Monocyte/Macrophage Dysfunctions Do Not Impair the Promotion of Myelofibrosis by High Levels of Thrombopoietin

This information is current as Orianne Wagner-Ballon, Hédia Chagraoui, Eric Prina, of September 28, 2021. Micheline Tulliez, Geneviève Milon, Hana Raslova, Jean-Luc Villeval, William Vainchenker and Stéphane Giraudier J Immunol 2006; 176:6425-6433; ;

doi: 10.4049/jimmunol.176.11.6425 Downloaded from http://www.jimmunol.org/content/176/11/6425

References This article cites 45 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/176/11/6425.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Monocyte/Macrophage Dysfunctions Do Not Impair the Promotion of Myelofibrosis by High Levels of Thrombopoietin1

Orianne Wagner-Ballon,*† He´dia Chagraoui,2* Eric Prina,2‡ Micheline Tulliez,§ Genevie`ve Milon,‡ Hana Raslova,* Jean-Luc Villeval,* William Vainchenker,* and Ste´phane Giraudier3*†

Several lines of evidence indicate that the megakaryocyte/platelet lineage is crucial in myelofibrosis induction. The demonstration that NOD/SCID mice with functionally deficient monocytes do not develop fibrotic changes when exposed to thrombopoietin (TPO) also suggests an important role for monocyte/macrophages. However, in this animal model, the development of myelofibrosis is dependent on the level of TPO. This study was conducted to investigate whether NOD/SCID mice exposed to high TPO levels Downloaded from mediated by a retroviral vector would be refractory to the development of bone marrow fibrosis. We show that TPO and TGF-␤1 in plasma from NOD/SCID and SCID mice engrafted with TPO-overexpressing hemopoietic cells reach levels similar to the ones reached in immunocompetent mice, and all animals develop a myeloproliferative disease associated with a dense myelofibrosis at 8 wk posttransplantation. Monocytes in NOD/SCID mice are functionally deficient to secrete cytokines such as IL-1␣ in response to stimuli, even under TPO expression. Surprisingly, the plasma of these mice displays high levels of IL-␣, which was demonstrated to originate from platelets. Together, these data suggest that completely functional monocytes are not required to develop my- http://www.jimmunol.org/ elofibrosis and that platelets are able, under TPO stimulation, to synthesize inflammatory cytokines, which may be involved in the pathogenesis of myelofibrosis and osteosclerosis. The Journal of Immunology, 2006, 176: 6425–6433.

ibrosis is a prominent clinical complication in several dis- proteases that inhibit enzymes involved in the degradation of the orders and occurs particularly in the lung, kidney, heart, or extracellular matrix. TGF-␤1 is secreted by numerous cell lin- F liver. In human hemopoietic tissues, myelofibrosis is less eages, but mainly by monocytes/macrophages and megakaryocytes frequent but is typically associated with two distinct clinical enti- (MKs)/platelets. Several lines of evidence obtained both from 4 ties: chronic idiopathic myelofibrosis (IM) with myeloid metapla- studies of patients with IM (5, 6) or murine models ending with sia and acute panmyelosis with myelofibrosis (1). Bone marrow myelofibrosis (7–9) are in favor of a crucial role of MK in my- by guest on September 28, 2021 fibrosis occurs as a cytokine-mediated secondary response to a elofibrosis induction. However, other studies suggested a central clonal malignant event originating in a multipotent hemopoietic role for the monocyte/macrophage lineage. This theory is based on stem cell (2, 3) and is characterized by excessive deposits of ex- two different observations: 1) Rameshwar et al. (10) reported that tracellular matrix proteins (4). In vivo and in vitro studies have monocytes from patients with IM are spontaneously activated and involved several cytokines in the aberrant stromal reaction, with a secreted abnormally TGF-␤1; and 2) Frey et al. (11) reported that strong emphasis on the pleiotropic cytokine TGF-␤1. Through fi- thrombopoietin (TPO) overexpression in SCID mice led to myelo- broblast stimulation, this cytokine is able to produce extracellular fibrosis, but not in NOD/SCID mice. Because NOD/SCID mice matrix, induce cell adhesion proteins, and enhance expression of differ from SCID mice by impaired mononuclear phagocyte func- tions (12, 13), the authors postulated that monocytes and macro- *Institut National de la Sante et de la Recherche Medicale (INSERM) U790, Pavillon phages must be required for promotion of myelofibrosis in TPO- de Recherche 1, Institut Gustave Roussy, Villejuif, France; †Laboratoire overexpressing mice. However, these experiments were performed ‡ d’He´matologie, Hoˆpital Henri Mondor, Cre´teil, France; Unite´Immunophysiologie et with a human TPO cDNA vectorized in an adenovirus construct Parasitisme Intracellulaire, Institut Pasteur, Paris, France; and §Laboratoire d’Anatomopathologie, Hoˆpital Cochin, Paris, France that leads to a relatively low elevation of plasma TPO. In the Received for publication September 29, 2005. Accepted for publication March present study, to ensure a high and sustained TPO level, we used 2, 2006. a retroviral vector encoding a mouse TPO cDNA to overexpress The costs of publication of this article were defrayed in part by the payment of page TPO in NOD/SCID, SCID, and immunocompetent wild-type charges. This article must therefore be hereby marked advertisement in accordance high with 18 U.S.C. Section 1734 solely to indicate this fact. (WT) mice. All TPO mice invariably developed a myelopro- 1 This work was supported by grants from INSERM and La Ligue Nationale contre liferative syndrome associated with a myelofibrosis and osteoscle- le Cancer E´ quipe Labellise´e 2000, fellowships from Association pour la Recherche rosis. At any time during the follow-up, TPO and TGF-␤1 plasma sur la Cancer and INSERM (Poste d’Accueil) (to O.W.-B.), and the Research Min- levels were substantially and similarly elevated in the three groups istry (to H.C.). of mice. Our data provide evidence that the monocyte/macrophage 2 H.C. and E.P. contributed equally to this work. lineage does not play a crucial role in the development of the 3 Address correspondence and reprint requests to Dr. Ste´phane Giraudier, Hoˆpital Henri Mondor, Laboratoire d’He´matologie, 51 Avenue du Mare´chal de Lattre de myelofibrosis induced by TPO. In addition, platelets and MKs Tassigny, 94000 Cre´teil, France. E-mail address: [email protected] when stimulated are able to synthesize IL-1␣ and thus to replace paris.fr or [email protected] monocytes in the synthesis of inflammatory cytokines, which 4 Abbreviations used in this paper: IM, idiopathic myelofibrosis; MK, megakaryocyte; TPO, thrombopoietin; WT, wild type; CFC, colony-forming cells; FISH, fluorescent may be involved in the pathogenesis of myelofibrosis and in situ hybridization. osteosclerosis.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6426 MYELOFIBROSIS IN TPOhigh NOD/SCID MICE

