J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

J Clin Pathol 1982;35:327-337

Combined immunological and histochemical analysis of skin and lesions in X

JA THOMAS,* G JANOSSY,* M CHILOSI,t J PRITCHARD,: JR PINCOTT§ From the *Department ofImmunology, Royal Free Hospital School ofMedicine, London NW3, the tInstitute ofHistopathology, University ofPadua, Verona, Italy, and the Departments oftHaematology and Oncology §Pathology, Hospitaljbr Sick Children, Great Ormond Street, London WCI

SUMMARY The immunological phenotype of the cells involved in skin and lymph node lesions from two cases of histiocytosis X (H-X) were analysed by immunofluorescence techniques using combi- nations of heterologous and monoclonal antisera to Ia-like and human cortical (HTA-1) determinant. These cells were also characterised by a new technique using simultaneous immunofluorescence and enzyme histochemistry for acid phosphatase (ACPase). The major type in the lesions was found to express the same Ia+, HTA-l+ phenotype as normalepidermalLangerhans' cells (LC) and was unreactive for ACPase. Additional cell types included Ia-, HTA-1- multinucleate giant cells and residual lymphoid populations These findings endorse previous concepts that H-X is a proliferation of abnormal LC and emphasise the heterogeneous nature of the cells involved in the disease.

Histiocytosis X (H-X) is a generic term encompassing In addition to routine histological staining a group of rare clinical disorders (Hand-Schuller- procedures, enzyme histochemistry has been used for Christian syndrome, Letterer-Siwe disease and many years to identify further various cell types in eosinophilic ) which show similar histo- histological preparations. Hitherto, immunological http://jcp.bmj.com/ logical features. The lesions in these diseases contain and histochemical techniques have been confined to granulomatous infiltrates of histiocytic cells with separate tissue sections as the optimal tissue process- abundant eosinophilic cytoplasm.' There is now ing for each method was considered to be different extensive evidence to show that these cells are and incompatible. In this study, combined immuno- morphologically and ultrastructurally similar to logical and histochemical markers have been used to epidermal Langerhans' cells (LC). Both cell types identify the proliferating cells in cutaneous and contain ultrastructurally distinct cytoplasmic bodies lymph node lesions from two cases ofhistiocytosis X. on October 1, 2021 by guest. Protected copyright. of Birbeck granules2 3 and are histochemically active The aims of the study were threefold. Combinations for adenosine triphosphtase (ATPase)4-5 and a- of heterologous and monoclonal antibodies to napthyl acetate esterase (ANAE).4-6 In addition, LC lymphoid and non-lymphoid cells were used in weakly express surface Fc IgG and C3 receptors7 double-labelled immunofluorescence (IF) studies on and contain abundant amounts of Ia-like antigen.8 frozen sections (i) to establish whether there is a Recent immunological studies have revealed surface phenotypic relation between HTA-1+, Ia+ LC and receptors for HTA-1 (human thymocyte antigen) on H-X cells, (ii) to characterise the relation between LC defined by monoclonal antibodies NA1/349 and H-X cells and other cell types and (iii) to compare the OKT6.'01' The expression of HTA-1 and Ia-like histochemical characteristics of different cells with antigen on LC provides an important new phenotype their immunological phenotype. (HTA-1+, Ia+) by which these cells may be identified in immunohistological analyses of normal and Material'and'methods pathological tissues. It is possible to identify the phenotypic expression of different immunological TISSUE PREPARATION markers on the same cell type using two colour A portion of each tissue biopsy (skin from case 1; immunofluorescence techniques.12 lymph node from case 2) was processed for routine Accepted for publication 21 September 1981 (haematoxylin and eosin), by 10 % formalin 327 J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

