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. 2011 Mar;89(3):423–432. doi: 10.1189/jlb.0810479

Neutrophil IL-10 suppresses peritoneal inflammatory monocytes during polymicrobial sepsis

Lee M Ocuin 1, Zubin M Bamboat 1, Vinod P Balachandran 1, Michael J Cavnar 1, Hebroon Obaid 1, George Plitas 1, Ronald P DeMatteo 1,1
PMCID: PMC3040467  PMID: 21106642

Using a depleting antibody, the authors demonstrate that neutrophils are dispensable for survival in polymicrobial sepsis and regulate inflammatory monocytes through IL-10.

Keywords: innate immunity, bacteria, cytokines, cecal ligation and puncture

Abstract

Septic peritonitis remains a major cause of death. Neutrophils and inflammatory monocytes are principal components of the innate immune system and are essential for defense against a range of microbial pathogens. Their role and interaction in polymicrobial sepsis have not been defined clearly. Using a murine model of CLP to induce moderate sepsis, we found that neutrophil depletion did not alter survival, whereas depletion of neutrophils and inflammatory monocytes markedly reduced survival. After neutrophil depletion, inflammatory monocytes had greater phagocytic capacity and oxidative burst, and increased expression of costimulatory molecules, TNF, and iNOS. Notably, peritoneal neutrophils produced IL-10 following CLP. Adoptive i.p. transfer of WT but not IL-10−/− neutrophils into septic mice reduced monocyte expression of TNF. In vitro experiments confirmed that monocyte suppression was mediated by neutrophil-derived IL-10. Thus, during septic peritonitis, neutrophils suppress peritoneal inflammatory monocytes through IL-10 and are dispensable for survival.

Introduction

Sepsis remains one of the leading causes of death in the intensive care unit, with mortality rates ranging from 30% to 70% [1]. Sepsis results from dysregulation of the immune response to infection, leading to systemic inflammation, acute lung injury, and multiorgan system dysfuction [2]. CLP is widely regarded as the most representative animal model of human polymicrobial sepsis [3]. Ischemia and necrosis of the cecum result in intra-abdominal spillage of intestinal contents and septic peritonitis. The host attempts to control the intra-abdominal infection, but ultimately, innate immune defenses are overwhelmed, and bacterial dissemination ensues, resulting in a massive systemic inflammatory response.

Neutrophils are a principal component of the innate immune system and provide a first line of defense against bacteria and other invading pathogens. Neutrophils are recruited rapidly to sites of inflammation or infection, and they possess a large number of antimicrobial functions, including phagocytosis of bacteria, release of antimicrobial peptides, and cytolysis via ROS generation [4]. The importance of neutrophils for microbial clearance in humans is illustrated by disorders resulting from neutrophil dysfunction or deficiency. Defective oxidative burst results in chronic granulomatous disease, and neutropenia induced by chemotherapy renders patients susceptible to opportunistic and potentially fatal bacterial or fungal infections [5]. Conversely, activated neutrophils can also mediate host tissue damage through the same mechanisms they use for pathogen clearance [5–8]. Several studies in mice have investigated the role of neutrophils in the host response to polymicrobial or monomicrobial infection using the depleting mAb to the myeloid differentiation antigen Gr1 (RB6-8C5) [7, 9–15]. Gr1 is an epitope expressed on the neutrophil-specific membrane protein Ly6G, as well as Ly6C, which is expressed on monocytes [16]. Therefore, αGr1 will deplete both cell types [17], and results from these studies are potentially confounded. The mAb αLy6G (1A8) reacts only with Ly6G and not Ly6C [16] and has been used to deplete neutrophils specifically [18], which allows for direct analysis of neutrophil function in various murine models.

