0022- 202X /85/8402-0l 00$02.00/ 0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 84 :100- 104 , 1985 Vol. 84, No.2 Copyright © 1985 by The Williams & Wilkins Co. Printed in U.S.A. Histologic Distribution of Staining by a Monoclonal Antibody ('1'-3) in Psoriasis and Occurrence of '1'-3 Antigen in Other Cutaneous Diseases

AARON M . 8TREFLING, M.D., A. MERRILL KNAPP, B.A., AND JONATHAN N. MANSBRIDGE, PH.D. Department of Dermatology, Stanford University School of M edicine, Stanford, California, and Psoriasis Research Institute, Palo Alto, California., U.S.A.

~-3 is a monoclonal antibody that recognizes a does not correlate with any single histologic characteristic. The 135,000 molecular weight structural component of ma­ antibody appears to define a unique feature in the keratinocyte turing keratinocytes in psoriasis (the 'lt-3 antigen) but response to certain pathologic conditions. It may, thus, be a fails to bind to any constituent of keratinocytes in nor­ valuable addition to the array of antibodies reacting with the mal epidermis. This paper describes the occurrence of different keratins [16- 19) and other structural components. the ~-3 antigen in a variety of dermatopathologic con­ The study described in this paper was undertaken to charac­ ditions using immunoperoxidase (biotin-avidin-peroxi­ terize the range of benign and malignant dermatologic condi­ dase) and immunofluorescence methods which show ex­ tions under which t he W-3 antigen is expressed. cellent concordance. In 35 of 36 specimens of psoriasis vulgaris, 'lt-3 anti­ body consistently immunolabels the cytoplasm of kerat­ MATERIALS AND METHODS inocytes above the basal layer. At the edges of psoriatic Patients admitted for psoriasis care and treatment of other cuta­ plaques, 'lt-3 antibody staining extends for a variable neous djseases to the Stanford University Hospital Outpatient Clinics distance into lesion-free epidermis. A similar pattern and the Palo Alto Veterans Administration Hospital Outpatient Clinics has been found in a certain number of other conditions comprised the patient population studied. The clinical criteria used to described in the paper, including squamous cell carci­ confirm the diagnosis of psoriasis included: (1) an established record noma and condyloma acuminatum, but not Darier's dis­ of cutaneous plaques clinically s uggestive of psoriasis; (2) bilateral ease, basal cell carcinoma, nor lamellar ichthyosis. In symmetry of lesions; (3) involvement of characteristic skin sites; (4) all but one condition, the outermost or basal layer of presence of accompanying nail changes; (5) family history of psoriasis. cells is never stained. The only disease in which the Specimens accepted from patients meeting the above clinical criteria lowermost cell layer is stained represented samples of plaques from many wfferent body locations. is a lichen planus-like Within 15 min after biopsy removal, each specimen was mounted in lesion. Tissue-Tek (Miles Laboratories, Naperville, Illinois) and slowly frozen The occurrence of ~-3 antigen cannot be correlated with Quik-Freeze (American Scientific Products, Sunnyvale, Cali for­ with any histologic feature of psoriasis such as acan­ nia). Fifty to 100 consecutive 4- to 6-,.,m sections of each specimen thosis, loss of the granular layer, or hyperproliferation. were cut. The frozen sections were appli ed to coated (0.5% gelatin and The antigen appears to be a unique keratinocyte con­ 0.05% KCr(SO,)z -1 2 H20) glass slides and stored at -7o·c. Consecu­ stituent which is expressed in certain pathologic condi­ tive 4- to 6-J.Lm sections were used to ensure hi stologic identity between tions and which is not detected by any other histologic controls and tested sections. The method of immunofluorescence has or immunophenotyping method. It is a