15/16: Inflammation I/II

Transcribed by Mandy Weil 5 September 2014
[General Pathology] Inflammation by R. Craig, DMD, PhD

[Dr. Phelan] Did you all get the message that the revised schedule is up for the
fall? The only difference in that schedule is that the exam is on Monday. And the
conferences for that week are cancelled. They will be on your schedule cause I
cant fix that. But there are no conferences. Having conferences the week of an
exam means that somebody has a whole bunch of you will have conferences the
morning of an examyoud rather be anyplace else than in a conference on
another subject. Nicely, there was an extra conference that was scheduled, that I
hadnt used and thats the one that was on the 27th. So i was able to move that and
free up that week of the exam from conferences. But those are the only changes.
Everything else should be exactly the same.

[ Slide 1] [Title Slide ]
[Dr. Craig] Can everyone hear me? I guess so.. Hello? It works. It works.
Alright.Okay. Good morning everyone. Ive been asked to be with you for the
next 6 sessions. Talk a little bit about inflammation and wound healing. And I
know youve already had your unit about inflammation with Dr. McCutcheon. Dr.
McCutcheon went over innate immunity and the prime effector of innate
immunity is the inflammatory response. So some of this going to be redundant.
Some of this might be a review. My intent is to try to present inflammation from
perhaps a bit of a clinical perspective because were going to deal with
inflammation on a daily basis in your practice. So with that, I should also tell you
that we have some learning aidsas you usually do with mefor these 6 hours. If
you get onto NYU courses you should be able to locate the power points and you
should be able to locate the lecture handouts. On the lecture handouts, as I usually
do, right in the front, theres a reference section and I usually divide references
into required, which means fair game on educational assessments, otherwise
known as exams. And then, non-required. Non-required are for those folks who
want to see some of the primary reference materials that I use. And i will be using
that in wound healing and also in the systemic effect of inflammation lectures. So,
having said that, when you get to the Robbins textbook, they write a lot on
inflammation and actually its at a fairly good level for us. The Robbins textbook is
really written for medical students. And medical students have different needs
than dental students. And so theres certain areas that Im gonna kinda expand
that are not covered in the Robbins textbook, and certain areas that Im not gonna
touch on at all. So when you read the Robbins textbook, and I highly suggest you
read the Robbins textbook, have the lecture handout by your side as a handy
guide. If Im not making a big deal about the 4 million cytokines that participate in
the inflammatory response, but only focus in on three, I would probably
concentrate on those three. Questions so far? I want you responsible for what the
resources are. Okay. So, um, lets talk a little bit about inflammation.

[Slide 2] [Outline]
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[Dr. Craig] Im gonna try to set the stage a little by talking about the host
defense against infection and I know Dr. McCutcheon has prepared you well for
that. And then were gonna talk about the clinical signs of inflammation. Because
this is kind of historically how people came to grips with this reaction. And then
were gonna divide our comments today and then the beginning of next
weekwe meet next Monday, is my understandinginto acute inflammation and
chronic inflammation. And this is how classically the inflammatory response has
been divided. Youll see how there is some artificiality based here. Well spend
most of our time talking about so called acute inflammation. And, um, well talk a
little bit about how edema is generated. How the body, how your patient recruits
out of the vascular compartment specific and appropriate cells of the innate
immune response into the site to deal with the challenges being presented. Were
also gonna talk a little bit about how phagocytes, things like macrophages,
monocytes and especially polymorphonuclear leukocytesotherwise known as
neutrophilshow they target, how they snarf down bacteria. Cause most of our
diseases in dentistry are bacterial diseases. And how they kill them. This is of
importance to us because as well see, as youll see in clinic too, the inflammatory
response comes at a really big price. Theres a lot of collateral damage. You know,
as I read the newspapers, I see thiscollateral damage. Well it really kind of
applies to the inflammatory response because a lot of the mechanisms that the
body uses to uncover pathogen also has bad effects for the host, especially if that
reaction carries on for a long time. Well talk a little bit about how, um, innate
immune cells recognize pathogens. I know Dr. McCutcheon probably talked to you
about Pathogen Associated Molecular Patterns (PAMPs). Well also talk about
damage associated molecular patternsso called inflammosome. And then well
talk about ways that you can resolve inflammation and theres some really neat
things on the future that youre gonna use clinically I predict that are gonna be
radically different than say what Ive done in my practice.

[Slide 3] [Host defenses against infection]
[Dr. Craig] So, as Dr. McCutcheon probably told you: theres three levels of host
response to infection and trauma. The first is physical barriers. The integument.
You know stratified squamous epithelium with keratin. Keratins really great.
Also, has acidic secretion, sebum, those sorts of things, which inhibit bacterial
colonization. Mucosal surfaces are those surfaces of the body that are specialized
for communication with the outside. Gastrointestinal tract for nutrition.
Respiratory for air exchange. So on and so forth. And those surfaces are really
susceptible to pathogenic challenge, to infection, because they dont have keratin.
Theyre kinda easy to breach. Most of them, except our area, have mucous. And one
of the things thats interesting about periodontitis is we have these teeth that
come from a calcified mesenchymal tissuepierces through that barrierand is
sitting out there in the gastrointestinal tract. And when we talk about
periodontitis in your next course, which is Diagnosis and Treatment of Oral
Diseases, i think in October, were gonna come back to that idea. So anyways, so,
the first layer is physical surfaces. You know in my practice, I shake the patients
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hand: Hello Mrs. Jones, how are ya? Sit her on a seat, and immediately Im past
physical barriers because I give a local anesthetic. Probably pick up a Bard Parker
blade, start doing surgery. So immediately Ive breached that. You know, I do clean
surgery, I dont do aseptic surgery like they do at the Langone Medical Center.
Right. So as you put your Bard Parker blade in, youre gonna drag in
bacteriasome really bad bacteriainto the site of the incision. So the patients
responsibility is to mount a really effective innate immune response. Were just
gonna briefly go over the innate immune response so its kinda firm in your mind
what that response is. And the focus of our attention is going to be the
inflammatory response, which is the prime effector of innate immunity. And then
Dr. McCutcheon ably talked about adaptive immunity. Im just gonna have a few
um comments to compare and contrast those two. And so this is kind of an
interesting idea. So innate immunity happens very, very quickly as you know. And
if it works, you dont even see signs or symptoms of much disease. If it doesnt
workthen you have pathology of quite often, quite significant pathology. The
medical profession has been very interested in those challenges that breach
innate immunity. So physicians in the medical profession have really focusd in on
this third level: adaptive immunity. Howe the patient mounts an adaptive
response. How you can help patients during that time. What are the mechanisms
involved. For years, folks didnt really focus in on innate immunity until Charlie
JanewayCharlie, like I know himCharles Janeway, who is diseased. Past chair
of Microbiology at Yale. Came up with the idea that, gee, weve been studying all
this immunology, but what really paves the way for the adaptive immune
response is the so-called hard-wired innate immune response. So now people are
really interested in the inflammatory response. And in the last 10-15 years or so,
the knowledges gained has really been unbelievable with lots of payoff in our
profession. Well talk more about that on Monday. So, innate immunity: ancient
form of host defense. Its not the most ancient, but it is an ancient form of host
defense. Um, and what characterizes the innate immune response is that your
successful

