• Myeloid cells;
  • Inflammation;
  • Receptors


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and discussion
  5. 3 Materials and methods
  6. Acknowledgements

Recruitment of myeloid cells during inflammatory reactions plays an important role in the propagation and resolution of inflammation. However, the identification and characterization of these cells in mice has been hampered by cellular heterogeneity at the functional and phenotypic level. We have defined criteria for the rapid flow-cytometric identification of monocytes (Mo), macrophages (MΦ), neutrophils (Neu) and eosinophils (Eos) in murine tissues using novel and established myeloid markers. These criteria were applied to the study of naive mice and mice with experimentally induced inflammation, both local and systemic, and also to a murine model of tumor progression. We show that the murine 7/4 antigen and the β-glucan receptor, Dectin-1, are particularly useful for the sub-division of myeloid cells into individual populations, even when inflammatory conditions modulate their surface expression. Furthermore, 7/4 expression allows distinction between Mo recently recruited to a site and the resident cells already present. These studies highlight the heterogeneity of the murine Mo/MΦ-lineage, define an extended phenotype for murine myeloid cells and greatly facilitate the ex vivo characterization of these cells during very different models of inflammation.








β-glucan receptor


MΦ mannose receptor


Forward scatter


Side scatter

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and discussion
  5. 3 Materials and methods
  6. Acknowledgements

Myeloid cells play a central role in inflammation. In the early acute stage, tissue resident macrophages (MΦ), granulocytes and mast cells, as well as the complement system and soluble inflammatory mediators, all contribute to the host response to injury. Later, MΦ recruited to the inflammatory lesion play a dominant role in the maintenance and resolution of inflammation 1. The ability to track leukocyte movements in various mouse models of inflammation has been invaluable in defining the roles of the selectins 2, integrins 3, Fc-receptors 4, complement 5 and chemokines 68 in the evolution of inflammatory cell recruitment and the subsequent propagation of inflammation.

Studies of murine inflammation often rely on laborious assessment of cellular infiltrates by differential cell counting or by indirect assay of cell recruitment [such as the presence of myeloperoxidase as a marker of neutrophils (Neu) recruitment], as there are currently no definitive markers available for the specific flow-cytometric identification of murine myeloid cells either in naive or immune challenged mice 59. Several markers for staining murine myeloid cells are readily available, which provide a limited ability to identify andcharacterize myeloid cells. CD11b is highly expressed on many myeloid cells, such as monocytes (Mo), Neu, eosinophils (Eos) and subsets of MΦ 10. However, CD11b is notably absent from the surface of many MΦ such as alveolar MΦ 11, several MΦ populations in the spleen and Kupffer cells in the liver 12, 13. Furthermore CD11b is expressed by NK cells 10, some dendritic cells (DC) 14 and subsets of lymphocytes. F4/80 is oneof the best characterized MΦ markers present on most tissue MΦ, but it is expressed at low levels on alveolar MΦ and Mo and not expressed by some subpopulations present in the splenic white pulp and the thymus 15. This marker, however, is also not unique to MΦ, its expression being detected on Eos16 and subsets of DC 17. The Gr-1 epitope, which is found on two receptors Ly-6G and Ly-6C has been useful in the initial identification of Neu, by their high expression 18, but it has now become apparent that many cell types (Neu, Mo, Eos, plasmacytoid pre-DC and subsets of T cells 1821) express this epitope. An additional useful but uncharacterized Neu marker, the polymorphic 7/4 antigen, has been shown to be expressed on Neu22 and subsets of immunologically activated MΦ 23, 24, and has been successfully used to deplete myeloid committed cells from murine bone-marrow 25, 26. We have recently identified the murine β-glucan receptor (βGR) 27 and using a novel mAb (2A11) demonstrated that βGR was predominantly expressed on Mo, Neu and MΦ 28, 29. Expression of βGR was notably absent from Eos, but was detected on some DC and at lower levels on Gr-1+ T cells and plasmacytoid DC 29. We have also generated monoclonal antibodies against the murine MΦ mannose receptor (MR) 30, which is mainly expressed by MΦ and selected endothelial cells in situ31. Although surface expression seems to be low, compared to the intracellular pool, we have been able to assess surface expression by FACS on several isolated MΦ populations 29.

