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Killing in the immune system

Ahmad Al-Kofahi

Ziad Al-Nasser

Thursday, 28/7/2011

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Chapter 21

The Doctor started the lecture by giving some Notes:

We will be doing review sessions over the internet on SHOWDINI twice aweek at around 9:00 pm when we get close to the final exam.

We will give you the schedule & which chapters we are going to review.

The second exam will be on August 13& what are said that the classes willend on August 14 are all RUMORS.

The Only difference in Ramadan is instead of having a 60-minute class it willbe 50 minutes.

Introduction

.We have other cells that operate with the help of the innate when they receive

the danger signal and they get involved in the battle against these micro organisms,

we talked about NK cells, and we talked about Eosinophils and how they play a role

as well as MAST cells, through the mediators that they produce;

Worms in particular, The cells that are involved with worms are MAST cells, they

have receptors for IgE antibodies ,and also the TH-2 are going to be activated; TH-2,

IL-4 and IL-5 and more IgE antibodies will be produced, more Eosinophiles IL-5

recruits Eosinophiles to come into the area , and Eosinophils they have almost the

same mediators as the MAST cells.

Viruses like Herpes virus-infected cells, in the specific immune response we need

T cytotoxic cells to kill those, and the T cytotoxic cells refuses to cooperate unless the

virus is carried through a class 1 MHC antigen then it will accept that.

Certain viruses are so evasive like Herpes viruses; when they enter the cell they

suppress the production of class 1 MHC antigen, so the antigen will not be presented

to the T cytotoxic cell, this is a very smart evasive mechanism. The role of NK cells

to get rid of Herpes virus-infected cells, and it encourages that cell to commit suicide.

The killing cell in the innate system mediated by MAST cells through the

receptors on their surface and the granules they produce, and those granules (vaso-

active amines , histamine bradykinins, prostaglandins ,leukotrienes, platelet activating

factors, chemotactic factors..) their main job is to get rid of parasites , they are in the

intestine ;they cause more smooth muscle contraction, they help in the peristalsis ,

they produce more mucus, they recruit Eosinophils as they come to the area and they

will kill those parasites, this is the acute reaction against parasites, if this process fails

it turns into chronic and we will see that in schistosomiasis; when we get infected

with Cercaria, acute infection will take place , but later when the worm lays down her

eggs and those get trapped in the liver and so on, what comes out of these eggs will

stimulate the chronic type of inflammation.

So MAST cells and Eosinophils are highly related and we will be talking about

those , those were designed originally to get rid of parasites REMEMBER THAT!

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Sometimes the MAST cells and Eosinophils in sequence are going to be activated

in response to environmental exogenous antigens that we should not be responding

against naturally, we call that hypersensitivity or allergy, this will stimulate the

MAST cells and recruit Eosinophiles to come into the area and all the granules they

have will inflect injury on us, simply we are going to have an inflammation where its

not needed like the angioneurotic edema that we have talked about .

The NK cells : those are innate cells , non-specific, they dont have T-cell

receptor, although they come from the lymphocytic progenitor line, they dont

develop in the thymus , they look like lymphocytes and they have some of their

antigens like CD2 which is present on all T-cells . but the NK cells produce IFN-

gamma and it can be affected by IFN-, and they have FC- receptor like the one

present on the macrophages , they are not phagocytic cells so you can see the

evolution pattern, they have FC receptors for immunoglobulins like macrophages , but

they are not phagocytic.. and they look like lymphocytes but they dont have the T-

cell receptor but they are not specific and they will not operate as lymphocytes , their

function is mainly mediated by other factors related to the FC receptors , they haveother receptors; the Killer immunoglobulin Like Receptor (KIR) and this will give an

inhibitory signal every time it binds to class 1 MHC as in immunology those cells are

specialized. So the NK cell will leave it to the T cytotoxic cell to kill it, if it fails; the

NK cell can sense that through the KIR then they can operate ,this is so interesting!

