Diagnostic significance of histiocyte-related markers in malignant histiocytosis and true histiocytic lymphoma

Interrelationships of immunologic and enzymatic markers of histiocytes have been studied in malignant neoplasms of histiocytic/monocytic origin and in differential diagnostically relevant, large cell non-Hodgkin's lymphomas. Cryostat sections required for demonstrating cell surface antigens by monoclonal antibodies are inadequate for studying cellular detail, enzymatic maturation by alpha-naphthyl acetate esterase (ANAE), and demonstrating the classical cytoplasmic markers of histiocytes like lysozyme, alpha-1-antitrypsin (AT), and alpha-1-antichymotrypsin (ACT). These markers have been compared in gently fixed and vacuum paraffin-embedded material. The reactivity for monoclonal anti-human monocyte 1 (Mo 1) has also been preserved by this method. Malignant histiocytosis (MH) is characterized by a heterogeneous cell population. The mature, ANAE-positive cells with macrophage morphology usually show a diffuse cytoplasmic positivity for AT and ACT. Lysozyme is moderately positive to negative in these cells, but it is more efficient than these markers in revealing smaller cells resembling monocytes by focal positivity in the cytoplasm. The expression of Factor XIIIa (F-XIIIa) is connected with the phagocytic activation of histiocytic cells. F-XIIIa positive cells usually form a minority of the neoplastic population in MH, but the large cytophagocytic marcophages are invariably positive. Reactive macrophages in large cell non-Hodgkin's lymphomas are characterized by a coexpression of ANAE, AT, ACT, lysozyme, F-XIIIa and Mo 1. Typical cases of true histiocytic lymphoma (THL) are made up of a homogeneous population showing the above mature, phagocytizing phenotype. In MH, Mo 1 and ANAE recognize different subpopulations. The reciprocal relation of these markers is an abnormal phenotypic feature. The results presented in this article prove the diagnostic value of ANAE and lysozyme in confirming the histiocytic differentiation of malignant cells. Monoclonal anti-human monocyte 1 is useful for identifying the immature component in MH. Factor XIIIa can be considered a functional marker of mature phagocytic histiocytes and an aid in the diagnosis of THL.     

Biology of the human malignant lymphomas. IV. Functional characterization of ten diffuse histiocytic lymphoma cell lines.

Ten consecutive diffuse histiocytic lymphoma (DHL) cell lines established in our laboratory were studied for the presence of Epstein-Barr virus (EBV) genomes, lysozyme, nonspecific esterase and other cytochemical reactions, phagocytic activity, cytoplasmic immunoglobulin light and heavy chains, and surface receptors to sheep erythrocytes, complement, and the Fc fragment of immunoglobulin. In agreement with previous studies performed on biopsy specimens, our results indicate that the diffuse histiocytic lymphomas, as a histopathologic entity, represent a heterogeneous group of neoplasms, the majority of which are B-lymphocyte in origin. The cell lines appear to fall into three categories based on the following criteria: 1) presence of monoclonal cytoplasmic immunoglobulins (B-lymphocytic type, 6/10 cell lines); 2) presence of non-specific esterase, phagocytic activity, and/or lysozyme (histiocytic type, 2/10 cell lines); and 3) absence of all lymphoid and histiocytic cell characteristics (null cell type, 2/10 cell lines). Despite the fact that many of the lymphoma patients had positive serologies to EBV antigens, all of the DHL cell lines were negative for the presence of EBV genomes. Both of the two B-lymphocytic type and one of the two histiocytic type lines tested were susceptible to infection with EBV, as indicated by synthesis of early antigen and also, in a small proportion of the infected cells, of viral capsid antigen. These prototypic DHL cell lines may permit the development of new criteria for the differential diagnosis and treatment of this highly malignant and diverse group of lymphomas.     

Pleomorphic reticulum cell sarcoma, monoclonal gammopathy and amyloidosis: an immunoperoxidase study.

