Clinical Immunology (2011) 138, 107–116

available at www.sciencedirect.com

Clinical Immunology
www.elsevier.com/locate/yclim

Synovial fluid-derived T helper 17 cells correlate

with inflammatory activity in arthritis, irrespectively
of diagnosis
Gaetano Zizzo, Maria De Santis, Silvia Laura Bosello, Anna Laura Fedele,
Giusy Peluso, Elisa Gremese, Barbara Tolusso, GianFranco Ferraccioli ⁎

Department of Rheumatology, Catholic University of the Sacred Heart, CIC, Via Moscati 31, 00168, Rome, Italy

Received 7 February 2010; accepted with revision 4 October 2010

Available online 5 November 2010

KEYWORDS Abstract We analyzed peripheral blood (PB) and synovial fluid (SF) mononuclear cells from 16
Th17; rheumatoid arthritis (RA), 9 spondyloarthritis (SpA), 3 microcrystal arthritis patients, to define the
Th1; presence of Th17 and Th1 and their relationship with inflammatory activity, and TCR-zeta chain and
TCR-zeta; ZAP-70 levels. Th17 were significantly higher in SF than in PB and more abundant in microcrystal
ZAP-70; arthritis patients compared to the other groups. Irrespectively of the diagnosis, SF Th17 percentages
Arthritis; correlated with joint (SF total leukocyte count, neutrophil percentage) and systemic (C reactive
Disease activity; protein [CRP], fibrinogen, erythrocyte sedimentation rate) inflammation markers. SF Th1
Inflammation; percentages directly correlated with inflammation and disease activity (CRP, swollen joint count
Microcrystal [SJC]) indices in SpA, but not in RA patients. These observations support the role of Th17 in the
pathogenesis of inflammatory arthritides. The TCR-zetadim lymphocytes in SF were found to produce
the highest amounts of cytokines including IL-17, whereas no ZAP-70 impairment was associated to
Th17.
© 2010 Elsevier Inc. All rights reserved.

Introduction as RA, multiple sclerosis, psoriasis, Crohn's disease and asthma

T helper (Th) 17 represent a new subset of effector CD4+ T
lymphocytes, having a clear inflammatory role. They are
implicated in triggering neutrophil-mediated acute reac-
tions [1–3], and many pathogens can stimulate a Th17 response
[4–7]. Th17 also seem to be critical in the development of
several chronic inflammatory and autoimmune diseases, such
⁎ Corresponding author. Fax: +39 06 353523.
E-mail address: gf.ferraccioli@rm.unicatt.it (G. Ferraccioli).
[8–13].
The discovery of Th17 broke the classical binomy Th1–Th2
[14] and brought into question some cornerstones of the
immunology, including the pathogenic role of IFN-γ and Th1
cells. Because of the reciprocal inhibition between Th1 and
Th17 differentiation pathways [15–17], Th1 might even have
a protective role, as suggested in some studies based on
animal models [16,18] and humans [9]. Nevertheless, CD4+ T
cells producing both IFN-γ and IL-17 (Th1/17) have been
described, primarily in humans [12,19].
Thanks to several studies performed on mutant and
cytokine-injected mice, the pathogenic role of Th17 in

1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2010.10.002
108 G. Zizzo et al.

animal models of inflammatory autoimmune diseases such as
arthritis [20–24], demyelinating encephalomyelitis [25], T cell-
mediated colitis [26] and psoriasis [27] is now unchallenged. In
humans instead, the actual importance of Th17 has not been
completely defined, due to the limited and often contradictory
results. With regard to human arthritis, the majority of the
studies focused on IL-17 expression in the joint rather than on
Th17 cells [9,23,24,28,29]; moreover, although many data
provide evidence that IL-17 can stimulate RA fibroblast-like
synoviocytes, osteoclasts, chondrocytes, and inflammatory
cells cultured in vitro [30], very few studies demonstrated a
direct relationship between IL-17 and articular damage in
arthritis patient series [9]. In addition, studies disagree with
each other about IL-17 levels and Th17 percentages in synovial
fluid (SF) and in peripheral blood (PB) [28,31,32], the
relationship between Th17 and disease activity [24,31,33,34],
the differences in IL-17 levels and Th17 percentages among
different types of arthritis [23,34,36]. We examined the
percentages of Th17 and Th1 in both SF and PB from patients
suffering from different types of arthritis, in order to look for
potential differences between SF and PB and on the basis of
diagnosis; furthermore, we verified the potential relationships
of Th17 and Th1 frequencies with systemic and joint inflam-
mation markers and with clinical disease activity indices.
Additionally, some studies have suggested that T cells with
low levels of TCR-zeta chain (TCR-zetadim) are increased in RA
synovial tissue [37–39], and would selectively migrate from PB
to the joint during disease flares [37]; also, a ZAP-70 defect
may cause autoimmunity [40,41], and a ZAP-70 mutant mouse
model (SKG) develops auto-reactive Th17 cells which cause a
RA-like arthritis [21]. The secondary aim of our study was to
investigate Th17 and Th1 cytokine production in relation to
TCR-zeta and ZAP-70 levels, in order to detect potential
differences in TCR-zeta and ZAP-70 expression between Th17
and Th1 and possibly identify lymphocyte subsets producing
higher amounts of cytokines.
Materials and methods
Patients
Twenty-eight out-patients with knee synovial effusion (16
suffering from RA, of whom 10 seropositive for rheumatoid
factor (RF) and/or anti-cyclic citrullinated peptide antibodies
(anti-CCP) and 6 seronegative; 9 suffering from SpA, of whom 6
from psoriatic arthritis, 1 from reactive arthritis, 2 from
undifferentiated SpA; 3 suffering from microcrystal arthritis,
of whom 2 from gout and 1 from pseudogout) referring to the
out-patient clinics of the Rheumatology Department, at the
Catholic University in Rome, were enrolled in the study
between January and September 2009. The demographic,
clinical, and serological characteristics of the patients are
described in Table 1. After obtaining an informed consent, the
patients underwent arthrocentesis using an echographic guide
and a 19 Gage needle. PB samples (35 mL) were also drowned.
SF and PB samples were collected in Ethylene Diamine
Tetraacetic Acid (EDTA) cuvettes.
Hemocytometric analysis was assessed on 1 mL of SF, diluted
1:3 with PBS, and on 7 mL of PB to obtain total leukocyte count
and total and percentage neutrophil, lymphocyte and mono-
cyte counts. The remaining samples immediately underwent
lymphomonocyte separation.
All the patients underwent clinical examination, including
swollen joint count (SJC) and tender joint count (TJC) of 44
joints; erythrocyte sedimentation rate (ESR), C reactive

