PATTERN-RECOGNITION RECEPTORS (INCLUDING TOLL-LIKE-RECEPTORS) AND CYTOKINES
Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to an antigen. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.

Unlike adaptive immunity, innate immunity does not recognize every possible antigen. Instead, it is designed to recognize a few highly conserved structures present in many different microorganisms. The structures recognized are called pathogen-associated molecular patterns and include LPS from the gram-negative cell wall, peptidoglycan, lipotechoic acids from the gram-positive cell wall, the sugar mannose (common in microbial glycolipids and glycoproteins but rare in those of humans), bacterial DNA, N-formylmethionine found in bacterial proteins, double-stranded RNA from viruses, and glucans from fungal cell walls. Most body defense cells have pattern-recognition receptors for these common pathogen-associated molecular patterns and so there is an immediate response against the invading microorganism. Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern-recognition receptors in the blood that function as opsonins and initiate the complement pathways. In all, the innate immune system is thought to recognize approximately 103 molecular patterns. All of this will be discussed in greater detail in upcoming sections.

The innate immune responses involve:

  • phagocytic cells (neutrophils, monocytes, and macrophages);
     

  • cells that release inflammatory mediators (basophils, mast cells, and eosinophils);
     

  • natural killer cells (NK cells); and
     

  • molecules such as complement proteins, acute phase proteins, and cytokines.

Examples of innate immunity include anatomical barriers, mechanical removal, bacterial antagonism, pattern-recognition receptors, antigen-nonspecific defense chemicals, the complement pathways, phagocytosis, inflammation, and fever. In the next several sections we will look at each of these in greater detail.

We will now take a closer look at pattern-recognition receptors, antigen-nonspecific antimicrobial body chemicals, and cytokines.

 

Pattern-Recognition Receptors (Including Toll-Like Receptors) and Cytokines

1. Pattern-Recognition Receptors

In order to protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns. In all, the innate immune system is thought to recognize approximately 103 molecular patterns.These include:

A. lipopolysaccharide (LPS) from the gram-negative cell wall;

B. peptidoglycan found abundantly in the gram-positive cell wall and to a lesser degree in the gram-negative cell wall ;

C. lipoteichoic acids found in the gram-positive cell wall;

D. mannose-rich glycans (common in microbial glycoproteins and glycolipids but rare in those of humans);

E. flagellin found in bacterial flagella;

F. pilin from bacterial pili;

G. bacterial and viral nucleic acid. Bacterial and viral genomes contain a high frequency of unmethylated cytosine-guanine dinucleotide sequences (a cytosine lacking a methyl or CH3 group and located adjacent to a guanine). Mammalian DNA has a low frequency of cytosine-guanine dinucleotides and most are methylated;

h. N-formylmethionine, an amino acid common to bacterial proteins;

i. double-stranded RNA unique to most viruses;

j. lipoteichoic acids, glycolipids, and zymosan from yeast cell walls; and

k. phosphorylcholine and other lipids common to microbial membranes.

To recognize these microbial molecules, various body defense cells have on their surface a variety of receptors called pattern-recognition receptors  capable of binding specifically to conserved portions of these molecules.

There are two functionally different classes of cell surface pattern-recognition receptors: endocytic pattern-recognition receptors and signaling pattern-recognition receptors.

1. endocytic pattern-recognition receptors

Endocytic pattern-recognition receptors are found on the surface of phagocytes and promote the attachment of microorganisms to phagocytes and their subsequent engulfment and destruction. They include:

A. mannose receptors

Mannose receptors bind to terminal mannose and fucose groups on microbial glycoproteins and glycolipids. (Human glycoproteins and glycolipids typically have terminal N-acetylglucosamine and sialic acid groups.)

B. scavenger receptors

Scavenger receptors bind to bacterial cell wall components such as LPS, peptidoglyan and teichoic acids. There are also scavenger receptors for certain components of other types of microorganisms.

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2. signaling pattern-recognition receptors

Signaling pattern-recognition receptors bind a number of microbial molecules: LPS, peptidoglycan, teichoic acids, flagellin, pilin, and DNA from bacteria; lipoteichoic acid, glycolipids, and zymosan from fungi; and double-stranded RNA and certain proteins and glycoproteins from viruses. These include toll-like receptors and CD14. Binding of microbial molecules to their receptor promotes the synthesis and secretion of intracellular regulatory molecules such as cytokines that a crucial to initiating innate immunity and adaptive immunity.

