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The DEL cell line isolated from a patient who died of malignant histiocytosis exhibits a reciprocal chromosomal translocation t(5;6)(5q35;6p21). The cells were analyzed for Ig(Jh), TCR beta-gene rearrangements and proto-oncogene expression pattern, using a panel of molecularly cloned probes that included c-fms, c-myc, c-myb, c-pim, c-fos, N-myc, c-sis, c-fgr as well as the virally derived probes v-ki-ras and v-src. Consistent levels of expression of c-fms, c-myc, c-myb, c-ki-ras and c-fgr were identified in cells from several in vitro passages as well as from the heterotransplanted tumors in nude mice. Transcripts homologous to the c-fos, c-src and c-sis were not observed. Southern blot study of DNA showed that the banding pattern of the screened proto-oncogenes was not altered. Furthermore, Southern blot analysis demonstrated monoallelic immunoglobulin heavy chain (IgJh) rearrangement but a normal germ-line configuration of the kappa light chain and TCR beta-genes. These results appear to imply that a T- or B-cell origin can be eliminated and that several activated proto-oncogenes, usually expressed in immature MPS cells (c-fms) and myeloblastic cells (c-fgr), may be implicated in the proliferative activity of the DEL cell line, the stem of which may be a primitive, ancestral myelomonocytic cell.

In vitro explantation of 38 fragments of eosinophilic granuloma of bones was attempted. A satisfactory growth was obtained in nearly 90% of cases. This short-term culture maintained the ultrastructural characteristics and, to a lesser extent, the cytochemical features of the Langerhans cells, confirming the Langerhans cell origin of this cell proliferation. In addition, this procedure was able to demonstrate an immunodependent erythrophagocytosis (3/3) and a preferential fixation of labelled precursors (Glycerol, choline of lipid metabolism as well as labelled dopamine (2/2). All attempts to obtain a permanent cell line and graft to nude mice (even irradiated) failed. Under the in vitro conditions, the Langerhans cells do not divide up but can be readily identified up to 2 or 3 weeks. The contrast between the evident in vivo proliferation and the in vitro quiescent state suggests that some undetermined growth-factors targeted to the Langerhans cell system are missing in our commonly used culture media. The in vitro culture procedure could be of some help to their identification.

The production of TNF-alpha and IL-1 alpha and beta molecules has been shown to be associated with the proliferation and activation of cells of the monocyte/macrophage series, the intermediate steps in the synthesis of these molecules have been less investigated. Unstimulated and TPA stimulated DEL cells (a CD30-positive, t(5;6)(q35;p21) malignant histiocytosis cell line) were used to study the expression of TNF-alpha and IL-1 genes and to evaluate, by nuclear run-on assay and biological measurements, the control of their transcription and the level of protein production. To refine this analysis, the effects of cycloheximide and actinomycin D were also evaluated in this investigation. Following TPA stimulation, transcription of TNF-alpha (constitutively present) increased threefold as early as 30 mins and started decreasing by 24h. Cycloheximide superinduced the expression of TNF-alpha mRNA and, accordingly, the release of its protein. By contrast, transcription of IL-1 molecules appeared de novo and did not result in a biologically detectable protein. Measurements of RNA half line after actinomycin D indicated that TNF-a and IL-1 alpha mRNAs are not as stable as that of IL-1 beta. These results indicate that, despite their common synergistic activity, the transcriptional and post-transcriptional mechanisms regulating the synthesis of TNF-alpha and IL-1 alpha and IL-1 beta involve different pathways.

