Download Full Text - Statistics

Relationship between Interleukin 4 and12 and chronic
periapical lesion size
1
Sattari M. DDS. PhD., 2Nazarinia H.DDS. MS., 3Khalili S. MSc., 4Asna-Ashari M.
DDS. MS., 5Kangarloo A. DDS. MS
1
Associate Professor, Dept. of Immunology, Medical School, Shahid Beheshti University of Medical Sciences,
Tehran- Iran
2
Specialist of Oral & Maxillofacial Radiology
3
Masterof Immunology, Dental Research Center, Shahid Beheshti University of Medical Sciences, Tehran- Iran
4
Professor, Dept. of Endodontics, Dental School, Shahid Beheshti University of Medical Sciences, Tehran- Iran
5
Associate Professor, Dept. of Endodontics, Dental School, Shahid Beheshti University of Medical Sciences, TehranIran
Abstract
Background and Aim: Chronic dental periapical lesions resulted from chronic inflammatory
response to the periapical tissues. Since T-helper (CD4+Y) cells are the prominent cells in these
lesions, so the aim of this study was to determine the correlation between the concentration of
two interleukins(IL-4 and IL-12) which are most important cytokines for differentiation of T
helper 2 and T helper 1 cells, respectively, and the diameter of chronic periapical lesions.
Materials & Methods: Thirty-eight chronic periapical lesions were collected which 18 periapical
lesions had diameter of ≥5 millimeter (case group) and 20 periapical lesions had the diameter <5
millimeter (control group). Tissue samples were cultured for 72 hours, then ELISA (Enzyme
linked immuno-sorbent Assay) was used for determining the concentration of IL-4 and IL-12 in
supernatant fluids. Mann-whitney U and Spearman correlation tests were used to analyze the data.
Results: IL-4 and IL-12 were found in all of the samples. There was no significant difference
between case and control groups regarding the concentration of IL-4, IL-12 and IL-4/IL-12. The
only significant correlation was between IL-4 and IL-6 concentration without any regard to the
diameter of lesions (P<0.001) (Spearman correlation coefficient= 0.593).
Conclusion: It is concluded that in chronic periapical lesions, probably T helper 1 and T helper 2
cells participate in active phases of inflammation and tissue damage equally. This could be
resulted from mixed population of bacteria in these lesions.
______________________________________________________________________________
Key words: T - helper, Chronic periapical lesions, Interleukin-4, Interleukin-12
Received on:April2007
Final revision:August 2008
1
Accepted on:September 2008
Introduction
In chronic periapical lesion, we run into immune reactions against bacteria causing infection and
destruction in tooth pulp. In some cases, these responses can destroy the periapical tissues rather
than protecting them
(1)
. Among defensive factors which play a role in destruction of tissue, are
cytokines such as IL - lα, IL – Iβ (2-4), IL-6 (5), IL-8
(6,7)
and TNE- a (4). These factors are active in
resorption of periapical areas. On the other hand it has been reported that some cytokines such as
IL-15 can be effective in controlling resorptions caused by above - mentioned cytokines
(1,8).
A
direct relationship between, cytokines related to T helper L such as IL - 12 and IF N-y and
cytokines resorbing the bone has also been obtained (9).As if in some researches, inhibitory effects
of IL-4 in resorbing bone have been alluded
(8)
It has also been reported that, in mice periapical
lesions, incidence of infection increases two weeks after infection, but decreases later.
(9)
About
the role of IL-12 , it has been pointed to the increase in production of this cytokine in periapical
lesions, but it is determined that unlike IL-4, it's level, does not reduce rapidly, and because of
this, the role of the T helper cells (in other words the role of cellular immunity cells) in chronic
periapical lesions has been considered more important, so that Kawashin and Stashenko (1999)
expressed that in response to bacterial infection, they have observed cytokinic lattice activity in
periapical area (with the advantage of cytokines derived for T helper 1 over others).
On the other hand, Robeiro Sobrinho et al (2002) stated that, in a mixed infection,
microorganisms such as Bifid bacterium, Nucleutum,Fusobacterium,Adolesintis and Clostridium
butyricum cause reduction in cytokines of T helper 1 cells, while microorganisms such as Gemella
morbillorum enhance the production of T helper 1 cytokines in endodontic infections in mice free
microbe (10).
Regarding the role of IL-4, conflicting findings have been reported, so that a research shows its
inhibitory effect on resorption of bone
on bone resorption
(8)
while another research implies lack of inhibitory effect
(1)
. In a research on 24 periapical lesions including 12 granulomas and 12
cysts, Walker et al (2000) expressed that T helper 1 cells excel at periapical lesions (11). Since IL-4
strengthens incidence of homoral immune responses and IL-12 strengthens incidence of cellular
immune responses(12) and regarding considerable presence of immunogloboline in chronic
periapical lesions(13-14) and recognition of increased sensitivity reaction type III or complex
immune disease in the pathogenesis of these lesions
(15)
, it is expected that IL-4 should have a
considerable concentration in the mentioned lesions.
