Conditional Macrophage Ablation Demonstrates That Resident Macrophages Initiate Acute Peritoneal Inflammation
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免疫学杂志 2005年第4期
Abstract
The role played by resident macrophages (M) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional M ablation strategy to determine the role of resident peritoneal M in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional M ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive M populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident M ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic M. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal M in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.
Introduction
Macrophages (M)7 are dispersed throughout the tissues and have an important role in innate immunity (1), apoptotic cell clearance (2), development (3), and morphogenesis (4, 5). Resident tissue M and dendritic cells are regarded as sentinels of the innate immune system. Various strategies, such as M depletion induced by administration of liposomal clodronate (6), have been used to examine M function in vivo. However, previous work using this method to study the role of resident peritoneal M in experimental peritonitis has produced conflicting results because they have suggested a key role (7), an inhibitory role (8), or no role at all (8). In the current study, we have used a conditional ablation strategy to clarify the role of resident M in the sensing of peritoneal injury and the regulation of neutrophil (PMN) infiltration.
A conditional M ablation strategy has advantages over the available naturally occurring and induced M-deficient mutant mice because the timing of M elimination can be chosen. Despite their limitations, nonconditional M-deficient mice have proven valuable for analysis. For example, the Csfmop/Csfmop (osteopetrosis) mouse is a naturally occurring mutant of the CSF-1 gene and exhibits a M deficiency at a level that permits viability in homozygotes (9). These mice have provided insight into M function during development (3). In addition, mice targeted at the PU.1 locus exhibit multiple defects in development of hemopoietic lineages including a complete absence of tissue M (10, 11). Although PU.1 mutation results in perinatal lethality, these mice have been used to demonstrate that mesenchymal cells are able to clear apoptotic cells during embryonic regression of interdigital tissues (12).
Previous strategies used to eliminate specific cell types in a living organism include the generation of transgenic lines that express diphtheria toxin A-chain (13, 14) or the ricin polypeptide (15). However, even low levels of unanticipated transgene expression can give unpredictable consequences (13). The alternative ablation strategy of killing thymidine kinase-expressing cells with gancyclovir (16, 17) only permits the elimination of proliferating cells. More recently, conditional M ablation has been achieved using transgenic expression of Fas under the control of the c-fms promoter coupled with drug-inducible Fas dimerization to induce cell death (18). Identification of the human receptor for diphtheria toxin (DT) (also known as heparin-binding epidermal growth factor (hbEGF) (19)) created an opportunity for a unique ablation strategy. The murine form of hbEGF binds DT poorly, but mouse cells can be rendered sensitive through transgenic expression of human hbEGF. In transgenic mice expressing human hbEGF lineage specifically, cell ablation results following toxin injection. In addition, DT is a protein synthesis inhibitor and kills both mitotic and terminally differentiated cells. This strategy has recently been used to generate transgenic mice in which hepatocytes (20) or dendritic cells (21) may be conditionally ablated. In the current report, we describe how we have used this strategy to generate a conditional M ablation mouse to assess the role of resident peritoneal M in the initiation of acute inflammation in experimental sterile peritonitis induced by Brewer’s thioglycolate (BTG). We demonstrate that resident peritoneal M are essential for PMN recruitment through M-dependent CXC chemokine production.
Materials and Methods
Transgenic construct
The CD11b promoter from coordinates –1704 to + 83 (22) was used to drive expression of the human hbEGF cDNA (19). Splicing and polyadenylation signals were provided by a region of the human growth hormone gene that had previously worked effectively with the CD11b promoter (22). The fusion protein between hbEGF and GFP was generated by continuing the open reading frame from the final residue of hbEGF with the first residue of GFP.
Transgenic mice
The CD11b-DTR construct was used to generate transgenic mice on the FVB/N background (23) using conventional techniques; transgene expression was detected using an RT-PCR assay. The primer sequences used were 5'-AAGATCCGCCACAACATCG for the forward primer and 5'-GCAGCTCTAGGTTGGATTTCTG for the reverse primer. Because the reverse primer sequence was taken from the base pairs flanking intron III of hGH, no PCR product could be amplified from genomic DNA.
Flow cytometry analysis
Bone marrow-derived M were prepared as previously described (4). Resident peritoneal cells were isolated by peritoneal lavage. Elicited peritoneal M were lavaged from the peritoneal cavity 4 days after i.p. injection of 1 ml of 3% BTG (Difco). For flow cytometric analysis, 2 x 106 cells were incubated for 10 min in FACS buffer containing rat anti-mouse FcR blocker CD16/CD32 (FcRIII/II). FITC- or PE-conjugated Abs at concentrations suggested by the supplier were then added, and the mixture was incubated at 4°C for 45–60 min. The cells were then washed and fixed in 2% formaldehyde before undergoing flow cytometry analysis. A fixed volume of whole blood was obtained by tail vein bleed into 3.9% sodium citrate. Blood was aliquoted into flow cytometry tubes, blocked, and stained as for peritoneal lavage cells. One milliliter of FACSLysis buffer (BD Biosciences) was then added to lyse erythrocytes, and samples were spun and processed by flow cytometry. The Abs used were anti-CD11b FITC conjugate (eBiosciences), anti-GR1 PE (eBiosciences), anti-B220 (mouse CD45R) FITC and PE conjugates (BD Pharmingen), mouse anti-CD3 PE conjugates (BD Pharmingen), and F4/80 allophycocyanin, FITC, and PE conjugates (Caltag and Serotec). Annexin VFITC conjugate (US Biological) and propidium iodide (PI; Sigma-Aldrich) were used to determine the levels of apoptosis and necrosis. A known amount of fluorescent Flow-Check fluorospheres (Beckman Coulter) was added to peritoneal lavage samples before analysis, and the ratio of cells to beads was used to calculate the absolute number of any cell type in peritoneal lavage fluid. Analyses were performed using a FACScan or FACSCalibur instrument.
Immunolabeling of tissues
Organs were fixed in 4% formaldehyde in PBS at 4°C overnight, and paraffin-embedded tissue sections were prepared according to conventional methods. Rehydrated sections were immersed in acetone for 10 min and rinsed in PBS, and a limited trypsin (Sigma-Aldrich) digestion was performed for 20 min. Sections were then washed in PBS, and endogenous peroxidase activity was quenched with a 30-min incubation in 0.3% H2O2 in methanol. Sections were then incubated with F4/80 mAb (Caltag Laboratories) at a 1/100 dilution. Further steps were performed according to the recommendations of the manufacturer of the VECTASTAIN Elite ABC (avidin/biotin complex) system used in the labeling (Vector Laboratories). Sections were then washed in water, lightly counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
Experimental peritonitis
Peritonitis was induced by i.p. injection of 1 ml of 3% BTG. Resident M ablation was induced in transgenic mice by i.p. injection of DT (25 ng/g body weight) 24 h before the administration of BTG, with DT-treated FVB/N wild-type (WT) mice serving as control. Mice underwent peritoneal lavage at various time points following BTG injection. In some experiments, peritonitis was induced by the injection of 0.2 mg of zymosan (Sigma-Aldrich) with peritoneal lavage being performed at 8 h. All experiments were performed in accordance with institutional and U.K. Home Office guidelines.
Adoptive transfer of peritoneal cells
Peritoneal lavage samples from groups of FVB/N WT mice were pooled, spun, and either resuspended in 1 ml of sterile PBS (M-rich peritoneal cells) or plated on tissue culture plastic for 2 h to deplete M by adhesion (M-depleted peritoneal cells). A total of 97 ± 2.8% of M was removed from the cell suspension. Four hours before induction of BTG peritonitis, M-rich or M-depleted peritoneal cells suspended in 1 ml of PBS were injected i.p. Experimental groups consisted of 1) CD11b-DTR transgenic mice depleted of resident peritoneal M by prior administration of DT, 2) DT-treated FVB/N WT mice, 3) M-depleted mice reconstituted with M-rich peritoneal cells, and 4) M-depleted mice reconstituted with M-depleted peritoneal cells. Groups 1 and 2 were injected with 1 ml of PBS 4 h before BTG administration as an injection control for the adoptive cell transfer procedure. Animals were sacrificed 8 h following the initiation of peritonitis.