Pleomorphic reticulum cell sarcoma, a histologic variant of the histiocytic lymphomas, presented as an abdominal mass in a 52-year-old woman. Extensive amyloid deposition was present within the tumor mass and an M-component (IgG Lambda) was identified in the serum. Direct immunoperoxidase staining of tissue sections demonstrated the same monoclonal immunoglobulin to be present within the neoplastic cells, presumptive evidence of their ability to both synthesize and secrete immunoglobulin. The presence of amyloid within this patient was probably the direct result of the tissue deposition of this monoclonal immunoglobulin and may be related to the amyloidogenic nature of Lambda light chains. Immunoglobulin production is a specific property of the B lymphocyte series. Transforming B lymphocytes and their differentiating progeny are engaged in active immunoglobulin synthesis and thus may be distinguished from morphologically identical but functionally distinct cells. The demonstration of cytoplasmic monoclonal immunoglobulin within these "malignant reticulum cells" strongly supports the assertion that at least some "histiocytic lymphomas" are neoplastic analogues of transformed B lymphocytes, and are not derived from phagocytic histiocytes, as previously believed.     

Induction of differentiation of the human histiocytic lymphoma cell line U-937 by retinoic acid and cyclic adenosine 3':5'-monophosphate-inducing agents

The monoblast-like human histiocytic lymphoma cell line, U-937, is induced to differentiate into monocyte-like cells by incubation with 0.1 to 1.0 microM retinoic acid (RA). These induced cells are phagocytic, reduce nitroblue tetrazolium, and show an increased hexose monophosphate shunt activity, consistent with monocyte-like cells. Prostaglandin E, cholera toxin, or dibutyryl cyclic adenosine 3':5'-monophosphate, all inactive alone, increased markedly the extent of RA-induced differentiation of U-937. These data suggest that the intracellular level of cyclic adenosine 3':5'-monophosphate is of importance for expression of the RA-induced effect. Responsiveness to RA seems to be limited to those leukemic myeloid cells blocked at a relatively late stage of maturation, like the promyelocytic HL-60 and the monoblast-like U-937 cell lines.     

Reactive histiocytosis in acute lymphoblastic leukemia and non-Hodgkin's lymphoma

An extensive hemophagocytic syndrome in the termination of one case of pre-T acute lymphoblastic leukemia (ALL) and another case of non-Hodgkin's lymphoma (NHL), are described. Since most of the proliferating cells were mature macrophages and these cells were limited in the mononuclear phagocytic system (MPS), it was determined to be a reactive histiocytosis rather than histiocytic medullary reticulosis (HMR) or malignant histiocytosis (MH). The pathogenesis of the HMR or MH-like syndrome in these patients is discussed, and it is considered that this might be a reaction of the bacterial sepsis related to their immunosuppressed state secondary to the pre-existing malignancies and/or the cytotoxic therapy. The literature was reviewed. Based on a proposal for differential diagnosis between reactive histiocytosis and MH (or HMR), the heterogeneity of HMR-like syndrome complicating the malignancies are clarified.     

The histogenesis of tumours induced in golden hamsters by murine sarcoma virus-Harvey (MSV-H)

Newborn golden hamsters were inoculated subcutaneously with a cell-free filtrate of cultured mouse 3T3 cells infected with murine sarcoma virus-Harvey (MSV-H). An electron microscopical study was made of the lesions that subsequently developed in these hamsters. Two weeks after inoculation, cysts developed eccentrically in the hila of lymph nodes draining the inoculation site. Type H (radial) virus-like particles were seen in the endothelial cells that lined the cysts and in the serous fluid that filled the cysts. During the 3–6 weeks after inoculation, nodules appeared in lymph nodes, subcutaneous and muscle fascia. They were composed of pleomorphic cells that interlocked with each other and resembled the fixed phagocytic histiocytes of normal lymph nodes. The pleomorphic interlocking cells infiltrated and replaced lymph nodes and perinodal tissues. Some lymph nodes were replaced by abnormal mast cells. During the 12–24 weeks after inoculation tumours were found that involved the paws or limb muscles. The predominant cells in these tumours were similar to the pleomorphic interlocking cells seen in early developing lesions. Similar cells were also found in lymph nodes draining the tumours. It is concluded that tumours induced in golden hamsters by MSV-H are mesenchymal tumours of histiocytic origin.     

