Regulation of steady-state neutrophil homeostasis by macrophages

Abstract

The timely clearance of apoptotic neutrophils from inflammation sites is an important function of macrophages; however, the role of macrophages in maintaining neutrophil homeostasis under steady-state conditions is less well understood. By conditionally deleting the antiapoptotic gene cellular FLICE-like inhibitory protein (C-FLIP) in myeloid cells, we have generated a novel mouse model deficient in marginal zone and bone marrow stromal macrophages. These mice develop severe neutrophilia, splenomegaly, extramedullary hematopoiesis, decreased body weight, and increased production of granulocyte colony-stimulating factor (G-CSF) and IL-1β, but not IL-17. c-FLIP(f/f) LysM-Cre mice exhibit delayed clearance of circulating neutrophils, suggesting that failure of macrophages to efficiently clear apoptotic neutrophils causes production of cytokines that drive excess granulopoiesis. Further, blocking G-CSF but not IL-1R signaling in vivo rescues this neutrophilia, suggesting that a G-CSF-dependent, IL-1β-independent pathway plays a role in promoting neutrophil production in mice with defective clearance of apoptotic cells.

Figure 1

c-FLIP is required for macrophage survival. (A) Expression of c-FLIP_L and c-FLIP_S isoforms in wild-type BMDM as determined by Western blot. (B,C) Macrophage death after in vitro deletion of c-FLIP. (B) BMDM from c-FLIP^f/f ER-Cre mice were cultured with ethanol EtOH or 4-hydroxytamoxifen (4-OHT) for 4 days. The frequency of dead cells after deletion of c-FLIP was quantified by staining with trypan blue. Cells were imaged in complete RPMI using a Zeiss Axovert 200 (20×/0.30 NA objective lens). Images were obtained using an AxioCam MRC camera and AxioVision Rel. 4.8 software. Black bars represent c-FLIP^f/f ER-Cre cells treated with EtOH and white bars represent c-FLIP^f/f ER-Cre cells treated with 4-OHT. (Triplicate values from a single experiment; **P < .01) (C) BMDM from c-FLIP^f/f or c-FLIP^f/f ER-Cre mice were cultured with EtOH or 4-OHT for 4 days. The number of live cells remaining after deletion of c-FLIP was quantified by trypan blue exclusion. Black bars represent c-FLIP^f/f cells and white bars represent c-FLIP^f/f ER-Cre cells. Triplicate values from a single experiment; ***P < .001. (D) Relative expression levels of c-FLIP_S and c-FLIP_L mRNA in surviving c-FLIP^f/f ER-Cre BMDM after treatment with 4-OHT. Expression levels were quantified by real-time PCR using Cyclophilin A as an internal control. Black bars represent c-FLIP^f/f ER-Cre cells treated with EtOH and white bars represent c-FLIP^f/f ER-Cre cells treated with 4-OHT. Triplicate values from a single experiment (**P < .01; ***P < .001; error bars represent standard deviations; NS, not significant).

Figure 2

Loss of macrophages in c-FLIP^f/f LysM-Cre mice. (A) Representative c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice show the decreased size observed in c-FLIP^f/f LysM-Cre mice. (B) Body weight of c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice at 6-8 weeks of age. Black bars represent c-FLIP^f/f mice and white bars represent c-FLIP^f/f LysM-Cre mice. Data were obtained in 4 independent experiments (n = 7-9; ***P < .001). (C) Wild-type, floxed, and deleted c-FLIP alleles in BMDM from c-FLIP^f/+, c-FLIP^f/f, c-FLIP^f/+ LysM-Cre, or c-FLIP^f/f LysM-Cre mice as detected by Southern blot. Film was scanned with a Canon CanoScan n122ou scanner. (D) Cytospins of Day 3 thioglycollate-elicited PEC samples from c-FLIP^f/f (left) and c-FLIP^f/f LysM-Cre mice (right). Original magnification 200×. Images were obtained using a Zeiss Axiovert 200 (20×/0.20 NA objective lens) with an AxioCam MRC camera and AxioVision Rel. 4.8 software. (E) Quantification of macrophages, neutrophils, and monocytes in Day 3 thioglycollate-elicited PEC. Data were obtained in 4 independent experiments (n = 5-6; NS, not significant; ***P < .001). (F) Absolute numbers of CD4⁺ or CD8⁺ T cells, B220⁺ B cells, CD11b⁺Ly6C^int neutrophils, SSC^loCD11b⁺Ly6C^hi inflammatory monocytes, and SSC^lo CD11b⁺ Ly6C^lo resident monocytes in peripheral blood as determined by flow cytometry. Data were obtained in 7 independent experiments. n = 8-10; NS, not significant; P < .05; **P < .01; ***P< .001. Error bars represent standard deviations.

