washing 3
times in Hanks’ balanced salt solution, the cells were resuspended in RPMI 1640 (Biochrom, Berlin, FRG), supplemented with L-glutamine (2 mM; Sigma, St. Louis, MO),
HEPES buffer (15 mM; Gibco, Eggenstein, FRG), penicillin/
streptomycin (100 IU/ml and 100 pg/ml, respectively;
Gibco), and either 10% fetal calf serum (FCS) (Gibco), 10%
pooled human AB serum, or 10% autologous serum.
Throughout the experiments, identical batches of AB serum
or FCS were used. These sera had been tested for low
toxicity and low background mitogenicity in proliferation
assays.
For the isolation of T cells, a single-gradient rosetting
procedure with neuraminidase-treated sheep red blood cells
was performed. This yielded an average of 90-95% mature
(CD3-positive) T cells. The non-T cell fraction contained
monocytes and B lymphocytes bearing the monocyte differentiation antigen CD14 ( - 5 0 4 0 % ) and the B cell antigen
CD20 (-30-40%), respectively.
Lymphocyte proliferation assay. Using 96-well microtiter plates (Nunc, Roskilde, Denmark) lo5 cells/well were
cultured in triplicate in RPMI 1640 medium supplemented as
described above, at 37°C in a humidified atmosphere with 5%
CO,. Cells were stimulated with whole (20) B burgdorferi
395
(strain PKo 2-85) (3) at a concentration of lo6 and lo5
bacteridwell (corresponding to 3 pg and 0.3 pg of protein
per well, respectively). The B burgdorferi strain PKo 2-85
had been isolated from the skin of a Bavarian patient with
ECM and was kindly provided by Dr. Vera Preac-Mursic
(Max-von-Pettenkofer-Institute, Munich, FRG) (3). The spirochetes were maintained in a modified Kelly medium at
33°C and subcultured every 4-6 days, as described previously (14).
Initial dose-response experiments had been conducted using lo3 to 2 x lo6 borreliae/well. In some samples,
maximum stimulation was reached with lo5 bacteridwell,
while in most, optimum proliferation was found with lo6
bacteridwell; there were no apparent correlations with the
diagnosis or with other clinical parameters. Therefore, in all
subsequent experiments, lo5 and lo6 borreliae/well were
used to achieve maximum stimulation.
Control cultures were incubated with ultrasonicated
T phagedenis (Behringwerke, Marburg, FRG). This spirochete is of the same family as B burgdorferi, and in Germany, it is routinely used to preabsorb sera to eliminate
cross-reacting antibodies prior to diagnostic serologic assays
for syphilis and for Lyme borreliosis. Initial dose-response
experiments showed that protein concentrations between 0.7
pg/well and 7 pg/well were optimal.
Parallel cultures were performed with medium alone,
with tetanus toxoid (10 pg/ml; Behringwerke), or with lipopolysaccharide (LPS) (10 pg/ml of Escherichia coli serotype
0128:B12; Sigma). Cells were cultured for 6-7 days. During
the final 18 hours of culture, 1 pCi of 3H-thymidine (specific
activity 2 Ci/mmole; Ammersham Buchler, Braunschweig,
FRG) was added to each well. Cells were harvested semiautomatically, and the proliferation was determined by measuring tritiated thymidine incorporation, using a liquid betascintillation counter (LKB Wallac, Turku, Finland). The
results are expressed as average disintegrations per minute
(quench correction by an internal standard) in triplicate
wells. The differences (A) in dpm were calculated by subtracting the control values in the absence of antigens or
mitogens from the maximum proliferation values in the
presence of the stimuli.
Preparation of IgG. Immunoglobulin G was prepared
from the serum of a patient with Lyme arthritis who had a
vigorous cellular immune response and a high titer of antibody to B burgdorferi (I: 1,024) in the immunofluorescence
test. To isolate the IgG, serum was mixed with DEAE-52
cellulose (Serva, Heidelberg, FRG), suspended in 0.01M
phosphate buffer, pH 7.5, at a concentration of 7 gm of
cellulose (wet weight) per ml of serum, and incubated at 4°C
for 2 hours. Samples were mixed repeatedly with an equal
volume of buffer. After sedimentation of the cellulose, the
supernatant containing the unbound IgG was collected.
Finally, the supernatant was sterilized by filtration (0.2-pm
pore size), and the IgG concentration was adjusted to 100
pg/ml by adding phosphate buffered saline solution (Gibco).