Materials and Methods pressed as the percentage of apoptotic MKs among the total MK Mice population. All procedures were approved by the local Institut Gustave Roussy ethics Production of bone marrow-derived monocytes committee. WT C57BL/6J mice (Janvier) were maintained at the Institute Bone marrow cells were harvested from femurs and tibia of age- and sex- Gustave Roussy animal facility under specific pathogen-free conditions. high SCID mutant mice (C57BL/6J-scid/scid) (14) (Charles River Laboratories) matched control or TPO mice (WT and NOD/SCID). Cells were cul- ϫ 6 and double NOD/SCID mutant mice (15) (NOD/LtSz-scid/scid obtained tured (1.6 10 cells/ml), as described previously (18), in DMEM sup- from J. Dick (University Health Network, Toronto, Ontario, Canada)) were plemented with antibiotics, 10% FBS, and 10% CSF-1-conditioned bred under sterile conditions and maintained in isolators. All mice used in medium (provided by M. Lebastard, Unite´Immunophysiologie et Parasit- this study have the same C57BL/6J genetic background. isme Intracellulaire, Institut Pasteur, Paris, France) with and without 10 U/ml IFN-␥. Bone marrow cells were cultured at 37°C in a humidified Infection procedure incubator (5% CO2) with an addition of fresh medium at day 3. After 5 days in culture, nonadherent cells were removed and centrifuged for 10 min Six- to 8-wk-old WT, SCID, or NOD/SCID male mice were used as bone at 400 ϫ g. Pellets were suspended in DMEM with antibiotics and 10% marrow donors. Seven- to 10-wk-old WT, SCID, or NOD/SCID female FBS by flushing through, successively, 25-, 27-, and 30-gauge needles, and mice were recipients. The infection procedure was performed as described single-cell suspensions were produced. Cells (2 ϫ 105 cells/ml) were previously (7, 16). Briefly, 4 days after 5-fluorouracil treatment (one in- seeded in fresh medium alone (DMEM supplemented with antibiotics, 10% jection of 150 mg/kg administered i.p.), total bone marrow cells were FBS, and 10% CSF-1-conditioned medium) or in fresh medium containing cocultured with 1 ϫ 106 MPZenTPO virus-producing GPϩE-86 cells in Escherichia coli LPS at 10 ␮g/ml. After a 24-h incubation period, culture DMEM supplemented with antibiotics, 20% FBS and murine IL-3 (100 supernatants were harvested and used for determination of IL-1␣ level. U/ml), murine IL-6 (20 ng/ml), and murine stem cell factor (20 ng/ml). All cytokines were purchased from R&D Systems. After 4 days, nonadherent Platelets and leukocytes preparations Downloaded from cells were harvested. An aliquot was used immediately in clonogenic pro- genitor assays to determine the percentage of infected colony-forming cells Mice were anesthetized and bled by cardiac puncture. Blood from age- and (CFC) as described previously (7, 16). The remaining cells were inoculated sex-matched control or TPOhigh mice (WT and NOD/SCID) was collected i.v. via the retro-orbital sinus into irradiated hosts (9.5 Gy for WT mice or onto 3.8% Na citrate (9 vol blood/1 vol citrate). Whole blood (500 ␮l) was 2.5 Gy for SCID and NOD/SCID mice, x-ray apparatus, single dose) in a mixed with 500 ␮l of buffer saline glucose citrate (BSGC) (129 mM NaCl, ϩ ratio of two donors per one recipient. Fifty-one recipients were studied. 13.6 mM Na3 citrate, 11.1 mM D glucose, 1.6 mM K2PO4, and 8.6 mM ϫ NaH2PO4) and spun at 400 g. Platelet-rich plasma was layered over a Determination of chimerism 10-ml Sepharose B column (Pharmacia), and pellets were used for leuko- http://www.jimmunol.org/ cyte RNA analysis after red cell lysis. To minimize leukocyte contamina- Two approaches were used in parallel. First, a PCR analysis was performed tion, only the upper (9 of 10) platelet-rich plasma was used for gel filtra- on myeloid colonies derived from the bone marrow of sacrificed animals at tion. Platelets were collected, adjusted at a concentration of 50 ϫ 106/ml, week 8 after transplantation, with primer sets corresponding to specific and pelleted by centrifugation. Pellets were used for protein or RNA anal- sequences on the Y chromosome and with actin primers as control (16). ysis. For protein analysis, platelets were lysed by three successive freezing/ The second approach was a fluorescent in situ hybridization (FISH) anal- thawings, suspended in 1 ml of BSGC, and centrifuged to remove platelet ysis performed on bone marrow cells from mice sacrificed at week 8 after debris. Supernatants were used to determine IL-1␣ level. transplantation. Briefly, 1 ϫ 105 bone marrow cells were cytospun and fixed with a 4% formaldehyde solution. Slides were successively incubated ELISA for 5 min in 0.1 M HCl and three times in a permeabilization solution containing 0.5% saponin and 0.5% Triton X-100 (Sigma-Aldrich), and then Plasma