Macrophages: Phagocytosis, Antigen Presentation, and Activation of Immunity

Abstract

Macrophages are phagocytes and one of the white blood cells discovered by Ilya I. Mechnikov in 1892. They engulf and digest foreign substances like pathogens and conduct antigen presentation, mature from haematopoietic stem cells in bone marrow, moving into blood vessels and become monocytes, and differentiate into macrophages in the tissue. Macrophages have intracellular granules called lysosome accumulating digestive enzymes. Their life span is several months and proliferates by cell division. There are three roles: First one is phagocytosis. Macrophages incorporate pathogens and work in natural immunity. In inflammation, macrophages aggregate after neutrophils recruitment and engulf pathogens into cellular phagosomes, fused with lysosomes and degrade. Second role is antigen presentation. Macrophages present fragment of digested foreign substances on cell surface MHC class II molecules and release cytokines. Dendritic cells and B cells are also APCs expressing MHC class II. CD4+ T cells recognize antigens presented on macrophages by using TCR. Only well-matched helper T cells via MHC class II-TCR interaction are activated. The third is activation of immunity. Cytokines produced by T cells activate macrophages and differentiate them into inflammatory M1 and wound-healing M2 macrophages.

1. Introduction

1.1 Development of macrophages

Macrophages are originated from variety of cells. In the early development, it depends on the tissues; however, macrophages are derived from yolk sac and replaced by the macrophages derived from liver and bone marrow [1]. Tissue resident macrophages are divided into two types, macrophages derived from circulating monocytes and having other origins including yolk sac, embryonic liver, and embryo near dorsal aorta-derived macrophages. In the adulthood, they are independently kept from their original monocytes. Tissue-specific macrophages differentiate from circulating monocytes by the ability of migration at the time of inflammation. Dendritic cells differentiate from monocytes as well as macrophages. Macrophages have variety of morphologies and phenotypes because they distribute in many organs and tissues. Instead of neutrophils that live only few days, the life span of the macrophages is several months. The diameter of the human macrophage is about 21 μm.

1.2 Differentiation and subtypes

Macrophages differentiate from premature M0 to M1 or M2 phenotypes depending on various factors from the signal transduction molecules, growth factors, transcription factors, and epigenetic or post-transduction changes to cytokines, cell adherence molecules, and metabolites [2]. Furthermore, macrophages change their activation state in response to microbes and microbial products like LPS. Recently, it is said that the classifications of macrophages are not easy because of the plasticity of the macrophages.

M1 macrophages are the so-called classically activated macrophages, pro-inflammatory macrophages, and killer macrophages. M1 macrophages produce high levels of IL-12 after the stimulation of LPS and IFN-γ. The feature of M1 macrophages is possessing specific pathways which converts arginine into “killer molecules” nitric oxide. M1 macrophage is the phenotype observed in early inflammation phase activated by IFN-γ, TNF, and damage-associated molecular patterns (DAMPS). They show high antigen presenting ability, producing high amounts of NO and reactive oxygen spices (ROS), showing increased expression of IL-12 and IL-23, and decreased IL-10 expression. In addition, M1 macrophages express high levels of MHC class II molecules, CD68, CD80, and Th1 cell-inducing chemokine CXCL9 and CXCL12 [3].

M2 macrophages are called alternatively activated macrophages and wound healing macrophages divided into M2a, M2b, M2c, and M2d phenotypes. They are a typical phenotype of tissue-resident macrophages and participate in constructive process including wound healing and tissue repair. These macrophages are stimulated by several factors including parasitic and fungal infection, immune complexes, apoptotic cells, macrophage colony stimulation factors (M-CSF), IL-13, TGF-β, and Th2 cytokine IL-4, and cytokines produced by Th2 cells like IL-25 and IL-33. Signal transduction pathways including STAT6, IRF4, PPARδ, and PPARδ are required for the differentiation of M2 macrophages. Generally, M2 macrophages produce low IL-1, IL-6, and TNF-α, whereas producing low IL-12. A typical feature of M2 macrophages is converting arginine to ornithine “repair molecules.” Ornithine is important for wound healing and required for vascular and endothelial regeneration. M2 macrophages are also important for clearance of pathogens, anti-inflammation, metabolism, wound healing, tissue regeneration, immune regulation, and progression of tumours. On the other hand, M2 macrophages induce tissue fibrosis in the lung and liver, and progressively stimulate tumour growth as tumour-related macrophages. M2 phenotypes are characterised by the expression of CD206, CD163, FIZZ1, and Ym1/2. There are four types of M2 macrophages a, b, c, and d. These are different by their cell surface markers, secreting cytokines, and biological function. However, the common feature of these M2 macrophages is the production of IL-10 [4].

