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

CONTENTS Inflammation Pigmentation Skin Aging

CONTENTS Inflammation Pigmentation Skin Aging irritation sensitization biochemistry Pigmentation Skin Aging

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

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

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

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

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

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

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

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

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

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

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

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

Langehans cell Allergen cytokine T cell Activated T cell

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

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

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

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

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

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

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

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

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

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)

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

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)

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

CONTENTS Inflammation Pigmentation anomalies melanogenesis Skin Aging

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

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

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)

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

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

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

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 TRP2 TRP1 PHEOMELANIN QUINONE-IMINE EUMELANIN

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)

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

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

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

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

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

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

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

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

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

CONTENTS Inflammation Pigmentation Skin Aging skin changes biochemical changes

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

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

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.

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

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)

UV DAMAGE AND OXIDATIVE STRESS

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

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

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

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)

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

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

Collagen Degradation A balance between MMP:TIMP

MMP ENZYMES TIMP ROS MMP COLLAGEN DEGRADATION

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

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

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

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

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

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

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