To characterize myeloid cells directly and to define new criteria for their rapid ex vivo identification we have utilized the above mentioned receptors and differentiation antigens in combination. Both 7/4 and βGR in conjunction with the more established myeloid markers CD11b, F4/80 and Gr-1 provide an efficient approach to the analysis of resting leukocyte populations from multiple tissues. Furthermore, the extended phenotypes that we have defined in this manuscript are retained during several models of local and systemic inflammation validating this approach for general myeloid cell analysis during immunological challenge. These studies highlight the significant heterogeneity of expression of these antigens within the Mo/MΦ-lineage, allow recently recruited Mo to be distinguished from other inflammatory and resident myeloid cells present in a lesion and form a basis for future characterization of myeloid cell subpopulations and the analysis of cell influx/efflux and death at the site of injury.

2 Results and discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and discussion
  5. 3 Materials and methods
  6. Acknowledgements

2.1 Evaluation of the expression of 7/4 and βGR by murine myeloid cells

To develop new approaches for the characterization of murine myeloid cells during steady-state and inflammatory conditions we first reassessed the expression of the murine myeloid differentiation antigen 7/4 22 and compared it with the recently described expression pattern of βGR, which exhibits predominantly myeloid expression with highest surface levels in naive animals found on Mo and Neu29. The murine 7/4 antigen is polymorphic and has been reported to be expressed on Neu22 and immune activated MΦ during infection 23, 24. Both of these antigens were compared with the more established myeloid antigens: F4/80, CD11b and Gr-1 (Ly-6G and Ly-6C).

We initially studied the surface expression of the murine 7/4 antigen and βGR on myeloid cells obtained from the peripheral blood, bone-marrow, and spleen of C57BL/6J and BALB/c mice (Fig. 1 and data not shown). Gr-1high Neu in the peripheral blood were found to express high levels of 7/4 and βGR (Fig. 1A). Gr-1int Mo, had higher surface expression of 7/4 (Fig. 1A) and βGR (as previously shown 29) than Neu. The identity of these surface 7/4highGr-1int cells as Mo was confirmed by their co-expression of F4/80 (Fig. 1A), demonstrating for the first time that 7/4 is not restricted to Neu. Similar 7/4 surface expression patterns were observed on Mo and Neu from the bone marrow (Fig. 1B) and spleen (Fig. 1C). We also assessed the expression of 7/4 in the polymorphic BALB/c (7/4) strain 22. Very weak immunoreactivity was observed on BALB/c Mo, but not on Neu (Table 1 and data not shown), indicating that 7/4 mouse strains are not totally deficient of antigen at the protein level with the differences a consequence of polymorphic expression or structure. Neu from the spleen and bone marrow both exhibited higher CD11b surface expression than those found in the peripheral blood, probably representing priming/activation of the neutrophils during isolation 32 or possibly differences in maturation.

The Gr-1 antigen itself is composed of two distinct antigens (Ly-6G and Ly-6C) and is expressed on Neu, Mo, subsets of MΦ, subsets of T cells, and plasmacytoid pre-DC 18, 19, 29. A rat IgM monoclonal antibody (clone AL-21) specific for mouse Ly-6C was included in the FACS staining of freshly isolated splenocytes to block the Gr-1 epitope on Ly-6C. This confirmed that neutrophils exhibit almost unique expression of Ly-6G (in naive animals) and that the majority of Gr-1 expression on other cell types is attributable to Ly-6C (Fig. 1D).

