Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells.
A common characteristic of malignant cells derived from patients with Hodgkin's disease (HD) is a high level of constitutive nuclear NF-kappaB/Rel activity, which stimulates proliferation and confers resistance to apoptosis. We have analysed the mechanisms that account for NF-kappaB activation in a panel of Hodgkin/Reed-Sternberg (H-RS) cell lines. Whereas two cell lines (L428 and KMH-2) expressed inactive IkappaBalpha, no significant changes in NF-kappaB or IkappaB expression were seen in other H-RS cells (L591, L1236 and HDLM-2). Constitutive NF-kappaB was susceptible to inhibition by recombinant IkappaBalpha, suggesting that neither mutations in the NF-kappaB genes nor posttranslational modifications of NF-kappaB were involved. Endogenous IkappaBalpha was bound to p65 and displayed a very short half-life. IkappaBalpha degradation could be blocked by inhibitors of the NF-kappaB activating pathway. Proteasomal inhibition caused an accumulation of phosphorylated IkappaBalpha and a reduction of NF-kappaB activity in HDLM-2 and L1236 cells. By in vitro kinase assays we demonstrate constitutive IkappaB kinase (IKK) activity in H-RS cells, indicating ongoing signal transduction. Furthermore, H-RS cells secrete one or more factor(s) that were able to trigger NF-kappaB activation. We conclude that aberrant activation of IKK's, and in some cases defective IkappaBs, lead to constitutive nuclear NF-kappaB activity, which in turn results in a growth advantage of Hodgkin's disease tumor cells. (+info)
Human immunodeficiency virus-associated Hodgkin's disease derives from post-germinal center B cells.
Human immunodeficiency virus-associated Hodgkin's disease (HIV-HD) displays several peculiarities when compared with HD of the general population. These include overrepresentation of clinically aggressive histologic types and frequent infection of Reed-Sternberg (RS) cells by Epstein-Barr virus (EBV). Recently, we have reported that the histogenesis of HD of the general population may be assessed by monitoring the expression pattern of BCL-6, a transcription factor expressed in germinal center (GC) B cells, and of CD138/syndecan-1 (syn-1), a proteoglycan associated with post-GC, terminal B-cell differentiation. In this study, we have applied these two markers to the study of HIV-HD histogenesis and correlated their expression status to the virologic features of this disease. We have found that RS cells of all histologic categories of HIV-HD consistently display the BCL-6(-)/syn-1(+) phenotype and thus reflect post-GC B cells. Although BCL-6(-)/syn-1(+) RS cells of HIV-HD express CD40, they are not surrounded by CD40 ligand-positive (CD40L+) reactive T lymphocytes, which, in HD of the general population, are thought to regulate the disease phenotype through CD40/CD40L interactions. Conversely, RS cells of virtually all HIV-HD express the EBV-encoded latent membrane protein 1 (LMP1), which, being functionally homologous to CD40, may contribute, at least in part, to the modulation of the HIV-HD phenotype. (+info)
Differential chemokine expression in tissues involved by Hodgkin's disease: direct correlation of eotaxin expression and tissue eosinophilia.
Hodgkin's disease (HD) is a lymphoid malignancy characterized by infrequent malignant cells surrounded by abundant inflammatory cells. In this study, we examined the potential contribution of chemokines to inflammatory cell recruitment in different subtypes of HD. Chemokines are small proteins that are active as chemoattractants and regulators of cell activation. We found that HD tissues generally express higher levels of interferon-gamma-inducible protein-10 (IP-10), Mig, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and eotaxin, but not macrophage-derived chemotactic factor (MDC), than tissues from lymphoid hyperplasia (LH). Within HD subtypes, expression of IP-10 and Mig was highest in the mixed cellularity (MC) subtype, whereas expression of eotaxin and MDC was highest in the nodular sclerosis (NS) subtype. A significant direct correlation was detected between evidence of Epstein-Barr virus (EBV) infection in the neoplastic cells and levels of expression of IP-10, RANTES, and MIP-1alpha. Levels of eotaxin expression correlated directly with the extent of tissue eosinophilia. By immunohistochemistry, IP-10, Mig, and eotaxin proteins localized in the malignant Reed-Sternberg (RS) cells and their variants, and to some surrounding inflammatory cells. Eotaxin was also detected in fibroblasts and smooth muscle cells of vessels. These results provide evidence of high level chemokine expression in HD tissues and suggest that chemokines may play an important role in the recruitment of inflammatory cell infiltrates into tissues involved by HD. (+info)
Identification of common germinal-center B-cell precursors in two patients with both Hodgkin's disease and non-Hodgkin's lymphoma.