The activity of this IgG preparation was verified in the
immunofluorescence assay, and the same titer was found
(compared with the initial serum level).
Indirect immunofluorescence assay for the detection of
antibodies to B burgdorferi. The indirect immunofluorescence
test was performed as described earlier (14). Briefly, 10-pl
KRAUSE ET AL
100-
0
,-
80-
E
-
4
60-
1
3
&
.
40-
D
E,
2
20-
0controts
Non-LB
ArlhriliS
Lyme
Borrellorls
Figure 1. Response of peripheral blood mononuclear cells to Borrelia burgdorferi in cultures containing autologous serum or fetal
calf serum (FCS).Patients with Lyme borreliosis and a few normal
donors (controls) with elevated proliferation to B burgdorferi had
maximum lymphoproliferative responses in cultures with autologous sera and marginal proliferation in cultures with FCS.Patients
with non-Lyme (non-LB) arthritides had much lower responses, and
in general, there were no significant differences (n.s.) in proliferation
between autologous sera and FCS cultures.
aliquots of a bacterial suspension containing lo7 borreliae/ml
were applied to each spot of multitest slides (Biomerieux,
Niirtingen, FRG) and air-dried at 22°C. Patient sera (25 pl)
were added after absorption with T phagedenis to eliminate
cross-reacting antibodies (starting with a 1: 16 dilution).
Binding of specific antibodies was detected by staining with
a polyvalent fluorescein isothiocyanate (F1TC)-conjugated
goat anti-human immunoglobulin (Biomerieux), used at a
1:200 dilution. For the detection of specific IgM antibodies,
all patient sera were additionally pretreated with rheumatoid
factor absorbent (Behringwerke) to prevent false-positive
reactions due to rheumatoid factor or false-negative results
because of competitive inhibition by specific IgG. Binding of
specific IgM was visualized by FITC-labeled goat antihuman IgM (Medac, Hamburg, FRG), diluted 1:25.
Statistical analysis. The stimulation values of each
group were compared by the Mann-Whitney U test when
appropriate. For the comparison of paired samples, Student's t-test was used.
0.0001). Cultures in allogeneic normal AB sera resulted in moderate levels of stimulation, which were
significantly lower than those in the autologous system
( P < 0.05) (Figure 2). The enhanced proliferation with
autologous serum was also evident in Lyme disease
patients who were seronegative in both immunofluorescence and Western blotting analysis. This effect was
even observed in the few normal donors who demonstrated significant T cell responses to B burgdorferi but
did not have detectable antibodies to the spirochetes
(Figures 1 a d 2). In contrast, patients with non-Lyme
arthritides did not show these differential responses and
generally had very low stimulation values (Figure 1).
Parallel investigations of unseparated PBMC, isolated T
lymphocytes, and the remaining cell fraction consisting
of monocytes and B lymphocytes demonstrated that the
responses were primarily attributable t6 the proliferation
of T cells (Table 2).
Further experiments using tetanus toxoid as
stimulus revealed that the effect that led to the enhanced T cell proliferation was not restricted to B
burgdorferi antigens. In Lyme borreliosis patients,
'"1
A
n
I-
- 1
pc o . 0 5
7
t
ao-
X
E
'0
-a
60-
aJ
x
m
L
n
3
aJ
.-S
40-
'0
E,
:
c
20-
n
RESULTS
Requirement of autologous serum for optimum T
cell stimulation. After kinetic and dose-response studies (see Patients and Methods), initial experiments
were performed to define the optimum culture conditions for maximum T cell stimulation in patients with
Lyme borreliosis. The results (Figure 1) demonstrate
that incubation with autologous serum elicited the
highest response, whereas incubation with FCS did
not allow for significant T cell proliferation (P <
0Autologous
Serum
AB Serum
Figure 2. Response of peripheral blood mononuclear cells (PBMC)
cultured in autologous serum or human AB serum. As with fetal calf
serum, there was significantly lower proliferation of PBMC cultured
in pooled human AB serum than in autologous serum. 0 = Lyme
borreliosis patients; 0 = normal donors.
