In Psoriasis and Occurrence of Ψ-3 Antigen in Other Cutaneo
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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.