Differential expression of receptors for interleukin-3 on subsets of CD34-expressing hematopoietic cells of rhesus monkeys

The target cell specificity of interleukin-3 (IL-3) was examined by flow cytometric analysis of IL-3 receptor (IL-3R) expression on rhesus monkey bone marrow (BM) cells using biotinylated IL-3. Only 2% to 5% of unfractionated cells stained specifically with the biotinylated IL-3 and most of these cells were present within the CD34+ subset. IL-3Rs were detected on small CD34dull/RhLA-DRbright/CD10+/CD27+/CD2-/++ +CD20- cells, which probably represent B-cell precursors. IL-3R+ CD34- BM cells, which were detected at low frequencies, consisted of small CD20dull/surface-IgM+/RhLA-DR+ cells. These cells represented immature B lymphocytes, whereas CD20bright mature B cells were IL-3R-. The highest IL-3R levels were detected on CD34dull/RhLA-DRbright blast-like cells. These cells differentiated into monocytes, neutrophils, and basophils after IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in vitro. The CD34bright/IL-3R- subset contained all clonogenic erythroid and myeloid progenitors (burst- forming unit-erythroid and colony-forming unit-culture), whereas CD34bright/IL-3Rdull cells differentiated into monocytes, neutrophils, and erythroid cells after shorter culture periods. This finding showed that IL-3R expression increases during monocyte and granulocyte differentiation. Results of three-color experiments indicated that IL- 3Rs are expressed on CD34bright/RhLA-DRbright cells as well as on CD34bright/RhLA-DRdull cells, with the latter population expression approximately twofold to threefold lower IL-3R levels. A large fraction (> 30%) of single-cell/well-sorted CD34bright/RhLA-DRdull cells formed multilineage colonies after 2 to 4 weeks of stimulation with IL-3, GM- CSF, Kit ligand, and IL-6. Individual colonies contained cells that still expressed CD34 as well as differentiated monocytes, granulocytes, and erythroid cells. These results confirmed that the CD34bright/RhLA- DRdull subset was enriched for immature, multipotent progenitor cells, whereas the CD34bright/RhLA-DRbright population mainly contained lineage-committed precursors. The results are consistent with the concept that IL-3Rs are induced at very early stages of hematopoiesis, as identified by high expression of CD34 and low expression of RhLA-DR. IL-3R expression continues to be low during differentiation into lineage-committed progenitors; gradually increases on differentiating progenitor cells for B cells, granulocytes, monocytes, and, possibly also, erythrocytes; but finally declines to undetectable levels during terminal differentiation into mature cells of all lineages in peripheral blood, with the exception of basophils.(ABSTRACT TRUNCATED AT 400 WORDS)     

The in vivo effects of recombinant human interleukin-3: demonstration of basophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in nonhuman primates

Recently human interleukin-3 (IL-3) produced by molecular cloning was characterized as a growth factor for basophils and eosinophils in human bone marrow cultures. Since we found a similar activity of the human factor on simian bone marrow cells, we investigated the in vivo effects of recombinant human (rh) IL-3 in healthy rhesus monkeys (n = 10). rh IL-3 was administered subcutaneously (SC) to monkeys at different doses (11, 33, and 100 micrograms/kg/d) for 14 days followed by subsequent rh GM-CSF administration (5.5 micrograms/kg/d SC) for another two weeks. During the second week of rh IL-3 administration monkeys responded with a twofold to threefold increase of WBCs caused by a dose-dependent elevation of basophils (up to 40% of WBCs) and eosinophils. rh IL-3 also induced a dose-dependent increase of histamine (up to 700-fold above normal values) in monkey blood cells. Administration of rh GM-CSF to rh IL-3 pretreated monkeys resulted in a twofold enhanced increase in WBCs (due mainly to eosinophils and neutrophils) compared with animals treated with rh GM-CSF alone. Simultaneous administration of both cytokines (100 micrograms/kg rh IL-3 + 5.5 micrograms/kg rh GM-CSF SC) to two separate monkeys for 14 days induced a WBC elevation similar to that observed in monkeys treated with rh GM-CSF alone. In conclusion, our results indicate that rh IL-3 is a differentiation factor for blood basophils and primes the hematopoietic system for subsequent rh GM-CSF actions.     

