Episode 49: The original recording is very long for this topic because it covers, inflammatory mediators, phagocytosis and opsonization, complement and arachadonic acid, and cluster designations.

Listen to the podcast here…


I. Acute Inflammation

A. Cardinal signs of inflammation

In the scenario with a bee sting: you will see redness (Tubor). The king of vasodilators is histamine and it vasodilates the arterioles. Therefore, histamine is responsible for the redness of acute inflammation (i.e., bee sting), and is working on arterioles. Now if we felt the area, it will be warm (Calor = heat), this is due to vasodilating the arterioles, which is caused by histamine. For example in endotoxic and septic shock, the skin is warm b/c you are vasodilated. Tumor is a raised structure caused by histamine. Histamine can lead to increased vessel permeability in the venules; is arterial thicker than venules? Yes. The venules are very thin; they basically have an endothelial cell with a basement membrane, all you have to is drill a hole through the BM and you are out. Therefore, increased vessel permeability occurs at the venule level, not the arterial level.

Histamine contracts the endothelial cells, and leaves the BM bare, leading to increased vessel permeability, producing an exudate, and swelling of tissue, hence tumor of acute  inflammation. The area may hurt (Dolor = pain) but histamine does not have anything to do with this. Bradykinin is part of the kininogen system between factor 11 and Hageman factor 12. So when you activate the intrinsic pathway, you automatically activate the kininogen system. When you activate factor 12 (Hageman factor), it will activate 11 and the whole kininogen system. The end product is bradykinin. ACE inhibits the degredation of bradykinin, therefore complicating the clinical condition with angioedema. Also inhibit metabolism of bradykinin, which increases vessel permeability, producing the angioedema (swelling of the tissues). How bradykinin produces cough is not really understood. Bradykinin and PGE2 cause pain (dolor) and is the only one out of the four Latin terms of acute inflammation that is not due to histamine release.

B. Steps involved in Acute inflammation

(this the normal sequence in acute inflammation):

1. Emigration:

Includes margination, pavementing, rolling, adhesion, and transmigration Neutrophils in  circulation start to become sticky b/c of adhesion molecule synthesis. Endothelial cells begin to synthesize adhesion molecules. Eventually, neutrophils will stick to endothelial cells, these steps are called pavementing or margination. Then neutrophils look for bare basement membrane on the venules and then they drill a hole through it via type 4 collagenase. Cancer cells also have type 4 collagenase, that’s how they metastasize. Cancer cells attach to endothelial via adhesion molecules, usually against laminin in BM, and they have collagensae to get through the BM, therefore, cancer cells are pretty much like a neutrophil when invading tissue.

2. Chemotaxis:

When they pass BM of small venules, they emigrate but they have to know what direction to go. They get directions in a process called directed chemotaxis. C5a and LT-B4 (leukotriene B4) are the chemotactic agents. These chemotactic agents are also involved in making adhesion molecules on neutrophils). Therefore, they make adhesion molecules AND give direction by acting like chemotactic agents.

3. Phagocytosis via opsonization:

a) Example: in an acute inflammation with staph aureus, the bacteria are being processed by opsonins, which immobilize the particles on the surface of the phagocyte. The two main opsonins are IgG and C3b. They help with phagocytosis.

b) Example of an opsonization defect: Brutons agammaglobinemia: an x-linked recessive dz, where all the immunoglobulins are missing, including IgG. Therefore, MCC death in these patients is due to infection because they cannot opsonize things. It produces hypogamma-globinemia, but the mechanism of infection is due to not having IgG to opsonize bacteria, therefore cannot phagocytose it. Bacteria are opsonized by IgG and C3b, which means that neutrophils must have receptors for those. In acute inflammation the main cell is neutrophil and in chronic inflammation the main cell is macrophage/monocyte (monocytes become macrophages).  These cells have to have receptors for these opsonins (IgG and C3b). Then they become phagocytosed or become phagolysomes. When they are phagocytosed, the lysosomes go to microtubules and empty their enzyme into this.

c) Example: In I-cell disease: in this dz, mannose residues cannot be phosphorylate in golgi apparatus therefore the enzymes are not marked with phosphorus, and the lysosome are empty.

4. Intracellular microbial killing:

a) Examples:

(1) Staph aureus in hottub surrounded by enzymes

(2)Chlamydia can get out of phagolysosome, mechanism unknown, but sometimes they have mucous and all kinds of things around them.

b) O2 dependent myeloperoxidase system is on the boards!! Molecular O2 is converted by NADPH oxidase, which is in the cell membrane of neutrophils and monocytes, but not macrophages. The most important cofactor is NADPH, which is synthesized in the pentose phosphate shunt. The enzyme responsible is glucose 6 phosphate dehydrogenase, which converts G6P into 6-phosphogluconate, generating NADPH and a neutralizing factor for free radicals (glutathione).

It is converting O2 into a free radical, superoxide. Superoxide has an unpaired electron giving off energy, which is called a resp burst, which can be measured by radiation detectors; and by a negative NBT dye test. In the NBT test, you have a test tube, add the colorless NBT dye; and if neutrophils and monocytes are working normally, they will phagocytose it, will have a respiratory burst, and the free radical O2 will cause the color to change to blue, indicating that the resp burst is working. If there is no color change, there is not a resp burst, therefore the pt has chronic granulomatous dz of childhood.

Free radical O2 is converted by SOD (it’s neutralizer) into peroxide. Peroxide itself could kill bugs, but it is used for another reason. Within the neutrophils and monocytes are reddish granules which are lysosomes, and are seen in the peripheral blood. Myeloperoxidase (one of the many enzymes in the granules) will catalyze the rxn. It will combine peroxide with chloride to from bleach. This is the most potent bactericidal mechanism – O2 dep myeloperoxidase system, which is in NEUTROPHILS and MONOCYTES but NOT in macrophages, b/c macrophages lose the system when they convert from monocytes to macrophages and they use lysosomes to kill. Macrophages of the CNS are microglial cells, so the reservoir cell for CNS/AIDS is the microbial cell. Outside the CNS, it is the dendritic cell; it is a macrophage located in the lymph nodes.

c) In G6PD deficiency…

Infection is the MC precipitation of hemolysis b/c there is no NADPH, therefore there is no functioning O2 dependent myeloperoxidase system, and therefore you are susceptible to infection, which will set of hemolysis of RBC’S.

d) Chronic granulomatous disease of childhood

X linked recessive dz where the mom gives the dz to the boy, and is an asymptomatic carrier, and they will transmit the dz to 50% of their son’s. In this dz, there is a deficient activity of NADPH oxidase, and the NBT dye test is negative (doesn’t show color of die), therefore no resp burst. Do they have superoxide? No. Peroxide? No. Myeloperoxidase? Yes. Chloride? Yes. Therefore, if they phagocytosed a bacteria that could make peroxide, and add it inside the phagolysosome, this is what the kid would need to kill the bacteria. These kids are missing PEROXIDE b/c there is no NADPH oxidase. ALL living organisms make peroxide (including ALL bacteria). However, not all bacteria contain catalase, which is an enzyme that breaks down peroxide. So, in chronic granulomatous dz, what can they and can’t they kill? Cannot kill staph, but can kill strep. Why? B/c staph is Coagulase and CATalase “+”; so, ie, if it’s staph. aureus and when it makes peroxide, it will also make catalase and neutralize it, therefore the child cannot kill staph, and will kill the kid. If it was a streptococcus organism that makes peroxide (does not have catalase therefore peroxide can be used by the child), it adds what kid really needed to make bleach, and the bacteria is then wiped out. Therefore, can kill strep and not staph!

e) Myeloperoxidase deficiency:

Do they have a resp burst? Yes b/c they have NADPH oxidase. Do they have peroxide? Yes. Do they have superoxide free radicals? Yes. Do they have chloride? Yes. Do they have myeloperoxidase? No. They have a normal resp burst and a normal NBT dye test, but they can’t kill the bacteria b/c they cannot make bleach. This is called a myeloperoxidase defect. Other types of defects:

(1)  opsonization defects with brutons (missing IgG), C3 def’s;

(2) chemotactic defects where cells do not respond to chemotaxis;

(3) microbiocidal defects, the defect in the ablility to kill bacteria, example: chronic granulmatous dz of childhood and myeloperoxidase deficiency are both microbiocidal dz, in that they cannot kill bacteria, but for different reasons. In myeloperoxidase def the problem is that they cannot make bleach (b/c of the missing enzyme), but do have resp burst, and is Autosomal recessive dz. In CGDz the problem is that they cannot make bleach either, but they have an ABSENT resp burst, and is a XLINKED recessive dz.

f) Child has an umbilical cord that doesn’t fall off when it should.

When it was removed and looked at histologically, they did not see neutrophils in the tissue or neutrophils lining the small vessels. This is an adhesion molecule defect or beta 2 integrin defect. Umblilcal cord needs to have an inflammatory rxn involving neutrophils; they have to stick in order to get out. Therefore, if the neutrophils can’t stick, they can’t get out, and then they can’t get rid of your umbilical cord – this is a classic adhesion molecule defect.

G. Chemical mediators:

1. Histamine: the king of chemical mediators of acute inflammation

a) What does it do to arterioles? Vasodilates

b) Venules? Increased vessel permeability

2. Serotonin:

a) What amino acid makes serotonin? Tryptophan

b) Is serotonin a neurotransmitter? Yes

c) In a deficiency, you get depression (also decreased NE)

d) a vasodilator and increases vascular permeability

3. Complement system:

Anaphylatoxins – C3a, C5a. Function: stimulate mast cells to release histamine, leading to vasodilation and increased vessel permeablility. They also play  a role in shock, b/c when there is inflammation the compliment system is activated, therefore there will be mast cells and histamine, therefore C3a, and C5a will both be there.

4. Nitric oxide

Made mainly in endothelial cells, and is a potent vasodilator. It is used for treating pulmonary hypertension. It has a big time role in septic shock.

5. IL-1

Associated with a fever, it is a pyrogen, therefore stimulates the hypothalamus to make PG’s, which stimulate thermoregulatory system to produce fever. Aspirin works by inhibiting the synthesis of prostaglandins thereby reducing the fever.

6. Arachidonic acid metabolites:

a) Corticosteroids inhibits Phospholipase A2, therefore do not release arachidonic acid from phospholipids, therefore not making PG’s or leukotrienes. This is the supreme antiflammatory agent b/c BOTH PG’s and leukotrienes are blocked by blocking phospholipase A2. Arachidonic acids make linoleic acid (omega 3), which is found in fish oils and walnuts. It is very good for you b/c it acts like aspirin, and blocks platelet aggregation, and that’s how omega 3 protects your heart.

b) Lipoxygenase pathway: Zileutin blocks 5-lipoxgenase, other drugs act by blocking the receptors, example: zirkufulast, etc. Leukotriene (LT) C4, D4, E4 (the slow reactor substances of anaphylaxis) seen in bronchial asthma. They are potent bronchoconstrictors; therefore it can be seen why zileutin works well in asthma b/c it blocks the leukotrienes, including these (LT-C4, D4, and E4). LT B4 is an adhesion molecule in chemotaxis.

c) Cyclooxygenase pathway: Aspirin blocks cycoloxygenase, irreversibly in platelets. PGH2: where everything seems to be derived from. PGI2: derives from endothelial cells, it’s also called prostacyclin synthase; is a vasodilator and inhibits platelet aggregation (exact the opposite of TxA2). Thrombaxane A2 (the enemy of PGI2) is made in the platelet; it’s a vasoconstrictor, a bronchoconstrictor, and promotes platelet aggregation. What drug blocks thrombaxane synthase and is used to stress testing for CAD? Dipyrramidal blocks the enzyme, TxA2 synthase, therefore does not have to perform a treadmill stress test, all you have to do is use the drug dipyrramidal. PGE2: vasodilator in kidney; keeps the ductus arteriosus patent in the baby’s heart; makes the mucous barrier in GI (stomach) thereby preventing ulcers; can cause dysmenorrhea woman and increased uterine contractility, and it an abortifactant, to get rid of fetal material.

d) COX 2-make sure you know how this works!

e) Corticosteroids blocks phospholipase A2, and it also decreases adhesion molecule synthesis, along with other steroids like epinephrine and NE. Decreased adhesion molecule synthesis, will lead to increased  neutrophils on CBC. In an inflammatory state, 50% neutrophils are stuck to the endothelial vessels, and the other 50% are circulating, therefore, decreasing adhesion molecule synthesis will lead to doubled WBC (b/c the 50% of neutrophils that were stuck are now circulating). Corticosteroids destroy B-cells b/c they are lymphocytotoxic. Mechanism: decrease WBC’s (B and T cells) via apoptosis; therefore, corticosteroids are the signal to activate the caspasases. Eosinophils, mainly seen in type one HPY rxn, corticosteroids decrease them. When on corticosteroids, the only thing that is increased is neutrophils, via decreased adhesion molecule synthesis.

Lymphocytes and eosinophils are decreased. Example: If have Addisons, do not have cortisol, therefore the neutrophil ct decreases and the eosinophil count will increase.

Example: a person with MI with an 18,000 CBC most of which are neutrophils.

Mechanism: Epinephrine decreases adhesion molecule sythesis and neutrophil count goes up.

D. Electron microscopy of inflammatory cells:

1. In lung, type II pneomocyte (black dots are lysosomes). Lamellar bodies – structures where lecithin and phosphotidyl choline is located; if ask where macrophage, is, will ask which makes surfactant.

2. Monocyte: single nucleus with a grayish cytoplasm – has scavaged; can form foam cell in atherosclerotic plaque b/c it has phagocytized oxidized LDL’s (which is a free radical); Vit E neutralizes oxidized LDL.

3. Lymphocyte – all nucleus and scant cyptoplasm, prob a T cell (60% of peripheral blood lymphocytes are T cells); ratio of helper to suppressor: CD4:CD8 is (2:1), therefore, more likely to be a Helper T cell, then a suppressor T-cell, and B cells (20%) are least likely.

4. RER looks like a thumbprint, have ribo’s on it, and likes to make proteins, like Ig’s (therefore it is a plasma cell). Multiple myeloma – has eccentrically located nucleus, cytoplasm is always sky blue, making  plasma cells ez to recognize. Plasma cells are derived from B cells, and located in the germinal follicle.

5. Granules – eosinophil (have a red color similar to color of RBC’s) – have crystals in the granules. Eosinophils are the only inflammatory cell that has crystals in the granules. They are called Charcot-Leiden crystals when it’s seen in the sputum of asthmatic patient. They are degenerated eosinophils in sputum of asthmatic, and have formed crystals that look like spear heads. Basophils have granules that are more purplish and darker, while basophils have darker colors.

6. Mech for killing invasive helminthes–Type II HPY–major basic protein is involved. Remember that shistotosome eggs are coated by IgE Ab’s. Eosinophils have IgE receptors; therefore, eosinophils hook into the IgE receptor and release chemicals; the main one released is major basic protein, which destroys the helminth, which is type II HPY, b/c it is a cell hooking into an Ab on the target cell. The effector cell is Type II HPY rxn is the eosinophils; don’t get confused with Type I HPY rxn where the effector cell is the MAST CELL, and they release histamine (an eosinophil chemotactic factor), therefore they are invited to area of type I HPY b/c they have histaminase and arylsulfatase, which neutralizes leukotrienes. The purpose of eosinophils in type I HPY is to knock off chemical mediators produced in rxn; however, when an  eosinophil kills an invasive helminth, it does so via type II HPY.

E. Cluster designations:

  • Helper t cell = CD4
  • Cytotoxic T cell = CD8
  • Marker for Ag recognition site for all T cells is CD3
  • Marker for histiocytes (including langerhan’s cells) is CD1
  • Marker for MC leukemia in children = CD10 (calla Ag); positive B-cell lymphoma
  • CD15 and 30 = RS cell
  • CD21, Only on B cells – Epstein barr virus; hooks into CD21 on B cells, and actually the atypical lymphocytes are not B-cells but T-cells reacting to the infected B-cells.
  • Burkitts is a B cell lymphoma
  • CD45 is found on all leukocytes, is a common antigen on everything

F. Fever – IL-1 is responsible and PGE2 (this is what the hypothalamus is making) which stimulates the thermoregulatory center. Fever is good! It right shifts the O2 dissoc curve. Why do we want more O2 in the tissues with an infection? B/c of O2 dependent myeloperoxidase system. Therefore, with antipyretics it’s bad b/c thwarting the mechanism of getting O2 to neutrophils and monocytes to do what they do best.  Also, hot temps in the body are not good for reproduction of bacteria/viruses.

II. Types of inflammation (scenarios)

A. post partum woman, with pus coming out of lactiferous duct – this is staph aureus – supperlative inflammation

B. Bone of child with sepsis, on top of the bone, was a yellowish area, and it was an abscess – osteomyelitis – staph. aureus; if the kid had sickle cell, it is salmonella; why at metaphysis of bone? B/c most of blood supply goes here, therefore, mechanism of spread is hematogenous (therefore comes from another source, and then it gets to bone).

C. Hot, spread over face – cellulitis due to strep (play odds!) group A pyogenes (called erysipilis, another name for cellulitis)

D. Diphtheria = psuedomembrane (corynebacterium diphtheria), a gram + rod, that makes an exotoxin, messing up ribosylation of protiens via elongation factor 2, the toxin damages mucosa/submucosa, producing a pseudomembrane; when bacteria doesn’t invade, produces a toxin that damages the membrane; clostridium difficile also does this. It also produeces a pseudomembrane and a toxin, which we  measure in stool to make the dx. Therefore, the answer is C. difficle.

E. Fibrinouis pericarditis, usually with increased vessel permeability; seen in:

(1)lupus, leading to friction rub; also seen in

(2) the first week of MI, and then again 6 weeks later in Dresslers’s syndrome,

(3) seen in Coxsackie

F. MC organism producing infection in third degree burns = pseudomonas auriginosa. Color of pus: green due to pyocyanin.

G. Basal cell layer on both sides of clot, proliferate, and go underneath it to clot. In a primary wound it’s usually sealed off in 48 hrs (ie appendectomy). Key to wound healing is prescence of granulation tissue. Fibronectin is a very important proteoglycan and is involved in the healing of the wound. Fibronectin is an important adhesion agent and chemotactic agent, inviting fibroblast in helping healing process. The granulation tissue starts at day 3 and is on its prime by day 5. If you ever picked at a scar and it bleed like mad and you try to stop it but it still bleed like mad, that’s granulation tissue. No granulation tissue means no healing of a wound.

Type of collagen in initial stage of wound repair = type 3; type 4 collagen seen in BM; type 1 – very strong tensile strength; seen in bone, skin, tendons, ligaments. After a few months, after months, the collagen type 3 is broken down by collagenases, and a metallic enzyme converts type 3 into type 1. Zinc is part of the metallic enzyme, this is why in a pt with zinc deficiency has poor wound healing b/c it screws up the collagenase (must replace type 3 with type 1). Max tensile strength after 3 months = 80%. MCC poor wound healing = infection.

H. Ehlers Danlos – defect in collagen due to syn/breaking down; have poor wound healing.

I. Marfan’s – defect in fibrilin; also have poor wound healing

J. Pt with scurvy – defect in hydroxylation of two aa’s – proline and lysine via ascorbic acid. Remember it’s a triple helix; what makes the triple helix stick together and increase tensile strength? Crossbridges. When you crossbridge things, they anchor into areas where you have hydroxylated proline and lysine. Therefore have weak abnormal collagen in scurvy b/c there are no crossbridges to attach, leading to not being able to heal wounds, hemorrhaging, hemarthroses….collagen has weak tensile strength b/c cannot crossbridge..

K. Granulation tissue with a lot of blood vessels due to lot of fibroblast G, with inflammatory cells from plasma cells and lymphocytes, necessary for wound healing (rich vascular tissue, which is absolutely essential for normal wound healing).

L. Keloid (hypertrophic scar) = excess in type 3 collagen deposition; which causes a tumor looking lesions, esp in blacks. In a white kid – keloid to due to third degree burns. In another example: in a chronically draining sinus tract of the skin, they tried to put antibiotics on it (didn’t work), there was an ulceration lesion at the orifice of this chronically draining tract, and nothing worked. What is it? The answer is  squamous cell carcinoma due to a lot of turnover; type 3 converted to type 1, and fibroblasts are involved. A lot of cell division occurring, which can presdispose to mutations and cancer, esp squamous cell cancer. Squamous cancer is imp b/c chronically draining sinus tracts, and predisposes to sqamous cell carcinoma. Hyperplasia predisposes to squamous cell carcinoma.

