Skin and Mucous Membranes

1st line defense

1. Mechanical protection

a. Epidermis: Physical barrier to entrance of microbes; replaced every 40 days

b. Mucous membranes: trap and filter out dust, pollutants, and microbes

Upper respiratory tract contains cilia which move inhaled dust and microbes trapped in mucus toward the throat

c. Lacrimal apparatus: Bathes the eye; dilute and wash away irritating substances and microbes; lysozyme

2. Chemical protection

a. Sebum: Secreted by sebaceous (oil) glands it forms a protective film over the skin

pH of skin is between 4.0 – 6.8 and serves to inhibit bacterial growth

The so called “acid mantle” is a mixture of oil, sweat and bacterial metabolic by products

b. Lysozyme: Enzyme capable of breaking down the walls of certain bacteria

Found in perspiration, tears, saliva, and nasal secretions

c. Gastric juice: pH of 1.2 - 3.0; mixture of HCL, pepsinogen and mucus; destroys bacteria and toxins

Chemical protection

a. Sebum: Secreted by sebaceous (oil) glands it forms a protective film over the skin

pH of skin is between 4.0 – 6.8 and serves to inhibit bacterial growth

The so called “acid mantle” is a mixture of oil, sweat and bacterial metabolic by products

b. Lysozyme: Enzyme capable of breaking down the walls of certain bacteria

Found in perspiration, tears, saliva, and nasal secretions

c. Gastric juice: pH of 1.2 - 3.0; mixture of HCL, pepsinogen and mucus; destroys bacteria and toxins

Nonspecific cellular and chemical defenses

2nd line defense

Includes

1. Phagocytosis by leukocytes

2. Immunological surveillance: Natural killer cells

3. Anti-microbial proteins (Interferon and Complement)

4. Fever

5. Inflammation 

PHAGOCYTOSIS

There are two general classes of phagocytic cells

Microphages: Neutrophils and eosinophils

Macrophages: Aggressive phagocytes that arise form the monocytes

Neutrophils

Also called polymorphonuclear leukocytes (PMN)

The most abundant leukocyte in blood (≈70%)

During times of acute infection the bone marrow initially releases stored PMN in response to cytokines

Typically the marrow stores 10 more neutrophils that are circulating in plasma

These short lived cells are highly mobile mature phagocytes which become active upon encountering microbes in tissue

These are the first phagocytes to arrive at the site of an injury

Neutrophils can phagocytize 5 –20 bacteria

Kill ingested bacteria via the respiratory or oxidative burst

Release of destructive chemicals such as H₂O₂ and Superoxide anions (0₂^-)

These chemicals are active against bacteria, fungi, and viruses but also the neutrophil

The functional life span of the PMN is about five days thus they are produced in large numbers

NOTE: In an acute active infection neutrophil destruction can exceed production and there is an increase in the number of immature neutrophils can be found in plasma

Denoted as a “shift to the left” [absence of nuclear segmentation in immature neutrophils (Band cells)]

Monocytes

Comprise about 5% of the leukocytes

Typically arrive at injury site within 10 – 12 hours after chemotatic signals reach bone marrow

Develop into the macrophages upon leaving the circulation and entering the tissue

This is accompanied by an increase in ribosomal activity to produce more lysosomes.

From the monocyte to the macrophage there is a 5 fold increase in size 

Macrophages

The primary phagocytes

Macrophages are selective and are generally not killed in the process of phagocytosis

Can ingest up to 100 microorganisms

Macrophages are found in nearly every tissue either as wandering or fixed macrophages

Wandering macrophages phagocytize and present antigens to helper T-cells

Fixed macrophages are named according to their location

Loose connective tissue - Histiocytes

Brain – Microglia

Kidney - Mesangial cells

Liver - Kupffer cells

Lungs – Alveolar macrophages

Bone – Osteoclasts         

Dendritic cells

play a role similar to macrophages in that they present to, and stimulate lymphocytes

However they express a larger number of MHC-II self antigens and are more proficient antigen presenting cells

