Elizabeth Raveche

Characterization of the Mouse IFN-λ Ligand-Receptor System: IFN-λs Exhibit Antitumor Activity against B16 Melanoma

Recently discovered type III IFNs (IFN-lambda) exert their antiviral and immunomodulatory activities through a unique receptor complex composed of IFN-lambdaR1 and interleukin-10 receptor 2. To further study type III IFNs, we cloned and characterized mouse IFN-lambda ligand-receptor system. We showed that, similar to their human orthologues, mIFN-lambda2 and mIFN-lambda3 signal through the IFN-lambda receptor complex, activate IFN stimulated gene factor 3, and are capable of inducing antiviral protection and MHC class I antigen expression in several cell types including B16 melanoma cells. We then used the murine B16 melanoma model to investigate the potential antitumor activities of IFN-lambdas. We developed B16 cells constitutively expressing murine IFN-lambda2 (B16.IFN-lambda2 cells) and evaluated their tumorigenicity in syngeneic C57BL/6 mice. Although constitutive expression of mIFN-lambda2 in melanoma cells did not affect their proliferation in vitro, the growth of B16.IFN-lambda2 cells, when injected s.c. into mice, was either retarded or completely prevented. We found that rejection of the modified tumor cells correlated with their level of IFN-lambda2 expression. We then developed IFN-lambda-resistant B16.IFN-lambda2 cells (B16.IFN-lambda2Res cells) and showed that their tumorigenicity was also highly impaired or completely abolished similar to B16.IFN-lambda2 cells, suggesting that IFN-lambdas engage host mechanisms to inhibit melanoma growth. These in vivo experiments show the antitumor activities of IFN-lambdas and suggest their strong therapeutic potential.

Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in non–New Zealand Black B cell and New Zealand Black–derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684–92]

Requirement for increased IL-10 in the development of B-1 lymphoproliferative disease in a murine model of CLL.

Malignant B-1 cells derived from NZB mice, a murine model of spontaneous autoimmunity and B cell lymphoproliferative disease, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for the expansion of malignant B-1 cells. In order to determine if elevated endogenous production of IL-10 was a required element for the malignant transformation of B-1 cells in NZB mice, backcross animals were studied for the linkage between elevated IL-10 expression and the presence of lymphoid malignancy. The phenotypes of aged (NZB x DBA/2)F1 x NZB animals were determined and a strong correlation was found between the elevated levels of IL-10 mRNA and the development of B-1 malignant clones. In contrast, an increased level of IL-10 message was not associated with elevated serum IgM or the presence of anemia or reticulocytosis which is mainly seen in response to autoantibody production. These results indicate that, at least in NZB, the autoimmunity and lymphoproliferation phenotypes are not linked genetically. IL-10 may enhance proliferation and the development of B-1 cell malignancy rather than antibody production by the B-1 cell subpopulation. Thus, IL-10 plays an important role in B-1 malignancies, and downregulation of IL-10 could be a likely site for intervention in B cell malignancies.

Investigation of promoter polymorphisms in the tumor necrosis factor-α and interleukin-10 genes in liver transplant patients

Background. Cytokines such as tumor necrosis factor (TNF)-&agr; and interleukin (IL)-10 play significant roles in the inflammatory and immune responses that mediate allograft rejection. The presence of a G→A polymorphism at position −308 in the promoter region of the TNF-&agr; gene increased its transcription 6- to 7-fold. A similar polymorphism at position −1082 of the IL-10 promoter results in decreased production of IL-10 protein. In this study we have determined whether the single nucleotide polymorphisms in the promoter regions of the TNF-&agr; and IL-10 genes can predict the outcome of the allograft in liver recipients. Methods. DNA was extracted from whole blood of liver recipients. The genotype of the patients was determined by polymerase chain reaction using sequence-specific primers. The level of TNF-&agr; and IL-10 protein was measured by ELISA after stimulation of peripheral blood mononuclear cells with concanavalin A. Results. There was significant correlation between acute cellular rejection and the presence of the −308A polymorphism (P <0.001), with 8 of 13 patients with the TNF-&agr; polymorphism having evidence of acute rejection. Cell stimulation studies revealed that the level of TNF-&agr; protein produced by patients with liver rejection was significantly higher than for patients without rejection (P =0.001). There were no strong associations between the presence of the IL-10 polymorphisms and rejection (P =0.71). Conclusions. This study adds to the understanding of the role of cytokine polymorphisms in liver transplants. The data suggest that cytokine promoter polymorphisms may be a risk factor associated with allograft rejection in the liver.

