Higher Elastase Activity Associated with Lower IL-1 s
GCF from Juvenile Systemic Lupus Patients
Carlos Marcelo S. Figueredoa,b/Alessandra Areasa/Flávio R.Sztajnbokc/Vivian Micelia/
Letícia A. Mirandab/Ricardo G. Fischera/Anders Gustafssonb
Purpose: Theour aim was to evaluate the expression of interleukin-18 (IL-18) and, interleukin-1-beta (IL-1b‚) and the amountof elastase activity in gingival crevicular fluid (GCF) from inflamed gingival sites in patients with juvenile systemic lupus ery-thematosus (JSLE), and compare these to the expression in GCF from inflamed sites in generally healthy controls. In addition,we related the local inflammation in periodontal tissues was related to systemic inflammation by the assessment of IL-18 lev-els in plasma.
Materials and Methods: GCF from 16 patients with JSLE and 14 controls were collected using a washing device. Elastase ac-tivity was measured with a specific substrate, and IL-18 and IL-1β were measured by ELISA.
Results: The percentage of visible plaque index, gingival bleeding index and attachment level were similar in JSLE and con-trols, while the percentage of probing depth greater or equal to 3 mm was significantly higher in the controls. The total amountof IL-1β and IL-18 in GCF were significantly decreased in JSLE, while the total amount and the percentage of free elastase ac-tivity were significantly higher in JSLE when compared with the controls. The plasma levels of IL-18 and the Eerythrocyte Ssed-imentation Rrate were significantly higher in JSLE patients.
Conclusion: We found more active elastase in GCF from inflamed sites in JSLE patients even in the presence of significantlylower levels of IL-18 and IL-1β. The increased elastase activity suggests a hyperactivity of neutrophils in JSLE, possibly gener-ated by a priming effect caused by the higher plasma levels of IL-18 found in these JSLE patients.
Key words: elastase, IL-18, IL1-b, juvenile systemic lupus erythematosus, periodontitis.
Oral Health Prev Dent 2008;1:xxx-xxx.
Submitted for publication: xxxxxxxx; accepted for publication: xxxxxxxxx.
Systemic Lupus Erythematosus (SLE) is a chronic sels and the brain (for review see Danchenko et al,
auto-immune disease, characterised by immune
2006). The incidence of juvenile SLE (JSLE) is esti-
responses directed to a great number of self antigens
mated between 6–20 cases per 100,000 children,
that mostly affects women in their second or third
and mainly occurs in girls and in children of non-Cau-
decade of life. SLE can affect various parts of the body,
casian origin (Hoschberg 1997, Stichweh et al, 2004).
including joints, skin, kidney, heart, lungs, blood ves-
The pathoaetiology of this chronic disease remains un-clear, but genetic, immunological and environmentalfactors have all been implicated. Patients with SLEhave been shown to have elevated plasma interleukin-18 (IL-18) concentrations (Tso et al, 2006).
a Department of Periodontology, Rio de Janeiro State University -
IL-18, formerly called interferon (IFN)-Á-inducing fac-
tor, is a pro-inflammatory cytokine related to the IL-1
b Division of Periodontology, Institute of Odontology, Karolinska Insti-
family that is produced by Kupffer cells, activated
macrophages, keratinocytes, intestinal epithelial cells,
Pediatric Rheumatologic Unit, Adolescent Hhealth Care Unit, Rio de
Janeiro State University - UERJ, Rio de Janeiro, Brazil
osteoblasts and adrenal cortex cells (Dinarello 1999).
Reprint requests: Carlos Marcelo da Silva Figueredo, Faculdade de
IL-18 is produced as a 24 kDa inactive precursor and
Odontologia – Secretaria de Pós-graduação e Pesquisa, Av. 28 de
is cleaved by the IL-1‚ converting enzyme (ICE, cas-
setembro, 157, Vila Isabel, Rio de Janeiro, RJ, Brasil, CEP: 20551-
pase-1) to generate a biologically active mature 18
030. Tel: + 55 21 25876255. Fax: + 55 21 25876255. E-mail: [email protected]
kDa moiety (Ghayur et al, 1997). It plays an important
role in innate immunity and it has been shown to in-
duce T-helper cells type I (Th1) and Th2 cytokines,
such as IL-4, IL-5, IL-10 and IL-13 (Hoshino et al, 1999).
