# Increased Responsiveness to Toll-Like Receptor 4 Stimulation in Peripheral Blood Mononuclear Cells from Patients with Recent Onset Rheumatoid Arthritis

Authors: M. L. Kowalski; A. Wolska; J. Grzegorczyk; J. Hilt; M. Jarzebska; M. Drobniewski; M. Synder; M. Kurowski
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Background. Cell signaling via Toll-like receptors (TLRs) leads to synovial inflammation in rheumatoid arthritis (RA). We aimed to assess effects of TLR2 and TLR4 stimulation on proinflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) from patients with recent-onset RA, osteoarthrosis (OA), and healthy control (HC).
Methods. PBMCs were stimulated with LPS, biglycan and cytokine mix. Cytokines were analyzed in supernatants with ELISA. Expression of toll-like receptors mRNA in leukocytes was analyzed using real-time qPCR.
Results. PBMCs from RA patients spontaneously produced less IL-6 and TNFα than cells from OA and HC subjects.
LPS increased cytokines' production in all groups. In RA patients increase was dramatic (30 to 48-fold and 17 to 31-fold, for respective cytokines) compared to moderate (2 to 8-fold) in other groups. LPS induced 15-HETE generation in PBMCs from RA (mean 251%) and OA patients (mean 43%), although  only in OA group, the increase was significant. TLR2 and TLR4 gene expressions decreased in response to cytokine mix, while LPS enhanced TLR2 expression in HC and depressed TLR4 expression in OA patients.
Conclusion. PBMCs from recent-onset RA patients are overresponsive to stimulation with bacterial lipopolysaccharide.
TLR expression is differentially regulated in healthy and arthritic subjects.


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## Body

## 1. Introduction

Rheumatoid  arthritis (RA)
is a complex disease  of yet unknown pathogenesis. Several factors  have been
proposed as potential triggers of inflammatory response
associated with RA rheumatoid arthritis, including microbes and their products [1, 2]. Products
of invading microbes may activate inflammatory cells via Toll-like receptors
(TLRs), which leads to immune and inflammatory responses. TLRs activation has
been linked to the pathogenesis of rheumatoid arthritis, and both TLR-2 and
TLR-4 are potentially critical receptors in the initiation and perpetuation of
the inflammatory cycle in arthritis [3].
It has been also demonstrated that endogenous
TLR4 ligands (e.g., heat shock proteins and extra domain A fibronectin) are
highly expressed in the joints of patients with RA [4–6].
Toll-like receptors are present on tissue
synoviocytes but also on peripheral blood monocytes which are recruited to the
site of inflammation and are
involved in the pathogenesis
of synovial inflammation [7–10]. Recent studies described
higher expression of TLR4 in leucocytes from
patients with ankylosing spondylitis and increased expression of TLR2 and TLR4
receptors in cartilage lesions occurring during OA suggesting that abnormal TLR
signaling may also be involved in the
pathogenesis of rheumatologic conditions other than rheumatoid arthritis [11, 12].IL-6 and TNFα as well as
IL-1 family cytokines and chemokines have been markedly elevated in synovial
fluid and synovial membranes from patients with RA, but not in patients with OA [13–15].
Moreover, in sera of RA patients spontaneous IL-1, -6, -8, -18, and TNFα production are significantly higher than in healthy
individuals and anti-TNFα
treatment decreases local and systemic expression of TLR2 and TLR4 receptors  [16, 17].Earlier studies documented
that peripheral blood mononuclear cells from RA patients generate significantly
more inflammatory cytokines and chemokines, although the difference between patients with new
onset and established rheumatoid arthritis could not be clearly determined [18–21].We aimed to assess the
effect of Toll-like receptors 2 and 4 stimulation on production of inflammatory cytokines and generation of 15-HETE by PBMCs from patients with newly diagnosed
RA in comparison with patients with osteoarthrosis and healthy controls. In
addition, we sought to determine in what way the stimulation of TLRs
(exemplified by TLR4 stimulation in our experiments) on leucocytes’ surface
might affect expression of Toll-like receptors themselves on the mRNA level.
Finally, we investigated the influence leucocytes’ stimulation with mix of strong
proinflammatory cytokines on expression of TLR2 and TLR4 at mRNA level.