We compared the observed Mo phenotype to that of splenic auto-fluorescent MΦ and MΦ from other tissues and inflammatory contexts (Table 1 and 28, 29). The splenic auto-fluorescent MΦ were found to be F4/ 80+MR+MHCIIintCD11clowCD11blow/–βGRlow/–7/4 (Table 1 and data not shown). This phenotype resembles the F4/ 80+MR+ immunohistochemical phenotype of ‘red-pulp MΦ' 12, 13, 31. Whilst the overall phenotype of Mo from multiple non-inflamed tissues was consistent, there was no common MΦ phenotype when comparing these splenic MΦ with MΦ from different tissues and inflammatory lesions (Table 1). 7/4 expression has been reported to be absent from tissue resident and thioglycollate elicited peritoneal MΦ 22, an observation which we have confirmed by FACS analysis (see below and data not shown). However, Mo express very high surface levels of 7/4 indicating that 7/4 expression may be a marker which is down-regulated during the differentiation of Mo into MΦ.

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Figure 1.  Comparison of 7/4 and βGR expression to that of Gr-1 and CD11b. (A) On peripheral blood leukocytes highest surface expression of both 7/4 and βGR is seen on Mo and both antigens are also highly expressed on Gr-1highNeu (as indicated by labeled regions). Expression of F4/80 by 7/4high cells confirms their identity as Mo. (B) A similar pattern of 7/4 surface expression is observed in the bone-marrow with the Gr-1int cells of the Mo/MΦ-lineage exhibiting the highest surface expression and Gr-1highNeu also having high expression. (C) In the spleen, a similar staining profile was also seen on Mo and Neu (see also Fig. 3). In all cases where not shown, Mo identity was confirmed by F4/80 labeling. (D) Gr-1 (Ly-6G/C) is expressed by multiple cell types in the spleen, but Ly-6G is largely restricted to Gr-1highNeu. FACS labeling was performed in the presence of rat IgM anti-Ly-6C to block this component of the Gr-1 epitope. Although slightly reduced, residual Gr-1 staining (‘Ly-6G’) was largely restricted to Neu.

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Table 1. Flow cytometric evaluation of myeloid cell phenotype from various mouse tissuesa)
 Surface expression of markersa)
Myeloid populationF4/80CD11bβGRMR7/4b)
  1. a) The table shows a comparison of data from this manuscript with our previously published data 28, 29. An arbitrary scoring system is used for each marker and ‘+/–’ denotes marginal if any expression. Expression data on murine Mo and Neu reflects a consistent phenotype found in bone marrow, peripheral blood and spleen of mice. ND, not done.

  2. b) Parentheses for 7/4 expression indicate immunoreactivity of mAb7/4 on cells from the 7/4 mouse strain, BALB/c (all other staining patterns are derived from 7/4+ C57BL/6 mice).

Splenic ‘auto-fluorescent’ Mϕ ++++/–+/–+++
Resident peritoneal Mϕ +++++++++++/–
Thioglycollate-elicited Mϕ ++++++++++++
Resident alveolar Mϕ +++++++ND

2.2 Analysis of marker expression by murine Eos

Murine Eos can be identified within the Gr-1intCD11b+ splenocyte fraction (Fig. 2A). This phenotype resembles that of Mo, however, they can be readily separated from Mo by their very high side scatter (SSC) (Fig. 2A). As previously reported 16, murine Eos consistently express F4/ 80 (Fig. 2A). We found that Eos expressed low levels of 7/4 on their surface (Fig. 2A), but as previously noted do not express βGR 29. Eosinophils were similarly readily identifiable in peritoneal inflammatory infiltrates recruited in response to thioglycollate by their F4/ 80+CD11b+SSCv.high phenotype, which distinguishes them from recruited monocytes and MΦ (Fig. 2B). The number of eosinophils identified in this way was confirmed by differential counting on cytospin preparations stained with Hema Gurr (BDH) (data not shown).

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Figure 2.  Flow cytometric characterization of Eos. (A) Eos freshly isolated from the spleen exhibit a similar phenotype to Mo (F4/80+CD11b+Gr-1int) but are separable from these cells by their very distinctive high SSC. The identity of the SSCv.highGr-1+CD11b+ cells (boxed) as Eos was confirmed by strong F4/80 staining. Eos were also found to express 7/4, albeit at much lower levels than Mo and Neu (staining shown obtained with biotinylated mAb7/4). (B) Eos can be similarly identified in inflammatory infiltrates by gating on F4/80+SSCv.high cells. The dot plot on the upper left represents the inflammatory infiltrate elicited 18 h after i.p. administration of 1 ml of 4% thioglycollate medium. Gating on the F4/80+ inflammatory cells (upper right panel) shows the SSChigh Eos (boxed) and MΦ. The dot plots on the lower left and right show the F4/80 and CD11b profiles for Eos and MΦ, respectively.

