Objectives:1. understand the multiple lines of defenses against microbial infection2. recognize the signs of acute inflammation when presented in a clinical context and describe

how each sign of acute inflammation is generated at a cellular/tissue level3. know why/how the alternate complement cascade is activated, what the important molecules

formed are, what their function is, and what the consequences of complement activation are4. understand how complement and phagocytosis are integrated processes5. compare and contrast PMNs and macrophages6. understand the importance of direct cell-cell contact in phagocytosis and the relationships

between capsules, opsonins, and phagocytosis

II. HOST DEFENSES

The immune system is composed of 2 arms innate = nonspecific – already in place, response is rapid, not as efficient adaptive = specific – must be induced, response is slower, highly efficient, enhances

nonspecific. Has “memory”

A. Nonspecific Defenses

1. Defenses against entry into the host (1st line defenses)

a.Physical defenses (examples)1. epithelial cells2. turbulence3. shedding, scraping, flushing (saliva, urine)4. muco-ciliary clearance (1-3 cm/hr)

b. Chemical defenses (exs)1. acids (e.g. gastric, fatty acids)2. enzymes (e.g. lysozyme in saliva, tears, perspiration, urine)3. other microbicidal chemicals (e.g. zinc, dermicidin)

c. Biological defenses (exs)1. normal microbiota – physical, competition, inhibitory substances2. immune defense cells and molecules

2. Defenses of the interior of the host (2nd line defenses)

Inflammation a. phagocytic cells b. nonspecific cytolytic cells c. complement cascade d. acute phase proteinse. interferons

Inflammation - a process that coordinates and regulates all aspects of non-specific interior defense.

Acute inflammation is characterized by:1. increased blood supply to the area2. increased capillary permeability3. accumulation of neutrophils

Signs of acute inflammation – 4 signs1.2.3.4.

Triggers of acute inflammation – 2 triggers

1. cell/tissue damage/injury à chemical “alarms”

2. cell wall components of bacteria (peptidoglycan, LTA, LPS)

How Signs of Acute Inflammation are Produced

1. release of Inflammatory Mediators (=IM; see table 9.3)

2. vaso-dilation & blood flow à

2. endothelial cells of vessels contract à

3. plasma leaks out of vessels & into tissues = exudation à

4. swelling à pressure on nerve endings à

bradykinin à

***Increased blood flow & capillary permeability - a mechanism for white blood cells and critical soluble factors to enter the tissues to combat microbial invaders.

The Critical White Blood Cells (WBC = leukocytes)

a. Phagocytes – professional engulfing cells2 main roles for phagocytes1. engulf and destroy foreign matter2. secrete chemicals (esp. cytokines)

2 main kinds of professional phagocytic cells1. polymorphonuclear leukocytes (= PMN = polymorph = neutrophils) – are granulocytes - 2. mononuclear leukocytes - monocytes & macrophages – are agranulocytes -

1. polymorphonuclear leukocytedominant cell type in early stages acute inflammatory responsemade in the bone marrow - 80 mil/min dominant WBC, ~5,000/ul of blood - ↑15,000-20,000/ul live for 2-3 days - function in anaerobic environmentsabundant cytoplasmic granules contain loads of antimicrobial enzymes and chemicals (acid

hydrolases, myeloperoxidase, lysozyme, lactoferrin)best with extracellular pathogens, esp. bacteria

2. mononuclear leukocyte majors players later in inflammatory process made in bone marrow in the blood - monocytes (~600/ul); in tissues - macrophages (~60,000) conc. in lung, liver, lymph nodes, spleen live for weeks or months fewer granules (acid hydrolases, peroxidase) one of the antigen presenting cells secrete lots of different proteins (incl. PG, IL-1, TNF) best with intracellular pathogens

Process of Phagocytosis – 6 steps

1. Activation - a. marginationb. pavementingc. diapedesis

2. Migration via chemotaxis

3-6. – illustrated in diagram3. Attachment

Pathogen-Associated Molecular Patterns

Microbe - PAMP – PRR – phagocyte Pattern Recognition Receptors

PAMP incl. LTA & LPS (table 9.2)

After PAMP-PRR interaction, macrophages secrete pro-inflammatory cytokines (TNF, IL-1)à enhance antigen-presentation à leads to activation of Th1. We’ll talk about this later.