Chemokine studies
Mice underwent peritoneal lavage at 1 and 3 h following the i.p. injection of BTG. Lavage fluid was centrifuged, aliquoted, and stored at –80°C until analyzed by ELISA for MIP-2 and keratinocyte-derived chemokine (KC) (R&D Systems). Chemokine production by peritoneal cell populations that had been depleted of either M or mast cells (MC) was also determined in vitro. Peritoneal cells were incubated with PE-conjugated anti-F4/80 Ab or an Ab to the MC marker c-kit (CD117) to stain M or MC, respectively. Peritoneal cells were then incubated with anti-PE-conjugated MACS magnetic beads, and M or MC were removed by passing the cells over a magnetic MACS column (Miltenyi Biotec). As a control, total peritoneal cells were incubated with an isotype control Ab followed by magnetic beads and subsequently passed over the magnetic MACS column. This method resulted in >97% depletion of M or MC. Control peritoneal cells and M- or MC-depleted peritoneal cells were then plated in 48-well plates (5 x 105 cells per well) and exposed to 1% BTG for 3 h. Peritoneal cell-conditioned supernatants were harvested, spun, and stored at –80°C until analyzed by specific ELISA for MIP-2 and KC (R&D Systems).
Statistical analysis
The Student t test with a tailed distribution or ANOVA was used to analyze data. A value of p < 0.05 was deemed statistically significant. Data are presented as mean ± SE.
Results
Generation of transgenic mice
Conditional ablation transgenic mice were generated using an established strategy (21) and a construct (designated CD11b-DTR) that used the CD11b promoter (22) to provide M expression specificity. CD11b-DTR expresses the DT receptor (alternatively named hbEGF (19)) (Fig. 1A) as a GFP fusion protein. The hbEGF-GFP construct conferred sensitivity to DT in transiently transfected murine cells indicating that it was functional (data not shown).
FIGURE 1. Structure and expression of the CD11b-DTR transgene. A, Schematic of the CD11b-DTR construct. The transcription start is indicated by the right-facing arrow and exons by shaded boxes. The DTR-eGFP fusion cDNA is inserted between the human CD11b promoter and the human growth hormone (hGH) sequences that provides splicing and polyadenylation signals. Oligonucleotides eGFP and hGH used for RT-PCR transcript detection are indicated by small arrows. B–E, RT-PCR expression analysis performed on BTG-elicited peritoneal cells (B), spleen cells (C), and bone-marrow derived M (D and E). Transgene mRNA amplification products were not evident in samples from WT mice or when reverse transcriptase was omitted but was detected in line 34 peritoneal and spleen cells (B and C). Line 34 bone-marrow derived M also exhibited normal expression of the M marker F4/80 (D) and persistent transgene expression (E).
Six transgenic lines were produced with the CD11b-DTR construct. Although the fluorescence signal from the hbEGF-GFP fusion protein was insufficient to permit detection of transgene expression by FACS analysis, a RT-PCR assay indicated that four lines exhibited detectable transgene expression in peritoneal cells and spleen with line 34 exhibiting high expression (Fig. 1, B and C). WT and CD11b-DTR-34 bone marrow-derived M expressed the M-specific gene F4/80 as expected (Fig. 1D), indicating that expression of the DT receptor was unlikely to have disrupted normal M differentiation. Transgene expression was also observed in day 4 and 8 CD11b-DTR-34 bone marrow-derived M (Fig. 1E). Because line CD11b-DTR line 34 showed the highest levels of transgene expression, further analysis was restricted to this line.
Transgenic M are killed by DT in vitro and in vivo
Treatment of BTG-elicited peritoneal M from CD11b-DTR-34 mice with concentrations of DT between 1 ng/ml to 1 mg/ml over a period of 48 h induced cell death at concentrations as low as 25 ng/ml. In contrast, M from WT mice or other transgenic lines were resistant (data not shown). We then asked whether i.p. injection of DT ablated resident peritoneal M in vivo. DT was injected at 25 ng/g mouse weight, and FACS analysis of peritoneal cells was performed 24 h later. Normal numbers of resident peritoneal M (F4/80 positive, CD11b positive, and Ly6C/G negative) were evident in WT mice receiving DT (Fig. 2A) as well as CD11b-DTR-34 transgenic mice injected with either PBS (Fig. 2B) or the inactive form of the toxin DTmut (Fig. 2C). However, CD11b-DTR-34 transgenic mice showed an almost complete absence of F4/80-positive peritoneal M after a single dose of DT (Fig. 2D). Administration of DT doses of 6.25 and 12.5 ng/g mouse weight resulted in M ablation of 72 and 82%, respectively, whereas lower doses resulted in <50% ablation. We therefore chose to use a dose of 25 ng/g body weight for the in vivo studies.
FIGURE 2. Flow cytometric analysis of peritoneal cell M killing by DT. A–D, Cells were removed from the peritoneal cavity by peritoneal lavage and labeled with PE-conjugated F4/80 Ab, and flow cytometric analysis was performed. A, WT mice injected with DT (25 ng/g body weight) show a normal percentage of peritoneal M. B and C, CD11b-DTR-34 mice receiving either PBS (B) or DTmut (C) have normal F4/80 profiles. D, In contrast, CD11b-DTR-34 mice treated with active DT show complete absence of F4/80-positive cells. E, In WT mice, injection of DT at 25 ng/g mouse weight does not affect either the small population of CD3+ T cells (upper-left quadrant) or the larger population of F4/80+ M (lower-right quadrant) in the peritoneal cavity. F, CD11b-DTR-34 mice receiving DT exhibit elimination of the F4/80+ population, whereas the CD3+ cells remain unaffected.
Time course of M elimination in vivo
We then examined the kinetics of M ablation in the peritoneal cavity following administration of a single dose of DT (25 ng/g body weight). The appearance of apoptotic and necrotic cells was monitored using Annexin VFITC and PI staining, respectively. Peritoneal lavages and flow cytometric analyses were conducted on a series of mice 4, 6, 8, and 12 h after DT injection (Fig. 3). After 6 h, 65% of the peritoneal population was annexin V positive, indicating a dramatic increase in early-stage apoptotic cells (Fig. 3A). The maximal numbers of PI-positive cells occurred 2 h later at 8 h after DT injection and represented 20% of the total peritoneal cells (Fig. 3B). The number of F4/80-positive cells was nearly zero at 12 h (Fig. 3C), and this corresponded to very low levels of PI-positive and annexin V-positive cells (A and B). These data suggest that DT induces sensitive cells to undergo apoptosis and that some of these dying cells then undergo secondary necrosis. All F4/80-positive cells were cleared by 12 h.
Restoration of the peritoneal M population occurs by day 4 following a single i.p. dose of DT (3.94 x 105 ± 1.7 x 105 M vs 5.8 x 105 ± 1.3 x 105; day 4 following DT treatment vs day 1 following PBS treatment; n = 5 per group, p > 0.05).
FIGURE 3. Time course of peritoneal M depletion. Cells were removed from the peritoneal cavity by peritoneal lavage. Mice were either injected with a single dose of DT (gray lines) or DTmut (black lines) at 25 ng/g mouse weight i.p., and the resident peritoneal population was assessed for the appearance of annexin V-positive cells (A), for labeling of cells with PI (B), and for presence of the M marker F4/80 (C). Flow cytometric analysis identified the labeled proportion of total cells over a 12-h time course following DT injection.
