Complement receptors. For efficient recognition of the target particle and initiation of phagocytosis, numerous receptors on the phagocyte membrane must interact with several IgG molecules on the opsonized particle. For this, receptors must have good mobility of the membrane 69 so that they can aggregate and get activated. However, free diffusion is not easy for most phagocytic receptors, because they are among other usually bigger transmembrane glycoproteins that cover the cell surface.
Phagocytic receptors are very short molecules compared to these longer glycoproteins; hence short receptors are obscured among a layer of large glycoproteins the glycocalyx , such as mucins, hyaluronan, and the membrane phosphatases CD45 and CD In addition, many large glycoproteins are tied to the cytoskeleton, and can interfere with the lateral diffusion of receptors on the cell membrane 15 , Removal of large glycoproteins from the membrane area of contact with the target particle is achieved by activated integrins.
Integrins, for example CR3, increase their affinity for their ligand after they receive an inside-out signal 71 , 72 from other receptors such as Fc receptors 73 , TLRs 74 , or CD44 Inside-out signaling leads to activation of integrins 66 , 76 via the small GTPase Rap1 Activated integrins extend their conformation and create a diffusion barrier that keeps larger glycoproteins, for example the phosphatase CD45, away from phagocytic receptors 16 Figure 3.
Figure 3. Cooperation among phagocytic receptors. Then, activated integrins also bind to the particle via complement fragment C3b , and form a diffusion barrier that excludes larger molecules, such as the transmembrane phosphatase CD This allows other Fc receptors to be engaged and increase the signaling for phagocytosis.
SFK, Src family kinases. Syk, spleen tyrosine kinase. When a particle is recognized by phagocytic receptors, various signaling pathways are activated to initiate phagocytosis.
Reorganization of the actin cytoskeleton and changes in the membrane take place resulting in a depression of the membrane area touching the particle, the phagocytic cup.
Then, pseudopods are formed around the particle until the membrane completely covers the particle to form a new phagosome inside the cell. The signaling mechanisms to activate phagocytosis are best-known for Fc receptors and for complement receptors 10 , 67 , 82 — For other phagocytic receptors, signaling pathways are just beginning to be investigated. These kinases phosphorylate tyrosines within the ITAM. Activated Syk, in turn, can phosphorylate multiple substrates and initiate different pathways that connect to distinct cellular responses such as phagocytosis 67 , 85 , 86 and transcriptional activation 86 Figure 4.
Phosphorylation of LAT induces docking of additional adaptor molecules such as Grb2 and Gab2 Grb2-associated binder 2 Phosphorylated active PI 3-K generates the lipid phosphatidylinositol-3,4,5-trisphosphate PIP 3 at the phagocytic cup 90 , This lipid also regulates activation of the GTPase Rac, and contractile proteins such as myosin. These second messengers cause calcium release and activation of protein kinase C PKC , respectively Figure 4.
Then, spleen tyrosine kinase Syk associates with phosphorylated ITAMs and leads to phosphorylation and activation of a signaling complex formed by the scaffold protein LAT linker for activation of T cells interacting with various proteins.
These second messengers cause calcium release and activation of protein kinase C PKC , respectively. The guanine nucleotide exchange factor Vav activates the GTPase Rac, which is involved in regulation of the actin cytoskeleton.
The enzyme phosphatidylinositol 3-kinase PI3K , which is recruited and activated by Syk, generates the lipid phosphatidylinositol-3,4,5-trisphosphate PIP 3 at the phagocytic cup. This lipid also regulates Rac activation, and contractile proteins such as myosin. P represents a phosphate group. ER, endoplasmic reticulum. IP 3 R, receptor calcium channel for inositoltrisphosphate.
Among complement receptors, CR3 integrin Mac-1 is the most efficient phagocytic receptor 66 , Active Rho in turn, promotes actin polymerization via two mechanisms Figure 5. First, Rho stimulates Rho kinase, which phosphorylates and activates myosin II Second, Rho can induce accumulation of mDia1 mammalian diaphanous-related formin 1 and polymerized actin in the phagocytic cup Also, mDia1 binds directly to the microtubule-associated protein CLIP at the phagocytic cup and provides a link to the microtubule cytoskeleton required for CR-mediated phagocytosis 96 , 97 Figure 5.