Growth inhibition of malignant CD5+B (B-1) cells by antisense IL-10 oligonucleotide

Malignant B-1 cells derived from NZB mice, a murine model of chronic lymphocytic leukemia, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for malignant B-1 cells. By addition of antisense oligodeoxynucleotides specific for IL-10 mRNA, we were able to dramatically inhibit the growth of leukemic B-1 cells in a time and dose dependent manner. Control cell lines which do not depend on IL-10 for growth were not affected. Antisense therapy targeted at the 5 region of the IL-10 mRNA not only resulted in inhibition of malignant B-1 cell proliferation but also inhibited IL-10 production by malignant B-1 cells. Because endogenous IL-10 gene activation is critical for B-1 cell expansion, inactivation of the endogenous IL-10 gene by IL-10 antisense rather than extracellular regulation of the IL-10 gene product should be successful in controlling the malignant growth.

Evidence of Borrelia Autoimmunity-Induced Component of Lyme Carditis and Arthritis

ABSTRACT We investigated the possibility that manifestations of Lyme disease in certain hosts, such as arthritis and carditis, may be autoimmunity mediated due to molecular mimicry between the bacterium Borrelia burgdorferi and self-components. We first compared amino acid sequences of Streptococcus pyogenes M protein, a known inducer of antibodies that are cross-reactive with myosin, and B. burgdorferi and found significant homologies with OspA protein. We found that S. pyogenes M5-specific antibodies and sera from B. burgdorferi-infected mice reacted with both myosin and B. burgdorferi proteins by Western blots and enzyme-linked immunosorbent assay. To investigate the relationship between self-reactivity and the response to B. burgdorferi, NZB mice, models of autoimmunity, were infected. NZB mice infected with B. burgdorferi developed higher degrees of joint swelling and higher anti-B. burgdorferi immunoglobulin M cross-reactive responses than other strains with identical major histocompatibility complex (DBA/2 and BALB/c). These studies reveal immunological cross-reactivity and suggest that B. burgdorferi may share common epitopes which mimic self-proteins. These implications could be important for certain autoimmunity-susceptible individuals or animals who become infected with B. burgdorferi.

Role of microRNA-15a in autoantibody production in interferon-augmented murine model of lupus ☆

MicroRNAs (miRNAs) are involved in the regulation of immunity via targeting of mRNA encoding immune response elements. In this report, alterations in the expression of microRNAs as autoantibody levels increase was investigated. The (NZB×NZW)F1 or B/W mouse model of systemic lupus erythematosus (SLE) naturally has increased autoantibodies with aging. IFNα (type I IFN) accelerates B/W disease, however, the effects of a related IFN, IFNλ, which is a type III IFN, is largely unknown. The purpose of the study was to investigate the relationship between IFN-accelerated disease, microRNAs, immunoregulatory B cell subsets and autoantibody production in the autoimmune-prone environment in vivo. B/W mice received osmotic pumps to chronically deliver IFNα and IFNλ for up to 16 weeks. Urine protein level was monitored weekly by urine strips and proteinuria was used as the disease marker. Splenic cells were taken for flow analysis of B cell subsets and levels of microRNAs determined. Plasma were analyzed for autoantibodies and microRNA levels. As a result of treatment, IFNα accelerated proteinuria in a dose dependent manner, while IFNλ single treatment did not show a significant effect, but greatly enhanced low dose IFNα effects in the combination treatment. Both the splenic cellular and plasma miR-15a were elevated in diseased compared to pre-diseased mice as well as autoantibody levels. Autoantibodies and miR-15a levels were significantly correlated. The immunosuppressive B subpopulation, B-10, was reduced following IFNα treatment. In addition in diseased mice, B-10 versus B-2 ratios were reduced in IFN-treated B/W compared to the control PBS treated group. In all B/W the miR-15a was highly expressed in the B-10 subset and this increased with disease development, suggesting that miR-15a has a specific negative effect on the B-10 subpopulation. In conclusion, our data support the involvement of elevated miR-15a in autoimmune disease development in B/W mice and suggest that decreasing this microRNA might be beneficial in B/W mice.