As None of the researches , done so far , has not evaluated the relationship between T helper 1 and
T helper 2 cytokines and inductive cytokines differentiating for the two cells with the size of
2
periapical lesions, so this research was designed to determine the correlation between
concentration of IL-4 and IL-12 and the diameter of chronic periapical lesions in patients admitted
to maxillaofacial and endodontic departments of dental school, Shahid Beheshti university of
medical sciences, private and state clinics of Tehran. In other words the aim of this study was to
find if it is possible to relate one of the pathogenic mechanisms in chronic periapical lesions and
their progress to disturbance of the balance between T helper l and T helper 2 cells.
Method and Materials:
In this analytical study, the samples were selected unrandomly among those adimitted to
maxillaofacial and endodontic departments of Shahid Beheshti university of medicals sciences,
private and state clinics of Tehran city. The samples were selected among people who were free
from malnutrition, infectious diseases, immune system defects, metabolic diseases and drug the
addiction during the last year , over smoking in the last two months , radiotherapy and intense
psychological anxieties during last one year, mental retardedness, pregnancy, menstruation, breast
feeding, they should also be free from taking antibiotics in the last 2 months; hormonal factors
during last one year, drugs strengthening immunity system in the last two months and drugs
inhibiting immune system during the last year. Based on diameter of chronic periapical lesions,
the samples were divided into two groups (control>5 mm, case 5mm), according to radiographic
diameter of lesions. The number of samples were 20 and 18 respectively. Totally 38 chronic
periapical lesions were studied. The samples were similar, regarding the age and gender of
patients. Average age of individuals in control and case group were 38.512.42 and 32.839.53
respectively. The samples of chronic lesions removed by operation were immediately placed in
sterile tubes containing a solution composed of %10 FCS4 (Bahar Afshan co, Tehran, Iran) + Gibco (10g/lit) JRMI-1640 (Touba Negin Tehran,lran)+Amphotricine B (5 mg/ml) + Gentamicine
sulphate ( 100 mg/ml) and were refrigerated at once.
Most of the provided tissues were tissue cultured at the end of the week so that the tissue samples
were washed in culture media containing (loglit PRMI-1640+10 FCS+2.5mg/ml) B-Amphotricin–
20 mg/ml, Gentamicin sulfate, then in another sterile petridish, using a scalpel, blood and
remnants of tissue were cleaned from the tissue. After that the samples were divided into several
pieces with 1 mm diameter Every piece was placed in each of the 96 parts of the tissue culture
plate (NUNC, Tube co, Tehran, Iran) which were covered by 300 µ lit of tissue culture .
Being divided after 72 hours culturing , the fluid over culture was extracted by Insulin syringe and
freezed under-20C in capped microtubes until final experiment. After collecting all the needed
samples, the fluid samples over culture were defrosted by ELISA method, and concentration of
3
IL-4, IL-6 and IL-12 was determined using the Bernard system kits (Nima - poyesh Teb, Tehran,
Iran). It is necessary to say that to determine the inflammatory activity of IL-6 ,concentration of
IL-6 was used as a determinant. SPSS 10. software was used for statistic analyses. Mann whitney U test was used to compare the amount of Inter-leukin with-in the two groups. Spearman
correlation test determined the difference between Interleukins.
The P value of < 0.05 was considered as significant.
Results:
In all samples , we faced all 3 kinds of Interleukins (IL-4, 6,12) In control group, OD5 relating to
IL-4 and IL-12 was 0.55  0.6 and 0 . 06  0.008 respectively.
In case group, OD relating to IL-4 and IL-12 was 0.0520.06 and 0.06011 respectively. OD
relating to IL - 6 in both control and case groups was 1.21.408 and 0.568±0.832 respectively.
The proportion of IL-4 to IL 12 (IL-4, IL-12) in both control and case group was about
0.9070.09 and 0.910.142 respectively. No significant statistical difference were observed
regarding concentration (OD) of IL4, IL 12, proportion of IL-4 to 1L-12 and concentration of IL-6
(P>0.05) It was also observed that there is not a significant correlation between concenteration of
IL-4, IL-12, proportion of IL-4 to IL-12 , concenteration of IL-6 and diameter of lesion (P>0.05).
Mean while, there was no significant correlation between concentration of IL-6 and IL-4, IL-12
(P>0.05). This study determined that regardless of the diameter of lesion there is a significant
correlation between IL-4 and IL-12 (Spearman correlation coefficient = 0.593).
