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HEMATOPOIESIS
From the Department of Adult Oncology, Dana-Farber
Cancer Institute and Department of Medicine, Harvard Medical School,
Boston, MA; Division of Oncology, Department of Internal Medicine,
Geneva University Hospital, Geneva, Switzerland; and Department of
Pathology, Massachusetts General Hospital, Boston, MA.
Studies of mice rendered deficient in granulocyte-macrophage
colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) have established unique roles for these cytokines in pulmonary homeostasis, resistance to infection, and antigen-specific T- and B-cell responses. In addition to these distinctive properties, however, GM-CSF and IL-3
also stimulate the development and activation of hematopoietic cells in
many similar ways, raising the possibility that each factor might
partially compensate for the other's absence in singly deficient mice.
To test whether endogenous GM-CSF and IL-3 mediate redundant functions
in vivo, we generated mice lacking both cytokines through sequential
gene targeting experiments in embryonic stem (ES) cells. Surprisingly,
doubly deficient animals, but not single knockouts, showed increased
numbers of circulating eosinophils. Doubly deficient mice, moreover,
developed weaker contact hypersensitivity reactions to haptens applied
epicutaneously than mice deficient in either factor alone. Together,
these findings delineate overlapping roles for GM-CSF and IL-3 in
hematopoiesis and immunity.
(Blood. 2001;97:922-928) Granulocyte-macrophage colony-stimulating factor
(GM-CSF) and interleukin-3 (IL-3) stimulate the proliferation,
differentiation, and activation of hematopoietic cells in vitro in many
similar ways.1 These overlapping functions reflect, at
least in part, the shared use of the Notwithstanding these similarities, mice rendered singly deficient in
GM-CSF or IL-3 manifest distinct phenotypes. Animals deficient in
GM-CSF display normal steady-state hematopoiesis, but develop a lung
disease resembling pulmonary alveolar proteinosis (PAP).4,5 The pathogenesis of PAP involves a reduction in surfactant clearance6 by defective alveolar
macrophages.7,8 The Although IL-3-deficient mice similarly display intact steady-state
hematopoiesis, unlike GM-CSF-deficient animals, they maintain normal
pulmonary homeostasis.25 Mice deficient in IL-3 mount attenuated mast cell and basophil responses to parasite infection that result in compromised worm expulsion.26 They also
show partial reductions in contact hypersensitivity reactions to
haptens applied epicutaneously.25 Mice rendered deficient
in Mice deficient in GM-CSF or IL-3 have been interbred with other
hematopoietic growth factor knockouts to uncover possible redundancies
of cytokine function in vivo.28 Mice lacking both GM-CSF
and granulocyte colony-stimulating factor (G-CSF), unlike either single
mutant, develop neutropenia early in life, resulting in increased
mortality.29 Mice deficient in both GM-CSF and macrophage
colony-stimulating factor (M-CSF) show more extensive pulmonary
pathology and a higher incidence of fatal bacterial pneumonia than
GM-CSF single knockouts.30 Mice deficient in both IL-3 and
c-kit signaling display more severe defects in mast cell
expansion and parasite resistance than either single
knockout.26 On the other hand, mice lacking both IL-3 and
mpl fail to develop further compromises in thrombopoiesis
when compared with single mpl knockouts.31
Because IL-3 signals through both Generation of GM-CSF/IL-3-deficient mice
Hematologic evaluation
Dendritic cells
Contact hypersensitivity Mice at least 7 weeks of age were sensitized epicutaneously on day 0 with 70 µL 4% 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone, Sigma, St Louis, MO) in acetone/olive oil (4:1) and challenged 5 days later on the ear with 20 µL 0.5% oxazolone or carrier only. To assess responsiveness to FITC (Sigma), mice were sensitized on day 0 with 400 µL 2.5% FITC in acetone/dibutyl phthalate (1:1) and challenged on day 6 with 40 µL 1.5% FITC. The hapten-specific increase in ear thickness at 24 hours was determined with a micrometer. Draining lymph nodes were harvested 24 to 48 hours after FITC application, processed to single cell suspension, and stained for major histocompatibility complex (MHC) II, B7-1, CD1d, and Ox40-ligand.36 For correction experiments, mice were injected intraperitoneally and subcutaneously with a total of 4700 ng GM-CSF and 810 ng IL-3, beginning 2 days before and finishing 2 days after sensitization ( 48 hours, 24 hours, 18 hours, 4 hours, 0 hours, +4 hours, +18 hours, +24 hours, +48 hours). This regimen involved more intensive dosing than previously examined in studies of
IL-3 singly deficient animals25 and was undertaken based on pilot experiments indicating an important dose-response effect. Cytokines were harvested from B16 cells engineered to secrete GM-CSF
and IL-3.37,38 Control supernatants were from wild-type B16 cells.
