Photobiology & Photo immunology

Anand

• UV Light 200-400 nm wavelength• 3 Types:- UVA, UVB & UVC

• 90% -UVA• 10%-UVB• UVC –Don’t reach earth surface

UV C

• 200 - 290 nm • <290 nm are absorbed by ozone • Strongly absorbed by DNA and therefore can be

lethal to viable cells of the epidermis• Care to avoid exposure of eyes & skin to UVC

radiation• Bactericidal:-Used for air & water purification• a.k.a Germicidal radiation

UVB

• 290 to 320 nm is mid-UV /sunburn spectrum • UVB -5% of the UV &0.5% of total radiation• Ordinary window glass blocks UVB• Most sunscreens efficiently reflect or absorb

these wavelengths, and SPF is primarily based on testing against this waveband

• And more effectively causes DNA damage & photocarcinogenesis.

• NB-UVB(311 nm) & Excimer laser (308 nm) are used to treat psoriasis

UVA

• UVA I (340–400 nm) & UVA II (320–340 nm)• UVA II more damaging to unsensitized skin • SPF≥ 45 some UVA protection• UVA causes photoageing & carcinogenesis

VL , Xrays ,Gamma rays…

• The visible spectrum (400–760 nm) is defined by the wavelengths that are perceived as color by the retina

X-ray and γ rays :-• In RT of tumors- ionize(Remove electrons) of

water molecules & produce ROS that damage DNA

Infrared

IR-A (760–1,440 nm) IR-B (1,440–3,000 nm) IR-C (3,000 nm–1 mm)

• IR-A causes skin damage • Recent studies -IR-A wavelengths can also be

therapeutic• Whereas IR-B and IR-C are felt as heat.

Uses of EM Radiations

• VL– in Blue-light ALA–photodynamic therapy• Several lasers • UVB Phototherapy• PUVA Photochemotherapy

• UV and/or visible radiation also from Fluorescent lights Incandescent bulbs Photocopy machines & Phototherapy lamps

Artificial Sources

• Incandescent Lamps• Fluoroscent lamps• Arc lamps• Woods lamp• Halide lamps

INCANDESCENT SOURCES

a)Conventional electric light bulbs- VR & IR• An electric current heats the filament &

emits -VR & IRb)Tungsten-halogen(flood lamps)- UVA & VRc) Quartz iodide lamps -UVA & UVB

ARC SOURCES

• Xenon lamps,medium- and high-pressure mercurylamps, fluorescent lamps & halide lamps

• In arc lamps, electrons are driven through a gas by a potential difference between two electrodes.

• The gaseous molecules are ionized and subsequently release EM radiation.

• Xenon arc lamps - UV & visible radiation• Used as solar simulator

Fluorescent lamps• Source of UVB & UVA lamps• Are modified low-pressure mercury arc lamps. • The inner surface of the glass tube is coated

with a phosphor, which absorbs the 254-nm radiation and reemits the energy at longer wavelengths.

• The chemical composition of the phosphor determines which wavelengths are reemitted.

• Emission peak at 311 nm (Philips TL01) • Used in phototherapy• Rx-Psoriasis,Vitiligo, AtopicDermatitis & PMLE

• Hot quartz Lamps – medium & high-pressure mercury lamps - sources of UVB

• Wood’s lamps- low-pressure, UVA emitting fluorescent lamps with a UVA-transmitting, visible-absorbing glass envelope

Halide Lamps

• Halide lamps emit a high-intensity continuum UVB along with UVA range

• Mainly as a UVB source

LASERS

• Monochromatic (single wavelength) radiation• Different lasers emit UV, visible Or infrared • Either continuous or pulsed source

• E …….Joules• Power(W)….. J/sec• Irradiance….. W/cm2

• Fluence/Dose of light…..Energy cm2

Spectral irradiance

• The irradiance delivered by a source as a function of wavelength is called the spectral irradiance and is expressed as units of irradiance per nanometer [(W/ cm2)/nm]

• A spectroradiometer is used to measure the spectral irradiance of a light source

• More wavelength means more penetration• UVA,VL & IR will reach upto S/c tissue• UVB upto mid dermis only• 10% of 300-nm & 50% of 350-nm reaches DEJ

• More energy – More erythema• More energy– More scattering• UVB scattered more easily than UVA

• 5%-10% of incident light -reflected by S.corneum.

