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Wednesday, 20/7/2011

Ziad Al-Nasser

Rafat Twalbeh

Phagocytosis

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Ra'fat Tawalbeh

Wednesday 20/7/2011

Good morning

The doctor started says that we will have review sections every week on

showdini.com

Yesterday we were talking about phagocytosis and the cells that are

involved in the phagcytosis process, as u remember we talked about the

innate immune system physical barriers; mucus membranes and mucus and

the complement. And then we start talking about phagocytosis;phagocytosis is very important mechanism of the immune system where it

involves non specific immune cells that can be activated sometimes by a

pattern recognition molecules like the lipopolysacrides for example. And

we said that we are dealing with two important cells in the phagocytosis

process:

1>neutrophils: in the daily bases we have 109 cells that come outfrom the bone marrow going into assigned areas called upon what

we call chemotactic factors, so neutrophiles normally not present

in tissues but they should be called upon chemotactic factors, and

these come from many different areas: mast cells, T helper cells

and macrophages. they have short half life; about six hours and

when they perform the phagocytosis they will die and form what

we call pus cells.

2>Macrophages: they are phagocytic cells normally present in tissuesand they can be mobilized as will, but normally they are present in

tissues.they can be activated be cytokines, pattern recognition

molecules, toll-like receptors and chemokine receptor. When they

are activated they can produce acute phase response IL-1, IL-6,

IL-8, TNF all of these are needed in the activation and

production of the complement and raising body temperature as we

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said yesterday IL-8 activates the bone marrow here to start

calling neutrophiles to call upon into the area and so on.

Macrophages they perform the killing process less efficiently compared

to neutrophiles because neutrophiles are so many and macrophagespresented in tissues and have to be activated.

And we call these macrophages based on their location in our body: Glial

cells in the brain, osteoclasts in the bone, mesinchymal cells in the

kidney, kuppfer cells in the liver, monocytes in the circulation and

langerhan cells in the skin. They are used in antigen presentation,

phagocytoses and they do not expire after the pahgocytic process and

they play a major role in giving us a danger signal that we have been

attacked, the warning signal can activate the adaptive immune system to

operate .

you can remember this

figure from the last lecture

this is the myeloid stem

cells and the effect of

growth factors in

differentiation and the

development of these

phagocytic cells .

here you can see the

hematopoietic stem cell

(CD34) that can give u the

myeloid series or the lymphoid series. here we require growth factorsfor differentiation to take place. the eosinophiles need IL-5 to

differentiate, IL-4 used for mast cells and we will talk about mast cells

in the non specific immunity and their role in inflammation at the same

time.

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These CSFs we can use them as medicine; so if somebody have

neutopenia and the bone marrow is not producing these phagocytic cells,

we can stimulate the bone marrow to produce these cells by these colony

stimulating factors like "linogastrine" and "phelogastrine" those are

synthetic CSF and we use them to stimulate the bone marrow.

When macrophages get stimulated they are called epithelioid cells or

multinucleated giant cells or if they are present in the alveoli we call

them alveolar macrophages.

So the phagocyte production by the bone marrow precursors came from

the HSC and the cytotocic drugs shut down the bone marrow so the

factory will shut down, so before we do radiation, we take the bone

marrow before the radiation and keep it in the freezer, then we do the

radiation and re-transplant the bone marrow we call it bone marrow auto

transplantation we make it, it is very easy and most of that is very

successful.

Neutrophile migration is controlled by chemotaxis, and they have to be

called upon into the area.

For example here we can see the G-CSF increase the bone marrow

production of the neutrophiles and then if you have the complement

activation (C3a, C5a) they call those to come into the area. the

activated tissue macrophages they will produce TNF, IL-1 and those will

increase the expression of the selectins and the integrins as u can see

they can bind to the endothelial lining through the ligand receptor and

then they have to pass through the diapedesis into the area and then

they have to be activated as well by IFN-, and we talk about how

INF- can activate macrophages to kill.

A student asked the doctor about IFN- if they can stimulate the

tumor cells, and the doctor said: yes, but at the same time all the killer

cells get activated also (macrophages NK cells) these activated can kill

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tumor cells, it does not matter if they increase in number as well you

have something to kill.

