Immune Defense against Bacterial Pathogens:
Innate Immunity
Host Defense Mechanisms
Humans are in continuous associations with microorganisms, including those that readily colonize the body surfaces (see The Bacterial Flora of Humans). It is relatively rare that these microorganisms cause damage to their host. In part, this is due to the effectiveness of the host defense mechanisms, which restrict invasion by normal flora (some of which may be potential pathogens), and which defend against non-indigenous microorganisms that are overt pathogens.
The outcome of an interaction between a human host and a microbe, whether it is a component of the normal flora or an exogenous pathogen, depends on specific properties inherent to both the host and the microbe. Sometimes, the host tolerates colonization by a parasite but restricts it to regions of the body where it can do no harm (e.g. Staphylococcus aureus on the nasal membranes or Streptococcus pneumoniae in the upper respiratory tract). If the parasite invades (i.e., breaches an anatomical barrier or progresses beyond the point of colonization), an infection is said to have occurred. If, as a result of infection, pathological harm to the host becomes evident, this is called an infectious disease.
The healthy animal defends itself against pathogens different stages. The host defenses may be of such a degree that infection can be prevented entirely. Or, if infection does occur, the defenses may stop the process before disease is apparent. At other times, the defenses that are necessary to defeat a pathogen may not be effective until infectious disease is well into progress.
The host defense mechanisms are mediated by the immune system. For our purposes, the term immunity refers to the relative state of resistance of the host to infectious disease. I will adopt the nomenclature used by my colleagues at University of South Carolina School of Medicine Microbiology and Immunology On-line to draw lines between the "types of immunity", particularly as it relates to to innate immunity and adaptive immunity.
The immune system is composed of two major subdivisions, the innate or nonspecific immune system and the adaptive or specific immune system. The innate immune system is a primary defense mechanism against invading organisms, while the adaptive immune system acts as a second line of defense. Both aspects of the immune system have cellular and humoral components by which they carry out their protective functions. In addition, there is interplay between these two systems, i.e., cells or components of the innate immune system influence the adaptive immune system and vice versa. The innate and adaptive immune systems differ in several ways. The adaptive immune system requires some time to react to an invading organism, whereas the innate immune system includes defenses that, for the most part, are constitutively present and mobilized immediately upon infection. Additionally, the adaptive immune system is antigen specific and reacts only with the organism that induced the response. The innate system is not antigen specific and reacts similarly to a variety of organisms. Finally, the adaptive immune system exhibits an immunological memory. It "remembers" that it has encountered an invading organism and reacts more rapidly on subsequent exposure to the same organism. The innate immune system does not exhibit a memory response. Cellular defense. This term is used to distinguish whether an immune response is mediated by a particular type of cell, as opposed to a non cellular defense which does not involve a specifically programmed cell. As stated above, a variety of tissue cells are involved in innate and adaptive immunity, hence the term cellular defense. These include neutrophils and macrophages, which are involved in phagocytosis, basophils and mast cells, which are involved in inflammation, and B cells and T cells which account for antibody mediated immunity and cell mediated immunity, respectively.
All these cells have their origin in the bone marrow (Figure 2). Myeloid progenitor (stem) cells in the bone marrow give rise to neutrophils, eosinophils, basophils, monocytes and dendritic cells, while lymphoid progenitor (stem) cells give rise to T cells and B cells. Macrophages and dendritic cells, which play a key role in innate and adaptive immunity, are derived from monocytes; and mast cells, which are fixed in tissues, develop from the same precrusor cell as circulating basophils. B cells are produced in bone marrow and released into the blood and lymphatic systems. B-cells can develop into plasma cells that secrete antibodies. Precursor T cells undergo differentiation in the thymus into two distinct types of T cells, CD4+ T helper cells, and the CD8+ cytotoxic T cells. Macrophages and dendritic cells function as one of several bridges between innate immunity and adaptive immunity, since they present antigens to immunocompetent T-cells, which initiates an immunological response.
Figure 2. Development of cells that participate in immunity.
Innate Immunity
Innate Immunity is a form of non specific host defense against invading bacteria. It is natural or "innate" to the host, depending, in part, on genetics. Innate defense mechanisms are contitutive to the host, meaning they are continually ready to respond to invasion and do not require a period of time for induction. The most important components of innate immunity are anatomical barriers, intact normal flora, tissue bactericides including complement, and ability to undergo inflammatory and phagocytic responses.Innate immunity provides the first line of defense against invading bacteria. The skin and mucous membranes provide physical and chemical barriers to infection. The normal bacterial flora antagonize colonization of body surfaces by nonindigenous bacteria. The internal tissues invariably contain bactericidal substances. The most noteworthy antibacterial substance is the enzyme lysozyme, which is present in mucus and all bodily tissues and secretions. If these barriers are penetrated, the body contains cells that respond rapidly to the presence of the invader. These cells include macrophages and neutrophils that engulf foreign organisms and kill them. Bacterial invasion is also challenged by the activation of complement in blood and tissues and the incitement of an inflammatory process which has the tendency to focus both the innate and adaptive immune defenses on the site of invasion. Categories of Innate or Nonspecific ImmunityThe first four categories are generally considered non cellular defenses. Inflammation and Phagocytosis are forms of cellular defense.
1. Differences in susceptibility to certain pathogens2. Anatomical defense 3. Tissue bactericides, including complement4. Microbial antagonism5. Inflammation (ability to undergo an inflammatory response)6. Phagocytosis
Differences in Susceptibility of Animal Hosts to Microbial Pathogens (Natural Immunity)
Natural immunity or resistance is based on the genetics of the host. There are two aspects: (1) resistance among all members of a species, called species resistance and (2) resistance within members of the same animal species, called individual resistance.
Species resistance
Certain animals are naturally resistant or non susceptible to certain pathogens. Certain pathogens infect only humans, not lower animals, e.g. syphilis, gonorrhea, measles, poliomyelitis. On the other hand, certain pathogens (e.g. canine distemper virus) do not infect humans. Shigella infects humans and baboons but not chimpanzees. Little information is available to explain these absolute differences in susceptibility to a pathogen but it could be due to:
Absence of specific tissue or cellular receptors for attachment (colonization) by the pathogen. For example, different strains of enterotoxigenic E. coli, defined by different fimbrial antigens, colonize human infants, calves and piglets by recognizing species-specific carbohydrate receptors on enterocytes in the gastrointestinal tract.
Temperature of the host and ability of pathogen to grow. For example, birds do not normally become infected with mammalian strains of Mycobacterium tuberculosis because these strains cannot grow at the high body temperature of birds. The anthrax bacillus (Bacillus anthracis) will not grow in the cold-blooded frog (unless the frog is maintained at 37o).
Lack of the exact nutritional requirements to support the growth of the pathogen. Naturally-requiring purine-dependent strains of Salmonella typhi grow only in hosts supplying purines. Mice and rats lack this growth factor in blood and pur- strains are avirulent. By injecting purines into these animals, such that the growth factor requirement for the bacterium is satisfied, the organisms prove virulent.
Lack of a target site for a microbial toxin. Most toxins produced by bacterial cells exert their toxic activity only after binding to susceptible cells or tissues in an animal. Certain animals may lack an appropriate target cell or specific type of cell receptor for the toxin to bind to and may therefore be nonsusceptible to the activity of the toxin. For example, injection of diphtheria toxin fails to kill the rat. The unchanged toxin is excreted in the urine. If a sample of the rat urine (or pure diphtheria toxin) is injected into the guinea pig, it dies of typical lesions caused by diphtheria toxin.
Individual resistance
There are many reasons why individuals of the same animal species may exhibit greater or lesser susceptibility to the same ineffective agent.
Age. Usually this relates to the development and status of the immunological system which varies with age. It may also be associated with changes in normal flora coincidental to developmental changes in the animal.
Sex. Usually this is linked to the presence and/or development of the sex organs. For example, mastitis and infectious diseases leading to abortion will obviously occur only in the female; orchitis would occur only in males. It could also be due to anatomical structure related to sex (bladder infections are 14-times more common in females than males), and possibly the effects of sex hormones on infections.
Stress. Stress is a complex of different factors that apparently has a real influence on health. Undue exertion, shock, change in environment, climatic change, nervous or muscular fatigue, etc. are factors known to contribute to increases in susceptibility to infection. The best explanation is that in time of stress the output of cortisone from the adrenal cortex is increased. This suppresses the inflammatory processes of the host and the overall effect may be harmful. There are also a number of relationships between stress-related hormones and the functioning of the immune defenses.
Diet, malnutrition. Infections may be linked with vitamin and protein deficiencies, and this might explain partly why many infectious diseases are more prevalent and infant mortality rates are highest in parts of the world where malnourishment is a problem. Also, overfed and obese animals are more susceptible to infection. Diets high in sucrose predispose individuals to dental caries.
Intercurrent disease or trauma. The normal defenses of an animal are impaired by organic diseases such as leukemia, Hodgkin's disease, diabetes, AIDS, etc. Frequently, inflammatory or immune responses are delayed or suppressed. Colds or influenza may predispose an individual to pneumonia. Smoking tobacco predisposes to infections of the respiratory tract. Burned tissue is readily infected by Pseudomonas aeruginosa.
