basic immunology

330 Pages·2015·2.85 MB·English

BASIC IMMUNOLOGY

A quick, mostly painless, usually apolitical, occasionally scurrilous,
almost Zen-like guide to the great mysteries of the field.

Ian Orme

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Every now and again I get a rush of blood to the head and decide it is time to update my teaching Notes.
Of course, having said that, I teach less and less these days as I become more infirm, babbling and
dribbling. So maybe this is a good time to squirt this stuff out one more time while I’m still coherent.

These Notes have a deep and important history. They began in 1987 when I first began to teach, and my
first attempt at providing a guide to MB342 students “Revenge of the Mutant Immunologists from Planet X”,
still holds a warm place in my heart, as does the award winning “Herrings from Betazoid ate my brain”. My
favorite of all, “Godzilla, Michael, and Lisa Marie*, Battle the Lords of the Death Planet [a treatise on the
influence of Japanese theater on current theories of immunology]” was of course a best-seller, but I’ve
been told that such silly titles project an image that I’m a bit crazy, European even, hence the current sober
title.

As before, these notes are full of stupid jokes which you should ignore. Ditto various ramblings and
rantings. There are certain passages relating to multiple occasions I got banged on the head on rugby
fields. Ignore these completely. In the latest opus I’ve put a big section on cancer therapy, a field racing
along right now.

Please note that in the last couple of versions I’ve covered the Wakefield disaster and the issue of
vaccination and autism, ending in the latest versions in which I’ve written about the big outbreak of measles
in Wales and England, and very recently in a Texas church. These notes are written for grown-ups such as
yourselves and I apologize in advance for some naughty words.

*Michael Jackson and Lisa Marie Presley [daughter of Elvis] had just gotten married at the time I produced
this particular opus. The marriage lasted a few hours [i.e. even shorter than first one for the part-carbon
part-silicon alien lifeform from planet Kardashian]. Kim has now married a foul-mouthed Rap grunter, and I
cannot wait for the next installment….

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1. FIND IT, EAT IT, FORGETTABBOUDIT….

We crawled from the primeval slime, replaced our gills with lungs, bent up on our hind legs to look for
predators across the African savannah, became Homo sapiens, and now Homo cellphonus.

We swim in a sea of bacteria and viruses. From Day One we’ve had to deal with this, because our
respiratory tract and our alimentary tract represent vast surface areas the more nasty bugs would love to
invade. But there is much more to it than that, because [despite the abject lies that horrendous frigging
Phillips Colon Health lady says on the commercials] we’ve developed a commensial relationship with many
bacteria, allowing them to live in our gut where they keep the other more pathogenic bugs at bay,
producing vitamins for us [like vitamin K], helping us digest food, and allowing us to expel vast amounts of
methane and sulfur filled gas at inopportune moments. Since the bacteria in us vastly outnumber us, we
are really a big bacterial colony with some human in it.

Our interactions with bacteria drive processes in the body. A good example [maybe, jury still out] is that
interactions with various bacteria probably drives and molds the type of responses we are capable of
making under certain circumstances, making us less likely to make a more inappropriate response. This
finger has been pointed at allergies, sweeping the Western World. People point to the fact that kids brought
up on farms, eating dirt so to speak, or kids that go to the human feed-lot, day care centers, have far lower
rates of allergy than those kids brought up in pristine conditions where they are perpetually followed around
their spotless house by Soccer Mom clutching the Clorox wipes with which little Johnny is smeared every
two or three minutes. Another example, perhaps not so useful, is the interaction between our immune
system and carbohydrates on bacterial cell walls in the gut, that lead to us generating antibodies that react
to the “blood group antigens” on red blood cells and in turn making blood transfusions much more exciting.

Unless heavily armed, a microbe has to get inside us to do any damage. So let’s start there, with barriers
and mucosal surfaces.