Table 1 Demographic, clinical and serological characteristics of the patients.

RA Seropos. RA Seroneg. RA SpA Microcryst.
Number of patients 16 10 6 9 3
Age (years, mean ± SD) 54.0 ± 15.2 53.2 ± 14.9 55.5 ± 16.9 47.3 ± 15.5 68.7 ± 19.5
Male (number, %) 4, 25% 1, 10% 3, 50% 7, 77.8% 3, 100%
Dis. duration (years, median) 12 a 11 12 1.5 4
Duration b 1 year (num, %) 3, 18.7% 2, 20% 1, 16.7% 4,44.4 % 1, 33.3%
ESR (mm/h, mean ± SD) 33.3 ± 18.9 26.5 ± 12.4 47.0 ± 23.5 17.1 ± 9.7 b 51.3 ± 41.0
CRP (mg/L, mean ± SD) 36.9 ± 54.1 12.0 ± 15.8 86.7 ± 70.8 c 15.7 ± 19.8 102.5 ± 93.9
Fibrinogen (mg/dL, mean ± SD) 442 ± 161 359 ± 70 637 ± 143 d 365 ± 25 779 ± 156
TJC (mean ± SD) 10.7 ± 12.7 9.8 ± 11.9 12.0 ± 15.7 4.3 ± 3.9 5.7 ± 6.0
SJC (mean ± SD) 7.7 ± 8.9 6.3 ± 6.4 10.7 ± 13.4 5.0 ± 3.7 3.0 ± 2.6
RAI (mean ± SD) 6.2 ± 5.0 6.0 ± 4.8 6.5 ± 6.3 3.6 ± 2.1 –
DAS44 (mean ± SD) 3.2 ± 1.4 3.0 ± 1.2 3.4 ± 1.9 – –
DAS-CRP (mean ± SD) 2.9 ± 1.4 2.7 ± 1.0 3.3 ± 2.0 – –
HAQ (mean ± SD) 1.3 ± 0.9 1.3 ± 0.8 1.3 ± 1.1 1.3 ± 1.0 –
Steroids (num, %) 11, 68.8% 7, 70% 4, 66.7% 5, 55.6% 0, 0%
DMARDs (num, %) 15, 93.8% 9, 90% 6, 100% 9, 100% 3, 100%
Biologic agents (num, %) 6, 37.5% 3, 30% 3, 50% 4, 44.4% 1, 33.3%
SF WC (units/μL, mean ×103 ± SD) 19 ± 17 18 ± 13 22 ± 24 28 ± 25 14 ± 11
SF neutrophils (% mean ± SD) 58.9 ± 29.0 51.3 ± 29.7 71.5 ± 25.0 50.8 ± 26.2 61.5 ± 51.3
Seropos.: seropositive; Seroneg.: seronegative; Microcryst.: microcrystal arthritis; SD: standard deviation; Dis.: disease.
a
p = 0.017 RA versus SpA disease duration.
b
p = 0.060 SpA versus RA ESR.
c
p = 0.031 seronegative versus seropositive RA CRP.
d
p = 0.028 seronegative versus seropositive RA fibrinogen.
Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis
protein (CRP), fibrinogen, RF, and anti-CCP, and clinical
diagnosis were recorded. In RA patients, the Disease activity
score on 44 joints (DAS44), DAS-CRP and Ritchie index (RAI)
were calculated, and the Health Assessment Questionnaire
(HAQ) was administered.
The study was approved by the local ethical committee
and was performed in compliance with the World Medical
Association's Declaration of Helsinki.
Lymphocyte culture and stimulation
SF and PB samples were diluted 1:2 and 1:1, respectively, with
Phosphate Buffered Saline (PBS) and underwent lymphomono-
cyte gradient separation through Ficoll-Hystopaque (Cederlane,
Ontario, Canada). The lymphomonocytes from SF and PB were
cultured in 48-well plates at the density of 2.5×106 cells/mL of
Rosewell Memorial Park Institute (RMPI)-1640 supplemented
with inactivated fetal calf serum 10%, penicillin 100 U/mL,
streptomycin 0.1 mg/mL, L-glutamine 2 mM and sodium pyru-
vate 1 mM (Invitrogen, Carlsbad, CA). Cells were incubated at
37°, in humidified atmosphere, at 5% of CO2, for 24 h, with
phorbol myristate acetate (PMA; 25 ng/mL) and ionomycin
(1 μg/mL) (PMA/i; Sigma-Aldrich, St Louis, MO) or anti-CD2,
anti-CD3, anti-CD28 biotinylated antibodies and beads conju-
gated with anti-biotin antibodies (T cell activation/expansion
kit, TCA; following the manufacturer's instructions; Miltenyi
Biotec, Bergisch Gladbach, Germany). Brefeldin A (10 μg/mL)
was added to cultures after 2 h. Preliminary experiments were
conducted in order to define the optimal PMA/i concentrations
and the period of cell stimulation. Executing the optimal
procedures, more than 95% of the cells were viable after 24 h of
culture, as they were double-negative for annexin-V and
propidium iodide staining.
Flow cytometer analysis
After stimulation, cells were collected and washed with PBS
(1500 rpm at 15° for 10 min). Five × 105 cells were resus-
pended in 100 μL of PBS per each flow cytometer panel. Cells
were incubated in the dark, at room temperature, for 20 min
with saturating concentrations of the following superficial
marker binding antibodies: anti-CD8-phycoerythrin texas red
