Eileen L. Thacker Dept. of Veterinary Microbiology & Preventive Medicine Associate Professor Extracted from The Pig Journal, Vol 52 Order your copy now View Vol. 52 here

The Pig Journal Immunology series comprises:

1. Immunology: The innate immune system
   The Pig Journal (2003) Vol. 52 p. 111-123.
2. Lymphocyte development and maturation
   The Pig Journal (2004) Vol. 53, p. 75-91.
3. The battle between the immune system and pathogens
   The Pig Journal (2004) Vol. 54, p 55-69.

Part 1: Immunology - The innate immune system

The immune response to pathogens and antigens is efficient, yet complicated. The goal of this 3 part series is to refresh your knowledge on how the immune system works, in addition to introducing you to new concepts. Immunology is a dynamic field. New knowledge of how this system protects the body against foreign invaders and yet does not destroy itself (usually) is being determined constantly.

As our ability to measure the immune response at both the cellular and molecular level increases, we begin to recognize how successful pathogens evade and circumvent the immune response in order to replicate and survive. Understanding the immune system and its response to a pathogen is required to develop successful intervention strategies for controlling and potentially eliminating the problematic pathogens we encounter in the field.

The two primary immune responses to micro-organisms and their antigens are generated by the innate and acquired or adaptive immune systems. Originally it was thought that these two systems functioned independently of each other. However, it is increasingly apparent that the two immune systems interact intimately. The two immune systems fulfill the different needs of the host to control micro-organisms.

The adaptive immune response is a specific response against a particular pathogen or antigen, lymphocytes are the primary effector cells, a memory response is generated, and the response increases with each exposure to the antigen.

The innate system is not as specific in its recognition of foreign antigens and organisms, has a number of different effector cells and molecules, and the response does not increase with repeated exposure.

The cells of the immune system originate in the bone marrow. The primary cell types include the myeloid cells; monocytes, macrophages, dendritic cells, neutrophils, eosinophils, and basophils and lymphocytes, of which there are two major types, the B and T lymphocytes.

Lymphocytes are the primary cell type of the adaptive immune response and will be discussed in more detail later. Lymphocytes mature in either the bone marrow (B cells) or thymus (T cells), which are known as primary lymphoid organs. The actual immune responses to antigens occur in the peripheral, or secondary lymphoid organs. The secondary lymphoid organs consist of lymph nodes, spleen, Peyer’s patches and various mucosal associated lymphoid tissues.

In the secondary lymphoid tissues, cells of the innate immune system trap antigens and present them to the lymphocytes that are constantly circulating through the lymphoid tissues. Lymphocytes are naïve until they encounter their specific antigen, after which they differentiate into the various effector cells capable of responding to their pathogen.

In most species, lymphocytes enter into the secondary lymphoid organs through the blood, circulate through the organ and if they do not encounter the appropriate antigen, leave through the lymphatic vessels. This circulation pattern is reversed in the pig, where the lymphocytes enter into the lymphoid organs through the lymphatic vessels and exit directly into the blood.

Most organisms encountered by the host do not cause disease under normal circumstances. Most are detected quickly by defense mechanisms of the innate immune system and infection prevented. The innate immune system is considered the first line of defense in protecting the host from invading organisms. Originally, it was thought to be non-specific, but recent research has demonstrated that is not the case. The innate immune response is immediate, followed by a number of early-induced responses that do not generate lasting immunity.

It is unknown how many micro-organisms are prevented on a daily basis from establishing an infection by the innate immune system. Only if the organism escapes and survives the innate immune response does an adaptive or acquired immune response occur, resulting in the generation of a specific immune response that will target that particular organism. The generation of the adaptive immune response is directed by the innate immune response, linking the two immune responses closely.

The innate immune system is composed of a number of different cell types, molecules, and defense mechanisms. Epithelial cells are the first barrier that prevents micro-organisms from infecting the host. This is true externally with skin and internally by the epithelial cells lining the various mucosal surfaces. These epithelial surfaces are more than just physical barriers; they also have other effector mechanisms, both physical in nature such as cilia and mucus on their surfaces that move potential pathogens out of the body, and molecular and chemical defense systems that include enzymes and antibacterial peptides.