## 2. Methods

### 2.1. Patients

Twenty one subjects
were recruited to the study, including 7 patients with rheumatoid arthritis (RA),
7 patients with osteoarthrosis (OA) and 7 healthy control (HC) subjects. RA and
OA were diagnosed according to American College
of Rheumatology
guidelines [22]. Severity of rheumatoid arthritis was assessed
employing the composite 28-joint disease activity score (DAS28).
Although no gold standard for assessment of RA activity exists, the DAS28 score
has been adopted by European rheumatologists as a daily practice tool [23, 24].
DAS28 scoring provides a numeric index, in which a score >5.1
implies high-disease activity, a score <3.2 indicates low disease activity, and
a score <2.6 indicates remission. Patients with RA were newly
diagnosed and had not received disease-modifying antirheumatic drugs (DMARDs) before inclusion into the
study and presented with moderate disease activity
according to DAS28 (mean index ± SD; 4,31 ± 1,32). Additional parameters used for
clinical characterization of patients included anti-cyclic citrullinated
peptide antibodies (aCCPs) as well as C-reactive protein (CRP) serum
concentration measurements. Serum aCCP presence is a marker of early phase RA. They
are more specific as a diagnostic tool than RF and are considered a good
predictor of severe erosive disease [25].
CRP elevation is the most useful index of the acute phase response being
specific, sensitive, and rising rapidly during 6 to 10 hours after the
initiation of an inflammatory process [26].The aCCPs were detected
in all RA patients and in none of patients without RA. The concentration of
C-reactive protein (CRP) in serum was increased in RA compared to OA patients. Control group consisted of young
healthy volunteers. A clinical characteristic of patients is presented in
Table1.Table 1
Clinical characteristic of patients.
Rheumatoid arthritisOsteoarthrosisHealthy controlFemale/male6 /17/06/1Age in years
(median; range)56 (35–68)59 (53–75)32 (27–55)aCCP in IU/mL (mean ± SD)68,76 ± 47,174,02 ± 5,79Not doneCRP mg/l (mean ± SD)12 ± 66 ± 0Not doneDAS28 (median; min-max)4,2 (1,96–6,27)n/an/aAll participants gave their written
consent after being fully informed about the purpose of the study, which was
previously approved by Local Bioethics Committee of the Medical University of
Lodz.


### 2.2. Purification of Peripheral Blood Mononuclear Cells

Blood samples (27 mL
per patient) were collected by peripheral venipuncture. Peripheral blood
mononuclear cells were purified by centrifugation on Histopaque-1077 (Sigma Aldrich,
St. Louis, Mo, USA).
Briefly, peripheral blood was diluted with phosphate buffered saline and
carefully layered onto Histopaque-1077 and centrifuged at 400 g for 30 minutes
at room temperature. The opaque interface was washed three times and finally
resuspended at a density of 106 cells/ml in RPMI-1640 medium (Sigma
Aldrich, St. Louis, Mo, USA) supplemented with 10% heat inactivated Fetal
Bovine Serum (Sigma-Aldrich, St. Louis, Mo, USA), antibiotic (penicillin,
streptomycin), and antimycotic (amphothericin B) solution (Sigma Aldrich, St.
Louis, Mo, USA).


### 2.3. Cell Stimulation

PBMCs were incubated with
LPS at a concentration of 1, 10, and 100 ng/mL, biglycan (BGN) at concentrations
of 0.1, 1, and 10 ng/mL and with cytokine mix (including 10 ng/mL of IL-1, 10 ng/mL
IL-6, and 100 ng/mL TNFα) for 24 hours. After centrifugation, supernatants were
harvested and analyzed for IL-6 or TNFα using ELISA kits (Bender MedSystems Inc.,  Burlingame, Calif, USA).


### 2.4. RNA Isolation and Reverse Transcription

Total RNA was isolated from leukocytes using Total RNA Mini kits
(A&A Biotechnology, Gdynia,
Poland) in
accordance with manufacturer’s instructions. Total RNAs were converted to
complementary DNA (cDNA) by random priming with M-MLV reverse transcriptase
(Promega, Madison, Wis, USA).
After reverse transcription, cDNA was stored at−20°C for further
analysis.


