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Hematopoietic Reconstitution After Lethal Irradiation and Bone Bone Marrow Transplantation (2000) 25, 427–433 2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells D Frasca1, F Guidi1, M Arbitrio1, C Pioli1, F Poccia2, R Cicconi2 and G Doria2 1Laboratory of Immunology, AMB-PRO-TOSS, ENEA CR Casaccia, Rome; and 2Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Summary: ative treatments of the recipient has been used to reconsti- tute hematopoiesis. The HPC capable of long-term reconsti- Lethally irradiated mice were grafted with syngeneic tution are quiescent and slowly cycling bone marrow cells bone marrow cells or left ungrafted. Mice of each group that form multiple colonies of mixed lineages only in the were injected with different hematopoietic cytokines presence of bone marrow stromal cell-derived cytokines.6 for 5 consecutive days starting immediately after Thus, the use of hematopoietic cytokines together with irradiation or left uninjected. The recovery of lymphoid bone marrow transplantation has been shown to hasten neu- tissues induced by hematopoietic cytokines 7 days after trophil and platelet recovery, suggesting that appropriate irradiation and bone marrow cell transplantation was combinations of these factors may be used to promote full comparable to that observed at days 21–28 in reconstitution of the hematopoietic compartment.7–9 irradiated, bone marrow-grafted, but cytokine-unin- In the present work, we have compared the effects of jected mice. IL-11 or IL-6, in combination with IL-3, hematopoietic cytokines, such as IL-3, IL-11, SCF, IL-6 was able to hasten thymus, spleen and blood cell num- and the IL-6 super-agonist K-7/D-6, on the repopulation of bers and functions. SCF also displayed a detectable lymphoid organs and tissues of mice irradiated with a lethal effect when used with IL-3. Conversely, the IL-6 super- dose of X-rays and reconstituted with syngeneic bone mar- agonist K-7/D-6 was able, when injected alone, to induce row cells. IL-3 is involved in blood cell formation, as it significant recovery of thymus, spleen and blood cells. stimulates the survival and the proliferation of pluripotent Thus, K-7/D-6 appears to be a most efficient cytokine stem cells and promotes colony formation by multipotential for fast reconstitution of lymphoid tissues after progenitor cells.10,11 IL-11 is a multifunctional hemato- irradiation and bone marrow transplantation. Bone poietic cytokine capable of supporting the maintenance of Marrow Transplantation (2000) 25, 427–433. multilineage hematopoiesis in long-term bone marrow cul- Keywords: hematopoietic cytokines; lymphocytes; X- rays tures, the proliferation of an IL-6-dependent cell line, and the formation of megakaryocyte, platelet and neutrophil colonies.12–14 SCF is a potent hematopoietic growth factor acting on both early stem cells and already differentiated Hematopoiesis is a process regulated by a complex network cells, particularly mast cells, in the bone marrow.15 IL-6 of soluble factors that stimulate the growth and differen- plays a primary role in hematopoiesis, as it regulates the tiation of hematopoietic progenitor cells (HPC).1,2 HPC differentiation of B cells, megakaryocytes and platelets.16,17 have two major characteristics: self-renewal ability and the K-7/D-6 carries amino acid substitutions in two distinct capacity to differentiate into all lineages of hematopoietic regions of the wild-type IL-6 that participate in the interac- cells. The proliferation and differentiation of HPC are tion with IL-6R and shows a 70-fold increased binding influenced to a large extent by interactions among various affinity as compared to the wild-type molecule.18,19 The cell types in the hematopoietic compartment and by hema- results herein provide evidence that it is possible to acceler- topoietic cytokines produced by stromal cells and lympho- ate the repopulation of lymphoid tissues after lethal cytes. Some of these factors have been identified, charac- irradiation and bone marrow transplantation by using differ- terized and their genes cloned. Experiments performed with ent combinations of hematopoietic cytokines, such as IL-3 recombinant molecules have shown that some hematopo- and IL-11, or IL-3 and IL-6. IL-3 and SCF gave only mar- ietic cytokines act in combination with others to stimulate ginal effects. K-7/D-6, conversely, was able even when proliferative and differentiative responses of HPC, but are used alone to induce a fast recovery of lymphoid tissues 3–5 devoid of detectable activity when used alone. after irradiation and bone marrow transplantation, suggest- Bone marrow transplantation performed after myeloabl- ing that it can be considered an excellent candidate for clinical applications. Correspondence: Dr D Frasca, Laboratory of Immunology, AMB-PRO- TOSS, ENEA