- Email: [email protected]
In situ demonstration of OKT 6-positive cells in cutaneous lymphoid infiltrates
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In situ demonstration of OKT 6-positive cells in cutaneous lymphoid infiltrates Euan M. McMillan, M . B . , M . R . C . P . , Rose Wasik, H.T., and Mark Alien Everett, M.D.
Oklahoma City, OK A monoclonal antibody, OKT 6, has been developed against an antigen which is expressed by immature T ceils as part of their normal intrathymic differentiation, but not by mature peripheral T cells. It was thought that a search for the expression of such an antigen might be worthwhile in prelymphomatous conditions. This communication describes the investigation of lymphocytic infiltrates of atrophic parapsoriasis, lymphomatoid papulosis, and a small group of miscellaneous skin conditions with OKT 6 and the immunoperoxidase technic. OKT 6-positive cells were identified in the dermis in varying numbers in four cases of atrophic parapsoriasis and in one case of lymphomatoid papulosis, but not in any of the other disorders. Positive epidermal staining was noted in all tissues examined. The pattern obtained suggested that epidermal dendritic cells may react with OKT 6. The findings indicate that OKT 6-positive cells may be found outside the thymus in certain conditions. The observations in epidermis cast doubt on the exact nature of the positively reacting cells observed in dermis, suggesting they may either be immature thymocytes or possibly Langerhans cells. (J AM ACAD DERMATOL5:274-279, 1981.)
Parapsoriasis
is a term used to cover a heterogeneous group of skin conditions with characteristic clinical manifestations. ~ One variant, at least, may, in a significant proportion of cases, develop into clinical mycosis fungoides. 2 The T cell nature of mycosis fungoides has been established by E rosetting technics on cells extracted from cutaneous infiltrates a and by the staining of cells in situ using a rabbit antiserum specific for human T cells in conjunction with the immunoperoxidase technic. 4 The development of mycosis fungoides in a significant proportion of cases From the Departments of Dermatologyand Pathology,Universityof Oklahoma, Health Sciences Center, Oklahoma City, OK. No reprints available. 274
of large-plaque (atrophic) parapsoriasis ~ suggests a relationship between the two conditions, and this suggestion has been enhanced by the demonstration of T cell predominance in parapsoriasis by the immunoperoxidase technic at light microscopic 5 and ultrastructural levels. 6 A fundamental question concerning the relationship between parapsoriasis and mycosis fungoides is whether the disease which becomes clinical mycosis fungoides is biologically malignant in its cellular properties ab initio or whether a progressive change takes place in the T cell population over a considerable period of time. Either way, it would be desirable from the clinical standpoint if cellular properties indicative of malignancy could be identified at an early stage. 0190-9622/81/090274+06500.60/0 © 1981 Am Acad Dermatol
Volume 5 Number 3 September, 1981
Methods presently being used to attempt this inelude assessment for deoxyribonucleic acid (DNA) content of the cell population 7 and cytomorphometry. s Another possible method for the early detection of malignant properties of the cell population may be assessment of the membrane properties of individual cells, e.g., for the expression of immature differentiation antigens. During normal differentiation, thymocytes acquire certain antigens and lose others. 9 Less mature thymicderived cells might therefore be detected by their expression of cell surface antigens characteristic for a particular stage of maturation. The development of monoclonal antibodies to the antigens associated with distinctive stages of T cell maturation has made the detection of immature T cells feasible 1° and has led to the recent demonstration that human T cell leukemias consist predominantly of clones of immature T cells. The cells involved belonged to early stages of thymic differentiation. 10 Preliminary attempts at in situ analysis of cutaneous infiltrates in one case of mycosis fungoides u and in five cases of large-plaque atrophic parapsoriasis 12 indicated the predominant cell type to be a mature T cell, in contrast to the previously mentioned findings in human T cell leukemia. Similar studies involving larger numbers of cases obviously must be performed in these conditions to determine if the pattern mentioned is typical. In the studies mentioned, 11'12 although T cell predominance was demonstrated, the presence of negatively staining cells was observed in the cutaneous infiltrates examined. It was suggested that this might in part be due to the presence of cells of non-T-lineage, e.g., B cells, monocytes, or the presence of immature T cells which do not express mature T cell antigens. Recently, a monoclonal antibody (OKT 6) has been developed which is directed against normal thymocytes at an earlier stage of intrathymic differentiation. 9 The antigen detected by this antibody is apparently lost as thymocytes mature and become ready for release into the peripheral circulation.10 This raises the question of whether cells reacting positively with OKT 6 are ever found outside the thymus, in physiologic or pathologic conditions. The latter possibility was recently
O K T 6 cells in cutaneous infiltrates
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Table I. Results of examination of dermal infiltrates with O K T 6 diagnosis ....Case No. [ 1 2 3 4 5 6 7 8
Parapsoriasis
''["Result
Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Lymphomatoid papulosis Pityriasis lichenoides chronica
--+ --+ +++ +++ + + + +--
Miscellaneous disorders 1 2 3 4
Discoid lupus e r y t h e m a t o s u s Actinic keratosis Halo n e v u s Lichen planus
---_+
_-_: Occasional cell found with equivocal staining; + + + : positive membranous staining in 5% to 10%; +: occasional cell identified with positive membranous staining; - : negative reaction.