TPO levels and IL-1␣ and IL-1␤ levels in plasma or supernatants by guest on September 28, 2021 washed in a solution containing 20% glycerol. After three freezing/thaw- were determined with the appropriate murine Quantikine Kits from R&D ing, slides were incubated in 20% glycerol, deshydrated with ethanol, in- Systems, according to manufacturer’s instructions. Sensitivity limits of the cubated in a 70% formamide solution at 75°C, and deshydrated again. A assays were 62.5, 4.69, and 7.8 pg/ml, respectively, for TPO, IL-1␣, and denatured rhodamine-labeled Y probe (purchased by QBiogene) was hy- IL-1␤. Human TGF-␤1 immunoassay, which detects only active forms of bridized overnight at 37°C in a humidified atmosphere. The slides were TGF-␤1, was used for circulating TGF-␤1 level determination (Quantikine incubated in a 60% formamide solution at 43°C, washed in SSC 0.1% Kit; R&D Systems). Samples were assayed after acidification (active and Tween 20, and counterstained with Vectashield and 4Ј,6Ј-diamidino-2-phe- latent forms) according to manufacturer’s instructions. The sensitivity of nylindole (Vector Laboratories). Numeration of Y-labeled cells was per- the assay was 31.2 pg/ml active TGF-␤1. formed on 500 cells for each slide using a fluorescence microscope (Nikon Eclipse 600). Real-time PCR for cytokine quantification Hematologic evaluation and histopathology Total RNA extraction from circulating platelets and leukocytes of age- and sex-matched control or TPOhigh WT mice was performed using either Blood from the orbital plexus was collected in citrated tubes at indicated TRIzol reagent (Invitrogen Life Technologies) or the RNeasy Mini kit times. Nucleated blood cells, hematocrit level, and platelet counts were (Qiagen), following the manufacturer’s recommendations. Total RNA was determined using an automated blood counter calibrated for mouse blood reverse transcribed in first-strand cDNA using random hexamers (Roche (MS9; Schloesing Melet). Differential cell counts were performed after Diagnostics) and Moloney Murine Leukemia Virus Reverse Transcriptase May-Gru¨nwald-Giemsa staining. Platelet-poor plasma was used for deter- (Invitrogen Life Technologies). mination of TPO, IL-1␣, IL-1␤, and TGF-␤1 levels. Eight weeks after PCR amplifications were conducted on the LightCycler (Roche Diag- transplantation, mice in each group were killed. Bones were excised and nostics) in duplicate or triplicate capillaries in a final volume of 11 ␮l. Two cleaned of soft tissue. One femur and one tibia were fixed in formaldehyde, microliters of cDNA along with primers (500 nM; Roche Diagnostics) and decalcified, and paraffin embedded. Spleen, liver, and pulmonary samples SYBG Green containing mix (QuantiTect SYBR Green Kit; Qiagen) were were fixed and embedded in the same manner. Sections (4.5 ␮m) were used per reaction. CD45-, CD11b-, IL-1␣-, IL-1␤-, and TGF-␤1-specific stained with H&E, periodic acid-Schiff, and Giemsa for overall cytology primers were obtained from published literature or designed by the Light- analysis. Reticulin fibers were revealed by silver staining according to Gor- Cycler Probe Design software (version 1.0; Idaho Technology). Identical don Sweet method. In parallel, 105 bone marrow, spleen, and blood cells thermal cycling conditions were used for all targets, allowing efficient syn- were grown in semisolid medium for CFC analysis as described previously thesis of all amplicons (Table I). Crossing Points were determined with the (16, 17). FitPoint method (LightCycler software, version 3.3) at a constant fluores- TUNEL analysis cent level in the early stage of the exponential phase of the reaction. Using the tool Gene Expression Macro from Bio-Rad, relative expression values To detect and quantify MK apoptosis, we performed in situ TUNEL anal- were expressed for each sample compared with a sample calibrator (a pool ysis on histological sections of bone marrow and spleen from control mice of platelets from control WT mice). The underlying principles and calcu- (WT, SCID, and NOD/SCID) or TPOhigh mice (WT, SCID, and NOD/ lations are described in studies by VandeSompele et al. (19). Briefly, the SCID). DNA strand breaks generated during apoptosis were identified us- Bio-Rad tool calculates a gene expression normalization factor for each ing In Situ Cell Death Detection Kit (POD; Roche) according to the man- sample based on the geometric mean of a user-defined number of house- ufacturer’s instructions. Samples were analyzed under a light microscope, keeping genes. To allow a better normalization between samples within and quantification of apoptotic MKs was performed. Results were ex- this study, three housekeeping genes were used, namely ␤-actin, GAPDH The Journal of Immunology 6427