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.     

Interleukin-3-induced downregulation of the expression of interleukin-2 receptor beta chain in human T cells

We examined the effect of interleukin-3 (IL-3) on human CD4+ cloned T cells, P607 and 1C2. By flow cytometric analysis, we found that IL-3 downregulated the surface expression of IL-2 receptor (R) beta chain in a dose-dependent manner but had little effect on that of IL-2R alpha chain. A simultaneous 125I-labeled IL-2 binding assay showed a decrease in the number of high-affinity, but not of low-affinity, IL-2Rs by IL-3. The downregulation of the IL-2R beta chain began 3 hours after culture initiation, increased further thereafter, and was completely inhibited by anti-IL-3 antibodies. Expression of mRNA for either alpha or beta chain was not reduced by IL-3, and this suggests that the reduction of surface beta chain expression was not caused by the reduction of beta chain mRNA. IL-3-accelerating internalization of IL-2R beta chain appeared to be one of the mechanisms for IL-3-induced downregulation of surface IL-2R beta chain expression. IL-3 alone increased the proliferation of T-cell clones but decreased the existing increment of their proliferation by IL-2. Accordingly, IL-3 may be one of the factors acting as a liaison between the hematopoietic and immune systems.     

Hematopoietic growth factors upregulate the p65 type II interleukin-1 receptor on bone marrow progenitor cells in vitro.

Having previously shown that interleukin-1 (IL-1) induces the expression of IL-1 receptors (IL-1Rs) on bone marrow (BM) cells in vivo through an indirect mechanism, we studied whether hematopoietic growth factors (HGFs) could induce the expression of IL-1R on BM cells in vitro. In vitro treatment of light-density murine BM (LDBM) cells with either IL-3, IL-6, granulocyte--colony-stimulating factor (CSF), or granulocyte-macrophage--CSF caused a 5- to 10-fold upregulation of IL-1R expression, whereas IL-1, IL-5, IL-7, and macrophage-CSF had no effect. Scatchard analysis showed one class of IL-1Rs on LDBM cells with an average of 66 +/- 20 sites per cells. After 24 hours of treatment with IL-3, the number of IL-1Rs increased to 413 +/- 125, without effecting the affinity. This effect required protein synthesis, but was independent of cell division. Purified lineage-negative progenitor cells (Lin-) did not express detectable levels of IL-1R, but 24 hours of treatment with IL-3, GM-CSF, and G-CSF stimulated IL-1--specific binding. Autoradiographic analysis of Lin- cells showed that IL-1R induction by IL-3 occurs on undifferentiated blast cells. Affinity labeling of Lin- cells treated with HGFs showed an increase in a 65-Kd IL-1 binding protein that did not bind or compete with an anti-type I IL-1R antibody, suggesting that these cells expressed type II IL-1R. These data suggest that IL-1 stimulation of myelopoiesis occurs by a mechanism involving IL-1R upregulation on hematopoietic progenitor cells by HGFs.     

Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture.

In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL-3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.     

Expression and factor-dependent modulation of the interleukin-3 receptor subunits on human hematopoietic cells

