Journal of the Formosan Medical Association (2013) 112, 352e357

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ORIGINAL ARTICLE

Modulation of serum smooth muscle antibody levelsby levamisole treatment in patients with oral lichenplanus

Kai-Ming Wu a, Yi-Ping Wang a,b, Hung-Pin Lin c,d, Hsin-Ming Chen a,e,Jean-San Chia a,b,e, Andy Sun a,b,*

aDepartment of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan University,Taipei, TaiwanbGraduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, TaiwancDepartment of Dentistry, China Medical University Hospital, College of Medicine, China Medical University,Taichung, Taiwand School of Dentistry, College of Medicine, China Medical University, Taichung, TaiwaneGraduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan

Received 19 February 2013; accepted 10 March 2013

KEYWORDSlevamisole;oral lichen planus;smooth muscleantibody

* Corresponding author. DepartmentE-mail address: andysun7702@yah

0929-6646/$ - see front matter Copyrhttp://dx.doi.org/10.1016/j.jfma.201

Background/Purpose: Serum autoantibodies have been found in patients with oral lichen pla-nus (OLP). This study evaluated whether OLP patients had significantly higher frequencies ofserum smooth muscle antibody (SMA) than healthy control individuals, and assessed whetherlevamisole treatment could modulate the serum SMA levels in OLP patients.Methods: This study used an indirect immunofluorescence technique to measure the baselineserum SMA levels in a group of 647 OLP patients and 53 controls. Ninety-five SMA-positive OLPpatients were treated with levamisole under a regular follow-up schedule, and their serum SMAlevels were measured after treatment.Results: The frequencies of serum SMA in patients with OLP (21.9%), erosive OLP (EOLP,21.6%), major EOLP (17.9%), minor EOLP (24.2%), and nonerosive OLP (24.4%) were all signifi-cantly higher than that (0%) in healthy controls (all p < 0.001). Of 142 SMA-positive OLP pa-tients, 95 were treated with levamisole under a regular follow-up schedule. Treatment withlevamisole for a period of 2e29 months (mean, 9.4 � 6.0 months) effectively reduced the highmean serum SMA titer (71.0 � 7.2) at baseline to an undetectable level (0) in all SMA-positiveOLP patients, regardless of different initial serum SMA titers.

of Dentistry, National Taiwan University Hospital, 1, Chang-Te Street, Taipei 10048, Taiwan.oo.com.tw (A. Sun).

ight ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.3.03.003

Smooth muscle antibody in oral lichen planus 353

Conclusion: There was a significantly higher frequency of serum SMA (21.9%) in OLP patientsthan in healthy controls. Treatment with levamisole for 2e29 months significantly reducedthe high serum SMA to an undetectable level, and significantly improved the signs and symp-toms in all treated OLP patients.Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

Introduction

Oral lichen planus (OLP) is a chronic inflammatory oralmucosal disease commonly found in the dental clinic. Pre-vious study showed involvement of both antigen-specificand nonspecific mechanisms in OLP. Antigen-specificmechanisms include antigen presentation by basal kerati-nocytes and antigen-specific keratinocyte killing by CD8þcytotoxic T lymphocytes. Nonspecific mechanisms comprisemast cell degranulation and matrix metalloproteinaseactivation in OLP lesions.1 Through mast cell/T cell in-teractions in OLP lesions, mast-cell-released cytokines,chemokines and matrix metalloproteinases can promoteT cell activation, migration, proliferation, and differentia-tion.2 Histologically, OLP is characterized by hyperkeratoticand acanthotic epithelium with liquefaction degenerationof basal epithelial cells, and a marked band-like sub-epithelial infiltrate of mononuclear cells that are predom-inantly CD8þ. CD4þ cells are observed predominantly inthe deep lamina propria.3 An increase in histocompatibilityleukocyte antigen (HLA)-D-related (DR)-positive CD3þ cellsin both the local lesional tissues and peripheral lympho-cytes also indicates T cell activation in OLP.4,5 The abovefindings suggest that OLP is a T cell-mediated inflammatorydisease.