### 2.5. Quantitative Real-Time PCR

Quantitative real-time PCR was performed using
ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif,
USA). We used iTaq SYBR Green Supermix With ROX (Bio-Rad
Laboratories, Inc., Hercules,
Calif, USA)
as suggested by the manufacturer. In brief, reactions were carried out in 20μl containing 1μl cDNA, 10μl 2 × SYBR Green supermix, and 200 nM of each
specific primer. The cycling parameters were 3 minutes at 95°C followed
by 45 cycles of denaturation at 95°C for 15 seconds and
annealing/extension at 60°C for 45 seconds. For relative
quantification, an n-fold differential expression has been calculated using 2−ΔΔCt method. Amplification of each target gene (TLR2 and TLR4) was normalized to
that of β-actin (housekeeping gene). Amplified products were checked for their
correct size by means of agarose gel electrophoresis. Specific primers used for
TLR2, TLR4, and β-actin amplification were described previously by
Gutiérrez-Cañas et al. [27] and
their sequences were as follows: TLR2 sense 5′GGA AGA ATC CTC CAA TCA GGC 3′;
TLR2 antisense 5′CTT CTG TGA GCC CTG AGG GA 3′; TLR4 sense 5′AGC CAC GCA TTC ACA GGG 3′; TLR4 antisense 5′CAT GGC TGG GAT CAG AGT CC 3′; β-actin sense 5′AGA
AGG ATT CCT ATG TGG GCG 3′;
β-actin antisense 5′CAT GTC GTC CCA GTT GGT GAC 3′.


## 2.1. Patients

Twenty one subjects
were recruited to the study, including 7 patients with rheumatoid arthritis (RA),
7 patients with osteoarthrosis (OA) and 7 healthy control (HC) subjects. RA and
OA were diagnosed according to American College
of Rheumatology
guidelines [22]. Severity of rheumatoid arthritis was assessed
employing the composite 28-joint disease activity score (DAS28).
Although no gold standard for assessment of RA activity exists, the DAS28 score
has been adopted by European rheumatologists as a daily practice tool [23, 24].
DAS28 scoring provides a numeric index, in which a score >5.1
implies high-disease activity, a score <3.2 indicates low disease activity, and
a score <2.6 indicates remission. Patients with RA were newly
diagnosed and had not received disease-modifying antirheumatic drugs (DMARDs) before inclusion into the
study and presented with moderate disease activity
according to DAS28 (mean index ± SD; 4,31 ± 1,32). Additional parameters used for
clinical characterization of patients included anti-cyclic citrullinated
peptide antibodies (aCCPs) as well as C-reactive protein (CRP) serum
concentration measurements. Serum aCCP presence is a marker of early phase RA. They
are more specific as a diagnostic tool than RF and are considered a good
predictor of severe erosive disease [25].
CRP elevation is the most useful index of the acute phase response being
specific, sensitive, and rising rapidly during 6 to 10 hours after the
initiation of an inflammatory process [26].The aCCPs were detected
in all RA patients and in none of patients without RA. The concentration of
C-reactive protein (CRP) in serum was increased in RA compared to OA patients. Control group consisted of young
healthy volunteers. A clinical characteristic of patients is presented in
Table1.Table 1
Clinical characteristic of patients.
Rheumatoid arthritisOsteoarthrosisHealthy controlFemale/male6 /17/06/1Age in years
(median; range)56 (35–68)59 (53–75)32 (27–55)aCCP in IU/mL (mean ± SD)68,76 ± 47,174,02 ± 5,79Not doneCRP mg/l (mean ± SD)12 ± 66 ± 0Not doneDAS28 (median; min-max)4,2 (1,96–6,27)n/an/aAll participants gave their written
consent after being fully informed about the purpose of the study, which was
previously approved by Local Bioethics Committee of the Medical University of
Lodz.


## 2.2. Purification of Peripheral Blood Mononuclear Cells

Blood samples (27 mL
per patient) were collected by peripheral venipuncture. Peripheral blood
mononuclear cells were purified by centrifugation on Histopaque-1077 (Sigma Aldrich,
St. Louis, Mo, USA).
Briefly, peripheral blood was diluted with phosphate buffered saline and
carefully layered onto Histopaque-1077 and centrifuged at 400 g for 30 minutes
at room temperature. The opaque interface was washed three times and finally
resuspended at a density of 106 cells/ml in RPMI-1640 medium (Sigma
Aldrich, St. Louis, Mo, USA) supplemented with 10% heat inactivated Fetal
Bovine Serum (Sigma-Aldrich, St. Louis, Mo, USA), antibiotic (penicillin,
streptomycin), and antimycotic (amphothericin B) solution (Sigma Aldrich, St.
Louis, Mo, USA).