Interleukin-3 (IL-3) regulates growth and differentiation of multipotential as well as lineage-committed progenitor cells. The human IL-3 receptor (IL-3R) consists of the alpha and common beta (beta c) subunits. The alpha subunit (IL-3R alpha) is specific for IL-3 and binds IL-3 with low affinity. In contrast, the beta c subunit does not bind any cytokine by itself, but forms a high-affinity receptor with IL- 3R alpha. As the same beta c subunit also forms high-affinity receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) with the respective cytokine-specific alpha subunit, the expression of the alpha subunits is responsible for specificity of cytokines. To examine the expression of IL-3R alpha, we have developed a monoclonal antibody (MoAb), N3A. N3A specifically bound to cells expressing IL-3R alpha and immunoprecipitated a 75 Kd glycoprotein, which became 43 Kd on N-glycosidase digestion. N3A and an anti-beta c antibody, CRS1, were used in double color fluorescence-activated cell sorter (FACS) staining with several lineage markers to see the IL-3R expression pattern in peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells. Both IL-3R subunits were expressed on myeloid cell lineages (CD13+, CD14+, CD15Lo, or CD33+). To further study the IL-3R expression on hematopoietic progenitor cells, the CD34+ populations were isolated from both BM and CB cells. Those populations showed positive staining profiles with the N3A MoAb and were weakly stained with the CRS1 MoAb. Furthermore, anti c-kit antibody staining of the CD34+ fraction from CB, but not from BM, showed two intensities and the IL-3R alpha expression seemed to be higher in a fraction of low c-kit expression. Because IL-1, IL-6, G-CSF, stem cell factor (SCF), interferon (IFN)- gamma, and tumor necrosis factor (TNF)-alpha are known to enhance IL-3- dependent colony formation, we have examined whether this enhancement could be correlated with upregulation of the IL-3R expression. Incubation of CD34+ cells with TNF-alpha for 2 days significantly increased the level of beta c and G-CSF increased the number of cells with high level expression of alpha, while other factors did not affect the IL-3R expression. Thus, different cytokines appear to have different mechanisms for enhancement of IL-3-dependent proliferation.     

Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils.

Splenic and bone marrow cells from normal mice, and from mice that have been polyclonally activated by injection of anti-IgD antibody, contain cells that produce interleukin 4 (IL-4) in response to crosslinkage of Fc epsilon receptors (Fc epsilon R) or Fc gamma R or to ionomycin. Isolated Fc epsilon R+ cells have recently been shown to contain all of the IL-4-producing capacity of the nonlymphoid compartment of spleen and bone marrow. Here, purified Fc epsilon R+ cells are shown to be enriched in cells that contain histamine and express alcian blue-positive cytoplasmic granules. By electron microscopy, the vast majority of cytoplasmic granule-containing cells are basophils; they constitute approximately 25% and approximately 50%, respectively, of Fc epsilon R+ spleen and bone marrow cells from anti-IgD-injected mice. The Fc epsilon R- populations contain cells that form colonies typical of mast cells. The Fc epsilon R+ populations also contain cells that, upon culture with IL-3, form colonies of alcian blue-positive cells, but (in contrast to colonies derived from Fc epsilon R- populations) the colonies are small, and all the cells die within 2-3 weeks. The Fc epsilon R+ cells synthesize histamine during a 60-hr culture with IL-3, while the Fc epsilon R- cells do not. These results indicate that IL-4-producing Fc epsilon R+ cells are highly enriched in basophils.     

Interleukin 3 and granulocyte/macrophage-colony-stimulating factor render human basophils responsive to low concentrations of complement component C3a.

Complement component C3a is an anaphylatoxin known to induce plasma exudation and smooth muscle contraction in tissues. The effects on inflammatory effector leukocytes, however, are poorly defined and controversial, being at best weak and occurring at very high C3a concentrations. Here, we examined the effect of C3a upon mediator release from human basophils, with and without pretreatment with interleukin 3 (IL-3), a hematopoietic growth factor recently found to profoundly modify the basophil response to various cell agonists. In the absence of cytokines, C3a, even at a concentration of 1 microM, was ineffective or only weakly stimulatory for basophil mediator release. However, when basophils were pretreated with IL-3 at concentrations of only 0.01-1 unit/ml, they became responsive to C3a, releasing large amounts of histamine and also generating leukotrienes. Surprisingly, almost optimal effects occurred with even very low C3a concentrations (1 nM). Another hematopoietic growth factor, granulocyte/macrophage-colony-stimulating factor (GM-CSF), was also found to render basophils capable of responding to C3a, but the effect was weaker than that of IL-3. C3a-induced histamine release and leukotriene generation occurred rapidly in IL-3-primed cells, being complete after 0.5 and 2 min, respectively. The rapid and strong degranulation response, occurring at very low concentrations of C3a, suggests the presence of a high-affinity C3a receptor on basophils, which might be inducible by cytokines. Our results demonstrate that, depending on the presence of IL-3 or GM-CSF, C3a is a potent basophil activator, and such a phenomenon could be of relevance in various inflammatory processes, especially hypersensitivity reactions.     

