Macrophage Biology Review

 

The Biology of Macrophages - An Online Review

Edition 1.1 May 2012
David A. Hume1
1The Roslin Institute and Royal Dick School of Veterinary Studies, University of Edinburgh, EH25 9PS, Scotland, UK

Contents

Preface

Macrophage Overview: Origin, Expression, and Functions

♦The Mononuclear Phagocyte System
♦Macrophage Tissue Expression
♦Macrophage Functions

The Mononuclear Phagocyte System and Macrophage Development

♦Differentiation in the Mononuclear Phagocyte System
♦Monocyte Subsets
♦Heterogeneity and Markers in the Mononuclear Phagocyte System
♦Transcriptional Regulation in the Mononuclear Phagocyte System
♦Macrophages in Embryonic Development and Maintenance of Tissue Macrophages by Local Proliferation

Macrophage Functions and Biology

♦Macrophage Recruitment and Chemotaxis
         >Chemokines
♦Endocytosis and Phagocytosis
♦Antigen Presentation to T lymphocytes and the Dendritic Cell Myth
        >Antigen Presentation
        >Dendritic Cells

Macrophage Activation and Transcriptional Networks

♦Macrophage Activation
♦Dynamic Networks in Macrophage Differentiation and Activation

Overview

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Preface

In 2008, Siamon Gordon wrote a historical essay on the life of Elie Metchnikoff, who received his Nobel prize a century earlier (Gordon, 2008).  The father of natural immunity, as Siamon described him, is credited with the discovery that antimicrobial defence requires the specific recruitment of specialized cells, the phagocytes, which are able to kill and eat potential pathogens. The larger of these specialized cells in mammals, the macrophages (or big eaters) are the subject of this review.

My interest in macrophages started during my PhD, which included a year spent at the Max Planck Institute for Immunobiology in Freiburg in the late 1970s.  This Institute was directed by its founder, Professor Otto Westphal, a pioneer in understanding the biochemistry of bacterial lipopolysaccharides (LPS), also known as endotoxins, which initiate much of the pathology of septicaemia.  We now know that LPS acts on macrophages to initiate a cascade of inflammatory processes that are essential for innate immunity. I was subsequently fortunate to spend a critical time with Siamon Gordon at the Sir William Dunn School of Pathology in Oxford, another organization with an important history at the centre of the development of antibiotics.  I have worked on the biology of macrophages ever since, and in fact, after some thirty years, I am still trying to make monoclonal antibodies against the CSF-1 receptor, a project that I undertook in Siamon’s laboratory. 

This review is a synthesis of reviews of macrophage biology that I have written over the past 20 years. With apologies to colleagues in the field, the review makes no attempt to provide comprehensive referencing, and refers exclusively to reviews by others for further reading where appropriate.

For further information, go to www.macrophages.com/reviews, where there is a curated database of reviews of the many different aspects of macrophage biology.

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Macrophage Overview: Origin, Expression, and Functions


The Mononuclear Phagocyte System

Macrophages as recognized by Metchnikoff can be found in all tissues, and as circulating cells called blood monocytes, which are around 20% of the peripheral blood mononuclear cell (PBMC) fraction, considerably less numerous than the other major phagocyte population, the polymorphonuclear cells, or neutrophilic granulocytes. Dale et al. 2008  have provided a historical overview of the distinct biology of these two lineages.  Monocytes are recruited into tissues in response to a very wide range of stimuli, with slower and rather distinct kinetics from the short-lived granulocytes.  The older literature considered that they were derived from mesenchymal cells, and shared biology with endothelial cells, leading to the widespread use of the term reticuloendothelial system (RES).

It was only in the 1960s that it became clear that inflammatory and tissue macrophages derived ultimately from bone marrow progenitors.  The concept of the mononuclear phagocyte system (MPS) was promoted by van Furth, Cohn and colleagues in the late 1960s and early 1970s.  The MPS was defined as a family of cells that includes committed precursors in the bone marrow, circulating blood monocytes and tissue macrophages in every organ in the body.

Until the early 1980s, macrophages resident within tissues, generally referred to as histiocytes, were recognized largely based upon morphology and location, notably the presence within them of the evidence of previous bouts of phagocytosis.  With the advent of monoclonal antibody technologies, numerous anti-macrophage antibodies were produced that bound selectively to surface antigens on macrophages of multiple species.  When those antibodies were used in combination, it became clear that they are not perfectly correlated with each other, so that the number of “subpopulations” of macrophages that can be defined is a function of the numbers of markers (Hume, 2006; Taylor and Gordon, 2005).

 Further reading:

  • Dale DC, Boxer L, Liles WC. The phagocytes: neutrophils and monocytes. Blood. 2008; 15;112(4):935-45. Pubmed link.
  • Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005; 5(12):953-64. Pubmed link.
  • Hume DA. The mononuclear phagocyte system. Curr Opin Immunol. 2006; 18(1):49-53. Pubmed link.
  • Hume DA. Differentiation and heterogeneity in the mononuclear phagocyte system. Mucosal Immuno. 2008; 1(6):432-41. Pubmed link.

