J. clin. Path., 1977, 30, 569-574

Immunohistochemical study of lichen planus

SAMI SHOUSHA AND JOHN SVIRBELY1

From the Department of Histopathology, Royal Free Hospital, London NW3 2QG

SUMMARY The distribution of IgM and IgG in 20 cases of lichen planus and five cases of non-

specific inflammation were studied by the unlabelled antibody-enzyme (PAP) technique. Routineparaffin sections were used. In lichen planus deposits of immunoglobulins were seen in and aroundepithelial cells, in colloid bodies, at epidermo-dermal junctions, and in some inflammatory cells. Allcases examined for IgM and eight out of 13 cases examined for IgG were positive. The peripheralmigrating epidermal cells were mostly negative. The application of the PAP technique to skin biopsyand the significance of the findings in lichen planus are discussed.

Various immunoglobulins and complement frac-tions have been demonstrated in lichen planus (LP)by immunofluorescent techniques (Baart de laFaille-Kuyper and Baart de la Faille, 1974;British Medical Journal, 1974; Abell et al., 1975).The recently introduced immunoperoxidase tech-nique may also be used to detect them (Burns, 1975).The test is done on ordinary paraffin-processedhistological sections and provides permanent stain-ing and exact localisation of the components. We re-port here the results of a study by this technique ofthe distribution of IgM and IgG in LP. We try toexplain the findings and relate them to the theorieson the pathogenesis of the disease. We also commenton the application of the immunoperoxidase tech-nique to skin biopsy.

Material and methods

Routine paraffin sections were prepared from tissuetaken from 25 patients suspected clinically of havingLP. The specimens for biopsy were from the skin in23 cases and from the mouth in two cases. They wereremoved in the Departments of Dermatology andOral Surgery of the Royal Free Hospital during thelast three years. The sections were deparaffinised inxylene and stained for IgM or IgG by the unlabelledantibody-enzyme (PAP) method described by Burns(1975). Fifteen cases were examined for IgM and 15for IgG, five cases being examined for both. Sections

"Medical student of the University of Maryland Schoolof Medicine, Baltimore, Maryland, USA, on a seniorelective post.Received for publication 14 October 1976

stained with haematoxylin and eosin were studied toconfirm the diagnosis.

Results

The diagnosis of LP was confirmed in 20 of the 23biopsies of skin (group A, Table). The criteria usedfor diagnosis were the presence of irregular acantho-sis with liquefaction degeneration of the basal layerand a band-like inflammatory mononuclear cellularinfiltrate in the upper dermis (Fig. 1). The 'migrating'spindle-shaped basophilic cells described by Markset al. (1973) were seen at the margins of most lesions.Five cases showed subepidermal bullae. Group Aincluded 11 females and 9 males. Their ages rangedfrom 9 to 76 years with an average of 46.The remaining five biopsies, including the two

oral biopsies (group B, Table), showed non-specifichistological features with varying combinations ofsubepithelial inflammatory cellular reaction, regularacanthosis, and an increase in melanin-containingcells. No degenerating basal cells and no 'migrating'cells were seen in any of these cases. This group in-cluded three females and two males. Their agesranged from 35 to 49 years with an average of 43.The immunological deposits were detected as

homogenous dark brown material in and aroundepithelial cells (Figs. 2, 3), in inflammatory cells(Figs. 3, 4), and at the epidermo-dermal junctions(Fig. 4). The number of stained cells and the intensityof the stain varied from case to case. Accordingly asemiquantitative method was used to indicate thedegree of positivity (Table). A similar method wasused to quantify mononuclear inflammatory cellsand melanin-containing cells.

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Table Findings in 20 cases of lichen planus (group A) and non-specific inflammation (group B)

Case No. Sex and age Inflammatory Dermal(years) cellular melanin-

infiltrate containingcells

Group A1234567891011121314151617181920Group B2122232425

M

FFM

M

M

FFFM

FM

M

M

F

F

F

M

F

F

M

F

65*45*61494924439

712568*5420*4573*2121652876

45+49±354935

7

IgM

Intra- and Epidermo-periepithelial dermal

junction

IgG

Inflammatory Intra- and Epidermo-cells periepithelial dermal

junction

+r

It tr

Note: + + +, -t -, and -= Pronounced, moderate, and mild dark brown deposits respectively. = Diffuse intercellular pale brown staining.- = Negative.*Cases with subepidermal bullae. tOral biopsy.

A homogenous pale brown interepithelial stainingwas obtained in a few LP and control cases (shown± in Table). The stain was diffuse, affecting everypart of the epidermis, in and outside the lesion, andwas thus considered non-specific.

Melanin granules appeared to cross-react with thestain and develop a brownish coloration. Yetmelanin as such could easily be identified by itsdistinct granular appearance and slightly darkercolour.

