Brazilian Journal of Oral Sciences Piracicaba Dental School - UNICAMP EISSN: 1677-3225 Vol. 10, Num. 2, 2011, pp. 83-87

Brazilian Journal of Oral Sciences, Vol. 10, No. 2, Apr-Jun, 2011, pp. 83-87

Cytokine profiles in the sera of Egyptian patients with oral pemphigus vulgaris

Hossam Eid¹, Basiouny El-Gamal²

¹PhD, Oral Medicine, Oral Diagnosis and Periodontology Department, Faculty of Dentistry, Suez Canal University, Egypt
²PhD, Clinical Biochemistry Department, College of Medicine, King Khalid University, Abha, Saudi Arabia
Correspondence to: Basiouny El-Gamal Department of Clinical Biochemistry College of Medicine, King Khalid University, Abha, Saudi Arabia Phone: +966 543175779 E-mail: basiouny_el_gamal@hotmail.com

Received for publication: January 01, 2011 Accepted: April 20, 2011

Code Number: os11017

Abstract

Cytokines have been suggested to play an important role in the pathogenesis of various inflammatory and autoimmune diseases, including the potentially fatal blistering disease, oral pemphigus vulgaris (PV). No data are currently available on the cytokine levels in the sera of Egyptian patients with oral PV.
Aim:The aim of this study was to measure the serum levels of some proinflammatory and antiinflammatory cytokines in Egyptian patients with PV.
Methods: Using highly sensitive ELISA kits, the levels of TNF-α , IL-2, IL-4 and IL-6 were measured in the sera of 10 patients affected with oral PV and 10 healthy subjects.
Results: Serum levels of TNF-α and IL-6 were found to be significantly higher in patients with oral PV than in healthy controls (p<0.001). On the other hand, no significant differences were observed in the levels of IL-2 and IL-4 between oral PV and control sera (p<0.05).
Conclusions: These data showed that TNF-α and IL-6 levels were significantly increased in the sera of Egyptian patients with oral PV and this might suggest its role in the pathogenesis of this disease.

Keywords: Pemphigus vulgaris, oral lesions, cytokines.

Pemphigus vulgaris (PV) is a chronic, vesiculobullous, mucocutaneous autoimmune fatal disease, characterized by the presence of antibodies against adhesionmolecules (desmoglein, Dsg3) present on the surface of keratinocytes, leading tothe loss of cellular adhesion or acantholysis and is typically associated with orallesions^1-5. Histologically, PV can be detected by indirect immunofluorescence assayof anti-Dsg3 antibodies in the sera⁶ and blister fluid⁷.

In fact, the oral mucosa is the first site to be involved in up to 70% of cases,and it is the only site to be affected in over 50% of patients⁸. Most patientsexhibit oral lesions at some time of the disease⁸. Clinically, the most commonsites of oral PV lesions are the labial and buccal mucosa or the edentulous ridges.Oral lesions are commonly characterized by the presence of vesiculobullous andulcerative lesions.

Recently, there has been increasing interest in the role of cytokines in thepathogenesis of various inflammatory and autoimmune diseases, including thepotentially fatal blistering disease, PV^1-2,4,8-13. Cytokines are regulatory compoundsproduced by cells of the immune system [T(H)1 and T(H)2] and act as intracellularmediators and control the immune and inflammatory reponses⁹. T(H)1 control thecell-mediated response and produce a number of proinflammatory cytokines, e.g.IL-1, IL-2, IL-6 and TNF-α which are counterbalanced by a number of antiinflammatory cytokines, e.g. IL-4 and IL-10 that areproduced by T(H)2 that participate in humoral response andantibody production. Recent studies point out atproinflammatory cytokines such as TNF-α , IL-1, or IL-6 as strong players involved in this process¹³. Moreover, experimental studies revealed that synergistic cooperationof pemphigus antibodies with IL-6 and TNF-α in the pathogenesis of PV.

There is a fairly strong genetic background to pemphigus vulgaris with linkage to HLA class II alleles. Certain ethnicgroups, such as Ashkenazi Jews and those of Mediterranean and South Asian origin are especially liable to PV^14-15. No data are currently available on the cytokine levels in thesera of Egyptian patients with oral VP. In the present study,we measured the serum levels of some proinflammatory cytokines (IL-2, IL-6, TNF-α ) and the antiinflammatory cytokine IL-4 in Egyptian patients with oral PV, in comparisonto healthy controls.

All participants were recruited from the Outpatient Clinics of the Department of Skin and Venereal Diseases,Faculty of Medicine, and the Department of Oral Medicine, Oral Diagnosis, and Periodontology, Faculty of Dentistry,Suez Canal University, Egypt. Ten patients with oral PVand 10 healthy controls were enrolled in the study after ethicalapproval by the Suez Canal University. A written informedconsent was taken from all participants prior to enrollment.

