JOURNAL OF PATHOLOGY, VOL. 180: 400-406 ( I 996)

THE PRESENCE OF CYTOKINES IN LANGERHANS' CELL HISTIOCYTOSIS

J A N H . DE GRAAF*, RIENK Y . J . TAMMINGAP, ANKE DAM-MEIRING", WILLEM A. KAME'S? AND WIM TIMENS*

*Dc~prirttiierit of' Ptitiiology arid tBeatrix Children's Hospital, CI2ildreri's Cancer Center, Univcwit! Hospitcil Gvoningm. The Netherlmrls

SUMMARY

Langerhans' cell histiocytosis (LCH) is characterized by an accumulation andlor proliferation of cells with a Langerhans' cell (LC) phenotype. The aetiology and pathogenesis of LCH are unknown; it is suggested that L C H is caused by an immunological dysregulation. Production of cytokines i\ a central feature of immunological regulation. LCH lesions and normal LCs were studied for the presence of cy tokines known to influence the functioning of LCs: IL-la, IL-lp, 1L-4, CM-CSF, IFN-y, TGF-a, TGF-/I, bFGF, and TNF-a. Cy tokines were abundantly present within LCH lesions; LCH cells stained for IL-10, IL-lp, IL-4, GM-CSF, TGF-a, TGF-/I, TNF-a, and IFN-y. Macrophages, lymphocytes, eosinophil granulocytes, and, surprisingly, multinucleated giant cells were also sources of cytokines. These results suggest that cytokines play a prominent role in the pathogenesis of LCH and may explain phenomena that often occur in LCH, such as osteolysis and fibrosis and the recruitment of typical inflammatory infiltrates. The results also suggest that a 'down-regulatory' signal is lacking in LCH, resulting in an accumulation andlor proliferation of abnormal LCs.

KC 'L woRD5-Langerhans' cell histiocytosis; histiocytosis X; cytokines; inflammation; multinucleated giant cells; mdcrophages; osteolysis; immunohistochemistry; double-staining; CDla

INTRODUCTION

Langerhans' cell histiocytosis (LCH) is characterized by an accumulation and/or proliferation of cells with a Langerhans' cell (LC) phen0type.I This is indicated by the expression of CDla and S-100 and the ultrastruc- tural presence of Birbeck granules in the LCH cells, features expressed only by epidermal LCs. Other cells may be present in LCH lesions, including lymphocytes, macrophages, eosinophil granulocytes, and multinucle- ated giants cells.

The disease predominantly occurs in children4 and the clinical presentation is variable. Solitary or multiple lesions of a single organ system can be found, most commonly of bone. I n the most severely affected patients, usually children under the age of 2 years, multisystem involvement may be present, with a high mortality rate.5 In these cases many sites can be affected, such as skin and mucosa, lymph node, lung, bone, liver, spleen, and bone marrow.5

The aetiology and pathogenesis of LCH are Ufiknown.' I t has been postulated that LCH is caused tysl an immunological dysregulation, causing LCs to fin&fate to and to accumulate at different sites.' LCH & produce interleukin (1L)-1, prostaglandin E2,h interferon-;! (IFN-y), and granulocyte/macrophage- colony stimulating factor (GM-CSF).',* LCH cells have additionally been found to produce tuniour necrosis factor-a (TNF-a) and IL-1p.9 General features of normal immunological regulation involve the expression of cellular adhesion molecules and the action of locally produced cytokines. We recently showed that LCH cells

Addressee for correspondence: Ian H. de Graaf, Department of Pathology. University Hospital Groningen. Oostersingel 63, 9713 EZ Groningen. The Netherlands.

CCC 0022-341 7/96/120400--07 c " 1996 by John Wiley & Sons, Ltd.

abnormally express adhesion molecules and that they may experience abnormal or insufficient activation when compared with normal LCs. We also found a variety of cellular adhesion molecules present on endothelial cells within the lesions.10."

Cytokines play an important role in regulating the expression of cellular adhesion molecules. Immunologi- cal dysregulation, such as is suggested to occur in LCH, may have effects on cytokine production by otherwise normal inflammatory cells, as well as by abnormal cells.

