ARTICLE
Auteur(s) : Luisa Vega1, Julia
Barbado1, Raquel Almansa1,2, Rocío
González-Gallego1, Lucía Rico2, Antonio
Jimeno1, Mercedes Nocito2, Raúl
Ortiz de Lejarazu2, Jesus F
Bermejo-Martin2
1Unidad de Enfermedades Autoinmunes, Servicio de
Medicina Interna, Hospital Clínico Universitario, Valladolid,
Spain
2Unidad de Investigación en Infección e Inmunidad &
Servicio de Microbiología e Inmunología, Hospital Clínico
Universitario, IECSCYL, Valladolid, Spain
accepté le 7 Decembre 2009
There is accumulative evidence for the key role of interferon α-
and interferon-inducible chemokines and their signalling pathways
in the pathogenesis of systemic lupus erythematosus (SLE) [1-4]. An
association between transcription levels of interferon-inducible
chemokines in peripheral blood leucocytes with disease activity,
degree of organ damage, and specific autoantibody patterns in SLE
has been recently described [1, 5]. With regard to the role of
cytokines in SLE, one of the interleukins most intensively
investigated in relation to lupus is TNF-α, given the fact that
anti-TNF-α drugs are now widely used in the treatment of rheumatoid
arthritis and Crohn’s disease. The role of TNF-α in lupus is
controversial. Its levels are increased in this disease, but while
there is evidence of disease improvement using short-term therapy
with anti-TNF-α antibodies, long-term therapy with infliximab, has
been associated with severe, adverse events [6, 7].
IL-10 levels are consistently high in the serum of patients
with this condition, and anti-IL-10 antibodies have been found
to improve disease in murine models of SLE [8, 9].
IL-10 induces polyclonal activation of B lymphocytes,
potentially inducing the secretion of autoantibodies.
Interferon-gamma-inducing monokines (IL-12, IL-18) are also
increased in serum from patients with lupus [10]. Although
IL-17 contributes to autoimmune disease in rheumatoid
arthritis and Crohn’s disease, its role in systemic lupus
erythematosus is far less clear [11-13]. In murine models of SLE,
the development of autoantibodies is dependent on IL-6, a
Th-17-related cytokine. As a consequence, a wide range of mediators
play a role in lupus, including Th1, Th2, Th17 cytokines, type
I and II interferons and chemokines [14]. Most of the work
quantifying cytokines and chemokines in lupus has been developed
using ELISAs, which has resulted in the description of the role of
a very limited number of mediators, making it difficult to obtain a
full picture of their part in this disease. For the same reason,
there is much left to understand concerning the regulatory
mechanisms, the relationships between these mediators, and the
influence of treatment on their secretion profiles. In the present
work, we studied 18, soluble immune mediators in plasma samples
from a cohort of patients diagnosed with SLE with different degrees
of disease activity, and also in a cohort of patients in complete
clinical remission, all receiving long-term, standard treatment
with immunomodulators, and using a multiplex system. This approach
allowed the simultaneously quantification of a large number of both
innate- and adaptive immunity-related mediators which are present
in very small amounts in plasma.
Methods and materials
Patients and control individuals
SLE patients (n = 57) were recruited at the Unit of Autoimmune
Diseases of our Hospital. A group of healthy volunteers of
similar age and comparable sex distribution working at the
University of Valladolid was recruited as the control (n = 10). The
SLE patients fulfilled the classification criteria of the American
College of Rheumatology for SLE [15]. For each patient, disease
activity and disease-related damage were assessed at the time of
blood sampling, using the SLE Disease Activity Index
2000 (SLEDAI-2K) [16] and the Systemic Lupus International
Collaborating Clinics/American College of Rheumatology Damage Index
(SDI) [17]. Fifteen patients were in complete clinical remission
(SLEDAI-2K score = 0) and the remaining 42 showed different
degrees of disease activity. Patients showing an SLEDAI index below
10 were classified as having mild/moderate disease (n = 13);
patients with an index of 10 or higher were classified as
having severe disease (n = 29), in accordance with the SLEDAI-2K
flare system. Consequently, four comparison groups were considered
in the analysis: 1) complete remission, 2) mild/moderate activity,
3) severe disease and 4) healthy controls. All of the patients
(with active or no active disease) were receiving immunomodulatory
therapy at the time of the sample collection. Patients were
classified into three groups depending whether the treatment
regimen included: a) non-steroidal anti-inflammatory drugs (NSAD)
b) steroids alone or associated with cloroquine and c)
immunosuppressors. Doses were as follows:
- – NSAID: naproxen, 1,500 mg/24 h; indomethacin, 200
mg/24 h; celecoxib, 400 mg/24 h; etericoxib, 120
mg/24 h;
- – average steroid dose: prednisone, 10 mg/24 h;
methylprednisolone, 10.6 mg/24 h; deflazacort, 7.5
mg/24 h; antimalarial: hydroxicloroquine, 350 mg/24 h;
cloroquine, 150 mg/24 h;
- – immunosuppressors: mycophenolate mofetil, 2
gr/24 h; azathioprine, 50 mg/24 h; cyclosporine, 100
mg/24 h; cyclophosphamide, 1,000 mg/month for six months;
methotrexate, 15 mg/week.
