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IL-1R Type I-Dependent Hemopoietic Proliferation Is Necessary for Inflammatory and Reactive Neutrophilia1

Yoshihiro Ueda,2 Derek W. Cain,2 Masayuki Kuraoka, Motonari Kondo, and Garnett Kelsoe3

Infections and inflammation trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as emergency granulopoiesis. The intrinsic factors that drive reactive neutrophilias and emergency granulopoiesis have ,been inferred but not demonstrated. Here, we show that alum cannot elicit reactive neutrophilias in IL-1R type I (IL-1RI)؊/؊ mice whereas other inflammatory responses, including eosinophilia and Ab production, remain intact. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by / progenitors and, surprisingly, multipotent progenitors and hematopoietic stem cells (HSC). Indeed, HSC and multipotent progenitor proliferative responses were most suppressed in IL-1RI؊/؊ mice, suggesting a critical role for their proliferation in inflammatory granulopoiesis. Whereas IL-1 drives increased HSC proliferation directly in vitro, IL-1RI expression by radiation-resistant host cells was both necessary and sufficient for alum-induced HSC, multipotent progenitor, and granu- locyte/macrophage progenitor proliferation and reactive neutrophilias in radiation chimeric mice. Thus, IL-1 plays a nec- essary, but indirect, role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis. The Journal of Immunology, 2009, 182: 6477–6484.

eutrophils are vital to innate (1, 2). Normally, as and , then band , and, physiologic numbers of mature neutrophils are main- finally, mature, segmented neutrophils (10). N tained by a “steady-state” granulopoietic pathway; acute Steady-state and emergency granulopoiesis can be distinguished infection or inflammation, however, trigger the mobilization of by separate dependencies on the C/EBP␣ and C/EBP␤ transcrip- stores from the marrow (BM)4 and into tion factors (CCAAT enhancer-binding proteins) (6, 11). Genetic inflammatory sites (3, 4). The result of this mobilization is a re- disruption of C/EBP␣ abolishes steady-state granulopoiesis (11), active neutrophilia immediately followed by accelerated or “emer- whereas C/EBP␤ deficiency leaves steady-state granulopoiesis in- gency” granulopoiesis in the BM (5, 6). tact but abrogates reactive neutrophilias (6). Neutrophils, like all other leukocytes, originate from self-renew- It is generally thought that emergency granulopoiesis is acti- ing, long-term (4) hematopoietic stem cells (HSC). By asymmet- vated by the increased expression of granulopoietic factors, IL-6, rical division, long-term HSC give rise to short-term HSC that IL-3, G-CSF, and GM-CSF, that induce granulocytic progenitors possess a limited capacity for self-renewal (7) and finally develop to proliferate (6, 12). Indeed, microbial infections increase serum into multipotent progenitors (MPP) (8). MPP produce lineage- GM-CSF and G-CSF coincidentally with the number of myeloid committed progenitors, including common myeloid progenitors progenitors in BM (13). In vitro, IL-6, IL-3, GM-CSF, and G-CSF (CMP) that differentiate into /erythroid progenitors promote proliferation and granulocytic differentiation by myeloid (MEP) or granulocyte/macrophage progenitors (GMP) (9). GMP progenitors (14–18). Finally, neutrophilias elicited by overexpres- produce neutrophils through a series of developmental stages, first sion of G-CSF, GM-CSF, or IL-3 are associated with increased C/EBP␤ transcription in GMP, suggesting that C/EBP␤ regulates

Department of Immunology, Duke University Medical Center, Durham, NC 27710 granulopoiesis through GMP (6). Despite these correlations, mice deficient for G-CSF, G-CSF Received for publication November 25, 2008. Accepted for publication March 10, 2009. and IL-6, or G-CSF and GM-CSF mount reactive neutrophilias (5, The costs of publication of this article were defrayed in part by the payment of page 19), and both steady-state and emergency granulopoiesis are intact charges. This article must therefore be hereby marked advertisement in accordance in mice deficient for the common ␤-chain of IL-3R/GM-CSF re- with 18 U.S.C. Section 1734 solely to indicate this fact. ceptor/IL-5R (20). Thus, IL-6, IL-3, GM-CSF, and G-CSF are dis- 1 This work was supported by National Institutes of Health Grants AI24335 and pensable for reactive neutrophilias and emergency granulopoiesis. AI56363 (to G.K.) and AI56123 and CA98129 (to M.K.). To understand emergency granulopoiesis, identification of indis- 2 Y.U. and D.W.C. contributed equally to this work. pensable factors is required. 3 Address correspondence and reprint requests to Dr. Garnett Kelsoe, Department of Immunology, Duke University, Durham, NC 27710. E-mail address: ghkelsoe@ We have observed that alum promotes granulopoiesis at the ex- duke.edu pense of B and have proposed that these lineages 4 Abbreviations used in this paper: BM, ; C/EBP, CCAAT enhancer-binding compete for developmental resources in the BM (10, 21). TNF-␣ protein; CFU-GEMM, CFU-granulocyte/erythroblast/macrophage/megakaryocyte; CFU- initiates these hematopoietic changes by lowering BM CXCL12 GM, CFU-granulocyte/macrophage; CGG, chicken ␥-globulin; CLP, common lymphoid ␣ progenitor; CMP, common myeloid progenitor; GMP, granulocyte/macrophage progen- expression and mobilizing BM ; TNF- alone, how- itor; HSC, ; IL-1RI, IL-1R type I; KO, knockout; MEP, ever, has little effect on granulopoiesis (21). Instead, TNF-␣ and megakaryocyte/erythroid progenitor; MPP, multipotent progenitor; NP, (4-hydroxy-3-ni- IL-1 synergize to reproduce alum’s effects on the BM (21), and trophenyl)acetyl; SCF, ; WT, wild type. that synergy implicated IL-1 as a central factor in emergency Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 granulopoiesis. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803961 6478 IL-1RI-DEPENDENT REACTIVE NEUTROPHILIA

Here, we show that alum does not elicit reactive neutrophilias not shown), preventing the discrimination of CMP and MEP (supplemental and emergency granulopoiesis in mice that lack functional recep- Fig. 2). tors for IL-1. These defects are not due to generally diminished HSC culture responses to alum’s inflammatory or adjuvant properties, as other HSC, MPP, and GMP (n ϭ 500) were cultured with recombinant IL-1␤ (1 responses, including eosinophilia and Ab production, remain intact ng/ml; PeproTech) or GM-CSF (1 ng/ml; PeproTech) in X-VIVO15 se- or are enhanced. Alum does not mobilize neutrophils from the BM rum-free medium (Cambrex) containing SCF (25 ng/ml; R&D Systems) to of IL-1R type I (IL-1RI)Ϫ/Ϫ mice nor does it elicit emergency promote HSC survival (24). Cultured cells were enumerated and charac- granulopoiesis as determined by accelerated granulopoietic output terized after 4 days. or proliferation by HSC, MPP, and GMP. These effects are indi- Quantification of mRNA rect, as neutrophil mobilization and emergency granulopoiesis in mRNA from cells (104–105) was precipitated in TRIzol (Invitrogen) and BM chimeric mice is determined by