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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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