4. Engulfment5. Phagosome-lysosome fusion and intracellular killing6. Expulsion of debris

Phagocytosis - Diagram

Role of Opsonins in Process of Phagocytosis - Diagram

Opsonin Rate of PhagocytosisMicrobe+phagocyte

nonepresent

phagosome membrane

Phagocyte Intracellular Killing Mechanisms

1. Oxygen-dependent killing (see box 9.2) – PMN and macrophages

“oxidative burst”Reduction superoxide anion O2

-

O2 hydrogen peroxide H2O2 Reactive OxygenNADPH NADP singlet oxygen 1O2 Intermediates (ROI)

hydroxyl radicals OH

Other reactive intermediates reactive nitrogen (nitric oxide =

NO) (macrophages) reactive chlorine (OCl) +

myeloperoxidase (PMN)

2. Oxygen-independent killing compounds – PMN and eosinophils acid hydrolases (PMN) cathepsin G (PMN cationic proteins (PMN, eosinophils) defensins (PMN) lactoferrin (PMN) - lysozyme (PMN, also macrophage) – peroxidase (eosinophils)

b. Nonspecific Cytolytic Cells

1. natural killer cells (NK)use - perforins + granzyme à apoptosisalso secrete TNFtarget - intracellular pathogens, primarily virusesalso – secrete -IFN that can activate macs to intracellular killing

2. basophils and mast cellscan be triggered to discharge cytoplasmic granulesrelease – histamine, heparin, anaphylactic factorstarget – parasites

3. eosinophils can be triggered to discharge cytoplasmic granulesuse - basic proteins, perforins, ROI à chemical burnstarget - large parasites (e.g. helminthes)

The Critical Soluble Factors

c. Complement - activation of the alternate cascade (= properdin pathway)

A group of 20 serum proteins – form an enzymatic cascade

C3 (most abundant)

C3a C3b

Factor B

C3bB

Factor D

C3bBb =

C3 C3

C3a C3b C3a C3b

C5

C5a C5b

C5b678 multiple C9

cell lysis

d. Acute Phase Proteins Plasma proteins – proteins that increase in concentration 2-100X during the acute phase of

an infection.

Ex. Many of the complement proteins

Ex. C-reactive protein (CRP) – produced by liver in response to pro-inflammatory cytokines (exs. IL-1, TNF)

uses pattern recognition to bind to bacteria promotes binding of C3b (opsonization) activates complement cascade

Opsonin Rate of PhagocytosisMicrobe+phagocyte

noneC3bCRP

e. Interferons – one of many kinds of “cytokines”, chemicals used in cell to cell communication

Cytokines: (see table 11.2) small secreted proteins that mediate and regulate inflammation, immunity, and

hematopoiesis act over short distances, short duration, and low conc. receptor binding à signal transduction

Interferons - interfere w/ viral replication

3 classes of Interferons:1. - produced by leukocytes2. - produced by fibroblasts and other cells3. - produced by T lymphocytes and NK (does NOT interfere with viral replication)

Integration of Nonspecific Defenses

1. Stimulusa. cell/tissue injury

inflammatory mediators releasedb. microbial surface polysaccharides

2. Within seconds to minutesa. acute inflammation begins

vaso-dilation of capillaries à increases blood flow to/volume at the site increased vascular permeability à exudation of plasma, proteins, and cells

b. complement activation C3a and C5a à mast cell degranulation

àmaintains vaso-dilation/vascular permeability C5a attracts phagocytes from vasculature C3b and C5b bind to cell surfaces

c. Acute phase proteins increase in concentration – CRP, complement, and IFN

3. Minutes to hoursa. PMNs arrive in huge number and encounter cells opsonized by C3b and CRP à

phagocytosis is enhanced.

4. Hours to daysa. Macrophages arriveb. NK arrive

SUMMARIZE – Nonspecific Defenses

List the Nonspecific Interior Defenses important against Bacteria

List the Nonspecific Interior Defenses important against Viruses

MacrophagePMN

CONCEPT CHECK – Nonspecific defenses

Bubble Map

Characteristics unique to PMN Shared characteristics Characteristics unique to macrophages

CONCEPT CHECK – Nonspecific defenses

Draw a concept map that illustrates the integration of complement and phagocytosis

CONCEPT CHECK - Nonspecific Host DefensesSteve, a college student, was backpacking in a remote wilderness region with some friends. While pitching a tent, he tripped and fell. In an attempt to break his fall, he extended his arms and sustained a puncture wound to his right palm. Although the wound was painful and bled for a short time, it didn’t appear to be serious, and Steve fell asleep that night unconcerned.By the next morning, however, Steve noticed that the tissues immediately surrounding his wound were red, swollen, and warm. A round area about 1 inch in diameter really looked abnormal compared to the rest of his hand. The affected area was also painful, especially when he touched it or bumped it. After hiking all day, the sore hand was even more painful, and a thick yellow discharge oozed from the open wound. Steve felt unusually tired, his body ached, and a brief chill made him aware that he was getting a fever. His friends helped him elevate his arm and applied warm compresses to his palm, hoping that he would feel better in the morning.1. List the nonspecific defenses against entry that are relevant to skin.