Levamisole is an immunomodulator that can modulateT cell-mediated immunity.6e11 Our previous studies havefound that levamisole can also modulate abnormal seruminterleukin (IL)-6, IL-8, tumor necrosis factor-a, antibasalcell antibody (anti-BCA), antinuclear antibody (ANA),antithyroglobulin antibody (TGA), and antithyroid micro-somal antibody (TMA) levels to normal in patients with OLPor erosive OLP (EOLP).6e11 The presence of serum auto-antibodies including anti-epithelial cell antibody, ANA,smooth muscle antibody (SMA), antimitochondrial anti-body, gastric parietal cell antibody (GPCA), TGA, TMA, andantidesmoglein 1 and antidesmoglein 3 antibodies hasbeen shown in several different groups of OLPpatients.9e17

Our previous studies found that the serum SMA-positive rates are 20%, 17%, 41%, and 23.9% in patientswith OLP, recurrent aphthous ulcerations (RAU), oralsquamous cell carcinoma (OSCC), and oral submucousfibrosis (OSF), respectively.18,19 In this study, we exam-ined the frequencies of serum SMA in a group of 647Taiwanese OLP patients and in 53 healthy control in-dividuals. We tried to assess whether there was a signif-icantly higher frequency of serum SMA in Taiwanese OLPpatients than in healthy controls, and whether levamisoletreatment could modulate serum SMA levels in OLPpatients.

Materials and methods

Patients and controls

The study group consisted of 647 OLP patients (111 men and536 women; age range, 21e88 years; mean age, 55.4 years)without lichen planus of other mucosal or skin surfaces. Thenormal control group consisted of 53 healthy controls(8 men and 45 women; age range, 21e83 years; mean age,54.7 years) without any oral mucosal or systemic diseases.All the patients and controls were seen consecutively,diagnosed, and treated in the Department of Oral Diagnosisof National Taiwan University Hospital from July 2004 toFebruary 2012. OLP patients with areca quid chewing habit,hypertension, and autoimmune diseases such as systemiclupus erythematosus, rheumatoid arthritis, Sjogren’s syn-drome, pemphigus vulgaris, and cicatricial pemphigoidwere excluded. In addition, none of them had taken anyprescription medication for at least 3 months prior toentering the study. The 647 OLP patients included 561 withEOLP (98 men and 463 women; age range, 21e88 years;mean age, 55.7 years) and 86 with nonerosive OLP (NEOLP;13 men and 73 women; age range, 24e83 years; mean age,53.2 years). They were selected according to the followingcriteria: (1) a typical clinical presentation of radiatinggrayish white Wickham striae, papules and plaques, sepa-rately or in combination (NEOLP), or erosion or ulcerationof the oral mucosa (EOLP); and (2) biopsy specimens char-acteristic of OLP, that is, hyperkeratosis or parakeratosis, aslightly acanthotic epithelium with liquefaction degenera-tion of the basal epithelial cells, a prominent band-likelymphocytic infiltrate in the lamina propria, and theabsence of epithelial dysplasia. EOLP was further dividedinto major and minor types according to previouslydescribed criteria.6e8

All OLP patients, regardless of their subtype, weretreated with levamisole that was administered at a dose of50 mg twice daily for patients with 30e50 kg body weight,or 50 mg three times daily for patients with 50e70 kg bodyweight, for 3 consecutive days at the beginning of each 2-week interval. Compliance was monitored by asking thepatients to record the time at which each drug was taken.Prior to the start of therapy, clinical data on all cases wererecorded according to a set protocol, and the patients wereexamined by the same dentist during each visit. Patientswere monitored once monthly to record clinical responsesafter treatment.

Blood samples were taken from OLP patients prior totreatment and from normal controls to measure the serumSMA, ANA, and hepatitis C virus antibody (HCVA) levels. TheANA and HCVA levels were concurrently determined to

Table 1 Frequencies of serum smooth muscle antibody(SMA) in different groups of oral lichen planus (OLP) pa-tients and 53 healthy controls.

