INFLAMMATION: Features, Signals for inflammation (pattern recognition, response, inflammatory chemokines, high endothelial venules), Extravasation ( cell adhesion molecules - selectin, mucin, integrin), sequence for neutrophile movement, lymphocytes ectravasation, inflammatory mediators (clotting, kinins, lipid derivatives), clinical application.

Inflammation is an instantaneous and vigorous immune response to a pathogen or foreign molecule; takes place at the skin surface which ultimately leads to neutralization of that foreign particle. The process of inflammation is characterized by several features, which are as follows -

1. Hallmarks of the inflammatory response

•    a. rubor (redness)
•    b. tumor (swelling)
•    c. calor (heat at tissue site)
•    d. dolor (pain)
•    e. functio laesa - loss of function.

VISIBLE INFLAMMATION

2. Cause by vascular changes  

•   a. endothelium leaks  
•   b. edema 
•   c. positive feedback loop of cell signaling
•   d. further changes in cell adhesion molecules on both cells and endothelium.

3. Medical Background 

• a. Long-time recognition that fever accompanies infection.   Lower fever, lower infection?
• b. Bleeding lowers fever.
• c. Giving blood is associated with lower heart disease (as are statins, aspirin and fish oils, all of which are anti-inflammatory and all of which tend to increase bleeding). 

 Signals for inflammation

 Pattern Recognition 

1. PAMPS (pattern-associated molecular proteins) - generally used to describe a variety of molecules used to identify pathogens.  The first group identified was the toll-like receptors, but many more types exist. 

2. DAMPS  (death or damage-associated molecular proteins) - molecules that are normal cell components but should be inside – cytoplasmic or nuclear proteins of membrane proteins belonging in the inner leaflet. 

PATTERN RECOGNITION LOCATIONS & TARGETS

Response: 

1. Activate complement, which in turn releases inflammatory signals (anaphylatoxins) 

2. Attracts neutrophils, macrophages and mast cells. 

Inflammatory Chemokines 

1. Smallish (90 to 130 amino acids), with conserved cysteines.

2. Receptors are seven-span membrane proteins. Cross reception common:  a given chemokine may bind more than one receptor and a specific receptor may bind more than on chemokine.

3. Binding activates G-proteins -

• a. large (heterotrimeric with αβγ subunits) – kicks off cytoskeletal changes –quick change in adhesiveness.
• b. Switches from active to inactive as it binds GTP and then hydrolyzes it to GDP.

4. G-proteins kick of several internal cascades: 

• a. hydrolyzes membrane phospholipids to IP3 and DAG.
• b. lets Ca2+ loose in the cytoplasm, polymerizing actin and promoting movement.
• c. kicks off Ras cascade that leads to activation of transcription factors.

G PROTEIN

GPCR MECHANISM

 The High-endothelial Venules 

1. found at the end of capillaries in lymph nodes, Peyr's patches, and tonsils (not spleen).
2. The endothelial lining here is composed of cells that do not look like flattened paving stones, but are thicker.
3. Most lymph cells that extravasate attach to these specific cells.
4. The HEVs specifically attract lymphocytes and do NOT attract neutrophils.
5. The HEV develops in response to foreign antigen exposure: you don't see this in mice raised in a germ-free environment and you don't see this in tissues that have the circulation to them blocked to that antigen does not enter.
6. The unique morphology is associated with the induced expression of specific selectins, mucin-like, and Ig CAMs, and they specifically allow different classes of lymphocytes to home to different organs and regions of the body. 

Extravasation

Cell Adhesion Molecules- Selectins and Mucins

Selectin Family, representation and ribbon diagram of lectin domain. 

a. membrane glycoprotein with lectin at amino terminus that binds carbohydrates.  Compare with complement lectin pathway 

b. specific for sialic acid (mucins have a lot of these) 

c. comes is L (Leukocyte), E and P (endothelium) versions d. initiate initial sticking of leukocytes to the endothelial wall.

SELCTIN (ENLARGED PORTION IS SHOWN ON THE RIGHT)

Mucin-Like Family 

a. protein part rich in serine and threonine (OH-containing R groups) 

b. LOTS of carbohydrate linked to these OHs. 

c. Carbohydrates have lots of sialic acid, interact with selectins 

d. The mucin-like versions of the endothelium interact with the selectins on the leukocytes and vice-versa.

