SKIN BIOLOGY
Alain KHAIAT, Ph.D.
Vice President R&D
Johnson & Johnson Asia Pacific
1
CONTENTS
• Inflammation
• Pigmentation
• Skin Aging
2
CONTENTS
• Inflammation
– irritation
– sensitization
– biochemistry
• Pigmentation
• Skin Aging
3
EPIDERMIS
The cells contained in the epidermis are:
• corneocytes
• keratinocytes
• Langerhans cells
4
DEJ
It is the site of adhesion of epidermis to
dermis, via:
• hemidesmosomes
• anchoring filaments (Kalinin)
• adhesive protein (Laminin)
• fibronectin
5
DEJ
Basal cell
Hemidesmosome
Lamina
lucida
Anchoring
filaments
Lamina densa
Anchoring
fibril
6
DERMIS
The dermis contains:
• fibrobalsts
• mast cells
• Langerhans cells
• lymphocytes and blood vessels
7
The skin is the interface between the organism
and its environment
Because it contains:
–
–
–
–
Langerhans cell
lymphocytes
blood vessels
masts cells
exogenous or endogenous stimuli will create
inflammation processes
8
INFLAMMATION
• Inflammation is the body’s general distress
response to biological, physical or chemical
causes of:
– irritation
– sensitization
– photosensitization
9
INFLAMMATION
• Clinically, inflammation has been defined
through 4 signs:
–
–
–
–
erythema
edema
pain
heat
10
IRRITATION
• Irritants are chemical, biological or physical
agents which can produce inflammation
• Irritation can be either objective or
subjective
• Objective irritation is characterized by the 4
signs mentioned. It is externally observable
• Subjective irritation is characterized by:
stinging, burning or itching
11
IRRITATION
• The result of insulting the skin is the release
of histamine by the mast cells in the
irritated area.
• Histamine is a potent vasodilator, it
produces the visible erythema and increased
vascular permeability (leaking of fluid =
edema), allowing cells (PMN=
polymorphonucleocytes) to migrate to the
area
12
SENSITIZATION
• Skin sensitization is the result of exposure
to sensitizers or allergens
• Skin sensitization is a delayed type
humoral immune response mediated by
the T cell
13
SKIN SENSITIZATION
• The sentitizing substance (hapten),
combines with a protein in the skin to form
the allergen
• The Langerhans cells in the stratum
germinativum interacts with the allergen
and migrates to the lymphoid gland
• It then “teaches” the T cells about the
allergen
14
SKIN SENSITIZATION
• Sensitized T cells migrate to the site and, on
contacting the allergen, liberate cytokines
• these cytokines attract leukocytes to the site
and appear to raise the temperature of the
area
15
Langehans
cell
Allergen
cytokine
T cell
Activated
T cell
16
CYTOKINES
• Cytokines are essential transmitters of
intercellular communication
• They have an inherent role in the regulation
of responses of the immune system
• Each cytokine has multiple functions
• More than one cytokine may mediate the
same, or very similar, function
17
CYTOKINES
• They form part of a complex cellular
signaling language
• They are proteins
18
T CELL RESPONSE
• TYPE 1: cell mediated response, essentially
to viruses, bacteria, protozoa, chemicals.
Th1 response leads to secretion of:
–
–
–
–
IL2
 IFN
TNF
IL12
19
T CELL RESPONSE
• TYPE 2: humoral response following
parasitic infection. Th2 releases:
–
–
–
–
–
IL4
IL5
IL6
IL10
IL13
20
T CELL RESPONSE
• The type of response is function of genes
and the environment.