SMA (þ) p*

Patientnumber

%

OLP (n Z 647) 142 21.9 <0.001Age �50 y (n Z 199) 40 20.1 <0.001Age >50 y (n Z 448) 102 22.8 <0.001Men (n Z 111) 21 18.9 <0.001Women (n Z 536) 121 22.6 <0.001

EOLP (n Z 561) 121 21.6 <0.001Major (n Z 234) 42 17.9 <0.001Minor (n Z 327) 79 24.2 <0.001

NEOLP (n Z 86) 21 24.4 <0.001Healthy controls (n Z 53) 0 0

EOLP Z erosive OLP; NEOLP Z nonerosive OLP.* Comparison between healthy control group and any other

groups by Chi-square test.

354 K.-M. Wu et al.

evaluate whether there was an association of serum SMApositivity with the serum ANA or HCVA positivity. To assesswhether the serum SMA levels in OLP patients were reducedafter treatment with levamisole, blood samples were ob-tained serially every 2e3 months until the serum SMA levelsbecame undetectable. Informed consent was obtained fromeach patient or control prior to blood sample collection. Thisstudywas reviewed and approved by the Institutional ReviewBoard at the National Taiwan University Hospital.

Determination of serum SMA and ANA levels

The circulating SMA and ANA were detected by the indirectimmunofluorescence technique with HEp-2 cells (for ANA;Medical & Biological Laboratories Co., Nagoya, Japan) andrat kidney (for SMA) as substrates as described previ-ously.15,19 In brief, HEp-2 cells or 5 mm-thick cryostat sec-tions of substrate tissues on slides were reacted withserially diluted OLP patients’ and controls’ sera in a moistchamber at room temperature for 30 minutes. The initialdilution of the sera was 1:20 with PBS. After washing, thesections were incubated with fluorescein isothiocyanate-labeled goat antihuman IgG antiserum (Boehringer Man-nheim Biochemicals, Indianapolis, IN, USA), which wasprediluted and supplied in a dropper vial by the manufac-turer and was ready-to-use in 30 minutes. The sectionswere washed again, mounted with buffered glycerine, andexamined with a fluorescence microscope (Olympus OpticalCompany Ltd., Tokyo, Japan). Sera were scored as positivewhen they produced fluorescence at a dilution of 160� ormore for ANA, and of 20� or more for SMA.

Determination of HCVA level

The serum HCVA was detected by an ARCHITECT anti-HCVassay (Abbott Laboratories, Abbott Park, IL, USA), asdescribed previously.20,21 This assay was an automatedchemiluminescent microparticle immunoassay based on theenzyme immunoassay principle. In contrast to the enzymeimmunoassay, where the antigeneantibody complexeswere detected by enzyme-labeled conjugate, the chemi-luminescent microparticle immunoassay used acridinium-labeled conjugate as the detection system. The resultingchemiluminescent reaction was measured as relative lightunits (RLUs). ARCHITECT Anti-HCV was designed to detectantibodies to structural and nonstructural proteins of theHCV genome (NS3, NS4, core). The ARCHITECT Anti-HCVassay cutoff value was calculated by the following for-mula: calibrator 1 mean RLU value �0.074 Z cutoff RLU.ARCHITECT Anti-HCV assay delivered specimen results as aratio of the specimen signal (in RLU) to the cutoff value (S/CO). S/CO ratios of �1.00 were considered reactive forHCVA; ratios <1.00 were nonreactive for HCVA.

Statistical analysis

The difference in frequency of serum SMA was comparedbetween any two groups by the Chi-square test. The serumlevels of SMA at baseline and after treatment werecompared to each other using a paired t test. The resultwas considered to be significant if p < 0.05.