(ECD) or anti-CD4-ECD (Beckman Coulter, Marseille, France)
for the cultures stimulated with PMA/i, and anti-CD3-
phycoerythrocyanin (PC) 5 or anti-CD56/CD14/CD16-PC5
(Beckman Coulter, Marseille, France) for the cultures stimu-
lated with TCA. Then, the cells were fixed and permeabilized
with Cytofix (BD Biosciences, San Diego, CA, US) and Perm 2
(BD Biosciences, San Jose, CA, US) following the manufac-
turer's instructions. Subsequently, the cells were incubated in
the dark, at room temperature, for 30 min with saturating
concentrations of the following intra-cellular marker binding
antibodies: anti-IFN-γ-fluorescein isothiocyanate (FITC) (R&D,
Minneapolis, MN, US) and anti-IL-17A-phycoerythrin (PE)
(eBiosciences, San Diego, CA). In 8 patients (4 suffering from
RA, of whom 3 RF and/or anti-CCP-positive, and 4 suffering from
SpA, of whom 2 PsA, 1 reactive arthritis and 1 undifferentiated
SpA), the following panels were also assessed: anti-ZAP-70-FITC
(Beckman Coulter, Marseille, France) or anti-TCR-zeta-FITC
(Novus Biologicals, Littleton, CO), with anti-IL-17A-PE or anti-
IFN-γ-PE (R&D, Minneapolis, MN, US). After washing, cells were
109
analyzed through the cytometer EPICS XL equipped with the
software EXPO32 (Beckman Coulter, Marseille, France) after
proper compensation.
Enzyme-Linked ImmunoSorbent Assay (ELISA) of
human IL-17
SF and PB IL-17 levels in 9 RA patients were determined by
ELISA following manufacturer's instructions (R&D, Minneapolis,
MN, US). The lower detection limit was 0.1 ng/mL.
Statistical analysis
Data were analyzed using SPSS 12.0 (SPSS, Chicago, IL, USA).
Mann–Whitney's or Wilcoxon's rank sum test, as appropriate,
were used to compare continuous variables. Spearman's rank
correlation was used to evaluate the relationship between
different disease parameters. A value of pb 0.05 was considered
statistically significant.
Results
Increased percentages of Th17 and Th1 in SF compared
to PB.
In the whole series of patients, Th17 (referred as IL-17-
producing CD4+ T cells) and Th1 were significantly higher in
SF than in PB after cell stimulation with PMA/i, regardless of
considering the SF and PB percentages of Th17 or Th1 as
coupled or uncoupled variables (Wilcoxon's test or Mann–
Whitney's test, respectively) (Table 2).
Additionally, we found a direct correlation between SF
Th17 and PB Th17 percentages (p 0.002, r +0.67, after PMA/i; p
0.035, r +0.53, after TCA); however, analyzing the patients on
the basis of the diagnosis, we found a correlation between SF
and PB Th17 percentages in RA (p 0.034, r +0.64, after PMA/i)
but not in SpA patients. Instead, there was no correlation
between SF and PB Th1 in the whole group of patients, nor in
RA and SpA subgroups.
Among SF Th17, about 30–40% of cells were also IFN-γ
positive (Th1/17) after PMA/i; this subset slightly varied
according to the diagnosis. However, very low percentages
of Th1/17 cells were detectable after TCA stimulation, and
they were nearly undetectable in PB (Table 2).
Similarly, Th1 percentages were significantly higher after
PMA/i compared to TCA stimulation, in both PB (p b 0.0001)
and SF (p 0.006) (Table 2), whereas there was no significant
difference among the total Th17 percentages obtained after
PMA/i or TCA stimulation.
IL-17, determined by ELISA in 9 RA patients, was detectable
only in 3 patients, in both SF and PB (data not shown).
Th17 in the different types of arthritis
Examining SF and PB Th17 and Th1 in the different subgroups of
patients according to the diagnosis, we found no significant
difference between RA and SpA patients (Table 2). Instead,
microcrystal arthritis patients showed higher SF Th17 percen-
tages compared to RA and SpA patients (Table 2; Fig. 1). This
subgroup of patients had the highest levels of inflammation
110 G. Zizzo et al.

