I. PHAGOCYTOSIS

A. Phagocytosis – a process by which certain cells (phagocytes) engulf microorganisms, other cell, cell debris, and foreign particles. While phagocytosis is a basic cell function, and is carried out by other body cells, only the professional phagocytes can ingest and kill microorganisms.

B. “The Professional Phagocytes”

1. Granulocytes –(polymorphonuclear leukocytes, polymorphs) – leukocytes with a lobed nucleus that contain cytoplasmic granules, particularly the neutrophil, the eosinophil or the basophil. Of these three types,the neutrophil is the most important phagocytic cell and is one of the “professional phagocyte”. The granules are lysosomes – membrane bound sacs containing enzymes and other materials.

a. Enzymes:

Peroxidase

alkaline phosphatase

acid phosphatase

ribonuclease

deoxyribonuclease

nucleotidases

glucuronidease

lysozyme

cathepsins

b. Proteins:

i. Cationic proteins – defensins – 30 –33 amino acids, rich in cysteine and arginine,and have a specific antibiotic-like activity by forming pores.

2. Lactoferrin – iron binding proteins

c. Interesting facts about polymorphs:

3 X 10¹⁰ polymorphs in normal human blood.

First cells to reach the sites of inflammation in the tissues.

Nondividing

Short-lived (days)

Each day about 10 11 normally disappear from the blood; this loss is balanced by production in the bone marrow.

There are enormous reserve waiting in the bone (3 X 10¹² polymorphs.)

Receptors for C3b and the Fc portion of antibody - opsonization

2. The Mononuclear Phagocytes – mononuclear leukocytes consisting of:

a. Monocytes – circulating precursors of macrophages that leukocytes that circulate briefly in the bloodstream

b. Macrophages – mononuclear leukocytes that have left the bloodstream to migrate in the tissues; strategically place thoroughout the body and fixed in the RES system – a collection fixed macrophages that are located in the liver, spleen, lymph nodes, and bone marrow.

Differences between polymorphs and macrophages:

Macrophages are distributed throughout the body

Not as numerous as polymorphs; no great reserves in the bone marrow.

They can exhibit great changes in surface shape and outline, but cannot move through tissues as well as polymorphs

They respond to different chemotactic mediators; have different content to their lysosomal enzymes - they cannot carry out the oxygen dependent killing.

They live much longer (months in man instead of days), and they can regenerate their granules once they are dumped.

They can generate reactive oxygen intermediates via the

NO pathway.

They have surface receptors for the Fc portion of Ig molecules and C3b receptors – that are important in opsonization.

They are secretory cells and liberate more than 60 different

products ranging from lysozyme to collagenase. Some of these are

the cytokines that stimulate the immune response.

They are important antigen presenting cells to the lymphocytes.

C. The process of phagocytosis

1. Migration (Diapedesis)

2. Attachment/Binding

a. By nonspecific receptors

b. After opsonization by:

i. complement fragment C3b

ii. antibody (IgG and IgM)

iii. both Antibody and C3b

3. Ingestion

4. Phagosome formation

b. Lysosome fusion

c. Phagolysosome

D. Microbial killing

1. Oxygen independent means

a. decreased pH

b. released from the granules (lysosomes):

i. defensins

ii. hydrolytic enzymes – lysosyme, proteases, nucleases, lipases

iii. lactoferrin

2. Oxygen dependent means - the “respiratory burst”

a. Enzymes that create reactive forms of oxygen

b. Reactive nitrogen intermediates (nitric oxide (NO), nitrite(NO2-), nitrate (NO3-)

E. Postkilling – the debris is released from the phagocyte by exocytosis

F. The importance of phagocytosis is illustrated by patients with:

1. Genetic Defects in Phagocytosis

2. Decreased neutrophil counts

3. Leukemia

 

II. How do pathogens deal with phagocytes

A. Extracellular strategies:

1. Inhibition of the production or mobilization of the phagocytes from the bone marrow

2. Inhibition of mobilization or chemotaxis of phagocytes

Ex. Toxins produced by S. aureus inhibit the locomotion of phagocytes; streptococcal streptolysins which kill phagocytes can suppress chemotaxis in lower concentrations; C. perfringens theta toxin can also inhibit chemotaxis. Examples of toxins as virulence factors!

3. Kill the phagocyte

Leukocidin – exotoxin produced by highly invasive pathogens such as staph, strep, and the clostridia that cause gas gangrene. Leukocidins kill the phagocyte by inducing lysosomal discharege into the cell cytoplasm.