 

Macrophage Tissue Expression

A particularly useful monoclonal antibody generated in Siamon Gordon’s laboratory was called F4/80 (the 80th hybridoma in the 4th attempted fusion). The use of F4/80 led to the recognition that macrophages in tissues are very numerous, and they occupy a precise anatomical niche in relation to other tissue cell types. The database at www.macrophages.com/macrophage-images contains a comprehensive set of colour images from the early characterization of F4/80 localisation by immunohistochemistry on fixed tissue sections.  One of the most striking things that emerged from these studies was the very large numbers of macrophages within tissues; they may well be the most numerous single cell type in the body, and the extensive ramification of processes throughout the tissues.  Macrophages have a particularly intimate relationship with epithelial and endothelial cells.  In simple epithelia, and throughout the capillary and lymphatic circulation, tissue macrophages spread along basement membranes; in stratified and pseudostratified epithelia such as skin, trachea and cervix, they are integrated within the epithelium.  Sinusoidal macrophages, such as those of liver, spleen and some endocrine organs have direct contact with the blood. But the separation by endothelium does not prevent pericapillary macrophages from extending processes into the lumen and sampling the blood contents. The ability of macrophages to extend processes across epithelia, and into lymphatic vessels has also been recognized.

Video:  Mac-Blue Marrow: Macrophages moving in the marrow of the 'MacBlue' transgenic mice, in which all macrophages express the cyan fluorescent protein-5. Video courtesy of Alexandre Boissonnas (Institut National de la Santé et de la Recherche Médical, Paris).
Download movie (right-click ->save as) 

Further reading/ information:

  • Macrophage images library available on macrophages.com
  • Hume DA, Halpin D, Charlton H, Gordon S: The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of endocrine organs. Proc Natl Acad Sci U S A 1984; 81:4174-4177. Pubmed link.
  • Gordon S, Crocker PR, Morris L, Lee SH, Perry VH, Hume DA. Localization and function of tissue macrophages. Ciba Found Symp. 1986;118:54-67. Pubmed link.

 

Macrophage Functions

We commonly think of macrophages as cells of the immune system, and forget their central function in many other aspects of embryonic development, homeostasis and wound repair. As an example, the macrophages of the epidermis, known as Langerhans cells, form the centre of so-called epidermal proliferative units and contribute to the control of proliferation and differentiation of keratinocytes. Those lining the surfaces of bone control osteoblast differentiation and calcification, and those in the embryo can control development and nephron endowment in the kidney. The central importance of macrophages in development is highlighted by the many systems affected by macrophage depletion in the CSF-1-deficient op/op mouse, including somatic growth, development of the pancreas and nervous system, and male and female fertility (Pollard, 2009; Gow, et al. 2010).  Resident macrophages become adapted to perform particular functions in different organs; so that brain macrophages (microglia) are very different from alveolar macrophages of the lung, Kupffer cells of the liver, or the largest tissue macrophage population, those lining the wall of the gut.

Macrophage expression and functions.
Click to view full size image and legend.


Further reading:

  • Pollard JW: Trophic macrophages in development and disease. Nat Rev Immunol 2009; 9:259-270. Pubmed link.
  • Gow DJ, Sester DP, Hume DA: CSF-1, IGF-1, and the control of postnatal growth and development. J Leukoc Biol. 2010; Sep;88(3):475-81. Pubmed link.

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The Mononuclear Phagocyte System and Macrophage Development


Differentiation in the Mononuclear Phagocyte System (MPS)

Committed cells within the mononuclear phagocyte lineage progress through a series of defined morphologically-distinct stages; a common myeloid progenitor shared with granulocytes giving rise to monoblasts, promonocytes and then monocytes which migrate into tissues (Hume, 2000). The production of mononuclear phagocytes from progenitor cells is directed by colony-stimulating factors, to some extent lineage restricted and hierarchical in their actions.  They include macrophage colony-stimulating factor (CSF-1), granulocyte macrophage colony-stimulating factor (GM-CSF) and fms-like tyrosine kinase 3 ligand (Flt3-ligand).  These growth factors instruct the common myeloid progenitor to adopt a macrophage fate (Stanley, 2009). Natural mutations in the CSF-1 gene provided the evidence that CSF-1 is required for the production of a substantial subset of tissue macrophages in the mouse (op/op) and rat (tl/tl); at the same highlighting the importance of tissue macrophages in many aspects of normal development (Pollard, 2009). Initially, analysis of the op/op mutation suggested that CSF-1 was not required for “DC”  (dendritic cell) development, but subsequent knockout of the CSF-1 receptor gene in the mouse produced an even more penetrant phenotype, perhaps due in part to the existence of a second ligand for the CSF-1 receptor, IL-34.  Both the CSF-1, and CSF-1R mutations do cause significant reductions in populations of CD11c-positive cells and their presumptive precursors. Injection of CSF-1 into mice expands the blood monocyte and tissue macrophage populations. A key discovery in the DC field was the observation that GM-CSF can promote the expansion/differentiation of bone marrow and blood monocytes into cells with potent APC activity. However, these cells are not equivalent to Steinman-Cohn DC; they are actively phagocytic and their gene expression profiles group them compellingly with other macrophages.
 
The cells of the MPS are produced from pluripotent progenitor cells in the bone marrow. These cells require combined stimulus from CSF-1 and factors including IL-1, IL-3, GM-CSF and interferon-gamma to produce colonies in semi-solid medium.  Early studies of progenitor cells demonstrated that the colony-forming cells lacked surface CSF-1 receptors, and the co-stimulatory factors probably act to induce the receptor on the cell surface.

The Mononuclear Phagocyte System (MPS).
Click to view full size image and legend.

