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Bacterial extract (OM-89) specific and non specific immunomodulation in

rheumatoid arthritis patients

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DOI: 10.1080/08916930600738425 · Source: PubMed

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 Autoimmunity, June 2006; 39(4): 299–306

Bacterial extract (OM-89) specific and non specific immunomodulation

in rheumatoid arthritis patients

ÉRIC TOUSSIROT1,2,4, ÉRIC ROBINET2,3, PHILIPPE SAAS2,3, JACQUELINE CHABOD2,

BENOı̂T AUGÉ1, GABRIEL COZMA5, PIERRE TIBERGHIEN2,3, JEAN ROUDIER6, &
DANIEL WENDLING1
1
Department of Rheumatology, University Hospital Jean Minjoz, Bd Fleming, F-25030 Besançon cédex, France, 2INSERM
U645 UPRES EA2240, Plateforme de Biomonitoring, Établissement Français du Sang, Bd Fleming, F-25020 Besançon,
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France, 3IFR133, Besançon, France, 4CIC Biotherapy, St Jacques Hospital, 25000 Besançon, France, 5OM PHARMA, 22
rue du Bois-du-Lan, 1217 Meyrin/Geneva, Switzerland, and 6Immuno-Rheumatology, INSERM E9940, F-13000 Marseille,
France

Abstract
The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the
immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell
For personal use only.

precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-g, TNF-a),
Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in
response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-
specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production
while TNF-a and IFN-g production tended to decrease during the study.
Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release
suggestive of an induced Tr1 response to OM-89.

Keywords: Oral tolerance, heat shock protein, rheumatoid arthritis, T regulatory cells, Interleukin-10

Introduction: (Th3) or T regulatory 1 (Tr1) cells. In this sense, a

Rheumatoid arthritis (RA) is a chronic joint inflam-
matory disease characterized by pain, swelling and
stiffness of joints. The disease involves the synovial
membrane, which is inflamed and characterized by
dense cellular infiltrates, mainly composed of macro-
phages, B and T cells. T cells play an important role in
sustaining the inflammation of RA, with a predomi-
nance of type 1 cells secreting IFN-g and TNF-a in
the synovium. This situation also concerns other T
cell-mediated autoimmune diseases such as multiple
sclerosis and diabetes mellitus. Data from experimen-
tal models of such autoimmune diseases suggested
that this type 1 activity can be inhibited by IL-4, TGF-
b and/or IL-10, i.e. cytokines from type 2, type 3
therapeutic approach targeting type 1 secreting cell
subsets is of particular interest in RA management [1].
RA is considered as an autoimmune disease but the
nature of the recognized autoantigen is still unknown.
Some autoantigens have been suspected such as type
II collagen which is present in cartilage. One of the
therapies that has been tried in RA is the induction of
oral tolerance (OT) which refers to the oral
administration of protein antigen and induces a state
of non- or hypo-responsiveness for the fed antigen
[2 – 4]. The mechanisms that lead to OT include active
cellular suppression via the generation of type 2 or type
3 cells, clonal anergy and/or clonal deletion [2,3,5].
In RA patients, oral administration of type II collagen
has been associated with clinical improvement in a

Correspondence: É. Toussirot, Department of Rheumatology, University Hospital Jean Minjoz, Bd Fleming, F-25030 Besançon cédex,
France. Tel: 33 3 81 66 82 41. Fax: 33 3 81 66 86 86. E-mail: eric.toussirot@ufc-chu.univ-fcomte.fr