Chronic lymphocytic leukaemia genetics overview

Although the familial aspect of chronic lymphocytic leukaemia (CLL) has been appreciated for decades, it is only with the recent confluence of improved molecular and gene technologies and world‐wide collaborative networks that accelerated progress has become apparent. In this summary we highlight selected themes in the genetics of CLL emphasizing the opportunities and challenges of this malignancy.

IL-10 production in a CD5+ B cell lymphoma arising in a CD4 monoclonal antibody-treated SJL mouse.

A majority of SJL mice develop spontaneous reticulum cell sarcomas (RCS) at about 1 year of age which can be transplanted into young SJL recipients. Previous studies have shown that RCS tumors are of B cell lineage, and that the development of these lymphomas and their subsequent growth depends upon host-derived T helper cell factors. In vivo treatment of SJL mice with anti-CD4 monoclonal antibody (mAb) prevents the development of the characteristic B lymphomas. Most of the mAb-treated animals were tumor free and had a significantly prolonged life span. However, one such CD4 mAb-treated mouse developed a transplantable IgM+ CD5+ B cell lymphoma (designated NJ101), which has not previously been described in SJL/J mice. NJ101 is clonal on the basis of a discrete non-germ line Ig heavy chain gene rearrangement by Southern blot analysis. Unlike the sIg- CD5- transplantable RCS-X cell line, the IgM+ CD5+ NJ101 lymphoma cells will grow in immuno-compromised hosts, such as irradiated recipients or in recipients treated with CD4 mAb in vivo. The RCS (B cell) lymphoma requires CD4+ T cells for progressive growth, whereas the growth of the CD5+ B lymphoma cells is enhanced by the removal of such cells. Thus, CD5+ B cell clonal development may be aided by the removal of regulatory T cells and/or the malignant CD5+ B cells may produce their own growth factors in an autocrine manner. Examination of IL-10 message by quantitative polymerase chain reaction techniques indicate that the CD5+ B lymphoma cells produce increased levels of IL-10 relative to normal LN cells or purified RCS lymphoma cells. These results suggest that two different types of B cell tumors, both of which can develop in SJL mice, have different growth requirements. Furthermore, treatment to prevent the occurrence of the characteristic RCS malignancy of SJL mice may lead to the development of another form of B cell neoplasia.

Genistein induces G2 arrest in malignant B cells by decreasing IL-10 secretion.

Chronic B cell malignancies are often chemoresistant and the development of new therapeutic modalities is a high priority. Many B cell malignancies have autocrine production of IL-10, which regulates B cell growth and differentiation. Here we demonstrated that the soy isoflavone genistein, a tyrosine kinase inhibitor, rapidly decreased IL-10 secretion followed by upregulation of IFN-gamma and inhibition of cell proliferation with predominantly G2 arrest. The antiproliferative effects of genistein could be reversed by the addition of exogenous IL-10. Genistein downregulated cdc25C and cdk1 as well as anti-apoptotic proteins survivin and Ian-5. After genistein withdrawal, the G2M arrested cells re-entered the cell cycle and underwent apoptosis, which was significantly augmented by fludarabine. We conclude that genistein can sensitize malignant B cells to the action of other chemotherapeutic agents by modulating the cytokine profile and controlling cell cycle progression.