Immunohistochemical characterization of two new monoclonal antibodies (LN-4, LN-5) reactive with human macrophage subsets and derived malignancies in B5-fixed, paraffin-embedded tissues

Two monoclonal antibodies (LN-4, LN-5) reactive to human macrophages in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by using deparaffinized cell extracts of peripheral blood mononuclear cells. Both monoclonal antibodies were initially identified on paraffin-embedded sections of hyperplastic lymph nodes by using the immunoperoxidase staining procedure. Specificity screens on normal human tissues show that LN-4 and LN-5 stain the cytoplasm of macrophages and histiocytes in hematopoietic organs including Kupffer's cells of the liver and Langerhans' cells of the skin. LN-4 also showed strong positivity with acini of the stomach, whereas LN-5 was positive with mantle zone B lymphocytes of the lymph node and spleen, spermatogonia, and chief cells of the stomach. Both antibodies were strongly reactive with cases of true histiocytic lymphoma but, except for infiltrating macrophages, were entirely negative in Hodgkin's disease and non-Hodgkin's lymphomas. In all cases of nodular sclerosis Hodgkin's disease, LN-4 was positive in macrophagelike cells present in the collagen bands surrounding the Hodgkin's lesions. Both monoclonal antibodies were also positive in macrophages and histiocytes present in a variety of benign lymphoid lesions including persistent generalized lymphadenopathy, Gaucher's disease, sinus histiocytosis, and dermatopathic lymphadenopathy. Because of their specificity for human macrophages, and their ability to stain B5-fixed, paraffin-embedded tissues, LN-4 and LN-5 are important new reagents for the diagnosis and classification of malignant and benign histiocytic lesions.     

Histiocytes and histiocytosis [see comments]

The term histiocyte refers to cells of either the macrophage or Langerhans cell lineages. The histiocytic disorders are characterized by the proliferation of cells of these lineages. With recent advances in knowledge of the developmental biology of histiocytic cells, it is now possible to formulate a reasonable catalogue of histiocytic diseases based on ultra-structural and phenotypic markers of cellular origins and molecular or chromosomal markers of malignancy. The catalogue includes the following groups of diseases. Nonmalignant reactive macrophage disorders include (1) macrophage storage diseases, (2) several benign proliferative macrophage disorders that predominantly involve skin and bone, and (3) several hemophagocytic syndromes that vary from indolent and benign to fulminant and fatal. In some of the latter disorders, viruses have been identified as the inciting stimulus. The malignant macrophage disorders include (1) acute monocytic leukemia and (2) chronic myelomonocytic leukemia. A rare disorder that gave rise to a permanent cell line with an anomaly of chromosomal segment 5q35 may also be an example of a histiocytic malignancy. The existence of a separate category of true histiocytic lymphoma of macrophage type is uncertain. Reactive Langerhans cell disorders include (1) congenital self-healing histiocytosis, (2) the many variants of eosinophilic granuloma, and (3) a related disorder designated as relapsing Langerhans cell histiocytosis that is characterized by a relapsing course and infiltration of bone and soft tissues by Langerhans cells. Presumptively neoplastic diseases of Langerhans and dendritic cells include (1) progressive Langerhans cell histiocytosis, a disease with prominent involvement of blood and BM as well as skin and viscera; (2) Langerhans cell lymphoma, and (3) dendritic cell lymphoma. However, clonality as a marker of malignancy has not been proven in these disorders.     