Th2
Th1
Genes
Environment
21
T CELL RESPONSE
• Allergic contact dermatitis is in its early
stages Th1 (IL2, IFN) becoming later Th2
(IL4). This explains why the reaction
decreases
• Atopic dermatitis is a Th2: IL4, IL5, IL6,
IL10, then IgE, mast cells growth,
eosinophil infiltration
22
UV B EFFECT
• UV B has been shown to suppress immune
reaction (induction phase only)
• UV B stimulates synthesis and release of
TNF- by keratinocytes which in turn
modifies the behavior and morphology of
Langerhans cells
23
TWO MECHANISMS
• Mast cells can respond directly to external trauma,
to antigen-IgE complexes on their surface or to
mediators generated from complement
(anaphylatoxins) by degranulating and releasing
vaso active mediators: histamins
• Langerhans cells interact specifically with Tlymphocytes and keratinocytes to initiate host
response to antigens
24
BIOCHEMISTRY OF
INFLAMMATION
• Phospholipids are the major raw material
and starting point for the arachidonic acid
pathway. Irritants increase the biosynthesis
of phospholipids
• Arachidonic acid is resident to the cell
membrane where it is the source of several
major biochemical pathways
25
Phospholipides
NSAID
Eugenol
Phospholipase A2
Steroids
Arachidonic Acid
Thromboxane
Cyclooxygenase
Acetylsalycilic
acid
12-Lipoxygenase
Prostaglandin G2
5-Lipoxygenase
Prostacyclin
Hydroperoxitetraenoic
Acid (HETE)
Prostaglandin H2
Leucotrienes
Prostaglandins
(PGE2, PGF2, etc)
26
ARACHIDONIC PATHWAY
• If arachidonic acid is acted upon by cyclooxygenase, prostaglandin G2 is generated. It
is itself converted into thromboxane or
prostacyclin or PGH2, the later then
generating the other members of the PG
family.
• Thromboxane stimulates platelet
aggregation and is a vasoconstrictor
27
ARACHIDONIC ACID
PATHWAY
• Prostacyclin inhibits platelet aggregation
and vasoconstriction
• Prostaglandins are non protein chemical
mediators: they are fatty acids
• 12-lipoxygenase transforms AA into HETE
• 5-lipoxygenase catalyses the production of
the leukotriens (eicosanoid family)
28
ANTI INFLAMMATORY
TESTS
• Cytokines secretion by PBL (human
peripheral blood lymphocytes) in culture
following addition of a stimulant
• IL 6 release by human fibroblasts
• Contact hypersensitivity in mouse (ear
edema), after application of Phorbol ester
• Ear edema in mouse following AA
inflammation
29
CONTENTS
• Inflammation
• Pigmentation
– anomalies
– melanogenesis
• Skin Aging
30
PIGMENTATION
Skin color is the result of:
• nature of the melanin
• where the melanin is concentrated, i.e.
quantity, type and distribution of
melanosomes (epidermis or dermis)
• skin vascularisation
31
PIGMENTATION ANOMALIES
1. Melanocytes proliferation is normal:
• Freckles: eumelanin zones on pheomelanin
backgrounds (skin areas exposed to the sun)
• Chloasma: pregnancy mask: hypersecretion
of melanin induced by hormonal factors and
amplified by the sun
32
PIGMENTATION ANOMALIES
• Diffuse brown melanosis: endocrine system
disorders or nutritional anomalies
• Hypermelanosis can follow cutaneous
inflammations:
– pigmentation of scars,
– caused by irritants combined with sun
(photosensitizers like bergamot oil)
33
PIGMENTATION ANOMALIES
2. Melanocytes do not proliferate correctly
• Lentigines: can be hereditary, appear
anywhere on the body
• Solar Lentigo: wider lesion than freckle,
occurs after serious sunburn
• Senile Lentigo: generally on the back of the
hand of older subjects, stimulated by solar
exposure
34
PIGMENTATION ANOMALIES
• Dubreuilh melanosis or malignant lentigo of
the elderly: large pigmented multi colored
stain, pre-cancerous
• Moles or Naevus: accumulation of
melanocytes in epidermis and dermis
• Malignant melanomas: cancerous tumors.