[Slide 4] [Innate immunity]
[Dr. Craig]
ancestors, the ones that lived long enough to actually contribute their genes to
the gene pool. Not the ones that didnt. But, the successful ones, they over time,
evolutionarily, learned to recognize certain immutable characteristics of
pathogens. It can be a structure or it can be a metabolic product that that pathogen
cant mutate away from. And so, you have a set, half from your mom, half from your
dad, of genes that encode for receptors that recognize various structures that
pathogens cant mutate away from. Pathogen associated molecular patterns. So
since, this is dentistry, lets focus in on bacteria. Tell me something bacteria do or
have that you as an eukaryote dont haveWhat do they do differently that you
can exploit(student response, inaudible)Hm? They have cell walls. Alright? Do
you have cell walls? No. You dont have cell walls. Youre a multicellular organism.
You use your integument to keep a constant osmotic pressure. Keep a constant pH.
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So, you have a plasma membrane, you dont have a cell wall. Bacteria dont have
that. So they have this structurally complexthat you learned about in
Microcell wall hat buffers them form radical changes in osmotic pressure,
radical changes in pH, those sorts of things. And on that cell wall, as you know
from Micro, theres a lot of interesting carbohydrates. One of which is mannose.
And I forget the derivative of mannosebut its mannose, right? When mannose is
presented on a surface within the body, there are receptors that recognize
mannose and theyll say Ack! Thats not self. It has to be a pathogenLPS,
endotoxinespecially in our fieldendotoxin really drives um both innate and as
you know, adaptive immunity. Right? Second stimulus in adaptive immunity.
Many points in adaptive immunity. So we got cell wall. What else do bacteria do
differently than you do? (silence) Not all at once!.
HahaDr. Craig! Youre so early and Im still you know, thinking about the
weekendHow do they do protein synthesis? What are the first 3 amino acids that
they synthesize with any protein thats gonna be exported? (student response)
Okay! So f-Met-Leu-Phe, so formulated Methionine, Leucine, Phenylalanine.
Bacteria, prokaryotes, because of their ribosomes, start everything that way. You
dont start your protein synthesis that way. You wouldnt be caught deadwell
actually thats a punsynthesizing that way. So, if there are any proteins or that
3-amino acid peptide present in the extracellular matrix, the immune system
knows. F-Met-Leu-PheOh! It has to be a prokaryote in the area. So thats another
pathogen associated molecular pattern. You also know that bacteria have different
adjuncts onto their DNA than you have. Right? Naked DNA itself is a bad thing.
Right? Should be in the nucleus. But especially when it has these different
adjuncts on it. Ugh..It has to be coming from a prokaryote. So you kinda get the
idea. That characterizes the innate immune response. Second characteristic of the
innate immune response is: once the host recognizes that Pathogen Associate
Molecular Pattern, it either tries to destroy the source and/or it starts to signal to
the outside that it needs recruits. Alright? So, the containment of that infection in
an innate immune response happens in the site of infection. Right? Happens in the
site of infection. And once recognized, theres a set of host effector mechanisms
that hopefully well get to at the end of today, that contain, or hopefully destroy
that pathogen. As opposed toDo i have a slide on this? Actually I dont. As
opposed to adaptive immunity.
What characterizes adaptive immunity? Right? The immune response is not
assembled at the site of infection. But its assembled at deep holes ((hard to
decipher this phrase; said when there is 1:42 of podcast remaining)) where the
body has anatomically set up to increase the concentration of antigen draining
from an area. Lymph nodes. Secondary lymphoid tissue. Right? Theres a set of
antigen presenting cells. What are those antigen presenting cells in secondary
lymphoid tissue? Dendritic cells. We got one. A person..is it Steinman? Just up
the street. Got the Nobel Prize two years ago for his work on Dendritic Cells.
Unfortunately passed away. Whats next? Macrophages. Whats the third? B
cells. Excellent. So Dr. McCutcheon did a good job.
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So being presented in various forms in secondary lymphoid tissue on the antigen
presenting cells are these wonderful bits of the pathogen. And then B and T cells
come in, right? Via the arterial circulation. Circulate through the secondary
lymphoid tissue and test the goodness of fit of their B cell receptor or T cell
receptor to bind with that piece of the pathogen. Those T cell receptors and B cell
receptors are generated by very unique genes. RAG 1 and RAG 2. Actually takes
pieces of genes in B cells and T Cells during development and cuts them and
splices them together. Almost in a Montecarlo stochasticby chancefunction to
generate genes that will encode for proteins that become the B cell and T cell
receptor that may or may not have affinity during that cells life for anything
outside of nature. So what characterizes the adaptive immune response is you
sequester all of the antigen in one place and you try to sort out from all of those B
and T cells that you have circulating in your body the few, the very few, the
selectright? its like the marinesthe very few that actually can bind with that
pathogen. And then you take those cells and you actually teach them to bind with
higher and higher affinity to make either effector T cells or plasma cells, which
makes antibody.
What blows my mind, well lots of things do as I get older, but, what really blows
my mind is this strategy that vertebrates have come up with, because adaptive
immunity is a characteristic of vertebrates, right? Helminth worms, those guys
dont have it. But vertebrates, backbone, weve got it. This strategy thats been
adapted will recognize pathogens that havent even evolved yet. Thats amazing!
Thats really powerful, you know. And that was all done by evolution, I guess. So,
anyways, thats the difference between innate and adaptive immunity. So lets
focus in on innate immunity. So The primary effector of innate immunity is
inflammation. Okay? The inflammatory response. Lets talk a little bit about the
inflammatory response. So the inflammatory response is the reaction of a tissue.
And its microcirculation to a pathogenic insult. And youll see that the insult can
be very broad.

[Slide 5] [The inflammatory response]
[Dr. Craig] So this is a very generalized reaction that the body has. Inflammation.
The primary objective of the inflammatory response is elimination or containment
of the insult and removal of damaged tissues. Thats what it says in the textbook.
But theres a number of complex diseases that well talk about when we get into
Diagnosis and Treatment of Oral Diseases class that are so called complex diseases.
And there are some diseases, like type 2 diabetes. where inflammation is a major
component, but it appears to be a derangement in lipid biosynthesis. And a few of
the things called the metabolic syndrome..So even, theres no real, insult there,
other than being overweight, that incites the inflammatory response. So the
inflammatory response is really kind of universal throughout vertebrate biology.
And as you know, you need to have inflammation in order to get an adaptive
immune response. So this is myI dont know whyI always get the most difficult
lectures to give. I wish I could just getI dont know, like taking alginate
impressions or something that was like, very simple and straightforward. I always
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get these complex things. So anyways, you can imagine that if inflammation is so
important for the survival of the species and theres been this complex interplay
over evolution between pathogens who want--

[Slide 6] [The inflammatory response]
[Dr. Craig] --to garner all the energy and resources that are in our bodies. And
our successful ancestors who are successful at thwarting those infections, so, you
know bacteria and viruses and other pathogens have sort of mutated and evolved
to get into us, and weve kinda mutated and evolved to come up with defenses
against it. And this has been going on for eons. You can imagine the complexity
that must be there. It is complex. So what Im gonna try to do is, Im gonna try to
distill out, in my best attempt, to give you the details of the inflammatory
response that are important to us at this moment as clinicians. But I want to give
you the idea that were entering into very complex ground. And our
understanding of inflammation is still developing. As I said, its an intense area of
investigation for the last 12-15 years or so. So during your clinical lifetimes, you
have to kinda keep up with this area because theres gonna be a lot of applications
for clinical dentistry and you dont want to be left out. So whats the clinical
significance? Inflammation is encountered daily in dental practice. Caries. Um,
pulpal disease. Periodontal disease. Inflammatory diseases, right? Many chronic
diseases have inflammatory components. As well talk about on Monday:
atherosclerosis, type 2 diabetes, Alzheimers disease, rheumatoid arthritis,
osteoarthritis. They all have a major component of an inflammatory response that
perhaps is not appropriate.

[Slide 7] [Clinical significance]
[Dr. Craig] Inflammation is an essential first step in wound healingso this is
gonna kinda like pave the way for our discussion next Friday on wound healing,
and uh finally, this has been the golden fleece in my little area: chronic
inflammation, perhaps from oral sources, may contribute to systemic diseases,
such as heart attack and stroke. Okay. So the Egyptians actually wrote about
inflammation. But the first person who is actually credited with it is the Roman
Celsius. My reference says..uh..second century source says first century. I dont
read Latin, so I really have a hard time with that. But anyways, hes usually
credited with classifying the four classic signs of an inflammatory response:
redness, heat, swelling and pain.

[Slide 8] [Classical (cardinal) hallmarks of inflammation]
[Dr. Craig] And this is uh, this periodontal patientwhoops, there goeshe
already lost a tooth to periodontitis, right? And if you look at this patients
tissues, you kinda see this redness here, right? And if you had an instrument, and
there is an instrument called the periotron, which you put, uh, thermal ____
((cannot decipher this word, 1:33:30 of podcast left)) on the core temperature of
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the patient and have like a little hand piece measuring device. And you can
actually put it into the sulci and youll actually measure the differences between
basal core temperature and peripheral temperature. Youll actually pick up sites
that have increased, you know, uh temperature. So you have Calorheat. Youve
got swelling in this area. This is not normal gingival architecture. The only thing
that you dont normally have, or usually have, is pain. Because most of this
inflammatory exudate, swelling and stuff, things that cause pain, have egress into
the oral cavity. But if this same response was further downsay in the apex, youd
have extreme pain. So um, periodontal inflammation has the 4 characteristics,
right. So what are the characteristics of an inflammatory response. The first thing
is to identify that there is an insult, whether it be pathogenic, whether it be you.
Whether it be something else that is considered an insult, and there are many
things. And the first thing that happens is generation of various soluble
inflammatory mediators. And were gonna talk quite a bit about what they are
because theyre wonderful targets for you in clinic, uh, to help control
inflammation for your patient.