Cytokines, including those previously investigated and others, will probably play a role in the pathogenesis of LCH, as cytokines in general play a role in the interplay between different cells. We therefore investi- gated tissue of LCH lesions for the presence of a number of cytokines known to influence the llinctioiiing of normal LCs.

MATERIALS AND METHODS

Patients Sections of formalin-fixed, paraffin-embedded tissue

were routinely stained with haematoxylin and eosin. All biopsies showed the presence of characteristic histio- cytes, macrophages, lymphocytes, and eosinophil granu- locytes. Multinucleated giant cells were seen in some cases. Frozen tissue (n= 15) from the ten patients with LCH was collected; in four cases, both primary and recurrent lesions were available.

Histopathological diagnosis was confirmed by stain- ing for CDla. Clinical data on the patients were collected and the extent of disease was stratified as follows: (I) single bone lesion, isolated skin disease, or solitary lymph node involvement; (11) multiple bone or skin lesions, or multiple lymph node involvement; and (111) multi-organ involvement.

ReccGvcd I4 Februarj I996 Accc~pterl 7 .June 1996

CYTOKINES IN LCH

Table I-Characteristics of the antibodies used

40 1

RESULTS

Antibody against:

CDla Interleukin-1 alpha Interleukin-1 beta Interleu kin-4 Basic fibroblast growth factor Granulocyte-macrophage CSF Interferon-gamma Transforming growth factor-alpha Transforming growth factor-beta Tumour necrosis factor-alpha

Abbreviation

CDI a IL-la IL-lP IL-4

bFGF GM-CSF

IFN-1, TGF-u TGF-P TNF-a

Source"

D 0 C G 0 G B 0 G E

*D: Dako, Glostrup, Denmark; 0: Oncogene Science, Cambridge, Massachusetts, U.S.A.; C: kindly provided by Dr Towbin, Ciba Geigy, Basel, Switzerland; G: Genzyme, Cambridge, Massachusetts, U.S.A.; B: Boehringer Mannheim, Mannheim, Germany; E: kindly provided by Dr Bosnian, Eurogenetics, Tessenderlo, Belgium.

Immunohistochemistvy

Immunohistology was performed on acetone-fixed frozen sections from tissue stored at - 80°C using a three-step immunoperoxidase method. Table I provides a summary of the antibodies used. Horseradish peroxidase-labelled rabbit anti-mouse immunoglobulin (Ig; Dako, Glostrup, Denmark) was used as the second- step reagent and peroxidase-labelled goat anti-rabbit Ig (Dako) was used as the third-step reagent. The peroxi- dase label was visualized using 3-amino-9-ethylcarbazol (AEC; Aldrich, U.S.A.), together with H,O,. Slides were counterstained with haematoxylin. To prevent non- specific background staining, the second-and third-step reagent dilutions contained 1 per cent normal human AB serum. Replacement of the primary antibodies by non-relevant antibodies of the same immunoglobulin isotype was used as negative control (see Table I for characteristics of the antibodies). Specimens of normal skin (n=3) and lymph nodes showing a dermatopathic reaction (n=3) were used as controls for normal epidermal LCs and activated LCs, respectively.

Indirect double immunojluovescence

Staining for IL-la, IL-lp, IL-4, transforming growth factor (TGF)-a, TGF-P, IFN-y, TNF-a, basic fibroblast growth factor (bFGF), and GM-CSF was evaluated with double-immunofluorescence. Sections were pre- incubated with normal goat serum diluted 10 per cent in phosphate-buffered saline. Binding of monoclonal anti- body T6 (Dako) of IgG2a subclass directed against CD1 a was visualized with anti-IgG2a fluorescein iso- thiocyanate (F1TC)-conjugated goat anti-mouse Ig. Binding of the other primary antibodies (all of IgG1 subclass) was Visualized with anti-IgG 1 rhodamine B isothiocyanate (TR1TC)-conjugated goat anti-mouse Ig. Double-staining was performed on sections of normal skin, dermatopathic lymph nodes, and LCH specimens.

Clinical data The patients were seven males and three females.

Ages at the time of diagnosis ranged from 6 months to 15 years (mean 4.3 years, median 2.4 months). Localized disease of a single-organ system was present in three patients (stage I). Multiple lesions of a single-organ system (stage 11) occurred in one patient and multi- system involvement (stage 111) was found in six patients. In four patients, residual lesions are present; the others currently have no evidence of disease.

