J dm Periodomol 1996: 23: 823-H25PnnU'J ifi Denmark . All rights reserved

Copyright © Munksgaard 1996

clinieal perjudontoloiy

Papillon-Lefevre syndromeAnalysis of peripheral blood lymphocyte subsets

Erhan FiratiP, Nuray Gurel^ andAhmet Efeoglu^'Department of Periodontology, School ofDentistry, ^Department of Virobgy andImmunology. Istanbul School of Medicine,University of Istanbul, Turkey

Firatli E, Giirel N, Efeoglu A: Papillon-Lefevre syndrome. Analysis of peripheralblood htnphocyte subsets. J Clin Periodontol 1996: 2S: 823-825, © Munksgaard,1996. '

Abstract. We have studied the peripheral blood lymphocyte populations in our 6patients (2 female and 4 male) with a mean age of 11,19 with Papillon-LefevreSyndrome (PLS) using adequate monoclonal antibodies and double colouredflow cytometry. Total B, T, CD4, CDS, CD29, CD45RA, NK, HLA-DR cells werestudied. Total B. T, CD4 and CDS lymphocytes were within normal limits. Wehave observed an increase in the CD29 lymphocytes and NK cells and a decreasein CD45RA lymphocytes. We think that these findings are important in explain-ing B lymphocyte activation and in the pathogenesis of the PLS.

Key words: Papillon-Lefevre syndromeimmunology; pathogenesis: flow cytometry;lympfiocytes

Accepted tor publication 8 November 1995

Rapid and advanced destruction ofperiodonta] tissues in the early child-hood has been reported in systemati-cally healthy children {Page et al. 1983)as well as in children with systemic dis-eases such as hypoposphatasia, neutro-penia, diabetes mellitus, leukocyte ad-hesion deficiency syndrome, Down'ssyndrome, and PLS (Watanabe 1990).Papillon & Lefevre (1924) described theassociation of palmar-plantar hyper-keratosis with precocious periodontaldisease that resulted in exfoliation ofprimary and permanent dentitions. Inthis entity, the periodontal tissues of thepatients reveal severely inflamed gin-giva, deep periodontal pockets, diffuseand extensive bone resorption,loosening and exfoliation of the teeth(Preus & Morland 1987).

The role of Gram-negative anaerobicbacteria in the aetiology and patho-genesis of periodontal lesions of thePLS was investigated (Newman et al,1975). The authors mentioned that thenumbers of gram negative anaerobicrods in lesion sites were higher than thecontrol sites from a patient with PLS.

The immunological status of the PLShas also been investigated in a limitednumber of cases (Djawari et ai. 1978,Sloan et al. 1984, Van Dyke et al. 1984,Preus & Morland 1987, Lu et al. 1987,Celenligil et al, 1992). Neutrophilchemotaxis was found to be normal in

some cases (Rateitschak-Pluss & Schro-eder 1984), while in other cases, a de-creased neutrophil chemotaxis was de-picted (Djawari et al. 1978. Van Dykeet al, 1984).

Lu et al. (1987) reported a case of Pa-pillon-Lefevre syndrome with decreasednumber of CD4 and CD8 T lympho-cytes revealing a reverse CD4/CD8ratio. Tosti et al, (1988) reported nor-mal values of CD4 T helper cells andincreased number of CD8 T-suppressorcells with a CD4/CD8 ratio of 0,88.Celenligil et al. (1992) mentioned thatthere is no difference in the T and Blymphocyte distribuUon but naturalkiller (NK) cells were significantly in-creased.

The aim of this study was to analysethe peripheral blood lymphocyte sub-populations in patients with PLS.

Material and MethodsPatient group

The patient group consisted of 6 pa-tients with PLS who were referred tothe Department of Periodontology,School of Dentistry, University of Is-tanbul during the period 1988-1994,Their mean age was 11.19 years. Theydid not use any medication includingantibiotics, anti-inflammatory, and hor-monal drugs that could have affectedthe periodontal tissues or immune re-

sponse during the previous 6 months.They had not received any periodontaltreatment during the previous 6months. All of the patients with PLSrevealed extensive loss of soft tissueattachment and alveolar bone, mobilityand migration of the teeth, Consangu-ity existed in all patients. Up to 10 mmof clinical attachment loss was ob-served, RadiographicaJ evaluation ofthe patients showed serious loss of al-veolar bone around the effected teeth.The eruption times of the deciduousand the permanent teeth were normal,but the deciduous teeth began to exfoli-ate a short time after eruption. Hali-tosis was also present in all patients. Inall of the 6 patients hyperkeratoticlesions that were typical for PLS werepresent on the hands, knees and feet.