Further reading:

  • Pollard JW: Trophic macrophages in development and disease. Nat Rev Immunol 2009; 9:259-270. Pubmed link.
  • Hume DA: Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression. Blood 2000; Oct 1;96(7):2323-8. Pubmed link.
  • Stanley ER: Lineage commitment: cytokines instruct, at last! Cell Stem Cell. 2009; 4;5(3):234-6. Pubmed link.
  • Hume DA, MacDonald KP. Therapeutic applications of macrophage colony-stimulating factor-1 (CSF-1) and antagonists of CSF-1 receptor (CSF-1R) signaling. Blood 2012; 23;119(8):1810-20. Pubmed link.

 

Monocyte Subsets

Monocytes in peripheral blood have been subdivided into subsets based upon certain surface markers. This is probably much more of a continuum of maturation with time in response to CSF-1 with the less mature cells (ly6C+ in mouse, CD14 hi/CD16 lo in human) more likely to be recruited in inflammation. During replenishment of blood monocyte pools following toxic liposome administration, the “inflammatory” ly6C(+) population appears first, supporting the view that two populations represent a differentiation sequence.
 
The key observation in the field was based upon monocyte adoptive transfer experiments, in which ly6C(+), CX3CR1(lo) monocytes were selectively recruited to the peritoneal cavity as thioglycollate-elicited exudate cells, where ly6C(-), CX3CR1(hi) cells were more inclined to replenish resident tissue MPS populations.  The immature ly6C(+) cells have a very short half-life, and can be depleted selectively without loss of the ly6C(-) population. So, even if they are able to differentiate, the normal fate of the “immature” cells may be to leave the circulation before they have an opportunity to become “mature” or “resident” type cells. The definition of the ly6C(-) monocytes as “resident” was confounded somewhat by visualisation of a subset of monocytes that patrol the endothelial surface. In response to a microbial or other challenge they extravasated rapidly.  The “resident” versus “inflammatory” definition must also take account of evidence that the monocyte subsets may possess different propensities to give rise to particular resident MPS populations, particularly in mucosal surface such as the lung and gut.  What is clearly the case is that blood monocytes are heterogeneous in terms of their expression of key molecules, chemokine receptors and cell adhesion molecules, that determine exactly how they will respond to stimuli that recruit them into tissues.  What is not yet clear is the extent to which the monocyte subsets can be further subdivided in terms of their effector functions and fates, and whether distinct populations are recruited to distinct stimuli and distinct locations.

Further reading:

  • Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of monocytes, macrophages, and dendritic cells. Science. 2010; 5;327(5966):656-61. Pubmed link.

 

Heterogeneity and Markers in the Mononuclear Phagocyte System

Although macrophages in tissues have many features in common, including extensive lysosomes, stellate morphology and location relative to epithelia, they are nevertheless extremely heterogeneous in terms of function and surface marker expression.  Our knowledge of this plasticity is most extensive for the mouse. Two different transgenic lines have been used to delineate the MPS; the CX3CR1-EGFP and csf1r-transgenes; MacGreen/MacBlue  (Hume, 2002).  An image database of the MacGreen mouse is found at www.macrophages.com/macrophage-images/transgenic-animals.

Another widely-studied MPS reporter gene is a CD11c-GFP/YFP transgene CD11c, along with several candidate endocytic receptors, including DEC-205, DC-SIGN, DCL1, and DC-HIL/gpnmb that probably function in antigen uptake, has been advocated as markers that distinguish DC from macrophages. CD11c in humans is a marker for the mononuclear phagocyte system and was subsequently shown to be an active complement receptor (CR4) that is induced during macrophage maturation.  Although CD11c is clearly not linked to APC function, and does not distinguish (the original definition of DC) Steinman-Cohn DC from phagocytes, the transgenic reporter nevertheless has utility in monitoring myeloid cell migration in vivo. 

Molecules expressed on the cell surface are of particular functional interest because they determine the ability of MPS cells to interact with pathogens, and with other cell types, to generate an appropriate innate and acquired immune response. With the possible exception of the CSF-1 receptor, which is cleaved selectively from the cell surface in response to TLR signaling, there are no markers that are expressed specifically and ubiquitously on all MPS cells. Surface markers of mouse MPS cells can be divided into two categories; those that are heterogeneously-expressed on individual cells within any one location and those that are enriched on defined functional subpopulations of cells in specific organs or locations within organs. Considering the latter category, Taylor et al., (2005), for example, highlight the distinct distributions of F4/80 and various scavenger receptors and C-type lectins between white pulp, red pulp, marginal zone, marginal zone metalophil and tingible body macrophages in the spleen. Examples of location-specific expression appear to become less well defined with more detailed observations.  For example, the langerin/CD207, was originally considered a marker for the macrophages of the epidermal layer of the skin, and appears to be necessary and sufficient for the formation of the Birbeck granules which are characteristic of these cells, is not restricted to the skin. Similarly, sialoadhesin, which was recently shown to be identical to the MOMA-1 antigen, was originally shown to be expressed most strongly on the marginal metalophilic macrophages of the spleen, and the macrophages that form the centres of hematopoietic islands. However, it is also expressed on subcapsular sinus macrophages in lymph nodes, most macrophages of intestinal lamina propria and Peyer’s patch and subsets in central nervous system and is widely-expressed on macrophages in the lung as well as uterus. If one considers the full set of surface markers that display heterogeneous expression in macrophages, which also includes the macrophage scavenger receptors, Fc receptors, CD36, CD14, SIRPalpha, TLRs, integrins, EGF-TM7 proteins (of which F4/80 is one), other Ig superfamily receptors (Siglecs) and multiple C-type lectins the set of combinations and subpopulations is essentially infinite.  This is especially true if one identifies subsets of cells on a FACS profile that express high or low levels of a marker.  At least some of this heterogeneity arises because of the stochastic nature of transcriptional regulation, so that there is a genuinely random assortment of surface markers.  One could take the view that this presents potential pathogens with a formidable arsenal of potential host defense in which every macrophage is unique, especially as we know that some receptors which have functional polymorphisms, such as TLR4, are expressed monoallelically in individual cells.