Antimicrobial peptides are important effectors in the innate immune system. They act primarily by damaging and forming pores in the bacterial membranes. These proteins play an important role by destroying bacteria prior to colonization. The antimicrobial proteins are produced by a number of different cell types, including epithelial cells and skeletal muscle. These antimicrobial proteins are also known as defensins. Many of them are cationic in nature and display broad-spectrum antimicrobial activity under conditions of low salt. Antimicrobial peptides have been isolated from all mammalian species, including pigs.

There are a number of families of cationic defensins designated as α, β, and θ-defensins. Of these, the β-defensins have been best characterized. The defensins from different species have variable abilities against different classes of microbes. In addition to the cationic proteins, surfactant proteins A and D, present in the respiratory tract, are also members of the defensin family. Instead of forming pores in bacterial membranes, surfactant proteins A and D increase the ability of phagocytic cells to engulf and destroy the organisms by binding to the pathogens surface. Coating micro-organisms with proteins to facilitate engulfment by phagocytes is called opsonization. Ongoing research is identifying an increasing number of these peptides. The possibility of using the various peptides as alternatives to antibiotics is an area of interest and ongoing research.

Phagocytic cells play a critical role in innate immunity. Ingestion of pathogenic micro-organisms accomplishes two essential immune functions. Firstly, phagocytosis of the organisms initiates their death by hydrolytic enzymes and oxidase complexes in the cell lysosomes. Secondly, the phagocytic cells bind pieces of the organisms to the major histocompatibility complex (MHC) I and II molecules, that migrate to the surface of the cell and are presented to lymphocytes, resulting in an adaptive immune response.

Neutrophils, monocytes, macrophages, B lymphocytes, and dendritic cells are all phagocytic cells. Macrophages, monocytes and dendritic cells are the primary or professional antigen presenting cells (APCs). B cells can also phagocytose and present antigens to the immune system, but to a lesser extent. It has been estimated that APCs can internalize the equivalent of 100% of their surface area within 30 minutes, without a reduction in exposed membrane surface. Thus, they are very efficient in engulfing foreign particles.

Macrophages are important, not only for phagocytosing and killing invading micro-organisms but, as APCs, are an important connection between the innate and adaptive immune systems. When micro-organisms successfully cross the epithelial cell barrier and replicate, they are quickly recognized by tissue macrophages. Tissue macrophages mature from monocytes that circulate in the blood. Neutrophils, while not APCs, are also important phagocytic cells and important in the innate immune response to pathogens. Both macrophages and neutrophils are capable of phagocytosing and destroying many pathogens without assistance from the adaptive immune system.

Specific receptors on neutrophils and macrophages recognize micro-organisms. Recognition and uptake of pathogens by macrophages induce the production of cytokines and chemokines that stimulate other phagocytic cells to migrate to the site of infection, increase phagocytosis, and induce production of toxic substances within the cell compartments to kill the micro-organisms. Specific receptors on phagocytic cells bind directly to the micro-organisms and further activate the cells. These receptors include mannose binding receptors that bind sugars found on microbial cell walls that differ in composition from the sugars on mammalian cells.

Another important receptor on the surface of macrophages recognizes and binds to IgG antibodies, resulting in increased phagocytosis of micro-organisms. There are a number of these important receptors on the surface of cells, known as toll-like receptors (TLR). These receptors are on a number of different cell types and are important in recognizing pathogens.

A cell surface receptor on macrophages, TLR 4, binds lipopolysaccharide (LPS) in conjunction with cell surface receptor, CD14, and a plasma protein called LPS-binding protein (LBP). LBP delivers the LPS, an important component in the membrane of gram negative bacteria, to the co-receptor system of CD14 and TLR 4 to activate macrophages to produce pro-inflammatory cytokines. Other TLRs bind other recognition patterns of pathogens resulting in activation of various immune cells.

In addition to LPS, which is associated with the cell membranes of gram negative bacteria, bacterial DNA contains significantly more unmethylated cytidine-guanine nucleic acid sequences (CpG nucleotides) than observed in mammalian cells. If enough CpG nucleotide sequences are present, phagocytosis and the production of pro-inflammatory cytokines is augmented, further activating macrophages and increasing inflammation. In addition to the receptors specific for micro-organisms, macrophages have receptors for a number of important cytokines, including interferon gamma (IFN-?) produced by natural killer (NK) cells. The binding of these cytokines by macrophages results in further activation, uptake of foreign materials and antigen presentation to lymphocytes, resulting in the induction of an adaptive immune response.