Histology Tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. They were then sectioned at 4 µm thickness and stained with hematoxylin and eosin. A semiquantitative scoring scheme for the intensity of contact hypersensitivity reactions was established as follows: trace, minimal edema, rare infiltrating lymphocytes or granulocytes, no epidermal changes; 1+, mild edema, focal infiltration of lymphocytes or neutrophils, no epidermal changes; 2+, easily visible edema, diffuse but scattered infiltration of lymphocytes, neutrophils, and eosinophils, foci of intraepidermal neutrophils; 3+, marked edema with numerous lymphocytes, many neutrophils and eosinophils, few intraepidermal abscesses; 4+, marked edema with numerous lymphocytes, neutrophils and eosinophils, many subcorneal and intraepidermal abscesses, focal keratinocyte necrosis.Statistics A one-way analysis of variance was used for statistical analysis. When significant differences were observed (P < .05), pairwise t tests were performed, using the Bonferroni correction for the multiple comparisons examined.
Generation of GM-CSF/IL-3-deficient mice Because GM-CSF and IL-3 are separated by only 14 kb on chromosome 11,3 doubly deficient mice could not be obtained by interbreeding single knockout animals. Thus, mice lacking both cytokines were generated through sequential gene targeting experiments in ES cells. A hygromycin cassette replacing exons 3 and 4 of the GM-CSF locus was introduced by homologous recombination into IL-3 heterozygous deficient ES cells25 (Figure 1A). Two correctly targeted clones gave rise to germline transmission following injection into C57Bl/6 blastocysts. Genotyping of progeny mice revealed that GM-CSF and IL-3 were disrupted on the same allele (Figure 1B). Heterozygous mutant mice were interbred to generate homozygous GM-CSF/IL-3-deficient animals. Mutant mice were obtained at the expected frequencies, remained clinically healthy throughout 18 months of observation, and were fertile. Supernatants of concanavalin A-stimulated splenocytes from mutant animals showed no immune-reactive GM-CSF or IL-3 protein as determined by ELISA (not shown), confirming the generation of a null allele. The mutant allele was back-crossed 9 generations onto Balb/c and C57Bl/6 backgrounds for detailed analysis. Additional studies are required to delineate whether the modest decrease in fertility of GM-CSF-deficient animals29,39 is influenced by the simultaneous ablation of IL-3.Pathology Complete pathologic examination of GM-CSF/IL-3-deficient mice revealed abnormalities restricted to the lungs. A progressive accumulation of surfactant in the intra-alveolar spaces and an extensive lymphoid hyperplasia around both airways and veins was observed. Alveolar macrophages demonstrated a marked increase in intracellular surfactant. These features were similar to those previously found in GM-CSF-deficient mice,4,5 and morphologic analysis did not reveal an exacerbation by the concurrent loss of IL-3. Tissue hematopoietic populations and lymphoid organs failed to disclose additional alterations.Hematopoiesis The hematocrits and total circulating white blood cell and platelet counts were normal in GM-CSF/IL-3-deficient mice. Unexpectedly, examination of stained blood smears revealed that circulating eosinophils were increased in doubly deficient mice, as compared to single knockouts and wild-type controls (Table 1). In contrast, circulating neutrophils, lymphocytes, and monocytes were not affected. Bone marrow-derived CFU-G, -M, -GM, and -GEMM were not altered in GM-CSF/IL-3-deficient animals, and bone marrow precursors did not show enhanced sensitivity to IL-5 in vitro (CFU-Eo in response to 10 ng IL-5 for +/+ mice: 15, 7, 15, 16.7; for/ mice: 14, 6.3, 16.7, 13.7. Colony sizes were