True histiocytic lymphoma: histopathologic, immunophenotypic and genotypic analysis

Five cases of true histiocytic lymphoma (THL) were analysed by immunophenotyping and immunoglobulin/T-cell receptor genotyping. These cases showed striking morphologic diversity but a strong degree of immunophenotypic homogeneity. The malignant cells reacted with multiple histiocytic markers including CD11c (Ki-M1, LeuM5), CD14, CD68 (Ki-M6) and Ki-M8; anti-HLA-DR and non-specific esterase staining was also found in all cases. The malignant cells did not express monoclonal immunoglobulin and did not react with the B- or T-cell monoclonal antibodies used except for those known to be cytoplasmically expressed in monocytes/histiocytes, such as CD4 and CD19; B- and T-cell staining was otherwise limited to background small lymphocytes. By genotypic analysis, three cases showed rearrangements: one with T beta, one with T beta and immunoglobulin heavy chain (JH) and one with both JH and light chain; the remaining two cases retained their immunoglobulin and T-cell receptor genes in germline configuration. The results not only suggest that certain subsets of the histiocyte/reticulum cell system may be capable of rearranging immunoglobulin or T beta genes while simultaneously expressing multiple histiocytic surface antigens but also demonstrate the necessity of using multiple histiocytic-specific monoclonal antibodies and cytochemical staining in diagnosing THL. Gene rearrangement studies must be interpreted in conjunction with immunophenotyping and morphology in the determination of cell lineage.     

Electron-microscopic aspects of Hodgkin's disease

Summary Lymph nodes from patients with Hodgkin's disease of the nodular sclerosis or mixed cellularity type were examined by electron microscopy to classify all the cells that occur in these types of lymphoma. Most of the cells showed morphological features that were the same, or nearly the same as those of cells of normal lymphoid tissue. These included typical interdigitating reticulum cells (IDC), histiocytic reticulum cells, so-called dark reticulum cells, and sinus macrophages. There were also small and medium-sized lymphocytes, immunoblasts, plasma cells, and plasma cell precursors resembling those seen in non-specific lymphadenitis. Germinal center cells, on the other hand, were present in negligible numbers.Special attention was paid to Hodgkin's (H) and Sternberg-Reed (SR) cells. This group of cells proved to be heterogeneous. The only common features were a large cell size, large nuclei, and a prominent nucleolus. Some of the H and SR cells resembled immunoblasts of normal lymphoid tissue. The cytoplasm of these cells contained numerous polyribosomes, and their heterochromatin was coarsely condensed at the nuclear membrane. Other H and SR cells were more similar to histiocytic cells or reticulum cells because of the large number of cell organelles (e.g., lysosome-like granules) and diffuse heterochromatin. Finally, cases of the nodular sclerosis type of Hodgkin's disease showed another cell type with some resemblance to IDC. The cells of this type are called lacunar cells because of their special light-microscopic appearance.Abbreviations H Hodgkin - IDC interdigitating reticulum cell - SR Sternberg-Reed     

Childhood Ki-1 lymphoma presenting with skin lesions and peripheral lymphadenopathy

We describe a large-cell lymphoma of activated lymphoid cells in six children and adolescents. The presenting clinical features of regressing skin lesions and peripheral lymphadenopathy, sinus infiltration of lymph nodes, and infrequent tumor cell erythrophagocytosis resulted in initial diagnoses of malignant or regressing atypical histiocytosis in five cases. Binucleate and multinucleate tumor cells, sometimes with prominent eosinophilic nucleoli, resembled Reed-Sternberg (RS) cells and occasionally were found in a cytoarchitectural milieu that was consistent with a diagnosis of Hodgkin's disease (HD). The tumor cells did in fact express the HD-associated antigen Ki-1, but unlike most types of HD, the RS-like cells expressed common leukocyte antigen and were negative for Leu-M1. A T cell origin for the malignant cells was demonstrated with monoclonal antibodies in two cases, by focal staining for nonspecific esterase in one case, and by rearrangement of the beta- chain genes for the T cell receptor in a fourth case. These studies provide further evidence that some cases previously interpreted as malignant or regressing atypical histiocytosis and some types of HD are actually T cell disorders.     

Cytochemical, immunologic, chromosomal, and molecular genetic analysis of a novel cell line derived from Hodgkin's disease

A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell.     

