1. Alain KHAIAT, Ph.D. Vice President R&D Johnson & Johnson Asia Pacific
SKIN BIOLOGY
Alain KHAIAT, Ph.D.
Vice President R&D
Johnson & Johnson Asia Pacific

2. CONTENTS
Inflammation
Pigmentation
Skin Aging

3. CONTENTS Inflammation Pigmentation Skin Aging irritation sensitization
biochemistry
Pigmentation
Skin Aging

4. EPIDERMIS The cells contained in the epidermis are: corneocytes
keratinocytes
Langerhans cells

5. DEJ It is the site of adhesion of epidermis to dermis, via:
hemidesmosomes
anchoring filaments (Kalinin)
adhesive protein (Laminin)
fibronectin

6. DEJ Basal cell Lamina lucida Anchoring filaments Lamina densa
Hemidesmosome
Lamina
lucida
Anchoring
filaments
Lamina densa
Anchoring
fibril

7. DERMIS The dermis contains: fibrobalsts mast cells Langerhans cells
lymphocytes and blood vessels

8. 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

9. INFLAMMATION
Inflammation is the body’s general distress response to biological, physical or chemical causes of:
irritation
sensitization
photosensitization

10. INFLAMMATION
Clinically, inflammation has been defined through 4 signs:
erythema
edema
pain
heat

11. 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

12. 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

13. 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

14. 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

15. 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

16. Langehans
cell
Allergen
cytokine
T cell
Activated
T cell

17. 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

18. CYTOKINES They form part of a complex cellular signaling language
They are proteins

19. T CELL RESPONSE
TYPE 1: cell mediated response, essentially to viruses, bacteria, protozoa, chemicals. Th1 response leads to secretion of:
IL2
 IFN
TNF
IL12

20. T CELL RESPONSE
TYPE 2: humoral response following parasitic infection. Th2 releases:
IL4
IL5
IL6
IL10
IL13

21. T CELL RESPONSE
The type of response is function of genes and the environment.
Th2
Th1
Genes
Environment

22. 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

23. 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

24. 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 T-lymphocytes and keratinocytes to initiate host response to antigens

25. 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

26. Hydroperoxitetraenoic
Phospholipides
Steroids
NSAID
Eugenol
Phospholipase A2
Arachidonic Acid
Acetylsalycilic
acid
Cyclooxygenase
Thromboxane
12-Lipoxygenase
Prostaglandin G2
5-Lipoxygenase
Prostacyclin
Hydroperoxitetraenoic
Acid (HETE)
Prostaglandin H2
Leucotrienes
Prostaglandins
(PGE2, PGF2, etc)

27. ARACHIDONIC PATHWAY
If arachidonic acid is acted upon by cyclo-oxygenase, 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

28. 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)

29. 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

30. CONTENTS
Inflammation
Pigmentation
anomalies
melanogenesis
Skin Aging

31. 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

32. 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

33. 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)

34. 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

35. 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

36. PIGMENTATION
All methods to reduce pigmentation on the market today have the objective to reduce melanogenesis

37. MELANOGENESIS PATHWAYS
L-TYROSINE
TYROSINASE
3,4-DIHYDROXYPHENYLALANINE
DOPA QUINONE
GSH CYCLISATION
GSH-DOPA LEUCODOPACHROME
3-S-CYSTEINYL DOPA DOPACHROME
INTERMEDIATE PDTS 5,6 DIHYDROXYINDOLE
TRP TRP1
PHEOMELANIN QUINONE-IMINE
EUMELANIN

38. 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)

39. 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

40. 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

41. Inflammatory Response
Pigmentation Formation Mechanism 1 UV
Variety of Causes
Variety of Responses 2
Irritation
Inflammatory Response
KERATINOCYTE
(Epidermis) 3
Hormone
MELANOCYTE
(Basal Layer)
Tyrosine
Melanin
Melanosome
FIBROBLAST
Tyrosinase
Dermis 1

42. Basic Structure of Skin
Stratum Corneum
Keratinocyte
Viable Epidermis
Melanocyte
Basal Layer
Dermis

43. 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

44. SUGGESTED MECHANISM Depigmentation Less Less eumelanin melanosome
transfer
Less eumelanin
produced,
lighter color
Melanosomes
accumulate
1. Less TRP-1 is made
tyrosinase not stable
2. More TRP-2 is made
shift to brownish
melanins
Negative
feed-back

45. 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

46. 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

47. PIGMENTATION TESTING Human volunteers tests: 3 months minimum,
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

48. CONTENTS Inflammation Pigmentation Skin Aging skin changes
biochemical changes

49. 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

50. 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

51. MANIFESTATION OF SKIN AGING
Dermis :
destruction of collagen and elastin fibers network
proteoglycans and glycoproteins are reduced
increase in elastin synthesis : elastosis
The dermo - epidermal junction flattens out and loses its waviness.
Less mechanical support : lower elasticity, higher fatigability.

52. PHOTOAGING 3 types of reactions to UV exposure:
Free Radicals, essentially due to UVA
Direct cell death, essentially due to UVB
MMP Enzymes

53. 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)

54. UV DAMAGE AND OXIDATIVE STRESS

55. DNA DAMAGE
UVA acts through oxidative stress forming “reactive oxygen species” (ROS) that will damage the DNA and lead to cancer

56. DNA DAMAGE
UVB impact on DNA in the cell creating damages which may lead to cancer: non-melanoma skin cancer (NMSC)

58. 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

60. p 53 GENE Plays an important role in:
blocking the cell cycle after exposure to DNA-damaging 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)

61. p 53 GENE
The induction of detectable levels of p53 in human epidermis after UV exposure is relevant to skin carcinogenesis

62. 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

63. Collagen Degradation A balance between MMP:TIMP

64. MMP ENZYMES
TIMP
ROS
MMP
COLLAGEN
DEGRADATION

65. 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

66. 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

67. ACTIVE PHOTOPROTECTION
Replenish antiox
system
Inhibition of
oxidative stress
ACTIVE
PHOTOPROTECTION
Reduce matrix
degradation
Quench
ROS

68. Irradiation of Epidermal Equivalents with Solar Spectrum UV
Solar Simulator
MM & TIMP-1

69. 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

70. UV Irradiation of Epidermal Equivalents
Model for assessing Photoprotective potential
Botanical ingredients
Fully formulated product

71. THANK YOU