Therapy against other diseases. Modern therapeutic procedures used in some diseases can render an individual more susceptible to infection. Under these conditions not only pathogens, but organisms of the normal flora and nonpathogens in the host's environment, may be able to initiate infection. Examples of therapeutic procedures that reduce the efficiency of the host's defenses are treatment with corticosteroids, cytotoxic drugs, antibiotics, or irradiation.
Anatomical Defenses
The structural integrity of the body surfaces, i.e., the skin and mucous membranes, forms an effective barrier to initial lodgment or penetration by microorganisms. The skin is a very effective barrier to bacterium, so that no bacterium by itself is known to be able to penetrate unbroken skin. Of course, a puncture, cut or scrape in the skin could introduce infectious bacteria. The mucous membranes are more vulnerable to penetration by infectious bacteria but still pose a formidable barrier of mucus and antimicrobial substances.The anatomical defenses are associated with all other aspects of noncellular immunity, including individual resistance, mechanical resistance, chemical resistance and resistance established by the normal flora (Figure 3)
Figure 3. Anatomical defenses associated with tissue surfaces
Skin. The intact surface of the healthy epidermis seems to be rarely if ever penetrated by bacteria. If the integrity of the epidermis is broken (by the bite of an insect, needle stick, abrasion, cut, etc.) invasive microbes may enter. The normal flora of the skin, which metabolize substances secreted onto the skin, produce end products (e.g. fatty acids) that discourage the colonization of skin by potential pathogens. Perspiration contains lysozyme and other antimicrobial substances.
Mucous membranes. Many are heavily colonized with bacteria in whose moist secretions they survive. These normal flora are restricted from entry and usually occupy any attachment sites that might otherwise be used by pathogens. The normal flora established on mucous membranes may antagonize non-indigenous species by other means, as well. Typically, mucus contains a number of types of anti-microbial compounds, including lysozyme and secretory antibodies (IgA). Sometimes phagocytes patrol mucosal surfaces (e.g. in the lower respiratory tract). Nonetheless, most infectious agents impinge on the skin or mucous membranes of the oral cavity, respiratory tract, GI tract or urogenital tract, and from these sites most infections occur. Damage to the epithelial cells caused by toxic products of these bacteria may play a role.
Respiratory tract. Fine hairs and baffles of the nares (nasal membranes) entrap bacteria which are inhaled. Those which pass may stick to mucosal surfaces of the trachea or be swept upward by the ciliated epithelium of the lower respiratory tract. Coughing and sneezing also eliminate bacteria. The lower respiratory tract (lung) is well protected by mucus, lysozyme, secretory antibody, and phagocytosis.
Mouth, stomach and intestinal tract. Microorganisms entering by the oral route, more than any other, have to compete with the well-adapted normal flora of the mouth and intestine. Most organisms that are swallowed are destroyed by acid and various secretions of the stomach. Alkaline pH of the lower intestine can discourage other organisms. The peristaltic action of the intestine ultimately flushes out organisms which have not succeeded in colonization. Bile salts and lysozyme are present, which kill or inhibit many types of bacteria.
Urogenital Tract. The flushing mechanisms of sterile urine and the acidity of urine maintain the bladder and most of the urethra free of microorganisms. The vaginal epithelium of the female maintains a high population of Doderlein's bacillus (Lactobacillus acidophilus) whose acidic end products of metabolism (lactic acid) prevent colonization by most other types of microorganisms including potentially-pathogenic yeast (Candida albicans).
Eyes (Conjunctiva). The conjunctiva of the eye is remarkably free of most microorganisms. Blinking mechanically removes microbes, the lavaging action of tears washes the surface of the eye, and lachrymal secretions (tears) contain relatively large amounts of lysozyme.
Microbial Antagonism
This refers to the protection of the surfaces afforded by an intact normal flora in a healthy animal, and it has already been mentioned in several contexts (See The Bacterial Flora of Humans). There are three main ways that the normal flora protect the surfaces where they are colonized:
Competition with non-indigenous species for binding (colonization) sites. The normal flora are highly-adapted to the tissues of their host. That is why they are there.
Specific antagonism against non-indigenous species. Members of the normal flora may produce very specific proteins called bacteriocins which kill or inhibit other (usually closely-related) species of bacteria.
Nonspecific antagonism against non-indigenous species. The normal flora produce a variety of metabolites and end products that inhibit other microorganisms. These include fatty acids (lactate, propionate, etc.), peroxides and antibiotics.
Figure 4. Enterococcus faecalis, also classified as Streptococcus faecalis. Occasionally there is invasion of the host by the normal flora, as evidenced by this blood culture. Enterococcus faecalis, blood culture. © Gloria J. Delisle and Lewis Tomalty, Queens University Kingston, Ontario, Canada. Licensed for use by ASM Microbe Library http://www.microbelibrary.org.
Antimicrobial Substances in Host Tissues
The body fluids and organized tissues of animals naturally contain a variety of antimicrobial agent that kill or inhibit the growth of microbes. The sources and activities of a variety of host antimicrobial substances are summarized in Table 1.
TABLE 1. ANTIMICROBIAL SUBSTANCES OF HOST ORIGIN PRESENT IN BODY FLUIDS AND ORGANIZED TISSUES
Substance
Common Sources
Chemical Composition
Activity
Lysozyme
Serum, saliva, sweat, tears
Protein
Bacterial cell lysis
Complement
Serum
Protein-carbohydrate lipoprotein complex
Cell death or lysis of bacteria; participates in inflammation
Basic proteins and polypeptides (histones, ß-lysins and other cationic proteins, tissue polypeptides)
Serum or organized tissues
Proteins or basic peptides
Disruption of bacterial plasma membrane
Lactoferrin and transferrin
Body secretions, serum, organized tissue spaces
Glycoprotein
Inhibit microbial growth by binding (withholding ) iron
Peroxidase
Saliva, tissues, cells (neutrophils)
Protein
Act with peroxide to cause lethal oxidations of cells
Fibronectin
Serum and mucosal surfaces
Glycoprotein
Clearance of bacteria (opsonization)
Interferons
Virus-infected cells, lymphocytes
Protein
Resistance to virus infections
Interleukins
Macrophages, lymphocytes
Protein
Cause fever; promote activation of immune system
Complement
Complement is considered as part of the innate immunity because of its role in inflammation, phagocytosis and bacterial killing. Complement may be activated by bacterial invasion, but also by reactions between antigens and antibodies, and therefore, it may play a role in adaptive immunity, as well.
Complement is an enzymatic system of serum proteins made up of nine major components (C1 - C9) that are sequentially activated during two pathways, the classical pathway and the alternative pathway, resulting in a variety of antibacterial defenses. Complement components play a part in phagocytic chemotaxis, opsonization and the inflammatory response, and may be involved in the lysis of certain bacteria, some viruses, and other microorganisms.
Complement is activated in the classical pathway by reactions between antibodies and antigens on the surface of a microbe. Some Immunoglobulins (i.e., IgG and IgM) can "fix complement" because they have a complement binding site on the Fc portion of the molecule. The reaction between IgG and Ag activates the complement and initiates a "cascade reaction" on the surface of the microbe that results in the principal effects of complement which are:
1. Generation of inflammatory factors, C3a and C5a, which focus antimicrobial serum factors and leukocytes into the site of infection.
2. Attraction of phagocytes. Chemotactic factors C3a and C5a attract phagocytes to the site.
3. Enhancement of phagocytic engulfment. C3b component on Ag - Ab complex attaches to C3b receptors on phagocytes and promotes opsonization of Ab-coated cells. C3b-opsonization is important when Ab is IgM because phagocytes have receptors for Fc of IgM only when it is associated with C3b.
4. Lysis of bacterial cells (lysozyme-mediated) or virus-infected cells. When C8 and C9 are bound to the complex, a phospholipase is formed that destroys the membrane of Ag-bearing host cells (e.g. virus-infected cells) or the outer membrane of Gram-negative bacteria. Lysozyme gains access to peptidoglycan and completes destruction of the bacterial cell.In addition to the classical pathway of complement activation, an alternative pathway (sometimes called the "properdin pathway") of complement activation exists, which is independent of immunoglobulins. Insoluble polysaccharides (including bacterial LPS, peptidoglycan and teichoic acids) can activate complement. This allows antibody-independent activation of the complement cascade that is thought to be important in initial (pre-antibody) defense against various types of infections caused by bacteria.Figure 5. The complement cascade, precipitated by certain antigen-antibody reactions (classical pathway) or by bacterial polysaccharides (alternative pathway), leads to four principal antimicrobial effects: 1. phagocytes are attracted to the site (POLYMORPH ACCUMULATION); 2. inflammatory agents re produced and/Or released from cells (INFLAMMATION); 3. microbes are opsonized to enhance uptake by phagocytic cells (PHAGOCYTOSIS); 4. Gram-negetive bacteria are lysed in the presence of lysozyme (LYSIS OF MICROBE).
