---start---

path
10/16/97
more dermpath
goldschmidt

basal lamina = 
basal membrane. epithelial layer attached to it by hemidesmosomes. two of 
those = complete desmosome --> seen in between the keratinocytes.

we're 
going to talk about proliferation in basal area and what controls it, the 
keratin filaments, the keratohyaline granules, the lamellar bodies, the 
formation of cornified cell envelopes, and functions of these structures. 
why? when you are in clinical situation, you have skin with too much 
keratin or not enough, too thick or too thin,and you need to know the 
pathogenic mechanisms. 

so remember this is a multilayered complex 
organ, is acheived by differentiation of cells from basal layer to 
surface layer. proliferation occurs mainly in basal cell layer. 
desquamation= sloughing of cells from skin surface. 

we used to consider 
epidermis as a fairly bland structure, esp the stratum corneum, which was 
considered as kind of saran wrap. that isn't the case anymore. it is a 
dynamic structure. it may function as saran wrap to say prevent loss of 
water, and prevent thigns from getting in, but normally cells do release 
some fluid, exfoliate, etc. 

so, first, we'll discuss the keratin. this 
is the intermediate fiber seen within the keratinocyte. keratins are the 
unique intermediate filament seen only in epi cells. they form a 
cytoskeleton to maintain the shape of the cell. most of these keratins 
are formed in basal and spiny layers. by the time you get to granular or 
cornified layer you don't make any more keratins. there are about 30 
known keratin molecules of varying sizes, and more are found yearly. each 
is made of two parts - an acidic and a basic part combined together to 
make keratin filament. acidic molecule is smaller and usually given # 1-8 
eg Ka 1-8 and the basic part, Kb, is given 9-20.... see p 10 in handout


normally the keratin expressed in basal cell is 50 and 58 KDa keratin. 
that's what is coded for, genetically. as cell migrates up - loses 
hemidesmosomal attachment to BM - a different gene is turned on, encoding 
for 56.5 KDa keratin. some hyperproliferative dzs cause different keratin 
to be made - eg, psoriasis in humans,causes thick skin, red areas on 
various parts of skin, where keratin is different. instead of normal 56 
and 66, you get a smaller acidic and basophilic keratin. changes ability 
of cells to migrate and differentiate within epidermis. 

next - how do 
epidermal cells, keratinocytes, actually stick together? two mechanisms 
are used. first is the desmosome, the intercellular bridges you can see 
on light microscopy. second is the adherence junction. the desmosome is a 
very extraordinarily complex structure, and they exist between all 
epithelial cells - not just in the skin. when we talk about intermediate 
filaments here, we're talkigna bout the keratin filament, which inserts 
into the inner plaque of the cell membrane. there are specific molecules 
which then attach the keratins to the deeper plaque - these are called 
desmoplakins. these are strange words, but when you get into clinics, you 
will see dz where animals are making auto-Ab against parts of the 
desmosome, so you need to know this. so the intermediate filament 
connects to inner plaque. then this connects to the deeper plaque. then 
you have the plaquoglobin. extending through plaquoglobin to the cell 
membrane and through the IC space, is the desmoglein, which meets in the 
intercellular space another desmoglein from the neighboring cell, and 
they join up in a zipperlike formation. this causes focal adhesions in 
multiple sites of your two keratiocytes by the desmoglein molecules. also 
you have similarly acting desmocollin molecules. if you had autoAb to the 
desmogleins, cells would separate from eachother, get rounded up, and 
you'd lose the integrity and contiguity of the epidermis. Dr G sees about 
6 of these a week. this is the major way these cells stay together. but 
not the only way. there are also "adherens junctions" like spot-welds, or 
buttons. desmosome = zipper, adherens junction is the button. desmosomes 
are better, and stronger, but the adherens junction is a good backup 
mechanism. nature enjoys redundancy. in skin there are two kinds of these 
junctions - those associated with epithelial caherins, e-cadherins, which 
occur throughout all areas of epidermis, and then there are focal 
adherent junctions at the basal area, where basal cell attaches to BM, 
and these are p-cadherin junctions. p=placental.  originally described in 
the cow. 

