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Phagocytosis
Rafat Twalbeh Ziad Al-Nasser Wednesday, 20/7/2011

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 out from 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 tissues and 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 macrophages presented 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 this cells

can is

remember myeloid factors and of

this stem of in the these

figure from the last lecture the and the effect

growth development

differentiation phagocytic cells . here you can

see stem

the cell

hematopoietic

(CD34) that can give u the myeloid series or the lymphoid series. here we require growth factors for 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.

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 and endothelial 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 from

macrophages they recruit neutrophiles , and causes expression of adhesive molecules (the sellictins and the integrens and adherens). Chemokines: Cytokines promote chemotaxis, IL-8 and adhesive molecules, and those adhesive molecules are so important for those to adhere, pass through the endothilum, and diapediesis Anaphylatoxins of C like C3a and C5a are chemokines.

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 macrophages to do . the

1- Chemokine and cytokine receptors and those are

: they are in the surface of phagocytes IL-8 ,complement ,chemokine ,

so important for the

anaphlatoxin . these will call the macrophages or phagocyte cells to come into the area . 2- Pattern recognition molecule ( Toll like receptors ) : they recognize pattern of 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 .

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 , the dead cells have to be taken out and they are taken out by these macrophages 5- Immunoglobulin receptors : they are macroghages is IgG antibody . 6- C-lectin receptors : we have receptors for carbohydrate antigens we call irrespective so important for the phagocytosis , have receptor on

opsonization , the most common immunoglobulin that

them C-lectin receptors , they bind sugars on bacteria , so the capsulated bacteria that have polysaccharide can bind into that receptors 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 And we have enzymes that gone inside the phagocytic cells . NADPH oxidase system where

called

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 : defensins and 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 we supposed to kill them. but if we failed to do that then the microorganism has more protection mechanism : like MTB which has specialized activate more macrophages to kill MTB some they have a capsule for example , the function of capsule is for 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 the bacteriocidal molecules that result from respiratory burst . Figure 20.7 p155 : mycolic acid that resist

phagocytosis , they require more action ,like interferons to

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 burst as you can see . Start with oxygen ,NADPH ,amino acid , chloride ions and look here to the reaction: myeloperoxidase lead to production of hypochlorus super oxide anion . H2O2 then if you have catalase you will have water and O2 . Nitric oxide synthetase necessary for vasodilation and vessels tone . from the surface stimulate the respiratory

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

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 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 is successful 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 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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and when you

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

macrophages could be

useful.( all of these are trials .the only thing they can do is to give

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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(the only explanation from the Dr about the figure above is that Listeria can escape from the phagolysosome into the cytoplasm so they can live in our body for long period of time).

When U have a chronic granulomatous myeloperoxidase deficiency

disease where U have

or NADPH oxidase deficiency ,and u do all

of tests and every thing is normal ( the # of neutrophils and so on ) then the only thing U need to do is to check whether these phagocytic cells does have a functioning respiratory burst or not, and we have a test for that we call it " Nitro blue tetrazolium test " which is simply a dye that can stain normal phagocytic cells so they look black in color ,while the ones that have defected respiratory burst will not take the dye (the dye will not be oxidized by the enzymes of the respiratory burst and they will look yellow in color) so this is the positive test for "Nitro blue tetrazolium test" that means the respiratory burst is not functioning , and this is the test we do to diagnose the chronic granulomatous diseases (very simple test and highly informative).

What about the inflammatory signaling .neutrophils and macrophages produce inflammatory mediators called prostaglandins and leukotrienes. IL-1 that comes from macrophages can activate inflammatory cells to produce prostaglandins ,and these prostaglandins can reset the hypothalamus setup (our body temperature regulator).

Remember what I have talked about, the acute infection ,pyogenic response,the ,macrophages neutrophils ,delyed which are and involved ,,chronic ,,,, inflammation acute phase clearance granuloma,

response ,the early warning signals ,they came from macrophages ( IL1, IL-6 , TNF ) the TNF increases fat metabolism so we get slimmer if we have chronic inflammation ,for example patient with TB U will see
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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 the bone 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 ( Tcytotoxic 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 IFNgama, 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 the eosinophils 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 and they 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 U get 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 crosslinked 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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