The primary functions of IL-18 include the induction ofIFN-γ and tumor necros factor α (TNF-α) in T cells and
The patient group comprised 16 adolescents (mean
natural killer (NK) cells (Tanaka et al, 2001), and the
age: 15.6, SD ± 2.7 years) who were attending the Pe-
up-regulation of Th1 cytokines including IL-2, granulo-
diatric Rheumatology Clinic (NESA), Rio de Janeiro
cyte macrophage colony stimulating factor (GMCSF)
State University (UERJ), Rio de Janeiro, Brazil. Diagno-
sis of the patients' SLE had previously been made by
Human neutrophils, both in circulation and in the
the same physician, in accordance with the classifica-
tissues, constitutively express the IL-18 receptor (IL-
tion of the American College of Rheumatology
18R) (Leung et al, 2001). IL-18 induces cytokine and
(Hoschberg, 1997). The median duration of disease
chemokine release from neutrophils, induces granule
was 4 years (range 1 to 10 years). Thirteen patients
release and enhances the respiratory burst (Leung et
were taking medication. Fourteen individuals (mean
al, 2001). The capacity to release cytokines and
age: 15.5, SD ± 1.5 years) with no signs of ongoing in-
chemokines was significantly enhanced in neutrophils
fections or inflammatory diseases were selected as
derived from rheumatoid arthritis sinovial fluid, indi-
controls. These persons were recruited among those
cating a differential response to IL-18 dependent up-
visiting the clinic for an annual medical check up. The
on prior neutrophil activation in vivo.
Ethics Committee of Pedro Ernesto University Hospital
Activated neutrophils release elastase, a serine pro-
(UERJ, Rio de Janeiro, Brazil) and the Regional Ethics
tease, which degrades elastin and several other func-
Committee in Stockholm, Sweden approved this study.
tionally and structurally important proteins in the peri-
All volunteers and parents/guardians gave written con-
odontium, including collagen, proteoglycans and base-
sent to participate. The patients answered a ques-
ment membrane components (Janoff, 1985). Several
tionnaire concerning their personal data.
studies have shown increased activity of this proteasein gingival crevicular fluid (GCF) from sites of peri-odontitis (Overall et al, 1987; Gustafsson et al, 1994;
Ingman et al, 1996; Meyer et al, 1997; Figueredo andGustafsson 1998). Elastase is released in an active
Periodontal and rheumatological clinical examinations
form but is normally rapidly inhibited by the protease
were performed. All tooth surfaces, except for third
inhibitors ·-1-antitrypsin (A1AT) and ·-2-macroglobulin
molars, of the selected patients were examined with a
(A2MG). Some studies have indicated that this inhibi-
type Williams probe (PCP10 Color Coded Probe, - Hu-
tion is less effective in inflamed periodontal tissues
Friedy Co., Chicago, EUAUSA), by the same calibrated
from patients with periodontitis, thereby allowing the
examiner. The variables registered were percentage of
elastase to remain active for a longer period of time.
visible plaque index (VPI) and giGengival bleeding in-
IL-1β a potent pro-inflammatory cytokine, has been
dex (GBI) (Ainamo & Bay 1975), pocket probing depth
considered a pivotal signalling substance involved in
(PD) and clinical attachment level (AL).
the up-regulation of matrix metalloproteinases anddown-regulation of tissue inhibitors (Page et al, 1997for review). Earlier studies have shown high levels of IL-
Clinical evaluation of rheumatological findings
1‚ in sites with periodontitis (Figueredo et al, 1999;Orozco et al, 2006). The role of IL-18 in periodontal dis-
The rheumatologic evaluation comprised a clinical ex-
ease has been less well studied. Orozo et al (2006)
amination and the measurement of the disease activ-
showed increased levels of GCF from inflamed sites of
ity through the Global Medical Evaluation (GME) in an
analogue visual scale (ranged from inactive to severe).
