Vladimir Lesnikov

Pro-apoptotic and anti-apoptotic effects of transferrin and transferrin-derived glycans on hematopoietic cells and lymphocytes

The aim of this study was to test the hypothesis that transferrin (Tf) has anti-apoptotic properties and thereby exerts a cytoprotective effect against tissue damage induced by irradiation and other cytotoxic modalities. This hypothesis was tested in several models, including in vitro human short-term marrow cultures, subpopulations of marrow cells, particularly, CD56(+) natural killer cells (and natural killer cell lines), and in vivo radioprotection of murine marrow cells. Reverse transcriptase polymerase chain reaction analysis was used for determination of cytokine mRNA. Preincubation of human marrow with Tf protected cells (except for a CD56(+) subpopulation) against cell death induced by gamma-irradiation, tumor necrosis factor-alpha (TNF-alpha), and agonistic anti-Fas monoclonal antibody. Deglycosylation of Tf abrogated this action of Tf; conversely, Tf-derived glycans (Tf-Gly) (but not glycans isolated from other proteins) mimicked the effects of the intact Tf molecule on apoptosis. Antibodies specific for the Tf receptor (CD71) did not block the effects of Tf or Tf-Gly on apoptosis. Determination of cytokine mRNA in the course of Fas-mediated apoptosis in the presence of Tf or Tf-Gly showed upregulation of mRNA for Fas ligand and TNF-alpha in CD56(+) and downregulation of these transcripts along with upregulation of mRNA for interleukin-10 in CD3(+) marrow cells. Under these conditions, a distinct increase in Fas-associated phosphatase-1 message was observed in CD3(+) cells that were protected by Tf or Tf-Gly against apoptosis. The in vitro data were confirmed in a murine in vivo model in which pretreatment of mice with Tf protected marrow cells against gamma-irradiation-induced cell death. These data suggest a role for Tf and particularly Tf-Gly in the regulation of programmed cell death, apparently via alterations in cytokine expression, and provide a basis for additional studies on the use of Tf in cytoprotective protocols.

Prevention of Fas-mediated hepatic failure by transferrin

Recent studies in lymphohemopoietic cells show that transferrin (Tf), a pivotal component of iron transport and metabolism, also exerts cytoprotective functions. We show here in a murine model that Tf interferes with Fas-mediated hepatocyte death and liver failure. The mechanism involves the downregulation of apoptosis via BID, cytochrome c, caspase-3 and caspase-9, and upregulation of antiapoptotic signals via Bcl-xL. The results obtained with iron-saturated Tf, Apo-Tf and the iron-chelator salicylaldehyde isonicotinoyl hydrazone indicate that the observed antiapoptotic effect of Tf was not mediated by iron alone. In conclusion, the data suggest that Tf has broader functions than previously recognized and may serve as a cytoprotective agent.

Transplantation of allogeneic T cells alters iron homeostasis in NOD/SCID mice

Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content. Iron deposition was accompanied by hepatocyte injury and intestinal villous damage. Feeding of low- or high-iron diet was associated with appropriate ferroportin 1 and hepcidin responses in mice given histocompatible T cells, whereas mice given histoincompatible T cells showed inappropriate up-regulation of duodenal ferroportin 1 and a loss of expression of hepatic hepcidin. These findings suggest that alloreactive T cell-dependent signals induced dysregulation of intestinal iron absorption, which contributed to liver iron overload after HCT.

Protection of human and murine hepatocytes against Fas-induced death by transferrin and iron

Recent studies in a murine model show that transferrin (Tf) interferes with Fas-mediated hepatocyte death and liver failure by decreasing pro-apoptotic and increasing anti-apoptotic signals. We show here in vitro in murine and human hepatocyte cell lines and in vivo in mice that Fas-induced apoptosis is modulated by exogenous Tf and iron. The results obtained with iron-free Tf (ApoTf), iron-saturated Tf (FeTf), and the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in its iron-free and iron-saturated (FeSIH) forms indicate that apoptosis-modulating effects of Tf are not mediated by iron alone. Both the Tf molecule and iron affect multiple aspects of cell death, and the route of iron delivery to the cell may be critical for the final outcome of cellular Fas signaling. Survival of hepatocytes ‘stressed’ by Fas signals can be manipulated by Tf and iron and may be a target for prophylactic and therapeutic interventions.

Upregulation of Interleukin-10 and Inhibition of Alloantigen Responses by Transferrin and Transferrin-Derived Glycans

Previous studies have shown that critically timed administration of transferrin (Tf) facilitates induction of immunologic unresponsiveness. Here, we determined in mixed leukocyte culture (MLC) and in concanavalin A (ConA)-driven cultures the effect of exogenous Tf and Tf-derived glycans (Tf-Gly) on lymphocyte proliferation. In cultures of human blood lymphocytes, Tf inhibited selectively alloantigen-driven proliferation in MLC, but not ConA-stimulated lymphocyte proliferation. Deglycosylation of Tf abrogated the inhibitory effect of Tf on alloantigen-induced lymphocyte proliferation, and, consistent with a role for glycans, an effect qualitatively and quantitatively similar to Tf was exerted by purified Tf-Gly. Glycans isolated from other proteins, for example, immunoglobulin G (IgG) or fibrinogen, failed to inhibit alloantigen-induced proliferation selectively. Rather, they suppressed lymphocyte proliferation in a non-specific manner. Determination of cytokines in MLC supernatant showed a downregulation of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-2, and IL-12 (p40), along with an upregulation of IL-10, a pattern entirely consistent with the observed effects of Tf and Tf-Gly on alloantigen-induced lymphocyte proliferation. The effect of Tf on MLC was directly IL-10-dependent. IL-10 levels were inversely correlated with lymphocyte proliferation and CD86 expression. Neutralization of IL-10 by anti-IL-10 monoclonal antibody (mAb) blocked the effect of Tf. The MLC-modulating effect of Tf (or Tf-Gly) was not dependent upon the Tf receptor CD71 but appeared to be mediated by a Gly-responsive receptor. These data suggest a role of Tf, and, in particular, Tf-Gly, in allo-interactions that is independent from the role of Tf in iron metabolism, and appears to involve co-stimulatory signals.

Transferrin fails to provide protection against Fas-induced hepatic injury in mice with deletion of functional transferrin-receptor type 2

We reported previously that Fas-induced hepatic failure in normal mice was attenuated or prevented by exogenous transferrin (Tf), particularly apoTf. Here we show in C57BL6J/129 mice with genetic inactivation of transferrin receptor 2 (TfR2Y245X), that Fas-induced hepatotoxicity (apoptosis; rise in plasma aspartate aminotransferase (AST) levels) was comparable to that in wild-type mice, but was not modified by pretreatment with Tf. Rises in plasma AST were preceded by a decline in serum iron levels. AST elevations and iron declines were more profound in female than in male mice. Female mice also showed higher baseline levels of Bcl-xL in hepatocytes, which declined significantly upon treatment with agonistic anti-Fas antibody. These data confirm the cytoprotective function of Tf, and show a novel property of TfR2. Both apoptotic Fas responses and cytoprotective effects of Tf were associated with significant shifts in plasma iron levels, which quantitatively differed between male and female mice.

A Preclinical Model of Double- versus Single-Unit Unrelated Cord Blood Transplantation

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained <or=1.7 x 10(7) TNC/kg. Recipients were given 9.2 Gy total-body irradiation (TBI) and DLA-matched unrelated cord blood with postgrafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with 1 dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (P = .03, log-rank test); neutrophil and platelet recovery was delayed (both P = .005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the nonengrafted unit facilitates engraftment of the dominant unit.