Presence of cytokines in LCH lesions

The LCH cells were identified by means of CDla staining (Figs 1 and 2); indirect double- immunofluorescence staining allowed confirmation of simultaneous expression of CDla and cytokines. A summary of the results is given in Table 11.

LCH cells contained IL-la in all lesions and IL-1/3 in all but two. In most cases, only a percentage of CDla- positive cells stained for these cytokines, varying from 10 per cent to virtually 100 per cent among the different cases, with more distinct staining for IL-ID.

Many cells positive for IL-4 were scattered through- out the lesions, probably representing eosinophil granulocytes. In addition, LCH cells were positive for IL-4 in 7 out of 15 lesions, although the staining was much weaker than in eosinophils. Only about 10 per cent of LCH cells within a lesion expressed this cytokine.

Expression of TGF-a was found in all but three lesions, in 25-75 per cent of LCH cells. In some cases, these cells were most striking at the borders of the lesions. TGF-P was found in LCH cells in all but three lesions. A distinct extracellular reticular staining pattern for TGF-@ was seen in several cases.

LCH cells stained for GM-CSF in all but one lesion (Fig. 2b). About 75 per cent of LCH cells expressed GM-CSF, although in a few cases only a few positive cells, about 10 per cent, could be identified. In several lesions a reticular extracellular staining pattern was identified, especially around the smaller vessels.

TNF-a was found in a substantial number of the LCH cells in 13 out of 15 lesions (Fig. lb).

IFN-y was demonstrated in 25-100 per cent of LCH cells in 12 of 15 lesions. In a few cases, distinct staining for IFN-y was found in endothelial cells of small vessels (Fig. 2c).

bFGF was generally absent from LCH cells, with the exception of LCH cells in one skin lesion. bFGF was found around vessels with a reticular staining pattern in a number of cases.

Many CD la-negative large cells within the lesions, probably macrophages, showed distinct staining for most cytokines. These cells generally demonstrated stronger expression of the cytokines than did LCH cells. They expressed IL-la and IL-lP, GM-CSF, TNF-a, TGF-a, IFN-y, and TGF-a. IFN-y was also seen in lymphocytes and TGF-/3 and bFGF were seen in fibroblasts.

402 J. H. DE GRAAF ET A L .

Fig. 1 --Expression of C D l a (A) and the presence of TNF-u (9) on LCH cells in the bone lesion of patient 10. As for most cytokines, the smaller multinucleated cells stained positive but the larger giant cells were negative for TNF-n (arrows). (Original magnification x 256)

Presence of cytokines in LCs in normal skin and dermatopathic reaction

LCs in normal skin and lymph nodes involved with dermatopathic reaction were evaluated with staining for CDla. Normal LCs in uninvolved skin and mucosa of some LCH lesions were also evaluated.

In normal epidermis and mucosa, 1L-la and IL-lp were demonstrated on LCs. Normal LCs also stained for GM-CSF, IFN-y, TGF:-a, TGF-P, bFGF, and TNF-a, but only a small percentage of normal LCs expressed these cytokines. In addition to LCs, other cells of the epidermis or dermis contained cytokines. Basal

keratinocytes expressed IL-la, and IFN-7 and TGF-a could also be demonstrated in keratinocytes. Cells containing bFGF and TNF-a were present in the papillary dermis. The nature of these cells was not determined.

LCs in lymph nodes with dermatopathic reaction expressed none of the cytokines investigated. Numerous macrophages expressing IL-1 a, IL-lB, IFN-y, TGF-a, and GM-CSF were found in the sinuses of these lymph nodes. Cells expressing IL-4 were scattered in lymph nodes, possibly eosinophil granulocytes, and T-lymphocytes. TGF-/3 and bFGF-positive cells were predominantly located in the trabeculae and

CYTOKINES IN LCH 403

Fig 2-LCH cells in the mucosal lesion of patient 5 , stained for CDla (A), GM-CSF (B), and IFN-y (C). Normal Langerhans’ cells in the mucosa also stained for both cytokines. (Original magnification x 160)

medullary cords. Endothelial cells weakly expressed The smaller multinucleated cells stained positive, but the TGF-a and IFN-I/. Staining for bFGF was found larger giant cells were negative for these cytokines. around the vessels.