Figure 3

Bone marrow stromal macrophages are lost and hematopoiesis is altered in c-FLIP^f/f LysM-Cre mice. (A) Total bone marrow cellularity and absolute numbers of F4/80⁺, F4/80⁺ CD11b^hi, and F4/80⁺ CD11b^lo bone marrow macrophage populations as determined by flow cytometry. Data were obtained in 6 independent experiments (n = 7-11; NS, not significant; *P < .05; **P < .01; ***P < .001). (B) Absolute numbers of B220⁺ B cells and CD11b⁺ Gr1⁺ neutrophils in bone marrow of c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice as determined by flow cytometry. Data were obtained in 3 independent experiments (n = 4-7; ***P < .001). (C) Representative bone marrow cytospins from c-FLIP^f/f (left) and c-FLIP^f/f LysM-Cre (right) mice showing immature (black arrowheads) and mature neutrophils (red arrowheads), lymphocytes (green arrowheads), monocytes (orange arrowhead), and macrophages (blue arrowhead). Original magnification 200×. Images were obtained using a Zeiss Axiovert 200 (20×/0.30 NA objective lens) with an AxioCam MRC camera and AxioVision Rel 4.8 software. (D-F) Absolute numbers of progenitor cells in bone marrow as determined by flow cytometry. Data were obtained in 2 independent experiments. n = 4; NS, not significant; **P < .01; ***P < .001. (D) Absolute numbers of Lin⁻ (CD3ϵ⁻CD4⁻CD8⁻CD11b⁻B220⁻) cells. (E) Absolute numbers of LSK (Lin⁻Sca-1⁺c-Kit⁺) cells, CMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^lo), and GMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^hi) populations. (F) Absolute numbers of MEP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁻ FcγR^lo) cells. In all panels, black bars represent c-FLIP^f/f mice and white bars represent c-FLIP^f/f LysM-Cre mice. *P < .05; **P< .01; ***P < .001. All error bars represent standard deviations.

Figure 4

c-FLIP^f/f LysM-Cre mice lack marginal zone macrophages and have splenomegaly and splenic extramedullary hematopoiesis. (A) Spleen weight (left) and cellularity of spleens lysed of red blood cells (right) in c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice. Black boxes represent c-FLIP^f/f mice and white boxes represent c-FLIP^f/f LysM-Cre mice. Data were obtained in 5 independent experiments (spleen weights) or 7 independent experiments (splenic cellularity). **P < .01; ***P < .001. (B) Loss of CD115⁺ ER-TR9⁺ marginal zone macrophages but not MOMA-1⁺ metallophilic macrophages as observed by immunofluorescent staining. Frozen spleen sections from c-FLIP^f/f (left) and c-FLIP^f/f LysM-Cre (right) mice were stained for CD115 (red) and B220 (green; top), or MOMA-1 (blue) and ER-TR9 (green; bottom). Original magnifications 100× (top), 200× (bottom). Images were obtained using a Zeiss Axiovert 200M (10×/0.30 NA objective lens, top; 20×/0.75 NA objective lens, bottom) with an AxioCam MRm camera and AxioVision Rel. 4.8 software. (C) Images of paraffin-embedded spleen sections from c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice stained with H&E. Original magnification 100×. Inset, image showing megakaryocytes in the spleen of a c-FLIP^f/f LysM-Cre mouse. Original magnification 400×. Images were obtained using a Zeiss Axiovert 200 (10×/0.25 NA objective lens or 40×/0.50 NA objective lens, inset) with an AxioCam MRC camera and AxioVision Rel. 4.8 software. (D) Absolute numbers of CD4⁺ or CD8⁺ T cells, B220⁺ B cells, CD11b⁺ Ly6C^int neutrophils, and total SSC^lo cells (left) or SSC^lo CD11b⁺ Ly6C^hi inflammatory monocytes and SSC^lo CD11b⁺ Ly6C^lo resident monocytes (right) in c-FLIP^f/f and c-FLIP^f/f LysM-Cre spleens as determined by flow cytometry. Data were obtained in 4-7 independent experiments (T cells, 5 experiments; B cells, 4 experiments; neutrophils and monocytes, 6 experiments, and SSC^lo cells, 7 experiments). n = 7-14; NS, not significant; ***P < .001. (E-G) Absolute numbers of progenitor cells in spleen as determined by flow cytometry. Data were obtained in 2 independent experiments. n = 4; *P < .05; **P < .01. (E) Absolute numbers of Lin⁻ (CD3ϵ⁻CD4⁻CD8⁻CD11b⁻B220⁻) cells. (F) Absolute numbers of LSK (Lin⁻Sca-1⁺c-Kit⁺) cells, CMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^lo), and GMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^hi) populations. (G) Absolute numbers of MEP (Lin⁻ c-Kit⁺ Sca-1⁻ CD34⁻ FcγR^lo) cells. In all panels, black bars represent c-FLIP^f/f mice and white bars represent c-FLIP^f/f LysM-Cre mice. *P < .05; **P < .01; ***P < .001. Error bars represent standard deviations.

