Presence of Epstein-Barr virus in Hodgkin''s disease is not exclusive to Reed-Sternberg cells.

Thirty-three cases of Hodgkin's disease (HD) have been studied for the presence of Epstein-Barr virus (EBV) using a novel nonisotopic in situ hybridization procedure, based on the detection of Epstein-Barr encoded RNAs with oligonucleotide probes. An intense and morphologically distinct nuclear staining, sparing the nucleolus was seen in a total of 12 cases (36%). In six of these cases, the signal was located to the Hodgkin and Reed-Sternberg cells (HR-S); in the other six positive cases, the signal was observed only in the non-neoplastic small lymphocytes. These lymphocytes were few in number and immunocytochemistry results were consistent with a B-cell phenotype. The presence of EBV in those cases characterized by nuclear staining of small lymphocytes was confirmed by the polymerase chain reaction (PCR) analysis. The authors report the detection of EBV in small lymphocytes in HD by in situ hybridization and discuss the implications of these findings in relation to the proposed etiologic association between EBV and HD.     

Hodgkin''s disease presenting with the histological features of Castleman''s disease

Three cases are reported in which an initial diagnosis of the plasma cell variant of Castleman's disease was made, but in which a second lymph node biopsy within a year showed evidence of Hodgkin's disease. Review of the initial biopsy indicated that atypical CD15 and CD30 positive cells were present in the initial biopsy. This illustrates the difficulty in making the diagnosis of Castleman's disease and suggests that the lymphoid reaction to the presence of Hodgkin's disease may result in similar histological appearances. The need for re-evaluation of the diagnosis of Castleman's disease in the face of persistent or recurrent disease is stressed.     

Phenotypic expression of Hodgkin''s and Reed-Sternberg cells in Hodgkin''s disease.

The phenotypic expression of Hodgkin's and Reed-Sternberg (H-RS) cells was determined by analysis with a panel of monoclonal antibodies and peanut agglutinin (PNA) by an immunohistochemical technique. Seven antibodies, including T200, anti-HLA-DR, anti-Leu 10, A1G3, anti-Tac, OKT9, and anti-Leu M1, were found to react with a great majority of H-RS cells. In some cases, H-RS cells also bound PNA. Other antibodies, including those highly specific for T cells (eg, Lyt 3) and B cells (eg, B1, anti-Leu 14) were consistently negative. The results argue against the derivation of H-RS cells from T or B lymphocytes. The H-RS cells were also negatively stained with antibodies which react with monocytes (OKM1, Mo-2, 63D-3), follicular dendritic cells (DRC-1), and natural killer/killer cells (Leu 7, Leu 11a, B73.1). The presence of Leu M1 and Tac in H-RS cells is of interest. Anti-Leu M1 positivity was seen in all 20 of Hodgkin's disease (HD) cases tested and should provide a very useful reagent for differential diagnosis of HD from other reactive and neoplastic conditions. Tac normally is present only on activated T cells. The presence of Tac in H-RS cells may reflect expression of T-cell growth factor receptor or a closely related protein during a stage of neoplastic transformation. Although the nature of the neoplastic cell of HD cannot be determined by these studies, they are consistent with an origin from interdigitating reticulum cells. Both H-RS cells and interdigitating reticulum cells have a similar antigenic phenotype (Leu M1+, T200+, HLA-DR+, Leu 10+, A1G3+, and OKT9+) and a similar pattern of lysosomal enzyme activity.     

Interleukin-6 and tumour necrosis factor alpha are expressed by keratinocytes but not by Langerhans cells.

The presence of human cytokines was examined in parallel skin biopsies and epidermal single cell preparations obtained from normal individuals. Using biotin-avidin-peroxidase and immunofluorescence techniques and antibodies against recombinant cytokines, a granular intercellular/membrane-associated staining for interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), but not IL-1 alpha or beta, was observed. An epidermal cytoplasmic staining pattern was also detected, which was most pronounced using the anti-rIL-6 antiserum. In the epidermal single cell preparations, membrane-associated staining was detected for both IL-6 and TNF alpha. Double staining revealed that CD1-positive Langerhans cells (LC) failed to express any of the examined cytokines. In vitro binding of rIL-6 or rTNF alpha to skin sections and epidermal single cell preparations indicated that the cell surface-associated IL-6 and TNF alpha originally demonstrated on keratinocytes were truly membrane-bound. Finally, co-cultivation of epidermal cells with an IL-6 responsive cell line, B9, and testing of epidermal cell supernatants in this assay, indicated that the in vivo membrane-bound IL-6 had biological activity.     