Specificity of M elimination in vivo
To test the specificity of M elimination, we examined CD3+ T cells in spleen and peritoneal cavity and B220+ B cells in the spleen. In this case, we injected two doses of DT at 25 ng/g at 48-h intervals and assessed ablation 24 h later. The F4/80-positive peritoneal population was unaffected in WT mice (Fig. 2E, lower-right quadrant), but was eliminated in CD11b-DTR-34 animals (G, lower-right quadrant). Despite complete peritoneal M elimination in CD11b-DTR-34 animals, the peritoneal CD3+ T cells were present in both WT and transgenic animals injected with DT (Fig. 2, E and F, upper-left quadrants). The relative increase in CD3-positive and double-negative cells in the DT-treated CD11b-DTR-34 animals is due to the plotting of equal numbers of detection events in the FACS analyses. B220+ and CD3+ populations in spleen were unaffected in either WT or CD11b-DTR-34 mice receiving two doses of DT (data not shown).
Because CD11b is expressed on both granulocytes and M, we asked whether both of these cell types were sensitive to DT. CD11b-DTR-34 mice were injected with BTG. DT (25 ng/g body weight) or PBS was injected 8 h after initiation of peritonitis with peritoneal lavage being performed 12 h later. Despite 90% M ablation (0.5 x 106 ± 0.15 x 106 vs 4.8 x 106 ± 0.23 x 106; DT injection vs PBS; p < 0.005), there was no difference in PMN number (2.3 x 106 ± 0.22 x 106 vs 1.6 x 106 ± 0.39 x 106; DT injection vs PBS; p > 0.05). In addition, flow cytometric analysis of whole blood performed 24 h following DT administration indicated that circulating PMN numbers were unaffected by DT administration (1.02 x 106 ± 0.18 x 106 PMNs/ml whole blood vs 0.97 x 106 ± 0.22 x 106; DT injection vs PBS; p > 0.05). In contrast, DT administration induced significant depletion of circulating monocytes (0.117 x 106 ± 0.059 x 106 monocytes/ml whole blood vs 0.52 x 106 ± 0.073 x 106; DT injection vs PBS; p < 0.05).
Differential deletion of M populations
We also asked whether DT injection could eliminate M in distant organs. Two doses of DT (25 ng/g) were administered IP at 48-h intervals, and the presence of F4/80-positive M in kidney, liver, and lung was analyzed 24 h later and in the ovary 16 h later (Fig. 4). The ovary was examined at 16 h, because there was evidence of some patchy ovarian necrosis present at 20 h. WT mice injected with DT and CD11b-DTR-34 homozygote mice injected with DTmut were unaffected (Fig. 4, top two rows). Both kidney and ovary of CD11b-DTR-34 homozygote mice injected with DT exhibited an absence of F4/80+ cells. In the kidney, mesangial and interstitial M were ablated in the absence of overt renal injury. However, hepatic sinusoidal M and alveolar M were unaffected, indicating that not all populations of tissue M were susceptible. However, the rapid elimination of M populations in the peritoneal cavity and kidney while leaving other cell populations intact establishes the basic validity of this approach to conditional cell ablation.
FIGURE 4. Effect of DT on tissue M populations. Micrographs showing the effect of DT treatment of WT mice (top row), DTmut treatment of CD11b-DTR mice (middle row), and DT treatment of CD11b-DTR mice (bottom row) upon the presence of F4/80-positive M in liver (left column), lung (center left column), kidney (center right column), and ovary (right column). Animals received two doses of DT or DTmut delivered at 48-h intervals. Liver, lung, and kidney were assessed 24 h after the second DT injection, whereas ovary was assessed at 16 h.
Resident M ablation reduces PMN influx and CXC chemokine responses during experimental peritonitis
We used the conditional ablation strategy to investigate the role of resident tissue peritoneal M in sensing perturbation of the microenvironment and subsequent initiation of acute peritoneal inflammation and PMN recruitment in experimental peritonitis. Resident M ablation markedly attenuated PMN infiltration following the administration of 3% BTG (Fig. 5A). We also performed M repletion studies with either M-rich or M-depleted peritoneal cells derived from WT mice. Reconstitution of DT-treated CD11b-DTR-34 homozygote mice with M-rich peritoneal cells 4 h before BTG treatment resulted in complete restoration of peak PMN infiltration at 8 h. In contrast, the administration of M-depleted peritoneal cells was ineffective (Fig. 5B). Previous work suggested that the nature of the inflammatory stimulus may determine the involvement of M in experimental peritonitis (8), and we therefore performed M depletion studies in zymosan peritonitis. We also found that depletion of resident peritoneal M resulted in a significant reduction in PMN infiltration 8 h following the induction of zymosan peritonitis (2.6 x 106 ± 8.8 x 105 PMNs vs 5.4 x 106 ± 3.7 x 105; DTR plus DT vs FVB/N WT controls plus DT; n = 6 per group; p < 0.05).
FIGURE 5. Resident M ablation attenuates peritoneal inflammation. WT and CD11b-DTR mice were injected i.p. with DT (25 ng/g body weight). One milliliter of 3% BTG was injected i.p. 24 h later with mice undergoing peritoneal lavage at various time points. A, Peritoneal cells were stained for the PMN marker GR1. Resident peritoneal M ablation induced a marked blunting of PMN infiltration of the peritoneal cavity. B, WT and three groups of CD11b-DTR mice were injected with DT (25 ng/g body weight). Four hours before i.p. injection of 3% BTG, two groups of M-depleted CD11b-DTR mice were reconstituted with either M-rich peritoneal cells (MR) or M-depleted peritoneal cells (MD). Administration of PBS served as control to the remaining groups. Mice underwent peritoneal lavage 8 h following administration of 3% BTG, and peritoneal cells were stained for GR1. *, p < 0.05.
In this model, we found peak levels of the PMN CXC chemokines MIP-2 and KC at the 1-h time point. Resident peritoneal M ablation before the initiation of BTG peritonitis markedly reduced the elevation in MIP-2 levels (148.5 ± 34.8 vs 1762.1 ± 153.5 pg/ml; M-depleted mice vs nondepleted mice: p < 0.00001). There was a slight, albeit statistically significant, difference in the much lower levels of MIP-2 between DT-treated and control mice at 3 h (204 ± 54 vs 74 ± 8 pg/ml; M-depleted mice vs nondepleted mice; p < 0.05). This suggests that the production of MIP-2 in vivo is predominantly M dependent. In addition, a 50% reduction in the level of KC was evident in M-depleted mice at the 1-h time point (1408.2 ± 322.5 vs 2467.5 ± 264.9 pg/ml; M-depleted mice vs nondepleted mice; p < 0.05). Interestingly, the levels of KC levels at 3 h are higher in M-depleted mice compared with control mice (1477 ± 400 vs 74 ± 8 pg/ml; M-depleted mice vs nondepleted mice; p < 0.01), thereby suggesting a source of KC other than resident M.
CXC chemokine responses are M dependent in vitro
Previous studies of peritoneal and dermal inflammation have implicated the MC as playing an important role in the initiation of PMN infiltration (8, 24). We therefore performed in vitro studies to determine the production of PMN chemokines by BTG-stimulated peritoneal cell populations that had been depleted of M or MC. Control peritoneal cells produced significant levels of MIP-2 and KC, which was not affected by MC depletion (Fig. 6). However, chemokine levels were dramatically reduced following the depletion of M, thereby indicating that chemokine production was completely M dependent with no involvement of MC (Fig. 6).
FIGURE 6. CXC chemokine production in response to BTG stimulation is M dependent and MC independent in vitro. Peritoneal cells were depleted of either M or MC by incubation with PE-conjugated anti-F4/80 or anti-c-kit (CD117) followed by incubation with anti-PE-conjugated magnetic beads and passage over a magnetic column (>97% depletion of M or MC). Incubation of total peritoneal cells with an isotype control Ab followed by magnetic beads and passage over the magnetic column served as control. Cells were then plated in 48-well plates (5 x 105 cells per well) and exposed to 1% BTG for 3 h. Peritoneal cell-conditioned supernatants were harvested, spun, and analyzed by specific ELISA for MIP-2 and KC. *, p < 0.005.