The group of patients with oral PV consisted of 6 malesand 4 females with mean age of 50.6 years (range: 45-60years). The selection and diagnosis of patients was based onthe history, clinical characteristics of oral PV lesions, thehistopathological specimens and the indirect immunofluorescence testing⁶ of PV. The duration of oral PV lesions rangedfrom 1-3 years. None of the patients had received any topicaland/or systemic treatment for the present illness at least 1month prior to study.

The control group consisted of 6 males and 4 femaleswith mean age of 48.4 years (range: 40-53 years). They werehealthy volunteers completely free from any local or systemicdiseases who were not taking any medication or contraceptives(in females).

Serum samples were collected from the individuals ofthe two groups and used to measure the levels of the cytokinesTNF-α , IL-2, IL-4 and IL-6 using commercially availableELISA kits (Quantikine, R & D, Minneapolis, MN, USA),according to manufacturer's procedure.

All results were expressed as mean ± SD. Differencesbetween two means were analyzed by Student's t-test. P values equal or less than 0.05 were considered as significant.

The most common sites of oral PV lesions found in the present study were the lip, buccal mucosa, palatal, ventralsurface of tongue, and gingival. Examples of palatal mucosa and buccal mucosa with oral lesions in patients with PV areshown in Figures 1 and 2, respectively.

Direct immunofluorescence testing revealed circulatingpemphigus antibodies in all patients with oral PV. Figure 3 shows an example of PV with intercellular deposition ofantibodies in stratum spinosum.

There was no statistically significant difference (p>0.05)between patients with oral PV and controls with respect tothe mean age.

Table 1 shows serum cytokines levels (pg/mL) ofTNF-α , IL-2, IL-4, and IL-6 in patients with oral PV andcontrols. In comparison with controls, patients with oralPV had 640% and 179% higher mean TNF-α and IL-6 levels (p<0.001 and p<0.001), respectively. On the otherhand, there were no significant differences in the levels of IL-2 and IL-4 of patients with oral PV compared withcontrols (p>0.05 and p>0.05), showing only 1% and4% increase, respectively.

Discussion

PV is a chronic, antibody mediated autoimmune diseasethat affects the skin and oral mucous membrane with distinct clinical, histopathological, and immunological features¹⁶. The exact cause of oral PV is unknown. Several studies supportthe immunological basis for the disease. Of importance todentist is the frequency with which oral lesions were theinitial presenting feature of oral PV as they often precedethe skin lesions by several months or may be the major, ifnot the only, manifestation in some patients⁸.

Serum cytokine levels were recently investigated in manyautoimmune disorders such as Lichen planus (LP) andrheumatoid arthritis⁶, and a relationship between serumcytokine levels and the clinical appearance of these diseases has been reported. Previous studies^6,17 have reported that,the increase of some cytokines levels, such as IL-1, IL-6, IL8 and TNF-α in the serum of patients with inflammatory andautoimmune diseases is well known, and these elevated cytokine levels seem to be important mediators restricting each disease^6,17. In the present study, the serum levels ofsome proinflammatory cytokines (IL-2, IL-6, TNF-α ) and the antiinflammatory cytokine IL-4 were measured in Egyptian patients with oral PV and compared with healthy controls.

Cytokine generation is regulated by other cytokines andalso by itself⁶. In cytokine network with autocrine andparacrine control, cytokine actions are usually balanced;however, cytokine imbalance occurs under pathologicalconditions and large amounts of cytokines are generated,which may be beneficial or harmful to the body¹⁷. Unbalanced cytokine actions are considered to be one of the immunopathogenesis mechanisms of autoimmune disorders⁶. Elevation of cytokine activities and unbalanced cytokinenetwork may induce oral mucosal lesions⁶. The identification of cytokine activities in patient's tissues and sera seems tobe advantageous for pathological analysis of oral diseases⁶.

The present study revealed a 640% increase in serumlevels TNF-α of patients with oral PV compared to controls.Increased serum levels of TNF-α were reported by manyauthors. Thus, Alecu et al.¹⁸ found increased TNF-α levels in the sera and blister fluid obtained from patients with PV.Similar results were obtained by D'Auria et al.^1,13 who showed increased levels and in situ expression of TNF-α . Also, Narbutt et al.² showed 72% increase in TNF-α levels in the sera of patients with PV compared to control. This indicates that, TNF-α may play a role in the disease process of PV.Like IL-1, TNF-α is also a key stone in the cytokine network.TNF-α is a cytokine involved in the majority of inflammatoryprocesses, and its increased activity is found in many skindiseases including psoriasis, SLE, or systemic sclerosis^19-20. TNF-α is released by cells under various stimuli includingbacterial infections or ultraviolet radiation. It plays a role inmany biological processes, enhances phagocytosis,cytotoxicity, and modulates activity of other cytokines suchas IL-1 and IL-4²¹.