Sample collection and mediator profiling
A blood sample was collected into a 10 ml EDTA tube. Plasma
was obtained after proper centrifugation and was immediately frozen
at - 80°C until evaluation. Mediators were analyzed in the
Infection and Immunity Research Unit of our Hospital using two
plates of a multiplex assay (Biorad TM, Hercules, CA, USA) on a
Luminex TM platform (Austin, TX, USA). IFN-α was measured using two
plates of an ELISA from Thermo TM. Detection limits (pg/mL) were as
follows: IL-2 (2.68); IL-4 (0.37); IL-6 (2.93);
IL-8 (1.77); IL-10(2.17); GM-CSF (7.55); IFN-γ (6.0); TNF-α
(6.62); IL-1β (2.54); IL-5(2.57); IL-7 (2.57);
IL-12p70 (2.74); IL-13 (3.02); IL-17(4.89); GCSF (1.94);
MCP-1 (2.23); MIP-1β (2.32), IFN-α (10.0). Measurements below
the level of detection were reported equal to the level of
detection. Levels of IFN-α, IL-1β, IL-6, IL-7 and
IL-13 in plasma were consistently below the limit of
detection, and were not included in further analyses.
Statistical analysis
The Mann-Whitney U test was used to assess the significance of
differences in the levels of mediators between groups. Associations
between mediator levels, laboratory parameters and severity scores
were studied calculating the Spearman-Karber correlation
coefficients.
Ethics
This study was approved by the Review Board of the Hospital Clínico
Universitario de Valladolid, Spain. Informed consent was obtained
from all study participants.
Results
Patients’ general characteristics are detailed in table 1. Disease duration in years (mean; min; max)
was (9.9; 0.2; 42.5) for those with active disease and (11.6; 2.0;
35.0) for those in complete remission. Treatment duration in months
was (mean; SD) (10.7; 3.7) at the time of sample collection for
those with active disease, and (19.2; 13.2) for those with “0”
points according to the SLEDAI score. All of the patients except
one showed positive antinuclear antibodies. The percentage of
patients showing positive autoantibodies were as follows: anti-DNA
antibodies (n = 34, 59.6%); Anti-Ro antibodies (n = 30, 52.6%);
anti-La antibodies (n = 7, 12.2%); anti-histone antibodies (n = 7,
12.2%); antiribosomal antibodies (n = 4, 7.0%); anti-Sm antibodies
(n = 5, 8.7%); anti-RNP antibodies (n = 5, 8.7%); anti-Jo
antibodies (n = 1, 1.7%); anticardiolipin antibodies, ACAs (n = 18,
31.5%). Six patients (10.5%) had positive anti-lupic coagulant.
When mediator levels were compared between patients and controls
on the basis of their SLEDAI score, the three groups of patients
(complete remission, mild-moderate and severe) showed higher levels
of the chemoattractant proteins MCP-1/CCL2, MIP1-β/CCL4,
IL-8/CXCL8 than the control (p < 0.05) (figure 1). Levels of
GM-CSF were increased compared to the control in the two groups of
patients with active disease (data not shown). Levels of MIP1-β and
IL-8/CXCL8 were higher in the mild/moderate group compared to
the severe group (figure
1). Conversely, levels of Th1, Th2, Th17 cytokines,
and type II interferon (IFN-γ) did not differ from those of the
control group. Interferon type I (IFN-α) could not be detected in
those patients with active disease, those in clinical remission or
in the controls.
Similar results were observed when the levels of mediators were
compared as regards the degree of chronic and irreversible tissue
damage, and as assessed by the SDI score (figure 1). Both groups of
patients (those with SDI scores of 1 to 2 and those with
scores above 2), showed significantly higher values for MCP-1/CCL2,
MIP1-β/CCL4, IL-8/CXCL8 and GM-CSF than the control (p <
0.05), but with no significant differences between them (figure 1).
Since the different immunomodulatory drug regimens could
theoretically influence the patterns of mediators observed,
patients were re-classified on the basis of treatment received.