Histiocytic differentiation in benign and malignant bone tumors

Summary In this study fresh frozen tissue samples of benign osseous tumors (five non-osteogenic fibromas, one fibrous dysplasia, one chondromyxoidfibroma), tumors of uncertain biological behaviour (eight cases of histiocytosis X, two giant-cell tumors), and of malignant intraosseous tumors (two malignant fibrous histiocytomas, two malignant histiocytosis, four osteosarcomas, one chondrosarcoma and two Ewing sarcomas) were studied with a panel of monoclonal antibodies reactive with monocyte/macrophages and various types of dendritic cells. In addition, tumors were further defined with a broad spectrum of antibodies against filamentous proteins and lymphocyte differentiation antigens. The specimens were stained with a triple-layer immunoalkaline phosphatase protocol. Tumors stained with these antibodies could be roughly divided into two groups. The first group comprised tumors with one predominant cell population reactive with one particular monoclonal antibody. In this group, cases of histiocytosis X were found to be consistently labelled with CD-1 antibodies. The giantcell tumors showed a very homogeneous staining with certain monocyte/macrophage antibodies (Ki-M8). Nevertheless, even in these tumors, heterogeneity was demonstrated by the occurrence of cells with monocytic differentiation in histiocytosis X and conversely by the occurrence of cells with differentiation antigens of the dendritic cell system in giant-cell tumors. An exception has to be made for the two cases of malignant histocytosis examined. These tumors were selectively labelled with antibodies against monocyte/macrophages (Ki-M8, IOM-1). The second group comprised tumors showing a high degree of heterogeneity demonstrated by the varying amounts of tumor cells reacting with the applied markers of the monocyte/macrophage and dendritic cell systems. In most cases it was difficult to ascribe labelled cells to the tumor cell population as opposed to an innocent bystander inflammatory cell population. This distinction was especially difficult in malignant fibrous histiocytomas underlining the current concept that these tumors are of primitive mesenchymal rather than true histiocytic origin.This study was supported by Deutsche Forschungsgemeinschaft and Hamburger Stiftung zur Förderung der Krebsbekämpfung     

Potentiation of fibroblast growth by nodular sclerosing Hodgkin''s disease cell cultures

Cell cultures were established from 8 lymph nodes replaced by nodular sclerosing Hodgkin's disease. Serum-containing and serum-free conditioned media from these cultures potentiated fibroblast growth and were found to be consistently more potent than fibroblast growth factor, 100 ng/ml, every other day. Both a proliferative response and transformation-like growth were observed using BALB/c 3T3 cells, human diploid fibroblasts, and human embryonic fibroblasts as target cells. The Hodgkin's disease growth factor(s) was not produced by fibroblasts or lymphocytes in the Hodgkin's cultures and was most potent when the Hodgkin's cultures had been enriched with Hodgkin's giant cells. Removal of normal macrophages decreased the proliferative activity but did not eliminate it or nonadherent growth of 3T3 cells in agar. Control cultures of 6 nonmalignant lymph nodes, a Lennert's lymphoma, a mixed cellularity Hodgkin's disease lymph node, and a malignant histiocytosis cell line suggested that among lymph node disorders, this feature may be relatively specific for nodular sclerosing Hodgkin's disease.     

A case of true malignant histiocytosis: identification of histiocytic origin with use of immunohistochemical and immunocytogenetic methods

We report here an autopsy case of true malignant histiocytosis. The patient was a 67-year-old woman who exhibited fever, wasting, hepatosplenomegaly, and progressive pancytopenia. The bone marrow aspiration disclosed hemophagocytosing cells, which resembled histiocytes. The molecular analysis did not show the clonal gene rearrangement of T-cell receptor or immunoglobulin heavy chain. Although the patient had been started on methylprednisolone pulse therapy and chemotherapy with etoposide, she died from cerebral hemorrhage. The autopsy specimens of spleen and liver showed extensive infiltration of atypical cells, for which histiocytic origin was identified with an immunohistochemical method using monoclonal antibodies against CD11c, CD68, macrophage colony-stimulating factor (M-CSF), M-CSF receptor, lysozyme, antitrypsin and alpha1-antichymotrypsin. Recent investigations have disclosed that in most cases diagnosed as malignant histiocytosis, hemophagocytosis was reactive and not evoked by histiocytic malignancy. True malignant histiocytosis, for which histiocytic origin is confirmed, is extremely rare.     