equivalent between +/+ and /mice). Enumeration of bone marrow
eosinophils by examination of both fixed core sections and cytospins of
marrow aspirates did not reveal differences between wild-type and
doubly deficient animals (percent eosinophils for +/+ mice: 2.3, 1.3, 2, 1.3; for / mice: 1.7, 1.3, 1.3, 1.7). Although no IL-5 was
consistently measurable in the blood (the sensitivity of ELISA was 25 pg/mL), interbreeding of GM-CSF/IL-3-deficient and  c-deficient mice
resulted in abrogation of the eosinophilia (Table 1), strongly
suggesting the participation of IL-5 in this response. Triply deficient
mice also demonstrated an unexpected reduction in circulating
lymphocytes.
To characterize hematopoiesis in GM-CSF/IL-3-deficient mice further, we lethally irradiated mutant animals and transplanted them with doubly deficient marrow. GM-CSF/IL-3-deficient mice achieved reconstitution that was comparable to wild-type controls, although there was a modest delay in the kinetics of leukocyte recovery (not shown), similar to that previously observed for GM-CSF-deficient animals.40 Dendritic cell development Recent studies have underscored the striking abilities of GM-CSF and IL-3 to stimulate the growth and differentiation of dendritic cells from hematopoietic precursors.41 However, we and others previously reported that mice deficient in GM-CSF or IL-3 maintained normal numbers of spleen and lymph node dendritic cells.4,25,42 Analysis of the spleens, thymi, and lymph nodes of GM-CSF/IL-3-deficient mice similarly revealed normal numbers of both myeloid- and lymphoid-type dendritic cells (not shown).In an effort to identify other factors that might contribute to
dendritic cell development in these animals, we implanted syngeneic
tumor cells engineered to secrete high levels of
flt3-ligand.35 These cells serve as an efficient vehicle
for the systemic administration of flt3-ligand, a cytokine that
dramatically augments dendritic cell numbers in wild-type
mice.43 By 14 days after injection, there was a marked
increase in splenocytes staining positive for CD11c and MHC II in both
mutant and wild-type animals, with an average of 25% positive cells
per spleen (Figure 2A,B). Because injection
of flt3-ligand-expressing tumor cells produced a 3- to 4-fold increase
in total spleen cellularity, a nearly 100-fold expansion of dendritic
cell numbers was accomplished in the absence of GM-CSF and IL-3.
Flt3-ligand-secreting tumor cells stimulated the generation of both
myeloid-type (CD8
Contact hypersensitivity To evaluate dendritic cell function in GM-CSF/IL-3-deficient mice, we compared the abilities of mutant and wild-type animals to develop contact hypersensitivity reactions to epicutaneously applied haptens. Contact hypersensitivity is a form of delayed-type hypersensitivity in which hapten-protein conjugates are presented by cutaneous dendritic cells, following their migration to regional lymph nodes, to hapten-specific CD4+ and CD8+ T lymphocytes.45-48 On secondary hapten challenge, sensitized T cells initiate a local inflammatory response.Although GM-CSF/IL-3-deficient mice were indistinguishable from
wild-type littermates in the initial reaction to oxazolone challenge
(data not shown), they exhibited a dramatically reduced response on
secondary challenge, as measured by ear swelling (Figure 3A). The degree of compromise was
significantly greater than that previously reported for IL-3-deficient
mice.25 Similar results were observed on both C57Bl/6 and
Balb/c backgrounds and when FITC was used as the hapten (not shown).
