Transcribed by Mandy Weil July 2, 2014

General Pathology - The Innate Immune Response by Dr. McCutcheon

[1] [The Inate Immune response]
[McCutcheon] Welcome to General Pathology. This is the immunology portion of
General Pathology. For those of you who dont know me, Im Dr. McCutcheon and
Im going to be taking you through the journey of the rest of the summer in this
class. I want to start by talking a little about this topic. You are all here, so that
means that you were very successful last year. Most of last year was a lot of
memorization and memorization got you where you needed to go. Unfortunately,
Dentistry is an essay profession. Okay? Your patient is going to walk in your room
and theyre going to sit in your chair and its up to you to figure out what youre
going to figure out in order to treat them. Okay? They dont come with a bib that
tells you what to look for. Its up to you to figure this out. Its up to you to look at
whatever complaint they have an diagnose the symptoms and that is not
something that goes along with memorization. So starting this year, youre going
to need to learn to do something different. Youre going to need to learn to
integrate information. You should have done it last year in Organ Systems, but
most of you probably didnt and you got away with it. Youre not gonna get away
with it if you continue to do that. Youre not going to learn to diagnose. So,
immunology is the first topic where theres memorization and theres a lot of it
and its not enough. Okay? You need to go beyond memorization and you need to
put pictures of the puzzle together. And the way I teach this course is I tell a story
and you know that you can understand the story when you can explain the story
to someone else. So its a good way to study this in groups cause you can talk
yourself into anything no matter how illogical, but your friends will say Oh no,
that doesnt make sense. Im helping you out posted along with every lecture is a
study guide. Some of the questions youre going to be able to look at the slides
and know the answers. Some of the study guide questions, youre going to have to
figure out the story to answer. When we do our conferences, our conferences will
be splitting into groups and then you the students will answer all of the questions
in the study guide. I do not give you the answers. I have given you all the
information that you need in lecture. So its up to you to put the answers together
and answer the questions in the study guide. Do this is in groups. Its really good
to study this stuff in groups. Okay? You learn a lot better from studying with your
friends. So, one of the things that Im after are concepts and to help me do this, Dr.
Phelan has actually given me an extra hour, so that when were working with a
difficult conceptand this lecture series is actually going to be one of the, if not
the most difficult things you learn in dental schoolIf you learn immunology, all
of pathology drops into place. Rather than trying to memorize a set of symptoms
that dont make any sense, youll know immunology and you can say Oh! thats
how this works! And then you understand the disease rather than try and
memorize the disease. And youre going to talk about B and T cells, youre going
to talk about cytokines in every single class that you have and youre going to
deal with these drugs, youre going to deal with these cytokines in every single
solitary patient that you treat. So its really worth doing the work now. And part
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Transcribed by Mandy Weil July 2, 2014

of the reason, we move immunology in the summer is because youre somewhat
less busy and youve had a whole month to rest so youre not as tired. Plus that
way, the test is just about immunology, cause traditionally, they tend to not study
the other subjects on the test. So, we did this to help you guys. So, I have extra
time, that frequently means that Im not going to end a lecture at the last slide. Ill
end when we run out of time. Ill pick that topic up at the next lecture. Ill finish
that topic. Ill start the next topic after that. So you know, be prepared, were
going to take the time that we need to go through the concepts. There are a lot of
details your book hasalthough we picked a book thats deliberately not detail
heavy. Parum. There still are details in there. When I want you to know a detail, I
will tell you specifically you have to know that. Okay? If there are details on the
slides and Im not talking about them, you need to know the CONCEPT, you dont
need to know the detail. And you are used to knowing details and a lot of people
try to do that. Its certainly isnt going to hurt you. But its not going to get you
where you need to go. Spend the time learning the concept. Putting this piece and
that piece and that piece together. A lot of you have never really practiced doing
that, so As we going through the study guidesyoull see that there are more
how? and why? questions as we get further on into the lecture series. How and
why are not things you can memorize. How and why are things you have to figure
out. Alright? One of the other things thats really important to do and if you talk
to the upperclassmen who did well in this classthey will tell you: stay caught
up. This is a building lecture series so everything we talk about now, youre
expected to understand that for the next lecture. So if you think youre going to
study for this two days before the test, Ill tell you now, youre going to flunk.
Alright? You cant do it. So stay caught up! and weve got this spread out so you
have time to do that. Alright so were going to pick up where we left off in May.
We learned about the lymphoid organs and now were going to talk about the
immune system itself. The order that Im telling this story is the order that a
lymph, an infection, a reaction to an infection occurs. So were going to start at the
beginning and were going to go through to the end. Now there are a couple times
where I have to drop out of that sequence and explain something before we can
move forward. But theyre very complicated B and T cell development and I didnt
want to start there because what a great way to lose your entire audience, to start
off with the most difficult thing you can talk about. So were gonna do that in
orderOne of the annoying things about this is that there are always things that
have happened that we havent talked about. Ive got those down to the minimum
number of places, but I cant get rid of all of them and I apologize in advance. Its
the nature of the beast. By the wayIts lovely to see you all here at 8 oclock in
the morning.


[2] [Immunology is the study]
[McCutcheon] So, Immunology is the study of the physiologic mechanisms that
humans and other animals use to defend their bodies from invasion by other
organisms. And they keyword in thator keywordsare physiologic and defend.
One of this courses pathology. And pathology as a rule is what happens when
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Transcribed by Mandy Weil July 2, 2014

physiologic immunity doesnt work. Ok? And then you go from an acute
inflammatory process to a chronic inflammatory process and all of the things that
are going wrong in a necrotic inflammatory process. Im going talk about normal.
Im going to talk about a physiologic immune response. And everybody who
follows be is going to talk about the things that happen when an immune response
goes wrong. and to understand this and to defendthis is your defense
mechanism. The Army, the Navy, the Marines and the Air Force put together And,
in order to know when were defending against, we need to define something. And
were going to define an antigen. An antigen is a part on a pathogenbacteria,
virus, fungi, parasite, some debris or ragweed pollenan antigen is the part of the
pathogen thats recognized by the specific receptors of a B cell, the antibodies, or
a T Cell. So the antigen--and Ill use the terms antigen and pathogen somewhat
interchangeably--but in fact, antigens are the spot on the pathogen that the
immune system recognizes. Antigens are the spot on the pathogen that the
immune system recognizes. Because we dont want our immune system to react
against us, so the antigen is something that is not found on a host cell. Cause
otherwise, youd have immunity against you and those are called autoimmune
disease and theyre all bad. So the antigen is a spot on the pathogen that is
different from anything on host and because its different, it can be recognized by
the immune system.

[3] [Unique Features]
[McCutcheon] We talked about this in May. There are several unique features of
the immune system that dont happen for other cells. Lymphocytes move. Their
job is to be in the circulation, to move around in the circulation, to crawl out of the
circulation and into the tissues. They move. Lymphocyte cells change their
function over time. Theyre secreted as inactive cells. They become activated, they
do their thing. Some of them stop being activated. Others have very short half
lives. Only 2-3 days and they die. Okay? Some of them have long half lives. They do
their thing, they can stop doing their thing. Theyre release as inactive cells for
both kinds of immunity, innate and adaptive. They have to be turned on and well
spend a fair bit of time talking about that this summer. And their functions are
specific and in May, I told you I would explain that and were gonna start
explaining that today and were gonna pick that up Friday the 11th.

[4] [Picture (Intrinsic epithelial barriers to infection]
[McCutcheon] Now before an immune response occurs, You have to get the
pathogen inside the body. Okay? So there are barriers that, while not officially
apart of the immune response, keep the immune system from needing to be active.
And the first barrier is fairly obviousits the physical barrier of your skin and
the endothelial cells line all of the cavities of the body that come into contact with
the outer world. Cause as long as youve got the pathogens out here, you dont
need an immune response. So before you can get an immune response going, you
have to break the physical barrier. There are some other barriers that help out. So
the next one is chemical. Your skin, saliva, tears, they secrete chemicals that help
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Transcribed by Mandy Weil July 2, 2014

degrade viruses and keep bacteria from growing. So low pH enzymes like pepsin,
there are small molecules called defensins and all of those help keep skin, bacteria
and any viruses from being able to attach and replicate. So theres a chemical
barrier. And then, surprisingly, theres a microbiological barrier. So any of you
whove always thought all pathogens, all bacteria are bad, youre wrong. and you
can tell that now because they have all these commercials for probiotic this and
probiotic that. You know what theyre giving you bacteria. Some bacteria keep you
healthy. One of the things to think about is, all these antibacterial soaps and
antibacterial this-es and antibacterial thats wiping off all of the bacteria on your
skin is not necessarily a good thing. You have bacteria on your skin that have
colonized and they filled the niches and they get all the resources in. If you wipe
them out, you can get something else that can colonize and now it can get to food
and resources and it can grow and may be bad for you. So part of our defense is
normal flora. For anybody who has taken a broad spectrum antibiotic like
tetracycline and then puked for the next week, you broke the microbiological
barrier and you allowed something else, you wiped out the normal flora in your
gut and you allowed something else to grow there that caused you to throw up or
gave diarrhea. So the three barriers are mechanical, chemical and microbiological.
And you have to break at least one of those before youre going to need an
immune response. Right? So youre not making an immune response against your
normal flora because you dont need to.