The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells

Ki-1 is a monoclonal antibody (raised against a Hodgkin's disease- derived cell line) that, in biopsy tissue affected by Hodgkin's disease, reacts selectively with Reed-Sternberg cells. The expression of Ki-1 antigen has been analyzed by immunocytochemical techniques in a wide range of human tissue and cell samples, including fetal tissue, malignant lymphomas (290 cases), and mitogen- and virus-transformed peripheral blood lymphocytes. The antigen was detectable on a variable proportion of cells in all cases of lymphomatoid papulosis and angio- immunoblastic lymphadenopathy and in 28% of the cases of peripheral T cell lymphomas (including lympho-epithelioid lymphomas). It was also expressed (more strongly) on tumor cells in 45 cases of diffuse large- cell lymphoma, most of which had originally been diagnosed as malignant histiocytosis or anaplastic carcinoma, because of their bizarre morphology. However, all of these cases lacked macrophage and epithelial antigens. Thirty-five cases expressed T cell-related antigens (associated in nine cases with the coexpression of B cell- related antigens), seven bore B cell-related antigens alone, and three were devoid of T and B cell markers. DNA hybridization with a JH specific probe showed a germline configuration in 11 cases of T cell phenotype, in two cases lacking T and B cell antigens, and in one case of mixed T/B phenotype, while rearrangement was found in two cases of clear B cell type and in one mixed T/B case. Expression of the Ki-1 antigen could be induced, together with interleukin 2 (IL 2) receptor, on normal lymphoid cells of both T and B cell type by exposure to phytohemagglutinin, human T leukemia viruses, Epstein-Barr virus, or Staphylococcus aureus. The results obtained indicate that Ki-1 antigen is an inducible lymphoid-associated molecule that identifies a group of hitherto poorly characterized normal and neoplastic large lymphoid cells. Tumors comprised solely of these cells show both morphological and immunological similarities to the neoplastic cells in Hodgkin's disease. This suggests that both disorders represent the neoplastic proliferation of activated lymphoid cells of either T cell or, less commonly, B cell origin. Disorders in which only a minority of cells express Ki-1 antigen (lymphomatoid papulosis, angio-immunoblastic lymphadenopathy, and certain T cell lymphomas) probably represent lesions in which only some of the abnormal cells have transformed into an "activation state." In direct support of this view is the finding that the Ki-1 expression in these lesions is accompanied by the expression of HLA-DR and IL 2 receptors.     

Sialosylated lewis x expression in CD30-positive anaplastic large-cell lymphomas

Summary The expression of sialosylated Lewis ^x (SLEX), a ligand for endothelial leukocyte adhesion molecule 1 in malignant lymphomas, was immunohistochemically examined, using the monoclonal antibody, CSLEX1, which specifically reacts with SLEX. It was expressed in 6 out of 64 non-Hodgkin's lymphomas, which consisted of 1 nasal large-cell lymphoma and 5 of 8 (62%) Ki-1-positive anaplastic large-cell lymphomas (ALCL). One nasal lymphoma positive for SLEX co-expressed a T cell marker, cluster of differentiation (CD) 5, and natural killer (NK) cell markers such as CD56 and CD16, indicating that SLEX⁺ nasal lymphoma cells are possibly malignant counterparts of SLEX⁺ NK cells. SLEX did not react with 30 B cell lymphomas or most Hodgkin's disease lymphomas, though it did with one lymphocyte predominance type. Although SLEX⁺ ALCL exhibit T cell markers in some cases, some ALCL expressing SLEX may represent histiocytic differentiation of the neoplastic cells. The lymphoma cells of ALCL were preferentially positive for SLEX, in contrast to Hodgkin's disease cells, and thus CSLEX1 in conjunction, with CD30 and CD15 should be of use for analyzing and making differential diagnoses of routine paraffin-embedded sections of ALCL.Abbreviations SLEX sialosylated Lewis ^x - ALCL anaplastic large-cell lymphomas     