In other cases, a particular combination of surface markers determines potential function (e.g. the antigen uptake, presenting and co-stimulatory molecules such as CD80, CD86 and CD40) but even in this case, different combinations may provide different T cell subsets with distinct signals. Surface markers such as the chemokine receptors and integrins must act in concert to determine recruitment and location in a tissue. There must be a degree of determination that ensures that certain sets of genes are co-expressed at the right time and place, and that in turn determines a cellular function.  The appropriate timing is an important issue, because much of the heterogeneity we see in tissue reflects the fact that macrophages within the tissue are in different stages of their cycle of life and death, migration, development and function, responding to an infinite combination of signals.  Many apparent subsets are clearly interconvertible and derived from a common progenitor.  So, surface marker expression cannot be taken as the sole indication of lineage, function or destiny amongst macrophages.

Further reading:

  • MacGreen mouse images available on macrophages.com 
  • Hume DA, Ross IL, Himes SR, Sasmono RT, Wells CA, Ravasi T: The mononuclear phagocyte system revisited. J Leukoc Biol 2002; Oct;72(4):621-7. Pubmed link.
  • Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, Gordon S: Macrophage receptors and immune recognition. Annu Rev Immunol 2005; 23:901-44. Pubmed link.

 

Transcriptional Regulation in the Mononuclear Phagocyte System

Studies of transcriptional regulation in cells of the mononuclear phagocyte system began in the early 1980s.  Such studies were significantly constrained in the first instance by the fact that macrophages recognize and respond to bacterial DNA.  Classical promoter transfection studies were difficult to achieve, because primary macrophages undergo apoptosis in response to DNA introduced into the cytoplasm.  Only recently, the receptor responsible for the response, AIM2, was identified by a number of groups.

A number of recent reviews cover the processes of transcriptional regulation during macrophage differentiation (Lawrence and Natoli, 2011; Ostuni and Natoli, 2011). Understanding of the process gained major impetus with the cloning of the transcription factor, PU.1 (aka Spi-1, or sfpi1) by Richard Maki and colleagues. A member of the Ets transcription factor family, and named for its binding to purine-rich sequence motifs, PU.1 was found to be restricted in its expression to macrophages and B lymphocytes. The level of both PU.1 mRNA and nuclear PU.1 protein are much higher in macrophages than B cells, and later studies confirmed high expression of PU.1 was essential to macrophage lineage commitment and macrophage-specific gene expression.

PU.1 has two apparent functions in macrophage transcriptional regulation.  Firstly, a specific subset of promoters that is active in macrophages, exemplified by that of the Csf1r gene, lacks either a TATA box or a CpG island, and instead contains repeats of a purine-rich motif that binds PU.1. Multimerised PU.1 sites alone can generate transcription initiation in macrophages; but PU.1 must act in concert with another member of the Ets transcription factor family.  To initiate transcription on such promoters, and to specify the transcription start site, PU.1 cooperates with Ewing sarcoma protein. The other major function of PU.1 appears to be to generate open chromatin around enhancers that are either constitutively, or potentially, activated in cells of the macrophage lineage and which can subsequently be occupied by other transcription factors.

PU.1 is certainly not the only factor one needs to consider to understand macrophage differentiation.  The phenotype of the PU.1 knockout, which certainly greatly reduces the numbers of all mature cells of the mononuclear phagocyte lineage, macrophages, DC and osteoclasts as well as other myeloid cells actually depends upon mouse genetic background. The PU.1 locus itself has a purine-rich TATA-less promoter, and at some point in myeloid lineage commitment, it must itself be transcriptionally activated. The numerous factors that interact with PU.1 include members of the runx, Ets, MITF, myb, myc, C/EBP, ATF, nuclear hormone receptor and IRF families.  In fact, around 2/3rds of all transcription factors encoded in the mammalian genome can be expressed in macrophages in some state of differentiation or activation. 

Further reading/ information:

  • Lawrence T, Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity.  Nat Rev Immunol 2011; 25;11(11):750-61. Pubmed link.
  • Ostuni R, Natoli G. Transcriptional control of macrophage diversity and specialization. Eur J Immunol 2011; 41(9):2486-90. Pubmed link.
  •  Heinz S, Glass CK. Roles of lineage-determining transcription factors in establishing open chromatin: lessons from high-throughput studies. Curr Top Microbiol Immunol 2012; 356:1-15. Pubmed link.
  • Escoubet-Lozach L, Benner C, Kaikkonen MU, Lozach J, Heinz S, Spann NJ, Crotti A, Stender J, Ghisletti S, Reichart D, Cheng CS, Luna R, Ludka C, Sasik R, Garcia-Bassets I, Hoffmann A, Subramaniam S, Hardiman G, Rosenfeld MG, Glass CK. Mechanisms establishing TLR4-responsive activation states of inflammatory response genes. PLoS Genet 2011; 7(12):e1002401. Pubmed link.
  • Microarray Expression Data from Macrophage Maturation and Polarization study: Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol 2006; 15;177(10):7303-11.
    Data available at: www.macrophages.com/expression-data-macrophage-maturation-and-polarization

Macrophages in Embryonic Development and Maintenance of Tissue Macrophages by Local Proliferation