To do nasty things to you bacteria and viruses have to first actually get inside you. To prevent this, plus to
hold all those organs inside, you are covered in a mechanical barrier called the skin. You’ve probably seen
it. It is actually the biggest organ in the body, about 10% of total body mass. The top part of the skin is the
epidermis. This consists of four layers of gradually dying skin cells, with the top layer [corneum] full of dead
cells and the molecule keratin which gives us our skin color [hence the name keratinocyte]. This
continuously sloughs off, taking with it bacteria that have attached [as well as those wonderful rejuvenating
creams my wife spends hundreds of dollars on]. Below is a much thicker layer, the dermis, which is mostly
connective tissue containing blood vessels, lots of sensory nerves, hair follicles, and sweat glands. The
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latter produce secretions that have a low pH, which most microbes do not enjoy. Underneath all this is
another layer, the hypodermis, full of fat cells and larger versions of blood vessels and nerves.

In the upper respiratory tract, the cilia in
the trachea beat in an upward motion Stratum corneum
Stratum lucidium
(the "ciliary escalator"), capturing Hair Stratum granulosum
follicle
Stratum germinativium
inhaled particles and driving them
upwards into the glottis, from whence
they are swallowed. In the gut,
assuming the ingested microbe is
Epidermis
sufficiently armor-plated to survive the
Stratum basale
extremely low pH of stomach acid, it will
Sweat
gland
pass into the intestines; here it may
Dermis
have difficulty in flourishing because of Sensory nerves
the competitive nature of the already
Connective tissue
established gut flora.

In the mucous membranes of the body, Hypodermis
bacteria may be destroyed by lysozyme,
an enzyme which breaks the bacterial Fat
Blood supply
peptidoglycan cell wall, as well as being
excluded from entering the tissues by binding by secretory IgA molecules [a class of antibodies].

A number of other factors in body fluids can also contribute to innate immunity. In semen, various
polyamines can be bactericidal. In the blood, viral infections result in the elaboration of interferons, proteins
which protect non-infected cells from the virus. Also found in the blood are iron-binding proteins, which
deprive microorganisms of molecular iron.

The skin is an effective barrier, but unfortunately you have to have various pipes in you so that you can
function. The biggest is your digestive tract [gut] which goes right through you from the mouth to the anus.
The others are the respiratory tract, so you can breathe, and the genitourinary tract, so that you can pee
and make babies. Each of these are potential targets for nasty things to get into, so they have multiple
defense systems, plus they are covered in gooey mucous that bugs have difficulty penetrating. For this
reason they are called “mucosal surfaces”.

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The mucosa differs from the skin in that while the skin is multi-layered the mucosa is a single layer of
epithelial cells. The mucosal surface of the gut is busy. It is constantly exposed to zillions of bacteria and
other potential pathogens for an entire lifetime. But it is a very effective barrier because of its design. The
epithelial cells it is made up of are very tightly stuck together so that water and other small molecules can
get through but larger molecules cannot. In addition it is covered with mucous, a gooey viscous material
containing glycoproteins [proper name glycocalx …”glyco-kay-lix”] full of antibodies and other nasty
antimicrobial substances. Of the antibodies [see below] “secretory IgA” dominates; there are 1011 cells in
the gut immune system making as much as 5 grams of this antibody each day.

Okay, so you are playing rugby and somebody tears your arm off. No…, that’s not a good example,
because you’d just go to the bar while somebody stitched it back on. Okay, you are bringing your flower
arranging diary up to date and the nasty paper gives you a paper cut. So what happens then…?

The first response is by platelets that leak into the wound. These aggregate at the ends of the damaged
blood vessels, change shape from discus shaped to spider like shaped cells, and then degranulate
releasing fibrinogen which converts to fibrin, stemming the massive blood flow said paper cut induced.
After a few hours or so, repair mechanisms get going in earnest. Keratinocytes, released from the wound
edges, migrate in and start to arrange in sheets. Cells on the edge of this express sticky molecules
[integrins] and extend lamellipodia, literally dragging themselves across the wound substratum. Activated
fibroblasts start to replace the damaged connective tissues below, and locally angiogenesis starts to
replace the capilliary bed.

As these processes get going, neutrophils arrive. Their job is simple, kill bugs. This they do by
phagocytosis and oxygen radical generation [some of which is released and can paradoxically contribute to
local cell damage]. In addition, neutrophils secrete DNA and histone which forms a matrix or net, trapping
bugs and preventing them from escaping into the blood. After a while macrophages arrive, clearing the
wound of debris, fibrin, and dead neutrophils [most of which self-destruct by programmed cell death, also
called apoptosis].