Results

There were 142 OLP patients (21.9%) with a serum SMA titer�20. The frequencies of serum SMA in different groups ofOLP patients and 53 healthy controls are shown in Table 1.The frequencies of serum SMA in patients with OLP (21.9%),EOLP (21.6%), major EOLP (17.9%), minor EOLP (24.2%), andNEOLP (24.4%) were all significantly higher than that (0%) inhealthy controls (all p < 0.001). In addition, OLP patientsaged �50 years or >50 years and male or female OLP pa-tients also had a significant higher frequency of serum SMAthan healthy controls (all p < 0.001; Table 1). The serumSMA positive rate was not associated with OLP patients’ ageand gender, type of OLP, type of EOLP, serum ANA-positivity, and serum HCVA-positivity (all p > 0.05;Table 2). Of 142 SMA-positive OLP patients, 95 were treatedwith levamisole under a regular follow-up schedule in ourdental clinic. Treatment with levamisole for a period of2e29 months (mean, 9.4 � 6.0 months) effectively reducedthe high mean serum SMA level (71.0 � 7.2) at baseline toan undetectable level (0) in all 95 SMA-positive OLP pa-tients, including those with EOLP, major or minor EOLP, andNEOLP (Table 3). Furthermore, when OLP patients weredivided into five different groups according to differentinitial serum SMA titers ranging from 1:20 to 1:320, all theserum SMA levels at baseline were effectively reduced to anundetectable level (0, all p � 0.002) after treatment withlevamisole for 2e29 months (Table 3). The 95 SMA-positiveOLP patients also had significant improvement in signs andsymptoms of OLP after levamisole treatment for 2e29months (such as a reduction in lesion size and pain causedby the lesion, healing of erosive or ulcerative lesions, andtransformation of EOLP into NEOLP).

Discussion

This study showed that the serum SMA-positive rate was21.9% for 647 OLP patients, 21.6% for 561 EOLP patients,

Table 2 Comparison of serum smooth muscle antibody(SMA) positivity between different groups of oral lichenplanus (OLP) patients.

SMA (þ)(n Z 142)

SMA (e)(n Z 505)

p

Patients’ age (y)�50 (n Z 199) 40 159 0.513>50 (n Z 448) 102 346

Patients’ genderMen (n Z 111) 21 90 0.471Women (n Z 536) 121 415

Type of OLPEOLP (n Z 561) 121 440 0.649NEOLP (n Z 86) 21 65

Type of EOLP (n Z 561)Major (n Z 234) 42 192 0.097Minor (n Z 327) 79 248

ANAPositive (n Z 136) 32 104 0.700Negative (n Z 511) 110 401

HCVAPositive (n Z 76) 19 57 0.591Negative (n Z 571) 123 448

ANA Z antinuclear antibody; EOLP Z erosive OLP;HCVA Z hepatitis C virus antibody; NEOLP Z nonerosive OLP.

Smooth muscle antibody in oral lichen planus 355

and 24.4% for 86 NEOLP patients. Our previous studydemonstrated the presence of serum SMA in 8.4% of 320 OLPpatients, 8.2% of 292 EOLP patients, and 10.7% of 28 NEOLPpatients.17 We suggest that the differences in the serumSMA-positive rate between these two groups of OLP pa-tients was due to different sample sizes in these twostudies. Although the serum SMA-positive rates weredifferent, NEOLP patients tended to have a slightly higherserum SMA-positive rate than EOLP patients in these twostudies. In the present study, we treated the OLP patients

Table 3 Serum titers of smooth muscle antibody (SMA) prior to(OLP) patients, including 33 with major and 48 with minor erosiv

Treatmentmodality

OLP type or SMA titer Duration of trea(mo)

Range Mea

Levamisole OLP (n Z 95) 2e29 9.4Levamisole EOLP (n Z 81) 2e29 9.3Levamisole Major (n Z 33) 2e29 9.8Levamisole Minor (n Z 48) 2e21 9.0Levamisole NEOLP (n Z 14) 3e25 10.0Levamisole SMA titer Z 20 (n Z 37) 2e16 6.6Levamisole SMA titer Z 40 (n Z 16) 2e21 9.6Levamisole SMA titer Z 80 (n Z 25) 2e29 11.6Levamisole SMA titer Z 160 (n Z 13) 3e25 12.0Levamisole SMA titer Z 320 (n Z 4) 9e18 12.8