suggested by the finding of a small percentage of OKT 6-positive cells in the cutaneous infiltrates of mycosis fungoides.ll The relationship between certain categories of parapsoriasis and mycosis fungoides already mentioned suggested that it would be worthwhile to examine cutaneous infiltrates of parapsoriasis for the presence of OKT 6-positive cells. The communication describes investigation of cutaneous lymphoid infiltrates of parapsoriasis using OKT 6 with the indirect immunoperoxidase technic. Tissues from patients with various types of parapsoriasis 1 were studied (large-plaque atrophic, pityriasis lichenoides chronica, lymphomatoid papulosis). As a comparison, tissues from a variety of skin disorders with a nonmalignant lymphocytic infiltrate were also studied (discoid lupus erythematosus, actinic keratosis, lichen planus, and halo nevus).
MATERIALS AND METHODS The monoclonal antibody used in this study was OKT 6 Thy, which is directed against human "common thymocytes" (Ortho ImmunobioLogy Ltd., Raritan, NJ). The nature of the tissues studied is given in Table I. Tissues from patients with a variety of dermatologic
276
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M c M i l l a n et al
Fig. 1. Dermal lymphoid infiltrate of a case of atrophic parapsoriasis showing OKT 6-positive cells. The surrounding negatively staining areas were packed with lymphoid cells when stained with hematoxylin and eosin. (Acetone fixation, indirect immunoperoxidase; x 64.) Fig. 2. Same patient as Fig. 1. Identical technic but with paraformaldehyde fixation. Equivocal staining pattern of dermal infiltrate. (Indirect immunoperoxidase; × 64.) Fig. 3. Positive epidermal staining with OKT 6 in a case of atrophic parapsoriasis. In certain areas the staining appears to have a dendritic pattern. (Indirect immunoperoxidase; x64.) Pig. 4. Control (primary antibody omitted). (Indirect immunoperoxidase; x64.) disorders other than parapsoriasis were also studied. In all conditions studied, the clinical diagnosis was supported by histologic confirmation on routine hematoxylin and eosin-stainedparaffin-embedded sections. Punch biopsies (3 mm) were removed from clinically affected areas and snap-.frozen in liquid nitrogen
with quenching in 2-methyl butane. Tissues were then stored at - 7 0 ° C in a Revco until used. Cryostat sections (5/z) were treated as follows: 1. Fixation in acetone or 3% paraformaldehyde in phosphate-buffered saline (PBS) at 4° C for 15 minutes. 2. PBS rinse for 30 minutes.
Volume 5 Number 3 September, 1981 3. Primary antibody (OKT 6, Ortho Immunobiology Ltd.), diluted 1 in 10 for 30 minutes. 4. PBS rinse for 30 minutes. 5. Secondary antibody: Peroxidase-conjugated,goat antimouse IgG (Tago, Inc., Burlingame,CA), dilution 1 in 10 for 30 minutes. 6. PBS rinse for 20 minutes. 7. TRIS HC1 buffer (pH 7.6) rinse for 10 minutes. 8. Applicationofdiaminobenzidine(DAB), 10 mg per 10 ml, in TRIS HCI buffer for 15 minutes,j3 9. Quick rinse in PBS to remove excess DAB. 10. Darkening of reaction product with 1% osmium tetroxide for 1 minute. 11. PBS rinse for 20 minutes. 12. Progressionfrom distilled water to xylene. 13. Mounting in Permount (Fisher Scientific Co., Fair Lawn, NJ). Negative controls consisted of: (1) omission of primary antibody; (2) application of peroxidase-conjugated goat antirabbit IgG instead of peroxidase-conjugated goat antimouse IgG as second-stage antibody; (3) DAB alone. Tissue embedded in paraffin and stained with hematoxylin and eosin was examined in all cases to determine if the pathologic findings were consistent with the clinical diagnosis. RESULTS The results are illustrated in Table I. In the dermal infiltrates examined, OKT 6-positive cells could be identified in four of six cases of atrophic parapsoriasis. These formed a significant proportion of the population in two cases, with approximately 5% to 10% of the population showing positive membrane staining. The presence of OKT 6-positive cells in a case of atrophic parapsoriasis is shown in Fig. 1. In the remaining two cases of atrophic parapsoriasis and in pityriasis lichenoides chronica, only occasional cells with equivocal positive membranous staining were identified. In lymphomatoid papulosis, occasional large cells with positive membranous staining were present in the mononuclear infiltrate. The results obtained with 3% paraformaldehyde were more variable and were more prone to show an equivocal or negative staining pattern. An example of this is illustrated in Fig. 2 in which the results may be compared with those in Fig. 1. In the miscellaneous disorders listed, no cells with unequivocally positive membranous staining were identified in dermal infiltrates.
