---start immuno 1.10.97----
handout: HYPERSENSITIVITY REACTIONS

TYPE I and II today.
in the book - ignore the "human" aspects of these reactions.

why do we say "hypersensitivity"?
these are reactions based on normal immunological principles that end up 
causing tissue injury since they take place in a n exaggerated fashion. 
these are OVERreactions. 

There are often mixed type reactions, not just straight I, II, III, or 
IV. But usually one type or another PREDOMINATES at a given time.

TYPE I "anaphylactic" reaction
Type II "cytotoxic" "cytolytic"
Type III "immune complex dz"
type IV "delayed" "t cell mediated"

need to know Ig class involved, cells involved, if there's complement 
involved, any chemical mediators, what Ag are involved: heterologous == 
foreign Ag, autologous == self Ag like thyroglobulin, Ig, DNA. 

TYPE I

[slide: bee sting on eye w/intense edematous locally that showed up w/in 
minutes due to type I rxn]

note that the individual has to be presensitized to have a rxn. First 
exposure does NOT cause a hypersensitivity. only REexposure. in type I, 
reexposure results in IMMEDIATE rxn w/intense swelling, edema, etc - can 
be quite serious if doesn't stay LOCAL. if whole vascular tree is 
involved, you can die in a matterof minutes.

the antigens involved here are often called "allergens" which are a 
foreign protein of a variety of types. pollens, weeds, grasses, insect 
material, but anyhow, MHC II is involved in processing this kind of Ag 
which comes in through major routes: inhalation, ingestion, dermal.

SENSITIZATION

MHCII will present the Ag on first exposure to the TH2 cells and the T 
cells make IL4 and IL5, causing B cells to make IgE (class switch). IgE 
almost immediately attaches through receptors onto mast cells and 
basophils and then it sits there waiting and waiting to meet new Ag. 
Almost all IgE is bound like this, very little is free in serum. Now mast 
cells are located along the CT of most blood vessels, mainly. Some 
species have more in GI tract, others have more in skin or lung, but they 
are always in CT along blood vessels. and the rxn you see is related to 
their location.

now, when you have RE EXPOSURE
antigen comes in again, crosslinks IgE, and causes degranulation of mast 
cells, releasing vasoactive substances, causing rxn in skin, bronchial 
tree, ....why? what's released from mast cells? HISTAMINE: doesn't last 
long in tissue but is a primary molecule responsible for these signs: it 
dilates blood vessels, increases vascular permeability - causes edema, 
swelling. it also increases mucus secretion and causes constriction of 
extravascular smooth muscle (eg, bronchiolar smooth muscle). This causes 
wheezing or asphyxia. 

clinical effects: asthma, eczema, hay fever..."anaphylactic shock"

know what IgE does, where it hangs out, and you need reexposure.

more about MAST CELLS:

there are other ways to degranulate a mast cell. 

-crosslinking IgE is one way
also: anaphylatoxins (C3a, C5a), bee venom (via above, but also 
directly), neutrophil basic protein, a wide range of chemicals, extreme 
heat, extreme cold, trauma (eg, hitting yourself with a hammer).

but we're talking right now about the antigen mediated crosslinking of 
IgE bound to the surface.

are all mast cells the same? 

mucosal mast cells: in respiratory, GI, urogenital tract: lives less than 
40 days. production is T cell dependent: thymectomized animal doesn't 
make these.
Have more FcE receptors. less histamine. Leukotriene:Prostoglandin ratio 
== 25:1
note that the leukotrienes and prostoglandins aren't preformed like the 
histamine, they are made after a rxn starts.