328 Thomas, Janossy, Chilosi, Pritchard, Pincott fixation, and paraffin wax embedding. The other specific rhodamine (tetramethyl rhodamine-isothio- portions to be used in the immunological and cyanate: TRITC) or fluorescein (fluorescein-isothio- histochemical studies were snap-frozen in isopentane cyanate: FITC) labelled second layer reagents were (2-methyl-butane: BDH) and liquid nitrogen and applied to the sections for a further 30 min. After stored at - 700C. Cryostat sections (6 p,m and 10 ,um) final rinsing in PBS pH 7-6, the sections were were mounted on to clean glass slides, air-dried for mounted in 1 % formol glycerol under a coverslip 12 h at room temperature (20°C) before fixation in and examined with a x 40 oil objective on a Zeiss cold ethanol (4°C) for 3 min. Unfixed cryostat 14 epifluorescence IV/Z microscope. Two distinctly sections were also successfully stored at - 20°C labelled cell populations could be examined simul- before fixation. taneously using a selective (red/green) filter attach- ment. ANTISERA The panel of heterologous and monoclonal antisera HISTOCHEMICAL TECHNIQUES are shown in Table 1. The specificities of these Enzyme histochemistry was performed on 10 ,um reagents have been previously characterised in cell cryostat sections prefixed in cold (4°C) calcium suspension and frozen sections of normal1213 and formalin for 3 min. Modifications of standard pathological12 tissues. Combinations of indirectly histochemical methods described by Chilosi et all5 labelled heterologous antisera to Ia-like antigen were used to demonstrate activity for ACPase, (chicken (C) anti-Ia), human T leucocyte antigen ANAE, ATPase and tartrate-resistant acid phos- (rabbit (R)anti-HuTLA)and directlylabelled antisera phatase (TRAP). to human immunoglobulin subclasses were used to identify B (Ia+, Ig+) and T (HuTLA+, Ig-) lympho- COMBINED IMMUNOFLUORESCENCE AND cytes. Cells expressing receptors for HTA-1 deter- HISTOCHEMICAL TECHNIQUES minant and Ia-like antigen were demonstrated by Ethanol-fixed (4°C) 6 ,um cryostat sections were monoclonal antibodies OKT6 or NA1/34 in combi- initially stained in the indirect IF test with TRITC- nation with C antiserum to Ia-like antigen. The labelled C-anti-Ia-like antigen or monoclonal histochemical staining reaction for acid phosphatase antibody to HTA-1 determinant (OKT6 or NA1/34). (ACPase) was used simultaneously with fluorescent After the final rinse in PBS pH 7-6, the sections were labelled antisera to HTA-1 or Ia-like antigen. immediately processed for the ACPase reaction using the modified Gomori metal salt procedure.1516 The IMMUNOFLUORESCENT TECHNIQUES IF sections were briefly (60 s) postfixed in 10% Direct and indirect IF techniques were used on 6 ,um formalin, rinsed in distilled water then immersed in http://jcp.bmj.com/ cryostat sections. Applications of 5-10 ,ui of freshly filtered incubation medium* at 37°C for appropriately diluted primary antisera were made 60 min. After two short washes in distilled water, to moist sections then incubated in a damp chamber at 20°C for 30 min. Excess antibody was removed by *0.1 M acetate buffer pH 5; 0'24% lead nitrate (Sigma); 3 % sodium P glycerosphosphate (Sigma). The medium was washing in phosphate-buffered saline (PBS) pH 7-6 incubated at 37'C until complete precipitation of lead at 20°C for 10 min. In the indirect IF test, species- phosphate (6-24 h), cooled and filtered before use. on October 1, 2021 by guest. Protected copyright.

Table 1 Immunological reagents used in the direct and indirect IF analysis ofcryostat sectivns First layer antibody* Second layer antibody* Reactivity pattern Reference Chicken (C) anti-Ia-like antigen (1/80) Sheep anti-C-IgG (Fab,) + + IDR cells, LC "veiled" 12, 13 TRITC (1/20) cells; + B cells; i Rabbit (R) antihuman T leucocyte antigen Goat (G) anti-R-IgG (Fab2) + ; peripheral T 12, 14 (HuTLA) (1/20) FITC (1/10) cells, Thy-ALL blasts FITC/TRITC conjugated antisera to whole None + B cells, plasma cells 12 human Ig, IgM, IgD, IgG, k, A raised in goat and burro Mouse (M) monoclonal antibodies to human thymocyte antigen HTA-1 NAI/34t G-anti-M-IgG-FITC (1/10) G-anti-M-IgGL-TRITC (1/10) + cortical thymocytes (90%) 9 - medullary thymocytes (5°%) 9, 10, 11 OKT6:(1/10) G-anti-M-IgG-FITC (1/10) J *Dilutions of antisera shown in brackets. tCulture supernatant. tAscitic fluid, J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