[5] [Picture (Adherence to epithelium)]
[McCutcheon] So this is one way to break the barrier and this is breaking the
physical barrier and this is the perfect slide for you as dentists. Thats your
fingerthe epithelium theyre showing you thereand youve just had an
explorer in somebodies mouth and your scraped a bunch of plaqueand whats in
plaque? Bacteria. And now youve stabbed that plaque-laden explorer into your
finger. You will do this at least once in your career. Ok? And since its somebody
elses bacteria, you may or may not have an immune response already against
them. If you dont, you now have to start an immune response. So the first thing
that happens in you break the physical barrier. You have to start an immune
response

[6] [Picture (The colon is colonized]
[McCutcheon] If you take antibiotics and you wipe out the normal flora in the
guy, you can allow something else, like Clostridium difficile to colonize. That is an
opportunistic pathogen. It causes nausea, vomiting and diarrhea. Because youve
wiped out the flora that kept all of the resources to themselves, this is now
allowed to grow up. You get sick. So two different ways to break a barrier. You
have to break the barrier before the immune response starts.

[7] [The Players]
[McCutcheon] So we talked about this in May. The Players. We have cells. The
first set of them, neutrophils, macrophages, dendritic cells, Natural Killer cells,
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Transcribed by Mandy Weil July 2, 2014

mast cells, eosinophils and basophils. These are all players in the innate immune
response. Mast cells, eosinophils and basophils are things were not really going
to talk about because they dont really have a huge role in a physiologic immune
response. They have a very large role in a lot of pathologic immune responses, so
other people are going to talk about them a lot more than I will. The last two on
the list are B Cells and T Cellsthose are part of the adaptive immune response
and they are antigen specific. And we will explain that starting today and
continuing on. But its not just cells that make up the immune response. Soluble
molecules. There are a series of proteins that live in the serum, you always have
them. They are called Complement. You have antibodies once youve had a B cell
reaction. Your macrophages, dendritic cells, neutrophils and T cells all secrete
cytokines and were going to start talking about those today. Chemokines are a
form of cytokine that have a specific ligand site and specific function. And then
you have various vasoactive mediators. Youre going to learn a lot more about a lot
of those from other people. But these are reactive oxygen species. The
prostaglandin pathway, things that are associated with causing pain and
inflammation. So this is our component. These are what were going to talk about
in the physiologic immune response.

[8] [Dichotomies]
[McCutcheon] There are some traditional dichotomies of how people divide up
the immune system. The two common, traditional dichotomies are the innate
immune response versus the adaptive immune response and the humoral immune
response versus the cell mediated immune response. So the innate immune
response is really talking about those macrophages, dendritic cells and
neutrophils and complement. Thats innate immunity. And then humoral, thats
immunity mediated by soluble factors, so complement and antibodies versus
immunity mediated by cells, mostly T Cells and macrophages. And Im gonna put
to you that these are not really good, theyre real dichotomies but they dont
explain how an immune response works. So theres a better way to think about
how an immune response works and were going to talk about that today.

[9] [Picture (Recognition mechanisms]
[McCutcheon] So what is innate immunity? Well, the real advantage of innate
immunity is that its fast. Within four hours of stabbing yourself in the finger
with that explorer, neutrophils are on site. Complement is there immediately.
Neutrophils show up within four hours. Macrophages and monocytes are not far
behind. Alright? So its very fast. In contrast, it takes at least a week to get your T
cells going. Five days at the fastest and it takes another couple days to get your B
cells going. So the first, beginning of that first week of an immune response, you
dont have B or T cells yet. Everything doing the work are innate immune cells,
mostly. They have a limited repertoire of things they can do. They can do the
things that they can do and thats the only thing that they can do. In contrast, B
and T cells have a number of different mechanism of dealing with pathogens and
unlike innate immunitywhere they do what theyre going to do, and if they do
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Transcribed by Mandy Weil July 2, 2014

it on a bacterium, it kills a bacterium, but if they do it on a host cell, it kills the
host cellthe variable part of the adaptive immune response is it pretty much
only kills the pathogen. It leaves the host cells in tact. I have to tell you, I have
allergies and so I start coughing while I talk; so Im going to keeping sipping
water. Um, innate immunity only recognizes a limited number of things and those
are all on bacteria. Alright? Do you guys know where viruses are made? Have you
had that microbiology yet? Okay? Viruses are made in host cells. So all of the
proteins made by a human host cell are going to be what kind of proteins? Take
a guess Human proteins! Yea! Do you want the immune system to be able to
recognize human proteins? No. So the innate immunity really doesnt recognize
anything on a virus because its made in a human cell. Innate immunity can
recognize some unique features of bacteria. In contrast, B and T cells, youll have
one or two that will recognize each and every single solitary bacteria and virus
and thats all you need to get enough adaptive immunity. So theyre very specific
and they dont recognize host unless somethings gone wrong. And then innate
immunity, it does what its gonna do and it cant change. And some of what it does
is against host cells and thats not good for you. Adaptive immunity gets better
over time. So the longer the infection goes on, the more efficient the immunity
becomes. And, in the end, (this is one of the reasons its not a good idea to split the
immune system up into innate and adaptive) is you have to have both things to
get rid of a pathogen. You have to have all of the innate immunity working and you
have to have all of the adaptive immunity working if you want to get rid of a
pathogen. If they dont all work right, the pathogen doesnt go away and you end
up with a chronic inflammatory disease. Does any dentist in this room want to tell
me a chronic inflammatory disease? I want a dentistthe key was dentist.
Periodontal disease. Okay? Periodontal disease. The patients in your office who
have periodontal disease have it because something isnt working right. Patients
in your office who dont have periodontal diseaseits because something in their
immune system is working right. Were beginning to figure out the things that go
wrong with people who have periodontal diseasewe dont know them all yet.
But its not the bacteria. Okay? People with periodontal disease have a different
bacterial profile than people that dont have it, but its because their immune
system couldnt get rid of them. People who dont have periodontal disease can
still have seen those bacteria but they didnt cause disease. So, its the host
response that causes periodontal disease.

[10] [Picture (Healthy skin is not)]
[McCutcheon] So what happened: you have your healthy skin, you get an owie
and you have some blood and dirt or bacteria, or something gets under your skin
so youve opened the physical barrier. And the beginning of the innate immune
response, it causes a change in the vasculature. You get fluid into the area. You get
cells into the area. The area becomes red, swollen, hot and painful. So ruber, tumor,
calor, dolor. The four symptoms of an infection. Somebody else is gonna tell you
this: so in Latin, rubor is red, tumor is swelling, calor is heat, dolor is pain. So
classic signs of inflammation: rubor, tumor, calor, dolorits because the immune
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Transcribed by Mandy Weil July 2, 2014

system has opened up the blood vessels, you have fluid, you have cells, it swells
things so that the redness is because the blood has leaked out. The swelling is
from the extra tissue. The heat is because youre starting to cause a fever and the
pain is because youre swelling the tissue and youre pushing on the nerves.