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Insufficient numbers of epidermal exocytic cells were present to make any definitive statements concerning these. However, positive staining of cells, which often appeared to have a dendritic pattern, was noted in the epidermis o f all tissues examined, whether affected by parapsoriasis or the miscellaneous disorders mentioned. This pattern was observed in areas affected and unaffected by lymphocytic exocytosis. An example of this epidermal staining pattern is illustrated from a case of atrophic parapsoriasis in Fig. 3. The results in dermis and epidermis of the negative controls (including omission of primary antibody) were negative (Fig. 4). The dendritic staining pattern observed was therefore not an effect mediated by nonspecific absorption o f secondary antibody to epidermal components. DISCUSSION The results obtained here indicate that OKT 6-positive cells may be found outside the thymus in certain pathologic conditions, OKT 6-positive cells being identified in the dermal infiltrates of atrophic parapsofiasis and lymphomatoid papulosis. The findings in atrophic parapsoriasis were, however, not uniform, with two patients exhibiting negative results, and the patients with positively staining cells in the dermis showing a varying quantity of these (Table I). The clinical heterogeneity of atrophic parapsoriasis, with a percentage of patients developing clinically overt mycosis fungoides, is well known. ~ Likewise, the development of mycosis fungoides from lesions diagnosed as lymphomatoid papulosis has been documented.14 These clinical observations, and the findings mentioned of OKT 6-positive cells in the dermal infiltrates of a proportion of patients with atrophic parapsoriasis and in one patient with lymphomatoid papulosis, make it tempting to postulate that these ceils represent immature T cells which might indicate biologic, if not clinically overt, malignancy. Two points against this contention should be raised. The number of miscellaneous skin disorders studied apart from parapsoriasis was small. A search for OKT 6-positive cells in the dermal infiltrates of other dermatoses where lymphoid malignancy does not normally supervene, e.g., contact dermatitis,
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McMillan et al
would be of interest. Of equal importance is the finding here of positive epidermal staining in parapsoriasis and the miscellaneous disorders studied. This was often noted to have a pattern which might be interpreted as dendritic, with long strands of peroxidase-positive material present between keratinocytes (Fig. 3). The fact that this was observed in a scattered, discrete pattern suggests staining of a subpopulation of cells other than keratinocytes, and the dendritic pattern observed suggests that epidermal dendritic cells may be staining positively with OKT 6. (Independent observations elsewhere, using the immunofluorescence technic, indicate OKT 6 positivity to be a property of Langerhans cells lZ.) This epidermal staining pattern could certainly be attributed to OKT 6 rather than to a nonspecific sticking of secondary antibody, as this pattern was not observed in negative controls in which OKT 6 was omitted, but secondary antibody applied with the same sequence of reagents. Furthermore, we have not observed this dendritic pattern with other monoclonal antibodies of the OKT series (OKT 3, 4, and 8), produced by Ortho Pharmaceuticals, Raritan, NJ, or with the Leu series (Leu 1, 2A, 3A), produced by Becton, Dickinson Facs Systems, Sunnyvale, CA. The possibility that this reaction observed between OKT 6 and epidermis is due to more than one specificity being present in the reagent, a problem often encountered with antisera raised by conventional technics, TM cannot be excluded, but it seems unlikely in view of the monoclonal nature of the antibody. This finding of OKT 6-positive components in epidermis is also of interest in view of the postulated relationship between thymus and epidermis. ~7 In view of the preceding findings, irnmunoelectron microscopic studies of epidermis and dermis using OKT 6 would obviously be of interest to determine the exact location and nature of these OKT 6-positive components. The superior results obtained with acetone fixation versus paraformaldehyde in the timing and concentrations used suggest that acetone may be a superior fixative for light microscopy in terms of preservation of this particular antigen. The findings here therefore illustrate one possible cause of variable results in the in situ identification of lym-