## 2.3. Cell Stimulation

PBMCs were incubated with
LPS at a concentration of 1, 10, and 100 ng/mL, biglycan (BGN) at concentrations
of 0.1, 1, and 10 ng/mL and with cytokine mix (including 10 ng/mL of IL-1, 10 ng/mL
IL-6, and 100 ng/mL TNFα) for 24 hours. After centrifugation, supernatants were
harvested and analyzed for IL-6 or TNFα using ELISA kits (Bender MedSystems Inc.,  Burlingame, Calif, USA).


## 2.4. RNA Isolation and Reverse Transcription

Total RNA was isolated from leukocytes using Total RNA Mini kits
(A&A Biotechnology, Gdynia,
Poland) in
accordance with manufacturer’s instructions. Total RNAs were converted to
complementary DNA (cDNA) by random priming with M-MLV reverse transcriptase
(Promega, Madison, Wis, USA).
After reverse transcription, cDNA was stored at−20°C for further
analysis.


## 2.5. Quantitative Real-Time PCR

Quantitative real-time PCR was performed using
ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif,
USA). We used iTaq SYBR Green Supermix With ROX (Bio-Rad
Laboratories, Inc., Hercules,
Calif, USA)
as suggested by the manufacturer. In brief, reactions were carried out in 20μl containing 1μl cDNA, 10μl 2 × SYBR Green supermix, and 200 nM of each
specific primer. The cycling parameters were 3 minutes at 95°C followed
by 45 cycles of denaturation at 95°C for 15 seconds and
annealing/extension at 60°C for 45 seconds. For relative
quantification, an n-fold differential expression has been calculated using 2−ΔΔCt method. Amplification of each target gene (TLR2 and TLR4) was normalized to
that of β-actin (housekeeping gene). Amplified products were checked for their
correct size by means of agarose gel electrophoresis. Specific primers used for
TLR2, TLR4, and β-actin amplification were described previously by
Gutiérrez-Cañas et al. [27] and
their sequences were as follows: TLR2 sense 5′GGA AGA ATC CTC CAA TCA GGC 3′;
TLR2 antisense 5′CTT CTG TGA GCC CTG AGG GA 3′; TLR4 sense 5′AGC CAC GCA TTC ACA GGG 3′; TLR4 antisense 5′CAT GGC TGG GAT CAG AGT CC 3′; β-actin sense 5′AGA
AGG ATT CCT ATG TGG GCG 3′;
β-actin antisense 5′CAT GTC GTC CCA GTT GGT GAC 3′.


## 3. Results

### 3.1. IL-6 Production by PBMCs in Response to Stimulation with LPS and Biglycan

Nonstimulated PBMCs from RA patients demonstrated a
significantly lower production of IL-6
as compared to OA patients (median value 48 times lower) and to control
subjects (median value 45 times lower) (Table2). Stimulation with LPS,
an agonist for TLR4 receptors, resulted in significant increase in IL-6
production in all 3 groups. However,
in cells from patients with RA, the
increase was dramatic (median 30, 48, and 44 folds over baseline after 1, 10,
and 100 ng/mL of LPS, resp.) as compared to only moderate (2 to 4 folds) increase
observed in cells from patients with OA
or healthy subjects (Figure 1).Table 2
Spontaneous and stimulated IL-6 production
by peripheral blood mononuclear cells from patients with rheumatoid arthritis
(RA), osteoarthrosis(OA), and healthy controls (HC); median values;P<.05*—significantly different from
nonstimulated; #—significant
difference between RA and HC ∧—significant
difference between RA and OA.
NonstimulatedStimulusLPS 1 ng/mLLPS 10 ng/mLLPS 100 ng/mLBGN 0,1 ng/mLBGN1# ng/mLBGN#10 ng/mLGroup of subjectsconcentration of IL-6 (ng/mL)RA0,56#⋀22,87*42,95*⋀46,72*2,0⋀1,24#⋀1,55#⋀OA27,243,6*88,47*75,1*22,633,0123,0HC25,4586,46*73,4799,11*21,15*50,8146,0Figure 1
Individual changes in IL-6 concentration in the supernatants of human leucocytes culture after stimulation with LPS at 100 ng/mL; log10 scale; HC-healthy controls, OA-osteoarthrosis, RA-rheumatoid arthritis;P*<.03, Wilcoxon’s test.Incubation of cells with
biglycan, the agonist for TLR2 receptors, resulted in small and nonsignificant
increase in IL-6 production in both RA patients (after all biglycan
concentrations) and AO patients (only after biglycan at 1 ng/mL). In healthy subjects, insignificant increases
were observed following 2 highest concentrations, and small decrease (22%) was
noticed after the lowest biglycan concentration.