LICHEN PLANUS

All the 11 cases stained for IgM and eight out of the13 cases stained for IgG were positive.IgM The positivity was strong in four cases,moderate in two, and mild in five. All the casesshowed intra- and periepithelial deposits. Onlyseven cases had deposits at the epidermo-dermaljunction. The cellular infiltrate included recognisableIgM-containing cells in only four cases. Seven caseshad dermal melanin-containing cells.IgG Three cases were strongly, one moderately,and four mildly positive. Three of the remaining fivecases showed a weak diffuse intercellular staining.Two cases were completely negative. The epidermo-

dermal junction showed positive deposits in onlyfour cases, of which three had epithelial deposits andone diffuse intercellular staining. The cellular in-filtrate included IgG-positive cells in only two cases.

Dermal melanin-containing cells were absent in twocases.

CORRELATIONS

The four cases examined for both immunoglobulinsshowed intraepithelial deposits of similar degree ofpositivity. The reaction at the epidermo-dermaljunction was similar in two cases and different in theother two. No correlation could be detected betweenthe immunoglobulin deposits and the severity of theinflammatory cellular reaction or the number ofmelanin-containing cells. All the five cases withbullae were positive for all the immunoglobulinstested. The cellular infiltrate in four of these includedimmunoglobulin-containing cells.

COLLOID BODIES AND MIGRATING CELLSThe colloid bodies always contained heavy immuno-globulin deposits. Almost all the 'migrating' cellswere negative. However, a positive cell was occa-sionally seen.

Inflammatorycells

570 Sami Shousha and John Svirbely

-r

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-r

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*Uattttl I a. r rFig. 1 Lichen planus: liquefactive degeneration of basal cells, and invasion of lower part of the epidermis withmononuclear in,flammatory cells. Note 'colloid' body with lymphocytes applied to border (arrow) (haematoxylinand eosin x 360).

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'...2 I - 4,.'

A~~~A,X,;tan D.; +c,;,s 4t 5:; +Ms

3#- > n-9 ;att.;*u

\+ ~Av .,a,,. s - t S

+* 5+*,t;

Fig. 2 Lichen planus: dark intra- andperiepithelial deposits ofIgG in suprabasal andprickle-ceIlllayers.Unlabelled antibody-enzyme (PAP) technique counterstained with celectine blue ( x 88).

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Fig. 3 Lichen planus: intra- and periepithelial deposits ofIgM in suprabasal and prickle-cell layers. Note negativedegenerating basal cells (B) and migrating spindle-shaped peripheral cells (M) (x 370).

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Fig4ichnpans:perlesonl' pidrmodemaldepsit o 1g (J. Bsa laer,basmet mmbrne,andeostsariterutdt it o lsin n ivaedbymooncla tflmmtoy els.To mal escls VcotiAeeeae g-oiieeihla n nlmaoycls( 4)

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Immunohistochemical study of lichen planus

CONTROL GROUPNone of the five cases showed any detectable epithe-lial deposits. However, one case (from oral mucosa)showed a weak diffuse intercellular staining for bothIgM and IgG, and another case showed positiveIgM epidermo-dermal deposits.

Discussion

Our results confirm previous studies of LP usingimmunofluorescence techniques (Baart de laFaille-Kuyper and Baart de la Faille, 1974; Abellet al., 1975) and establish the immunoperoxidasemethod as anewand useful means ofinvestigating skindiseases. By being able to do the test on old, storedparaffin sections and by providing a permanent re-cord of the exact location of the positive areas withineasily identifiable counter-stained surroundings themethod has great potentialities in advancing the useof immunological studies in the diagnosis and under-standing of the pathogenesis of skin diseases and incomparing the results with those of other conven-tional methods used in studying skin. The positivereaction was always easy to identify as dark browndeposits, quite distinct from the reaction obtainedwith melanin granules.The possibility of the epithelial stained material

being something other than immunoglobulins-namely, keratohyalin or products of melanin-wasexcluded because: (1) the positivity was alwayslimited to the diseased areas, (2) cases in group Bwith non-specific inflammation associated withacanthosis and hyperkeratosis were negative, (3)there was no correlation between the number ofdermal or epidermal melanin-containing cells andthe results of the stain, and (4) the deposits demon-strated by immunofluorescence in LP (Baart de laFaille-Kuyper and Baart de la Faille, 1974; Abellet al., 1975) occupied similar sites.The identification of IgM in all LP cases examined

and of IgG in only some accords with the findings ofthe previous authors. However, in our cases thedeposits appeared in more epidermal cells and infewer epidermo-dermal junctions. This might be dueto severe degeneration and inflammation in ourcases. Colloid bodies are always strongly positiveand the degree of positivity might be related to thedegree of degeneration. The epidermo-dermal junc-tion is usually only positive in the normal lookingareas next to the lesions (Fig. 4). Such normal look-ing areas were absent in most of our cases withnegative dermo-epidermal junction.The diagnostic significance of immunoglobulin

deposits in LP was questioned by Abell et al. (1975),but it seems from our results that they might be of

help in differentiating LP from other clinically andhistologically related conditions. Further studies onother skin diseases should clarify this point.The deposits can, however, be of great help in

understanding the pathogenesis of the disease. Thesmall areas of basal cell degeneration without in-flammatory cells that were sometimes seen appearedto be always associated with heavy dermo-epidermalimmunoglobulin deposits. Possibly the first changethat occurs after the change in the antigenic characterof basal cells (Sarkany and Gaylarde, 1971) or theirbasement membranes (Abell and Ramnarain, 1975)is the appearance of immunoglobulins at these siteseven before the arrival of any inflammatory cells.This might be achieved by diffusion from bloodvessels. Such diffusion was demonstrated in eczemaby immunofluorescence (Welbourn et al., 1976).The diffused globulins spread in all directions butbecome especially concentrated at the epidermo-dermal junction and on collagen fibres. The pre-sence of an intact basal cell layer seems to hinder theupward diffusion of the globulins. When the basallayer is much destroyed the immunoglobulindiffuses freely upwards and the epidermo-dermalimmunoglobulin condensation disappears from theseareas.The distribution of the intact epidermal immuno-

globulin-containing cells corresponds with the dis-tribution of the large eosinophilic cells found byBlack and Wilson-Jones (1972) to have a muchdepressed respiratory enzyme activity. These cellsconstitute the main bulk of the acanthotic areasoverlying the basal erosion and inflammation. A re-lation between the presence of immunoglobulins,depression of respiratory enzymatic activity, and thedecreased epidermal cell turnover suggested byseveral authors (Black, 1972) is highly probable.The absence of any immunoglobulin deposits in

almost all the 'migrating' spindle-shaped cells seen atthe margins of some lesions supports the idea thatthese are physiologically and immunologicallynormal cells derived from the adjacent unaffectedcells in an attempt at replacing the destroyed onesand limiting the spread of the disease reaction(Marks et al., 1973).The presence of heavy deposits of immuno-

globulins in colloid bodies confirms the results ofUeki (1969) and Baart de la Faille-Kuyper andBaart de la Faille (1974). They were mostly seen inareas with heavy lymphocytic infiltration and some-times in close contact with them (Fig. 1). Probablylymphocytes play a role either in the formation orthe final disposal of theses bodies. The presence ofdetectable immunoglobulins in only a minority ofthe inflammatory cells accords with the findings ofWalker (1976) that most of the inflammatory cells in

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574

LP are T-lymphocytes, with only a few B-lympho-cytes and macrophages.

In conclusion, immunological reactions involvingimmunoglobulins and lymphocytes seem to play an

important role in the sequence of events that lead tothe development of lichen planus lesions. These re-

actions can be demonstrated on ordinary paraffinsections by the PAP technique and could be of helpin the diagnosis of clinically and histologicallydoubtful lesions.

We thank Professors G. B. D. Scott and P. J.Scheuer for their valuable suggestions for the manu-script, Miss Jill Vernon for typing, and Mr PaulBates for photography.

References

Abell, E., Presbury, D. G. C.. Marks, R., and Ramnarain,D. (1975). The diagnostic significance of immuno-globulin and fibrin deposition in lichen planus. BritishJournal of Dermatology, 93, 17-24.

Abell, E. and Ramnarain, D. (1975). Epidermal antigensin lichen planus. British Joutrnal of Dermatology, 92,631-636.

Baart de la Faille-Kuyper, E. H. and Baart de laFaille, H. (1974). An immunofluorescence study oflichen planus. British Jouirnal of Dermatology, 90, 365-371.

Sami Shousha and John Svirbelv

Black, M. M. (1972). The pathogenesis of lichen planus.British Journal of Dermatology, 86, 302-305.

Black, M. M. and Wilson-Jones, E. (1972). The role ofthe epidermis in the histogenesis of lichen planus.Arch. Derm, 105, 81-86.

British Medical Journal (1974). Lichen planus someprogress. (Editorial). British Medical Journal, 2, 643-644.

Burns, J. (1975). Background staining and sensitivity ofthe unlabelled antibody-enzyme (PAP) method. Corn-parison with the peroxidase labelled antibody sand-wich method using formalin fixed paraffin embeddedmaterial. Histochemistry, 43, 291-294.

Marks, R., Black, M., and Wilson-Jones, E. (1973).Epidermal cell kinetics in lichen planus. British Journalof Dermatology, 88, 37-45.

Sarkany, I. and Gaylarde, P. M. (1971). Ultrastructuraland light microscopic changes of the epidermo-dermal junction. Trans. St. Johns Hosp. derm. Soc.(Lond.), 57, 139-142.

Ueki, H. (1969). Hyaline bodies in subepidermal papillae.Arch. Derm1, 100, 610-617.

Walker, D. M. (1976). Identification of subpopulationsof lymphocytes and macrophages in the infiltrate oflichen planus lesions of skin and oral mucosa. BritishJournal of Dermatology, 94, 529-534.

Welbourn, E., Champion, R. H., and Parish, W. E.(I1976). Hypersensitivity to bacteria in eczema. I.Bacterial culture, skin tests and immunofluorescentdetection of immunoglobulins and bacterial antigens.British Jouirnal of Dermatology, 94, 619-632.

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