The RT-PCR-identified Fas and FasL expression and apoptosis in mouse hepatoma MH-22a and histiocytic sarcoma J-774 clonal lines coupled with in vitro cultivation with syngenic splenocytes

Malignant growth is associated with various patterns of interaction between tumor cells and those of the body immune system, interaction between Fas-receptor (Fas) and Fas-ligand (FasL) expression being one of them. These mechanisms were simulated in vitro using the main cell populations from murine hepatoma MH-22a, histiocytic sarcoma J-774 and their clonal lines obtained from cocultivation of tumor cells and syngenic splenocytes. Fas and FasL expression was identified by the RT-PCR method while apoptosis--by electrophoresis of low molecular DNA fractions and clonogenic survival.     

Immunohistochemical and ultrastructural studies on disseminated skin lesions of midline malignant reticulosis

Midline malignant reticulosis (MMR), a disease included in the lethal midline granuloma, is histologically characterized by a mixture of lymphoid cells and atypical reticulum cells. Recent investigations of the nature of proliferating cells in MMR have suggested different conclusions, i.e., that the lesion is a true histiocytic, B- or T-cell. Two cases of MMR are presented, on which extensive laboratory studies were carried out. The results showed that the atypical reticulum cells were negative when stained immunohistochemically with monoclonal antibodies for T- or B-cells, diffusely stained by reactions for acid phosphatase and alpha-naphthyl acetate esterase, positively stained with human lysozyme and alpha-1-antitrypsin, and possess abundant cytoplasm containing primary lysosomes, polylysosomes and residual bodies. These findings indicate the true histiocytic nature of the proliferating cells in MMR.     

Establishment and characterization of a human histiocytic lymphoma cell line (U-937).

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.     

Role of proteins in Fas-mediated apoptosis in tumor cells and lymphocytes co-cultured in vitro

The investigation deals with the role of Fas, FasL, RIP, caspase 3, and PARP taking part in Fas-mediated apoptosis, and contributing to in vitro interaction of hepatoma MH-22a and histiocytic sarcoma J-774 in mice with syngenic splenocytes. Protein expression was identified by means of indirect immunofluorescence. There were two patterns of interaction of tumor cells and splenocytes: apoptosis occurred either in 80% or in an insignificant number of tumor cells. In the latter case, high Fas expression was identified before and when it dropped after the experiment. FasL expression in tumor cells often peaked before the experiment and then it decreased after contact with lymphocytes. That mechanism was reversed in splenocytes: contact with tumor cells boosted expression. RIP, caspase 3 and PARP expression was very low and failed to show until the experiments on both patterns of cells were undertaken. After the experiments, it either remained latent or soared up. In the latter case, simultaneous expression of all proteins took place both in tumor cells and lymphocytes. A second battery of experiments demonstrated maximum rates of apoptosis both of tumor cells and splenocytes. However, the situation was different: Fas expression intensified in both patterns of cells after their interaction which was followed by post-experimental drop in RIP, caspase 3, and PARP expression in tumor cells; hence, the importance of perforin/granzyme-mediated apoptosis which occurred at the early stages of tumor growth in the midst of interaction with immune system cells. That pattern of apoptosis was highly cytotoxic. It is suggested that Fas-mediated apoptosis or any other receptor-sensitive pathway might take place during tumor progression, i.e. at a stage when tumor is most susceptible to change.     

Heterogeneity of cloned cell lines established from a transplantable rat malignant fibrous histiocytoma.