SD Z standard deviation; SEM Z standard error of the mean.* Comparison of serum SMA titers between patients at baseline and

with levamisole and found that all different initial serumSMA titers in different groups of OLP patients could bereduced to an undetectable level after treatment for 2e29months, although OLP patients with lower SMA titersneeded shorter treatment periods to render the SMA titersto an undetectable level. Our previous studies have alsoshown the disappearance of serum anti-BCA in all three ofsix anti-BCA-positive EOLP patients, the disappearance ofserum ANA in three ANA-positive EOLP patients, partialreduction or absence of serum TGA in 36 of 41 TGA-positiveOLP patients and of serum TMA in 46 of 48 TMA-positive OLPpatients, and complete regression of serum ANA in all 79OLP patients after levamisole treatment for differentperiods.9e11 In addition, treatment with levamisole for0.5e7.5 months can significantly reduce the abnormallyhigh serum IL-6, IL-8, and tumor necrosis factor-a levels tonormal in patients with OLP or EOLP.7,8 The most importantfact is that the reduction of autoantibody or cytokine levelsafter levamisole treatment is always accompanied by sig-nificant improvement of signs and symptoms in OLPpatients.6e11 These findings suggest that levamisole treat-ment decreases the abnormal serum autoantibody andcytokine levels to normal in OLP patients, and is an effec-tive treatment modality for OLP.

Previous studies have demonstrated an overall incidenceof 21e82% for serum autoantibodies in several differentgroups of OLP or EOLP patients.10e17 The frequencies ofserum TGA and TMA are 21.6% and 24.5% in 278 OLP pa-tients, respectively.10 The positive rate of serum ANA is23.2% in 583 OLP patients.11 Serum anti-BCA is discovered in34 (54%) of 63 OLP patients.12 Serum autoantibodiesincluding rheumatoid factor, ANA and SMA are found in 27%of 30 OLP patients and in 9% of 23 controls.13 The presenceof circulating antibodies to epithelial antigen was shown in57% of 14 HCVA-positive OLP patients and in none of 14HCVA-negative patients.14 Carrozzo et al15 demonstratedthe presence of serum autoantibodies, including ANA, SMA,antimitochondrial antibody, GPCA, and antithyroid anti-body in 41% of 27 HCVA-positive OLP patients and in 52% of

or after treatment with levamisole in 95 oral lichen planuse OLP (EOLP), and 14 with nonerosive OLP (NEOLP).

tment Serum titers of SMA (fold ofdilution of serum)

p*

n � SD At baseline After treatment

Mean � SEM Mean � SEM

� 6.0 71.0 � 7.2 0 <0.001� 5.9 67.4 � 7.4 0 <0.001� 6.8 80.0 � 11.3 0 <0.001� 5.3 58.8 � 9.7 0 <0.001� 6.3 91.4 � 24.0 0 0.002� 3.7 20 0 <0.001� 4.7 40 0 <0.001� 7.4 80 0 <0.001� 7.1 160 0 <0.001� 4.5 320 0 <0.001

after levamisole treatment by paired t test.

356 K.-M. Wu et al.

23 HCVA-negative patients. Lukac et al16 showed, byenzyme linked immunosorbent assay, significantly higherconcentrations of circulating autoantibodies to desmoglein1 and desmoglein 3 in 32 EOLP patients compared with 50healthy controls. Indirect immunofluorescence alsorevealed significantly higher frequencies of desmogleinautoantibodies in EOLP patients (82%, 18/22) than inhealthy controls (5%, 1/20).16 Our previous study demon-strated an overall incidence of 60.9% for the presence ofserum autoantibodies in a large group of 320 OLP patients,and significantly higher frequencies of serum ANA, GPCA,TGA, and TMA in OLP patients than in healthy controls.17

The above findings demonstrate significantly higher fre-quencies of serum autoantibodies in OLP patients than inhealthy controls and also indicate that OLP carries a highpotential toward an autoimmune disease.