Combined immunological and histochemical analysis ofskin and lymph node lesions in histiocytosis X 329 the enzyme reaction was developed in 05 % yellow no radiological lung changes. Excision biopsy of a ammonium sulphide (Sigma) for 2 min. The sections left upper cervical lymph node showed the histo- were rinsed finally in distilled water and mounted logical features consistent with histiocytosis X. under a coverslip in 1 % formol glycerol. Both the Treatment with , prednisolone and brown enzyme reaction product and TRITC-labelled antibiotics was followed by a rapid and marked antibodies could be easily examined under the clinical improvement. The lymphadenopathy, media- fluorescence microscope using phase contrast and stinal mass and skeletal lesions resolved within three the appropriate (green) fluorescence filter channel. months of starting therapy. At 12 month follow-up, Precautions were taken to ensure that the same having continued on an intermittent regimen of enzyme reaction patterns were obtained in the vinblastine and steroids, the patient was well and combnied IF/enzyme tests as were demonstrated on developing normally. sections which had been treated separately for IF or ACPase activity. Separate sections were stained for Results IF followed by ACPase localisation using either the pararosaniline16 or lead salt methods. Photo- HISTOLOGICAL PREPARATIONS graphs of marked areas on the slide were taken after (HAEMATOXYLIN AND EOSIN) each procedure which were later superimposed to In case 1, the skin (Fig. la) showed marked narrow- show identical areas of ACPase activity. ing of the epidermis with focal areas of large, round cells containing eccentric nuclei infiltrating the PATIENTS dermis and epidermis. These cells were accompanied by scanty numbers of small . In case 2, Case I the normal lymph node (Fig. lb) architecture was An 8-year-old boy presented in April 1980 with virtually replaced by a profuse, mixed cell infiltrate polydipsia and polyuria following a short influenza- consisting of irregular histiocytic cells with abundant like illness. was diagnosed and he eosinophilic cytoplasm, moderate numbers of plasma was treated with 1-diamino-8-D-arginine vaso- cells, lymphocytes and occasional . The pressin (DDAVP). Five months later in September histiocytic cells showed wide morphological variation 1980, he developed severe breathlessness, weight loss from bizarre multinucleate giant cells (containing and general lassitude. He was readmitted to hospital from 6 to 12 nuclei) to smaller, irregular mono- and on examination he was noted to have generalised nuclear forms with vesicular and folded nuclei. Small areas of depigmnentation (face, axillae, trunk and aggregates of residual lymphocytes were clearly knees) as well as a papular eruption over the back. present in the tissue apparently compressed by the http://jcp.bmj.com/ There was no lymphadenopathy or hepatospleno- abnormally proliferating cell population. Mitotic megaly. Chest x-ray revealed a right pneumothorax figures were occasionally recognised in both biopsies and reticular changes in both lungs. No bone lesions but these were too infrequent to substantiate a were apparent on skeletal survey. The diagnosis of malignant process. histiocytosis X was confirmed by skin biopsy and a right pleurectomy was performed. Postoperatively DEMONSTRATION OF IF AND HISTOCHEMICAL the child was treated with vinblastine and predniso- MARKERS ON CELL POPULATIONS IN H-X on October 1, 2021 by guest. Protected copyright. lone and his condition markedly improved. At the SKIN (CASE 1) latest follow-up (July 1981) the patient was well and continues on a modified regimen of vinblastine, and Immunological analysis DDAVP to control the diabetes insipidus. The dermal infiltrate of large, round cells showed strong reactivity for Ia-like antigen (Fig. 2a). These Case 2 cells were situated in the upper dermis particularly A 6-month-old baby boy of Turkish Cypriot origin along the dermoepidermal border. Scanty numbers of presented in March 1980 with failure to thrive, smaller Ia+ round cells were present in the epidermis. swellings behind the ears and apparent bone pain. In addition to the Ia+ round cell population, there On examination he had marked bilateral lymph- were occasional large, Ia+ irregular cells in the adenopathy (cervical, parotid, submandibular, post- dermis. auricular) and hepatosplenomegaly. In addition there Using monoclonal antibodies OKT6 or NA1/34 was a petechial lesion on the left foot and papular (both detecting HTA/1 antigen) in combination with lesions over the scalp. An x-ray examination showed C-anti-Ia-like antigen, all the Ia+ epidermal cells and multiple lytic areas throughout the skeleton (skull, the majority (> 70%) of the Ia+ dermal round cells femora, tibulae, humeri, lumbar vertebrae, ribs) as expressed the HTA-1 determinant (Fig. 2b). Con- well as a large anteromediastinal shadow. There were spicuous numbers of HTA-1+, Ia+ round cells were J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

330 Thomas, Janossy, Chilosi, Pritchard, Pincott

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*3s * *;dWB. *:. f§ 9 Fig. I Histiocytosis X (Haemtatoxylin and * Pe- ... F ..S:|5 eosin paraffin wax sections). (a) Skin case 1: Sk X ,, .,!e :s fbcal infiltrate of large round cells in the x ":: Xi dermis and epidermis (arrows) 480. 4 (b) Lymph node case 2: diffuse infiltrate of mixed cell types replaces the normal lymph nodes .1 architecture. The main cell type consists of large cells with eosinophilic cytoplasm (arrows) and multinucleate giant cells (aster isks). Other cells include residual lymphoid aggregates (L) x 480. http://jcp.bmj.com/ on October 1, 2021 by guest. Protected copyright.

present at the dermoepidermal border. Not all the cells were smaller than either of the HTA-1+, la+ and cells in the infiltrate, however, expressed the HTA-1+, Ia+, HTA-I - cell types but were not morphologically la+ phenotype. The large, dermal irregular cells were or immunologically identifiable as T lymphoblasts. Ia+ but HTA-1-. Small numbers of HTA-1- Ta- None of the cells in the skin reacted with anti- round mononuclear cells were also observed. These immunoglobulin reagents and virtually no T J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

Combined immunological and histochemical analysis ofskin and lymph node lesions in histiocytosis X 331

Fig. 2 Histiocytosis X (cryostat section). Skin (case 1) stained with (a) C-anti- Ia-like antigen (TRITC) in combination with (b) monoclonal antibody to HTA-J determinant (OKT6-FITC). Numerous Ia+, HTA-I+ round type I cells (arrows) are present in the dermis, along the dermoepidermal border and in the epidermis. Ia+, HTA-J- irregular type II cells (big arrows) are situated in the dermis as well as occasional Ia-, HTA-I+ mononuclear cells (star). http://jcp.bmj.com/ on October 1, 2021 by guest. Protected copyright.