IL-1RI expression on radia- reverse transcribed with SuperScript II (Invitrogen). Quantitative PCR am- tion-resistant, nonhematopoietic cells in the host. We conclude that plifications of cDNA were performed (iCycler thermal cycler; Bio-Rad IL-1RI provides an indispensable signal that induces secondary Laboratories) with SYBR Green PCR Master Mix (Applied Biosystems) factors to initiate and sustain reactive neutrophilias and emergency using primers specific for IL-1RI, GM-CSF receptor, and ␤-actin cDNA: Ј Ј Ј granulopoiesis. IL-1RI forward, 5 -CTGAGGTCTTGGAGGGACAG-3 , and reverse, 5 - TCCTTCCTGGATGAGAGCAT-3Ј; GM-CSF receptor forward, 5Ј-GA CACGAGGATGAAGCACTG-3Ј, and reverse, 5Ј-GAGGTCCTTCCT Materials and Methods GAGGGTCT-3Ј; and ␤-actin forward, 5Ј-AGCCATGTACGTAGCCAT Mice CC-3Ј, and reverse, 5Ј-CTCTCAGCTGTGGTGGTGAA-3Ј. Amplification parameters were: initial denaturation at 94°C for 10 min; amplification cycle, C57BL/6 (CD45.2), congenic CD45.1 (B6.SJL-Ptprca Pep3b/BoyJ) (21), denaturing at 94°C for 10 s, anneal/extension at 60°C for 45 s. Relative gene Ϫ/Ϫ tm1Imx and congenic IL-1RI mice (B6.129S7-Il1r1 /J) (22) were from expression was calculated by the comparative CT (threshold cycle) method of ϫ ␤ ⌬ The Jackson Laboratory. (C57BL/6 C57BL/6.CD45.1)F1 mice were the manufacturer (Applied Biosystems) normalized to -actin message; CT bred locally. Mice were housed in specific pathogen-free conditions at the values were determined by subtracting CT (target) from CT(␤-actin).Ex- Duke University Animal Care Facility and with sterile bedding, water, and pression levels relative to ␤-actin were defined as 2Ϫ⌬⌬CT. food. All studies were approved by the Duke University Institutional An- imal Care and Use Committee. Serum Ab measurements Immunization NP-specific serum Abs were quantified as described (25). Mice were immunized with one injection of (4-hydroxy-3-nitrophenyl)ac- ␥ ␮ Adoptive reconstitutions etyl (NP)8-chicken -globulin (CGG) (60 g) in alum (21). ϫ (C57BL/6 C57BL/6.CD45.1)F1 mice were sublethally irradiated (600 rad) (26) and reconstituted with equal numbers (5 ϫ 106) of congenic ϩ/ϩ Ϫ/Ϫ Cell suspensions from blood and BM were stained with FITC-, PE-, PE- C57BL/6.CD45.1 (IL-1RI ) and C57BL/6 (IL-1RI ) BM cells to Texas Red-, biotin-, allophycocyanin-, allophycocyanin-Cy7-, PE-Cy5-, generate mixed chimeric mice. Reciprocal chimeras were generated 3 and PE-Cy7-conjugated mAbs specific for mouse B220, IgM, CD11b, similarly (IL-1RI-deficient C57BL/6 irradiated (C57BL/6.CD45.2/ 3 CD34, CD4, CD8, TER119, Gr-1, CD117, Sca-1, IL-7R␣,Fc␥RII/III, Flt3, CD45.1)F1 and (C57BL/6.CD45.2/CD45.1)F1 into irradiated Ϫ/Ϫ or Ly-6G (BD Biosciences or eBioscience); and PE-conjugated 7/4 mAb C57BL/6 (IL-1RI )). To control for any effects of hematopoietic 3 3 (AbD Serotec). Streptavidin-PE-Texas Red or streptavidin-Texas Red (BD reconstitution in these knockout (KO) wild-type (WT) and WT KO 3 3 Biosciences) were used to identify biotinylated mAbs. Propidium iodide chimeras, homologous (WT WT and KO KO) animals were created (Sigma-Aldrich) labeling identified dead cells. Labeled cells were analyzed as well. Donor and recipient cells in mixed and reciprocal chimeras or sorted with FACSVantage SE or LSR II flow cytometers (BD Bio- were distinguished by CD45.1 and CD45.2 expression; chimerism was sciences); data were analyzed with FlowJo software. LSK cells, Flt3Ϫ LSK determined by the CD45.1/CD45.2 ratio of blood leukocytes. cells, and Flt3ϩ LSK cells were sorted from BM suspensions following enrichment with CD117 microbeads (Miltenyi Biotec). Statistics Paired data were analyzed by Student’s t test. CFU complete Flt3Ϫ LSK cells (100) from naive or immunized (day 2) mice were cultured Results in methylcellulose media (StemCell Technologies) with mouse stem Alum-induced neutrophilias require IL-1RI cell factor (SCF), IL-3, IL-6, and human erythropoietin. Later (8 days), colonies were counted and typed as CFU-granulocyte/erythroblast/mac- IL-1 is an important component to inflammatory responses elicited rophage/megakaryocyte (CFU-GEMM), CFU-granulocyte/macrophage by mineral salts (27) and synergizes with TNF-␣ to increase neu- (CFU-GM), burst-forming unit-erythroblast, CFU-macrophage, and trophil production in BM (21). To determine the role of IL-1RI in CFU-granulocyte by microscopy. reactive neutrophilias, we injected C57BL/6 (BL/6) and congenic Ϫ/Ϫ Limiting dilution assays IL-1RI mice with alum/Ag (21) and followed changes in blood leukocyte numbers over 8 days. Ϫ ϭ Flt3 LSK cells were sorted from BM pools from naive (n 4) and In BL/6 mice, alum elicited a biphasic neutrophilia; neutrophil immunized (day 2; n ϭ 4) mice. Cultures containing 1 (n ϭ 72), 2 (n ϭ Ϫ Ͼ 48), or 8 (n ϭ 12) Flt3 LSK cells were made by sorting onto OP9 stromal (supplemental Fig. 1) numbers rose ( 2-fold) 1 day after immu- cell layers in 96-well plates with 10 ng/ml IL-7 and 10 ng/ml Flt3 ligand nization, returned to naive levels on day 2, and again rose Ϸ5-fold (R&D Systems) (23). After 14 days, wells containing CD45ϩCD11bϪ above controls on days 4 and 8 postimmunization (Fig. 1). Alum ϩ ϩ B220 CD19 cells (23) were scored positive for production. also modulated the numbers of inflammatory (28) and BrdU labeling (supplemental Fig. 1), with initial decreases 1 day after immunization, followed by steady increases that continued to day Mice were injected i.p. with 1 mg of BrdU; 6 h after injection, BM cells 8 (7-fold increase; Fig. 1). were harvested and labeled to identify specific populations (supplemental Ϫ/Ϫ Fig. 2).5 Labeled BM cells were fixed, permeabilized, and treated with The blood of naive BL/6 and IL-1RI mice contain identical DNaseI to expose incorporated BrdU using a commercial (BD Bio- numbers of neutrophils, inflammatory monocytes, and eosinophils sciences); cells were then stained with FITC-labeled anti-BrdU for flow (29) (Fig. 1). Alum did not elicit neutrophilia in IL-1RIϪ/Ϫ mice, cytometric analysis. These preparations denatured the CD34 epitope (data as blood neutrophil numbers were not significantly changed at any time point after immunization (Fig. 1). Inflammatory Ϫ Ϫ 5 The online version of this article contains supplemental material. responses were also abrogated, but IL-1RI / mice did mount The Journal of Immunology 6479

FIGURE 1. IL-1RI is required for alum-induced neutrophilia. Periph- eral blood cells of BL/6 and IL-1RIϪ/Ϫ mice were harvested after immu- nization (1–8 days) with NP8-CGG/alum. Neutrophils, inflammatory monocytes, and eosinophils were enumerated by flow cytometry (supple- mental Fig. 1). The mean (ϮSEM) numbers of cells/ml of blood from BL/6 (F; n ϭ 4–7) and IL-1RIϪ/Ϫ (E; days 1 and 2, n ϭ 2, others, n ϭ 4–5) mice are shown. Significant differences from naive controls are indicated p Յ 0.01) and IL-1RIϪ/Ϫ (†, p Յ 0.05; ††, p Յ ,ءء ;p Յ 0.05 ,ء) for BL/6 0.01) mice. robust eosinophilias that matched BL/6 controls (Fig. 1). Thus, alum induces inflammatory neutrophilias and monocytoses via an IL-1RI-dependent pathway, while induction of eosinophilia is IL-1RI-independent.