CT mast cells: ubiquitous: lives more than 40 days. not so T cell 
dependent.
Fewer IgE receptors, MORe histamine. Leukotriene: prostoglandin ratio == 
1:40


so, how does this all work? how is a mast cell triggered? crosslinking of 
IgE by antigen causes INFLUX OF CALCIUM. enzyme machinery kicks in, you 
have an increase in cytoplasmic cGMP, which causes an increase in 
functional cellular activity and release of preformed histamine granules.

norepi, ach: stimulate mast cells.

inhibition of mast cells: beta andrenergic receptors, histamine type II 
receptors. you get an elevation of cAMP which causes inhibition.
epi, histamine - inhibit mast cells.
now, epi also constricts blood vessels and decreases the edema being 
formed, so it's a good tx for anaphylaxis.

note that cAMP tends to downregulate cGMP and vice versa.
mast cells have huge numbers of receptors and they don't always work the 
same way. you can have stimulatory and inhibatory receptors activated at 
same time...depends what happens to cAMP and cGMP levels what happens to 
cell.

mast cell derived mediators.

histamine
eosinophilic chemotactic factor

these two are preformed factors released from granules. the eosin.c.f is 
one reason why we get a huge influx of eos when we have inflammatory 
reactions.

leukotrienes
prostoglandins/thromboxanes

these two are "newly formed" during a rxn. leukotrienes form in 
lipoxygenase pathway, prosto's form in cyclooxygenase pathway. you end up 
with components which function similarly to histamine, but these are 
being formed continuously during the rxn, and these are responsible for 
the prolonged parts of rxn (swelling, asthma) continuing more than 24 hrs 
after the initial rxn long after the histamine is mostly gone. cause 
bronchoconstriction, vasodilation. etc.

so knwo that some are already there, and others formed later, and resp 
for different phases of rxn.

recall production of IL10 by TH2 cells inhibits prod of TH1 cells, 
reducing IFNg production, reducing macrophage activation, and causing the 
IL4 and IL5 formed by TH2 clls to activate mast cells, eos, and B cells, 
promoting IgE formation. 

if more stimulation of TH1 cells, can inhibit TH2 cells, can cut down on 
TH2 effects, prevent allergic response: allergy shots attempt this.

IL10 decreases Ag presentation to TH1 cells also, preventing their 
activation. 

RISK OF ALLERGY:

why are some individuals more sensitive than others?

in humans, children with no atopic parents - 14% have atopy
if one parent has atopy, 30%
if both parents, 50% of kids are atopic.

same thing is seen w/monozygotic and dizygotic twins. monozygotic have 
same level of sensitivity, dizygotic may or may not. so there's clearly a 
genetic predisposition.

what is really being controlled genetically is total level of IgE being 
produced, and the specificity to some allergen, and whether you are hyper 
or hyporesponsive. eg, most people who have allergies are allergic to 
ragweed - 80% of people with ragweed allergies have a certain 
histocompatibility type.  also, individuals known to be hyperresponsive 
will hyperrespond to almost any antigen they get. these people also 
correlate with a specific tissue type.

among the normal population: some people are "low responders" to antigen, 
some people are "high responders" - and these high responders may also 
hyperrespond to things like pollen, grasses, etc, which you really don't 
want. it's best to be in middle.

how to test for allergy: 
in humans.... skin prick test

they take dilutions of allergen in saline and they put it ID 
if you are allergic, within 20 min you will have local edema and 
erythema. "wheal and flare" rxn. the wheal is the elevated bleb, and is 
due to edema. the flare is the result of increased vasodilation. these 
immediate responses go down after an hour or two. there is also a late 
phase, 5 or more hours later, lasting about 24 hrs, which is due to the 
newly formed mediators eg leukotrienes, prostoglandins.

RAST radioallergosorbent test

-solid phase: add antigen
-block disk w/nonspecific protein
-add IgE (test serum) (will bind to Ag if present - note, level is 
elevated in allergic individuals, even though IgE isn't normally found in 
the serum.)
-wash
-add anti IgE (labelled ligand)
-wash and count

so can use a number of different antigens to find out what specific IgE 
is present.
similar to ELISA.

now, systemic anaphylaxis has different signs in different species and 
diff target organs.