Control group

The control group consisted of 6- ageand sex-matched volunteers. Theirmean age was 11.35 years. They werehealthy in oral and systemic aspects.Periodontal clinical and radiographicalperiodontal examination revealed nosigns of clinica! attachment loss or al-veolar bone resorption. The controlsubjects had not used any kind of drugsthat affect the periodontal tissues orimmune response during the last 6months. They did not use any kind of

824 Firatli et al

prosthesis or orthodontic appliances,had not interproximal decay or fillings,and were not missing more than 2 teethin each quadrant. They had not re-ceived any periodontal treatment dur-ing the previous 6 months. None of thennembers of the control group were sib-lings.

Sera collection

Peripheral blood was sampled between9:00 a,m, and 12:00 pm noon by veni-puncture of the antecubital vein, after12 h fasting. Samples were collectedinto commercial glass tubes, one ofwhich contained Na hepann; and theothers were allowed to clot at roomtemperature and all were analysed atthe same day. Sera were neither lipemicnor icteric, Haemolytic sera were ex-cluded.

Glucose was determined with a speci-fic glucose oxidase method adapted toan RA-XT random access, discrete ana-lyser. Total protein and albumin weredetermined with the bluret andbromcresol green methods, respectively,also adapted to the RA-XT analyser.Fructosamine and HbAlc were deter-mined according to the methods de-scribed before (Firatli et al,1994). Theblood cell counts were made by H-2analyser,

IgG. IgA. IgM. IgGi .IgG2, lgG3.IgG4. C3c. C4, AH50. CH50 and circu-lating immune complex values were de-termined by radial immune diffusionmethod using commercial plates*.

Flow cytometric analyses

iOO ml of heparinized blood was incu-bated with 10 ml of appropriate doublecoloured Coulter cyto-stat antibodiesdonated by Coulter Electronics®**,After 10 min, incubation at room tem-perature, the tubes were centrifuged ina Q-Prep** and flow cytometric analy-sis was performed using the Epics Pro-file**, Using monoclonal antibodies asdual markers: Tl 1RD1/B4FITC, T4R-D1/T8FITC. T4FITC/4B4RD1, T4Fi-TC/2H4RD1. T8FITC/NKH-lRDi. T3-RD1/13FTIC double coloured analysiswas performed. The "Ai of antibodypositive cells were calculated in an im-munosubstraction programme.

* Behringwerke GmbH. Marburg, Germany** Coulter Electronics. Hieleh, USA,

Table I. Lymphocyte populations of the Papillon-Lefevre syndrome (PLS) and control groups

Antibody

PLS group(1=6)

mean±sd

Control group

mean±sd Test

Ti lT3T4T8T42H4T44B4NKH-IB413

81,n±3,4376,67±3,9347,33±4,9326,50±6,54i7,50±3,1525,17±5,1912,33±3,2712,17 + 4.241,67±6,82

80,82 ±3,2775,7Oi4,2847,50±3,5725,85±5,6721,32 + 3,0321,40±5,32

8,5±2,78ll,90i4,50

l,58±4,90

* ;!<0,05.

Statistical analyses

The peripheral blood lymphocytecounts of the PLS group were comparedstatistically to those of the control groupby using Mann Whitney 6-test by usinga computer program. The results wereexpressed as a significant level of

Results

The values for the PLS and control sub-jects for the routine biochemistry were mnormal range. Mean values of Igs andcoplement were compatible with theirage and growth period. The values forthe PLS patients Tl 1, T3. T4. T8. B4.13cells were not significantly different #Behringwerke GmbH. Marburg. Ger-many, § Coulter Electronics. Hieleh.USA, from those of the controls (Table1), T4 4B4 lymphocytes were higherwhile T4 2H4 lymphocytes were lowerthan the control group (p<0,05). As a re-suit of this situation Thl/Th2 ratio wasincreased {p<0.05). The numbers of NKcells were also higher than the controls(p<0.05).

Discussion

Various cases of PLS have been de-scribed in the previous literature andspecific immunologic mechanismsagainst bacteria or bacterial productshave been proposed for the study of PLS,We found that the NK cells and CD29lymphocytes were elevated whileCD45RA lymphocytes were decreased.Due to these changes in Th subsets, Th 1/Th2 ratio was elevated. We were not ableto find any significant alterations in theperipheral blood B, T, T4, T8. HLA-DRlymphocytes and CD4/CD8 ratio,

Lu et al. (1987) reported a case of PLSwith decreased number of CD4 and CDS

lymphocytes. In contrast to the results ofLu et al. (1987). Tosti et al. (1988) andCelenligil et al, (1992) reported normalvalues of CD4 and CD8 lymphocytesand CD4/CD8 ratio. The results were de-rived from 1 or 2 cases in the previouspapers. Our study was the first attemptto demonstrate a statistical significancefor lymphocyte analysis, (^elenligil et al,(1992) reported that HLA-DR andIL2R cells may indicate an absence ofactivated cells in the systemic circula-tion. We found no difference of HLA-DR cells in our PLS and control groups.

