CCR2-dependent recruitment of macrophages by tumor-educated mesenchymal stromal cells promotes tumor development and is mimicked by TNFα.

Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. These tumor-resident MSCs are known to affect tumor growth, but the mechanisms are largely unknown. We found that MSCs isolated from spontaneous lymphomas in mouse (L-MSCs) strikingly enhanced tumor growth in comparison to bone marrow MSCs (BM-MSCs). L-MSCs contributed to greater recruitment of CD11b(+)Ly6C(+) monocytes, F4/80(+) macrophages, and CD11b(+)Ly6G(+) neutrophils to the tumor. Depletion of monocytes/macrophages, but not neutrophils, completely abolished tumor promotion of L-MSCs. Furthermore, L-MSCs expressed high levels of CCR2 ligands, and monocyte/macrophage accumulation and L-MSC-mediated tumor promotion were largely abolished in CCR2(-/-) mice. Intriguingly, TNFα-pretreated BM-MSCs mimicked L-MSCs in their chemokine production profile and ability to promote tumorigenesis of lymphoma, melanoma, and breast carcinoma. Therefore, our findings demonstrate that, in an inflammatory environment, tumor-resident MSCs promote tumor growth by recruiting monocytes/macrophages.

Brief report: interferon-gamma induces expansion of Lin(-)Sca-1(+)C-Kit(+) Cells.

The balance between Th1 and Th2 cells is critical for homeostasis of the immune system. Th1 cells can also regulate hematopoietic progenitor cell homeostasis by production of oncostatin M. Here we show that Th1 cell products, but not those of Th2 cells, caused a rapid expansion of lineage(-)Sca-1(+)C-kit(+) (LSK) cells in vivo and in vitro. Among Th1 cytokines, interferon-gamma (IFNgamma) was found to play a major role in this expansion by activating the expression of Sca-1 in lineage(-)Sca-1(-)C-kit(+) cells. This process was dependent on IFNgammaR1 signaling and the STAT1 pathway. Furthermore, those IFNgamma-induced LSK cells had a higher proliferation potential than control LSK cells. In addition, while the overall production of colony-forming units in bone marrow was decreased after IFNgamma treatment, the sorted LSK cells could give rise to a higher yield of colony-forming units. Finally, the IFNgamma-induced hematopoiesis was biased toward the differentiation of myeloid lineages. Therefore, our findings demonstrated a novel role of IFNgamma in activating hematopoietic progenitor cells and provide a new insight into the clinical application of interferon.

Mutagenesis in vivo in T cells of p21-deficient mice.

Mice that are deficient in p53 exhibit an early onset of multiple types of tumors, especially thymic lymphoma. However, it remains unclear to what extent each of the p53-regulated pathways exerts its tumor suppressor activity. p21(Cip1/Waf1), acting down stream of p53, is a major G1/S checkpoint protein that restricts cell cycle progression into S phase in the presence of DNA damage. While at old ages p21-/- mice have a higher incidence of many types of tumors than p21+/+ mice, they are more resistant to thymic lymphomagenesis. In this study, we characterized mutagenesis in vivo in T cells of p21-deficient mice, using loss of heterozygosity (LOH) at Aprt locus as an indicator. We found that the spontaneous Aprt mutant frequency in T cells of p21-/- mice is lower than that in p21+/+ mice. The mutational spectra, however, are similar, with mitotic recombination being the predominant pathway. In contrast to the remarkable induction of LOH events in T cells of p53-/- mice exposed to X-rays, LOH in T cells of p21-/- mice is not significantly induced by X-rays. Correspondingly, lymphoid cells of p21-/- mice are more sensitive to IR-induced apoptosis than those of p21+/+ mice, in contrast to the radioresistance of p53-deficient lymphocytes. Reduction in mutation load in T cell lineages may contribute to the suppression of thymic lymphomagenesis in p21-/- mice.

X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells.

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here, we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase beta, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks, to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally regulated phenotypes, such as mitotic index.

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin.

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

Co-detection of PTH/PTHrP receptor and tartrate resistant acid phosphatase in osteoclasts.

Serial sections of rat metaphyses were prepared from paraffin embedded tissue blocks and analyzed in sets of three. The central section was stained for tartrate resistant acid phosphatase (TRAP) in order to identify osteoclasts, one adjacent section was immunostained with an affinity purified antibody to a 15 amino acid sequence unique to rat PTH/PTHrP receptor, and the other adjacent section in the set served as an immunostaining control. This allowed each of the 110 osteoclasts examined to be identified by TRAP and to be tested for the presence or absence of PTH/PTHrP receptor. All antibody solutions and rinses contained 1% donkey serum and 0.5% Tween 20 to ensure antibody integrity and good rinsing procedure. Confocal microscopy was used to evaluate fluorescence intensity of the immunostained osteoclasts. Pixel intensities of 58 osteoclasts from young (4 month) rats and 52 osteoclasts from old (15 month) rats were obtained. Pixel intensities were similar (P = 0.89) for both young and old animals. However, the number of PTH/PTHrP receptor deficient osteoclasts was greater for the older animals (14.29% vs. 7.24%). This provides direct evidence of PTH/PTHrP receptors in osteoclasts.