Discussion
Previous analyses (20, 21) and the experiments we describe here show that expression of human hbEGF (19) in mouse cells can confer sensitivity to DT in vivo, and that, as a consequence, injection of DT will kill cells that express hbEGF. Our data indicate that M populations in the peritoneal cavity and kidney can be rapidly killed or eliminated while leaving other cell populations intact, and this establishes the basic validity of this approach to conditional cell ablation. We noted that hepatic and alveolar M populations were unaffected, and it may be the case that a higher dose of DT may have ablated these cells. However, we found that mice could become unwell with doses of DT >25 ng/g body weight, and we therefore did not use doses >25 ng/g body weight in this study. It is pertinent that, despite PMN expression of CD11b, the administration of DT did not induce the death of recruited or circulating PMNs, indicating that PMNs are insensitive to DT, potentially as a result of their lower level of protein synthesis.
We used the conditional ablation strategy to investigate the role of resident peritoneal M in the initiation of acute peritoneal inflammation following the administration of BTG. Previous work has indicated that leukotrienes derived from resident peritoneal M are involved in the development of early vascular permeability in sterile peritonitis (25). Although early work in rat models of peritonitis implicated the resident peritoneal M in the orchestration of PMN recruitment (26, 27, 28), more recent studies have produced conflicting results (7, 8). Indeed, studies by Ajuebor et al. (8) suggest that resident M depletion inhibits PMN influx in LPS-induced inflammation, has no effect in BTG peritonitis, and augments PMN influx in zymosan peritonitis. In the latter model, it is proposed that M-derived IL-10 inhibits PMN recruitment. Conversely, work by Knudsen et al. (7) using clodronate-induced depletion of peritoneal M in a rat model of sterile peritonitis demonstrated that PMN infiltration was M dependent.
In this study, administration of DT resulted in a dramatic 98% M ablation that markedly blunted PMN infiltration, thereby indicating a key role for the resident M in the orchestration of acute peritoneal inflammation in this experimental model. It is important to note that the reduced PMN infiltration in DT-treated mice was not attributable to a systemic neutropenia, because PMNs were not sensitive to DT and the number of circulating PMNs in DT-treated mice was comparable with that of PBS-treated mice at 24 h. The importance of the resident M was reinforced by experiments involving the adoptive transfer of nontransgenic peritoneal M following DT-mediated M ablation and before the initiation of peritonitis. The presence or absence of M in the transferred peritoneal cell population directly correlated with the restoration of the PMN influx, thereby suggesting that the M exerts a critical role in this process. In addition, we found that depletion of resident M also significantly reduced PMN infiltration in zymosan peritonitis, thereby suggesting that the sensing function of the resident M may be stimulus independent.
The magnitude of the M depletion may explain the apparent discrepancy between these results and the study by Ajuebor et al. (8). Administration of a single dose of DT induced 98% M ablation, whereas three doses of liposomal clodronate resulted in >85% M depletion in the study by Ajuebor et al. (8). M are a potent source of chemokines and cytokines, and it may be the case that, in certain circumstances, a relatively small population of residual M may exert significant biological effects. Although administration of liposomal clodronate may exert marked biological effects despite depletion of only 80% of M (29), it may be necessary to deplete almost all peritoneal M to delineate their roles as sentinel cells.
Although peritoneal M may produce myriad mediators capable of recruiting PMNs (30, 31), we examined the effect of M ablation upon the level of CXC chemokines in this model. Our data suggest that the initiation of PMN infiltration is mediated by resident peritoneal M-dependent production of chemokines previously documented to play a role in orchestrating PMN recruitment in BTG peritonitis (32, 33) and in other inflammatory situations (34, 35, 36). MC are also a rich source of proinflammatory and vasoactive mediators and have been documented to play an important role in PMN recruitment during inflammation of the peritoneum (8) as well as other sites such as the skin (24).
We found that resident M ablation markedly reduced the peak level of MIP-2 and significantly blunted the level of KC at 1 h. However, partial inhibition (50%) of KC production at 1 h and the persistent elevation of KC at 3 h in M-depleted mice suggest that KC may be produced by other cells within the peritoneum. The persistent elevation of KC also suggests that M may play a role in the negative regulation of KC production by non-M cells. In this context, it is pertinent that our in vitro data indicate that both KC and MIP-2 production by peritoneal cells obtained by peritoneal lavage is almost entirely dependent upon M, because peritoneal cells depleted of M produced minimal levels of chemokines. These findings suggest that peritoneal cells retrievable by peritoneal lavage are not the source of KC detected in our in vivo study. This interpretation of the data is consistent with recent work in a wound model (37) demonstrating MIP-2 expression by inflammatory cells and KC expression by resident tissue cells such as endothelial cells and fibroblasts. Peritoneal mesothelial cells undoubtedly participate in peritoneal inflammation and can produce chemokines and cytokines (38), and it may be the case that mesothelial cells contribute to the KC production evident in this study. Despite this, however, we found that PMN infiltration is still markedly blunted despite the persistent presence of KC at 3 h.
We also examined the potential interaction between M and MC by performing in vitro studies of peritoneal cells that had been depleted of M or MC before stimulation with BTG. Interestingly, depletion of MC had no significant effect upon the production of KC and MIP-2 following BTG stimulation, suggesting that chemokine production in this model was M dependent and MC independent.
In conclusion, this work has used a novel model of conditional M ablation to dissect the role of resident peritoneal M in the initiation of acute peritoneal inflammation. Our data indicate a key role for the resident M in sensing peritoneal irritation and orchestrating PMN infiltration in BTG and zymosan peritonitis. This proinflammatory function is predominantly mediated by production of the potent PMN CXC chemokine MIP-2 and, to a lesser extent, KC. Although previous work has implicated the involvement of other cells such as MC, our study suggests that resident M are critically important producers of PMN chemokines and act to orchestrate PMN recruitment in murine BTG peritonitis. We also anticipate that CD11b-DTR transgenic mice will be valuable for studying other M functions in vivo in a variety of different biological contexts. The option of being able to choose the time and, with local toxin injection, perhaps the locality of ablation offers a number of advantages over other cell ablation systems.
Acknowledgments
We thank Leon Eidels for providing the cDNA to human hbEGF, Daniel Tenen for the CD11b-based transgene construct, and Michael Clay for technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The Lang Laboratory is supported by grants from the National Institutes of Health (RO1 EY10559, EY11234, EY12370, and EY14102) and by funds from the Abrahamson Pediatric Eye Institute Endowment at Children’s Hospital Medical Center of Cincinnati. J.H. is in receipt of a Wellcome Trust Senior Research Fellowship in Clinical Science (Grant 061139). J.-F.C. is supported by the Canadian Institutes of Health Research. J.S. is supported by the Wellcome Trust (Program Grant 064487).
2 J.-F.C., M.P., S.V., S.W., J.H., and R.A.L. contributed equally to this manuscript.
3 Current address: Departments of Radiation Oncology and Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016.
4 Current address: Regeneron Pharmaceuticals, Tarrytown, NY 10591.
5 Current address: Molecular Pathogenesis Program, Skirball Institute for Biomolecular Medicine, 540 First Avenue, New York, NY 10016.
6 Address correspondence and reprint requests to Dr. Jeremy Hughes, Phagocyte Laboratory, Medical Research Council Center for Inflammation Research, University of Edinburgh, Teviot Place, Edinburgh, U.K., EH8 9AG. E-mail address: jeremy.hughes@ed.ac.uk
7 Abbreviations used in this paper: M, macrophage; PMN, neutrophil; DT, diphtheria toxin; hbEGF, heparin-binding epidermal growth factor; BTG, Brewer’s thioglycolate; WT, wild type; PI, propidium iodide; KC, keratinocyte-derived chemokine; MC, mast cell.
