Host conditioning with 5-fluorouracil and kit-ligand to provide for long-term bone marrow engraftment.

Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 microg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 10(7) congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 x 10(7) or 3 x 10(7) donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 +/- 2.7), compared with 5-FU alone (18.5 +/- 2.6, P < .0005), or saline controls (17.8 +/- 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 microg/dose) was added to a reduced dose of KL (12.5 microg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

Radioprotection of bone marrow stem cell subsets by interleukin-1 and kit-ligand: implications for CFU-S as the responsible target cell population.

Various cytokines have been reported to have radioprotective effects on the bone marrow. Of these, c-kit-ligand (KL) and interleukin-1 (IL-1) have the most dramatic effect when given prior to total body irradiation (TBI). Given simultaneously, KL and IL-1 demonstrated a strong effect on increasing the LD50/30 of mice. In this case the LD50/30 of C57BL/6 mice was 1.25 (1.14-1.38) times higher (10.08 Gy [confidence interval (c.i.): 9.62-10.56] vs. 8.05 Gy [c.i.: 7.64-8.42]) when KL (120 micrograms/kg) and IL-1 (40 micrograms/kg) were injected subcutaneously at 20 hours before TBI. It was also investigated whether the combined effects of KL and IL-1 resulted in changes in the intrinsic radiation sensitivity of different bone marrow subsets. Therefore, mice were irradiated and the survival of bone marrow subsets was determined at 4-6 hours after TBI by using the CFU-S assay and the competitive repopulation assay. The CFU-S subset displayed an increased Dzero value in KL and IL-1-treated mice (0.88 Gy vs. 0.72 Gy) and the protection factor was 1.22, close to the factor found for the hemopoietic syndrome (LD50/30). It may therefore be concluded that CFU-S are the target cell population involved in hemopoietic death. Additional protection of the more primitive stem cell subset with long-term repopulation ability (LTRA) could not be shown from the data we obtained with the competitive repopulation assay. Both Dzero and the extrapolation number (n) were increased, but not significantly. These data suggest that radioprotection by cytokines is caused mainly by the decreased radiation sensitivity of the CFU-S subset, although earlier subsets may also be protected (but to a lesser extent).

Hematopoietic stem cells in the blood after stem cell factor and interleukin-11 administration: evidence for different mechanisms of mobilization.

Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony-forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU-S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine-induced pathways for mobilization of primitive stem cells.

Mechanisms generating functionally heterogeneous macrophages: chaos revisited.

Macrophage populations exhibit a wide range of antigenic and functional phenotypes, including cytokine production, response to immunomodulatory stimuli, and clearance of pathogens. The expanding clinical exploitation of recombinant growth factors and cytokines with the potential to regulate the production and function of peripheral macrophage populations necessitates an increased understanding of the mechanisms by which functionally distinct macrophage populations arise as well as the ramifications of macrophage heterogeneity. The present review summarizes recent data which supports multiple mechanisms by which heterogeneous macrophage populations arise: 1) differential signals experienced within diverse tissue microenvironments; 2) developmentally-staged expression of specific functions; 3) clonal variation of myeloid progenitor cells; and 4) alternate hematopoietic stimulation. These data show that the above processes are not mutually exclusive and that each likely contributes to the observed heterogeneity of peripheral macrophage populations.

Utilization of polymerase chain reaction for clonal analysis of gene expression.

The reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool when studying gene expression in a limited number of cells. RT-PCR was first described by Veres et al. (1) and numerous accounts have since followed. Classic studies of gene expression have utilized Northern blot analysis to monitor expression of transcripts in response to developmental and activational signals. Problems associated with using the Northern blot technique include the necessity for an abundant number of cells, the limited numbers of genes that can be analyzed on a single blot, and that, usually, only one gene can be monitored at a time. Finally, little information on which cells within the cell population being analyzed are expressing the gene(s) is provided by Northern blot analyses. For example, on a Northern blot it is virtually impossible to determine whether all cells contain a transcript for a specific gene(s) or whether all cells exclusively express the particular gene of interest. In situ hybridization has been used in conjunction with Northern blot analyses to determine which cells in a population are expressing a particular gene. However, this technique is limited by low sensitivity and by the fact that usually only one gene can be monitored at a time. RT-PCR alleviates these problems because specific transcripts can routinely be detected in RNA quickly isolated (2) from 5-10 cells (3-5). The principle of clonal analysis by RT-PCR is to use 50-100 cells that are clonally derived from a single progenitor cell. RNA is then isolated from individual clones, reverse-transcribed into cDNA, and amplified with transcript-specific primers. Two steps are involved in RT-PCR: 1. Isolation and reverse-transcription of the specific transcripts or total mRNA into cDNA. 2. Amplification of specific sequences of transcripts by PCR. During the PCR step, primers used for amplification of specific sequences are designed in order to amplify sequences of predetermined size. In addition, it is also useful to design primers that span across introns to assure that contaminating DNA is not being amplified. There can be numerous technical difficulties associated with amplifying cDNA sequences from limited transcripts obtained from low cell numbers. We have used an internal nested primer that results in increased specific amplification with decreased background amplification to overcome this problem (4). In demonstrating the RT-PCR technique, we describe in this chapter the approach we have developed to molecularly phenotype colonies of bone marrow-derived macrophage obtained using hematopoietic growth factors (5).