A single monoclonal antibody identifies T-cell lineage of childhood lymphoid malignancies

Immunophenotyping studies with monoclonal antibodies have revealed the heterogeneity of childhood acute lymphoblastic leukemia (ALL) and non- Hodgkin's lymphoma (NHL). The lymphoid malignancies of T-cell lineage are particularly heterogeneous and, until now, no single monoclonal antibody has been found to identify all cases of T-ALL and T-NHL. A monoclonal antibody, 4H9, recognizes an antigen of 40,000 molecular weight on normal and malignant T cells. Thirty-six cases of childhood T- ALL and T-NHL were tested, and in all cases, the malignant blast cells were reactive with 4H9, whereas malignant cells from 61 cases of non-T ALL and NHL were not reactive with 4H9. Monoclonal antibody 4H9 is a sensitive and specific reagent for the identification of childhood T- cell ALL and NHL and should be extremely useful in immunophenotyping studies of lymphoid malignancies.     

Human lymphoid cells express epithelial membrane antigen. Implications for diagnosis of human neoplasms

Epithelial membrane antigen (EMA) is generally assumed to be expressed only on epithelial cells. However, EMA was also found on reactive and neoplastic plasma cells, on some non-Hodgkin's lymphomas (particularly cases of T-cell lymphoma and "malignant histiocytosis"), and on Reed-Sternberg cells in cases of lymphocyte-predominant Hodgkin's disease. EMA was also induced on normal blood lymphocytes by exposure to human T-lymphotropic virus type II or phytohaemagglutinin. The specificity of these aberrant reactions was confirmed by the use of three different monoclonal anti-EMA antibodies (E29, HMFG2, and LICR.LON/M8), and by showing that EMA in lymphoid tissue is similar in molecular weight to EMA in human milk. These results indicate that additional anti-epithelial antibodies (eg, anti-cytokeratins) should be used in conjunction with anti-EMA for tumour diagnosis.     

Immunohistological analysis of Hodgkin's and Sternberg-Reed cells: Detection of a new antigen and evidence for selective IgG uptake in the absence of B cell,T cell and histiocytic markers

Summary To help clarify the origin and nature of Hodgkin's (H) and Sternberg-Reed (SR) cells, three different sets of experiments were performed. First, it was shown that cytoplasmic , , and occur not only in H and SR cells, but also in polymorphic tumor cells of epithelial, neurogenic, and lymphoid origin. Furthermore, human IgG that was injected i.v. into rats penetrated many rat liver cells, whereas injected human ₁-antitrypsin did not. Second, staining of frozen section revealed that H and SR cells lack surface immunoglobulin and peripheral T-cell antigen. Third, an antiserum raised against the L 428 cell line (derived from Hodgkin's disease) and absorbed with human serum and normal cells did not react with any cells of tonsil tissue (lymphoid cells, histiocytes, and interdigitating reticulum cells), whereas it reacted strongly with the L 428 cell line cells and with H and SR cells of 10 different cases. In all ten cases, the antiserum stained the surface of H and SR cells; in two cases, it also stained the nucleoli and some chromatin spots in H and SR cells.The results obtained in these experiments indicate that H and SR cells are not closely related to lymphoid cells, histiocytes, or interdigitating reticulum cells. The findings also suggest that H and SR cells express one or more antigens that have not yet been detected on or in normal cells.Abbreviations H Cell Hodgkin's cell - SR cell Sternberg-Reed cell - C cytoplasmic - Ig immunoglobulin Supported by the Deutsche Forschungsgemeinschaft, SFB 111, Project D8, and Di 184/6, by the Schleswig-Holsteinische Krebsgesellschaft, and the Kind Philipp Stiftung     

