Demonstration of α1-antitrypsin and α1-antichymotrypsin in cutaneous histiocytic infiltrates and a comparison with intracellular lysozyme

Clinical a n d l a b o r a t o r v s t u d i e s Demonstration of a rantitrypsin and a l-antichymotrypsin in cutaneous histiocytic infiltrates and a comparison with intracellular lysozyme Francisco A. Kerdel, B.Sc., M . B . , E m y r W. Morgan, B.Sc., Colin A. Holden, B . S c . , M.B., M . R . C . P . , and Donald M. MacDonald, M . A . , M.B., M.R.C.P.

London, England otrAntitrypsin and acantichymotrypsin are intracellular enzymes found in cells of the monoeyte-histiocyte-macrophage series. Their presence in cutaneous infiltrates is demonstrated by employing a peroxidase-antiperoxidase technic on paraffin-embedded sections. A comparative study of the enzymes with intracellular lysozyme has been carried out to assess their individual efficacy as markers of histiocytes in cutaneous infiltrates. (J AM ACAD DERMATOt. 7:177-182, 1982.)

In b e n i g n skin conditions the variable morp h o l o g y o f histiocytes ~ renders their differentiation f r o m other mononuclear cells difficult. Of greater importance is the distinction between malignant histiocytic and lymphocytic tumors, which is often m a d e practically impossible in hematoxy l i n - e o s i n - s t a i n e d specimens by the similarity b e t w e e n poorly differentiated cells of either derivation. In a previous study," lysozyme was shown to be a reliable marker for cutaneous histiocytic infiltrates despite variable labeling of individual cells .:3,4 o~t-Antitrypsin, when demonstrated intracellularly, provides evidence of a histiocytic derivation, a-6 and recently in lymphoid tissue o~1From the Laboratoryof AppliedDermatopathology,Guy's Hospital. Accepted for ptzblicationNov. 20, 198t. Reprint requests to: Dr. D. M. MacDonald, Departmentof Dermatology, Guy's Hospital, St, Thomas' St., London. SE1 9RT, England.

0190-9622182/080177+06500.60/0 © 1982 Am Acad Dermatol

antichymotrypsin was localized in cells of the monocyte-histiocyte-macrophage series. 7 The purpose of this study is to determine the relative value of anti-al-antitrypsin and a n t i - a t antichymotrypsin to identify histiocytes in cutaneous infiltrates and to compare them with antilysozyme. MATERIALS AND METHODS

Cutaneous sections. Paraffin-embedded lesional cutaneous sections showing evidence of conditions listed in Table I were obtained from stored paraffin-embedded blocks after an examination of hematoxylineosin-stained sections had confirmed the histopathologic diagnosis. The skin disorders studied were selected on the basis of a presumed prominent histiocytic component. Antisera. Commercially available antisera were obtained from DAKO, Mercia Brocades, Ltd., Weybridge, England. The specificity of the antisera, which were mainly of the IgG class and which were obtained from animals immunized with pure antigen, was improved by liquid- or solid-phase absorption of un177

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oq-Antitl3~psin and ~-antichymotrypsin

wanted nonspecific antibodies. Cross-immunoelectrophoresis was performed as a further check of specificity (DAKO). 1. Rabbit antihuman o~l-antitrypsin antiserum was used in a dilution of 1 : 10 in 1 : 5 normal swine serum. 2. Rabbit antihuman cerantichymotrypsin antiserum was used in a dilution of 1:20 in 1:5 normal swine serum. 3. Rabbit antihuman lysozyme antiserum was used in a dilution of 1 : 20 in 1 : 5 normal swine serum. 4. Swine antirabbit antiserum was used in a dilution of 1 : 10 in 1 : 5 normal swine serum in conjunction with oq-antitrypsin studies and in a dilution of 1:20 in 1:5 normal swine serum with oqantichymotrypsin and lysozyme studies. 5. Rabbit peroxidase-antiperoxidase (PAP) complexes were used in a dilution of 1:100 in 1:5 normal swine serum. 6. Swine antimouse antiserum (Nordic Immunological Laboratory, London, England) was used in a dilution of 1:10 in phosphate-buffered saline (PBS) solution, 7. Normal rabbit serum was used in a dilution of 1:10 in PBS solution. Controls. 1. Primary antiserum was replaced by incubating with 1 : 10 normal rabbit serum. 2. Secondary antiserum was replaced by incubating with 1:10 swine antimouse antiserum. 3. Endogenous peroxidase activity was detected by incubating with 3,3'-diaminobenzidine and hydrogen peroxide alone. A modification of the basic method described by Mason and Taylor ~ was employed. Briefly,.serial 4-/x paraffin sections were mounted on separate glass slides.