397
INDUCTION OF T CELL PROLIFERATION BY B BURGDORFERI
Table 2. Proliferation of unseparated peripheral blood mononuclear cells (PBMC), isolated T cells, and B celldmonocytes from
patients with Lyme borreliosis in response to stimulation with B
hurgdorferi*
Patient
PBMC
T cells
B celldmonocytes
1
2
3
4
5
34,200
57,400
65,500
132,600
137,300
33,300
34,900
43,900
92,000
114,300
7,200
2,900
3,900
12,100
22,400
* Values are differences in dpm, calculated by subtracting the
control value in the absence of antigens or mitogens from the
maximum proliferation values in the presence of the stimuli; background dpm range of unstimulated cells was 1,1604,100.
significantly higher responses were obtained in the
presence of autologous serum than in the presence of
FCS, except for 1 patient who did not respond to
tetanus toxoid (Figure 3 ) . Interestingly, in the control
group, the markedly higher levels of proliferation in
autologous serum were detected only in those 4 individuals who exhibited vigorous responses to tetanus
,---
200-
-
1
Autologous
Serum
D
o
n.s.
4-
FCS
Autologous
Serum
p<0.051
FCS
160-
r
Y
E
U
a_
120-
Y
m
I
Q
3
a,
.-
80-
9
5
8
400
0I
I
I
Controls
Lyme Borreliosis
Figure 3. Response of peripheral blood mononuclear cells stimulated with tetanus toxoid and cultured in autologous sera or fetal calf
serum (FCS). Patients with Lyme borreliosis had higher proliferation values in cultures with autologous sera than in cultures with
FCS, except for 1 patient whose cells were unresponsive to tetanus
toxoid. Only the 4 control subjects whose cells vigorously responded to tetanus toxoid had higher values in the presence of
autologous serum. Two controls had equivalent proliferation values
under both conditions, and 1 had maximum responses in cultures
with FCS. In both groups of subjects tested, significant responses to
tetanus toxoid were obtained in FCS and in autologous sera. n.s. =
not significant.
I
Controls
I
Non-LB
Arthritis
I
Lyme
Borreliorir
I
Figure 4. Response of peripheral blood mononuclear cells to Borrelia burgdorferi and Treponema phagedenis in patients with Lyme
borreliosis, those with non-Lyme borreliosis (non-LB) arthritides,
and normal donors (controls). T cell reactivity to B burgdorferi was
significantly higher than that to T phagedenis only in patients with
Lyme borreliosis. The patients with non-Lyme arthritides and the
controls had similar or, sometimes, even weaker responses to B
burgdorferi than to T phagedenis. n s . = not significant.
toxoid. Among the remaining control subjects with
moderate T cell proliferation, 2 showed equivalent
proliferation values under both test conditions, and in
1 donor, incubation of cells with FCS allowed for the
maximum response. Of special interest, in contrast to
B burgdorferi, tetanus toxoid induced a significant T
cell response even in FCS in both groups of subjects.
B burgdorferi induction of proliferation of T cells
from patients with Lyme borreliosis. All subsequent
experiments were carried out using autologous sera
unless otherwise indicated. Figure 4 demonstrates that
the T cell responses of patients with Lyme borreliosis
were primarily induced by B burgdorferi, as documented by parallel experiments in which Tphagedenis
was used as the alternative antigen. In these experiments, stimulation of PBMC by B burgdorferi resulted
in significantly higher thymidine incorporation compared with that by Tphagedenis ( P < 0.006). In contrast,
patients with non-Lyme arthritides and normal donors in
general showed similar and, sometimes, weaker responses to B burgdorferi than to T phagedenis.
PBMC from both patient groups and from the
normal donors showed a moderate proliferative response to T phagedenis antigens. However, proliferation of cells from Lyme borreliosis patients in response to T phagedenis was significantly increased
compared with that of cells from patients with non-
KRAUSE ET AL
398
Lyme arthritides (P < 0.03), but not in comparison
with that of cells from normal controls ( P = 0.08).
Additional experiments examined a possible
influence of LPS in the T cell proliferation assay. The
results demonstrated that LPS induced only a rnoderate response of cells from patients with Lyme borreliosis (mean 8,700 dpm; 22 patients) as well as cells
from normal donors (mean 10,844 dpm; 15 subjects),
while cells from patients with non-Lyme arthritides
showed only minimal proliferation (mean 3,914 dpm;
20 patients).