Resident macrophags at the site of infection secrete cytokines and

chemokines which stimulate: neutrophil production, neutrophil andendothelial expression of selectins and intgrins, neutrophil adherence to

endothelium in local vessels, and finally chemotaxis to the site of

infection . these cytokines and chemokines have to bind with receptors

on the surfaces of the concerned phagocytic cells. Unlucky that those

receptors are also for HIV viruses and now we have two receptors for

HIV viruses: CD4 and the chemokines receptors, I will tell you more

about these receptors and how we call them like CXCR 4 and CCR 5.

Phagocyte recruitment:summarization

Macrophages they are Resting unless stimulated. Neutrophiles will die in six hours if they are not stimulated, so

Neutrophiles never present in normal tissues. And you should

remember that of the CSF, if a patient have Neutrophiles in the

CSF then he have a bacterial infection, and when you have

Neutrophiles then we have acute infection and pus forming cells,and when we have chronic inflammation then we have macrophages

and granuloma

Macrophages recruit neutrophiles so the IL-8 come frommacrophages they recruit neutrophiles , and causes expression of

adhesive molecules (the sellictins and the integrens and adherens).

Chemokines: Cytokines promote chemotaxis, IL-8 and adhesivemolecules, and those adhesive molecules are so important forthose to adhere, pass through the endothilum, and diapediesis

Anaphylatoxins of C like C3a and C5a are chemokines.

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Receptors on macrophages

Every thing in immunology are dealing with attachment , proteins and receptors

and this is how these molecule interact with each other to perform the specific

function that we want in defence.

Receptors in macrophages are so important to you to know them and how these

receptors they can manipulate and perform the function that we want the

macrophages to do .

1- Chemokine and cytokine receptors : they are in the surface of phagocytesand those are so important for the IL-8 ,complement ,chemokine ,

anaphlatoxin . these will call the macrophages or phagocyte cells to come into

the area .

2-Pattern recognition molecule ( Toll like receptors ) : they recognize patternof molecules like lipopolysaccharide on the surface of so many bacteria(gram

negative ) , when that happened the macrophages get stimulated and giving

the warning signal through the cytokines , we have I think 9 of these

receptors ( in the book : we have at least 10 receptors ) each one of them

can be target and the cell can be stimulated .

Figure 20.6 p155 :

These the toll-like receptors I was telling you about , we have toll-like

receptors 2,3,4,5,7, ,9 I dont know why we dont have 1 or 6 or 8 then the

DR read the table .

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nowadays we can activate TLR-9 by ummethylated cytisone and guanine

(CpG) synthetically and we can make those in the lab we can inject that in our

body and macrophages will be activated , enhance the acute phase response .

If I want to activate macrophages I can also inject antigens of

Mycobacterium tuberculosis (MTB) , and the antigen of MTB are well known

for the granuloma that they make in the lung and in the kidney and so on .

And I told you that we use the BCG vaccine (Bacillus Calmette-Gurin ) that

we use to vaccinated against MTB , we inject that in the urinary bladder for

the treatment of bladder cancer and what the BCG is going to do ?? it is

going to stimulate the macroghages , activated macrophages ,non specifically

they can go and attack tumor cells and clear that.

3-Complement receptors : in order to enhance the phagocytosis , opsonization ,they have C3b receptor .

4-Receptors for apoptotic cells: we cant keep cells for ever in our body , thedead cells have to be taken out and they are taken out by these macrophages

5-Immunoglobulin receptors : they are so important for the phagocytosis ,opsonization , the most common immunoglobulin that have receptor on

macroghages is IgG antibody .

6-C-lectin receptors : we have receptors for carbohydrate antigens we callthem C-lectin receptors , they bind sugars on bacteria , so the capsulated

bacteria that have polysaccharide can bind into that receptors irrespective

to that sugars if it is lectin (non specific) .

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.

Action of macrophages :

They kill through the respiratory burst and it is biochemical pathway

hexose monophosphate shunt that gone inside the phagocytic cells .

And we have enzymes called NADPH oxidase system where

myeloperoxidase is the most common enzyme that we see there .