potentially valu­ been described previously (15]. For immunoperoxidase staining by the avidin-biotin-peroxidase able addition to the panel of antibodies available for method [20- 24], the tissue sections selected for study and consecutive characterizing epithelial cells. control sections (previously stored at -70. C) were thawed, dried, fixed in cold acetone (-20. C) for 10 min and endogenous peroxidase-reduced by incubation with 0.3 % hydrogen peroxide. Sections were incubated With the development of antibodies capable of reacting spe­ successively with nonimmune serum (undiluted horse serum, GIBCO, cifically with a variety of cellular components, a widely appli­ Grand Island, New York) for 30 min, 60 nanonormal 'i'-3 antibody, cable immunophenotyping system, analogous to that available biotinylated goat ant1mouse lgM (Cappel Laboratories, Cochranville, for lymphocytes [1 -5], is developing [6-9]. The expression of Pennsylvania), 5 J.Lg/ml, and with aviwn-conjugated peroxidase (Vector intermediate filament classes has been found to be character­ Laboratories, Burlingame, California), 93 l'g/ml. All dilutions were istic of the tissue of origin of a tumor in a number of cases and made with 1% horse serum in phosphate-buffered saljne (PBS). All has been used for recognition of metastases [9- 11]. In t he case incubations were of 1-h length at room temperature in a humidjtied of the chamber. Two 15-min PBS washes followed each incubation. The lymphocyte immunophenotyping antibodies, it has been peroxidase reaction was developed in fresh ly made 3,3' -dimethylamino­ found that the use of a panel of antibodies provides a great deal benzidine (Sigma Chemical Company, St. Louis, Missouri), 1 mg/ml, more diagnostic information than the use of one or a small in 0.3 % hydrogen peroxide in PBS and filtered through a 0.2-J.Lm number [12- 14). Applying similar reasoning, it would be valu­ Millipore filter. After 15 min, the sections were washed thoroughly in able to extend the range of markers available for typing non­ distilled water and counterstained for 45 s in hematoxylin. F inally, all lymphoid tissues. sections were dehydrated in graded alcohols, cleared in xy lene, and The W-3 antibody, which was selected on the basis of its mounted in Permount. ability to stain psoriatic but not normal epidermis, binds to a The 'it-3 antibody was origi nally selected on its ability to stain p triton-insoluble component of keratinocytes in certain experi­ soriatic but not normal epidermis (immunofluorescence and immu­ noperoxidase techniques). It is an IgM. Supernatant from 'i'-3 hybri­ mental and dermatologic lesions [15] . The range of conditions doma cultures grown in 0.5 % horse serum, concentrated by ammonium sulfate precipitation, gave a single precipitin line with antimouse lgM Manuscript received June 14 , 1984; accepted for publication August in Ouchterlony double diffusion plates [25 ]. No lines were observed in 22, 1984. control wells containing horse serum alone. The hybridoma producing NIH Training Grant #AM07422-03 and the Psoriasis Research In­ 'it-3 antibody has been recloned 3 times and there has been no change stitute. detected in immunoreactivity among different lots. Reprint requests to: Aaron M. StreOing, M.D., Psoriasis Research In controls for the studies described below, we have found no staining Institute, P.O. Box V, Stanford, Cali fornia 94305. of normal epidermis in 16 specimens from nonpsoriatic individuals nor Abbreviations: in 10 biopsies taken at least 2 em away from psoriatic lesions. No IJt -3- PBS: phosphate-buffered saline like staining has been observed in experiments where psoriatic skin

100 Feb. 1985 '.lt-3 STAINING OF PSORIASIS AND OTHER CUTANEOUS DISEASES 101