Hodgkin and Sternberg-Reed cells--cell origin, cell marker, cell function

The controversy in literature with regard to the origin of Hodgkin's and Reed-Sternberg cells persists, however only two conceptions seem to be plausible at present: the first is based on the relation to histiocytic elements, the second postulates lymphocytic precursors. The significance of surface-markers of these malignant cells in cryostat-sections and in cell-cultures and the relevance of their functional properties are discussed with respect to pathophysiology, clinical appearance, diagnosis and prognosis of Hodgkin's disease. The authors present two tendencies in the classification of malignant lymphomas based on the present knowledge achieved especially by monoclonal antibodies: the first includes aspects of integration between non Hodgkin's lymphomas and Hodgkin's disease, illustrated by the Ki-1-lymphoma, the second is related to separation of entities of the group of Hodgkin's lymphomas (for example the nodular paragranuloma). The aetiopathogenesis of Hodgkin's disease is considered as a causal trinity of virus infection, genetic determination and immunologic predisposition.     

B lymphocyte antigens in the differential diagnosis of human neoplasia.

Human B lymphocyte antigens (HBLA) were detected with fluorescent-labeled antibodies on malignant cells of 102 patients with Hodgkin disease and other lymphomas, plasma cell myeloma, and nonlymphoreticular neoplasms including carcinomas of the breast, lung, and ovary, soft tissue sarcomas, and neuroblastoma. HBLA were present in Hodgkin disease and other lymphomas of B cell or histiocyte derivation. They were absent in plasma cell myeloma and nonlymphoreticular neoplasms. Absorption studies revealed that malignant T cells had smaller amounts of HBLA, usually not detected by immunofluorescence. Expression of HBLA was dependent on both cell differentiation and origin. Detection of HBLA enabled immunologic distinction of Reed-Sternberg and other lymphoma cells from morphologically similar cells of nonlymphoreticular origin. The rapidity, reproducibility, and economy of the immunofluorescence test make this a useful clinical tool for the differential diagnosis of lymphoma from other malignant disorders in man.     

Increased sensitivity of T cells to regulation by normal suppressor cells persists in long-term survivors with Hodgkin's disease

We have studied the function of T cells in the peripheral blood obtained from long term survivors with Hodgkin's disease in order to determine the sensitivity of those T cells to normal suppressor cell immunoregulatory mechanisms. Concanavalin A-activated suppressor cells from normal donors suppressed the proliferation of lymphocytes obtained from 11 patients (56.8 +/- 3.5 percent) and from 28 allogeneic normal control subjects (39.8 +/- 2.7 percent [p less than 0.001]). When suppressor monocytes from the normal donors were studied, the mean proliferation of lymphocytes from 19 patients was suppressed 76.3 +/- 4.8 percent whereas proliferation of lymphocytes from 26 normal donors was suppressed 46.6 +/- 4.4 percent (p less than 0.0001). There was no tendency for the increased sensitivity to suppression that was observed in either assay system to return to normal as the patients' disease free interval increased from 1.5 years to 12 years. Furthermore, long-term survivors with diffuse histiocytic lymphoma, who had been treated with comparable chemotherapy, had normal sensitivity to the suppressor monocytes (45.1 +/- 3.8 percent). In Hodgkin's disease, the persistent increased sensitivity of T cells to two different normal immunoregulatory cells suggests that the response of the T cell to regulatory signals may be an important cause of the depressed cellular immunity observed in Hodgkin's disease and a clue to the etiology of the disease.     