Figure 5. Complement receptor signaling for phagocytosis. The complement receptor 3 CR3 integrin binds the complement molecules iC3b deposited on the target particle, and activates a signaling pathway that leads to activation of the GTPase Rho. Then, active Rho induces actin polymerization via two mechanisms.
Rho also promotes accumulation of mDia1 mammalian diaphanous-related formin 1 , which stimulates linear actin polymerization. In addition, mDia1 binds directly to the microtubule-associated protein CLIP providing a link to the microtubule cytoskeleton.
Phagocytosis initiates when phagocytic receptors engage ligands on the particle to be ingested. Then, receptors activate signaling pathways that change the membrane composition and control the actin cytoskeleton, resulting in the formation of membrane protrusions for covering the particle.
Finally, these membrane protrusions fuse at the distal creating a new vesicle that pinches out from the plasma membrane. This new vesicle containing the ingested particle is the phagosome. During phagosome formation the membrane changes its lipid composition. These changes have been revealed by elegant fluorescence imaging techniques 3 , , and involve the formation and degradation of different lipid molecules on the phagosome membrane in an orderly fashion.
The decline in PI 4,5 P 2 is important for particle internalization, probably by facilitating actin disassembly Together with the changes in lipid composition, the plasma membrane also changes by remodeling the actin cytoskeleton in order to generate the membrane protrusions that will cover the target particle.
Important steps for pseudopodia formation are recognized. First, the cortical cytoskeleton gets disrupted. Second, pseudopodia are formed by F-actin polymerization. Third, at the base of the phagocytic cup, actin gets depolymerized while the membrane phagosome is sealed at the distal end to form the phagosome When phagocytosis is initiated, the membrane-associated cortical cytoskeleton is altered by the action of coronins F-actin debranching proteins , and cofilin and gelsolin F-actin-severing proteins.
Coronin 1 concentrates at the nascent phagosome and debranches F-actin leaving linear fibers that can be severed by cofilin and gelsolin.
The activity of these enzymes is controlled by their binding to phosphoinositides, such as PI 4,5 P 2 , resulting in their association with or separation from actin filaments , Rho also promotes accumulation of mDia1, which produces long straight actin filaments at the phagocytic cup , Figure 5. Together, these changes help extend membrane protrusions that completely cover the target particle.
The final step for phagosome formation involves fusion of the membrane protrusions at the distal end to close the phagosome. Just before the phagosome is completed, F-actin disappears from the phagocytic cup. It is thought that removal of actin filaments from the phagocytic cup may facilitate curving of the membrane around the particle The mechanism for removing F-actin involves termination of actin polymerization and depolymerization of existing filaments.
Both steps seem to be controlled by PI 3-K. Inhibition of this enzyme blocks actin depolymerization at the phagocytic cup and stops pseudopod extension In support of this model, it was found that inhibition of PI 3-K led to an increase of activated GTPases at the phagocytic cup 94 , Thus, its disappearance at the phagocytic cup , promotes pseudopod extension It seems that myosins, actin-binding proteins , use their contractile activity to facilitate phagosome formation.
In macrophages, it was shown that class II, and IXb myosins were concentrated at the base of phagocytic cups, while myosin Ic increased at the site of phagocytic cup closure, and myosin V appeared after phagosome closure During pseudopod extension, a tight ring of actin filaments moves from the bottom toward the top of the phagocytic cup squeezing the particle to be ingested This contractile activity is dependent of myosin light-chain kinase MLCK.