Therefore, the aim was to evaluate the expression of
The Systemic Lupus International Collaborating Clinics
IL-18 and IL-1‚ and the amount of elastase activity in
(SLICC) evaluation and the Systemic Lupus Erythe-
GCF from inflamed gingival sites in patients with JSLE,
matosus Disease Activity Index (SLEDAI) were used to
and compare these to the expression in GCF from in-
measure SLE damage and activity, respectively. All
flamed sites in generally healthy controls. In addition,
evaluations were performed by the same paediatric
the local inflammation in periodontal tissues was re-
lated to systemic inflammation by the assessment of
The JSLE patients were sub-divided according to the
disease activity. A patient with a SLEDAI score differ-
ent from zero was considered to have active disease.
Thirteen of the 16 participating patients took im-
munosuppressant drugs, prednisone, chloroquine
In the gingival fluid, IL-1‚ was measured using enzyme-
and azathioprine, and five of them also took non-
linked immunosorbant assay (ELISA), as reported ear-
steroidal anti-inflammatory drugs (NASIDs).
lier (Figueredo et al, 1999). A monoclonal antibodyagainst IL-1‚ (MAB 601, R & D Systems, Minneapolis,MN, USA) diluted 1:125 in carbonate buffer was coat-
ed onto microtitre plates (Nunc Maxisorb, Nunc a/s,Roskilde, Denmark) at 4°C overnight. Samples and
Samples were taken from five to six deep pockets or
standards were diluted in PBS, pH 7.4. The microtitre
the most inflamed sites. GCF was collected with an in-
plates were washed with PBS containing 0.05% poly-
tracrevicular washing device modified from Salonen
oxyethylene sorbitan monolaurate (Tween® 20, Sigma
and & Paunio (1991). The sites to be sampled were
Chemical, St Louis, MO, USA). After washing with
isolated with cotton rolls and dried gently with an air
PBS/Tween (4 times with 300 μl) the plates were
syringe. Supragingival plaque was carefully removed
blocked with 1% human serum albumin (HSA) for 1
before sampling. Each pocket selected was washed
hour at room temperature. The samples were washed
five times with 5μl of phosphate buffer saline (PBS)
as above and 100 μl of standard (2 pg/ml to 200
during continuous aspiration. The samples from the
pg/ml) and undiluted samples were added respec-
same type of site in each person were pooled, diluted
tively. The plates were incubated at 37°C for 45 min
with PBS to a volume of 1 ml ml and immediately cen-
followed by washing. The detection antibody (BAF 201,
trifuged at 3000 g for 10 min. The supernatant was
R & D Systems, Minneapolis, MN, USA) was diluted
collected and frozen at 70°C, pending analysis.
1:250 in PBS and incubated for 45 min. After washing,
For the Bblood samples, Twenty millilitersa total of
streptavidin diluted 1:200 was added to the plates
20 ml of venous peripheral blood were was collected
and incubated further at 37°C for 20 min. The plates
from each patient and control subject and stored in he-
were once again washed and the undiluted substrate
was added (TMB, Sigma Chemical, St. Louis, MO,USA). The reaction was stopped with 1M H2SO4 after15 minutes and the absorbency was read at 450 nm
IL-18 was measured in GCF and in plasma with com-
The granulocyte elastase substrate S-2484 (L-pyroglu-
mercially available ELISA kits (IL-18, MBL, Nagoya,
tamyl-L-prolyl-L-valine-p-nitroaniline, MW 445.5 Da,
Japan), according to the manufacturer instructions. To-
Heamatochrome Diagnostica AB, Mölndal, Sweden)
tal amounts of IL-1‚ and IL-18 were expressed as pg/ml.