Alum-induced inflammatory granulopoiesis is IL-1RI-dependent The absence of alum-induced neutrophilias in IL-1RIϪ/Ϫ mice im- plied a defect in emergency granulopoiesis (5, 6). To determine the role of IL-1RI in emergency granulopoiesis, we immunized BL/6 FIGURE 2. Ϫ/Ϫ IL-1RI is required for alum-induced emergency granulopoi- and IL-1RI mice and followed the dynamics of HSC, MPP, esis. A, BM cells of BL/6 mice and IL-1RIϪ/Ϫ mice were harvested after CMP, and GMP (supplemental Fig. 2) populations, as well as the immunization (1–8 days) and labeled to identify HSC, MPP, CMP, GMP, primitive and mature neutrophil compartments (supplemental Fig. primitive neutrophils, and mature neutrophils (supplemental Figs. 1 and 2). 1) in BM. To estimate any changes in proliferation rates, we in- Average cell numbers (ϮSEM) from BL/6 (F) and IL-1RIϪ/Ϫ (E) mice are jected mice i.p. with BrdU 6 h before sacrifice and compared the shown (n ϭ 3–11, each point). B, BL/6 and IL-1RIϪ/Ϫ mice were immu- frequencies of BrdUϩ cells in each cell compartment from naive nized at various times then injected i.p. with 1 mg of BrdU 6 h before tissue and immunized mice (30). harvest. BM cells were labeled to identify hematopoietic progenitor com- Ϫ Ϫ partments and then fixed, exposed to DNase, and stained with anti-BrdU Immunization of BL/6 mice expanded the HSC (Flt3 Lin Sca- ϩ ϩ ϩ Ϫ ϩ mAb. Frequencies of BrdU cells in each progenitor compartment were 1 c-Kit (Flt3 LSK)) and MPP (Flt3 LSK) compartments 1 ϩ determined by flow cytometry. Average frequencies (ϮSEM) of BrdU day after immunization. HSC (supplemental Fig. 2) (7) numbers HSC, MPP, CMP/MEP, and GMP from BL/6 (F; n ϭ 3–9) and IL-1RIϪ/Ϫ rose to 150% of naive controls, remained elevated through day 4, (E; n ϭ 3–5) mice are shown. Significant differences from naive controls p Յ 0.01) and IL-1RIϪ/Ϫ (†, p Յ ,ءء ;p Յ 0.05 ,ء) and then returned to naive levels by day 6 (Fig. 2A). Similarly, are indicated for BL/6 MPP numbers (supplemental Fig. 2) (8) increased to 200% of con- 0.05; ††, p Յ 0.01) mice. trols 2 days after immunization and returned to normal by day 4 (Fig. 2A). Alum immunization did not increase CMP numbers in BL/6 Increased numbers of HSC and MPP were accompanied by in- mice; rather, CMP numbers fell on day 1 but then recovered and creased BrdU uptake. Frequencies of BrdUϩ HSC and MPP in- remained at naive levels (Fig. 2A). In contrast to the substantial creased 3.5- and 2.5-fold, respectively, within 1 day of immuni- increases in the frequencies of BrdUϩ HSC and MPP, BrdU la- zation and gradually returned to naive levels by day 4 (Fig. 2B), beling of CMP/MEP (LinϪc-KitϩSca-1ϪFc␥RII/IIIϪ) rose only indicating that alum induces HSC and MPP proliferation. modestly and returned to normal by day 4 (Fig. 2B). 6480 IL-1RI-DEPENDENT REACTIVE NEUTROPHILIA

Unlike CMP, GMP numbers increased significantly 1 and 2 days gency granulopoiesis and enhanced production of inflammatory after immunization and returned to normal by day 4 (Fig. 2A). monocytes. Increased GMP numbers correlated with increased (2-fold) BrdU Comparable HSC and MPP from naive and immunized mice uptake 1 and 2 days after immunization, indicating that GMP, like HSC and MPP, are targets of alum-induced proliferative signals IFN-␥ and TNF-␣ can induce Sca-1 expression by lineage-com- (Fig. 2B). mitted progenitors, causing them to mimic the LSK phenotype Early proliferation by HSC, MPP, and GMP was followed by (31). Thus, the rapid increases in HSC and MPP numbers after increased numbers of primitive neutrophils (myelocytes and meta- alum immunization could be only apparent, an artifact of prolif- myelocytes; supplemental Fig. 1). This neutrophil compartment eration by committed progenitor cells induced to express Sca-1. To grew Ͼ2-fold by day 4 after immunization and remained signifi- exclude this possibility, we analyzed LSK cells from naive and cantly elevated through day 8 (Fig. 2A). The effect of alum on the immunized mice for expression of Fc␥RII/III and IL-7R␣, mole- mature neutrophil compartment in BM was more complex. Ini- cules not expressed by HSC and MPP but characteristic of GMP tially, mature neutrophils were mobilized from the BM by alum; 1 and common lymphoid progenitors (CLP), respectively (9, 23). day after immunization, the numbers of mature BM neutrophils LSK from naive and immunized (day 2) mice were phenotypically fell by 85% (Fig. 2A), coincident with the first peak of neutrophilia identical, and neither expressed Fc␥RII/III or IL-7R␣ (Fig. 3, A (Fig. 1). This loss of mature neutrophils from the BM was soon and B), indicating that alum does not cause GMP or CLP to mimic the LSK phenotype. reversed and followed by significant, 2-fold increases over naive Ϫ controls at days 4–8 (Fig. 2A). This increase in BM neutrophils Next, we compared the ability of HSC (Flt3 LSK cells) from coincided with the second and sustained wave of neutrophilia naive and immunized mice to generate myeloid, erythroid, and multilineage colonies in methylcellulose cultures (32). We cul- (Fig. 1). Ϫ We conclude that neutrophilic responses to alum are similar to tured 100 Flt3 LSK BM cells from naive and immunized (day 2) those elicited by infection (3, 4); that is, both begin with the mo- mice with SCF, IL-3, IL-6, and erythropoietin (32). If inflamma- tion induces myeloid-committed progenitors to mimic the LSK bilization of BM neutrophil stores and are sustained by inflamma- Ϫ tory granulopoiesis. Unexpectedly, GMP were not the most prim- phenotype, then the Flt3 LSK compartment would contain a itive hematopoietic cells to proliferate in response to alum. HSC lower frequency of cells capable of generating multilineage colo- nies (CFU-GEMM) but a higher frequency that would produce and MPP proliferation increased coincidentally with that of GMP, myeloid colonies (CFU-GM) compared with naive controls. After suggesting that all three compartments are sensitive to inflamma- 8 days in culture, both Flt3Ϫ LSK cell cohorts