(HuTLA+) lymphocytes were identified in the frozen (Fig. 3b), ACPase and granular, cytoplasmic TRAP+ sections. activity.

Histochemical analysis DEMONSTRATION OF IF AND HISTOCHEMICAL The round cell infiltrate in the dermis and epidermis MARKERS ON CELL POPULATIONS IN H-X showed distinct membrane localisation of ATPase LYMPH NODE (CASE 2) (Fig. 3a) but demonstrated weak cytoplasmic reactivity for ANAE (Fig. 3b) and ACPase. These Immunological analysis ATPase+, ANAE l, ACPase l round cells were A wide variety of cells expressed variable amounts of unreactive with TRAP. By contrast, the infrequent Ia-like antigen. Four main cell types could be Ia+ irregular cells showed absent or weak localisation identified on the basis of Ia-like antigen expression of ATPase but contained diffuse, cytoplasmic ANAE (Fig. 4a-d and Table 2). J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

332 Thomas, Janossy, Chilosi, Pritchard, Pincott

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...... -.-,.*, LR(O'...Q.. b .X Fig. 3 Histiocytosis X (cryostat section). Skin case 1: enzyme histochemical demonstration of(a) ATPase and (b) ANAE. (a) Most cells in the dermis and epidermis show strong ATPase activity and (b) weak ANAE activity although afew strongly ANAE+ irregular cells (arrows) are scattered throughout the dermis.

(i) The majority of the infiltrating cell population ally observed but these were much less conspicuous consisted of round or asymmetrical large cells in the lymph node than in the skin biopsy. Very few expressing moderate amounts of Ia-like antigen T (HuTLA+) lymphocytes were identified.These were pattern the tissue and constituted which appeared as a fine, granular staining scattered throughout http://jcp.bmj.com/ (Fig. 4a). On phase contrast microscopy, these cells < 2% of the total cell population. were agranular and sometimes contained one or two As immunofluorescence for Ia-like and HTA-1 characteristically folded nuclei. could be combined with ACPase, the (ii) Diffuse Ia-like antigen was present on a smaller foregoing main cell types were also classified by population of cells with distinct irregular or stellate enzyme histochemistry (Fig. 5a, b and Table 2). None outlines produced by long processes (Fig. 4a). of the round or asymmetrical Ia+, HTA-1+ cells (iii) In contrast to these two cell types, the bizarre (type I) exhibited strong ACPase activity. The multinucleate giant cells were conspicuous by their irregular Ia+, HTA-1- cells (type 11) showed focal on October 1, 2021 by guest. Protected copyright. total absence or very weak expression of Ia-like areas of moderate ACPase deposition (Fig. 5a, b antigen. The morphology of these cells could be insets) and the majorityof Ia-,HTA-1 -multinucleate identified on phase contrast microscopy and were giant cells (type JII) were strongly ACPase+. A few distinguished as "black holes" in the fluorescent ACPase+ giant cells however showed weak ex- tissue preparations (Fig. 4c). pression ofIa-like antigen(HTA-1-; Fig. 5a, b insets). (iv) Collections of membrane Ia+ small mononuclear A further cell type was revealed by combined IF lymphocytes constituted the fourth main cell type. and histochemical analysis. Low numbers of small These were surface membrane immunoglobulin round Ia+, HTA-1- mononuclear cells contained (SMIg+) B cells (Fig. 4a). prominent cytoplasmic ACPase+ "dots" or granules. When the sections were stained for HTA-1 These cells were morphologically different from the determinant (Fig. 4b, d) prominent clusters of large Ia+, HTA-1- (type II) cells which were much more Ia+, HTA-1+ round or asymmetrical cells (type I) prominent in the tissue. were observed throughout the tissue. These Ia+, HTA-1+ cells contrasted with large irregular Ia+, Histochemical analysis HTA-1- cells (type II) and with confluent and Multinucleate giant cells exhibited strong or scattered areas of la-, HTA-1- giant cells (type III). moderate ACPase and ANAE activity. The large Cells with the Ia-, HTA-1 + phenotype were occasion- round cells with characteristically folded nuclei were J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

Combined immunological and histochemical analysis ofskin and lymph node lesions in histiocytosis X 333 http://jcp.bmj.com/ on October 1, 2021 by guest. Protected copyright.