The names for our “white blood cells” was dreamed up histologists, so blame them. Hence you will see in
text books the terms mononuclear cells, which refer to the round nuclei of lymphocytes and monocytes,
and the term polymorphonuclear granulocytes (or "polys"), which refers both to the appearance and major
properties of neutrophils, eosinophils, and basophils.

In the circulating blood neutrophils predominate, comprising about 65% of total leukocytes. Lymphocytes
come next, at about 25%, followed by smaller numbers of eosinophils (4-5%), monocytes (3%), and
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basophils (1%). These proportions can change dramatically in pathological states, such as during
infections. [Actual percentages can differ widely from animal to animal as well.....]

The neutrophil is a circulating end-point cell, which comprises the bulk of leukocytes [60-70% of total
leukocytes in humans, less in mice]. They are contuously made in the bone marrow from myeloid
precursors, a process stimulated by granulocyte colony stimulating factor [G-CSF] and controlled by IL-17
made by T cells and IL-23 made by macrophages [all explained later]. Neutrophils are normally short-lived
cells, dying by apoptosis after about 12-24h. They never divide and synthesize very little protein or RNA.
They are continually produced by the bone marrow by a tightly controlled process of myelopoiesis.
Neutrophils have an
Neutrophil
irregular, indented
nucleus (although it
often looks like it has
several pieces of Irregular
Small
shaped
nuclei scattered bluish
dark
around the cell, granules
staining
in
electron microscopy
nucleus
cytoplasm
indicates that these
are all connected
together as a single
body). The cytoplasm contains a large number of secretory vesicles and granules, the latter of which
contain two major ingredients. The first is a large package of hydrolytic enzymes, and the second, enzymes
capable of generating an array of molecular species of oxygen, collectively called toxic oxygen radicals, as
well as nitric oxide, a potent anti-microbial agent.

The function of neutrophils is to phagocytose, and then biochemically fry, bacteria and other infectious
agents. With its array of hydrolases and highly oxidative radicals, the cell is well equipped for this purpose.
Neutrophils are short-lived [less than a day unless triggered] and then die by apoptosis [programmed cell
death]. They are considered “resting” cells, a jolly good idea considering the jello-making molecules tucked
safely away in their granules.

The neutrophil is a ravenous beast. It must patrol the whole body in search of prey, knowing full well that its
life will be brief otherwise. Live bacteria, dead host cells, it doesn’t actually matter. We are born to kill. Of
course, given that our mucosal and skin surfaces have several trillion bacteria [sorry Clorox Wipes Soccer
Mom] chances are good we will get a chance to blast out some splendidly lethal oxygen radicals before we
ourselves blow up in a blaze of glory. Nor do we have to continually get blasted around in the blood if we
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don’t want to; it is equally honorable to sequester in the lungs or liver sinusoids and just lie in wait. Several
hundred thousand puff adders can’t be wrong.

One of the first questions posed about professional phagocytes like neutrophils was “how on earth do they
actually know what to eat”? Now we are realizing that they possess a huge array of receptor systems
capable of recognizing all sorts of microbial molecules, and in addition molecules released by our own cells
when they are damaged.

We’ll get to this later but our pals studying the fruit fly, Drosophila, identified lots of cool genes, with
competing labs all coming up with crazier and crazier names to label them. One gene, that they called Toll
[German for light beer I was once told] was able to detect molecular “patterns” expressed by nasty fungi
who enjoy nibbling on said flies. So, guess what, when we looked for similar genes in humans we found a
whole bunch of them, now called the Toll-like receptors, or “pattern recognition molecules”. These can see
all sorts of stuff like bacterial peptidoglycan, or viral DNA sequences.