Immunopathology of Hodgkin''s disease. Characterization of Reed-Sternberg cells with monoclonal antibodies.

The cellular origin of the Reed-Sternberg cells of Hodgkin's disease is controversial. The authors studied 14 cases of Hodgkin's disease (nodular sclerosis, 9; mixed cellularity, 3; lymphocyte predominant, 2), utilizing a panel of 16 monoclonal antibodies, including 5 new monoclonal antibodies defining differentiation antigens of the monocyte/macrophage system. Reed-Sternberg cells were found to react with antibodies to Ia-like (HLA-DR) determinants (14 of 14 cases), Leu M1, an antigranulocyte antibody (11 of 14 cases), and rarely B-1, an antibody defining an antigen expressed on human B lymphocytes (2 of 14 cases). Reed-Sternberg cells did not react with any of 5 antibodies to differentiation antigens of the monocyte/macrophage system (MoP9, MoS39, MoR17, MoU26, MoU50). In contrast, reactive histiocytes in the Hodgkin's disease infiltrates stained strongly. The findings are evidence against the monocyte-macrophage origin of Reed-Sternberg cells and support the view that the Reed-Sternberg cells of Hodgkin's disease derive from other cell types, such as interdigitating reticulum cells, or as yet uncharacterized cells which do not share antigens of the monocyte/macrophage system.     

Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin''s disease.

The immunophenotype of the Reed-Sternberg cells in Hodgkin's disease is heterogeneous among different cases; this heterogeneity has contributed to the continuing uncertainty regarding the normal counterpart of the Reed-Sternberg cell. In this study, the authors demonstrate coexpression of the B-cell marker, CD20, and the granulocyte associated antigen, CD15, by Reed-Sternberg cells in three of 20 cases of nodular sclerosis and mixed cellularity Hodgkin's disease using a double-labelling technique in one case and staining of serial sections in three cases. Additionally, the authors found that expression of CD20 occurred more often in tumors with a monomorphous proliferation of mononuclear and binucleate Hodgkin's and Reed-Sternberg cells, without numerous eosinophils or polymorphonuclear neutrophils. In contrast, expression of CD15 by Reed-Sternberg cells was associated with a greater granulocyte infiltrate. The presence or absence of fibrosis, plasma cells, and histiocytes did not correlate with antigen expression. These results suggest that there may be a continuum of antigen expression by Reed-Sternberg cells, with some cells expressing CD20, some CD15, and others expressing both antigens; cells coexpressing both CD15 and CD20 may represent an unstable intermediate in the process of antigen switching. The possibility that antigen expression by the neoplastic cells in a given case may modulate depending on the background infiltrate could explain the heterogeneity of immunophenotype among cases of Hodgkin's disease.     

Expression of B-cell antigens by Hodgkin''s and Reed-Sternberg cells.

Twenty frozen and 55 paraffin sections of lymphnode specimens from 55 patients with pretreatment Hodgkin's disease (nodular sclerosis Hodgkin's disease, n = 45; mixed cellularity Hodgkin's disease, n = 10) were studied by immunohistochemistry and molecular analysis to determine the phenotype of Hodgkin's and Reed-Sternberg cells (HRS). In all cases the HRS cells were CD45-, and CD30+, and in 43/55 (78%) cases they were CD15+. In 48/55 cases (87%) HRS cells were reactive with at least one B-cell marker (CD19, CD20, CD22, CDw75, MB2), 8/55 cases (14.5%) showed reactivity (mainly cytoplasmic) of a subpopulation of HRS cells with the T-cell markers CD3 and beta F1. All cases that expressed T-cell antigens were also reactive with at least one B-cell marker. In frozen sections, a minority of HRS cells in each case studied showed cytoplasmic positivity for bcl-2 protein. Rearrangement of immunoglobulin heavy chain genes was detected in one case and of T-cell receptor beta chain genes in none. The authors were unable to confirm previous reports of bcl-2 gene rearrangement in Hodgkin's disease. The results strongly support a B lymphocytic origin of HRS cells.     