Because phagocytosis is the first host response to most bacterial and fungal pathogens, many pathogens have developed mechanisms to circumvent their uptake in order to survive. Some species of pathogens, such as yersinia or listeria, utilize phagocytosis to enter and infect macrophages. Following uptake, these pathogens survive and multiply within the phagocytic cells. Other bacteria and fungi have developed large cell walls or slime layers that prevent engulfment of the organisms. Some micro-organisms produce products that are toxic to the phagocytic cells. Bacteria that do not have mechanisms to prevent phagocytosis and killing by macrophages and neutrophils require increased numbers to overwhelm the immediate innate response resulting in infection. Overall however, phagocytic cells are very effective in recognizing and destroying invading micro-organisms.

Activation of macrophages results in the release of cytokines that induce inflammation. Inflammation has a number of essential roles in fighting micro-organisms. Attraction of additional effector molecules and cells to the site by cytokines and chemokines further augments the control and killing of the micro-organisms. Macrophages produce a physical barrier and promote the healing and repair of the injured tissue. Inflammation is characterized by pain, redness, heat and swelling at the infection site. Most of these changes are due to changes in the local blood vessels.

Following infection, blood vessels dilate, resulting in an increased blood flow to the area. At the same time, the velocity of the blood flow slows. Endothelial cells lining the blood vessels express adhesion molecules to bind circulating phagocytic cells and allow them to migrate to the site of infection and inflammation. Permeability of the blood vessels is increased, resulting in oedema. Neutrophils are usually the first cells attracted to the site, followed by monocytes, which further differentiate into tissue macrophages.

In later stages of inflammation, other cells such as eosinophils and lymphocytes may also be attracted to the area. A variety of inflammatory mediators, including prostaglandins, leukotrienes and tumor necrosis factor-alpha (TNF-a) are produced by activated inflammatory cells.

Complement and its components are important mediators of the innate immune system. The complement system is made up of specific plasma proteins that react with one another to opsonize pathogens and induce inflammation to assist in fighting pathogens. Most of the complement proteins are enzymes that are distributed throughout the body fluids and tissues and require cleavage by other enzymes for activation. This results in a cascade that is triggered by enzymes, with each enzyme cleaving another. This allows a very small number of enzymes to activate a system that amplifies as it progresses.

In order to prevent this powerful system from overwhelming the host, many regulatory mechanisms are in place at each step of the cascade. The complement system aids in protecting and controlling infection by activating large numbers of complement proteins that bind to pathogens and facilitate their opsonization by phagocytic cells. In addition, small fragments produced during the cleavage of the complement proteins act as chemokines and recruit more phagocytes to the area. Finally, the terminal components of complement bind together and attach to the bacterial membranes, resulting in the formation of pores and the lysis of the bacteria.

Complement can be activated by three distinct pathways. The three pathways converge to generate the same set of effector complement molecules. The first pathway involves the binding of the first protein in the complement cascade (C1q) directly to the surface of pathogens and activates the classical pathway. The mannan-binding lectin pathway (MB-lectin pathway) is initiated by binding of mannan-binding lectin, a serum protein, to mannose-containing carbohydrates on bacteria or viruses that activates the complement cascade. The alternative pathway is initiated when a spontaneously activated complement component binds directly to the surface of a pathogen. Each of these pathways generate C3 convertase which activates the complement cascade, producing equivalent products and terminating in the membrane-attack complex, which creates a pore in the cell membrane of pathogens causing their death.

In addition to opsonization and pore formation by the primary components of complement, small fragments produced during the cleavage of the complement proteins can initiate local inflammation. All induce smooth muscle contraction and increased vascular permeability. The inflammation induced recruits antibodies, complement, and phagocytic cells to the site of the infection. The increased fluid in the tissues hastens the movement of pathogen-bearing APCs to the secondary lymphoid tissues, contributing to the prompt initiation of the adaptive immune response. When these small complement fragments are produced in large amounts they induce generalized circulatory collapse, producing a shock-like syndrome similar to that induced by IgE and anaphylaxis.

It is not within the scope of this article to discuss the complement cascade in detail, nor is it of great interest to most practitioners. Historically, it was thought that the membrane attack complex was the most important mediator of activated complement. However, further research has demonstrated that facilitating the uptake and destruction of pathogens by phagocytic cells through complement binding is the primary mechanism used to control pathogens and infection by the complement system. While the complement cascade is an important aspect of the innate immune system, recent research has demonstrated that there are important interactions and communication between the complement system and the adaptive immune system.

