The Elusive Hodgkin Cell

Scand J Haematol(l981) 26,353-358

ANNOTATION

The Elusive Hodgkin Cell

i

0036-553X/81/050353-06 $02.50/0 0 1981 Munksgaard, Copenhagen

The past few years have seen several im- portant studies pertaining to the classification of Hodgkin cells. However, it is still a curious fact that - almost uniquely among haematological malignancies - the origin of the malignant cell in Hodgkin’s disease re- mains an enigma.

Several explanations may be offered for this state of ignorance: the tissue in Hodg- kin’s disease is extremely heterogenous, and the specific cells are difficult to isolate for further study; none of the methods used to classify cells are absolutely specific. Even more discouraging, however, is the fact that no clear-cut answers’can be given to such fundamental questions: are certain cells specific for Hodgkin’s disease, and is Hodg- kin’s disease a true malignancy?

Most investigators consider that the Reed-Sternberg cells and the mononuclear Hodgkin cells described by Lukes & Butler (1966) are the characteristic cells in Hodg- kin tissue, although it is rather unusual that tumor cells constitute less than 10% of the tumor mass, as is the case in this extraordinary neoplasm. Still, although the presence of these ‘Hodgkin cells’ is considered a prerequisite for the diagnosis, the most characteristic of these cells, the Reed-Stern- berg cells, are not specific for Hodgkin’s disease. Thus, Reed-Sternberg cells have been described in such disorders as mono- nucleosis (Lukes et a1 1969, Strum et a1 1970, Agliozzo & Reingold 1971), rubeola, thymoma, carcinoma and various haematological malignancies (Strum et a1 1970).

Evidence that Hodgkin’s disease is a malignant disease of clonal origin stems main- ly from cytogenetic studies. Chromosome analysis has shown a remarkable aneu- ploidy, mostly in the range of hypotetra- ploidy; in a review Kaplan (1980) enumer- ates at least 100 chromosome studies, 40 of which demonstrated marker chromosomes, and at least half of these showed clonal distribution of the marker chromosomes. Studies using the method of glucosed- phosphate-dehydrogenase isoenzymes for the demonstration of clonality have, to our knowledge, not been published. Studies of DNA-synthesis based on the incorporation of 3H-thymidine after in vitro incubation of tissue samples or in vivo injection in lymph nodes have clearly demonstrated the pro- liferative capacity of both the mononuclear Hodgkin cell and the Reed-Sternberg cell (Peckham & Cooper 1973, Schiffer 1973).

On balance, then, it is likely that Hodg- kin’s disease is a malignant disorder derived from a special cell type, although the very special accumulation of the other cellular elements in Hodgkin tissue may give rise to many speculations on the pathogenesis of this neoplasm, especially concerning the possibility of cell-cell interactions between the different cell types. In the following we shall not consider this further but limit our- selves to a discussion of studies concerned with the classification of mononuclear Hodgkin cells and Reed-Sternberg cells. For the sake of clarity we shall consider the different studies grouped according to the

354 NIELS EBBE HANSEN & HANS KARLE

methods used to solve the riddle, even if of course several methodological overlappings are found.

Electron microscopy Although in light microscopy Hodgkin cells may resemble macrophages, electron micro- scopic studies have demonstrated similarities to lymphocytes and a lack of features usually found in macrophages (Dorfman et a1 1973, Glick et a1 1976, Hayhoe et a1 1978). There are abundant ribosomes in the cells, indicating an active protein synthesis, but no organelles concerned with phagocytosis (lysosomes, endocytic vesicles) or granula. Even Carr (1975), who concluded that Hodgkin cells were probably derived from macrophages, found only a few lysosomes in the cells. Scanning electron microscopy has identified a large cell surrounded by clusters of lymphocytes and morpholog- ically distinct from these. However, these studies have not been able to identify these large cells further (Braylan et a1 1974, Schnitzer & Mead 1975).