A. toll-like receptors (TLRs)

A series of signaling pattern-recognition receptors known as toll-like receptors (TLRs) play a major role in innate immunity and the induction of adaptive immunity.

Different combinations of TLRs appear in different cell types and seem to appear in pairs. Different TLRs directly or indirectly bind different microbial molecules. For example:

1. TLRs found on cell surfaces:

a. TLR-1/TLR-2 pairs bind uniquely bacterial lipopeptides and glycosylphosphatidylinositol (GPI)-anchored proteins in parasites;
b. TLR-2/TL6 pairs bind lipoteichoic acid from gram-positive cell walls and zymosan from fungi;
c. TLR-2 plays a role in binding peptidoglycan fragments (glycopeptides);
d. TLR-4/TLR-4 pairs bind lipopolysaccharide from gram-negative cell walls;
e. TLR-5* binds bacterial flagellin;
 

2. TLRs found in the membranes of the endosomes used to degrade pathogens:

a. TLR-3* binds double-stranded viral RNA;
b. TLR-7* binds uracil-rich single-stranded viral RNA such as in HIV;
c. TLR-8* binds single-stranded viral RNA;
d. TLR-9* binds unmethylated cytosine-guanine dinucleotide sequences (CpG DNA) found in bacterial and viral genomes.

*other member of pair is unknown

The binding of a microbial molecule to its TLR transmits a signal to the cell's nucleus inducing the expression of genes coding for the synthesis of intracellular regulatory molecules called cytokines. The cytokines, in turn, bind to cytokine receptors on other defense cells.

Many of the TLRs, especially thost that bind to bacterial and fungal cell wall components, stimulate the transcription and translation of cytokines such as interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8) that trigger innate immune defenses such as inflammation, fever, and phagocytosis in order to provide an immediate response against the invading microorganism. Most of the TLRs that bind to viral components trigger the synthesis of cytokines called interferons that block viral replication within infected host cells. Cytokines such as interleukin-6 (IL-6) that promotes B-lymphocyte activity and interleukin-12 that promotes T-lymphocyte activity are also produced.

TLRs also participate in adaptive immunity by triggering various secondary signals needed for humoral immunity (the production of antibodies) and cell-mediated immunity (the production of cytotoxic T-lymphocytes and additional cytokines). Without innate immune responses there could be no adaptive immunity.

a. T-independent (TI) antigens allow B-lymphocytes to mount an antibody without the requirement of interaction with T4-lymphocytes. The resulting antibody molecules are generally of the IgM isotype and do not give rise to a memory response. There are two basic types of T-independent antigens: TI-1 and TI-2. TI-1 antigens are pathogen-associated molecular patterns such as lipopolysaccharide (LPS) from the outer membrane of the gram-negative cell wall and bacterial nucleic acid. These antigens activate B-lymphocytes by binding to their specific toll-like receptors rather than to B-cell receptors. Antibody molecules generated against TI-1 antigens are often called "natural antibodies" because they are always being made against bacteria present in the body.

b. The activation of naive T-lymphocytes requires co-stimulatory signals involving the interaction of accessory molecules on antigen-presenting cells (APCs) with their corresponding ligands on T-lymphocytes. These co-stimulatory molecules are only synthesized when toll-like receptors on APCs bind to pathogen-associated molecular patterns of microbes

B. CD14

CD14 is found on monocytes, macrophages, and neutrophils and promotes the ability of TLR-4 to respond to LPS and peptidoglycan. Interaction of these molecules with CD14 and TLR-4 leads to an elevated synthesis and secretion of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-alpha, and PAF. These cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway

c. NODs

NOD (nucleotide-binding oligomerization domain) proteins, including NOD1 and NOD2, are cytostolic proteins that allow intracellular recognition of peptidoglycan components.

1. NOD1 recognizes peptidoglycan containing the muramyl dipeptide NAG-NAM-gamma-D-glutamyl-meso diaminopimelic acid, part of the peptidoglycan monomer in common gram-negative bacteria and just a few gram-positive bacteria.

2. NOD2 recognizes peptidoglycan containing the muramyl dipeptide NAG-NAM-L-alanyl-isoglutamine found in practically all bacteria.

As macrophages phagocytose either whole bacteria or peptidoglycan fragments released during bacterial growth, the peptidoglycan is broken down into muramyl dipeptides. Binding of the muramyl dipetides to NOD1 or NOD2 leads to the activation of genes coding for proinflammatory cytokines in a manner similar to the cell surface toll-like receptors.