We call that the CD 94 receptor or the NKG2 receptor.

The mechanism of NK cell killing , if it was through the ADCC mechanism the

outcome is the production of what we call cytolysin or perforin or granzyme ;

perforins function exactly like the C9 of the complement; it interferes with the

function of the cytoplasimic membrane, then an enzyme will be produced called

granzyme, it activates the Caspases which are involved with the apoptosis ,it activatesDNases which destroy the DNA and the cell will commit suicide and die.

You can see that NK cells and T cytotoxic cells perform the same function.

The same thing with Intracellular bacteria, viruses or parasites are killed by NK

cells, T cytotoxic cells and phagocytes.

And of course T-helper cell will

provide help.

What if we have large parasites orworms like the multi-cellular parasite in

the GIT and Resp tract ? they are killed by

MAST cells and Eosinophiles; the MAST

cell enhances the secretions & motility,

and the Eosinophils try to kill the parasite

,the MAST cells operates in clearing the

parasite from the tract.

Some of the antigen of the parasite

will be secreted and then will be absorbed,

and those are going to be taken bymacrophages and they provide that to T-

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helper cells , and the T-helper cells will provide cytokines that will activate the B-

cells, and here the cytokines are mainly of IL- 4 & IL-5.

T-helper 2 are going to shut down T-helper 1,and the T-helper 2 will give the

isotype switching into the IgE antibodies which are going to bind to the MAST cells,

so the bound MAST cells for the first time we call them sensitized and when theantigen is produced it will bind by cross-linking to those IgE antibodies making the

MAST cells degranulate which causes smooth muscle contraction, massive secretions

of cells that will cause the parasite to be excreted easily, and of course IL-5 will

recruit Eosinophiles to come to the area and those are also going to participate in the

killing process.

So the response to parasites is no longer a problem in the developed world, they

are mainly related to hygiene. Many of those parasites can be transmitted byarthropods like malaria and so on, and those are very common in the 3

rdworld

country, they are variable in sizes; they could be so small like bacteria, and they can

be up to 10 meters in length! Imagine a worm of 10 meters can inhabit your intestine!!

So they vary here in sizes but the immune response is almost the same here, many of

those parasites are so evasive they can stay in our bodies for a long long period of

time.

In parasitic infections we are talking about MAST cells and Eosinophils , and the

first thing when you do CBC and differential , when you see Eosinophilia you have

to think of a parasitic type of an infection or allergies and its very very important to

do the differential , its very important to remember

These are the mast cells, they are large cells filled with granules and these

granules they have the vasoactive amines. The granules as we said are responsible for

all signs and symptoms of inflammation, and they can be present in the skin and in the

mucus membranes as well. There is a bit difference between the ones that are found in

the skin and the ones that are found in the mucus membrane but the outcome of

vasoactive amines or the mediators is the same. On the surface of the mast cells there

is an Fc receptor (called Fc receptor 1) for epsilon () heavy chain that is presented in

IgE antibodies which is the responsible for the activation of mast cells. The Fc1

receptor will bind with IgE with any specificity so if we have many types of parasite

that will secrete different types of specific IgE antibodies all of these different types

will activate the mast cells by cross linking with the receptor on the surface because

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the IgE will bind through the Fc portion (the constant heavy chain portion) which is

the same in all of the IgE antibodies. The cross linking between the IgE and their

receptors on the surface of the mast cells will generate signals that will cause the

degranulation to occur.

Again and again. Not just IgE antibodies activate mast cells; this is a rule ofthumb. Mast cells if they are cross-linked with an antigen (we call it allergen)

this will cause degranulation, and we will see some rapid mediator that can be

produced and late mediators that will be sensitized later through what we call the

arachidonic acid metabolism. Certain drugs they can stimulate the degranulation of

mast cells, one of the most important examples of drugs causing hypersensitivity-like

conditions through stimulating the mast cells directly without production of antibodies

is morphine. Morphine has receptors on the surface of mast cells and some patients

will react to it through activating the mast cells. Some patients after physical exercise

mast cells could be activated and will develop what we call urticaria, and some may

develop asthma following severe exercise. So we have to remember the granules and

the degranulation process that is responsible for all inflammatory processes.