Although no pathologic differences between GM-CSF/IL-3-deficient and
wild-type mice were noted in untreated skin or skin at the
sensitization site, marked differences were apparent in skin at the
challenge site (Figure 4A-C). In wild-type
animals, the inflammatory response was characterized by an intense
cellular infiltrate consisting primarily of neutrophils, lymphocytes,
and eosinophils, which was associated with substantial dermal edema, hyperkeratosis, and focal intraepidermal abscesses (4+, see
"Materials and methods" for description of semiquantitative scoring
scheme). GM-CSF/IL-3-deficient animals, in contrast, generated a
dramatically less intense cellular infiltrate with much less edema and
little keratinocyte activation (trace to 1+). IL-3-deficient mice
displayed intermediate reactions (2+) and GM-CSF-deficient animals
developed strong reactions (3+), but these were reduced compared to
wild-type controls (not shown).
To delineate whether the compromise in contact hypersensitivity reflected a defect during the priming phase of the response, we injected doubly deficient mice with GM-CSF and IL-3 protein at the time of initial hapten application. Remarkably, the administration of these factors resulted in complete reconstitution of the attenuated secondary reaction, as measured both by ear swelling (Figure 3B) and pathologic analysis, where the intensity and character of the corrected response were indistinguishable from wild-type levels (Figure 4D,E). These findings formally establish a dual requirement for GM-CSF and IL-3 during hapten sensitization. To explore this requirement further, we analyzed the dendritic cells that migrated to the draining lymph node following FITC application in doubly deficient and wild-type animals. Similar numbers of FITC-positive cells were found in both groups, and these cells showed comparable staining for CD11c, MHC II, B7-1, and CD1d (Ox40-ligand was not detected). Additional studies are required to identify which features of dendritic cells are compromised by the absence of GM-CSF and IL-3.
The generation of GM-CSF/IL-3-deficient mice has provided a system to test the hypothesis that these molecules mediate redundant functions in vivo. The experiments presented here definitively establish overlapping roles for these cytokines in both hematopoiesis and immunity. Previous studies demonstrated that IL-3/ Because recent investigations have highlighted the abilities of GM-CSF and IL-3 to stimulate dendritic cell development,41 we quantified these cells in the spleen, thymus, and lymph nodes of GM-CSF/IL-3-deficient animals. However, as in our previous studies of mice singly deficient in GM-CSF4 or IL-3,25 dendritic cell numbers were not altered in the doubly mutant mice. These results suggest that GM-CSF and IL-3 are either not involved in steady-state dendritic cell development or are components of a larger network of redundant cytokines. In this context, flt3-ligand is likely to play a decisive role, based on its ability to increase dendritic cells in GM-CSF/IL-3-deficient mice by nearly 2 logs. Indeed, a recent report of flt3-ligand knockout mice demonstrated a substantial reduction of dendritic cell numbers in the spleen, thymus, and lymph nodes.49 Although GM-CSF/IL-3-deficient mice maintained normal dendritic cell numbers, these animals were markedly compromised in priming contact hypersensitivity reactions. The degree of impairment significantly exceeded that observed in GM-CSF or IL-3 single knockouts, establishing a dual requirement for both cytokines in this response. Although the administration of GM-CSF and IL-3 protein at the time of hapten sensitization reversed the defect, we have not yet identified the specific pathway compromised in GM-CSF/IL-3-deficient mice. Unlike CCR7 knockout mice,50 which fail to mount contact hypersensitivity reactions due to the lack of dendritic cell migration from the skin to the draining lymph nodes, GM-CSF/IL-3-deficient animals display normal numbers of hapten-loaded dendritic cells in the draining nodes. Moreover, although B7 family members51,52 and Ox40-ligand36 also contribute to hapten-specific priming, we were unable to detect differences in these molecules between doubly deficient animals and wild-type controls. Additional experiments are required to elucidate the mechanisms underlying defective hapten sensitization in the absence of GM-CSF and IL-3. Our own studies have revealed that vaccination with irradiated
tumor cells engineered to secrete GM-CSF and, to a lesser extent IL-3,
stimulate potent, specific, and long-lasting antitumor
immunity.37,38,53 Although we have not yet examined the
susceptibility of GM-CSF/IL-3-deficient mice to tumor induction,
recent investigations have established a striking inverse correlation
between the ability to generate contact hypersensitivity reactions to
polycyclic hydrocarbons and susceptibility to the carcinogenic effects
of these agents.54 These results raise the intriguing
possibility that GM-CSF and IL-3 may, like IFN-
We thank Glenn Begley (WAIMR) and Lorraine Robb (WEHI) for
providing
Submitted August 1, 2000; accepted October 24, 2000.