ISSN 0891-6934 print/ISSN 1607-842X online q 2006 Taylor & Francis

DOI: 10.1080/08916930600738425
 300 É. Toussirot et al.
preliminary open label study [6], but data from
placebo-controlled studies did not confirm these
beneficial therapeutic effects [7 – 9].
Oral administration of the Escherichia coli bacterial
extract OM-89 is used to treat RA [10]. This slow-
acting anti-rheumatic drug has been shown to
ameliorate clinical parameters of RA [10]. It has also
been shown that this compound contains heat shock
proteins (hsp) of 60 and 70 kD [11]. Heat shock
proteins are highly conserved proteins which are
suspected to play a role in the onset or the control of
autoimmune diseases [12]. In RA, both cellular and
humoral reactivity to hsp has been demonstrated.
Moreover, adjuvant arthritis is an animal model of RA
which is induced by immunization with purified heat-
killed Mycobacterium tuberculosis in Freund’s incom-
plete adjuvant [13]. In this model, the Mycobacterium
tuberculosis antigen has been identified to be hsp 60 kD
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[14]. Furthermore, modulation of adjuvant arthritis
has been possible by oral or nasal administration of
hsp 60 [15,16].
Oral administration of OM-89 has been shown to
improve adjuvant arthritis and hsp 60 and 70 kD were
suspected to play a key role in this arthritis inhibitory
effect. However, the precise mode of action of OM-89
is not fully elucidated. Thus, in this study, we aimed to
For personal use only.
better delineate the immunological changes induced
by OM-89 in RA patients. Because OT is the probable
mechanism of action of OM-89, we evaluated T cell
proliferative response and cytokine release to this
antigen after its oral administration.
Patients and methods
Patients
Patients with RA were recruited from our outpatient
rheumatology department between 1999 and 2000.
Inclusion criteria were: a diagnosis of RA according to
the 1987 criteria of the American College of
Rheumatology [17]; an age between 18 and 75; a
disease duration of at least 6 months; a clinically active
disease with at least 6 swollen and 6 tender joints; a
morning stiffness $ 45 min; a prednisolone dosage
# 10 mg/ day for one month before the trial and during
the trial; an erythrocyte sedimentation rate (ESR)
$ 25 mm/h during the month before the trial; and the
patient’s written consent. Patients were required to
discontinue treatment with disease-modifying anti-
rheumatic drugs (DMARDs) one month before
entering the study. No intra-articular injections of
corticosteroids were allowed during the trial. Women
of childbearing potential were required to have a
negative pregnancy test result at baseline and to be
using an effective contraceptive measure. All patients
were allowed to be treated with non steroidal anti-
inflammatory drugs (NSAIDs) during the trial.
Patients were excluded from the study if they
had renal insufficiency, myocardial insufficiency,
disturbance of liver enzymes, human immuno-
deficiency virus infection or other chronic infection,
past history of E. coli toxic-infectious shock.
All the patients in this trial had previously received a
tetanus vaccine.
Study design
This was an open label trial of 6 months duration. The
planned study size was 12 patients. The study protocol
was approved by the Besançon Ethic Committee
(France).
Clinical and laboratory assessments
All patients were assessed at baseline (time of
inclusion) and at months 1, 3 and 6. The following
variables were recorded at each visit: number of swollen
joints (28-joint count) and tender joints (28-joint
count), patient global assessment of pain using a visual
analogue scale (VAS: 100 mm), patient’s and
physician’s assessment of disease activity (evaluated
semi-quantitatively as follow: 0 ¼ inactive; 1 ¼ mild;
2 ¼ moderate; 3 ¼ severe; 4 ¼ very severe disease),
duration of morning stiffness, health assessment
questionnaire (HAQ) (at baseline and month 6),
ESR and CRP levels, rheumatoid factors (nephelo-
metry) and antinuclear antibodies (indirect immuno-
fluorescence, at baseline and month 6), HLA class II
typing (sequence specific primed polymerase chain
reaction-PCR, at baseline). Immunological assess-
ments as well as a complete blood cell count, urinalysis,
levels of serum electrolytes, glucose, creatinine,
asparate and alanine aminotransferases were obtained
at each evaluation.
Study medication
OM-89 is a lyophilized extract with immunomodulat-
ing properties of selected E. coli strains. Each capsule
contains as active principle 24 mg of the bacterial
extract (Subreumw, code name OM-8980, OM
PHARMA, Meyrin/Geneva, Switzerland) consisting
mainly of acidic proteins, peptides and amino acids
[18]. Patients were treated with one capsule daily,
taken in the morning on an empty stomach for
6 months.
Immunological assessments
T cell precursor frequency. To determine the T cell
reactivity specific for OM-89 and the control protein
(Tetanus toxoid; TT) (Massachusetts Biologic
Laboratories, Jamaica Plain, MA, USA), we used a T
cell proliferation assay and a limiting dilution analysis
without previous bulk culture, as previously described
[19]. Peripheral blood mononuclear cells (PBMC) were
isolated by centrifugation through Ficoll–Hypaque
 Immunomodulation in rheumatoid arthritis patients 301