Tumor necrosis factor alpha is an autocrine growth regulator during macrophage differentiation.

Previous experiments have revealed the expression of tumor necrosis factor alpha (TNF-alpha) transcripts in all murine bone marrow-derived macrophage colonies isolated from days 5 through 9 of differentiation in vitro. These results implicated a role for TNF-alpha gene expression during macrophage differentiation. Antisense oligomers to the initiation region of the TNF-alpha message were used to inhibit its expression, thus allowing the role of TNF-alpha gene expression in controlling the differentiation of macrophages to be determined. Results showed that TNF-alpha regulated the proliferation of macrophages during differentiation. Cells isolated on day 3 were exclusively vulnerable to the effects of blocking TNF-alpha gene expression, displaying a 30% increase in proliferation over control cells or sense oligomer-treated cells. Thus, in the absence of TNF-alpha gene expression, cells maintained proliferation instead of undergoing terminal differentiation. Exogenous TNF-alpha was capable of rescuing day 3 antisense-treated cells, therefore maintaining normal levels of proliferation. In contrast, blocking interleukin 1 beta gene expression by antisense oligonucleotide treatment had no effect on proliferation. Addition of exogenous recombinant murine or human TNF-alpha decreased the total cell number 25-50% regardless of whether cells were grown in medium containing colony-stimulating factor 1 (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These results suggested that exogenous TNF-alpha suppressed proliferation of early hematopoietic progenitors, whereas endogenous TNF-alpha regulated proliferation of macrophage progenitors. The number of differentiated, adherent macrophages on day 5 of differentiation in vitro was increased by TNF-alpha treatment of GM-CSF-induced macrophages but was suppressed in CSF-1-induced macrophages. These findings suggest that distinct TNF receptor expression and/or signaling is induced in differentiating macrophages stimulated with either growth factor.

Macrophage heterogeneity occurs through a developmental mechanism.

The versatility and importance of macrophages in host defense and homeostasis have long been recognized. Anatomically, macrophages isolated from various tissues manifest extreme differences in shape, in metabolic and functional activities, and in the expression of macrophage-specific markers. To determine the mechanisms responsible for generating macrophage heterogeneity, we have employed the reverse transcription-polymerase chain reaction to molecularly phenotype colonies of bone marrow-derived macrophages during differentiation in vitro. By utilizing this method, results have revealed a hierarchal expression of macrophage-associated genes. Tumor necrosis factor alpha was expressed in all colonies analyzed suggesting an important role for this molecule during macrophage differentiation. Predominant colony phenotypes observed were unique for (i) the period of differentiation and (ii) the growth factor with which they were derived (either colony-stimulating factor 1 or granulocyte-macrophage colony-stimulating factor). Exogenous stimulation of the cultures with either bacterial lipopolysaccharide or interferon-gamma led to predictable phenotypic transitions. These results suggest that macrophage heterogeneity is generated through differentiation-related mechanisms and that generated macrophage phenotypes are then maintained by systemic environmental constraints.

Clonal analysis of gene expression by PCR.

A method for determining the heterogeneity of gene expression from isolated macrophage colonies is described. Clones derived using specific growth factors were isolated by soft agar cloning and used as sources of mRNA for analysis. Gene expression was monitored by the reverse transcription polymerase chain reaction method to permit a number of specific genes to be simultaneously amplified. The technique described here involves the use of internal primers in addition to the usual two external primers typically used in gene amplification. Such internal primers greatly increased the accuracy and precision of amplification by adding an extra constraint such that the gene of interest was reliably amplified while decreasing background noise. This method permits gene expression to be monitored in cell colonies, thereby allowing the extent of phenotypic heterogeneity in developing macrophages (or other cell types) to be determined. This technique may also be useful in gene expression studies involving other cell types under the influence of specific growth factors.

Induction of serum colony-stimulating activity (CSA) following dimethylnitrosamine (DMN) exposure: effects on macrophage differentiation.

Dimethylnitrosamine (DMN) exposure in vivo affects hematopoiesis at the level of CFU-GM and CFU-M precursor cells. This effect on hematopoiesis has been shown to be an indirect consequence of DMN exposure; therefore, we examined serum from animals exposed to DMN in vivo for the presence of hematopoietic growth factor activity. Serum obtained from animals exposed to DMN in vivo supported colony formation in normal bone marrow stem cells whereas serum obtained from untreated or vehicle-exposed animals failed to support colony formation. Differential staining of the cells which arose following the in vitro culture of normal bone marrow cells with serum from DMN-exposed animals demonstrated the presence of cells of the monocytic and granulocytic lineages. Pre-treatment of serum from DMN-exposed animals with anti-CSF-1 antibodies prior to in vitro culture had no affect on either cell number, cell phenotype or colony-stimulating activity, suggesting the presence of GM-CSF. Administration of serum from DMN-exposed animals to naive recipient animals resulted in increased percentages of both blood-borne monocytes and neutrophils, mimicking the profile observed in DMN-exposed animals. Studies using oligonucleotide-directed PCR demonstrated the presence of GM-CSF transcripts in the livers obtained following DMN exposure. These results demonstrate the presence of a serum-borne activity(ies) following DMN exposure, most likely GM-CSF, which is capable not only of enhancing macrophage and granulocyte hematopoiesis but also of increasing the numbers of these two cell types in the blood.