Supported by the Swiss National Science Foundation (N.M., S.G.), the Swiss Cancer League (S.G.), the Cancer Research Institute/Partridge Foundation, and CA74886 (G.D.). G.D. is a Clinical Scholar of the Leukemia and Lymphoma Society. S.G. and N.M. should be considered equal first authors.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Glenn Dranoff, Dana-Farber Cancer Institute, Dana 510E, 44 Binney St, Boston, MA 02115; e-mail: glenn_dranoff{at}dfci.harvard.edu.
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H. Iwasaki, C. Somoza, H. Shigematsu, E. A. Duprez, J. Iwasaki-Arai, S.-i. Mizuno, Y. Arinobu, K. Geary, P. Zhang, T. Dayaram, et al. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation Blood, September 1, 2005; 106(5): 1590 - 1600. [Abstract] [Full Text] [PDF]
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G. Terszowski, C. Waskow, P. Conradt, D. Lenze, J. Koenigsmann, D. Carstanjen, I. Horak, and H.-R. Rodewald Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E) Blood, March 1, 2005; 105(5): 1937 - 1945. [Abstract] [Full Text] [PDF]
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 E. Sitnicka, C. Brakebusch, I.-L. Martensson, M. Svensson, W. W. Agace, M. Sigvardsson, N. Buza-Vidas, D. Bryder, C. M.Cilio, H. Ahlenius, et al. Complementary Signaling through flt3 and Interleukin-7 Receptor {alpha} Is Indispensable for Fetal and Adult B Cell Genesis J. Exp. Med., November 17, 2003; 198(10): 1495 - 1506. [Abstract] [Full Text] [PDF]
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 O. G. Ribeiro, D. A. Maria, S. Adriouch, S. Pechberty, W. H. K. Cabrera, J. Morisset, O. M. Ibanez, and M. Seman Convergent alteration of granulopoiesis, chemotactic activity, and neutrophil apoptosis during mouse selection for high acute inflammatory response J. Leukoc. Biol., October 1, 2003; 74(4): 497 - 506. [Abstract] [Full Text] [PDF]
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J. Iwasaki-Arai, H. Iwasaki, T. Miyamoto, S. Watanabe, and K. Akashi Enforced Granulocyte/Macrophage Colony-stimulating Factor Signals Do Not Support Lymphopoiesis, but Instruct Lymphoid to Myelomonocytic Lineage Conversion J. Exp. Med., May 19, 2003; 197(10): 1311 - 1322. [Abstract] [Full Text] [PDF]
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T. Enzler, S. Gillessen, J. P. Manis, D. Ferguson, J. Fleming, F. W. Alt, M. Mihm, and G. Dranoff Deficiencies of GM-CSF and Interferon {gamma} Link Inflammation and Cancer J. Exp. Med., May 5, 2003; 197(9): 1213 - 1219. [Abstract] [Full Text] [PDF]
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A. G. Jegalian, A. Acurio, G. Dranoff, and H. Wu Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3 Blood, April 1, 2002; 99(7): 2603 - 2605. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(IFN-
, CD1d, CD3
, CD4, NK1.1, B7-1, B7-2, and
CD40 in the presence of blocking antibodies against Fc
, CD11b+) and
lymphoid-type (CD8