Table I. Primers and amplicons for real-time PCR analysisa

Amplicons

Gene Name Accession no. Source Primer Sequences (5Ј-3Ј) Tm Length (bp)

GAPDH NM_001001303 Lean et al. (21) FW CggATTTggCCgTATTgg 83.4 218 RV ggTCTCgCTCCTggAAgATg ␤-actin NM_007393 Colle et al. (20) FW ggACTCCTATgTgggTgACgAgg 84.8 366 RV gggAgAgCATAgCCCTCgTAgAT ␣ IIb/CD41 NM_010575 Probe Design FW AgTTCTCCgTCTATgCAg 82.5 123 RV CTTgACTTgCgTTTAggg IL-1␣ NM_010554 Probe Design FW gTATgCCTACTCgTCgg 80.7 235 RV ggTggCAATAAACAgCTC IL-1␤ NM_008361 Probe Design FW AggCAggCAgTATCAC 81.4 167 RV CACACCAgCAggTTATC TGF-␤1 NM_011577 Probe Design FW gCggACTACTATgCTAAAgA 82 243 RV gTAACgCCAggAATTgT CD11b NM_008401 Probe Design FW TTTAggAgCACCTCgg 82.8 223 RV ACACACTgACACCTgg CD45 NM_011210 Probe Design FW TATgACATCATCgCCAgC 79.2 127 RV CTggACggACACAgTT Downloaded from a Accession nos. of housekeeping and target genes along with characteristics of primers and amplification products are depicted. The LightCycler run protocol used in this study included a denaturation and Taq polymerase activation program (95°C for 15 min), an amplification program repeated 40 times with a temperature transition rate of 20°C/s (denaturation step, 95°C for 10 s; annealing step, 54°C for 30 s; elongation step, 72°C for 25 s with a single fluorescent measurement), a melting curve program (72–95°C with a heating rate of 0.1°C/s and a continuous fluorescent measurement), and finally a cooling step to 35°C. FW, Forward primer; RV, reverse primer; Tm, melting temperature is the temperature, where 50% of the DNA is single stranded. http://www.jimmunol.org/ ␣ (21), and integrin IIb/CD41. Then, taking into account the PCR efficien- iments were performed with a total of 51 hosts engrafted. As cies for each target mRNA as described in the mathematical model devel- shown from PCR analysis, the percentage of colonies demonstrat- oped by M. W. Pfaffl (22), the algorithm provided values for each sample ing the integrated TPO cDNA was comparable in the three groups relative to the sample calibrator. of mice (Table II). Plasma levels of TPO were determined in the Statistical analysis three groups of engrafted mice over time using an ELISA. Six Results are presented as mean Ϯ SEM, and data were analyzed with the weeks postengraftment, TPO concentration in plasma was 1,000- two-tailed Student t test. to 10,000-fold higher than normal. Thereafter, the magnitude of the increase was comparable in the three groups (Fig. 1). by guest on September 28, 2021 Results high Hemopoietic chimerism Hematological parameters are similar in all TPO mice high The different x-ray irradiation protocols used for SCID and NOD/ Platelet numbers in TPO WT mice increased over 8 wk, achiev- ing values 4-fold higher than control WT mice. TPOhigh SCID SCID mice compared with WT may lead to different levels of high chimerism and subsequently different pathological processes in he- mice present the same features. In TPO NOD/SCID mice, mopoietic tissues. We analyzed the host chimerism in sex-mis- platelet numbers increased less significantly with barely a 2-fold matched transplantations using two approaches: PCR on Y chro- increment over controls (Fig. 2A). Macroplatelets were observed in mosome (donors were males, recipients were females) on bone all mice for which blood smears were performed. As previously marrow-derived myeloid colonies, and FISH on Y chromosome reported, no correlation between TPO levels and platelet numbers analysis on whole nucleated bone marrow cells. Chimerism levels was observed (17). Nucleated blood cells were increased in all were similar in TPOhigh WT, SCID, and NOD/SCID mice when groups of mice (Fig. 2B) due to a striking increment in mature studied on myeloid colonies 8 wk posttransplantation (69 Ϯ 18% polymorphonuclear neutrophils in association with immature my- (n ϭ 3 mice), 62 Ϯ 15% (n ϭ 4 mice), and 67 Ϯ 16% (n ϭ 5 eloid precursor cells (data not shown). Leukocyte numbers were more elevated in TPOhigh WT and SCID mice than in TPOhigh NOD/SCID mice), respectively). Moreover, studies by FISH on marrow cells high gave identical results as those from PCR. mice, without significant difference. TPO WT and NOD/SCID mice became progressively anemic; however, TPOhigh SCID mice spontaneously corrected this anemia over time (Fig. 2C). Retroviral transfer efficiency To obtain a highly elevated and long-lasting overexpression of a Table II. Analysis of transduction efficiency in progenitor cells injected mouse TPO molecule in immunodeficient SCID and NOD/SCID to WT, SCID, and NOD/SCID micea mice, we used a previously described retroviral gene transfer pro- tocol in which lethally irradiated hosts are hematologically repop- Bone Marrow Cells TPO/Actin Transduced (%) ulated with transduced stem cells (7). Because SCID and NOD/ SCID mice could not be heavily irradiated before bone marrow WT 20/23 87 SCID 65/73 89 transplantation (23), and to overcome a possible risk of a low NOD-SCID 66/74 89 chimerism, 10 times more cells (6–8 ϫ 106 cells) were engrafted a into 2.5 Gy-irradiated SCID and NOD/SCID mice as compared WT, SCID, and NOD/SCID pooled bone marrow cells were transduced and used to reconstitute 10 WT, 19 SCID, and 22 NOD/SCID. Each pool was plated at the end 5 with 6 ϫ 10 cells injected in 9.5 Gy-irradiated WT mice. Trans- of the infection protocol, and CFC-derived colonies were picked from methylcellu- duction efficiencies were determined at the end of the infection lose. Samples were analysed by PCR with specific primers for the viral TPO gene and with actin primers to ascertain the presence of material. No significant differences protocol before transplantation in myeloid progenitor-derived col- were observed between the three groups of mice with more than 80% transduced onies. For each mouse genotype, two independent infection exper- progenitor cells. 6428 MYELOFIBROSIS IN TPOhigh NOD/SCID MICE