Acute promyelocytic leukemia (APL) is thought to be caused by the t(15,17) translocation that fuses the PML gene to that of the retinoic acid receptor alpha (RAR alpha) and generates a PML/RAR alpha fusion protein. Yet, paradoxically, APL cells are exquisitely sensitive to retinoic acid (RA), as they terminally differentiate upon RA exposure. In this report, we have examined the expression of PML and PML/RAR alpha in normal and APL cells. By immunofluorescence or immunocytochemistry, we show that PML has a speckled nuclear pattern of expression that contrasts with that of PML/RAR alpha (mostly a micropunctuated nuclear pattern or a cytoplasmic localization). The APL- derived cell line NB4 that expresses both the PML and PML/RAR alpha genes also shows the fine micropunctuated nuclear pattern, suggesting a dominant effect of the fusion protein over the localization of wild- type PML. RA treatment of NB4 cells or clones expressing PML/RAR alpha gradually leads to a PML pattern before apparent morphologic maturation. In 14 untreated APL patients, the PML-reactive proteins were cytoplasmic (by immunocytochemistry) or both cytoplasmic and nuclear with a micropunctuated pattern (by immunofluorescence). Strikingly, in 4 patients, after 1 to 2 weeks of RA therapy, the speckled nuclear PML pattern reappeared concomitant with the onset of differentiation. These results establish that fusion of PML to RAR alpha results in an altered localization of PML that is reverted upon RA treatment. This observation, which highlights the importance of PML, is likely to be a key to unravelling the molecular mechanism of both leukemogenesis and RA-induced differentiation of APL.

Following exposure to phorbol ester (TPA), DEL cell line, a human malignant histiocytosis (MH) cell line, is able to differentiate along a macrophage phenotype and thus it provides a suitable model for analyzing the sequential and differential gene expression associated with monocyte/macrophage differentiation. C-myc, c-myb, c-fos, c-sis and c-fms expression were determined by Northern analysis at various times following TPA treatment. The results showed that TPA down-modulated the constitutive expression of c-myc, c-myb, and c-fms, mRNA to low but still detectable levels. Conversely, TPA-induced differentiation resulted in transient appearance of c-fos, whereas no change in the level of c-sis and actin transcripts were observed. Thus, the c-fms and c-sis genes appear to be regulated in a specific manner in this malignant histiocytosis derived cell line. Furthermore, these investigations demonstrated a constitutive CSF-1 gene expression which transiently increased at mRNA and also at protein level as evaluated by a murine bone marrow CFU bioassay. Through this drug-induced modulation, the DEL cell line offers an additional model for studying some of the subtle interrelations existing between a growth factor (CSF-1) and its receptor (c-fms) in the monocyte/macrophage system.

The clinicopathological data on 20 cases of malignant histiocytosis (MH) collected over a period of 30 years at the Hôpital des Enfants Malades (Paris) are reported. Childhood MH was characterized by disseminated, frequently tender lymphadenopathy (19/20), skin (8/20), bone (6/20), and soft tissue localizations (7/20). These features were usually accompanied by fever, deterioration of general condition, and hematological abnormalities including anemia, thrombocytopenia, and occasionally fibrinopenia. These manifestations were clinically suggestive of a diagnosis of a severe neoplastic blood disease, although this hypothesis was not entertained for a long time because of the initial absence of abnormal cells in the blood and bone marrow. MH was characterized by the proliferation of large "histiocyte-like," usually mononucleated cells. When suitable material was available, MH cells appeared to react positively with acid-phosphatase, alpha-naphthyl acetate esterase (ANAE), alpha-antichymotrypsin, and antibodies directed against EMA, HLA DR, CD25, CD30, CD68, and CD71. No B- and T-cell antigens (except for one case) have been detected. Due to the frequent abundance of accompanying granulocytes, lymphoid, and plasma cells, and the presence of areas of necrosis, an initial correct diagnosis of MH was often difficult to establish on skin (four cases), bone (two), and soft tissue (three) biopsies. In lymph nodes, the sinusoidal and perifollicular topography of cell proliferation represented a highly reliable morphological feature. A permanent cell line (DEL) was obtained from a pleural effusion showing a t(5;6)(q35;p21) translocation and a monoallelic immunoglobulin (IgjH) rearrangement and consistent levels of expression of c-fms, c-myc, c-myb, c-ki-ras and c-fgr. Since an identical 5q35 breakpoint has been reported in four other MH cell lines with a comparable phenotype and in several isolated published cases, this chromosomal abnormality provides a highly valuable argument for individualizing an authentic malignancy of the mononuclear phagocyte system (MPS) in childhood, among the rather heterogeneous group of the CD30+ anaplastic large cell lymphomas.

