Bio 141 Lecture Guide Unit 2: IIIA,B,C. Nonspecific Body Defenses

The universal principle of molecular recognition. Biological molecules interact by recognizing and binding with one another in a highly specific manner. Pairs of molecules that interact in this way are called receptors or binding sites and ligands respectively. Specific regions of atoms (molecular domains) on a receptor molecule have the characteristic of binding or attaching (docking) specifically to unique molecular domains on specific ligands. Following this binding something happens depending on the system involved. In this chapter we will see how the defense system of our bodies recognizes certain molecules of pathogens which in turn triggers a specific series of defensive reactions that defend us from many pathogens. Cartoon from Sigma Chemical Co.

The defense mechanisms humans have evolved to combat microorganisms can be characterized as:

1) Physical (mechanical,)
2) Chemical,
3) Inflammatory.
4) Biological

I. Anatomical Barriers and Mechanical Removal

Anatomical barriers: intact barriers that prevent the entry of many microbes.

skin: consists of the epidermis (the protein keratin and layers of epithelial cells) and the dermis. The dermis is bound to underlying muscles by connective tissue.
mucous membranes: line body cavities that open to the exterior such as the respiratory tract, the gastrointestinal tract, and the genitourinary tract. Mucous membranes are composed of an epithelial layer, which secretes mucous, and a connective tissue layer.
bony encasements: protect vital organs from injury and entry of microbes.

Mechanical removal

mucous and cilia: Mucous traps microorganisms and contains various antimicrobial agents. Cilia propels mucous and trapped microbes upwards towards the throat where it is swallowed. This is called the tracheal toilet.
cough and sneeze reflex: removes mucous and trapped microbes.
vomiting and diarrhea: removes pathogens and toxins in the gastrointestinal tract.
physical washing action of body fluids: urine, tears, saliva, perspiration, bleeding, etc.

a) Bacterial Antagonism by Normal Flora
Bacteria that are normal body flora keep potentially harmful opportunistic microorganisms in check and also inhibit the colonization of pathogens by:

producing metabolic products (fatty acids, bacteriocins, etc.) that inhibit the growth of many pathogens;
adhering to target host cells thus covering them and preventing pathogens from colonizing;
depleting nutrients essential for the growth of pathogens; and
nonspecifically stimulating the immune system.

Destruction of normal bacterial flora by the use of broad spectrum antibiotics may result in superinfections or overgrowth by antibiotic resistant opportunistic normal flora. The yeast Candida, which causes infections such as vaginitis and thrush, and the bacterium Clostridium difficile, which causes potentially severe antibiotic-associated colitis, are opportunistic flora normally held in check by normal flora bacteria. Antibiotic-associated colitis is especially common in older adults. It is thought that C. difficile survives the exposure to the antibiotic by sporulation. After the antibiotic is gone, the endospores germinate and C. difficileovergrows the intestinal tract and secretes toxin A which has a cytotoxic effect on the cells. Fortunately, C. difficile does respond to the antibiotic vancomycin so antibiotic-associated colitis is treatable.

Physical defense mechanisms useful to the human include:

i) Skin. Skin (the largest organ in the body--20% of total body weight) is very thick and tough. Further,
    the surface layer of skin made of dead cells which have a low water activity, are cool, and easily
    abrade.
ii) Hair and eyelashes. Hair physically protects the surface of the head, and maybe the arms and legs,
    and prevents cuts, etc., from occurring to skin under the hair, air in armpits and the groin area tends to
    keep skin surfaces from rubbing on one another and provides a "cushion" to allow air movement.
iii) Saliva and Phlegm. Mucous produced by tissues in the mouth nose and throat serve to wash
    microorganisms from the surface of the tissue.

Chemical defense mechanisms:

i) Lysozyme. Found in tears and serves to hydrolyze peptidoglycan on bacterial cells.
ii) Low pH of the vagina. Sugars secreted in vaginal fluids serve as a substrate for lactic acid (and
    other acid) producing bacteria. The acid(s) produced protect the vagina from infection by dangerous
    microorganisms.
iii) Immune system. An incredibly complex system of cellular and humoral components which the body
    uses to respond to microbial invasion in a highly specific fashion.