Inflammation
Of all the defense mechanisms in the animal host, the inflammatory response may be the most important for dealing with microbial infection. Inflammation is necessary for the proper functioning of all the host defenses, including the immune defenses, because it focuses all circulating antimicrobial factors on the site of infection. These include phagocytes, lymphocytes, antibodies, complement and other antimicrobial components of plasma. However, inflammation is also an important aspect of bacterial pathogenesis since the inflammatory response induced by a microbe can result in considerable damage to the host and, therefore, be part of the pathology of microbial disease.
Inflammation is a tissue reaction to infection or injury, the characteristic symptoms of which are redness, swelling, heat and pain. These are sometimes called the cardinal signs of inflammation. The redness is due to increased blood flow to the area of injury. The swelling (edema) is due to increased extravascular fluid and phagocyte infiltration to the damaged area. The heat is due to the increased blood flow and the action of pyrogens (fever-inducing agents). The pain is caused by local tissue destruction and irritation of sensory nerve receptors. If a whole organ or tissue is involved, loss of function may occur.
Inflammation can be induced by certain immunological reactions, tissue damage, or the entry of an injurious agent (microbial or nonmicrobial). Certain bacterial cells and/or their products (e.g. structural components or toxins) can induce an inflammatory response. Inflammation increases the blood supply and temperature in the inflamed tissues, which favors maximal metabolic activity of the leukocytes, and lowers the pH slightly, which tends to inhibit the multiplication of many microorganisms.
Inflammation is mediated by mast cells in fixed tissues (the GI tract, respiratory tract, conjunctiva), giving rise to a localized response; or by basophils in the blood giving rise to a systemic response. The response may be initiated in a variety of ways. Some of the main events involved in the induction and maintenance of an inflammatory response during a microbial infection are summarized below.
(1) The inflammatory response is triggered by pathogen invasion or tissue injury. Injured and dying cells release cytoplasmic constituents which lower the pH in the surrounding extracellular environment.
(2) The increased acidity activates an extracellular enzyme kallikrein which in turn activates bradykinin.
(3a) Bradykinin binds to receptors on the capillary walls opening junctions between cells to allow leakage of plasma components collectively referred to as the inflammatory exudate.
Increased capillary permeability allows leukocytes to pass from the vessels into tissues (this process is called diapedisis). The first cells to appear, and the most dominant, are neutrophils, which are actively phagocytic. The other components of the inflammatory exudate and their functions are described in Table 2 below).
(3b) Bradykinin also binds to mast cells of the connective tissue that are associated with the small vessels of most tissues. This initiates other events that are associated with the process of inflammation.
Initially, in the mast cell, there is a rapid influx of Ca++, intracellular cAMP levels drop, and mediator-rich lysosomal granules migrate to the cell surface, fuse with the cell membrane, and discharge their contents (preformed mediators of inflammation such as histamine, heparin, etc.) to the exterior by exocytosis
The change in mast cell permeability activates an enzyme, phospholipase A2 to synthesize a substance called arachidonic acid. This compound can be acted upon subsequently by the cyclooxygenase pathways or lipooxygenase pathways of the mast cell leading to new synthesis of prostaglandins, leukotrienes, and other mediators of inflammation. These substances contribute to the inflammatory exudate.
TABLE 2. FUNCTION OF COMPONENTS AND CELLS IN THE INFLAMMATORY EXUDATE
Component
Function
Bradykinin, histamine, leukotrienes, serotonin, prostaglandins
Inflammatory Agents (IA) which act on the vascular system to produce increased blood flow and permeability
Fibrin: (formed from fibrinogen in plasma)
coagulates and may localize an invading pathogen
Lysozyme
causes lysis of bacterial cell walls
Complement
various activities increase the inflammatory response and lead to increased phagocytosis and complement-mediated lysis of cells
Antibodies (in immune individuals)
block colonization by pathogens; neutralize microbial toxins or viruses; opsonize pathogens making them more susceptible to phagocytosis; activate complement
Pyrogens, including endogenous pyrogen (Interleukin 1)
cause fever acting on the thermo-regulatory control centers in the hypothalamus. (Interleukin-1, which is produced by macrophages, also promotes activation and mitosis of B-cells and T-cells)
Neutrophils
migrate to focus of infection and ingest and destroy foreign agents by phagocytosis
Macrophages
engulf and destroy infective agents, process antigenic components and convey them to lymphocytes
Immunocompetent lymphocytes (B-cells and T-cells)
for direct participation in immunological responses (AMI and CMI)
The overall effect of an inflammatory reaction is to recruit various cells and components to the actual site of microbial invasion. Many of these cells and plasma components have a direct role in defense against the intruding microorganism. These include neutrophils (phagocytes which engulf and destroy the microbes); macrophages and lymphocytes which are the cells necessary to initiate immunological responses against the pathogen; pre-existing antibodies which can neutralize microbial pathogens or their toxins; and plasma components such as lysozyme, complement and fibrin, which have a variety of antimicrobial activities.
Phagocytic Defenses
When invading parasites penetrate the tissues the inflammatory response, previously described, is immediately brought into play. Part of this response leads to the recruitment of phagocytes to the site of inflammation. Phagocytes are a class of cells which are capable of ingestion (engulfment) and destruction of microorganisms that are responsible for inciting the inflammatory response. First to accumulate around the invaders and initiate the phagocytic process are neutrophils. Later, local and blood-borne macrophages also migrate to the tissue site and initiate phagocytosis. Neutrophils (also known as polymorphonuclear leukocytes, polymorphism or Pans) and macrophages are sometimes referred to as professional phagocytes for their roles in this process.
Properties of Neutrophils
Neutrophils have their origin in multi-potential stem cells in the bone marrow. They differentiate in the marrow and are released in a mature form, containing a full complement of bactericidal agents. They are short-lived cells which constitute 30-70% of the circulating white blood cells (leukocytes).
During differentiation in the marrow (2-3 days) the nucleus of the cell becomes multilobed (hence the name polymorphonuclear leukocyte), cell division ceases, and mitochondria and endoplasm reticule disappear from the cytoplasm. At the same time the cell becomes motile and actively phagocytic. Cytoplasmic granules are formed from the Geology apparatus. These granules are called lysosomal and contain the various bactericidal and digestive enzymes which can destroy bacterial cells after engulfment. The contents of lysosomal granules include lysozyme, cationic proteins, acid hydrolyses, protease's, peroxidase and lactoferrin. Neutrophils also contain large stores of glycogen. Since they derive most of their metabolic energy from glycolysis, they can function efficiently in anaerobic environments.
Some additional properties of neutrophils are:
-Only half the neutrophils in human circulation are detectable in the blood; the rest adhere to vessel walls.
-For every circulating neutrophils, approximately 100 near mature cells are held in reserve in the bone marrow pool.
-Once a neutrophils enters the tissues, intestinal tract or respiratory tract, it never returns to the circulation.
Properties of Macrophages
Macrophages (also called mononuclear phagocytes) also arise from bone marrow stem cells which give rise to monocytes which develop into monocytes that are released into the blood stream. Monocytes make up 3-7% of the circulating white blood cells. The monocytes is actively phagocytic and bactericidal. Within 2 days or so, the blood stream monocytes (sometimes called wondering macrophages) emigrate into the tissues where they settle down, enlarge and become fixed macrophages (tissue histiocytes), which also have phagocytic potential. Macrophages are more active in phagocytosis than monocytes and develop many more granules containing hydrolytic enzymes. New macrophages can develop by cell division under inflammatory stimuli, but most macrophages are matured blood monocytes.
The total pool of macrophages is referred to as the system of mononuclear phagocytes. The system is scattered throughout connective tissue, basement membranes of small blood vessels, liver sinusoids, spleen, lung , bone marrow and lymph nodes. Monocytes from the blood migrate into virtually every organ in the body where they mature into fixed macrophages. In the lymph nodes, macrophages function as scavengers to remove foreign material from the circulation.
Compared to neutrophils, macrophages are long-lived cells. As phagocytes, neutrophils play a more important role in the acute stages of an infection, while macrophages are principally involved in chronic types of infections. Neutrophils circulate in the blood stream, and during an acute inflammatory response they migrate through the endothelial cell junctions as part of the inflammatory exudate. They migrate to the focus of the infection and ingest or phagocytose foreign agents, Neutrophils which have become engorged with bacteria usually die and largely make up the material of pus. Macrophages, which are also attracted to the area during an inflammatory response, are slower to arrive and become increasingly involved in chronic infections. They, too, are actively phagocytic and will engulf and destroy foreign particles such as bacteria. However, macrophages have another indispensable function in host defense: they "process" the antigenic components of infective agents and present them to lymphocytes, a process that may usually required for the initiation of immunological responses of the host. Macrophages and related dendritic cells are among an elite corps of antigen-presenting cells or APC's.
The Phagocytic Process
Phagocytosis and destruction of engulfed bacteria involves the following sequence of events:
1. Delivery of phagocytic cells to the site of infection
2. Phagocytic adherence to the target
3. Ingestion or engulfment of the target particle
4. Phagolysosome formation
5. Intracellular killing
6. Intracellular digestion (and egestion, in the case of macrophages)
These steps involved in the phagocytic process in macrophages are illustrated below.