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The sections were dewaxed, rehydrated, and incubated with 0.5% solution of hydrogen peroxide in methanol to block endogenous peroxidase activity. :~ Incubation with 0.1% trypsin at 37 ° C, 1°,1~ followed by incubation with 1:5 normal swine serum, was undertaken to reduce background staining, s,12 The sections were then incubated with the primary antiserum (rabbit antihuman a~-antitrypsin, or rabbit antihuman %-antichymotrypsin, or rabbit antihuman lysozyme) and diluted in 1:5 normal swine serum in order to further reduce background staining, t~ Following incubation with swine antirabbit immunoglobulin and PAP, the use of 3,3'-diaminobenzidine and hydrogen peroxide TM al/owed the localization of the horseradish peroxidase activity to be revealed. Finally, the lesions were counterstained with Mayer's hemalum for 45 seconds before mounting in DPX. The sections were then examined blind by two independent observers to compare the results obtained by using the three different primary antisera. The following criteria were specifically observed: 1. The intensity of staining was scored on a scale of 1+ to 3+. 2. The percentage of positively labeled ceils was estimated by counting the proportion of stained cells in ten equivalent high-power fields for each specific enzyme marker, and the average of the two independent observations was calculated. The morphology of the positive cells was recorded. RESULTS Results are s u m m a r i z e d in T a b l e I. Cells that labeled positively for ~ v a n t i t r y p s i n , oq-antichymotrypsin, and l y s o z y m e were seen to stain with a fine brown granularity evenly distributed throughout the cytoplasm (Fig. 1). T h e nuclei charac-

Fig. 1. A, al-Antitrypsin. The majority of cells composing the sarcoidal granuloma show positive cytoplasmic labeling of mild intensity. B, al-Antichymotrypsin. Universal positive labeling of moderate intensity of the constituent cells of granuloma. C, Lysozyme. Intense staining of cells of the granuloma. Some cells show absence of labeling. (Positive Kveim test immunoperoxidase counterstained with Mayer's hemalum; × 110.) Fig. 2. Granuloma annulare. The palisading granuloma comprises predominantly positively labeled cells. (al-Antichymotrypsin immunoperoxidase counterstained with Mayer's hemalum; ×50.) Fig. 3. Histiocytoma. The constituent cells show absence of labeling. Positively labeled histiocytes are present at the periphery of the lesion beneath the epidermis. (oqAntichymotrypsin immunoperoxidase counterstained with Mayer's hemalum; ×50.) Fig. 4. Lupus erythematosus. Sparse, positively labeled cells. (Lysozyme immunoperoxidase counterstained with Mayer's hemalum; × I I0.)

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Table I Intenslty/percentage of positive cells per 10 high-power

Condition

Sarcoidosis Positive Kveim test Lupus vulgaris South American leishmaniasis Granuloma annulare Mycosis fungoides Eczema Lupus erythematosus Acute lichen planus Histiocytoma