(Amersham, Les Ulis, France) and washed three times
in RPMI 1640 medium (BioWhittaker, Verviers,
Belgium). After the isolation procedures, cell viability
was always greater than 95% viable by trypan blue dye
exclusion. Cells were cultured in 96-well plates in 200 ml
of RPMI 1640 medium supplemented with 1%
L -glutamine, 100 U/ml penicillin, 10 mg/ml
streptomycin and 10% self serum. For each subject, we
measured the proliferative response in 10 wells
containing 4 £ 105, 2 £ 105, 105 or 5 £ 104 cells in
the presence of OM-89 (10 mg/ml), TT (10 mg/ml) or no
antigen. Cell proliferation was assessed by 3[H]
thymidine (Amersham) incorporation after five days of
culture: 1 mCi of 3 [H] thymidine was added to each well
18 h before cell harvesting on glass fiber filters. Positive
wells were defined as having counts per minute (cpm)
higher than the mean plus 2 standard deviations of the
cpm obtained for the 10 control wells without antigen. T
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the study were included in the evaluation. Results
were given as mean ^ SEM (median). Due to the
small patient sample size in this study, results were
examined using non-parametric test (Wilcoxon paired
test) to evaluate the difference between baseline and
the following visits. Using this paired test, we
examined the difference between baseline and the
follow-up time points and thus the dropouts were not
included in the analysis. Friedmann test was used for
evaluating differences in cytokine production between
PBMC cultures with OM-89, TT or without
stimulating antigen. A p value below 0.05 was
considered significant.
Results
A total of 13 patients were screened for the study, of
whom 12 met the inclusion criteria and were enrolled.

cell precursor frequencies were calculated from the
percentage of negative wells as previously described [19].
Polyclonal T cell proliferation assay. To test the polyclonal
proliferative responses of circulating T cells, PBMCs
were stimulated with 1 mg/ml phytohemagglutinin A
(PHA; Murex, Chatillon, France) in triplicate using 3
For personal use only.
All these 12 patients had active disease. The
characteristics of the enrolled patients are listed in
Table I. Eight patients completed the 6 month period.
Three patients had to be withdrawn from the study
because of a severe flare of the disease (1 at month 3
and 2 at month 4). Another dropout (at month 2) was
related to a protocol violation in relation with a
corticosteroid dosage increase in response to disease

different cellular concentrations: 2 £ 105, 105 or worsening. No side effects were observed in all 13
5 £ 104 cells. Cell stimulation was assessed by 3 [H] patients.
thymidine (Amersham) incorporation after five days of
culture. The mitogenic stimulation was calculated by
Response to the treatment
cellular stimulation index (SI, calculated as the mean
cpm response of triplicate samples in the presence of A significant decrease in the tender joint score at
PHA divided by the mean cpm response without month 3 as compared to baseline was observed
stimulating antigen). The mean background responses ( p ¼ 0.01). There was no statistically significant
(i.e. without stimulation) for the 3 cellular difference in the other relevant clinical variables
concentrations ranged from 1427 ^ (SEM) 98 and
10467 ^ 6103 cpm.
Table I. Demographic and clinical characteristics of the rheuma-
toid arthritis patients.
Cytokine production by PBMCs. After 3 washes, PBMCs
Mean ^ SEM (median)
were distributed in a 96-well microplate as described
above and incubated (2 £ 105 cells/well) with OM-89, Age (year) 62.9 ^ 1.6 (61.5)
TT or without stimulating antigen in 10 wells for each [range: 57.2– 74.7]
condition. Cells were cultured at 378C, 5% CO2 and Disease duration (year) 11.8 ^ 5.4 (10.5)
95% relative humidity. The culture supernatants were Sex (male/female) 3/9
Extra-articular disease Nodules: N ¼ 3
collected after 24 h (for TNF-a and IL-10 assays), 48 h Vasculitis: N ¼ 1
(for IL-4 assay) or 5 days (for IFN-g assay) and then Sicca syndrome: N ¼ 8
stored at 2808C until testing. These time points were Positive rheumatoid factor 11/12
found in prior studies by our group as optimal (data not HLA-DRB1* HLA-DRB1* 01: N ¼ 6
shown). Determination of TNF-a, IL-4, IL-10 HLA-DRB1* 04: N ¼ 6
Prednisolone treatment 12/12
and IFN-g in the supernatants were done by Previous DMARD treatments None ¼ 0
ELISA (Diaclone, Besançon, France) following the (multiple medications possible)
manufacturer’s instructions. Gold salts ¼ 7
Hydroxychloroquine ¼ 10
Sulfasalazine ¼ 7
Statistical analysis Methotrexate ¼ 9
Cyclosporine ¼ 3
Analysis was performed on the intent-to-treat popu- Thiopronine ¼ 6
lation and thus all the patients who were enrolled in
 302 É. Toussirot et al.