Mast cells

Mast cells can be present in the tissue, the mucus membranes, and on the skin.they are large and have a lot of granules and enzymes, and those granules and

enzymes mediate inflammation.

On their surface, they have receptors for the binding of the IgE antibodies; we callthem the Fc R1. These receptors are occupied by the Fc portion of IgE, then the

mast cells are sensitized. Any IgE antibody with any specificity can bind to those

receptors, so the most important point here in the activation of the mast cells is

that those receptors have to be cross linked in order for the signal to pass through,

and then degranulation takes place. I.e. the allergen (the antigen that mediates the

IgE secretion) cross links the IgE antibodies and then degranulation takes place.

Mast cells can be stimulated by other factors like: Drugs (such as morphine),opiates, physical exercise in certain people, changing of temperature and some

other factors that could stimulate the mast cells & may cause what we callurticaria.

Mast cells have receptors for C3a and C5a (anaphylatoxins), those can stimulatethe mast cells to degranulate and to produce vasoactive amines (substances

containing amino groups, such as histamine or serotonin).

Mast cells have enzymes called Tryptase and Chymotrypsin, they are responsiblefor the production of the mucus, and late they become mediators for the activation

of the arachidonic acid pathway.

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Arachidonic acid pathway branches:1. The Cyclo-oxygenase pathway that gives prostaglandins and thromboxanes

2. The lipooxygenase pathway that gives leukotrienes and platelet-activating

factor.

The cyclo-oxygenase pathway gives you prostaglandins and thromboxanes, bothof which inhibit TH1 response. They also cause vasodilatation, increase in

vascular permeability, and bronchial smooth muscle contraction.

Those mediators -especially thromboxanes- are inhibited by the action of Aspirin(salicylic acid) which can interfere with their processes, and thats why the

salicylic acid is considered as an anti-inflammatory drug.

Most of the pain killers; the non steroidal ones, work at many different levelsinhibiting the thromboxanes, and other mediators.

The lipooxygenase pathway gives you Leukotrienes which are usually one of thelate mediators, and all of them have the same functions: smooth muscle

contraction, vasodilatation, increase in vascular permeability, and edema.

The lipooxygenase pathway also gives platelet-activating factor; which functionsas a chemotactic factor, activates eosinophils and neutrophils, and induces mucus

secretion and smooth muscle contraction.

All those mediators will cause expulsion of these parasites outside, and alsostimulate the production of eosinophils by inducing chemotactic factors that call

eosinophils to come into the infected area. For example Eotaxin (a chemotactic

factor from epithelial cells) and leukotrienes will attract the eosinophils to come

into the area to inflict more injury to the parasite and to get rid of them from our

body. By the way, eosinophils have the same mediators as the mast cells except

for histamine.

Prostaglandins and histamine cause smooth muscle contraction, edema (the fluidcomes out from the endothelial cells into the area), itching, hyperemia, and

flushing of fluids into the area. this is all because of vasoactive amines, and these

are the signs and symptoms of inflammation.

Mast cells has unknown precursor cells, and IL-3 and IL-4 are needed for theirproduction. IL-3 is a major interleukin that stimulates the bone marrow to produce

more precursor cells, and then homes mature cells to the Submucosa, the skin, and

connective tissues.

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Mast cells have granules that in order to be degranulated they require cross likingof IgE receptors and the FcRI.

Mast cells will bind many IgE antibodies with different Ag specificities, becausethe Fc portion is only needed, not the hypervariable region of the

immunoglobulin.

After degranulation, preformed mediators that are already there, and latemediators that require the activation of the arachidonic acid metabolism pathway,

will be secreted, and the outcome of that will lead to the signs and the symptoms

of inflammation.

the contents of the granules:1- Enzymes: such as Tryptase, Chymotrypsin, and those are responsible for

mucous secretion, smooth muscle contraction, complement activation and

kinin production.