### 3.2. TNFα Production by PBMCs in Response to Stimulation with LPS and Biglycan

Nonstimulated PBMCs from RA patients generated significantly
less TNFα as compared to OA
patients (mean 12 times less) and
control subjects (mean 10 times less) (Table 3). Incubation with LPS induced an
increase in TNF-α production in all 3
groups. However, similarly to IL-6, the increase in TNFα production was significantly more intense after
LPS at 10 and 100 ng/mL (resp., 31 and 29 folds over the baseline) as compared
to that observed in cells from patients with OA (mean 3 to 8 folds increase for
different LPS concentrations) or healthy subjects (mean 4- to 7-fold increase for
different LPS concentrations) (Figure 2). Stimulation with biglycan induced a
significant increase in TNFα production only in PBMCs from healthy subjects in
response to the highest concentration.Table 3
Spontaneous and stimulated TNFα
production by peripheral blood mononuclear cells from patients with
rheumatoid arthritis (RA), osteoarthrosis
(OA), and healthy control (HC); P<.05*—significantly different from nonstimulated; #—significant difference between RA and HC ∧—significant difference between RA and
OA.
NonstimulatedLPS 1 ng/mLLPS 10 ng/mLLPS 100 ng/mLBGN 0,1 ng/mLBGN 1 ng/mLBGN 10 ng/mLGroup of subjectsConcentration of TNFα (ng/mL)RA0,05⋀#0,72⋀#2,02*2,84*0,1⋀#0,260,07⋀#OA0,594,71*2,48*2,62*0,840,450,71HC0,492,59*1,98*1,94*0,630,650,68*Figure 2
Individual changes in TNFα concentration in the supernatants of human leucocytes culture after stimulation with LPS 100 ng/mL; log10 scale; HC-healthy controls; OA-osteoarthritis; RA-rheumatoid arthritis; p*=.04; p**=.027.


### 3.3. 15-HETE Generation by PBMCs in Response to Stimulation with LPS and Biglycan

15-HETE  was generated by unstimulated PBMCs cells
from both RA and OA patients in similar amounts. Stimulation with LPS increased 15-HETE generation in 5 RA patients
(range 27–320%) while
decreased in 2 patients (32% and 34%, resp.). In OA patients, LPS induced a significant (P<.005)
increase in 15-HETE production (mean increase 43%; range 20–175%) (Figure 3). Incubation with biglycan did not affect 15-HETE generation.Figure 3
Changes in 15-HETE release into mononuclear cells supernatants after stimulation with 100 ng/mL LPS in individual patients OA-osteoarthritis; RA-rheumatoid arthritis;p*<.01.


### 3.4. Effect of Cytokines and LPS on TLR2 and TLR4 Gene Expression in PBMCs

Expression of TLR2
mRNA in nonstimulated, cytokine
mix-stimulated, or LPS stimulated cells was similar in RA, OA and HC subjects (Table4(a)). Mean expression of TLR-4 mRNA in
nonstimulated as well as in LPS or
cytokine-mix stimulated cells was similar
in patients with RA and OA, although
in both groups, it was
significantly  lower than
in cells from HC.Table 4
Relative
quantification of the TLR2 and TLR4 mRNA expression in PBMCs from patients with rheumatoid
arthritis (RA), osteoarthrosis (OA), and healthy control (HC).
(a)
Target gene
expression is normalized to housekeeping gene expression and presented asn-fold of the expression in HC group (2−ΔΔCT).
*denotes significant difference in expression comparing with HC (P<.03).
NonstimulatedCytokine mixLPSTLR2HC111RA2.0452.9150.036OA1.2181.660.074TLR4HC111RA0.008*0.2250.0009*OA0.004*0.044*0.00002*(b)
Target gene
expression after stimulation with
cytokine mix or LPS is normalized to housekeeping
gene expression and presented asn-fold
of the expression in non stimulated cells from patients from every group (2−ΔΔCT).
Significant differences in expression compared to nonstimulated cells are
marked with P*<0.02;P#<0.01.
NonstimulatedCytokine mixLPSTLR2HC10.26817.753*RA10.3820.316OA10.365#1.071TLR4HC10.00006*0.1RA10.2121.498OA10.093#0.092*Incubation with cytokine
mix tended to decrease both TLR2 and
TLR4 expressions although the decrease was significant for
both receptors in OA patients and for TLR4 in
HC subjects (Table4(b)). TLR2 gene expression was significantly increased by LPS in healthy controls, while significant suppression of TL4 mRNA
expression was observed after LPS in
cells from patients with OA.