The concept of the mononuclear phagocyte system was based largely upon the demonstration that bone marrow cells, and their progeny, blood monocytes, can give rise to tissue macrophages in the steady state in an adult mouse.  The bone marrow only becomes the major source of blood cells after birth in a mammal.  During embryonic development, the first active phagocytic cells are formed in the yolk sac, independently of the blood islands which are the first site of production of red blood cells, and these phagocytic cells actually migrate into the head in advance of the formation of a circulatory system. The yolk-sac derive phagocytes do not pass through a monocyte stage, and are not affected by mutation of the macrophage-specific transcription factor, PU.1. Later in development, around 10.5 days post conception (dpc) in the mouse, the first definitive haemopoietic cells form in an area called the aorta-gonad-mesonephros (Robin et al., 2003), and thereafter populate the liver which is the major source of macrophages and other blood cells for the remainder of development.  Macrophages in the developing embryo are involved in many aspects of morphogenesis and organogenesis, and examined closely, can be seen to be full of the corpses of dying cells.  Their function has been reviewed in Lichanska et al. 1999.  It is common in the developing embryo to see macrophages that are actively phagocytic undergoing cell division within tissues.

Developing footpad in the MacBlue mouse embryo.
Click to view full size image and legend.

Recent studies have suggested that in at least some organs, the phagocytes that infiltrate during embryonic development are able to self-renew and are maintained into adulthood by local proliferation.  For example, the majority of the macrophages of the lung and brain remain of recipient origin long after a bone marrow transplant in mice.  In certain kinds of inflammatory process, haemopoietic stem cells and much less mature committed precursors may be mobilized from the bone marrow (Schroeder and DiPersio, 2012).  These cells may proliferate locally in an inflammatory site where appropriate growth factors are produced at high levels. 

Further reading/ information:

  • Robin C, Ottersbach K, de Bruijn M, Ma X, van der Horn K, Dzierzak E. Developmental origins of hematopoietic stem cells. Oncol Res 2003;13(6-10):315-21. Pubmed link.
  • Lichanska AM, Browne CM, Henkel GW, Murphy KM, Ostrowski MC, McKercher SR, Maki RA, Hume DA. Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1. Blood 1999; 1;94(1):127-38. Pubmed link. 
  • Schroeder MA, DiPersio JF.  Mobilization of hematopoietic stem and leukemia cells.  J Leukoc Biol 2012; 91(1):47-57. Pubmed link.
  • Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, Kierdorf K, Prinz M, Wu B, Jacobsen SE, Pollard JW, Frampton J, Liu KJ, Geissmann F. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 2012; 6;336(6077):86-90. Pubmed link.
  • Lichanska AM, Hume DA. Origins and functions of phagocytes in the embryo. Exp Hematol 2000; 28(6):601-11. Pubmed link.

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Macrophage Functions and Biology

 

Macrophage Recruitment and Chemotaxis

Monocytes are recruited into tissues in response to a very wide range of different stimuli.  Where a pathogen is involved, they are commonly preceded by neutrophils, which release a range of toxic agents designed to kill extracellular pathogens.  The macrophage then has the task of clearing both the dead pathogens and the dead neutrophils. To enter a tissue, the monocyte in peripheral blood must adhere to the vessel wall, cross the endothelial cell barrier, and then migrate towards the stimulus; a process known as chemotaxis. The process of recruitment of neutrophils and macrophages involves the resident macrophages which act as sentinels. They responds to local stimuli by producing cytokines that make the endothelial cells more sticky (through the increased expression of cell adhesion molecules such as P-selectin) and so-called chemokines, that promote the directed migration of inflammatory cells. Monocytes may also migrate towards increasing concentrations of molecules that produced by microorganisms themselves, by damaged tissues, or by the activation of the complement or clotting cascades which release bioactive peptides such as C5a.  One example of a microbial chemoattractant is N-formyl-methionyl peptides; which are unique to bacteria because this is the initiating amino acid at the N terminus of all bacterial proteins.

Video:  Movement of GFP expressing monocytes from the MacGreen mouse through live blood vessels
Download movie
(right-click ->save as)  

Chemokines

Chemokines are a family of small peptides, subdivided into classes based upon the core cysteine motifs that form disulphide bonds to fold the molecule; CC chemokines has two adjacent cysteines whilst in CXC chemokines, there is an intervening amino acid. A single chemokine, CX3CL1 has three intervening amino acids.  Chemokine receptors are classified accordingly, CCR, CXCR and CX3CR families.  The large majority of receptor that control chemotaxis fall into the class of G protein coupled receptors (GPCRs). Agonist occupation of GPCRs stimulates a change in conformation of the receptor, which couples the receptor to a so-called G-protein and promotes the exchange of GDP for GTP on the α-subunit. The GTP-bound α-subunit dissociates from the βγ-subunit; the free subunits then regulate effector enzymes positively or negatively, ultimately leading to a biological response, in this case, directed cell migration.  As reviewed by Lattin et al. (2007), macrophages are remarkably adapted to respond to a wide range of different signals coupled to GPCRs, with very high levels of expression of many of the downstream signaling and feedback control mechanisms.  Expression of specific chemokine receptors (e.g. CCR2 and CX3CR1) on different populations of monocytes provides a mechanism for their differential recruitment in response to different signals.

Further reading:

  • Lattin J, Zidar DA, Schroder K, Kellie S, Hume DA, Sweet MJ. G-protein-coupled receptor expression, function, and signaling in macrophages. J Leukoc Biol. 2007 Jul;82(1):16-32. Pubmed link.
  • Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immunol. 2008;9(9):949-52. Pubmed link.
  • Nature Immunology Focus Issue: Leukocyte Trafficking. Nat Immunol. September 2008; 9(9):947-1083. Nature link.