Elevated T cell responses to B burgdorfen' in
patients with Lyme borreliosis. The lymphoproliferative response to stimulation with B burgdorferi was
strikingly elevated in patients with Lyme borreliosis
(mean 64,750dpm) compared with the normal donors
(mean 19,700 dpm; P < 0.0001)and, especially, the
non-Lyme arthritis patients (mean 11,600 dpm; P <
0.0001)(Figure 5). There was no apparent difference in
T cell proliferation between seropositive patients and
the 4 seronegative patients. No correlation was observed between proliferation values and disease
stages. Comparison of the responses of T cells from
non-Lyme arthritis patients and normal donors revealed significantly reduced proliferation of cells from
the arthritis patients (P < 0.006). It is of interest,
however, that nearly all normal donors and most of the
non-Lyme arthritis patients showed a moderate T cell
response to B burgdorferi and a few subjects in both
groups showed very high proliferation values.
Decline of T cell proliferation after treatment of
Lyme borreliosis. In 6 patients with Lyme borreliosis
(4with arthritis and 2 with stage I1 disease) who were
treated successfully with doxycycline or ceftriaxone,
T cell proliferation and titers of antibody to B burgdorferi (by immunofluorescence assay) were evaluated
prospectively. The corresponding data were obtained
in parallel at the beginning of therapy and 2 months
later, at which time, there were no clinical signs of
active disease. Serologic indicators of inflammatory
activity (erythrocyte sedimentation rate, C-reactive
protein level, serum protein levels) showed normal
values for all 6 patients. As shown in Figure 6, the
cellular immune response had markedly declined after
successful therapy, but proliferation values remained
significantly elevated above normal levels (Figure 5).
Antibody titers decreased 4-fold or more in 4 patients,
2 of whom became seronegative (titer <1:16), but
titers remained stable in 2 individuals.
140
120
100
0
80
?
i
P
60
.
.
..
40
20
.
a
0
I
C o nt r o I s
~~
I
Non-LB
Arthritis
I
Lyme
Borreliosis
Figure 5. Cellular immune response of peripheral blood mononu
clear cells to Borrelia burgdorferi in patients with Lyme borreliosis,
those with non-Lyme borreliosis (non-LB) arthritides, and normal
donors (controls). T cell proliferation was strikingly increased in
Lyme borreliosis patients, even in seronegative patients ( O ) ,compared with that in normal donors and, especially, that in non-Lyme
arthritis patients. The non-Lyme arthritis group also had a significantly reduced response compared with the controls. Bars show
group means.
Effect of different Lyme borreliosis sera and IgG
on T cell proliferation. To further investigate the
different effects of serum factors on T cell proliferation
stimulated by B burgdorferi, IgG was isolated from the
serum of a patient with Lyme borreliosis who had a
high antibody titer (1:1,024) and a vigorous T cell
response to Borrelia antigens. This IgG preparation
was then added to cultures containing pooled human
AB serum (final concentration 6-60 pg of IgG/ml).
Maximum proliferation values in parallel cultures with
autologous serum, AB serum alone, or AB serum
supplemented with IgG were compared. The addition
INDUCTION OF T CELL PROLIFERATION BY B BURGDORFERI
Lymphocyte
Prol#teral#on Assay
lmmunollvorercence
Table 3. T cell proliferative responses after incubation in autologous sera and allogeneic sera from patients with Lyme borreliosis*
Tefl
140 7
132.
~
~~
~
Autologous serum
Allogeneic serum
(source)
39,700
26,800
26,800
9,000
15,300
19,600 (AZM)
470 (AZM)
5,000 (FP)
5,900 (AZM)
23,200 (SD)
3,900
3,900
2,400
6,800
6,300
7,800 (SD)
2,800 (SB)
3,500 (AZM)
1,900 (SD)
3,299 (SD)
125
Subject
g
4
JB
m
FP
Betore
Afler
Therapy
Before
Alter
Therapy
Figure 6. T cell proliferation values and IgG antibody titers to
Borrelia burgdorferi before and 2 months after antibiotic therapy in
6 patients with Lyme borreliosis. The cellular immune response
declined significantly after therapy, but stimulation values remained
markedly elevated above normal levels. Immunofluorescence test
(IFT) titers decreased significantly (by at least 2 titers) in 4 patients,
and 2 patients became seronegative (0 = titer <1:16). The patients’
initials are shown.
of IgG did not lead to an increase in lymphocyte
proliferation (Figure 7).