Monocytes possess similar antimicrobial functions as neutrophils. Murine monocytes are divided into two main subsets: a CX3CR1hiCCR2–Gr1– subset recruited to noninflamed tissues, which differentiate into tissue macrophages, and a short-lived CX3CR1lowCCR2+Gr1+ subset that is recruited to sites of inflammation [19]. The CX3CR1lowCCR2+Gr1+ subset is also known as inflammatory monocytes and can be characterized further by intermediate expression of the integrin CD11b and high expression of the membrane protein Ly6C [20]. In addition, these cells secrete large amounts of TNF and generate reactive nitrogen species through expression of iNOS, both of which play key roles in host defense [20]. Inflammatory monocytes emigrate from the bone marrow to sites of inflammation by a CCR2-dependent mechanism. CCR2−/− mice are more susceptible to monomicrobial infection with Listeria monocytogenes, Toxoplasma gondii, and Mycobacterium tuberculosis [20]. However, the role of inflammatory monocytes in polymicrobial sepsis has not been defined clearly.

In this study, we investigated the contribution of neutrophils and inflammatory monocytes in CLP by using the neutrophil-specific depleting antibody αLy6G, as well as αGr1, which depletes neutrophils and monocytes. Neutrophil depletion alone did not alter survival, but the concomitant depletion of monocytes markedly increased mortality. We showed that neutrophils suppress inflammatory monocyte function through an IL-10-mediated mechanism in vitro and in vivo. In mice depleted of neutrophils alone, this suppression was abolished, leading to enhanced monocyte function. These data suggest that up-regulation of monocyte function compensated for the lack of neutrophils and their contribution to the antimicrobial response, enabling equivalent control of infection and survival.

MATERIALS AND METHODS

Animals and procedures

Eight- to 12-week-old male WT C57BL/6J (CD45.1+ and CD45.2+) and IL-10−/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). CCR2-GFP+/− mice on a C57BL/6J background were a gift from Dr. Eric Pamer (Sloan-Kettering Institute, New York, NY, USA). CLP was performed as described [14] with modifications. Briefly, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) by i.p. injection. A midline laparotomy was performed, and the cecum was located and exteriorized. The distal third of the cecum was ligated with a 4-0 silk suture and then punctured once with an 18-gauge needle. A small amount of stool was extruded to ensure patency. The incision was closed in two layers, and the skin was stapled with metallic clips. All mice received 1 ml normal saline s.c. following abdominal closure. Mice subjected to sham laparotomy underwent the same procedure without CLP. Animals had full access to water and chow before and after CLP. Mice were monitored every 8 h for 7 days to determine survival. At the end of the observation period, mice were killed by carbon dioxide inhalation. All animals were maintained in a pathogen-free animal housing facility at Memorial Sloan-Kettering Cancer Center (New York, NY, USA). All procedures were approved by the Institutional Animal Care and Use Committee.

Cell depletion

Mice were injected i.p. with 500 μg αGr1 antibody (RB6-8C5; Monoclonal Core Facility, Sloan-Kettering Institute), αLy6G antibody (1A8; BioXCell, West Lebanon, NH, USA), or their respective isotype controls (control IgG, rat IgG2b, or rat IgG2a; eBioscience, San Diego, CA, USA), 24 h and 2 h before CLP.

Cell isolation and adoptive transfer

Peritoneal cells were isolated by injecting 5 ml PBS i.p. and then aspirating the fluid 1 min later. The peritoneal washings were then filtered through a 100-μM filter to remove debris. Blood was obtained by cardiac puncture. Bone marrow cells were obtained from the tibia and femur. WT inflammatory monocytes were isolated from the bone marrow of CCR2-GFP+/– reporter mice by flow cytometric sorting of the GFP+ cell population [21]. Cell purity was >93% by FACS analysis, and cell viability was >97% by trypan blue exclusion. WT and IL-10−/− neutrophils were isolated from bone marrow by immunomagnetic beading of Ly6G+ cells, according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity was routinely >96% by FACS analysis and cell viability >97% by trypan blue exclusion. CD45.2+ WT (107) or IL-10−/− neutrophils were injected i.p. into CD45.1+ WT mice 6 h following CLP.