[Slide 9] [Components of the inflammatory response]
[Dr. Craig] And then those inflammatory mediators work on the endothelial cells
of the microvasculature within that site. Right? So especially the arteriole,
capillary and post-capillary endothelium are gonna be major players in regulation
of inflammation. And then finally, under the control of these inflammatory
mediators released into the local environment, theres gonna be first movement of
fluid from the vascular space to the extravascular space generating edema,
generating lymph. Increase lymph flow. And then finally those endothelial cells
are also gonna have a specialized mechanism for picking up the appropriate
leukocytes, white blood cells, that are passing through in the circulation and
recruiting them out of the vascular compartment into the extravascular
compartment to deal with the challenge. So those are the three components:
generation of the inflammatory mediators, change in those endothelial
cellsthose endothelial cells are gonna become really important. And then,
finally, the movement of fluid and cells, regulated by those endothelial cells, from
the vascular to the extravascular space.
Okay sohow many people went to Parochial school? Just one? Okay so I
remember when I was maybe about, I dont know..fifth grade or something like
thatand I wasnt a very good student. And I remember we were having this
lecture or something, some class thing and the girls gym class was outside, and it
was kind of a nice day. So, I was kinda like watching the girls gym class and not
really paying attention to what I was supposed to be paying attention to

[Slide 10] [Acute inflammation: physical signs]
[Dr. Craig] and I had my hands out and all of a sudden this yardstick came out
of the heavens, and Sister Brigid brought me back into the classroom with this
WHACK. And I remember kinda sitting there. At first it was painful. But I could see
the indentations of the yardstick. And the first thing that happened was the entire
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area across my hands became kind of white. So, the first thing that happens during
an inflammatory responseand this is under control of innervation of the small
arteriolesis just a transient vasoconstriction. Perhaps its there to try to control
hemorrhage in really severe lacerations. And then, as Im watching and the
painit was a great rush, I remember the pain was just an unbelievable rush. Im
looking and all of a sudden that area starts to get red again or hyperemic, right?
Hyperemia. So both the arterioles and the venues in the area start to relax and
open up, so theres more blood going into the area. That gives you that red
appearance and also in time the increase flow of blood to the area increases the
temperature of the area. And then, finally, after about, I dont know, 5-10 minutes,
something like thatI remember because the girls gym class was going back
inthere was a lot of swelling that occurred in the area. So, theres a number of
people whose names are kinda classically associated withso this is the so-called
triple response first described by Lewis is 1924, thats not important for us.

[Slide 11] [Gingival health]
[Dr. Craig] Alright. So heres, just to drive it home, so heres a picture of so
called gingival health. The gingiva mimics the cement enamel junction. If you push
on this tissue, it doesnt blanch. Its really nicely, um, conforms to the underlying
alveolar bone.

[Slide 12] [Gingival inflammation]
[Dr. Craig] As opposed to this poor gentleman, everything is red, everythings
inflamed. This is a case of gingivitis, so you can kinda see.

[Slide 13] [Cellular events and inflammation]
[Dr. Craig] So anyways, now were beginning to focus down and were going to
leave the clinical, right. What you see chair side. And now were gonna begin to
focus down first at the cellular level, and then were gonna go into the molecular
level because theres a lot of therapeutic targets here. So, classically, inflammation
has been divided basically by the histopathology of these two readtions into
something called acute inflammation and chronic inflammation. Again, as we begin
to understand more about inflammation, the demarcation between acute and
chronic is becoming less and less clear.

[Slide 14] [Cellular events in inflammation: acute inflammation]
[Dr. Craig] So, here we have a little diagram lifted from your text. So up here,
this is a normal capillary bed, arteriole, venule draining, a nice capillomeric plexus
here. You may have some sentinel (?) cells in this connective tissue, dendritic
cells, right? And some macrophages kinda sitting there waiting for, um, to detect
if theres any pathogenic challenge in the area. And if there is a pathogenic
challenge then, especially the macrophages start to release vasoactive mediators.
And it goes to the arterioles and the venules and it tells those smooth muscles to
start to relax. Alright? So this increases blood flow in this area. But, because of
fluid dynamics, if you take a pipe and you keep the flow constant, or the pressure
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going in there constant, and all of a sudden you dilate it: now youve got a bigger
volume. So now the flow starts to go slower. So even though you have more blood
in the area, flow starts to decrease. And sometimes this flow is so slow that the
erythrocytes begin to lose their oxygen content. Youll learn in clinic that
inflamed gingiva is sorta cyanotic. It turns, like, this bluish red color and thats
because of the changes in vascularity during inflammation. The next thing that
occurs, is theres increased vascularity because the endothelias cells that are
lining this microvasculature begin to contract and expose the underlying
basement membrane. And basement membrane works like an ultrafilter. So
anything in blood, which is below the molecular weight cutoff, will percolate out
of the vascular into the extravascular space. Another thing that happens during
inflammation is that endothelial cells start to release mediators to platelets and
platelets begin to clump together and degranulate. So intravascular platelet
activation. And then, these endothelial cells, later on will start to express adhesion
molecules that will select out appropriate cells of the innate immune system. So
early on, say its a bacterial challenge, it will be polymorphonuclear leukocytes,
like in this little cartoon. Later on it may need monocytes or other cells such as T
and B cells into the area.

[Slide 15] [Cellular events in inflammation: acute inflammation]
[Dr. Craig] So this is sort of a schematic of inflammation. What does this actually
look like? So heres a slide that was generously donated by the Dr. Phelan
Research Foundation, I think, years ago. So anyways, this is what
histopathologically acute inflammation looks like. And what you see hereyou
know, you guys dont have to look at micrograph slides anymore so what you guys
are probably seeing is a bunch of dots, huh? But if you look at the dots real
carefully, youll see that theyre like individual cells and this dark stuff is the
nuclei. And youll notice that these nuclei are kinda like multi-lobed. Alright?
Theyre polymorphonuclear leukocytes. So this whole field is almost completely
populated by polymorphonuclear leukocytes. This is the classic presentation of
acute inflammation.

[Slide 16] [Cellular events in inflammation: Chronic inflammation]
[Dr. Craig] Chronic inflammation. So chronic inflammationaccording to the
textbookusually follows acute inflammation. If acute inflammation hasnt able to
resolve the challenge. But acute inflammation by insidiously develop in areas of
malignancy. Viral infections, parasitic infectionsthose sorts of stuff. So, you
dont have to start an inflammatory response with a so called chronic
inflammatory response. And what characterizes chronic inflammation is the
cellular infiltrate. The inflammatory cellular infiltrate. So in acute inflammation its
polymorphonuclear leukocytes. In chronic inflammation, its much more divers. So
youll see mononuclear cells and their derivatives: macrophages. Youll also see
small lymphocytes. Right? And you may even see plasma cells. In fact, when we
talk about periodontitis, the characteristic lesion of established periodontitis are
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plasma cells. Actually antigen-specific-antibody-secreting plasma cells, which was
always a bit of a question mark in folks minds, up until recently.

[Slide 17] [Cellular events in inflammation: Chronic inflammation]
[Dr. Craig] So heres the histopathology of a chronic inflammatory lesion. And
again, if you look at the pepper grains (chuckle), alrightthe individual cells. All
of the cells that have that multilobularthe nuclei, rather, dont have that multi
lobular appearance any longer. They look more like small lymphocytes that have
been populating this area.

[Slide 18] [ Possible outcomes of the inflammatory response]
[Dr. Craig] Okay, so what are the possible outcomes of the inflammatory
response? And so this is a classic term from pathology: so-called resolution. So if
everything works well, right, the innate immune response works well, identifies
the pathogen. The correct innate immune cells are recruited into the area, they
destroy the pathogen. Everything goes back to where it was before: you get
resolution. Right? And thats always been, at least is perio, thats been kind of like
the golden fleece. We wanna be able to get regeneration and you kinda know from
Craniofacial Biology course, thats indeed possible. Unfortunately a lot of times
what happens is scarring. So, perhaps, resolution of the inflammatory response is
not 100%. And, you have inappropriate expression of type I collagen and other
genes associated with the extracellular matrix. So the anatomy is not truly
reinstated. Some of the function may be, but perhaps the anatomy is not.You end
up with something called fibrosis which can have very serious clinical sequelae. So
much so, that the NIH floated a very big program project last year looking for
grants that will study in depth fibrosis after wounding. And then what also can
happen is that that lesion can progress from an acute inflammatory response into a
chronic inflammatory response. In fact, there are diseases where components of an
acute inflammatory response coexist with chronic inflammatory responses and
theyre associated with excessive extracellular matrix degradation. And
periodontitis fits into that very, very well.

[Slide 19] [Stimuli for acute inflammation]
[Dr. Craig] Okay. What are the stimuli for an acute inflammatory response? So,
from Dr. McCutcheon, you probably learned that you can kinda loosely divide
pathogenic challenges into four different types, right? Um, bacteria, prokaryotes,
viruses, fungi and then this grab-bag of protozoans and worm-type parasites.
These guys are all, the individual entities are smaller than a human cell. These
guys are bigger than a human cell. And you should know by now that certain arms
of the adaptive immune response have evolved to deal with these components.
Alright? But however, the inflammatory response is more broad than just dealing
with infections. It also can sense if there is tissue that has died, become necrotic or
tissue that is on its way to dying. And well talk a little about that. Uh, foreign
bodies, sutures, are really notorious for setting up inflammatory responses. Um,
talc, from surgical gloves, really good at setting up inflammatory responses.
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Immune reactions, that youll learn about more when this course talks about auto
inflammatory disease, autoimmune diseases. And then of course, trauma. And most
of us in this room will inflict trauma everyday on our patients in the name of
surgery, alright? So surgery is nothing really more than, hopefully, controlled
trauma.