TNF-α is generated from macrophages, T and B cellsand endothelial cells¹⁷. The important inducers of TNF-α production include viruses, IL-1, immune complex, endotoxinand TNF-α itself⁶. The TNF-α plays a major role in cellmediated cytotoxicity being able to induce cytotoxic T-cell differentiation, enhances monocyte cytotoxicity, andstimulates lymphokine activated natural killer cells²². TNF-α has marked effects on epithelial cells. It is cytotoxic at highconcentration and anti-proliferative at lower concentration²². Prolonged release of TNF-α has been implicated in epithelialcell damage. So in the view of the enhanced cytotoxicity and epithelial cell lysis reported in PV, TNF-α seems to be implicated in the pathologic process of PV.

The increased serum levels of TNF-α may be attributedto the activation of macrophages during recognizing theantigenic epitopes and presenting them to T-lymphocytes.Moreover, mononuclear leukocytes might be stimulated tomigrate into the submucosal area to produce cytokinesincluding TNF-α ²³. Alternatively, the predominant cellularsources of TNF-α are the mast cells, macrophages, monocytesand endothelial cells. These cells can secrete TNF-α into the circulation, thus the TNF-α serum levels reach an appreciableamount that could play a significant role in the immunopathogenesis of PV, and in the same time gets reflected in the patient's serum²². Consequently, it seems that, in PV theantigen-antibody complex formation within the epitheliallayer may induce increased cytokine release including TNF-α , which enhances the epithelial cell damage.

The IL-6 is a multifunctional cytokine which is animportant mediator in host response to injury and infection.It stimulates the production of acute phase protein by livercells²⁴. Previous studies^24-25, reported that, the production ofIL-6 may be a response to many inducers such as TNF-α , IL4, IL-3, and some viruses. Moreover, the production of IL-6 by epithelial cells is increased in many inflammatoryautoimmune diseases such as Lichen planus and psoriasis^19,26. It seems likely therefore that, IL-6 plays an important role inimmunopathogenesis of a number of immunoinflammatoryskin diseases, and since several inflammatory skin diseasesalso affect the oral mucous membrane such as LP, SLE, and PV, it is reasonable to detect high serum level of IL-6 inpatients with active PV oral lesions²⁴.

The results of the present study revealed a markedincrease of IL-6 serum levels (179%) in patients with oralPV compared with those of the control group. As much as 72% increase in serum IL-6 levels was reported by others inactive stage of PV². The elevated IL-6 serum levels in patientswith active PV oral lesions seem to be compatible with thehigh levels of IL-6 generated and released in acuteinflammatory conditions. The high IL-6 serum levels inpatients with PV may be attributed to the elevated serumlevels of TNF-α which is one of the strong inducers forgeneration and release of IL-6²⁴. There are wide variety ofcells that are responsible for generation and release of IL-6including lymphocytes, endothelial cells and epithelial cells.Therefore, IL-6 seems to be generated and released into thecirculation in acute and chronic immunoinflammatory as wellas autoimmune disorders^17,24. Since PV is a chronic inflammatory mucocutaneous disease, the higher IL-6 serumlevels in patients with oral PV seem to be a reasonable finding.

The findings of the present study revealed a slight, nonsignificant elevation of IL-4 serum levels in patients withoral PV compared with those of control. However, Satyam etal.⁹ reported significantly higher levels of serum IL-4 inpatients with PV compared to healthy controls and suggestedthat this increase shows the induction of T(H)2 cells in thepathogenesis of PV. Also, Keskin et al.¹¹ reported elevatedlevels of IL-4 in serum of patients with PV and showed reduced levels of this cytokine to the control values followingtreatment with high-dose, long term systemic corticosteroidswith or without immunoglobulins. The IL-4 has a wide rangeof biological functions that include activation ofimmunoglobulin synthesis, T-cell proliferation, and T-cell adhesion to endothelial cells. Generation of IL-4 in some patients with autoimmune disorders seems to be likely inthese diseases.

In the present study, it could not be confirmed anysignificant changes in IL-2 serum levels in patients with oralPV when compared to the healthy controls. However, Satyamet al.⁹ reported decreased levels of IL-2 in the sera of patientswith PV.

In conclusion, the present findings showed that TNF-α and IL-6 levels were significantly increased in the sera ofEgyptian patients with oral PV, probably suggesting its rolein the pathogenesis of this disease.

We acknowledge the help and guidance of ProfMohmoud Kandeel of Oral Diagnosis, Oral Medicine &Periodontology Department, Faculty of Oral & DentalMedicine, Cairo University, Egypt.

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