Table 2 SF and PB percentages of Th17, Th1/17 alone and Th1 in the whole cohort and in the subgroups of patients according to
the diagnosis.
SF PB
Th17 Th1/17 alone Th1 Th17 Th1/17 alone Th1
All the patients
PMA/i 1.3 ± 1.9 a 0.4 ± 0.5 29.9 ± 16.1 a,b 0.4 ± 0.5 0.1 ± 0.1 13.5 ± 8.6 b
TCA 1.2 ± 2.0 0.1 ± 0.3 12.7 ± 12.1 0.5 ± 0.9 0.1 ± 0.2 4.7 ± 8.1
RA
PMA/i 1.1 ± 1.0 0.4 ± 0.5 29.7 ± 15.8 0.4 ± 0.5 0.1 ± 0.1 12.8 ± 7.9
TCA 1.0 ± 1.3 0.2 ± 0.3 12.7 ± 11.8 0.6 ± 1.2 0.1 ± 0.3 6.1 ± 9.4
Seropositive RA
PMA/i 0.9 ± 1.2 0.4 ± 0.7 36.7 ± 16.8 0.4 ± 0.7 0.1 ± 0.1 12.3 ± 7.8
TCA 0.5 ± 1.1 0.1 ± 0.3 15.1 ± 15.7 0.2 ± 0.3 0 3.3 ± 2.2
Seronegative RA
PMA/i 1.4 ± 0.8 0.4 ± 0.3 19.1 ± 9.1 0.4 ± 0.2 0.1 ± 0.1 13.7 ± 7.9
TCA 1.6 ± 1.5 c 0.2 ± 0.3 9.2 ± 7.3 1.2 ± 1.8 0.2 ± 0.5 11.0 ± 15.0
SpA
PMA/i 0.7 ± 0.6 0.3 ± 0.3 31.6 ± 16.3 0.3 ± 0.3 0 13.8 ± 11.4
TCA 0.9 ± 1.3 0.2 ± 0.3 14.2 ± 14.3 0.1 ± 0.2 0 0.8 ± 0.3
Microcr. arthr.
PMA/i 5.5 ± 4.1 d 0.8 ± 0.7 25.0 ± 28.7 0.9 ± 0.5 0.2 ± 0.1 17.3 ± 5.6
TCA 7.9 0.2 3.5 0.3 0 5.4
Data are expressed as mean ± standard deviation. Microcr. arthr.: microcrystal arthritides.
a
p b 0.05 SF versus PB Th1 or Th17 percentages (Wilcoxon's test or Mann–Whitney's test).
b
p b 0.005 PMA/i versus TCA-stimulated SF and PB Th1 percentages.
c
p b 0.05 seronegative versus seropositive RA-derived SF Th17 percentages.
d
p b 0.05 microcrystal arthritis versus RA and p b 0.05 versus SpA-derived SF Th17 percentages.