[11] [Location, Location, Location (I)]
[McCutcheon] Now, in the end, so we have the innate versus the adaptive; we
have the humoral versus the cell mediated immunity. Here is the real dichotomy
in the immune system: its where the pathogen lives. Pathogens can live outside
the cell in the extracellular space; pathogens can live inside a cell. Theres
nowhere else. Outside the cell, inside the cell, there are no other options. And the
way the immune system works depends on whether or not youre trying to get
rid of something thats living in the extracellular space or something thats living
inside a cell. And thats really where the dichotomy comes in. And so for
extracellular, you have additional proteins that play a role against extracellular
pathogens that dont work against things that live inside host cells. For
intracellular pathogens: you have an additional cell that comes into play that does
not play a role in extracellular pathogens. And except for the couple of extra
proteins, and extra cell, everything else is the same. Okay? Everything else is the
same. So the way to think about this is: where does the pathogen live? Because
that tells you what you need to get rid of it.

[12] [Location, Location, Location (II)]
[McCutcheon] So, because we have these two different places for pathogens to
live, we have two different places for an immune response to start. So for
extracellular, the immune system starts with complement, those germ proteins
complement, those extra proteins that are part of an extracellular response. And
toll-like receptors on macrophages, dendritic cells and neutrophils. So one of the
principles is: in order for an immune cell to recognize something, there has to be
a receptor. No receptor no recognitionno activity. There has to be a receptor.
For the intracellular pathogens, theyre inside a cell. So are proteins floating
around in the serum or extracellular spaces going to be able to get to something
inside a cell? No. So complement plays very little role in an intracellular pathogen.
To get rid of it, you need a different kind of cell. So intracellular response starts
with a dendritic cell. Thats DC stands for dendritic cell.

[13] [Extracellular First Responders]
[McCutcheon] So for extracellular pathogens: the first thing that comes into play
is complement and if we have them, and we probably have them, its antibodies.
And then its these three cells: neutrophils, macrophages and dendritic cells are
the cells that are resident in the tissue. Theyre call Langerhans cells. Dr. Wishe
probably called them tissue macrophages. Theyre really dendritic cells. Now, I
said we have antibodies. So how is it that if we have a bacterium that weve never
seen before we have an antibody? In May, I talked about a subset of B Cells called
CD5 B cells or B1 B cells. These B cells live in the tissues.
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Transcribed by Mandy Weil July 2, 2014


[14] [Complement]
[McCutcheon] Alright.. Ill pick that up in a minute.So complement.
Complement is a series of proteins that are found in the serum. They exist in
inactive forms. The inactive forms get converted to active forms usually be
previous complement pieces. Does anyone remember the song The Hole in the
Bottom of the Sea? So(Singing in italics) Theres a hole in the bottom of the sea,
Theres a log on the hole in the bottom of the sea and then you end up eventually
with: Theres a flea on the tail of the frog on the log in the hole in the bottom of the
sea.
Thats how complement works. Were going to start off with a piece and then
were going to put a piece on that and then were going to put a piece on the piece
on that and eventually we end up with something that will get rid of bacteria.

[15] [Picture (Classical pathway)]
[McCutcheon] There are three ways to start complement. The classical pathway
begins with antibody. Were gonna talk about where that comes from in a minute.
You always have antibodies. After youre a week old you have antibodies. The
alternative pathway does not start with antibody and it leaves off the first several
proteins of the classic pathway. The alternative pathway can always be activated. It
actually is always being activated and then you have ways to shut it down. These
two pathways are not exclusive. They can run simultaneously. Once the classical
pathway gets to the convergence point, it can drive the alternative pathway to
become more effective. The lectin pathway is similar to the alternative pathway
and were not gonna talk about that.

[16] [Picture (B-1 cell binds bacterial . . .)]
[McCutcheon] So B1 B cells are CD5 B cells. They live in the tissues. They can
make antibodies without any T cell help and they only make a very limited
specificity of antibodies. They recognize bacterial carbohydrate residues. So every
species of streptococcus has a similar carbohydrate residue. Once you make an
antibody against a carbohydrate residue of one kind of streptococcus, it will be
against all other streptococcus. Once you make an antibody against one kind of
staphylococcus carbohydrate it will work against all staphylococcus
carbohydrates. Now the staphylococcus antibody wont bind to the streptococcus
and the strep wont bind to the staph, but the staph will bind any staph; the strep
will bind any strep. Early on, you know just within weeks of when youre born,
you get these antibodies and these are IgM antibodies. Theyre HUGE. They have
really low binding efficiencies and it doesnt matter because one IgM antibody can
be a landing pad for the first component of the classical component pathway. So
you only need one and it doesnt have to stick on very long.

[17] [Picture (C1r, C1q, C1s)]
[McCutcheon] So this is the first piece of complement. Its called C1. It has
componentswe dont care. Okay? Details you dont have to memorize. You do
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have to know about C1. C1 binds to an IgM molecule, the IgM is this big thing. Its
a landing pad. C1 sits down on the landing pad. Once the C1 is bound, it activates
itself.

[18] [Picture (Pentameric IgM molecule. . .)]
[McCutcheon] C1 can also bind to IgG, a different isotope of antibody. Except, it
takes about 1,000 IgGs to scatter themselves around on a bacterium in order for
five of them to be in the right position for the C1 to bind. So one IgM; 1,000 IgG.
C1 binds, it activates itself.

[19] [C1 Converts C2 and C4]
[McCutcheon] It starts recruiting in other factors of complement. So the next
piece of complement that binds is not C2, but, C4. The complement pieces were
numbered in the order they were found, which then turned out not to be the
order that they acted. So C1 binds C4 and it cleaves C4 into two pieces. The big
piece binds, the little piece goes away. Okay? C1 also cleaves C2. The big piece
binds, the little piece goes away. Alright? C1 binds to IgM, it cleaves itself and
becomes active. It starts converting. We call it a convertase because it converts C2
and C4. It binds C4, the little piece goes away. It converts C4, the little piece goes
away, the big piece binds. It converts C2, the little piece goes away, the big piece
binds. The C4/C2 pieces now become a convertase. They convert C3. The little
piece goes away, the big piece binds. Okay? So the two bound pieces of
complement, C4 and C2, become a C3 convertase. They convert C3, the little piece
goes away, the big piece binds.

[20] [Activation of C3]
[McCutcheon] So, here we have our C4/C2 piece. They convert C3. The little piece
goes away, the big piece binds. Now C3 is the convergence point between the
classical and the alternative pathway because not only can C3 be cleaved by C4/C2,
it can also spontaneously cleave. And so you can end up with C3 bindingThe C3b
piecethe bound piecebinding to an alternative protein called Factor B, B as in
boy. B gets cleaved into two pieces. The little piece goes away, the big piece binds.
Now, in the alternative pathway, C3b, big B, little B, that also is a C3 convertase.
Okay, so you can have the classical pathway C4/C2 cleaving C3; you can have C3
cleaving itself spontaneously; you can have C3 plus factor B cleavingC3 plus
factor B that gets cleaved forming C3 convertases. So at this point in time, you
have three things that all cleave C3. So now you get lots and lots and lots and lots
of C3 cleaved. The C3b bindingand C3b is the piece you have to remember,
okay? You need to remember C1 and you need to remember C3C3b is the only
complement piece that plays a role in viral infections. And remember, it can cleave
spontaneously, so thats how it gets there. C3b binds and its going to do two
things

[21] [Complement Activation]
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[McCutcheon] So, before we talk about the two things C3b does, were going to
look at a movie. The sound worked perfectly when I did this yesterday. And its
not working at all.I just rearranged my slides so sorry about that.
So C3 turns into C3a and C3b. C3a is a chemotaxin. It starts recruiting
cellsNeutrophilsto the site of infection. C3b binding causes opsonization.
Opsonization is Greek. It means to coat with butter. So weve coated the bacterium
with butter so that would make it very easy for what? What are the cells that
are showing up at this point? Macrophagesand what does macrophage mean?
Big eater. And so whats going to be eating? Something thats coated with
butter. So what this means is that when C3b is bound, theres a receptor on
macrophages and neutrophils thats a high affinity receptor. So now its very easy
for the macrophage and neutrophil to eat, phagocytose, the bacterium. So C3b, it
can cleave itself, so you get this massive auto-loop of ramping up complement. And
that can work in both the alternative and the classical pathways. Its an opsonin so
it makes it easy for cells with receptors to eat whatever the C3b is stuck to. And
then, eventually, once you get C3b, youre going to end up with end stage of
complement, which is the Membrane Attack Complexor the Big MAC Attack.

[22] [Picture (Fixation of complement)]
[McCutcheon] So C3 cleaves. It coats the bacterium. Just so you know, this is
ridiculous not in scale. If this were in scale, the C3b would be this tiny little blip
that you couldnt see. So its not the same size as a bacterium.