Flow cytometry and cell sorting

Flow cytometry and cell sorting were performed on a FACSAria (BD Biosciences, San Jose, CA, USA). FcRs were blocked with 1 μg αFcγRIII/II antibody (2.4G2; Monoclonal Core Facility, Sloan-Kettering Institute)/106 cells. Neutrophils were defined as CD11bhiLy6Ghi, and inflammatory monocytes were defined as CD11bintLy6Chi in WT mice or as GFP+ in CCR2-GFP+/– reporter mice. Cells were stained with fluorochrome-conjugated antibodies to CD11b (M1/70), Ly6G (1A8), Ly6C [AL-21 (BD Biosciences) or HK1.4 (Abcam, Cambridge, MA, USA)], CD80 (B7-1), CD86 (B7-2), and CD210 (IL-10R; 1B1.3a; BD Biosciences). Intracellular cytokine analysis was performed following treatment of isolated peritoneal cells with Brefeldin A (1 μg/μl/106 cells; BD Biosciences) for 4 h at 37°C. Cells were fixed and permeabilized according to the manufacturer's instructions (Cytofix/Cytoperm kit, BD Biosciences). The following antibodies were used for intracellular staining: PE-conjugated anti-mouse TNF (MPC-XT22; BD Biosciences) and purified polyclonal rabbit anti-mouse iNOS (Millipore, Billerica, MA, USA). FITC-conjugated polyclonal goat anti-rabbit IgG (Abcam) was used as the secondary antibody for iNOS staining. Appropriate isotype control antibodies were used.

Assays for phagocytosis and oxidative burst

Phagocytic activity was determined by measuring the uptake of opsonized, FITC-labeled Escherichia coli by flow cytometry using the Phagotest kit, and measurement of oxidative burst as determined by the conversion of dihydrorhodamine-123 to its oxidized form, rhodamine-123, was determined by flow cytometry using the Phagoburst kit, according to the manufacturer's protocol (Orpegen Pharma, Heidelberg, Germany).

Measurement of cytokines and CFUs

Serum, peritoneal cell-free supernatant, and culture supernatant were analyzed for cytokines using a CBA (mouse inflammation kit, BD Biosciences). Aerobic bacterial CFUs were determined by plating serial dilutions of blood or peritoneal fluid on BHI agar plates, followed by 18 h of culture at 37°C.

In vitro coculture assays

To model polymicrobial sepsis in vitro, we used heat-killed (80°C for 60 min) cecal contents from WT mice suspended in PBS at 20 mg/ml. WT inflammatory monocytes (5×104) were cultured for 24 h at 37°C, alone or together with 105 WT or IL-10−/− neutrophils in a total volume of 200 μl. In certain wells, cells were stimulated with heat-killed stool (200 μg/ml). In other experiments, WT peritoneal cells were isolated 12 h following sham laparotomy or CLP. Bulk peritoneal cells or those depleted of neutrophils or inflammatory monocytes by cell sorting were plated in 96-well plates in a total volume of 200 μl and cultured in media (RPMI 1640 containing 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 10% FCS; Media Preparation Core Facility, Sloan-Kettering Institute). Supernatant was harvested at 24 h.

Statistics

Results are expressed as mean ± sem. Statistical significance was determined by the two-tailed Student's t test, one-way ANOVA, two-way ANOVA, and log-rank test as appropriate, using statistical software (Prism 5.0; GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was considered significant.

RESULTS

Neutrophil depletion does not alter survival during sepsis

We performed CLP in WT mice and found that neutrophils and inflammatory monocytes were recruited to the perito-neum over the first 12 h (Fig. 1A and B). Prior studies about the role of neutrophils in single pathogen infections and polymicrobial sepsis have used the nonspecific antibody αGr1 [7, 9–13], which depletes neutrophils as well as monocytes. To ascertain the contribution of neutrophils alone during moderate polymicrobial sepsis, we performed CLP in WT mice pre-treated with αLy6G. Surprisingly, mice treated with αLy6G had similar survival to mice treated with control IgG, whereas nearly all mice treated with αGr1 died (Fig. 1C). A more severe model of CLP [14] resulted in 100% mortality in all groups (data not shown). Peritoneal cells from septic mice treated with αLy6G or αGr1 were virtually devoid of neutrophils (Fig. 1D). The proportion of peritoneal inflammatory monocytes at 6 h (data not shown) and 12 h (Fig. 1D) was not affected by treatment with αLy6G but was diminished greatly by αGr1. We also performed manual differential counting of peritoneal cells and confirmed neutrophil-specific depletion with αLy6G and neutrophil and inflammatory monocyte depletion with αGr1 (data not shown). Treatment of mice with αLy6G did not change the kinetics of inflammatory monocyte recruitment to the peritoneum following CLP when compared with control IgG (data not shown). Twelve hours following CLP, mice treated with αLy6G had similar levels of serum inflammatory cytokines and bacterial counts in the blood and peritoneum as mice treated with control IgG, and mice treated with αGr1 had higher serum inflammatory cytokines and higher bacterial loads (Fig. 1E and F). Taken together, our data show that neutrophils are dispensable for survival of murine polymicrobial sepsis.