• Reflected,Scattered• Absorbed by chromophores in various layers

• Specular reflectance is relatively constant for all visible wavelengths and accounts for the surface appearance of skin

• “Glossy” if the surface is smooth, wet, or oily. • “Rough” if irregular.• Moisturizers applied to the skin make the skin

look shiny

Application

• Skin with scales-as in psoriasis- scatters more• During phototherapy-Apply emmolients• Allows more of the effective wavelengths to

penetrate into the viable tissue.

• Melanin, which absorbs relatively uniformly over the visible wavelengths in epidermis will

decrease the remittance• The greater overall melanin content the lesser

light will be remitted back to the observer.• Hemoglobin within the dermis absorbs mainly

blue spectrum and remits red • This explains melasma & vitiligo discolouration

Vitamin D

• Chick independently observed that sunlight would cure rickets just as well as cod-liver oil

• Hess -cholesterol in skin is activated by UV-irradiation and rendered antirachitic

• Steps in the vitamin D pathway- elucidated by Velluz

• Vitamin D- Promotes• 1)Absorption of Ca & Phosphorus from the intestines• 2)Reabsorption of calcium in the kidney

• Vitamin D – Inhibits• 1)Proliferation of T-cells &• 2)Maturation of dendritic cells • 3)Keratinocyte function.

• Rickets in children and Osteomalacia in adults • Contributes to osteoporosis.

BIOCHEMICAL PATHWAY

• Provitamin D3 (7-dehydrocholesterol)• Previtamin D3• Isomerizes to vitamin D3, entering the circulation on a

binding protein • Joins with dietary D2 (ergocalciferol) & D3 (cholecalciferol)

absorbed from the gut• Liver- passive hydroxylation -25-hydroxyvitamin D3 (calcidiol)• Kidneys- 1,25-hydroxyvitamin D3 (calcitriol)• Bound to a carrier- vitamin D-binding & transported to

various target organs.

• Most effective production in 295 nm -315 nm, (most responsible for photocarcinogenesis)

• 10–15 min ,Twice in a week • Face, arms, hands, or back without sunscreen• Vitamin D production decreases >70 yrs • More melanin in skin = More exposure

• We make 10,000–25,000 IU of vitamin D upon at one MED exposure

• Within 20 min of sun exposure in fair-skinned individuals (1–3 hrs for pigmented skin)

• Sun protection recommendations may interfere with vitamin D production

• Low vitamin D levels & Parkinson’s disease

• Excess Vit-D with-DM ,Multiple sclerosis , HTN M.I, affective disorder,chronic pain, PAD, Memory loss

• Preventing colorectal Ca , breast & prostate Ca

Vit D

• Optimal level in blood 30ng/mL • 1,000 IU daily 10,000 IU weekly 50,000 IU monthly• Levels < 15 ng/ml – prescribe 50,000 IU Vitamin D weekly for 8 weeks and then switches to standard maintenance doses

• Persistently low levels, despite several attempts at correction …..Malabsorption & referral to a gastroenterologist

• A trial of UVB light therapy may be considered to improve vitamin D status

• High body mass index (BMI) with Vit D deficiency require higher doses

Photoproducts

Stimulate cellular signal transduction pathways leading to Biochemical changes

Proliferation, Secretion of cytokines, and Apoptosis

Acute skin responses.