[23] [Picture (Fixation and action of the C3 . . . )]
[McCutcheon] So weve got C3b all over the pathogen and it doesnt matter if its
C3b that got there because it was cleaved by the C4/C2 complex or if it was
cleaved by the the C3bB complex. As long as its there, its an opsonin. So now
macrophages have receptors That can bind to the C3b. Theyre high affinity
receptors. The pathogen gets engulfed.

[24] [Activation of C5]
[McCutcheon] You ready? The C4b2a3b becomes a convertase. It cleaves C3 and
C5. Okay? So the 423 piece cleaves C3 and C5. In the alternative pathway, you get
the BC3btwo of them, C3b, C3b plus B piece, becomes a convertasecleaves C3
and C5. Ill say it again. So, we had our convertase for C3 which was 4 and 2. They
cleave C3. The C3b falls in with the 4 and the 2. So now we have a 423 piece that
cleaves both C3 and C5. Classic Pathway: 423 cleaves C3 and C5. Alternative
Pathway: C3b plus C3b plus the B piece, so two C3bs and a B, cleaves C3 and C5.
So weve got both the C42 cleaving C3; Weve got the C3b-B piece cleaving C3;
Now we have the 423 piece cleaving C3 AND the 33B piece cleaving C3and C5.
So we have a massive amount of C3b being cleaved. And of complement of the only
piece if you have deficiencies in C3b. Thats the only thing we really see disease
associated with. And youre never gonna see those people because they dont live
long enough to have teeth. Its a very rare mutation but its quite lethal.
10
Transcribed by Mandy Weil July 2, 2014

Okay, C5a is an anaphylatoxin so it goes off and it starts recruiting inflammatory
mediators and its about 1,000 times more potent than C3a. So 3a and 5a go off and
do other things and theyre good at it. So this is how you help start recruiting the
neutrophils and the macrophages to the site of infection. Remember, complement
is in the serum. The minute you start breaking capillary walls, complement is
where the infection is. Okay? cause the serum comes out of the capillary, the
complement is thereoff it goes. Within, you know, a minute, this starts working.
So, we have the C5 convertase of 423 cleaving C5 (it also can cleave C3). We have
the alternative pathway convertase BC3bC3b cleaving C5, also C3. Once C3 gets
into play, the MAC attack is going to occur. So C5 gets cleaved, C5, the big piece
binds the little piece goes away.

[25] [Picture (pathogen)]
[McCutcheon] When the big piece binds and the little piece goes away, it
recruits the next three pieces: C6, C7 and C8. They dont get cleaved, they bind
together and they insert themselves onto the membrane and they insert
themselves onto the membrane of the bacterium. So 5 recruits 6, 7 and 8. Doesnt
matter how we got the 5if it came from alternative or classical pathway. 5 is 5 is
5. It makes the 6, 7 and 8 bind together. And then once they insert into the
membrane, that causes the polymerization of C9. You get 10-15 C9 pieces that
polymerize and they form a pore. The pore pokes all the way through the bacterial
membrane and if theres a big hole in the bacterial membrane, what happens?
All the inside stuff rushes outside and the bacterium is dead. Alright? So thats
the MAC.
Now as cool as the MAC attack is, in reality, if you dont have it, the only bacteria
((NOTE: ?--she names something, I THINK this is what she is referring to))
leishmaniasis listerium monocytogeni So every other bacterium in the planet,
you can get rid of without a MAC attack.

[26] [Complement]
[McCutcheon] Now, try the movie So when I did this last night, the sound was
fine and when we tried to do this today, there was no sound so I may have to
narrate this for you. If she does talk, wait.go backOkay thats not gonna
work.Okay, shes not gonna talk. If she does talk, she mispronounces lots of
words. OkayWell, its running on my computer. UmOr were not gonna watch
movies unless I can figure out how this works

[27] [Functions of C3]
[speaker] Sorry about that. Ill get the guys back in here at break and well get
the movies running. So C1 is important, and Ill talk about why in a minute. C3b if
you dont have C3b, youll be dead. C3b is important because its an opsonin and
macrophages and monocytes have receptors. Dendritic Cells have receptors.
Neutrophils have receptors and most importantly, B lymphocytes have receptors.
Without C3b, you dont get an antibody response. And so the only piece of
complement that plays a role in an intracellular infection is C3b. And the reason
11
Transcribed by Mandy Weil July 2, 2014

why isnt for the macrophages, monocytes and neutrophils, its to get the B cells
going. If you dont have C3b bound to that virus, you dont get an antibody
response. Alright? In truth, the pore of complement is bigger than a virus. So if
you form that MAC attack complex, the virus would just go through it.
IgMbigger than a virus. So youre never going to be able to get C1 to bind,
because the virus would just sit inside the IgM and nothing would happen. Okay?
So complement has NO role against viral infections, except for C3b. Intracellular
pathogens, you have to have C3b. Nothing else. Extracellular pathogens: you want
all of it. And then you need C3 as the C3/C5 convertase. But you get this massive
expansion of the immune response beau se every C3 convertase, be it the C42 or
the C3Bb piece, can cleave 1,000 C3 molecules. And every one of those C3bs that
binds can then form a convertase that can cleave another 1,000 C3 molecules. So
you get a massive expansion of the complement system.

[28] [Picture (Chart: CR1, CR2)]
[McCutcheon] Okay so briefly, there always has to be a receptor. There are
actually several receptors that recognize several forms of C3. So we have C3this
i is the inactivated C3. C3d is a breakdown product and thats part of the B cell
receptor. You dont need to memorize that. You need to know there are receptors
but you dont need to memorize which one is where.

[29] [Picture (The terminal complement)]
[McCutcheon] Functions of the other factors, we talked about them: C5 forms the
membrane attack complex. 6,7 and 8 bind together. 9 polymerizes and pokes a
hole.

[30] [Complement must be controlled]
[McCutcheon] Now, complement is very powerful. Anyone with glomerular
nephritis, that was caused by complement binding to things bound on the kidney
endothelial walls and it destroys the endothelium and it get replaced with scar
tissue and the kidney doesnt work. Alright? Because complement is powerful, it
is very tightly regulated. So for every factor that can do something, we have
several inactivating factors. You need to know about these.

[31] [Picture (Activated C1r and C1s)]
[McCutcheon] There are two forms of inactivating factors. Soluble inactivating
factors will inactivate complement no matter what its found on. Host cells have
membrane inactivating factors that will prevent complement from activating on
host cells, but it will not prevent complement from acting on bacterial cells. So the
first factor is C1 inhibitory factor. C1 inhibitory factor inhibits C1it stops C1
from being a convertase. People who have defects in C1 inhibitory factor have
hereditary angioedema, a disease you will deal with as dentists. What happens is
their lips and tongue swell and they cant breathe. Thats caused by a mutation in
the C1 inhibitory factor.

12
Transcribed by Mandy Weil July 2, 2014


[32] [Picture (Formation and action) ]
[McCutcheon] The rest of the factors are pretty much directed at C3not
surprisingly. So, you have soluble factor, um, so we have C3b bindingokayand
then we can have factor D inhibit this, so we get an inactive C3b.

[33] [Picture (Inactivation of the classical) ]
[McCutcheon] We have C4-binding protein that prevents C4 from cleaving C3.
C4-binding protein inactivates the C3 convertase. We have Factor H that does the
same thing that does the same thing with the C3bBb convertase. So Factor H binds
to the C3b and makes it inactive. Okay so C4-binding protein inactivates the C42
C3 convertase. Factor H inactivates the C3bBb convertase.

[34] [Picture (DAF dissociates C3 convertases. . .)]
[McCutcheon] So those are our soluble factors. Membrane, we have DAF, which
inactivates the C3b by binds to the C3b and displaces the Bb, so its no longer
C3 convertase.
We have MCP, which cleaves off a portion of the C3, so its no longer active. This
inactive form of C3b

[33] [*back to previous slide*]
[McCutcheon] Even though its inactive, there are still receptors that bind
thatso it can still act as an opsonin. It just cant cleave anymore. So the
inactivating factors prevent the C3b from being a cleaving agent. They do not
prevent it from being an opsonin. Nicely designed system.