Also we have nitric synthetase , it makes nitric oxide(NO) and and NO

is important for the integrity and the tone of blood vessels , where we

have NO we have vasodilation , and we know that body builders use NO

to increase the blood flow into the muscles and so on.

The activation of respiratory burst lead to production of bacteriocidal

molecule most common one is called hydrogen peroxide (H2O2) super

oxide anion , hypochlorite (HOCL) , nitric oxide (NO) and many other

free radical like hydroxy (OH) those have strong oxidizing ability that

they can destroy the bacteria easily .

Beside that we have proteolytic enzymes that present in granules, we

also have anti-proteases like 1_antitrypsin that neutralizes almost all

proteolytic enzymes that are produce from macrophages . if you dont

have 1_antitrypsin then the destruction is going to take place by

proteolytic enzymes , this is one of the mechanism of developing what

we call emphysema one of the chronic obstructive pulmonary disease

COPD.

Other substances are released into the phagosome including : defensinsand lactoferrins.

Defensins they can produce holes in the cytoplasmic membrane like those

of the complement system and destroy the pathogens at the same time .

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Lactoferrin : binds to iron , iron is needed for the growth of bacteria

so iron will be no more available , so bacteria can suffer and die

because of that.

Some pathogens are evasive means when we phagocytose organisms wesupposed to kill them. but if we failed to do that then the

microorganism has more protection mechanism :

like MTB which has specialized mycolic acid that resistphagocytosis , they require more action ,like interferons to

activate more macrophages to kill MTB

some they have a capsule for example , the function of capsule isfor antiphagocytic , so what the body has to do to encounter this?? to produce antibody against the capsule so antibody bind to the

capsule then macrophages come bind to the antibody through the

Fc receptors as opsonin and then help in the phagocytosis process.

Some bacteria can produce catalase enzyme that counter thebacteriocidal molecules that result from respiratory burst .

Figure 20.7 p155 :

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as you can see here the signaling process when these microorganism bind

through opsonin and how the respiratory burst is going to function inside

and get rid of pathogen . the signal trigger the granules to fuse with

lysosome then the oxidative burst has to be activated .

Figure 20.8 p156 :

this is the respiratory burst ,hexose monophosphate shunt , NADPH

oxidase system , signals from the surface stimulate the respiratory

burst as you can see .

Start with oxygen ,NADPH ,amino acid , chloride ions and lookhere to the reaction:

myeloperoxidase lead to production of hypochlorus super oxideanion .

H2O2 then if you have catalase you will have water and O2 . Nitric oxide synthetase necessary for vasodilation and vessels

tone .

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So if you have any thing that is missing here through this process then

you will be immunocompromised , we have a disease called chronic

granuloma disease , when you have defect of NADPH oxidase system

mainly myeloperoxidase deficiency , the number of phagocytic cells will

be normal the only problem that those phagocytic cells they cant kill

they are dysfunctional and this is a genetic problem we can use gene

therapy for treatment .

Figure 20.9 p156 :

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as you can see here the role of cytokines , cytokines can come from the

T helper when it become activated but you need the warning signal from

macrophages. Chemokines , TNF , and IL-1 stimulate adherence and

chemotaxis ,,,, macrophages mature into multinucleate cells and

epithelioid cells under the influence of interferon ,,,,,expression of

costimulatory molecule and cytokines ,,,,make macrophages good antigen

presenting cells.

Figure 20.10 p157 :

so you can see here how the innate and the adaptive immune system

they can work together through this cytokine network that comes from

the innate immune system and act on the adaptive immune system to

perform the function of killing .

One of the drugs that we use to suppress the immune system is the

anabolic steroids ( corticosteroids ,cortisol , hydrocortisone ) . so we

keep telling those body lifters who take anabolic steroids so their

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muscles become huge ( anabolic steroids increase protein synthesis

within cells especially in muscles ) that their immune system is going to

be suppressed , it also affect sexual functions ( anabolic steroids

suppress natural sex hormones ) so you can see these huge people but

most of them are sexually non-function . it also interferes with the

metabolism of fat we can see the moon face appearance , "buffalo neck"

,steroids also cause osteoporosis so they are dangerous drugs specially if

you take them for a long time .

