Gunnar Mueller

Expression and Function of Homing‐Essential Molecules and Enhanced In Vivo Homing Ability of Human Peripheral Blood‐Derived Hematopoietic Progenitor Cells after Stimulation with Stem Cell Factor

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the α4β1 and α5β1 integrins very‐late antigen (VLA)‐4 and VLA‐5 on peripheral blood‐derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA‐4 and VLA‐5 on CD34+/c‐kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient‐derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long‐term culture‐initiating cell. The improvement of the homing abilities of SCF‐stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic‐scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.

Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease

Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day −1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-α and IFN-γ levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.

Safety and feasibility of long term administration of recombinant human granulocyte-colony stimulating factor in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuronal disease resulting in a loss of the upper and lower motor neurons and subsequent death within three to four years after diagnosis. Mouse models and preliminary human exposure data suggest that the treatment with granulocyte-colony stimulating factor (G-CSF) has neuro-protective effects and may delay ALS progression. As data on long-term administration of G-CSF in patients with normal bone marrow (BM) function are scarce, we initiated a compassionate use program including 6 ALS patients with monthly G-CSF treatment cycles. Here we demonstrate that G-CSF injection was safe and feasible throughout our observation period up to three years. Significant decrease of mobilization efficiency occurred in one patient and a loss of immature erythroid progenitors was observed in all six patients. These data imply that follow-up studies analyzing BM function during long-term G-CSF stimulation are required.

Steroid treatment alters adhesion molecule and chemokine expression in experimental acute graft-vs.-host disease of the intestinal tract

OBJECTIVE Acute graft-vs.-host disease (aGVHD) is a major complication after allogeneic bone marrow transplantation (allo-BMT) that is characterized by high morbidity and mortality. Systemic treatment with steroids has been the mainstay of first-line therapy of aGVHD, although controlled experimental data in this context are limited. MATERIALS AND METHODS Using a haploidentical murine BMT model, steroid effects on hepatic and intestinal inflammation during aGVHD have been investigated. Lethally irradiated B6D2F1 mice received bone marrow cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. RESULTS Intraperitoneal administration of prednisolone (2 mg/kg body weight every day) early after onset of GVHD from day +10 until day +42 resulted in reduced clinical GVHD severity and improved survival of allogeneic recipients. Although the liver was barely affected by prednisolone treatment, aGVHD-related histopathologic injury of the gastrointestinal tract was strongly reduced in association with diminished expression of interferon-γ, tumor necrosis factor, CXCL 9-11, CCL2-3, mucosal addressin cell adhesion molecule-1, and intercellular adhesion molecule-1. Prednisolone-induced reduction of adhesion molecule expression in the gut manifested earlier than seen for cytokines or chemokines. Interestingly, when starting steroid treatment on day +28, the course of GVHD was unchanged and no major differences in cyto- or chemokine expression in gastrointestinal tract or liver on day +42 were seen. CONCLUSIONS When started early after GVHD onset, prednisolone-related beneficial effects can affect aGVHD target organs differently, involving divergent regulation of inflammation and leukocyte migration. Specifically, a change in adhesion properties between leukocytes and endothelial cells in the gastrointestinal tract may be one of the initial steps in a cascade of steroid-related aGVHD-attenuating events.

Differentiation of hematopoietic progenitor cells towards the myeloid and B-lymphoid lineage by hepatocyte growth factor (HGF) and thrombopoietin (TPO) together with early acting cytokines.