Four cloned cell lines, MT-7, MT-8, MT-9 and MT-10, were established from a transplantable malignant fibrous histiocytoma (MFH) of F344 rats to investigate the histogenesis of the tumor. Cells of MT-7, MT-9 and MT-10 had fine structures characteristic of histiocytes, such as numerous cell processes, many lysosomes and well-developed cytoplasmic organelles. They stained positively for histiocytic lysosomal and antigenic markers. In addition, MT-9 cells contained microfilaments and well-developed RER in their cytoplasm, suggesting that they may be facultative fibroblasts. MT-8 cells stained weakly for histiocytic markers and had scant cytoplasmic organelles. They were identified as undifferentiated mesenchymal cells. The tumors induced in syngeneic rats by inoculating MT-7 or MT-10 consisted of a mixture of the pleomorphic, myxoid and storiform types of MFH, and those by MT-9 were of the storiform type. Cells forming these tumors stained positively for histiocytic markers. Tumors induced by MT-8 consisted of undifferentiated cells negative to these stainings. The histogenesis of MFH is surmised to be related to various differentiation stages shifting from undifferentiated cells to histiocytic cells capable of acting as facultative fibroblasts.     

Heterogeneity of Cloned Cell Lines Established from a Transplantable Rat Malignant Fibrous Histiocytoma

Four cloned cell lines, MT-7, MT-8, MT-9 and MX-10, were established from a transplantable malignant fibrous histiocytoma (MFH) of F344 rats to investigate the histogenesis of the tumor. Cells of MT-7, MT-9 and MT-10 had fine structures characteristic of histiocytes, such as numerous cell processes, many lysosomes and well-developed cytoplasmic organelles. They stained positively for histiocytic lysosomal and antigenic markers. In addition, MT-9 cells contained microfllaments and well-developed RER in their cytoplasm, suggesting that they may be facultative fibroblasts. MT-8 cells stained weakly for histiocytic markers and had scant cytoplasmic organelles. They were identified as undifferentiated mesenchymal cells. The tumors induced in syngeneic rats by inoculating MT-7 or MT-10 consisted of a mixture of the pleomorphic, myxoid and storiform types of MFH, and those by MT-9 were of the storiform type. Cells forming these tumors stained positively for histiocytic markers. Tumors induced by MT-8 consisted of undifferentiated cells negative to these stainings. The histogenesis of MFH is surmised to be related to various differentiation stages shifting from undifferentiated cells to histiocytic cells capable of acting as facultative fibroblasts.     

Immunohistochemical study on cutaneous histioproliferative lesions

Immunohistochemical examinations were performed using five kinds of histiocytic markers [S100 protein, lysozyme, non-specific cross reacting antigen with carcinoembryonic antigen (NCA), alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1-antitrypsin (alpha 1-AT)] in biopsied tissues from histiocytosis X, juvenile xanthogranuloma, xanthoma tuberosum, xanthoma disseminatum, reticulohistiocytic granuloma and multicentric reticulohistiocytoma, all of which have been classified as histiocytic proliferative disorders. Our results suggested that xanthomatous lesions of the skin to be composed of the histiocytic proliferation of two different cell lineages, i.e. S100+lyso-NCA- T-zone histiocytes and S100-lyso+NCA+ tissue macrophages. Only lesions of histiocytosis X were composed of the former cells. It is suggested that these markers will be useful in determining the delineation of the histiocytic system on the basis of functional heterogeneity.     

