Production of tumor necrosis factor-alpha and lymphotoxin by cells of Hodgkin''s neoplastic cell lines HDLM-1 and KM-H2.

The production of tumor necrosis factors (TNF) from cells of two Hodgkin's Reed-Sternberg (H-RS) lines, HDLM-1 and KM-H2 was examined. The culture supernatant from these two types of H-RS cells exerts a cytotoxic effect on L929 cells. Both tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are responsible for this activity. This was confirmed by the presence in the cells of proteins and m-RNAs of TNF-alpha and TNF-beta, as determined with immunoperoxidase staining and Northern blot hybridization. Approximately 20% of HDLM cells and 5% of KM-H2 cells were positively stained by a monoclonal anti-TNF-alpha antibody, and this staining was inhibited by preabsorption of the antibody with recombinant TNF-alpha. Staining with anti-TNF-beta, however, showed an intense reaction in more than 60% of HDLM-1 cells, but only in 5% to 10% of KM-H2 cells. The abundant expression of TNF-beta in HDLM-1 cells is consistent with approximately 10 times the TNF activity in HDLM-1-conditioned medium as compared with that of KM-H2. The rich secretion of TNF-beta in HDLM-1 cells was also validated by the inhibition of most of the TNF activity in HDLM-1-conditioned medium with anti-TNF-beta antibody, and by the presence of abundant TNF-beta mRNA in HDLM-1 cells. The reason for the abundant production of TNF-beta in HDLM-1 cells is not yet known, but may be attributable to a chromosomal abnormality in the 6p21 region. The expression of TNF-alpha, but not TNF-beta, by H-RS cells was demonstrated in lymph nodes from patients with Hodgkin's disease. The capacity of H-RS cells to secrete TNF as well as other cytokines, such as interleukin-1, colony-stimulating factors, and transforming growth factors, may contribute to the unique clinical and histopathologic alterations in patients with Hodgkin's disease.     

Cellular localizations and processing of the two molecular forms of the Hodgkin-associated Ki-1 (CD30) antigen. The protein kinase Ki-1/57 occurs in the nucleus.

The Ki-1 antibody not only detects a Hodgkin-associated membrane molecule of 120 kd (Ki-1/120 = CD30), but also reacts with an independently synthesized molecule of 57 kd (Ki-1/57) that only occurs intracellularly. Hodgkin's disease-derived cell lines L428 and L540 contain both Ki-1-reactive antigens, whereas others, e.g., U266/Bl myeloma cells, only express the intracellular Ki-1/57. The present immunoelectronmicroscopic analysis detected the Ki-1/57 antigen of U266/Bl cells not only in the cytoplasm, but also in association with the nuclear envelope, chromatin structures, and nucleoli. This Ki-1/57-specific type of labeling also was observed in L428 and L540 cells that, in contrast to U266/Bl cells, showed an additional staining of cell membranes and cytoplasmic vesicles. These results were confirmed by two independent methods: 1) cytocentrifuge preparations of isolated nuclei of L540 cells showed a spotted Ki-1-specific labeling, 2) immunoprecipitations demonstrated that the Ki-1/57, but not the Ki-1/120 antigen, was transferred into the nuclei of L540 and U266/Bl cells, whereas the Ki-1/120 antigen with its 90-kd precursor remained in the non-nuclei fraction of L540 cells.     

Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin's disease

Vascular endothelial growth factor (VEGF) is involved in tumour angiogenesis, an important process for the growth and metastatic potential of solid tumours. Numerous studies have demonstrated up-regulation of VEGF at both mRNA and protein level in various tumours and a correlation with advanced stage and prognosis has been demonstrated in some cases. Limited information exists about its role in lymphoid malignancies and in particular, Hodgkin's disease. The present study examined the immunohistochemical expression of VEGF using the monoclonal antibody VG1 in a series of 61 cases of Hodgkin's disease, including both classical Hodgkin's disease and the nodular lymphocyte predominance variant, and correlated these results with microvessel density, using an anti-CD31 monoclonal antibody. In 41 cases (70.6%) of classical Hodgkin's disease and one of the three cases of nodular lymphocyte predominance Hodgkin's disease, the neoplastic Reed-Sternberg and Hodgkin cells expressed VEGF. The staining observed was cytoplasmic, either diffuse or with a focal paranuclear distribution. Macrophages were always positive, while reactive lymphocytes showed occasional positivity. A variable amount of strong extracellular staining was also observed in the tissue stroma and intravascular plasma staining was prominent. There was no statistically significant relationship between VEGF expression and the subtype of Hodgkin's disease or microvessel density. In vitro studies using the Reed-Sternberg cell lines L428 and KM-H2 were also performed in both normoxia and hypoxia and VEGF protein production was assessed by flow cytometry (FACS), immunoassay of cell culture supernatant, and RT-PCR. Analysis by FACS demonstrated a subset of cells in both cell lines reacting with VG1 and analysis of secreted VEGF (pg/ml per 1x10(6) cells) in cell culture supernatant confirmed the normoxic production in both cell lines and significant hypoxic induction (p<0.005). Additionally, both cell lines expressed VEGF mRNA, as demonstrated using the RT-PCR method. In conclusion, neoplastic cells express VEGF in Hodgkin's disease, as is the case in solid tumours, and this expression may be induced by hypoxia. The presence of VEGF in reactive macrophages and in the extracellular matrix might facilitate tumour progression.     

Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulating T lymphocytes. Inhibition studies using monoclonal antibodies indicated similar contributions within HD line-T cell MLR to that documented in human tonsil DC-T cell MLR. The HD lines produced no detectable interleukin-1 (IL-1) by biological or immunological analysis. Moreover they stimulated allogeneic T lymphocytes in the presence of anti-IL-1 antibodies. Thus although IL-1 mRNA can be detected in both HDLM-2 and KM-H2 by polymerase chain reaction, these lines, and L428, share with DC the ability to stimulate allogeneic T lymphocytes in an IL-1-independent manner [corrected]. HD lines, particularly L428, may provide a standardized, reproducible, IL-1-independent model for dissection of the co-stimulatory requirements of the human primary MLR.     

Characterization of the interleukin-1beta-converting enzyme/ced-3-family protease, caspase-3/CPP32, in Hodgkin's disease: lack of caspase-3 expression in nodular lymphocyte predominance Hodgkin's disease.

Apoptosis (programmed cell death) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cysteine proteases, is an important downstream mediator of several complex proteolytic cascades that result in apoptosis in both hematopoietic and nonhematopoietic cells. Previous studies have demonstrated that caspase-3 is commonly expressed in classical Hodgkin's disease (CHD); however, the biological significance of its expression in Hodgkin's disease is unknown. In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodgkin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, we investigated the role of caspase-3 in CD95 (Fas)-mediated apoptosis in three CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease. Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell lines, HS445, L428, and KMH2. Whereas caspase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody induced marked apoptosis that was significantly inhibited by pretreatment with the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant increase in caspase-3 activity as determined by an enzyme colorimetric assay was detected in HS445 and L428 cells after 48 hours of CD95 stimulation. In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apoptosis. These data demonstrate caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting these tumor cells may be resistant to apoptotic stimuli dependent on caspase-3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity.     

Expression of the matrix metalloproteinase 9 in Hodgkin's disease is independent of EBV status.

BACKGROUND: In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix. AIM: To test whether the expression of MMP-9 in Hodgkin's disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin's disease cell line, L428. METHODS: MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin's disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkin's disease cell line, L428. RESULTS: MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkin's disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkin's disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin's disease cell line. Induction of LMP-1 expression in the Hodgkin's disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography. CONCLUSIONS: These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkin's disease tumours and by the Hodgkin's disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.     

