Th1 and Th2 cytokine profile in patients with early onset periodontitis and their healthy siblings.

Early onset periodontitis (EOP) is a chronic inflammatory periodontal disease with a strong genetic link affecting individuals aged 17 to 25. In the familial studies we tested the hypothesis about the role of Th1 and Th2 cytokines in the pathogenesis of EOP disease. The study involved 6 individuals with EOP disease and their 6 siblings with healthy periodontium. Actinobacillus actinomycetemcomitans (A. a), a bacterium typical for EOP, was detected in all people studied. Th1 and Th2 cytokine production was measured after in vitro stimulation. Peripheral blood mononuclear cells (PBMC) were isolated and cultivated for 24 h and 7 days with PWM, A. a. or Escherichia coli. The levels of IL-4, IFN-gamma, IgA, IgG and IgM were measured by ELISA methods. After in vitro stimulation of PBMC, a significantly higher production of IL-4 and significantly lower production of IFN-gamma were found in the group of patients compared with their healthy siblings. The increased level of IL-4 in patients was in good agreement with an increased level of IgM after stimulation of lymphocytes with E. coli. These results support Seymour's hypothesis according to which patients with progressive disease primarily activate Th2 lymphocytes while non-susceptible individuals activate Th1 lymphocytes.


Introduction
Early onset pe riodontitis (EOP) is a chronic inflammatory periodontal disease affecting individuals age d 17 to 25. 1  Pe riodontal disease is caused by bac te ria in dental plaque, and evide nce is accumulating that spec ific periodontal pathoge ns are assoc iated w ith the progre ssive form of the disease. 1 ,2 How e ver, some individuals harbour the se spe cific bacteria but do not appear to show e videnc e of disease progression. Patient susc eptibility is most like ly genetically determine d, although a numbe r of local and environme ntal factors are thought to influenc e disease ex p re ssion. 3 Immunological me chanisms have be en implicate d in the pathoge nesis of periodontal disease for over 30 ye ars. In the 1970s, attention foc used on the participation of peripheral blood T lymphocyte s in response to the action of dental plaque bacteria and on their role in the de velopment of inflammation. 4 Sinc e then many pape rs have be en published that c oncern the relationship betw ee n peripheral blood mononuclear ce lls (PBMC) blastic transformation and periodontitis . 5 -7 The signific anc e of the de ntal plaque bacteria on p olyclonal B lymphocyte activation w as re ally only studied late r. 7,8 Results show ed that both spe cific and non-spe cific mechanisms of B lymphoc yte activation can p lay a role in the pathogene sis of pe riodontal disease and that the y can be highly influe nce d by the tre atment. 8 -1 3 At the same time, immunological studie s clearly established the B c ell/plasma c ell dominance of the periodontitis le sion. 11 ,12 Both specific and non-spec ific ac tivation are re gulated by T lymphocytes. Base d on the c once pt of functional Th1 /Th2 and Tc 1 /Tc 2 subsets, 1 4,1 5 a model for the pathogene sis of p eriodontal dise ase has be en construc te d. 1 6,1 7 Ac cording to the hypothe sis of Se ymour e t a l., 1 6 p atie nts w ith p rogre ssive disease activate mainly Th2 lymphocyte s in response to an antigenic stimulus w hereas patients w ith the slow type of the disease ac tivate Th1 lymphocyte s. In the latter re sponse it is proposed that a loc al ex pansion of the B ce ll population w ould re sult, on the one hand, in the production of IL-1 and henc e subse que nt tissue de struction and, on the other hand, in the production of spec ific antibodies. It is furthe r proposed that if protec tive antibody is produc ed it could result in the elimination of the organism and disease progression w ould c ease. Production of antibody that is non-prote ctive w ould, how e ve r, re sult in continuous connec tive tissue breakdow n.
Our pre sent study conc erns the e ffec t of short-term cultivation of pe ripheral blood lymphoc ytes w ith pokew ee d mitogen (PWM), A. a . and Es ch e rich ia co li on the formation of IL-4 and IFN-gamma in patie nts w ith early onse t pe riodontitis and their healthy siblings. A. a . has be en sugge ste d to play a pathoge nic role in the de velopment of this dise ase . 1 In our previous studies w e have show n that p olyclonal activation of B ce lls w ith a suspension of E. c o li le ads to high produc tion of immunoglobulins by PBMC of patients w ith periodontitis. 18 The results should contribute to finding the origins of the susc eptibility to early onset pe riodontitis.