M2a macrophages are activated by IL-4 or IL-13. IL-4 induces the expression of the mannose receptor (CD206). Upregulation of IL-10, TGF-β, CCL17, CCL18, and CCL22 induces cell proliferation, cell repair, and endocytosis of M2a macrophages.

Immune complex, toll-like receptor (TLR) and their ligands, and IL-1β activate M2b macrophages. When activated, these subtypes of macrophages produce both proinflammatory and anti-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-10. M2b macrophages work on immune response and regulation of inflammation. High IL-10-producing and low IL-12-producing M2b macrophages are the so-called regulatory macrophages (Mreg). Mregs are recently focused on their ability to induce regulatory T cells (Treg) [5].

M2c macrophages are activated by glucocorticoid, IL-10, TGF-β, and inactivated macrophages. The feature of M2c macrophages is high expression of anti-inflammatory IL-10, TGF-β, CCL16, CCL18, and tyrosine-protein kinase MER (MerTK), which enhance phagocytosis activity.

TLR antagonist, IL-6, and adenosine activate M2d macrophages. Adenosine induces the expression of IL-10 and vascular endothelial growth factor (VEGF) and enhances angiogenesis and tumour progression.

M2 macrophages are important for the stability of blood vessels because they produce VEGF-A and TFG-β. In acute lesion, macrophages change their phenotype from M1 to M2; however, these changes will be lost in chronic lesion. This dysregulation results in insufficient M2 macrophages and induces the deficiency of growth factor. The lack of growth factors and anti-inflammatory cytokines from M2 macrophages and excess production of proinflammatory cytokines from M1 macrophage prevent sufficient repair of wound healing. Normally, depletion of neutrophils by apoptosis after eating debris and pathogens induces the switch of macrophages from M1 to M2, but inflammation is unnecessary at that time. Then, M1 macrophages cannot eat apoptosis-inducing neutrophils, and this phenomenon increases the numbers of macrophages and inflammation because of the dysregulation [6].

2. Classification of macrophages by the tissue

2.1 Adipose tissue macrophages: Adipose tissue

Macrophages exist in body fat and increase in case of obesity.

2.2 Monocytes: bone marrow, blood

The largest white blood cells in the blood. They develop into macrophages and dendritic cells.

2.3 Kupffer cells: liver

Kupffer cells exist in the liver and also known as stellate macrophages. Kupffer cells were named after Karl Wilhelm von Kupffer. They work as the first defence against gut bacteria and endotoxin in the liver.

2.4 Alveolar macrophages: pulmonary alveoli

Macrophages exist in alveoli and bronchus. Alveolar macrophages have high activity to get rid of dusts and microbes in the lung.

2.5 Microglia: central nerve system

A family of glial cells with different origin from other family of cells. Most of glial cells developed from ectoderm; however, alveolar macrophages are developed from mesoderm and haematopoietic stem cells. Microglia have phagocytic activity in the nerve and participate in the repair of neural tissue after the tissue damage.

2.6 Hofbauer cells: placenta

Eosinophilic histocytes found in the placenta, often seen in early pregnancy, named after J. Isfred Isidore Hofbauer. Hofbauer cells are considered as a type of macrophage.

2.7 Intraglomerular mesangial cells: kidney

Intraglomerular mesangial cells exist in basement membrane surrounded by glomerular capillaries. They are considered as a type of fibroblast.

2.8 Osteoclasts: bone

Osteoclasts are the specialist of absorbing or destroying bone in the process of bone regeneration. They are usually polygonal giant cells with 5–20 nuclei, but sometimes mononuclear osteoclast can be found. Bone marrow-derived monocyte progenitors differentiate into osteoclasts. The marker of osteoclasts is tartrate-resistant acid phosphatase. On the other hand, the marker of osteoblast is alkaline phosphatase.

2.9 Langerhans cells: skin

Langerhans cells are named after Paul Langerhans. Usually, they are regarded as dendritic cells other than macrophages.

2.10 Epithelioid cells: granulomas

Activated macrophages similar to epithelial cells. They have a thin eosinophilic cytoplasm with small granules and nucleus less dense than lymphocytes. They are found in granulomatous inflammation and participate in arthritis.

2.11 Red pulp macrophages (sinusoidal lining cells): red pulp in spleen

Macrophages found in red pulp in spleen are necessary for the blood homeostasis by depleting damaged or aged red blood cells with the phagocytosis.