Figure 6. Phagocytosis by a Macrophage. A bacterium, which may or may not be opsonized, is engulfed by the process of endocytosis. The bacterium is ingested in a membranous vesicle called the phagosome. Digestive granules (lysosomes) merge with phagosome, release their contents, and form a structure called the phagolysosome. The killing and digestion of the bacterial cell takes place in the phagolysosome. The macrophage egests debris while processing the antigenic components of the bacterium, which it returns to its surface in association with MHC II for antigen presentation to TH cells.
Delivery of phagocytic cells to the site of infection
The delivery of phagocytic cells, monocytes or neutrophils, to the site of microbial infection involves two processes:
Diapedisis: the migration of cells across vascular walls which is initiated by the mediators of inflammation (kinins, histamine, prostaglandins, etc.)
Chemotaxis. Phagocytes are motile by ameboid action. Chemotaxis is movement of the cells in response to a chemical stimulus. The eventual concentration of phagocytes at a site of injury results from chemotactic response by the phagocytes which is analogous to bacterial chemotaxis. A number of chemotactic factors (attractants) have been identified, both for neutrophils and monocytes. These include bacterial products, cell and tissue debris, and components of the inflammatory exudate such as peptides derived from complement.
Phagocytic adherence
Phagocytosis is initiated by adherence of a particle to the surface of the plasma membrane of a phagocyte. This step usually involves several types of surface receptors on the phagocyte membrane. Three major receptors on phagocytes recognize the Fc portion of IgG: one is for monomeric IgG and the others are for antigen-crosslinked IgGs. Another receptor binds a complement factor C3b. Other phagocyte receptors bind fibronectin and mannose-terminated oligosaccharides. Under certain circumstances of infection, bacteria or viruses may become coated or otherwise display on their surfaces one or another of these substances (i.e., IgG, C3b, fibronectin or mannose). Such microbes are said to be opsonized and such substances as IgG or complement C3b bound to the surface of microbes are called opsonins. (Opsonin comes from a Greek word meaning "sauce" or "seasoning": they make the microbe more palatable and more easily ingested by the phagocyte.) Opsonins provide extrinsic ligands for specific receptors on the phagocyte membrane, which dramatically increases the rate of adherence and ingestion of the pathogen. Opsonized bacteria can be cleared from the blood by phagocytes; many types of non opsonized bacteria cannot be cleared.
Less firm attachments of a phagocyte to a particle can take place in the absence of opsonization. This can be thought of as nonspecific attachment which might be due to net surface charge on the phagocyte or particle and/or hydrophobicity of the particle.
Lastly, in the absence interaction beween the phagocyte and microbe surfaces, a phenomenon called surface phagocytosis exists: a phagocyte can simply trap an organism against a surface and initiate ingestion. Surface phagocytosis may be an important pre-antibody defense mechanism which may determine whether an infection will become a disease and how severe the disease will become.
Ingestion
After attachment of the phagocyte to its target, some sort of signal generation, which is poorly understood, results in physical or chemical changes in the cell that triggers ingestion. Ingestion is an engulfment process that involves infolding or invagination of the cell membrane enclosing the particle and ultimately releasing it into the cytoplasm of the cell within a membrane vesicle. The end result of ingestion is entry of the particle enclosed in a vesicle derived from the plasma membrane of the cell. This structure is called the phagosome.
Toll-Like Receptors
Macrophages, dendritic cells, and epithelial cells have a set of transmembrane receptors that recognize different types of molecular determinants associated with both pathogenic and non pathogenic bacteria. Foremost among these are Toll-like receptors (TLRs).
In macrophages and dendritic cells, a pathogen is exposed to a TLR when it is engulfed within the phagosome membrane. Depending on which TLR it binds to will determine what the response will be. In this way, the TLRs identify the nature of the pathogen and turn on a response appropriate for dealing with it, generally by expression of various cytokines. Humans have 12 different TLRs, each of which specializes in a slightly different response to a pathogen (be it a bacterium, virus or protozoa).
For example TLR-2 binds to the peptidoglycan of Gram-positive bacteria such as streptococci and staphylococci; TLR-3 binds to double-stranded RNA; TLR-4 is activated by the lipopolysaccharide (endotoxin) in the outer membrane of Gram-negative such as Salmonella and E. coli; TLR-5 binds to the flagellin of motile bacteria like Listeria; TLR-6 forms a heterodimer with TLR-2 and responds to peptidoglycan and certain bacterial lipoproteins. TLR-7 binds to the single-stranded RNA genomes of viruses such as as influenza, mumps and measles.
In all these cases, binding of the pathogen to the TLR initiates a signaling pathway that leads to the activation of a transcription factor that turns on cytokine genes such as those for tumor necrosis factor-alpha (TNF-α), Interleukin-1 (IL-1), and chemotactic attractants that attract white blood cells to the site. These effector molecules lead to inflammation at the site. Even before these late events occur, the binding of Gram-positive bacteria to TLR-2 and Gram-negative bacteria to TLR-4 enhances phagocytosis and the fusion of the phagosomes with lysosomes.
Formation of the phagolysosome
The phagosome migrates into the cytoplasm and collides with lysosomal granules which explosively discharge their contents into the membrane-enclosed vesicle (phagosome). Membranes of the phagosome and lysosome actually fuse resulting in a digestive vacuole called the phagolysosome. Other lysosomes will fuse with the phagolysosome. It is within the phagolysosome that killing and digestion of the engulfed microbe take place. Some of the microbicidal constituents of the lysosomes of neutrophils and macrophages include lysozyme, cationic proteins, various proteases and hydrolyases and peroxidases. The killing processes are confined to the phagolysosome, such that none of the toxic substances and lethal activities of the phagocytes are turned against themselves.
Intracellular killing of organisms
After phagolysosome formation the first detectable effect on bacterial physiology, occurring within a few minutes after engulfment, is loss of viability (ability to reproduce). The exact mechanism is unknown. Inhibition of macromolecular synthesis occurs later. By 10 to 30 minutes after ingestion many pathogenic and nonpathogenic bacteria are killed followed by lysis and digestion of the bacteria by lysosomal enzymes. The microbicidal activities of phagocytes are complex and multifarious. Metabolic products, as well as lysosomal constituents, are responsible. These activities differ to some extent in neutrophils, monocytes and macrophages.
The microbicidal activities of phagocytes are usually divided into oxygen-dependent and oxygen-independent events.
Oxygen-independent activity
Lysosomal granules contain a variety of extremely basic proteins that strongly inhibit bacteria, yeasts and even some viruses. A few molecules of any one of these cationic proteins appear able to inactivate a bacterial cell by damage to their permeability barriers, but their exact modes of action are not known. The lysosomal granules of neutrophils contain lactoferrin, an extremely powerful iron-chelating protein, which withholds potential iron needed for bacterial growth. The pH of the phagolysosome may be as low as 4.0 due to accumulation of lactic acid, which is sufficiently acidic to prevent the growth of most pathogens. This acidic environment apparently optimizes the activity of many degradative lysosomal enzymes including lysozyme, glycosylases, phospholipases, and nucleases.
Oxygen-dependent activity
Liganding of Fc receptors (on neutrophils, monocytes or macrophages) and mannose receptors (on macrophages) increases their O2 uptake, called the respiratory burst. These receptors activate a membrane-bound NADPH oxidase that reduces O2 to O2- (superoxide). Superoxide can be reduced to OH. (hydroxyl radical) or dismutated to H2O2 (hydrogen peroxide) by superoxide dismutase. O2-, OH., and H2O2 are activated oxygen species that are potent oxidizing agents in biological systems which adversely affect a number of cellular structures including membranes and nucleic acids. Furthermore, at least in the case of neutrophils, these reactive oxygen intermediates can act in concert with a lysosomal enzyme called myeloperoxidase to function as the myeloperoxidase system, or MPO.
Myeloperoxidase is one of the lysosomal enzymes of neutrophils which is released into the phagocytic vacuole during fusion to form the phagolysosome. Myeloperoxidase uses H2O2 generated during the respiratory burst to catalyze halogenation (mainly chlorination) of phagocytosed microbes. Such halogenations are a potent mechanism for killing cells.
When the NADPH oxidase and myeloperoxidase systems are operating in concert, a series of reactions leading to lethal oxygenation and halogenation of engulfed microbes occurs.
Intracellular digestion
Dead microbes are rapidly degraded in phagolysosomes to low molecular-weight components. Various hydrolytic enzymes are involved including lysozyme, proteases, lipases, nucleases, and glycosylases. Neutrophils die and lyse after extended phagocytosis, killing, and digestion of bacterial cells. This makes up the characteristic properties of pus.
Macrophages egest digested debris and allow insertion of microbial antigenic components into the plasma membrane for presentation to lymphocytes in the immunological response.
Figure 7. Phagocytosis of Streptococcus pyogenes by a macrophage. CELLS alive!
Bacterial Defense Against Phagocytosis
Pathogenic bacteria have a variety of defenses against phagocytes. In fact, most successful pathogens have some mechanism(s) to contend with the phagocytic defenses of the host. These mechanisms will be discussed in detail later as part of the determinants of virulence of pathogens. However, in general, pathogens may resist phagocytosis by:
Evading phagocytes by growing in regions of the body which are not accessible to them
Avoiding engulfment by phagocytes after contact
Being able to kill phagocytes either before or after engulfment
Being able to survive inside of phagocytes (or other types of cells) and to persist as intracellular parasites
Host Defense Mechanisms
Humans are in continuous associations with microorganisms, including those that readily colonize the body surfaces (see The Bacterial Flora of Humans). It is relatively rare that these microorganisms cause damage to their host. In part, this is due to the effectiveness of the host defense mechanisms, which restrict invasion by normal flora (some of which may be potential pathogens), and which defend against non-indigenous microorganisms that are overt pathogens.