4. Inhibit phagocytosis

Many pathogens have cell surface structures that inhibit phagocytosis

Ex.'s: M proteins on the surface of streptococci

Polysaccharide capsule of pneumococcus, N. meningitidis, H. influenzae, anthrax and plague

The enterics – virulent strains of E. coli and S. typhi have thin polysaccharide capsules (called K antigens)

Bacteria with certain types of LPS (O antigens) – with long polysaccharide chains that make them appear smooth when grown on culture media

Evidence that staphlococci that produce coagulase and coat themselves with fibrin are resistant to phagocytosis.

Other reasons mechanical ones: highly motile G – bacteria, or the sheer size may be a problem – phagocytes attempting to engulf the growing hyphae of fungi (macrophages cooperate and form syncytial giant cells to handle these large objects)

Host counter defense to this strategy?Opsonization by C3b or Ab.

B. Intracellular Strategies

1. Phagocytosis with inhibition of lysosomal fusion, e.g M. tuberculosis

2. Capture in the phagosome but an early escape into the cell cytoplasm by lysis of the the phagosomal membrane. - Shigella and Listeria species (Listeria produce a pore-forming toxin which is necessary for escape; surface bound phospholipase may also play a role)

3. Inhibit the phagocytic respiratory burst in the neutrophils– L. pneumophila

4. Resistance to killing and digestion in phagolysosomes with multiplication, e.g.Mycobacteria, Brucella, S. typhimurium

III. INFLAMMATION

A localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute, or wall off both the injurious agent and the injured tissue. It is characterized in the acute form by the classical signs of pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (functio laesa). Histologically, it involves a complex series of events, including dilatation of arterioles, capillaries, and venules, with increased permeability and blood flow; exudation of fluids, including plasma proteins; and leukocytic migration into the inflammatory focus. (Dorland's Illustrated Medical Dictionary, 26th ed. 1981. W.B. Saunders Co., Philadelphia)

A. Inflammatory barriers – tissue damage and infection induce leakage of vascular fluid, containing serum proteins with antibacterial activity, and influx of phagocytic cells into the affected area. Phagocytes internalize, kill and digest whole microorganisms.

B. Participants in Inflammation

1. Inflammatory cells: Neutrophils, Macrophages, Eosinophils, Mast Cells, Basophils, Lymphocytes, Platelets

Emphasize the role of the professional phagocytes- the (neutrophils (which arrive 1st) and the macrophages (which arrive 2nd).

2. Soluble proteins in tissue, blood, lymph and body fluids:

a. enzymes of the complement system:

b. enzymes of the kinin, blood clotting and fibrinolytic systems

3. Chemical mediators

C. Clinical Signs of Inflammation

1. rubor (redness)

2. calor (heat)

3. tumor (swelling/edema)

4. dolor (pain)

5. functio laesa (loss of function)

D. Major events that occur during an inflammatory response:

1. Vasodilation - increase in the diameter of the blood vessels (resulting in an increase in the volume of blood to the area and a reduction in the flow of blood).

2. Increase in blood vessel permeability

3. Extravasation of phagocytes from the capillaries into the tissue-

a. margination- phagocytes adhere to the endothelial wall

b. diapedesis/extravasation- phagocytes squeeze between the endothelial cells into the tissue

c. chemotaxis –directed migration of the phagocytes to the site of injury (drawn by various chemotactic substances (chemokines) - inflammatory mediators

d. phagocytes collect at the site of injury, phagocytize, and kill the pathogens.

If the infection continues, and more neutrophils are needed- supplied from the bone marrow reserves and then by increased bone marrow production(stimulated by colony stimulating factor).

The patients peripheral blood count will show elevated total WBC's and the differential will “shift to the left” as more immature band cells appear in the bloodstream.

There are serious consequences if something goes wrong and the bone marrow supplies are exhausted. A fall in circulating neutrophils (neutropenia) during a bacterial infection is an ominous sign.

E. Formation of a fibrin clot that walls off the injured area.

F. How does the inflammatory response to viruses differ from the response to bacteria???

Viruses produce inflammatory products in tissues - necrotic host cell materials or antigen-antibody complexes, but they are less potent than bacterial products.

The acute inflammatory response is of shorter duration. Neutrophils (polymorphs) are replaced by macrophages.

G. What triggers inflammation? Inflammation is initiated by a complex series of interactions involving a variety of cells and chemical mediators. Where do these chemical mediators come from?