Figure 1. Neutrophil depletion alone does not alter survival of WT mice following CLP.

Figure 1.

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WT mice underwent CLP, and peritoneal cells were isolated at serial time-points. (A) Gating strategy for peritoneal neutrophils and inflammatory monocytes 12 h following CLP. CD11bhiLy6Ghi and CD11bintLy6Chi peritoneal cells were sorted by FACS, and cytospins were performed to determine cell morphology. (B) Time course of recruitment of neutrophils (PMNs) and inflammatory monocytes (IMs) to the peritoneum following CLP. (C–F) Mice were injected i.p. with αLy6G, αGr1, or control IgG, 24 h and 2 h prior to undergoing CLP. (C) Mice were monitored every 8 h for 7 days to determine survival (n=18–20 mice/group pooled from three independent experiments with similar results). (D) Depletion of neutrophils (upper row) and inflammatory monocytes (lower row) 12 h after CLP in mice treated as indicated by the column title. (E) Serum cytokines were determined by CBA 12 h following CLP. (F) Serial dilutions of blood or peritoneal lavage fluid obtained 12 h after CLPs were cultured on BHI agar plates, and the number of bacterial colonies was counted. (A and D) Data are representative of at least three experiments. (B, E, and F) Data represent mean ± sem and are pooled from three independent experiments with similar results; n = 13–15 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001.

Peritoneal inflammatory monocytes express higher levels of TNF and iNOS than peritoneal neutrophils

To understand the mechanism for how animals compensated for the lack of neutrophils, we first determined the function of peritoneal neutrophils and inflammatory monocytes during sepsis in WT mice. TNF is a pleiotropic cytokine that is critical for cell trafficking, inflammation, and host defense against various pathogens [22]. TNF plays a beneficial role in the elimination of pathogens [22, 23] but also mediates many of the pathological sequelae of septic shock [24, 25]. We isolated peritoneal cells from WT mice 12 h following sham laparotomy or CLP and cultured them in vitro (Fig. 2A). Using cell sorting, removal of monocytes from bulk peritoneal cells of septic mice decreased supernatant TNF levels. Removal of neutrophils from bulk peritoneal cells led to a slight decrease in supernatant TNF, but this level did not reach statistical significance. Intracellular cytokine analysis of peritoneal cells from WT mice 12 h following CLP confirmed that inflammatory monocytes expressed higher levels of TNF than neutrophils (Fig. 2B), and inflammatory monocytes comprised >50% of all TNF+ cells in the peritoneum (Fig. 2C). Compared with neutrophils, monocytes also expressed higher levels of TNF at 6 h and more IL-6 at 6 h and 12 h following CLP, as measured by intracellular cytokine staining (data not shown).

Figure 2. Peritoneal inflammatory monocytes express higher levels of TNF and iNOS than peritoneal neutrophils following CLP.

Figure 2.

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WT mice underwent sham laparotomy or CLP, and peritoneal cells were isolated 12 h later. (A) Bulk peritoneal cells were cultured at 106 cells/ml for 24 h at 37°C. In some wells, neutrophils or inflammatory monocytes were removed by sorting (Bulk minus PMNs or Bulk minus IMs). Supernatant TNF concentration was determined by CBA. We confirmed that cell sorting removed >95% of neutrophils or inflammatory monocytes from bulk peritoneal cells (data not shown). (B–E) Peritoneal cells were cultured with Brefeldin A for 4 h, followed by intracellular cytokine analysis for TNF and iNOS. Representative isotype control FACS plots are depicted. (F) Peritoneal neutrophils and inflammatory monocytes were assessed for phagocytic capacity (upper row) and oxidative burst (lower row). Data are representative of three experiments with similar results, and data in bar graphs are pooled and represent mean ± sem; **P < 0.01. MFI, Mean fluorescence intensity; SSc, side scatter.

