J Clin Periodontol 2000; 27: 69–73 Copyright C Munksgaard 2000Printed in Denmark . All rights reserved

ISSN 0303-6979

Case ReportRongkun Liu, Caifang Cao,Huanxing Meng and Zhihui TangLeukocyte functions in 2 cases ofDepartment of Periodontology, School ofStomatology, Beijing Medical University,Beijing, ChinaPapillon-Lefevre syndrome

Liu R, Cao C, Meng H, Tang Z: Leukocyte functions in 2 cases of Papillon-Lefevre Syndrome. J Clin Periodontol 2000; 27: 69–73. C Munksgaard, 2000.

AbstractAim: To investigate the role of leukocytes in the pathogenesis of Papillon-Lefevresyndrome (PLS).Methods: Peripheral blood polymorphonuclear neutrophils (PMNs), monocytes(MNs) and gingival crevicular fluid (GCF) were obtained from 2 cases of PLSwith typical features. The chemotaxis of PMNs and MNs were evaluated using amodified Boyden chamber. The adherence of PMNs was determined by adherenceof PMNs to petri dishes. Interleukin-8 (IL-8) in GCF was detected by sandwichELISA. Elastase activity in GCF was measured with a low molecular weight sub-strate (S-2484) specific for granulocyte elastase.Results: PMNs from both patients showed depressed chemotactic response toFMLP and IL-8. Total amounts of IL-8 in GCF from the 2 patients were muchhigher than those of the normal controls. Elastase activity was not significantlydifferent from that of the controls. The adherence of PMN and the chemotaxis

Key words: Papillon-Lefevre syndrome;of MN in the 2 patients were normal.polymorphonuclear leukocyte chemotaxis;Conclusion: The depressed chemotactic response of PMN leads to decreased re- monocytes; interleukin-8; elastase

cruitment of PMN and/or release of lysozyme from PMN in the diseased gingi-val tissue, increasing the susceptibility of PLS patients to periodontal infection. Accepted for publication 15 March 1999

Papillon-Lefevre syndrome (PLS) isconsidered to be transmitted as anautosomal recessive trait affecting 1 to 4persons per million (Gorlin et al. 1964).Since Papillon and Lefevre first de-scribed the syndrome in 1924, morethan 200 cases have been reported up to1995 (Hattab et al. 1995). While someatypical cases have been reported(Schroeder et al. 1983, Bullon et al.1993, Fardal et al. 1998), PLS is char-acterized by diffuse palmar-plantar hy-perkeratosis and severe, rapid peri-odontal destruction with many clinicalsimilarities to prepubertal periodontitis(PPP) and rapidly progressive peri-odontitis (RPP) (Gorlin et al. 1964, Cao1979, Bimstein et al. 1990, Firatli et al.1996a, Boutsi et al. 1997).

Intensive work in recent years on theetiology and pathogenesis of PLS sug-gested that the alteration of the host de-fense response is essential for the devel-opment of PLS. The impairment of

polymorphonuclear leukocyte (PMN)chemotaxis has been commonly re-ported (Djawari 1978, Van Dyke et al.1984, Preus et al. 1988, Bullon et al.1993, Firatli et al. 1996b); other authorsreported normal PMN chemotaxis(Lyberg 1982, Schroeder et al. 1983).Chemoattractants stimulate the directedmigration of PMN as well as the acti-vation of PMN functions includingphagocytosis of micro-organisms, bac-tericidal superoxide radicals, and releaseof proteolytic enzymes (Boulay 1990).Whether the depressed chemotactic re-sponse of PMN shown in vitro could af-fect the recruitment and release of lyso-zyme of PMN in the diseased gingivaltissue of PLS patients, has not pre-viously been reported. Pedersen (1987)reported that PMNs respond diverselyto different chemoattractants. Most ofthe previous studies used N-formyl-me-thionyl-leucyl-phenylalanine (FMLP)and zymosan to determine the PMN

chemotaxis of PLS patients. Interleukin-8 (IL-8), an important chemokine fromhost cells (Baggiolini et al. 1989), has notyet been used in such studies. Somestudies showed that PPP and RPP wereassociated with defective adherence ofPMN or reduced chemotaxis of mono-cytes (MN) (Page et al. 1985, Katsuragiet al. 1994). Because of the similar clin-ical features, we attempted to verifywhether PLS patients and PPP and RPPpatients have comparable chemotacticand adherence responses.

In this study, peripheral blood andgingival crevicular fluid (GCF) werecollected from 2 PLS patients who hadbeen diagnosed at the Department ofPeriodontology, School of Stomatology,Beijing Medical University. The chemo-tactic response of PMN to FMLP andIL-8, GCF IL-8 level, and elastase ac-tivity were measured. The adherencerate of PMN and chemotaxis of MNwere also tested.