Hematopoietic progenitor cells from allogeneic bone marrow transplant donors circulate in the very early post-transplant period

Despite the therapeutic efficacy of allogeneic bone marrow transplantation (allo-BMT), circulating hematopoietic progenitor cells after bone marrow transplantation have not been well characterized. In the present study, we focused on these 'post-transplant circulating progenitor cells (PTCPC)' which may be on their way to bone marrow. We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just before transplantation), 1 (8-15 h after the completion of transplantation), 2, 3, 5, 7, 10, 14, 17, 21, 28 and 35 after allo-BMT in five transplant patients using a standard methylcellulose assay. In addition, high proliferative potential colony-forming cells (HPP-CFC) of the harvested donor bone marrow (BM) and day 1 PB of recipients were assayed in five patients. The origin of HPP-CFC from day 1 PB was analyzed by polymerase chain reaction of a DNA region containing a variable number of tandem repeats. The replating potential of these HPP-CFC was evaluated by a secondary colony assay. The proportion of CD38negative cells among CD34+ cells in the harvested BM and day 1 PB was evaluated by two-color flow cytometric analysis. The number of CFU-GM on day 1 ranged from 6 to 73/10 ml PB, and became undetectable on day 5. The reappearance of PTCPC was observed on day 14, along with hematopoietic recovery. The proportion of HPP-CFC among myeloid colonies from day 1 PB was significantly higher than that from harvested BM (44.3+/-10.4% vs 11.3+/-2.1%, respectively, n=5, P=0.0030). These HPP-CFC from day 1 PB were confirmed to be of donor origin. More than 90% of these HPP-CFC had replating potential. Two-color flow cytometric analysis revealed that the proportion of CD34+CD38negative cells was significantly higher in day 1 PB than in the harvested BM (61.0+/-16.5% vs 9.3+/-3.5%, respectively, n=7, P=0.0002). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following allo-BMT.     

骨髓中表达白细胞介素5受体 mRNA的CD+34 细胞与支气管哮喘气道炎症的关系

目的　探讨支气管哮喘 (简称哮喘 )小鼠骨髓 (BM)中表达CD+ 34 与白细胞介素 5受体(IL 5RmRNA+ )的造血细胞 (CD+ 34 IL 5RmRNA+ 细胞 )在气道炎症中的作用。方法　以卵白蛋白(OVA)及生理盐水致敏并激发Balb/c小鼠 ,建立各哮喘及对照组 (A组 )模型。分别于OVA及生理盐水首次激发后 1、6、12、2 4、48h处死小鼠 ,取支气管肺泡灌洗液 (BALF)、外周血 (PB)及BM标本备用。测定BALF中嗜酸性粒细胞 (EOS)、PB中有核细胞及EOS计数及BM中有核细胞数 ;用流式细胞仪分别测定PB及BM中CD+ 34 细胞占相应有核细胞的比例并推算其相对计数 ;用免疫组化结合原位杂交法分别标记骨髓细胞CD+ 34 抗原及IL 5RmRNA ,定位BM中CD+ 34 IL 5RmRNA+ 细胞并计数其占CD+ 34 细胞的比例。结果　 (1)OVA激发后 6h组 ,BALF中EOS计数为 (2 67± 1 0 0 )× 10 5/L ,与A组 [(0 46±0 0 6)× 10 5/L]比较差异有显著性 (P <0 0 1) ;OVA激发后 12h组 ,BALF中EOS、PB中EOS计数分别为 (7 74± 1 98)× 10 5/L、(2 91± 0 64 )× 10 8/L ,与A组 [(0 46± 0 0 6)× 10 5/L、(1 43± 0 3 7)× 10 8/L]比较 ,差异有显著性 (P均 <0 0 1) ;OVA激发后 2 4h组 ,BALF中EOS、PB中EOS计数分别为 (19 43±3 69)× 10 5/L、(3 93± 0 5 1)× 10     

Characterization of CD34+ subsets derived from bone marrow, umbilical cord blood and mobilized peripheral blood after stem cell factor and interleukin 3 stimulation