Figure 5

Neutrophilia in c-FLIP^f/f LysM-Cre mice is G-CSF–dependent. (A) Expression of G-CSF, IL-1β, IL-17, and MIP-1α in serum of 4-week-old c-FLIP^f/f (black bars) or c-FLIP^f/f LysM-Cre (white bars) mice as determined by multiplex cytokine assay. Data presented were obtained in a single experiment, and are representative of 2 independent experiments. n = 3; NS, not significant; **P < .01; ***P < .001. (B-D) In vivo inhibition of G-CSF and IL-1 signaling. (B) Absolute numbers of circulating neutrophils and monocytes in c-FLIP^f/f (black bars) or c-FLIP^f/f LysM-Cre (white bars) mice before and after combination treatment with anti-G-CSF antibody and IL-1Ra. (C-D) Absolute numbers of bone marrow B cells, neutrophils, and progenitor cells (C), or splenic neutrophils, inflammatory monocytes, and progenitor cells (D) in c-FLIP^f/f (black bars) or c-FLIP^f/f LysM-Cre (white bars) mice after 15 days of combination treatment with anti-G-CSF antibody and IL-1Ra. Data were obtained in 2 independent experiments. (n = 4-7; NS, not significant; *P < .05. (E-F) Frequency of neutrophils in peripheral blood of c-FLIP^f/f (black bars) or c-FLIP^f/f LysM-Cre (white bars) mice treated daily beginning at 4 weeks of age with neutralizing anti–G-CSF antibody (E) or IL-1Ra (F). Data were obtained in a single experiment (n = 5 (E) or n = 3-5 (F); NS, not significant; *P < .05; ***P < .001). (G) Body weight of c-FLIP^f/f (black bars) or c-FLIP^f/f LysM-Cre (white bars) mice treated daily beginning at 4 weeks of age with IL-1Ra. Data were obtained in a single experiment. n = 3-5; NS, not significant; *P < .05; **P < .01; ***P < .001. Error bars represent standard deviations.

Figure 6

Neutrophil clearance is delayed in c-FLIP^f/f LysM-Cre mice. (A) Kinetics of neutrophil production and clearance in c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice. Mice were injected intaperitoneal with BrdU. Blood samples were taken at time points from 24-192 hours postinjection, and the absolute number of BrdU⁺ neutrophils was quantified by flow cytometry (top). Bottom, absolute number of BrdU⁺ neutrophils normalized to the maximum number of BrdU⁺ neutrophils observed in each mouse strain. Data were obtained in 3 independent experiments. Triplicate values from a single experiment (n = 3-6). (B) Accumulation of annexin V⁺ 7-AAD⁺ neutrophils in the spleens of c-FLIP^f/f LysM-Cre mice. Data were obtained in 2 independent experiments (n = 6-7). (C) Levels of lactate dehydrogenase in plasma of c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice. (n = 6; ** P < .01) In all panels, black bars or boxes represent c-FLIP^f/f mice and white bars or boxes represent c-FLIP^f/f LysM-Cre mice. Error bars represent standard deviations.

Figure 7

Neutrophilia, splenomegaly, decreased body size, and increased G-CSF production are secondary to the loss of macrophages in c-FLIP^f/f LysM-Cre mice. (A) c-FLIP^f/f recipient mice were lethally irradiated before transfer of congenically marked bone marrow. Transferred bone marrow was either c-FLIP^f/f, c-FLIP^f/f LysM-Cre, or a 1:1 mixture of c-FLIP^f/f and c-FLIP^f/f LysM-Cre. (B-G) Posttransfer analysis of bone marrow chimeric mice. Wild-type (WT) chimeric mice are represented by black boxes, knock-out (KO) chimeric mice are represented by white boxes, and mixed chimeric mice are represented by gray boxes. In mixed chimeric mice, cells of WT donor origin are represented by black boxes and cells of KO donor origin are represented by white boxes. Data in B-F were obtained in a single experiment (NS, not significant; *P < .05; **P < .01; ***P < .001). (B) Absolute numbers of thioglycollate-elicited PEC macrophages. Recipient mice were injected intaperitoneally with thioglycollate. PEC samples were harvested 3 days after injection, and differential counts were performed on cytospins. (C) Absolute numbers of neutrophils in peripheral blood of recipient mice as determined by flow cytometry. (D) Absolute numbers of inflammatory monocytes in peripheral blood of recipient mice as determined by flow cytometry. (E) Body weight of recipient mice 17 weeks after transfer. (F) Spleen weight of recipient mice 17 weeks after transfer. (G) Cytokine levels in plasma of recipient mice 2 weeks after transfer. Data were obtained in a single experiment. (n = 4-5). *P < .05; **P < .01; ***P < .001. All error bars represent standard deviations.