Fig. 4 Histiocytosis X (cryostat section). Lymph node (case 2) stained with combinations of (a)-(c) C-anti-Ia-like antigen (TRITC) and (b)-(d) monoclonal antibody to HTA-1 determinant (OKT6-FITC). (a), (b). In most areas the cell infiltrate consists ofdense clusters ofIa+, HTA-1+ round type I cells (arrows) with a few Ia+, HTA-1- irregular type II cells (big arrows) and Ia-, HTA-1- multinucleate giant type III cells (asterisk). Residual lymphoid areas (L) contain membrane Ia+ (HTA-J-) B cells. (c), (d). Other areas of the same tissue contain prominent numbers ofIa-, HTA-1- multinucleate giant type III cells (asterisk) and scanty la+, HTA-I+ type I cells (arrows). generally unreactive or weakly reactive for ANAE, Discussion ACPase and ATPase. Additional ANAE+, ACPase+ cell types included irregular and round mononuclear The two cases presented in this study showed cells with either cytoplasmic granules or diffuse typical (though different) clinical features associated deposition of enzyme reaction products. Only a with histiocytosis X. Histologically characteristic small proportion of multinucleate giant cells (30%) lesions in the skin and lymph node biopsies con- showed ATPase and TRAP activity. firmed the diagnosis in both patients. In the skin, the J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

334 Thomas, Janossy, Chilosi, Pritchard, Pincott Table 2 Immunological and histochemical characteristics ofthe major cell types in H-X lymph node (case 2) Cell types Ia-like antigen HTA-1 antigen SMlg* ACPase ATPase ANAE TRAP Round/asymmetrical cells + + - - t + - i (type I) (granular) Irregular/stellate cells + - - + + i + (type II) (diffuse) (focal) Multinucleate giant cells - - - ++ ++++ +t + ++ +t (type HI) Residual B lymphocytes + - + - + (type IV) *Surface membrane immunoglobulin. tATPase and TRAP demonstrated in 30 % multinucleate giant cells. http://jcp.bmj.com/ on October 1, 2021 by guest. Protected copyright.

Fig. 5 Histiocytosis X (cryostat section). Lymph node case 2: combined histochemical and immunofluorescence analysis with (a) monoclonal antibody to HTA-J antigen (NAI/34-TRITC), (inset a) C-anti-Ia-like antigen (TRITC) and (b) ACPase. A cluster ofHTA-I+ (Ia+) round (type I) cells do not exhibit ACPase activity (arrows). Intense ACPase reactions are present in large and medium sized HTA-1- (Ja-) giant type III cells (asterisk). Insets (a), (b) show a single la+, (HTA-I-) irregular type II cell containing localised ACPase+ reactionproducts (asterisk). A with weak expression ofIa-like antigen and moderate ACPase activity is also identified (big asterisk). relatively homogeneous population of large round The main feature of this study is the demonstration cells with kidney shaped nuclei were similar in that many of the cells in the skin and lymph node morphology and distribution to previous descrip- express the same la+, HTA-1+ phenotype as normal tions of H-X cell infiltrates.5 17 18 epidermal LC. This finding demonstrated by com- J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