markers, although the statistical significance could not be
achieved because of the low number of patients (Table 1).
Analyzing the RA patients on the basis of serum autoantibody
positivity, we found that the seronegative (RF and anti-CCP-
negative) RA patients had significantly higher SF Th17 percen-
tages compared to the seropositive RA patients (RF and/or anti-
CCP-positive) (Table 2). Also in this case, it could be at least
partially explained by the differences in disease activity among
the patients, as suggested by the presence of significantly
higher inflammation markers (CRP and fibrinogen) in the
seronegative compared to the seropositive RA patients
(Table 1).
We detected no difference in Th17 percentages between
early and long-standing RA patients; additionally, we found
no differences on the basis of treatment (data not shown).
Relationships with inflammatory activity indices
Considering all the patients, Th17 percentages showed a
significant direct correlation with CRP level (p 0.001, r +0.65,
after PMA/i; p 0.003, r +0.65, after TCA; Fig. 2A) fibrinogen
level (p 0.002 , r +0.74, after PMA/i; p 0.005, r +0.75, after
TCA; Fig. 2B) and ESR (p 0.031, r +0.44, after PMA/i). SF Th17
percentages directly correlated with SF total leukocyte count
(p 0.015, r +0.48, after PMA/i; p 0.006, r +0.56, after TCA;
Fig. 2C) and more strongly with SF neutrophil percentage count
(pb 0.0001, r +0.80, after PMA/i; p 0.004, r +0.60, after TCA;
Fig. 2D). We observed, instead, an inverse correlation between
SF Th17 and SF lymphocyte (p b 0.0001, r −0.70, after PMA/i; p
0.003, r −0.60, after TCA) and monocyte percentage count
(pb 0.0001, r −0.76, after PMA/i; p 0.008, r −0.55, after TCA).
Analyzing patients according to the diagnosis, these correla-
tions were substantially kept in RA patients; in particular, SF
Th17 percentages correlated with CRP level (p 0.001, r +0.78,
after PMA/i; p 0.011, r +0.66, after TCA), fibrinogen (p 0.031, r +
0.56, after PMA/i), SF neutrophil percentage count (p 0.003, r +
0.71, after PMA/i; p 0.007, r +0.67, after TCA) and SF total
leukocyte count (p 0.031, r +0.56, after PMA/i; p 0.038, r +0.67,
after TCA).
In the SpA patient subgroup, we found that SF Th17
percentages correlated with ESR (p 0.015, r +0.90, after TCA)
and SF neutrophil percentage count (p 0.013, r +0.82, after
PMA/i).
In the whole group of patients, SF percentage of Th1 did
not show any correlation with inflammation markers;
however, only in the SpA patients, SF Th1 directly correlated
with CRP (p 0.023, r +0.82, after PMA/i).
Although PB percentages of Th17 and Th1 did not show any
correlation with inflammation markers in all the patients taken
together (data not shown), we observed that PB Th17
percentages correlated with CRP level (p 0.017, r +0.84,
after TCA) and with SF total leukocyte count (p 0.029, r +0.81,
after TCA) in the seropositive RA patients. In addition, PB Th1
percentages inversely correlated with disease duration (p 0.03,
r −0.65, after TCA) in the RA patients.
Additionally, we looked for potential correlations between
SF and PB Th17 and Th1 and clinical disease activity indices in
RA and SpA patients. In the seronegative RA patients, we found
that SF Th17 percentages showed a trend towards a significant
correlation with TJC, RAI, DAS44, DAS-CRP, HAQ (p 0.051, r +
0.95 for all these parameters, after PMA/i). In the seropositive
RA patients, we found an inverse correlation between SF Th1
Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis 111

Figure 1 SF and PB-derived Th17 and Th1 from arthritis patients. Flow cytometry analysis of Th17 and Th1 from the synovial fluid
(SF) and the peripheral blood (PB) of four patients suffering from different types of arthritides, after 24 h stimulation with PMA and
ionomycin (PMA/i) or T cell activation kit (TCA). Data are representative of each diagnostic group.

percentages and both RAI (p 0.039, r −0.78, after TCA) and DAS
(p 0.053, r −0.75, after TCA). In the RA patients, we observed
an inverse correlation between SF Th1 percentages and SJC (p
0.053, r −0.60, after TCA) and between PB Th1 and HAQ (p
0.014, r −0.81, after TCA); instead, in the SpA patients, we
found a significant direct correlation between SF percentages
of Th1 and SJC (p 0.019, r +0.84, after PMA/i).
We could not perform the statistical analysis in the
subgroup of microcrystal arthritis patients because of the
low number of subjects.
We did not find any statistical significance between SF or PB
Th1/17 taken alone and inflammatory activity indices, and
Th17 showed the same statistical relations whether Th1/17
were included or not in the analysis (data not shown).
SF and PB levels of TCR-zeta and ZAP-70 in Th17 and
Th1
found that both SF TCR-zetadim Th17 and Th1 produced
significantly higher amounts of cytokine (IL-17 and IFN-γ,
respectively) compared to Th17 and Th1 expressing bright and
intermediate levels of TCR-zeta (TCR-zetabright and TCR-
zetaint Th cells), respectively, at the single cell level (p
0.043 in all cases; Figs. 3A–B); that was not valid considering
PB-derived Th cells (Table 3).
We also examined ZAP-70 levels in Th17 and Th1 from SF
and PB. We found no significant difference in ZAP-70 MFI
values between Th17 and Th1 and between SF and PB-
derived Th17 (Table 3). Instead, Th1 expressing low level of
ZAP-70 (ZAP-70dim Th1) were found more frequently in PB
rather than in SF (p 0.038; Table 3). SF ZAP-70dim Th1 were
found to produce significantly higher amounts of IFN-γ
compared to Th1 expressing bright and intermediate levels
of ZAP-70 (ZAP-70bright and ZAP-70int Th1) (p 0.043 in both
cases; Figs. 3C–D and Table 3).

