Interleukin-18 expression induced by Epstein-Barr virus-infected cells.

Human Epstein-Barr virus (EBV)-negative Burkitt lymphomas cells usually grow as malignant subcutaneous tumors in athymic mice, but these tumors regress when the Burkitt cells are injected in conjunction with EBV-positive lymphoblastoid cells or when the Burkitt cells are transfected with the EBV latent membrane protein-1 (LMP-1) gene. Tumor regression is mediated, in part, by murine interferon gamma (IFN-gamma) and the IFN-gamma-induced murine chemokine IFN-gamma-inducible protein-10 (IP-10). The mechanisms by which EBV-LMP-1 promotes the expression of IFN-gamma has remained unclear. Here we show that murine interleukin (IL)-18 was consistently expressed in regressing Burkitt tumors but was either expressed at low levels or absent from progressively growing Burkitt tumors. By immunohistochemical methods, IL-18 protein was visualized in regressing but not in progressively growing Burkitt tumors. In contrast, IL-12 p35 and IL-12 p40 were only rarely expressed in regressing Burkitt tumors. In splenocyte cultures, EBV-infected lymphoblastoid cells and LMP-1-transfected Burkitt cells promoted the expression of IL-18 but not the expression of IL-12 p35 and IL-12 p40. A neutralizing antibody directed at murine IL-18 reduced murine IP-10 expression induced by EBV-immortalized cells in splenocyte cultures. These results provide evidence for IL-18 expression in response to a viral latency protein and suggest that IL-18 may play an important role as an endogenous inducer of IFN-gamma expression, thereby contributing to tumor regression.

Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12.

Solid tumors are dependent on preexisting vasculature and neovascularization for their growth. Successful cancer therapies targeting the tumor vasculature would be expected to block the existing tumor blood supply and to prevent tumor neovascularization. We tested the antitumor activity of experimental therapy with 2 distinct antiangiogenic drugs. Vasostatin inhibits endothelial cell growth and neovascularization, and interleukin-12 (IL-12) targets the tumor vasculature acting through interferon-gamma (IFN-gamma) and the downstream chemokines interferon-inducible protein-10 (IP-10) and monokine induced by IFN-gamma. Individually, vasostatin and IL-12 produced distinct efficacy profiles in trials aimed at reducing tumor growth in athymic mice. In combination, these inhibitors halted the growth of human Burkitt lymphoma, colon carcinoma, and ovarian carcinoma. Thus, cancer therapy that combines distinct inhibitors of angiogenesis is a novel, effective strategy for the experimental treatment of cancer. (Blood. 2000;96:1900-1905)

Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth.

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

Interleukin-18, interferon-gamma, IP-10, and Mig expression in Epstein-Barr virus-induced infectious mononucleosis and posttransplant lymphoproliferative disease.

T cell immunodeficiency plays an important role in the pathogenesis of posttransplant lymphoproliferative disease (PTLD) by permitting the unbridled expansion of Epstein-Barr virus (EBV)-infected B lymphocytes. However, factors other than T cell function may contribute to PTLD pathogenesis because PTLD infrequently develops even in the context of severe T cell immunodeficiency, and athymic mice that are T-cell-immunodeficient can reject EBV-immortalized cells. Here we report that PTLD tissues express significantly lower levels of IL-18, interferon-gamma (IFN-gamma), Mig, and RANTES compared to lymphoid tissues diagnosed with acute EBV-induced infectious mononucleosis, as assessed by semiquantitative RT-PCR analysis. Other cytokines and chemokines are expressed at similar levels. Immunohistochemistry confirmed that PTLD tissues contain less IL-18 and Mig protein than tissues with infectious mononucleosis. IL-18, primarily a monocyte product, promotes the secretion of IFN-gamma, which stimulates Mig and RANTES expression. Both IL-18 and Mig display antitumor activity in mice involving inhibition of angiogenesis. These results document greater expression of IL-18, IFN-gamma, Mig, and RANTES in lymphoid tissues with acute EBV-induced infectious mononucleosis compared to tissues with PTLD and raise the possibility that these mediators participate in critical host responses to EBV infection.

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.

MIP-1alpha expression in tissues from patients with hemophagocytic syndrome.

Hemophagocytic syndrome (HPS) is a clinicopathologic syndrome that can be precipitated by a variety of causes and is characterized by a systemic activation of macrophages, which are induced to undergo phagocytosis. Chemokines play an important role in the inflammatory cell recruitment into tissues. We examined the expression of chemokines and cytokines in tissues exhibiting histologic evidence of HPS in a variety of settings: peripheral T-cell lymphoma, three patients; nasal T/NK cell lymphoma, one patient; subcutaneous panniculitis-like T-cell lymphoma, one patient; and chronic EBV infection, one patient. Compared with control tissues, we found elevated macrophage inflammatory protein-1alpha (MIP-1alpha) and interferon-gamma (IFN-gamma) expression, but not macrophage-derived chemotactic factor (MDC) or TNF-alpha, in tissues of patients with HPS irrespective of the cause or setting. MIP-1alpha can promote macrophage chemotaxis and IFN-gamma promotes macrophage activation. Elevated expression of IP-10 and monokine induced by IFN-gamma (Mig) was also detected in tissues exhibiting features of HPS, providing an explanation for the occurrence of chemoattraction of T-cells and NK cells. Immunohistochemical analysis of tissues with evidence of phagocytic activity in that site showed MIP-1alpha characteristically localized to endothelial cells of blood vessels and splenic sinuses, lymphocytes, and macrophages. These results provide evidence for MIP-1alpha chemokine expression in tissues from patients with HPS and suggest that MIP-1alpha may play an important role in the pathogenesis of the hemophagocytic syndrome.

Expression of human herpesvirus-8 oncogene and cytokine homologues in an HIV-seronegative patient with multicentric Castleman's disease and primary effusion lymphoma.

Human herpesvirus-8 (HHV-8) has been described in association with two lymphoproliferative disorders: one benign, multicentric Castleman's disease (MCD), and one malignant, primary effusion lymphoma (PEL). The factors that lead to malignant transformation of lymphoid cells are unknown, although most cases of PEL also are positive for EBV, suggesting a role for EBV as a cofactor in malignant transformation. We encountered a rare case of an HHV-8-associated MCD, followed by the development of an HHV-8-positive pleural PEL and a gastric large cell lymphoma in an HIV-seronegative male patient. The lesions were negative for Epstein-Barr virus (EBV). The combination of these diverse HHV-8-associated lymphoproliferative disorders in a single patient afforded us the ability to study potential differences in gene expression in these conditions. HHV-8 DNA was demonstrated by PCR in lymphoid tissues involved by MCD and PEL. By reverse transcriptase-PCR, HHV-8-related transcripts, including vG-coupled protein receptor, vbcl2, vcyclin D, vIL-6, vMIPI, and vMIPII, were detected in the PEL from the pleural cavity and the gastric lymphoma, whereas these transcripts, except for vIL-6, were not detected in a lymph node biopsy with MCD. Expression of hIL-10 was weak in the PEL from the pleural cavity, and expression of hIL-6 was undetectable in all three lesions. These data suggest that vIL-6 may be integral to the pathogenesis of MCD, whereas other viral transcripts that encode oncogene and chemokine homologues are important for HHV-8 tumorigenicity.