Received for publication July 14, 2004. Accepted for publication December 6, 2004.
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The role played by resident macrophages (M) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional M ablation strategy to determine the role of resident peritoneal M in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional M ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive M populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident M ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic M. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal M in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.
Introduction
Macrophages (M)7 are dispersed throughout the tissues and have an important role in innate immunity (1), apoptotic cell clearance (2), development (3), and morphogenesis (4, 5). Resident tissue M and dendritic cells are regarded as sentinels of the innate immune system. Various strategies, such as M depletion induced by administration of liposomal clodronate (6), have been used to examine M function in vivo. However, previous work using this method to study the role of resident peritoneal M in experimental peritonitis has produced conflicting results because they have suggested a key role (7), an inhibitory role (8), or no role at all (8). In the current study, we have used a conditional ablation strategy to clarify the role of resident M in the sensing of peritoneal injury and the regulation of neutrophil (PMN) infiltration.
A conditional M ablation strategy has advantages over the available naturally occurring and induced M-deficient mutant mice because the timing of M elimination can be chosen. Despite their limitations, nonconditional M-deficient mice have proven valuable for analysis. For example, the Csfmop/Csfmop (osteopetrosis) mouse is a naturally occurring mutant of the CSF-1 gene and exhibits a M deficiency at a level that permits viability in homozygotes (9). These mice have provided insight into M function during development (3). In addition, mice targeted at the PU.1 locus exhibit multiple defects in development of hemopoietic lineages including a complete absence of tissue M (10, 11). Although PU.1 mutation results in perinatal lethality, these mice have been used to demonstrate that mesenchymal cells are able to clear apoptotic cells during embryonic regression of interdigital tissues (12).
Previous strategies used to eliminate specific cell types in a living organism include the generation of transgenic lines that express diphtheria toxin A-chain (13, 14) or the ricin polypeptide (15). However, even low levels of unanticipated transgene expression can give unpredictable consequences (13). The alternative ablation strategy of killing thymidine kinase-expressing cells with gancyclovir (16, 17) only permits the elimination of proliferating cells. More recently, conditional M ablation has been achieved using transgenic expression of Fas under the control of the c-fms promoter coupled with drug-inducible Fas dimerization to induce cell death (18). Identification of the human receptor for diphtheria toxin (DT) (also known as heparin-binding epidermal growth factor (hbEGF) (19)) created an opportunity for a unique ablation strategy. The murine form of hbEGF binds DT poorly, but mouse cells can be rendered sensitive through transgenic expression of human hbEGF. In transgenic mice expressing human hbEGF lineage specifically, cell ablation results following toxin injection. In addition, DT is a protein synthesis inhibitor and kills both mitotic and terminally differentiated cells. This strategy has recently been used to generate transgenic mice in which hepatocytes (20) or dendritic cells (21) may be conditionally ablated. In the current report, we describe how we have used this strategy to generate a conditional M ablation mouse to assess the role of resident peritoneal M in the initiation of acute inflammation in experimental sterile peritonitis induced by Brewer’s thioglycolate (BTG). We demonstrate that resident peritoneal M are essential for PMN recruitment through M-dependent CXC chemokine production.
Materials and Methods
Transgenic construct
The CD11b promoter from coordinates –1704 to + 83 (22) was used to drive expression of the human hbEGF cDNA (19). Splicing and polyadenylation signals were provided by a region of the human growth hormone gene that had previously worked effectively with the CD11b promoter (22). The fusion protein between hbEGF and GFP was generated by continuing the open reading frame from the final residue of hbEGF with the first residue of GFP.
Transgenic mice
The CD11b-DTR construct was used to generate transgenic mice on the FVB/N background (23) using conventional techniques; transgene expression was detected using an RT-PCR assay. The primer sequences used were 5'-AAGATCCGCCACAACATCG for the forward primer and 5'-GCAGCTCTAGGTTGGATTTCTG for the reverse primer. Because the reverse primer sequence was taken from the base pairs flanking intron III of hGH, no PCR product could be amplified from genomic DNA.
Flow cytometry analysis
Bone marrow-derived M were prepared as previously described (4). Resident peritoneal cells were isolated by peritoneal lavage. Elicited peritoneal M were lavaged from the peritoneal cavity 4 days after i.p. injection of 1 ml of 3% BTG (Difco). For flow cytometric analysis, 2 x 106 cells were incubated for 10 min in FACS buffer containing rat anti-mouse FcR blocker CD16/CD32 (FcRIII/II). FITC- or PE-conjugated Abs at concentrations suggested by the supplier were then added, and the mixture was incubated at 4°C for 45–60 min. The cells were then washed and fixed in 2% formaldehyde before undergoing flow cytometry analysis. A fixed volume of whole blood was obtained by tail vein bleed into 3.9% sodium citrate. Blood was aliquoted into flow cytometry tubes, blocked, and stained as for peritoneal lavage cells. One milliliter of FACSLysis buffer (BD Biosciences) was then added to lyse erythrocytes, and samples were spun and processed by flow cytometry. The Abs used were anti-CD11b FITC conjugate (eBiosciences), anti-GR1 PE (eBiosciences), anti-B220 (mouse CD45R) FITC and PE conjugates (BD Pharmingen), mouse anti-CD3 PE conjugates (BD Pharmingen), and F4/80 allophycocyanin, FITC, and PE conjugates (Caltag and Serotec). Annexin VFITC conjugate (US Biological) and propidium iodide (PI; Sigma-Aldrich) were used to determine the levels of apoptosis and necrosis. A known amount of fluorescent Flow-Check fluorospheres (Beckman Coulter) was added to peritoneal lavage samples before analysis, and the ratio of cells to beads was used to calculate the absolute number of any cell type in peritoneal lavage fluid. Analyses were performed using a FACScan or FACSCalibur instrument.
Immunolabeling of tissues
Organs were fixed in 4% formaldehyde in PBS at 4°C overnight, and paraffin-embedded tissue sections were prepared according to conventional methods. Rehydrated sections were immersed in acetone for 10 min and rinsed in PBS, and a limited trypsin (Sigma-Aldrich) digestion was performed for 20 min. Sections were then washed in PBS, and endogenous peroxidase activity was quenched with a 30-min incubation in 0.3% H2O2 in methanol. Sections were then incubated with F4/80 mAb (Caltag Laboratories) at a 1/100 dilution. Further steps were performed according to the recommendations of the manufacturer of the VECTASTAIN Elite ABC (avidin/biotin complex) system used in the labeling (Vector Laboratories). Sections were then washed in water, lightly counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
Experimental peritonitis
Peritonitis was induced by i.p. injection of 1 ml of 3% BTG. Resident M ablation was induced in transgenic mice by i.p. injection of DT (25 ng/g body weight) 24 h before the administration of BTG, with DT-treated FVB/N wild-type (WT) mice serving as control. Mice underwent peritoneal lavage at various time points following BTG injection. In some experiments, peritonitis was induced by the injection of 0.2 mg of zymosan (Sigma-Aldrich) with peritoneal lavage being performed at 8 h. All experiments were performed in accordance with institutional and U.K. Home Office guidelines.
Adoptive transfer of peritoneal cells
Peritoneal lavage samples from groups of FVB/N WT mice were pooled, spun, and either resuspended in 1 ml of sterile PBS (M-rich peritoneal cells) or plated on tissue culture plastic for 2 h to deplete M by adhesion (M-depleted peritoneal cells). A total of 97 ± 2.8% of M was removed from the cell suspension. Four hours before induction of BTG peritonitis, M-rich or M-depleted peritoneal cells suspended in 1 ml of PBS were injected i.p. Experimental groups consisted of 1) CD11b-DTR transgenic mice depleted of resident peritoneal M by prior administration of DT, 2) DT-treated FVB/N WT mice, 3) M-depleted mice reconstituted with M-rich peritoneal cells, and 4) M-depleted mice reconstituted with M-depleted peritoneal cells. Groups 1 and 2 were injected with 1 ml of PBS 4 h before BTG administration as an injection control for the adoptive cell transfer procedure. Animals were sacrificed 8 h following the initiation of peritonitis.