[35] [Picture (On microbial cells) ]
[McCutcheon] And then finally, host cells have a protein called CD59 that
prevents the polymerization of C9. So you prevent the C3, you prevent the pore
from forming on host cells. It does nothing for bacterial cells because bacterial
cells dont have human host cell proteins on them. So the pores can still form on
bacterium. They cannot form on host cells.

[36] [Complement starts inflammation]
[McCutcheon] So, C3 and C5 are anaphylatoxins. They alter the vascular
endothelium and were going to show pictures of that in the next hour. They
recruit inflammatory cells.

[37] [Picture (Anaphylatoxins act on blood vessels) ]
[McCutcheon] So, C3 and C5, what they cause is they cause the capillaries to
become leaky. That allows more complement to go out of the capillary into the
inflamed, infected area. It also allows anything else in the plasma. So any
antibodies that are around can get out, into the infected area. And then it allows
cells to diapedese in between the endothelial cell walls so that the neutrophils
and the monocytes can get out of the capillary and into the tissue. Any reason why
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Transcribed by Mandy Weil July 2, 2014

these cells are going in and out of capillaries? Why not arteries? Why cant
they go in and out of an artery? Somebody mumble really loudly Theyre too
thick! What are they thick with? Collagen..whats the thing that arteries have that
veins dont? Starts with an M.Muscle! Yea. Youre not gonna have something be
able to crawl through thick collagen and several layers of muscle cells. So the
inflammatory cells get out of the circulation into the tissues through capillaries
and really, really, really small venues and its not 8:50Ill give you a ten minute
break and see if I cant get AV to come help.

[38] [The Initial Response to Extracellular Bacteria]
[McCutcheon] Alrighthaving argued with my computer So initial response
to the extracellular bacteriaIt starts with complement. Second after youve
disrupted the capillary beds, the inactive proteins start becoming active by
cleaving themselves or cleaving other things. C3b is an opsonin for any cells that
have receptors. Cells that dont have receptors, it doesnt do anything. Always
have to have a receptor. C3a and C5a are inflammatory mediators. So seconds after
youve poked yourself with that explorer, you now have signals that start
changing the endothelium and telling the neutrophils and monocytes to show up.
In the blood, monocytes and monocytes. When they migrate out into the tissues,
they differentiate into macrophages. Okay?

[39] [Pathogens Reside in Different Compartments]
[McCutcheon] So, thats an extracellular response. Now, for purposes of this class,
were going to saywhen I say bacterium, Im talking about something that lives
outside a cell. When Im talking about a virus, Im talking about something that
lives inside a cell. There are obligate intracellular pathogens that are bacteria.
Okay? So im using virus as the model. it does not mean there cannot be bacteria
that reside inside the cell. If there is bacteria that reside inside the cell, it is dealt
with the same way a virus would be dealt with. If the something lives outside the
cell, its dealt with as an extracellular pathogen. If it lives inside the cell, its dealt
with as an extracellular pathogen. Ill say bacterium as extracellular and virus
meaning intracellularbut thats a shorthand, thats not inclusive. Okay?

[40] [Picture (Site of infection. . .) ]
[McCutcheon] Typical extracellular pathogens, viruses can also be outside the
cell and you know this, although you may not know that you know this. Because
when you came here, they drew blood and they did what? They titer-ed you. What
does that mean? They looked at antibodies. And what disease did they look at
antibodies for? Pardon? Yep. Measles, mumps, chicken pox. Okay, what type
of pathogens are measles, mumps, chicken pox? Theyre all viruses. So theyre
looking to see if you have antibodies against a virus. And knowing the state of
your immunity is by knowing how good your antibodies are against a virus. So
even though viruses live primarily in the cell, there are periods when theyre out
and about in the extracellular space and thats when the antibodies bind them. If
you dont have antibodies, you are not going to live. Im telling you this now,
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Transcribed by Mandy Weil July 2, 2014

were going to talk about it over and over and over. You have to have antibodies
against a virus.
When things are in extracellular spaces: its complement, antibodies and
phagocytosis. When things are inside, so viruses, things like kvidia ((???)), things
like microbacterium, microbacterium only live inside vesicles inside cellsthen
you have to have T lymphocytes and macrophages. So extracellular, you have extra
molecules, complement Intracellular, you have extra cells. CD8 T cells. And
thats the difference. Everything else is both extracellular and intracellular. You
need both innate and adaptive to get rid of extracellular and intracellular
pathogens. You need both humoral and CMI immunity to get rid of extracellular
and intracellular pathogens. The difference: you have some extra proteins in the
beginning of an extracellular response. You dont have those proteins, you have an
extra cell at the end of an intracell response. Everything else, same. So the real
dichotomy is where something lives.

[41] [Extracellular Pathogens]
[McCutcheon] So how do we recognize bacteria? Ive told you, it has to be a
receptor. It turns out that bacteria make some proteins and carbohydrates and
fatty acids that are different than anything a mammalian cell can make. Theyre
different than anything a mammalian cell can make. We call these things
Pathogen Associated Molecular Patterns or PAMPs. And if a bacterium can make
something that is not host, then the host can make a receptor that will specifically
bind to the bacterium but wont bind to host. If the bacteria has something thats
the same as the host, you cant make a receptor against it, because then your
immune cells would act against you. Youd have nasty, awful autoimmune disease.
So the bacteria are only recognized by things unique to them. So whats the first
amino acid in all proteins? Methionine. And bacteria, they have the first three
amino acids identical: methionine, leucine, phenylalanine. Where all of our
proteins start with a Met, theirs start with Met, Leu, Phe. Not only to they start off
with methioninethey can formulate it. We cant formulate methionine. So
theres this unique structure: formulates methionine, leucine, phenylalanine that
start every bacterial protein. Since thats not something found in host, you can
make a receptor that will recognize that. So f-Met-Leu-Phe glycopolysaccharide is
found on every gram negative bacteria. Its not something mammalian cells can
make. If the bacterium can make something that humans cant make, we can make a
receptor against it. So, theres a receptor against glycopolysaccharide. Bacterium
can do different things with carbohydrates. They can make glycan structures and
mannose structures. Human cells cannot do those things. Because human cells or
mammalian cells will never have glycan or mannose, you can make receptors
against them. We call these receptors toll-like receptors. These receptors reside
primarily on neutrophilsthat funny nucleated thingand macrophages. Theyre
not very high affinity, but they still work. Anything that has an f-Met-Leu-Phe, an
LPS, glycan or mannose, a neutrophil and a macrophage will have a receptor
against one or more of those things that allows the neutrophil or macrophage
monocyte to bind to the pathogen and then it can eat it. Phagocytosis.
15
Transcribed by Mandy Weil July 2, 2014


[42] [No title Video]
[McCutcheon] Cross your fingers
Ach! I keep setting it on duplicate, and then it goes backIll play later Okay
duplicateAhh!
So this is a neutrophil. This is in the bloodstream. These are RBCs. That are
staphylococcus that have been introduced into the bloodstream. The neutrophil
has receptors that recognize the staphylococcus. The staphylococcus are being
bounced around by Brownian motion Eventually the neutrophil catches
upphagocytosis. So, thats toll-like receptors.

[43] [Toll Receptors]
[McCutcheon] So, toll-like receptors. There are ten, theyre a family of ten
receptors. If you can have extracellular pathogens, where will you have toll-like
receptors? Says so on the slideon the cell membrane.
If you can have intracellular pathogens, where do you have to have toll-like
receptors? Says so on the slide You have to have receptors inside the cell. So
somewhat unusually, you have toll receptors both on the cell membrane and in the
endosomes.

[44] [Cartoon of TLR Receptors]
[McCutcheon] So this is a cartoon of toll-like receptors. You need to know there
are toll receptors. You need to know there are things they bind against. You do not
need to know the names of the receptors that bind the things. So you have some
on the cell membrane; you have some that are in vesicles. Notice that that in the
vesicle part, the membrane end is in the vesicle and the cytoplasmic domain is in
the cytoplasm.

[45 [Picture (Structure of Toll-like)]
[McCutcheon] So this is the real structure of toll-like receptors. This is the
pathogen recognition domain out in the extracellular space. And this is the
cytoplasmic domain and that stands for Toll Isle-1 Receptor Domain.

[46] [Picture (Sensing microbial products) ]
[McCutcheon] We can see our toll-like receptor recognizing something on
bacterium. A different one recognizing something different on bacterium. We can
see toll-like receptors that are against single stranded and double stranded RNA.
DNA and RNAwhich is what viruses are made up of.