Combined immunological and histochemical analysis ofskin and lymph node lesions in histiocytosis X 335 bined IF analysis confirms previous evidence that striking lack of HuTLA+ T cells. H-X cells may be related to LC. Abundant amounts The second feature of this study is the demon- ofIa-like antigenshavebeen demonstrated on normal stration that IF reagents can be successfullycombined LC by IF810 and immunoelectronmicroscopic with ACPase enzyme histochemistry on frozen studies.19 Recently, cortical thymocyte antigen tissue sections without loss of reactivity in either HTA-1 has also been demonstrated on LC defined system. Using these techniques separately on adjacent by monoclonal antibody OKT6.1011 It has been tissue sections, the enzyme reactions and immunolog- shown that both OKT6 and NA1/34 monoclonal ical findings were strikingly variable in both skin and antibodies react with HTA-1 antigen although with lymph node biopsies. However, these features could different epitopes on the same molecule.10 The fact be closely related to each other using the combined that these two independently produced monoclonal IF and histochemical technology. Demonstration of antibodies react with LC indicates that this is not a Ia+, HTA-1+ cells in the skin with weak ACPase and chance cross-reaction between single epitopes on two ANAE activity but moderate amounts of ATPase unrelated molecules but rather that these cells supports previous histochemical studies.5 24Similarly carry at least part of the same HTA-1 molecule. Thus in the lymph node, the Ia+, HTA-1+ cells were the Ia+, HTA-1 + phenotype provides an important unreactive or weakly reactive for ACPase, ATPase new marker for LC in addition to the well established and ANAE. ln contrast, the multinucleate giant morphological criteria of ultrastructurally defined cells were Ia-, HTA-1- but were strikingly ACPase+ Birbeck granules2 and histochemical demonstration and showed variable amounts ofANAE and ATPase. ofATPase.4 5 Other minor cell populations includingIa+, HTA-1-, Most of the proliferating cells in the skin expressed ACPase+ (type II) irregular cells and small numbers the Ia+, HTA-1+ phenotype although a few irregular of Ia+, HTA-1-, ACPase+ round cells were similar forms exhibited Ia-like antigen alone. It was in- to interdigitating reticulum cells and tissue macro- teresting to observe the presence of many Ia+, phages. These various immunological and enzyme HTA-1+ cells in the dermis. This contrasts with a patterns more clearly define the heterogeneous variety of dermatological conditions such as mycosis nature of the cells involved in H-X. It is not known fungoides (MF), lichen planus (LP) and graft-versus- whether the predominating Ia+, HTA-1+, ACPase- host disease (GvHD) in which the dermis is in- cells and the other cell types including Ia-, HTA-1 -, filtrated by numerous strongly Ia+ but HTA-1- ACPase+ giant cells are derived from the same cell irregular cells. The Ia+, HTA-1+ cells in these dis- lineage or whether the latter cell type represents a orders (MF, LP, GvHD) are primarily restricted to reactive element stimulated by the aberrant Ia+, the epidermis and Ia+, HTA-1+ cell types constitute HTA-1 + population. http://jcp.bmj.com/ only a small proportion (5-20%) of the dermal Ia+ The phenotypic similarity between Ia+, HTA-1+ (HTA-l-) populations.1020 H-X cells and normal Ia+, HTA-1+ LC confirms In contrast to the skin, the lymph node showed a previous observations that H-X cells contain ultra- more heterogeneous infiltrate. Again, there were structural bodies which are indistinguishable from numerous Ia+, HTA-1+ cells throughout the tissue the in LC.3 These findings support which is an uncommon finding in normal or stimu- the concept that H-X represents an abnormal lated lymph nodes. Likewise, lymph nodes from proliferation of LC.25 There is much evidence to on October 1, 2021 by guest. Protected copyright. patients with MF or Sezary syndrome contain less show that normal epidermal LC function as special- than 10-15% of Ia+, HTA-l+ cell types within the ised immunological accessory cells to initiate cleate giant cells, often described in H-X lesions,'7 2122 immune responses in allergen-stimulated states.26 Ia+ non-lymphoid population. The bizarre multinu- These -derived cells27 are capable of were Ia-, HTA-1-. Only a weak expression of trapping antigen and migrating through dermal Ia-like antigen was observed in the peripheral lymphatics to regional lymph nodes to present cytoplasm of some giant cells. Characteristically, the antigen to effector T cells.28 LC also resembles a nodal infiltrates in H-X effacethenormalarchitecture variety of other Ia+ bone marrow derived macro- and are accompanied by a variety of normal cell phages including interdigitating reticulum cells (IDR) types.2' 22 Although typical in T-dependent areas of peripheral lymphoid tissue28 were not present in this lymph node, numerous and "veiled" cells of the dermal afferent lymphatics.29 plasma cells and moderate numbers of eosinophils Clearly a spectrum of these types as well as tissue were observed. Residual areas of B lymphocytes are involved in H-X lesions. maintained the same phenotype (Ia+, IgM+, IgD+, It was not surprising to find that only scanty mixed K+, A+ light chains) as immunocompetent B numbers of T lymphocytes were present in the skin cells in primary nodules and follicle mantles of and lymph node lesions. Immunological abnor- normal lymphoid tissue.23 There was, however, a malities such as combined and J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