Chemokine studies
Mice underwent peritoneal lavage at 1 and 3 h following the i.p. injection of BTG. Lavage fluid was centrifuged, aliquoted, and stored at –80°C until analyzed by ELISA for MIP-2 and keratinocyte-derived chemokine (KC) (R&D Systems). Chemokine production by peritoneal cell populations that had been depleted of either M or mast cells (MC) was also determined in vitro. Peritoneal cells were incubated with PE-conjugated anti-F4/80 Ab or an Ab to the MC marker c-kit (CD117) to stain M or MC, respectively. Peritoneal cells were then incubated with anti-PE-conjugated MACS magnetic beads, and M or MC were removed by passing the cells over a magnetic MACS column (Miltenyi Biotec). As a control, total peritoneal cells were incubated with an isotype control Ab followed by magnetic beads and subsequently passed over the magnetic MACS column. This method resulted in >97% depletion of M or MC. Control peritoneal cells and M- or MC-depleted peritoneal cells were then plated in 48-well plates (5 x 105 cells per well) and exposed to 1% BTG for 3 h. Peritoneal cell-conditioned supernatants were harvested, spun, and stored at –80°C until analyzed by specific ELISA for MIP-2 and KC (R&D Systems).
Statistical analysis
The Student t test with a tailed distribution or ANOVA was used to analyze data. A value of p < 0.05 was deemed statistically significant. Data are presented as mean ± SE.
Results
Generation of transgenic mice
Conditional ablation transgenic mice were generated using an established strategy (21) and a construct (designated CD11b-DTR) that used the CD11b promoter (22) to provide M expression specificity. CD11b-DTR expresses the DT receptor (alternatively named hbEGF (19)) (Fig. 1A) as a GFP fusion protein. The hbEGF-GFP construct conferred sensitivity to DT in transiently transfected murine cells indicating that it was functional (data not shown).
FIGURE 1. Structure and expression of the CD11b-DTR transgene. A, Schematic of the CD11b-DTR construct. The transcription start is indicated by the right-facing arrow and exons by shaded boxes. The DTR-eGFP fusion cDNA is inserted between the human CD11b promoter and the human growth hormone (hGH) sequences that provides splicing and polyadenylation signals. Oligonucleotides eGFP and hGH used for RT-PCR transcript detection are indicated by small arrows. B–E, RT-PCR expression analysis performed on BTG-elicited peritoneal cells (B), spleen cells (C), and bone-marrow derived M (D and E). Transgene mRNA amplification products were not evident in samples from WT mice or when reverse transcriptase was omitted but was detected in line 34 peritoneal and spleen cells (B and C). Line 34 bone-marrow derived M also exhibited normal expression of the M marker F4/80 (D) and persistent transgene expression (E).
Six transgenic lines were produced with the CD11b-DTR construct. Although the fluorescence signal from the hbEGF-GFP fusion protein was insufficient to permit detection of transgene expression by FACS analysis, a RT-PCR assay indicated that four lines exhibited detectable transgene expression in peritoneal cells and spleen with line 34 exhibiting high expression (Fig. 1, B and C). WT and CD11b-DTR-34 bone marrow-derived M expressed the M-specific gene F4/80 as expected (Fig. 1D), indicating that expression of the DT receptor was unlikely to have disrupted normal M differentiation. Transgene expression was also observed in day 4 and 8 CD11b-DTR-34 bone marrow-derived M (Fig. 1E). Because line CD11b-DTR line 34 showed the highest levels of transgene expression, further analysis was restricted to this line.
Transgenic M are killed by DT in vitro and in vivo
Treatment of BTG-elicited peritoneal M from CD11b-DTR-34 mice with concentrations of DT between 1 ng/ml to 1 mg/ml over a period of 48 h induced cell death at concentrations as low as 25 ng/ml. In contrast, M from WT mice or other transgenic lines were resistant (data not shown). We then asked whether i.p. injection of DT ablated resident peritoneal M in vivo. DT was injected at 25 ng/g mouse weight, and FACS analysis of peritoneal cells was performed 24 h later. Normal numbers of resident peritoneal M (F4/80 positive, CD11b positive, and Ly6C/G negative) were evident in WT mice receiving DT (Fig. 2A) as well as CD11b-DTR-34 transgenic mice injected with either PBS (Fig. 2B) or the inactive form of the toxin DTmut (Fig. 2C). However, CD11b-DTR-34 transgenic mice showed an almost complete absence of F4/80-positive peritoneal M after a single dose of DT (Fig. 2D). Administration of DT doses of 6.25 and 12.5 ng/g mouse weight resulted in M ablation of 72 and 82%, respectively, whereas lower doses resulted in <50% ablation. We therefore chose to use a dose of 25 ng/g body weight for the in vivo studies.
FIGURE 2. Flow cytometric analysis of peritoneal cell M killing by DT. A–D, Cells were removed from the peritoneal cavity by peritoneal lavage and labeled with PE-conjugated F4/80 Ab, and flow cytometric analysis was performed. A, WT mice injected with DT (25 ng/g body weight) show a normal percentage of peritoneal M. B and C, CD11b-DTR-34 mice receiving either PBS (B) or DTmut (C) have normal F4/80 profiles. D, In contrast, CD11b-DTR-34 mice treated with active DT show complete absence of F4/80-positive cells. E, In WT mice, injection of DT at 25 ng/g mouse weight does not affect either the small population of CD3+ T cells (upper-left quadrant) or the larger population of F4/80+ M (lower-right quadrant) in the peritoneal cavity. F, CD11b-DTR-34 mice receiving DT exhibit elimination of the F4/80+ population, whereas the CD3+ cells remain unaffected.
Time course of M elimination in vivo
We then examined the kinetics of M ablation in the peritoneal cavity following administration of a single dose of DT (25 ng/g body weight). The appearance of apoptotic and necrotic cells was monitored using Annexin VFITC and PI staining, respectively. Peritoneal lavages and flow cytometric analyses were conducted on a series of mice 4, 6, 8, and 12 h after DT injection (Fig. 3). After 6 h, 65% of the peritoneal population was annexin V positive, indicating a dramatic increase in early-stage apoptotic cells (Fig. 3A). The maximal numbers of PI-positive cells occurred 2 h later at 8 h after DT injection and represented 20% of the total peritoneal cells (Fig. 3B). The number of F4/80-positive cells was nearly zero at 12 h (Fig. 3C), and this corresponded to very low levels of PI-positive and annexin V-positive cells (A and B). These data suggest that DT induces sensitive cells to undergo apoptosis and that some of these dying cells then undergo secondary necrosis. All F4/80-positive cells were cleared by 12 h.
Restoration of the peritoneal M population occurs by day 4 following a single i.p. dose of DT (3.94 x 105 ± 1.7 x 105 M vs 5.8 x 105 ± 1.3 x 105; day 4 following DT treatment vs day 1 following PBS treatment; n = 5 per group, p > 0.05).
FIGURE 3. Time course of peritoneal M depletion. Cells were removed from the peritoneal cavity by peritoneal lavage. Mice were either injected with a single dose of DT (gray lines) or DTmut (black lines) at 25 ng/g mouse weight i.p., and the resident peritoneal population was assessed for the appearance of annexin V-positive cells (A), for labeling of cells with PI (B), and for presence of the M marker F4/80 (C). Flow cytometric analysis identified the labeled proportion of total cells over a 12-h time course following DT injection.