Absorption Spectra

• A chromophore will take up only a specific wavelength of photon

Absorption maxima of various Chromophores

• Absorption maxima means the wavelength which has the greatest probability of absorption

• Purines & Pyrimidines- 260 nm• Amino acids-290 nm• Hemoglobin -410• Bilirubin-450• Melanin –UV& VL ( No distinct value)• Psoralens -UVB

Ground stateSinglet excited state (a few nanoseconds) PhotoproductsInternal conversion Fluorescence Intersystem crossing Triplet excited statePhosphorescenceIntersystem crossing

Applications

• The heat generated by internal conversion is responsible for the effects of pulsed lasers

•Photo Products

• The photoproduct molecule produced by rearrangement of the bonds in the chromophore

Eg:- pyrimidine dimer (CPD) in DNA

• The term quantum yield indicates the likelihood that one of these processes occurs

• Excited state chromophore transfers its energy to O2 ROS• These ROS initiates inflammation in sunburn &

drug phototoxicity.

• Cell Membranes• ROS Phospho Lipase A2

• • Arachidonic Acid• cox lox • • Eicosanoids

• Photosensitization:-• Tetracyclines,FQ, Psoralens & Dyes • Delayed erythema & inflammation• ROS will oxidize Lipids in membranes Amino acids in proteins & Guanine in nucleic acids• The oxidized products will produce erythema• Topical antioxidants can modulate it.

• PG &NO appear to be the major mediators Eg:- PGE2, PGF2α & PGE3 levels increased

• Inhibiting cox with indomethacin used for UVB induced erythema

• UV induced signs of inflammation1. Erythema:- Increased blood flow 2. Temperature:-Increased3. Swelling (increased vaso permeabilty) &4. Pain (released mediators)

UVA UVB

Immediate erythema, Deep red Starts after 3-5 hrs, Bright red

Increases melanin production and transfer of melanosomes to keratinocytes

Increases number and activity of melanocytes

Less protective from sunburns More protective(Increases ‘t’ of s.corneum)

Not much effect in Vit D synthesis Major role

Less photo ageing and carcinogenesis More chance

UVA MED in fair skin is 30–75 J/cm2 UVB MED for fair skin is about 30 mJ/cm2

HISTOLOGY- UVB

• “sunburn cells,” (apoptotic keratinocytes)appear in the epidermis at 30 min after a 3 MED exposure.

• Intercellular edema & Dermal changes persist to 72 hrs

• Endothelial cell swelling, perivenular edema & degranulated mast cells

• Neutrophils influx peaks at 14 hrs.

UVA -HISTOLOGY“Sunburn cells” :-Not apparentThe major histological changes after UVA occur in the dermis

•Epidermal intercellular edema 48hrs• LCs decreased over 48 hours•A lymphocytic infiltrate was also apparent throughout the dermis•In the dermis, Neutrophils present for at least 48 hours

• DNA is one of the important chromophores that initiate pathways leading to UV-induced inflammation

• X. pigmentosum patients prolonged UVB induced erythema that can be reduced by treatment with a DNA repair enzyme

Sunburn

• Production of inflammatory mediators & cytokines causes sunburn response

• The time in the sun required to produce sunburn is strongly influenced by many factors including skin pigmentation, season, geographical location, cloud cover and time of day.

SLIP, SLOP, SLAP

Photo immunology

• UV light induces Cancer via1) DNA damage2) Mutation3)Immunosuppression

• Uv induced tumors have a highly antigenic phenotype

• Treatment with low doses of UVB, led to failure of tumor rejection(Due to immunosuppression)

• Various researches been done in this field • Those are very complex & the results are

contradictory

Immunosuppression• 2 types: • (1) local immunosuppression in which the• immune response to antigens applied at the

irradiated site is impaired• (2) systemic immunosuppression in which the

immune response to antigens applied to unexposed sites is impaired

• Mice with the genetic loci for Lps & Tnf are more susceptible to UVB-induced immunosuppression

Actions of UV light1. DNA is a chromophore for UVB radiation

produces CPD- -causes UV-induced immunosuppression

2. CPD in Langerhan cells & immunosuppression3. PAF receptor activation causes

immunosuppression4. Aberrant receptor clustering-

Immunosuppression5. Changes in cell membrane6. UCA mediated immunosuppression

1)Pyrimidine dimers(CPD)

• DNA is the direct target for UVB radiation• And produces Pyrimidine dimers(CPD)• This will cause UV induced immunosuppression

2) CPD s in LC

• UVB CPDs in antigen-presenting cells (APCs) and impair their antigen presenting capacity.