1. Tissue damage activates four enzymatic systems found in plasma including:

a. The kinin system – produces bradykinin – a small peptide which increases vasodilation and vascular permeability and stimulates PAIN receptors in the skin (the 4^th clinical sign of inflammation)

b. The clotting system - fibrinopeptides formed from this system increase vascular permeability

c. The fibrinolytic system (breaks down fibrin clots) – the endproduct of this system - plasmin activates the complement pathway

d. Activation of the complement pathway generates complement split products that mediate inflammation:

i. C3a , C4a, and C5a - anaphylatoxins- bind to the mast cells and cause them to release histamine (increases vascular permeability and smooth muscle contraction). Mast cells also synthesize prostaglandins and leukotrienes which increase vascular permeability.

ii. C3a and C5a- chemotaxins - calling all neutrophils!

2. The neutrophils (arrive on the scene first) phagocytize invading bacteria and release chemical mediators:

a. macrophage inflammatory protein - calls in the macrophages!

3. Macrophages arrive, phagocytose bacteria, become "activated" and release:

a. Prostaglandins and leukotrienes – lipid inflammatory mediators which increase vascular permeability, increase vasodilation, and cause neutrophil chemotaxis.

b. Cytokines :

1. Interleukin 1 (IL-1)

2. Interleukin 6 (IL-6)

3. Interleukin 8 (IL-8)

4. Tumor necrosis factor (TNF)

4. Microbial surfaces act directly as chemotaxins drawing neutrophils and macrophages into the injured area.

H. The Systemic Inflammatory Response: This localized inflammatory response is accompanied by a systemic response known as the acute-phase response:

1. the induction of fever

2. leukocytosis

3. production of corticosteroids

4. acute-phase proteins by the liver including the C-reactive (activates complement resulting in the opsonization by C3b)

Many of the systemic effects are due to the action of IL- 1, Il-6, and TNF. Symptoms: headache, muscle pains, fever, anemia; proteins are broken down to provide amino acids needed by multiplying cells and to synthesize Ig's and acute phase proteins.

I. The Location of Acute Inflammation In The Body

1. abscess/ boil

2. lymphadenitis

3. dissemination

J. Chronic Inflammation: Chronic inflammation – if the microbe is not successfully cleared from the tissues (or in allergies and autoimmune diseases, and cancer). Hallmarks of chronic inflammation are the accumulation and activation of macrophages carrying out delayed type hypersensitivity:

1. granulomas – tumor like masses with a central area of activated macrophages surrounded by activated lymphocytes. The center of the granuloma – contains multinucleated giant cells formed by the fusion of activated macrophages, surrounded by large, modified macrophages (epitheloid cells).

IV. Complement– REVIEW

V. NONSPECIFIC AND SPECIFIC DEFENSES AGAINST VIRUSES

A. Non-Specific Defenses

1. Interferons

a. What are interferons?

b. What is their specificity?

c. Induction

d. Mechanism of action

2. Phagocytosis and Inflammation: Unlike bacteria, macrophages rather than neutrophils that are important in phagocytosis. Viruses produce inflammatory products in tissues in the form of necrotic host materials or antigen-antibody complexes, that are less potent than bacterial products at inducing inflammation and and the inflammatory reponses last for a shorter period. If the alternative complement pathway is activated – extracellular viruses can be:

a. Lysed by the MAC

b. Opsonized by C3b, phagocytosed, and killed

3. Natural Killer Cells

a. NK cells are large granulated lymphocytes that make up 5- 10% of peripheral blood lymphocytes.

b. They are important in the early responses to viruses; they also kill tumor cells

c. Their activity is stimulated by alpha and beta interferon and IL-12.

d. During viral infections, alpha and beta interferons increase, then NK cells (within 3 days). (Before CTL responses)

e. Mechanism of killing similar to CTL's – granules with perforin and granzymes; aptoptosis of target cells.

f. Unlike CTL's NK do not express CD3, TCR's, and they are not MHC restricted. They do not show immunologic memory. The mechanism of killing of NK cells is similar to CTL's.

g. NK cells can work in concert with antibody to kill virus-infected cells by Antibody Dependent Cell-Mediated Toxicity (ADCC). Antibodies first bind to the viral antigens on the surface of the infected cell via the Fab region, then NK cells can bind to Fc region of the antibody molecules. The NK cells then release lytic enzymes, perforins, etc. which kill the virus-infected cell.

4. Fever

5. Mucociliary clearance in the respiratory tract

6. Other factors:

a. age

b. nutrition

c. compromising treatments such as immunosuppression or chemotherapy

B. Specific Defenses

1. Active Immunity

a.For extracellular virus particles:

i. Neutralization -antibody binds with extracellular viruses and prevents their binding to susceptible target cells.

ii. Antibody can activate the classical complement pathway mediating lysis of extracellular virions.

b. For intracellular viruses:

i. Antibody + NK cells – lyse virus infected cells

ii. Cytotoxic T- lymphocytes (cellular immunity) – lyse virus-infected cells.

2. Passive Immunity