MOST domestic species, except dog, main target organ is the lung, so you 
have edema in lung, airway constriction, severe dyspnea, collapse, and 
death unless treated promptly w/epi

in DOG, major target organ is vascular system in abdomen/GI tract. 
tremendous pooling of blood in viscera, so ends up with a hypovolemia, 
engorgement of hepatic veins, visceral hemorrhage, and death unless 
treated promptly with epi.

now...could you get a system rxn after local exposure to skin? yes. why? 
well, antigen can get into blood vessel and be distributed through entire 
body very quickly, or can sometimes just be absorbed very quickly.

effects of hyposensitization therapy:
allergists are happy, because patients rarelydie, but always come back. 
so to treat allergies....yougive repeated injections of small amounts of 
allergen, the idea being that maybe you can trigger a competing Ab 
response, like an IgG response, that will drive down the IgG response, 
and there is some clinical correlation in responders, a number of people 
show an increased level of IgG and a decrease in "lymphotoxin?". now why 
is a competing Ab effective? if you primarily bind allergen to IgG, it 
isn't available to bind with the IgE! oh, this sounds great. i gotta get 
me some of these allergy shots again :) [seriously, i had them before and 
they worked]

also some feel that you're driving the TH1 cell response and minimizng 
the TH2 response via this method. 
what is DESENSITIZATION? if you suspect patient is allergic to something, 
say penicillin, and you need to give that drug, you can't just give it, 
but you can give very small amount of it and monitor response, and if you 
do that over a day, you can gradually increase the amount - the idea 
being that the repetitive small amts of Ag will "use up" the IgE on the 
mast cells, and you may get some small rxns but not a big rxn. so where 
hyposensitization aims to reduce allergy signs, desensitization is used 
in the short term to "use up" the IgE.

----break-----

now. is there a benefit to immediate hypersensitivity/type I rxn?
look at it evolution-wise. many folks believe IgE response was a 
protective mechanism in response to somme parasites.

schistosome: worm of gi tract. this is a pretty antigenic item. some 
epitopes trigger IgE response. so if you have IgE response to this 
schistosome, they end up getting bound to mast cells and basophils - and 
now there isn't really a "reexposure" because there is actually a 
continuous presence...but you end up getting a release of the granules 
and vasoactive amines, and chemotactic factors. the increase in vascular 
permeabilitya and other factors calls in eosinophils and other Ig types 
to do their job on the parasite - this can kill the parasite, so it is a 
protective mechanism...the Eosinophil has both low and high affinity FcE 
receptors, and it has FcG receptors, so it can crosslink the Ab that's 
already bound to the parasite, and cause antibody dependent cytotoxicity. 
the eosinophilic granules contain major basic protein. this is released 
and damages the membrane of the parasite.

so mast cell attracts eos, eos crosslink IgE or IgG on schistosome, and 
ADCC kills schistosome. don't worry about complement. main thing is know 
that eos can via ADCC damage parasite membrane, and release of MBP also 
plays a role. so the IgE response to parasites is an important protective 
mechanism.

[slide: skiing basset hounds with big ears]

in dogs and cats, skin allergy is most common allergy. about 30% of skin 
probs in dogs are due to allergy and of those about 2% are actually food 
allergy. you can also have inhalant allergic dermatitis, caused by 
something they breathe in. Some breeds are predisposed: dalmations, 
terriers: get inhalant dermatitis.
5-10% of animals w/food allergy have GI signs eg diarrhea. respiratory 
allergens tend to be pollens, dust, etc. in horses, the primary allergic 
situation is respiratory. they can get COPD which is believed to be 
primarily a type I rxn caused by repetitive rxn to hay, dust, fungal 
proteins, etc. you can get allergic rxn to vaccines. in these cases you 
dont' usually respond to the intended antigen, but often what happens is 
that in the production of the vaccine there is a contaminant of some kind 
- cell used to grow the bacterium, antibiotic, whatever. so SUBSEQUENT 
vaccinations can induce an anaphylactic response. so tell owners to watch 
animal and bring it back if there is a problem. main skin allergy in dog 
is to FLEAS. the saliva of fleas is really the main allergy that you see 
in animals.