Journal of the American Academy of Dermatology
phocytes using antibodies directed against certain antigens, namely, choice of fixative. We are presently investigating this aspect of fixation on technical results obtained with monoclonal O K T 6 by examining the effect of various fixatives on the demonstration of OKT 6-positive cells in thymus. In a recent study of lymphoid infiltrates in largeplaque (atrophic) parapsoriasis with monoclonal antibodies directed against mature peripheral T cells and T cell subsets, ~'z it was suggested that one cause for negatively staining cells might be heterogeneity of the cell population, with the presence of cells of non-T-lineage or T cells of a less mature stage of differentiation which do not express mature differentiation antigens. The results obtained here indicate that the lymphoid infiltrates of parapsoriasis are heterogeneous with respect to their expression of T cell-associated antigens. The staining pattern obtained in the epidermis of various non-prelymphomatous conditions, as well as parapsoriasis, suggests that OKT 6 positivity cannot necessarily be equated with only immature T cells, but possibly also with dendritic cells (e.g., Langerhans cells?). This observation casts doubt on the exact nature of the OKT 6-positive cells found in the dermal infiltrates of large-plaque (atrophic) parapsoriasis and lymphomatoid papulosis. In other words, are these cells lymphocytes, Langerhans cells, or some other cell type expressing T-associated antigens? The problem then also arises of what significance these cells have in terms of progression to clinically manifest lymphoma, whatever their lineage happens to be. Answers to questions of this kind can be obtained only by further examination of benign and cutaneous lymphoid infiltrates, with possible sequential analysis in individual patients, as well as a more detailed analysis of the cell types involved by immunoelectron microscopy. We especially thank Dr. David Mason, Department of Hematology, John Radcliffe Infirmary, Oxford, England, who suggested the use of acetone as a fixative.
REFERENCES 1. Everett MA, Headington JT: "Parapsoriasis." J C E Dermatol, pp. 12-24, 1978. 2. Samman PD: The natural history of parapsoriasis en
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3. 4. 5.
6. 7.
8.
9. 10.
plaque (chronic superficial dermatitis) and pre-reticulotic poikiloderma. Br J Dermatol 87:405-411, 1972. Edelson RL, Smith RW, Frank MM, Green I: Identification of subpopulations of mononuclear cells in cutaneous infiltrates. J Invest Dermatol 61:82-89, 1973. Chu AC, MacDonald DM: Identification in situ of T lymphocytes in dermal and epidermal infiltrates of mycosis fungoides. Br J Dermatol 100:177-189, 1979. McMillan EM, Wasik R, Martin D, Everett MA: Identification of " T " cells in parapsoriasis infiltrates using an anti-human " T " cell serum and the immunoperoxidase technique. Arch Dermatol. (In press.) McMillan EM, Wasik R, Martin D, Everett MA: lmmuno-electron microscopy of " T " cells in large plaque parapsoriasis. J Cutan Pathol. (In press.) Van Vloten WA, Van Duijn P, Schaberg A: Cytodiagnostic use of Feulgen-DNA measurements in cell imprints from the skin of patients with mycosis fungoides. Br J Dermatol 91:365-371, 1974. Meijer CJLM, Vanderloo EM, Van Vloten WA, Vander Velde EA, Scheffer E, Cornelisse CJ: Early diagnosis of mycosis fungoides and S6zary's syndrome by morphometric analysis of lymphoid cells in the skin. Cancer 45:2864-2871, 1980. Reinherz EL, Schlossman SF: Regulation of the immune response-inducer and suppressor T lymphocyte subsets in human beings. N Engl J Med 303:370-373, 1980. Reinherz EL, Kung PC, Golstein G, Levy RH, Schloss-
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11.
12. 13.
14. 15.
16. 17.