70 Liu et al.

Material and MethodsClinical status of the PLS patients

Case no. 1A 10-year-old girl, having spon-taneously lost a tooth, was referred tothe Department of Periodontology. Atthe age of 4 years she began to displayscaly, fissured, and hyperkeratoticlesions in the palmar-plantar regions.Her deciduous teeth began to exfoliatewith gingival swelling and pain. At theage of 6 years, the child had lost all herdeciduous teeth. After the permanentteeth erupted, the gingival swelling andpain repeated and the teeth became mo-bile. Intraoral examination revealedthat the right mandibular 1st molar wasmissing. There was severe gingival in-flammation, mobility, and extensive lossof soft tissue attachment, with gingivalrecession and probing depths up to 10mm in the erupted teeth. Radiographicevaluation showed severe loss of al-veolar bone around the affected teeth.Palmar and plantar surfaces presentedwith roughness, fissured and hyperkera-totic lesions. The child was systemicallyhealthy with no familial history of PLS.

Case no. 2A 31-year-old man was diagnosed withPLS 20 years ago by one of the authors(Cao 1979). Several months after hisbirth, the plantar region began to showscales and hyperkeratosis. Plantar hy-perkeratosis exacerbated gradually andthe skin lesions spread to palms, knees,and elbows. At age of 4 years, his de-ciduous teeth became mobile and ex-foliated. 1 year later, the child was com-pletely edentulous. At 7 years of age,the permanent teeth started to eruptand the cycle of destructive peri-odontitis repeated. The patient was di-agnosed and thoroughly treated withscaling and root planing at the age of11 years. 1 year later, the periodontaldestruction worsened significantly. Helost most of his permanent teeth, andseveral sequential sets of removabledentures were made to fit the develop-ment of his jaws. Finally at the age of20 years, all the diseased teeth exceptthe newly erupted lower right 3rd molarwere extracted and a set of final den-tures made. At a recall visit in 1997, thissingle surviving abutment tooth wasstable with probing depth of 2–4 mmand attachment loss of 4–6 mm. Hyper-keratosis of palmar and plantar sur-faces still existed although much lesssevere, and the patient was also gener-

ally healthy with no familial history ofPLS.

Neither patient had received anyperiodontal treatment during the pre-vious year. During the 6 months pre-vious to this study, they did not use anymedication including antibiotics, anti-inflammatory, or hormonal drugs thatcould affect the immune response.

Control subjects

Control subjects for PMN chemotaxisand adherence test consisted of 2 age-matched volunteers. A 2nd group ofcontrol subjects for measuring crevic-ular fluid IL-8 and elastase activity were11 volunteers (male 6, female 5) withmean age of 29.3 years old (19–35). Allcontrol subjects were periodontally andsystemically healthy. During the past 6months, they had not used any kind ofmedications that could affect the im-mune responses.

PMN and MN preparation

6 ml of heparinized peripheral bloodwere collected simultaneously from thepatients and healthy controls at 8:00–10:00 am by venipuncture of the ante-cubital vein after 10 h of fasting. PMNand MN were isolated by Ficoll-Hy-paque gradient centrifugation. Con-taminated erythrocytes were lysed withGey’s lysing solution. Leukocytes werethen washed 2¿ in Hanks’ balancedsalt solution (HBSS) and resuspendedin MEM medium at concentrations of1¿106 cells/ml for PMN and 2¿106

cells/ml for MN. Viability of these cellswas found to be greater than 98% bytrypan blue dye exclusion test.

Measurement of PMN and MN chemotaxis

PMN and MN chemotaxis were deter-mined in a 12-well micro chemotaxisassembly (Neuro Probe INC, Mary-land, USA). 100 ml of PMN suspen-sions (1¿106 cells/ml) were placed inthe upper compartment of thechamber, separated from the lowercompartment by a 3 mm-pore polycar-bonate membrane (25 mm¿80 mm,PVP Free, Poretics Corp, USA). Thelower compartment contained eitherFMLP (10ª8 M, Sigma) or rhIL-8(100ng/ml, Molecular ImmunologyLab, Beijing Medical University) aschemoattractants. After incubation in5% CO2 at 37æC for 45 min, the mem-brane was removed and cells on top of

the membrane were wiped off. Thenthe membrane was fixed in methanoland followed by Giemsa stain. PMNchemotaxis was determined by count-ing the number of migrated leukocyteson the distal surface of the filter, in 10randomly selected high power micro-scopic fields (¿600) for each well. Allsamples were run in duplicate and theaverage calculated. Values from blankwells (no attractant in the lowerchamber) were subtracted. The assayof MN chemotaxis was run similarly,except that cell suspensions were2¿106 cells/ml and 5 mm-pore filters(25 mm¿80 mm, Standard, PoreticsCorp) were employed. Only FMLP(10ª7 M, Sigma) was used aschemoattractant; the incubation timewas 2 h.