336 Thomas, Janossy, Chilosi, Pritchard, Pincott impaired cell mediated immune responses have been 1979 ;9 :520-10 associated in some cases of H-X.30 Recently, 10Thomas JA, Janossy G, Graham-Brown RAC, Kung PC, Goldstein G. The relation between T cell subsets and abnormal immune function has been demonstrated Ia-like antigen positive non-lymphoid cells in early in a series of H-X patients who lacked T cell H2 stages ofcutaneous T cell (mycosis fungoides) receptors.3' In normal tonsil, inducer T cells (OKT4+, J Invest Dermatol 1982;78:(in press). form close contact with Ia+ IDR cells.13 1 Fithian E, Kung PC, Goldstein G, et al. Reactivity of OKT8-) Langerhans' cells with hybridoma antibody. Proc Natl Similar T cell-Ia+ cell contacts are demonstrated in AcadSci USA 1981 ;78:2541-4. MF where Ia-like antigens are strongly expressed on 12 Janossy G, Thomas JA, Pizzolo G, etal. Immunohistological the proliferating non-lymphoid elements.10 Despite diagnosis of lymphoproliferative diseases by selected increased numbers of Ia+ cells in H-X lesions, the combinations of antisera and monoclonal antibodies. BrJ Cancer 1980;42:1-20. lack of T cells suggest either a loss of normal T 13 Janossy G, Tidman N, Selby WS, Thomas JA, Granger control or a reactive local T cell de- SM. Human T lymphocytes of inducer and suppressor ficiency. It will be interesting to see if the reported type occupy different microenvironments. Nature 1980; beneficial effects of thymic hormonal extracts3l show 287:81-4. 14 Janossy G, Thomas JA, Bollum F, et al. The human any influence on the T cell deficiency in the lesions. thymic microenvironment: an immunohistologic study. J It is clear from this studythat the Ia+, HTA-1 +cells Immunol 1980;125:202-12. in H-X express the usual immunohistochemical 11 Chilosi M, Pizzolo G, Menestrina F, et al. Enzyme histo- characteristics of Ia+, HTA-1 + LC but have abnormal chemistry on normal and pathological paraffin embedded patterns in lymphoid tissues. Am J Clin Pathol 1981 ;76:(in press). migratory the skin, lymph node2l and 16 Lojda Z, Gossrau R, Schiebler TH. Enzyme histochemistry: bone marrow (unpublished observations). The a laboratory manual. Berlin, New York: Springer- reduction of intracellular enzymes, particularly Verlag, 1979. ACPase, suggests that these cells are non-functional 17 Gianotti F, Caputo R. Skin ultrastructure in Hand- types derived from the LC In addition the Schuller-Christian disease: report on abnormal Langer- lineage. hans' cells. Arch Dermatol 1969;100:342-9. aberrant features of these cells might be influenced 18 Wolf HH. Subtle clues to diagnosis of skin diseases by by the T lymphoid system. electron microscopy: Langerhans' cell granules in histiocytosis X. Am J Dermatopathol 1979;1 :77-81. JA Thomas is supported by MRC grant No 19 Rowden G, Phillips TM, Lewis MG, Wilkinson RK. Target role of Langerhans' cells in mycosis fungoides. G/978/443. J Pritchard issupportedbytheLeukaemia Transmission and immunoelectron microscopic studies. Research Fund. J Cutan Pathol 1979;6:364-82. 20 Lampert IA, Janossy G, Suitters AJ, et al. Immunological References analysis of graft versus host disease in skin. Lancet 1981;

ii:1352. http://jcp.bmj.com/ Lichtenstein L. Integration of of 21 Williams JW, Dorfman RF. Lymphadenopathy as the bone "Letterer Siwe disease" and "Schuller-Christian initial manifestation of histiocytosis X. Am J Surg disease" as related manifestations of a single nosological Pathol 1979;3:405-21. entity. Arch Pathol 1953 ;56:84-102. 22 Imamura M, Muroya K. Lymph node ultrastructure in 2 Birbeck MS, Breathnach AS, Everall JD. An electron- Hand-Schuller-Christian disease. Cancer 1971 ;27:956- microscopic study of basal melanocytes and high level 64. clear cells (Langerhans' cells) in vitiligo. JInvest Dermatol 23 Halper JP, Knowles DM, Wang CY. la antigen expression 1961 ;37:51-63. by malignant : correlation with coventional

3Basset F, Turiaf J. Identification par la microscopie lymphoid markers. 1980;55:373-82. on October 1, 2021 by guest. Protected copyright. -lectronique de particules de nature probablement 24 Leder LD. Subtle clues to diagnosis by histochemistry. virale dans les lesions granulomateuses d'une histiocytose Histiocytosis X. Am J Dermatopathol 1979;1:365-9. X pulmonaire. C R Acad Sci (Paris) 1965;261:3701-3. 25 Nezelof C, Basset F, Rousseau MF. Histiocytosis X. 4 Berman B, France DS. Histochemical analysis of Histogenic arguments for a Langerhans' cell origin. Langerhans' cells. Am J Dermatopathol 1979;1 :215-2 1. Biomedicine 1973 ;3:259-79. 5Elema JD, Poppema S. Infantile histiocytosis X (Letterer- 26 Silberg-Sinakin I, Baer RL, Thorbecke GJ. Langerhans Siwe disease). Investigations with enzyme histochemical cells: A review of their nature and emphasis on their and sheep-erythrocyte rosetting techniques. Cancer 1978; immunologic functions. Prog Allergy 1978;24:128-35. 42:555-65. 27 Katz SI, Tamaki K, Sachs DH. Epidermal Langerhans' 6Jaubert F, Barbey S, Nogues C, Monnet JP, Grun M, cells are derived from cells originating in the bone Nezelof C. X positivity for non-specific marrow. Nature 1979;282:324-6. esterase. J Histochem Cytochem 1980;28:45-6. 28Thorbecke GJ, Silberg-Sinakin 1, Flotte TJ. Langerhans' 7 Stingl G, Wolff-Schreiner ECH, Pichler WJ, Gschnait F, cells as macrophages in skin and lymphoid organs. J Knapp W, Wolff K. Epidermal Langerhans' cells bear Invest Dermatol 1980;75:32-43. Fc and C3 receptors. Nature 1977;268:245-6. 29 Kelly RM, Balfour BM, Armstrong JA, Griffiths S. 8Rowden G, Lewis MG, Sullivan AK. Ia antigen expression Functional anatomy of lymph nodes. II. Peripheral on human epidermal Langerhans' cells. Nature 1977 ;268: lymph-bornemononuclearcells. Anat Rec 1978 ;190:5-21. 247-8. 30 Nesbit ME, O'Leary M, Dehner LP, Ramsay NKC. The McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre JW, and the histiocytosis syndromes. Am J Milstein C. A human thymocyte antigen defined by a Pediatr Oncol 1981 ;3:141-50. hybrid myeloma monoclonal antibody. Eur J Immunol 3 Osband ME, Lipton MJ, Lavin P, et al. Histiocytosis X. J Clin Pathol: first published as 10.1136/jcp.35.3.327 on 1 March 1982. Downloaded from