produced identical tory signals. Ϫ Ϫ numbers of multilineage colonies (Fig. 3C). Furthermore, the fre- In IL-1RI / mice, immunization with alum/Ag neither mobi- quency of myeloid colonies arising from Flt3Ϫ LSK cells of im- lized mature neutrophils nor expanded the HSC, MPP, or GMP munized mice was actually lower than naive controls (Fig. 3C). compartments in BM. HSC numbers were not changed by immu- Ϫ ϩ Flt3 LSK cells from both naive and immunized mice produced nization (Fig. 2A). The frequency of BrdU HSC did not rise 1 day virtually no single lineage colonies (Ͻ1 granulocyte, macrophage, after immunization, but it did exhibit a transient increase on day 2 Ϫ or erythroid colony/100 Flt3 LSK) (Fig. 3C). These results dem- that was significantly attenuated compared with BL/6 controls Ϫ onstrate that alum does not enrich the Flt3 LSK compartment (Fig. 2B). Likewise, MPP numbers in the BM of IL-1RIϪ/Ϫ mice with myeloid-committed progenitors. were not affected by immunization (Fig. 2A). Frequencies of Ϫ ϩ We also compared the lymphoid potential of Flt3 LSK cells BrdU MPP transiently increased 1 day after immunization but from naive and immunized mice by determining their capacity to returned to naive levels by day 2 (Fig. 2B). These observations generate B-lineage cells in OP9 cultures containing indicate that alum-induced expansions of the HSC and MPP com- IL-7 and Flt3 ligand (23). The frequencies of Flt3Ϫ LSK cells from partments in the BM are IL-1RI-dependent. Ϫ/Ϫ immunized (1/3.6) and naive (1/4.6) mice that supported B-lineage In contrast to BL/6 mice, GMP numbers in IL-1RI mice did development were virtually identical (Fig. 3D); the equivalent lin- not increase after immunization; instead, GMP numbers gradually eage potentials of both Flt3Ϫ LSK cohorts identify these cells fell, becoming significantly depleted by day 6 (Fig. 2A). This de- as HSC. cline was accompanied by reduced BrdU uptake, as BrdUϩ GMP were increased only on day 1 and then returned to naive levels Alum retains robust inflammatory and adjuvant properties in Ϫ Ϫ (Fig. 2B). Thus, alum coincidentally elicits IL-1RI-dependent pro- IL-1RI / mice liferation in the HSC, MPP, and GMP compartments of BM. IL-1 is a potent proinflammatory and IL-1RI is expressed The impaired proliferation by HSC, MPP, and GMP in immu- by many cell types (33), raising the possibility that the absence of Ϫ/Ϫ nized IL-1RI mice was followed by almost no change in the alum-induced neutrophilias in IL-1RI-deficient mice is not a spe- numbers of neutrophils in BM (Fig. 2A). Whereas immunization cific effect but a general suppression of all inflammatory responses. eventually (day 8) produced a modest but significant increase in We think this not to be the case. First, alum elicits eosinophilias the numbers of primitive neutrophils, the mature neutrophil com- in IL-1RIϪ/Ϫ mice (Fig. 1) that are equal to or greater than the partment of BM was neither depleted by mobilization nor ex- eosinophilic responses of BL/6 mice (Fig. 4A). Second, alum mo- panded by proliferation (Fig. 2A). The absence of alum-induced bilizes pre-B cells from the BM of BL/6 and IL-1RIϪ/Ϫ mice with Ϫ Ϫ neutrophilias in IL-1RI / mice results from defective neutrophil equal efficiency (21). In immunized BL/6 and IL-1RIϪ/Ϫ mice, mobilization and abrogation of inflammatory granulopoiesis. pre-B cell numbers in BM fell Ͼ50% by day 4 (Fig. 4B). Recovery Ϫ Ϫ Alum’s failure to elicit reactive monocytoses in IL-1RI / mice of these inflammatory losses was more rapid in IL-1RIϪ/Ϫ mice, (Fig. 1) suggested defective monocyte output; although immuni- perhaps due to the lack of competition for growth resources in the zations did not significantly alter the numbers of inflammatory absence of expanded granulopoiesis (10) (Fig. 4B). Finally, IL- monocytes in the BM of BL/6 mice, in IL-1RIϪ/Ϫ mice the num- 1RIϪ/Ϫ mice immunized with NP-CGG in alum mounted charac- bers of inflammatory monocytes fell to 25% of controls by day 4 teristic and robust serum Ab responses (Fig. 4C). before returning to normal (day 6; Fig. 2). Thus, IL-1RI controls Given that alum induces strong eosinophilic responses, mobi- inflammatory myelopoietic pathways necessary for both emer- lizes BM pre-B cells, and acts as a potent adjuvant in IL-1RIϪ/Ϫ The Journal of Immunology 6481

FIGURE 4. Robust inflammatory responses in IL-1RIϪ/Ϫ mice. BM cells of BL/6 mice and IL-1RIϪ/Ϫ mice were harvested after immunization (1–8 days) and labeled with mAbs to identify eosinophils (supplemental Fig. 1) and pre-B cells (10). Average numbers (ϮSEM) of eosinophils (A) and pre-B cells (B) from BL/6 (F) and IL-1RIϪ/Ϫ (E) mice are shown (n ϭ 3–11, each point). Significant differences from naive controls are indicated p Յ 0.01) and IL-1RIϪ/Ϫ (†, p Յ 0.05; ††, p Յ ,ءء ;p Յ 0.05 ,ء) for BL/6 ␭ϩ ␬ϩ 0.01) mice. C,NP16-BSA binding and serum Ab was determined in BL/6 (closed symbols) and IL-1RIϪ/Ϫ (open symbols) mice (n ϭ 2, each ␮ point) before and after immunization with 60 gofNP8-CGG/alum. NP- specific serum ␭ (circles) and ␬ (squares) Abs were quantified (mean Ϯ SD) by ELISA. FIGURE 3. LSK cells from naive and immunized mice have equivalent lineage potential. A, Sca-1 and c-Kit staining of BM LinϪ cells from naive Ϫ/Ϫ and immunized (2 days after injection of NP8-CGG/alum) mice. B, Anal- IL-1RI HSC, MPP, and GMP proliferate in immunized BL/6 ysis of LSK cells from naive (filled histograms) and immunized (open hosts histograms) mice for Fc␥RII/III and IL-7R␣ expression, compared with expression by GMP (PIϪLinϪc-KitϩSca-1ϪCD34ϩFc␥RII/IIIϩ; broken Although LSK cells express IL-1RI and proliferate in response to lines, left histogram) and CLP (PIϪLinϪIL-7R␣ϩc-KitlowSca-1low; broken IL-1␤ in vitro (supplemental Fig. 3), IL-1 could modulate hema- lines, right histogram), respectively. C, Erythroid and myeloid potential topoiesis indirectly (33). To determine whether IL-1RI expression from single Flt3Ϫ LSK cells was determined by methylcellulose assay. by