2- Histamine: and the main effect of histamine as you know is smooth musclecontraction and increasing vascular permeability. so the area will be

edematous and this what happens when we have allergy and hay fever;

stiffness of the nose, sneezing, and all of that. The only thing we can do here is

to give anti-histamine, in many time the Anti-histamine could help but not

cure, because there are other mediators that can be secreted such as:

prostaglandins, leukotrienes.

Histamine is the most important one of them all, but its not enough. somechemotactic signals bring eosinophils to come into the area, and the itching will

give you a sign that you have smooth muscle contraction, inflammation, and mostimportantly a parasitic type of injury. The itching by the way is mainly mediated

by smooth muscle contraction of the skin.

In mast cells we have cytokines like TNF. In addition to its known functions; ithelps in adhesion, homing, endothelium activation and diapedesis, and it helps

also in the inflammation.

Most of the cytokines are produced in an immune response only if they areneeded. when they are not needed then the cell will stop producing them, so they

are not produced all the time

Activation of TH2 cells by IL-4 will induce more IL-4 production that will causeisotype switching to the IgE antibodies. It will also block the action of TH1 cells

(that produce IL-6 and IL-10) because we dont need TH1 cells here (parasitic

infection). So the action of TH1 will be blocked by IL-4 and IL5, and vice versa,

and that represents the balance between TH1 and TH2 cells.

In addition to IL-4 and IL-5; IL-3 is produced and stimulates the bone marrow toproduce more of these cytokines to bring more eosinophils into the area.

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So as mentioned above, there are preformed mediators released immediately (oneof them is the histamine), and late mediators requiring the activation of the

arachidonic acid metabolism pathways.

The preformed mediators such as proteolytic enzymes (Tryptase and

Chymotrypsin) induce mucus membrane secretions, or they help to expel theparasite. Histamine also causes smooth muscle contraction, vasodilatation, and so

on.

Cytokines like TNF, IL-4, IL-3, and IL-5, their functions as you know areactivating endothelium to enhance diapedesis, TH2 cell activation and stimulating

eosinophils production and activation. You should remember all of these things.

Response according to location:

- Mucosal Mast cells: mainly respond by Leukotrienes and eosinophils which of

course will be produced, activating TH2 cells but not TH1 cells.

- Connective Tissue mast cells: they have more Histamine. And this is the

difference between connective tissue mast cells and mucosal mast cells, the 1st

one has a rapid effect on the connective tissue while the mucosal is mainly

delayed, but after all, the effect will be the same.

Eosinophils

Eosinophils are called by the Eotaxin and other chemotactic factors.

They have all the vasoactive amine mediators found in the mast cells excepthistamine, and granules of eosinophils contain toxic substances.

The Precursor cells under the effect of IL-3 and IL-5 will produce eosinophils, andthe chemotactic factors such as Eotaxin (from the epithelial cells and leukotrienes)

will attract the eosinophils to the sites that need them.

Cross linking of IgE to FcRI activates the eosinophils, and then they inflict injuryto the parasite.

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In hypersensitivity or allergy, hypereosinophilia can be noted, so eosinophilia isjust an indicator that mast cells are activated, not necessarily a parasitic infection,

so hypereosinophilia should be treated by finding the causative agent.

They have peroxidases and they produce hypochlorus ions and the hypocloride as

you know -like the ones produced in the phagocytic cells- they cause destructionto the microorganisms and parasites.

Other mediators are also found, such as the major basic protein, which also causedamage to the parasites, also theres the cationic protein, and all of these have a

very strong oxidative effect that cause damage to the parasites, and the cationic

protein also acts as neurotoxin which damages the CNS of the parasites.

These toxins and proteins are seen in the immediate or type-1 hypersensitivity,where there is stimulation of mast cells. Remember that stimulation of mast cells

always lead to eosinophilia.