The outcome of an interaction between a human host and a microbe, whether it is a component of the normal flora or an exogenous pathogen, depends on specific properties inherent to both the host and the microbe. Sometimes, the host tolerates colonization by a parasite but restricts it to regions of the body where it can do no harm (e.g. Staphylococcus aureus on the nasal membranes or Streptococcus pneumoniae in the upper respiratory tract). If the parasite invades (i.e., breaches an anatomical barrier or progresses beyond the point of colonization), an infection is said to have occurred. If, as a result of infection, pathological harm to the host becomes evident, this is called an infectious disease.
The healthy animal defends itself against pathogens different stages. The host defenses may be of such a degree that infection can be prevented entirely. Or, if infection does occur, the defenses may stop the process before disease is apparent. At other times, the defenses that are necessary to defeat a pathogen may not be effective until infectious disease is well into progress.
The host defense mechanisms are mediated by the immune system. For our purposes, the term immunity refers to the relative state of resistance of the host to infectious disease. I will adopt the nomenclature used by my colleagues at University of South Carolina School of Medicine Microbiology and Immunology On-line to draw lines between the "types of immunity", particularly as it relates to to innate immunity and adaptive immunity.
The immune system is composed of two major subdivisions, the innate or nonspecific immune system and the adaptive or specific immune system. The innate immune system is a primary defense mechanism against invading organisms, while the adaptive immune system acts as a second line of defense. Both aspects of the immune system have cellular and humoral components by which they carry out their protective functions. In addition, there is interplay between these two systems, i.e., cells or components of the innate immune system influence the adaptive immune system and vice versa. The innate and adaptive immune systems differ in several ways. The adaptive immune system requires some time to react to an invading organism, whereas the innate immune system includes defenses that, for the most part, are constitutively present and mobilized immediately upon infection. Additionally, the adaptive immune system is antigen specific and reacts only with the organism that induced the response. The innate system is not antigen specific and reacts similarly to a variety of organisms. Finally, the adaptive immune system exhibits an immunological memory. It "remembers" that it has encountered an invading organism and reacts more rapidly on subsequent exposure to the same organism. The innate immune system does not exhibit a memory response. Cellular defense. This term is used to distinguish whether an immune response is mediated by a particular type of cell, as opposed to a non cellular defense which does not involve a specifically programmed cell. As stated above, a variety of tissue cells are involved in innate and adaptive immunity, hence the term cellular defense. These include neutrophils and macrophages, which are involved in phagocytosis, basophils and mast cells, which are involved in inflammation, and B cells and T cells which account for antibody mediated immunity and cell mediated immunity, respectively.
All these cells have their origin in the bone marrow (Figure 2). Myeloid progenitor (stem) cells in the bone marrow give rise to neutrophils, eosinophils, basophils, monocytes and dendritic cells, while lymphoid progenitor (stem) cells give rise to T cells and B cells. Macrophages and dendritic cells, which play a key role in innate and adaptive immunity, are derived from monocytes; and mast cells, which are fixed in tissues, develop from the same precrusor cell as circulating basophils. B cells are produced in bone marrow and released into the blood and lymphatic systems. B-cells can develop into plasma cells that secrete antibodies. Precursor T cells undergo differentiation in the thymus into two distinct types of T cells, CD4+ T helper cells, and the CD8+ cytotoxic T cells. Macrophages and dendritic cells function as one of several bridges between innate immunity and adaptive immunity, since they present antigens to immunocompetent T-cells, which initiates an immunological response.
Figure 2. Development of cells that participate in immunity.
Innate Immunity
Innate Immunity is a form of non specific host defense against invading bacteria. It is natural or "innate" to the host, depending, in part, on genetics. Innate defense mechanisms are contitutive to the host, meaning they are continually ready to respond to invasion and do not require a period of time for induction. The most important components of innate immunity are anatomical barriers, intact normal flora, tissue bactericides including complement, and ability to undergo inflammatory and phagocytic responses.Innate immunity provides the first line of defense against invading bacteria. The skin and mucous membranes provide physical and chemical barriers to infection. The normal bacterial flora antagonize colonization of body surfaces by nonindigenous bacteria. The internal tissues invariably contain bactericidal substances. The most noteworthy antibacterial substance is the enzyme lysozyme, which is present in mucus and all bodily tissues and secretions. If these barriers are penetrated, the body contains cells that respond rapidly to the presence of the invader. These cells include macrophages and neutrophils that engulf foreign organisms and kill them. Bacterial invasion is also challenged by the activation of complement in blood and tissues and the incitement of an inflammatory process which has the tendency to focus both the innate and adaptive immune defenses on the site of invasion. Categories of Innate or Nonspecific ImmunityThe first four categories are generally considered non cellular defenses. Inflammation and Phagocytosis are forms of cellular defense.
1. Differences in susceptibility to certain pathogens2. Anatomical defense 3. Tissue bactericides, including complement4. Microbial antagonism5. Inflammation (ability to undergo an inflammatory response)6. Phagocytosis
Differences in Susceptibility of Animal Hosts to Microbial Pathogens (Natural Immunity)
Natural immunity or resistance is based on the genetics of the host. There are two aspects: (1) resistance among all members of a species, called species resistance and (2) resistance within members of the same animal species, called individual resistance.
Species resistance
Certain animals are naturally resistant or non susceptible to certain pathogens. Certain pathogens infect only humans, not lower animals, e.g. syphilis, gonorrhea, measles, poliomyelitis. On the other hand, certain pathogens (e.g. canine distemper virus) do not infect humans. Shigella infects humans and baboons but not chimpanzees. Little information is available to explain these absolute differences in susceptibility to a pathogen but it could be due to:
Absence of specific tissue or cellular receptors for attachment (colonization) by the pathogen. For example, different strains of enterotoxigenic E. coli, defined by different fimbrial antigens, colonize human infants, calves and piglets by recognizing species-specific carbohydrate receptors on enterocytes in the gastrointestinal tract.
Temperature of the host and ability of pathogen to grow. For example, birds do not normally become infected with mammalian strains of Mycobacterium tuberculosis because these strains cannot grow at the high body temperature of birds. The anthrax bacillus (Bacillus anthracis) will not grow in the cold-blooded frog (unless the frog is maintained at 37o).
Lack of the exact nutritional requirements to support the growth of the pathogen. Naturally-requiring purine-dependent strains of Salmonella typhi grow only in hosts supplying purines. Mice and rats lack this growth factor in blood and pur- strains are avirulent. By injecting purines into these animals, such that the growth factor requirement for the bacterium is satisfied, the organisms prove virulent.
Lack of a target site for a microbial toxin. Most toxins produced by bacterial cells exert their toxic activity only after binding to susceptible cells or tissues in an animal. Certain animals may lack an appropriate target cell or specific type of cell receptor for the toxin to bind to and may therefore be nonsusceptible to the activity of the toxin. For example, injection of diphtheria toxin fails to kill the rat. The unchanged toxin is excreted in the urine. If a sample of the rat urine (or pure diphtheria toxin) is injected into the guinea pig, it dies of typical lesions caused by diphtheria toxin.
Individual resistance
There are many reasons why individuals of the same animal species may exhibit greater or lesser susceptibility to the same ineffective agent.
Age. Usually this relates to the development and status of the immunological system which varies with age. It may also be associated with changes in normal flora coincidental to developmental changes in the animal.
Sex. Usually this is linked to the presence and/or development of the sex organs. For example, mastitis and infectious diseases leading to abortion will obviously occur only in the female; orchitis would occur only in males. It could also be due to anatomical structure related to sex (bladder infections are 14-times more common in females than males), and possibly the effects of sex hormones on infections.
Stress. Stress is a complex of different factors that apparently has a real influence on health. Undue exertion, shock, change in environment, climatic change, nervous or muscular fatigue, etc. are factors known to contribute to increases in susceptibility to infection. The best explanation is that in time of stress the output of cortisone from the adrenal cortex is increased. This suppresses the inflammatory processes of the host and the overall effect may be harmful. There are also a number of relationships between stress-related hormones and the functioning of the immune defenses.
Diet, malnutrition. Infections may be linked with vitamin and protein deficiencies, and this might explain partly why many infectious diseases are more prevalent and infant mortality rates are highest in parts of the world where malnourishment is a problem. Also, overfed and obese animals are more susceptible to infection. Diets high in sucrose predispose individuals to dental caries.
Intercurrent disease or trauma. The normal defenses of an animal are impaired by organic diseases such as leukemia, Hodgkin's disease, diabetes, AIDS, etc. Frequently, inflammatory or immune responses are delayed or suppressed. Colds or influenza may predispose an individual to pneumonia. Smoking tobacco predisposes to infections of the respiratory tract. Burned tissue is readily infected by Pseudomonas aeruginosa.
