Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo

Abstract

Human Burkitt lymphoma cell lines give rise to progressively growing subcutaneous tumors in athymic mice. These tumors are induced to regress by inoculation of Epstein-Barr virus-immortalized normal human lymphocytes. In the present study, analysis of profiles of murine cytokine/chemokine gene expression in Burkitt tumor tissues excised from the nude mice showed that expression of the murine alpha-chemokine interferon-inducible protein-10 (IP-10) was higher in the regressing than in the progressive Burkitt tumors. We tested the effects of IP-10 on Burkitt tumor growth in nude mice. Inoculation of established Burkitt tumors either with crude preparations of murine IP-10 or with purified human IP-10 caused visible tumor necrosis in a proportion of the animals, although no complete tumor regressions were observed. Constitutive expression of murine IP-10 in Burkitt cells reduced their ability to grow as subcutaneous tumors, and caused visible tumor necrosis in a proportion of the animals. Histologically, IP-10-treated and IP-10-expressing Burkitt tumors had widespread evidence of tumor tissue necrosis and of capillary damage, including intimal thickening and vascular thrombosis. Thus, IP-10 is an antitumor agent that promotes damage in established tumor vasculature and causes tissue necrosis in human Burkitt lymphomas established subcutaneously in athymic mice.

Figure 1

Cytokine and chemokine mRNAs in regressing and progressive Burkitt tumors revealed by RT-PCR analysis. Tissue fragments were obtained from regressing and progressive Burkitt tumors established in athymic mice. Positive controls were derived from murine macrophages incubated with 1 mg/ml lipopolysaccharide for 6 hr (IP-10, IL-12, Mig, and JE/MCP-1); murine splenocytes incubated with 4 mg/ml concanavalin A for 3 hr (IFN-γ); murine splenocytes from a 3-day mixed lymphocyte reaction (RANTES, MIP-1α, and MIP-1β). Total cellular RNA, isolated from tumor and control tissues, was subjected to RT-PCR analysis. After normalization to G3PDH, the signals derived from cytokine expression in regressing versus progressive Burkitt tumors, expressed as ratios, were as follows: IP-10, 8.2; IFN-γ, 10.6; IL-12 p35, 15.6; IL-12 p40, 4.0; Mig, 12.8; JE/MCP-1, 2.3; RANTES, 8.1; MIP-1α, 2.8; MIP-1β, 2.7.

Figure 2

Gross and microscopic morphology of progressive and regressing Burkitt lymphoma tumors. (A–D) Tumor tissue from a mouse injected subcutaneously with CA46 Burkitt cells. (E–H) Tissue from a tumor induced by subcutaneous injection with CA46 Burkitt cells and subsequently injected with LCL (VDS line) weekly for 3 weeks. (I–L) Tissue from a tumor induced by subcutaneous injection with CA46 Burkitt cells and subsequently injected daily with human IP-10. (M–P) Tumor tissue from a mouse injected subcutaneously with murine IP-10-expressing CA46. (A, E, I, and M) Gross morphology of Burkitt tumors showing viable looking tumor tissue (A) and regressing tumors with extensive central necrosis surrounded by viable tumor (E, I, and M) (no magnification). (B, F, J, and N) microscopic morphology of Burkitt tumors extending to the epidermidis; in B viable-looking tumor tissue, and in F, J, and N the interface between necrotic (top) and viable (bottom) tumor tissue are shown. (C, G, K, and O) Higher power magnification of viable tumor tissue (C) and of the interface of necrotic (top) and viable (bottom) tumor tissue (G, K, and O). A prominent macrophage infiltration is noticeable in G. (D, H, L, and P) Higher power magnification of viable tumor tissue with patent capillaries (containing red cells) in D, and capillaries occluded with thrombi at various stages of reorganization (H, L, and P). (B, F, J, and N, ×5; C, G, K, and O, ×10; D, H, L, and P, ×20.)

Figure 3

Effect of murine IP-10 expression on Burkitt lymphoma tumorigenesis. (A) Northern blot analysis of murine IP-10 mRNA expression in CA46-IP-10 clones 11/3, 11/4, and 11/9, vector control transfected and CA46 pCEP4 clones 1 and 3, and murine IP-10 expressing CHO cells. The RNA (15 mg) was run on a 1.2% agarose gel, transferred to nitrocellulose membrane, and hybridized for murine IP-10 and murine GAPDH. (B) Tumor growth in BALB/c nu/nu mice inoculated subcutaneously with murine IP-10-expressing and control CA46 Burkitt cells. Murine IP-10 expressing CA46 clones 11/3 (▪), 11/4 (▴), and 11/9 (•) and control CA46 clones 1 (□) and 3 (○) (10⁷ cells in 0.2 ml culture medium), were each inoculated subcutaneously in irradiated (400 rad) BALB/c nu/nu mice. Tumor size was measured weekly. Each data point represents the mean tumor size of four or five mice. Because of large tumor size and mouse mobility impairment, mice injected with CA46-pCEP4 clones 1 and 3 were killed at 7 weeks.