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Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major βchains, Hgbβ ma KO or Hgbβ mi KO. Hgbβ mi is the most prominent fetal Hgbβ chain, with Hgbβ ma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from Hgbβ ma KO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from Hgbβ mi KO mice. Following immunization in vivo with ovalbumin in alum, Hgbβ ma KO mice produced less IgE than Hgbβ mi KO mice, suggesting that in the absence of Hgbβ mi KO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbβ ma or Hgbβ mi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbβ chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbβ ma or anti-Hgbβ mi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in Hgbβ mi KO mice.

CD200 acts through its receptor (CD200R) to inhibit excessive inflammation. The role of CD200-CD200R1 interaction in tumor immunity is poorly understood. In this study, we examined the role of CD200-CD200R1 interaction in the progression and metastasis of highly aggressive 4THM murine-breast carcinoma using CD200 transgenic (CD200(tg)) and CD200R1 knock-out (CD200R1(-)(/-)) BALB/c mice. 4THM cells induce extensive visceral metastasis and neutrophil infiltration in affected tissues. CD200 overexpression in the host was associated with decreased primary tumor growth and metastasis, whereas lack of CD200R1 expression by host cells was associated with enhanced visceral metastasis. Absence of CD200R1 expression led to decreased tumor-infiltrating-cytotoxic T cells and increased the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. In contrast, CD200 overexpression led to increased tumor-induced interferon-γ and IL-10 response and decreased TNF-α and IL-6 release. Neutrophil infiltration of tissues was markedly decreased in CD200(tg) animals and increased in CD200R1(-/-) mice. These findings are contradictory to what has been reported in the EMT6 mouse breast-cancer model. Other distinguishing features of tumor elicited by EMT6 and 4THM cell injections were also examined. Visceral tissues from mice bearing EMT6 tumors showed a lack of neutrophil infiltration and decreased IL-6 release in CD200R1(-/-) mice. EMT6 and 4THM cells also differed in vimentin expression and in vitro migration rate, which was markedly lower in EMT6 tumors. These results support the hypothesis that CD200 expression can alter immune responses, and can inhibit metastatic growth of tumor cells that induce systemic and local inflammatory response. Increasing CD200 activity/signaling might be an important therapeutic strategy for treatment of aggressive breast carcinomas.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Abstract: Previous reports from our group have established that the fetal ovine gamma globin chain (Hbγ) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFα, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbγ and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbγ and LPS in TNFα induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFα production which could be inhibited independently both by anti-Hbγ and by anti-MIF, and optimally by a combination of these reagents. [Copyright &y& Elsevier]

Abstract: We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFα, TGFβ and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned γ-chain of human or sheep fetal hemoglobin, but not cloned α- or β-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, γ-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction. [Copyright &y& Elsevier]