b) Nonspecific Antimicrobial Body Chemicals

Hydrochloric acid and enzymes in gastric secretions destroy microbes which are swallowed.
Lysozyme in tears, mucous, saliva, plasma, tissue fluid, etc., breaks down peptidoglycan in bacteria causing osmotic lysis.
Beta-lysins in blood serum disrupt the cytoplasmic membrane in a variety of bacteria.
Lactic and fatty acids in perspiration and sebaceous secretions inhibit microbes on the skin.
Complement proteins: Produced in the plasma and serum as a result of the complement pathways, these proteins are responsible for a) inflammation; b) sticking microorganisms to phagocytes; c) chemotaxis of phagocytes; and d) lysis of membrane-bound cells displaying foreign antigens (discussed later in Unit 3).
Cytokines: A wide variety of intercellular regulatory proteins produced by many different cells in the body which ultimately control every aspect of body defense. Cytokines activate and deactivate phagocytes and immune defense cells, increase or decrease the functions of the different immune defense cells, and promote or inhibit a variety of nonspecific body defenses. Examples include interleukins (IL), tumor necrosis factors (TNF), colony-stimulating factors (CSF), chemokines, and interferons (INF).
Chemokines: Cytokines that promote inflammation by enabling white blood cells to adhere to the inner surface of blood vessels, migrate out of the blood vessels into the tissue, and be chemotactically attracted to the injured or infected site. Examples include IL-8, macrophage inflammatory proteins (MIP), and monocyte chemotactic proteins (MCP).

Interferons: (alpha and beta) cytokines that prevent viral replication, activate a variety of cells important in body defense, and exhibit some anti-tumor activity.

NONSPECIFIC BODY DEFENSES

These are general mechanisms (cellular functions and biochemical pathways) which become engaged after any kind of injury and protect against all pathogens in general.

Non-specific host defense mechanisms are distinct from specific host defense mechanisms, which rely upon the specific recognition of the pathogen by lymphocytes. specific immunity is based upon the antibodies made by B-cells and upon the activities and cytokine secretions of T-cells. B-cells and T-cells have receptors, which recognize molecules on the invading organisms. Each b-cell and t-cell has a unique receptor. There are millions of specificities.

A. Phagocytosis
Before we look at phagocytosis in some detail we need to first become familiar with the various cells in both the bloodstream and in the tissues and organs of the body that will play a role in body defense.

Defense Cells in the Blood: The Leukocytes
All leukocytes (white blood cells or WBCs) are critical to body defense. There are normally between 5,000-10,000 leukocytes per cubic millimeter (mm3) of blood and these can be divided into five major types: neutrophils, basophils, eosinophils, monocytes, and lymphocytes. The production of colonies of the different types of leukocytes (leukopoiesis) is induced by various cytokines known as colony stimulating factors or CSFs.

The five types of leukocytes fall into one of two groups, the polymorphonuclear leukocytes and the mononuclear leukocytes.

Polymorphonuclear leukocytes (granulocytes) have irregular shaped nuclei with several lobes and their cytoplasm is filled with granules containing enzymes and antimicrobial chemicals. They include the following:

Neutrophils
* Neutrophils are the most abundant of the leukocytes, normally accounting for 54-75% of
    the WBCs. An adult typically has 3,000-7,500 neutrophils/mm3 of blood but the number
    may increase two- to three-fold during active infections.
* Neutrophils are important phagocytes.
* Their granules contain various agents for killing microbes. These include lysozyme
    (breaks down peptidoglycan), lactoferrin (makes iron unavailable to bacteria), acid
    hydrolase (degrades cellular proteins), and myeloperoxidase (catalyzes reactions that
    produce lethal oxidants, including hypochlorous acid, free chlorine, hydrogen peroxide,
    and hydroxyl radicals). These agents kill microbes intracellularly during phagocytosis
    but are also often released extracellularly where they kill not only microbes but also
    surrounding cells and tissue, as will be discussed later under phagocytosis.
* They release the enzyme kallikrein which catalyzes the generation of bradykinins.
    Bradykinins promote inflammation by causing vasodilation, increasing vascular
    permeability, and increasing mucous production. They are also chemotactic for leukocytes
    and stimulate pain.
* They release enzymes which catalyze the synthesis of prostaglandins from arachidonic
    acid in cell membranes. Certain prostaglandins promote inflammation by causing
    vasodilation and promoting capillary permeability. They also cause constriction of
    smooth muscles, enhance pain, and induce fever.

Eosinophils
Eosinophils normally comprise 1-4% of the WBCs (50-400/mm3 of blood).
* Their granules contain destructive enzymes for killing infectious organisms. These
enzymes include acid phosphatase, peroxidases, and proteinases.
* The substances they release defend primarily against fungi, protozoa, and parasitic worms,
pathogens that are too big to be consumed by phagocytosis.