We characterized CD34+ cells purified from bone marrow (BM), mobilized peripheral blood (PB) and cord blood (CB) and we tried to establish correlations between the cell cycle kinetics of the CD34+CD38- and CD34+CD38+ subpopulations, their sensitivity to SCF and IL-3 and their expression of receptors for these two CSFs. At day 0, significantly fewer immature CD34+CD38- cells from CB and mobilized PB are in S + G2M phases of the cell cycle (respectively 2.0 +/- 0.4 and 0.9 +/- 0.3%) than their BM counterpart (5.6 +/- 1.2%). A 48-h incubation with SCF + IL-3 allows a significant increase in the percentage of cycling CD34+CD38- cells in CB (19.2 +/- 2.2%, P < 0.0002) and PB (14.1 +/- 5.5%, P < 0.05) while the proliferative potential of BM CD34+CD38- progenitors remains constant (8.6 +/- 1.0%, NS). CD123 (IL-3 receptor) expression is similar in the three sources of hematopoietic cells at day 0 and after 48-h culture. CD117 (SCF receptor) expression, although very heterogeneous according to the subpopulations and the sources of progenitors evaluated, seems not to correlate with the difference of progenitor cell sensitivity to SCF nor with their proliferative capacity. Considering the importance of the c-kit/SCF complex in the adhesion of stem cells to the microenvironment, several observations are relevant. The density of CD117 antigen expression (expressed in terms of mean equivalent soluble fluorescence, MESF) is significantly lower on fresh PB cells than on their BM (P < 0.017) and CB (P < 0.004) counterparts, particularly in the immature CD34+CD38- population (560 +/- 131, 2121 +/- 416 and 1192 +/- 129 MESF respectively); moreover, when PB and BM CD34+CD38- cells are stimulated for 48 h with SCF + IL-3, the CD117 expression decreases by 1.5- and 1.66-fold, respectively. This reduction could modify the functional capacities of ex vivo PB and BM manipulated immature progenitor cells.     

Type beta transforming growth factors promote interleukin-3 (IL-3)-dependent differentiation of human basophils but inhibit IL-3-dependent differentiation of human eosinophils.

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.     

Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients

We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC- ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients.     

Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype.

The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells. Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes. To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR). All subsets were positive by PCR, but only minimal MDR1 mRNA was detected in monocytes and granulocytes. Significant efflux of Rhodamine-123 (Rh-123), a measure of P-gp function, was detected in CD4+, CD8+, CD14+, CD19+, and CD56+ cells but not in granulocytes. Next, PCR-analysis was performed on FACS-sorted bone marrow (BM) cells to assess MDR1 expression in different maturational stages. Precursors (CD34+), early and late myeloid cells (CD33+/CD34+, CD33+/CD34-) as well as lymphocytes of the B-cell lineage (CD19+/CD10+, CD19+/CD10-) expressed the MDR1 gene. BM monocytic cells (CD33++/CD34-) were negative, and a very weak signal was detected in erythroid cells (glycophorin A+). Significant Rh-123 efflux was found in CD34+, CD10+, CD33+, and CD33++ BM cells, but not in glycophorin A+ cells. We conclude that PB and BM lymphocytes, PB monocytes, BM progenitors, and immature myeloid cells, but not late BM monocytes, erythroid cells, and PB granulocytes, express MDR1 mRNA and a functional P-gp. These results have to be taken into account when MDR1 expression is determined in tumor samples containing normal blood cells.     

Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver

Fetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.     

In vivo interleukin-1 (IL-1) administration indirectly promotes type II IL-1 receptor expression on hematopoietic bone marrow cells: novel mechanism for the hematopoietic effects of IL-1.