[Slide 20] [Tissue injury or infection]
[Dr. Craig] Okay, so this is sort of like a forest, not a trees slide here. This is
gonna be kinda like our outline for the next few minutes. So up here, we have
tissue injury or infectionits very, very wide category of things that can uh
precipitate an inflammatory response. And then we said: the first thing that
happens in an inflammatory response is the production of inflammatory
mediators. And those mediators come in 2 flavors. They can either act on
endothelial cells to increase the edema, increase dilation of the arterioles and the
venues in the area and also increase the permeability of that microvasculature,
which allows fluid to move from the vascular to extravascular space: producing
edema. And well probably end up talking about this in this first hour.
And then the second hour: the second form of inflammatory mediatorsand some
of these will actually do both things, as well seeare chemotactic factors. So
theyre factors that are released locally in the challenged environment, which tell
or signal the endothelial cell, gee, I really need to have some neutrophils into this
area if its a bacterial infection. And well talk a little bit about these and these
should be kind of familiar to you from Dr. McCutcheons lecture. This results in
inflammatory cell recruitment and activation. And depending upon the type of cell
thats recruited, youll either end up with acute inflammation or chronic
inflammation.
So, um, the list of vasoactive mediators goes from here approximately down to the
center of the earth. So Ive only highlighted the ones that are targets for the drug
industry, that you will be using to help manage your patients. Or, ones that have
kind of interesting nuances that will probably be targets in the future. So
everything here is clinically relevant. And Ive done kind of the same thing over
here. Chemotactic factors dont go to the center of the Earth. They probably only
go 100 miles down or so. But, theres an enormous amount of chemotactic factors
and were just gonna talk about the ones that are perhaps more clinically
significant to us. So just get a sense that youre getting a view, sort of a
tip-of-the-iceberg kinda thing.

[Slide 21] [Images]
[Dr. Craig] So! What does this actually looks like? So heres a little cartoon.
Heres a normal venule or capillary cross-section, right? You got some
erythrocytes, theyre forming rouleaux. They form, because of fluid dynamics.
Here, this area will have some friction as fluid flows through it. And so, the fastest
flow is gonna be in the center of the capillary. In addition, endothelial cells have a
net negative charge and erythrocytes have a net negative charge. Theses
electrostatic repulsion. Sot his kind of keeps everything in the center of the flow.
11

And its the fastest. Anyways, if you look, this little venule here is lined by living
endothelial cells and where they touch one another, theres a tight junction. So
heres a transmission electron micrograph. So heres the cytoplasm of one
endothelial cell. Heres the cytoplasm of a second endothelial cell. And heres this
little tight junction uniting the two of them. And all of a sudden if there is a
vasoactive amines that are being released into the area. Theres receptors for the
vasoactive amines on the basal surface of those endothelial cells.Those endothelial
cells recognize that. They activate their cytoskeletal network and they begin to
contract. Um, we had a researcher that would culture endothelial cells in culture
plates. And if you added stuff to it, you could actually see them contract in culture.
It was kinda neat. But thats what they do in situ here. And whats happening in
this situation is that as their tight junctions disperse, and they expose the
underlying basement membrane, right? Now fluid from the vasculature can leak
out into the surrounding area, causing edema. So since the basement membrane is
in tact, and you can kinda think of itWhat kind of collagen is there? In basement
membranes? student: four Four! Excellent. Remember, when you see me, its
gonna be a collagen question. Alright. So anyways, type IV collagen forms a
mesh-like array, right? It has a molecular weight cutoff, just like an ultrafiltration
membrane. So, low molecular weight molecules are gonna get out. High molecular
weight molecules are not gonna get out. So early on, during an acute inflammatory
response, the edema is called either an ultrafiltrate of blood or a transudate.
Alright? And, if thats enough to take care of the response if this is a very mine
response indeed, then these cells will reestablish their connections and the edema
generation will stop. So, heres an electron micrograph of one endothelial cell.
Heres another endothelial cell. Here, the gap junctions have been opened up
exposing this underlying, um, basement membrane. If, uh, you have say a heat
injury, and you actually damage the endothelial cells, this extravasation of fluid
into the extravascular space will continue. And so you get continued generation of
inflammation.

[Slide 22] [Inflammatory vasoactive mediators]
[Dr. Craig] Ao lets talk a little bit more about the actually mediators. So you can
kind of divide the mediators into two forms, right. This is the innate immune
response, so these are kinda generated very quickly, if not already pre-formed. So
those in the green box are either already pre-formed in cells or are made very
quickly de novo in cells. And these are the guys were gonna talk about.
And then theres another group of vasoactive mediators that are pre-formed or
very quickly synthesized in the vascular compartment, and well talk a little bit
about those too. So, um, your book starts off with histamine and serotonin. Im
gonna start off with prostaglandin. So were gonna start off with these guys cause
theyre real important in our profession. The generation of any or all of these
components is going to act to increase permeability through contraction of those
endothelial cells, resulting in edema.

[Slide 23] [Cell-derived mediators: arachidonic acid]
12

[Dr. Craig] So, lets talk a little bit about arachidonic acid. So, this is a real
ancient signaling pathway based on membrane lipids. And its not just for
inflammation, unfortunately. Its used throughout the body for various things.
Controlling permeability to kidneys and things like that. So, under the
appropriate stimulus, many, many cells, if not all cells, in the extracellular matrix,
by activating a protein called phospholipase A2 can take
phosphatidylcholineWheres phosphatidylcholine found? -Cell membranes.
Excellent. Theres a lot of it right. You can take phosphatidylcholine and make
arachidonic acid. Another enzyme, phospholipase C can take phosphatidyl inositol
and also make arachidonic acid. So once the cell pool of arachidonic acid begins to
increase, it can either go in one of two pathways. Or it can go in both, depending
upon the inciting molecules or depending upon the cell type.
The cycloxygenase pathway takes arachidonic acid, and this is the black box, so
there are several things in here that we dont need to know, okay. And it can
generate 2 different classes of molecules. One class of molecules, derived form
lipid, are called prostaglandins and literally, the list of prostaglandins that have
been isolated, really goes way, way down off this screen. Were only gonna ask you
to remember twoonly two. But realize, the list is long. Alright? Prostaglandin
E2 is synthesize early in an inflammatory response. It is used as a mediator for
many different things. One of the things it does is it increases edema in the area. It
also sensitizes nerve endings, pain, okay. And then, its synthesized macrophages,
monocytes, resident cells that have been damaged. Endothelial cells will also make
prostaglandins. And then, later on in the inflammatory response, during
resolution, endothelial cells will shift their synthesis and start making another
prostaglandinthat Im gonna hold you toProstaglandin I2. And this one is sort
ofIts not totally true, but for our discussions, were going to discuss it as sort of
being the antagonist of Prostaglandin E2. Okay? Were gonna come back to
Prostaglandin E2 many times during our discussions. Especially during
periodontitis. The cycloxygenase pathway can also take a arachidonic acid and
make a whole class of lipid derived mediators called thromboxanes. The only one
Im gonna ask you to remember isThromboxane A2. Everything in these red boxes,
by the way, are gonna increase edema. Increase endothelial permeability. But
Thromboxane A2 also has another function. Its synthesized by platelets and is
very important in platelet aggregation and establishing the platelet plug, one of
the first steps in hemostasis. And its 8:52. At this point in time, perhaps this is a
good time to stop, and well come back and finish up in the next hour. Ill take
questionsmy throat is beginning to go so Have an 8 minute break.
[Same slide after the break]
Okay everyonesoum, what we need to remember or what Im asking you to
remember, out of the enormous shopping list of arachidonic acid metabolites: for
at least for Cycloxygenase is that Cycloxygense makes both prostaglandins and
thromboxanes depending on the situation and the cell type. Uh, PGE2: early on,
big player in periodontitis. Sensitizes nerve endings, painful, that sort of stuff. Uh,
PGI2 synthesized mainly by vascular endothelial cells. Later on in the
inflammatory response promotes resolution. So its sort of like the antagonist in a
13

way to PGE2. Thromboxanes made my platelets. Right. Not only are these
vasoactive mediators but they also participate in the reactions that allows
platelets to adhere. Right. Form platelet plug. Arachodonic acid can also be taken
down the Lipoxygenase pathway into a series of compounds that I cant even
pronounce. So HETES is one of the end products and its very active vasoactive
mediator, promotes edema. Uh, it can also be made into something called
Leukotriene A4 (LTA4). And one of the products of Leukotriene A4 is Leukotriene
B4 (LTB4). And not only is this a vasoactive mediator, this also functions as a very
powerful chemoattractant, especially for neutrophils. And LTA4 can also be
metabolized into Leukotriene C4, D4 and E4. And these 3 together are classically
called the slow reacting substances of anaphylaxis. Cause theyre elaborated, um,
they were first described as being elaborated later in anaphylactic reactions, um,
through mast cells. But theyre also important. And these guys also play a role in
diseases such as asthmas and those sorts of things. So, um, this is sort of a scaled
down pathway chart of arachidonic acid metabolism. But, if youre really
interested in controlling inflammation and controlling pain and youre in the
pharmaceutical industry, youd look at this as sort of like the happy hunting
ground. Its fairly easy to synthesize some of the inhibitors. In fact, some of the
inhibitors were known hundreds of years before they were actually synthesized.