HSC, MPP, and GMP is required for their alum-induced pro- Ϫ Flt3 LSK cells (100 cells) were sorted from naive and immunized (day 2) liferation, we generated chimeric mice by reconstituting irradiated mice and seeded in methylcellulose medium containing SCF, IL-3, IL-6, ϫ (C57BL/6 C57BL/6.CD45.1)F1 mice with equal numbers of and erythropoietin. The numbers and types of colonies were determined by BM cells from C57BL/6.CD45.1 (WT) and IL-1RIϪ/Ϫ C57BL/6 optical microscopy 8 days later. The bars show the average numbers of (KO; CD45.2) donors. Donor and host hematopoietic cells were colonies classified as CFU-GEMM (multilineage), CFU-GM (myeloid lin- eage), and the total number of CFU-G, -M, and -E (CFU-G/M/E; single identified by CD45 allelism. We determined BrdU incorporation lineage). The SDs of the total numbers of colonies are shown. D, B cell pro- by donor HSC, MPP, and GMP 2 days after immunization, when duction by Flt3Ϫ LSK cells from naive (F) and immunized (E) mice. Multiple IL-1RI-dependent proliferative defects are maximal (Fig. 2B). If wells of 1, 2, and 8 Flt3Ϫ LSK cells from naive and immunized (day 2) mice IL-1RI expression by HSC, MPP, or GMP is required for their were cultured on OP9 stromal cell layers in the presence of IL-7 and Flt3L. proliferation in response to alum, then WT progenitor cells should The frequencies of wells with B cell growth were determined by the presence respond to immunization but KO cells should not. ϩ Ϫ ϩ ϩ of CD45 CD11b CD19 B220 cells after 14 days in culture. We determined the frequencies of WT and KO hematopoietic cells in the blood of mixed and reciprocal chimeric mice 4 wk after mice, we conclude that the absence of inflammatory neutrophilias reconstitution. All chimeras exhibited a modest excess of KO leu- and monocytoses in IL-1RIϪ/Ϫ mice represents specific effects on kocytes (51 Ϯ 6% KO vs 40 Ϯ 7% WT; p Յ 0.05); all chimeras neutrophils and monocytes. had similar numbers of blood leukocytes (data not shown). 6482 IL-1RI-DEPENDENT REACTIVE NEUTROPHILIA

inflammasome (34). Taken together, these observations suggested that IL-1␤ might be the intrinsic activating signal for emergency granulopoiesis. Whereas myeloid cell numbers in naive IL-1RIϪ/Ϫ and C57BL/6 mice are indistinguishable (Figs. 1 and 2), alum/Ag elic- ited neither the pronounced reactive neutrophilias and inflamma- tory monocytic responses nor the emergency granulopoiesis in IL- 1RIϪ/Ϫ mice (Fig. 2) that characterized C57BL/6 controls. To our knowledge, this is the first demonstration of a single, indispensable cytokine/receptor pathway for emergency granulopoiesis (5, 20). FIGURE 5. Alum induces IL-1RI-deficient HSC, MPP, and GMP to A modest increase in the number of primitive neutrophils was ϫ proliferate in chimeric mice. A, Irradiated (C57BL/6 C57BL/ observed in the BM of IL-1RIϪ/Ϫ mice 8 days after immunization ϫ 6 6.CD45.1)F1 mice were reconstituted with equal numbers (5 10 )of Ϫ Ϫ (Fig. 2A). In contrast to the rapid induction of eosinophilia and C57BL/6.CD45.1 (WT/CD45.2) and IL-1RI / (KO/CD45.2) BM cells. mobilization of BM lymphocytes (Fig. 4), this late neutrophilic Afterward (4 wk), chimeras were immunized with NP8-CGG/alum and 2 days later were injected i.p. with 1 mg of BrdU. Six hours after BrdU response comes well after reactive neutrophilias of congenic con- injection, BM cells were harvested and the frequencies of BrdUϩ HSC, trols, and it may represent another (chronic?) pathway for in- MPP, and GMP (supplemental Fig. 2) were determined by flow cytometry. creased neutrophil production. Average frequencies (ϮSD) of BrdUϩ WT (CD45.1) and KO (CD45.2) Alum, a common vaccine adjuvant, promotes inflammation and HSC, MPP, and GMP from naive (open symbols; n ϭ 4) and immunized immunogenicity via the Nalp3 inflammasome and the production ϭ (closed symbols; n 3) mice are shown. To generate reciprocal chimeras, of the proinflammatory IL-1␤, IL-18, and IL-33 (34, 35). ϫ (B) irradiated (C57BL/6 C57BL/6.CD45.1)F1 mice were reconstituted Ϫ/Ϫ 3 Nonetheless, abrogation of neutrophilia and emergency granulo- with KO (C57BL/6.IL-1RI ) BM cells (KO WT) or (C) irradiated Ϫ/Ϫ C57BL/6 IL-1RIϪ/Ϫ mice were reconstituted with (C57BL/6 ϫ C57BL/ poiesis in IL-1RI mice was a specific defect, as other inflam- 3 matory responses, reactive eosinophilia (Figs. 1 and 4A), mobili- 6.CD45.1)F1 BM cells (WT KO). Four weeks later, chimeric mice were immunized and analyzed as for A. Average (ϮSD) frequencies of BrdUϩ zation of BM pre-B cells (Fig. 4B), and robust Ab production (Fig. HSC, MPP, and GMP from naive (open symbols; n ϭ 4) and immunized 4C), remained intact. IL-3 and IL-5 promote eosinophilia (36), and (closed symbols; n ϭ 4) mice are shown. Significant differences between mobilization of BM B-lineage cells depends on TNF-␣ (21). Thus, p Յ 0.01. other alum-induced inflammatory responses are fully active in ,ءء ;p Յ 0.05 ,ء .groups are indicated IL-1RIϪ/Ϫ mice. ϩ Emergency granulopoiesis is generally thought to represent in- In naive chimeras, the frequencies of BrdU WT and KO HSC, creased GMP proliferation (6), and we observed significant in- MPP, and GMP were equivalent, but labeling of HSC and MPP Ϫ Ϫ creases in GMP numbers 1 and 2 days after immunization accom- was elevated compared with naive BL/6 and IL-1RI / controls ( p Յ 0.05; compare Figs. 2 and 5). Increased frequencies of panied by increased BrdU uptake (Fig. 2). Additionally, however, BrdUϩ HSC and MPP in chimeras may reflect ongoing hemato- we found that in C57BL/6 mice, alum simultaneously induced pro- poietic replenishment. Immunization of chimeric mice equally in- liferation by HSC and MPP (Fig. 2); HSC and MPP are themselves creased (Ϸ150%) BrdU labeling of WT and KO HSC, MPP, and the immediate targets of inflammatory signals. GMP (Fig. 5), and these increases matched those of BL/6 controls In contrast to control mice, alum elicited no significant increases Ϫ/Ϫ (Fig. 2). Robust proliferative responses by IL-1RI-deficient hema- in