Epstein-Barr viral genome in lymph nodes from patients with Hodgkin''s disease may not be specific to Reed-Sternberg cells.

A possible etiologic role for Epstein-Barr virus (EBV) in Hodgkin's disease (HD) was investigated by probing for EBV genome in 52 biopsy specimens involved with HD and 43 hyperplastic lymph node specimens. Using dot-blot hybridization (Bam HIW probe), Southern blot hybridization (Xho I probe), and polymerase chain reaction analyses, 27%, 27%, and 58% of the nodes with HD were positive for EBV genome, respectively, as compared to 16%, 14%, and 43% in the hyperplastic lymph nodes. Clonal and nonclonal episomal EBV and linear replicating EBV genome were present in both conditions. Immunoglobulin heavy chain gene rearrangements were found in two clonal and two nonclonal EBV-positive HD cases, but not in the lymphoid hyperplasia cases. These findings and other recent reports showing EBV genome in benign lymphoid cells by in situ hybridization in Hodgkin's disease suggest that the characteristics of EBV infection in HD could be explained by the reactive cellular milieu, especially in the setting of defective immunity. The identification of EBV genome in Reed-Sternberg cells may, therefore, be a nonspecific phenomenon.     

T and B lymphocytes and Reed-Sternberg cells in Hodgkin''s disease lymph nodes and spleens.

Lymphoid cells from twenty-four untreated Hodgkin's disease biopsies were examined for spontaneous sheep erythrocyte and sensitized ox erythrocyte rosette formation for the identification of T cell and cells with Fc and C3 receptors and surface immunoglobulin. Compared with normal tissues mean T-lymphocytes values were elevated in both involved lymph nodes and uninvolved spleens from Hodgkin's patients. Lymphocytes bearing C3 receptors were correspondingly reduced in these tissues. Involved spleen T-cell values fell within the normal range. In normal tissues the sum of lymphocytes with surface immunoglobulin and sheep erythrocyte receptors fell in the range 89-108%. In six biopsies of Hodgkin's tissue the sum was outside the normal range (121-142%). This observation is compatible with surface immunoglobulin-coated T cells. Surface marker characteristics and intracellular immunoglobulin studies of small lymphocytes, lymphoblasts and Hodgkin's cells suggested that the neoplastic cells were of B lymphocyte origin.     

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin''s disease is frequently associated with CR2 (EBV receptor) expression

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.     

Interleukin-1 beta, but not interleukin-1 alpha, is required for T-cell-dependent antibody production

Interleukin-1 (IL-1) consists of two molecules, IL-1 alpha and IL-1 beta, and IL-1 receptor antagonist (IL-1Ra) is a natural inhibitor of these molecules. Although the adjuvant effects of exogenously administered IL-1 in the humoral immune response are well known, the roles of endogenous IL-1 and the functional discrimination between IL-1 alpha and IL-1 beta have not been elucidated completely. In this report, we investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Both primary and secondary antibody production against T-dependent antigen, sheep red blood cells (SRBC), was significantly reduced in IL-1 alpha/beta-/- mice, and was enhanced in IL-1Ra-/- mice. The intrinsic functions of B cells, such as antibody production against type 1 T-independent antigen, trinitrophenyl-lipopolysaccharide and proliferative responses against mitogenic stimuli, were normal in IL-1 alpha/beta-/- mice. The proliferative response of T cells and cytokine production upon stimulation with anti-CD3 monoclonal antibody were also normal, as was the phagocytotic ability of antigen-presenting cells (APCs). However, SRBC-specific proliferative response and cytokine production of T cells through the interaction with APCs were markedly impaired in IL-1 alpha/beta-/- mice, and enhanced in IL-1Ra-/- mice. Moreover, we show that SRBC-specific antibody production was reduced in IL-1 beta-/- mice, but not in IL-1 alpha-/- mice. These results show that endogenous IL-1 beta, but not IL-1 alpha, is involved in T-cell-dependent antibody production, and IL-1 promotes the antigen-specific T-cell helper function through the T-cell-APC interaction.     