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2.3 Characterization of myeloid cell heterogeneity during inflammation

To evaluate the usefulness of these markers in the measurement of inflammatory cell infiltrates we studied three different and established experimental models of inflammation. Systemic inflammation was induced by intravenous administration of 5 μg LPS. Fig. 3A shows FACS analysis of murine splenocytes 24 h after challenge. LPS injected mice exhibit a marked increase in Gr-1+CD11b+ cells in the spleen. CD11bhigh splenocytes were gated and then separated into Mo/MΦ and Neu by expression of 7/4 and βGR. Both Mo/MΦ and Neu were expanded after systemic LPS exposure (Fig. 3A). Although the receptors expressed by the expanded myeloid cell populations remained similar to the ones expressed in naive populations, expression of 7/4 and βGR was reduced on the surface of Mo/MΦ cells and F4/80 was markedly increased compared to control animals (Fig. 3A). A reduction in Neu βGR surface expression was also evident after LPS challenge. The variations in the level of βGR, 7/4 and F4/80 on Mo/MΦ after LPS challenge highlight the potential for increased myeloid cell heterogeneity as a direct result of marker regulation. These alterations could be due to receptor regulation, Mo maturation or a complex interplay of secondary factors induced by LPS in vivo. In the case of βGR, we have seen a down modulation of surface receptor expression induced by LPS, on isolated MΦ in vitro (J. Willment et al., in submission). A preliminary study of F4/80 expression after in vitro challenge of isolated MΦ with LPS identified an increase in surface antigen (data not shown). Both of these observations are consistent with the above described in vivo phenotype.

This method was also used to assess the recruitment of myeloid cells in a syngeneic AKR tumor progression model, in order to test the conservation of the myeloid cell phenotypes in different inflammatory models. A marked increase in the number of Gr-1+CD11b+ splenocytes has been previously observed in this model 33. Gating on CD11bhigh splenocytes in a similar way to Fig. 3A above showed that these cells could also be readily separated into Mo/MΦ and Neu by significantly higher surface expression of 7/4 and βGR on the Mo/MΦ and that both of these populations are expanded during tumor progression (data not shown). This model of tumor progression differed from systemic LPSchallenge in that antigen levels on the cells were not directly affected compared to the unchallenged animals (data not shown). The Gr-1-F4/80+βGR+7/4low/– splenocytes identified in these studies (Fig. 3A and data not shown) also express CD11c and MHCII (data not shown) and hence could represent the subsets of DC, which are known to express F4/80 and CD11b 17, or possibly distinct populations of MΦ.

We next examined the local recruitment of myeloid cells during the early stages of inflammation using the well-established intraperitoneal Zymosan administration model 9, 34 with the aim of identifying recently recruited Mo in the inflammatory infiltrate. Peritoneal cells were recovered up to 4 h after administration of Zymosan and characterized by flow cytometry (Fig. 3B and C). Few 7/4+ cells were present in the peritoneum of naive mice, but by 2 h after administration there was a marked influx of 7/4+Gr-1+ cells. By 4 h after Zymosan administration, these 7/4+ inflammatory cells (mostly Gr-1highF4/80 Neu) included Gr-1intF4/80+ Mo (Fig. 3B). Resident peritoneal MΦ (F4/80v.high), disappeared from the peritoneal cavity during this challenge as reported 3537 and this most likely reflects migration out of the peritoneal cavity to the draining lymph nodes 36. The early phase of the inflammatory myeloid response to i.p. administration of Zymosan assessed in this way is summarized graphically in Fig. 3C. These studies show that myeloid cell subsets can be rapidly identified by flow cytometry and that recently recruited Mo can be specifically identified amidst complex cell mixtures with shared antigen expression.