Combined immunological and histochemical analysis of skin and lymph node lesions in histiocytosis X 337 Demonstration of abnormal immunity, T cell histamine Requests for reprints to: Dr JA Thomas, Department of receptor deficiency, and successful treatment with Histopathology, Royal Marsden Hospital, Sutton, Surrey, thymic extract. N Engl J Med 1981 ;304: 146-53. England.

The February 1982 Issue THE FEBRUARY 1982 ISSUE CONTAINS THE FOLLOWING PAPERS Quantitative bone histology in 38 patients with Circulating CK-MB and CK-BB isoenzymes after advanced renal failure JB EASTWOOD gastrointestinal surgery SWEI H TSUNG Quantitative analysis of non-Hodgkin's lymphoma Total CK activity and isoenzyme patterns in normal CEDRIC R ABBOTT, ROBERT W BLEWITT, COLIN C BIRD and neoplastic tissue of gastrointestinal tract Detection of surface immunoglobulins of human SWEI H TSUNG lymphoid cells: a comparative study of live and fixed Serum glutamate dehydrogenase is not a reliable cells using a direct immunoperoxidase procedure marker of liver cell necrosis in alcoholics WJ GUY LAURENT, MARIE FRANrOISE GOURDIN, FELIX JENKINS, SB ROSALKI, Y FOO, PJ SCHEUER, E REYES NEMESANSZKY, S SHERLOCK Immunological study of human lungs by immuno- Diagnosis of Legionella pneumophila infections by peroxidase technique BERNARD FOX, SAMI SHOUSHA, means offormalised yolk sac antigens TG HARRISON, KEITH R JAMES, GILLIAN C MILLER AG TAYLOR Practical use of a word processor in a histopathology Failure of the fluorescent antibody reaction to laboratory JAMES C BRIGGS, NASSIF BN IBRAHIM, identify penicillinase-producing gonococci SHEENA IAN MACKINTOSH, DAVID NORRIS A WAITKINS, R DAWN ANDERSON origin of Reed-Stemnberg cells: an Osteomyelitis and septic arthritis caused by Kingella immunohistochemical study sV PAYNE, DH WRIGHT, Kingae JM DAVIS, MM PEEL KJM JONES, MA JUDD Lysosomal localisation of parallel tubular arrays in Swabs and swab-transport media kits in the isolation chronic lymphocytic leukaemia of T cell origin: an of upper respiratory bacteria PW ROSS, CG http://jcp.bmj.com/ ultrastructural cytochemical study FREDE'RIQUE CUMMING, H LOUGH CAPRON, JEAN ,YVES PERROT, CLAUDE BOUCHEIX, Comparative study of three tests (dye test, indirect MICHEL REYNES, VIVIANE TRICOTTET-PACZYNSKI, ALAIN haemagglutination test, latex agglutination test) for BERNADOU, JAQUES DIEBOLD the detection of antibodies to Toxoplasma gondii in Iron granules in plasma cells MARGARET K COOK, human sera ALAN H BALFOUR, DG FLECK, HUW PA MICHAEL MADDEN HUGHES, D SHARP on October 1, 2021 by guest. Protected copyright. chemotaxis: multiple assay screening Appraisal of Anotox, a new anerobic atmospheric using a raft technique U JAYASWAL, S ROPER, detoxifying agent for use in anaerobic cabinets S ROATH JS BRAZIER Bone marrow processing and cryopreservation DC Technical methods LINCH, LJ KNOTT, KG PATTERSON, DA COWAN, PG Tissue carbohydrate identification by the use of HARPER lectins KP WEST, HA PLATTS, A FLETCHER, F WALKER A method for transmission and scanning electron Cell structure and percent viability by a slide centri- microscopy of undecalcified human bone marrow fuge technique MARGARET G FITZGERALD, CS biopsy specimens using cryofracture F KUTO, HOSKING T NAGAOKA, M HAYASHI, Y HIRASAWA, H TOKUHIRO Ascorbate status and fibrinogen concentrations after Letters to the Editor cerebrovascular accident R HUME, BD VALLANCE, MM MUIR Book Reviews Copies are still available and may be obtained from the PUBLISHING MANAGER, BRITISH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, LONDON WC1H 9JR, price £5-00, including postage