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man SF: Discrete stages of intrathymic differentiation: Analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage. Proc Natl Acad Sci USA 77: 1588-1592, 1980. McMillan EM, Martin D, Wasik R, Everett MA: In situ demonstration of "T" cells and "T" cell subsets in mycosis fungoides using monoclonal antibodies. (Submitted for publication.) McMillan EM, Wasik R, Everett MA: In situ demonstration of "T" cell subsets in atrophic parapsoriasis. J AM ACAD DERMATOL.(In press.) Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubule of the mouse kidney. Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291302, 1966. Fine RM, Meltzer HD, Rudner EJ: Lymphomatoid papulosis eventuating in mycosis fungoides. South Med J 67:1492-1497, 1974. Fithian E, Kung P, Goldstein G, Rubenfeld M, Fenoglio C, Edelson R: Reactivity of Langerhans cells with hybridoma antibody. Proc Natl Acad Sci USA 78:25412544, 1981. Heyderman E: Immunoperoxidase technique in histopathology: Applications, methods and controls. I Clin Pathol 32:971-978, 1979. Safai B, Good RA: Lymphoproliferative disorders of the T cell series. Medicine 59:339, 1980.
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In situ demonstration of OKT 6-positive cells in cutaneous lymphoid infiltrates Euan M. McMillan, M . B . , M . R . C . P . , Rose Wasik, H.T., and Mark Alien Everett, M.D.
Oklahoma City, OK A monoclonal antibody, OKT 6, has been developed against an antigen which is expressed by immature T ceils as part of their normal intrathymic differentiation, but not by mature peripheral T cells. It was thought that a search for the expression of such an antigen might be worthwhile in prelymphomatous conditions. This communication describes the investigation of lymphocytic infiltrates of atrophic parapsoriasis, lymphomatoid papulosis, and a small group of miscellaneous skin conditions with OKT 6 and the immunoperoxidase technic. OKT 6-positive cells were identified in the dermis in varying numbers in four cases of atrophic parapsoriasis and in one case of lymphomatoid papulosis, but not in any of the other disorders. Positive epidermal staining was noted in all tissues examined. The pattern obtained suggested that epidermal dendritic cells may react with OKT 6. The findings indicate that OKT 6-positive cells may be found outside the thymus in certain conditions. The observations in epidermis cast doubt on the exact nature of the positively reacting cells observed in dermis, suggesting they may either be immature thymocytes or possibly Langerhans cells. (J AM ACAD DERMATOL5:274-279, 1981.)
Parapsoriasis
is a term used to cover a heterogeneous group of skin conditions with characteristic clinical manifestations. ~ One variant, at least, may, in a significant proportion of cases, develop into clinical mycosis fungoides. 2 The T cell nature of mycosis fungoides has been established by E rosetting technics on cells extracted from cutaneous infiltrates a and by the staining of cells in situ using a rabbit antiserum specific for human T cells in conjunction with the immunoperoxidase technic. 4 The development of mycosis fungoides in a significant proportion of cases From the Departments of Dermatologyand Pathology,Universityof Oklahoma, Health Sciences Center, Oklahoma City, OK. No reprints available. 274
of large-plaque (atrophic) parapsoriasis ~ suggests a relationship between the two conditions, and this suggestion has been enhanced by the demonstration of T cell predominance in parapsoriasis by the immunoperoxidase technic at light microscopic 5 and ultrastructural levels. 6 A fundamental question concerning the relationship between parapsoriasis and mycosis fungoides is whether the disease which becomes clinical mycosis fungoides is biologically malignant in its cellular properties ab initio or whether a progressive change takes place in the T cell population over a considerable period of time. Either way, it would be desirable from the clinical standpoint if cellular properties indicative of malignancy could be identified at an early stage. 0190-9622/81/090274+06500.60/0 © 1981 Am Acad Dermatol
Volume 5 Number 3 September, 1981
Methods presently being used to attempt this inelude assessment for deoxyribonucleic acid (DNA) content of the cell population 7 and cytomorphometry. s Another possible method for the early detection of malignant properties of the cell population may be assessment of the membrane properties of individual cells, e.g., for the expression of immature differentiation antigens. During normal differentiation, thymocytes acquire certain antigens and lose others. 9 Less mature thymicderived cells might therefore be detected by their expression of cell surface antigens characteristic for a particular stage of maturation. The development of monoclonal antibodies to the antigens associated with distinctive stages of T cell maturation has made the detection of immature T cells feasible 1° and has led to the recent demonstration that human T cell leukemias consist predominantly of clones of immature T cells. The cells involved belonged to early stages of thymic differentiation. 10 Preliminary attempts at in situ analysis of cutaneous infiltrates in one case of mycosis fungoides u and in five cases of large-plaque atrophic parapsoriasis 12 indicated the predominant cell type to be a mature T cell, in contrast to the previously mentioned findings in human T cell leukemia. Similar studies involving larger numbers of cases obviously must be performed in these conditions to determine if the pattern mentioned is typical. In the studies mentioned, 11'12 although T cell predominance was demonstrated, the presence of negatively staining cells was observed in the cutaneous infiltrates examined. It was suggested that this might in part be due to the presence of cells of non-T-lineage, e.g., B cells, monocytes, or the presence of immature T cells which do not express mature T cell antigens. Recently, a monoclonal antibody (OKT 6) has been developed which is directed against normal thymocytes at an earlier stage of intrathymic differentiation. 9 The antigen detected by this antibody is apparently lost as thymocytes mature and become ready for release into the peripheral circulation.10 This raises the question of whether cells reacting positively with OKT 6 are ever found outside the thymus, in physiologic or pathologic conditions. The latter possibility was recently
O K T 6 cells in cutaneous infiltrates
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Table I. Results of examination of dermal infiltrates with O K T 6 diagnosis ....Case No. [ 1 2 3 4 5 6 7 8
Parapsoriasis
''["Result
Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Large plaque (atrophic) Lymphomatoid papulosis Pityriasis lichenoides chronica
--+ --+ +++ +++ + + + +--
Miscellaneous disorders 1 2 3 4
Discoid lupus e r y t h e m a t o s u s Actinic keratosis Halo n e v u s Lichen planus
---_+
_-_: Occasional cell found with equivocal staining; + + + : positive membranous staining in 5% to 10%; +: occasional cell identified with positive membranous staining; - : negative reaction.
suggested by the finding of a small percentage of OKT 6-positive cells in the cutaneous infiltrates of mycosis fungoides.ll The relationship between certain categories of parapsoriasis and mycosis fungoides already mentioned suggested that it would be worthwhile to examine cutaneous infiltrates of parapsoriasis for the presence of OKT 6-positive cells. The communication describes investigation of cutaneous lymphoid infiltrates of parapsoriasis using OKT 6 with the indirect immunoperoxidase technic. Tissues from patients with various types of parapsoriasis 1 were studied (large-plaque atrophic, pityriasis lichenoides chronica, lymphomatoid papulosis). As a comparison, tissues from a variety of skin disorders with a nonmalignant lymphocytic infiltrate were also studied (discoid lupus erythematosus, actinic keratosis, lichen planus, and halo nevus).
MATERIALS AND METHODS The monoclonal antibody used in this study was OKT 6 Thy, which is directed against human "common thymocytes" (Ortho ImmunobioLogy Ltd., Raritan, NJ). The nature of the tissues studied is given in Table I. Tissues from patients with a variety of dermatologic
276
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M c M i l l a n et al
Fig. 1. Dermal lymphoid infiltrate of a case of atrophic parapsoriasis showing OKT 6-positive cells. The surrounding negatively staining areas were packed with lymphoid cells when stained with hematoxylin and eosin. (Acetone fixation, indirect immunoperoxidase; x 64.) Fig. 2. Same patient as Fig. 1. Identical technic but with paraformaldehyde fixation. Equivocal staining pattern of dermal infiltrate. (Indirect immunoperoxidase; × 64.) Fig. 3. Positive epidermal staining with OKT 6 in a case of atrophic parapsoriasis. In certain areas the staining appears to have a dendritic pattern. (Indirect immunoperoxidase; x64.) Pig. 4. Control (primary antibody omitted). (Indirect immunoperoxidase; x64.) disorders other than parapsoriasis were also studied. In all conditions studied, the clinical diagnosis was supported by histologic confirmation on routine hematoxylin and eosin-stainedparaffin-embedded sections. Punch biopsies (3 mm) were removed from clinically affected areas and snap-.frozen in liquid nitrogen
with quenching in 2-methyl butane. Tissues were then stored at - 7 0 ° C in a Revco until used. Cryostat sections (5/z) were treated as follows: 1. Fixation in acetone or 3% paraformaldehyde in phosphate-buffered saline (PBS) at 4° C for 15 minutes. 2. PBS rinse for 30 minutes.