Not surprisingly given their job, neutrophils express most of these [they only lack TLR3 and TLR7]. Until
recently, it was thought TLRs could just see molecules of foreign origin, but now we know they can see
host molecules, called damage associated molecular patterns [DAMPs] or “alarmins”. In my very last rugby
match I attempted to tackle this behemoth and he stuck his shoulder into my stomach and completely lifted
me off the ground [and I’m hardly small]; my serum DAMPs went up significantly at that point. Prominent
DAMPs are the heat shock proteins [hsp60 for instance], hyaluronan, precipitated uric acid, and heparin.
Others include the nuclear protein high-mobility group protein B1 [HMGB1], which can be seen by TLR2/4
complexes when it is released by damaged cells, which leads in turn to neutrophils pumping out oxygen
radicals and pro-inflammatory cytokines. New evidence suggest that ATP, released by damaged cells, can
also act as a DAMP, and that in addition to TLRs, these can be seen by a family of 22 proteins belonging to
the NOD family [NRLs, NOD-like receptors]. NODs form “inflammasomes” when triggered by ATP and
other molecules; these are big molecular complexes that trigger caspase activity and IL-1 production [IL-1
is an example of a cytokine], driving inflammation. It is now thought that IL-1 is the central molecule driving
inflammation and cell recruitment, triggering signaling pathways in turn, and resulting in the establishment
of chemoattactant gradients that neutrophils can follow.

Bacterial endotoxin, LPS, has long been used in lots of lab studies as a potent neutrophil activator, doing
so we now realize via TLR4, but it is now known this only triggers neutrophil survival for a few hours, and
other factors then kick in. If anything, it is now thought that LPS slows cell movement in chemoattractant
gradients by counteracting the effects of IL-8, an event counterbalanced in turn by IL-33. No, I don’t
understand this either….
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In the addition to the Tolls and NLRs, further constitutively expressed receptors include various C-type
lectin receptors, as well as retinoic acid inducible gene-I [RIG-1] and melanoma differentiation associated
protein-5 [MDA-5], which can detect microbial dsRNAs. Bacterial DNA is also detected specifically by TLR9.

Neutrophils also express receptors for the Fc bit of the IgG type of antibodies. There are three types of
FcR, and cell triggering via these can drive chemotaxis. Problem is, often as not this recruits neutrophils
into joints, and is a contributor to the pathogenesis of rheumatoid arthritis. In fact, when you develop RA,
neutrophils are the first to turn up.

In this regard, a major property of neutrophils is their ability to extravascate, that is, to leave blood vessels
to go sites of bacterial implantation. If we take a scratch in the skin as an example, the local tissue damage
results in the release of prostaglandins, as well as histamine, a small molecule belonging to a group of
materials called vasoactive amines. This material diffuses away from the wound, and has the property of
relaxing blood vessels (vasodilation) hence increasing the permeability of the local capillary bed. Not only
does this allow fluid to accumulate, but neutrophils then also move out of the blood vessel and towards the
wound site. This movement is not random, however, because the neutrophils have receptors for a large
variety of chemoattractant molecules and they follow gradients of these until they reach the source, a
process known as chemotaxis.

A wound in the tissues creates local
inflammation. Local blood vessels “Chemotaxis”
respond by putting up adhesion

molecules on their surface

Once through and into the tissues
the cells can attack and destroy

any bacteria that have gotten in

After binding, the neutrophils
squeeze through tiny gaps
between the blood vessel
Neutrophils have receptors for

wall cells
these molecules
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We now know that neutrophils and all the other leukocytes express "adhesion" or "homing" markers [I like
to call them “sticky molecules”] that recognize complimentary ligands expressed by inflamed blood vessel
endothelial cells. When these ligands are engaged the cell slows down in the blood vessel and rolls along
the endothelial surface as it tags more and more ligands. One way to think of the overall process is to
imagine tumbleweed rolling across the prairie and catching a fence line. Yee hoo cowboy.

Once the cell has come to a halt even tighter stickies and ligands come into play allowing a process that
results in the cell squeezing through the junction between two adjacent endothelial cells. Once in the
tissues the cells follow gradients as above, but they also use a third array of stickies that allow them to
pass through the tissue extracellular matrix, and well as chemokine receptors that bind to chemokine
molecules immobilized on the tissue matrix surface and act as a very specific road map.

In the circulation, neutrophils are half asleep, with very few receptors up on their surface. However when
sticky molecules bind to their ligands on the surface of the blood vessel wall, the cell wakes up, it becomes
activated, and its life span increases to 4-5 days or more. At the molecular level, stickies trigger a surge in
intracellular Ca++, and this causes an organelle called the “secretory vesicle” to fuse to the cell plasma
membrane. The vesicle contains a laundry list of receptors, most of them further stickies [integrins] and
chemokine receptors and various soluble factors. This sets up the neutrophil for firm binding to the
inflamed blood vessel endothelium.