Liposomal doxorubicin-induced toxicity: Depletion and impairment of phagocytic activity of liver macrophages

Doxorubicin entrapped within conventional liposomes (200 nm in diameter; lip-Dox) has major toxic effects on liver macrophages of the rat for a considerable period of time following i.v. administration, with respect to both specific phagocytic capacity and cell numbers. At different time-points after injection of lip-Dox or free doxorubicin, radiolabeled, negatively charged, “empty” test liposomes were injected. Phagocytic capacity was determined by isolating the liver macrophages and measuring the amount of macrophage-associated radioactivity. Four subfractions of liver macrophages of different cell-size and with intrinsically different phagocytic capacity were isolated. Twenty-four hours after injection of lip-Dox, the phagocytic capacity of the larger-sized liver macrophages was strongly decreased. The relatively low intrinsic phagocytic capacity of the smaller-sized macrophages was only slightly impaired. Phagocytic capacity after injection of lip-Dox was nearly restored to control values after 14 days. Blood clearance of Klebsiella pneumoniae bacteria after pre-treatment with lip-Dox was strongly decreased. Pre-treatment with the free drug and/or placebo liposomes had no effect on phagocytic and bacterial blood-clearance capacity. A major depletion of the liver macrophage population was observed, as revealed by both macrophage isolation and histology. Only 2 weeks after injection of lip-Dox, the number of cells had returned to that seen in control animals. In view of the important host-defense functions of the liver macrophages, especially in the control of tumor growth and infection, the findings reported here should be taken into consideration when lip-Dox is to be administered in anti-tumor therapy. © 1995 Wiley-Liss, Inc.     

Neutral glycosphingolipid expression in B-cell neoplasms

The expression of neutral glycosphingolipids (GSL) in 37 B-cell neoplasms [7 acute lymphocytic leukemia (ALL), 5 Burkitt's lymphoma (BL), 7 chronic lymphocytic leukemia (CLL), 5 diffuse, poorly differentiated lymphoma (DPDL), 6 diffuse histiocytic lymphoma (DHL), 3 hairy-cell leukemia (HCL), and 4 multiple myeloma (MM)] was examined. Patterns of expression of simple (GlcCer, LacCer) and globo-series GSL (Gb3, Gb4) were found for each tumor type. In addition, pre-B ALL expressed the neo-lacto series GSL, paragloboside, which was not significantly seen at later stages of maturation. As a group, leukemias expressed about 10 times higher ratios of simple GSL to Globo-series GSL as compared to lymphomas, regardless of stage of differentiation. Significant amounts of GSL of other series were not found except in one CLL which contained asialo-GM2. GSL phenotype in these cells was not grossly affected by cell genotype since pre-B ALL containing Philadelphia chromosome t(9q;22q) translocations were similar to other ALL; and DHL with t(8q;14q) translocations had GSL patterns similar to other DHL samples and dissimilar to GSL patterns found in Burkitt's lymphomas with t(8q;14q). Differences in GSL expression among the different types of B-cell neoplasm suggested that GSL patterns form a phenotypic map that may complement the traditional glycoprotein immunophenotypic map and contribute to our understanding of the biology of these diseases and B-cell differentiation.     

Differential expression of markers in extensive and restricted Langerhans Cell Histiocytosis (LCH)

Langerhans cell histiocytosis (LCH) represents a poorly defined pathologic entity characterized by diverse clinical appearence and falling into two major categories namely a restricted and an extensive disease. Since the outcome and the course of the disease is variable, we postulated that this might be reflected by the phenotype of the Langerhans cells. We have selected 11 adult restricted cases and 10 extensive childhood cases and compared the phenotype of LCH cells by immunohistochemistry on paraffin sections. Morphometric analysis indicated a significantly higher expression of histiocytic (CD68, S-100, lysozyme) markers in the adult restricted cases compared to the extensive form of the disease. Both groups were equally positive for LCH marker CD1a and negative for T cell marker CD4. On the other hand, HLA-DR expression was significantly higher in LCH cells of the extensive childhood cases suggesting higher activation. These data suggest that LCH cells have a different phenotype in the extensive childhood and restricted adult LCH where the latter is characterized by a more differentiated histiocytic phenotype.     