Basophils
* Basophils normally make up 0-1% of the WBCs (25-100/mm3 of blood).
* Basophils release histamine which promotes inflammation by causing vasodilation,
increasing capillary permeability, and increasing mucous production.

Mononuclear leukocytes (agranulocytes) have compact nuclei and have no visible cytoplasmic granules. The following are agranulocytes:

Monocytes
* Monocytes normally make up 2-8% of the WBCs (100-500/mm3 of blood).
* Monocytes are important phagocytes.
* Monocytes become macrophages and dendritic cells when they leave the blood and
    enter tissues. Macrophages and dendritic cells are very important in phagocytosis and
    aid in the immune responses (see below). They produce a variety of cytokines that
    play numerous roles in body defense.

Lymphocytes
* Lymphocytes normally represent 25-40% of the WBCs (1,500-4,500/mm3 of blood).
* Lymphocytes mediate the specific immune responses
* Only a small proportion of the body's lymphocytes are found in the blood. The majority
are found in lymphoid tissue.
* Lymphocytes circulate back and forth between the blood and the lymphoid
system of the body.
* There are 2 major populations of lymphocytes:

B-lymphocytes (B-cells) mediate humoral immunity (antibody production). Generally 10-20% of the lymphocyte are B-lymphocytes. They become antibody-producing plasma cells.
T-lymphocytes (T-cells) mediate cellular immunity and regulate the immune responses. Generally 60-80% of the lymphocytes are T-lymphocytes. Based on biochemical markers on their surface, there are two major classes of T-lymphocytes:

T4-lymphocytes (T4-helper cells and inflammatory T4-lymphocytes) have CD4 molecules on their surface;
T8-lymphocytes (T8-suppressor cells and cytotoxic T-lymphocytes [CTLs]) have CD8 molecules on their surface.

Although not white blood cells, platelets (thrombocytes) are another formed element in the blood. They promote clotting by sticking together after becoming activated and forming platelet plugs to close up damaged capillaries. They also produce vasodilators to promote inflammation.

Defense Cells in the Tissue: Macrophages, Dendritic Cells, and Mast Cells

Macrophages
When monocytes leave the blood and enter the tissue, they become activated and differentiate into macrophages. Those that have recently left the blood during inflammation and move to the site of infection through positive chemotaxis are sometimes referred to as wandering macrophages.

In addition, the body has macrophages already stationed throughout the tissues and organs of the body. These are sometimes referred to as fixed macrophages.

Many fixed macrophages are part of the lymphoreticular (reticuloendothelial) system. They are found supported by reticular fibers (along with B-lymphocytes and T-lymphocytes) in lymph nodules, lymph nodes, and the spleen where they filter out and phagocytose foreign matter such as microbes.

Similar cells derived from stem cells, monocytes, or macrophages are also found in the liver (Kupffer cells), the kidneys (mesangial cells), the brain (microglia), the bones (osteoclasts), and the lungs (alveolar macrophages).

Macrophages actually have a number of very important functions in body defense including:

killing of microbes, infected cells, and tumor cells by phagocytosis;
processing antigens so they can be recognized by T-lymphocytes during the immune responses. Macrophages, as well as the dendritic cells mentioned below, that process antigens through phagocytosis and present them to T-lymphocytes are often termed antigen-presenting cells or APCs. They will be discussed in detail in Unit 3.
secreting proteins called cytokines (sometimes called monokines when produced by monocytes or macrophages) that play a variety of roles in nonspecific body defense. (Cytokines refer to hormone-like proteins secreted by one cell which then bind locally to other cells and influence their activity.) For example, macrophage-produced cytokines promote inflammation and induce fever, increase phagocytosis and energy output, promote sleep, activate resting T-lymphocytes, attract and activate neutrophils, and stimulate the replication of endothelial cells to form capillaries and fibroblasts to form connective scar tissue. Four important cytokines that macrophages produce are tumor necrosis factor-alpha (TNF-), interleukin-1 (IL-1), and interleukin-6 (IL-6).

Dendritic cells
Dendritic cells are cells with numerous pseudopodia-like projections and are located in the follicles of the lymphoid tissue (follicular dendritic cells) as well as the connective tissue of the skin and mucous membranes (Langerhans' cells). They are derived from bone marrow progenitor cells and from monocytes.