The first signs are degeneration of existing
naevus or Dubreuilh melanosis
35
PIGMENTATION
• All methods to reduce pigmentation on the
market today have the objective to reduce
melanogenesis
36
MELANOGENESIS
PATHWAYS
L-TYROSINE
TYROSINASE
3,4-DIHYDROXYPHENYLALANINE
TYROSINASE
DOPA QUINONE
GSH
CYCLISATION
GSH-DOPA
LEUCODOPACHROME
3-S-CYSTEINYL DOPA
DOPACHROME
INTERMEDIATE PDTS
TRP2
PHEOMELANIN
5,6 DIHYDROXYINDOLE
TRP1
QUINONE-IMINE
EUMELANIN
37
MELANOGENESIS
INHIBITION
• Inhibition of the production of active
tyrosinase in the ribosomes: placental
extract
• Inhibition of the transfer of tyrosinase to
pre-melanosomes by interrupting
glycosylation (tunicamycine, glucosamine)
• Elimination of inflammatory reactions
(flavonoids, tannins, etc)
38
MELANOGENESIS
INHIBITION
• Inhibition of tyrosinase: Kojic acid,
ascorbic acid, etc. EDTA or Phytic acid
(since tyrosinase requires Cu++)
• Inhibition of the formation of eumelanin: by
adding glutathion and glutathion reductase
transforming GSSG into GSH, promote the
formation of glutathion DOPA leading to
pheomelanin
39
PIGMENTATION
• Melanin is formed in the Melanocytes,
where it is stored in the melanosomes
• Melanocytes extend arms to transfer
melanosomes into the keratinocytes
• It is the keratinocytes charged with the
melanosomes that constitute the dark spots
on the skin
40
Pigmentation Formation Mechanism
1
2
Irritation
UV
• Variety of Causes
• Variety of Responses
Inflammatory Response
KERATINOCYTE
(Epidermis)
3
Hormone
Tyrosine
Melanin
MELANOCYTE
(Basal Layer)
Melanosome
Tyrosinase
FIBROBLAST
Dermis
41
Basic Structure of Skin
Stratum Corneum
Keratinocyte
Viable Epidermis
Melanocyte
Basal Layer
Dermis
42
PIGMENTATION
• A novel approach has recently been
published: blocking the transfer of
melanosomes from the melanocyte to the
keratinocytes
• Accumulation of charged melanosomes
inhibits melanin synthesis
43
SUGGESTED MECHANISM
Depigmentation
Less
melanosome
transfer
Less eumelanin
produced,
lighter color
1. Less TRP-1 is made
tyrosinase not stable
2. More TRP-2 is made
shift to brownish
melanins
Melanosomes
accumulate
Negative
feed-back
44
NOVEL MECHANISM
• Protease Activated Receptor (PAR-2) is
expressed in keratinocytes. PAR-2 is
activated by trypsin
• By inhibiting PAR-2, one probably blocks
the keratinocyte-melanocyte interaction
• TRP1 (tyrosinase-related protein) decreases
leading to less Eumelanin
45
PIGMENTATION TESTING
• Tyrosinase activity in solution: mushroom,
mouse or human tyrosinase are used with
different results
• S91 melanoma cells in culture
• Keratinocytes-Melanocytes co culture
• Guinea pig ear: 15 days treatment
• Microswine spotted model: 6-8 weeks
46
PIGMENTATION TESTING
• Human volunteers tests:
– Chromameter® : L measure
– Mexameter® : evaluation of melanin and
redness
– Photography : visible, UV with data analysis
3 months minimum,
changes, so far, are not very significant
against placebo
47
CONTENTS
• Inflammation
• Pigmentation
• Skin Aging
– skin changes
– biochemical changes
48
MANIFESTATIONS OF SKIN
AGING
• Epidermis :
– reduction in cell renewal rate
– thickening of stratum corneum
– decrease in barrier efficiency : increase in
TEWL and hyperkeratosis
– ridges are flattened out and intercellular spaces
enlarged
– pigmentation problems : actinic lentigines