## 3.1. IL-6 Production by PBMCs in Response to Stimulation with LPS and Biglycan

Nonstimulated PBMCs from RA patients demonstrated a
significantly lower production of IL-6
as compared to OA patients (median value 48 times lower) and to control
subjects (median value 45 times lower) (Table2). Stimulation with LPS,
an agonist for TLR4 receptors, resulted in significant increase in IL-6
production in all 3 groups. However,
in cells from patients with RA, the
increase was dramatic (median 30, 48, and 44 folds over baseline after 1, 10,
and 100 ng/mL of LPS, resp.) as compared to only moderate (2 to 4 folds) increase
observed in cells from patients with OA
or healthy subjects (Figure 1).Table 2
Spontaneous and stimulated IL-6 production
by peripheral blood mononuclear cells from patients with rheumatoid arthritis
(RA), osteoarthrosis(OA), and healthy controls (HC); median values;P<.05*—significantly different from
nonstimulated; #—significant
difference between RA and HC ∧—significant
difference between RA and OA.
NonstimulatedStimulusLPS 1 ng/mLLPS 10 ng/mLLPS 100 ng/mLBGN 0,1 ng/mLBGN1# ng/mLBGN#10 ng/mLGroup of subjectsconcentration of IL-6 (ng/mL)RA0,56#⋀22,87*42,95*⋀46,72*2,0⋀1,24#⋀1,55#⋀OA27,243,6*88,47*75,1*22,633,0123,0HC25,4586,46*73,4799,11*21,15*50,8146,0Figure 1
Individual changes in IL-6 concentration in the supernatants of human leucocytes culture after stimulation with LPS at 100 ng/mL; log10 scale; HC-healthy controls, OA-osteoarthrosis, RA-rheumatoid arthritis;P*<.03, Wilcoxon’s test.Incubation of cells with
biglycan, the agonist for TLR2 receptors, resulted in small and nonsignificant
increase in IL-6 production in both RA patients (after all biglycan
concentrations) and AO patients (only after biglycan at 1 ng/mL). In healthy subjects, insignificant increases
were observed following 2 highest concentrations, and small decrease (22%) was
noticed after the lowest biglycan concentration.


## 3.2. TNFα Production by PBMCs in Response to Stimulation with LPS and Biglycan

Nonstimulated PBMCs from RA patients generated significantly
less TNFα as compared to OA
patients (mean 12 times less) and
control subjects (mean 10 times less) (Table 3). Incubation with LPS induced an
increase in TNF-α production in all 3
groups. However, similarly to IL-6, the increase in TNFα production was significantly more intense after
LPS at 10 and 100 ng/mL (resp., 31 and 29 folds over the baseline) as compared
to that observed in cells from patients with OA (mean 3 to 8 folds increase for
different LPS concentrations) or healthy subjects (mean 4- to 7-fold increase for
different LPS concentrations) (Figure 2). Stimulation with biglycan induced a
significant increase in TNFα production only in PBMCs from healthy subjects in
response to the highest concentration.Table 3
Spontaneous and stimulated TNFα
production by peripheral blood mononuclear cells from patients with
rheumatoid arthritis (RA), osteoarthrosis
(OA), and healthy control (HC); P<.05*—significantly different from nonstimulated; #—significant difference between RA and HC ∧—significant difference between RA and
OA.
NonstimulatedLPS 1 ng/mLLPS 10 ng/mLLPS 100 ng/mLBGN 0,1 ng/mLBGN 1 ng/mLBGN 10 ng/mLGroup of subjectsConcentration of TNFα (ng/mL)RA0,05⋀#0,72⋀#2,02*2,84*0,1⋀#0,260,07⋀#OA0,594,71*2,48*2,62*0,840,450,71HC0,492,59*1,98*1,94*0,630,650,68*Figure 2
Individual changes in TNFα concentration in the supernatants of human leucocytes culture after stimulation with LPS 100 ng/mL; log10 scale; HC-healthy controls; OA-osteoarthritis; RA-rheumatoid arthritis; p*=.04; p**=.027.