Production of lymphotoxin, a bone-resorbing cytokine, by cultured human myeloma cells

Myeloma cells destroy bone by producing an osteoclast-stimulating factor that has chemical and biological characteristics similar to the bone-resorbing activity present in the supernatants of activated leukocyte cultures. Recently, a number of bone-resorbing leukocyte cytokines have been identified, including interleukin-1, lymphotoxin, and tumor necrosis factor. We have examined the products of human myeloma cells for the presence of these bone-resorbing cytokines. In a tumor cell line derived from a patient who had myeloma with osteolytic bone lesions and hypercalcemia, we found that the myeloma cells induced bone-resorbing activity and cytotoxic activity in vitro. Most of the bone-resorbing activity and all cytotoxic activity were suppressed by neutralizing antibodies to lymphotoxin. The myeloma cells expressed both lymphotoxin and tumor necrosis factor mRNA, but no tumor necrosis factor could be detected in the cell-culture medium. Interleukin-1 mRNA was not detected in the myeloma cells, and biologic activity of interleukin-1 was not measurable in the medium harvested from the cultured cells. The bone-resorbing activity induced by recombinant tumor necrosis factor and recombinant interleukin-1 was not affected by treatment with the lymphotoxin antibodies. When lymphotoxin was infused subcutaneously into normal mice (10 micrograms per day for three days), their plasma calcium levels increased. We also evaluated four established cell lines derived from three other patients with myeloma, and found a similar pattern of lymphotoxin expression in each. It appears that production of the bone-resorbing cytokine lymphotoxin is related to osteoclastic bone destruction and hypercalcemia in patients with myeloma.     

T Cell-Mediated Production of Tumour Necrosis Factor-α by Monocytes

The aim of this study was to examine the tumour necrosis factor (TNF)-alpha production by peripheral blood mononuclear cells activated by mitogens. Considerable amounts of TNF-alpha, ranging from 1.0 to 5.0 ng/ml, were present in the supernatants of cultures of human peripheral blood mononuclear cells (PBMC), stimulated with either the anti-CD3 monoclonal antibody OKT3 or the lectin phytohaemagglutinin (PHA). The amount of TNF-alpha secreted in the supernatant was closely correlated to the degree of T cell proliferation in such cultures, as measured by [3H]TdR incorporation. In the absence of proliferating T cells the mitogens did not induce secretion of TNF-alpha by monocytes. Supernatants of proliferating T cells were shown to induce TNF-alpha production by monocytes. The macrophage-activating factor gamma interferon (IFN-gamma) was also shown to induce, in the absence of endotoxin, TNF-alpha secretion by monocytes in a dose-dependent manner. The induction of TNF-alpha production by supernatants of proliferating T cells could largely be abrogated by passaging the supernatants on an anti-IFN-gamma column before adding them to the monocytes. It is therefore concluded from this study that the production of TNF-alpha by monocytes can be induced by proliferating T cells and that this induction can largely be attributed to the T cell lymphokine IFN-gamma.     

Interleukin-1-like activity constitutively generated by Hodgkin derived cell lines. I. Measurement in a human lymphocyte co-stimulator assay

Culture supernatants (CS) from Hodgkin derived cell lines have previously been shown to contain colony stimulating activity (CSF) for human cord blood cells, fetal bone marrow and fetal liver cells. In this study 3-day CS from four Hodgkin lines (L428, L538, L540, L591) and two sublines (L428KS, L428KSA) were examined for interleukin (IL) activity. None of the tested CS supported the growth of an IL-2 dependent murine T-cell line, suggesting that the Hodgkin lines do not produce significant amounts of IL-2. When crude 3-day CS from the various lines were assayed for IL-1-activity in the conventional murine thymocyte costimulator assay no or only borderline IL-1-activity was detectable. However, concentrated CS from L428KS exhibited IL-1-activity also in this assay as did lipopolysaccharide (LPS) induced human IL-1. Surprisingly, crude 3-day CS from all Hodgkin cell lines were capable of fully replacing the accessory cell requirement in ConA-induced lymphoproliferation assays of heavily monocyte-depleted human blood lymphocytes. The monocyte-depleted lymphocyte populations were obtained by 1 X g sedimentation at a sedimentation rate of 30.2 to 38.8 mm/hr (fraction IIIa and IIIb). These cells responded poorly to the T-cell mitogen ConA at 10 micrograms/ml and produced no IL-2. Addition of irradiated, autologous monocytes or of CS from the various Hodgkin cell lines quantitatively restored the ConA responsiveness and induced significant IL-2 production in the monocyte-depleted lymphocyte population, suggesting that Hodgkin lines constitutively secrete IL-1 or IL-1-like activity. A preliminary biochemical characterization (heat and pH stability, molecular weight range of 13-24 KD) supports the notion that the accessory cell replacing activity present in CS of Hodgkin cell lines is a type of human IL-1.     