[Slide 24] [Cell-derived mediators: arachidonic acid metabolite inhibition]
[Dr. Craig] So, um, lets take the cycloxygenase pathway first. As it turns out,
there are two forms of cycloxygenase, the enzyme. COX1 and COX-2. And it was
sorta thought, up until a few years ago, that COX-1 was constitutively expressed in
many parts of the body. And that COX-2 was only associated with inflammatory
cells and it was only induced, its synthesis was only induced during times of
inflammation. So thats kinda an interesting insight. And it has turned out that
there are a class of drugs called COX-2 inhibitors that came out of this finding. But
preceding COX-2 inhibitors were aspirins. So, Native Americans used to take
willow bark and chew on it if they had pain, either headaches, or arthritis, or what
have you. And, something in willow bark was very effective at inhibiting
inflammation and inhibiting pain. And it took folks at the Bayer company in
Germany, during the height of organic chemistry explosion, in the late 1800s to
actually synthesize aspirin, the active ingredient in willow bark. And what aspirin
does is it places an acetyl group and irreversibly acetylates both COX-1 and
COX-2. And so this is a non-competitive irreversible inhibition. And as soon as that
acetyl group is placed on COX-1 or COX-2, that knocks out this pathway and then
everything downstream is inhibited. Alright? So aspirin, a wonderful drug. I wish
I had that patent. I would not be here before you if I had that patent! Cause not
only does it do this, it does other things quite well, and well come back to aspirin
several times.
So, consequently, If youre knocking out cycloxygenase 1 and 2, right? And, you
happen to be a platelet, this knocks out thromboxane A2. Why? Well the COX-1
and COX-2 that you have has been irreversibly inhibited. And you dont have a
nucleus, cause youre a platelet. So, you cant synthesize more, right? So patients
14

who are chronically taking aspirin are at a risk for hemorrhage. And so you wanna
determine that in your dental patients. And well talk about low dose aspirin on
Monday, but theres a lot of folks out there taking low dose aspirins. Uh, aspirin
has other problems though, because its knocking out COX-1 that has a lot of good
effects. It regulates renal flow, uh blood flow, to the kidneys. Its used as a
signaling system in many parts of the body. Folks are really interested in
knocking out COX-2 because this is the one thats induced and it was thought it
was only being expressed in inflammatory cells. So, wouldnt it be wonderful if we
could knock out COX-2 and leave COX-1 in tact? And the Merck Sharpe & Dohme
folks were actually the first ones to crystallize the enzyme COX-2. I remember
sitting at a research seminary and, you know, computer visualization at the time
was just kinda new and there was the active site. And then they synthesized this
molecule that fit into the active site. It was really kinda neat to see. And that was
developed into a drug called Vioxx. I loved Vioxx when it was available. Theres
another one called Celebrex out there also. And Vioxx, the story of Vioxx is kinda
neat. So anyways, you know, to get a drug through they have to go through Phase
1, 2 and 3 trials by the FDA. And you have to satisfy the Federal Drug
Administration that you have an effective, safe drug before you can market it. And
Merck Sharpe & Dohme did diligence. Their phase 3 trials had, I believe, over
9.000 patients. And it was considered to be a safe, and very effective drug. The
problem is phase 3 trials were, at that time, pure randomized control clinical
trials, where you take a defined patient population with a defined randomization
schedule. Give them half the drug, half something else. Look at the outcome. It was
effective. But folks that we kinda see in our practices, are not medically healthy or
not as medically simplistic as the ones in these randomly controlled clinical trials.
Right? We see folks who are at risk for heart attack and stroke. We see folks who
have type 2 diabetes. We see patients who have Alzheimers disease, all of which
were excluded from the phase 3 trials from Merck Sharpe & Dohme. So after a few
years, people at the CDC, Center for Disease Control, started to realize that Gee,
there was this increase in heart attacks from people taking Vioxx. So,
consequently, Vioxx now, I believe its been taken off the market. And, its kinda
sad because Vioxx was really really neat. It was very, very specific for
inflammation associated with inflammatory disease. Um, okay.Because of the
problems with aspirin, because it irreversibly acetylates both COX-1 and COX-2,
the pharmaceutical industry has been really, very diligent in developing
Non-Steroidal Anti-Inflammatory DrugsNSAIDS. And also theres been a big
move now to get away from narcotics, especially in the dental profession. And so
Non-Steroidal Anti-Inflammatories are really big now. They, as a class,
non-competitively inhibit COX-1 and COX-2 by various methods. One that I really
like is Ibuprofen. So the majority of my surgical patients get preloaded with 600
mg of Ibuprofen before their surgery. And its really neat because I can knock
down these pro-inflammatory mediators. These vasoactive mediators, so I can
decrease inflammation. I can also use them as analgesics. So, its kinda a powerful
drug to use. On the lipoxygenase side, theres several drugs that are being
developed. One that you might come into contact with in the clinic is Zileutin. Its
15

a principal used for asthma patients. But it knocks down lipoxygenase, which
knocks down the incidence of these slow reacting substances of anaphylaxis. If
youre anticipating a very large surgery, say, multiple dental implants or
craniofacial surgery, those sorts of things, um, there are folks in our profession,
oral surgeons, periodontists, those types, who may pre-medicate a day before,
patients with corticosteroids. Corticosteroids inhibit phosholipase A2. So if you do
that, you knock down the pool of arachidonic acid and you knock everything down.
Right. The problem with doing that is that a lot of these mediators are real
important for stimulating an inflammatory response. And, you know, the
inflammatory response is there for a good reason, right? To control pathogen, and
help in its elimination. So, um, use of things like corticosteroids, which knock
everything down, you have to be very careful in that now you have a patient who
may be very susceptible to post-surgical infections.

[Slide 25 and 26] [Cell derived mediators: Platelet activating factor and Cell
derived mediators: Platelets]
[Dr. Craig] Okay. A few more. Platelet activating factors. So its synthesized from
membrane phospholipids by our friend Phospholipase A2. You put corticosterioids
in, on a patient, youre gonna knock this down also. And, um, what does it do? It
stimulates platelets to aggregate and release their granules of pre made factors;
one of which is serotonin, which is really important. And it also primes innate
immune cells for activity, a function that Dr. McCutcheon probably touched on.
And its synthesized by endothelial cells, inflammatory cells and cells that have
become injured in the area.
Plateletsreal important..and there are a number of strategies to try to increase
the number of platelets and their products in surgical areas. Youll learn about
platelet rich plasma. In fact, we have recombinant-human-platelet-derived growth
factor for use. Tends to be a little expensive. But, uh, theyre being used in the
clinic. So, platelets have several different vesicles and they have different names.
You dont have to remember whats in the vesicles. Just that dense granules
release serotonin, a very reactive vasoactive mediator. Calcium, to help with blood
clotting. ADP. Um, alpha granules release cationic proteins that begins to
neutralize that negative charge between cells that are in the vasculature. And the
endothelial cells release fibrinogen, coagulation proteins and well be talking a lot
about platelet derived growth factor. Even in this early stage of inflammation, the
groundwork is already being set for wound healing. Platelet derived growth
factor is one of the factors that is very active in the early stages of the wound
healing cascade.
They also have lysosomal vesicles that release acid hydrolyases that are
antimicrobial. And then we talked about thromboxane A2, promotes platelet
adhesion and aggregation.

[Slide 27] [Cell derived mediators: Mast cells and baseophils]
[Dr. Craig] Mast cells and basophils. So Mast cells have wonderful receptors for
the constant domain of IgE. And its thought that mast cells probably evolved, or
16

one of their functions was to evolve to take care of pathogens that were larger
than a single cell. So, things like parasites, helminth worms, those sorts of things.
And, if the IgE is bound to its antigen, then the mast cells release their granules.
And inside those granules is histamine. Right? Serine proteases, chemotactic
factors for neutrophils and eosinophils. Usually will result in extreme contraction,
violent contraction to try to dislodge the pathogen from the mucosal surface. But
mast cells also function in other reactions against non-parasitic infections. Also, if
the receptors are bound, then the slow reacting substances of anaphylaxis are
synthesized and released into the area. Okay?