has been treated with supernatants from hybridomas which did not basal layer staining in this condition could be accounted for by produce "'-3 antibody. melanin granules. This conclusion that \lt-3 does not stain the Tissue sections fixed in formalin, glutaraldehyde, or methanol could basal cells was confirmed by examination of companion sec­ not be stained by 'I1 ·3, demonstrating the instability of the "'-3 antigen tions stained by immunofluorescence methods (data not in these solvents. shown). RESULTS Squamous Cell Carcinoma Histopathologic Survey Fig li shows a well-differentiated squamous cell carcinoma and stained by Table I lists the cutaneous diseases surveyed with nests and prongs of abnormal squamous cells projecting monoclonal antibody. immunoperoxidase techniques with \lt-3 downward into the dermis. Several rounded nests have concen­ Psoriasis Vulgaris tric layers of parakeratosis at their cores (keratin pearls). \lt-3 antibody immunolabels most of the malignant squamous cells We have studied 36 patients with typical psoriatic plaques of except the outermost cell layer and the parakeratotic cores. In which 35 showed staining with w-3 antibody. The biopsy shown companion hematoxylin and eosin-stained sections, many of in Fig la-c was taken from the margin of a well-established the mitoses occur in the outermost layer of cells, but we have plaque of an untreated patient. Fig la-c demonstrates the found a number of mitotic figures in the inner layers of the pattern of immunoperoxidase staining with \lt-3 antibody squamous cell nests which are stained by 'li-3 antibody. within the lesion, at its margin, and 3 mm from the edge of the clinically visible plaque. Condyloma Acuminatum Within the plaque, \lt-3 antibody uniformly labels the cyto­ p lasm of the keratinocytes above the basal layer. Neither the One case has been studied to date, a patient who was a graft basal layer nor the stratum corneum is stained. A similar recipient receiving immunosuppressive therapy. Fig lj demon­ pattern is seen at the border of the lesion. In the adjacent strates a large connective tissue core surmounted by an epider­ nonacanthotic epidermis, \lt-3 antibody staining is present out mis which shows papillomatosis and regular acanthosis. The to the edge of the biopsy, at least 3 mm from the visible plaque. granular layer is focally preserved. 'li-3 antibody stains the However, the number of labeled keratinocytes decreases, stain­ keratinocytes above the basal layer, disappearing near the ing being lost first from the most superficial layers of the hyperkeratotic stratum corneum. epidermis until it remains only in keratinocytes immediately above the basal layer. DISCUSSION \lt-3 antigen (or a cross-reactive antigen) can be detected Lichen Planus-like Lesions both in established psoriatic lesions as large as plaques and The biopsy was taken from a flat, violaceous papule. The developing lesions as small as a 2-mm papule. It is also found section shown in Fig le,f displays hypergranulosis, acanthosis, in clinically "lesion-free" skin immediately adjacent to a plaque, a heavy mononuclear dermal infiltrate, and is clearly stained which histologically shows changes suggestive of impending by 'li-3 antibody. The pattern of 'li-3 antibody staining in this development of visible psoriasis. As a subcellular marker, \lt-3 condition is interesting in that it includes the epidermal cell antigen can thus be identified before there is clinical evidence layer adjacent to the dermis in some regions and shows peri­ or unequivocal histologic findings of psoriasis. nuclear distribution. The occurrence of \lt-3 antigen in other dermatologic condi­ tions, however, shows no correlation with any simple histo­ Acanthotic Epidermis Overlying Dermatofibroma pathologic feature or combination of features of psoriasis. In Fig lg,h shows a dermatofibroma occupying almost the whole