Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation

Cytokines play an important role in the regulation of homeostasis and inflammation. A de-regulated cytokine function can subsequently promote chronic inflammation. This is supported by clinical evidence showing the beneficial effect of inhibiting TNF-alpha through injection of antibodies and soluble receptor in disorders such as rheumatoid arthritis and Crohn's disease. Systemic anti-TNF-alpha therapy however is associated with infectious complications. We therefore suggest a concept for the local deposition of therapeutically active agents into areas of inflammation or malignancy, based on the use of hematopoietic storage and secretory granules as delivery vehicles. Hematopoietic cells are induced to express the therapeutically active protein and to store it in the secretory lysosomes. The cells migrate into a tumour or site of inflammation, where the cells become activated and release the contents of their secretory lysosomes resulting in the local delivery of the therapeutically active protein. In support of this concept, gene transfer and granule loading can be achieved using the soluble TNF-alpha receptor (sTNFR1) after cDNA expression in hematopoietic cell lines. Endoplasmic reticulum (ER)-export can be facilitated by the addition of a transmembrane domain, and constitutive secretion can be prevented by incorporating a cytosol-sorting signal resulting in secretory lysosome targeting. The sTNFR1 is released from the transmembrane domain by proteolytic cleavage and finally, regulated sTNFR1-secretion can be triggered by a calcium signal. In vivo investigations are currently determining the feasibility of local protein delivery at sites of inflammation.     

Diverse isoforms of colony-stimulating factor-1 have different effects on the development of stroma-dependent hematopoietic cells

Maintenance and differentiation of hematopoietic stem and progenitor cells are controlled by complex interactions with the stroma microenvironment. Stroma-cell interactions can be supported by locally expressed membrane-spanning cell-surface (cs) growth factors. CSF-1 is expressed by stroma as a soluble glycoprotein, as proteoglycan, or as a membrane-spanning cs glycoprotein. CSF-1 regulates the survival, proliferation, and differentiation of mononuclear phagocytes. Whereas the biological role of soluble CSF-1 is well characterized, the function of the membrane-spanning cell-surface CSF-1 (csCSF-1) remains unclear. To analyze the biological significance of csCSF-1 in vitro, we used an epithelial cell line to ectopically express the different CSF-1 isoforms. In co-cultures of CSF-1 transduced epithelial cells with primary, early hematopoietic progenitor cells we examined whether interaction between csCSF-1 and its receptor mediates cell proliferation, self-renewal, or differentiation. csCSF-1 induces long-lasting proliferation of stimulated cells and furthermore supports self-renewal. Ectopic secretion of soluble CSF-1 does not permit long-term growth of progenitor cells but induces differentiation of monocytes into macrophages. Previously, we showed that the soluble and cs isoforms of stroma-encoded SCF differently affect the development of hematopoietic cells. Cell-surface SCF (csSCF) promotes self-renewal of stimulated cells whereas soluble SCF causes clonal extinction. These results and those presented here for CSF-1 provide evidence for diverse functions of the isoforms of the ligands SCF and CSF-1 for two tyrosine kinase receptors of the subclass III both regulating hematopoiesis on stroma.     

Effects of sodium butyrate on the differentiation of pancreatic and hepatic progenitor cells from mouse embryonic stem cells

Recently significant progress has been made in differentiating embryonic stem (ES) cells toward pancreatic cells. However, little is known about the generation and identification of pancreatic progenitor cells from ES cells. Here we explored the influence of sodium butyrate on pancreatic progenitor differentiation, and investigated the different effects of sodium butyrate on pancreatic and hepatic progenitor formation. Our results indicated that different concentration and exposure time of sodium butyrate led to different differentiating trends of ES cells. A relatively lower concentration of sodium butyrate with shorter exposure time induced more pancreatic progenitor cell formation. When stimulated by a higher concentration and longer exposure time of sodium butyrate, ES cells differentiated toward hepatic progenitor cells rather than pancreatic progenitor cells. These progenitor cells could further mature into pancreatic and hepatic cells with the supplement of exogenous inducing factors. The resulting pancreatic cells expressed specific markers such as insulin and C‐peptide, and were capable of insulin secretion in response to glucose stimulation. The differentiated hepatocytes were characterized by the expression of a number of liver‐associated genes and proteins, and had the capability of glycogen storage. Thus, the current study demonstrated that sodium butyrate played different roles in inducing ES cells toward pancreatic or hepatic progenitor cells. These progenitor cells could be further induced into mature pancreatic cells and hepatocytes. This finding may facilitate the understanding of pancreatic and hepatic cell differentiation from ES cells, and provide a potential source of transplantable cells for cell‐replacement therapies. J. Cell. Biochem. 109: 236–244, 2010. © 2009 Wiley‐Liss, Inc.     