## 3.3. 15-HETE Generation by PBMCs in Response to Stimulation with LPS and Biglycan

15-HETE  was generated by unstimulated PBMCs cells
from both RA and OA patients in similar amounts. Stimulation with LPS increased 15-HETE generation in 5 RA patients
(range 27–320%) while
decreased in 2 patients (32% and 34%, resp.). In OA patients, LPS induced a significant (P<.005)
increase in 15-HETE production (mean increase 43%; range 20–175%) (Figure 3). Incubation with biglycan did not affect 15-HETE generation.Figure 3
Changes in 15-HETE release into mononuclear cells supernatants after stimulation with 100 ng/mL LPS in individual patients OA-osteoarthritis; RA-rheumatoid arthritis;p*<.01.


## 3.4. Effect of Cytokines and LPS on TLR2 and TLR4 Gene Expression in PBMCs

Expression of TLR2
mRNA in nonstimulated, cytokine
mix-stimulated, or LPS stimulated cells was similar in RA, OA and HC subjects (Table4(a)). Mean expression of TLR-4 mRNA in
nonstimulated as well as in LPS or
cytokine-mix stimulated cells was similar
in patients with RA and OA, although
in both groups, it was
significantly  lower than
in cells from HC.Table 4
Relative
quantification of the TLR2 and TLR4 mRNA expression in PBMCs from patients with rheumatoid
arthritis (RA), osteoarthrosis (OA), and healthy control (HC).
(a)
Target gene
expression is normalized to housekeeping gene expression and presented asn-fold of the expression in HC group (2−ΔΔCT).
*denotes significant difference in expression comparing with HC (P<.03).
NonstimulatedCytokine mixLPSTLR2HC111RA2.0452.9150.036OA1.2181.660.074TLR4HC111RA0.008*0.2250.0009*OA0.004*0.044*0.00002*(b)
Target gene
expression after stimulation with
cytokine mix or LPS is normalized to housekeeping
gene expression and presented asn-fold
of the expression in non stimulated cells from patients from every group (2−ΔΔCT).
Significant differences in expression compared to nonstimulated cells are
marked with P*<0.02;P#<0.01.
NonstimulatedCytokine mixLPSTLR2HC10.26817.753*RA10.3820.316OA10.365#1.071TLR4HC10.00006*0.1RA10.2121.498OA10.093#0.092*Incubation with cytokine
mix tended to decrease both TLR2 and
TLR4 expressions although the decrease was significant for
both receptors in OA patients and for TLR4 in
HC subjects (Table4(b)). TLR2 gene expression was significantly increased by LPS in healthy controls, while significant suppression of TL4 mRNA
expression was observed after LPS in
cells from patients with OA.