Table II. Initial and follow-up results of clinical and laboratory parameters in the study (results are given as mean ^ SEM (median).

Assessment Baseline (N ¼ 12) Month 1 (N ¼ 12) Month 3 (N ¼ 11) Month 6 (N ¼ 8) p

Swollen joint 9.75 ^ 0.7 (10) 10.3 ^ 1.0 (10) 8.7 ^ 0.8 (9) 7.25 ^ 1.3 (5.5) NS
score (0 –28)
Tender joint 10.8 ^ 1.7 (7) 8.25 ^ 1.8 (6.5) 6.1 ^ 1.8 (6) 6.25 ^ 2.6 (5.5) Month 3 vs Baseline
score (0 –28) p ¼ 0.01
Pain (VAS: 0– 100 mm) 64.1 ^ 7.3 (69) 63.0 ^ 8.1 (61.5) 64.1 ^ 6.9 (71) 55.5 ^ 7.4 (56.5) NS
Patient global 2.7 ^ 0.3 (3) 2.6 ^ 0.2 (3) 2.4 ^ 0.2 (3) 2.6 ^ 0.3 (3) NS
score (0 –4)
Physician global 2.3 ^ 0.2 (2) 2.2 ^ 0.2 (2) 2.2 ^ 0.2 (2) 1.7 ^ 0.2 (2) NS
score (0 –4)
Morning 180 ^ 27.4 (180) 132.9 ^ 25.9 (136) 150 ^ 33.6 (120) 79.3 ^ 19.1 (60) NS
stiffness (min)
HAQ (0–3) 1.89 ^ 0.24 ND ND 2.09 ^ 0.2 NS
ESR (mm/h) 48.3 ^ 6.3 (46.5) 49.4 ^ 7.5 (47) 56.8 ^ 9.3 (48) 40.4 ^ 10.5 (28.5) NS
CRP (mg/l) 35.9 ^ 7.9 (34.5) 40.4 ^ 9.0 (34.5) 34 ^ 6.4 (29) 29.5 ^ 10.8 (18) NS
Hemoglobin (g/dl) 12.5 ^ 0.5 (12.7) 12.0 ^ 0.5 (12.4) 11.4 ^ 0.6 (12.4) 12.3 ^ 0.6 (12.8) Months 1 and 3
vs baseline: p ¼ 0.03
and p ¼ 0.04
Neutrophils (109/l)
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5.7 ^ 0.6 (5.1) 7.9 ^ 0.7 (8.1) 7.7 ^ 0.9 (6.7) 7.6 ^ 1.4 (7.0) NS
Lymphocytes (109/l) 1.6 ^ 0.1 (1.6) 1.2 ^ 0.1 (1.0) 1.25 ^ 0.2 (1.0) 1.5 ^ 0.2 (1.4) Months 1 and 3
vs baseline p ¼ 0.02
and p ¼ 0.04

ESR, erythrocyte sedimentation rate. VAS, visual analogue scale.

