Phagocytosis pdf - D. Dhafer

Thi-Qar University.                                                                                                                    Immunology
College of medicine                                                                                                                 Lect: 7
Microbiology Department                                                                                                       Dr Dhafer Alghezi

Phagocytosis

Phagocytosis (“phago”=eat, “cyte”=cell) is a specific form of endocytosis and represents a

cellular process used to ingest and eliminate particles larger than 0.5 μm in diameter,

including microorganisms, foreign substances, and apoptotic cells. Cells that perform

phagocytosis are called a

phagocytes.

Phagocytes:

They are protected the body by ingesting harmful foreign particles, bacteria, and dead cells.

Phagocytes are called "professional" or "non-professional" depending on how effective they

are at phagocytosis. The professional phagocytes are responsible of removing

microorganisms and of presenting antigens to lymphocytes in order to activate an adaptive

immune response. They include many types of white blood cells such as neutrophils,

monocytes, macrophages, mast cells, and dendritic cells. In contrast, fibroblasts, epithelial

cells, and endothelial cells can also accomplish phagocytosis with low-efficiency and are thus

described as non-professional phagocytes. These cells cannot ingest microorganisms, but are

important in eliminating dead cells and maintaining homeostasis.
The main difference between professional and non-professional phagocytes is that the

professional phagocytes have molecules called receptors on their surfaces that can detect

harmful objects, such as bacteria, that are not normally found in the body. Numerous

receptors are involved in phagocytosis such as complement receptors and Fc receptors are

particularly important for the recognition and phagocytosis of opsonized microbes and other

solid matter.

Other receptors, including opsonin receptors, scavenger receptors, and Toll-like

receptors, are also important in the uptake of many pathogenic microorganisms. Opsonin

receptors increase the phagocytosis of bacteria that have been coated with immunoglobulin G

(IgG) antibodies or with complement. Scavenger receptors bind to a large range of molecules

on the surface of bacterial cells, and Toll-like receptors—so called because of their similarity

to well-studied receptors in fruit flies that are encoded by the Toll gene—bind to more

specific molecules. Binding to Toll-like receptors increases phagocytosis and causes the

phagocyte to release a group of hormones that cause inflammation.
The general activities of phagocytes are summaries below:
(1)

To survey the tissue compartments and discover microbes, particulate matter (dust,

carbon particles, antigen-antibody complexes, and injured or dead cells).

(2)

To ingest and eliminate these materials.

(3)

To extract immunogenic information (antigens) from foreign matter.

The three main types of phagocytes are neutrophils, monocytes, and macrophages.

1. N

eutrophil

The neutrophil is the primary phagocyte that arrives early at the site of inflammation, usually

within 90 minutes of injury. The neutrophils’ cytoplasmic granules contain enzymes and

other antibacterial substances that are used in destroying and degrading the engulfed

particles. The neutrophil count in the blood often increases greatly during the inflammatory

process, especially with bacterial infections. Increased neutrophil count in the blood is called

neutrophilia. Neutrophils are also a primary component of pus.

2. Eosinophils

The second major polymorphonuclear granulocyte. Eosinophils leave the bone marrow in a

relatively immature state and move directly to the spleen, where they reach maturity. They

spend short time circulating in the blood stream, then they migrate into tissues. Eosinophils

are attracted to sites of parasitic infections and antigen-antibody reactions, though they play

only a minor phagocytic role. The granules of eosinophils contain a protein that is highly

toxic to large parasitic worms that cannot be phagocytized.

3. Monocytes

The monocytes are the largest of the white blood cells. They help to destroy the causative

agent, aid in the signaling processes of specific immunity, and serve to resolve the

inflammatory process. The monocytes, which migrate in increased numbers into the tissues in

response to inflammatory stimuli, mature into macrophages. This process is marked by an

increase in size and by enhanced development of lysosomes and other organelles.
Macrophages, king of phagocytes, can be classified into fixed (adherent to tissue) or

wandering macrophage. The fixed macrophage concentrates in specific areas that are more

vulnerable to intruders like the lungs or the intestine, whereas, wandering macrophages travel

throughout both blood and lymph streams to perform their job. Specialized macrophages

called histiocytes migrate to a certain tissue and remain there during their lifespan. Examples

are alveolar macrophages (lung), the Kupffer cells in the liver, Langerhans cells in the skin

and macrophages in the spleen, lymph nodes, bone marrow, kidney, and brain. Other

macrophages do not reside permanently in a particular tissue and drift nomadically

throughout the RES. Not only are macrophages dynamic scavengers, but they also process

foreign substances and prepare them for reactions with B and T lymphocytes.

Mechanisms of Phagocytic (steps of phagocytosis)

Step 1: Activation of the Phagocyte
Resting phagocytes are activated by inflammatory mediators such as bacterial products

(bacterial proteins, capsules, LPS, peptidoglycan, teichoic acids, etc.), complement proteins,

inflammatory cytokines, and prostaglandins. As a result, the circulating phagocytes produce

surface glycoprotein receptors that increase their ability to adhere to the inner surface of

capillary walls, enabling them to squeeze out of the capillary and be attracted to the site of

infection.
Step 2: Chemotaxis of Phagocytes (for wandering macrophages, neutrophils, and

eosinophils
Chemotaxis is the movement of phagocytes toward an increasing concentration of some

attractant such as bacterial factors (bacterial proteins, capsules, LPS, peptidoglycan, teichoic

acids, etc.), complement proteins (C5a), chemokines (chemotactic cytokines such as

interleukin-8), fibrin split products, kinins, and phospholipids released by injured host cells.

Some microbes, such as the influenza A viruses,

Mycobacterium tuberculosis

, blood invasive

strains of

Neisseria gonorrhoeae

, and Bordetella pertussis have been shown to block

chemotaxis.
Step 3: Attachment of the Phagocyte to the Microbe or Cell
Attachment of microorganisms is necessary for ingestion. Attachment may be unenhanced or

enhanced.
a. Unenhanced attachment

: It is the innate recognition of pathogen-associated molecular

patterns or PAMPs - components of common molecules such as peptidoglycan, teichoic

acids, lipopolysaccharide, mannans, and glucans common in microbial cell walls but not

found on human cells - by means of endocytic pattern-recognition receptors, such as

scavenger receptors and mannose receptors, on the surface of the phagocytes.

Enhanced attachment: It is the attachment of microbes to phagocytes by way of an

antibody molecule called IgG, the complement proteins C3b and C4b produced during the

complement pathways and acute phase proteins such as mannose-binding lectin (MBL)

and C-reactive protein (CRP). Molecules such as IgG, C3b, and mannose-binding lectin

(MBL) that promote enhanced attachment are called opsonin and the process is also

known as opsonization. Enhanced attachment is much more specific and efficient than

unenhanced.

c. Extracellular trapping with neutrophil extracellular traps (NETs): In response to

certain pathogen associated molecular patterns such as LPS, and certain cytokines such as

IL-8, neutrophils release DNA and antimicrobial granular proteins. These (NETs) bind to

bacteria, prevent them from spreading, and kill them with antimicrobial proteins.

Some microorganisms are more resistant to phagocytic attachment.

● Capsules can resist unenhanced attachment by preventing the endocytic pattern

recognition receptors on phagocytes from recognizing the bacterial cell wall

components

and

mannose-containing

carbohydrates.

Streptococcus

pneumonia

activates the classical complement pathway, but resists C3b opsonization,

and complement causes further inflammation in the lungs.

● Some capsules prevent the formation of C3 convertase. Without this enzyme, the

opsonin C3b and C4b, as well as the other beneficial proteins are not produced.

● Some capsules simply cover the C3b that does bind to the bacterial surface and prevent

the C3b receptor on phagocytes from making contact with the C3b. This is seen with

the capsule of

Streptococcus pneumoniae

● An outer membrane molecule of

Neisseria gonorrhoeae

called Protein II and the

M-protein of

Streptococcus pyogenes

allow these bacteria to be more resistant to

phagocytic engulfment. The M-protein of S. pyogenes, for example, binds factor H of

the complement pathway and this results in the degradation of the opsonin C3b by

factor I and the formation of C3 convertase. S. pyogenes also produces a protease that

cleaves the complement protein C5a.

Step 4: Ingestion of the Microbe or Cell by the Phagocyte
Following attachment, polymerization and then depolymerization of actin filaments send

pseudopods out to engulf the microbe and place it in an endocytic vesicle called a

phagosome.
During this process, an electron pump brings protons (H+) into the phagosome. This lowers

the pH within the phagosome to 3.5 - 4.0 so that when a lysosome fuses with the phagosome,

the pH is correct for the acid hydrolases to effectively break down cellular proteins. The

acidification also releases defensins, cathelicidin, and bacterial permeability inducing protein

(BPI), peptides and enzymes that can kill microbes, from a matrix and enabling their

activation.
Intracellular microbes, such as viruses and bacteria that invade host cells, can also be

engulfed once they enter the cytosol of the cell by a process called autophagy. A

membrane-bound compartment called an autophagosome grows around the microbe and the

surrounding cytosol and subsequently delivers it to lysosomes for destruction.
A. Pathogenic

Yersinia

, such as the one that causes plague, contact phagocytes and, by means

of a

type III secretion system, deliver proteins which depolymerize the actin

microfilaments needed for phagocytic engulfment into the phagocytes

B. Some

bacteria,

Mycobacterium

tuberculosis,

Salmonella

and

Listeria

monocytogenes

can block autophagy.

Step 5: Destruction of the Microbe or Cell
Phagocytes contain membranous sacs called lysosomes produced by the Golgi apparatus that

contain various digestive enzymes, microbicidal chemicals, and toxic oxygen radicals. The

lysosomes travel along microtubules within the phagocyte and fuse with the phagosomes

containing the ingested microbes and the microbes are destroyed.
Some bacteria are more resistant to phagocytic destruction once engulfed.

a. Some bacteria, such as

Legionella pneumophilia

and Mycobacterium species, cause the

phagocytic cell to place them into an endocytic vacuole via a pathway that decreases their

exposure to toxic oxygen compounds.
b. Some bacteria, such as

Salmonella

, are more resistant to toxic forms of oxygen and to

defensins (toxic peptides that kill bacteria).

c. Some bacteria, such as

Shigella flexneri

and the spotted fever Rickettsia, escape from the

phagosome into the cytoplasm prior to the phagosome fusing with a lysosome
There are 2 killing systems in neutrophils and macrophages: the oxygen-dependent

system and the oxygen-independent system.
1.

The oxygen-dependent system: production of reactive oxygen species (ROS)

The cytoplasmic membrane of phagocytes contains the enzyme oxidase which converts

oxygen into superoxide anion (O2-). This can combine with water by way of the enzyme

dismutase to form hydrogen peroxide (H2O2) and hydroxyl (OH) radicals.
In the case of neutrophils, but not macrophages, the hydrogen peroxide can then combine

with chloride (Cl2-) ions by the action of the enzyme myeloperoxidase (MPO) to form

hypochlorous acid (HOCL), and singlet oxygen. In macrophages, nitric oxide (NO) can

combine with hydrogen peroxide to form peroxynitrite radicals. (In addition to ROS and NO,

macrophages secrete inflammatory cytokines such as TNF-alpha, IL-1, IL-8, and IL-12 to

promote an inflammatory response.)
These compounds are very microbicidal because they are powerful oxidizing agents which

oxidize most of the chemical groups found in proteins, enzymes, carbohydrates, DNA, and

lipids. Lipid oxidation can break down cytoplasmic membranes. Collectively, these oxidizing

free radicals are called reactive oxygen species (ROS).
Oxidase also acts as an electron pump that brings protons (H+) into the phagosome. This

lowers the pH within the phagosome so that when lysosomes fuse with the phagosome, the

pH is correct for the acid hydrolases, like elastase, to effectively break down cellular proteins.
In addition to phagocytes using this oxygen-dependant system to kill microbes intracellularly,

neutrophils also routinely release these oxidizing agents, as well as acid hydrolases, for the

purpose of killing microbes extracellularly. These agents, however, also wind up killing the

neutrophils themselves as well as some surrounding body cells and tissues.
2. The oxygen-independent system
Some lysosomes contain defensins , cationic peptides that alter cytoplasmic membranes;

lysozyme, an enzyme that breaks down peptidoglycan, lactoferrin, a protein that deprives

bacteria of needed iron; cathepsin G, a protease that causes damage to microbial membranes;

elastase, a protease that kills many types of bacteria; cathelicidins, proteins that upon

cleavage are directly toxic to a variety of microorganisms; bactericidal permeability inducing

protein (BPI ), proteins used by neutrophils to kill certain bacteria by damaging their

membranes; collagenase ; and various other digestive enzymes that exhibit antimicrobial

activity by breaking down proteins, RNA, phosphate compounds, lipids, and carbohydrates.