In this manuscript we have studied the heterogeneity of expression of the murine pattern recognition receptor βGR and the differentiation antigen 7/4 on myeloid cells in the adult mouse, both unstimulated and during inflammatory responses. We have shown that the undefined 7/4 antigen, previously reported to be restricted to Neu22 and some MΦ in immunological challenged mice 23, 24 is also expressed on peripheral Mo, at higher surface levels than Neu, and, albeit at much lower levels, on Eos. Whilst these studies highlight the lack of definitive myeloid cell subset-specific markers in the mouse, we show that combination of the established myeloid antigens with either 7/4 or βGR permits rapid and simple flow cytometric separation of Mo from Neu, a principle which applies to myeloid cells in the bone marrow or periphery. Eos, which exhibit similar labeling patterns to Mo with respect to the conventional antigens used (F4/80, Gr-1 and CD11b), can be distinguished from Mo by their 7/4lowβGRSSChigh phenotype. The extended marker phenotype of murine myeloid cells is summarized in Table 1 and defined in naive mice as: Neu, Gr-1highCD11b+ 7/4+βGR+SSChigh; Mo, F4/80+Gr-1intCD11b+7/4highβGR+; Eos, F4/80+CD11b+7/4lowβGR-SSCv.high.

The majority of tissue MΦ, such as those resident in the peritoneum or elicited after thioglycollate treatment (both of which are characterized by an F4/80+CD11b+ phenotype, with the latter being separated from Eos as described in Fig. 2) and those in the spleen, are essentially 7/4 (this study and data not shown). This indicates that 7/4 expression on naive Mo is lost as they differentiate to MΦ. However, as mentioned, 7/4 expression has been reported on some MΦ in immunologically challenged mice 23, 24. These cells could possibly represent activated populations of MΦ that are able to express the 7/4 antigen or recently emigrated Mo or Mϕ-like cells with a more immature phenotype. Identification of the 7/4 antigen would enhance our understanding of its expression and how this relates to its function.

These studies represent a coherent and rapid approach to the simultaneous identification and phenotypic characterization of multiple myeloid cells in naive and immunologically challenged mice.We have defined an extended phenotype for murine myeloid cells which greatly facilitates the evaluation of inflammatory cell infiltrates, and permits identification of recently recruited 7/4+ Mo in inflammatory lesions. This analysis has also highlighted the marked surface heterogeneity of cells of the Mo/MΦ-lineage, which most likely represents specialization of function dependent on both tissue of residence and inflammatory context, and forms a foundation for the future exploration of this heterogeneity ex vivo.

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Figure 3. Characterization of myeloid cell heterogeneity during inflammation. (A) 24 h after i.v. LPS, gating on CD11bhigh splenocytes allows simple identification of Gr-1high7/4+βGR+ Neu, Gr-1int7/4highF4/80+βGRhigh Mo/MΦ and F4/80+7/4lowβGR- Eos. After systemic LPS challenge a marked expansion is evident in the number of CD11b+Gr-1+ splenocytes. These cells are predominantly of the Neu and Mo/MΦ phenotype. Percentages reflect prevalence of the indicated myeloid subsets of total splenocytes (left panels). The other plots are gated on CD11b+ splenocytes. All profiles are representative examples. In this example, of the CD11b+ splenocytes 37.8% are Neu and 22.7% are Mo prior to challenge and 61.2% and 26.21% are Neu and Mo, respectively post-challenge (derived from the gated regions). (B) Zymosan challenge (i.p.) of C57BL/6 mice resulted in a depletion of resident peritoneal MΦ from the peritoneal cavity (F4/80high ‘res MΦ’). 7/4+ inflammatory cells (boxed in left panels) recruited into the peritoneal cavity could be divided into Neu and recently recruited Mo, respectively (central panels, gated on 7/4+ cells). F4/80 expression by Mo was confirmed with isotype control antibody (data not shown). Among the 7/4low– cells (right panels) were Eos identified by their expression of F4/80 and high SSC (not shown). (C) Graphical summary of the composition of inflammatory infiltrate after i.p. Zymosan challenge Data represent the results of analysis of cells pooled from three mice at each time-point and are representative of two experiments.