It has been demonstrated that the in-creased NK cell properties may indicatea natural host defense response to mi-crobial antigenic stimulation (Lindeman1991), The mean numbers of the NKcells were reported to be higher than thecontrols a case with PLS (Celenligil et al.1992),

It was reported that the CD4 moleculeplays a distinct functional role in trig-gering the activation mechanisms of dif-ferent subsets of T4 cells (Takeuchi et al.1987). The 2H4 and 4B4 antigens definereciprocal subsets of T cells involved inproviding suppressor inducer function(T4 2H4) and helper function (T4 4B4)to B cells (Rudd et aI,1987),Clement etal, (1988) mentioned that all CD4 cellshave the potential to function as helperceils when fully differentiated. However,it is also possible that only some cellswith the CD45R phenotype acquire thisability. The T4 2H4 subset, while not di-rectly suppressive, can induce cellswithin the T8 subset to suppress Ig pro-duction by B cells in response to PWMand specific antigen (Rudd et al.l987).The T4 4B4 subset of T-helper lympho-cytes can provide help to B cells. Thesesubsets within the T4 subpopulation ap-pear to be differentially triggered byvarious stimuli to elicit their immunefunctions (Rudd et al, 1987), Investi-

Immunology of Papillon-Lefevre syndrome 825

gations of peripheral blood T lympho-cytes demonstrated the presence oflymphocytes with specific immune re-sponses for periodontopalhic bacteria inperipheral blood (Mahanonda etal,1991), h was suggested that, whileThl lymphocytes enhance the peri-odontal disease symptoms, the Th2lymphocytes oppose functions in thepathogenesis of the periodontitis (Taub-man et al 1994), The mechanism sug-gested activation of macrophages bytFN-}" produced by Thl celis to producetL-l causing increased bone resorptionand decreased bone formation, Th2 cellscan produce IL-4, tL-5, iL-6 and IL-10that are essential for B-cell production ofantibody. Thus, the nature of the host re-sponse to periodontal microflora can bemodulated by different subsets of T cellsto produce a destructive and a protectiveimmune response (Taubman et al,1994).The mechanism of the effect of local fac-tors that might induce a systemic effecton immunoregulation is still unknown.The only statistically significant differ-ences were the increase in the CD29 andNK cells, the decrease in CD45R cells,and CD29/CD45 ratio due to this phen-omenon in patients with Papillon Lefev-re syndrome. These alterations may ex-plam the nature of the host response inpatients with PLS, We think that thesefindings may be helpful in explainmg thesystemic immune response in the pa-tients with PLS,

Zusammenfassung

Das Papillim-Lefevre Syndiom. .4nalyseperi-phcrer Vntergrupper der Bhtt LympJwzytenWir haben die peripheren Blut Lvmphozyten-populationen hei 6 Patienten (2 weiblichenund 4 mannliehen, in dem ,'\ltersmittel von11,19 JahrenI mit diagnostiziertem Papillon-Lefevre Syndrom (PLS), mit Hilfe von ada-quaten monoklonalen Antikorpern und dop-pelter gefarbter Flow-Zytometrie untersucht.Samtliehe B, T CD4, CDS, CD29, CD45RA,NK, HLA-DR Zellen wurden untersucht,Alle B,T, CD4 und CDS Lymphozyten hieltensich in normalen Grenzen, Wir haben einenAnstieg der CD29 Lymphozyten und der NKZallen und eine Verminderung der CD45RALymphozyten beobachtet, Wir meinen, daBdiese Befunde zur Erklarung der Aktivierungder B Lymphozyten und filr die Pathogeneseder PLS von Bedeutung sind.

Resume

Syndrome de Papilioii-Lefevre. Analyse dessnu.s-groupes iytnphocytaires dans le sang pe-ripheriqueLes populations lymphocytaircs dans le sang

peripherique ont ete etudiees chez quatrehommes et deux femmes ayant une moyennedage de 11,2 ans et presentant le syndrome dePapillon-Lefevre, en utilisant des anticorpsmonocSonaux adequats et une eytometrie parflux a coloration double. Les cellules B, T,CD4, ies CDS, les CD29, !es CD45RA, les NKet les HLA-DR ont ete etudies, Les lympho-cytes B, T CD4 et CD8 etaient dans des limitesnormales, II y avait une augmentation deslymphocytes CD29 et des cellules NK, et unediminution des lymphocytes CD45RA, Cesdecouvertes sont importantes pour expliquerractivation des lymphocytes B et la pathoge-nese du syndrome de Papilion-Lefevre,

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Address:

£. FirattiDepartment of PeriodontologySchool of DentistryUniversity of Istanbul(^apa 34390 IstanbulTurkey

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