Presence of cytokines in multinucleated giant cells DISCUSSION

Multinucleated giant cells were present, especially in lesions of bone. Both CDla-positive and CDla-negative multinucleated giant cells were found within the lesions. These giant cells showed variable staining for IL-la and IL-lp, with most pronounced staining for IL-la. They also contained TGF-a, TGF-/3, IFN-y, GM-CSF, IL-4, and TNF-a. TGF-/3 and IFN-y staining was variable.

In this study, a variety of cytokines have been dem- onstrated to be abundantly present in the LCH lesions. Although the precise effects of cytokines in vivo are difficult to determine, it is likely that the cytokines found in LCH lesions participate in characteristic events and may have distinct effects on the LCH cells, as well as on other inflammatory cells within the lesions.

404 J. H. DE GRAAF ET A L

Fig. 2-C

Table 11-Expression of cytokines on LCH cells

Age at Patient diagnosis No. Sex years HS* Lesion Localization CDla IL-la IL-b IL-4 TGF-a TGF-P GM-CSF IFN-7: TNF-a bFGF

1 M 1.75 I A 2 M 5.5 I A 3 M 15 I A 4 M 4 11 A 5 M 0.5 111 A

B C

6 F I 111 A 7 F I 111 A

B 8 M 2.5 111 A 9 M 2.5 I l l A

B 10 F 4.5 III A

B

Bone (radius) Bone (L2) Bone (humerus) Bone (scapula) Bone (palate) Adenoid Mucosa Skin Bone (mandible) Bone (mandible) Bone (skull) Lymph node Bone (mandible) Bone (skull) Mucosa

+ + + + + + + + + + + - + + + + + + + + + + + + + - + + + + + - + + + + + - - + + + + + + - + + + t + + + + + + + + + + + + - + ++ + - + - +

+ ++ + + -

++ + + - + + + + + + + -

+ + + + + + + + + + + + + + + +

-

-

-

+ + + + + -

+ + + + + + + + +

+ + + + + -

+ + +

+ + + -

+ + -

+ + +

*HS: Stratification according to the Histiocyte Society (see text) Staining: + +, strong; f . weak to moderate; - , negative.

LCH lesions of bone are often characterized by oste- olysis. In LCH lesions, IL-la and IL-ID, and other cytokines related to bone resorption, such as T N F - C ~ , ~ ~ ' ~ are abundantly present and may activate osteoclastic bone resorption, providing an explanation for the devel- opment of osteolytic lesions. Although the exact nature of the multinucleated giant cells that characterize LCH lesions of bone is unknown, these cells may originate from activation of macrophages under such IL-I influ- ence. In addition, IFN-y stimulation has been reported to enhance the IL-1 secretory capacity of LCH cells,6 further contributing to the osteolytic capacity of LCH lesions. It should be mentioned that IFN-y has been reported to be a marker of LCH cells in the skin." We

found IFN-y-positive LCH cells in lesions of skin, bone, and lymph node, suggesting that IFN-7 is not a specific marker of LCH cells in the skin alone.

The presence of TGF-P in LCH lesions could have important implications for the development of fibrosis. Fibrosis is often prominent in end-stage LCH lesions; pulmonary lesions are often fibrotic and the develop- ment of diabetes insipidus in LCH of the central nervous system (CNS) has also been related to fibrosis. 14.1s

TGF-jl is identified as a major mediator of fibrosis.16 Although we could not investigate LCH lesions of lung or CNS in this study, prominent production of TGF-p by LCH cells, macrophages, and giant cells could be an important mediator of fibrosis in susceptible tissue.

CYTOKINES IN LCH 405

Additional cytokines such as TGF-a, GM-CSF, bFGF, and platelet-derived growth factor have roles in wound healing and f i b r o ~ i s , ' ~ , ~ ~ and could thus contribute to the development of this fibrosis in LCH.