Production of reactive nitrogen species via the enzyme iNOS is another important mechanism by which inflammatory cells are able to kill invading pathogens [20]. Mice deficient in iNOS have higher mortality following CLP [26]. We found that 12 h following CLP, inflammatory monocytes but not neutrophils had increased iNOS expression (Fig. 2D). As with TNF expression, inflammatory monocytes made up the majority of iNOS-expressing cells in the peritoneum after CLP (Fig. 2E). In contrast, phagocytosis and oxidative burst in neutrophils and monocytes were similar after sham laparotomy and CLP (Fig. 2F). We also found that neutrophils and inflammatory monocytes produced similar levels of ROS after sham laparotomy and up-regulated ROS production to a similar degree 6 h following CLP (data not shown).

Neutrophil depletion increases the function of peritoneal inflammatory monocytes

Thus far, we had established that selective neutrophil depletion did not alter the proportion of inflammatory monocytes in the peritoneum or the kinetics of their recruitment (Fig. 1D, and data not shown). Futhermore, peritoneal monocytes from nondepleted mice following CLP produced more TNF and iNOS and had comparable phagocytosis and oxidative burst as neutrophils (Fig. 2). These findings raised the possibility that increased monocyte function accounted for the similar survival that we observed after the selective depletion of neutrophils (Fig. 1C). In fact, we found that peritoneal inflammatory monocytes from mice depleted of neutrophils consistently demonstrated increased CD80 and CD86 expression, suggesting a higher state of activation (Fig. 3A). Peritoneal inflammatory monocytes from septic mice treated with αLy6G had enhanced phagocytic capacity and increased ROS production (Fig. 3B). Interestingly, peritoneal fluid contained similar TNF levels after selective neutrophil depletion with αLy6G compared with nondepleted mice (Fig. 3C). Accordingly, a higher percentage of peritoneal monocytes in septic mice depleted of neutrophils expressed TNF and almost twofold more TNF on a per-cell basis, as demonstrated by intracellular cytokine analysis (Fig. 3D). Similarly, a higher percentage of peritoneal monocytes expressed iNOS, and expression also increased nearly twofold on a per-cell basis after CLP in mice depleted of neutrophils compared with those treated with control IgG (Fig. 3E).

Figure 3. Depletion of neutrophils increases peritoneal inflammatory monocyte function after CLP.

Figure 3.

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WT mice were injected with αLy6G or control IgG, 24 h and 2 h prior to sham laparotomy or CLP. Twelve hours later, peritoneal cells were harvested, and inflammatory monocytes were analyzed. (A) CD80 expression (upper row) and CD86 expression (lower row) on inflammatory monocytes. (B) Inflammatory monocyte phagocytic capacity (upper row) and oxidative burst (lower row). (C) Peritoneal cells were harvested 12 h after CLP in mice treated with αLy6G or αGr1, and the concentration of TNF in cell-free peritoneal lavage fluid was determined by CBA. (D and E) Inflammatory monocytes from control IgG- or αLy6G-treated mice were compared for intracellular expression of TNF and iNOS based on isotype staining. (A, B, D, and E) Data are representative of at least three independent experiments with similar results, and data in bar graphs are pooled and represent mean ± sem. (C) Data are mean ± sem pooled from three experiments with similar results; n = 13–15 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001.