Specificity of M elimination in vivo
To test the specificity of M elimination, we examined CD3+ T cells in spleen and peritoneal cavity and B220+ B cells in the spleen. In this case, we injected two doses of DT at 25 ng/g at 48-h intervals and assessed ablation 24 h later. The F4/80-positive peritoneal population was unaffected in WT mice (Fig. 2E, lower-right quadrant), but was eliminated in CD11b-DTR-34 animals (G, lower-right quadrant). Despite complete peritoneal M elimination in CD11b-DTR-34 animals, the peritoneal CD3+ T cells were present in both WT and transgenic animals injected with DT (Fig. 2, E and F, upper-left quadrants). The relative increase in CD3-positive and double-negative cells in the DT-treated CD11b-DTR-34 animals is due to the plotting of equal numbers of detection events in the FACS analyses. B220+ and CD3+ populations in spleen were unaffected in either WT or CD11b-DTR-34 mice receiving two doses of DT (data not shown).
Because CD11b is expressed on both granulocytes and M, we asked whether both of these cell types were sensitive to DT. CD11b-DTR-34 mice were injected with BTG. DT (25 ng/g body weight) or PBS was injected 8 h after initiation of peritonitis with peritoneal lavage being performed 12 h later. Despite 90% M ablation (0.5 x 106 ± 0.15 x 106 vs 4.8 x 106 ± 0.23 x 106; DT injection vs PBS; p < 0.005), there was no difference in PMN number (2.3 x 106 ± 0.22 x 106 vs 1.6 x 106 ± 0.39 x 106; DT injection vs PBS; p > 0.05). In addition, flow cytometric analysis of whole blood performed 24 h following DT administration indicated that circulating PMN numbers were unaffected by DT administration (1.02 x 106 ± 0.18 x 106 PMNs/ml whole blood vs 0.97 x 106 ± 0.22 x 106; DT injection vs PBS; p > 0.05). In contrast, DT administration induced significant depletion of circulating monocytes (0.117 x 106 ± 0.059 x 106 monocytes/ml whole blood vs 0.52 x 106 ± 0.073 x 106; DT injection vs PBS; p < 0.05).
Differential deletion of M populations
We also asked whether DT injection could eliminate M in distant organs. Two doses of DT (25 ng/g) were administered IP at 48-h intervals, and the presence of F4/80-positive M in kidney, liver, and lung was analyzed 24 h later and in the ovary 16 h later (Fig. 4). The ovary was examined at 16 h, because there was evidence of some patchy ovarian necrosis present at 20 h. WT mice injected with DT and CD11b-DTR-34 homozygote mice injected with DTmut were unaffected (Fig. 4, top two rows). Both kidney and ovary of CD11b-DTR-34 homozygote mice injected with DT exhibited an absence of F4/80+ cells. In the kidney, mesangial and interstitial M were ablated in the absence of overt renal injury. However, hepatic sinusoidal M and alveolar M were unaffected, indicating that not all populations of tissue M were susceptible. However, the rapid elimination of M populations in the peritoneal cavity and kidney while leaving other cell populations intact establishes the basic validity of this approach to conditional cell ablation.
FIGURE 4. Effect of DT on tissue M populations. Micrographs showing the effect of DT treatment of WT mice (top row), DTmut treatment of CD11b-DTR mice (middle row), and DT treatment of CD11b-DTR mice (bottom row) upon the presence of F4/80-positive M in liver (left column), lung (center left column), kidney (center right column), and ovary (right column). Animals received two doses of DT or DTmut delivered at 48-h intervals. Liver, lung, and kidney were assessed 24 h after the second DT injection, whereas ovary was assessed at 16 h.
Resident M ablation reduces PMN influx and CXC chemokine responses during experimental peritonitis
We used the conditional ablation strategy to investigate the role of resident tissue peritoneal M in sensing perturbation of the microenvironment and subsequent initiation of acute peritoneal inflammation and PMN recruitment in experimental peritonitis. Resident M ablation markedly attenuated PMN infiltration following the administration of 3% BTG (Fig. 5A). We also performed M repletion studies with either M-rich or M-depleted peritoneal cells derived from WT mice. Reconstitution of DT-treated CD11b-DTR-34 homozygote mice with M-rich peritoneal cells 4 h before BTG treatment resulted in complete restoration of peak PMN infiltration at 8 h. In contrast, the administration of M-depleted peritoneal cells was ineffective (Fig. 5B). Previous work suggested that the nature of the inflammatory stimulus may determine the involvement of M in experimental peritonitis (8), and we therefore performed M depletion studies in zymosan peritonitis. We also found that depletion of resident peritoneal M resulted in a significant reduction in PMN infiltration 8 h following the induction of zymosan peritonitis (2.6 x 106 ± 8.8 x 105 PMNs vs 5.4 x 106 ± 3.7 x 105; DTR plus DT vs FVB/N WT controls plus DT; n = 6 per group; p < 0.05).
FIGURE 5. Resident M ablation attenuates peritoneal inflammation. WT and CD11b-DTR mice were injected i.p. with DT (25 ng/g body weight). One milliliter of 3% BTG was injected i.p. 24 h later with mice undergoing peritoneal lavage at various time points. A, Peritoneal cells were stained for the PMN marker GR1. Resident peritoneal M ablation induced a marked blunting of PMN infiltration of the peritoneal cavity. B, WT and three groups of CD11b-DTR mice were injected with DT (25 ng/g body weight). Four hours before i.p. injection of 3% BTG, two groups of M-depleted CD11b-DTR mice were reconstituted with either M-rich peritoneal cells (MR) or M-depleted peritoneal cells (MD). Administration of PBS served as control to the remaining groups. Mice underwent peritoneal lavage 8 h following administration of 3% BTG, and peritoneal cells were stained for GR1. *, p < 0.05.
In this model, we found peak levels of the PMN CXC chemokines MIP-2 and KC at the 1-h time point. Resident peritoneal M ablation before the initiation of BTG peritonitis markedly reduced the elevation in MIP-2 levels (148.5 ± 34.8 vs 1762.1 ± 153.5 pg/ml; M-depleted mice vs nondepleted mice: p < 0.00001). There was a slight, albeit statistically significant, difference in the much lower levels of MIP-2 between DT-treated and control mice at 3 h (204 ± 54 vs 74 ± 8 pg/ml; M-depleted mice vs nondepleted mice; p < 0.05). This suggests that the production of MIP-2 in vivo is predominantly M dependent. In addition, a 50% reduction in the level of KC was evident in M-depleted mice at the 1-h time point (1408.2 ± 322.5 vs 2467.5 ± 264.9 pg/ml; M-depleted mice vs nondepleted mice; p < 0.05). Interestingly, the levels of KC levels at 3 h are higher in M-depleted mice compared with control mice (1477 ± 400 vs 74 ± 8 pg/ml; M-depleted mice vs nondepleted mice; p < 0.01), thereby suggesting a source of KC other than resident M.
CXC chemokine responses are M dependent in vitro
Previous studies of peritoneal and dermal inflammation have implicated the MC as playing an important role in the initiation of PMN infiltration (8, 24). We therefore performed in vitro studies to determine the production of PMN chemokines by BTG-stimulated peritoneal cell populations that had been depleted of M or MC. Control peritoneal cells produced significant levels of MIP-2 and KC, which was not affected by MC depletion (Fig. 6). However, chemokine levels were dramatically reduced following the depletion of M, thereby indicating that chemokine production was completely M dependent with no involvement of MC (Fig. 6).
FIGURE 6. CXC chemokine production in response to BTG stimulation is M dependent and MC independent in vitro. Peritoneal cells were depleted of either M or MC by incubation with PE-conjugated anti-F4/80 or anti-c-kit (CD117) followed by incubation with anti-PE-conjugated magnetic beads and passage over a magnetic column (>97% depletion of M or MC). Incubation of total peritoneal cells with an isotype control Ab followed by magnetic beads and passage over the magnetic column served as control. Cells were then plated in 48-well plates (5 x 105 cells per well) and exposed to 1% BTG for 3 h. Peritoneal cell-conditioned supernatants were harvested, spun, and analyzed by specific ELISA for MIP-2 and KC. *, p < 0.005.