Materials and methods
Tw o ye ars be fore be ginning this study the group of 47 patients w ith EOP w as collected from a se t of 9225 young (16 -19 ye ars ) individuals at the Institute of De ntal Re search. These patie nts me t the basic clinic al diagnosis c riterion of pe riodontitis: the y had at least one pe riodontal pocke t deep er than 3 mm, situated in the re gion of the molars or incis ors, inflammation of gingiva and the presenc e of the suspec t pathoge nthe bac te rium A. a .-in the sulcular region. There w ere no individuals w ith syste mic dise ase or pregnant w ome n in the group of patie nts.
During the familial study 19 w e found that among the total of 47 patie nts and the ir 54 siblings only 6 patients had siblings w ith he althy pe riodontium and presenc e of A. a . in sulc ular region. These familie s w ere include d in this study.
In agreeme nt w ith Helsinki c onve ntions (1964 ), informed c onsent for blood c ollec tion for immunologic al ex amination w as obtained from all pe rsons ex amined.

Clinical examination
The ex amination of patients at the be ginning of therapy included the de te rmination of gingival inflammation using the gingival index (GI), 20 ascertaining the depth of the pocke ts w ith the calibrated probe and X-ray. This w as follow ed immediately by conse rvative treatme nt. The dental calc ulus w as remove d, unsatisfactory fillings or crow ns w ere restore d and correc t tooth brushing te chnique w as demonstrate d. All patients w e re thoroughly instruc te d in oral hygie ne including te eth cle aning and gingival massage . The y w e re also w arned that oral hygie ne is the decis ive factor in periodontitis the rap y. The con-se rvative part of the the rap y laste d 4 w e eks and w as follow ed by a 10-day te tracycline treatme nt. Tw o w eeks afte r the antibiotic therapy the patie nts w ere invite d for a c ontrol clinical ex amination, w ith further che ck-ups at 3-and then 6-month intervals.
This study reports on the re sults of an immunologic al ex amination 2 ye ars afte r the start of the therapy.

Bacterial suspension and mitogens
A. a . w as isolate d from a subgingival locality from the patients of the ex pe rimental group. Bacte rial culture w as incubate d for 48 h in Brain He art Infusion (Ox oid ) medium and then inactivated for 20 min at 120°C. The bac te rial suspe nsion w as w ashe d 4 times w ith physiological saline, centrifuged at 600 g and adjuste d to the require d conce ntration (10 9 c ells /ml) w ith RPMI 1640 tissue c ulture me dium.
The susp ension of heat-killed bac te ria of the E. co li 086 strain w as obtained from the De partme nt of Immunology, Institute of Mic robiology, Acade my of Sc ie nce s of the Czech Re public, and diluted to the ex pe rimentally optimal conce ntration (10 9 c ells /ml) w ith RPMI medium.
PWM (Phy to la c ca a m e rica n a mitogen, Sigma ) and ConA (Concanavalin A, Sigma ) w e re dilute d to the ex pe rimentally optimal c onc entration (PWM 2 m g /ml, PWM 2 m g /ml+ ConA 10 m g /ml) w ith RPMI 1640 tissue culture medium.

Stimulation of lymphocytes to cytokine and immunoglobulin production
Mononuclear ce lls is olated from the periphe ral blood of the patie nts and the ir siblings w ith healthy periodontium w ere w ashe d, dilute d w ith a suppleme nted tissue c ulture medium RPMI 1640 to a conc entration of 10 6 /ml, and c ultivate d w ith mitogens and he at-killed bacteria (A. a . 10 8 /ml and E. c o li 10 8 /ml), as described e arlie r. 2 1 Supernatants w ere take n afte r 24 h and afte r 7 days, w hich w e re then stored at -20°C until further assay.