HSC, MPP, and GMP numbers in IL-1RI mice (Fig. 2A) and topoietic cells demonstrate an intermediate signal that determines significantly attenuated proliferative responses by these compart- HSC, MPP, and GMP proliferation. This intermediate, trans-act- ments (Fig. 2B). The lack of increases in HSC, MPP, and GMP Ϫ/Ϫ ing signal could be produced by the IL-1RIϩ hematopoietic com- numbers in IL-1RI mice after immunization (Fig. 2A), despite partment of mixed chimeras or, as IL-1RI is widely expressed (33), modest but significant increases in proliferation (Fig. 2B), raises it could come from radiation-resistant host cells of hematopoietic the possibility that IL-1RI-dependent signals act not only to sustain or nonhematopoietic origin. high levels of progenitor proliferation but also to promote progen- To determine the origin of this proliferation signal, we generated itor survival. These observations indicate an unexpected role for KO3WT and WT3KO BM chimeras. Four weeks after recon- HSC and MPP proliferation in the induction of emergency stitution, ϳ60% of blood leukocytes were derived from donor cells granulopoiesis. in both chimera types. Chimeric mice were immunized and the Curiously, proliferation and expansion by the HSC and MPP frequencies of BrdUϩ HSC, MPP, and GMP were measured 2 days compartments in immunized C57BL/6 mice are associated with later. In KO3WT chimeras, immunization significantly increased increased numbers of GMP but not CMP (Fig. 2A). If GMP arise BrdU labeling of progenitors compared with naive chimera con- from CMP, why do we not detect an expansion in this compart- trols (Fig. 5B). In contrast, the frequencies of BrdUϩ HSC, MPP, ment as well? One possibility is that inflammation accelerates and GMP did not increase after immunization of WT3KO chi- CMP differentiation, such that the increased CMP production by meras (Fig. 5C). We conclude that it is IL-1RI expression/activity MPP is obscured by a very rapid CMP to GMP differentiation. in a radiation-resistant, host compartment that determines the re- Alternatively, inflammation may cause some fraction of MPP to active proliferation of HSC, MPP, and GMP. differentiate into GMP directly, a phenomenon suggested by Ado- lfsson et al. (8). The latter hypothesis is consistent with our failure Discussion to observe increased numbers of MEP and CLP after alum immu- Infections elicit neutrophilias that are sustained by a distinct, emer- nization (data not shown). Inflammatory signals elicited by alum gency granulopoietic program that is activated by ill-defined in- appear to accelerate specifically (10, 21) (Figs. 2 and trinsic signals (6). We previously noted that reactive neutrophilias 4, A and B). Given that immunization did not alter the lineage induced by alum (Fig. 1) are mimicked by the coadministration of potential of Flt3Ϫ LSK HSC (Fig. 3, C and D), this hematopoietic TNF-␣ and IL-1␤ (10, 21). More recently, alum has been shown to specialization likely represents alteration of external cues that in- be a potent inducer of IL-1␤ secretion by activation of the Nalp3 fluence hematopoietic lineage decisions (10, 21). The Journal of Immunology 6483

Although HSC and MPP express IL-1RI and proliferate in re- capable of mounting inflammatory eosinophilias despite attenuated sponse to IL-1 in vitro (supplemental Fig. 3), IL-1RI expression by proliferation of HSC, MPP, and GMP implies that a more differ- HSC, MPP, and GMP is not required for their proliferation in entiated progenitor responds to inflammatory signals that increase response to alum (Fig. 5). In mixed chimeras, proliferation by IL- production. This observation is consistent with the work 1RIϪ/Ϫ HSC, MPP, and GMP was identical to that of IL-1RI- of Iwasaki and colleagues, who showed that eosinophilias elicited bearing cells (Fig. 5). IL-1/IL-1RI signals are therefore necessary by helminth infections are supported by expansions of a committed for the induction of reactive neutrophilias and emergency granu- eosinophil progenitor compartment but not CMP or GMP (41). lopoiesis by alum, but they must act indirectly to drive prolifera- The requirement for IL-1RI in alum-induced neutrophilias and tion in the HSC, MPP, and GMP compartments. emergency granulopoiesis emphasizes the central role of IL-1 in IL-1 induces the production of many growth factors and inflam- innate immune responses (27, 42, 43). Alum drives IL-1␤ and matory mediators (33); among these, IL-3, IL-6, G-CSF, and GM- IL-18 secretion by activating the Nalp3 inflammasome (27, 34, CSF have been implicated as agents of emergency granulopoiesis 35), and these cytokines have been proposed to be the agents of (6, 12). However, these cytokines are dispensable for robust granu- alum’s several inflammatory properties including adjuvanticity lopoietic responses to inflammation (5, 20). Consequently, these (34). Instead, our work indicates that the effects of secreted IL-1␤ dispensable cytokines cannot be the intermediate cues that drive may be focused on neutrophilic responses, as alum-induced eosin- IL-1RI-dependent emergency granulopoiesis in alum-immunized ophilia, pre-B cell mobilization, and Ab responses remain intact in mice. Given that HSC, MPP, and GMP appear to be the immediate IL-1RIϪ/Ϫ mice (Fig. 4). Indeed, our findings are consistent with targets of alum-induced, IL-1RI-dependent proliferation (Fig. 2), the seemingly paradoxical findings by Eisenbarth et al. (34), show- IL-1RI signals may act through intermediate hematopoietic factors ing that the alum’s adjuvanticity is abrogated in mice defective for that induce proliferation by both HSC and committed progenitors, components of the Nalp3 inflammasome, but intact in animals such as IL-11 (37, 38) or thrombopoietin (39). lacking MyD88, a crucial signaling component of the IL-1 and Alternatively, the emergency granulopoiesis induced by alum IL-18 receptors (44). Thus, alum must stimulate humoral re- could be driven by a density-dependent feedback between