particular, it is not uniformly associated either with loss of the ofthe dermis, overlayed by acanthotic epidermis. \lt-3 antibody granular layer or epidermal hyperproliferation, two major char­ immunolabels keratinocytes in the lower half of the epidermis acteristics of psoriasis [26]. In our lichen planus-like lesion and throughout the area of acanthosis (Fig. lg). This dermal neo­ condyloma acuminatum, for instance, ~-3 antibody staining is plasm is accompanied by minimal inflammatory infiltrate. The present together with the granular layer. With respect to ker­ basal cells are heavily pigmented and the dusty granular ap­ atinocyte growth rate, lamellar ichthyosis is generally hyper­ pearance of the melanin could resemble w-3 antibody staining. proliferative [27] but, in the case we examined, showed no w-3 Careful comparison of the immunoperoxidase-stained sections antibody staining. By contrast, lichen planus, which generally (Fig lg) with controls (Fig lh) indicated that, indeed, all the shows a proliferation rate which is less than normal [28], was stained by \lt-3 antibody. Expression of \lt -3 antigen by a cell, however, does not preclude mitosis. In squamous cell carci­ TABLE I. Occurrence of 1/;-3 antibody staining in cutaneous diseases by noma, we were able to detect mitoses within the region of cells the immunoperoxidase method stained with w-3 antibody. In addition, the intensity of staining 1/;·3 Cases by 'li-3 antibody shows no relationship to the density of inflam­ Psoriasis + 10/10 matory infiltrate. Although we have not obtained a \lt-3 anti­ Squamous cell carcinoma + 3/3 body stained specimen with no inflammation at all, the dermis Keratoacanthoma + 1/1 adjacent to the dermatofibroma described above shows minimal Condyloma acuminatum + 1/1 inflammation but the overlying epidermis is still stained by \lt- Irritated seborrheic keratosis + 1/ 1 3 a ntibody. In Darier's disease, we found no \lt-3 antibody Tape-stripped skin + 1/ 1 staining, but an appreciable infiltrate. Blister injury + 1/ 1 The staining of lesion-free psoriatic skin immediately adja­ Lichen planus-like lesion + 1/ 1 cent to a psoriatic plaque shows two patterns. In a pinpoint pilaris 1/ 1 Pityriasis rubra + lesion, staining diminishes rapidly with 0.5 mm of the visible Basal cell carcinoma 4/4 Actinic porokeratosis 1/1 margin of the lesion. In a well-established lesion, it persists for Nontraumatized normal epidermis 10/10 a number of millimeters beyond the lesion and is lost first from Lesion-free epidermis in psoriatics 6/6 the most superficial layers, remaining in the immediately su­ Epidermis overlying dermal lesions: prabasal layer for a considerable distance. The reason for the Dermatofibroma + 2/2 variation in these -It -3 antibody staining patterns is not known, Basal cell carcinoma + 4/4 but they are reminiscent of results obtained during wound Hemangioma + 1/ 1 healing experiments (unpublished results). Following epidermal 102 STREFLlNG, KNAPP, AND MANSBRIDGE Vol. 84, No.2 Feb. 1985 '11-3 STAINING OF PSORIASIS AND OTHER CUTANEOUS DISEASES 103 stripping, w-3 antibody staining 6. MukaiK, Rosai J: Application of immunoperoxidase techniques in injury, for instance by tape rgical Pathology, vo l 1. Edited of the stratum spinosum, surgical pathology, P rogress m Su appears within 24 h in all levels by CM Fenogli o, M Wolff. New York, Masson Publishi ng, 1980, reaches a peak at 48 h, and t hen disappears first from the most pp 15-49 superficial l ayers, finally leaving a s ingle s uprabasal l ayer 7. Battifora H, Sun T-T, Ba llU RM, Rao S: The use of antikeratin e t hat the abrupt disappear­ antiserum as a diagnostic tool. Hum Pathol 11:635-641 1980 stained. It is tempting to conclud , Farmer ER, S~a ll EA, he induc­ 8. Weiss RA, Guill et GYA, Freedberg IM ance of w-3 antige n in a pinpoi nt papule represents t We1ss MM, Sun T-T: T he use of monoclonal antibody to keratin tion of its expression in rapidly advancing lesions, while t he in human epidermal disease: alterations in immunohistochemical gradual loss from the more superficial regions of the spinous staining pattern. J Invest Dermatol 81 :224-230, 1983 markers: an over­ outside an establi shed plaque results from an attempt to 9. Osborn M: Intermediate filaments as histologic layer view. J Invest Dermatol 81 (suppl):104s- 107s, 1983 repress w- 3 antigen expression. 