Further experiments examining the effects of
various seropositive patient sera on T cell response
’0°1
D
\
0
l-
X
-
a
399
Patients with Lyme
borreliosis
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Normal donors
Control 6
Control 7
Control 8
Control 9
Control 10
* Values are differences in dpm, calculated by subtracting the
control value in the absence of antigens or mitogens from the
maximum proliferation values in the presence of the stimuli. The
serum source shows the patients’ initials, whose proliferative responses were as follows: AZM 77,500 dpm, FP 65,000 dpm, SB
35,000 dpm, and SD 15,000 dpm.
were performed. PBMC from patients with Lyme
disease and from normal donors were cultured simultaneously in medium containing 10% autologous or
allogeneic serum derived from Lyme borreliosis patients. Table 3 demonstrates that in cells from 4 of the
5 patients studied, incubation with allogeneic sera
resulted in a markedly lower cell proliferation compared with that with autologous sera. In 1 patient’s
cells, incubation with allogeneic serum led to a higher
T cell response. In normal donors, Lyme borreliosis
patient sera did not induce enhanced proliferation,
which indicates that the autologous serum effect is not
due to the presence of a nonspecific factor which
enhances T cell stimulation (Table 3).
at
m
Y
c
DISCUSSION
n
a
0-J
I
I
1
Autologous
Serum
AB Serum
AB Serum
+ IgG
Figure 7. T cell responses after incubation in autologous serum,
pooled human AB sera, and AB serum supplemented with 6-60
pgiml of IgG from a seropositive patient with Lyme borreliosis (only
maximum proliferation values are shown). Addition of IgG did not
result in an increase in lymphocyte proliferation.
The following major points emerged from the
present investigation. 1) Autologous serum was required for optimal T cell stimulation. 2) No evidence
was obtained, as yet, that this enhanced stimulation
was mediated by immunoglobulins, since the increased proliferation was also observed in cells from
seronegative patients. Moreover, an isolated IgG fraction did not enhance stimulation. 3) Patients with
Lyme disease showed a strikingly elevated T cell
responsiveness, which was mostly specific for B burgdorferi. In contrast, the minor T cell stimulation seen
in normal donors and patients with non-Lyme arthritides appeared to be primarily directed against anti-
400
genic determinants also present on nonpathogenic
spirochetes and/or on lipopolysaccharide.
The initial experiments outlined in the present
investigation focused on the establishment of optimum
culture conditions. The results obtained demonstrated
a strikingly enhanced T cell proliferation toward B
burgdorferi in cultures with autologous serum, while
there was no proliferation or only marginal proliferation in cultures with FCS and only moderate responses
in cultures with allogeneic normal sera. Considerable
effort was made to investigate the nature of these
factors. Even though immunoglobulins appeared to be
the most likely sources (22), an isolated IgG fraction
derived from a patient with vigorous T cell proliferation did not elicit elevated T cell responses. Moreover,
the enhancing effect of autologous serum was evident
in patients who were seronegative, as tested by immunofluorescence and Western blot analysis.
However, the initial data obtained in the
present investigation do not rule out the involvement
of immunoglobulins in mediating elevated T cell response to B burgdorferi. In this respect, recent data
presented by Schutzer et al (23) appear to be of
particular importance. Those authors demonstrated
the sequestration of antibodies to B burgdorferi in
immune complexes in seemingly “seronegative” patients. After dissociation of these complexes, reactivity of antibodies was demonstrated both in immunofluorescence and in Western blot analyses. With
regard to the present investigation, it appears possible
that initially complexed antibodies that were not detectable by conventional assays opsonized the live
bacteria used (22) and helped to enhance T cell responsiveness. Moreover, experiments performed in the
present study, using different patients’ sera in “crossover” T cell cultures, demonstrated that in the majority of patients, autologous sera were significantly more
effective in enhancing T cell responses. These data
would strongly suggest a possibly immunoglobulinmediated effect of sera obtained from patients with
Lyme disease.