2.12 Intestinal macrophages: intestine

Macrophages specifically evolved in intestinal environment. Intestinal macrophages do not induce inflammation to coexist with intestinal microbiome. They do not excrete proinflammatory cytokines such as IL-1, IL-6, and TNF-α. TGF-β produced by surrounding environment changes these macrophages from proinflammatory phenotype to non-inflammatory phenotype. Intestinal macrophages conduct phagocytosis, but they do not produce cytokines after phagocytosis nor express receptors for LPS, IgA, and IgG.

2.13 Others

Sinus histiocytes: lymph nodes

Tissue macrophages leading to giant cells: connective tissue

Peritoneal macrophages: peritoneal cavity

LysoMac: Peyer’s patch

3. Function of macrophages

3.1 Phagocytosis

Macrophages are one of the three professional phagocytes with other phagocytes including granulocytes (eosinophils, neutrophils, and basophils) and dendritic cells (DC). Phagocytosis is the process that microorganisms entering the host and recognised by phagocytes and incorporated and destroyed. This process starts after the interaction with pathogen-specific receptors (usually pathogen-specific sugar or lipid structures) on phagocytes and the surface molecular on pathogens. Typical phagocyte receptors are dectin-1 and mannose receptor (CD206), and both are the family of members of c-type lectin. Dectin-1 expressed on macrophages and neutrophils connects with glucose polymers on the cell walls of fungus. On the other hand, CD206 expressed on macrophages and DCs connects with variety of ligands on fungus, bacteria, and virus. Generally, macrophages exist in all of tissues and monitor potential pathogens with amoebic motility. Most of macrophages are strategically placed where microbes invade or debris accumulate [7].

After starting interaction with pathogens, phagocytic plasma membrane in macrophages engulfs pathogens into phagosomes, large membrane-enclosed endocytic vesicles (endosomes). Phagosomes enclose pathogens, merged with lysosomes containing antimicrobial peptides and enzymes, and form phagolysosomes. Toxic peroxides like superoxide radicals in phagolysosomes kill and digest pathogens after acidification and enzymic processes. Macrophages ultimately digest over 100 bacteria through digestive compounds in their lifetime. However, some bacteria have resistant properties to these digestive methods. Mycobacterium tuberculosis survives within the macrophages through inhibiting the fusion to phagosomes. To reproduce themselves, Salmonella enterica serovar Typhi induces phagocytosis to incorporate into macrophages, inhibits lysosomal digestion, and triggers apoptosis of macrophages. Furthermore, leishmaniasis causes Leishmania parasitises in macrophages.

3.2 Activation of natural immunity

Macrophages participate in natural immunity by engulfing and digesting pathogens. They protect hosts from the infections and damages through phagocytosis [8]. Macrophages are the first defence against pathogens working with neutrophils and are specific phagocytes with long life. After the invasion of pathogens, neutrophils are firstly recruited to the site of infection, die after phagocytosis of pathogens, and generate neutrophil traps (NETs). Then, macrophages are recruited and digest NETs after approximately 48 hours later. Recruited macrophages digest pathogens and dead cells through phagocytosis. Finally, they initiate immune responses through releasing factors like TNF-α to recruit other immune cells such as lymphocytes.

3.3 Adaptive immunity and antigen presentation

Macrophages are the most important antigen-presenting cells (APC) as well as dendritic cells (DC), which have important roles in the initiation of immune responses. Furthermore, they produce strong modification factors and chemical substances, such as enzymes, complement proteins, and IL-1. At the same time, macrophages activate to seek microorganisms and tumour cells through their lymphokine receptors.

Antigen-digested macrophages present pathogen antigens to helper T cells, most of which are protein molecules expressed on the surface of pathogens. Antigen presentation is conducted by MHC class II molecules (MHCII) on the surface of macrophages presenting antigens incorporated. Antibody production attaching to the pathogenic antigens starts from plasma B cells after the antigen presentation by APCs and making macrophages easy to adhere to cell membrane of pathogens the so-called opsonisation.

In lymph nodes, antigen presentation via macrophages through MHCII stimulates Th1 cells to start proliferation. B cells recognise same unprocessed antigen by the cell surface antibodies and then incorporate and process them through endocytosis. MHCII molecules on the surface of B cells present processed antigens. T cells that recognise antigen-MHCII complex with co-stimulatory factor CD40-CD40L help B cells to produce antibodies. Then, macrophages incorporate opsonised pathogens by antibodies and eliminate them from the body. However, regarding phagocytosis, recently dendritic cells are more focused than macrophages.

Macrophages provide another defence pathway against fungus and parasites. After the recognition of specific antigen on the cell surface, activated T cells differentiate into effector cells and produce lymphokines. Produced lymphokines stimulate macrophages to more offensive form.