Interleukin-6 production by thyroid epithelial cells. Enhancement by interleukin-1.

Interleukin-1 is a potent inhibitor of thyroglobulin and cAMP production in human thyroid cells and the inhibitory effect is enhanced by tumor necrosis factor-alpha and interferon-gamma. In the present study secondary cultures of human thyroid cells produced interleukin-6 and the production was significantly increased after exposure of the cells to recombinant interleukin-1 alpha and -1 beta. This increase was dose-dependent and concomitant of the IL-1 induced decrease in cAMP and thyroglobulin production. Both tumor necrosis factor-alpha and -beta also augmented interleukin-6 production, but less potently than interleukin-1. Interferon-gamma did not affect the production of interleukin-6. The rat thyroid cell line FRTL-5 produced interleukin-6 spontaneously, and the production was enhanced after addition of recombinant interleukin-1 beta. A pathogenetic role of interleukin-6 in autoimmune thyroid disease is suggested.     

Interleukin-10 is an unequivocal Th2 parameter in the rat, whereas interleukin-4 is not

Exposure of Wistar rats to the immunotoxic compounds hexachlorobenzene (HCB), bis(tri-n-butyltin)oxide, and benzo(a)pyrene was previously found to affect mRNA expression of interleukin (IL)-2, IL-2R alpha-chain, and interferon (IFN)-gamma, the prototypic Th1 cytokine. In contrast, the mRNA expression of IL-4, the prototypic Th2 cytokine, was unaffected. This latter finding suggested that the IL-4 mRNA expression may not be an unequivocal parameter for Th2 responses in the rat. In order to obtain such a parameter the present study was performed, consisting of two types of experiments. Expression and production of IL-4 as well as IL-10, a second Th2 cytokine, were measured. First, Lewis (Th1 prone) and Brown Norway (BN; Th2 prone) rats were exposed to HCB. Exposure was previously found to increase the serum immunoglobulin (Ig)E levels, an IL-4-dependent response, in BN but not Lewis rats, and in Lewis rats to aggravate experimental allergic encephalomyelitis (EAE), severity being inversely related to IL-10 levels. Secondly, BN rats were infected with Trichinella spiralis, an infection previously found to induce IL-4 production. HCB exposure did not affect IL-4 mRNA expression in either strain, while IL-4 production was decreased in Lewis and unaffected in BN rats. In Lewis rats both the mRNA expression and the production of IL-10 were decreased. The T. spiralis infection induced IL-4 and IL-10 mRNA expression, as well as IL-10 production. In contrast, the IL-4 production was strongly reduced. Thus, both the IL-10 mRNA expression and production correlated with the EAE development and T. spiralis infection. In HCB exposed Lewis rats and T. spiralis infected BN rats the IL-4 mRNA expression correlated with IgE levels and T. spiralis infection, respectively, whereas the IL-4 production lacked correlation in all cases. Collectively, these results suggest that IL-10 is an unequivocal Th2 parameter in the rat, whereas IL-4 is not.     

Leu-M1--a marker for Reed-Sternberg cells in Hodgkin''s disease. An immunoperoxidase study of paraffin-embedded tissues.