[47] [Picture (A complex of TLR4)]
[McCutcheon] I have been telling classes for years that they did have to know
anything about 2nd messengers until we had drugs against 2nd messengers. And
you guys drew the short straw. Theres now a drug against them, so were going
to learn about them. All of the immune receptors work through a 2nd messenger
system. So something is going to bind the cytoplasmic domain. You go through a
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Transcribed by Mandy Weil July 2, 2014

cascade kinase. You dont need to know the specifics of that. You end up turning on
a transcription factor that translocates into the nucleus and that transcription
factor turns on cassettes of immune related genes that give you cytokines.
All receptors, all immunology receptors, work through a 2nd messenger system.
The receptor binds to what it binds to, that phosphorylates a 2nd messenger in
the cytoplasm, you go through a cascade kinase chain and you end up turning on a
transcription factor. THat transcription factor translocates into the nucleus, it
binds to DNA and it causes the transcription of groups. Cassettes of related genes.
So different transcription factors turn on different sets of cytokine genes.

[48] [Toll Receptors]
[McCutcheon] Toll Receptors are critical in an immune response because they
dictate the end result. SO the Toll receptors that you turn on and the proportion of
cytokines that get turned on help drive you to have primarily whats called a
humoral response driven by B cells or a cell-mediated response driven by TH1 T
cells. And that all starts at the beginning depending on the toll receptors that
bind. So, you activate the different toll like receptors, you go through a 2nd
messenger system, that turns on transcription factors. The transcription factors
then drive the differentiation of the various T cells depending on what cytokines
are secreted.

[49] [Macrophage Activation (TLR)]
[McCutcheon] So when macrophages, the neutrophils, get to the site of infection,
they are end-stage cells. They eat bacteria. Thats all they do. They do that for 2-3
days. They die. Theyre replaced. Okay? Without neutrophils, you cannot keep the
pathogen at bay long enough to get anything else in there to do the deed. So
anybody who has gone through chemotherapy, theyre in isolation until their
neutrophils come up enough that they can defend against common bacteria. So
neutrophils are the first thing thats there. The function of the innate immune
response is to keep the pathogen whittled down enough so that the adaptive
immune response can show up. The function of antibiotics is to help the innate
immune system keep the pathogen down enough so that the adaptive immune
response can show up. Antibiotics will never cure you of anything if your adaptive
immune response cannot show up, youre going to die. Doesnt matter what
antibiotics they put you on. So innate immunity is there simply to keep bacteria
from overwhelming you. A lot of bacteria have a 40 minute replication cycle. If you
started off with one and divided it in 40 minutes to two, and 40 minutes later its
divided to four. At the end of 24 hours, you have a million-something. Okay? So
the function of the neutrophils and eventually the macrophages, is to keep those
numbers down. Its the adaptive immune response that actually gets rid of it.
When a macrophage is activated through toll-receptors, it does two things. Its
very phagocytic, so it can eat things and it starts secreting cytokines.

[50] [Picture (On sensing)]
17
Transcribed by Mandy Weil July 2, 2014

[McCutcheon] Before we talk about the cytokines the macrophage secretesand
these are it--

[51] [General Principles of Cytokine Biology]
[McCutcheon] Were gonna talk about cytokine biology in general. So
cytokineshow many people know what one is? Okaytwo. Cytokines are small
soluble molecules, they have a myriad of functions. Originally they were
discovered being secreted by white blood cells, leukocytes. So a lot of the early
ones were named IL for leukocyte, interleukin because they were secreted by
white blood cells and they thought they acted on white blood cells. So interleukin:
between white blood cells. We now know that almost any cell in the body can
secrete cytokines given the right stimulus. Any cell in the body can be acted on
cytokines provided that they have a receptor. So, all cytokines are pleiotropic.
They have many, many, many functions. Some of them have more functions than
others, but they all have more than one. All cytokines, all of the functions of
cytokines (except one, and well talk about that) are redundant. So cytokine X can
do this, cytokine Y can do this, cytokine Z can do this. What that tells you is that
that function is so critical that you have to have more than one thing do it. So all
cytokines, their functions, except for one, are redundant. More than one cytokine
causes that function to happen. They have really broad activities. And the more we
know about immunology, the more broad the activities are. One of the things that
were discovering is that pretty much all diseases have something to do with the
immune response. Even disease that you wouldnt have thought have anything to
do with the immune response. And a lot of the effects dont particularly have
anything to do with the immune system. A lot of them do, but some of them dont.
And then they come in families. What that means is that there are groups of
structurally related proteins that generally have similar redundant functions. So
they have many, many, many functions. All but one of those functions are done by
more than one cytokine. They have very broad activity, including non-immune
effects. And they come in families. All of these act through 2nd messengers. They
now have their first drug against a 2nd messenger and well talk about that.

[52] [Examples]
[McCutcheon] So, the two major families of cytokines are pro-inflammatory
cytokinesthings that ramp up inflammation. And, anti-inflammatory cytokines.
Things that calm down inflammation. And then, the third family of cytokines are
the chemokines. Chemokines all have a cysteine binding motif. So the part of the
ligand, the chemokine, that binds a receptor has some kind of a cysteine and
theyre named after their cysteine motif. So pro-inflammatory IL1, TNF-alpha,
Interferon-gamma and IL6 are the major players in the pro-inflammatory
cytokines. Theres also IL17 and related proteins that are also pro-inflammatory
cytokines. The major players of anti-inflammatory cytokines are IL4, IL10 and
TGF-beta and then the more recently discovered IL22 function.
Now, one of the things that has happened over the past several years is theres
been kind of a sea change in the way we understand cytokine biology. The old way
18
Transcribed by Mandy Weil July 2, 2014

was, you knocked it in or you knocked it out of a mouse. And then, you said it does
this, that, this, that, this and that. Well, because some of these cytokines are so
structurally related and the functions are redundant, a lot of times, when you
knock something out, the functions still happen because the other cytokines that
have the redundant properties were still there. And the other thing is that, and
this is gonna come as a shock to you guys, mice are not people. Okay? And
especially with cytokines, the functions in mouse are not the same thing as the
functions in human. So you knock it out in a mouse and you say Oh! It does this.
And then you knock it out in human cells and nothing happens, or something
completely different happens. So trying to use the mouse knock-in, knock-out
model to explain what cytokines do is really kinda inadequate. So weve gone to
broader categories. And experiments nowadays, if you knock something out of a
mouse, you have to go prove that it does the same thing in humans before you can
go anywhere with it. So the science has really changed in the past 5-10 years.

[53] [Receptors]
[McCutcheon] All cytokines work through a receptor. Receptors like the
cytokines themselves, come in families. So pro-inflammatory cytokines generally
have very similar looking receptors. The receptors have widely variable structure.
Okay? And then, what in part explains the redundancy are the shared chains. So,
these three chains can be shared between main cytokine receptors and because
theyre =shared that means youre activating the same 2nd messenger, which
means you turns on the same transcription factor, which means you turn on the
same cassette of genes. And thats why the properties are redundant.

[54] [Picture (A,B,C)]
[McCutcheon] Looking at these, we can see that IL2, which is pro-inflammatory,
IL4, which is anti-inflammatory, IL15, which is pro-inflammatory and IL21, which
is pro-inflammatory all share this gamma-C chain. So the difference is that IL2 has
a different IL2 alpha and IL2 beta chains compared to IL4, which has an IL4 alpha
chain. And then IL7 has a different chain, so its an IL7. Of these, you need to know
that they share a chain. I want you to remember this structure because were
going to about it when we talk about T cell activation. The rest of these: you
should know that theres receptors. And so each receptor is specific for a specific
cytokine but with shared chains that means that you can have the same 2nd
messenger systems activated. So, cytokines have a combination of unique
cytokines, unique chains and a shared chain. You need to know the structure of the
IL2 receptor. The rest of them you just need to know that they have specific
chains. Each cytokine has a specific chain. And a shared chain.