We analyzed TCR-zeta levels in CD3+ and CD4+ T cells from PB Discussion

and SF of 8 (4 RA and 4 SpA) patients. We found that CD3+ TCR-
zetadim T cells were significantly more abundant in SF
compared to PB (7.0 ± 2.6% versus 3.3 ± 1.0%; p 0.05); that
was even more significant considering only CD4+ TCR-zetadim T
cells (5.8 ± 2.2% versus 2.3 ± 0.2%; p 0.014). Then, we studied
TCR-zeta levels in relation to cytokine production in CD4+ T
cells after PMA/i stimulation. We observed that TCR-zetadim T
cells were able to produce not only IFN-γ but also IL-17.
However, we failed to demonstrate significant differences in
terms of TCR-zeta MFI values between Th17 and Th1 and
between SF and PB Th17 or Th1 (Table 3). Nevertheless, we
Our study demonstrates that, in patients suffering from
inflammatory arthritides (RA, SpA, and microcrystal
arthritides), small but not negligible amounts of IL-17-
producing CD4+ T cells are detectable in SF and PB,
significantly more abundant in SF compared to PB. To date,
very few studies quantified both SF and PB Th17 and compared
them in different types of arthritides. Moreover, they led to
contradictory results. Yamada et al. found that Th17 are more
frequent in PB than in SF and reasoned that Th17 might have
only a minor role in RA pathogenesis [31]. Instead, Shahrara et
112 G. Zizzo et al.

A B
200 900

800

Fibrinogen (mg/dL)
150
CRP (mg/L) 700

600
100
500

400
50
300 p= 0.002
p= 0.001
r= +0.65 r= +0.74
0 200
-0.1 0 0.1 1 10 -0.1 0 0.1 1 10

SF Th17+Th1/17 (% Log) SF Th17+Th1/17 (% Log)

C D
SF total leukocyte count (unit/ µL)

70000 100.0

60000

SF neutrophils (%)
80.0
50000
60.0
40000

30000 40.0
20000
20.0
10000
p= 0.015 p< 0.0001
0 r= +0.48 0.0 r= +0.80
-0.1 0 0.1 1 10 -0.1 0 0.1 1 10

SF Th17+Th1/17 (% Log) SF Th17+Th1/17 (% Log)

Figure 2 Direct correlations of SF percentages of Th17 with systemic and joint inflammation markers. Synovial fluid (SF)
percentages of Th17 significantly correlated with C reactive protein (CRP) (A) and fibrinogen (B) levels, with SF total leukocyte count
(C) and SF neutrophil percentage (D). Values of Th17 were obtained after 24 h PMA/i stimulation, include Th1/17 percentages and are
expressed in logarithmic scale (Log).

Table 3 TCR-zeta chain and ZAP-70 in SF and PB Th17 and Th1.

SF PB
Th17 Th1 Th17 Th1
bright
TCR-zeta (%) 60.8 ± 13.7 59.0 ± 4.9 41.7 ± 22.7 61.4 ± 24.4
TCR-zetaint (%) 32.0 ± 13.9 34.5 ± 6.7 35.0 ± 11.0 35.6 ± 23.2
TCR-zetadim (%) 7.6 ± 1.6 7.1 ± 3.6 24.6 ± 15.1 3.6 ± 2.0
TCR-zeta MFI 127.3 ± 46.1 121.7 ± 8.2 94.7 ± 35.6 131.0 ± 60.0
Cytokine MFI in TCR-zetabright 112.1 ± 69.9 208.7 ± 102.9 98.7 ± 60.6 118.6 ± 17.6
Cytokine MFI in TCR-zetaint 112.4 ± 115.4 226.8 ± 142.8 65.3 ± 35.1 119.9 ± 19.6
Cytokine MFI in TCR-zetadim 364.7 ± 367.6 a 451.3 ± 317.9 b 85.4 ± 43.4 221.1 ± 74.6
ZAP-70bright (%) 32.9 ± 34.0 26.9 ± 29.2 9.4 ± 15.1 7.6 ± 8.7
ZAP-70int (%) 48.2 ± 29.0 61.8 ± 22.1 71.4 ± 9.9 62.4 ± 11.4
ZAP-70dim (%) 19.8 ± 16.5 12.6 ± 9.0 19.0 ± 17.0 30.8 ± 14.6 c
ZAP-70 MFI 81.4 ± 46.0 73.0 ± 36.0 53.6 ± 29.6 41.7 ± 16.7
Cytokine MFI in ZAP-70bright 72.3 ± 34.1 110.0 ± 59.4 17.6 ± 19.9 75.5 ± 57.1
Cytokine MFI in ZAP-70int 67.3 ± 19.9 132.4 ± 51.1 42.9 ± 8.3 112.8 ± 42.6
Cytokine MFI in ZAP-70dim 214.7 ± 283.6 198.1 ± 90.2 d 316.1 ± 490.8 181.2 ± 61.0
Data are expressed as percentages (%) or mean ± standard deviation. Data were collected after T lymphocyte stimulation with PMA/i from
8 (4 RA and 4 SpA) patients. int: intermediate.
a
p b 0.05 TCR-zetadim versus both TCR-zetabright and TCR-zetaint IL-17 MFI in SF Th17.
b
p b 0.05 TCR-zetadim versus both TCR-zetabright and TCR-zetaint IFN-γ MFI in SF Th1.
c
p b 0.05 PB versus SF ZAP-70dim Th1.
d
p b 0.05 ZAP-70dim versus both ZAP-70bright and ZAP-70int IFN-γ MFI in SF Th1.
Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis 113