FIGURE 1. TPO quantification in plasma. Results are presented as the mean Ϯ SEM of four to 10 animals per experimental group. Values in control WT, SCID, and NOD/SCID were 0.78 Ϯ 0.04, 0.56 Ϯ 0.07, and 1.05 Ϯ 0.08 ng/ml, respectively. Results of statistical analysis with the two-tailed Student t test are as follows: SCID and NOD/SCID mice vs WT .p Ͻ 0.001 ,ءء p Ͻ 0.05 and ,ء ;mice Downloaded from

The number of progenitor cells in bone marrow, spleen, and blood was studied at week 8 posttransplantation in the three groups of mice. Bone marrow progenitor numbers were 3- to 5-fold de- high

creased in all TPO mice in comparison to controls, parallel to http://www.jimmunol.org/ decrease of total bone marrow cell number. All TPOhigh mice de- veloped a splenomegaly. Probably due to the myeloproliferative disorder, spleen CFC increased achieving values 22-fold higher than control in TPOhigh WT mice, 17-fold higher than control in TPOhigh SCID mice, but only 2-fold higher than control in TPOhigh NOD/SCID mice. Finally, blood-circulating CFC were 30- to 1,500-fold increased at 8 wk posttransplantation (Table III). These data indicate that overproduction of TPO in hosts repop- ulated with WT, SCID, and NOD/SCID hemopoietic cells resulted by guest on September 28, 2021 in a similar myeloproliferative syndrome. A similar feature was observed at the histological examination. However, some hemato- logical differences were observed. A lower increase in all cell types was observed in TPOhigh NOD/SCID mice compared with high TPO WT and SCID mice. FIGURE 2. Blood parameters and TGF-␤1 level of TPOhigh mice. Evo- A B C high lution in platelet number ( ), leukocyte number ( ), hematocrit ( ), and Fibrosis on femur and spleen sections is similar in all TPO mice TGF-␤1 level (D)inTPOhigh mice. Results are presented as the mean Ϯ Histological sections revealed a massive hyperplasia of maturing SEM of four to 10 animals per experimental group. Some WT mice dis- ϫ 9 granulocytic cells and dysmorphic MKs found in large clusters. played major leukocytosis (233 10 /l), explaining large SEM. For the ␤ ␤ Erythroblasts were rare. Increased reticulin fibers were noted in TGF- 1 level (D), bars indicate TGF- 1 levels after acidification of the samples. Values in control WT, SCID, and NOD/SCID were 7.9 Ϯ 2.0, bone marrow samples at 8 wk postengraftment. No differences Ϯ Ϯ high 9.4 1.4, and 8.7 2.1 ng/ml, respectively. No spontaneously active were observed between the TPO WT, SCID, or NOD/SCID TGF-␤1 was detected before acidification of the samples. Results of sta- high mice (Fig. 3). Osseous cortex was a bit thicker in TPO NOD/ tistical analysis with the two-tailed Student t test are as follows: SCID and high .p Ͻ 0.001 ,ءء p Ͻ 0.05 and ,ء ;SCID than in TPO SCID or WT, but some bone trabeculae NOD/SCID mice vs WT mice were seen in the marrow cavity in the three lineages. In spleen samples, hyperplasia of the red pulp was observed without white pulp in TPOhigh SCID and NOD/SCID. MKs were extremely abundant as well as neutrophils. Numerous MKs were observed in SCID). Percentages of apoptotic MKs are illustrated in Table IV. large sheets in TPOhigh WT in the red pulp and in TPOhigh NOD/ Apoptosis of bone marrow and spleen MKs was increased in all SCID (more frequently than in TPOhigh SCID mice). Apoptotic TPOhigh mice in comparison to controls. Representative sections of figures of MKs were often seen in TPOhigh WT and SCID mice. spleen from WT control (Fig. 3G) and TPOhigh WT (Fig. 3H) mice Erythroid cells were observed. Similar reticulin deposits were seen are shown in Fig. 3. 8 wk posttransplantation in all TPOhigh mice analyzed. Increased plasma TGF-␤1 level high MK apoptosis is increased in TPO mice In this TPOhigh mouse model, fibrosis development has been re- To study whether myelofibrosis could be the consequence of a ported to be a direct consequence of high TGF-␤1 level in blood dysmegakaryopoiesis, we studied whether apoptotic MKs were in- and bone marrow fluids (17). TGF-␤1 level was thus measured in creased in TPOhigh mice. Apoptotic MKs were detected using the three groups of TPOhigh mice at different times posttransplan- TUNEL analysis on histological sections of both bone marrow and tation. As reported before in an immunocompetent background spleen from control and TPOhigh mice (WT, SCID, and NOD/ (16), TGF-␤1 levels were invariably increased in plasma as soon The Journal of Immunology 6429