BM sponses independently of the cytokines presently associated with neutrophils and their progenitors; that is, BM neutrophils suppress the Nalp3 inflammasome. the proliferation and differentiation of hematopoietic progenitor cells. Inflammatory mobilization of mature neutrophils from the Acknowledgments BM would relax this suppression, activating HSC, MPP, and GMP We thank T. M. Holl, D. Liao, and A. Patel for assistance. proliferation. A similar hypothesis was offered previously to ex- plain the stability of steady-state neutrophil pools (40). If so, ab- Disclosures Ϫ/Ϫ rogation of emergency granulopoiesis in IL-1RI mice could be The authors have no financial conflicts of interest. the result of defective mobilization by BM neutrophils and the continuation of suppression (Fig. 2A). Consistent with this feed- References back hypothesis, alum neither mobilized BM neutrophils (data not 1. Engle, W. D., and C. R. Rosenfeld. 1984. in high-risk neonates. shown) nor induced HSC, MPP, and GMP proliferation in J. Pediatr. 105: 982–986. WT3KO chimeras (Fig. 5C). In contrast, all chimeras generated 2. Gessler, P., R. Luders, S. Konig, N. Haas, P. Lasch, and W. Kachel. 1995. Neo- 3 3 natal neutropenia in low birthweight premature infants. Am. J. Perinatol. 12: in IL-1RI-sufficient hosts (WT plus KO WT, KO WT; Fig. 5, 34–38. A and B) exhibited robust neutrophil mobilization (data not shown) 3. Quie, P. G. 1980. The phagocytic system in host defense. Scand. J. Infect. Dis. and progenitor proliferation after immunization with alum. 24(Suppl.): 30–32. 4. Burg, N. D., and M. H. Pillinger. 2001. The neutrophil: function and regulation The linkage of neutrophil mobilization and progenitor prolifer- in innate and humoral immunity. Clin. Immunol. 99: 7–17. ation points out a caveat to interpreting our and earlier studies of 5. Basu, S., G. Hodgson, H. H. Zhang, M. Katz, C. Quilici, and A. R. Dunn. 2000. “Emergency” granulopoiesis in G-CSF-deficient mice in response to Candida emergency granulopoiesis. All previous reports relied solely on albicans infection. Blood 95: 3725–3733. blood neutrophil counts to define emergency granulopoiesis (5, 6); 6. Hirai, H., P. Zhang, T. Dayaram, C. J. Hetherington, S. Mizuno, J. Imanishi, our observations highlight an intrinsic limitation to this type of K. Akashi, and D. G. Tenen. 2006. C/EBP␤ is required for “emergency” gran- ulopoiesis. Nat. Immunol. 7: 732–739. analysis, as it fails to discriminate between mobilization and emer- 7. Yang, L., D. Bryder, J. Adolfsson, J. Nygren, R. Mansson, M. Sigvardsson, and gency granulopoietic output. S. E. Jacobsen. 2005. Identification of LinϪSca1ϩkitϩCD34ϩFlt3Ϫ short-term Earlier, we proposed that developing neutrophils and B cells hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloa- blated transplant recipients. Blood 105: 2717–2723. compete for specific resources in the BM such that the expansion 8. Adolfsson, J., R. Mansson, N. Buza-Vidas, A. Hultquist, K. Liuba, C. T. Jensen, of one lineage results in a compensatory diminution of the other D. Bryder, L. Yang, O. J. Borge, L. A. Thoren, et al. 2005. Identification of Flt3ϩ (10). Alum mobilizes B-lineage cells from the BM and effects a lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121: 295–306. central lymphopenia that coincides with the period of emergency 9. Akashi, K., D. Traver, T. Miyamoto, and I. L. Weissman. 2000. A clonogenic granulopoiesis (10). In IL-1RIϪ/Ϫ mice, alum depleted the BM of common myeloid progenitor that gives rise to all myeloid lineages. Nature 404: 193–197. pre-B cells but the recovery of this compartment was significantly 10. Ueda, Y., M. Kondo, and G. Kelsoe. 2005. Inflammation and the reciprocal faster than in control mice (Fig. 4B). As alum does not expand production of and lymphocytes in bone marrow. J. Exp. Med. 201: granulopoiesis in IL-1RIϪ/Ϫ mice, our competition model predicts 1771–1780. 11. Zhang, D. E., P. Zhang, N. D. Wang, C. J. Hetherington, G. J. Darlington, and that vacated niches/resources normally occupied by GMP and D. G. Tenen. 1997. Absence of granulocyte colony-stimulating factor signaling primitive neutrophils might be available to accelerate the restora- and neutrophil development in CCAAT enhancer binding protein ␣-deficient tion of the B-lineage compartments of the BM. mice. Proc. Natl. Acad. Sci. USA 94: 569–574. 12. Walker, F., H. H. Zhang, V. Matthews, J. Weinstock, E. C. Nice, M. Ernst, The increased availability of developmental niches in the BM of S. Rose-John, and A. W. Burgess. 2008. IL6/sIL6R complex contributes to emer- immunized IL-1RIϪ/Ϫ mice may also explain the enhanced pro- gency granulopoietic responses in G-CSF- and GM-CSF-deficient mice. Blood 111: 3978–3985. duction of eosinophils compared with immunized C57BL/6 mice 13. Cheers, C., A. M. Haigh, A. Kelso, D. Metcalf, E. R. Stanley, and A. M. Young. (days 6 and 8; Fig. 4A). On the other hand, neutrophils likely 1988. Production of colony-stimulating factors (CSFs) during infection: separate mitigate alum-induced inflammation, and impaired neutrophilic re- determinations of macrophage-, granulocyte-, granulocyte-macrophage-, and Ϫ/Ϫ multi-CSFs. Infect. Immun. 56: 247–251. sponses in immunized IL-1RI mice may prolong inflammatory Bull. Ϫ Ϫ 14. Metcalf, D. 1978. In vitro cloning of hemopoietic cells. 65: responses and enhance eosinophilias. That IL-1RI / mice are 417–419. 6484 IL-1RI-DEPENDENT REACTIVE NEUTROPHILIA

