About InVivoMAb anti-mouse F4/80
The CI:A3-1 monoclonal antibody reacts with mouse F4/80 also known as EMR1. F4/80 is a 160 kDa membrane glycoprotein belonging to the EGF-TM7 family. F4/80 is expressed on most tissue macrophages including peritoneal, lung, gut, thymus and splenic red pulp macrophages, as well as Kuffer cells, Langerhans cells, bone marrow stromal cells, and a subset of dendritic cells. F4/80's cognate ligand has not been identified however studies in mice have shown that F4/80 is important for cytokine production and induction regulatory T cells.
InVivoMAb anti-mouse F4/80 Specifications
|Isotype||Rat IgG2b, κ|
|Recommended Isotype Control(s)|
|Recommended Dilution Buffer|
|Immunogen||C57BL/6 mouse thioglycollate stimulated peritoneal macrophages|
|Sterility||0.2 μM filtered|
|Production||Purified from tissue culture supernatant in an animal free facility|
|Molecular Weight||150 kDa|
|Storage||The antibody solution should be stored at the stock concentration at 4°C. Do not freeze.|
InVivoMAb anti-mouse F4/80 (Clone: CI:A3-1)
Paul, S., et al. (2019). “Natural killer T cell activation increases iNOS(+)CD206(-) M1 macrophage and controls the growth of solid tumor.” J Immunother Cancer 7(1): 208. PubMed
BACKGROUND: NKT cells play an important role in anti-tumor immunity. Alpha-galactosylceramide (α-GalCer), a synthetic glycolipid is presented to natural killer T (NKT) cells by most antigen-presenting cells through CD1d molecules leading to activation of NKT cells. However, the precise mechanisms of how α-GalCer-activated NKT regulate the polarization of the macrophages and effector T cells in the solid tumor are not studied adequately. METHODS: We induced solid tumor in C57BL/6 mice by subcutaneous injection of B16F10 cell line (1 X 10(6) cells) and monitored the tumor growth. Animals were given an intraperitoneal injection of α-GalCer (2 μg/injection) in 200 μl PBS on day + 1, + 5, + 10, + 15, and + 20 (with respect to tumor cell injection). Immune cells were characterized using flow cytometry and immunofluorescence staining. NK cells, Gr1(+) cells, and F4/80(+) macrophages in the mice were depleted by intravenous injection of cell-specific antibodies. Statistical analysis was performed using Student’s t-test or one-way ANOVA. RESULTS: Our results showed that intratumoral NKT cells have a lower frequency of CD69, CD25, CD122, and IFN-γR expression; produced less inflammatory cytokines such as IFN-γ, TNF-α, and GM-CSF; higher frequency CD62L(+) NKT cells; and also showed reduced proliferation as compared to the splenic NKT cells. Mice treated with α-GalCer showed a significantly increased frequency of IFN-γ-producing NKT cells, CD8(+) T cells, and effector Th1 cells. Depletion of NK cells in α-GalCer-treated mice showed a lower frequency of IFN-γ-producing CD4(+) and CD8(+) T cells in the tumor and prevented the α-GalCer-induced tumor growth. NKT cell activation with α-GalCer treatment significantly increased the iNOS(+)CD206(-) M1-macrophages and reduced the iNOS(-)CD206(+) M2-macrophages in the spleen and tumor, and depletion of F4/80(+) macrophages prevented the α-GalCer-induced reduction in the tumor growth. CONCLUSIONS: We showed that activation of NKT cell with α-GalCer modulates the frequency of M1-macrophages and effector Th1 cells in the secondary lymphoid tissues and tumor microenvironment and inhibit tumor growth. The finding suggests that activation of NKT cells with α-GalCer may provide an effective anti-cancer outcome.
Wang, E. C. E., et al. (2019). “A Subset of TREM2(+) Dermal Macrophages Secretes Oncostatin M to Maintain Hair Follicle Stem Cell Quiescence and Inhibit Hair Growth.” Cell Stem Cell 24(4): 654-669.e656. PubMed
Hair growth can be induced from resting mouse hair follicles by topical application of JAK inhibitors, suggesting that JAK-STAT signaling is required for maintaining hair follicle stem cells (HFSCs) in a quiescent state. Here, we show that Oncostatin M (OSM), an IL-6 family cytokine, negatively regulates hair growth by signaling through JAK-STAT5 to maintain HFSC quiescence. Genetic deletion of the OSM receptor or STAT5 can induce premature HFSC activation, suggesting that the resting telogen stage is actively maintained by the hair follicle niche. Single-cell RNA sequencing revealed that the OSM source is not intrinsic to the hair follicle itself and is instead a subset of TREM2(+) macrophages that is enriched within the resting follicle and deceases immediately prior to HFSC activation. In vivo inhibition of macrophage function was sufficient to induce HFSC proliferation and hair cycle induction. Together these results clarify how JAK-STAT signaling actively inhibits hair growth.
Wang, W., et al. (2018). “RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer.” Cancer Cell 34(5): 757-774 e757. PubMed
Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCII(hi)TNFalpha(+)IFNgamma(+) immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.