Two Distinct Mechanisms of Cytotoxity Mediated by Herpes Simplex Virus-Specific CD4+Human Cytotoxic T Cell Clones

The present study was undertaken to elucidate the mechanisms responsible for the cytotoxicity of herpes simplex virus (HSV)-specific CDA4⁺human cytotoxic T lymphocyte (CTL) clones, focusing on perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-β). Two HSV-specific CDA4⁺CTL clones, which expressed both perforin and membrane-bound LT, exerted HSV-specific cytotoxicity and cytotoxicity against LT-sensitive L929 cells. These CDA4⁺CTL clones lysed HSV-infected cells directly in an HLA class II-restricted manner and did not exhibit "bystander killing." The culture supernatants of these clones stimulated with HSV antigen showed no cytotoxicity against HSV-infected cells or L929 cells, suggesting that adhesion to target cells is essentail to their antigen-specific and antigen-nonspecific cytotoxicities. The cytotoxicities of these clones against HSV-infected autologous cells were inhibited by an anti-CD3 monoclonal antibody but not by an anti-LT antibody. Conversely, their cytotoxicities against L929 cells appeared to be partially inhibited by the anti-LT antibody but not by the anti-CD3 monoclonal antibody. Furthermore, target cell DNA fragmentation induced by these CDA4⁺CTL clones was apparently observed in L929 cells but only faintly detected in HSV-infected autologous cells. L929 cell DNA fragmentation was also inhibited by adding the anti-LT antibody to CDA4⁺CTL cultures. these data suggest that some CDA4⁺CTL possess at least two cytolytic mediators, i.e., perforin and membrane-bound LT simultaneously, and can exert both antigen-specific and antigen-nonspecific cytotoxicity via two distinct mechanisms, necrosis and apoptosis.     

Highly sensitive enzyme immunoassay for human lymphotoxin (tumor necrosis factor β) in serum

We have developed a rapid, simple and highly sensitive ‘sandwich’ enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor β) in serum. The assay performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor does not cross-react even at 10⁷-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the qunatification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.     

Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin''s disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies

In order to elucidate the origin of the Hodgkin's and Reed-Sternberg cells, the expression of immunoglobulin kappa- and lambda light chain mRNA in 23 cases of nodular sclerosing and two cases of mixed cellularity Hodgkin's disease was examined by in situ hybridization using biotinylated oligonucleotide probes and compared with immunohistochemical staining with mono- and polyclonal antibodies against immunoglobulin kappa- and lambda light chains. No hybridization signals were seen in Hodgkin's or Reed-Sternberg cells in any of the cases. Polyclonal staining with polyclonal anti-immunoglobulin light chain antibodies was seen in Hodgkin's and Reed-Sternberg cells in 12 cases of nodular sclerosis and in two cases of mixed cellularity and with monoclonal antibodies in three cases of nodular sclerosis, but in no cases of mixed cellularity. In all cases, there was polyclonal labelling of plasma cells with both the oligonucleotide probes and the antibodies. In five cases, the Hodgkin's and Reed-Sternberg cells were also stained with one of the B-cell antibodies L26, MB2 or LN1. Lack of mRNA signals in Hodgkin's and Reed-Sternberg cells might indicate that these cells in Hodgkin's disease of the nodular sclerosis subtype are either not B-cell derived or they are early B-cells (precursor B-cells) not yet able to produce immunoglobulin light chain mRNA, at least not at a level detectable by in situ hybridization. Immunohistochemical staining of Hodgkin's and Reed-Sternberg cells, however, with antibodies against immunoglobulin kappa and lambda light chains may be explained by cellular uptake of the light chains, but the difference in reactivity between poly- and monoclonal antibodies cannot be explained at present.     