Neutrophils suppress inflammatory monocytes through an IL-10-dependent mechanism

Neutrophils have been shown to secrete IL-10 in response to infection with Mycobacterium bovis [4]. IL-10 is a regulatory cytokine whose principal function is to limit the extent of inflammatory responses [27]. Blockade of IL-10 in mice increases mortality from endotoxemia [28] and CLP [29, 30]. Given our finding that peritoneal inflammatory monocytes from neutrophil-depleted mice had increased function, we hypothesized that neutrophil-derived IL-10 may regulate peritoneal inflammatory monocyte function during polymicrobial sepsis. Peritoneal neutrophils isolated from WT mice 12 h following CLP demonstrated a tenfold up-regulation of IL-10 secretion compared with peritoneal neutrophils isolated 12 h after sham laparotomy (Fig. 4A). In addition, peritoneal inflammatory monocytes expressed the IL-10R 12 h following sham laparotomy or CLP (Fig. 4B). To ascertain whether neutrophil-derived IL-10 could suppress inflammatory monocyte function, we cultured WT inflammatory monocytes from unmanipulated mice with WT or IL-10−/− neutrophils and heat-killed stool. WT neutrophils produced the majority of the IL-10 (Fig. 4C). WT monocytes cultured alone with heat-killed stool produced large quantities of TNF and IL-6, which were reduced in the presence of WT but not IL-10−/− neutrophils (Fig. 4C). Thus, neutrophil-derived IL-10 decreased inflammatory monocyte cytokine production after polymicrobial stimulation in vitro. To prove the relevance of these findings in vivo, we injected CD45.2+ WT or IL-10−/− neutrophils i.p. into WT CD45.1+ mice 6 h after CLP. Six hours later, peritoneal inflammatory monocytes from mice receiving WT neutrophils expressed lower levels of TNF by intracellular cytokine analysis compared with monocytes from mice given IL-10−/− neutrophils (Fig. 4D). Thus, neutrophil-derived IL-10 suppressed peritoneal inflammatory monocyte function in vivo in the setting of polymicrobial sepsis.

Figure 4. Neutrophil IL-10 suppresses inflammatory monocyte function.

Figure 4.

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WT mice underwent sham laparotomy or CLP, and peritoneal cells were harvested 12 h later. (A) Peritoneal neutrophils were sorted by flow cytometry and cultured for 18 h. Supernatant IL-10 was determined by CBA. (B) Inflammatory monocytes were assessed for IL-10R expression. (C) WT GFP+ bone marrow monocytes were isolated from CCR2-GFP+/− mice by flow cytometric cell sorting, stimulated with heat-killed stool, and cultured alone or in combination with WT or IL-10−/− bone marrow neutrophils, isolated by immunomagnetic beading for Ly6G, for 24 h. Supernatant IL-10, TNF, and IL-6 were determined by CBA. (D) WT CD45.1+ mice underwent CLP. Ly6G+ neutrophils were isolated from the bone marrow of CD45.2+ WT and IL-10−/− mice by immunomagnetic beading. Six hours later, 107 CD45.2+ WT or IL-10−/− neutrophils were injected i.p., and mice were killed 12 h following CLP. Peritoneal inflammatory monocytes were assessed for expression of intracellular TNF. (A–C) Data are representative of at least two independent experiments with similar results. (A, C, and D) Data represent mean ± sem. (D) Three to five mice/group were used. Peritoneal cells from respective groups were pooled prior to intracellular cytokine analysis. One of three experiments with similar results is shown (P<0.01); *P < 0.05; **P < 0.01; ***P <0.001.

DISCUSSION

Neutrophils have long been considered a vital component of host defense. However, using a depleting antibody specific for neutrophils, we have shown that neutrophils are not essential for survival in a murine model of polymicrobial sepsis. The Gr1 epitope is expressed on the neutrophil-specific membrane protein Ly6G, as well as Ly6C, which is expressed on monocytes [4, 18–20, 31]. Fleming et al. [16] showed that the RB6-8C5 mAb to Gr1 detected Ly6G and Ly6C, whereas the 1A8 mAb reacted only with Ly6G and not Ly6C. In our hands, we found that following CLP, peritoneal CD11bintLy6Chi inflammatory monocytes express intermediate levels of Gr1 (data not shown). Therefore, αGr1 will bind to and deplete inflammatory monocytes in addition to neutrophils when used in vivo. Data from prior studies of neutrophils in infection and sepsis may be confounded by the use of the nonspecific αGr1 antibody [4, 7, 9–15]. Hoesel et al. [7] reported that administration of αGr1 prior to CLP increased bacteremia and liver and renal dysfunction, whereas depletion starting at 12 h following CLP actually improved liver and renal function and increased survival. The low dose of αGr1 (25 μg) used in their study was purported to deplete neutrophils selectively. However, we found that as little as 10 μg αGr1 depleted >50% of inflammatory monocytes from the spleen (Supplemental Fig. 1). This discrepancy could be a result of our route of injection of antibody (i.p. vs. i.v.) or because we used flow cytometry to analyze cellular depletion, whereas past studies have used manual differential counting. Furthermore, we found no difference in survival whether αLy6G was given prior to CLP or 12 h after CLP when compared with control IgG (Fig. 1C and Supplemental Fig. 2).