Similarly to the results obtained regarding degree of severity,
increases in MCP-1/CCL2, MIP1-β/CCL4, IL-8/CXCL8, GM-CSF over
control levels were observed in the three treated groups, with no
significant differences between them (figure 1). No differences
were found between the three treatment groups for the SLEDAI
score.
Patients with active disease and ACAs, showed significantly
higher levels of IL-8 and MIP-1β than those with no ACAs.
Median levels of these mediators were respectively 2.3-fold and
1.7-fold the median of those with no ACAs: 21.6 pg/mL versus
9.3 pg/mL in the case of IL-8 and 131.3 pg/mL versus
74.9 pg/mL in the case of MIP-1β.
Interestingly, when associations between MCP-1/CCL2,
MIP1-β/CCL4, IL-8/CXCL8, GM-CSF were studied on the basis of
2-by-2 comparisons in the group of patients with active
disease, significantly positive associations were observed between
all of them (figure
2). MCP-1/CCL2, MIP1-β/CCL4, IL-8/CXCL8 showed also
positive correlations in the group of patients with non-active
disease (figure
3). On the other hand, none of these mediator levels
correlated with the SLEDAI, the SDI scores or with disease
duration.
Table 1 Patient characteristics. Data are shown as mean
(SD)
|
Disease activity
|
Treatment
|
|
Complete remission (n = 15)
|
Mild-moderate (n = 13)
|
Severe (n = 29)
|
NSAID (n = 11)
|
Steroids (n = 19)
|
Immuno- suppressor (n = 12)
|
|
Sex (M/F)
|
5/10
|
3/10
|
4/25
|
1/12
|
5/20
|
6/13
|
|
Age
|
34,0 [12.7]
|
37.6 [11.9]
|
41.4 [13.3]
|
34.6 [8.5]
|
42.7 [14.2]
|
35.5 [12.7]
|
|
Age at diagnosis
|
22.3 [9.1]
|
29.9 [10, 6]
|
32.7 [12.4]
|
24.6 [7.9]
|
33.4 [13.8]
|
26.8 [9.7]
|
|
SLEDAI
|
0 [0]
|
4.6 [2.2]
|
16.6 [6.3]
|
10.6 [7.9]
|
8.8 [8.3]
|
9.2 [10.2]
|
|
SDI
|
2.7 [1.7]
|
2.5 [1.4]
|
3.1 [1.9]
|
2.3 [1.4]
|
2.9 [2.1]
|
3.1 [1.3]
|
Discussion
The roles of both innate and adaptive immunity mediators have been
extensively demonstrated in the pathogenesis of lupus. Currently
available multiplex systems allow the simultaneous detection of a
large number of immune mediators. This technology, when applied to
the study of a complex disease such as SLE, provides us with a
broad picture of the ongoing immune response taking place at a
given moment. The patients studied in our work suffered from
different degrees of disease severity, requiring long-term
treatment with standard immunomodulatory drugs commonly employed in
the treatment of lupus (NSAIDs, steroids, cloroquine and
immunosuppressors). We also included a group of patients showing
complete clinical remission for comparison purposes. Interestingly,
regardless of the degree of disease activity, none of the drug
combinations employed was able to normalize the plasma levels of a
group of chemotactic factors which participates in the innate
response to pathogens: MCP-1/CCL2, MIP1-β/CCL4 and IL-8/CXCL8.
Conversely, none of the levels of the adaptive immunity-related
mediators measured (Th1, Th2, Th17 cytokines, IFN-γ) differed
from those found in the healthy controls. Similarly, IFN-α, a key
initiator of innate immunity, was not detected in the plasma of the
patients. It has been suggested that IFN-α plays a central role in
SLE. IFN-α stimulates the production of chemokines such as
MCP-1 and IL-8 [5]. TNF-α, in turn, is able to induce, via
NF-kB, the secretion of MIP-1β [18]. The absence of increased
levels of both IFN-α and TNF-α in the plasma of patients with lupus
suggests that other factors are inducing the secretion of MCP-1,
IL-8 and MIP-1β. Alternatively, IFN-α and TNF-α could be
playing a role in this disease, but at levels below the limits of
detection of the measurement methods employed here. The third
possibility is that both cytokines could be increased at the local
rather than systemic level.
MCP-1/CCL2, MIP1-β/CCL4 and IL-8/CXCL8 can be induced
by a wide variety of stimuli, including growth factors, bacterial
and viral products [3]. The increased levels of GM-CSF found in the
plasma of patients with active lupus is consistent with the
findings of Willeke et al., who described an increased
frequency of GM-CSF-secreting PBMC in these patients [19]. The
absence of increased levels of GM-CSF in those patients in clinical
remission could reflect the success of treatment in controlling the
secretion of this mediator.