Interestingly, the effects observed are not specific for Lyme borreliosis sera and are not restricted to
B burgdorferi antigens. Additional experiments were
performed using tetanus toxoid as a T cell stimulus. In
all patients with Lyme borreliosis whose cells responded to tetanus toxoid, but only in those normal
donors whose T cells showed vigorous responses,
significantly higher proliferation values were obtained
with incubations in autologous sera compared with
incubations in FCS. Moreover, one normal donor
KRAUSE ET AL
showed maximum responses to tetanus toxoid in cultures with FCS, while this could not be demonstrated
in borreliosis patients. Of particular importance, however, was the observation that in contrast to B burgdorferi in Lyme borreliosis patients, as well as in
normal donors, tetanus toxoid induced a strong T cell
proliferation even in the presence of FCS. These data
and the finding that patients’ sera did not induce an
enhanced proliferation of normal T cells are strong
evidence against the presence of a mitogenic or antigenic factor in Lyme borreliosis sera that might have
induced a nonspecific T cell proliferation independently of the specific antigen B burgdorferi. The
intricate interplay between T cells, antigens, and
autologous sera appears to be much more complex,
and experiments are currently being performed in our
laboratory to further investigate the serum factors that
lead to an enhanced antigen-driven T cell response.
In any event, the effects of different sera appear
to explain the divergent findings published so far on T
cell responses in patients with Lyme disease. Thus,
only weak or moderate T cell responses were found in
assays using FCS (16,17) while considerably higher
stimulation values have been described in experiments
using human sera (18-20). In contrast, strong responses have been found in T cells obtained from
tissue sites, i.e., synovial fluid (16) and cerebrospinal
fluid (24,25). However, these extravascular T cells
usually represent a different lymphocyte subset, with
characteristics of helperhnducer cells that are readily
activated by antigens (26). Therefore, serum factors
may not be that important in assays of extravascular T
cells. On the other hand, in the present study, there
was a pronounced T cell responsiveness to LPS
present in the synovial fluids from patients with RA
and reactive arthritides, which confounded the results
of the stimulation assays and, therefore, did not allow
for the discrimination of T cell responses readily seen
in blood cells (data not shown).
Using the conditions outlined above, there was
a strikingly elevated response of T cells from the
peripheral blood of patients with Lyme disease compared with that of cells from normal donors and, of
particular importance, compared with that of cells
from patients with non-Lyme arthritides. This elevated responsiveness did not correlate with different
stages of disease and occurred regardless of the presence or absence of detectable antibodies to B burgdorferi. In general, T cell response was rather vigorous
and was comparable to the reaction elicited by common antigens such as tetanus toxoid (Figures 3 and 5 ) ,
INDUCTION OF T CELL PROLIFERATION BY B BURGDORFERZ
which indicates a high frequency of T cell precursors
in the blood of Lyme disease patients (20,27). Work is
in progress in our laboratory to determine the precursor frequencies in autologous sera.
The increased T cell response of patients with
Lyme disease was primarily attributable to the pathogen itself and was not due to cross-reactions with other
spirochetes such as T phagedenis or with LPS. In
contrast, the T cell responses seen in the other populations studied (28) were principally induced by the
latter agents, which are common antigens in humans.
Thus, on the humoral side, in nearly all normal donors
and patients with non-Lyme arthritides, a reaction
against the 41-kd (flagellin)protein, which is shared by
nonpathogenic and pathogenic spirochetes, has been
demonstrated (refs. 10, 29, and Schoerner C: unpublished observations).
Of particular interest was the poor cellular
reaction of patients with non-Lyme arthritides, a
group that represents the main challenge in the differential diagnosis of 1,yme arthritis (6,8,10). The cellular
responses of patients with non-Lyme arthritides were
significantly lower than those of even the normal
donors. The reasons for these poor reactions are not
clear. However, it is likely that in these diseases,
antigen-reactive cells home to extravascular sites,
particularly the joints, and possibly deplete this cell
population from the peripheral blood (23). These circumstances might possibly allow for the use of lymphocyte transformation assays as additional diagnostic
aids in differentiating patients with Lyme arthritis from
those with other arthritides (13,15). Moreover, as
suggested by the followup studies showing a decrease
in T cell reactivity after successful antibiotic treatment, it will be of interest to investigate whether a
reduction in cellular reactivity can be used as a marker
of diminished disease activity in Lyme disease.
Overall, the data obtained strongly suggest that
lymphocyte transformation assays performed in autologous serum provide not only a means of studying host
defense mechanisms in Lyme borreliosis, but also may
represent important tools in the diagnosis and prospective evaluation of patients with Lyme disease.
ACKNOWLEDGMENTS
We thank Dr. U. Schoenherr, Department of Ophthalmology. University of Erlangen-Nuernberg, FRG, for
referring patients to our clinic, Kenate Meyer for performing
the B hurgdorfrri cultures, Christine Zima for skillful technical assistance, and Dieter Landes for statistical analysis.
40 1
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