Albacker, L. A., et al. (2013). “TIM-4, expressed by medullary macrophages, regulates respiratory tolerance by mediating phagocytosis of antigen-specific T cells.” Mucosal Immunol 6(3): 580-590. PubMed
Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80(+) lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Blockade of TIM-4 inhibited the phagocytosis of antigen-specific T cells by TIM-4 expressing lymph node medullary macrophages, resulting in an increase in the number of antigen-specific T cells and the abrogation of respiratory tolerance. Moreover, specific depletion of medullary macrophages inhibited the induction of respiratory tolerance, highlighting the key role of TIM-4 and medullary macrophages in tolerance. Therefore, TIM-4-mediated clearance of antigen specific T cells represents an important previously unrecognized mechanism regulating respiratory tolerance.
Tittel, A. P., et al. (2012). “Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice.” Nat Methods 9(4): 385-390. PubMed
Transgenic mice expressing the diphtheria toxin receptor (DTR) in specific cell types are key tools for functional studies in several biological systems. B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J (CD11c.DTR) and B6.Cg-Tg(Itgax-DTR/OVA/EGFP)1Gjh/Crl (CD11c.DOG) mice express the DTR in CD11c(+) cells, allowing conditional depletion of dendritic cells. We report that dendritic-cell depletion in these models caused polymorphonuclear neutrophil (PMN) release from the bone marrow, which caused chemokine-dependent neutrophilia after 6-24 h and increased bacterial clearance in a mouse pyelonephritis model. We present a transgenic mouse line, B6.Cg-Tg(Itgax-EGFP-CRE-DTR-LUC)2Gjh/Crl (CD11c.LuciDTR), which is unaffected by early neutrophilia. However, CD11c.LuciDTR and CD11c.DTR mice showed late neutrophilia 72 h after dendritic cell depletion, which was independent of PMN release and possibly resulted from increased granulopoiesis. Thus, the time point of dendritic cell depletion and the choice of DTR transgenic mouse line must be considered in experimental settings where neutrophils may be involved.
Winkler, I. G., et al. (2012). “Hematopoietic stem cell mobilizing agents G-CSF, cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation.” Leukemia 26(7): 1594-1601. PubMed
The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM. In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.
Bedoret, D., et al. (2009). “Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice.” J Clin Invest 119(12): 3723-3738. PubMed
The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.
Joffre, O., et al. (2008). “Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes.” Nat Med 14(1): 88-92. PubMed
A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.
Lloyd, C. M., et al. (2008). “Three-colour fluorescence immunohistochemistry reveals the diversity of cells staining for macrophage markers in murine spleen and liver.” J Immunol Methods 334(1-2): 70-81. PubMed
Macrophages have traditionally been identified in murine tissues using a small range of markers, typically F4/80, CD68 and CD11b. However many studies have suggested that substantial heterogeneity exists in macrophage populations, and no single marker, nor even pair of markers, can necessarily identify all the populations. Further, many of the key monoclonal antibodies have been raised in the same species, making it difficult to combine them in histochemical studies. Here we have optimised a triple colour immunofluorescent staining protocol, utilising an anti-FITC technique, to allow antibodies to macrophage markers to be used simultaneously. We highlight the substantial heterogeneity of cells in both normal liver and spleen that stain for F4/80, CD68, CD11b, and CD11c. Using diet-induced steatohepatitis as a model of liver inflammation, we show that CD11b is expressed by newly migrating macrophage precursors, but is an unreliable marker for macrophage precursors when used alone because it is also expressed by migrating neutrophils. In healthy livers CD11c expression is a unique feature of a population of cells immediately surrounding the sinusoids. However, during hepatic inflammation CD11c can also be co-expressed by other cells, including both infiltrating cells and F4/80+ cells within the liver parenchyma. While no one marker alone is sufficient to account for all macrophage populations, we confirm that F4/80 marks the majority of the tissue-resident macrophages in both the liver and the spleen, although F4/80- populations that are positive for CD68, CD11b, or CD11c also exist. Distinguishing between tissue macrophages and dendritic cells with these markers remains problematic.
Pull, S. L., et al. (2005). “Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury.” Proc Natl Acad Sci U S A 102(1): 99-104. PubMed
We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate. This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals. Analysis of conventionally raised C57BL/6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd88(-/-) bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response. Studies of B6 Csf1(op/op) (lacking macrophages) mice, Rag1(-/-) mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary. GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base. GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding. Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile “cellular transceivers” that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs.
Warschkau, H. and A. F. Kiderlen (1999). “A monoclonal antibody directed against the murine macrophage surface molecule F4/80 modulates natural immune response to Listeria monocytogenes.” J Immunol 163(6): 3409-3416. PubMed
Whole spleen cell cultures from SCID mice release high levels of IFN-gamma when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (MPhi) and NK cells: HKL-stimulated MPhi release TNF-alpha and IL-12, which together activate NK cells for IFN-gamma release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the MPhi surface glycoprotein F4/80. HKL-induced IL-12, TNF-alpha, and IFN-gamma in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when MPhi and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced MPhi/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the MPhi surface glycoprotein F4/80.