How these anabolic steroids suppress the immune system ??? they

affect the chemotaxis , so if you inject somebody with corticosteroids

chemotactic factor will not be produce , there will be no longer acute

phase response , no more IL-1 ,IL-8,TNF,prostaglandin .

So every time we have to use steroids we should balance between the

benefits and the side effects .

In medicine they call these drugs as magic medicine , any disease they

dont know its etiology they give corticosteroids , they suspect it to be

autoimmune disease.

Figure 20.11 p 158 :

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we have talked about the vital signs and relationship with infection , if

you remember the pulse , temperature , blood pressure and when you

have so many neutrophils that involved in the process and production of

NO .

how lipopolysaccride can introduce the endotoxic shock and blood will

flow to the area and blood pressure will fall while the temperature will

sometimes be up and the pulse here will be upTemperature and pulse

they go hand on hand ..In severe cases when you have septic shock the

pressure will go down and even temperature will go down (hypothermia )

and this is very bad sign that we have to take care of .

If the patient has septic shock then he/she is dead until prove

otherwise .

to reverse this condition of septic shock (where the tone of blood

vessels is weak ,the blood pressure is down and so on) we can do many

things here . For example : blocking the NO production by macrophages,

endothelium and smooth muscle but it' s effective in animals not in

human (not that much we can do ,may be the only thing sometimes that

we give "catecholamines " to increase the blood pressure and the tone of

the blood vessels and so on ,but blocking the production of NO issuccessful in animals not in humans . Also we can block the TNF with

monoclonal antibody. We do that a lot in autoimmune diseases in which a

lot of TNF will be produced ,they need monoclonal antibodies or soluble

receptors to neutralize the excessive amounts of TNF .but this process

is ineffective because it's given too late (the damage has already

occurred and whatever U do it will not be effective).And finally

recombinant bacteriocidal/permeability increasing protein (BPI) binding to

endotoxin and preventing it from activating macrophages could beuseful.( all of these are trials .the only thing they can do is to give

the patient catecholamines .(box 20.1)

If I need to stimulate the macrophages ,the CPG (unmethylated cytosine

and guanine) is one example, it's a treatment of bladder cancer ,we

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can use it bind with TLR9 to activate the macrophages . Also we can use

another drug called "imiquimod" that will bind to the TLR7 which can be

also activated by viruses and so on . Macrophages produce the IL-12 in

response to TLR stimulation. IL-12 induces the differentiation of TH1

and TH2 .A nieve T cell will develop mainly into TH1 in this situation. So

anything stimulates the macrophages to produce the IL-12 will modify

the naieve T cell to develop into TH1 cells ,and TH1 cells will produce

IFN- that will activate the macrophages as well.

We talked about evasiveness of micro organisms,we said that

encapsulation resists phagocytosis, so our body has to produce Igs for

that .(U have to read this slide because the Dr skipped it) :-

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him losing weight coz of the TNF that is produced by the macrophages,

and of course fever all the time.

Remember the phagocytic defects , chronic granulomatous disease

,neutropnia ,drug cytotoxic therapy ,radiation therapy, shutdown of thebone marrow .

Molecular recognition by the innate and adaptive immune system they

integrate and help each other ,autoimmunity could develop with the

adaptive immune system while in the innate it doesn't take place . also

remember that the innate system alerts the adaptive system, and the

alert comes from the acute phase response (IL-1,IL-6,IL-8,TNF as

well at the same time).

Molecular recognition in the adaptive and innate systems:

(the Dr skipped it so read it from the slide )

By this CH20 has been finished ,so lets start CH21 with Dr Ziad

el naser

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So we will continue with the killing of the immune system, we talked

about phagocytic cells (neutrophils and macrophages) and how those could

integrate with the adaptive immune system by the cytokines network

that we have talked about to perform the function .we still have other

innate cells that perform killing . worms, parasites -in particular- we

get rid of them by mast cells ( that can get rid of these parasites by

vasoactive amines they have) and the basophils as well at the same time

;so we get rid of parasite mainly by mast cells.