It seems that the squeezing action of the phagocytic cups pushes extra-particle fluid out of the phagosomes. Myosin X is also recruited to phagocytic cups in a PI 3-K-dependent manner, and seems to be important for pseudopod spreading during phagocytosis At the same time, myosin Ic, a subclass of myosin I, concentrates at the tip of the phagocytic cup, implicating it in generating the contraction force that closes the opening of phagocytic cups in a purse-string-like manner Myosin IX also appears in phagocytic cups similarly to myosin II , Thus, it is believed that myosin IX is involved in the contractile activity of phagocytic cups.
However, it is also possible that myosin IX functions as a signaling molecule for the reorganization of the actin cytoskeleton. Finally, myosin V appears on fully internalized phagosomes. Because class V myosins are involved in vesicular transport in other cell types , it is possible that myosin V is responsible for phagosome movement rather than formation of phagosomes Video microscopy experiments have shown that newly formed phagosomes remain within the periphery of the cells for a while, hence it is likely that myosin V mediates the short-range slow movement of newly formed phagosomes Consequently, the described roles of myosins during phagosome formation are: myosin II is involved in phagocytic cup squeezing, myosin X and myosin Ic are responsible for pseudopod extension and phagocytic-cup closing, respectively, myosin IX may activate Rho to direct actin remodeling, and myosin V controls the short-range movement of new phagosomes.
Once internalized the new phagosome transforms its membrane composition and its contents, to become a new vesicle, the phagolysosome, that can degrade the particle ingested.
This transformation is known as phagosome maturation, and consists of successive fusion and fission interactions between the new phagosome and early endosomes, late endosomes, and finally lysosomes 4 , The new phagosome combines with early endosomes 3 in a process that involves membrane fusion events regulated by the small GTPase Rab5 , Rab5 recruits the molecule EEA1 early endosome antigen 1 , promoting the fusion of the new phagosome with early endosomes EEA1 functions as a bridge between early endosomes and endocytic vesicles , and promotes recruitment of other proteins, such as Rab7 , Although, the new phagosome combines with several endosomes it does not increase in size because at the same time vesicles, named recycling endosomes, are removed from the phagosome Figure 6.
Figure 6. Phagosome maturation. The nascent phagosome gets transformed into a microbicidal vacuole, the phagolysosome, by sequential interactions with vesicles from the endocytic pathway. The process can be described in three stages of maturation: early A , late B , and phagolysosome C. In this process, composition of the membrane changes to include molecules that control membrane fusion, such as the GTPases Rab5 and Rab7.
The phagolysosome becomes increasingly acidic by the action of a proton-pumping V-ATPase and acquires various degradative enzymes, such as cathepsins, proteases, lysozymes, and lipases scissors. As phagosome maturation proceeds, Rab5 is lost, and Rab7 appears on the membrane Then, Rab7 mediates the fusion of the phagosome with late endosomes At the same time, there is an accumulation of V-ATPase molecules on the phagosome membrane. Also, lysosomal-associated membrane proteins LAMPs and luminal proteases cathepsins and hydrolases are incorporated from fusion with late endosomes 4 , Figure 6.
At the last stage of phagosome maturation, phagosomes fuse with lysosomes to become phagolysosomes 3. The phagolysosome is the fundamental microbicidal organelle, equipped with sophisticated mechanisms for degrading microorganisms. First, phagolysosomes are very acidic pH as low as 4. This last reaction is catalyzed by the enzyme myeloperoxidase In addition, the phagolysosome contains several hydrolytic enzymes, such as cathepsins, proteases, lysozymes, and lipases, which contribute to degrade ingested microorganisms Figure 6.
Phagocytosis is not an isolated cell response. It usually occurs together with other cell responses, including formation of reactive oxygen species ROS , , secretion of pro-inflammatory mediators , degranulation of anti-microbial molecules , , and production of cytokines Cell responses associated to phagocytosis can be controlled by parallel signaling pathways triggered by the same phagocytic receptors.
Phagocytosis and associated cell responses can also be controlled by partially overlapping signaling pathways. For instance, antibody-dependent phagocytosis, in macrophages involves the signaling molecules Syk, PI 3-K, PKC, and ERK, but it is independent of an increase in cytosolic calcium concentration , Most phagocytes have relatively low levels of phagocytosis at resting conditions. However, during inflammation, phagocytes are exposed to a variety of activating stimuli, which increase phagocytosis efficiency.
These stimuli include bacterial products, cytokines, and inflammatory mediators. The signaling induced by these stimuli leads to increased stimulation of molecules involved in phagocytosis. For example, leukotriene B4 increases Syk activation and in consequence antibody-dependent phagocytosis Phagocytosis efficiency can also be regulated by cell differentiation. For example, monocytes have a lower phagocytic capacity than neutrophils and macrophages, but can enhance their phagocytic capacity upon cell differentiation 1 , The capacity of monocytes to phagocytize diverse targets changes with their state of differentiation.
IgG-opsonized particles are phagocytized better by mature macrophages than by undifferentiated monocytes Similarly, the efficiency of complement-mediated phagocytosis depends on monocyte differentiation , How the process of monocyte-to-macrophage differentiation enhances phagocytic capacity is still unknown.
It is possible that during cell differentiation the molecular machinery for phagocytosis gets rearranged. However, during monocyte-to-macrophage differentiation the enzymes PI 3-K and ERK are recruited in an orderly fashion for efficient phagocytosis Similarly, PLA2 is also implicated in regulation of phagocytosis.
During phagocytosis, various PLA2 isoforms participate in releasing arachidonic acid from membrane triglyceride lipids. Thus, during monocyte-to-macrophage differentiation important signaling enzymes are reorganized in order to achieve enhanced phagocytosis.
Phagocytosis is a fundamental process for the ingestion and elimination of microbial pathogens and apoptotic cells. All types of cells can perform phagocytosis, but specialized cells called professional phagocytes do it much more efficiently. Phagocytosis is vital, not only for eliminating microbial pathogens, but also for tissue homeostasis. Because there are different types of phagocytic cells and they can ingest a vast number of different targets, it is evident that phagocytosis involves diverse mechanisms.
For other phagocytic receptors, we are just beginning to describe the signaling pathways they use to activate phagocytosis. Today, we have a better understanding on the process of phagosome maturation, but there are still many gaps in our knowledge of the signaling pathways regulating this process.
Similarly, the resolution of the phagolysosome, after degradation of the ingested particle, is a topic that requires further research. Many important questions remain unsolved. For example, how different phagocytic receptors on the same cell work together? An improved understanding of phagocytosis is essential for future therapeutics related to infections and inflammation.
EU-Q prepared the reference list, made the figures and reviewed the manuscript. CR conceived the issues which formed the content of the manuscript and wrote the manuscript.
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Nimmerjahn F, Ravetch JV. Curr Top Microbiol Immunol. Rosales C. Front Immunol. Ravetch JV, Bolland S. It transports nutrients as well as wastes throughout the body. Various compounds, including proteins, electrolytes , carbohydrates, minerals, and fats, are dissolved in it.
The formed elements are cells and cell fragments suspended in the plasma. The three classes of formed elements are the erythrocytes red blood cells , leukocytes white blood cells , and the thrombocytes platelets. Erythrocytes, or red blood cells, are the most numerous of the formed elements. Erythrocytes are tiny biconcave disks, thin in the middle and thicker around the periphery.
The shape provides a combination of flexibility for moving through tiny capillaries with a maximum surface area for the diffusion of gases. The primary function of erythrocytes is to transport oxygen and, to a lesser extent, carbon dioxide. Leukocytes, or white blood cells, are generally larger than erythrocytes, but they are fewer in number. Even though they are considered to be blood cells, leukocytes do most of their work in the tissues.
They use the blood as a transport medium. Some are phagocytic , others produce antibodies ; some secrete histamine and heparin , and others neutralize histamine. Leukocytes are able to move through the capillary walls into the tissue spaces, a process called diapedesis. In the tissue spaces they provide a defense against organisms that cause disease and either promote or inhibit inflammatory responses. There are two main groups of leukocytes in the blood.
The cells that develop granules in the cytoplasm are called granulocytes and those that do not have granules are called agranulocytes.
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