 

Endocytosis and Phagocytosis

All mammalian cells are able to take up macromolecules and particles from the extracellular environment through fluid-phase or receptor-mediated uptake processes (Doherty and McMahon, 2009).  The macrophages are “professional” phagocytes.  They are exceptional compared to other cells in the scale of membrane movement devoted to endocytosis, the diversity of receptors used for receptor-mediated uptake, the specific adaptation to internalize larger particles rapidly and the focus on degradation of the internalized materials. Phagocytosis, generally defined as the uptake of particles around 1µ or greater in diameter, is a function that is conserved from the simplest eukaryotic organisms such a slime moulds which feed on bacteria.  Quite apart from the functions in immunity in adult life, phagocytosis is used during mammalian embryonic development to eliminate cells undergoing programmed cell death (Lichanska et al., 1999) and key molecules involved have been identified in model organisms such as C.elegans and D.melanogaster based upon the persistence of cellular corpses.
 
The precise process of phagocytosis depends upon the particle being internalized, its size and whether it controls its own fate (Aderem and Underhill, 1999; Underhill and Ozinsky, 2002).  In broad terms, the uptake process usually requires receptor-mediated contact around the full circumference of the particle, signaling to promoter membrane extension and polymerization of the underlying actin cytoskeleton, and subsequent maturation of the internalized vacuole (the phagosome) to fuse with lysosomes and initiate particle degradation.
 
Phagocytosis is a front-line defense against pathogen attack, so almost by definition,a pathogen is an infectious agent that avoids being killed by phagocytosis.  Some microorganisms produce anti-phagocytic capsules, other produce toxins that are specifically toxic to macrophages. Many pathogens exploit macrophages as their preferred site of replication, promoting their own internalization and either preventing phagosome-lysosome fusion, permitting such fusion and resisting destruction, or escaping into the cytoplasm through rupture of the uptake vesicle.

Phagocytosis is a process that requires a mechanism for self-nonself discrimination (or in the case of recognition of dead cells and debris, a mechanism for distinguishing that material has passed its use-by date). Macrophages possess numerous receptors that allow direct recognition of particles based upon novel sugars, lipids, protein sequences and concentrations of charge that are unique to pathogens (so-called pathogen-associated molecular pathogens).  Particles may also be recognized indirectly if they are coated with opsonins such as specific antibodies or complement components.

The process of phagocytosis and degradation requires the concerted actions of hundreds of gene products. With the advent of cDNA microarray technologies, and access to very large datasets comparing cell types and tissues, it has become possible to identify the genes that make a phagocyte distinctive. They include all of the digestive enzymes found in lysosomes (a mini-stomach within the cell), the components of the proton pump that acidifies the lysosome, and the apparatus needed to make and move the lysosome and internalize particles.

Genes expressed in phagocytic cells. As shown in Hume et al., 2010. Reproduced courtesy of Elsevier. Copyright Licence No: 2910701186331.
Click
to view full size image and legend.

 

Further reading:

  • Doherty GJ, McMahon HT. Mechanisms of endocytosis. Annu Rev Biochem 2009; 78:857-902. Pubmed link.
  • Lichanska AM, Browne CM, Henkel GW, Murphy KM, Ostrowski MC, McKercher SR, Maki RA, Hume DA. Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1. Blood 1999; 1;94(1):127-38. Pubmed link.
  • Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 1999;17:593-623. Pubmed link.
  • Underhill DM, Ozinsky A. Phagocytosis of microbes: complexity in action. Annu Rev Immunol 2002; 20:825-52. Pubmed link.

 

Antigen Presentation to T lymphocytes and the Dendritic Cell Myth

 

Antigen Presentation

The innate immune system and the acquired immune system mediated by T and B lymphocytes are linked by the fact that T lymphocytes do not respond directly to soluble antigen. A historical perspective of the discovery of the requirement for specialized antigen-presenting cells was provided by one of the leaders in this field, Emil Unanue (2002).  The fundamental finding, now generally-accepted, is that antigens derived from extracellular sources must be taken up, processed, and presented to T lymphocytes, bound into the cleft formed by class II MHC.  The process of uptake, processing and presentation via a specialized compartment is now well understood (Unanue, 2002; Trombetta and Mellman, 2005;   Jutras and Desjardins, 2005). The early literature on antigen presentation considered that macrophages were the cells responsible.  Most tissue macrophages express class II MHC on their surface, it is further inducible by T cell products (notably interferon gamma) and is expressed at highest levels on the macrophages recruited in response to an immune stimulus.   Recognition of the antigen-MHCII complex by the T cell receptor is not sufficient to trigger T cell activation; this requires a second co-stimulatory signal from the APC in the form of specific cytokines and coreceptors, each of which can also be expressed inducibly in phagocytic cells.

Further reading:

  • Unanue ER. Perspective on antigen processing and presentation. Immunol Rev. 2002;185:86-102. Pubmed link.
  • Trombetta ES, Mellman I. Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol. 2005; 23:975-1028. Pubmed link.
  • Jutras I, Desjardins M. Phagocytosis: at the crossroads of innate and adaptive immunity. Annu Rev Cell Dev Biol. 2005; 21:511-27. Pubmed link.