Cytochemical studies These have given somewhat conflicting results, although again a lymphocyte origin is favored. Thus, whereas Braunstein et a1 (1962) showed that Reed-Sternberg cells were positive for non-specific esterase, favoring a macrophage origin of the cell, neither Dorfman (1961) nor Hayhoe et a1 (1978) could confirm this finding. Dorfman noticed a general lack of hydrolytic en- zymes in Hodgkin cells, which agrees well with the structural lack of an apparatus concerned with phagocytosis. Hodgkin cells were mostly PAS negative (Hayhoe et a1 1978).

Immunochemical studies The demonstration that intracellular immunoglobulin (nearly always IgG, rarely IgA; IgM, IgD and IgE have never been demonstrated) was found in Hodgkin cells (Garvin et a1 1974, Payne et a1 1976) led initially to the assumption that Hodgkin cells were derived from B-lymphocytes. However, the subsequent observation that both kappa and lambda chains were found within the same Hodgkin cells is not com- patible with clonal Ig production by lymphocytes and is not found in other B-lymphocyte disorders (Taylor 1976, Papadimi- triou et a1 1978, Landaas et a1 1977, Pop- pema et a1 1978).

An alternative would be that polyclonal Ig was phagocytosed (or pinocytosed) by the Hodgkin cells, which then in this respect would resemble macrophages. This, however, would be difficult to reconcile with the ultrastructural studies which showed a lack of an apparatus for phagocytosis.

Ultrastructural localization of intracellular Ig has given conflicting results. Bernuau et a1 (1979) and Reynes et a1 (1979) found Ig in close approximation with polyribosomes which would be expected if Ig was produced within the cell. Poppema (1978), on the other hand, found Ig without any associa- tion to any protein synthesizing organelle and therefore believed that Ig had entered the cell through a possibly defect cell membrane.

These studies leave us with the unhappy alternative that either Hodgkin cells produce polyclonal Ig within one cell, which would, indeed, be extraordinary, or that Ig enters the cell from the outside, although the cells do not possess the apparatus for phagocytosis.

The intracellular presence of other proteins has also been examined, especially

THE ELUSIVE HODGKIN CELL 355

lysozyme which is produced in macrophages (and in myeloid precursors). Lysozyme is usually considered a reliable macrophage in- dicator. Taylor could not demonstrate lysozyme in Hodgkin cells (Taylor 1976) and Papadimitriou et a1 (1978) found intracellular lysozyme in only 1 out of 44 patients. However, Hansen et a1 (1981) found lysozyme in Hodgkin cells in 10 out of 33 patients, and intracellular lysozyme was also demonstrated by Ree et a1 (1980), who also found lysozyme around Hodgkin cells pre- sumably resulting from lysozyme excretion by the cells. The fact that lysozyme posi- tivity was not a constant finding may be related to technical, physiological and pathological (dedifferentiation) factors; thus it is known that macrophages produce varying amounts of lysozyme according to the degree of stimulation and differentia- tion. Other proteins have been found in- constantly in Hodgkin cells. Papadimitriou et a1 (1978) found intracellular albumin and alpha-l-antichymotrypsin in 12 out of 44 patients, whereas Kadin et a1 (1978) found neither albumin nor fibrinogen in Hodgkin cells. In a recent study Resnick & Nachman (1981) found fibrinectin in Hodgkin cells, which supports the macrophage origin, whereas they failed to find unspecific proteins like factor VIII antigen, albumin, fibrinogen, alpha-2-macroglobulin, anti- thrombin 111, and ceruloplasmin.

Functional studies An inherent difficulty in the immunohistochemical studies is that they were carried out on fixed tissue treated with different techniques. Kadin et a1 (1978), however, performed immunological studies on freshly isolated Hodgkin cells and achieved re- markably clear-cut results. They showed that in all cases Hodgkin cells had intra-

cytoplasmic Ig of both kappa and lambda types, and that the cells also had surface Ig of the same type. Furthermore, they showed that Hodgkin cells would ‘inter- nalize’ aggregated exogenous Ig and im- mune complexes, and they confirmed the presence of Ig Fc receptors and complement receptors on the cell surface. Since they could not demonstrate albumin and fibrinogen in the cells they postulated that the cellular uptake of Ig was ‘non-random’, dependent on the presence of surface Ig receptors. They concluded that Hodgkin cells were most likely derived from macrophages.