15. Koike, K., E. R. Stanley, J. N. Ihle, and M. Ogawa. 1986. Macrophage colony 1997. Absence of IL-1 signaling and reduced inflammatory response in IL-1 type formation supported by purified CSF-1 and/or 3 in serum-free culture: I receptor-deficient mice. J. Immunol. 159: 2452–2461. evidence for hierarchical difference in macrophage colony-forming cells. Blood 30. Schittek, B., K. Rajewsky, and I. Forster. 1991. Dividing cells in bone marrow 67: 859–864. and incorporate bromodeoxyuridine with high efficiency. Eur. J. Immunol. 16. Souza, L. M., T. C. Boone, J. Gabrilove, P. H. Lai, K. M. Zsebo, D. C. Murdock, 21: 235–238. V. R. Chazin, J. Bruszewski, H. Lu, K. K. Chen, et al. 1986. Recombinant human 31. Zhang, P., S. Nelson, G. J. Bagby, R. Siggins, 2nd, J. E. Shellito, and Ϫ ϩ ϩ granulocyte colony-stimulating factor: effects on normal and leukemic myeloid D. A. Welsh. 2008. The lineage c-kit Sca-1 cell response to Escherichia coli cells. Science 232: 61–65. bacteremia in Balb/c mice. Stem Cells 26: 1778–1786. 17. Donahue, R. E., J. Seehra, M. Metzger, D. Lefebvre, B. Rock, S. Carbone, 32. Ling, K. W., K. Ottersbach, J. P. van Hamburg, A. Oziemlak, F. Y. Tsai, D. G. Nathan, M. Garnick, P. K. Sehgal, D. Laston, et al. 1988. Human IL-3 and S. H. Orkin, R. Ploemacher, R. W. Hendriks, and E. Dzierzak. 2004. GATA-2 GM-CSF act synergistically in stimulating hematopoiesis in primates. Science plays two functionally distinct roles during the ontogeny of hematopoietic stem 241: 1820–1823. cells. J. Exp. Med. 200: 871–882. 18. Caracciolo, D., S. C. Clark, and G. Rovera. 1989. Human interleukin-6 supports 33. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87: granulocytic differentiation of hematopoietic progenitor cells and acts synergis- 2095–2147. tically with GM-CSF. Blood 73: 666–670. 34. Eisenbarth, S. C., O. R. Colegio, W. O’Connor, F. S. Sutterwala, and 19. Hibbs, M. L., C. Quilici, N. Kountouri, J. F. Seymour, J. E. Armes, R. A. Flavell. 2008. Crucial role for the Nalp3 inflammasome in the immunos- A. W. Burgess, and A. R. Dunn. 2007. Mice lacking three myeloid colony- timulatory properties of aluminium adjuvants. Nature 453: 1122–1126. stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce 35. Li, H., S. Nookala, and F. Re. 2007. Aluminum hydroxide adjuvants activate ␤ and granulocytes and mount an inflammatory response in a sterile model of peri- caspase-1 and induce IL-1 and IL-18 release. J. Immunol. 178: 5271–5276. tonitis. J. Immunol. 178: 6435–6443. 36. Yamaguchi, Y., T. Suda, J. Suda, M. Eguchi, Y. Miura, N. Harada, A. Tominaga, 20. Nishinakamura, R., A. Miyajima, P. J. Mee, V. L. Tybulewicz, and R. Murray. and K. Takatsu. 1988. Purified interleukin 5 supports the terminal differentiation 1996. Hematopoiesis in mice lacking the entire granulocyte-macrophage colony- and proliferation of murine eosinophilic precursors. J. Exp. Med. 167: 43–56. stimulating factor/interleukin-3/interleukin-5 functions. Blood 88: 2458–2464. 37. Hangoc, G., T. Yin, S. Cooper, P. Schendel, Y. C. Yang, and H. E. Broxmeyer. 21. Ueda, Y., K. Yang, S. J. Foster, M. Kondo, and G. Kelsoe. 2004. Inflammation 1993. In vivo effects of recombinant interleukin-11 on myelopoiesis in mice. controls B lymphopoiesis by regulating chemokine CXCL12 expression. J. Exp. Blood 81: 965–972. Med. 199: 47–58. 38. Lemoli, R. M., M. Fogli, A. Fortuna, M. R. Motta, S. Rizzi, C. Benini, and S. Tura. 1993. Interleukin-11 stimulates the proliferation of human hematopoietic 22. Glaccum, M. B., K. L. Stocking, K. Charrier, J. L. Smith, C. R. Willis, ϩ ϩ Ϫ Ϫ CD34 and CD34 CD33 DR cells and synergizes with stem cell factor, in- C. Maliszewski, D. J. Livingston, J. J. Peschon, and P. J. Morrissey. 1997. Phe- terleukin-3, and granulocyte-macrophage colony-stimulating factor. Exp. Hema- notypic and functional characterization of mice that lack the type I receptor for tol. 21: 1668–1672. IL-1. J. Immunol. 159: 3364–3371. 39. Young, J. C., E. Bruno, K. M. Luens, S. Wu, M. Backer, and L. J. Murray. 1996. 23. Kondo, M., I. L. Weissman, and K. Akashi. 1997. Identification of clonogenic Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion Cell ϩ ϩ ϩ Ϫ common lymphoid progenitors in mouse bone marrow. 91: 661–672. of CD34 progenitor cells from single CD34 Thy-1 Lin primitive progenitor 24. Domen, J., and I. L. Weissman. 2000. Hematopoietic stem cells need two signals cells. Blood 88: 1619–1631. to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the 40. Layton, J. E., H. Hockman, W. P. Sheridan, and G. Morstyn. 1989. Evidence for other. J. Exp. Med. 192: 1707–1718. a novel in vivo control mechanism of granulopoiesis: mature cell-related control 25. Chen, Z., S. B. Koralov, M. Gendelman, M. C. Carroll, and G. Kelsoe. 2000. of a regulatory growth factor. Blood 74: 1303–1307. Ϫ/Ϫ Humoral immune responses in Cr2 mice: enhanced affinity maturation but 41. Iwasaki, H., S. Mizuno, R. Mayfield, H. Shigematsu, Y. Arinobu, B. Seed, impaired antibody persistence. J. Immunol. 164: 4522–4532. M. F. Gurish, K. Takatsu, and K. Akashi. 2005. Identification of eosinophil lin- 26. Congdon, C. C., N. Gengozian, and T. Makinodan. 1956. Agglutinin production eage-committed progenitors in the murine bone marrow. J. Exp. Med. 201: in normal, sublethally irradiated, and lethally irradiated mice treated with mouse 1891–1897. bone marrow. J. Immunol. 77: 250–256. 42. Chen, C. J., H. Kono, D. Golenbock, G. Reed, S. Akira, and K. L. Rock. 2007. 27. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock, Identification of a key pathway required for the sterile inflammatory response K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate triggered by dying cells. Nat. Med. 13: 851–856. the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: 43. Chen, C. J., Y. Shi, A. Hearn, K. Fitzgerald, D. Golenbock, G. Reed, S. Akira, 847–856. and K. L. Rock. 2006. MyD88-dependent IL-1 receptor signaling is essential for 28. Henderson, R. B., J. A. Hobbs, M. Mathies, and N. Hogg. 2003. Rapid recruit- gouty inflammation stimulated by monosodium urate crystals. J. Clin. Invest. ment of inflammatory monocytes is independent of neutrophil migration. Blood 116: 2262–2271. 102: 328–335. 44. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, 29. Labow, M., D. Shuster, M. Zetterstrom, P. Nunes, R. Terry, E. B. Cullinan, K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results T. Bartfai, C. Solorzano, L. L. Moldawer, R. Chizzonite, and K. W. McIntyre. in loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150.