[55] [Macrophage Cytokines]
[McCutcheon] So, macrophage cytokines. So IL1, also, although its not listed in
your textbook, IL18 is a part of the IL1 family. And it does the same things that IL1
does. And if you knock IL1 out IL18 does it anyway. So, it activates soluble
inflammatory mediators. It does a couple of things. It goes off, and it starts
19
Transcribed by Mandy Weil July 2, 2014

activating locally on the prostaglandin pathways. So you start getting these small
molecules that can cause pain and inflammation. It activates the vascular
endothelium; ll show you a picture of that in a minute. That does several things.
First of all, activated vascular endothelium changes shape. IT changes the
receptors that are expressed on the activated vascular endothelium. It puts up the
receptor for the chemokine CXCL8. And its a concentration gradient. So the closer
you get to the site of infections, the more receptor for CXCL8. And so eventually
that stops the incoming cells from rolling. They stop long enough that they can
crawl in between the tissues. So IL1 does those things. IL1 causes the production
of gamma interferon. When we get cells that can secrete gamma interferon. IL1
also has a host of systemic effects. Well talk about those in a minute. And IL1 can
activate the resident dendritic cells and cause them to go from an immature tissue
phenotype to a mature phenotype.
TNF-alpha. Redundancy. It activates the same soluble inflammatory mediators that
IL1 can activate. TNF-alphaalso activates the vascular endothelium. It causes the
same shape changes in the endothelium. It causes the same change in the
receptors on the endothelium. And it puts the CLCX8 on the endothelium. Unique
function of TNF-alpha: TNF-alpha, in addition to activating the vascular
endothelium, TNF-alpha causes the venues to occlude. TNF-alpha, and only
TNF-alpha, causes the venules to occlude. Okay? Weve opened up the capillary
beds, weve clamped off the venules. Wheres all that fluid going to go?
Capillaries are open. Say it loudIts gonna go into the lymph. All of that
fluid ends up into the lymphatics. And where do the lymphatics drain into? They
drain into the lymph nodegood for you. Okay, so weve got the infection. Weve
got all this bacteria and good replicating. And weve closed off the venous return.
Okay? So now can the bacteria spread throughout the body? No. They all get
forced up into the lymph node. And whats one of the functions of the lymph node?
Its a meeting place. Whats another function?Its meeting placepardon?
It traps things. So all of the bacteria that are at the site of infectionThey can be
at the site of infection or they can end up in the lymph node. They dont get out
into the body, most of the time. Okay? So TNF-alpha and IL1, they open up the
capillary beds. TNF-alpha and only TNF-alpha occlude the venules. So the blood
does not go back out of the site of infection carrying pathogen. It all gets forced
into the lymph. TNF-alpha, it causes IL1 production. Okay? TNF-alpha has the same
systemic effects as IL1, TNF-alpha can cause dendritic cells to go from immature to
mature phenotypes. TNF-alpha, directly, this stands for tumor necrosis factor. You
learned what necrosis is. Tumor necrosis factor can cause cell death. IL1 doesnt do
that. So if you look, you can see that these have similar overlapping, redundant
functions, but that theyre not identical. So, its important to realize that. Just
because you have redundant functions doesnt mean that TNF-alpha and IL1 do the
same things. They do some of the same things, they do some unique things.

[56] [histological slide I]
[Mccutcheon] Does anybody wanna grab the pointer and point at, um, an
endothelial cell in this normal capillary? Be brave. Whats the worst thing thats
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Transcribed by Mandy Weil July 2, 2014

gonna happen? Capillarieswhere are the endothelial cells? ((throws clicker))
OopsIts under your seat. Oh, without any batteries. This was not my best
ideaOkay, top button is the laser. Yes, exactly, thats the vascular endothelium.
Nowpush the right arrow

[57] [histological slide II]
[McCutcheon] Now wheres the vascular endothelium? Exactly.. Those flat cells
in the previous slidepush the previous slide ..((back to #56).. So the flat cells
here, flat connected, flat connected, next slide((back to #57))Big puffy cell.
Big puffy cell. Big puffy cell. Space. Thats activated vascular endothelium. Okay, so
the cells round up, and they lose their hemidesmosomes and desmosomes. So
there are big spaces now in between the capillary cells, the cells that line the
capillary bed. And if there are big spaces, whats gonna happen to all the fluid in
all the capillaries? Lets give him a hand. ((clapping)) Thank you very much. So
if there are big spaces in between the capillary cells, whats gonna happen to all
the stuff in the capillaries? Its gonna end up in the tissue. Whats gonna happen to
cells that come into the area? Theyre gonna have an easy time of getting from the
capillary into the tissue. So thats activated endothelium. So the endothelium
changes shape. Plus the endothelium has changed their receptors. Theyve
changed their receptors. Theyve put up the receptor for CXCL8. When you have
enough CXCL8 receptor, the cells following that concentration gradient come to a
dead stop. That allows them the time to migrate into the tissue.

[58] [Chart (Molecular basis *mutations)]
[McCutcheon] So, I said that immunology is now the cause of many diseases.
These are diseases that have something to do with IL1. Sometimes we know what
they have to do, sometimes we dont. But if you treat these people with a drug that
treats IL1, you make the disease go away. So some of these make sense. Arthritis is
an autoimmune disease. Weve known that for a long time. How about diabetes?
How many people have you thought that type II, not type I, type II diabetes has a
autoimmune component. There was a clinical trial in Europe. They treated people
with one round of an anti-IL1 drug and they caused remission of type II diabetes.
The last I heard, it had been going on for over a year. So, um, you know, vitiligo.
Who thought that vitiligo would have an autoimmune component? How about high
blood pressure? Cancer? We knew cancer. So many, many, many of the disease that
youre going to seeyoure not going to treat these people. Youre dentists, not
physicians.But theyre going to come in and theyre gonna be on biological
drugs against cytokines because many, many diseases have a cytokine component.
All sorts of things that we never thought the immune system had anything to do
with. We were wrong.

[59] [Picture (Local infection with)]
[McCutcheon] So, TNF-alpha, and only TNF-alpha, can occlude blood vessels. And
what does that mean? So you stabbed your finger with an explorer, right? So
youve got localized inflammation in your finger. And you know that may spread a
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Transcribed by Mandy Weil July 2, 2014

little bit to the surrounding hand. So the TNF-alpha and IL1 have opened up the
capillary beds. And so youve got lots of fluid, and lots of complement and
antibodies and cells all moving into the area of the infection. And then TNF-alpha
has occluded the venules so all of the blood, everything that went off into the
extracellular space, now has to go into the lymphatics, and they end up in the axial
lymph nodes. Okay? What would happen if there were bacteria all over the body?
So you have a systemic infectionokay? IL1 and TNF-alpha open up the capillary
beds throughout the body. What happens to you when your capillary beds open
up throughout the body? Theres a massive drop in blood pressure.Thats
called? Anaphylactic shock. Okay, so first you go into anaphylactic shock. And what
do you think happens to all your major organs if youve occluded all the blood
vessels in them? Yeahthey die. So you die. Alright. Thats why TNF-alpha and
only TNF-alpha does this. This one function is so potent that if it happens
widespread, its death. Its massive organ failureits quick, painful and you cant
stop it. So TNF-alpha in a localized area is fantastic. TNF-alpha spread throughout
the body is death. Only TNF-alpha occludes blood vessels.

[60] [Picture (Selectin-mediated adhesion)]
[McCutcheon] Red blood cells get pushed through the blood system by the beat
of the heart and the forces from the heart beat. White blood cells roll along the
walls. And they always roll along the walls. So there are receptors on the capillary
walls, on the endothelium, and then there are ligands on the white blood cells
bind to the receptors. This is a very low affinity interaction. The push from the
blood flow causes the white blood cells to roll along the walls. This is how all
white blood cells move throughout the body all the time. When you have an
infection, you change these receptors and that allows only certain white blood
cells to migrate to the site of infections. Neutrophils are one of them, macrophages
are the other. And then the adaptive cells, the B cells and the T cells that are
specific to the infection can go to the site of infection. The rest of them dont.
When you get enough CXCL8 receptor, that stops the rolling interaction long
enough to allow the cell to diapedese through the endothelial walls, which should
be puffier than they are in this cartoon. And then, that causes the neutrophils and
macrophages to go to the site of infection. So one of the causes of juvenile
periodontitis and I know they changed the name, and I dont know what they
changed it to. What did they change the name to?One of the causes of juvenile
periodontitis is the mutation in CXCL8. So that it saturates out too soon. So you
have the bacteria here, and you have neutrophils that get to here and instead of
going all the way out to here to diapedese where the infection is, they diapedese
here and they go to work here. And since what theyre going to work here on, A,
isnt getting rid of the bacteria over here, and B, the only thing thats there are
host cells, what they do is, they kill a lot of host cells. So, juvenile periodontitis is
an autoimmune disease. There are other causes, but thats one of them.