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity.

Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE.     

Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis.

Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.     

Expression of cytokine genes in hematological malignancies

A substantial number of leukemic blast colonies were formed when conditioned medium of human bladder carcinoma cell line 5637 was added as a stimulator. Recombinant colony-stimulating factor (CSF) also stimulated leukemic blast cell proliferation, leading to colony formation. Furthermore, serum CSF levels in some patients with acute myelogenous leukemia (AML), as detected by sensitive enzyme-linked immunosorbent assay (ELISA), were high. These observations prompted us to study further the expressions of hematopoietic growth factor genes. Granulocyte-macrophage CSF (GM-CSF) mRNA was detected in the leukemic blast cells from about 30% of patients with AML by Northern blot analysis using strict hybridization conditions with or without in vitro blast cell enrichment. These findings suggest that the expression of cytokine genes including the GM-CSF gene reflects in vivo phenomena, although no clear relationship between expression of the genes and serum CSF level has been established. Gene encoding tumor necrosis factor alpha (TNF-alpha), lymphotoxin (LT) and transforming growth factor beta (TGF-beta) were sometimes expressed in some malignant hematological cell lines and also some fresh leukemic cells.     

Monoclonal antibodies to human tumor necrosis factors alpha and beta: application for affinity purification, immunoassays, and as structural probes

Monoclonal antibodies were produced against two structurally related tumor necrosis factors (TNFs), TNF-alpha (previously called tumor necrosis factor) and TNF-beta (previously called lymphotoxin). The potential of these antibodies for the purification of TNFs, the development of specific immunoassays, and for defining the antigenic and functional domains of these cytokines was investigated. None of the monoclonal antibodies cross-reacted with both TNF-alpha and TNF-beta, or reacted with synthetic peptides which represented several of the regions of homology between these cytokines. Neutralizing monoclonal antibodies were utilized as immunoadsorbents to purify recombinant TNF-alpha and TNF-beta from E. coli lysates. TNFs purified by this method were greater than 98 percent pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited specific activities that were the same as TNFs isolated from natural sources using conventional chromatographic techniques. In addition, specific ELISA assays were developed that could detect less than 1 ng/ml of TNF-alpha or TNF-beta, and in contrast to bioassays, could discriminate between these related cytokines.     

Nodular sclerosing, mixed cellularity and lymphocyte-depleted variants of Hodgkin''s disease are probable dendritic cell malignancies.

The normal counterpart of the Reed-Sternberg cell and its mononuclear variant, collectively referred to as Hodgkin's cells (HC), remains controversial. The possibility that HC are malignant dendritic cells was tested by using a panel of 38 monoclonal antibodies to phenotype the cells from 16 cases of Hodgkin's disease (HD), excluding lymphocyte-predominant HD, and the Hodgkin's cell line L428. The results were then compared with the known phenotype of human dendritic cells. HC stained strongly for HLA Class I and Class II antigens. The leucocyte common antigen was weakly expressed in most cases. Expression of T and B cell markers was unusual, with the exception of the CD40 antigen which was found on a majority of HC. HC commonly expressed the CD11a, CR4 (CD11c), CD15, CD18 and a number of activation antigens but did not stain with a variety of macrophage-specific antibodies. The antigenic phenotype of L428 and the HC of case material were similar. This immunocytological analysis failed to support a lymphocyte or macrophage origin for HC. Instead the antigenic phenotype of the Reed-Sternberg cell and its mononuclear variant more closely resembles that of dendritic cells than of any other haemopoietic cell normally resident in lymph nodes.