(swollen joint score, pain, patient and physician global (i.e. the E. coli extract OM-89) without however
scores, morning stiffness and HAQ as well as in reaching the statistical significance ( p ¼ 0.08), fol-
For personal use only.

inflammation laboratory parameters (ESR, CRP
levels). Similarly, no significant variation in bio-
chemical measurements, liver enzymes and platelets
was observed. Hemoglobin levels decreased signifi-
cantly at months 1 and 3 ( p ¼ 0.03 and p ¼ 0.04,
respectively) but returned to baseline values at the end
of the study. Similarly, lymphocyte counts decreased
lowed by a return to baseline values at month 6
(Figure 2). However, the frequency of T cells specific
for the control antigen (i.e. TT), significantly
decreased at month 3 as well ( p ¼ 0.045), before
SI PHA 2 x 105 cells / well
Stimulation Index (SI) (mean ± SEM)

at the same times ( p ¼ 0.02 and p ¼ 0.04) and tended 18

SI PHA 105 cells / well
to return to initial values at month 6 (Table II). 16 SI PHA 5 x 104 cells / well
14 * p < 0.05

Oral OM-89 treatment induced a decrease in non specific 12

polyclonal T cell reactivity
Polyclonal T cell reactivity in response to PHA was
first analyzed using 3 different PBMC concentrations
(2 £ 105, 105 and 5 £ 104) (Figure 1). When
compared to the polyclonal T cell reactivity before
treatment, we observed a progressive decline of the
response, which was most obvious at month 3. This
was attested by a decrease of SI which was significant
at month 1 using 2 £ 105 cells/well ( p ¼ 0.04) and at
month 3 for the 3 cellular concentrations (2 £ 105
cells/well: p ¼ 0.02; 105 cells/well: p ¼ 0.02; and
5 £ 104 cells/ well: p ¼ 0.04). After month 3,
polyclonal T cell reactivity to PHA increased without
reaching the initial values.
To determine whether this decrease of T cell
reactivity was linked to an inhibition of T cells specific
for the fed antigen, T cell precursor frequency for
E. coli extract OM-89 was evaluated before and after
treatment. We observed at month 3 a decrease of the
frequencies of T cells specific for the fed antigen
10
8
6
4
*
2 *
* *
0
Baseline 1 3 6
Months
Figure 1. Stimulation index (SI) of PBMC cultures with
phytohemagglutinin. SI of PBMC cultures with PHA were
evaluated at baseline (before starting the treatment by OM-89 oral
administration) and during the treatment at months 1, 3 and 6. The
SI was calculated as the ratio of the mean cpm obtained in triplicate
cultures with PHA with the triplicate culture without antigen
(see Methods section). The mean background responses (i.e.
without stimulation) for the 3 cellular concentrations (2 £ 105, 105
and 5 £ 104 cells/well) ranged from 1427 ^ (SEM) 98 and
10467 ^ 6103 cpm. Results are expressed as means ^ SEM of SI
obtained from each patient (SI of PBMC for X patients were
analyzed). Statistical significant differences between month 3 or 1
and baseline are indicated with asterisks. Number of patients was 12
at baseline, 12 at 1 month, 11 at month 3 and 8 at month 6.
 Immunomodulation in rheumatoid arthritis patients 303

T cells specific for OM-89 Background

T cell precursor frequencies (x 10–6)(mean ± SEM) T cells specific for Tetanus toxoid 2000 ***
OM - 89
10 1800 Tetanus toxoid
* p < 0.05

IFNγ (pg/ mL) (mean ± SEM)