Table III. CFC analysis of TPOhigh mice 8 wk postengraftment with MPZen-TPO-bone marrow cellsa

Tissue

Bone marrow Spleen Blood

Nucleated cells Mice Cells ϫ 106/femur CFC ϫ 103/femur Weight, mg CFC/106 cells ϫ 106/ml CFC ϫ 102/ml

Control WT 14.8 Ϯ 2.4 21.7 Ϯ 3.4 91 Ϯ 990Ϯ 31 6.6 Ϯ 0.7 Ͻ1 Control SCID 12.2 Ϯ 2.0 14.9 Ϯ 0.8 52 Ϯ 3 150 Ϯ 39 3.5 Ϯ 0.4 1 Ϯ 0.1 Control NOD/SCID 10.3 Ϯ 1.0 17.9 Ϯ 2.7 54 Ϯ 5 753 Ϯ 195 2.1 Ϯ 0.4 4.5 Ϯ 1.6 TPOhigh WT 3.1 Ϯ 1.0 4.4 Ϯ 0.8 894 Ϯ 141 1950 Ϯ 579 84.9 Ϯ 23 1146 Ϯ 627 TPOhigh SCID 1.1 Ϯ 0.1 3.5 Ϯ 1.2 384 Ϯ 63 3033 Ϯ 501 100 Ϯ 31 2335 Ϯ 1450 TPOhigh NOD/SCID 5.1 Ϯ 0.9 5.2 Ϯ 0.3 366 Ϯ 52 1126 Ϯ 147 32.3 Ϯ 4.3 140 Ϯ 25

a Values are means Ϯ SEM of data from three controls and three TPOhigh mice in each group. Progenitor cells numbers (CFC) were calculated from the number of colonies obtained from 105 cells grown in semisolid medium.

as 3 wk after transplantation (data not shown). These levels in- regulate IL-1␣. Using this approach, we failed to detect IL-1␤ in

creased with time, leading to a 2- to 4-fold higher level than in these platelet lysates. Downloaded from control mice after 6 wk (Fig. 2D). Such an increase in TGF-␤1 high level was also noticed in TPOhigh SCID mice but was detected Transcriptional regulation of IL-1␣ and TGF-␤1inTPO later in the TPOhigh NOD/SCID mice background (2-fold higher mice level than normal at 8 wk only). Quantitative RT-PCR on Sepharose column-purified platelets from control WT and TPOhigh WT mice was performed to determine Increased plasma IL-1␣ level ␣ whether TPO overexpression could be responsible for IL-1 and http://www.jimmunol.org/ Because it has been reported that TGF-␤1 is regulated by IL-1 in TGF-␤1 protein overexpression. IL-1␣, IL-1␤, and TGF-␤1 quan- human IM (24), and that NOD/SCID monocytes have a major tification was performed on two independent WT platelet pools defect in the synthesis of IL-1 (12), we investigated the IL-1␣ and used as “calibrator” and “control” (pools were performed to lower IL-1␤ levels in these mice. IL-1␣ and IL-1␤ were both quantified interindividual variations, and because RNA from nonthrombocy- in plasma with specific ELISA. Surprisingly, we found a dramatic themic mice was obtained in low quantity) and in five individual increase (up to 8-fold the normal value) in IL-1␣ levels in all TPOhigh WT mice, TPOhigh1–TPOhigh5. Fold changes are ex- TPOhigh mice analyzed at 6 and 8 wk posttransplantation (Fig. 4A). pressed relative to calibrator. The normalization factor used for However, IL-1␤ was detected only 8 wk posttransplantation (data each sample was based on the geometric mean of three housekeep-

␤ ␣ by guest on September 28, 2021 not shown). ing genes ( -actin, GAPGH, and IIb). To check potential leukocyte contamination, we used CD45- TPO overexpression does not correct the monocyte defect of and CD11b-specific primers (two highly expressed genes in puri- NOD/SCID mice fied leukocytes from the same animals). No transcript was de- To test whether TPO overexpression could correct the IL-1 syn- tected, illustrating a very low presence or absence of leukocyte. A thesis defect of NOD/SCID monocytes, monocytes were cultured 4- to 12-fold overexpression of IL-1␣ was found in the five from control WT, control NOD/SCID, TPOhigh WT, and TPOhigh TPOhigh mice (Fig. 6). This observation was strictly correlated to NOD/SCID bone marrow isolated cells in the presence of CSF-1 the protein overexpression determined in platelet extracts from the with or without IFN-␥. After 5 days, LPS was added (10 ␮g/ml), same animals. A slight increase in TGF-␤1 was also found in these and 24 h later, the culture supernatants were harvested and assayed mice. According to the lack of IL-1␤ protein, no increase in IL-1␤ for IL-1␣ level. Monocytes from NOD/SCID mice secreted less transcript was found. IL-1␣ in response to IFN-␥ stimulation compared with WT mono- cytes as described previously (12). TPO overexpression did not im- Discussion prove IL-1␣ secretion, indicating that monocyte dysfunction in NOD/ IM is a myeloproliferative disease known as a stem cell disorder SCID mice was not corrected by TPO overexpression (Fig. 5). with involvement of both the myeloid and the lymphoid lineage (3), whereas the associated stromal reaction in the bone marrow ␣ high Platelets are a major source of IL-1 in TPO mice environment is considered reactive and cytokine mediated (2). The Lindeman et al. (25) have reported that human platelets synthesize current pathogenetic hypothesis is that clonal proliferation of MKs and secrete IL-1␤ in response to activation stimuli. Because we or monocytes or both leads to collagen fibrosis and new bone for- observed an increase in IL-1␣ level in TPOhigh NOD/SCID mice mation called osteosclerosis. Development of mouse models has that have deficient monocytes, we hypothesized that platelets from provided important insights into the pathophysiological mecha- these mice could also synthesize high levels of IL-1␣ in response nisms of myelofibrosis with special regard to the role of MK. The to TPO. two most important mouse models are characterized by either sys- Platelets from control WT and NOD/SCID mice or from temic overexpression of TPO (7, 11, 26–28) (TPOhigh mice) or TPOhigh WT and NOD/SCID mice were collected and lysed. Ly- MK lineage restricted under expression of the transcriptional fac- sates (50 ϫ 106 platelets/ml) were harvested and assayed for IL-1␣ tor GATA-1 (8) (GATA-1low mice). These models display hyper- concentration. Levels of IL-1␣ in platelets from control WT mice plasia of MK, a result of TPO-driven proliferation in the TPOhigh were low and below the threshold of detection in control NOD/ mice (29), and impaired MK proliferation and maturation in the SCID mice (Fig. 4B). However, when platelets were collected GATA-1low mice (30). However, the observation that TPO over- from TPOhigh WT and NOD/SCID mice, a marked elevation in expression in SCID mice (T and B cell deficient) (14) led to my- IL-1␣ content in platelets was detected, indicating that TPO could elofibrosis, but not in NOD/SCID mice (T and B cell as well as 6430 MYELOFIBROSIS IN TPOhigh NOD/SCID MICE