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3 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and discussion
  5. 3 Materials and methods
  6. Acknowledgements

3.1 Tissue and cell preparation

All mice used in this study were between 8 and 12 weeks of age and were C57BL/6J (bred within our own colonies) or, in the case of the tumor model, AKR (which were obtained from Harlan, Zeist,The Netherlands). Animals were kept and handled in accordance with institutional guidelines. Splenocytes were harvested by standard methods using a combination of digestion with "Liberase BlendzymeII" in RPMI (Roche Molecular Biochemicals) and mechanical dissociation. Femurs were collected and fresh bone marrow was flushed from within using Liberase Blendzyme II and incubated for 10 min at 37oC to disaggregate cells. Enzymatic activity was quenched with RPMI:20%FCS, erythrocytes lysed with Gey's hypotonic solution and cell debris removed by centrifugation through 100% FCSat 300×g.

Peripheral blood was collected by cardiac puncture into 0.1 volume of 100 mM EDTA, pH 8.0. Cells were harvested by centrifugation and resuspended in 50 volumes of Gey's solution to lyse erythrocytes. Peripheral blood leukocytes were then recovered by centrifugation through FCS as described above.

3.2 Induction of inflammation

To induce sterile peritonitis, mice were injected intraperitoneally with 4% thioglycollate broth (BD) or 0.5 mg of Zymosan A particles (Molecular Probes) up to 4 days prior to peritoneal lavage. After humane sacrifice, inflammatory cells were collected by peritoneal lavage with ice-cold 5 mM EDTA in PBS. Resident peritoneal cells were collected in the same way from untreated animals.

Systemic LPS challenge was achieved by a single i.v. injection of 5 μg of LPS from Salmonella typhimurium (Sigma) in PBS. Splenocytes were harvested as described above 24 h after the treatment.

3.3 Murine tumor model

The BW-Sp3 tumor cell line, which was derived from the spontaneous BW5147 T lymphoma (AKR origin) by in vitro and in vivo passages as described 38, was injected subcutaneously (2×106 cells in PBS per mouse). Mice with naturally progressing tumors 33 were identified after 20 days and were assayed for an expansion of CD11b+Gr-1+ myeloid cells as reported 33.

3.4 Flow cytometry

FACS of surface antigen expression was performed according to conventional protocols at 4oC in the presence of 2 mM NaN3. Cells were blocked with 5% heat-inactivated rabbit serum; 0.5% BSA; 5 mM EDTA and 10 μg/ml 2.4G2 (anti-FcγRII and III) prior to the addition of primary antibodies. Biotinylated antibodies were detected using streptavidin-allophycocyanin (BD PharMingen). Cells were fixed with 1% formaldehyde in PBS prior to analysis on a BD FACScalibur with Cell Quest software. All FACS profiles shown in this report are representative of individual mice from reproducible experiments except for analysis of peripheral blood and induction of peritonitis where three to six mice were pooled for each time point to reduce biological variation.

The following antibodies were used in this study: F4/80-PE (Serotec), Gr-1-PE (anti-Ly6C/G; BD PharMingen), 5C6-FITC (Serotec; anti-CR3/CD11b 39), AL-21 (rat IgM anti-Ly-6C;BD PharMingen) 7/4-FITC/biotin (Serotec; rat IgG2a ‘anti-neutrophil’ 22), 2A11-biotin (rat IgG2b anti-βGR 28), 5D3-biotin (rat IgG2a anti-MR 30) and irrelevant isotype-matched control antibodies.


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results and discussion
  5. 3 Materials and methods
  6. Acknowledgements

We would like to thank the staff of our animal facilities for the care of the animals used in these studies and Dr. Jo Miller for critical reading of the manuscript. We would also like to thank Simon Wong and Delyth Reid for help with the production of mAb 2A11 and mAb 5D3. This work was funded by the Wellcome Trust and the Medical Research Council.

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