The present study showed a significantly higher serumSMA positive rate in NEOLP, major EOLP, and minor EOLPpatients. Our previous studies found the presence ofserum SMA in 20%, 17%, 41%, and 23.9% of patients withOLP, RAU, OSCC, and OSF, respectively.18,19 Myofibroblastsidentified by a-smooth muscle actin antibody are detec-ted in OSCC and OSF lesions22e24 as well as in woundhealing and fibrocontractive diseases such as livercirrhosis and renal fibrosis.25 EOLP and RAU are oralmucosal diseases characterized by multiple recurrences oforal ulcers followed by wound healing. Repeateddestruction of oral mucosal tissues by the disease processof OLP, RAU, OSCC, and OSF may cause the release ofsmooth muscle antigens from myofibroblasts and bloodvessel wall into the local tissues and blood circulation.These smooth muscle antigens are phagocytosed andprocessed by macrophages and B cells, and in turn, pre-sented to helper/inducer T cells in the oral mucosal le-sions, regional lymph nodes, and blood circulation of OLP,RAU, OSCC, and OSF patients. With the help of T cells,antigen-specific activated B cells thus produced highlevels of SMA in the local oral mucosal lesions and bloodcirculation of OLP, RAU, OSCC, and OSF patients. Inaddition, our recent study showed that 26.3%, 21.3%,24.4%, and 12.8% of 320 OLP patients are GPCA-, TGA-,TMA-, and HCVA-positive, respectively.17 This study alsodemonstrated an HCVA-positive rate of 11.7% in 647 OLPpatients. GPCA-positive, TGA/TMA-positive, and HCVA-positive OLP patients may also have underlying autoim-mune atrophic gastritis, autoimmune thyroid disease, andchronic liver disease, respectively.26e29 Therefore, thesepatients may have more smooth muscle antigens releasedin the local gastric, thyroid and liver tissues and bloodcirculation. By similar mechanisms, these released smoothmuscle antigens may stimulate the antigen-specific acti-vated B cells to produce more SMA. The locally-producedSMA in the interstitial fluid may diffuse into blood capil-laries or be drained into the lymphatic vessels, and finallyreach the blood circulation. The locally-secreted SMA,together with those produced in the blood circulationeventually give rise to significantly higher levels of serumSMA in OLP patients than in healthy controls. The disap-pearance of the serum SMA after levamisole treatment-induced healing of the OLP lesions in the present studyalso suggests that the principal smooth muscle auto-antigens may come from the local OLP lesions.

It has been well established that chronic HCV infectionplays important roles in the production of noneorgan-spe-cific autoantibodies, including SMA. Serum SMA is detected33e37% of HCV-infected patients.29 An SMA-positive rate of25% is found in 175 Indian patients with chronic liver dis-ease.30 In addition, polyclonal B cell activation by persis-tent HCV infection has been proposed as anothermechanism for the production of autoantibodies.31 Thefindings from these three studies also indicate the contri-bution of chronic HCV infection or liver disease to theproduction of SMA.

This study showed a significantly higher frequency ofserum SMA (21.9%) in OLP patients than in healthy controls.Treatment with levamisole for 2e29 months could signifi-cantly reduce the high serum SMA concentrations to unde-tectable levels, and significantly improve signs andsymptoms of OLP patients. Therefore, we conclude thatlevamisole treatment can modulate the high serum SMAtiter to undetectable levels in OLP patients, and is aneffective treatment modality for OLP.

Acknowledgments

This study was supported by research grants of NSC 95-2314-B002-202 and NSC 99-2314-B002-142-MY3 from theNational Science Council, Taipei, Taiwan.

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