Therapy against other diseases. Modern therapeutic procedures used in some diseases can render an individual more susceptible to infection. Under these conditions not only pathogens, but organisms of the normal flora and nonpathogens in the host's environment, may be able to initiate infection. Examples of therapeutic procedures that reduce the efficiency of the host's defenses are treatment with corticosteroids, cytotoxic drugs, antibiotics, or irradiation.
Anatomical Defenses
The structural integrity of the body surfaces, i.e., the skin and mucous membranes, forms an effective barrier to initial lodgment or penetration by microorganisms. The skin is a very effective barrier to bacterium, so that no bacterium by itself is known to be able to penetrate unbroken skin. Of course, a puncture, cut or scrape in the skin could introduce infectious bacteria. The mucous membranes are more vulnerable to penetration by infectious bacteria but still pose a formidable barrier of mucus and antimicrobial substances.The anatomical defenses are associated with all other aspects of noncellular immunity, including individual resistance, mechanical resistance, chemical resistance and resistance established by the normal flora (Figure 3)
Figure 3. Anatomical defenses associated with tissue surfaces
Skin. The intact surface of the healthy epidermis seems to be rarely if ever penetrated by bacteria. If the integrity of the epidermis is broken (by the bite of an insect, needle stick, abrasion, cut, etc.) invasive microbes may enter. The normal flora of the skin, which metabolize substances secreted onto the skin, produce end products (e.g. fatty acids) that discourage the colonization of skin by potential pathogens. Perspiration contains lysozyme and other antimicrobial substances.
Mucous membranes. Many are heavily colonized with bacteria in whose moist secretions they survive. These normal flora are restricted from entry and usually occupy any attachment sites that might otherwise be used by pathogens. The normal flora established on mucous membranes may antagonize non-indigenous species by other means, as well. Typically, mucus contains a number of types of anti-microbial compounds, including lysozyme and secretory antibodies (IgA). Sometimes phagocytes patrol mucosal surfaces (e.g. in the lower respiratory tract). Nonetheless, most infectious agents impinge on the skin or mucous membranes of the oral cavity, respiratory tract, GI tract or urogenital tract, and from these sites most infections occur. Damage to the epithelial cells caused by toxic products of these bacteria may play a role.
Respiratory tract. Fine hairs and baffles of the nares (nasal membranes) entrap bacteria which are inhaled. Those which pass may stick to mucosal surfaces of the trachea or be swept upward by the ciliated epithelium of the lower respiratory tract. Coughing and sneezing also eliminate bacteria. The lower respiratory tract (lung) is well protected by mucus, lysozyme, secretory antibody, and phagocytosis.
Mouth, stomach and intestinal tract. Microorganisms entering by the oral route, more than any other, have to compete with the well-adapted normal flora of the mouth and intestine. Most organisms that are swallowed are destroyed by acid and various secretions of the stomach. Alkaline pH of the lower intestine can discourage other organisms. The peristaltic action of the intestine ultimately flushes out organisms which have not succeeded in colonization. Bile salts and lysozyme are present, which kill or inhibit many types of bacteria.
Urogenital Tract. The flushing mechanisms of sterile urine and the acidity of urine maintain the bladder and most of the urethra free of microorganisms. The vaginal epithelium of the female maintains a high population of Doderlein's bacillus (Lactobacillus acidophilus) whose acidic end products of metabolism (lactic acid) prevent colonization by most other types of microorganisms including potentially-pathogenic yeast (Candida albicans).
Eyes (Conjunctiva). The conjunctiva of the eye is remarkably free of most microorganisms. Blinking mechanically removes microbes, the lavaging action of tears washes the surface of the eye, and lachrymal secretions (tears) contain relatively large amounts of lysozyme.
Microbial Antagonism
This refers to the protection of the surfaces afforded by an intact normal flora in a healthy animal, and it has already been mentioned in several contexts (See The Bacterial Flora of Humans). There are three main ways that the normal flora protect the surfaces where they are colonized:
Competition with non-indigenous species for binding (colonization) sites. The normal flora are highly-adapted to the tissues of their host. That is why they are there.
Specific antagonism against non-indigenous species. Members of the normal flora may produce very specific proteins called bacteriocins which kill or inhibit other (usually closely-related) species of bacteria.
Nonspecific antagonism against non-indigenous species. The normal flora produce a variety of metabolites and end products that inhibit other microorganisms. These include fatty acids (lactate, propionate, etc.), peroxides and antibiotics.
Figure 4. Enterococcus faecalis, also classified as Streptococcus faecalis. Occasionally there is invasion of the host by the normal flora, as evidenced by this blood culture. Enterococcus faecalis, blood culture. © Gloria J. Delisle and Lewis Tomalty, Queens University Kingston, Ontario, Canada. Licensed for use by ASM Microbe Library http://www.microbelibrary.org.
Antimicrobial Substances in Host Tissues
The body fluids and organized tissues of animals naturally contain a variety of antimicrobial agent that kill or inhibit the growth of microbes. The sources and activities of a variety of host antimicrobial substances are summarized in Table 1.
TABLE 1. ANTIMICROBIAL SUBSTANCES OF HOST ORIGIN PRESENT IN BODY FLUIDS AND ORGANIZED TISSUES
Substance
Common Sources
Chemical Composition
Activity
Lysozyme
Serum, saliva, sweat, tears
Protein
Bacterial cell lysis
Complement
Serum
Protein-carbohydrate lipoprotein complex
Cell death or lysis of bacteria; participates in inflammation
Basic proteins and polypeptides (histones, ß-lysins and other cationic proteins, tissue polypeptides)
Serum or organized tissues
Proteins or basic peptides
Disruption of bacterial plasma membrane
Lactoferrin and transferrin
Body secretions, serum, organized tissue spaces
Glycoprotein
Inhibit microbial growth by binding (withholding ) iron
Peroxidase
Saliva, tissues, cells (neutrophils)
Protein
Act with peroxide to cause lethal oxidations of cells
Fibronectin
Serum and mucosal surfaces
Glycoprotein
Clearance of bacteria (opsonization)
Interferons
Virus-infected cells, lymphocytes
Protein
Resistance to virus infections
Interleukins
Macrophages, lymphocytes
Protein
Cause fever; promote activation of immune system
Complement
Complement is considered as part of the innate immunity because of its role in inflammation, phagocytosis and bacterial killing. Complement may be activated by bacterial invasion, but also by reactions between antigens and antibodies, and therefore, it may play a role in adaptive immunity, as well.
Complement is an enzymatic system of serum proteins made up of nine major components (C1 - C9) that are sequentially activated during two pathways, the classical pathway and the alternative pathway, resulting in a variety of antibacterial defenses. Complement components play a part in phagocytic chemotaxis, opsonization and the inflammatory response, and may be involved in the lysis of certain bacteria, some viruses, and other microorganisms.
Complement is activated in the classical pathway by reactions between antibodies and antigens on the surface of a microbe. Some Immunoglobulins (i.e., IgG and IgM) can "fix complement" because they have a complement binding site on the Fc portion of the molecule. The reaction between IgG and Ag activates the complement and initiates a "cascade reaction" on the surface of the microbe that results in the principal effects of complement which are:
1. Generation of inflammatory factors, C3a and C5a, which focus antimicrobial serum factors and leukocytes into the site of infection.
2. Attraction of phagocytes. Chemotactic factors C3a and C5a attract phagocytes to the site.
3. Enhancement of phagocytic engulfment. C3b component on Ag - Ab complex attaches to C3b receptors on phagocytes and promotes opsonization of Ab-coated cells. C3b-opsonization is important when Ab is IgM because phagocytes have receptors for Fc of IgM only when it is associated with C3b.
4. Lysis of bacterial cells (lysozyme-mediated) or virus-infected cells. When C8 and C9 are bound to the complex, a phospholipase is formed that destroys the membrane of Ag-bearing host cells (e.g. virus-infected cells) or the outer membrane of Gram-negative bacteria. Lysozyme gains access to peptidoglycan and completes destruction of the bacterial cell.In addition to the classical pathway of complement activation, an alternative pathway (sometimes called the "properdin pathway") of complement activation exists, which is independent of immunoglobulins. Insoluble polysaccharides (including bacterial LPS, peptidoglycan and teichoic acids) can activate complement. This allows antibody-independent activation of the complement cascade that is thought to be important in initial (pre-antibody) defense against various types of infections caused by bacteria.Figure 5. The complement cascade, precipitated by certain antigen-antibody reactions (classical pathway) or by bacterial polysaccharides (alternative pathway), leads to four principal antimicrobial effects: 1. phagocytes are attracted to the site (POLYMORPH ACCUMULATION); 2. inflammatory agents re produced and/Or released from cells (INFLAMMATION); 3. microbes are opsonized to enhance uptake by phagocytic cells (PHAGOCYTOSIS); 4. Gram-negetive bacteria are lysed in the presence of lysozyme (LYSIS OF MICROBE).
Inflammation
Of all the defense mechanisms in the animal host, the inflammatory response may be the most important for dealing with microbial infection. Inflammation is necessary for the proper functioning of all the host defenses, including the immune defenses, because it focuses all circulating antimicrobial factors on the site of infection. These include phagocytes, lymphocytes, antibodies, complement and other antimicrobial components of plasma. However, inflammation is also an important aspect of bacterial pathogenesis since the inflammatory response induced by a microbe can result in considerable damage to the host and, therefore, be part of the pathology of microbial disease.