• The damage persists for several days, and the damaged cells migrate from the skin to lymph nodes

• Topical application of photolyase-containing liposomes to UVB-exposed sites prevents UVB-induced immunosuppression

3) PAF Receptor activation

• UVB radiation can lead to lipid peroxidation of cell membrane lipids. (By ROS)

• Phosphatidylcholine, can be oxidized to (PAF) “like” lipids that bind to PAF receptors • PAF receptor activation stimulates a variety of

downstream effects, including the synthesis of immunosuppressive cytokines (ie IL-10)

• Resulting in immunosuppression

4) Aberrant receptor clustering

• UVB exposure leads to clustering & internalization of cell surface receptors for EGF, TNF &IL-1 in the absence of the respective ligands

• Such aberrant receptor clustering may subvert signaling pathways normally used by growth factors and cytokines, eventually contributing to the immunological response mainly immunosuppression

5) Changes in cell membrane

• Cytoplasmic tryptophan is a UVB chromophore • The resulting tryptophan photoproduct bind to

arylhydrocarbon receptor (AhR)= c-src + Hsp90• AhR complex dissociates and the Hsp 90

translocates into the nucleus whereas c-src translocates to the cell membrane

• C-SRC causes various changes in cell membrane

6) UCA Mediated

• An extracellular chromophore that mediates UVB induced immunosuppression is UCA(Urocanic acid)

CELLULAR EVENTS INVOLVED INPHOTOIMMUNOSUPPRESSION

• UVB radiation of keratinocytes induces IL-1, IL-6, IL-8, TNF-α & PGE2 • TNF-α &IL-10 - immunosuppressive cytokines

• UVB Stimulates Th2 cells & T-suppressor cells & Suppresses Th 1 cells

IL -10

• The key immunosuppressor cytokine• IL-10 is a - Th2 cytokine • Impairs Th1 cytokines production• Thus impairs Th1 mediated cellular reactions• IL-10 inhibits the antigen-presenting capacity

of LCs• PAF and its receptor enhance its production

• IL-12 can reverse the IL-10 production• IL-12 can restore UV-impaired CHS• IL-12 -enhance the repair of UVB -induced DNA

photoproducts

Langerhan cell action• LCs, 2%–5% of epidermal cells• DC subset which originates from bone marrow

precursors • Ingest antigen in the skin, but lack costimulatory

capacity• After antigen uptake, LCs migrate to the DLN• At l.node, LC s will express high levels of MHC molecules

& costimulatory molecules -B7.1,B7.2 & ICAM1

• UV Light Antigen uptake ,MHC II & ICAM-1

• UV radiation causes disappearance of LC from irradiated sites

• UV exposure results in reduced surface expression of costimulatory molecules B7.1, B7.2 & ICAM-1

• UVB-irradiated LCs activate preferentially CD4+ Th2 cells

• UVB induced hapten-specific tolerance -i.e. resensitization with the same hapten at later time points

• CD8+ T cells - important mediators of UVB-induced immunosuppression

• UV-exposed mice are capable to suppress CHS by producing IL-10

• In addition, T cells that express (CTLA)-4 on their surface also causes immunosuppression

• CTLA-4 is an important molecule in immune regulation in UVB induced immunosuppression

ULTRAVIOLET RADIATION EFFECTSON INNATE IMMUNITY

• Suppresses adaptive immune response & Promotes Innate immunity

• UV radiation will induce innate immune response by increasing the production of the antimicrobial peptides, human β-defensin (HBD)-2, -3, ribonuclease 7 in skin

• This may explain why T-cell-mediated immune reactions are suppressed on UV exposure but not host defense reactions against bacterial attacks.

CONCLUDING REMARKS

• Photoimmunosuppression-A protective method

Modern approaches to prevent immunosuppression• Broad-spectrum UV filters,• Liposomally encapsulated DNA repair enzymes• Taurine uptake • IL-12• Antioxidants & Osmolytes

Thank You

Acknowledgement

Thomas.B.Fitzpatrick(1919-2003)