Discussion:
Since IL-4 increases, the incidence of homoral immune responses and IL-4 increase the incidence
of cellular immune responses
chronic periapical lesions
(12)
(13-14)
and regarding considerable existence of immunogloboline in
and the increased type III sensitivity reaction in the pathogenesis
of the lesions is determined, it can be concluded that, considering the role of T helper 2 cells in
homoral immune responses, in the lesions and specially bigger lesions having more tissue
destruction, the balance between T helper I and T helper 2 cells has been violated in favor of T
helper 2 cells.
In this case, as IL-4 has an important role in differentiating T helper 2 cells from T helper 0 cells
and IL-2 plays an important role in distinguishing T helper 1 cells from T helper 0 ones, it is
expected IL-4 to have higher concenteration in comparison with IL-12 and concenteration of
bigger lesions to be higher than that of smaller ones.
4
With performing this study it was determined that there is not any significant correlation between
the diameter of lesions and IL-4 or IL-12 (P>0.05) and or proportion of IL-4 to IL-12. There was
also no significant correlation between the above Interleukins and IL- 6 as an important
inflammatory cytokine (P>0.05). This research showed that , regardless of the diameter of the
lesion there is a significant correlation between IL- 4 and IL-12 (P<0.001) (Spearman correlation
coefficient = 0.593). This may be due to the same role of both T helper 1 and T helper 2 cells in
the pathagenesis of chronic periapical lesions. It should be said that, significant differences
between lesions having diameter of <5mm and or <5mm was not observed from IL-6 point of
view (P>0.05). It may imply that inflammatory activity is likely to happen equally in both two
kinds of lesions.
In 1999, Kavashima and Stashenko expressed that they faced cytokinic activity in periapical area,
in response to bacterial infection; 6 cytokines derived from T helper 1 excels in this case
(9)
. The
difference between the findings of current study and the result of the above study can be attributed
to using different animal models in studing periapical lesions and the number of samples and
diameter of lesions in the above mentioned research was not determined. Meanwhile, the
researchers are going to survey the correlation between IL- 4, IL-12 and the diameter of lesions.
Robeiro Sobrinho etal (2002) stated that in a mixed infection, microorganisms such as Bifido
bacterium adolescentis, Fusobacterium nucleatum and Clostidium butyricum reduce the cytokines
of T helper 1 cells while microorganisms such as Gemella morbillourm strengthen the production
of T helper 1 Cytokines in endodontic infections in mice free germ (10).
As it is clear there is no conflict between the findings of above research and those of the current
one. According to the current study there is no correlation between IL-4 and IL-12. (Spearman
correlation coefficient = 0.593).This matter shows that, in chronic periapical lesions both T helper
1 cells and T helper 2 may have equally been active, this may be due to presence of various
micro-organisms or their derivatives.
Studing on 24 apical lesions including 12 granulomas and 12 cysts, Walker etal (2000) stated that
in periapical lesion, T helper 2 cell has advantage over the other one
(11)
. The difference between
the findings of the mentioned and current research maybe due to difference in number of samples
and in the current research, the cystic cases were not determined. Meanwhile, two different
laboratory methods have been used in these two studies. Diversity of microorganisms relating to
lesion can also be another factor in justifying this difference.
De Sa etal (2003) stated that T F N and IL-6 have a probable role in periapical granuloma
5
pathogenesis. In 1999, Barkhordar et al. reported that IL-6 is locally produced and released in
inflamed tissue of pulp and lesions
(17)
. Like previous researches , there was a similarity between
the findings of these two researches, because IL-6 was observed in all the samples of the current
research.
In 1994, Matsuo et al perceived that the level of IL-1a in exudates relating to bigger lesions is
higher that of smaller ones, but in regard to IL-1β they did not observe such a difference (18). Also
in current research, with regard to IL-6 which is an important inflammatory cytokine of lesion
such as IL - 1β, no significant difference was found between bigger and smaller lesions (P> 0.05).
Although IL-1β, which had a higher level in larger lesions, is considered as an inflammatory
cytokine, and is mostly produced by T cells (and not macrophages) and exists as membranous and
not free.
Conclusion:
Totally it is concluded that T helper 1 and 2 cells probably have an active contribution in chronic
periapical lesions IL-6, as an important cytokine, can play a role in pathogeneses of these lesions,
so that it can both induce bone resorption through activating osteoblast and osteoclast and
destruction of soft tissue through activating collagens. Despite the hypothesis of the current study,
there was not any significant correlation between IL-4 or IL-12 and the diameter of the lesion
(P>0.05). This can be resulted from presence of different microorganisms in chronic periapical
lesions, a group of them can violate the balance between T helper 1 cells and T helper 2 cells in
favor of T helper 1 cells and another group can change the balance in favor of T helper 2 cells .
In order of validity of this hypothesis, more researches in this field and evaluation of cystic cases
are needed to be done.
Acknowledgements:
We would like to thank the esteemed professors and assistants of endodontic department , Shahid
Beheshti University of medical sciences who helped us to perform this study.