PMN adherence test

Patient and control PMN in MEM me-dium suspension (1¿106/ml) wereplaced in petri dishes (60¿15 mm) andincubated in 5% CO2 for 1 h at 37æC.The supernatant was collected and non-adherent cells were counted. Experi-ments were performed in duplicate andthe average calculated. The % of PMNadherence was calculated according tothe following formula:

% PMN adherence Ωno. seeded PMN – no. nonadherent PMN

no. seeded PMN¿100.

GCF sample collection and elution

The GCF of 24 tooth sites from caseno. 1, 4 sites on the only existing toothfrom case no. 2, and 29 tooth sites from11 control subjects were collected.Briefly, after the sampled tooth was iso-lated with cotton rolls, supragingivaldental plaque was removed gently andthe tooth surface dried with air. Crevic-ular fluid was collected onto pre-weigh-ed 2¿10 mm filter paper strip (What-man 3MM chromatography paper,Whatman Ltd, Maidstone, UK). Thestrip was placed into the gingival sulcusuntil mild resistance was felt and re-mained for 30 s. Then the strip was re-moved and placed in a pre-weighed Ep-pendorf tube and sealed. After eachtube with the strip was re-weighed, theywere stored at ª70æC until assayed. Onthe day of assay, 170 ml of PBS (pH 7.4)containing 0.1% bovine serum albuminand Tween-20 (PBS-BSA-T) were added

Papillon-Lefevre 71

to the tube. Then, the GCF sample wasrecovered from the strip by centrifugalelution technique. 100 ml of each eluatewas used for IL-8 testing and 42 ml ofthe same GCF eluate were used for elas-tase activity measuring.

Assay of IL-8

GCF IL-8 was measured by sandwichELISA using a commerically availablekit (Immunology Dept of the FourthMilitary Medical University, China).The sensitivity of the assay was 156 pg/ml. Each sample was tested in dupli-cate. 50 ml of GCF eluate/well wereplaced in the 96-well microtiter plate(Nunc Maxisorp, Denmark) and the as-say performed as indicated by themanufacturer. Absorbance was read at410 nm. IL-8 concentration in GCFeluate was obtained using a microplatereader (Bio-Rad 450, USA) interfacedwith a computer. The total amount ofIL-8 was then calculated by multiplyingIL-8 concentration in GCF eluate andtotal GCF elution volume.

Elastase activity measurement

Elastase activity was measured usingthe method described by Gustafsson etal. (1992). Briefly, granulocyte elastasesubstrate S-2484 (L-pyroglutamyl-L-propyl-L-valine-p-nitroaniline, Chro-mogenix AB, Molndal, Sweden) wasdissolved in dimethylsulfoxide to 8mmol/l and the working solution ad-

Table 1. Leukocyte chemotaxis and PMN adherence rate in PLS patients and control subjects

PMN chemotaxis MN PMNFMLP IL-8 chemotaxis adherence

(10ª8 M) (100 ng/ml) FMLP (10ª7 M) rate (%)

case no. 1 4.3* 19.3 39.5 92.1case no. 2 6.7 5.7 37.1 95.4control no. 1 18.7 52.6 37.7 94.5control no. 2 17.3 49.7 36.7 93.0

* Average of duplicates.

Table 2. Clinical parameters, IL-8 and elastase activity (mean∫SD) in GCF from controlsubjects and PLS patients

Control sites Patient sites(nΩ29) (nΩ28) p-value

probing depth (mm) 1.71∫0.59 6.69∫3.32attachment loss (mm) 0 7.76∫4.23bleeding index 0.17∫0.38 2.83∫0.56GCF volume (ml) 0.24∫0.22 1.26∫1.25total IL-8 (pg/site) 50.62∫91.11 291.34∫111.58 ∞0.001elastase activity (abs/site) 0.07∫0.05 0.04∫0.02 0.09

P: Mann-Whitney U-test.

justed to 2 mmol/l by dilution with PBS(pH 9.20). 42 ml of GCF eluate weremixed with 28 ml of substrate in a 96-well microtiter plate (Nunc Maxisorp,Denmark) and incubated at 37æC for 2h. Absorbance at 405 nm was measuredusing a microplate reader (Bio-Rad 450,USA). The recorded absorbance repre-sents the hydrolysis of elastase in aGCF sample for 2 h and is presented asarbitrary units per site (Abs/site).

