In Vivo and In Vitro Analysis of IL-10 in the NZB Leukemic Model

Background: The B-1 malignancy, CLL has been associated with a failure to undergo apoptosis and increased endogenous IL-10 production. This study was undertaken to identify IL-10 effects in the NZB murine model of CLL. Materials and Methods: Antisense IL-10 was employed in vitro and in vivo to decrease IL-10 protein. Following treatment, cells were analyzed for alterations in cell cycle and RNA was studied for alterations in gene expression. Additional in vivo studies employed NZB mice in which the IL-10 gene was deleted. Results: IL-10 (-/-) knockout NZB mice overwhelmingly failed to develop leukemia. In vitro antisense IL-10 treatment resulted in a G2/M block and apoptosis and in vivo treatment with antisense IL-10 increased the survival of mice. Microarray analysis indicated a significant role for IL- 10 in cell cycle regulation via cdc25C up-regulation and decreased p47 phox redox activity. Conclusion: In summary, IL- 10 is a critical survival factor for malignant B cells via anti- apoptotic and cell cycle effects. CLL is characterized as a malignant expansion of B-1 cells, a subset of B cells which express features normally found on other hematopoietic cells, i.e. CD5 (T lymphocytes) and Mac-1/CD11b (macrophages) (1). B-1 cells are found predominantly in the peritoneum and clonal expansion of B-1 cells in the peritoneum has been implicated in CLL, the most common leukemia in the western world (2). B-1 cells compose a minor fraction of splenic B cells and are not normally present in lymph nodes. In contrast, they are the main B cell population in the peritoneal and pleural cavities. (1). The role of IL-10 in cancer has been somewhat controversial. In one report, apoptosis was observed after treating B-CLL samples with IL-10 in vitro (3). Other laboratories have since found IL-10 in B-CLL samples and did not find IL-10 to be associated with apoptotic induction (4-7). Although initially termed "cytokine synthesis inhibitory factor" (CSIF) and characterized as a product of T helper 2 (Th2) cells that down-regulated interferon gamma in mice (8), IL-10 is now well known to have complex effects on many cell types (see review) (9). When analyzing CLL patients by ELISA, serum IL-10 levels were higher in CLL patients (median 5.04 pg/ml) than in normal volunteers (median undetectable, n=55) and increased IL-10 was an independent prognostic factor for decreased survival (10). In order to examine the role of IL-10 in the protection of malignant cells from apoptosis, the NZB strain of mice, a murine model of CLL (2), was employed. Previous studies of the NZB have established that IL-10 is produced in an autocrine manner by the malignant B-1 cells in NZB (11- 16). As a source of NZB malignant B-1 cells, an in vitro established line, LNC, (11) was used. LNC has previously been shown to express high levels of IL-10 and following in vivo transfer to result in a fatal B-1 malignant expansion. In contrast to wild-type, NZB IL-10 (-/-) knockout mice overwhelmingly fail to develop leukemic symptoms (12). Microarray analysis of DNA can provide unprecedented clues into biological processes with great accuracy (17). Therefore, in this manuscript RNA was obtained from: untreated, antisense IL-10 or scrambled oligo-treated LNC, an NZB malignant B-1 cell line, and employed in microarray experiments. In this report, several new pathways influenced by IL-10 are elucidated.