  

2. How were Steve’s skins defenses against entry overcome?   

3. List the nonspecific interior defenses that would become activated in this case of bacterial invasion.

  

4. What are the signs in the case history that Steve is experiencing an inflammatory process? List them.

  

5. Describe the mechanisms at the cellular/tissue level that cause each of the signs of inflammation listed in 4 above?

Supplemental information FYI

Saliva (modified from Wikipedia) - Produced in salivary glands, human saliva is 98% water, but it contains many important substances, including electrolytes, mucus, antibacterial compounds and various enzymes It is a fluid containing:

Water Electrolytes: (sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate,

iodine) Mucus. Mucus in saliva mainly consists of mucopolysaccharides and glycoproteins; Antibacterial compounds (thiocyanate, hydrogen peroxide, and secretory IgA) Epidermal growth factor or EGF Various enzymes. There are three major enzymes found in saliva. o α-amylase - starts the digestion of starch and lipase fat. o lingual lipaseo Antimicrobials that kill bacteria:

Lysozyme Salivary lactoperoxidase Lactoferrin

o Proline-rich proteins (function in enamel formation, Ca2+-binding, microbe killing and lubrication)

o Minor enzymes Cells: Possibly as much as 8 million human and 500 million bacterial cells per mL. The

presence of bacterial products (small organic acids, amines, and thiols) causes saliva to sometimes exhibit foul odor.

Opiorphin, a newly researched pain-killing substance found in human saliva.

Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are a family of enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is abundant in a number of secretions, such as tears, saliva, human milk, and mucus. It is also present in cytoplasmic granules of the polymorphonuclear neutrophils (PMN).

Urine is approximately 95% water. The other components of normal urine are the solutes that are dissolved in the water component of the urine. These solutes can be divided into two categories according to their chemical structure (e.g. size and electrical charge).

Organic molecules These include: Urea – Makes up 2% of urine. Urea is an organic compound derived from ammonia and

produced by the deamination of amino acids. The amount of urea in urine is related to quantity of dietary protein.

Creatinine - Creatinine is a normal constituent of blood. It is produced mainly as a result of the breakdown of creatine phosphate in muscle tissue. It is usually produced by the body at a fairly constant rate (which depends on the muscle mass of the body).

Uric acid - Due to its insolubility, uric acid has a tendency to crystallize, and is a common part of kidney stones.

Other substances/molecules - Example of other substances that may be found in small amounts in normal urine include carbohydrates, enzymes, fatty acids, hormones, pigments, and mucins (a group of large, heavily glycosylated proteins found in the body).

Ions These include: Sodium Potassium Chloride Magnesium Calcium Ammonium Sulfates Phosphates

Blood (modified from Wikipedia)Blood accounts for 8% of the human body weight. The average adult has a blood volume of roughly 5 liters (1.3 gal), composed of plasma and several kinds of cells; these formed elements of the blood are erythrocytes (red blood cells; RBC), leukoytes (white blood cells; WBC), and thrombocytes (platelets). By volume, the red blood cells constitute about 45% of whole blood, the plasma about 54.3%, and white cells about 0.7%.

Cells:One microliter of blood contains:

4.7 to 6.1 million (male), 4.2 to 5.4 million (female) erythrocytes. The proportion of blood occupied by red blood cells is referred to as the hematocrit, and is normally about 45%.

4,000–11,000 leukocytes. White blood cells are part of the immune system; they destroy and remove old or aberrant cells and cellular debris, as well as attack infectious agents and foreign substances.

200,000–500,000 thrombocytes: Platelets are responsible for blood clotting (coagulation). They change fibrinogen into fibrin. This fibrin creates a mesh onto which red blood cells collect and clot, which then stops more blood from leaving the body and also helps to prevent bacteria from entering the body.

Plasma:About 55% of whole blood is blood plasma, a fluid that is the blood's liquid medium, which by itself is straw-yellow in color. The blood plasma volume totals of 2.7–3.0 liters (2.8–3.2 quarts) in an average human. It is an aqueous solution containing 92% water, 8% blood plasma proteins, and trace amounts of other materials. Plasma circulates dissolved nutrients, such as glucose, amino acids, and fatty acids (dissolved in the blood or bound to plasma proteins), and removes waste products, such as carbon dioxide, urea, and lactic acid.

Other important components include: Serum albumin Blood-clotting factors (to facilitate coagulation) Immunoglobulins (antibodies) lipoprotein particles Various other proteins Various electrolytes (mainly sodium and chloride)

The term serum refers to plasma from which the clotting proteins have been removed. Most of the proteins remaining are albumin and immunoglobulins.

Constitution of normal bloodParameter Valuehematocrit 45 ± 7 (38–52%) for males

42 ± 5 (37–47%) for femalespH 7.35–7.45base excess −3 to +3PO2 10–13 kPa (80–100 mm Hg)PCO2 4.8–5.8 kPa (35–45 mm Hg)HCO3 21–27 mMoxygen saturation Oxygenated: 98–99%

Deoxygenated: 75%