was dissolved in dimethyl sulfoxide to 8 mmol/l andthe working solution was made to 2 mmol/l by dilutionin PBS. The alkaline phosphatase substrate, p-nitro-
phenol phosphate (Janssen Chimica, Geel, Belgium),was diluted to 2.7 mmol/l in diethanolamine buffer,
In the gingival fluid, IL-1b‚ was measured using en-
pH 10.0. A total of 100 Ìl of sample was mixed with 67
zyme-linked immunosorbant assay (ELISA), as report-
Ìl of substrate in a 96-well microtitre plate (Nunc Max-
ed earlier (Figueredo et al, 1999). ShortlyA, a mono-
isorp, Nunc, Roskilde, Denmark). The mixture was in-
clonal antibody against IL-1β (MAB 601, R & D Sys-
cubated at 37°C and the absorbency at 405 nm was
tems, Minneapolis, MN, USA) diluted 1:125 in car-
read after 2 h in a spectrophotometer (Millenia Kinet-
bonate buffer was coated onto microtitre plates (Nunc
ic Analyzer, Diagnostic Product Corporation, Los An-
Maxisorb, Nunc a/s, Roskilde, Denmark) at 4°C
geles, CA, USA). The elastase activity was expressed in
overnight. Samples and standards were diluted in
an arbitrary unit mAbs. To inhibit elastase activity, 10
PBS, pH 7.4. The microtitre plates were washed with
Ìl of 0.1% A1AT was added to 90 Ìl of sample and in-
PBS + containing 0.05% polyoxyethylene sorbitan
cubated with agitation for 15 min at room tempera-
monolaurate (Tween® 20, Sigma Chemical, St Louis,
ture. After inhibition, the samples were tested for elas-
MO, USA). After washing with PBS / Tween (4 times
tase activity, as described above. The elastase activi-
with 300 μl) the plates were blocked with 1% human
ty inhibited by A1AT was regarded as deriving from free
serum albumin (HSA) for 1 hour at room temperature.
elastase and the remaining activity as deriving from
The samples were washed as above and 100 μl of
standard (2 pg/mL ml to 200 pg/mLml) and undiluted
Table 1 Percentual mean (± standard deviation) of visible plaque index (VPI), gingival bleeding inde n
with pocket depth (PD) ≥ 3 mm and sites with proximal attachment loss (AL) ≥
and in the active and inactive subgroups.
JSLE: juvenile systemic lupus erythematosus; AL: presence of at least 1 proximal site with AL ≥ 2 mm.
* JSLE versus control, Mann-Whitney test, p < 0.05; ** JSLE active versus JSLE inactive, Mann-Whitney test, p < 0.05; *** JSLE active versus JSLE inac-tive, Mann-Whitney test, p < 0.01.
samples were added respectively. The plates were in-
lar in JSLE and controls while the percentage of prob-
cubated at 37°C for 45 min followed by washing. The
ing depthPD greater or equal to 3 mm was higher in the
detection antibody (BAF 201, R & D Systems, Min-
neapolis, MN, USA) was diluted 1:250 in PBS was and
The total amount of IL-1β and IL-18 in GCF were sig-
incubated for 45 min. After washing, streptavidin, di-
nificantly decreased in JSLE when compared to con-
luted 1:200 was added to the plates and incubated
trols (p = 0.05 and 0.02, respectively), while the total
further at 37°C for 20 min. The plates were once again
amount and the percentage of free elastase activity
washed and the undiluted substrate was added (TMB,
were significantly higher in JSLE when compared with
Sigma Chemical, St. Louis, MO, USA). The reaction was
controls (p = 0.03 and 0.001,, respectively, y) (Table 2).
stopped with 1M H2SO4 after 15 minutes and the ab-
The plasma levels of IL-18 and erythrocyte sedi-
sorbency was read at 450 nm in a spectrophotometer.
mentation rate (ERS) were significantly higher in JSLE
IL-18 was measured in GCF and in plasma with a
patients (p = 0.04 and 0.03, respectively, ) (Table 2).
commercially available ELISA kits (IL-18, MBL, Nagoya,
The SLEDAI median was 48 (range from 0 to 36) and
Japan), according to the manufacturer instructions. To-
the SLICC median was 0 (range from 0 to 8). IL-18 in
tal amounts of IL-1β‚ and IL-18 were expressed as
plasma did not correlate with ERS or with the rheuma-
tological evaluation using the SLEDAI scale.
The unit of analysis was the individual and the signifi-
The percentage of visible plaque index and gingival
cance was set at 5%. Mann-Whitney and Spearman’s
bleeding index were higher in active compared with in-
correlation were applied as indicated in the text/tables.
active JSLE patients, while the percentage of probing
SPSS 8.0 software was used to analyzse the data.
depth (PD) greater or equal to 3 mm,, and attachmentlevel (AL) greater or equal to 2mm was similar withinbetween them (Table 1).
The total amount of IL-1β‚ and IL-18, and the total
amount and the percentage of free elastase activity in
GCF were similar in active and inactive JSLE patients.