MUCIN STRUCTURE

Cell Adhesion Molecules- Igs and Integrins

 I-CAM - Immunoglobulin Super-Family.

a. endothelium has several versions.

b. mucosa has another, which also has a mucin-like domain.

c. bind to integrin on leukocytes.

d. inflammation increases their expression.

IMMUNOGLOBULIN SUPER FAMILY RECEPTOR

Integrin Family – varied binding partners

a. Heterodimer ( and  chains) α β 

b. Expressed in leukocytes 

c. Different integrins bind to different immunoglobulin CAMs (cell specificity) 

d. Can also bind to fibronectin 

e. Cytoplasmic portion can interact with cytoskeleton and signaling proteins such as the tyrosine kinases, Fyn and Lck.

INTEGRIN BINDING TO EXTRACELLULAR MATRIX (ORANGE)

Sequence

1. rolling - attachment by low affinity P selectins (yellow) on the endothelium to mucin-like molecules on the neutrophil (purple).  Because the connections are loose, they tend to break and the neutrophils kind of roll along, attaching to one endothelial cell after the next as it is swept along by the flow of blood.  

NEUTROPHIL EXTRAVASATION

 2. activation – the “stick and release” from the rolling response along the endothelium triggers chemokine (IL-8) release by the endothelium.  The neutrophils activate G-protein cytoplasmic pathways via the 7-span receptor.

•  We looked at these in connection with T cell activation, but they also form membrane compartment to organize other interactions as well. Will return to this. 
•  The mechanical pulling and tugging causes the endothelium to release a chemokines. 

3. arrest and adhesion - The G-proteins activate integrins, changing their conformation, and increasing their affinity for Ig-related CAMs. This nails down the neutrophils.  Neutrophils cannot bind to non-inflamed endothelium. 

• The chemokine binds to a 7-span chemokine receptor. 
• This receptor activates a G protein and sets off an internal signaling cascade. 
• The integrins change conformation, enter the lipid rafts, deploy and stick Ig tightly to the Ig CAMs of the endothelium  
• Some of the first proteins affected are those that associated with the inner side of the plasma membrane and connect to the cytoskeleton.

NEURTOPHIL EXTRAVASATION INTERACTION

4. transendothelial migration - the arrested neutrophil then finds the gap between two adjacent endothelial cells and squeezes through it. 

• The cytoskeletal changes lead to the neutrophils changing shape and moving by amoeboid motion.
• The neutrophil forms a leading wedge and ootches through a gap between the endothelial cells (recall that inflammation makes the endothelium somewhat leaky).

 Lymphocyte Extravasation, Trafficking, and Homing 

While the processes that allow the lymphocyte to leave the blood vessel  (rolling,activation, arrest, and migration) are similar, the mechanisms that control exactly where they lymphocyte will under go extravasation are more complex and involve specific homing signals.

LYMPHOCYTE EXTRAVASATION & HOMING

1. Naïve lymphocytes Lymphocytes migrate in and out of secondary lymph organs where they contact presenting sentinel dendritic cells.  Recall that the chance of any one lymphocyte recognizing any one antigen are miniscule (1 in 105), so the cells run repeated loops through secondary organs and in and out of circulation, essentially kissing frog after frog and looking for the rare prince. 

• T cells, for example are attracted by inflammatory signals and cruise into sites of active infection, where they may be activated by antigen presented by innate cells fighting the infection. 
• T cells may also be activated by dendritic cells in the lymph nodes in spleen.  In that case the dendritic cells also communicate the site of the infection by activating specific T-cell surface receptors for cytokines and CAMs characteristic of particular tissues.  For example retinal/retinoic acid is produced by the gut and only gut-homing T cells have receptors for it.  For skin, it’s vitamin D.  

2. Effector and Memory Cells

• Effector cells tend to head to the site of infection. 
• Memory cells tend to head to the tissues that initially had the infection. 
• Cells are directed by a combination of cytokines and cell surface receptors (above) but can also be retrained, which is necessary if an infection spreads. 
• T cells, in particular, are programmed to home to a specific site because they express a specific combination of cytokine and CAM receptors. 