[Slide 28] [Image]
[Dr. Craig] Heres a little diagram of the mast cell being stimulated. So heres the
EM. And heres a little cartoon. Here are the receptors for the constant domain of
the IgE antibody. If a ligand is bound on IgE antibody, the granules are released.
So you can kinda see all these dense granules being released into the area. Sort of
like a claymore mine for those of you who have been in the military.

[Slide 29] [Cell derived mediators: Endothelial cells]
[Dr. Craig] And endothelial cells. So, theyre the regulators of local tissue
perfusion, late in the inflammatory response, for our discussion. They synthesize
Prostaglandin A2, I2 rather (said with 41:19 remaining in podcast). Um, they also
have nitric oxide, a product of which is a very, very potent vasodilator. Its also
used in bactericidal reactions that well talk about more, probably on Monday.
They also synthesize a protein called Endothelin, which is another vasodilator.
Kinda getting the idea that theres multiple overlapping and redundant mediators
that are being released. And they also release something called Procoagulation
tissue factor in response to endotoxin, LPS or interleukin 1, or TNF-alpha. Um,
so-called pro-inflammatory cytokines that well talk more about very, very soon.

[Slide 30] [Cell derived mediators: Macrophages, inflammatory cells]
[Dr. Craig] Okay so macrophages and inflammatory cells, when their receptors
for PAMPs become bound, they can do several things. One of the most important
things is that genesum, and were gonna talk more about something called the
NF-kappa-B pathway (?)genes associated with the pathway are activated. And
one of the product is something called Tumor Necrosis Factor alpha. Kinda of an
unfortunate name for this cytokine. Uh, it not only increases vascular
permeability, but it also induces expression of endothelial cell adhesion molecules
and also has systemic effects. It usually is found in association with 2 other
cytokines: Interleukin 1, which does similar things to TNF-alpha, and IL-6, which
is similar, it also activates something called the acute phase response, that well
talk about more on Monday.

[Slide 31] [Inflammatory vasoactive mediators]
[Dr. Craig] Okay, so now, weve kinda gotten through the green box. These are
all cell-associated. Youre either pre-packaged or it needs to be synthesized de
17

novo during an acute inflammatory response. What about factors that are present
in the circulation? Present in blood. And theres gonna be three of them. We have
the clotting factorsso, clotting and inflammation are very, very tightly linked
because evolutionarily, if one had a laceration, it was usually not done in an
operating room situation. But was done outdoors with lots of pathogenic
challenge. That first layer of defense has been breached and so theres usually
gonna be a pathogenic challenge associated with a laceration. So it makes sense
that inflammation and uh, clotting, are linked. Um, and youve already talked about
complement, I hope, with Dr. McCutcheon. Complement. Well talk a little bit
about complement in something called the Kallikren-kinin System.

[Slide 32] [Blood-derived vasoactive mediators]
[Dr. Craig] Okay. So clotting/fibrin formation, kinin and complement are the
three interrelated cascade systems that are present in blood. So, uh, as you will
learn in this course, if you havent learned already: clotting is a complex cascade of
reactions. But its initiated by many, many different things. One of the best things
to initiate the clotting cascade is exposed naked type I collagen. Right. And that
sort of makes sense. Collagen is found extracellularly, but not within the
vasculature. And, if all of a sudden components of the blood are starting to see
exposed type I collagen, with its very unique structure, things arent good.
Alright. So its probably a good idea to institute the clotting cascade. Large
negatively charged surfaces: endotoxin/LPS can activate the clotting cascade. And
other endogenous expressed enzymes, such as plasmin and trypsin can also
activate the clotting cascade. We will learn that theres two arms: intrinsic and
extrinsic to clotting. Thats not important to our discussion at this point in time.
Whats important is: activation is something called Factor XII, otherwise called the
Hageman factor. And once that factor is activated, many things can occur within
that site. Kinda linking inflammation and blood clotting cascade. So one of the
things that can happen is the clotting cascade, you get, um, the conversion of
fibrinogen to fibrin. And that stabilizes that initial platelet plug in the
areawhich is a good thing. Um, Factor XII can also activate the Kallikren Kinin
Cascade, ending up with Brady Kinin and other kinds of molecules derived from
that cascade. These guys are also very active vasoactive mediators. They increase
inflammation. They also increase pain. And youll learn more about that in your
endodontics lectures. Uh, Factor XII takes plasminogen and makes plasmin, an
enzyme. And plasmin can activate complement through the alternate pathway and
generate anaphylotoxinsthat well talk about in the next slideincreasing
edema in the area. It can also, later on in the cascade, begin to degrade that initial
fibrin clot. As it turns out, fibrin degradation products themselves also tend to
stimulate inflammation in the area. So the idea that you should be getting at this
point in time is that theres this complex interwoven series of factors all
converging on endothelial cells to increase edema into the area.

[Slide 33] [Blood-derived vasoactive mediators: Hageman factor activation]
18

[Dr. Craig] And last, but not least, for blood products, complementIm not
gonna spend a lot of time on complement because I assume that Dr. McCutcheon
has done that before me. I just want to point out several things that are important
to inflammation. So complement is a group of over 20 proteins that are
synthesized in the liver. In a cascade, oops..get outta herethat are synthesized in
a cascade fashion. And there are four functions that complement activation
accomplishes. The first is targeted cell opsonization. And thats like, real
important. Why is that real important? Well. Most receptors for PAMPs bind
non-covalently with their ligands. Alright? They bind non-covalently with their
ligands. In fact antibody binds non-covalently with its antigen. Right? So, if its
non-covalent, that means theres an equilibrium. Theres an on/off. Right? Very
few places during an inflammatory response do you get a covalent tagging of a
structure during these reactions. And this one is the exception. So, through the
generation of a phosphodiester bond, complement component C3b is covalently
attached, hopefully to the bacterial cell surface. And now you have a permanent
covalently bound tag to identify that cell for phagocytosis. Alright? Thats a really
important function of complement. Something called the MAC Attack. Membrane
Attack Complex effects, hopefully, bacterial cell lysis. Some components diffuse
away from the complex thats build up during complement activation. And one of
those are C5a which acts as a chemoattractant for neutrophils. And then, C5a, 3a
and 4a together work on endothelial cells to increase, um, the amount of edema in
the area. And as you know, complement activation can occur through three
pathways: classical, lectin and alternative.

[Slide 34] [Plasma-derived vasoactive mediators: complement activation]
[Dr. Craig] Did Dr. McCutcheon tell you why they call it complement?Anyone
know? So, the story goesup the street somewhere, I think it was at Cornell. This
was like, before WWII. They were immunizing, I forget the experimental animal,
and I dont even remember the bug. But they would take heat-killed
whatever-the-bug-was, immunize the bunny rabbit. Make believe its a bunny
rabbit. And they would take the blood from the immunized bunny rabbits and put
it in with a live bacteria. And the bacteria would be lysed. And, if they took the
blood from a non-immunized bunny rabbit, against this bacterium, the bacterium
would not be lysed. Ah! So this is an immunology lab. So what were gonna do is
were going to do is purify all the antibody. As it turns out, its frickenly easy to
purify antibody from blood. So they did that. And when they reacted from the
immunized bunny rabbit, um, the antibody, purified antibody, with a bacteria, it
didnt lyse the cells. Only if you added back in components from the blood back
into the antibody/bug mixture did you get cell lysis. Hence, there was something
in the blood that complemented the binding of the antibody to allow lysis to
occur. Hence, thats why its called complement. Right? And, unfortunately,
because that was the first series of experiments that were done, they perhaps, the
latest evolved pathwayso called classical pathway, which you would think
would be the earliest pathway evolutionarily, based on the nomenclatureuh,
thats how it kinda got its name. Okay, so binding of the antibody to the cell
19

surface generates a series of reactions that result in the generation of a C3
convertase. Perhaps the earliest is the so-called Alternate pathway. And Im sure
Dr. McCutcheon went over the various ways that the Alternate pathway can be
activated, but that also results in a C3 convertase. And then, finally, theres a
Lectin Binding Pathway. And, remember we said that mannose is expressed on
bacterial cell membranes? And its a PAMP. Well, we make a protein called a
mannose-binding protein in the liverpart of the acute phase response. Its also
present. And binding of the mannose binding protein with its lectin will also
generate the C3 convertase. Everyone know what a lectin is, by the way? A lectin
is a protein thats specific for a 3-dimensional structure on a carbohydrate.
Alright? And youll see lectins over and over again. But anyways, I wanted to make
sure you had that. So, no matter which of the three pathways, as soon as you
generate a C3 convertase, then its off to the races, Alright? So, the convertase
takes complement component 3, splits it into two pieces. C3b gets a thiodiester
bond covalently bound to the surface, hopefully of the bacterium. C3a diffuses
away, goes to endothelial cells, tells them lets get more edema into the area. That
complex then takes C5, okay C4 has been used up in therethey kinda got mixed
up in the ordertakes C5. The same thing happens. C5b stays with the complex
on the cell surface. C5a diffuses away. This is really good for neutrophil
chemotaxis. So on and so forth, on the surface of the bacterial cell to assemble a so
called Membrane Attack Complex and lysis of the cell by establishing a pore
through the bacterial cell membrane. So activation of complement is sort of like a
watershed event during inflammation. Cause, not only do you detect and tag that
pathogen, youre also amplifying the acute inflammatory response to that area.
Now youve set up a gradient that cells can follow to get into the site of activity.
Youve also generated uh inflammation, edema, and not only help clear pathogen,
but also, as the lymph is drained from that area to begin to set up an adaptive
immune response. Youve helped identify cells to be phagocytosed by the cells
that being called through this covalent tag. There are receptors for immobilized
C3b on phagocytic cells. And also, this is probably the least important, youve also
begun to destroy the bacteria thats present in the area through generation of the
Membrane Attack Complex.