Volume 5 Number 3 September, 1981 3. Primary antibody (OKT 6, Ortho Immunobiology Ltd.), diluted 1 in 10 for 30 minutes. 4. PBS rinse for 30 minutes. 5. Secondary antibody: Peroxidase-conjugated,goat antimouse IgG (Tago, Inc., Burlingame,CA), dilution 1 in 10 for 30 minutes. 6. PBS rinse for 20 minutes. 7. TRIS HC1 buffer (pH 7.6) rinse for 10 minutes. 8. Applicationofdiaminobenzidine(DAB), 10 mg per 10 ml, in TRIS HCI buffer for 15 minutes,j3 9. Quick rinse in PBS to remove excess DAB. 10. Darkening of reaction product with 1% osmium tetroxide for 1 minute. 11. PBS rinse for 20 minutes. 12. Progressionfrom distilled water to xylene. 13. Mounting in Permount (Fisher Scientific Co., Fair Lawn, NJ). Negative controls consisted of: (1) omission of primary antibody; (2) application of peroxidase-conjugated goat antirabbit IgG instead of peroxidase-conjugated goat antimouse IgG as second-stage antibody; (3) DAB alone. Tissue embedded in paraffin and stained with hematoxylin and eosin was examined in all cases to determine if the pathologic findings were consistent with the clinical diagnosis. RESULTS The results are illustrated in Table I. In the dermal infiltrates examined, OKT 6-positive cells could be identified in four of six cases of atrophic parapsoriasis. These formed a significant proportion of the population in two cases, with approximately 5% to 10% of the population showing positive membrane staining. The presence of OKT 6-positive cells in a case of atrophic parapsoriasis is shown in Fig. 1. In the remaining two cases of atrophic parapsoriasis and in pityriasis lichenoides chronica, only occasional cells with equivocal positive membranous staining were identified. In lymphomatoid papulosis, occasional large cells with positive membranous staining were present in the mononuclear infiltrate. The results obtained with 3% paraformaldehyde were more variable and were more prone to show an equivocal or negative staining pattern. An example of this is illustrated in Fig. 2 in which the results may be compared with those in Fig. 1. In the miscellaneous disorders listed, no cells with unequivocally positive membranous staining were identified in dermal infiltrates.
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Insufficient numbers of epidermal exocytic cells were present to make any definitive statements concerning these. However, positive staining of cells, which often appeared to have a dendritic pattern, was noted in the epidermis o f all tissues examined, whether affected by parapsoriasis or the miscellaneous disorders mentioned. This pattern was observed in areas affected and unaffected by lymphocytic exocytosis. An example of this epidermal staining pattern is illustrated from a case of atrophic parapsoriasis in Fig. 3. The results in dermis and epidermis of the negative controls (including omission of primary antibody) were negative (Fig. 4). The dendritic staining pattern observed was therefore not an effect mediated by nonspecific absorption o f secondary antibody to epidermal components. DISCUSSION The results obtained here indicate that OKT 6-positive cells may be found outside the thymus in certain pathologic conditions, OKT 6-positive cells being identified in the dermal infiltrates of atrophic parapsofiasis and lymphomatoid papulosis. The findings in atrophic parapsoriasis were, however, not uniform, with two patients exhibiting negative results, and the patients with positively staining cells in the dermis showing a varying quantity of these (Table I). The clinical heterogeneity of atrophic parapsoriasis, with a percentage of patients developing clinically overt mycosis fungoides, is well known. ~ Likewise, the development of mycosis fungoides from lesions diagnosed as lymphomatoid papulosis has been documented.14 These clinical observations, and the findings mentioned of OKT 6-positive cells in the dermal infiltrates of a proportion of patients with atrophic parapsoriasis and in one patient with lymphomatoid papulosis, make it tempting to postulate that these ceils represent immature T cells which might indicate biologic, if not clinically overt, malignancy. Two points against this contention should be raised. The number of miscellaneous skin disorders studied apart from parapsoriasis was small. A search for OKT 6-positive cells in the dermal infiltrates of other dermatoses where lymphoid malignancy does not normally supervene, e.g., contact dermatitis,
278
McMillan et al