Statistical analysis

Mean values of clinical parameters(GCF volume, Probing Depth, Attach-ment Loss, and Bleeding Index) at GCFsampling sites were calculated. Differ-ences of total IL-8 and elastase activitybetween patients and controls were ana-lyzed using the Mann-Whitney U-test.

ResultsPMN and MN chemotaxis

Chemotactic responses of PMN andMN from the 2 patients and 2 controlsare presented in Table 1. PMN from bothpatients showed depressed chemotacticresponse to both FMLP and IL-8. MNchemotaxis from both patients did notdiffer significantly from controls.

PMN adherence rate

The % of PMN adherence in the 2 pa-tients and the 2 age-matched controlsubjects are also shown in Table 1. No

significant difference was found be-tween patients and controls.

IL-8 and elastase levels in GCF

Table 2 shows clinical parameters of theGCF sampled sites, and IL-8 and elas-tase levels from the 2 patients and 11controls. IL-8 was detected in all 28samples (100%) from the 2 patients andin 11 of 29 samples (37.9%) from con-trols. Both patients demonstrated a sig-nificantly higher IL-8 level than that ofcontrols. Elastase activity of both pa-tients was not significantly differentfrom the controls.

Discussion

PMN plays a crucial role in the hostdefense mechanism against virulentbacteria. Any factor that could sup-press recruitment of PMN to infectedareas, as well as the activation ofPMN, may increase the susceptibilityto infection. Numerous studies havedemonstrated that severe and rapidperiodontal breakdown may be associ-ated with defective PMN function,more specifically with the chemotaxisof these cells (De Nardin 1996). Theliterature has been controversial onwhether chemotaxis of the PMNsfrom PLS patients is depressed. Thepresent study showed that in both ourpatients, the chemotactic response ofPMN to FMLP and to IL-8 was de-pressed. Since elastase is PMN-speci-fic, and substrate S-2484 is a specificsubstrate for granulocyte elastase thatwould not be hydrolyzed by otherproteases to any significant extent(Kramps et al. 1983), elastase activityin GCF tested by the method in thepresent study is dependent on thenumber of PMN as well as theamount of elastase locally releasedfrom PMN. Cross-sectional studieshave shown that GCF elastase wassignificantly elevated at sites with peri-odontitis (Gustafsson et al. 1992, Ar-mitage et al. 1994, Giannopoulou etal. 1994, Eley & Cox 1996). In thepresent study, GCF elastase activity inperiodontitis sites from the PLS pa-tients was not significantly differentfrom that of the controls. It suggestedthe depressed chemotactic response ofPMN reduced recruitment and/or re-lease of lysozyme of PMN in the in-flamed gingival tissue.

The molecular events that govern thedirected migration and activation of

72 Liu et al.

PMN is initiated by chemoattractantsbinding to specific cell-surface receptorsthat are coupled G proteins (Synder-man & Pike 1984, Smith et al. 1985,Boulay et al. 1990). Therefore, the ab-normalities of agonist, receptor, or Gprotein transduction pathway may in-fluence PMN chemotaxis and acti-vation in tissue. IL-8 differs from classi-cal chemoattractants such as FMLP inseveral aspects: it is produced by a widevariety of host tissue cells; it is highlyselective for PMN; it has long-lastingeffects; and it has an amplification ef-fect because of its role in the cytokinenetwork (Peveri et al. 1988, Colditz etal. 1989). These features indicate thatIL-8 is a major host mediator for PMNinfiltration and activation in diseasedtissues. Results of the present study re-vealed that PLS patients had higher IL-8 levels in GCF compared to healthycontrols, and also higher levels thanthose of RPP and adult periodontitis(AP) patients detected at the same time(data not presented). This might be aregulatory response to the depressedchemotaxis of PMN. The result sug-gested that the reduced accumulation oractivation of PMN in inflamed gingivaltissue is not associated with the agonist.It is more likely related to abnormalbinding of the agonist-receptor or tosignal transduction. Depressed chemo-taxis of PMN to both FMLP and IL-8also indicated a common mechanism.

It has been reported that peripheralleukocytes of PLS patients have higherexpression of CD11b molecules (Firatliet al. 1996a). Abnormal expression ofadhesion molecules in leukocytes maylead to aberration of adherence. How-ever, the present study failed to show anyaberration of adherence of PMN in PLSpatients. Defective chemotaxis of MNwas reported to be associated with somesevere, rapidly progressive periodontitis(Page et al. 1985). We found that chemo-taxis of MN in PLS patients was normal.