Inflammation is a tissue reaction to infection or injury, the characteristic symptoms of which are redness, swelling, heat and pain. These are sometimes called the cardinal signs of inflammation. The redness is due to increased blood flow to the area of injury. The swelling (edema) is due to increased extravascular fluid and phagocyte infiltration to the damaged area. The heat is due to the increased blood flow and the action of pyrogens (fever-inducing agents). The pain is caused by local tissue destruction and irritation of sensory nerve receptors. If a whole organ or tissue is involved, loss of function may occur.
Inflammation can be induced by certain immunological reactions, tissue damage, or the entry of an injurious agent (microbial or nonmicrobial). Certain bacterial cells and/or their products (e.g. structural components or toxins) can induce an inflammatory response. Inflammation increases the blood supply and temperature in the inflamed tissues, which favors maximal metabolic activity of the leukocytes, and lowers the pH slightly, which tends to inhibit the multiplication of many microorganisms.
Inflammation is mediated by mast cells in fixed tissues (the GI tract, respiratory tract, conjunctiva), giving rise to a localized response; or by basophils in the blood giving rise to a systemic response. The response may be initiated in a variety of ways. Some of the main events involved in the induction and maintenance of an inflammatory response during a microbial infection are summarized below.
(1) The inflammatory response is triggered by pathogen invasion or tissue injury. Injured and dying cells release cytoplasmic constituents which lower the pH in the surrounding extracellular environment.
(2) The increased acidity activates an extracellular enzyme kallikrein which in turn activates bradykinin.
(3a) Bradykinin binds to receptors on the capillary walls opening junctions between cells to allow leakage of plasma components collectively referred to as the inflammatory exudate.
Increased capillary permeability allows leukocytes to pass from the vessels into tissues (this process is called diapedisis). The first cells to appear, and the most dominant, are neutrophils, which are actively phagocytic. The other components of the inflammatory exudate and their functions are described in Table 2 below).
(3b) Bradykinin also binds to mast cells of the connective tissue that are associated with the small vessels of most tissues. This initiates other events that are associated with the process of inflammation.
Initially, in the mast cell, there is a rapid influx of Ca++, intracellular cAMP levels drop, and mediator-rich lysosomal granules migrate to the cell surface, fuse with the cell membrane, and discharge their contents (preformed mediators of inflammation such as histamine, heparin, etc.) to the exterior by exocytosis
The change in mast cell permeability activates an enzyme, phospholipase A2 to synthesize a substance called arachidonic acid. This compound can be acted upon subsequently by the cyclooxygenase pathways or lipooxygenase pathways of the mast cell leading to new synthesis of prostaglandins, leukotrienes, and other mediators of inflammation. These substances contribute to the inflammatory exudate.
TABLE 2. FUNCTION OF COMPONENTS AND CELLS IN THE INFLAMMATORY EXUDATE
Component
Function
Bradykinin, histamine, leukotrienes, serotonin, prostaglandins
Inflammatory Agents (IA) which act on the vascular system to produce increased blood flow and permeability
Fibrin: (formed from fibrinogen in plasma)
coagulates and may localize an invading pathogen
Lysozyme
causes lysis of bacterial cell walls
Complement
various activities increase the inflammatory response and lead to increased phagocytosis and complement-mediated lysis of cells
Antibodies (in immune individuals)
block colonization by pathogens; neutralize microbial toxins or viruses; opsonize pathogens making them more susceptible to phagocytosis; activate complement
Pyrogens, including endogenous pyrogen (Interleukin 1)
cause fever acting on the thermo-regulatory control centers in the hypothalamus. (Interleukin-1, which is produced by macrophages, also promotes activation and mitosis of B-cells and T-cells)
Neutrophils
migrate to focus of infection and ingest and destroy foreign agents by phagocytosis
Macrophages
engulf and destroy infective agents, process antigenic components and convey them to lymphocytes
Immunocompetent lymphocytes (B-cells and T-cells)
for direct participation in immunological responses (AMI and CMI)
The overall effect of an inflammatory reaction is to recruit various cells and components to the actual site of microbial invasion. Many of these cells and plasma components have a direct role in defense against the intruding microorganism. These include neutrophils (phagocytes which engulf and destroy the microbes); macrophages and lymphocytes which are the cells necessary to initiate immunological responses against the pathogen; pre-existing antibodies which can neutralize microbial pathogens or their toxins; and plasma components such as lysozyme, complement and fibrin, which have a variety of antimicrobial activities.
Phagocytic Defenses
When invading parasites penetrate the tissues the inflammatory response, previously described, is immediately brought into play. Part of this response leads to the recruitment of phagocytes to the site of inflammation. Phagocytes are a class of cells which are capable of ingestion (engulfment) and destruction of microorganisms that are responsible for inciting the inflammatory response. First to accumulate around the invaders and initiate the phagocytic process are neutrophils. Later, local and blood-borne macrophages also migrate to the tissue site and initiate phagocytosis. Neutrophils (also known as polymorphonuclear leukocytes, polymorphism or Pans) and macrophages are sometimes referred to as professional phagocytes for their roles in this process.
Properties of Neutrophils
Neutrophils have their origin in multi-potential stem cells in the bone marrow. They differentiate in the marrow and are released in a mature form, containing a full complement of bactericidal agents. They are short-lived cells which constitute 30-70% of the circulating white blood cells (leukocytes).
During differentiation in the marrow (2-3 days) the nucleus of the cell becomes multilobed (hence the name polymorphonuclear leukocyte), cell division ceases, and mitochondria and endoplasm reticule disappear from the cytoplasm. At the same time the cell becomes motile and actively phagocytic. Cytoplasmic granules are formed from the Geology apparatus. These granules are called lysosomal and contain the various bactericidal and digestive enzymes which can destroy bacterial cells after engulfment. The contents of lysosomal granules include lysozyme, cationic proteins, acid hydrolyses, protease's, peroxidase and lactoferrin. Neutrophils also contain large stores of glycogen. Since they derive most of their metabolic energy from glycolysis, they can function efficiently in anaerobic environments.
Some additional properties of neutrophils are:
-Only half the neutrophils in human circulation are detectable in the blood; the rest adhere to vessel walls.
-For every circulating neutrophils, approximately 100 near mature cells are held in reserve in the bone marrow pool.
-Once a neutrophils enters the tissues, intestinal tract or respiratory tract, it never returns to the circulation.
Properties of Macrophages
Macrophages (also called mononuclear phagocytes) also arise from bone marrow stem cells which give rise to monocytes which develop into monocytes that are released into the blood stream. Monocytes make up 3-7% of the circulating white blood cells. The monocytes is actively phagocytic and bactericidal. Within 2 days or so, the blood stream monocytes (sometimes called wondering macrophages) emigrate into the tissues where they settle down, enlarge and become fixed macrophages (tissue histiocytes), which also have phagocytic potential. Macrophages are more active in phagocytosis than monocytes and develop many more granules containing hydrolytic enzymes. New macrophages can develop by cell division under inflammatory stimuli, but most macrophages are matured blood monocytes.
The total pool of macrophages is referred to as the system of mononuclear phagocytes. The system is scattered throughout connective tissue, basement membranes of small blood vessels, liver sinusoids, spleen, lung , bone marrow and lymph nodes. Monocytes from the blood migrate into virtually every organ in the body where they mature into fixed macrophages. In the lymph nodes, macrophages function as scavengers to remove foreign material from the circulation.
Compared to neutrophils, macrophages are long-lived cells. As phagocytes, neutrophils play a more important role in the acute stages of an infection, while macrophages are principally involved in chronic types of infections. Neutrophils circulate in the blood stream, and during an acute inflammatory response they migrate through the endothelial cell junctions as part of the inflammatory exudate. They migrate to the focus of the infection and ingest or phagocytose foreign agents, Neutrophils which have become engorged with bacteria usually die and largely make up the material of pus. Macrophages, which are also attracted to the area during an inflammatory response, are slower to arrive and become increasingly involved in chronic infections. They, too, are actively phagocytic and will engulf and destroy foreign particles such as bacteria. However, macrophages have another indispensable function in host defense: they "process" the antigenic components of infective agents and present them to lymphocytes, a process that may usually required for the initiation of immunological responses of the host. Macrophages and related dendritic cells are among an elite corps of antigen-presenting cells or APC's.
The Phagocytic Process
Phagocytosis and destruction of engulfed bacteria involves the following sequence of events:
1. Delivery of phagocytic cells to the site of infection
2. Phagocytic adherence to the target
3. Ingestion or engulfment of the target particle
4. Phagolysosome formation
5. Intracellular killing
6. Intracellular digestion (and egestion, in the case of macrophages)
These steps involved in the phagocytic process in macrophages are illustrated below.
Figure 6. Phagocytosis by a Macrophage. A bacterium, which may or may not be opsonized, is engulfed by the process of endocytosis. The bacterium is ingested in a membranous vesicle called the phagosome. Digestive granules (lysosomes) merge with phagosome, release their contents, and form a structure called the phagolysosome. The killing and digestion of the bacterial cell takes place in the phagolysosome. The macrophage egests debris while processing the antigenic components of the bacterium, which it returns to its surface in association with MHC II for antigen presentation to TH cells.