Multiple marker studies on a malignant fibrous histiocytoma with primary cutaneous localization

Continuing controversy exists concerning a possible relation between neoplastic cells of malignant fibrous histiocytoma (MFH) and the mononuclear phagocyte system. The aim of this study was to investigate the membrane and cytoenzymatic phenotype of a primary cutaneous MFH, storiform pleomorphic type, and to compare these data with ultrastructural observations. Cytoplasmic proteins (acid phosphatase, non specific esterase, alpha-1 antitrypsin, and lysozyme) suggestive of a mononuclear phagocyte origin were demonstrated in varying amounts in neoplastic cells infiltrating the dermis. Consistent with these data, two (LeuM3 and OKM5) out of four (OKM1 and LeuM1) monoclonal antibodies directed against mononuclear phagocyte antigens stained most of the neoplastic cells. Class II MCH antigens (DR and DQ) were variably expressed on distinct groups of neoplastic cells, suggesting different activation/differentiation states. The results favor the view that the present case of primary cutaneous MFH was of mononuclear phagocyte origin. However, the observed phenotypic profile was expressed on neoplastic cells irrespective of their ultrastructural morphology (histiocytic or fibroblastic). Together with previous data in the literature, the latter finding corroborates the view that distinction between these two cell types in MFH is likely to reflect divergent growth and differentiation patterns rather than histogenesis.     

Microcinematographic and electron microscopic study of the death of human tumor cells induced by histiocytic cells

The cells from a malignant fibrous histiocytoma were enzymatically isolated and cultured in vitro. The cultures were observed by microcinematography and with an electron microscope after fixation and embedding. The interactions between histiocytic and tumor cells resulted in tumor cell death. The microcinematographic study revealed that filiform projections of the histiocytic cell protrude toward the tumor cell surface making contacts for varying periods of time. The tumor cell then contracts and almost simultaneously some granules (lysosomes?) appear in the tumor cell cytoplasm. These granules eventually fuse with each other thus increasing in volume. The resulting granulation heads for the periphery of the tumor cell at a site previously touch by a projection of a histiocytic cell; at this site the membrane bursts and the tumor cell dies leaking amorphous cytoplasmic material. Before bursting the tumor cell does not show any noticeable morphological changes.     

Cytologic and ultrastructural studies of a rare breast carcinoma with osteoclast-like giant cells

An unusual case of breast carcinoma with osteoclast-like giant cells (OGCs) was reported, for which the diagnosis was made by aspiration biopsy. The OGCs appeared to derive from large mononuclear cells, probably via their cellular fusion. The mononuclear cells had abundant lysosome-like granules, endoplasmic reticulum, and mitochondria. They were thought to be histiocytic cells, though active phagocytosis could not be demonstrated. Histologically, the tumor showed a pattern of well differentiated ductal adenocarcinoma, of which the stroma was crowded with histiocytic cells, OGCs, and lymphocytes. Marked hemorrhage and fibrosis were also seen. The tumor cells tended to be distorted and greatly diminished in accord with the accumulation of OGCs.     

Is there a role for fibronectin upon true histiocytic lymphoma progression?

Cell interaction with extracellular matrix is a crucial event for various biological processes, including tumor progression. Although not exclusively, these interactions are frequently mediated by bidirectional signaling receptors known as integrins. Using a human histiocytic lymphoma-derived cell line (U-937), we evaluated the effects of ECM proteins and their integrin-type receptors in the regulation of cell attachment, proliferation, migration and survival. Fibronectin induces higher cell attachment in vitro when compared to laminin. Fibronectin also promotes a decrease in cell migration but do not modulate cell proliferation and death. Pre-incubation of U-937 cells with VLA-5 antagonistic peptides inhibited attachment of the cells to fibronectin-coated substrates. In a second vein, we observed that lymph node specimens obtained from diagnosed patient for true histiocytic lymphoma had greater deposition of fibronectin (but not laminin) around malignant clones. These results suggest that fibronectins play a relevant role in the establishment and progression of true histiocytic lymphoma cells.