e-cadherins junctions are also found between other cells in 
epidermis - as follows. you have e-cadherins b/w keratinocytes and 
langerhans cells, maintaining position of langerhans cells. also b/w 
keratinocyte and melanocye, and keratinocyte and merkel cell. desmosomes 
do not exist between keratinocytes and these other cell types - only 
between the epidermal cells. 

the next thing to discuss is the granular 
cell layer, which contains a specific dark blue granule called a 
keratohyaline granule. yesterday in lab some people saw two types of 
granules. there are blue ones up in superficial epidermis -> 
keratohyaline granules. then, associated with hair follicles were some 
red granules called trichohyaline granules, which have similar function. 
we're just talking about keratohyaline granules now. these granules 
contain profillagrin, a high molecular wt protein. in the granular layer 
this protein becomes fillagrin which causes keratin filaments to bind 
eachother and to form these larger, cable like structures. the fillagrin 
kind of sits between the filaments, bundling them into larger, thicker, 
stabler bundles. we need this because as cells reach the surface, they 
will be the cells exposed to the environment, to all the insults out 
there. need to be tough, dense, etc. the other thing that happens is we 
see formation in cornified layer of cornified cell envelope (CE). this is 
an insoluble membrane that forms on the inside of the normal cell 
membrane. the CE actually takes the place of normal membrane. this is 
done by a calcium dependent enzyme. as keratinocytes reach upper layers, 
membrane becomes more permeable, calcium enters cell, transglutaminase is 
activated, and it takes keratolinin, involucrin, and loricrin molecules 
and sticks them together on the outside of the cell, so now you're left 
with this CE on the surface of the cell, and an outer membrane which is 
being destroyed. within the cytoplasm of the cell you have these very 
thick keratin fibers (cables) that are bound by the fillagrin. as a 
result of all this somethign really unique happens. we lose the nucleus. 
because it can't be nourished any longer. so as we go from basal to 
spinous layer, we still see nuclei, and as we reach granular layer, 
nuclei are fading,and in the cornified layer, nuclei are gone completely. 
we don't know  how it does it, but under normal circumstances, the 
stratum corneum is an anucleate structure. 

finally, the lamellar 
bodies, aka Odland bodies. these are phospholipid bodies. they are made 
in upper spinous layer and in granular layer. they are enclosed within a 
membrane which fuses with cell membrane and releases contents into the 
intercellular space between two cells. this material is a very potent 
lipid and phospholipid material. it is high in cerumides, and 
cholesterol. what it does is it provides some waterproofing b/w the 
individual keratinocytes. so you have on the surface of the skin what is 
lovingly referred to as the brick and mortar phenomenon, where bricks = 
keratinocytes in stratum corneum, and mortar = phospholipid secreted from 
lamellar bodies, which surrounds individual cells. provides waterproofing 
and is also important b/c if there is dysfunction there is too much 
adhesion of cells to eachother and more pathology. 

does this remind you 
of anything? any other place where a phospholipid is secreted to the 
outside of the cell? the lung. surfactant lines the alveoli in a similar 
way. 
so that's differentiation

now, proliferation. a simple statement: 
the rate of basal cell mitotic activity is directly proportional to the 
desquamation of corneocytes from top of skin. you can increase the number 
of cells undergoing mitosis, or decreasing cell cycle time, or decrease 
time it takes to get from bottom to top. in the normal beagle dog (there 
is no such thing as normal beagle but whatever)  - in beagles in the lab, 
it takes about 22 days to get from basal layer to cornified layer. so 
what influences this process? see p 12 handout. there is a list of 
factors which control this. arachadonic acid and metabolites of that. 
calcium ions, cytokines, vitamin A and retinoids. 

arachadonic acid - an 
unsaturated FA on the membrane of the keratinocyte (as well as in 
membrane of inflammatory cells). REMEMBER this. it is released when 
phospholipase A2 breaks link between it and epidermal lecithin. 
phospholipase A2 can be released by physical injury (eg, simply 
scratching skin), UV light B, some but not all retinoic acids - and you 
have to know, no one understands how retinoic acids act. one does one 
thing, one does another thing, we don't have to know which is doing 
which. histamine, bradykinin, (from mast cell, dr G's favorite cell) and 
prostaglandins and calcium.

after arachadonic acid is released it is 
metaboliezd into mediators -->cyclooxygenase and lipooxygenase pathways. 
leukotrienes, thromboxanes and prostaglandins. 