Monoclonal antibody to Leu-M1, a granulocyte-related differentiation antigen, represents a highly effective reagent for detection of diagnostic Reed-Sternberg (R-S) cells and variants in paraffin-embedded tissues of Hodgkin's disease. In 69 of 73 cases of Hodgkin's disease (41 nodular sclerosis, 25 mixed cellularity, 4 lymphocyte predominance, and 3 lymphocyte depletion types), R-S cells were strongly immunoreactive for Leu-M1. Four cases of lymphocyte predominance Hodgkin's disease (nodular) were uniformly nonreactive for Leu-M1. In most of the positive cases (57/69, 83%), the majority (60-90%) of R-S cells and variants exhibited immunoreactivity for Leu-M1. A characteristic staining pattern included granular and/or vesicular cytoplasmic immunoreactivity, often with a prominent globular paranuclear reaction product, and membrane staining with highly irregular cytoplasmic borders. Evaluation of B-cell (37 specimens), T-cell (20 specimens), and true histiocytic (3 specimens) neoplasms and a case of mastocytosis revealed immunoreactivity for Leu-M1 only in 1 B-cell and 4 T-cell malignancies. The staining patterns in these cases, however, clearly differed from that observed for R-S cells. Studies of nonneoplastic lymphoid tissues (38 total) demonstrated that lymphoid cells were typically nonreactive; histiocytes revealed variable reactivity for Leu-M1. Occasional histiocytes of the sinusoidal network of lymph nodes, particularly in toxoplasmic lymphadenitis, exhibited a staining pattern (membranous/cytoplasmic/paranuclear) similar to that observed for R-S cells. Leu-M1 represents a potentially helpful diagnostic discriminant in the assessment of Hodgkin's disease and its distinction from non-Hodgkin's lymphomas and other lymphoid proliferations.     

Lymphocyte functional antigens stabilize agglutination between Reed-Sternberg cells and T cells, but are not responsible for homotypic binding of Hodgkin's Reed-Sternberg cells.

The neoplastic (Hodgkin's Reed-Sternberg [H-RS]) cells in Hodgkin's disease (HD) are known for their unique capacity to form rosettes with unprimed T cells. Recently, a family of leukocyte-adherence molecules (LFA-1 and LFA-2) has been identified on T lymphocytes. The molecules bind to intercellular-adhesion molecules (ICAMs) and to LFA-3, respectively, which are associated with antigen-presenting cells. In this study, the authors examined whether these molecules are responsible for the homotypic and heterotypic agglutination that occurs in the cultured H-RS cells HDLM-1, HDLM-1d, and KM-H2. Despite their similar expressions of LFA-3 and ICAM-1, the different H-RS cells tested showed different growth patterns in culture. HDLM-1 cells grew singly, whereas HDLM-1d and KM-H2 cells grew in clumps. The HDLM-1 cells formed clumps when mixed with peripheral-blood T lymphocytes, cells of two lymphoblastic T-cell lines (MOLT-3 and MOLT-4), and cells of two monocyte lines (ML-1 and U-937). The addition of anti-LFA and ICAM-1 antibodies to cultures did not result in disassembly of the homotypic clusters of HDLM-1d or KM-H2 cells and it did not cause any significant changes in the size of heterotypic clusters or in the timing of cluster formation of HDLM-1 cells with other types of cells. In all studies, the cell clusters formed during homotypic and heterotypic aggregation were disassembled only minimally by cell shearing with pipetting. The disaggregation by pipetting was slightly more prominent in the presence of antibodies than was that of control cultures. However, in no case did the use of monoclonal antibodies (MAbs) and cell shearing cause complete disaggregation of homotypic and heterotypic clusters. The result seems to suggest that binding between H-RS cells and T cells and between H-RS cells and monocytes is not mediated primarily by LFAs and ICAMs, but that the binding may be strengthened in the presence of these molecules.     

Interleukin-4 downregulates interleukin-6 production in human peripheral blood mononuclear cells

We report that recombinant human interleukin-4 (IL-4) downregulates interleukin-6 (IL-6) production by human peripheral blood mononuclear cells (PBMC). PBMC were preincubated for up to 24 hr in the presence of IL-4 (100 U/ml) and then activated with lipopolysaccharide B Escherichia coli 026:B6 (LPS, 10 micrograms/ml), recombinant human tumor necrosis factor-alpha (TNF-alpha, 200 U/ml), or Concanavalin A (Con A, 10 micrograms/ml). Although all these signals induced IL-6 production, IL-4-treated cells produced significantly reduced levels of IL-6 protein. This effect was dose and time dependent. We conclude that IL-4 is a potent downregulatory modulator of IL-6 expression in human PBMC.