Abstract:  Objectives: The effect of stem cell factor (SCF), flt3‐ligand (FL), and interleukin (IL)‐3 (SF3) in combination with hepatocyte growth factor (HGF), thrombopoietin (TPO), and Hyper‐IL‐6 on maintenance and differentiation of early human peripheral blood‐derived progenitor cells was investigated. Methods: Single sorted CD34+ 38− cells were cultured with various combinations of these growth factors in order to identify the most effective cytokine combination. Then, lineage‐depleted cells were stimulated for 7 d in bulk culture before they were assessed by flow cytometry and in functional assays. Results: The highest number of clones in the single‐cell assay was obtained after culture with SF3 + TPO + HGF. Cell expansion with SF3 + TPO + HGF yielded an increase of the total cell number (11‐fold), the number of CD34+ cells (sevenfold), colony forming cells (CFC; 13‐fold), granulocytes (CD15/66b+; 45‐fold) and B‐cells (CD19/20+; 55‐fold). However, the number of long‐term culture initiating cells (LTC‐IC) decreased from 779 ± 338 per 1 × 105 CD34+ cells on day 0 to 253 ± 115 on day 7. In parallel, the number of pluripotent mouse repopulating cells decreased by the factor 11, and no significant change in the proportion of human myeloid or lymphoid cells found in the mouse bone marrow was noted. Conclusion: The observation that mature cells of different lineages are generated and that transplantable multipotent hematopoietic cells are lost during culture suggests the differentiation of early hematopoietic progenitors toward lineage committed cells by the tested cytokines. The detection of cells expressing B‐lymphoid markers after culture indicates a possible role in the propagation of B‐cells.

Preventive Azithromycin Treatment Reduces Noninfectious Lung Injury and Acute Graft-versus-Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.

The absence of donor-derived IL-13 exacerbates the severity of acute graft-versus-host disease following allogeneic bone marrow transplantation

Acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (allo‐BMT) predominantly involves a Th1‐type cytokine response. Interestingly, the Th2‐cytokine, Interleukin‐13 (IL‐13), produced by alloreactive donor T cells in vitro was recently shown to correlate with clinical aGVHD severity. Using an established mouse model, we show that the systemic cytokine milieu following allo‐BMT with IL‐13−/− donors is characterized by decreases in serum Th2 cytokines and an increase in serum TNFα, and ultimately correlates with higher aGVHD mortality compared to allogeneic controls. In vitro studies further demonstrate that both exogenous and T cell‐derived IL‐13 can regulate TNFα production by macrophages following lipopolysaccharide stimulation. Thus, donor‐derived IL‐13 may have a role in modulating inflammatory cytokine release that is associated with aGVHD. Pediatr Blood Cancer 2008;50:911–914.

Splenic pooling and loss of VCAM-1 causes an engraftment defect in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34+ were significantly reduced at early time points in myelofibrosis patients, whereas CD34+CD38− and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34+ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.

Detection and quantification of functionally defined hematopoietic progenitor cells and tissue specific mRNA within the peripheral blood of myeloma patients after administration of granulocyte colony-stimulating factor and erythropoietin.

Objective:  Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony‐stimulating factor (G‐CSF) plus/minus erythropoietin (EPO).

SCF modulates organ distribution and hematopoietic engraftment of CB-derived pluripotent HPC transplanted in NOD/SCID mice.

BACKGROUND During the engraftment process of transplanted HPC, the beta 1 integrins play an important role. An increased expression and adhesive function of these integrins has been shown in hematopoietic cell lines and peripheral blood-derived HPC after stimulation with SCF. In this study, we investigated the influence of SCF on the engraftment capability and tissue distribution of cord blood (CB) cells transplanted into NOD/SCID mice. METHODS CB-derived mononuclear cells were injected i.v. into 40 sublethally irradiated NOD/SCID mice with or without the addition of 10 microg SCF/ mouse. Six weeks later, BM, liver, kidneys, brain and testicular tissue were analyzed for the prevalence of human cells. RESULTS The mean proportion of human CD45+ CD71+ cells within the BM of all engrafted mice receiving SCF in addition to the cells was 1.7-fold higher than in the respective controls. By immunohistochemical staining, human cells were found in liver and kidneys of the engrafted animals, but not in neural tissues or testicles. In the kidneys, the proportion of human cells rose significantly from 0.07 +/- 0.3% to 0.24 +/- 0.05% with treatment with SCF, compared with untreated controls. Single human cells in the liver additionally stained positive for human albumin, indicating organ-specific differentiation of the transplanted cells. DISCUSSION Our results indicate that stimulation with SCF modulates the tissue distribution of the progeny of the transplanted cells and improves the hematopoietic engraftment potential of transplanted CB cells.