 

Dendritic Cells

The field of antigen presentation became somewhat confused by the discovery in the early 1970s, by Steinman and Cohn, of a distinct cell type they referred to as the dendritic cell (DC). It became further confused by the loose use of the term “dendritic cell (DC)” to be synonymous with antigen-presenting cell (APC). The DC as originally defined was a non-phagocytic cell that was particularly active at stimulation in the allogeneic mixed lymphocyte reaction.  The original DCs are most likely derived from the network of interdigitating cells within T cell areas of lymphoid tissues. Purification of these cells from spleen or lymph node led to substantial enrichment of APC activity; and the contrast was made with the phagocytic “macrophage fraction” which lacked APC activity. The fundamental problem with the interpretation from my viewpoint is that the macrophages are a mixture of suppressive and stimulatory cells derived from different parts of the organ. The purification of the so-called DCs simultaneously removes the suppressive macrophages, which exert a dominant effect in the unphysiological context of an in vitro assay in a round bottom tissue culture well.  There is still really no compelling evidence that the function of the Steinman-Cohn DC as an APC is unique.

The myth evolved further to propose that DCs are a separate cell lineage uniquely able to present antigen to naïve T cells. DCs were originally identified in lymphoid tissues, but were subsequently isolated from peripheral organs such as the gut lamina propria again clearly distinguished from active phagocytes/macrophages.  Subsequently, it was found that monocytes could differentiate into “DCs” and that APC activity could be elicited in active phagocytes grown from bone marrow or peripheral blood monocytes in the growth factor, GM-CSF.

What is now generally accepted is that the classical, non-phagocytic, DC identified by Steinman and Cohn, and the tissue macrophage, share a common committed progenitor in the bone marrow and they share responsiveness to the major macrophage growth factor, CSF-1. The Steinman-Cohn DC isolated from tissues, unlike the macrophage, retains expression of the alternative growth factor receptor, Flt3, which is also expressed upon pluripotent hematopoietic progenitor cells. These cells are expanded substantially in vivo and in vitro in response to Flt3-ligand.  They most probably seed T cell areas in response to Flt3-ligand produced constitutively by T lymphocytes.  Indirect evidence, based upon a lysM-cre-mediated lineage tracer, suggests that Steinman-Cohn DC have never passed through a mature monocyte intermediate. The confusion in the literature evaporates if we accept that classical, monocyte-derived macrophages can also acquire APC function for naïve T cells and the only clear functional and differentiation dichotomy is between phagocytic (macrophages) and non (or much less)-phagocytic APC (Steinman-Cohn DC). A substantial proportion of the actively phagocytic, resident tissue macrophages associated with epithelia/mucosal surfaces express class II MHC, and are able to stimulate naïve T cells, sometimes more directed towards tolerance/suppression.

Further reading:

  • Hume DA. Macrophages as APC and the dendritic cell myth. J Immunol. 2008; 1;181(9):5829-35. Pubmed link.
  • Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ. Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol. 2010; 10(6):453-60. Pubmed link.

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Macrophage Activation and Transcriptional Networks

 

Macrophage Activation

A number of groups have advocated sub-classification of the activation states seen in recruited macrophages, broadly into M1 and M2, or classically-activated and alternatively-activated (Mantovani et al., 2004; Mantovani et al., 2005; Gordon, 2003).  Classical macrophage activation refers specifically to the broad class of activation observed in response to challenge by microorganisms.  Classically-activated macrophages are strongly positive for class II-MHC, and adapted to kill microorganisms and tumour cells and present antigen to T lymphocytes. The classical macrophage activating factor, produced by stimulated Th1 lymphocytes and NK cells, is interferon-gamma (Schroder et al., 2004).  The alternatively-activated macrophage is associated with Th2 activation and the cytokine interleukin -4 (Gordon, 2003). 

The T cell products are, of course, only part of the story of macrophage activation.  Macrophages respond directly to pathogen-associated molecular patterns (PAMPs).  They recognize them through the plasma membrane and cytoplasmic receptors such as the Toll-like receptors and intracellular receptors of the NLR family.  Shizuo Akira and colleagues have dissected the functions of numerous of the receptors by generating null mutations in mice, and have produced a number of excellent reviews of the signaling pathways that lie downstream of these receptors (Kawai and Akira, 2009; Kawai and Akira, 2010; Takeuchi and Akira, 2010). The receptors themselves are under strong evolutionary selection and are commonly associated with variation in disease susceptibility between individuals.  Classical macrophage activation, involving a synergistic interaction between interferon-gamma and a pathogen molecule such as LPS, is just one of the numerous interactions that occurs between distinct stimuli. 

Like the Th1/Th2 dichotomy, the M1/M2 distinction blurs on the boundaries when one compares distinct types of stimulus and individual cells. A more sustainable view could consider the range of macrophage phenotypes as the spectrum of colours on a colour wheel (Mosser and Edwards, 2008).  Macrophages could be classified as “red”, “yellow” and “blue”, but every combination and shade is possible and they can be inter-converted.  This kind of plasticity may ensure that the number of “subsets” than can be defined is infinite: a function of the number of markers.

Colour wheel of macrophage activation as proposed by Mosser and Edwards, 2008. Reproduced courtesy of Nature Publishing Group. Copyright Licence No: 2910231412816.
Click
to view full size image and legend.


The activation of macrophages leads to the production of a wide-range of hormone-like molecules called cytokines, that are important for orchestration of an appropriate inflammatory and acquired immune response, but can also initiate much of the pathology of disease.  These molecules include tumor necrosis factor-a, interleukin-1 and interleukin-6. Together they contribute to initiation of systemic responses including fever, known collectively as the acute-phase response. These responses contrive to initiate sickness behaviour and to make the body less conducive to pathogen replication.  In extremus, they are themselves the cause of life-threatening complications seen in septic shock; multiple organ failure and disseminated intravascular coagulation.  For this reason, cytokines have been attractive targets for therapeutic interventions.