This still leaves us with the difficult question how we should interpret the findings that Hodgkin cells may take up material from the surroundings even if ultrastructural studies have indicated that the cells do not possess the necessary build-up for phagocytosis. ‘Internalization’ of blood cells by Hodgkin cells has been demonstrated for both erythrocytes, lymphocytes and granu- locytes (Quaglino et a1 1977, Brooks 1979, Shervin & Margolick 1979). The term emperipolesis has been suggested meaning the migration of lymphocytes over the surface membrane into the cytoplasm of other cells. This problem of internalization is far from settled, but at present caution is war- ranted before uptake of cells or other material from the surroundings is taken as evidence that the cells in question are macrophages. The complexity of this question ap- pears also from the findings that a potential of ‘internalization’ has also been described in leukaemic B-lymphocytes (Catovsky et a1 1977), ALL lymphocytes (Foadi et a1 1978), hairy cells (Marmont et a1 1978), and Ty lymphoma cells (Kadin et a1 1981).

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Cell culture studies During the past few years 3 long term cell culture studies of Hodgkin cells have ap- peared (Boecker et a1 1975, Kaplan & Gart- ner 1977, Long et a1 1977). In the first study Boecker et a1 cultured cells in diffusion chambers from 2 patients with pleural ef- fusion. They found that their cultured cells retained the morphological appearance of mononuclear Hodgkin cells and Reed-Stern- berg cells and had chromosome abnormalities. Since the cells had surface Ig even after 50 d of culture the authors concluded that the Hodgkin cells were derived from B-lymphocytes.

The conclusion of two subsequent studies (Kaplan & Gartner 1977, Long et a1 1977) was, however, that their cultured Hodgkin cells had macrophage characteristics and no lymphocyte markers, although the results of the two studies were not identical. In the study by Kaplan & Gartner the cultured cells (from Hodgkin spleens) had the morphological appearance of Hodgkin cells, displayed chromosome abnormalities and had invasive properties after heterotransplantation. The cells were adherent, could phagocytose, had FC and complement receptors and produced lysozyme. Especially as far as the latter feature is concerned, however, some macrophages were present in the culture, which somewhat weakens this otherwise powerful argument for the macrophage origin of the cultured cells. The cultured cells lacked several lymphocyte markers. In the study by Long et a1 (1977) the cells were not adherent, could not phagocytose, but did produce lysozyme and were esterase positive. These cells also lacked lymphocyte markers.

It is difficult to reconcile the findings of these 3 culture studies, and it is difficult to believe that they have cultured the same

cell type. The cell culture approach, however, is exciting and is likely to yield more conclusive results in the future. In fact, it has now been demonstrated that the cell lines studied by Long et a1 (1977) were not derived from Hodgkin cells, but stemmed from con- taminating owl monkey cells (Harris 1981).

Conclusion It may be stated that if Hodgkin cells are derived from a well defined cell type the Hodgkin cells must be modified to a con- siderable extent compared to the theore- tical normal counterparts, either by losing characteristics, by acquiring new features, or by a combination of both. Otherwise it is difficult to explain why methods which in other haematological disorders have been successful in classifying malignant cells have failed and produced so many conflicting results in Hodgkin’s disease.

At present the trend of emerging results favor a macrophage origin, whereas it has been impossible to establish a lymphocyte parenthood. However, we are still left with observations which do not fit with this con- cept, and at this junction it may be pertinent to question the relevancy of the search for a parent cell. Alternative speculations could be e.g. that the Hodgkin cells were derived from a very primitive common stem cell which might have properties from more than one cell type, or that the cells are hybrids derived from 2 different cell types.

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NIELS EBBE HANSEN, M.D. & HANS KARLE, M.D. Division of Haematology Department of Medicine Hvidovre Hospital DK-2650 Hvidovre, Denmark

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The Elusive Hodgkin Cell