Figueredo_

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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 Publication
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- Publication
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 Publication
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- Publication
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.
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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 1. Ainamo J, Bay I. Problems and proposals for recording gingivi- tis and plaque. Int Dent J 1975; 25: 229-235.
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idase activity, but if the primed cell also receives an ac- Effects of antirheumatic agents on cytokines. Semin Arthritis tivating stimulus, the ensuing oxidase activity is greater than that in non-primed, activated cells (Hallet 5. Brandolini L, Sergi R, Caselli G, Boraschi D, Locati M, Soz- and Lloyds, 1995). Greater chemotaxis and degranu- zani, S, Bertini, R. Interleukin-1 beta primes interleukin-8-stimulated chemotaxis and elastase release in human lation have also been demonstrated when neutrophils neutrophils via its type I receptor. Eur Cytokine Netw 1997; 8: are primed with cytokines, such as TNF-α (Bajaj et al, 1992), IL-1β (Brandolini et al, 1997) and IL-18 (Hewins 6. Brustolim D, Ribeiro-dos-Santos R, Kast RE, Altschuler EL, et al, 2006). Therefore, we it is hypothetsizsed that the Soares MB. A new chapter opens in anti-inflammatory treat-ments: the antidepressant bupropion lowers production of higher neutrophil activity found in JSLE might be re- tumor necrosis factor-alpha and interferon-gamma in mice.
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