Discussion
Previous analyses (20, 21) and the experiments we describe here show that expression of human hbEGF (19) in mouse cells can confer sensitivity to DT in vivo, and that, as a consequence, injection of DT will kill cells that express hbEGF. Our data indicate that M populations in the peritoneal cavity and kidney can be rapidly killed or eliminated while leaving other cell populations intact, and this establishes the basic validity of this approach to conditional cell ablation. We noted that hepatic and alveolar M populations were unaffected, and it may be the case that a higher dose of DT may have ablated these cells. However, we found that mice could become unwell with doses of DT >25 ng/g body weight, and we therefore did not use doses >25 ng/g body weight in this study. It is pertinent that, despite PMN expression of CD11b, the administration of DT did not induce the death of recruited or circulating PMNs, indicating that PMNs are insensitive to DT, potentially as a result of their lower level of protein synthesis.
We used the conditional ablation strategy to investigate the role of resident peritoneal M in the initiation of acute peritoneal inflammation following the administration of BTG. Previous work has indicated that leukotrienes derived from resident peritoneal M are involved in the development of early vascular permeability in sterile peritonitis (25). Although early work in rat models of peritonitis implicated the resident peritoneal M in the orchestration of PMN recruitment (26, 27, 28), more recent studies have produced conflicting results (7, 8). Indeed, studies by Ajuebor et al. (8) suggest that resident M depletion inhibits PMN influx in LPS-induced inflammation, has no effect in BTG peritonitis, and augments PMN influx in zymosan peritonitis. In the latter model, it is proposed that M-derived IL-10 inhibits PMN recruitment. Conversely, work by Knudsen et al. (7) using clodronate-induced depletion of peritoneal M in a rat model of sterile peritonitis demonstrated that PMN infiltration was M dependent.
In this study, administration of DT resulted in a dramatic 98% M ablation that markedly blunted PMN infiltration, thereby indicating a key role for the resident M in the orchestration of acute peritoneal inflammation in this experimental model. It is important to note that the reduced PMN infiltration in DT-treated mice was not attributable to a systemic neutropenia, because PMNs were not sensitive to DT and the number of circulating PMNs in DT-treated mice was comparable with that of PBS-treated mice at 24 h. The importance of the resident M was reinforced by experiments involving the adoptive transfer of nontransgenic peritoneal M following DT-mediated M ablation and before the initiation of peritonitis. The presence or absence of M in the transferred peritoneal cell population directly correlated with the restoration of the PMN influx, thereby suggesting that the M exerts a critical role in this process. In addition, we found that depletion of resident M also significantly reduced PMN infiltration in zymosan peritonitis, thereby suggesting that the sensing function of the resident M may be stimulus independent.
The magnitude of the M depletion may explain the apparent discrepancy between these results and the study by Ajuebor et al. (8). Administration of a single dose of DT induced 98% M ablation, whereas three doses of liposomal clodronate resulted in >85% M depletion in the study by Ajuebor et al. (8). M are a potent source of chemokines and cytokines, and it may be the case that, in certain circumstances, a relatively small population of residual M may exert significant biological effects. Although administration of liposomal clodronate may exert marked biological effects despite depletion of only 80% of M (29), it may be necessary to deplete almost all peritoneal M to delineate their roles as sentinel cells.
Although peritoneal M may produce myriad mediators capable of recruiting PMNs (30, 31), we examined the effect of M ablation upon the level of CXC chemokines in this model. Our data suggest that the initiation of PMN infiltration is mediated by resident peritoneal M-dependent production of chemokines previously documented to play a role in orchestrating PMN recruitment in BTG peritonitis (32, 33) and in other inflammatory situations (34, 35, 36). MC are also a rich source of proinflammatory and vasoactive mediators and have been documented to play an important role in PMN recruitment during inflammation of the peritoneum (8) as well as other sites such as the skin (24).
We found that resident M ablation markedly reduced the peak level of MIP-2 and significantly blunted the level of KC at 1 h. However, partial inhibition (50%) of KC production at 1 h and the persistent elevation of KC at 3 h in M-depleted mice suggest that KC may be produced by other cells within the peritoneum. The persistent elevation of KC also suggests that M may play a role in the negative regulation of KC production by non-M cells. In this context, it is pertinent that our in vitro data indicate that both KC and MIP-2 production by peritoneal cells obtained by peritoneal lavage is almost entirely dependent upon M, because peritoneal cells depleted of M produced minimal levels of chemokines. These findings suggest that peritoneal cells retrievable by peritoneal lavage are not the source of KC detected in our in vivo study. This interpretation of the data is consistent with recent work in a wound model (37) demonstrating MIP-2 expression by inflammatory cells and KC expression by resident tissue cells such as endothelial cells and fibroblasts. Peritoneal mesothelial cells undoubtedly participate in peritoneal inflammation and can produce chemokines and cytokines (38), and it may be the case that mesothelial cells contribute to the KC production evident in this study. Despite this, however, we found that PMN infiltration is still markedly blunted despite the persistent presence of KC at 3 h.
We also examined the potential interaction between M and MC by performing in vitro studies of peritoneal cells that had been depleted of M or MC before stimulation with BTG. Interestingly, depletion of MC had no significant effect upon the production of KC and MIP-2 following BTG stimulation, suggesting that chemokine production in this model was M dependent and MC independent.
In conclusion, this work has used a novel model of conditional M ablation to dissect the role of resident peritoneal M in the initiation of acute peritoneal inflammation. Our data indicate a key role for the resident M in sensing peritoneal irritation and orchestrating PMN infiltration in BTG and zymosan peritonitis. This proinflammatory function is predominantly mediated by production of the potent PMN CXC chemokine MIP-2 and, to a lesser extent, KC. Although previous work has implicated the involvement of other cells such as MC, our study suggests that resident M are critically important producers of PMN chemokines and act to orchestrate PMN recruitment in murine BTG peritonitis. We also anticipate that CD11b-DTR transgenic mice will be valuable for studying other M functions in vivo in a variety of different biological contexts. The option of being able to choose the time and, with local toxin injection, perhaps the locality of ablation offers a number of advantages over other cell ablation systems.
Acknowledgments
We thank Leon Eidels for providing the cDNA to human hbEGF, Daniel Tenen for the CD11b-based transgene construct, and Michael Clay for technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The Lang Laboratory is supported by grants from the National Institutes of Health (RO1 EY10559, EY11234, EY12370, and EY14102) and by funds from the Abrahamson Pediatric Eye Institute Endowment at Children’s Hospital Medical Center of Cincinnati. J.H. is in receipt of a Wellcome Trust Senior Research Fellowship in Clinical Science (Grant 061139). J.-F.C. is supported by the Canadian Institutes of Health Research. J.S. is supported by the Wellcome Trust (Program Grant 064487).
2 J.-F.C., M.P., S.V., S.W., J.H., and R.A.L. contributed equally to this manuscript.
3 Current address: Departments of Radiation Oncology and Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016.
4 Current address: Regeneron Pharmaceuticals, Tarrytown, NY 10591.
5 Current address: Molecular Pathogenesis Program, Skirball Institute for Biomolecular Medicine, 540 First Avenue, New York, NY 10016.
6 Address correspondence and reprint requests to Dr. Jeremy Hughes, Phagocyte Laboratory, Medical Research Council Center for Inflammation Research, University of Edinburgh, Teviot Place, Edinburgh, U.K., EH8 9AG. E-mail address: jeremy.hughes@ed.ac.uk
7 Abbreviations used in this paper: M, macrophage; PMN, neutrophil; DT, diphtheria toxin; hbEGF, heparin-binding epidermal growth factor; BTG, Brewer’s thioglycolate; WT, wild type; PI, propidium iodide; KC, keratinocyte-derived chemokine; MC, mast cell.
Received for publication July 14, 2004. Accepted for publication December 6, 2004.
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