Secretory production of human granulocyte colony-stimulating factor in Escherichia coli.

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes. In this study, secretory production of hG-CSF in the periplasmic space of Escherichia coli using the Bacillus sp. endoxylanase signal peptide was examined. For the efficient expression of hG-CSF gene, the first five codons at the N-terminal were altered based on the E. coli high-frequency codon database. The hG-CSF gene fused to the endoxylanase signal sequence was expressed using an inducible trc promoter. However, recombinant E. coli cells were completely lysed after induction with 1 mM isopropyl-beta-D-thiogalactopyranoside. Insertion of a small oligopeptide (13 amino acids) containing the histidine hexamer and factor Xa cleavage site between the signal peptide and the mature hG-CSF protein allowed successful secretion of hG-CSF into the periplasm without cell lysis. Among the several E. coli strains examined, E. coli BL21(DE3) and E. coli MC4100 allowed production of hG-CSF to the highest levels (20-22% of total proteins) with the secretion efficiencies greater than 98%. The circular dichroism spectra showed that the conformation of purified hG-CSF is almost identical to native hG-CSF.     

Design of novel granulopoietic proteins by topological rescaffolding

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

De novo designed cytokines that activate the G-CSF receptor show that the receptor-binding information can be encoded onto stable, miniaturised protein scaffolds that possess potent granulopoietic activity; such novel proteins provide for ideal candidates for protein-based therapeutics.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

NF-kappaB family proteins participate in multiple steps of hematopoiesis through elimination of reactive oxygen species

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.     

Combination of drugs elevating extracellular adenosine with granulocyte colony-stimulating factor promotes granulopoietic recovery in the murine bone marrow after 5-fluorouracil treatment.

Combined administration of drugs elevating extracellular adenosine, namely dipyridamole and adenosine monophosphate, together with granulocyte colony-stimulating factor was shown to enhance granulopoietic recovery in the bone marrow of mice treated with 5-fluorouracil. Enhanced regeneration was found both at the level of hematopoietic progenitor cells for granulocytes and macrophages and in the compartment of morphologically recognizable granulocyte precursors. The results might have positive clinical impact. The adjunct use of drugs elevating extracellular adenosine might reduce the cost expenditure of therapy with granulocyte colony-stimulating factor.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.     

Delineation among eight major hematopoietic subsets in murine bone marrow using a two-color flow cytometric technique

BACKGROUND: Many methods have been developed specifically for identifying hematopoietic progenitor cells in murine bone marrow, but few methods allow rapid identification of multiple bone marrow populations. We describe a new, simple method for identifying simultaneously eight populations in murine bone marrow with two-color flow cytometry and phenotypically define these populations. METHODS: Bone marrow was stained with anti-Ly-6C and anti-B220 (CD45R) in one fluorochrome and wheat germ agglutinin (WGA) in another fluorochrome. The eight populations identified in this way were defined further primarily by four-color flow cytometry. RESULTS: Six of the eight populations were characterized phenotypically as containing erythroid, granulocytic, mast, early B, mature B, and stem cell populations. Two additional populations with phenotypic characteristics of partially differentiated precursor cells also were identified. One population was Ly-6C/B220+ and WGA-. It also expressed markers associated with early B, T, and/or dendritic cell differentiation. The second population was Ly-6C(hi)WGA(hi)Mac-1+ and was negative for numerous other lineage-specific and precursor markers. Its morphology suggested monocytic differentiative potential. CONCLUSIONS: A two-color flow cytometric assay profiles six bone marrow populations with identifiable phenotypes and two additional unique, putative hematopoietic precursor populations.     

Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-beta 1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-beta 1 costimulation (basic cytokine combination GM-CSF plus TNF-alpha, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-beta 1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-beta 1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-beta 1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-beta 1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.