References
1. Sasaki H, Hou L, Belani A, Wang CY, Uchiyama T, Muller R, Stashenko P: IL-10, But not
IL-4, suppresses infection-stimulated bone resorption in vivo. J Immuno 2000;165:36263630.
2. Wang CY, Stashenko P: The role of interleukin-1 alpha in the pathogenesis of periapical
bone destruction in a rat model system. Oral Microbial Immunol 1993;8:50.
6
3. Tani-Ishii N, Wang CY, Stashenko P: Immunolocalization of bone resorptive cytolkines in
rat pulp and periapical lesions following surgical pulp exposure. Oral Microbial Immunol
1995;10:213.
4. Wang CY, Tani-Ishii N, Stashenko P: Bone-resorptive cytokine gene expression in
periapical lesions in the rat. Oral Microbial Immunol 1997;12:65.
5. Balto K, Sasaki H, Stashenko P: Interleukin-6 deficiency increase inflammatory bone
destruction. Infect Immun 2001;69:744-750.
6. Shimauchi H, Takayama S, Narikawa-Kiji M, Shimabukuro Y, Okada H: Production of
interleukin-8 and nitric oxide in human periapical lesions. J Endod 2001;27:744-752.
7. Lin SK, Hong CY, Chang HH, Chiang CP, Chen CS, Jeng JH, et al: Immunolocalization of
macrophage and transforming growth factor-beta in induced rat periapical lesions. J Endod
2000; 26:335-340.
8. Rinacho JA, Zarrabeitia MT, Gonzalez-Macias J: Interleukin-4 modulates osteoclast and
inhibits the formation of resorption pits in mouse osteoclast cultures. Biochem. Biophys Res
Commun 1993;196:678.
9. Kavashima N, Stashenko P: Expression of bone-resorptive and regulatory cytokines in
murine periapical inflammation. Arch Oral Boil 1999;44:55.
10. Ribeiro Sobrinho AP, de Melo Maltos SM, Farias LM, de Carvalho MA, Nicoli JR, de
Uzeda M, et al: Cytokine production in the response to endodotic infection in germ-free
mice. Oral Microbial Immunol 2002;17:344-353.
11. Walker KF, Lappin DF, Takahashia K, Hope J, Macdonald DG, Kinane DF: Cytokine
expression in periapical granulation tissue as assessed by immunohistochemistry. Eur J Sci
2000;108:195-201.
12. Goldsby RR, Kindh TJ, Osborne BA: Kuby Immunology. 4th Ed. Freeman: New York
2000;Chap12:320-321.
13. Miossec P, Chomarat P, Dechanet J, Moreau JF, Roux JP, Delmas P, et al: Interleukin-4
inhibits bone resorotion through an effect on osteoclast and proinflammatory cytokines in an
ex vivo model of bone resorption in rheumatoid arthritis. Arthritis Rheum 1994;37:1715.
14. Bizzarri C, Shioi A, Teitelbaum SL, Ohara J, Harwalkar VA, Erdmann JM, et al:
Interleukin-4 inhibits bone resorption and acutely increases cytosolic ca in murine
osteoclasts. J Biol Chem 1994;269:13817.
15. Torabinejad M, Eby WC, Naidorf IJ: Inflammatory and immunological aspects of the
pathogenesis of human periapical lesions. J Endod 1985;11:479-488.
7
16. De Sa AR, Pimenta FJ, Dutra WO, Gomez RS: Immounolocalization of interleukin-4,
interleukin-6 and lymphotoxin alpha in dental granulomas. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2003;96:356-360.
17. Barkhordar RA, Hayashi C, Hussain MZ: Detection of interleukin-6 in human dental pulp
periapical lesion: Endod Dent Traumatol 1999;15:26-27.
18. Matsuo T, Ebiso S, Nakanishi T: Immunoglobulins in periapical exudates of infected root
canals: Correlations with the clinical findings of the involved teeth. Endod Dent Traumatol
1995;11:95-99.
19. De Rossi A, Rocha LB, Rossi MA: Interferone-gamma, interleukin 10, intercellular
Adhesion Molecule-1, and Chemokine Receptor 5, but not interleukin-4, Attenuate the
development of periapical lesions. J Endod 2008;34: 31-38.
20. Yamasaki M, Morimoto T, Tsuji M, Akhihiro I, Maekawa Y, Nakamura H: Role of IL-12
and helper T-lymphocytes in limiting periapical pathosis. J Endod 2006;32:24-29.
21. Brekalo Prso I, Kocjan W, Simic H, Brumini G, Pezelj–Ribaric S, Borcic J, Ferreri S, et al:
Tumor Necrosis factor-Alpha and interleukin-6 in human periapical lesions. Mediators of
inflamm 2007;1:38210.
8