Movement of neutrophils from the blood across the endothelial cell barrier is called diapedesis. Years ago,
the first low resolution photos of this seemed to be suggesting the neutrophil somehow pushed its way
through the endothelial cell, which made little sense until we realized they were moving through the
junctions between cells.

When neutrophils are exposed to chemoattractants they undergo cell shape polarization creating a leading
edge at the front, and a trailing end or uropod. This is controlled by a kinase signaling system consisting of
the C90 isoform of phosphatidylinositol 4-phosphate 5-kinase and signaling via a clathrin dependent
vesicular pathway. This in turn drives the formation of Rac-dependent lamellipodia on the front, needed to
play touchy feely with the endothelial surface so that they can ferret out the nearest junction.

Initial neutrophil recruitment involves a sticky called P-selectin, which is stored inside endothelial cells [in
vesicles called Weibel-Palade bodies] and E-selectin, which is made de novo on demand. These get
pushed up to the apical cell membrane so they can wave hello to passing neutrophils via ligands on these
cells [PSGL-1, ESL-1, and others including neutrophil expressed L-selectin, CD44, and some other
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sialylated/glycosylated ligands]. This triggers “tethering and rolling”, the first step in neutrophil [and other
white blood cells for that matter] recruitment mechanisms. The affinity of binding by these ligands is very
high, which is needed to counteract the hemodynamic shear stress and allow cell rolling, thus preventing
the cell heading off at high speed towards your toenails.

The end of the rolling process is a state of tight stationary adhesion. As mentioned above, integrins, more
specifically 2-integrins, are the key mediators. These molecules are dimeric, non-covalently linked type I
transmembrane glycoproteins. They use variable chains [designated CD11a, CD11b, or CD11c] and a
common chain [CD18]. Part of the molecule is cytoplasmic, and it is thought that this bit stabilizes the
cytoskeleton when the integrin is bound, allowing internal signaling and cell activation. The stickies
mentioned above fall into this family; LFA-1 is CD11a/CD18, and Mac-1 [originally discovered as the
receptor for complement component 3] is CD11b/CD18. These integrins can in fact bind to a fairly broad
range of blood vessel stickies, but principally ICAM on the endothelial surface. In their resting state the
integrins appear to be bent over, but when activated they erect to an extended state, providing a high
affinity binding site; the two chains also slide away from each other, probably pushed apart by the fluid
shear force pumping by. These then bind strongly to integrin molecules on the neutrophil and slow down
their rolling further until it ceases. When ICAM is ligated there are multiple signals, including Src, pyk2,
small GTPases, and triggering of the actin cytoskeletoin. Another molecule involved is FAK [and no, I’m not
sure what this actually stands for, but the review I read was obviously by the guys who found FAK and so it
is obviously the most important thing since sliced bread].

It is now thought that the 2 integrins [CD11a, CD11b, and CD18] on the neutrophils flip “inside-out”, an
event that seriously increases their avidity/affinity, so that the cell skids to a halt as it starts to bind to their
counterpart ligand on the endothelial surface, ICAM-1, with secondary interactions occurring between their
 2 molecules and VCAM-1 on the endothelial surface. Well, almost, because now they begin to crawl
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towards the heart of the gradient. The shearing force of the blood flow pushes the crawling neutrophils, as
well as elongating their cell shape. As a result they very quickly find a junction between adjacent
endothelial cells on the blood vessel wall.

There seems to be a “guidance hierarchy” as well. Early on in this process neutrophils seem to respond
more strongly to chemokines like IL-8 and MIP-2, but then, once crawling happily, look instead for signals
from fMLP and the complement fragment C5a. This seems to involve switches in complex signaling I won’t
bore you with.

The tight junctions between endothelial cells are discontinuous, and at these points neutrophils
extravascate. At this point the neutrophil starts to push down through this junction. At first we thought this
was a combination of brute force and enzyme release breaking down the junction molecular basis. But new
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