1600 * p < 0.05
** p < 0.01
8 1400 *** p < 0.005
1200
6 1000 *** * **
800

4 600
400
*
2 200
0
Baseline 1 3 6
0 Months
Baseline 1 3 6
Months Figure 3. IFN-g production by PBMC stimulated with OM-89,
TT or without antigen. The culture supernatants of PBMC
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Figure 2. T cell precursor frequencies specific for OM-89 and TT.
The frequencies of T cells specific for OM-89 (fed antigen) and TT
(control antigen) were evaluated at baseline (before OM-89 oral
treatment) and after 1, 3 and 6 months of drug administration. The
OM-89 and TT specific T cell responses were evaluated at each time
point by a direct limiting dilution analysis (as described in Methods
section according to Ref. 19). T cell precursor frequencies for the
tested antigens were obtained by measuring the proliferative
responses of PBMC cultured in 10 wells at 4 £ 105, 2 £ 105, 105
and 5 £ 104 cells/well in the presence of OM-89, TT or no antigen.
For personal use only.
incubated with OM-89, TT or no antigen were collected after 5
days and at each time point of the study, i.e. at baseline and after 1, 3
and 6 months of oral OM-89 administration. IFN-g production was
evaluated using ELISA. Results (pg/ml) are given as means ^ SEM.
Statistical significant differences between the 3 cultures
(spontaneous, OM-89 and TT IFN-g production) are indicated
with asterisks. Number of patients was 12 at baseline, 12 at 1 month,
11 at month 3 and 8 at month 6.

antigen stimulation during the study period

The T cell precursor frequencies were calculated from the
(Figures 3 – 6).
percentage of negative wells. Results are given as means ^ SEM of
T cell precursor frequencies. Statistically significant difference When the levels of cytokine release by PBMCs
between month 3 and baseline is indicated with asterisk. Number of stimulated by OM-89 were compared to those
patients was 12 at baseline, 12 at 1 month, 11 at month 3 and 8 at obtained with control antigen TT and those obtained
month 6. in the absence of stimulation, higher levels of IFN-g,
increasing again without however reaching pre- TNF-a and IL-10 production were obtained with
treatment values. Overall, oral OM-89 treatment was
associated with decline of non specific T cell reactivity
that was most pronounced at month 3 after treatment. *
7000 Background
OM- 89
TNFα (pg/mL) (mean ± SEM)

6000 Tetanus toxoid

Oral OM-89 treatment resulted in an antigen-specific * * p < 0.05
5000
decrease in PBMC type 1 and type 2 cytokine production,
while IL-10 production was maintained at high levels 4000
*
Then, changes in the PBMC-cytokine production 3000
induced by OM-89 were analyzed: IFN-g production
by PBMCs cultured with OM-89 tended to progress- 2000
ively decrease over the study period (month 3 as
1000
compared to baseline: p ¼ 0.07) and then remained
stable until the end of the study (Figure 3). The same 0
changes were observed for TNF-a without significant Baseline 1 3 6
differences between the different follow-up visits and Months
baseline (Figure 4). Interleukin-4 concentrations
Figure 4. TNF-a production by PBMC stimulated with OM-89,
decreased progressively during the study with a nadir TT or without antigen. The culture supernatants of PBMC
occurring at month 3 (as compared to baseline: incubated with OM-89, TT or no antigen were collected after 24 h
p ¼ 0.04), while concentrations of IL-10 in the and at each time point of the study, i.e. at baseline and after 1, 3 and
supernatants of PBMCs stimulated with OM-89 6 months of oral OM-89 administration. TNF-a production was
evaluated using ELISA. Results (pg/ml) are given as means ^ SEM.
remained stable (no difference between baseline and
Statistical significant differences between the 3 cultures
the follow-up visits) as shown in Figures 5 and 6. We (spontaneous, OM-89 and TT TNF-a production) are indicated
did not observe any changes in spontaneous cytokine with asterisks. Number of patients was 12 at baseline, 12 at 1 month,
production or in cytokine production after control 11 at month 3 and 8 at month 6.
 304 É. Toussirot et al.

for IFN-g while disappearing for TNF-a. In particu-

0.40 Background
OM - 89 lar, we observed a very high-level of IL-10 production
0.35 Tetanus toxoid as compared to the other cell cultures ( p , 0.005 at
IL-4 (pg/ mL) (mean ± SEM)