Inflammatory Mediators 

 Clotting 

1. Clotting both up-regulates, and is up-regulated by, inflammation.  This is why chronic inflammation can lead to strokes and cardiac infarcts. 

2. Initiated by platelets and RBCs contacting damages surfaces (collagen fibrils) and breaking open. 3. This releases and activates Hageman factor, with several consequences. 

4. The clotting cascade ends with prothrombin activation, which clips fibrinogen into fibrin and peptides. 

5. The fibrin forms the clot. 

6. The released peptides promote inflammation.  

7. Hageman’s factor also activates the fibrinolytic system, which dismantles the clot using the enzyme plasmin. 

8. Plasmin also produces inflammatory peptides and activates complement.

 Kinins 

BRADYKININ

  1. Specifically leads to pain, in addition to increased endothelial permeability and smooth muscle contraction. 
2. Cascade initiated by Hageman factor.
3. Also involve proteolysis.
4. Leads to clipping of kininogen to bradykinin. (ACE inhibitors).

 Lipid derivatives- these often depend of activations of specific enzymes. 

1. Membrane phospholipids play a structural role but also serve as the raw material for generating a lot of signaling compounds.

2. Phospholipids have two fatty acids linked to glycerol and the glycerol links up via a phosphate to a polar group (ethanoleamine or inositol for example). 

3. Depending on what you begin with and where you cut it, and how you modify the results, you can get an array of different potent signaling molecules. 

4. Arachidonic acid- derived from one to the fatty acids. 

5. Act on it with enzymes called cyclooxygenases (COX2 inhibitors) and you can get 

           a. thromboxane b. prostaglandins 

6. Act on it with lipoxygenase and get a variety of leukotrienes. 

7. In a separate origin pathway, phospholipids can provide the raw material for Lyso-PAF, which is converted to PAF (Platelet Activating Factor).

LIPID DERIVATIVES

CLINICAL APPLICATION

 Disease 

1. Systemic Acute-Phase Response – defense from something like flu or bacterial disease – acts more globally- if your toe infection turns into sepsis, for example. 

  a. Compromised by malnutrition or starvation 

  b. IL-6, TNF, IFN γ 

  c. Act on the hypothalamus to induce fever and CRP release, which in turn causes the pituitary to release of ACTH which causes the adrenals to produce steroids (cortisol etc.).  

  d. The liver responds with the release of acute-phase proteins (a general term) - activates complement. 

  e. increased leukocyte production and activation, especially neutrophils 

2. Chronic Inflammatory Response – IFNγ and TNFa, transcription factor NF-κB 

  a. persistent infection (gum disease), autoimmune response, cancer, chronic injury. 

  b. old age, obesity, diet high in trans-fats, triglycerides (diet high in sugar) wrong gut flora, diabetes, sleep disorders. 

  c. contributes to disease processes, including cancer and cardiovascular diseases 

  d. Increased clotting 

  e. fibrosis - a type of scar tissue formed when chronic inflammation leads to excess production of fibroblasts and collagen 

  f. granuloma (also called tubercle) - an attempt to wall off the problem with macrophages and specialized TH cells.

 Anti-inflammatories - 

              Braking the System- Hey, that drug sound familiar! 

1. Antibodies used to block leukocyte extravasation: 

    a. antibodies to integrins 

    b. antibodies to CAMS 

2. Corticosteroids 

    a. Pregnisone, cortisone, dihydrocortisone 

    b. High doses block adrenals and many immune functions 

    c. Side effects – mood swings, edema, glaucoma, increased susceptibility to infections 

3. NSAIDs 

    a. Aspirin (acetylsalicylic acid), Advil (ibuprofen) Aleve (naproxin) are OTC 

    b. prevent prostaglandin production from arachidonic acid. Celebrex specifically blocks COX-2 

    c. NOT Tylenol/acetaminophen- acts on brain to raise pain threshold and lower hypothalamic thermostat). 

4. Cooling - Interferes with inflammatory process (recall that fever is often a helpful part of the response to infection)  

  a. used to prevent excessive tissue damage following injury or stroke- exercise raises levels of CRP and other inflammatory indicators 

  b. used during surgery 

  c. used to cut down on symptoms of autoimmune disease multiple sclerosis – chilling via hands to allow athletic competition.