Studies in NZB IL-10 knockout mice of the requirement of IL-10 for progression of B-cell lymphoma.

NZB mice develop an age-related malignant expansion of a subset of B cells, B-1 cells, with autocrine production of IL-10. IL-10, a pleiotropic cytokine with anti-inflammatory properties, is a potent growth and survival factor for malignant B cells. To further examine the in vivo requirement for IL-10 in the development and expansion of malignant B-1 clones in NZB mice, we developed a strain of homozygous IL-10 knockout (KO) mice on an NZB background. The NZB IL-10 KO mice develop peritoneal B-1 cells with approximately the same frequency as heterozygous and wild-type littermates. In contrast, the development of malignant B-1 cells in the peripheral blood and spleen, observed in wild-type NZB, rarely occurred in the NZB IL-10 KO. Phenotypic analysis of surface marker expression in splenic B cells indicated that, in contrast to the NZB with malignant B-1 splenic lymphoma, the surface marker expression of NZB IL-10 KO splenic B cells indicated that the majority of the B cells were typical B-2 cells. In the absence of IL-10, spontaneously activated B cells and antiapoptotic gene expression were reduced and lymphoma incidence was decreased. These results indicate that IL-10 is a critical factor for the progression of this B-cell malignant disease.

Interleukin-4 up-regulates mouse mammary tumor virus expression yet is not required for in vivo virus spread.

The mouse mammary tumor virus (MMTV) superantigen induces T-cell production of cytokines, such as interleukin-4, which in turn increase MMTV transcription. However, interleukin-4 is not required for in vivo virus spread, because mice lacking interleukin-4 or the STAT6 transcription factor showed wild-type infection of lymphoid and mammary tissue. In spite of this, mammary tumor incidence was decreased in STAT6 null mice.

Cell cycle effects of IL-10 on malignant B-1 cells.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

Comparison study for identifying promoter allelic polymorphism in interleukin 10 and tumor necrosis factor alpha genes.

Cytokines such as tumor necrosis factor (TNF)-alpha and Interleukin (IL)-10 play significant roles in autoimmunity and transplantation tolerance. Allelic polymorphisms that occur in the regulatory regions of these cytokine genes are closely associated with acute and chronic transplant rejection. The presence of a G-to-A polymorphism at position -308 in the promoter region of the TNF-alpha gene can increase transcription six- to sevenfold. Likewise, the G-A polymorphism at position -1082 of the IL-10 promoter results in lower levels of IL-10 protein. Accordingly, a genotype that dictates the production of high levels of TNF-alpha with low IL-10 capabilities is most likely to generate an inflammatory environment that is less receptive to the transplant. The potential for determining a patient's haplotype before transplantation may be an effective way of monitoring the post-transplant status of such patients. A variety of methodologies that address the detection of mutations have been used both in research and clinical diagnostic tests. This study analyzes the genetic variations in cytokines using two methodologies: the traditional allele-specific oligonucleotide (ASO) polymerase chain reaction (PCR) and the newer and more flexible Invader technology. The sensitivity and specificity of the Invader assay for simultaneous investigation of multiple targets makes it a useful tool in such analyses.

Antisense IL-10 abrogates the inhibitory effects of IL-10 production by transfected tumor cells.

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a variety of downregulatory effects on immunologic and inflammatory processes. Ectopic tumor expression of IL-10 inhibited tumor growth, and local administration of antisense IL-10 significantly reversed the effects of IL-10 transfection in P815 mastocytoma. Tissue inhibitors of metalloproteinase (TIMPs) have been associated with decreased tumorigenesis and reduced metastasis, and TIMPs were increased in the region surrounding P815/IL-10 tumors and reduced in antisense IL-10-treated mice. In addition, the antisense IL-10 group had the largest tumor volume and poorest survival when compared with the P815/IL-10 control or sense groups. In summary, our data suggest that, in a mouse model, antisense IL-10 has substantive effects in reducing IL-10 translation and inhibiting IL-10-mediated TIMP upregulation, and, by doing so, allows IL-10-transfected mastocytoma to grow unchecked. Thus, ectopic tumor expression of IL-10 inhibits tumor growth, and antisense IL-10 administration in vivo reverses this protective effect.

Effects of cord blood transfer on the hematopoietic recovery following sublethal irradiation in MRL lpr/lpr mice.

The potential of cord blood (CB) to serve as a rich source of stem cells and stem cell factors is receiving increasing attention. In addition, perhaps because of the early ontogeny of these cells or the lack of surface antigens, cord blood stem cells do not appear to require close identity with the recipient. In the present pilot study, we investigated the presence of a hematopoiesis enhancing effect (HEE) by assaying the ability of human cord blood cells to augment hematopoiesis across a species barrier. For these experiments, autoimmune-prone MRL-Ipr/Ipr mice were exposed to sublethal levels of irradiation and cord blood administration to study the role of factors present in human cord blood in augmenting the rate of lymphopoiesis. This strain was chosen because of the increased presence of peripheral T and B subpopulations, namely the B-1 and CD4/CD8 double negative T-cell subpopulations, which do not arise directly from bone marrow precursors, but rather accumulate with age. MRL-Ipr/Ipr mice were sublethally irradiated and reconstituted with syngeneic bone marrow (BM) cells or with human cord blood cells or peripheral blood mononuclear cells (PBMC), or were left unreconstituted. At 2 weeks post-treatment, lymphoid populations in the spleen and lymph nodes were studied as a measure of hematopoiesis. Factors present in cord blood were able to augment hematopoiesis over that which occurred endogenously. At 2 weeks postirradiation, recipients of BM cells displayed the fastest rate of peripheral lymphoid recovery, nonreconstituted mice showed the slowest lymphoid recovery, and recipients of cord blood recovered their lymphoid populations at an intermediate rate. Similarly, myelopoiesis was increased in irradiated SJL/J recipients of human cord blood. Thus, human cord blood cells appear to produce/induce factors that may act as an adjunct to increase stem-cell activity.

Effect of human cord blood transfer on survival and disease activity in MRL-lpr/lpr mice.

The MRL-lpr/lpr mice have a genetic defect and by 6 months of age usually die after developing severe autoimmune disease and massively enlarged lymph nodes (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991). Similarly as in some genetic diseases in humans, these mice, if given an appropriate marrow transplant from a mouse not having the defect, will have partial correction of the disease process and an extension of life (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991; Lenarsky, C., Kohn, D. B., Weinberg, K. I., and Parkman, R., Hematol. Oncol. Clin. N. Am. 4, 589-603, 1990). Utilizing human umbilical cord blood as a donor source for marrow transplantation, we have been able to obtain significant correction of the MRL-lpr/lpr genetic defect and double the life span. By 11 months of age, 5 months beyond their usual life span, both the animals receiving congenic marrow (MRL-+/+) and the animals with human cord blood had mild lymphadenopathy, a decrease in double-positive T cells, and an increase in double-negative T cells. Granulomatous vasculitis could be identified in the animals receiving human cells and could not be found in the animals receiving MRL-+/+ marrow.