Figure 3 TCR-zeta chain and ZAP-70 levels in SF Th17 and Th1. Synovial fluid (SF) Th17 (A) and Th1 (B) were divided, according to
TCR-zeta chain levels, in three subgroups (TCR-zetabright, TCR-zetaint and TCR-zetadim). Although most cells were TCR-zetabright or
TCR-zetaint, TCR-zetadim Th17 and Th1 produced significantly higher amounts of IL-17 or IFN-γ, respectively, compared to TCR-
zetabright and TCR-zetaint Th cells, at the single cell level (see text and Table 3). Additionally, Th17 (C) and Th1 (D) were divided,
according to ZAP-70 levels, in three subgroups (ZAP-70bright, ZAP-70int and ZAP-70dim). Similarly, SF-derived ZAP-70dim Th cells
produced higher amounts of cytokines compared to ZAP-70bright and ZAP-70int, at the single cell level, although statistical significance
was reached only for ZAP-70dim Th1 (see text and Table 3). Data were obtained after 24 h PMA/i stimulation and are representative of
eight different patients (4 RA and 4 SpA).

al., in agreement with our results, showed a higher level of
Th17 in SF compared with PB in RA patients [28].
Considering the patients according to the diagnosis, we
observed that RA and SpA patients had comparable amounts of
Th17. Instead, SF level of IL-17 was reported to be significantly
higher in SpA than in RA patients [35], and one study showed
higher PB Th17 in SpA compared to RA patients [32], although
other authors did not confirm the latter results [31].
The microcrystal arthritis patients herein analyzed showed
the highest percentages of Th17 in SF. Instead, previous
studies reported significantly lower SF levels of IL-17 in
microcrystal arthritis compared with RA patients [23,36]. So
far, microcrystal arthritides have been considered as innate
immunity disorders, due to the activation of Toll-like receptors
and NALP3 inflammosome by monosodium urate (MSU) and
calcium pyrophosphate dihydrate crystals [42,43]. Recently,
some authors found that MSU, especially in the crystalline
form, up-regulates CD25 and CD70 surface expression in
human T lymphocytes in vitro, suggesting that microcrystals
can directly contribute to activate T cells [44]. The detection
of Th17 in microcrystal arthritis patients provides new insights
in the pathogenesis of microcrystal arthritides and fits well
with microcrystal-induced T cell activation and NALP3-
mediated IL-1 release; in fact, IL-1 is essential for Th17
development in both mice [20,22] and humans [3,45].
Analyzing the RA patients based on serum RF and/or anti-
CCP positivity, we observed that the seronegative RA patients
presented higher SF Th17 compared to the RA seropositive
patients. That might be related to the preponderant role of T
cells in seronegative RA pathogenesis, whereas in seropositive
RA the autoantibody production would suggest a major
contribution from B lymphocytes and plasma cells. However,
the number of seronegative RA patients analyzed does not
allow to draw final conclusions about this concern.
Furthermore, differently from the majority of the studies
performed to date, we considered the inflammation markers
and the disease activity indices of the patients for a better
interpretation of the results. In fact, we found that SF Th17
percentages directly correlated with articular (SF total
leukocyte count and SF neutrophil percentage) and systemic
inflammation markers (CRP, fibrinogen, and ESR) in the whole
group of patients. The correlation between SF Th17 and SF
114
neutrophil percentage count could be explained by the key
role of IL-17 in neutrophil chemotaxis into the joint space, as
observed in animal models [46,47]. Besides, the close
relationship found between SF Th17 and CRP level may be
consistent with the essential role of IL-6 in promoting CRP gene
expression [48]; actually, IL-6 induces Th17 differentiation and
is produced by Th17 themselves [49]. That can explain the
observation that the microcrystal arthritis patients had higher
average levels of inflammation markers compared with RA and
SpA patients; similarly, the seronegative RA patients had
significantly higher inflammation markers in comparison with
the seropositive RA patients. Thus, the differences in Th17
frequencies among the patient subgroups could, at least
partially, be related to the inflammatory status in the single
patient rather than to the type of arthritis.
Analyzing patients on the basis of their diagnosis, we
observed that in both RA and SpA patients SF Th17 directly
correlated with joint (SF total leukocyte count and neutrophil
percentage in RA; neutrophil percentage in SpA) and systemic
(CRP and fibrinogen in RA; ESR in SpA) inflammation markers.
In this regard, to our knowledge, this is the first cytofluori-
metric study that directly supports the role of SF Th17 in
inflammatory activity in RA and SpA. However, we did not
observe significant correlations between Th17 and clinical