FIGURE 3. Studies of myelofibrosis and apoptosis on histological sections of femur and spleen. Three mice of each group were killed 8 wk after transplantation. Representative sections are shown. Longitudinal femur sections stained by H&E from TPOhigh mice WT 2 (A), Downloaded from SCID 2 (C), and NOD/SCID 2 (E) show the hyperplasia of MK and granulocytes. Silver staining of longitudinal femur sections from mice WT 2 (B), SCID 2 (D), and NOD/SCID 2 (F) reveals similar myelofibrosis with re- ticulin fibers surrounded MKs. Original magnification, ϫ250. Sections of spleen from WT control (G) and TPOhigh WT (H) after TUNEL technology. Apoptotic http://www.jimmunol.org/ MKs are stained (black arrow). Original magnification, ϫ600. by guest on September 28, 2021

multiple other defects in innate immunity including NK and mono- driven by the IgH promoter (33) developed myelofibrosis and os- cyte/macrophage function (12, 13)), suggested an important role teosclerosis with the same plasma TPO level as the former model. for the monocyte/macrophage lineage (11). We hypothesized that The TPOhigh and GATA-1low mice develop a myeloproliferative insufficiently high TPO levels, and not lack of a specific cell pop- disorder featuring numerous aspects of the human disease includ- ulation, was the reason why NOD/SCID mice failed to develop ing dysmegakaryopoiesis (5). Histological analyses at the time of myelofibrosis. myelofibrosis occurrence showed very large-sized platelets with In this study, we demonstrated, using a retroviral vector, that empty granules and dysmorphic MK with pycnotic nuclei and in- high TPO levels induce myelofibrosis in the NOD/SCID mice sim- creased emperipolesis (34). Recently, it has been demonstrated ilar to the one developed by WT or SCID mice. All of these mice that a unique and acquired mutation of JAK2 (JAK2V617F)isas- developed a very similar disease, but, strikingly, SCID mice had sociated with BCR/ABL-negative myeloproliferative disorders in- only a mild anemia. This demonstrates that T and B cells (deficient cluding IM (35–38). When the JAK2V617F is expressed in mice, it in SCID and NOD/SCID mice) as well as monocyte/macrophage leads to the development of a polycythemia vera, which progresses (deficient in NOD/SCID mice) are probably not directly involved to myelofibrosis (C. Lacout, D. F. Pisani, M. Tulliez, F. Moreau- in the development of myelofibrosis in this murine model. In fact, Gachelin, W. Vainchenker, and J. L. Villeval, submitted for pub- both level and site of TPO overexpression appear to influence my- lication). The fact that a constitutively active JAK2 may be asso- elofibrosis development (31). For example, first-described trans- ciated with IM in human suggests that alteration in the TPO/c-mpl genic mice overexpressing TPO (32) using a liver-specific apoli- signaling may be at the origin of this dysmegakaryopoiesis. It is poprotein E promoter did not exhibit myelofibrosis or possible that in the TPOhigh mice, a long-lasting stimulation leads osteosclerosis, whereas recently described TPO transgenic mice to a Mpl traffic defect and an altered signaling (S. Constantinescu, The Journal of Immunology 6431

Table IV. TUNEL analysis of apoptotic megakaryocytes in bone marrow and spleen of TPOhigh mice 8 wk postengraftment with MPZen-TPO-bone marrow cellsa

Mice Bone Marrow (%) Spleen (%)

Control WT 3/164 (1.8) 0/27 (0) Control SCID 6/226 (2.7) 6/315 (1.9) Control NOD/SCID 7/486 (1.4) 3/85 (3.5) TPOhigh WT 32/992 (3.2) 52/1478 (3.5) TPOhigh SCID 42/716 (5.9) 67/783 (8.6) TPOhigh NOD/SCID 63/981 (6.4) 57/862 (6.6)

a Apoptosis of MK was detected using TUNEL analysis on histological sections of both bone marrow and spleen from control and TPOhigh mice. Results are presented as number of apoptotic stained MKs per number of total MKs (percentage of apo- ptotic MK).