## 4. Discussion

This study was aimed at investigation of the significance of TLRs and
their stimulation in contribution to development of a chronic inflammatory
process present in patients with rheumatoid arthritis. We have demonstrated that peripheral blood mononuclear
cells from patients with newly detected
RA spontaneously generated significantly smaller amounts of interleukin-6 and
TNFα as compared to monocytes from OA patients or healthy subjects and that stimulation with LPS resulted in
dramatically higher augmentation of cytokine production in cells from RA
patients as compared to OA patients. Low-basal production of cytokines is an unexpected
finding, since these two cytokines are considered to play a significant role in
the pathogenesis of joint inflammation in RA; TNFα and IL-6 may stimulate
collagenase production, increase bone resorption and inhibit cartilage
regeneration targeting fibroblasts, osteoclasts, and chondrocytes [28, 29].
Accordingly, previous studies demonstrated increased serum concentrations and
enhanced generation of both cytokines by mononuclear cells from patients with
RA [17–21]. Low baseline cytokine production in circulating mononuclear cells may be explained by the fact that we have
studied patients with early detected RA; it is possible that at this stage of
the disease, cells with high potential for cytokine production have been
recruited to the site of inflammation (synovium) and only less active ones are
available in the peripheral circulation. The most striking
observation is a dramatic difference in response to stimulation with lipopolysaccharides
between mononuclear cells from RA as compared to OA and healthy controls. Although
in patients with OA and healthy controls LPS
induced significant (2-7-fold)
increase in production of both cytokines, the increases in monocytes from RA patients were dramatic: 17 to 31 folds for TNFα and 30 to 48 folds for IL-6. These observations indicate that
monocytes from patients with early detected RA, in spite of having low-baseline
production of cytokines demonstrate high
potential for cytokine generation upon stimulation with LPS. LPS is thought to activate
cells through specific TLR4 receptor which triggers a complex signaling cascade
leading to release of inflammatory mediators potentially contributing to development of
synovial inflammation. It has been previously demonstrated that TLR2 and TLR4
ligands enhance cytokine expression in synovial fluid macrophages from RA patients and induce
catabolic responses in chondrocytes from OA patients [12, 30]. Our observation is in line with putative role of TLR4 receptors in the development
of inflammatory responses in rheumatoid arthritis, suggesting that even in
patients with newly detected and not yet pharmacologically treated RA, circulating
inflammatory cells (monocytes) can be easily triggered via TLR4 stimulation to
generate proinflammatory cytokines.15-HETE is an arachidonic acid metabolite  generated by
reticulocyte-type B synoviocytes
in human rheumatoid
arthritis  with potential  modulatory effect on inflammation [31, 32]. We have demonstrated for the first time
that stimulation of TLR4  but not
TLR2 receptors increases
generation of 15-HETE  by mononuclear cells from both RA and OA
patients. Although LPS  consistently increases 15-HETE generation in cells from 7
of 7 OA patients, in 4 of 7 patients with RA, the
enhancement in 15-HETE generation by LPS was much higher. Further studies are necessary to elucidate the
potential role of TLR4-triggered 15-HETE generation in the pathogenesis of RA and OA.Higher responsiveness
of monocytes from RA subjects to LPS as compared to cells from OA subjects and
healthy controls could be explained by increased expression of TLR4 receptors
on cell surface. Increased expression of
Toll-like receptors on synoviocytes derived from the site of rheumatoid inflammation
has been reported [33, 34].
However, information on TLRs expression on peripheral blood monocytes is less
consistent and studies using
either cytofluorometry to detect TLR2 and TLR4 protein or RT-PCR to detect
receptor transcript reported variable results [29, 30].In
this study, we used quantitative real-time PCR
technique to asses TLR2 and TLR4 expressions in
mononuclear cells. Expression of those 2 types of TLRs was investigated in
nonstimulated cells as well as under stimulatory conditions. We have chosen LPS—a TLR4 agonist—and mix of potent proinflammatory cytokines as
stimulants for this part of our experiments. Although the level of expression
of TLR2 gene was not significantly different among three groups, expression
of TLR4 mRNA was significantly decreased
in cells from RA or OA
patients as compared of healthy controls. However, these data have to be interpreted
with caution since mRNA was isolated
from total leukocyte population and its measurement
might not reflect receptor expression
in  monocytes, which are primarily targeted by TLR agonists. Moreover, recent studies demonstrated
dissociation of mRNA expression of Toll-like receptors from  the
modulation of TLR-mediated responses, suggesting that factors other than receptor expression may be responsible for increased responsiveness
of RA monocytes to TLR ligands [26, 35].  RT-PCR technique allowed us to analyze TLR mRNA expression in response to stimulation with cytokine mix (IL1, TNFα and IL-6) and LPS. While cytokine mix tended to decrease Toll-like receptors
gene expression in all groups studied,
LPS had more variable effects: enhancement of TLR2 receptors was observed in healthy
controls while significant depression of TLR4 expression was
noticed in patients with osteoarthritis.
These observations confirm that TLR
agonist  similarly to endogenous cytokines may modulate
expression of Toll-like receptors on
leukocytes and suggest that this
modulation may vary in cells derived from  patients with different form of rheumatic pathology or from healthy persons.In
conclusion, our study demonstrated a significant overresponsiveness of
peripheral blood mononuclear cells from patients with recently diagnosed
rheumatoid arthritis to stimulation with a bacterial product, lipopolysaccharide. Regulation of TLR expression by cytokine and TLR agonist may differ in
healthy subjects and patients with arthritides.



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