In many patients, LCH lesions of bone tend to follow a chronic course. Our study suggests that in these cases an important role may be played by multinucleated giant cells and macrophages. We found multinucleated giant cells, characteristically present in LCH lesions of bone, staining for a variety of cytokines. This finding suggests that multinucleated giant cells are a prominent and active component of bone lesions, instead of passive by-standers, by virtue of their cytokine production. Multinucleated giant cells could represent a long-lasting influence on the micro-environment of bone lesions, allowing LCH cells and other cells, such as macrophages and eosinophils, to accumulate continuously and to proliferate, contributing to the chronic course of these lesions. Since the number of LCH cells may decrease in regressive lesions, and macrophages and multinucleated giant cells may become the predominant cells within the lesions,I4 an important role for these cells in chronic lesions is further suggested.

It is thought that GM-CSF and 1L-1 are essential for LCs to acquire a full antigen-presenting phen~ type . '~ GM-CSF also cooperates with TNF-a in the generation of LCs from CD3Cpositive haematopoietic progeni- tors.20,21 We found expression of GM-CSF in LCH cells, confirming previous observation^.^ We also found TNF-u to be abundantly present in LCH lesions; the immunohistochemical localization suggests that LCH cells, amongst others, produce this cytokine. The presence of IL-1, GM-CSF, and TNF-a in LCH lesions may have important implications for LCH cells. The presence of these cytokines suggests that LCH cells may provide themselves with an optimal micro-environment to generate their specific phenotype and to prolong their viability, possibly by creating autocrine loops.

The expression of cellular adhesion molecules, and subsequently the tissue distribution of specific cells, is also regulated by cytokines. Cytokines found in the lesions play a role in the migration, recruitment, and distribution of LCs. GM-CSF has been identified as important in the recruitment of L C S . ~ ~ 24 It is likely that the presence of GM-CSF within LCH lesions is import- ant in the distribution of LCH cells. TNF-u augments the migratory capacity of normal LCs from the epi- dermis to draining lymph node^.^^,^^ TNF-a, IL-la, and IL-1,B also mediate on LCs the expression of cellular adhesion molecules that are essential for migration.27 These cytokines may have a role not only in the migration of LCH cells, but also in the recruitment of inflammatory cells. Endothelial cells in LCH lesions express a variety of inducible adhesion molecules, such as ICAM-1, VCAM-1, and E- and P-~electin. '~ The presence of IL-1 and TNF-a, known to influence the expression of ICAM-1 on endothelial cells, could explain this e x p r e s ~ i o n . ~ ~ , ~ ~ The expression of VCAM-1 could in part be explained by the presence of IL-1, which together with TNF-a is a strong inducer of VCAM-1 on endothelium.3O VCAM-1 is used by eosinophils to trans- migrate through tissue,31 and this transmigration may be

augmented by IL-l,B and T N F - u , ~ ~ providing a mechan- ism to explain the abundant presence of these cells. IFN-y present in the endothelial cells of many LCH lesions may have an up-regulatory effect on the expression of adhesion molecules.

LCH cells also express TGF-a. Little is known of the effects of TGF-a on Langerhans' cells, but it has a wide range of tissue-specific functions including the promo- tion of epithelial p r~ l i f e ra t ion .~~ LCH cells proliferate within the lesion^;^^^^^ further studies should determine whether TGF-a is partly responsible.

It is of special interest that the pattern of cytokines in LCH cells resembles that observed in normal epidermal LCs. In contrast, LCs in lymph nodes involved with dermatopathic reaction do not stain for any of the cytokines investigated. Epidermal LCs have important functions in the immune system of the skin. After activation, possibly by antigen or by products of epider- mal cells, LCs migrate through the dermis and the afferent lymph to draining Here the LC presents antigen to T cells in the paracortical T-cell area, with subsequent T-cell activation and ~ ro l i f e ra t ion .~~ It seems likely that cytokines are involved in the initial activation and migration of normal LCs. In the normal situation, this production is probably down-regulated once LCs start to migrate, providing an explanation for the limited presence of cytokines in lymph nodes with dermatopathic reaction. The abundant presence of cytokines in LCH cells and LCH lesions may thus indicate that a 'down-regulatory' signal is lacking in LCH, resulting in the characteristic accumulation and/or proliferation of LCs.

ACKNOWLEDGEMENTS

We thank Mr S. Noorman for assistance in preparing the microphotographs. This study was supported by the Histiocytosis Association of America and the Groningen Foundation for Pediatric Oncology.

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