[Slide 35] [Diagram]
[Dr. Craig] And so heres kinda like a diagram that just illustrates that. Alternate
pathway: heres the bacteria convergence on that C3 convertase, right? Generation
of C3b. Covalent tag tagging that bacteria for phagocytosis. Classical pathway uses
antibody. As well see on Monday, there are other things that can stimulate the
Classical pathway other than antibody. Um, that also generates a C3 convertase.
Lectin pathway: mannose binding protein activates a pathway again, culminating
in the C3 convertase. And its off to the races from there. Inflammation.
Chemotaxis. Cell-directed phagocytosis, generation of the Membrane Attack
Complex and lysis of the bacterium.

[Slide 36] [Plasma-derived vasoactive mediators: complement effector functions]
20

[Dr. Craig] So, weve gotten through this side: so, now we have kind of an idea of
how is the inflammation generated. And how do we get important things that are
in the blood, like pre-form antibody if this persons actually seen the challenge
before. But more importantly, getting complement from the vascular compartment
into the extravascular compartment cause activation of complement is really a
watershed event. How do we get specific cells into that site? So now were going
to go to this side of this diagram and were only gonna talk about a few
chemotactic factors. Uh, C5a, which youve actually already talked about. Weve
also talked about arachidonic acid metabolism. Leukotriene B4. Uh,
f-Met-Leu-Phereally important. And weve touched already on chemokines and
lets kinda talk about how these interact

[Slide 37 and 38] [Tissue injury or infection and Inflammatory cell recruitment:
Endothelial/leukocyte cell interactions]
[Dr. Craig] Okay, so if you actually look at an area thats undergoing
inflammation, if you look at the microvasculature under a microscope and when I
was a dental student, we actually had something called lab, where we actually
kinda did this with a frog. It was kinda neat, you know? Well, it wasnt too neat for
the frog. But the frog lived. So, it was nice. But, youd anesthetize a frog and put
him under a microscope. And as it turns out for this species of frog, and I forget
which one it was, but the skin in between the individual digits was really thin,
and you could kinda like thin it out and you could see the blood going through the
individual small vessels. And then you could put various drugs on the area. And,
because frog skin doesnt have a lot of, uh is very permeable, doesnt have a lot of
keratin, you get very quick absorption, so you can see changes in blood flow. And
one of the things you can actually see, if you use vasoactive mediators, such as
Prostaglandin E2, those sorts of things. You can sorta see is not only is the area
becoming hyperemic, filled with blood, and because of the increased vascular
caliber, right, blood is flowing through slower, cause its under constant pressure.
But you also start to see: some cells start to become more located along the
endothelial cells and these are polymorphonuclear leukocytes for the most part.
And histologists call that margination. Another synonym that youll see in books
is pavementation also. So as these polymorphonuclear leukocytes are kind of
rolling along in the circulation, the endothelial cells start to express receptors
that are specific for sugars. Right, theyre lectinstheyre called selectins, right. P
and E selectins are being expressed by the endothelial cell and L-selectin is
expressed by the polymorphonuclear leukocyte. And these selectins are specific
for the Sialyl-x-Lewis carbohydrates that are on the polymorphonuclear
leukocytes. They used to be called addressins. Selectins and addressins. Every cell
has an address and its on its Sialyl-X-Lewis carbohydrate. So anyways, this allows
the endothelial cells to begin to select out of the cells that are passing by in the
blood flow, the ones they want. And they want these polymorphonuclear
leukocytes. By these interaction with selectins. Next, if youre looking at the frog,
you can see further down in the stream, all of a sudden cells are becoming very,
very closely associated and very tight. Theyre not rolling around the endothelial
21

cell tube any longer. And thats done by expression of integrins by the
polymorphonuclear leukocyte, which bind to cell adhesion molecule 1, thats
being expressed by the endothelial cells. So now, you have tight adherence. And
then, finally, these endothelial cells kinda migrate to the gaps between thethe
polymorphonuclear leukocytes, rathermigrate to the gaps that have formed
between the endothelial cells. And they start to transmigrate across the basement
membrane into the extracellular matrix. So as theyre trying to do that, right, they
can get past the cells alright, cause the cells have moved off to the side. But, they
come up against the basement membrane. And basement membrane, being tight
for collagen and other basement membrane associated proteins, laminin, those
sorts of things, alright? Uh, this is associated with matrix metalloproteinase
(MMP) gene expression. Especially ones that are associated with the membrane.
So they chew their way through the basement membrane and out into the
extracellular matrix. And I think I have a little cartoon of that. Yes, we have a little
cartoon.

[Slide 39] [Inflammatory cell recruitment: Endothelial/leukocyte cell
interactions]
[Dr. Craig] So heres the inside of a small vessel, and heres those endothelial
cells have been induced because of pro-inflammatory cytokine release and also
pro-inflammatory mediators, such as PGE2. And these little guys sense that, and
they start to express selectins. These selectins are specific for Sialyl-X modified
carbohydrates on the leukocytes, in case monocyte (? mumbles at 19:55 left on
podcast) And, then, expression of integrins, which bind the intercellular adhesion
molecule 1 (ICAM-1) thats being expressed on these little guysIncreasing the
tethering of this neutrophil. And now the neutrophil comes through and it has to
degrade its way through the underlying basement membrane and then it goes
out into the extracellular matrix. And starts to follow the pathway to the pathogen
using diffusion gradients of things like f-Met-Leu-Phe or Interleukin-8, those
sorts of things. Now, in doing this, it takes awhile for these endothelial cells to
resynthesize the basement membrane. So now you dont have an ultrafiltrate in
this area any longer. You actually have a hole in the filter. So now, larger molecular
weight proteins can come into this area. So, later on during inflammation, the
edema that was originally characterized as a transudate or an ultrafiltrate of blood
now becomes an exudate. Has higher molecular weight proteins associated with it,
has polymorphonuclear leukocytes associated with it. Changes character from a
clear straw fluid to a milky, thick, sometimes almost caseous kinda exudate.

[Slide 40] [Inflammatory cell recruitment: Chemotactic factors]
[Dr. Craig] And, this is just a photomicrograph of what my frog looked like. So,
heres some vascular channels, right? And erythrocytes inside the vasculature
being lined with polymorpholucular leukocytes. And all these little guys out here
are all PMNs that have been recruited to the extravascular space by things like
C5a, uh, bacterial products, leukotriene B4 and chemokines, such as interleukin 8.

22

[Slide 41] [Mechanisms of inflammatory cell killing]
[Dr. Craig] Okay, so, now weve got the polymorphonuclear leukocyte out into
the extravascular space. And now we kinda have an idea, through receptors for
PAMPs and also receptors for things like C5a and receptors for leukotriene B4 that
start to migrate up this concentration gradient towards where the infection
actually is. And so what do they do when they get there? So heres a
polymorphonuclear leukocyte. Umm, its a transmission electron micrograph and
you can kinda see one of its little podocytes is beginning to to lift up and trying to
get down on the basement membrane between these two endothelial cells. And
this is the cell nucleus. Its cut in cross section. Its a lobated nucleus so thats why
it looks like two, its really just one. But inside the polymorphonuclear leukocyte
are all these little vesicles. These dense vesicles. And they all contain interesting
substances that are gonna be released into the area of or fuse with phagosomes to
destroy bacteria. Okay? So, on the outside of a neutrophil, you can kinda think of a
neutrophil as sort of like, I dont know, a policeman in a way or..some kinda dooms
day machine for bacteria. And they are studded with receptors for all sorts of
things. Uh, bound immunoglobulin, C5a, C3b, especially that, C3b, uh arachidonic
acid, metabolites, pro-inflammotry cytokines.