Interleukin-1 (IL-1) has profound stimulatory effects on hematopoiesis but the mechanism(s) of action remain unknown. The direct action of IL-1 on hematopoietic progenitor cells requires the presence of a specific IL-1 receptor (IL-1R). In this report, we tested the effect of in vivo IL-1 treatment on the expression of IL-1R on bone marrow (BM) cells. Injection of mice with IL-1 results in a marked upregulation of IL-1R on light-density BM cells as on a subpopulation enriched for myeloid precursors. Pretreatment of mice with anti-type I IL-1R antibody (35F5), which has been shown to prevent the radioprotective effect of IL-1, also blocked IL-1-induced IL-1R expression on BM cells. This antibody did not directly bind and block IL-1 binding to the type II IL-1R expressed on hematopoietic cells, suggesting that IL-1R upregulation by IL-1 is indirect. It is therefore possible that IL-1 acts on type I IL-1R-expressing accessory cells such as stromal cells or T cells to induce production of hematopoietic growth factors (HGFs). In support of this, granulocyte colony-stimulating factor administration can induce the increase of IL-1R on BM cells. Thus, the increased expression of IL-1R on hematopoietic BM cells by IL-1 is indirect, probably mediated in part through endogenous HGF production. These results also suggest that the restorative hematopoietic effect of IL-1 occurs through both indirect and direct mechanisms.     

Enrichment of murine bone marrow natural suppressor activity in the fraction of hematopoietic progenitors with interleukin 3 receptor-associated antigen.

Natural suppressor (NS) activity has been identified in several sites of active hematopoiesis. In this study we characterized NS activity in murine bone marrow (BM) using monoclonal antibodies (mAbs) to interleukin 3 (IL-3) receptor-associated antigen (IL-3RAA) and various cytokines that exert a strong influence on hematopoiesis or lymphocyte interaction. NS activity of BM cells of relatively low density was enhanced by IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). When the BM cells were separated into IL-3RAA+ cells and IL-3RAA- cells, the IL-3RAA+ cells demonstrated potent NS activity, whereas IL-3RAA- cells had either no or weak NS activity. The IL-3RAA+ cells showed non-T- and non-B-cell phenotype and had high affinity to wheat germ agglutinin (WGA), a marker for hematopoietic progenitors. In assays for hematopoietic activity, it appeared that the early differentiating progenitors (day 8 spleen colony-forming units [CFU-S], granulocyte-macrophage colony-forming units [CFU-GM]) were enriched in the IL-3RAA+ cell population, whereas more immature multipotent progenitors (day 12 CFU-S, granulocyte erythrocyte macrophage megakaryocyte colony-forming units [CFU-GEMM]) were contained in the IL-3RAA- cell population. Both suppressor cells and IL-3RAA+ cells spontaneously developed from the IL-3RAA- cell population. These findings suggest that NS cells in murine BM are early hematopoietic progenitors and are probably committed to the myeloid lineage. Hybridoma cells established between the IL-3RAA+ cells and BW5147 cells produced suppressor factor(s). This finding suggests that the NS cells produce soluble mediator(s) that may be responsible for their suppressive action.     

Characterization of peripheral blood stem cells in mice

Peripheral blood stem cells (PBSCs) were mobilized in mice by treatment with cytosine-arabinoside on day 0, followed by the administration by injection of granulocyte colony-stimulating factor for 4 days. There were remarkable increases in the numbers of cells with lineage-negative (Lin-) c-kit+ markers, cells with colony-forming unit-cell (CFU-C) and colony-forming unit-spleen (CFU-S) activities, and cells with marrow- repopulating ability (MRA) in the extramedullary sites (the spleen, peripheral blood, and liver) on day 5, whereas the number of these immature hematopoietic cells decreased in the bone marrow (BM) on day 5. This finding suggests the mobilization of immature hematopoietic cells from the BM to the extramedullary sites. Three-color flow cytometric analyses showed that CD4 antigen was not expressed on the Lin-Sca-1+ cells in the mobilized PB cells (PBCs), although CD4lo cells were found in those of normal BM cells. Lin-c-kit+ cells in the mobilized PBCs contained more cells with immature phenotypes (Sca-1+, Thy1.2lo, CD71-, and Rh123dull) than in normal BMCs, indicating an alteration of the hierarchical composition of the Lin-c-kit+ cells. The Lin-c-kit+Sca-1+ cells in the mobilized PBCs had similar CFU-C and CFU- S activities to those in normal BMCs. Electron microscopic studies of these cells in the mobilized PBCs showed that only 10% to 20% of these cells had a thin rim of cytoplasm with poorly developed organelles. Allogeneic transplantation [B6 --> C3H] of PBSCs showed long-term reconstituting activity across the major histocompatibility complex barrier 24 weeks after transplantation, although longer observation is necessary.