– decrease in skin immune system
49
MANIFESTATIONS OF SKIN
AGING
• Sebaceous glands :
– reduction in sebum secretion (hormones
influenced)
• Sweat glands :
– less active
• HLP film :
– thinning of film means less protective barrier
50
MANIFESTATION OF SKIN
AGING
• Dermis :
– destruction of collagen and elastin fibers
network
– proteoglycans and glycoproteins are reduced
– increase in elastin synthesis : elastosis
51
PHOTOAGING
3 types of reactions to UV exposure:
• Free Radicals, essentially due to UVA
• Direct cell death, essentially due to UVB
• MMP Enzymes
52
FREE RADICALS
Free radicals or ROS (reactive oxygen
species) can lead to breakage of important
molecules:
• DNA (mutations, renewal failure, cell
death)
• collagen, elastin, GAG (skin firmness)
• lipids (membrane or structural)
53
UV DAMAGE AND
OXIDATIVE STRESS
DNA effects
Matrix effects
DNA fragmentation
MMP : TIMP ratio
UV
damage
Membrane effects:
ROS
Lipids peroxidation
Hydroperoxides
Enzymatic systems
SOD
Glutathion peroxidase
Heme oxidase
54
DNA DAMAGE
• UVA acts through oxidative stress forming
“reactive oxygen species” (ROS) that will
damage the DNA and lead to cancer
55
DNA DAMAGE
• UVB impact on DNA in the cell creating
damages which may lead to cancer: nonmelanoma skin cancer (NMSC)
56
57
UVB DAMAGE
• Following structural changes in DNA, there
is an altered expression of oncogenes and
tumor suppression genes, such as p53
• NMSC show a high incidence of mutation
in p53 gene
58
59
p 53 GENE
Plays an important role in:
– blocking the cell cycle after exposure to DNAdamaging agents e.g. UV, in order to allow for
repair before duplication
– or killing the cell to avoid multiplication of
damaged cells (formation of sunburn cells)
60
p 53 GENE
The induction of detectable levels of p53 in
human epidermis after UV exposure is
relevant to skin carcinogenesis
61
Collagen & Photodamage
• Major structural component of ECM
– 70% of the dry weight of skin
• Collagen degradation is believed to play a
role in formation of wrinkles
62
Collagen Degradation
A balance between MMP:TIMP
MMP
TIMP
63
MMP ENZYMES
TIMP
ROS
MMP
COLLAGEN
DEGRADATION
64
MMP ENZYMES
• Collagenases (1 to 4) are specific to various
collagen,
• Gelatinases (A & B) are non specific
• Stromelysins (1-3) specific of fibronectin,
laminin, collagen IV, etc.
• Elastase: elastin
• etc
65
MEMBRANE EFFECTS
With age, reduction in membrane fluidity
leading to less efficient exchanges:
– intrinsic: reduction in the methylation of PE
into PC
– extrinsic: lipid peroxides
Methyl donors will restore membrane fluidity
66
ACTIVE PHOTOPROTECTION
Replenish antiox
system
Inhibition of
oxidative stress
ACTIVE
PHOTOPROTECTION
Reduce matrix
degradation
Quench
ROS
67
Irradiation of Epidermal Equivalents
with Solar Spectrum UV
10-4
Solar Simulator
10-5
W/cm
2
10-6
10-7
10-8
10-9
10-10
10-11
260
280
300
320
340
360
380
Wavelength (nm)
MM & TIMP-1
68
400
420
UV Irradiation of Epidermal
Equivalents
• Markers of damage
– MMP-1 induction
– TIMP-1 induction, but to a lesser extent than
MMP-1
– MMP:TIMP imbalance
• Protection provided by
– Sunscreens
– Anti-oxidants
69
UV Irradiation of Epidermal
Equivalents
• Model for assessing Photoprotective
potential
– Botanical ingredients
– Fully formulated product
70
THANK YOU
71