[Dr. Craig] So they can recognize and dock with their targets. And once they
dock with their targets, histopathologically, there are several classes of vesicles
that are present in PMNs. I dont want you to remember the various classes. All I
want you to remember is whats present. So they contain things like acid
hydroxylases. Alright. So that would be good to kill bacteria. But they alsoand
heres some phosphorylase A2, which weve already talked aboutand heres some
myloperoxidase, and were gonna talk about how it generates reactive oxygen
species to blow holes in bacteria. But also we find things like MMP2 and 9, alright?
So, gelatinases. Theyre not there kill bacteria. And they also contain lysozyme,
and thats good, that gets rid of bacteria. Lactoferrin. Thats good. It chelates iron
so that bacteria cant replicate as fast. But also has collagenase MMP-8. Alright?
That destroys the host. And tertiary granules: cathepsin and more gelatinase.
Alright? So, it seems like these bacteria come in, or these polymorphonuclear
leukocytes come in armed to get rid of bacteria by both enzymatic and oxidative
means. But, they also come armed with stuff to get rid of extracellular matrix.
Yes?
Student question (cant hear) I believe so. Yea, Ill have to check Um..where
were we?
So whats the reason for having matrix metalloproteinases present in these cells?
(inaudible student response) Yea so, if you can kind of envision whats
happening is that youve got a relatively large cell thats climbing on the
extracellular matrix thats made of collagen and other proteins and embedded in
this matrix are relatively small cells, such as bacteria. And so that extracellular
matrix has to be resorbed in order to uncover the pathogen. Alright? And hence,
thats why there are matrix metalloproteinases present. So, while phagocytosis
23

and engulfment and killing, bacterial cell killing is occurring, theres also release
of matrix metalloproteinases to liquify that extracellular matrix and liberate the
pathogens that may be incorporated. So in order to actually get to the bacteria,
theres some collateral damage. Okay. Polymorphonuclear leukocytes are
damaging their own tissue. So theres two mechanisms

[Dr. Craig] Two generalized mechanisms for killing of bacteria by
polymorphonuclear leukocytes. One includes oxygen, called oxidative
mechanisms. And one does not, so non-oxidative mechanisms. So lets talk a little
bit about oxidative. And you can actually measure this. Something called
respiratory burst. PMNs actually use molecular oxygen. Not for respiration, but
they actually generate reactive oxygen species and you can actually measure this.
So one of the things that happens is that inside the phagosome of a PMN, theres
an enzyme called NADP oxidase (12:20 remainingslide says NADPH oxidase),
and what that enzyme does is it takes molecular oxygen and converts it into a
superoxide ion. And then, theres a second enzyme called superoxide dismutase,
which takes the superoxide anion and forms hydrogen peroxide gee, I use
hydrogen peroxide, alright? Bust holes in membranes. And then theres another
enzyme, myleoperoxidase, which takes the hydrogen peroxide and makes
something called hyperchlorous acid, which is really effective at blowing holes in
membranes and denaturing proteinsall sorts of things it can do against
biomolecules. And if thats not enough, in the presence of iron, hydrogen peroxide
can also form hydroxyl radicals. Alright. All of which act to destroy the bacteria.

[Slide 44] [Mechanisms of inflammatory cell killing Reactive oxygen (ROS) and
nitrogen (iNOS) species]
[Dr. Craig] And heres a little cartoon of that. So heres a tagged, uh, covalently
tagged, in this case, bacterium. Heres a receptor for that tag. Immobilized C3b. Uh,
this becomes uh, podia begin to move around that docked, bound molecule, uh,
becomes a phagosome a phagosome can be bound with other vesicles that contain
enzymes, which will non-oxidatively degrade the bacterium. On this end, the use
of oxygen. So, heres the generation of the active oxygen species. Also, nitric oxide
can also be generated into a peroxide, nitrous peroxide molecule, which is also
very effective at destroying bacteria.

[Dr. Craig] So, there are also NON-oxidative methods of killing bacteria by
neutrophils. Not using oxygen, per se. Um, lysosomal hydroxylases have a broad
profile of specificities against both gram positive and gram negative. Theres
something called bactericidal permeability increasing protein, which
permeablizes gram negative bacteria. Defensins, that we talked a little bit about
with mucosal immunity, PMNs also have that. Lyses cell membranes. Lysozyme
attacks gram positive cell wall. And lactoferrin chelates iron that they cant
replicate. So, we have both oxidating and non-oxidative means of killing. Um, but,
24

theres some collateral damage involved, right? So in order to uncover the
pathogen, you have matrix metalloproteinases that are liquefying the area so that
PMNs can dock with and destroy the bacteria.

[Slide 46] [Mechanisms of host tissue destruction in inflammation]
[Dr. Craig] You also have relatively non-specific ways of killing bacteria.
Generating reactive nitrogen species, generating reactive oxygen species; are not
really specific for bacteria, but theyll also kill host cells very easily. So there are
several different ways of generating collateral damage. So the release of reactive
oxygen and nitrogen species decrease tissue perfusion because there tends to be
blood stasis in the area if the inflammatory response takes a long time. Expression
of MMPs and local bone resorption.

[Slide 47 and 48] [Matrix metalloproteinase family]
[Dr. Craig] And lets just finish up, I guess, with the matrix metalloproteinses. So
just to remind you, theres a whole family of matrix metalloproteinases that are
out there. And, as it turns out, its really difficult to cleave type I collagenthat
rope like molecule, only has that one hinge region. And it turns out theres two
MMPs: MMP 1 and MMP 8 that invertebrates have the ability to cleave that hinge
region. Collagenase 1 is constitutively expressed in all cells. MMP 8 is induced in
inflammatory cells. There are gelatinases that take denatured collagen and
degrade them. As it turns out, there are two of them also. MMP9 tends to be
associated with connective tissue cells, but also tends to be associated with
inflammatory cells.

[Slide 49] [Periodontitis is associated with MMP-8 expression and localized
bone resorption]
[Dr. Craig] And as it turns out, periodontitis is associated with an inflammatory
lesion. So what has increased expression of MMP 8. And MMP 8, MMP 9, um, the
MMPs associated with inflammatory lesions are the ones associated with
extracellular matrix loss in periodontitis.

[Slide 50 and 51] [Localized bone resorption: Osteoimmunology]
[Dr. Craig] And, lets just finish up with bone resorption and then we can start
on targeting next week. So, theres a couple things that are kinda odd that people
couldnt explain up until the last few years or so. So when I was a dental student,
and you will learn this too, there are several stages of the periodontal lesion
described by Page and Schroeder. And the one thats actually associated with bone
loss was something called the established lesion and if you look at the
inflammatory cell infiltrate in the established lesion, its mostly plasma cells.
What are plasma cells doing there? Right? Then, theres a group that showed that
bone formation is always coupled with bone resorption. So you have to have bone
respiration before you have bone formation. So whats happening here? So, theres
this idea of osteoimmunology that we touched on in Craniofacial Biology. And
perhaps the easiest way to review this is to go right to a cartoon
25

[Slide 52] [Picture ]
[Dr. Craig] So, in this cartoon, the cell is regulating this bone resorption in this
area. This is an osteoblast stromal cell. But for our purposes, make believe this is
an alveolar bone cell, or perhaps, even better still, periodontal fibroblasts. And its
an area of inflammation. So in comes mononuclear cells that have been recruited.
And this periodontal cell expresses something called monocyte colony stimulating
factor. And theres a receptor for it on the monocyte. Once that ligand is bound,
then this monocyte starts to express RANK receptor. Receptor associated with
NF-kappa-B. Once that occurs, then this regulatory cell starts to express
RANK-ligand, which binds with RANK. And tells this mononuclear cell to begin to
fuse to become an activated osteoclast and begin to resorb this area of bone
matrix. Youll also remember that theres another factor that can be expressed.
OPG, Osteoprotegrin, which is a decoy receptor. So it binds RANK-ligand so that
the ligand cant bind to the receptor. So, its really, bone resorption is a mix of how
much OPG is being expressed and how much RANK-ligand is being expressed. And
as it turns out, those plasma cells, and also T cells that are part of the established
lesion of periodontitis,theyre a really good source of RANK-ligand. So thats
probably one of the reasons why those antigen specific cells are present.
So at this point, weve gotten through the collateral damage thats associated with
the inflammatory response. Next week, were gonna finish up with how cells are
targeted to the area. How they detect, rather, the pathogen. Well talk a little bit
about the inflammatory response, and then well talk about systemic effects of
inflammation next Monday.
Have a good weekend.
26

15/16: Inflammation I/II

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15/16: Inflammation I/II

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Transcribed by Mandy Weil 5 September 2014

[General Pathology] Inflammation by R. Craig, DMD, PhD

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