each visit) (Figure 6). Such differences were not

0.30
observed at any time for IL-4 production (Figure 5).
0.25

0.20 Discussion
*
0.15
In this study, patients with active RA were treated orally
0.10 with the E. coli bacterial extract OM-89. As previously
0.05 observed, its safety profile was satisfactory with no
serious side effects [10]. In accordance to the results of
0.00 previous trials (reviewed in Ref. 10), OM-89 was not
Baseline 1 3 6
Months
associated with disease exacerbation [10]. Safety
laboratory parameters did not significantly change
Figure 5. IL-4 production by PBMC stimulated with OM-89, TT except hemoglobin levels and lymphocyte counts, but
or without antigen. The culture supernatants of PBMC incubated both returned to baseline values at study end.
with OM-89, TT or no antigen were collected after 48 h and at each Among the clinical variables, we observed a
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time points of the study, i.e. at baseline and after 1, 3 and 6 months
significant improvement in tender joint score at
of oral OM-89 administration. IL-4 production was evaluated using
ELISA. Results (pg/ml) are given as means ^ SEM. Statistical month 3. This moderate clinical response, as well
significant differences for IL-4 production by OM-89 stimulated as the mild variation in inflammatory laboratory
cells between month 3 and baseline is indicated with asterisk. parameters (ESR and CRP), could be explained by the
Number of patients was 12 at baseline, 12 at 1 month, 11 at month 3 fact that our patients had severe disease, as showed
and 8 at month 6.
by the inclusion criteria. Another explanation could be
the low sample size, which did not permit to reach
significant level for most variables. However, previous
For personal use only.

OM-89 as compared to TT or to the background placebo-controlled studies have demonstrated that

production ( p , 0.05 at baseline and at months 1 and
3 for these 3 cytokines and at month 6 for IFN-g and
IL-10) (Figures 3, 4 and 6). Such a difference
persisted throughout the study period for IL-10, less so
***
1000
IL-10 (pg/ mL) (mean ± SEM)
OM-89 is effective in RA, with significant improve-
ment in clinical measures which was almost consist-
ently evident after 3 months of treatment [18,20].
Thus, our results on clinical and biological parameters
of RA agreed in part with those of previous studies.
The major findings in this study included changes in
Background T cell reactivity as evidenced by a decline in polyclonal
OM - 89 T cell reactivity and in T cell precursor frequencies
***
Tetanus toxoid
specific for OM-89 and TT. These modifications were
*** p < 0.005
most pronounced at month 3. However, subsequently,