Cytokines produced by activated macrophages effect the immune responses, both locally and systemically. Cytokines can further influence the cells that produced them (autocrine), adjacent cells (paracrine), or distant cells (endocrine), depending on their ability to enter the circulation and how long they survive (half life). Most cytokines have very short half lives, consisting of a few hours. Cytokines are proteins produced by cells to communicate with each other. Chemokines compromise a specific group of cytokines that function to attract other cells to the area of infection. Different cells produce different chemokines or cytokines under different stimuli.

A number of important pro-inflammatory cytokines are produced by macrophages in response to pathogens and include interleukin (IL)-1, IL-6, IL-12 and TNF-α. In addition, the chemokine, IL-8, a strong neutrophil attractant, is often produced. The cytokines produced are dependent on the mechanism used by the pathogen to activate the macrophage. Depending on the specific receptors activated, and the effect of pathogen uptake on the APC, different cytokines are produced that will affect both the amount of inflammation produced and the type of adaptive immune response induced.

Typically, the first cells attracted to the infection site in large numbers are neutrophils responding to IL-8 produced by macrophages. Their number peaks within the first 6 hours. Monocytes appear later in the inflammatory response and differentiate into macrophages and dendritic cells, which produce cytokines, further activating and directing the immune response.

The quantity produced, as well as the ratio between the different cytokines produced by the APCs determines the type of adaptive immune response induced. TNF-α is an important pro-inflammatory cytokine produced by activated macrophages. Production of TNF-α by macrophages induces dendritic cells and macrophages to migrate to the regional secondary lymphoid tissues, initiating an adaptive immune response to the micro-organisms. TNF-α also assists in containing local infection by stimulating endothelial cells to produce proteins that cause blood clots to form in the local blood vessels, cutting blood flow to the area.

This local clotting aids in preventing the systemic spread of pathogens through the blood. In addition, the fluid that leaks into the tissues through the permeabilized blood vessels in the early stages of infection carry more phagocytic cells that continue to engulf the micro-organisms. These phagocytic cells then migrate to the regional lymph nodes where they present the antigens to the lymphocytes and initiate an adaptive immune response. However, if an infection becomes systemic, the increased levels of TNF-α can become problematic. Septic shock and death due to disseminated intravascular coagulation (DIC) result from elevated levels of systemic TNF-α.

The kinetics of TNF-α production illustrates a fact that is true of many of the mediators of immunity; if a little bit is good, more is not necessarily better. Because of the strong impact by cytokines on the immune system, many regulatory mechanisms are in place to control their levels through degradation by enzymes that ensure that cytokines have very short half lives.

The cytokines produced by activated macrophages also can have broad systemic effects that further contribute to host defense. Fever, induced by IL-1 and IL-6, is generally beneficial to the host, as most pathogens grow optimally at lower temperatures. Increased temperatures also protect the host cells from the effects of TNF-α. Production of acute-phase proteins by the liver are also induced by IL-1, IL-6 and TNF-α. Several of the acute phase proteins mimic the action of antibodies, although lack their specificity, and bind to bacterium as opsonins. C-reative protein is an acute phase protein that activates the classical complement cascade in a similar manner as antibodies.

A second acute phase protein is the mannan-binding lectin, discussed earlier, that also initiates the complement cascade. In addition, surfactants A and D, discussed as defensins, are also produced as acute-phase proteins. Acute phase proteins can be produced in response to any stimulus that induces production of IL-1, IL6 or TNF-α.

Infection by viruses induces the production of interferon (IFN)-α and IFN-β. These proteins are named for their ability to interfere with viral replication. IFN-α and IFN-β are Type I interferons that are distinct from IFN-?, which will be discussed in more detail later. Production of IFN-α and IFN?beta; are thought to occur in response to the presence of double-stranded RNA, which is not found in mammalian cells.

Either IFN-α and/or IFN-β are secreted by cells following viral infection and bind to receptors on neighbouring cells to prevent their infection by the virus. These cytokines block viral replication in either infected or neighbouring cells by causing the cells to produce a number of inhibitory proteins. In addition, interferons impact the adaptive immune response by increasing the number of MHC class I molecules on the cell surface. This increases the ability of T lymphocytes to recognize virus infected cells. Interferons also activate natural killer (NK) cells to kill virus-infected cells and release cytokines.