Dendritic cells have a morphology distinct from macrophages and are so named for the spine like projections of their plasma membranes; Similar to neuronal dendrites 

Follicular Dendritic cells

Found in the lymph nodes and display antigen for recognition by the B cells

May differ in lineage from the peripheral dendritic cells 

Dendritic cells

Capture antigen in peripheral tissues

PROCESS OF PHAGOCYTOSIS

Chemotaxis: The process of attraction or migration

Phagocytes are attracted to area of tissue injury by chemotaxis

Chemical mediators which direct chemotaxis are called chemotaxins

Sources of chemotaxins

Bacterial byproducts

Toxins

LPS

Peptidoglycan

Host cell release reactions

Interferon

Complement proteins: C₃a and C₅a

C₅a is a powerful neutrophil chemoattractant

C₅a can also bind to, and activate, mast cells and basophils

Mast cell degranulation

Release of TNF by mast cells increases the migration of neutrophils into an area

Injured tissue can even promote the release of neutrophils from the red bone marrow via the release of leukocytosis inducing factor

Chemotactic signals are effective up to 100 um away

No tissue area is greater than 30 –50 um away from the nearest capillary

Chemotactic signals result in:

Margination: The adherence of blood-borne neutrophils and and monocytes to cell adhesion molecules (CAMs) on the endothelial lining

Damaged tissue and pathogen by-products cause up-regulation of CAMs on the endothelial cell wall

Diapedesis: Amoeboid like emigration of the leukocyte through endothelial pores

Chemotaxins bind to protein receptors on leukocyte membrane to direct movement via calcium entrance and activation of cellular contraction

Chemotactic signals also result in the synthesis and release of multiple cytokines and chemokines

Cytokine + chemotaxis =Chemokine 

Chemokines

promote migration of leukocytes

Note: different chemokines result in the diapedesis of different leukocytes

E.g. – IL-8 (CXCL8) binds CXCR1 and CXCR2 on the neutrophil and allows for active margination and diapedesis  

Cytokines

activate and direct the cellular response to antigenic challenge and immune responses 

Toll-like receptors (TLR)

A type of pattern recognition receptor (PRR)

The TLR is a membrane spanning protein that recognizes a specific component of a microbe

Together the TLRs can recognize and respond to almost any pathogen and modulate the bodies’ response

In humans there are 10 known toll-like receptors 

Tumor Necrosis Factor (TNF)

TNF is released primarily by LPS activated macrophages

TNF promotes acute inflammatory responses to Gram-negative bacteria

TNF recruits neutrophils and monocytes to site of infection

TNF binding to endothelial cells stimulates them to express adhesion molecules

Note: In high concentrations TNF can result in severe drops in blood pressure, disseminated intravascular coagulation and apoptotic cell death

Process of margination and diapedesis

Integrins are cell adhesion molecules that can be subdivided into two different classes

Beta 2 – Enable the leukocytes to adhere to endothelial cells

Beta 1 – Enable the leukocyte to adhere to the extracellular matrix preventing their uptake into the lymphatic capillaries

Note: IL – 8, released by the macrophage, allows the integrin to change from a low affinity to a high affinity state

Adherence

Attachment of the phagocyte to the invading microorganisms

Opsonization makes adherence more efficient as the surface of the microorganism is coated by complement proteins and antibodies

Allows for receptor mediated endocytosis

Phagocytes have receptors for:

1. Components of the immune response

a. CR1: Binds to C3b fragment

b. FcR: Binds to the constant region of the antibody

2. Components of the bacterial cell wall

a. scavenger receptors: Bind to LPS and lipoteichoic acid

b. mannose receptors: Bind to complex carbohydrates on the microbial surface

Phagolysosome

Formed as the phagocytes engulfs the microbe within a membrane bound phagosome

The phagocyte has two means by which to kill

a. Non-oxidative killing: involves lysosomes

Lysosomal vesicles fuse with the phagosome releasing:

Lysozyme – cleaves peptidoglycans

Defensins - disrupt the membrane and lyse the microbes

Hydrolytic digestive enzyme - chemically break down the microbial remains

b. Oxidative killing: Termed the Respiratory (oxidative) burst

NADPH oxidase, an enzyme present in the cell membrane of the phagosome, reduces oxygen to superoxide free radicals

These lethal oxidants have a lethal effect on the ingested microbe

Note: Neutrophils are much less lethal in the absence of oxygen

IMMUNOLOGICAL SURVEILLANCE 

Natural killer cells: Population of lymphocytes which have the ability to recognize and kill abnormal tissue cells

They are not phagocytic but rather kill by lysing cells which do not display MHC-I molecules

Via release of perforin, which perforates the cell membrane, and granzymes which cause the cell to undergo apoptosis

NK cells express:

Killer activating receptors: Used for nonspecific recognition

Killer inhibitory receptors: Interact with MHC-I

NK will lyse abnormal cells not displaying MHC-I molecules

NK activation is enhanced by exposure to LPS, interferon alpha, and interferon beta

Process of Immunological Surveillance

NK cells encounter a cell with abnormal proteins on its membrane

NK attaches to target cell

If cell does not display MHC 1 complex NK releases vesicles of perforin via exocytosis

Perforins create pores in the membrane of the targeted cell so that it can no longer control it’s internal environment and undergoes lysis

Perforins do not affect the NK cell membrane 

ANTI-MICROBIAL PROTEINS (Interferon and Complement proteins) 

Interferons (INF'): Released by activated lymphocytes and virus infected body cells

Once produced by the cell they are released and diffuse to uninfected neighboring cells where they bind to surface receptors.

Two effects of INF

i. INF binding to surface receptors stimulates uninfected cells to synthesize antiviral proteins that inhibit or interfere with viral replication

Virus is able to enter cell but INF restricts viral replication at the ribosome

Viruses can cause disease only if they can replicate within body cells

ii. INF's also stimulate and activate macrophages and NK cells against host or malignant cells 

b. Complement system: Consists of a group of special proteins in blood plasma

These constitute about 10% to 15% of the plasma proteins

When activated they enhance (complement) certain immune, allergic and inflammatory reactions

Kill bacteria and certain other microbes by disrupting the plasma membrane resulting in rupture of the microbe

There are three converging pathways that activate C₃ cleaving it into two fragments that result ultimately in rupture of the microbe (Classical; Alternate; Lectin)

Classical pathway- Linked to immune system because it depends on formation of antigen-antibody complex

Rapid activation as compliment proteins bind to antibodies already attached to its antigen

Involves IgG and IgM

Alternate pathway- Certain polysaccharides on the surface of the microbe activate factors B, D, and properdin (factor P) which converts C₃

Slower and less effective

Functions of the Complement proteins

a. C₃a à Activation of inflammation

Vasodilaton  increases blood flow to area

Histamine release from: mast cells, platelets, basophils  increases permeability

b. C₃b fragment à Opsonization

C3b binds to surface of microbe to promote phagocytosis of microbe 

Phagocytic cells have cell surface receptors for C₃b

c. Cytolysis - Activated complement proteins, C₅b – C_9, form a membrane attack complex (MAC) that open pores in the plasma membrane

Leads to microbial rupture

d. Chemotaxic agents - Activated compliment proteins serve as to attract phagocytes to site of invasion

C_3a and C_5a serve as a chemoattractant for neutrophils and macropahges 

FEVER

Speeds up body reactions that aid repair

Faster enzymatic reactions

Increases the activity of neutrophils

Inhibits the growth of some microbes

Intensifies the effects of interferons

Hypothalamus normally regulates body temp to keep heat production and heat loss in balance

Fever results as macrophages ingest microbes and release interleukin -1 which acts as a pyrogen

IL-1 circulates to the hypothalamus, which in turn allows for secretion of prostaglandins which reset the hypothalamic thermostat at a higher temp

INFLAMMATION

Serves to:

Temporarily repair injured tissue

Prevent spread of infectious material or microbes

Mobilize phagocytes to

Affront pathogens

Clear the area of cellular debris

Set the stage for repair

Cardinal signs of acute inflammation

Redness: r/t relaxation of vascular smooth muscle cells

Edema

r/t relaxation of vascular smooth muscle cells

r/t Contraction of endothelial cells

Note: Decrease blood plasmaà increased localized viscosity

Slows flow to allow for increased margination

Heat: r/t relaxation of vascular smooth muscle cells

Note: Heat increases rate of enzymatic reactions and phagocytic activity

Pain: Chemical and mechanical stimulation of pain receptors

Local tissue injury leads to activation of arachidonic acid à COX 2 --> sensitizes receptors to painful stimuli. E.g. – Bradykinin (chemical) and edema (mechanical)

Inflammation is a response to tissue injury that is both produced by, and results in the release of, chemical mediators

Mediators of Acute Inflammation 

Relaxation of vascular smooth muscle cells: Mechanisms of vasodilation

Histamine

Released from mast cells, platelets, and basophils results in the vasodilation of local arterioles à increased blood flow

Histamine activation of H₁ receptors on vascular smooth muscle cells à decreased Ca²⁺ influx à VSM relaxation à vasodilation à increased blood flow à ↑ HPc

Note: in the bronchioles histamine binds to H1 receptors on smooth muscle to produce bronchiolar constriction

Bradykinin

Activated during tissue inflammation

Stimulates vasodilation by increasing NO synthesis

Small amounts injected locally into tissue result in local edema

Nitric oxide: produces relaxation of vascular smooth muscle

Nitric oxide is a lipophilic gas released from healthy endothelial cells,

Acts as a local paracrine on VSM, with a half life of approximately six seconds

Normal blood flow sheer force stimulates nitric oxide release by healthy endothelial cells (along with Prostacyclin PGI₂)

NO produces vasodilation in VSM by blocking IP₃

IP₃ stimulates Ca²⁺ from the endoplasmic (sarcoplasmic) reticulum 

Contraction of endothelial cells

Mechanisms of increased permeability

Histamine

Activates H₁ receptors on endothelial cells à contraction à enlarged intracellular clefts à increased capillary permeability

Increased permeability à ↑ OPif and ↓ OPc à edema

Clotting factors contribute to ↑ OPif but heparin prevents formation at site

Clot may form around site isolating pathogens

Decrease blood plasmaà increased localized viscosity

Slows flow to allow for increased margination

Substance P

Increases vessel permeability

Produced by endothelial cells and macrophages

Stimulates mast cell release of histamine

Leukotrienes

Stimulate contraction of endothelial cells

Chemical signals serve to provide chemotaxis

Chemotaxis of leukocytes 

Release of histamine and TNF by mast cells

Secretion of cytokines by neutrophils

Chemotaxis increases marginationà diapedesis of neutrophils and monocytes à phagocytosis of dead cells, infectious agents and affected matrix

Activation of APC à activation of 3^rd line defense

Cytokine stimulation of fibroblasts à walling of area with fibers à further prevent spread and reinforce clot

Lymphatic system

Allows the lymphocytes to monitor the approximately 2-3 L/day of lymph that is formed in the interstitial spaces. Lymph returning from tissue spaces is monitored for microbes and microbial byproducts

Lymph nodes provide areas of small volume but high lymphocyte density

Increases the likelihood of antigen encounters; Filter out more than 99% of the antigens in lymph

Dendritic cells present fragments of these antigens to B and T cells

System design increases the likely hood of encounter with a particular antigen.

Antigen will likely encounter one of the 10,000 or so T cells that have TCR specificity for that particular antigen

Antigen will likely encounter one and the 10,000 or so B cells that have BCR specificity for that particular antigen

MALT – Mucosal associated lymphoid tissue

Collection of lymphoid tissues that is associated with and monitors the mucosal surface epithelium

Consists of individual lymphocytes, and structures such Peyer’s patches, tonsils, and vermiform appendix which consist of aggregates of lymphocytes.

Note: The mucosal epithelial surface area is approximately 400 square meters, compared to only 2m² for the epidermal surface area