10. Osborn M, Weber K: Intermediate fi laments: cell-type-specific The pattern of w-3 antibody staining fou nd in suprabasal markers in different iation and pathology. Cell 31:303- 306, 1982 maturing cells is retained in keratinocyte-derived tumors. Squa­ 11 . Madri J, Barwick K: An immunohistochemical study of nasophar­ and show sparing of yngeal neoplasms using keratin antibodies: epithelial versus non­ mous cell carcinomas are immunolabeled Surg Pathol 6:143- 149, 1982 cell carcinomas are not epit hel ial neoplasms. Am J the outermost layer of cells. Basal 12. Wood G, Deneau D, Miller R, Levy R, Hoppe R, Warnke R: labeled, presumably because they never express a maturation Subtypes of cutaneous T -cell lymphoma defined by expression pathway involving '1'-3 antigen. of leu-1 and Ia. Blood 59:876-882, 1982 tion in which we have fo und W- 3 13. Wood G, Burke J, Horning S, Doggett R, Levy R, Warnke R: The The only pathologic condi neity of cutaneous is lichen planus. immunologic and clinicopathologic heteroge antige t, in the lowermost layer of the epidermis lymphomas other than mycosis fungoides. Blood 62:464- 472 The occurrence of -11 -3 antigen and, t herefore, presumably 1983 ' maturing keratinocytes in this region supports t he proposal 14. Li C-Y: Immunocytochemical techniques for identifying leukemias. olves a destruction of basal Mayo Clin Proc 59:185-188 1984 that the etiology of lichen planus inv idence for an alternative epithelial 15. Mansbridge J, Knapp A, Stre'tling A: Ev cells [29]. '1'-3 antibody staining in this case identifies pathway of keratinocyte maturation in psoriasis from an antigen cells expressing this keratinocyte maturation marker, migrating found 111 psona t1c but not normal epidermis. J Invest Dermatol to fill the area of basal cell destruction. 83:296- 301, 1984 a unique feature of t he 16. Sun T-T, Eichner R, Nelson WG, T seng SCG, Weiss RA, Jarvinen 'It -3 antibody appears to recognize lecular markers for ns which is not M, Woodcock-Mitchell J: Keratin classes: mo keratinocyte under certain pathologic conditio different types of epithelial differentiation. ,J Jnvest Dermatol detected by any other histologic o r immunophenotyping 81(suppl):109s- 115s, 1983 method. It is thu a potentially va luable addition to the anti­ 17. Viae J, Reano A, B rochi er J, Staquet MJ, Thivolet. J: Reactivity markers currently available for char­ pattern of monoclonal anti keratin ant ibody (KLJ ). J Invest bodies and morphologic 1983 [30], the AE Dermatol 81:351- 354, acterizi ng epitheli al t issues, e.g., antivimentin 18. La_ne EB,_ Klymkowsky M..Y: Epitheli al tonofi laments: investigat­ series [16], antibodies against basement membrane components mg the1r form and functiOn using monoc lonal antibodies. 