tof No. Cases studied

oq-Antitrypsin Intensity I Percentage

oq-Antichymotrypsin Intensity [ Percentage

4 3 3 3

+/+ + +/+ + + +/+ +

48 3l 58 20

+ + + +/+ + + + + + + + +

53 30 65 34

2 3 4 2 2 7

+ ++ + + + + Negative

30 26 34 22 Few

+ + + +/+ + + + + + + + + Negative

32 29 33 37 Few

teristically stained blue. The granulomas of cutaneous sareoidosis, positive Kveim tests, lupus vulgaris, South American leishmaniasis, and granuloma annulare (Fig. 2) consisted of numerous positively labeled histiocytes, epithelioid cells, and giant cells. The cellular components of histiocytoma were generally negative, but scattered positively labeled histiocytes were detected on the periphery of the lesions (Fig. 3). The epidermal infiltrate of mycosis fungoides comprised approximately 20% of positively labeled histiocytes, but no cells having the dendritic morphology of Langerhans cells were positively labeled. The remainder of the infiltrate was composed of unlabeled mononuclear cells presumed to be of lymphocytic origin. The number of positive bistiocytes in eczema varied between 10% and 40%. Sections from patients with lupus erythematosus were characterized by the presence of histiocytes among many unlabeled inflammatory cells (Fig. 4). Sections from patients with acute liche n planus were largely devoid of positive cells, howe ver. In most conditions studied, the labeling for ~1 antitrypsin, c~rantichymotrypsin, and lysozyme appeared to parallel each other; however, the intensity and the proportion of positively labeled cells in equivalent fields was different for each enzyme. Incubation with the different antisera also

fields Lysozyme Intensity

+ + + +

+ + + +

+ + + +

+ + + + + + + +/+ + + + +/+ + + ++ + Negative

Percentage

50 29 53 25 29 21 27 16 Few

produced a variable degree of nonspecific background staining. ~l-Antitrypsin. Sections incubated with anti%-antitrypsin labeled with an intensity of l + and on occasion 2 + (Fig. 1, A). On the sections where the labeling was less intense, the presence of moderate, nonspecific background staining made the localization of the enzyme sometimes difficult. Histiocytic cells were more consistently positive in mixed inflammatory conditions than in granulomatous conditions (Table I), where many negative epithelioid and giant cells could be identified. oz~-Antichymotrypsin Incubation with anti-oq-antichymotrypsin (Fig. 1, B) produced a 2 + intensity of labeling and allowed the identification of the highest percentage of positive cells (Table I). In particular, the proportion of positively labeled epithelioid cells and giant cells localized in granalomas was greater when this antiserum was employed. However, background staining, particularly in keratinocyte cytoplasm, rendered differentiation of epidermal histiocytic cells more difficult. Lysozyme The lowest proportion of positive cells was visualized using antilysozyme (Fig. 1, C) in spite of a 3+ intensity of labeling of individual histio-

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cytes. In the granulomatous conditions studied, the labeling was variable, with some epithelioid and giant cells showing weak or absent staining, which made their identification difficult even though background staining was minimal. DISCUSSION

Results indicate that the histiocytic nature of the mixed inflammatory and granulomatous infiltrates studied can readily be demonstrated immunohistochemically by employing antisera against a~antitrypsin, acantichymotrypsin, and lysozyme. In general the three antisera labeled a similar population of cells, but the results demonstrate minor differences in intensity and pattern of labeling. Although the high intensity of staining obtained with antilysozyme allowed easy identification of solitary histiocytes, the variable staining of epithelioid and giant cells was a problem. This inconsistency was not apparent when cells were incubated with anti-al-antichymotrypsin. Indeed, o~:antichymotrypsin was the best marker for dermal histiocytic infiltrates, but the presence of nonspecific background staining because of the primary antibody, especially in the epidermis, rnade the identification of histiocytes in this region particularly difficult. The moderate background staining found on incubating with anti-c~-antitrypsin made the localization of epidermal histiocytes possible and allowed their differentiation from Langerhans cells, which do not contain these enzymes. ~ However, its low intensity of labeling and a variability similar to lysozyme in labeling granulomas offset this advantage. The differences encountered in intensity, pattern, and background staining suggest that the use of a single antiserum may not be ideal for demonstrating histiocytes in cutaneous infiltrates. However, the use of antisera against all three intracellular enzymes employed on serial sections provides a complementary and highly accurate means of identifying cells of histiocytic derivation. The only condition of presumed histiocytic origin in which the three antisera failed to demonstrate positively labeled cells was histiocytoma, which supports other studies casting doubt on the histiocytic origin of these cells .'2,"~,t7