The absence of increased plasma levels of IFN-α and adaptive
immunity mediators in patients with active lupus and in those in
remission could also be due to a modulatory effect exerted by the
treatment administered to these patients. Further studies
collecting sequential samples are necessary to clarify this
particular aspect, however previous reports demonstrating increases
in IFN-α, IL-6, TNF-α, IL-10, IL-17 and IL-12 [5, 8-14, 20-22]
in the plasma of patients with lupus supports a down-modulatory
effect exerted by the treatment, at least on these cytokines. On
the other hand, our results, which contradict previous reports
involving Th1, Th2 and IL-10 levels, could be explained on the
basis of the timing of the sample collection, which used to be
early in the course of a flare. The samples in our study were
collected well after treatment instigation, by which time, cytokine
levels in our patients might have been greatly modified.
The increase in MCP-1/CCL2, MIP1-β/CCL4 and
IL-8/CXCL8 levels over control values was observed in those
patients in clinical remission, in those with mild/moderate
disease, as well as in the severely affected ones (based in the
SLEDAI score). Increases in these chemokines were also observed in
those patients with and without chronic tissue damage (based in the
SDI score), indicating the active role of these mediators in all
the stages of the disease. This is reinforced by the absence of any
association between chemokine levels, SLEDAI/SDI scores and
duration of the disease. A major finding of this study is the
evidence of a positive correlation between MCP-1/CCL2,
MIP1-β/CCL4 and IL-8/CXCL8 in both active and inactive
lupus, pointing somehow towards a coordinated regulation of their
secretion in this disease. This coordinated secretion might
indicate a synergistic role in the pathogenesis of the disease,
since these four molecules mediate a chemotactic activity inducing
cell migration towards inflammatory sites. The significance of the
markedly higher levels of MIP1-β and IL-8 in the mild/moderate
patients compared to severe patients, and of the increase in
IL-8 in those patients in remission compared to patients with
severe disease, is difficult to interpret, since these chemokines
were increased, compared to control values, in all the patient
groups. Studies involving a larger number of patients would help to
clarify this particular issue.
An association of anti-Sm or anti-RNP autoantibodies with
chemokine mRNA levels has previously been described [5]. It was not
possible to consider this variable in our analysis given the low
number of patients positive for these autoantibodies in our study
population. However, we were able to compare the levels of
mediators in those patients with and without anticardiolipin
antibodies. Here we describe, for the first time, significantly
higher levels of IL-8 and MIP-1b in patients with positive
ACAs. The relationship between ACAs and chemokines has not been
studied in depth. The presence of ACAs is known to be linked to
thrombosis and endothelial damage. Whether chemokines such as
IL-8 are at the origin of the vascular damage seen in patients
with ACAs or, conversely, whether their levels rise as a
consequence of it, remains to be elucidated.
In conclusion, a coordinated secretion of both CC chemokines
(MCP-1/CCL2, MIP1-β/CCL4) and CXC chemokines (IL-8/CXCL8) was
observed in SLE patients receiving long-term treatment with
immunomodulators. This was seen in absence of detectable levels of
IFN-α in plasma, and with normal levels of TNF-α and
adaptive-immunity mediators, regardless of disease severity. These
results indicate the existence of a basal, pro-inflammatory state
in patients with lupus, even in the absence of symptoms. This might
serve as a “substratum” or initiator of the immunological events
taking place during a flare of the disease. Our results also
highlight a failure of the standard drug regimens employed in the
treatment of lupus in targeting these innate immunity
pro-inflammatory molecules. Addition of specific chemokine
inhibitors to the classical drug regimens might contribute to
improve the clinical response to treatment [23].
Acknowledgments
The authors thank the Nursing Team of the Internal Medicine Service
of our Hospital, who kindly collected the samples. The authors
would also like to thank Epifanio Ramos, Ana Loma and Concha Nieto
for performing autoantibody detection. This work was possible
thanks to a grant obtained from “Caja de Burgos” (“Premios de
Investigacion Biomédica”). Jesus F Bermejo-Martin, R. Almansa and
L. Rico are supported by “Fondo de Investigaciones Sanitarias”,
FIS, Ministry of Science and Innovation, Spain, EMER07/050,
“Programa para favorecer la incorporación de grupos de
investigación en las Instituciones del Sistema Nacional de Salud,
EMER07/050” and “proyectos de investigación en salud” PI081236.
Disclosure. None of the authors has any conflict of
interest to disclose.
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