In conclusion, the present study indi-cates that depressed chemotactic re-sponse of PMN in PLS patients sup-presses recruitment of PMN and/or re-lease of lysozyme in diseased gingivaltissue. This deficiency increases the sus-ceptibility of PLS patients to peri-odontal infection. The molecular basisof the chemotaxis deficiency is probablyat the receptor level or in the post-re-ceptor pathway.

Acknowledgments

This work was supported by grant.9501020 from the Ministry of Edu-

cation, China. We are most grateful toDrs. Peter Milgrom and Joan Hiltner,University of Washington, for their as-sistance in preparing the manuscript.

Zusammenfassung

Leukozytenfunktion in 2 Fällen von Papillon-Lefevre-SyndromZwei typische Fälle von Papillon-Lefevre-Syndrom (PLS) wurden untersucht, um dar-an die Rolle der Leukozyten in der Pathoge-nese dieser Erkrankung zu studieren. Bei denPatienten wurden polymorphkernige neutro-phile Granulozyten (PNGs) aus dem peri-pheren Blut, Monozyten (MNs) und Sulkus-flüssigkeit (SF) gewonnen. Die Chemotaxisder PNGs und MNs wurde in der modifi-zierten Boyden-Kammer untersucht. Die Ad-härenz der PNGs wurde durch deren Adhä-renz an Petrischalen bestimmt. Interleukin-8(IL-8) in der SF wurde durch Sandwich-ELI-SA bestimmt. Die Elastaseaktivität in der SFwurde mit einem für Granulozyten spezifi-schen Substrat für geringes Molekularge-wicht (S-2484) gemessen. Für die Bestim-mung von Chemotaxis und Adhärenz derPNGs wurden 2 gesunde Kontrollprobandenund für die IL-8-Aktivität 11 Probanden un-tersucht. Die PNGs beider Patienten zeigteneine reduzierte Chemotaxis auf FMLP undIL-8. Die Gesamtmenge von IL-8 in der SFbeider Patienten war viel höher als bei dengesunden Kontrollprobanden. Die Elastase-aktivität unterschied sich nicht im Vergleichzu den Kontrollprobanden. Die Adhärenzder PNGs und die Chemotaxis der MNs beibeiden Patienten war normal. Die Ergebnisseweisen darauf hin, daß die reduzierte chemo-taktische Reaktion der PNGs zu einer redu-zierten Einschleusung von PMGs und/oderverminderten Freisetzung von Lysozym ausden PMGs in das erkrankte Gewebe führtund somit die Empfänglichkeit der Patientenfür eine parodontale Infektionen erhöhtwird.

Resume

Les fonctions du leucocyte dans deux cas desyndrome de Papillon-LefevrePour etudier le role des leucocytes dans la pa-thogenese du syndrome de Papillon-Lefevre(PLS), deux cas de PLS avec des caracteristi-ques typiques sont presentes. Les polymor-phonucleaires neutrophiles du sang periphe-rique (PMN), les monocytes (MN) et le flui-de creviculaire gingival (GCF) ont eteobtenus des deux patients. La chemotaxiedes PMN et des MN a ete evaluee en utilisantune chambre de Boyden modifiee. L’adheren-ce des PMN a ete determinee par l’adherencedes PMN aux boıtes de Petri. L’interleucine-8 (IL-8) dans le GCF a ete detectee par ELI-SA en sandwich. L’activite elastase dans leGCF a ete mesuree avec un substrat de faiblepoids moleculaire (S-2484) specifique pourl’elastase du granulocycte. Les PMN desdeux patients accusaient une reponse de che-

motactisme diminuee au FLMP et au IL-8.Les quantites totales de IL-8 dans le GCFdes deux patients etaient beaucoup plus im-portantes que celles trouvees dans descontroles normaux. L’activite de l’elastasen’etait significativement pas differente de cel-le des controles. L’adherence des PMN et lachemotaxie des MN chez les deux patientsetaient normales. Ces donnees indiquent quela reponse affaiblie du chemotactisme desPMN reside dans un recrutement diminue dePMN ou une liberation de lysozyme duPMN dans le tissu gingival malade ou lesdeux, qui augmenterait la susceptibilite despatients PLS a la maladie parodontale.

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Address:

Rongkun LiuDepartment of PeriodontologySchool of StomatologyBeijing Medical UniversityBaiShiQiao Road 38., 100081, BeijingChina

e-mail: liurk/pku.edu.cn