*** ***
T cell reactivity to PHA as well as frequencies of T
800 cells specific for the tested antigens increased,
suggesting secondary mechanisms that reverse the T
600 cell hyporeactivity.
Other findings included a strong and persistent IL-
400 10 production by PBMCs cultured with OM-89 and a
progressive decline in type 1 cytokines, TNF-a and
200 IFN-g, during the study. Remarkably, levels of IL-10
production by PBMCs stimulated with OM-89
0 remained stable during the study, despite a decrease
Baseline 1 3 6 in the frequency and thus in the number of T cells
Months specific for OM-89.
The immunomodulatory properties of OM-89 have
Figure 6. IL-10 production by PBMC stimulated with OM-89,
TT or without antigen. The culture supernatants of PBMC been well studied and for instance, it is known that this
incubated with OM-89, TT or no antigen were collected after 24 h compound is able to enhance in vitro PBMC cytokine
and at each time point of the study, i.e. at baseline and after 1, 3 and production (IL-1, TNF-a, IL-2, IFN-g) and it has
6 months of oral OM-89 administration. IL-10 production was various other effects including both humoral and
evaluated using ELISA. Results (pg/ml) are given as means ^ SEM.
cell-mediated immune mechanisms [18]. Its anti-
Statistical significant differences between the 3 cultures
(spontaneous, OM-89 and TT IL-10 production) are indicated rheumatic effects have been demonstrated in animal
with asterisks. Number of patients was 12 at baseline, 12 at 1 month, models of arthritis as it can suppress adjuvant arthritis
11 at month 3 and 8 at month 6. in rats [21]. It contains hsp60 as demonstrated by
 Immunomodulation in rheumatoid arthritis patients
the staining of OM-89 with hsp60- specific mono-
clonal antibodies by Western blot [22]. Proliferative T
cell responses to bacterial hsp60 and hsp70 from
mycobacteria and E. coli have been observed in rats
immunized with OM-89, suggesting the presence
of such hsp [23]. Thus, it has been hypothesized that
the arthritis inhibitory effect of OM-89 could be
related to the generation of hsp-specific regulatory
T cells after oral administration [11].
Heat shock proteins are suspected to be implicated
in the physiopathology of autoimmune diseases. In
this sense, T cell reactivity to mycobacterial and E. coli
hsp60 and 70 has been detected in the synovial tissue
of RA patients [12]. Heat shock proteins may play a
protective role by inducing regulatory T cells which
may suppress autoimmunity [1]. In animal models of
arthritis, oral treatment with mycobacterial hsp60 or
hsp70 has been found to lead to the protection against
Autoimmunity Downloaded from informahealthcare.com by INSERM on 03/11/13
induction of the disease by a mechanism of OT
[15,24]. The mechanisms explaining such effects were
thought to be related to the induction of T cells cross-
responsive to self hsp. Moreover, in rat adjuvant
arthritis, it has been demonstrated that these cells
produced IL-10 as well IL-4 and TGF-b, which may
contribute to the inhibition of the inflammatory
process. Conversely, IFN-g production was usually
For personal use only.
reduced [16].
In our study, we observed that OM-89 was capable
of inducing high IL-10 release in PBMC cultures as
compared to other cytokines releases. We identified
monocytes as the major source of IL-10. Indeed,
immunomagnetic cell sorting was performed to isolate
CD4 þ T, CD8 þ T, CD19 þ B cells and CD14 þ
monocytes. These cells from four healthy subjects
were stimulated by OM-89 and IL-10 release was
detected by ELISA only in monocytes (data not
shown). Moreover, we observed during the study
period a progressive decline in IFN-g and TNF-a
production by OM-89-stimulated PBMCs. We also
observed a decrease in IL-4 release by OM-89
stimulated PBMCs, although, for all Ag tested, IL-4
production remained low. This progressive decline in
cytokine production could be related to the generation
of regulatory T cells. Several different CD4 þ subsets
of regulatory T cells have been recently identified,
including IL-10-producing Tr1 cells, TGF-b-secret-
ing type 3 T (Th3) cells and CD4 þ CD25 þ
suppressive Tr cells. CD4 þ CD25 þ suppressive Tr
cells are defined by their phenotypic markers and exert
their regulatory effects by direct cell contact with
effector cells, as well as by generating other regulatory
T cells such as Tr1 cells [25,26]. Immunophenotypic
analysis of a limited number of samples (unstimulated
PBMCs) in 3 patients at month 6 compared
to baseline samples was unable to reveal an
increased number of circulating CD4 þ CD25 þ
(and CD45RO þ CD69-CD40L-) regulatory T cells
(data not shown). Furthermore, we did not observe
305
significant changes in lymphocyte activation markers
(CD69, CD45RO and HLA-DR) between baseline
and month 6 in these patients. Nevertheless, the
strong IL-10 production by OM-89-stimulated
PBMCs associated with a decrease in type 1 (IFN-g,
TNF-a) and type 2 (IL-4) cytokines suggested the
involvement of IL-10-producing Tr1 cells. Such an
involvement of Tr1 cells in the immunomodulatory
effects of oral OM89 is further strengthened by the
observation of marked expression of IL-10 mRNA in
Peyer’s patches of OM-89-orally treated mice [27].
In conclusion, in this open-label study using an
orally administered E. coli bacterial extract, changes in
the levels of immune responses were observed as
demonstrated by a non specific decrease in T cell
reactivity to various antigens. In addition, OM-89 was
capable of inducing a strong and sustained antigen-
specific IL-10 production by PBMCs, while type 1
and type 2 cytokines progressively decreased,
suggesting a shift in the type 1 and type 2 cytokine
profile to the generation of Tr1 cells.
Acknowledgements
The authors thank Giancesare Gamba (PhD) for his
statistical assistance, Patricia Caillon (OM
PHARMA) for her contribution, Agnès Lienard
(Établissement Français du Sang, Besançon, France)
for flow cytometry analysis and Patricia Mercier
(Établissement Français du Sang, Besançon, France)
for cytokine cellular source analysis. This work
was supported by a grant from OM PHARMA,
Meyrin/Geneva, Switzerland.
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