4. Role of macrophages in tissue regeneration and homeostasis

4.1 Wound healing

Macrophages have significant roles in wound healing. By 2 days after the injury, they replace neutrophils and become the dominant cells in the place where injured. Monocytes are attracted to the wound site by the growth factors released from platelets and other cells and then enter the site from bloodstream through the blood vessel wall. The number of monocytes in the injured sites peaks at 1.5 days. At the site of injury, monocytes mature into macrophages. In addition, spleen contains half the numbers of macrophages as a spare, and they are sent to the wound sites when injured [9].

The main role of macrophages is conducting phagocytosis to the microbes and injured tissues. In addition, protease released from macrophages induces tissue necrosis. After 3 to 4 days of injury, macrophages secrete variety of factors including cytokines, which proliferate and attract cells involved in wound healing. Under the stimulation of low oxygen environment, macrophages induce and generate accelerating factors of angiogenesis. These factors stimulate cells to promote the growth of epithelial cells, create granulation tissues, and form new extracellular matrix. Then, macrophages direct the next stage of wound healing via the secretion of these factors.

4.2 Muscle regeneration

There are two waves in muscle regeneration by macrophages. The first wave is the increased population of phagocytes after the damage of muscular fibre with development of rhabdomyolysis and muscle membrane inflammation by the use of muscle. This population peaks after 24 hours of recruitment to the muscle damage and rapidly decreases after 48 hours. The second wave is non-phagocytic macrophages distributes near the close region of regenerative fibres. These cells peak at 2 to 4 days, and the numbers of the cells remain increased for few days whilst muscle tissue is reconstituted. The first groups of cells do not have any benefits for muscle repair, but second croups are beneficial. They release soluble factors related to the muscle growth, differentiation, repair, and regeneration [10].

4.3 Foot regeneration

In salamanders, macrophages participate in not only consume debris but also a typical regeneration of limbs. Depletion of macrophages in salamanders resulted in the failure of limb regeneration [11].

4.4 Macrophages related with the maintenance of homeostasis

All of tissues have residential macrophages interacting with stromal and functional tissue. These macrophages are unmovable, protecting tissues from inflammatory injuries and provide essential factors to support tissue physiological functions [12].

4.5 Maintenance of pigments

Melanophages, a tissue-resident macrophages, absorb pigments from organ specific or exogenous out-cellular environment. In contrast to melanocytes, melanophages only accumulate melanin incorporated from lysosome-like phagosomes. This phenomenon occurs by which melanophages conduct phagocytosis of tissues from dead skin macrophages. This occurs because melanophages conduct phagocytosis of tissue from dead skin macrophages.

4.6 Nerve-associated macrophages

Nerve-associated macrophages are macrophages related to neurone. They have elongated morphology and stretch up to 200 μm.

5. Macrophages in disorders

5.1 Pathogen hosting macrophages

Generally, macrophages destroy pathogens by phagocytosis. However, some pathogens live in macrophages by interrupting this phagocytosis processes. This phenomenon hides pathogens from immune system and provides them environment to reproduce themselves. Tuberculosis-inducing mycobacterium and Leishmania species are well known [13].

5.2 Heart diseases and cardiovascular diseases

Macrophages are main cause in the onset of progressive plaque lesions in atherosclerosis. Residential M2 macrophages incorporate oxidised LDL in the cells and become form cells which clogging blood vessels. In addition, both M1 and M2 macrophages participate in the progression of atherosclerosis. M1 macrophages enhance atherosclerosis through inflammation induction. M2 macrophages eliminate cholesterol, but incorporated oxidised cholesterol induces apoptotic form cells from macrophages [14].

On the other hand, macrophages are recruited to the place where tissue regeneration is required after acute myocardial infraction, removing apoptotic cells and debris.

5.3 Tissue fibrosis

M2 macrophages induce tissue fibrosis by the production of TGF-β in the damaged lung and liver.

5.4 HIV

Macrophages participate in HIV infection. In addition to CD4+ T cells, macrophages become the storage of reproductive virus. Gp120 protein on HIV couples with chemokine receptor CCR5 to invade into cells.

5.5 Cancer

Some macrophage subtypes participate in the progression of cancer. Cancer-related macrophages participate in tumour cell growth and invasion, progression of angiogenesis, and suppression of anti-tumour immune cells.

5.6 Obesity

Proinflammatory macrophages in fat tissues participate in obesity-related complications such as insulin resistance and type-2 diabetes.

5.7 Inflammatory bowel disease (IBD)

Macrophages participate in inflammatory bowel diseases (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC). In healthy intestine, macrophages suppress the inflammation; however, in the patients with IBD, the numbers and diversity of macrophages change and cause adverse effects on the onset of disorders.