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a, This !00% parakeratotic zo ne of a FIG 1. lmmunoperoxidase staining of selected dermatologic lesions by 'it -3 monoclonal ant ibody. disappea rs just beneath the psoriatic plaque shows uniform cytoplasmic staining of the maturing keratinocytes in the epidermis. "'-3 staining ary between t he c linically parakeratotic scale. The basal cell layer i s not stained (hematoxylin, X 100). b, T he approx imate hi stologic bound ical arrow. In thi s hi stologic visible edge of a psoriatic plaque and the immediately adjacent "clinically lesion-free skin " is demarcated by a vert n-free skin"). Concurrently, t here is border zo ne, the intensity of '11 -3 staining progressively diminishes from left to right (v isible plaque--> "lesio he midportion of t he stratum a lso a rapid loss of 'it -3 positivity beginning with the most superficial acanthocytes and progressing downward to t plaque, there is a patchy loss of the granular malpighii (hematoxylin, X 100). c, Three millimeters from the clinically visible edge of the psoriatic above the basal cell layer accompanied by mild acanthosis. "'-3 staining is now perceptible in only a few layers of keratinocytes immediately a psoriatic plaque (a). There is sparing layer (hematoxylin, X 100). d, '11 -3 staining of a pinpoint psori atic papul e (2 mm) is s imilar to that seen in lin, x 100). e and(, Li chen of the basal cell layer and rapid loss of '1'-3 positivity in t he immediately adjacent (0.5 mm) epidermis (hematoxy losis (top, e) and staining of the p lanus-like lesion. The pattern of '11 -3 staining is both cytopla mic and perinuclear. Note the zone of hypergranu and increased pigmentation lowermost cell layer of t he epidermis where active destruction is occurring (hematoxylin, x 290). g and h, Acanthosis '11 -antibody. "'-3 staining is of the basal ce ll layer induced by an underlying dermatofibroma. g, stained with 'l'-3 antibody; h, co ntrol without (compare control section present uniformly t hrough the lower half of t he t hickened stratum malpighii, but not in the pigmented basal cell layer 1 in the epidermis and downward h) (hematoxylin, x 100). i, A well-differentiated squamous ce ll carci noma show ing \f -3 immunolabeling j, representative frond from a infilt rating nests and prongs. The keratin pearls and t he outer cell layer are not stained (hematoxylin, x 100). A 1 parakeratotic surface and is condyloma acuminatum shows uniform cytoplasmi c staining by 'it -3 antibody. \) -3 staining diminishes near the absent in the basal cell layer (hematoxylin, X 100). 104 STREFLrNG, KNAPP, AND MANSBRIDGE Vol. 84, No.2 27. Rand R, Baden H: The icht hyoses: a review. J Am Acad Dermatol Dermatol 106:267- 273, 1982 8:285- 305, 1983 33. Oseroff AR, Roth R, Lipman S, Morhenn VB: Use of a murine 28. Ebner H, Gebhart W , Lassman H. Jurec ka W: The epidermal cell monoclonal antibody which binds to mali gnant kerat inocytes to proliferation in lichen planus. Acta Derm Venereol (Stockh) detect tumor cells in microscopically controlled surgery. J Axn 57:133- 136, 1977 Acad Dermatol 8:616-619, 1983 29. Marks R, Black M , Wilson J ones E: Epidermal cell kinetics in 34 . Murphy G, Flynn T, Rice R, Pinkus G: lnvolucrin expression in li chen planus. Br J D ermatol 88:37- 45, 1973 normal and neoplastic human skm: a marker for keratinocyte 30. Mahrle G, Bolling R, Osborn M, Weber K: Intermediate fil aments differentiation. Lab Invest 50:42A, 1984 of t he vi ment in and prekeratin type in human epidermis. J Invest 35. Watt FM: Involucrin and other markers of keatinocyte terminal Dermatol 81:46- 48, 1983 differentiation. J Invest Dermatol 81 (suppl):100s- 103s, 1983 31. de Moragas JM, Winkelmann RK, J ordon RE: Immunofluores­ 36. Fyrand 0: Studies on fib ronectin in t he skin. I. Indirect immuno­ cence of epit heli al skin tumors, II. Basement membrane. Cancer fluorescence studies in normal human skin. Br J Dermatol 25: 1404- 1407, 1970 101:263- 270, 1979 32. Weber F, Krieg T , Muller PK, Kirsch E, Timpl R: Imrnunof1u ­ 37. Fyrand 0: Studies o n fibronectin in the s kin. II. Indirect immu­ orescent localization of type IV coll agen and laminin in human nofluorescence studies in psoriasis vulgaris. Arch Dermatol R es skin and its a pplication in junctional zone pathology. Br J 266:33- 41, 1979