in the dog, and that's 
the only species wehre this has really been studied, the inflammatory 
mediators of allergy are known to be leukotriene B2, prostaglandin E2, 
and 12 HETE. 

the favorite tx of skin disease is steroids. dr. G though 
before that this was a bad thing to do without knowing the underlying 
etiology and pathogenesis. however, now that his wife has a very allergic 
jack russell terrier, he thinks it is better to just give the steroids. 
you can assume the owners will do whatever they need to do, but if you 
give it the pred, it won't scratch and that's the bottom line. steroids 
promote the formation of another protein called lipomodulin or 
macrocortin which inhibits phospholipase A2, which prevents formation of 
the other mediators.

calcium: important in proliferation and 
differentiation and degradation. low in basal cells, high in granular 
cells. influx of Ca into cytoplasm occurs in upper granular layer, 
resulting in mitotic arrest (if happens in basal layer), exocytosis of 
secretory granules, activation of transglutaminase leading to 
crosslinking of cornified envelope proteins. so you get reduced 
proliferation and differentiation. also if you get increased 
intracellular Ca in lower granular layer, you get premature terminal 
differentiation, and parakeratotic hyperkeratosis (retention of nucleus). 
in the cornified layer, it causes abnormal desquamation and abnormal 
barrier function -> fluid loss and bacteria nd yeast can invade.


cytokines: large family. new ones coming along all the time. act through 
receptors. can have autocrine where target is cell. paracrine, where 
target is neighbor cell, or endocrine, where target is distant. transient 
and self limiting. not usually stored as preformed molecules - need gene 
transcription to get it. are receptor mediated, high affinity. potent at 
low concentrations. see handout.

most important cell is keratinocyte. it 
liberates a lot of cytokines-> IL1 which activates other keratinocytes 
and acts on the endothelial cells to make them stickier, promoting 
inflammatory response and neutrophils adhereing and migrating in. we have 
lots of other less important ones. TNFa, acts on endothelium. TGFa acting 
on vessels in dermis, inducing angiogenesis -> important when you do 
surgery b/c when you incise skin and get cytokine release it's going to 
act on underlying vasculature, which is what you want - you will need 
granulation tissue to fill in defect. the other important cells are 
langerhans cells which also make IL1. can get some proliferation of these 
cells within epidermis with colony stimulating factors. 
in the dermis, 
monocytes and mphages are seen around the vessels, and they can release 
another large group of cytokines. also, cytokines act upon them from 
adjacent cells. lymphoid cells present in perivascular tissue. most 
important is the cytokines liberated from teh mast cells. in most 
animals, mast cells are not present around vessels in young dogs. they 
get there from blood, get out through vessels, and attach around the 
vessels. they liberate a variety of cytokines including IL5 which is 
eosinophilotactic, adn causes eos to come into the area. you can't see 
mast cells degranulating in allergic dermatitis as an actual event. but 
you see the release of cytokines results in influx into dermis of 
eosinophils. eos around vessels in dermis are a hallmark of allergic 
disease. 

other structures in dermis to think about are endothelial 
cells which liberate cytokines and respond to cytokines. anything causing 
leukocyte adhesion, eg IFNg and TNFa is a major participant in acute infl 
response. with acute infl we get adhesion of WBC to endothelial cells by 
these adhesion molecules, we get cells migrating out via diapedesis into 
dermis. if inflammation exists in epidermis, WBC migrate up into there to 
do the dirty work of cleaning it up. we see this clinically as the 
formation of pustules. so if you don't have endothelial adhesion 
molecules expressed, you don't get the pustule formation. 

vitamins: vit 
D acts by modulating calcium homeostasis. strongly inhibits cell 
proliferation and induces terminal differentiation. 

retinoids: we don't 
know how they do what they do. are said to regulate growth and 
differentiation. in GENERAL they inhibit differentiation, suppress CE 
formation and fillagrin expression. but dose dependent and also dependent 
on type of retinoic acid given to animal. we don't often use retinoids in 
vet med. are used in people w/cystic acne. we have used them in a single 
dz called cutaneous LSA and probably there, these are forming a highly 
impenetrable barrier to the neoplastic cells which then can't get into 
epidermis.

---end---