 

d. secreted pattern recognition receptors

In addition to the cell surface pattern-recognition receptors there are also secreted pattern-recognition receptors. These bind to microbial cell walls and enable them to be recognized by the complement pathways and phagocytes. For example, mannan-binding lectin is synthesized by the liver and released into the bloodstream where it can bind to the carbohydrates on bacteria, yeast, some viruses, and some parasites. This, in turn, activates the lectin complement pathway and results in the production of C3b, a molecule that promotes the attachment of microorganisms to phagocytes.

 

2. Cytokines

Cytokines are low molecular weight, soluble proteins that are produced in response to an antigen and function as chemical messengers for regulating the innate and adaptive immune systems. They are produced by virtually all cells involved in innate and adaptive immunity, but especially by T helper (Th) lymphocytes. The activation of cytokine-producing cells triggers them to synthesize and secrete their cytokines. The cytokines, in turn, are then able to bind to specific cytokine receptors on other cells of the immune system and influence their activity in some manner.

Cytokines are pleiotropic, redundant, and multifunctional.

  • Pleiotropic means that a particular cytokine can act on a number of different types of cells rather than a single cell type.

  • Redundant refers to to the ability of a number of different cytokines to carry out the same function.

  • Multifunctional means the same cytokine is able to regulate a number of different functions.

Some cytokines are antagonistic in that one cytokine stimulates a particular defense function while another cytokine inhibits that function. Other cytokines are synergistic wherein two different cytokines have a greater effect in combination than either of the two would by themselves.

There are three functional categories of cytokines:

1. cytokines that regulate innate immune responses,
2. cytokines that regulate adaptive Immune responses, and
3. cytokines that stimulate hematopoiesis.

Cytokines that regulate innate immunity are produced primarily by mononuclear phagocytes such as macrophages and dendritic cells although they can also be produced by T-lymphocytes, NK cells, and other cells. They are produced primarily in response to pathogen-associated molecular patterns such as LPS, peptidoglycan monomers, teichoic acids, and double-stranded DNA. Most act on leukocytes and the endothelial cells that form blood vessels in order to promote and control early inflammatory responses.

Examples include:

a. Tumor necrosis factor-alpha (TNF-alpha)

TNF-alpha is the principle cytokine that mediates acute inflammation. In excessive amounts it also is the principal cause of systemic complications such as the shock cascade. Functions include acting on endothelial cells to stimulate inflammation and the coagulation pathway; stimulating endothelial cells to produce selectins and ligands for leukocyte integrins during diapedesis; stimulating endothelial cells and macrophages to produce chemokines that contribute to diapedesis, chemotaxis and the recruitment of leukocytes; stimulating macrophages to secrete interleukin-1 (IL-1) for redundancy; activating neutrophils and promoting extracellular killing by neutrophils; stimulating the liver to produce acute phase proteins, and acting on muscles and fat to stimulate catabolism for energy conversion. In addition, TNF is cytotoxic for some tumor cells; interacts with the hypothalamus to induce fever and sleep; stimulates the synthesis of collagen and collagenase for scar tissue formation; and activates macrophages. TNF is produced by monocytes,macrophages, dendritic cells, Th1 cells, and other cells.

b. Interleukin-1 (IL-1)

IL-1 function similarly to TNF in that it mediates acute inflammatory responses. It also works synergistically with TNF to enhance inflammation. Functions of IL-1 include promoting inflammation; activating the coagulation pathway, stimulating the liver to produce acute phase proteins, catabolism of fat for energy conversion, inducing feverand sleep; stimulates the synthesis of collagen and collagenase for scar tissue formation; stimulates the synthesis of adhesion factors on endothelial cells and leukocytes for diapedesis; and activates macrophages. IL-1 is produced by monocytes, macrophages, dendritic cells, and a variety of other cells in the body.