The histiocytic or lymphoid origin of human malignant histiocytosis is currently a subject of debate. The aim of this study was to investigate the in vitro effects of 12-O-tetradecanoylphorbol-13-acetate used as a differentiation inducer on the CD30, t(5;6)(q35;p21) DEL cell line, taken to be a reliable representative of the human malignant histiocytosis cell line. Treatment of DEL cells with 33 nM 12-O-tetradecanoylphorbol-13-acetate for 6-24 h resulted in cell surface attachment (up to 80%), decrease in dividing ability, enhancement of nitro blue tetrazolium reducing capacity (from 8 to 42%), occurrence of a limited immunodependent phagocytosis, and transient increase in expression of tumor necrosis factor alpha gene and in production of tumor necrosis factor alpha protein, whereas tumor necrosis factor beta remained undetectable. From these data, we can conclude that the malignant histiocytosis DEL cell line is not of lymphoid origin but stems from a myelomonocyte lineage.

Summary Expression of HLADR, I, i blood group antigen and T6 antigen were studied in Histiocytosis X cells and pulmonary alveolar macrophages using double labelling immunofluorescence technique or immuno-peroxydase procedure. Alveolar macrophages express simultaneously HLADR and i blood group antigen. Histiocytosis X cells, caracterized by HLADR and T6 antigens, and by their ultra-structural marker do not express i antigen. These results confirm the hypothesis that histiocytosis X cells constitute a specialized sub-population of the mononuclear phagocyte system.

Summary Histiocytosis X cells were demonstrated to react with T 6 antigen as well as with the M 1 and I 1 markers of monocytes using immuno-electron microscopy and double labeling immunofluorescence technique. The data confirm the close relationship existing between histiocytic X cells, Langerhans cells and dendritic cells, and suggest to consider the T 6 antigen either as an early differentiation marker of thymocytes or as a functional marker of Mononuclear Phagocyte System subpopulations.

Summary The ultrastructural and histochemical exploration of histiocytes X done by the means of Concanavalin A (Con A), peanut agglutinin (PNA) and BSPT demonstrate that the plasma membrane of histiocytes X shared some properties with all the other cells and is also specialized. The rod part of the X body has the same properties as the plasma membrane while its vesicular part differs and is closer to the inner membrane system of the cell. In consequence it is suggested that the rod part of the X body is a specialized pathway or shuttle for receptor linked glycoprotein exchanges in highly specialized cells.

Lytic activity of eosinophilic granuloma and other tumours was studied in vitro on collagen substrate. Collagen degradation was measured through the release of hydroxyproline-rich peptides into the medium. The in vitro lytic action was at a maximum in the case of EG and was correlated with the presence of histiocytic cells.

Histochemical study combined with differential extractions of lipids from biopsy fragments in three cases of eosinophil granuloma reveals a great amount and variety of lipidic categories existing in histiocytosis X cells : sterids, fatty-acids, triacylglycerols, phosphoglycerids, cholino-phospholipids, sphingomyelins, glycolipids. Comparison of the lipidic content of histiocytosis X cells and that of normal histiocytes, showed a preponderance of cholesterol esters in histiocytosis X contrary to the normal macrophages and alteration in phospholipidic control of cholesterol esterification. This study confirms that the histiocytosis X cells have an active and probably preferential lipid metabolism, explaining the frequent xanthomatous changes of the lesions and the intense production of various cytoplasmic membranous structures.