Dendritic cells function in processing antigens, presenting those antigens to T-lymphocytes, and producing cytokines similar to the macrophages mentioned above. Dendritic cells are considered to be the most potent antigen-presenting cells (APCs) in the body.

Mast cells
Mast cells, found throughout the connective tissue of the skin and mucous membranes, carry out the same functions as basophils. They release histamine which promotes inflammation by causing vasodilation, increasing capillary permeability, and increasing mucous production. Mast cells are the cells that usually first initiate the inflammatory response (discussed later in this unit).

An Overview of Phagocytic Defense
* Infection or tissue injury stimulates cells such as mast cells and basophils to release
    vasodilators to initiate the inflammatory response. As a result of vasodilation and
    increased capillary permeability, phagocytic white blood cells (neutrophils,
    monocytes/macrophages, eosinophils) and other white blood cells enter the tissue
    around the injured site and are chemotactically attracted to the area of infection. In other
    words, inflammation allows phagocytes to enter the tissue and go to the site of
     infection.
    Neutrophils are the first to appear and are later replaced by macrophage.
* Lymph nodules are unencapsulated masses of lymphoid tissue containing lymphocytes
    and macrophages. They are located in the respiratory tract, the liver, and the
    gastrointestinal tract and are collectively referred to as mucosa-associated lymphoid
    tissue or MALT. Examples include the adenoids and tonsils in the respiratory tract
    and the Peyer's patches on the small intestines. Organisms entering these systems can be
    phagocytosed by fixed macrophages anddendritic cells and presented to
    lymphocytes to initiate the immune responses.
* Tissue fluid (plasma which has left the blood vessels and entered body tissues and
    organs) picks up microbes and then enters the lymph vessels as lymph. Lymph vessels
    carry the lymph to regional lymph nodes. Lymph nodes contain many reticular fibers
    that support the fixed macrophages and dendritic cells as well as everchanging
    populations of circulating B-lymphocytes and T-lymphocytes. Microbes picked up by
    the lymph vessels are filtered outand phagocytosed in the lymph nodes by these
    fixed macrophages and dendritic cells and presented to the circulating
    T-lymphocytes to initiate immune responses. The lymph eventually enters the circulatory
    system at the heart to maintain the fluid volume of the circulation.
* The spleen contains many reticular fibers that support fixed macrophages and dendritic
    cells as well as everchanging populations of circulating B-lymphocytes and
    T-lymphocytes. Blood carries microorganisms to the spleen where they are filtered out
    and phagocytosed by the fixed macrophages and dendritic cells and presented to
    the circulating T-lymphocytes to initiate immune responses.
* As mentioned above under fixed macrophages, there are also specialized macrophages
    and dendritic cells located in the brain (microglia), lungs (alveolar macrophages), liver
    (Kupffer cells), kidneys (mesangial cells), bones (osteoclasts), and skin and mucous
    membranes (Langerhans' cells).

Steps in Phagocytosis
a) Activation
Resting phagocytes are activated by inflammatory mediators such as bacterial products, complement proteins, proinflammatory cytokines, and prostaglandins. As a result, the phagocytes produce surface glycoprotein receptors that increase their ability to adhere to surfaces and recognize microbes. They also exhibit increased metabolic and microbicidal activity (production of ATPs, lysosomal enzymes, lethal oxidants, etc.).

b) Chemotaxis(for wandering macrophages and neutrophils)
Chemotaxis is the movement of phagocytes toward an increasing concentration of some attractant such as bacterial factors (bacterial proteins, capsules, cell wall fragments, endotoxin), complement components (C3a, C5a, C5b67), chemokines (chemotactic cytokines such as interleukin-8 secreted by various cells), 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.

c) Attachment
Attachment of microorganisms is necessary for ingestion and may be:
* unenhanced - non-specific attachment to a variety of microbes by means of glycoprotein
    receptors on the surface of the phagocytes; or
* enhanced - attachment by way of the antibodies IgG and IgA or the complement protein
    C3b. Molecules such as IgG, IgA and C3b which promote enhanced attachment are
    called opsonins and the process is called opsonization. Enhanced attachment is much
    more specific and efficient than unenhanced.
* Organisms with capsules, such as Streptococcus pneumoniae, Neisseria meningitidis,
    and Hemophilus influenzae, may initially block attachment of microorganisms; the
    exotoxin protein A produced by Staphylococcus aureus blocks opsonization with IgG.

d) Ingestion
After attachment, the plasma membrane of the phagocyte invaginates, by means of the contractile proteins actin and myosin on the inner membrane surface pulling against rigid microtubules in the cytoplasm, and pinches off. This places the ingested organism in a membranous sac called a phagosome.