Further reading:

  • Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M: The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004; 25:677-686. Pubmed link.
  • Mantovani A, Sica A, Locati M: Macrophage polarization comes of age. Immunity 2005; 23:344-346. Pubmed link.
  • Schroder K, Hertzog PJ, Ravasi T, Hume DA: Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 2004; 75:163-189. Pubmed link.
  • Gordon S: Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23-35. Pubmed link.
  • Mosser DM, Edwards JP: Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008; 8:958-969. Pubmed link.
  • Kawai T, Akira S: The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2009; 4:317-37. Pubmed link.
  • Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010; 5:373-84. Pubmed link.
  • Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010; 140(6):805-20. Pubmed link.

 

Dynamic Networks in Macrophage Differentiation and Activation

The process of macrophage activation, regardless of the nature of the stimulus, involves a radical change in gene expression, with numerous genes increasing or decreasing their expression with time. To understand how transcriptional regulation is achieved in macrophages, or any other system, it is worthwhile to start to look at the entire system.  Looking at one gene at a time can be rather like gazing at the individual brush strokes of an impressionist painting.  System-wide views of transcriptional networks have been greatly expedited by the advent of genome-scale technologies. There are a number of features of the response of macrophages to a stimulus such as bacterial lipopolysaccharide that are shared by many other biological systems

  1. There is a sequential cascade of gene regulation.  In the case of LPS-stimulated macrophages, the early response genes, which include a number of classical inflammatory cytokines such as TNF-alpha, have been shown to be subject to regulation primarily at the level of transcription elongation from poised RNA pol II complexes.  Later response genes are regulated by autocrine factors, including TNF and type 1 interferon, and regulated transcription factors.

  2. The numbers and magnitudes of regulation/expression of induced genes are almost precisely balanced by the numbers of repressed genes.  In a sense, this is obvious, because the total amount of mRNA per cell does not change radically, but the overall mRNA abundance profile is maintained. Amongst the repressed genes are numerous transcriptional repressors that would otherwise block the response to LPS, and genes involved in other pathways.

  3. Many of the early response genes, both induced and repressed, return to the pre-stimulation level with time; the response is in some measure self-limiting even in the continued presence of the agonist.  Amongst the LPS-inducible genes are numerous additional feedback-regulators, which we have referred to as “inflammation suppressor genes” (Wells et al. 2005).

TLR-4 signalling pathways. Click to view full size image and legend.

In broad terms, we might think of the control of macrophage activation like an automatic vehicle, in which forward progress requires the release of the brake as well as the application of the accelerator, and in which the accelerator links back to the reapplication of the brake. 

The signaling pathway from the receptor, TLR4, leading to transcriptional activation, has been described in considerable detail; the major focus has been on the transcription factor complex NF-kappaB, and on the interferon-regulated factors (IRFs) which are induced in part by autocrine interferon-beta.  Genome-scale analysis by the FANTOM consortium revealed that some 2/3rds of annotated transcription factors are expressed in primary macrophages.  A relatively small number of expressed transcription factors are very highly-connected to others, whereas others occupy peripheral niches within the network with relatively few direct connections.  It is important to recognize that because this is a single network, every transcription factor in the network is connected in some way to every other factor via multiple paths.  So, the system, in this case macrophage activation, will respond in some way to perturbation of any of the components of the network.

The set of genes express by macrophages responding to a microbial challenge evolves rapidly across species. Comparative analysis of the underlying regulatory networks of human and mouse macrophages was expedited by the availability in both species of data that precisely defined transcription start sites, so that we were able to look sequences of the promoters of regulated genes in both species, including the numerous genes that were induced in one species and not the other.  The list of transcription factor binding sites over-represented in the promoters, and their relative over-representation, was identical between the two species, suggesting that in both species the promoters sample a common transcriptional milieu. In essence, the network architecture is conserved. However, a pairwise comparison of individual promoters, revealed that there was very little conservation of individual elements between the species, even though we did not specify direct alignment. This is consistent with a model in which gain and loss of individual motifs, including the TATA box, is a significant driver of evolution, few individual motifs/binding sites have indispensable functions.

Further reading:

  • Wells CA, Ravasi T, Hume DA. Inflammation suppressor genes: please switch out all the lights. J Leukoc Biol. 2005; 78(1):9-13. Pubmed link.
  • Hume DA, Wells CA, Ravasi T. Transcriptional regulatory networks in macrophages. Novartis Found Symp. 2007; 281:2-18. Pubmed link. 
  • Medzhitov R, Horng T. Transcriptional control of the inflammatory response. Nat Rev Immunol. 2009; 9(10):692-703. Pubmed link.

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Overview

The mononuclear phagocyte system is a complex system that can be viewed in different ways. It can be unified based upon a common origin of the cells from pluripotent progenitors, the co-expression of certain markers, dependence upon growth factors especially CSF-1 and common location and morphology of the cells of the system.  It can be divided into essentially infinite subsets because of the plasticity of gene expression profiles, and the incredible diversity of different stimuli that MPS cells can encounter.  As in so many intellectual pursuits, whether one sees MPS subsets as useful constructs depends in some measure upon whether you are a lumper, or a splitter and arguments for splitting are generally more contentious.  In facing that challenge, it is worth remembering that the MPS functions as a System within individual organs and indeed within the whole body; a diverse population of interacting cells.