disease activity indices, but only a positive trend towards TJC,
RAI, DAS44, DAS-CRP and HAQ in seronegative RA patients.
PB Th17 correlated with SF Th17 percentages in the whole
group of patients and in the RA patients, but not in SpA.
Moreover, only in the RA patients (at least in the seropositive
group), PB Th17 correlated with inflammation markers (CRP,
SF total leukocyte count). This fact is consistent with the
results reported by Shen et al., who found a correlation
between PB Th17 and CRP in RA but not in SpA patients [33].
As regards Th1, we found a direct correlation of SF Th1
percentages with joint and systemic inflammation (SJC and
CRP) in SpA, but not in RA patients. Indeed, other studies
have even supported a protective role of Th1 and IFNγ in
both RA patients [9] and RA animal models [16,18].
Differences in results from several studies on Th17 could be
partially due to the different methods used for lymphocyte
stimulation: PMA/i for 4–5 h [28,31]; PMA/i after pre-
incubation with IL-23 for 18 h [28]; IL-15 for 72 h [50]; and
anti-CD3/CD28 for 24 h [34]. The concentrations of PMA and
of ionomycin were also quite variable. Certainly, ex vivo
stimulation is more an indication rather than a definitive
evidence of the in vivo cytokine expression. Nevertheless,
we used two methods in parallel for T cell stimulation to
confirm the results. After repeated attempts, in order to
assess the optimal stimulation procedures, we found that
both TCA (anti-CD3/CD28/CD2) and PMA/i stimulations for
24 h led to congruous and comparable amounts of Th17, in
both SF and PB. Instead, Th1 amounts were significantly
higher after stimulation with PMA/i rather than with TCA, as
previously reported [51].
Presumably because PMA/i better stimulate IFNγ produc-
tion, SF Th1/17 were more recognizable in the SF after PMA/i
rather than TCA stimulation. At any rate, we did not find any
correlation between Th1/17 taken alone and inflammatory
activity indices, and Th17 correlations were substantially kept
whether Th1/17 were included or not. Moreover, Th1/17 were
often undetectable in the PB, in agreement with other studies
[34].
G. Zizzo et al.
Finally, we analyzed TCR-zeta and ZAP-70 levels in Th17
and Th1 from SF and PB, in order to notice potential
differences among these cells. Chronically stimulated
lymphocytes undergo TCR-zeta down-regulation and become
TCR-zetadim lymphocytes; TCR-zetadim are increased in RA
synovial tissue [37–39], and would selectively migrate from
PB to the joint during disease flares [37]. In agreement with
previous studies, we found a significantly higher frequency of
TCR-zetadim lymphocytes in SF compared to PB. Additionally,
we demonstrated that TCR-zetadim Th cells not only produce
IFN-γ [37], but also IL-17; moreover, these cells can produce
significantly higher amounts of IL-17 or IFN-γ compared to
TCR-zetabright and TCR-zetaint Th cells. However, we failed
to detect significant differences in TCR-zeta levels between
Th17 and Th1, thus we could not provide evidence that Th17
and Th1 might be differentially associated to chronic
stimulation.
It has been observed that a ZAP-70 defect can determine
autoreactivity, by affecting both positive and negative
thymic selection and impairing the formation of thymic and
peripheral Tregs [41,42]. Moreover, the ZAP-70 mutant SKG
mice develop auto-reactive Th17, of which stimulation and
expansion result in a Th17-mediated arthritis, that clini-
cally and serologically resembles RA [21]. However, in our
study, we did not observe a defect in the levels of ZAP-70 in
SF Th17 compared to SF Th1. Nevertheless, we did find a
considerable subset of T lymphocytes with low levels of ZAP-
70, but they basically produced high amounts of IFNγ. Instead
of a primary defect, it is likely that ZAP-70 undergoes a
progressive down-regulation — similar to what would happen
with TCR-zeta expression — in chronically stimulated T cells;
in fact, the hypomorphic mutations in the human ZAP-70
gene described so far resulted in immunodeficiency rather
than autoreactivity [52].
In conclusion, our study demonstrates that both Th17 and
Th1 lymphocytes are present in PB and mainly in SF derived
from patients with inflammatory arthritides (RA, SpA, and
microcrystal arthritis), and that Th17 percentage directly
correlates with inflammatory activity, suggesting a key role
of Th17 in the pathogenesis of these diseases. This study also
supports the hypothesis that TCR-zetadim lymphocytes could
contribute to joint inflammation by releasing high amounts
of cytokines including IL-17, whereas no ZAP-70 impairment
is apparently associated to Th17 development in arthritis
patients.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We would like to thank Prof. A. Fattorossi and Dr. A.
Battaglia, for their precious support.
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