Some bacteria, such as pathogenic Yersinia, secrete proteins that depolymerize actin and prevent phagosome formation.

e) Destruction
Phagocytes contain membranous sacs called lysosomes(produced by the Golgi apparatus) which contain various hydrolytic enzymes and microbicidal systems. The lysosomes fuse with the phagosomes and the microorganisms are killed and digested.

Some bacteria are more resistant to phagocytic destruction once engulfed.

Bacteria such as Legionella pneumophilia and Mycobacterium species cause the phagocytic cell to place them into an endocytic vaculole via a pathway that decreases their exposure to toxic oxygen compounds.
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.
Bacteria such as species of Salmonella, Mycobacterium, Legionella, and Chlamydia block the vesicular transport machinery that enables the phagosome to fuse with the lysosome.
Bacteria such as pathogenic Mycobacterium and Legionella pneumophilia prevent the acidification of the phagosome which is needed for effective killing of microbes by lysosomal enzymes.

Some bacteria are able to kill phagocytes.

Bacteria such as Staphylococcus aureus and Streptococcus pyogenes produce the exotoxin leukocidin which damages the membranes of the lysosomes resulting in the phagocyte being killed by its own enzymes.
Bacteria, such as Shigella and Salmonella, induce macrophage apoptosis, a programmed cell death.

If the phagocyte is overwhelmed with microorganisms, the phagocyte will empty the contents of its lysosomes by a process called degranulation in order to kill the microorganisms or cell extracellularly. These released lysosomal contents, however, also kill surrounding host cells and tissue. Most tissue destruction associated with infections is a result of this process. This is discussed more later under chronic inflammation.

The phagocyte will also empty the contents of its lysosomes for extracellular killing if the cell to which the phagocyte adheres is too large to be engulfed.

There are 2 killing systems in neutrophils and macrophages: the oxygen-dependent system and the oxygen-independent system.

1) The oxygen-dependent system

The cytoplasmic membrane of phagocytes contains the enzyme oxidase which converts oxygen into superoxide anion (O₂^2-). 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. 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. Neutrophils also produce acid hydrolases that 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 as mentioned above.

2) Oxygen-independent system

Some lysosomes contain cationic proteins that alter cytoplasmic membranes, lysozyme which breaks down peptidoglycan, lactoferrin which deprives bacteria of needed iron, and various digestive enzymes which exhibit antimicrobial activity by breaking down proteins, RNA, phosphate compounds, lipids, and carbohydrates.

(2.) INFLAMMATION
This is a general response mounted by the body to virtually any insult. (If you recall your own bodies response to a recent cut, puncture wound, or burn you will be able to appreciate each of the active components of the inflammatory response.)
i) Reddening --> ii) Swelling ---> iii) Heat ---> iv) Pain

· A complex reaction triggered by any damage to the body. Can be provoked by infectious
    agents, physical agents, and by certain immune pathways.
· Symptoms include: pain, redness, swelling, heat and loss of function.
· Purpose is to destroy the invader, limit damage and repair the damage.
· There are a number of biochemical mediators including histamine, prostaglandins,
    leukotrienes, complement and kinin.
· These promote vasodilation and increased vascular permeability as well as phagocyte
    and lymphocyte chemotaxis and activation.
· Eventually cytokines and hormones stimulate tissue regeneration.

(3.) FEVER
Activated macrophages release interleukin-1 (IL-1). This cytokine resets the hypothalamus thermostat and the body temperature increases. Higher temperature may increase the metabolic rate of white cells as well as slow down the growth of some pathogens. Fever stimulates the release of transferin an iron binding protein.

(4.) COMPLEMENT ACTIVATION
· Function of activated complement:
    a.) destroys cells, b.) stimulates inflammation, c.) stimulates chemotaxis d) estimulates
         phagocytosis.
· Complement is activated by two different cascades:
    The classical pathway and the alternative pathway
    Both pathways activate C3 to form C3a (inflammatory mediator) and C3b (opsonin and
    enzyme which then activates C5)

· C5 is broken down by C3b to form C5a (inflammatory mediator and leukocyte attractor)
and C5b (activates C6, C7, C8 and C9 to form the membrane attack complex (MAC)
which forms the transmembrane channel in the target cell and leads to cytolysis).