III. Chronic inflammation

A. Difference in Immunoglobulins:

1. Acute Inflammation: IgM = main Ig first, and then IgG.

IgM = main Ig; need a lot of complement components in healing process; IgM is the most potent activator, and have activation of complement pathway (all the way for 1-9); IgM has 10 activating sites (pentamer). IgG can activate the classical system, but does NOT go passed C3 and stops and does not go onto C5-9. After 10 days, there is isotype switching, and the mu heavy chain is spliced out (mu chain defies specificity of an Ig); it splices in a gamma heavy chain, and IgG is made via isotype switching

2. Chronic inflammation: IgG (as main Ig – IgM is coverted to IgG immediately)

B. Difference in Cell Types:.

1. Acute inflammtions = neutrophil

2. Acute allergic reactions= eosinophils (mast cells are in tissues)

3. Viral infections = lymphocytes are the main inflammatory cells

4. Chronic inflammations = monocytes/macrophages are imp. And see a lot of plasma cells and lymphocytes; do not see pus-exudative (this is in acute inflamm – increased vessel permeability, and increased emigration of neutrophils into interstitial tissue, a protein rich fluid with >3 grams/dL, with a protein rich fluid = pus). Example: Cholecystis.

C. Type IV Hypersensitivity Reaction:

Another example: Granuloma = chronic inflammation (never acute); ie caseious necrosis in someone with TB; roundish, pink, multinucleated giant cells = granulomas; pathogenesis = type IV hypersentistivity reaction – delayed HPY. The main actors are cytoxic T cells; when they kill neoplastic, virally infected cells, these are also type IV HPY (no Ab’s involved). Poison ivy = type IV HPY. Back to TB infection, aleovlar macrophage phagocytoses it, and there is lymphohemotogenous spread; meanwhile the macrophage is processing the Ag. Then after weeks, it presents it to helper T cells. Therefore, the key players in Type IV hypersensitivity rxn are macrophages which process that Ag and presents that Ag via class II MHC sites to the helper T cells. These helper T-cells release cytokines: gamma IFN and macrophage inhibitory factor. Gamma IFN will activate the macrophage to kill the TB, Cryptococcus, histoplasmosis, etc. Therefore the gamma IFN is the trigger to active the macrophage; macrophage cannot kill without the activation from gamma IFN; b/c systmemic fungi and TB have lipid in the cell wall, this leads to caseous necrosis. All the pink staining cells are ‘epthiloid’, which are activated macrophages (which have been activated by gamma IFN); when they die, they die in style – they fuse together and form multinucleated giant cells (like their ‘gravestone’). Therefore, epitheloid cells are fused macrophages; black dots are helper T cells.

There are two types of helper T-cell:

a. Subset 1: involved in Type IV (delayed type) HPY; macrophages have IL-12; when it is secreted, the subset 1 helper T cells are presented with the antigen; then, subset 1 become MEMORY T cells. IL-12 is involved in activating the memory of subset 1 helper t cells. Most people in their primary dz usually recover with no problems, but the granulomas can calcify, as seen on x-ray. A calcified granuloma is not dead b/c they are resistant to dying. Therefore, most cases of secondary TB are due to reactivation TB. Granulomas necrosis is due to reactivation. “+” PPD (purified protein derivative) – injected into the skin; the macrophage of the skin is a langerhan’s cell (histiocyte) (marker: CD 1) – which have birbeck granuleslook like tennis rackets on EM. They phagocyotose the Ag (the PPD), and process it very quickly; they present it to helper subset 1, which has memory of previous exposure. Therefore, it hooks in the MHC class II Ag sites (as all immune cells do), and once the Ag (PPD processed by the langerhan’s cell) is presented, the  helper T cell releases the cytokines producing the inflammatory rxn with induration called the “+” PPD.

Correlation: older people usually don’t host a very good Type IV hypersensitity rxn: they have a less response to “+” PPD; therefore have to do a double test on them. In pt with AIDs, may not get any rxn. They don’t have enough helper T-cells therefore don’t have granuloma formation. Macrophage inhibitory factor keeps macrophages in that area; therefore, with HIV, b/c the helper t cell ct is decreased, you don’t form granulomas at all. Therefore, they will have Mcobacterium avium-intracellulariae all over the body without granulomas b/c helper T cells are decreased. When you do “+” PPD, 5 mm induration is enough to say it’s positive.

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