Natural killer (NK) cells are T lymphocytes that are members of the innate immune system. NK cells are activated by interferons and cytokines produced by activated macrophages. NK cells were initially identified by their ability to kill tumour cells without prior sensitization, making them members of the innate immune system. NK cells kill infected cells by releasing cytotoxic granules onto the surface of the bound target cells, resulting in cell lysis. In addition, effector proteins produced by the NK cells penetrate the cell membrane and induce programmed cell death (apoptosis).

NK cell activation receptors have been identified that recognize specific virus-encoded molecules, making them important cells for controlling virus infections. In addition to activation through binding of viruses to receptors on NK cells, the presence of IFN-α, IFN-β, IL-12, IL-15 and IL-18 produced by infected cells, activated macrophages, or dendritic cells can stimulate NK cells to produce cytokines including IFN-γ, TNF-α, as well as various chemokines that attract other inflammatory cells and direct the adaptive immune response. Immunologists, for many years, considered the innate immune system less important than the adaptive immune response. However, as more is learned about the interaction between the innate and adaptive immune systems, we are recognizing its importance in not only controlling the initial invasion by micro-organisms, but in directing and activation of the adaptive immune system. Activation of the innate immune system is an important tool used in vaccination. To make effective vaccines using killed and inactivated organisms, use of adjuvants is typically required.

The adjuvants activate effectors of the innate immune system in order to activate the adaptive immune response to the antigens in the vaccine. Uptake of the vaccine antigens by macrophages and dendritic cells is required for activation of an adaptive immune response. This requires the use of salts, oils or other formulations to attract macrophages, induce inflammation and the cytokines that direct the migration of the APCs to the secondary lymphoid tissues and activation of the adaptive immune response. As our understanding of the innate immune system increases, our ability to further refine adjuvants to enhance and direct the adaptive immune response to vaccine antigens will improve.

The innate immune system is crucial for survival. If any part of the innate immune system is absent, infection with secondary pathogens, especially bacteria, occurs. The goal of the innate immune response to a pathogen is to either clear the infection or control it until an adaptive response can develop. The adaptive immune response uses many of the same effector mechanisms used by the innate immune system, but is able to target them with greater precision.

Thus antigen-specific T cells further activate the microbicidal and cytokine-secreting properties of macrophages harbouring pathogens, while antibodies activate complement, act as direct opsonins for phagocytic cells and stimulate NK cells to kill infected cells. In addition, the adaptive immune response produces cytokines and chemokines, in a similar manner to the innate immune system, resulting in further inflammation and influx of phagocytic cells, antibodies and effector lymphocytes to the site of infection.

This was a brief overview of the innate immune system. As our understanding of the innate immune system increases, our ability to manipulate it and use it in intervention strategies increases. Examples of ongoing research include the use of antimicrobial peptides as antibiotics and growth promotants and the use of cytokines as adjuvants to direct the immune response to a vaccine. The innate immune system is no longer considered uninteresting to immunologists and its activation is typically the key to the development of an effective adaptive immune response.

Glossary

Acute phase proteins: Proteins that are produced by the liver in the early phases of host defense against infection. Part of the innate immune system.

Antigens: Any foreign substance that can induce an immune response.

Apoptosis: Programmed cell death with characteristic morphology and specific physiological pathways.

Chemokines: Small pro-inflammatory chemo-attractant cytokine molecules that attract and activate cells, especially phagocytic cells and lymphocytes.

Cytokines: Proteins produce by cells that mediate cellular interaction, regulate cell growth and affect the behavior of other cells. They regulate the immune response.

Effector cells: A cell that is able to effect an immune response. They can mediate the removal of pathogens without further differentiation or proliferation.

Interferons: Cytokines that can induce cells to resist viral replication. Some interferons play an important role in immune regulation.

Interleukins: Proteins that act as growth and differentiation factors for the cells of the immune system. The term used to name some of the specific cytokines.

Major Histocompatibility Complex (MHC): MHC class I molecules present peptides generated in the cytosol of cells to CD8+ T cells and MHC class II molecules presents peptides degraded in intracellular vesicles to CD4+ T cells.

Natural killer cells (NK cells): Large, granular non-T, non-B lymphocytes which kill tumor and cells infected with intracellular pathogens, such as viruses. They are important in the innate immune system.

Opsonization: The alteration or coating of the surface of a pathogen that facilitates phagocytosis. Antibody and complement opsonize bacteria for destruction by neutrophils and macrophages.

Pathogens: Micro-organisms that cause disease.

Source: Pig Journal - March 2004