The sequelae of septic shock are often attributed to the overproduction of TNF, which is produced by nearly all leukocytes [32]. Administration of high doses of TNF causes signs and symptoms indistinguishable from septic shock [33]. In vivo neutralization of TNF using antibodies directed against TNF prevented shock and mortality from lethal doses of E. coli [34] and LPS [35]. However, TNF is also important for fighting pathogens by activating neutrophils and monocytes, promoting their adherence and migration to sites of infection [32]. TNF, produced in proper amounts by the appropriate cells, is essential for promoting resistance to pathogens [22]. Mice deficient for TNF production in neutrophils and macrophages demonstrated higher mortality from infection with L. monocytogenes [22]. Neutralization of TNF in CLP has been shown to increase mortality if performed during the first 8 h after induction of sepsis [23]. Therefore, the amount and cellular source of TNF are important in shaping the immune response to infection. We found that selective depletion of neutrophils did not alter systemic (Fig. 1E) or local (Fig. 3C) TNF levels. However, depletion of neutrophils and inflammatory monocytes resulted in higher serum levels of TNF (Fig. 1E) and lower levels of i.p. TNF (Fig. 3C). High systemic TNF levels likely resulted from uncontrolled infection and widespread activation of inflammatory cells. The reduction of i.p. TNF was consistent with the depletion of inflammatory monocytes, which we found to be the predominant cells producing TNF in the peritoneum (Fig. 2A–C).

Neutrophils have demonstrated regulatory properties in the setting of hemorrhagic shock by down-regulating IL-6 within the liver and lungs [36]. Neutrophils are recognized as being able to modulate macrophages in sterile inflammation and single pathogen infection models [37, 38], but the effects of neutrophils on inflammatory monocytes have not been reported previously. Daley et al. [37] showed elevated levels of TNF and superoxide anion release from wound macrophages when neutrophils and presumably inflammatory monocytes were depleted with αGr1 in the polyvinyl alcohol sponge wound model of inflammation. The authors concluded that an unidentified soluble factor released from neutrophils was responsible for this suppression. Our data indicate that selective neutrophil depletion increased inflammatory monocyte antimicrobial function within the peritoneum, as measured by activation phenotype, phagocytic capacity, oxidative burst, and a twofold increase in expression of TNF and iNOS (Fig. 3). Thus, enhanced function of inflammatory monocytes may have compensated for neutrophil depletion in our model of septic peritonitis, ultimately resulting in equivalent bacterial clearance and survival as in nondepleted mice.

IL-10 is an immunoregulatory cytokine produced by multiple cell types [39]. Monocyte function is regulated in part by the autocrine effects of IL-10 [4]. In fact, we found that monocytes cultured in the presence of heat-killed stool up-regulated TNF and IL-6 production approximately sixfold compared with when αIL-10 was added (data not shown). Zhang et al. [4] reported that neutrophil-derived IL-10 down-regulated TNF expression by macrophages, DCs, and CD11b+CD115+ monocytes within the lung during infection with M. bovis. Inflammatory and resident monocytes are included within this CD115+ population [20], and conclusions about the Ly6Chi subset of inflammatory monocytes cannot be made definitively. In their model, depletion of neutrophils with αLy6G increased mycobacterial burden within the lung. In contrast to their findings, we showed that depletion of neutrophils did not affect bacterial burden locally or systemically (Fig. 1F). Thus, the importance of neutrophils depends on the type and location of infection. We demonstrated neutrophil IL-10 production in response to polymicrobial stimulation in vivo and in vitro (Fig. 4A and C). Our in vitro data show that inflammatory monocyte function is suppressed by neutrophil IL-10 (Fig. 4C). We were also able to demonstrate this relationship in vivo through adoptive transfer of WT or IL-10−/− neutrophils into WT mice following CLP (Fig. 4D).