any questions? no? ok. so that was type I immediate hypersensitivity, 
anaphylaxis, caused by release of vasoactive substances from mast celsl, 
causing drug allergies, hay fever. end of that.

ok, now we need to learn this phrase of a nepalese dialect from Doc 
Weber:

Mir sand doch ned deppert (meer sahnd dock ned daypert)

we'll find out about this later. Dr weber claims to have been born in 
katmandu. yeah right,someone commented. anyway, he'll come back to that, 
and he hopes the phrase is in the note service.

moving on to TYPE II reactions. simplest of all. basically involved a 
specific Ag/Ab interaction, complement fixation, and cell lysis. involves 
mostly IgM, and IgG, complement fixation on cell surface, and cell lysis. 
so most of the time, the tissue where this  is most readily seen is in 
the peripheral blood, because we can measure those cells more readily. a 
common thing we find is lysis of RBCs or platelets causing an anemia or 
ITP (idiopathic thrombocytopenic purpura). A neutrophil lysis would cause 
a neutropenia. these are caused by isoantigens or hapten modified 
autoantigens. this rxn can also occur in thyroid, adrenal, pancreas, 
elsewhere - but hard to quantify there.
most common way this occurs is due to hapten of a drug or a virus 
attaching to RBC surface. the immune system starts making Ab against the 
virus or hapten and the Ab will attach per SOP to the RBC bound Ag,a nd 
then complement comes along per SOP and lysis takes place - because the 
complement cascade is occuring ont he surface. now, the reaction is not 
against the rbcs, per se - this is called IMMUNE MEDIATED HEMOLYTIC 
ANEMIA and is mediated by either IgM or IgG. you can also have an 
AUTOIMMUNE HEMOLYTIC ANEMIA, where the body starts to recognize some 
aggregate of the rbc membrane itself as foreign - same process, except 
you are making Ab actually against the patient's own red cells. in the 
first case, the anemia disappears when the drug or virus is cleared, 
where the AIHA is repetitive and recurrent. this AIHA is relatively 
infrequent.

now, in horses, we see HDN hemolytic dz of newborn. horses have 7 major 
blood group Ag. major breeding farms type horses now. the groups causing 
probs are Aa positive or Qa positive. 

Say you have Aa neg mare with Aa positive foal. normally, the rbc from 
fetus don't get into mare until during parturition, some vessels rupture, 
and blood from fetus gets into mare, sensitizing mare to make Ab against 
Aa pos. in first pregnancy,, not a big deal. foal is born and gets 
colostrum before mare has a chance to make a lot of Ab. but if mare is 
bred again to same stud, and has another Aa pos foal, the mare is making 
high levels of Ig, and the foal can die from drinking the colostrum - 
will die 12-24 hrs after nursing depending on Ab level. foal will be 
icteric and dead w/in 24 hrs. icteric b/c lots of rbc lysis.
the colostrum, btw, must be ingested w/in first 12 hrs for foal to absorb 
Ig via GI tract. to test for this (not that you need to) you would do a 
direct coombs test on foal blood. coombs reagent in animals is generally 
antiserum against IgG, IgM and complement. made in rabbit or goat. if you 
see clumping/agglutination when you add the antisera, you know you've 
crosslinked and you have a positive test.

in humans (don't need to knwo) you have rh pos and neg. main thing is as 
fetus grows,mom is gradually sensitized and IgG can cross the placenta as 
we know, so the Ab is transferred to the fetus, and you end up with a 
very sick baby being born. so human hemolytic dz of newborn type thing is 
different.

if you have an incompatible transfusion, you have a similar type rxn. 

how do some of these Ab in type II rxn participate in autoimmune dz?
myasthenia gravis: normally, you have ACH receptor on muscle, and ACH 
comes to receptor and activates. with this dz, you have anti ACH receptor 
Ab which binds to receptor but doesn't activate it, this leads to varying 
degrees of muscle weakness depending on amt of Ab in body. this is a type 
II hypersensitivity rxn despite lack of cell lysis.

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