Regulation of fetal hemoglobin expression during hematopoietic stem cell development and its importance in bone metabolism and osteoporosis.

We have shown that an altered tissue redox environment in mice lacking either murine beta Hemoglobin major (HgbßmaKO) or minor (HgbßmiKO) regulates inflammation. The REDOX environment in marrow stem cell niches also control differentiation pathways. We investigated osteoclastogenesis (OC)/osteoblastogenesis (OB), in bone cultures derived from untreated or FSLE-treated WT, HgbßmaKO or HgbßmiKO mice. Marrow mesenchymal cells from 10d pre-cultures were incubated on an osteogenic matrix for 21d prior to analysis of inflammatory cytokine release into culture supernatants, and relative OC:OB using (TRAP:BSP, RANKL:OPG) mRNA expression ratios and TRAP or Von Kossa staining. Cells from WT and HgbßmaKO mice show decreased IL-1ß,TNFα and IL-6 production and enhanced osteoblastogenesis with altered mRNA expression ratios and increased bone nodules (Von Kossa staining) in vitro after in vivo stimulation of mRNA expression of fetal Hgb genes (Hgbε and Hgbßmi) by a fetal liver extract (FSLE). Marrow from HgbßmiKO showed enhanced cytokine release and preferential enhanced osteoclastogenesis relative to similar cells from WT or HgbßmaKO mice, with no increased osteoblastogenesis after mouse treatment with FSLE. Pre-treatment of WT or HgbßmaKO, but not HgbßmiKO mice, with other molecules (rapamycin; hydroxyurea) which increase expression of fetal Hgb genes also augmented osteoblastogenesis and decreased cytokine production in cells differentiating in vitro. Infusion of rabbit anti- Hgbε or anti- Hgbßmi, but not anti-Hgbα or anti- Hgbßma into WT mice from day 13 gestation for 3 weeks led to attenuated osteoblastogenesis in cultured cells. We conclude that increased fetal hemoglobin expression, or use of agents which improve fetal hemoglobin expression, increases osteoblast bone differentiation in association with decreased inflammatory cytokine release.

A fetal sheep liver extract reverses age-related increments in spontaneous and induced cytokine production by indirect environmental effects.

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.

An antibody specific for interleukin-6 reverses age-associated changes in spontaneous and induced cytokine production in mice.

A number of quantitative and qualitative changes in the pattern of cytokine production have been reported to accompany the process of ageing in laboratory animals and in human populations, including an increase in serum levels of interleukin-1 (IL-1) and IL-6, as well as increased concanavalin A (ConA)-stimulated production of IL-4, IL-10 and transforming growth factor-beta (TGF-beta), and decreased production of IL-2 from cultured spleen cells. Increased IL-1 and IL-6 production is a feature of splenic adherent cells and peritoneal exudate cells taken from aged mice and stimulated with lipopolysaccharide in vitro. We have asked whether the altered production of lymphocyte-derived cytokines (IL-4, IL-2, TGF-beta) is itself a function of a primary alteration in IL-1/IL-6 production (from macrophage/monocytes) by infusing monoclonal antibodies to these cytokines prior to harvesting cells from aged mice and stimulating the cells in vitro. Anti-IL-6, but not anti-IL-1, reversed the age-associated alteration in lymphocyte cytokine production. The general pattern of cytokine production in aged mice is of a type-2 cytokine type, and thus these data are consistent with the idea that increased production of IL-6 in aged animals is causally implicated in this age-associated polarization to type-2 cytokine production.