would be of interest. Of equal importance is the finding here of positive epidermal staining in parapsoriasis and the miscellaneous disorders studied. This was often noted to have a pattern which might be interpreted as dendritic, with long strands of peroxidase-positive material present between keratinocytes (Fig. 3). The fact that this was observed in a scattered, discrete pattern suggests staining of a subpopulation of cells other than keratinocytes, and the dendritic pattern observed suggests that epidermal dendritic cells may be staining positively with OKT 6. (Independent observations elsewhere, using the immunofluorescence technic, indicate OKT 6 positivity to be a property of Langerhans cells lZ.) This epidermal staining pattern could certainly be attributed to OKT 6 rather than to a nonspecific sticking of secondary antibody, as this pattern was not observed in negative controls in which OKT 6 was omitted, but secondary antibody applied with the same sequence of reagents. Furthermore, we have not observed this dendritic pattern with other monoclonal antibodies of the OKT series (OKT 3, 4, and 8), produced by Ortho Pharmaceuticals, Raritan, NJ, or with the Leu series (Leu 1, 2A, 3A), produced by Becton, Dickinson Facs Systems, Sunnyvale, CA. The possibility that this reaction observed between OKT 6 and epidermis is due to more than one specificity being present in the reagent, a problem often encountered with antisera raised by conventional technics, TM cannot be excluded, but it seems unlikely in view of the monoclonal nature of the antibody. This finding of OKT 6-positive components in epidermis is also of interest in view of the postulated relationship between thymus and epidermis. ~7 In view of the preceding findings, irnmunoelectron microscopic studies of epidermis and dermis using OKT 6 would obviously be of interest to determine the exact location and nature of these OKT 6-positive components. The superior results obtained with acetone fixation versus paraformaldehyde in the timing and concentrations used suggest that acetone may be a superior fixative for light microscopy in terms of preservation of this particular antigen. The findings here therefore illustrate one possible cause of variable results in the in situ identification of lym-
Journal of the American Academy of Dermatology
phocytes using antibodies directed against certain antigens, namely, choice of fixative. We are presently investigating this aspect of fixation on technical results obtained with monoclonal O K T 6 by examining the effect of various fixatives on the demonstration of OKT 6-positive cells in thymus. In a recent study of lymphoid infiltrates in largeplaque (atrophic) parapsoriasis with monoclonal antibodies directed against mature peripheral T cells and T cell subsets, ~'z it was suggested that one cause for negatively staining cells might be heterogeneity of the cell population, with the presence of cells of non-T-lineage or T cells of a less mature stage of differentiation which do not express mature differentiation antigens. The results obtained here indicate that the lymphoid infiltrates of parapsoriasis are heterogeneous with respect to their expression of T cell-associated antigens. The staining pattern obtained in the epidermis of various non-prelymphomatous conditions, as well as parapsoriasis, suggests that OKT 6 positivity cannot necessarily be equated with only immature T cells, but possibly also with dendritic cells (e.g., Langerhans cells?). This observation casts doubt on the exact nature of the OKT 6-positive cells found in the dermal infiltrates of large-plaque (atrophic) parapsoriasis and lymphomatoid papulosis. In other words, are these cells lymphocytes, Langerhans cells, or some other cell type expressing T-associated antigens? The problem then also arises of what significance these cells have in terms of progression to clinically manifest lymphoma, whatever their lineage happens to be. Answers to questions of this kind can be obtained only by further examination of benign and cutaneous lymphoid infiltrates, with possible sequential analysis in individual patients, as well as a more detailed analysis of the cell types involved by immunoelectron microscopy. We especially thank Dr. David Mason, Department of Hematology, John Radcliffe Infirmary, Oxford, England, who suggested the use of acetone as a fixative.
REFERENCES 1. Everett MA, Headington JT: "Parapsoriasis." J C E Dermatol, pp. 12-24, 1978. 2. Samman PD: The natural history of parapsoriasis en
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plaque (chronic superficial dermatitis) and pre-reticulotic poikiloderma. Br J Dermatol 87:405-411, 1972. Edelson RL, Smith RW, Frank MM, Green I: Identification of subpopulations of mononuclear cells in cutaneous infiltrates. J Invest Dermatol 61:82-89, 1973. Chu AC, MacDonald DM: Identification in situ of T lymphocytes in dermal and epidermal infiltrates of mycosis fungoides. Br J Dermatol 100:177-189, 1979. McMillan EM, Wasik R, Martin D, Everett MA: Identification of " T " cells in parapsoriasis infiltrates using an anti-human " T " cell serum and the immunoperoxidase technique. Arch Dermatol. (In press.) McMillan EM, Wasik R, Martin D, Everett MA: lmmuno-electron microscopy of " T " cells in large plaque parapsoriasis. J Cutan Pathol. (In press.) Van Vloten WA, Van Duijn P, Schaberg A: Cytodiagnostic use of Feulgen-DNA measurements in cell imprints from the skin of patients with mycosis fungoides. Br J Dermatol 91:365-371, 1974. Meijer CJLM, Vanderloo EM, Van Vloten WA, Vander Velde EA, Scheffer E, Cornelisse CJ: Early diagnosis of mycosis fungoides and S6zary's syndrome by morphometric analysis of lymphoid cells in the skin. Cancer 45:2864-2871, 1980. Reinherz EL, Schlossman SF: Regulation of the immune response-inducer and suppressor T lymphocyte subsets in human beings. N Engl J Med 303:370-373, 1980. Reinherz EL, Kung PC, Golstein G, Levy RH, Schloss-
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