Delivery of phagocytic cells to the site of infection
The delivery of phagocytic cells, monocytes or neutrophils, to the site of microbial infection involves two processes:
Diapedisis: the migration of cells across vascular walls which is initiated by the mediators of inflammation (kinins, histamine, prostaglandins, etc.)
Chemotaxis. Phagocytes are motile by ameboid action. Chemotaxis is movement of the cells in response to a chemical stimulus. The eventual concentration of phagocytes at a site of injury results from chemotactic response by the phagocytes which is analogous to bacterial chemotaxis. A number of chemotactic factors (attractants) have been identified, both for neutrophils and monocytes. These include bacterial products, cell and tissue debris, and components of the inflammatory exudate such as peptides derived from complement.
Phagocytic adherence
Phagocytosis is initiated by adherence of a particle to the surface of the plasma membrane of a phagocyte. This step usually involves several types of surface receptors on the phagocyte membrane. Three major receptors on phagocytes recognize the Fc portion of IgG: one is for monomeric IgG and the others are for antigen-crosslinked IgGs. Another receptor binds a complement factor C3b. Other phagocyte receptors bind fibronectin and mannose-terminated oligosaccharides. Under certain circumstances of infection, bacteria or viruses may become coated or otherwise display on their surfaces one or another of these substances (i.e., IgG, C3b, fibronectin or mannose). Such microbes are said to be opsonized and such substances as IgG or complement C3b bound to the surface of microbes are called opsonins. (Opsonin comes from a Greek word meaning "sauce" or "seasoning": they make the microbe more palatable and more easily ingested by the phagocyte.) Opsonins provide extrinsic ligands for specific receptors on the phagocyte membrane, which dramatically increases the rate of adherence and ingestion of the pathogen. Opsonized bacteria can be cleared from the blood by phagocytes; many types of non opsonized bacteria cannot be cleared.
Less firm attachments of a phagocyte to a particle can take place in the absence of opsonization. This can be thought of as nonspecific attachment which might be due to net surface charge on the phagocyte or particle and/or hydrophobicity of the particle.
Lastly, in the absence interaction beween the phagocyte and microbe surfaces, a phenomenon called surface phagocytosis exists: a phagocyte can simply trap an organism against a surface and initiate ingestion. Surface phagocytosis may be an important pre-antibody defense mechanism which may determine whether an infection will become a disease and how severe the disease will become.
Ingestion
After attachment of the phagocyte to its target, some sort of signal generation, which is poorly understood, results in physical or chemical changes in the cell that triggers ingestion. Ingestion is an engulfment process that involves infolding or invagination of the cell membrane enclosing the particle and ultimately releasing it into the cytoplasm of the cell within a membrane vesicle. The end result of ingestion is entry of the particle enclosed in a vesicle derived from the plasma membrane of the cell. This structure is called the phagosome.
Toll-Like Receptors
Macrophages, dendritic cells, and epithelial cells have a set of transmembrane receptors that recognize different types of molecular determinants associated with both pathogenic and non pathogenic bacteria. Foremost among these are Toll-like receptors (TLRs).
In macrophages and dendritic cells, a pathogen is exposed to a TLR when it is engulfed within the phagosome membrane. Depending on which TLR it binds to will determine what the response will be. In this way, the TLRs identify the nature of the pathogen and turn on a response appropriate for dealing with it, generally by expression of various cytokines. Humans have 12 different TLRs, each of which specializes in a slightly different response to a pathogen (be it a bacterium, virus or protozoa).
For example TLR-2 binds to the peptidoglycan of Gram-positive bacteria such as streptococci and staphylococci; TLR-3 binds to double-stranded RNA; TLR-4 is activated by the lipopolysaccharide (endotoxin) in the outer membrane of Gram-negative such as Salmonella and E. coli; TLR-5 binds to the flagellin of motile bacteria like Listeria; TLR-6 forms a heterodimer with TLR-2 and responds to peptidoglycan and certain bacterial lipoproteins. TLR-7 binds to the single-stranded RNA genomes of viruses such as as influenza, mumps and measles.
In all these cases, binding of the pathogen to the TLR initiates a signaling pathway that leads to the activation of a transcription factor that turns on cytokine genes such as those for tumor necrosis factor-alpha (TNF-α), Interleukin-1 (IL-1), and chemotactic attractants that attract white blood cells to the site. These effector molecules lead to inflammation at the site. Even before these late events occur, the binding of Gram-positive bacteria to TLR-2 and Gram-negative bacteria to TLR-4 enhances phagocytosis and the fusion of the phagosomes with lysosomes.
Formation of the phagolysosome
The phagosome migrates into the cytoplasm and collides with lysosomal granules which explosively discharge their contents into the membrane-enclosed vesicle (phagosome). Membranes of the phagosome and lysosome actually fuse resulting in a digestive vacuole called the phagolysosome. Other lysosomes will fuse with the phagolysosome. It is within the phagolysosome that killing and digestion of the engulfed microbe take place. Some of the microbicidal constituents of the lysosomes of neutrophils and macrophages include lysozyme, cationic proteins, various proteases and hydrolyases and peroxidases. The killing processes are confined to the phagolysosome, such that none of the toxic substances and lethal activities of the phagocytes are turned against themselves.
Intracellular killing of organisms
After phagolysosome formation the first detectable effect on bacterial physiology, occurring within a few minutes after engulfment, is loss of viability (ability to reproduce). The exact mechanism is unknown. Inhibition of macromolecular synthesis occurs later. By 10 to 30 minutes after ingestion many pathogenic and nonpathogenic bacteria are killed followed by lysis and digestion of the bacteria by lysosomal enzymes. The microbicidal activities of phagocytes are complex and multifarious. Metabolic products, as well as lysosomal constituents, are responsible. These activities differ to some extent in neutrophils, monocytes and macrophages.
The microbicidal activities of phagocytes are usually divided into oxygen-dependent and oxygen-independent events.
Oxygen-independent activity
Lysosomal granules contain a variety of extremely basic proteins that strongly inhibit bacteria, yeasts and even some viruses. A few molecules of any one of these cationic proteins appear able to inactivate a bacterial cell by damage to their permeability barriers, but their exact modes of action are not known. The lysosomal granules of neutrophils contain lactoferrin, an extremely powerful iron-chelating protein, which withholds potential iron needed for bacterial growth. The pH of the phagolysosome may be as low as 4.0 due to accumulation of lactic acid, which is sufficiently acidic to prevent the growth of most pathogens. This acidic environment apparently optimizes the activity of many degradative lysosomal enzymes including lysozyme, glycosylases, phospholipases, and nucleases.
Oxygen-dependent activity
Liganding of Fc receptors (on neutrophils, monocytes or macrophages) and mannose receptors (on macrophages) increases their O2 uptake, called the respiratory burst. These receptors activate a membrane-bound NADPH oxidase that reduces O2 to O2- (superoxide). Superoxide can be reduced to OH. (hydroxyl radical) or dismutated to H2O2 (hydrogen peroxide) by superoxide dismutase. O2-, OH., and H2O2 are activated oxygen species that are potent oxidizing agents in biological systems which adversely affect a number of cellular structures including membranes and nucleic acids. Furthermore, at least in the case of neutrophils, these reactive oxygen intermediates can act in concert with a lysosomal enzyme called myeloperoxidase to function as the myeloperoxidase system, or MPO.
Myeloperoxidase is one of the lysosomal enzymes of neutrophils which is released into the phagocytic vacuole during fusion to form the phagolysosome. Myeloperoxidase uses H2O2 generated during the respiratory burst to catalyze halogenation (mainly chlorination) of phagocytosed microbes. Such halogenations are a potent mechanism for killing cells.
When the NADPH oxidase and myeloperoxidase systems are operating in concert, a series of reactions leading to lethal oxygenation and halogenation of engulfed microbes occurs.
Intracellular digestion
Dead microbes are rapidly degraded in phagolysosomes to low molecular-weight components. Various hydrolytic enzymes are involved including lysozyme, proteases, lipases, nucleases, and glycosylases. Neutrophils die and lyse after extended phagocytosis, killing, and digestion of bacterial cells. This makes up the characteristic properties of pus.
Macrophages egest digested debris and allow insertion of microbial antigenic components into the plasma membrane for presentation to lymphocytes in the immunological response.
Figure 7. Phagocytosis of Streptococcus pyogenes by a macrophage. CELLS alive!
Bacterial Defense Against Phagocytosis
Pathogenic bacteria have a variety of defenses against phagocytes. In fact, most successful pathogens have some mechanism(s) to contend with the phagocytic defenses of the host. These mechanisms will be discussed in detail later as part of the determinants of virulence of pathogens. However, in general, pathogens may resist phagocytosis by:
Evading phagocytes by growing in regions of the body which are not accessible to them
Avoiding engulfment by phagocytes after contact
Being able to kill phagocytes either before or after engulfment
Being able to survive inside of phagocytes (or other types of cells) and to persist as intracellular parasites