BACKGROUND: Hodgkin's disease and non-Hodgkin's B-cell lymphoma occasionally occur in the same patient. The identification of a common precursor of the two types of lymphoma would show definitively that Reed-Sternberg cells originate from B cells. METHODS: We studied lymphomas from two patients, one with a composite lymphoma (classic Hodgkin's disease and a follicular lymphoma in the same lymph node) and the other with a T-cell-rich B-cell lymphoma that was followed by classic Hodgkin's disease. Single Reed-Sternberg cells and non-Hodgkin's lymphoma cells from frozen sections were micromanipulated. The rearranged immunoglobulin variable-region genes (V genes) of the heavy and light chains were amplified by the polymerase chain reaction from genomic DNA and sequenced. RESULTS: In both patients, the Reed-Sternberg cells were related clonally to the non-Hodgkin's lymphoma B cells. The V genes carried somatic mutations (a hallmark of germinal-center B cells and their descendants). In both patients, some somatic mutations were shared by the Reed-Sternberg and non-Hodgkin's lymphoma cells, whereas other somatic mutations were found exclusively in one or the other cell type. CONCLUSIONS: In two patients with classic Hodgkin's disease and non-Hodgkin's B-cell lymphoma, we identified a common B-cell precursor, probably a germinal-center B-cell, for both lymphomas. This finding suggests that the two types of lymphoma underwent both shared and distinct transforming events and provides proof of the B-cell derivation of Reed-Sternberg cells in classic Hodgkin's disease. (+info)
High expression of the CC chemokine TARC in Reed-Sternberg cells. A possible explanation for the characteristic T-cell infiltratein Hodgkin's lymphoma.
Hodgkin's lymphoma is characterized by the combination of Reed-Sternberg (R-S) cells and a prominent inflammatory cell infiltrate. One of the intriguing questions regarding this disease is what is causing the influx of T lymphocytes into the involved tissues. We applied the serial analysis of gene expression (SAGE) technique on the Hodgkin's lymphoma-derived cell line L428 and on an Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell line. A frequently expressed tag in L428 corresponded to the T-cell-directed CC chemokine TARC. Reverse transcription polymerase chain reaction analyses demonstrated expression of TARC in nodular sclerosis (NS) and mixed cellularity (MC) classical Hodgkin's lymphomas but not in NLP Hodgkin's lymphoma, anaplastic large-cell lymphomas, and large-B-cell lymphomas with CD30 positivity. Two of five cases of T-cell-rich B-cell lymphoma (TCRBCL) were TARC positive. RNA in situ hybridization (ISH) showed a strong signal for TARC in the cytoplasm of R-S cells, and immunohistochemical staining confirmed the presence of the TARC protein in the R-S cells of NS and MC Hodgkin's lymphomas. The lymphocytic and histiocytic (L&H)-type cells of nodular lymphocyte predominance Hodgkin's lymphoma and the neoplastic cells of non-Hodgkin's lymphomas with the exception of two cases of TCRBCL did not stain for TARC. TARC is known to bind to the CCR4 receptor, which is expressed on activated Th2 lymphocytes. The immunophenotype of lymphocytes surrounding R-S cells is indeed Th2-like, and by RNA ISH these lymphocytes showed a positive signal for the chemokine receptor CCR4. The findings suggest that production of TARC by the R-S cells may explain the characteristic T-cell infiltrate in classical Hodgkin's lymphoma. (+info)
An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line.
The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). The resulting immunotoxin Ki-4(scFv)-ETA' specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies. (+info)
Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.
Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies. (+info)
Reed-Sternberg cell genome expression supports a B-cell lineage.
The malignant Reed-Sternberg cell of Hodgkin's disease, first described a century ago, has resisted in-depth analysis due to its extreme rarity in lymphomatous tissue. To directly study its genome-wide gene expression, approximately 11,000,000 bases (27,518 cDNA sequences) of expressed gene sequence was determined from living single Reed-Sternberg cells, Hodgkin's tissue, and cell lines. This approach increased the number of genes known to be expressed in Hodgkin's disease by 20-fold to 2,666 named genes. The data here indicate that Reed-Sternberg cells from both nodular sclerosing and lymphocyte predominant Hodgkin's disease were derived from an unusual B-cell lineage based on a comparison of their gene expression to approximately 40,000,000 bases (10(5) sequences) of expressed gene sequence from germinal center B cells (GCB) and dendritic cells. The data set of expressed genes, reported here and on the World Wide Web, forms a basis to understand the genes responsible for Hodgkin's disease and develop novel diagnostic markers and therapies. This study of the rare Reed-Sternberg cell, concealed in its heterogenous cellular context, also provides a formidable test case to advance the limit of analysis of differential gene expression to the single disease cell. (+info)