What about viruses?!!they are attacked either by the adaptive ( T-

cytotoxic cells) or by the innate ( natural killer cells NK cells ) ,and U

will see what really determines what the body will usewhat are the

differences between the NK cells and the T cytotoxic cells . the NK

cells are non specific cells ,no memory is involved , they produce IFN-

gama, and they get activated by IFN-gama ,,, although the NK cells

and the lymphocytes originate from the same progenitor cell ,we really

dont know where those were developed to give out as NK cells,, by the

way they have common things such as CD2 which is found on the surface

of NK cells and on the surface of T-lymphcytes , so they must be

related , but they dont have TCR so we used to call them "large

granular lymphocytes" (they are not Lymphocytes) .

Why do herpes virus - infected cells favor NK cells ???because herpes

virus suppresses the expression of MHC molecules . the role of thumb

here : any activity requires MHC presentation _specially with class 1 _

it's going to favor the T-cytotoxic cells ,,any activity where MHC is not

involved it's going to favor the NK cells ,,,,why ???? we will see in just

a minute how the molecular mechanisms can favor this pathway or that

,,,and how the ADCC mechanism works ( ADCC: antibody dependent

cellular cytotoxicity) this is mechanism that NK cells use to kill virally

infected cells or tumor cells, but why tumor cells favor the NK cells?!!

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Coz many tumors suppress the production of MHC molecules the same as

the herpes-virus infected cells (evasiveness) so NK cells get activated.

So the outcome here of that whether NK cells or T cytotoxic cells is

apoptosis (programmed cell death).

So we R talking about Mast cells ,we will till U more about mast cells,

eosinophils (mast cells can recruit eosinophils into the area ),, and U will

see that all the vasoavtive amines that are present in the mast cells are

present in the eosinophils except the histamine,, so we will see

eosinophils also in the site of parasitic infection ,,,NK cells for the

virally infected cells and tumor cells where MHC is suppressed .

Look at this slide :

If U have a worm which is long and sometimes it could reach to one

meter and last long periods in our body , its multicellular ,,and as we

said that multicellular parasites that live in the gut or the respiratory

tract are killed by mast cells and eosinophils . mast cells can kill a

parasite indirectly through the vasoactive amines and recruiting theeosinophils into the area which can kill the parasite by the cationic

proteins and many proteolytic enzymes they produce, and activating

proteins that will lead to a massive production of mucus by "trypsin and

"chymotrypsin" dose will produce a lot of mucus. The products of mast

cells (prostaglandin ,histamines and so on ) cause smooth muscle

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contraction, vasodilatation ,lots of fluid into the area ,mucus production

so the parasite in the gut will be washed out .

So this is what really happens; if we have a parasite in the GIT or the

RST ,some of the proteins of the parasite will be released and work as

T-dependent antigen and stimulate the TH2 to produce IL-4 ,IL-5 .

IL-4 favors B cells to switch into IgE antibody production ,IL-5 will call

upon eosinophils to come into the area. So IgE will be produced andthey will bind to the surface of mast cells . mast cells are present in

the skin ,the sub mucosa or tissues in general and they are full of

granules ,these granules contain many inflammatory mediators, the most

important one is the histamine ,we have heparin, prostaglandin,

leukotrienes , platelet activating factor and we have some chemotactic

factors as well and U will see that many of those are pre-formed

(already there) while some could develop later, and this so important to

understand the hypersensitivity that could occur immediately when Uget exposed to the antigen and the responses that could occur may be

one hour or eight hours after , because those late mediators could

develop.

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What is really interesting here about parasites is that the antibodies

that have developed are going to sensitize the mast cells ,so mast cells

here on the surface they have what's called "Fc R epsilon 1 and 2 " ,so

they have receptors for IgE antibodies ,so U can see here the IgE

antibodies they bind by their Fc portion . So now we say that this mast

cell is sensitized, then if the IgE on the sensitized mast cells are cross-

linked degranulation of mast cells will take place and mediators that we

have talked about will get out and do their function.

The end

Done by : R2f@ Tawalbeh.

Big thank to my brothers : Ihab theep and Sa3d Kan3an for their help.

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