Charcot-leyden crystals : Which are simply Killed eosinophils. They areindicative of a disease involving eosinophilic inflammation or proliferation, such

as is found in allergic reactions and parasitic infections. They are often seen

pathologically in patients with bronchial asthma.

From robbins : charcot-leyden crystals collections of crystalloids made up of

eosinophils proteins .

Treatment methods for hypersensitivity:1- Receptors for Leukotrienes or histamines can be blocked in a tissue (such as

bronchial smooth muscles) by anti-histamines. Sometimes anti-histamines are

not enough, so more blockage of other mediators is needed and that is

difficult.

2- By stabilizing mast cells which will prevent mast cells from producing itsmediators, which can be done by corticosteroids, and thats why we use them

in emergency.

3- Catecholamines, they stimulate and receptors.

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drugs that are given in emergencies are given in large doses, so if anybody comesto the clinic that has an anaphylactic shock caused by a sting of a bee or a wasp, or

major allergies against anything, the drugs of choice that are given are:

Catecholamine, adrenalin, high dose hydrocortisone to stabilize the mast cells, and

corticosteroids to inhibit the synthesis of the mast cell mediators. Cromoglycate

can also be given which may stabilize mast cells, and the cromoglycate is a drugthat sometimes you can use for prophylaxis (like in bronchial asthma and hay

fever), and this will block the receptors on the surface of the mast cells, so it

stabilizes the mast cells. but the action of corticosteroid is so effective in

suppressing the immune system.

A patch test is a method used to determine if a specific substance causes allergicinflammationof the skin. It is intended to produce a local allergic reaction on a

small area of your back where the diluted chemicals were planted.

Natural Killer cells

NK cells perform killing. they look like lymphocytes, itlooks like the progenitor cells. they are CD2+ like

lymphocytes but it doesnt have the TCR. they have

lots of granules and hence they are called large granular

lymphocytes, and also they have receptors for the Fc

portion of Gamma 3 IgG in particular.

the NK cell has a receptor called KIR (killer

immunoglobulin like receptors), and another one calledNKG2/CD49, and they can perform the killing, those

are present on other lymphocytes and NK cells

constituting 5-15 % of the total number of the lymphocytes that are present

circulating in the secondary lymphoid organs.

NK cells work against virally infected cells, as well as tumor cells especially theones that lack the presentation of class 1 MHC-antigen complex.

The function of the NK cells is exactly the same as T-cytotoxic cells (CTL), butthe surrounding conditions are different.

CTLs are presented by class 1 MHC antigenthrough the TCR, while the NK cell doesnt

have this property, in fact they can sense the

presence of the MHC antigens through KIR

receptors and if the MHC antigens are there

they leave the function to T-cytotoxic cells,

but the mechanism of killing when they are

activated is the same.

Both types of cells are activated byinterferon- that comes from macrophages

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and T H1 cells, so the cytokines that are produced; their function is to kill virally

infected cells.

if you have a virus like influenza for example presented with class 1 MHC antigento the TCR on the CTL, so here the CTL is going to be activated and it will kill

the virally infected cells with influenza, but if the cell is infected with the HSV(Herpes simplex virus) , and HSV has the ability to suppress the production of

class 1 MHC-antigen because its so evasive, so it doesnt have presentation to

CTLs, so there will be no killing by CTLs, so HSV infected cells now can be

killed by the NK cells, and the NK cell have receptors ( KIR receptors). The

function of the KIR is to sense the presence of class 1 MHC, if theres lack of

MHCs then the NK cell will be activated.

Killing the virally infected cells stimulates the adaptive immune response by theproduction of cytokines like interferon- like. NKs and CTLs are not phagocytic

cells, they have similarity with macrophages that they have receptors for the Fc

Gamma 3 -this is the only similarity between them-.

NK cells function when we have evasive viruses like: HSV, or in tumors forexample sometime mutation could take place and will mutate the cell not to

produce MHC antigens. we have so many viruses like: HSVs which are so many

like Type 1 and 2, CMV (cytomegalovirus), and EBV (Epstein-Barr virus), so all

of those can be killed by the NK cells.