CR Casaccia, Via Anguillarese, 301, 00060 S Maria di Galeria, Rome, Italy Received 23 August 1999; acccepted 14 October 1999 Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 428 Materials and methods cytokines were certified to be devoid of detectable endo- toxin. The biological activities of IL-3,10,11 IL-11,12–14 Animals SCF15 and IL-616,17 are widely described elsewhere. The doses of cytokines injected and the regimen of one injection Congenic male and female C57BL/6 Thy1.1 or Thy1.2 per day for 5 consecutive days starting immediately mice, bred and maintained in our animal facilities, were after irradiation have been determined in previous used at the age of 3 to 6 months. Normal and treated mice experiments.20,21 were distributed six per group. Cell culture Irradiation procedure Mice were individually tested. Mice were killed by decapi- C57BL/6 Thy1.1 mice were total body irradiated with 9.5 tation, their thymuses and spleens were aseptically removed Gy in a lucite chamber, 7–28 days before sacrifice. This and single cell suspensions prepared. Cell cultures were dose of X-rays is 100% lethal as it kills all mice within 30 performed in microtissue culture plates (Falcon 3040, days. The X-ray machine (Stabilipan, Siemens, Germany) Oxnard, CA, USA) in RPMI 1640 medium supplemented was operated at 250 kV, 15 mA, 0.5 mm Cu filtration, dose with 10% fetal calf serum (Flow Laboratories, Irvine, UK), rate 1.4 Gy/min in air, focus distance 50 cm. 2mml-glutamine (GIBCO), 10 ␮g/ml gentamycin (Shering, Kenilworth, NY, USA) and 2 × 10−5 m 2-mercap- Bone marrow transplantation toethanol. This supplemented medium is hereafter referred to as complete medium. Bone marrow cells were harvested from C57BL/6 Thy1.2 mice by gently flushing their femurs with RPMI 1640 medium (GIBCO, Grand Island, NY, USA). Cells were Thymocyte mitotic response to Concanavalin A (ConA) centrifuged, resuspended in phosphate-buffered saline The number of cells per thymus was determined and then (PBS) and counted in a hemocytometer. From 105 to 107 single thymocyte suspensions were cultured in triplicate at ° cells in 0.2 ml were intravenously injected into C57BL/6 37 C in a 5% CO2 humidified incubator, for 48 h. Each Thy1.1 recipient mice, immediately after irradiation. Alter- culture contained 106 cells in 0.2 ml complete medium natively, T cells were removed from the bone marrow cell alone or containing 2 ␮g ConA. Cultures received 18.5 kBq suspension by negative selection using the Macs system. of tritiated thymidine (specific activity 1.739 GBq/mmol) Briefly, bone marrow cells were washed with PBS and (Amersham International, Amersham, UK) in 20 ␮l, 4 h incubated for 20 min on ice with goat anti-mouse Thy1.2 before harvesting with an automated cell harvester MoAb-coated MicroBeads (Miltenyi Biotech, Bergisch- (Micromate 196; Packard, Meriden, CT, USA). Radio- Gladbach, Germany). One hundred ␮l of MicroBeads were activity was measured in a Matrix 96 direct betacounter used for magnetic staining of 107 cells. After incubation (Packard) and expressed as c.p.m. per culture. with MicroBeads, cells were washed with PBS, resus- pended in 500 ␮l and applied to the top of a prefilled and washed A2 column (Miltenyi), provided with a G22 needle Cytofluorimetric analysis of thymocytes for flow regulation. Columns were washed with 2 ml of The reagents used to detect Thy1.1 and Thy1.2 antigens PBS. The negative fraction was collected and analyzed by were anti-Thy1.1 fluorescein isothiocyanate (FITC)-conju- cytofluorimetry for the presence of T cells. gated and anti-Thy1.2 phycoerythrin (PE)-conjugated MoAbs (Becton Dickinson Immunocytometry Systems, San 6 ␮ Cytokine treatment Jose, CA, USA). Thymocytes (10 cells/100 l) were incu- bated with 4 ␮l of a titrated mixture of the two MoAbs for Irradiated mice were injected subcutaneously, once a day 30 min on ice and finally washed with PBS. Samples were for 5 consecutive days starting immediately after analyzed using a FACStar Plus (Becton Dickinson Immu- irradiation, with 0.1 ml PBS, supplemented with 0.1% bov- nocytometry Systems) flow cytometer with standard optical ine serum albumin, alone or containing 8 ␮g of murine configuration. The argon laser was tuned at 488 nm, 200 recombinant IL-3 (320 ␮g/kg/day), and/or 5 ␮g of human mW power output. Fluorescence was collected in log mode. recombinant IL-11 (200 ␮g/kg/day), and/or 5 ␮g of rat After color compensation adjustment, data were acquired recombinant SCF, and/or 5 ␮g of human recombinant wild- in list mode with the FACStar Plus Research Software type IL-6 (200 ␮g/kg/day), and/or 5 ␮g of K-7/D-6. IL- (Becton Dickinson Immunocytometry Systems). Low for- 3, provided by Genzyme (Cambridge, MA, USA), had a ward scatter signals were gated out and at least 10000 biological activity of 7 × 106 U/mg.
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