This study does not rule out the possibility that in addition to IL-10, there are other neutrophil-mediated mechanisms of suppression of inflammatory monocytes in our model. It is well established that ingestion of apoptotic cells can suppress the proinflammatory phenotype of effector immune cells, including macrophages [40–43]. In a model of systemic listeriosis, Holub et al. [38] demonstrated that apoptotic neutrophils suppressed the proinflammatory response after being ingested by Kupffer cells. Neutrophils are short-lived cells with a half-life of 6–10 h and upon activation, undergo spontaneous apoptosis [44]. We have confirmed that neutrophils recruited to the peritoneum following CLP undergo apoptosis, and the absolute number and percentage of apoptotic neutrophils increase during the first 24 h after CLP (data not shown). Inflammatory monocytes recruited to the peritoneum may ingest these apoptotic neutrophils as part of their function in clearing the peritoneum of bacteria and devitalized tissue, thereby suppressing their inflammatory phenotype. In fact, when we adoptively transferred CFSE-labeled neutrophils i.p. 6 h following CLP, we found that 10% of peritoneal inflammatory monocytes were CFSE+, 12 h following CLP (data not shown). Neutrophils also secrete other soluble mediators such as PGE2 and adenosine, which have been shown to modulate macrophage TNF production [37]. MDSCs are a heterogenous group of cells that include neutrophils and also express Ly6G [45]. MDSCs accumulate in the spleens of septic mice and suppress T cell function [46], however they have not been described within the peritoneum of septic mice following CLP. It is possible that in addition to neutrophils, other subsets of MDSCs contribute to the regulation of inflammatory monocytes within the peritoneum and that these cells were depleted in mice treated with αLy6G.

This study does not address whether neutrophils can compensate for an absence of inflammatory monocytes during CLP. CCR2−/− mice have defective recruitment of Ly6Chi monocytes to sites of infection and demonstrate increased mortality following infection with T. gondii, L. monocytogenes, and M. tuberculosis [20]. However, it has been shown that CCR2 also mediates neutrophil recruitment [47, 48]; therefore, the CCR2−/− mouse or global blockade of CCR2 may not be the ideal way to study the contribution of inflammatory monocytes in CLP. Currently, there is no commercially available reagent for the specific depletion of Ly6Chi monocytes.

One of the cornerstones in the treatment of sepsis is to control the source of infection [49]. We characterize an interaction previously unrecognized by which neutrophils suppress peritoneal inflammatory monocytes through secretion of IL-10. Several studies have examined the role of neutrophils in infection using αGr1 for depletion [7, 9–15], and results have been variable. Some studies conclude that neutrophils are harmful, and other studies conclude that they are essential. In contrast, our data indicate that neutrophils are dispensable for survival in a clinically relevant model of sepsis.

Supplementary Material

Supplemental Figures
supp_89_3_423__index.html (698B, html)

ACKNOWLEDGMENTS

This work was supported by National Institute of Health grants AI70658 and DK068346 (R.P.D.). We are grateful to Mesruh Turkekul of the Molecular Cytology Core Facility (Sloan-Kettering Institute) for technical assistance.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

α
anti
BHI
brain-heart infusion
CLP
cecal ligation and puncture
MDSC
myeloid-derived suppressor cell

AUTHORSHIP

L.M.O. designed and performed all experiments and wrote the manuscript. Z.M.B., V.P.B., M.J.C., H.O., and G.P. contributed to the experimental design, execution of the experiments, and provided editorial comments to the preparation of the experiments. R.P.D. is the principal investigator and contributed to all aspects of experimental design and manuscript preparation.

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