Ultrastructural and immunohistochemical analysis of six specimens revealed for the first time a small number of Langerhans cells in non-neoplastic bronchial epithelium. These cells were usually found either interspersed perpendicularly between columnar epithelial cells or just above the basal lamina with extending cytoplasmic processes. Usually few Birbeck granules, especially located in the Golgi region, were present throughout the cytoplasm. Immunohistochemical studies were performed on semi-thin sections with a polyclonal anti-S-100 protein. The population of S-100 positive cells represented about 1% of all the epithelial cells. Not all ultrastructurally identified Langerhans cells were shown to be positive for S-100 antigen. Our results suggest that Langerhans cells could be a constant cellular constituent for the normal bronchial epithelium. The exact function of Langerhans cells in the respiratory epithelium remains to be investigated, but they may have an immunologic function, such as antigen presentation to T lymphocytes.

Using 14C glycerol and 3H glycerol, in a study of lipid metabolism of two cases of eosinophilic granuloma of the bone, the authors point out that this precursor is preferentially but not electively incorporated by mononuclear cells. This fixation particularly concerns the big cells with numerous lipid droplets and few X bodies. The precursor is essentially involved in neutral lipid synthesis. As is the case with common macrophages, this lipid synthesis might express various states of cellular maturation and ageing.

The Langerhans cell system including the Langerhans cell (LC), the indeterminate epidermal cell, the lymphoid interdigitating cell, and the lymph veiled cell is nowadays considered as a very peculiar subpopulation of the mononuclear phagocyte system. The authors have tried in this review to point out the main salient features, the qualitative and quantitative variations of these cells during physiological, experimental and pathological processes.

Immunohistochemical study of 7 cases of histiocytosis X (HX) and of histologically normal skin shows that HX Langerhans cells react with Bl.49.9 monoclonal antibody which recognizes the Interleukin-2 receptor, whereas normal skin Langerhans cells do not. This reactivity suggest activation of HX Langerhans cells.

The anatomo-clinical investigation of a series of 12 cases of AIDS in infants, over two years in Hôpital des Enfants Malades (Paris) allows the following observations to be made: the frequency of materno-foetal contaminations (8/12); the relative shortness of the incubation period (3-6 months); the variable degree of the atrophy of the lymphoid tissues; the absence of vascular proliferation; the interest of demonstrating viral proteins associated with LAV virus (p 18) in imprecisely identifiable cells of lymphoid tissue; the severity of the thymic alterations, which are characterized less by a lymphocyte depletion than by a loss of the maturation of the epithelial tissue, which still preserves its hormone secretory properties.

Alveolar macrophages of non smoking and smoking human adults reduce BSPT salt spontaneously. The staining obtained is located on three cell membrane systems: the endoplasmic reticulum, the Golgi apparatus and the nuclear envelope. Methylene blue MB inhibits BSPT reduction. The smokers alvelolar macrophages have less positivity than those of the non smokers. The endogenous cell substrate revealed in this work is the initial common pathway of two different oxidative chains bounded to the microsome. One acts with cytochrome P 450 for chemical detoxification by hydroxylation, the other one acts with cytochrome B5 for lipid oxidation or peroxidation and both may be connected with the cell bactericidal system.

We have investigated the secretory function of cell suspensions from bone eosinophilic granulomas surgically collected in two patients with histiocytosis X. Unseparated cell preparations spontaneously produced interleukin 1 (IL-1) and prostaglandin E2 (PGE2). In order to ascertain that this secretion was due to the characteristic Langerhans cell-like histiocytosis X cells predominantly found in the bone lesions, we have purified T6+ cells by the use of a fluorescence-activated cell sorter. Such highly purified cell preparations were found to secrete IL-1 and PGE2 spontaneously in culture. Stimulation with endotoxins and treatment with interferon gamma (IFN gamma) revealed an intense IL-1 secretory function of histiocytosis X cells. Since both IL-1 and PGE2 are able to induce bone resorption in vitro, our findings are compatible with the hypothesis that histiocytosis X cells are responsible for the typical osteolytic lesion observed in histiocytosis X through the local secretion of these two mediators.