unpublished observation). This may secondarily imply transcrip- tion factors such as GATA-1, which are implicated in MK sur- FIGURE 4. IL-1␣ quantifications. A, IL-1␣ quantification in plasma. vival, proliferation, and differentiation. In favor of this hypothesis, Results are presented as the mean Ϯ SEM of four to 10 animals per ex- Downloaded from high it has been shown by Vannucchi et al. (39) that in TPO and perimental group. Values in control WT, SCID, and NOD/SCID were 73 Ϯ GATA-1low mice, TPO, GATA-1, and TGF-␤1 are linked in an 8, 68 Ϯ 9, and 29 Ϯ 4 pg/ml, respectively. Results of statistical analysis upstream-downstream relationship. with the two-tailed Student t test are as follows: SCID and NOD/SCID p Ͻ 0.001. B, IL-1␣ quantification ,ءء p Ͻ 0.05 and ,ء ;However, how a high level of TPO can induce myelofibrosis mice vs WT mice development remains unknown. TPO could regulate synthesis and in platelets. Platelets from control WT and NOD/SCID mice or from TPOhigh WT and NOD/SCID mice were collected and lysed by three freez- release of cytokines or induce a dysmegakaryopoiesis. In this http://www.jimmunol.org/ ϫ 6 study, we show that TPO overexpression directly or indirectly in- ing/thawing. The lysates (50 10 platelets/1 ml) were harvested and assayed for IL-1␣ levels. Results are presented as the mean Ϯ SEM of duces apoptosis of MKs in bone marrow and spleen from TPOhigh three animals per experimental group. IL-1␣ platelet levels were low for mice using TUNEL technology, regardless of genetic background. WT mice and under detection threshold for NOD/SCID mice. However, This result suggests that the main mechanism of TPO-induced my- TPOhigh mice, particularly NOD/SCID, showed marked elevation in IL-1␣ elofibrosis is related to a dysmegakaryopoiesis. platelet levels. Results of statistical analysis with the two-tailed Student t In this study, we were able to demonstrate that TPO induced an test are as follows: TPOhigh mice vs control mice. increased synthesis of IL-1␣ by platelets. Indeed, numerous cyto- kines are involved in myelofibrosis and osteosclerosis, including TGF (TGF-␤1), platelet-derived growth factor, basic fibroblastic by guest on September 28, 2021 growth factor, and IL-1. Experiments in TPOhigh mice have estab- In addition, there is strong evidence that IL-1 is implicated in the lished the pivotal role of the hemopoietic cell derived-TGF-␤1in regulation of the bone mass. Notably, IL-1 is able to induce os- the promotion of myelofibrosis (17) and the role of the stromal teoprotegerin synthesis (42) by stromal cells, inhibiting the devel- osteoprotegerin produced by the microenvironment in the promo- opment of osteoclasts and consequently inducing the development tion of osteosclerosis (16). Accordingly, we found elevated TGF-␤1 plasma levels in all TPOhigh mice analyzed and a slight increase of the TGF-␤1 transcript in platelets. Thus, TGF-␤1 ap- pears to originate from platelets. The slight increase of its tran- script could be explained by the 2- to 4-fold higher thrombocytosis displayed by the TPOhigh mice. Moreover, we found increased IL-1␣ plasma levels in all mice analyzed, including in the mono- cyte-deficient NOD/SCID. This means that IL-1␣ may have an- other cellular origin than monocytes in this model. There is strong evidence that platelets have transcripts for both IL-1␣ and IL1-␤ (40) in humans and are innate, inflammatory cells able to release inflammatory cytokines after activation (25). To clearly determine whether platelets may be involved in this excess level of IL-1␣ in TPOhigh mice, we studied the presence of the IL-1␣ protein and transcript in platelet extracts from these mice. We found high lev- FIGURE 5. IL-1␣ secretion by bone marrow-derived monocytes in re- els of IL-1␣ protein and transcript in TPOhigh mice, indicating that sponse to IFN-␥ and LPS. Monocytes from control WT and NOD/SCID platelets (and probably MKs) can replace the monocyte/macro- mice or from TPOhigh WT and NOD/SCID mice were cultivated in CSF-1 phage function to produce IL-1␣ when TPO levels become suffi- with or without IFN-␥. After 5 days, the culture medium was replaced with ciently high. However, we cannot exclude that nonhemopoietic fresh medium containing LPS (10 ␮g/ml). After an additional 24-h incu- cells could be also involved in this synthesis of IL-1␣. The role of bation period, the cultured supernatants were harvested and assayed for IL-1␣ levels. Monocytes from NOD/SCID mice secreted less IL-1␣ in IL-1 in human IM is not completely solved. It has been suggested ␥ ␬ response to IFN- stimulation compared with WT monocytes as described that IL-1 and probably the NF- B pathway play a role in the patho- previously (12). TPO overexpression does not improve IL-1␣ secretion. ␤ genesis of IM (10). IL-1 may indirectly regulate the TGF- 1 syn- Results are presented as the mean Ϯ SEM of three animals per experi- thesis and, consequently, the development of myelofibrosis as sug- mental group. Bone marrow cells from each mouse were assayed sepa- gested by Rameshwar et al. (24), and we recently have shown that rately in duplicate culture. Results of statistical analysis with the two-tailed the NF-␬B pathway is also activated in MK from IM patients (41). Student t test are as follows: CSF-1 with IFN-␥ vs CSF-1 alone. 6432 MYELOFIBROSIS IN TPOhigh NOD/SCID MICE

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