The same results were observed with the plasma lev-
The percentage of visible plaque indexVPI, gingival
bleeding indexGBI and attachment levelAL were simi-
The present study showed more unbound elastase,
corresponding to still active elastase in the GCF sam-ples from inflamed gingival sites in patients with JLSE,when compared with inflamed sites in the control pa-
tients. Elastase activity in GCF samples has been con-
vincingly associated with inflammation and tissue de-
struction in periodontal disease Figueredo and
Gustafsson, 1998; Loos and Tjoa, 2005. Armitage etal, 1994 showed that sites with high levels of elastaseare at a significantly greater risk for progressive boneloss as assessed by digital subtraction radiography.
Elastase is released from the cells in an active form
but under normal circumstances the body has effec-
tive ways of inhibiting this potentially damaging en-zyme. Remaining active elastase in the gingival pock-et could be due to hyperactive neutrophils generatinglarge amounts of reactive oxygen species, which caninactivate the most abundant protease inhibitor in
A1AT. This increased concentration of active elastase
in the gingival pocket could indicate that patients with
viation) in JSLE and control groups, and JSL
JLSE are at a greater risk of tissue degradation and inthe long- term clinical attachment loss.
In contrast to elastase, lower levels of IL-18 and IL-
1β were observed in the patient group. Several stud-
ies have shown that IL-1β is increased in inflamed pe-
riodontal tissues as compared to controls (Figueredo
et al, 1999; Faizuddin et al, 2003; Hou et al, 2003,Faizuddin et al, 2003). Increased levels of IL-1‚ havebeen strongly associated with high neutrophil activity
(Drugarin et al, 1998; Figueredo et al, 2000), where-
as the role of IL-18 in periodontal disease has been
less studied. Johnson and Serio (2005) evaluated IL-
18 in gingival biopsies and observed higher concen-
trations in deeper pockets (≥ 6mm). The lower levelsof IL-1β and IL-18 in GCF from JSLE patients could re-
sult from the use of various different anti-inflammato-
ry drugs. The JSLE patients were prescribed several
medications according to their clinical manifestation
of the disease. Some of these medications, such
as prednisone, chloroquine and azathioprine have animmunosuppressive effect (for a review, see Barrera
et al, 1996). Prednisone and azathioprine have been
shown to lower the concentration of IL-1b‚ in a mouse
lipopolysaccharide (LPS) induced inflammation mod-
el (Brustolim et al, 2006). Jang et al, (2006) showedthat chloroquine-mediated inhibition of TNF-α, IL-1β
and IL-6 synthesis occurred through different modes in
LPS-stimulated human monocytes/macrophages.
Wozniacka et al, (2006) showed that after three -
months of chloroquine therapy, the mean level of IL-6,
Table 2 Mean concentration of Inflammator
IL-18 and TNF-α decreased significantly in the serum.
Taken together, it is reasonable to believe propose that
ophils in JSLE could be someho n
in the current study, the local production of IL-1β and
IL-18 in the gingiva was affected by the systemic use
of IL-18 found in these JSLE patients.
of the anti-inflammatory drugs mentioned above.
The plasma IL-18 concentration, in contrast to that
in GCF, was higher in JSLE than in controls. This mightindicate that the plasma IL-18 has an effect on local
neutrophil activity. This hypothesis is supported byWyman et al, (2002) who found that IL-18, even at
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Tennessee Outcomes for Alcohol and Drug Services (TOADS) Project Questionnaire (July 2005 Version) CLIENT - 1 ST INTERVIEW - COVER SHEET A1a: Basic Demographics A1b: Interview Final Status _____________________________________________ (2) Client Case #: ______________________________ (3) Date of Admission to Facility: ______/_______/_______ (5) Agency Code ( 5 DIGITS):
PATIENT’S NAME_________________________ AGE_______ DATE OF BIRTH___________ EXPLAIN BRIEFLY WHAT SYMPTOMS BRING YOU TO THIS OFFICE: ARE ANY OF YOUR PRESENT PROBLEMS DUE TO INJURY? Yes____, No___ ARE YOU RIGHT-HANDED [ ] OR LEFT-HANDED [ ]? PAST MEDICAL HISTORY: 1. HAVE YOU EVER HAD: (Check the appropriate boxes and list year to the right) 2. PLEASE LIST IN CHRONOLOGICAL ORDER ALL HOSP