ce~-Antio3,psin alzd oq-antichymotl37)sin

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In the present study, anti- a rantitrypsin, anti- a ~antichymotrypsin, and antilysozyme have been shown to label histiocytic ceils in benign cutaneous infiltrates. Other investigators have demonstrated the value of anti-c~l-antitrypsin and antilysozyme in the characterization of systemic l y m p h o m a s ? -c~ The presence of al-antitrypsin has also been demonstrated in Reed-Sternberg cells, thus suggesting their possible histiocytic derivation.~'~s The differentiation of cutaneous histiocytic and B lymphocyte lymphomas with attendant prognostic and management implications may be greatly facilitated by the application of this technic employing the three different antisera. REFERENCES

1. Bonvalet D, Civatte J: Histiocytosis histopathology. G Ital Dermatol Venereol 115:51-58, 1980. 2. Kerdel FA, Morgan EW, MacDonald DM: lmmunohistochemical demonstration of lysozyme in cutaneous histiocytic infiltrates. Clin Exp Dermatol, July, 1982. 3. Isaacson P, Wright DH: Anomalous staining patterns in immunohistologie studies of malignant lymphoma. J Histochem Cytochem 27: t 197-1199, 1979. 4. Isaacson P, Wright DH, Judd MA, Mephan BL: Primary gastrointestinal lymphomas: A classification of 66 cases. Cancer 43:1805-1819, 1979. 5. Isaacson P, Wright DH, Judd MA, Jones DB, Payne SV: The nature of the immunoglobulin-eontaining cells in malignant iymphoma. J Histochem Cytochem 28:761770, 1980. 6. Taylor CR: Immunohistologic studies of lymphoma: Past, present and future. J Histochem Cytochem 28: 777-787, 1980. 7. Papadimitriou CS, Stein H, Papacharalampous NX: Presence of alpha- 1-antiehymotrypsin and alpha-l-antitrypsin in haematopoietic and lymphoid tissue cells as revealed by immunoperoxidase method. Pathol Res Pract 169:287-297, 1980. 8. Mason DY, Taylor CR: The distribution of muramidase (lysozyrne) in human tissue. J Clin Pathol 28:124-132, 1975. 9. Streefkerk JG: inhibition of erythroeyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J Histochem Cytochem 20:829-830, 1972. 10. Heyderman E: Immunoperoxidase technique in histopathology: Apparatus, methods and controls, J Clin Pathol 32:971-978, 1979. 11. Mason DY, Bibeffeld P: Technical aspects of lymphoma immunohistology. J Histoehem Cytochem 28:731-745, 1980. 12. Burns J: Background staining and sensitivity of the unlabeled antibody-enzyme (PAP) method: Comparison with the peroxidase labeled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43:291-294, 1975.

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13. Taylor CR: Immunoperoxidase technique: Practical and theoretical aspects. Arch Pathol Lab Med 102:113-121, 1978. 14. Graham RC, Kamovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidneys: Ultrastrvctural cytochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966. 15. Holden CA, Kerdel FA, Macdonald DM: Lack of lysozyme, eq-antitrypsin and ozl-antichymotrypsin in normal Langerhans cells: Differentiation from cells of

Journal of tile American Academy of Dermatology

the monocyte-histiocyte-macrophage series. Acta Derm Venereol (Stockh). (Submitted for publication.) 16. Burgdorf WH, Duray P, Rosai J: Immunohistochemical identification of lysozyme in cutaneous lesions of alleged histiocytic nature. Arch Dermatol 115: 1347, 1979. 17. Burgdorf WH, Moreland A: Immunohistochemical staining for lysozyme in dermatofibromas. Arch Dermatol 116:1392, 1980. 18. Poppema S: The diversity of the immunological staining pattern of Sternberg-Reed cells. J Histochem Cytochem 28:788-79 1, 1980.