Assay of cytokines and immunoglobulins in culture media
Cy to k ine a s s a y. Cytokine s IL-4 and IFN-gamma w ere assaye d in the tissue culture supe rnatants using comme rcial Immunote ch France kits. The te chnique is base d on the sandw ich method in w hic h monoclonal antibody is used as a binding antibody, and another biotinylate d monoclonal antibody is use d for de te c tion. This se cond antibody reacts w ith the streptavidin-peroxidase c onjugate . The binding e nzymatic ac tivity is dete rmined by adding a chromoge n (w ithout TMB), and the c olour intensity is me asure d on an SLT Spe ctra II microre ader.

En zy m e lin ke d im m u n o s o rbe n t a s s a y o f im m u n o g lo bu lin s ELISA a s s a y o f im m u n o g lo bu lin s .
Polyclonal antibodie s Q-Sw aHu-IgM, Q-Sw aHu-IgG or Q-Sw aHu-IgA (Se vac , Prague ) w ere use d as the binding antibodie s. Afte r inc ubation and w ashings, control sera w ith defined immunoglobulin contents and the supernatants w e re applied. Follow ing inc ubation and w ashing, perox idase-labelled antibodies against human immunoglobulin (Q-Sw aHu-IgM, Q-Sw aHu-IgG or Q-Sw aHu-IgA) w ere adde d. A 1-h inc ubation at room tempe rature w as follow e d by w ashing the plate s and the redox re ac tion w ith OPD in phosphate ELISA buffe r (pH = 6.2 ) w as de ve lope d using hydrogen pe rox ide . The reaction w as stoppe d w ith 1 M sulphuric acid and re ad on an SLT Spec tra II ELISA reade r. Individual samples w e re evaluate d in ng /ml using the KIME E program.

Statistical evaluation
Student's pair t-te st at a probability le vel of P= 0.05 w as used to evaluate the signific anc e of diffe renc es betw e en patients and their siblings. The results are presente d as mean ± SE. Table 1. The IFN-gamma formation w as found to be significantly higher in he althy siblings than in the patients after stimulation by A. a . and E. co li. On the othe r hand, a signific antly higher formation of IL-4 w as de te c te d in patients after stimulation by A. a . and E. co li as compare d w ith their healthy siblings.

Cytokine produc tion by PBMC afte r a 24-h stimulation by mitogen and bac te ria suspensions is show n in
The re lease of immunoglobulins after a 7-day stimulation of PBMC by mitogen and bac te ria suspensions is show n in Table 2. Compared to their siblings, patients ex hibite d a significantly highe r IgM le vel after polyclonal stimulation w ith E. c o li. The formation of IgA w as signific antly higher in the patie nts than in their siblings in cultures stimulate d by both PWM and ConA. No significant differenc es w ere found after stimulation of PBMC by other mitoge ns and in unstimulated c ulture s. No significant differenc es w ere found in the le vel of IgG.