[61] [Video slide]
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Transcribed by Mandy Weil July 2, 2014

[McCutcheon] - So, lets cross our fingersIf this doesnt work, Ill skip it and try
it later.AchOh! Okay sothis is a zebrafish. They poked a hole in the fin of the
zebrafish. This is a vessel, and this is a capillary bed. And the reason it looks like
that is because the red blood cells are moving so quickly that you cant visualize
them. So here is a neutrophil thats crawled out of the extracellular space and its
moving up towards the puncture. So these are fibroblasts that are in the tissue.
But all of the neutrophils, they dont just move anywhere. They only move to the
site of infection. And theyre following that CXCL8 concentration gradient, so they
only go to where the infection issee them crawling out?

Alrightlets try this one So well do the cartoonSo the white blood cells
(those look like monocytes) are moving through the blood vessels. As you start to
change the receptors on the endothelium, that causes the white blood cells now to
start sticking. ((Video zooms in to depict sticking mechanism)) Um, you dont
need to know that. So the white blood cells are rolling along. You change the
receptorsNowsee this has hit enough CXCL8 receptor that it stuck and they
diapedese though the tissues and they migrate towards the infection So looking
at this in real time. This is a vein. Okay, this is an artery and againnothings
going to come out of an artery because of that muscular wall. So we can see the
cells rolling along the in veinand the reason this is moving up and down is
because the mouse is breathing. So we can see the cells rolling alongthat is not
gonna diapedese (mumbling indistinctly) So these have diapedesed out.
Theyre moving toward the site of infection. This one is stuck. Now theyve
clamped the blood vessels a bit so that theyve slowed down the circulation so you
can seeand you can see how this is pulsing. Thats the artery. The veinthe
pressure isnt as strong, so its more of a slow flow. But the millions of red blood
cells in hereyou can see how crowded they are. And theyre just being pushed
along. The white blood cells are crawling along the endothelial cell walls. They
always crawl. They get moved forward, but they crawl

[62] [Macrophage cytokines]
[McCutcheon] So in addition to IL1 and TNF-alpha, macrophages secrete IL6. IL6
primarily has systemic effects. IL6 can help with B cell activation in the right
places. And then, IL6 helps B cells make more antibodies once the B cells are
activation.
CXCL8, it used to be called IL8, so if youre reading older literature and they say
IL8, theyre talking about CXCL8. So this means that cysteine, any amino acid,
cysteine-leucine is the binding motif for the receptor. Okay? If I ask you the
question on the test and you say IL8, its wrong. The new name is CXCL8. It is
chemotactic. So, the chemokines are all chemotactic. It calls the cells, primarily
neutrophils and macrophages, to come to the site of infection, where the
macrophage is secretingum, CXCL8. And then, along with TNF-alpha, it helps
activate the neutrophils that get there. The neutrophils get there. So, the
neutrophils get there; they have CXCL8 receptorswe knew that. And they have
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Transcribed by Mandy Weil July 2, 2014

receptors for TNF-alpha. And those two things cause the neutrophil to become
highly phagocytic. They start eating everything in sight.
And then IL12 activates a member of the innate immune cells called the natural
killer cell. And then IL12 also will drive activated T cells to become TH1 T cells,
and well talk about that several lectures from now.

[63] [Selected IL1 and TNF-alpha Non-Immune Effects]
[McCutcheon] Non-immune effects. Femtomole. Does anyone know how many
zeroes are in a femtomole? How about millimole? Close. How about milli-mol? 6?
Nanomole? 9. Picomole? 12. Femtomole? 10 to the minus 15. IL1 activates
osteoclasts in the Femtomole range. So its something like four molecule of IL1
will make an osteoclast turn on. TNF-alpha also activates osteoclasts, but its in the
nano mol range, so you need a million times more. But whats one of the functions
of TNF-alpha? Thats one of themwhats the function related to this? What does
TNF-alpha cause the production of? IL1. So even thought TNF-alpha isnt very
good at activating osteoclasts comparatively, it can make lots of IL1, which is really
good. So, one of the disease pathways for rheumatoid arthritis, inflammatory
bowel diseases is IL1. So, there are drugs now, either against IL1 agonists, and for
the IL1 receptor or against TNF-alpha. And one set of those will cause good
remission of people with rheumatoid arthritis and inflammatory bowel disease.
And you hear the commercials for these all the time. Humira. Remicaide.
Infliximab. These are all drugs that block IL1 or TNF-alpha. And in some people,
blocking TNF-alpha, even though thats not the thing that activates osteoclastsit
prevents the production of IL1 and that works really well So as a dentist, you
are now looking at the cause of all the bone loss that youre going to deal with.

[64] [Picture (IL-1/IL-6/TNF-alpha)]
[McCutcheon] So the systemic effects. IL1, TNF-alpha and IL6 go off to the liver.
They cause production of acute phase proteins. C-reactive protein. And then the
lectins. And that helps activate the alternative. And then the lectin binding
pathways of the complement system. So, the liver makes proteins that help
activate complementJust in case it wasnt going well enough on its own. They go
to the bone marrow endothelium, and that gets the neutrophils up out of the bone
marrow so that they go off to the site of infection, and there theyre phagocytic.
They go to the hypothalamus. And they cause fever. So when youre sick, the fever
is not caused by the pathogen. The fever is caused by IL1, IL6 and TNF-alpha. The
higher your body temperature, the less well bacteria and viruses can replicate.
They cause fat and muscle to metabolize, and that causes you to shiver, and that
keeps the bacteria from replicatedand viruses. So those are the systemic effects
of IL1, IL6 and TNF-alpha.
IL6, the other systemic effects of IL6, are lymphocyte activation and antibody
production.

[65] [Summary]
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Transcribed by Mandy Weil July 2, 2014

[McCutcheon] So in summary, our extracellular pathogens, an immune response
to extracellular pathogens begins with complement. Theres always IgM from the
B1 B cells. So you have both the classical and the alternative complement pathways
in play. That causes inflammation that gives you the vascular changes that allows
other cells to get into the area and prevents the bacterium from getting out of the
area, except for the efferent lymphatic, which is going to drain into the lymph
node where the bacteria will be trapped and either eaten by macrophages or acted
on by other cells. Neutrophils eliminate the bacteria and then the macrophages
and dendritic cells secrete cytokines. Macrophages are phagocytic. Theyre not
fully functional yet. It takes a TH1 T cell to make them fully functional. The
dendritic cells are going to pull up stakes. Theyre going to follow the pathogens
off to the lymph node and thats where well begin the adaptive immune response,
starting with the dendritic cells.

[66] [Intracellular Pathogens]
[McCutcheon] Intracellular Pathogens. Intracellular Pathogens start by infecting
dendritic cells. Now, one of the unique things about a dendritic cell is that its
capable of being infected by all types of viruses. So viruses, generally speaking
are tissue specific. The HIV virus can infect T cells, CD4 T cells and some kinds of
macrophages. Rhinovirus can infect nasal epithelium. Okay? Uniquely, dendritic
cells can be infected by any type of virus. Its a unique property. What happens
when the dendritic cell is infected by the virus..so it starts off with dendritic cells
being infected by the virus, and then the C3b piece of complement, you have to
have that on the virus when its in the extracellular space because you have to
have that to get B cells activated. So this where the immune response starts for
intracellular pathogens. They infect the dendritic cells. And then you have the C3b
pieces which are then for the B cells.

[67] [Tissue Macrophages=Dendritic Cells]
[McCutcheon] So, these are dendritic cells in epithelium. The red stuff, thats the
body of the cell. You can see some of the dendritic cell arms. So there are lots of
dendritic cells in your epithelium because epithelium is one of the places where
you break the barrier and you get pathogens.

[68] [A Dendritic Cell]
[McCutcheon] A scanning micrograph of a dendritic cell, so again you can see
how many arms it has.

[69] [Intracellular Pathogens]
[McCutcheon] Andwe will pick upIll start the intracellular pathway again on
Friday the 11th, so well finish the last few slides of this, and then well go into
our next lecture. So, have a fantastic 4th of July. Studywhich is not what you
want to hearand Ill see you a week from Friday.

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