c. Chemokines

Chemokines are a group of cytokines that enable the migration of leukocytes from the blood to the tissues at the site of inflammation. They increase the affinity of integrins on leukocytes for ligands on the vascular wall during diapedesis, regulate the polymerization and depolymerization of actin in leukocytes for movement and migration, and function as chemoattractants for leukocytes. In addition, they trigger some WBCs to release their killing agents for extracellular killing and induce some WBCs to ingest the remains of damaged tissue. Chemokines also regulate the movement of B-lymphocytes, T-lymphocytes, and dendritic cells through the lymph nodes and the spleen. Certain chemokines have also been shown to suppress HIV, probably by binding to the chemokine receptors serving as the second binding factor for HIV on CD4+ cells. When produced in excess amounts, chemokines can lead to damage of healthy tissue as seen in such disorders as rheumatoid arthritis, pneumonia, asthma, adult respiratory distress syndrome (ARDS), and septic shock. Examples of chemokines include IL-8, MIP-1a, MIP-1b, MCP-1, MCP-2, MCP-3, GRO-a, GRO-b, GRO-g, RANTES, and eotaxin. Chemokines are produced by many cells including leukocytes, endothelial cells, epithelial cells, and fibroblasts.

d. Interleukin-12 (IL-12)

IL-12 is a primary mediator of early innate immune responses to intracellular microbes. It is also an inducer of cell-mediated immunity. It functions to stimulate the synthesis of interferon-gamma by T-lymphocytes and NK cells; increases the killing activity of CTLs and NK cells; and stimulates the differentiation of naive T4-lymphocytes into interferon-gamma producing Th1 cells. It is produced mainly by macrophages and dendritic cells.

e. Type I Interferons

Interferons modulate the activity of virtually every component of the immune system. Type I interferons include more than 20 types of interferon-alpha, interferon-beta, interferon omega, and interferon tau. There is only one type II interferon, interferon-gamma.

Type I interferons, produced by virtually any virus-infected cell, provides an early innate immune response against viruses. Interferons induce uninfected cells to produce enzymes capable of degrading mRNA. These enzymes remain inactive until the uninfected cell becomes infected with a virus. At this point, the enzymes are activated and begin to degrade both viral and cellular mRNA. This not only blocks viral protein synthesis, it also eventually kills the infected cell. They also promote body defenses by enhancing the activities of CTLs, macrophages, dendritic cells, NK cells, and antibody-producing cells.

Type I interferons also induce MHC-I antigen expression needed for recognition of antigens by CTLs; augment macrophage, NK cell, CTL, and B-lymphocyte activity; and induce fever. Interferon-alpha is produced by T-lymphocytes, B-lymphocytes, NK cells, monocytes/macrophages; interferon-beta by virus-infected cells, fibroblasts, macrophages, epithelial cells, and endothelial cells.

f. Interleukin-6 (IL-6)

IL-6 functions to stimulate the liver to produce acute phase proteins; stimulates the proliferation of B-lymphocytes; and increases neutrophil production. IL-6 is produced by many cells including T-lymphocytes, macrophages, monocytes, endothelial cells, and fibroblasts.

g. Interleukin-10 (IL-10)

IL-10 is an inhibitor of activated macrophages and dendritic cells and as such, regulates innate immunity and cell-mediated immunity. IL-10 inhibits their production of IL-12, co-stimulator molecules, and MHC-II molecules, all of which are needed for cell-mediated immunity. IL-10 is produced mainly by macrophages, and Th2 cells.

h. Interleukin 15 (IL-15)

IL-15 stimulates NK cell proliferation and proliferation of T-lymphocytes. IL-15 is produced by various cells including macrophages.

i. Interleukin-18 (IL-18)

IL-18 stimulates the production of interferon-gamma by NK cells and T-lymphocytes and thus induces cell-mediated immunity. It is produced mainly by macrophages.

 

3. Harmful Effects Associated with Pattern-Recognition Receptors and Cytokine Production

There are a number of harmful effects that are known to occur as a result of either an overactive or an underactive innate immune response. These include:

a. Sepsis (Systemic Inflammatory Response Syndrome or SIRS) from an Overactive Innate Immune Response

Cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interleukin-8 (IL-8) are known as proinflammatory cytokines because they promote inflammation. Some cytokines, such as IL-8, are also known as chemokines  . They promote an inflammatory response by enabling white blood cells to leave the blood vessels and enter the surrounding tissue, by chemotactically attracting these white blood cells to the infection site, and by triggering neutrophils to release killing agents for extracellular killing. In addition to promoting an inflammatory response, these same cytokines activate the complement pathways   as well as the coagulation pathway  .

  • Inflammation is the first response to infection and injury and is critical to body defense. Basically, the inflammatory response is an attempt by the body to restore and maintain homeostasis after injury. Most of the body defense elements are located in the blood, and inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around an injured or infected site. The release of proinflammatory cytokines eventually leads to vasodilation of blood vessels. Vasodilation   is a reversible opening of the junctional zones between endothelial cells   of the blood vessels and results in increased blood vessel permeability. This enables plasmathe liquid portion of the blood, called plasma, to enter the surrounding tissue. The plasma contains defense chemicals such as antibody molecules, complement proteins, lysozyme, and defensins. Increased capillary permeability also enables white blood cells to squeeze out of the blood vessels and enter the tissue. As can be seen, inflammation is necessary part of body defense. Excessive or prolonged inflammation can, however, cause harm as will be discussed below.
     
  • As mentioned in a previous section, products of the complement pathways   lead to more inflammation, opsonization of bacteria, chemotaxis of phagocytes to the infected site, and MAC lysis of gram-negative bacteria.
     

The products of the coagulation pathway   lead to the clotting of blood to stop bleeding, more inflammation, and localization of infection.
 

At moderate levels, inflammation, products of the complement pathways, and products of the coagulation pathway are essential to body defense. However, these same processes and products when excessive, can cause considerble harm to the body.

When there is a minor infection with few bacteria present, low levels of cell wall components are present. This leads to moderate cytokine production with the results being primarily beneficial. However, in the case of a severe infection with very large numbers of bacteria present, high levels of cell wall components are present. This leads to excessive cytokine production with the results causing damage to the body.

This excessive inflammatory response is referred to as Systemic Inflammatory Response Syndrome or SIRS. Death is a result of what is called the shock cascade. The sequence of events is as follows:

This is seen during septicemia  , a condition where bacteria enter the blood and cause harm. There are approximately 750,000 cases of septicemia per year in the U.S. and the mortality rate is between 20% and 50%. Over 210,000 people a year in the U.S. die from septic shock. Approximately 45% of the cases of septicemia are due to gram-positive bacteria, 45% are a result of gram-negative bacteria, and 10% are due to fungi (mainly the yeast Candida).

b. People with an underactive form of TLR-4, the toll-like receptor for bacterial LPS, have been found to be five times as likely to contract a severe bacterial infection over a five year period than those with noemal TLR-4.

c. Most people that die as a result of Legionnaire's disease have been found to have a mutation in the gene coding for TLR-5.

d. People with the autoimmune disease systemic lupus erythematosis have an altered form of TLR-9 that reacts with the body's own DNA.

e. Mutations in the gene coding for NOD2 that prevent the NOD2 from recognizing muramyl dipeptide make a person more susceptible to Crohn's disease, an inflammatory disease of the large intestines.

4. Therapeutic Possibilities

Researchers are now looking at various ways to either artificially activate TLRs in order to enhance immune responses or inactivate TLRs to lessen inflammatory disorders. Examples of agents being evaluated in clinical studies include:

a. TLR-4 and TLR-9 activators: as vaccine adjuvants to activate the immune system.
b. TLR-7 activator: as an antiviral against hepatitis C.
c. TLR-4 inhibitor: as an antisepsis agent against SIRS.
d. General TLR inhibitors: to treat autoimmune disorders.
 

A number of human cytokines produced by recombinant DNA technologies are now being used to treat various infections or immune disorders. These include:

1. recombinant interferon alfa-2a (Roferon-A): a cytokene used to treat Kaposi's sarcoma, chronic myelogenous leukemia, and hairy cell leukemia.
2. peginterferon alfa-2a (Pegasys) : used to treat hepatitis C (HCV).
3. recombinant interferon-alpha 2b (Intron A): a cytokine produced by recombinant DNA technology and used to treat Hepatitis B; malignant melanoma, Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia, warts, and Hepatitis C.
4. peginterferon alfa-2b (PEG-Intron; PEG-Intron Redipen): used to treat hepatitis C (HCV).
5. recombinant Interferon alfa-2b plus the antiviral drug ribavirin (Rebetron): used to treat hepatitis C (HCV).
6. recombinant interferon-alpha n3 (Alferon N): used to treat warts.
7. recombinant iInterferon alfacon-1 (Infergen) : used to treat hepatitis C (HCV).
8. G-CSF (granulocyte colony stimulating factor): for reduction of infection in people after myelotoxic anticancer therapy for solid tumors.
9. GM-CSF (granulocyte-macrophage colony stimulating factor): for hematopoietic reconstruction after bone marrow transplant in people with lymphoid cancers.

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