胃原发性恶性淋巴瘤临床病理、免疫组化及超微结构研究

目的　探讨胃原发性恶性淋巴瘤的临床病理特点。方法　对 3 2例胃原发性非霍奇金恶性淋巴瘤的临床病理、免疫组化及超微结构进行观察。结果　 3 2例恶性淋巴瘤原发于胃底 3例 ,胃体 7例 ,胃角 8例 ,胃窦14例。全部病例做免疫组化染色 ,证实B细胞性淋巴瘤 3 1例 (96 9% ) ,T细胞性淋巴瘤 1例 (3 1% )。另外 ,对11例非霍奇金恶性淋巴瘤和 6例胃未分化癌进行了对比电镜观察 ,发现二者的超微结构有明显的差异。结论　绝大多数胃原发性恶性淋巴瘤为B细胞来源 ;免疫组化和超微结构观察对本病的诊断和鉴别诊断具有十分重要的意义。     

Reed-Sternberg cells in Hodgkin's cell lines HDLM, L-428, and KM-H2 are not actively replicating: lack of bromodeoxyuridine uptake by multinuclear cells in culture

We compared the proliferation of mononuclear and multinuclear cells in four Hodgkin's cell lines, HDLM-1, HDLM-1d, L-428, and KM-H2, by examining their capacity to incorporate bromodeoxyuridine (BrdUrd) into nuclei. Approximately 5% of all cells in HDLM-1 cultures had two or more nuclei, a characteristic of Reed-Sternberg (RS) cells. Unlike mononuclear Hodgkin's (H) cells, these RS cells exhibited no uptake, or only minimal uptake of BrdUrd, suggesting that they did not replicate actively. Cytogenetic study showed that 25% of the HDLM-1 cells contained a tetraploid (4X) set of chromosomes with a characteristic two-peak distribution. Following treatment of HDLM-1 cells with phorbol ester, the percentages of 4X cells and RS cells increased to 50% and 12%, respectively. This increase in RS cells was not likely to be due to cell fusion as shown by the absence of hybridization of BrdUrd- positive and -negative nuclei. Phorbol ester has a short-term effect of blocking the exit of cells from G1 into S phase, but no effect on the transition from S phase to G2/M phase. The block is more prominent in 2X cells than in 4X cells, which may explain the increase in percentage of 4X cells in phorbol ester-treated cultures. In addition, phorbol ester induced the differentiation of H-RS cells, which was accompanied by loss of the marker HeFi-1 from the cell surface. Approximately one third of the RS cells did not express HeFi-1, or expressed only minimal amounts. The findings led us to the following conclusions: (1) The 4X cells probably are formed from 2X H cells as a result of disturbed cytokinesis, but not a cell fusion. (2) A considerable number of 4X cells were H cells, because the number of 4X cells consistently exceeded that of RS cells. (3) Since mitotic figures are extremely rare in RS cells and these cells did not show active BrdUrd uptake, the increased number of RS cells must also be a consequence of disturbed cytokinesis of H cells or a result of nuclear transformation (twisting, convolution, or separation of the nucleus) in H cells. (4) Most RS cells lose their proliferating capacity and some RS cells may undergo further differentiation. Uptake of BrdUrd and phorbol ester induction were also studied on the other three H-RS cell lines, HDLM-1d, L-428, and KM-H2, with results similar to those for HDLM-1.     

Immunoglobulin gene rearrangements and expression in diffuse histiocytic lymphomas reveal cellular lineage, molecular defects, and sites of chromosomal translocation

We have examined the immunoglobulin gene configurations in cell lines from eight patients with diffuse histiocytic lymphoma in order to establish the cellular lineage and stage of differentiation of these lymphomas. The presence of heavy and light chain gene rearrangements as well as heavy chain class switching in seven cells placed these tumors within the B cell lineage. In contrast, one cell (SU-DHL-1), which lacks B cell-restricted surface antigens, retained germline heavy and light chain loci, indicating that it may represent a true histiocyte or uncommitted cell. Truncated RNAs for both the heavy and light chain immunoglobulins were responsible for the lack of surface immunoglobulin in the SU-DHL-2 cell line. Another cell line (SU-DHL-6), which possesses a t(14;18)(q32;q21) translocation, demonstrated an unexpected recombination within its heavy chain gene locus that may be the interchromosomal breakpoint.