Discussion
Evidenc e base d on mic robiologic al, immunologic al and animal studie s has show n that some forms of periodontal disease in adults can remain stable ove r many ye ars and not endange r the life of the de ntition, w hereas othe r forms, de spite ex te nsive treatme nt, continue to break dow n, leading ultimately to tooth loss. 22 The regulatory ac tivity of T lymphoc ytes and the identification of w hich c ytokine s are pre sent have bee n studied inte nsively in orde r to she d some light on the pathoge nesis of pe riodontal disease. It has bee n show n that individuals susce ptible to periodontal dise ase pre dominantly activate Th2 lymphocyte s, w hile non-susc eptible individuals activate Th1 lymphoc ytes. 16 The ve rification of this hypothesis re quired a correc t selec tion of patie nts. Patie nts w ith early onset periodontitis (susce ptible individuals ) and their siblings w ith healthy periodontium (non-susc eptible individuals ), both ex hibiting prese nce of A. a ., se em to be ideal for the study of the role of Th1 and Th2 lymphoc ytes in the pathoge nesis of the disease. De spite the similar ge netic backgrounds, similar age and similar hygie ne habits in families, w e found different produc tion of cytokines after in vitro stimulation.
The majority of immune responses oc cur loc ally rathe r than systemic ally w ithin a small are a of tissue: therefore most authors have conce ntrated on the T ce lls isolated from inflame d tissues of patients. 16 ,23 -2 6 This tissue can be obtaine d during surgical treatme nt of se riously affec te d patients. Lymphocyte s isolate d from peripheral blood of patie nts are rare ly used. 27 -2 9 The use of PBMC is favourable for analysing the transformation of the immune response during the disease's re mission, w hen it is not possible to use cells from the tissue.
PBMC w e re c apable of ex pressing mRNA for IL-2, IL-4, IL-5, IL-6, IL-10, IL-13 and IFN-gamma  follow ing stimulation by ConA for 2 h. 2 9 Ce lls is olated from affe cted tissue only ex pressed IL-10, IL-6, IL-13 and IFN-gamma. It see ms that PBMC w ere capable of producing a w hole spectrum of cytokines, but after homing of T ce lls into inflame d tissue, the y w ere only c apable of ex pre ssing either Th1 or Th2 c ytokine s. By immunohistoche mical analysis a higher perce ntage of IL-4 and IL-6 produc ing c ells w as found in the tissues affec te d by inflammation. 23 Follow ing in vitro stimulation by Po rp hy ro m o n a s g ing iva lis and Fu s o ba c te rium nu c le a tu m , most lymphocytes obtained from biopsie s of periodontitis affec te d tissues w e re of the Th2 phenotype , i.e. the y formed high amounts of Il-4 and only small amounts of IL-2 and IFN-gamma. 16 Manhart e t a l. 2 4 proved that alte re d IL-2 /IL-4 ac tivities w ere founded in le sion of patients w ith early onset pe riodontitis.
After activation by PHA, CD8+ CD28+ c ells isolate d from inflame d lesions of p atie nts w ith adult periodontitis 25 ,26 produc ed high amounts of IFN-gamma and w ere show n to have c ytotox ic ac tivity, w hereas CD8+ CD28-c ells produc ed predominantly IL-4 and IL-5 and the y show e d a suppressive e ffec t.
We found a significantly higher formation of IL-4 and signific antly low e r formation of IFN-gamma after E. c o li stimulation in patients w ith EOP as compare d w ith their siblings. These results corroborate the finding of a significantly highe r count of PBMC that produce IL-4 afte r P. g ing iva lis and Lip opolysaccharide (LPS) stimulation, w hich w as de te c te d by Aoyagi e t a l. 30 in patie nts w ith adult periodontitis.
As a B cell grow th fac tor, IL-4 stimulates the prolife ration and diffe re ntiation of B lymphocytes and induc es the ex pression of MHCII class II antige ns on B lymphoc ytes. It also stimulate s the antigen-sp ecific antibody re sponse , w hich has a p rote ctive character and w ill probably be ac tivated in patients in the re sting stage. 16 , 3 1 On the other hand, polyclonal activation probably le ads to the p rogre ssion of the disease.
A higher production of imunoglobulins after the polyclonal stimulation of B lymphoc ytes w as found in patients compared w ith their siblings. Compared to their siblings, patients ex hibite d a significantly higher IgM level after polyclonal stimulation w ith E. c o li. The formation of IgA w as significantly highe r in the patients than in their siblings in cultures c o-stimulate d by PWM and ConA.
Prediction of clinical disease ac tivity in patients is not ye t possible , and only the measure ment of past activity is c urre ntly available . The establishme nt of a me ans for classifying patie nts as susce ptible or nonsusc eptible is therefore nece ssary to define p atie nt se le ction more pre cise ly. 31 The pathogenic mechanisms involved in the de ve lopment of EOP have yet to be e luc idate d, but the pre vale nt produc tion of IL-4 by the patients' pe ripheral blood lymp hocyte s suggests that Th2 c ells, and probably B ce lls, and their products may play an important role in the pathogenesis of this dise ase.