NK cells are seen in the bone marrow but not in the thymus, although NK cellshave some similarity with T-cells like having CD2. So they have a connection, the

same killing mechanism as the T-cytotoxic cells, the same precursor cell as the Tcell.

NK cells signaling occurs by receptor binding, we mean by that the Fc portion,and the KIR receptors that sense the presence and the absence of the MHC

antigens.

So the cytokines -early in viral infections- that stimulate the NK cells are: INF-,IL-12, and these cytokines are produced from macrophages, and when the

adaptive system is responding, T-cells produce: TNF, IL-12 and IL-2, and those

cytokines are also responsible for the activation of NK cells.

The main mechanism of killing by the NK cells is the ADCC (antibody-dependent cellular cytotoxicity): the NK cells cannot kill unless the antigen is

bound to an anti body, and this antibody is bound on the NK cells Fc receptor; for

example if you have a virally infected cell or a tumor cell and theres an antibody

bound on that cell surface having a free Fc portion, then the NK cell will come

and bind that antibody on its surface using the Fc receptor, then the NK cell will

be activated.

Activated NK cells produce:1- Perforin: a protein that acts like the complement system causing pores in the

cytoplasmic membrane, causing water and electrolytes imbalance which

damages the cell.

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2- Granzyme: which activates the caspase pathway from inactivated caspases toactivated ones, then DNases will be activated which cause DNA destruction,

then the cell will commit suicide (apoptosis).

3- FasL: which is not usually expressed unless the Killer cell is activated.

T-Cytotoxic cells have the same killing mechanism as the NK cells but theactivation of CTLs is different, CTLs recognize the antigen with a MHC antigen

presented on an infected cell, and then perforin and granzyme are produced.

If The NK cell senses that there are MHC antigens on the cell surface through itsKIR, then this will give a negative signal and the NK cell wont be activated. but

if there were no MHC antigens, the KIR wont produce the negative signal and a

positive signal is produced, and activation occurs.

So if the infected cell expresses MHC antigen it will be killed by the CTL, whileif it doesnt; the killing is processed by the NK cell.

In the uterus there are plenty NK cells that provide protection against viruses andvirally infected cells for both the uterus and the fetus.

In both NK cells and CTL the Fas-fasL and the TNF-TNF receptor interactionshave the same mechanism of killing by inducing death domains which also induce

transcription factor that end up in forcing the cell to commit suicide.

Apoptosis is reversely proportional to the amount of gene products that inhibitapoptosis such as bcl-2 gene products. So if you have a tumor activating bcl-2

genes then those cells wont die and will proliferate forever.

IL-2 can activate bcl-2 genes, so IL-2 is going to facilitate cell growth, and thatplays a major role in memory cells which are sustained for a long period of time

under the effect of IL2.

The KIR recognizes MHC class I, while KMG2/CD94 binds to non-classicalHLA-E.

When Natural killers bind to Non-classical MHC, they can't bind to classical ones.

Lymphokines activated killer cells: NK cells that have been taken from a patientthen exposed to INF- in vitro which activates them, and then the cells are

returned back to the patient to produce better killing.

CTLs and NK cells produce INF- which activates them and -at the same time-activates TH1 cells which produce more INF-, so more activation will occur.

The immune system is shaped by apoptosis

The doctor started reading the following table without any extra information!!

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fratricide principle: activated cells sometimes produce FasL so they can bindto Fas, so those immune cells having the FasL can interact with each other so

they can convince each other to commit suicide, and this principle is important

in the immune response regulation, so when theres excess activated T cells

like in autoimmune disease, these cells will express FasL, and afterwards they

will commit suicide.

Apoptosis in the immune system is important at many different sites: T-cells, autoreactive T-cells, responding T-cells, B-cells, any cell, malignant cell as stated in

the table above.

Special thanks to my friends: