Skin barrier responses to moisturizers - DiVA portal172597/... · 2009-05-28 · 11 INTRODUCTION THE STRUCTURE AND BARRIER FUNCTION OF THE SKIN Terrestrial life would not be possible

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 381

Skin barrier responses tomoisturizers

IZABELA BURACZEWSKA

ISSN 1651-6206ISBN 978-91-554-7296-2urn:nbn:se:uu:diva-9300

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To my Family

'Well, in OUR country,' said Alice, still panting a little,

'you'd generally get to somewhere else–if you ran very fast

for a long time, as we've been doing.'

'A slow sort of country!' said the Queen. 'Now, HERE, you

see, it takes all the running YOU can do, to keep in the

same place. If you want to get somewhere else, you must

run at least twice as fast as that!'

Lewis Carroll, “Through the Looking-Glass”

PAPERS INCLUDED

This thesis is based on the following papers, which will be referred to in the text by their

Roman numerals:

I. Buraczewska I., Lodén M. Treatment of surfactant-damaged skin in humans with

creams of different pH values. Pharmacology 2005; 73:1–7.

II. Buraczewska I., Berne B., Lindberg M., Törmä, H. and Lodén M. Changes in skin

barrier function following long-term treatment with moisturizers, a randomized

controlled trial. Br J Dermatol 2007; 156:492–8.

III. Buraczewska I., Berne B., Lindberg M., Lodén M. and Törmä, H. Effect of a long

term-treatment with moisturizers on the keratinocyte differentiation and desquamation.

Arch Dermatol Res, in press.

IV. Buraczewska I., Berne B., Lindberg M., Lodén M. and Törmä, H. Moisturizers change

the mRNA expression of enzymes synthesizing skin barrier lipids. Manuscript.

Reprints were made with permission from the publishers.

TABLE OF CONTENTS

Introduction .............................................................................................................................. 11�

The structure and barrier function of the skin ..................................................................... 11�

Skin structure .................................................................................................................. 11�

Stratum corneum as the skin barrier ............................................................................... 13�

Intercellular lipids of the stratum corneum..................................................................... 15�

Enzymes and non-enzymatic regulatory proteins of the skin barrier .................................. 15�

Corneocyte formation ..................................................................................................... 15�

Desquamation ................................................................................................................. 16�

Formation of extracellular lipid matrix of stratum corneum .......................................... 17�

Nuclear hormone receptors and lipoxygenases .............................................................. 18�

Skin pH................................................................................................................................ 19�

Moisturizers and the skin barrier ......................................................................................... 21�

Chemistry of moisturizers ................................................................................................... 22�

Methods used for evaluation of the skin barrier function.................................................... 23�

Aim of the research.............................................................................................................. 24�

Materials and Methods ............................................................................................................. 25�

Volunteers............................................................................................................................ 25�

Test moisturizers ................................................................................................................. 25�

Experimental design ............................................................................................................ 27�

Paper I ............................................................................................................................. 27�

Paper II............................................................................................................................ 27�

Papers III and IV............................................................................................................. 27�

Evaluations .......................................................................................................................... 28�

Calculations and statistics.................................................................................................... 28�

Results ...................................................................................................................................... 30�

Paper I.................................................................................................................................. 30�

Paper II ................................................................................................................................ 30�

Paper III ............................................................................................................................... 31�

Paper IV............................................................................................................................... 33�

Discussion and conclusions...................................................................................................... 37�

Choice of test moisturizers .................................................................................................. 38�

Effect of 7-week use of test preparations on the skin barrier .............................................. 39�

Possible explanations of observed effects ........................................................................... 39�

Lipids .............................................................................................................................. 40�

Water............................................................................................................................... 40�

Emulsifiers and polymers ............................................................................................... 41�

Humectants ..................................................................................................................... 41�

Occlusion ........................................................................................................................ 42�

pH .................................................................................................................................. 43�

Molecular changes induced by Hydrocarbon and Complex creams ................................... 44�

Effect of Hydrocarbon cream on the skin barrier ........................................................... 44�

Nuclear receptors and lipoxygenases.............................................................................. 44�

Enzymes and proteins involved in keratinocyte differentiation and desquamation ....... 45�

Conclusions ......................................................................................................................... 47�

Future perspectives................................................................................................................... 48�

Svensk sammanfattning (summary in Swedish) ...................................................................... 51�

Acknowledgments.................................................................................................................... 52�

References ................................................................................................................................ 54�

ABBREVIATIONS

ACACB acetyl-CoA carboxylase beta

ACSL1 acyl-CoA synthetase long-chain family member 1

ACTB actin, beta (�-actin)

ALOX12B arachidonate 12-lipoxygenase, 12R type

ALOXE3 epidermal arachidonate lipoxygenase 3

ARCI autosomal recessive congenital ichthyosis

CDKN1A cyclin-dependent kinase inhibitor 1A

cDNA complementary deoxyribonucleic acid

CoA Coenzyme A

DAPI 4’-6-diamidino-2-phenylindole

FASN fatty acid synthase

FLG profilaggrin

GBA glucocerebrosidase, beta; acid (�-glucocerebrosidase)

HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)

HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMG-CoA synthase 1)

IL1A interleukin-1�

IVL involucrin

KLK5 kallikrein 5

KLK7 kallikrein 7

mRNA messenger ribonucleic acid

NMF natural moisturizing factor

o/w oil-in-water

PBS phosphate-buffered saline

PCA pyrrolidone carboxylic acid

PPARA peroxisome proliferator-activated receptor alpha (PPAR-�)

PPARB peroxisome proliferator-activated receptor beta (PPAR-�)

PPARG peroxisome proliferator-activated receptor gamma (PPAR-�)

QRT-PCR quantitative real-time polymerase chain reaction

RT reverse transcription

RXRA retinoid X receptor alpha (RXR-�)

SLS sodium lauryl sulfate

SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal (acid sphingomyelinase)

SPTLC2 serine palmitoyltransferase, long chain base subunit 2 (serine palmitoyl-

transferase 2)

TEWL transepidermal water loss

TGM1 transglutaminase 1

UGCG UDP-glucose ceramide glucosyltransferase

11

INTRODUCTION

THE STRUCTURE AND BARRIER FUNCTION OF THE SKIN

Terrestrial life would not be possible without protection from dehydration and harmful

factors. The skin gives us such protection, but it is more than an impenetrable shield: it is a

dynamic and complex tissue, mediating a multiplicity of functions. It provides a physical

permeability barrier, hampering excessive water and electrolyte loss from inside-out, and

protects from external chemical, microbial, and mechanical insults. The skin plays an

essential role in thermoregulation, absorbance of ultraviolet radiation, sensation and

sociosexual communication. Since it is our largest organ, its immunological performance as

well as its ability to repair wounds and regenerate itself, is of vital importance for the entire

body.1,2

Skin structure

The skin consists of three distinctive layers, the subcutis, dermis, and epidermis (Figure 1).

The lowest of these layers, the subcutis (hypodermis, subcutaneous fat), is built of adipose

tissue, which helps to cushion and insulate the body. This layer serves as energy storage and

allows for skin mobility over underlying structures.1,2

The dermis constitutes the principle mass of the skin. Its main component, extracellular

matrix (ground substance), attracts and retains water due to presence of strongly hygroscopic

molecules, proteoglycans. Dermis is crossed with nerve and vascular networks and embraces

epidermal appendages such as hair, sweat glands and sebaceous glands. It contains various

cell types, such as fibroblasts, macrophages, mast cells, and transient circulating cells of the

immune system. The dermis is tightly connected to the uppermost layer of the skin, the

epidermis, by the basement membrane, which is the main part of the dermal–epidermal

junction.1,2

The epidermis, a constantly self-renewing stratified structure, is built mostly of keratinocytes,

which account for at least 80% of its total cells. Therefore, the properties and functioning of

keratinocytes determine the condition of the epidermis. The remaining cell types are

melanocytes, Langerhans cells, Merkel cells, and various cells of the immune system. Due to

the differentiation state of keratinocytes, layers of epidermis are classified into the stratum

basale, stratum spinosum, stratum granulosum and stratum corneum (Figure 2).1,2

12

Figure 1 – Skin layers. From: Gilberg S., Tse D. Atlas of Ophthalmology, Edited by Richard

Parrish II, David T. Tse, 2000, Current Medicine Group LLC, with permission.

Figure 2 – Layers of epidermis. From Gilberg S., Tse D. Atlas of Ophthalmology. Edited by

Richard Parrish II, David T. Tse, 2000, Current Medicine Group LLC, with permission.

13

Stratum corneum as the skin barrier

Keratinocytes in the epidermis migrate from stratum basale with a proliferative cell type,

through stratum spinosum, to the stratum granulosum, where cornification, a process of

terminal differentiation, is initiated; transforming keratinocytes into flat anucleated cells

called “corneocytes”. Cornification involves degradation of organelles, organization of keratin

bundles inside the cell, and formation of a cornified envelope around it.1,2 This is

accompanied by secretion of lamellar bodies, organelles containing a mixture of lipids,

mainly polar ones: glucosylceramides, sphingomyelin, and phospholipids, and also

cholesterol, as well as numerous enzymes. After secretion at the stratum granulosum/stratum

corneum interface, polar lipids are enzymatically converted into non-polar products:

ceramides and free fatty acids (reviewed by Feingold3). Lipids derived from lamellar bodies

are assembled into lamellar structures surrounding the corneocytes.1,2

As a result, the stratum corneum consists of layers of corneocytes, which are densely packed

with keratin and embedded in extracellular lipid matrix. Corneocytes are connected to each

other by corneodesmosomes, and are gradually removed from the skin surface by

desquamation, making place for new cells coming from underneath. Process of keratinocyte

turnover in epidermis is highly organized in space and time, so that proliferation and

differentiation of keratinocytes are in balance.1,2

The stratum corneum, though only 10–20 �m thick, is the most essential layer of epidermis

from the perspective of its barrier properties and protection outside-in and inside-out. The

structure of the stratum corneum can therefore be compared to a wall made of bricks

(corneocytes) and mortar (intercellular lipids). This “brick and mortar” model was initially

presented by Michales et al.4 in 1975. The skin barrier models presented later: stacked

monolayer model,5 mosaic domain model,6 single gel-phase model,7 and sandwich model,8

can be regarded as extensions of the brick and mortar model (a concise summary of those

models is presented by Norlén9). They accept the two-compartment structure of the stratum

corneum and focus on explanation of molecular arrangements of intercellular lipids. Although

the organization of the stratum corneum and its components is not completely understood, the

development of new techniques, such as cryo-electron microscopy and cryo-electron

tomography of vitreous skin sections (reviewed by Norlén10), gives new insight into its

structure.

14

The function of the skin as a barrier can be regarded as protective, or even defensive, in

nature, and it is localized mostly in the stratum corneum. Corneocytes and extracellular lipid

matrix fulfill various protective functions, which often co-localize and/or are linked together

(Table 1) (reviewed by Elias11,12). In dermatological research, overall protective properties of

the stratum corneum and epidermis are often referred to as the “skin barrier”, while their

performance is known as the “skin barrier function”. However, it is important to realize that

depending on the research design and the evaluation methods, the exact meaning of the term

“skin barrier” may vary. For example, some studies may focus more on the permeability

barrier to water from inside the skin to the outside, while others investigate selective

absorption of a substance from outside into the epidermis.

Table 1 – Multiple protective function of outer epidermis*

Function Principal compartment Biochemical basis

Permeability Extracellular matrix of SC Ceramides, cholesterol, nonessential fatty acids in proper ratio

Antimicrobial Extracellular matrix of SC Antimicrobial peptides, free fatty acids, sphingosine

Antioxidant Extracellular matrix of SC Cholesterol, free fatty acids, vitamin E, redox gradient

Cohesion (integrity) � desquamation Extracellular matrix of SC Intercellular DSG1/DSC1

homodimers

Mechanical or rheological Corneocyte �-glutamyl isopeptide bonds

Chemical (antigen exclusion) Extracellular matrix of SC Hydrophilic products of

corneodesmosomes

Psychosensory interface Extracellular matrix of SC Barrier lipids

Neurosensory Stratum granulosum Ion channels, neurotransmitters

Hydration Corneocyte Filaggrin proteolytic products, glycerol

Ultraviolet light Corneocyte Trans-urocanic acid (histidase activity)

Initiation of inflammation (first-degree cytokine activity)

Corneocyte Proteolytic activation of pro-interleukin-1�/�

*Modified from Elias12, published with permission; SC = stratum corneum; DSG1 = desmoglein 1; DSC1 = desmocollin 1

15

Intercellular lipids of the stratum corneum

Lipids of the continuous extracellular lipid matrix of the stratum corneum are predominantly

ceramides, cholesterol, cholesteryl esters (cholesterol esters with fatty acids) and free fatty

acids, in an estimated molar ratio 37:32:15:16, respectively.13 Moreover, small quantities of

cholesterol sulfate14 and glucosylceramides15 are present as well. Lipids are organized into

lamellar phases, oriented approximately parallel to the surface of the corneocytes (reviewed

by Bouwstra et al.16). The detailed lamellar organization of intercellular lipids is still not

completely known, as data vary, depending on the method used.17,18 Their lateral organization

is also under investigation, since it determines the permeability of the stratum corneum: lipids

are organized mostly in orthorhombic and hexagonal packing, with the first type being the

least permeable arrangement, while the latter is more permeable.19,20 It has been shown that in

normal skin, lipids of stratum corneum have mostly orthorhombic packing, although

hexagonal packing and blends of hexagonal and orthorhombic packing are also present. By

contrast, patients with atopic dermatitis and lamellar ichthyosis have been found to have

predominantly hexagonal organization of intercellular lipids.20

ENZYMES AND NON-ENZYMATIC REGULATORY PROTEINS OF THE

SKIN BARRIER

The formation of components of stratum corneum, corneocytes and intercellular lipids, as

well as degradation of corneodesmosomes, which results in desquamation, is regulated by

several key enzymes and non-enzymatic proteins. Abnormalities in their expression, structure,

or activity may lead to impairment of the skin barrier, which is often found in various skin

disorders, such as atopic dermatitis, psoriasis and disorders of keratinization.

Corneocyte formation

Among the most important proteins in the process of cornification are transglutaminase 1,

involucrin and filaggrin. Transglutaminase 1, together with transglutaminase 3, crosslinks

various proteins into a mechanically and chemically resistant structure named the “cornified

envelope”, formed around each corneocyte. Involucrin is one of the first proteins linked

during this process and acts as a scaffold for other proteins, e.g., loricrin, trichohyalin, small

proline-rich proteins, cystatin �, and elafin (reviewed by Candi et al.21 and Ishida-Yamamoto

et al.22). Reduced level of involucrin and its altered distribution in epidermis was found in

patients with atopic dermatitis.23 Transglutaminase 1 is also probably involved in the

16

formation of ester bonding between involucrin, possibly also other proteins, and �-

hydroxyceramides, forming a lipid envelope of about 5 nm around the cornified envelope,

which acts as a frame for attaching other intercellular lipids.24 Mutations in gene of

transglutaminase 1 result in enzyme deficiency, and are found in patients with lamellar

ichthyosis and congenital ichthyosiform erythroderma (nonbullous) (reviewed by Richard25).

Keratohyalin granules are small organelles that are visible in stratum granulosum by ordinary

light microscope. They are composed primarily of profilaggrin and loricrin.1 Profilaggrin is

cleaved into filaggrin, which aggregates keratin filaments into tight bundles and causes the

collapse of filament network of keratinocytes, resulting in a flattened cell (reviewed by Candi

et al.21). Later, filaggrin undergoes degradation into free amino acids, pyrrolidone carboxylic

acid (PCA) and urocanic acid.26 Amino acids and PCA are the main components of the bulk

of natural moisturizing factor (NMF), helping to maintain an appropriate hydration level of

the stratum corneum (reviewed by Rawlings et al.27,28). The importance of filaggrin for the

skin barrier function has been demonstrated in recent studies, which link atopic dermatitis,29

chronic irritant contact dermatitis,30 and ichthyosis vulgaris31 to mutations in the gene of

profilaggrin. Individuals with mutation of FLG have been shown to have less NMF, which

may be one of factors predisposing them to dry skin disorders.32 Moreover, FLG mutations

are connected to a higher risk of developing asthma among patients with atopic dermatitis and

severe asthma in individuals without eczema.33

Another essential protein in the cell cycle is cyclin-dependent kinase inhibitor 1A, which has

a broad functionality in skin biology. Together with other inhibitors of the same family, this

protein is involved in cycle progression through the G1 phase into the S phase and in gene

expression regulation. Differentiation of squamous epithelia, including the epidermis, is

associated with increased expression of cyclin-dependent kinase inhibitor 1A (reviewed by

Weinberg et al.34). Increases in both its mRNA and protein levels have been found in psoriatic

plaques, and also after application of irritants and tape stripping.35

Desquamation

Desquamation of corneocytes from the stratum corneum surface, terminating the keratinocyte

life cycle, is mediated by enzymes belonging to the group of proteases, among which

kallikrein 5 and kallikrein 7 (previously known as stratum corneum tryptic enzyme (SCTE)

and stratum corneum chymotryptic enzyme (SCCE), respectively) are currently assumed the

most crucial.36-39 In normal skin, kallikrein 5 and 7 are found in the stratum granulosum and

17

corneum, as well as in the skin appendages.37,38 In transgenic mice with pathologic skin

changes, such as increased epidermal thickness, hyperkeratosis, dermal inflammation, and

severe pruritus, kallikrein 7 was expressed also in the suprabasal epidermal layers.40

Moreover, increased protein expression of kallikrein 5 and 7 was found in stratum corneum of

lesional skin of psoriasis vulgaris, while non-lesional psoriasis skin had no such increase,41

and also in stratum corneum obtained from patients with atopic dermatitis, which had none or

only very mild lichenification.42 These findings suggest an involvement of kallikreins in

various skin disorders, including inflammatory reactions. Kallikrein 7 has also been found to

be affected by the humidity of the environment: the low relative humidity decreased activity

of kallikrein 7 in excised animal skin due to a diminished water concentration in the upper

stratum corneum, reversible by application of a moisturizer or a humectant (glycerin).43

Formation of extracellular lipid matrix of stratum corneum

So far, nine classes of ceramides have been identified in the human stratum corneum, and they

differ from each other with regard to their head group architecture. They can contain

sphingosine, phytosphingosine, or 6-hydroxysphingosine as a base. Ceramides 1, 4, and 9 are

unique, as they contain linoleic acid. The ceramides, especially ceramide 1, have an important

role in the organization of stratum corneum lipids (reviewed by Bouwstra et al.16).

All types of ceramides are synthesized de novo from palmitoyl-CoA and serine, where serine

palmitoyltransferase is a rate-limiting enzyme in this process.1,44 In order to be transported to

stratum corneum in lamellar bodies, ceramides are converted to glucosylceramides and

sphingomyelin, by enzymes UDP-glucosylceramide synthase and sphingomyelin synthase,

respectively.45,46 After extrusions from lamellar bodies, enzymes �-glucocerebrosidase and

serine palmitoyltransferase transform them to ceramides (reviewed by Feingold3).

Deficiency in ceramides or abnormalities in enzymes involved in their formation has been

found in atopic dermatitis, where a significant reduction in the quantity of ceramides has been

observed in both lesional and non-lesional skin.23,47,48 This has been associated with a

decreased activity of acid sphingomyelinase23 and increased activity of glucosylceramide

deacylase,48 but no changes were found in activity of �-glucocerebrosidase.49 The level of

ceramides is also decreased in psoriasis, probably due to decreased levels of serine

palmitoyltransferase, and the severity this disease has been reported to correlate with the level

of this enzyme.50 In psoriasis, mRNA and protein expression of �-glucocerebrosidase has

been decreased in non-lesional skin and increased in lesional skin.51 Except for their structural

18

function, ceramides are also involved in a number of cellular processes including apoptosis,

the cell cycle, and cellular differentiation (reviewed by Ruvolo52). Recently, a role for

ceramides was proposed in the formation of the epidermal pH gradient, as acquiring free fatty

acids from ceramides may contribute to acidification of the stratum corneum.53

HMG-CoA synthase and reductase enzymes are involved in the synthesis of cholesterol in

epidermis, with acetyl-CoA as a substrate.1 The importance of cholesterol synthesis for the

barrier function and recovery has been demonstrated after acute and chronic skin barrier

disruption in mice, by acetone, tape stripping and essential fatty acid-deficiency diet, which

significantly increased the mRNA expression of HMG-CoA reductase and synthase.54 The

same type of barrier damage increased also activity HMG-CoA reductase.55 Topical

application of lovastatin, an inhibitor of HMG-CoA reductase, has been reported to impede

barrier recovery.56 Cholesterol seems to be important for the organization of intercellular

lipids (reviewed by Bouwstra et al.16). Moreover, cholesterol sulfate has a significant role in

desquamation (reviewed by Elias et al.57).

Acetyl-CoA serves as a substrate for synthesis not only of cholesterol, but of fatty acids as

well, involving fatty acid synthase in this process.1 The mRNA expression of fatty acid

synthase was shown to increase after barrier disruption by in mice, as also does the expression

of another lipid-processing enzyme, acetyl-CoA carboxylase beta.54 Interestingly, when

murine skin is occluded just after tape stripping, mRNA expression of acetyl-CoA

carboxylase beta, fatty acid synthase, HMG-CoA reductase and synthase is decreased, which

suggests that their expression is regulated by the skin barrier function itself, not by a non-

specific response to damage.54

Nuclear hormone receptors and lipoxygenases

Recently, the significance of nuclear hormone receptors, namely peroxisome proliferator-

activated receptors PPAR-�, PPAR-� and PPAR-�, and retinoid X receptor alpha (RXR-�),

for the normal skin barrier formation has been demonstrated. PPARs are transcription factors

involved in keratinocyte differentiation and proliferation and lipid synthesis. Their activation

was shown to increase expression of proteins essential in formation of cornified envelope:

involucrin, loricrin, and transglutaminase 1, stimulate synthesis of epidermal lipids and

formation of lamellar bodies, as well as to increase activity of lipid-processing enzymes.

PPARs are also involved in anti-inflammatory processes and cutaneous carcinogenesis.

Therefore, their performance is essential for the skin barrier formation and function. They

19

may play an important role as drug targets for such skin diseases as psoriasis and atopic

dermatitis, and also in skin cancer (reviewed by Feingold,3 Schmuth et al.,58 and Sertznig et

al.59). RXR-� is a receptor for the vitamin A metabolite, 9-cis retinoic acid, and is the most

abundant retinoid X receptor in the epidermis.60 It heterodimerizes with PPARs, and it has

been shown that these heterodimers act as signal transducers in different signaling pathways.61

The exact function of RXR-� for the skin barrier formation and function remains not

completely understood, but studies on mice show that mutation of RXR-� induces

hyperproliferation and abnormal differentiation of epidermal keratinocytes.60,61

The formation of the skin barrier lipids can be regulated at the transcriptional level by

substances acting as ligands to the PPARs. Enzymes believed to generate endogenous ligands

for these receptors have recently been described.62 Mutations in the coding regions of two of

these enzymes, lipoxygenases arachidonate 12-lipoxygenase, 12R type and epidermal

arachidonate lipoxygenase 3, were discovered in patients with autosomal recessive congenital

ichthyosis (ARCI), characterized by a defect water diffusion barrier in the skin.63,64 The exact

functions of those two lipoxygenases still remain unknown, but it seems that they may be

connected to lipid metabolism of the lamellar granule contents or intercellular lipid layers, as

well as formation of cornified envelope (reviewed by Akiyama et al.65).

SKIN PH

The pH value of the skin has been investigated since the end of 19th century. The acidic nature

of the skin surface was first mentioned by Heuss66 in 1892. In 1928, Schade and

Marchionini67 coined the term “acid mantle” of the skin (“Säuremantel”). Since then, many

studies have been carried out aiming to explain the mechanism of pH gradient formation and

its importance for the skin and its barrier function, but to this day, this issue is not completely

understood.

It is important to realize that the term “skin pH” is not completely correct, as pH values

should refer only to diluted aqueous solutions (�0.1 mol/kg),68 while the epidermis is a dense

structure containing only about 20–30% of water in the stratum corneum and about 70%

water in deeper layers.69,70 Moreover, various residues located on the skin surface may

influence the readings. Therefore, what is actually measured is pH of the “(extractable) water-

soluble constituents of skin”, and that measured pH of the skin is not the pH in a precise

analytical–chemical sense.71 Consequently, instead of “pH of the skin”, terms such as “pH on

the skin” or “apparent pH” have been proposed to be more appropriate.71 However, despite

20

the mentioned considerations, it is widely accepted to use the terms “skin pH” or “pH of the

skin” and these expressions are also used in this thesis.

Several studies show that the pH value on the surface of healthy, undamaged skin of adults is

slightly acidic, about 5, varying between 4 and 6. A mixture of various substances secreted on

the skin surface with sweat, sebum and NMF, such as lactic acid, butyric acid, PCA, amino

acids, and free fatty acids, helps to shift the surface pH towards acidic values. In addition,

ingredients of exogenous origin, such as metabolites of cutaneous microflora (e.g., free fatty

acids) and cosmetic products may be present. However, it seems that pH on the skin surface

depends mainly on processes taking place in deeper layers of the epidermis (reviewed by

Parra et al.,72 Rippke et al.,73 and Fluhr et al.74).

Below the skin surface, in the epidermis, pH increases from acidic values in the upper layers

of the stratum corneum to near-neutral values of around 7.4 in viable epidermis, forming a

gradient through the stratum corneum.75-77 The exact course of this gradient is still uncertain.

Measurement of tape-stripped human skin with a glass electrode has shown a gradual

decrease in pH towards the skin surface.75-77 Visualization of hydrogen ions in human skin

biopsies demonstrated that pH decreases sharply at the stratum corneum/granulosum

interface, but later slightly increases within stratum corneum, and then decreases again

towards the skin surface.78 However, recent investigation with fluorescence lifetime imaging

microscopy (FLIM) suggests that the observed pH is in fact an average from two distinct pH

gradients formed by two types of acidic microdomains, each of them localized in one

compartment of stratum corneum: corneocytes or extracellular lipid matrix.79 As the result,

the average pH of the stratum corneum decreases towards the surface, due to an increase in

the ratio of acidic to neutral regions.79

Protons forming the pH gradient are generated most likely by several mechanisms and

perhaps not all of them have been identified yet. Two endogenous mechanisms are currently

believed to be the most important for acidification of the epidermis: formation of free fatty

acids from phospholipids through the action of secretory phospholipase A2 (PLA2) and

exchange of protons for sodium ions by non-energy-dependent sodium-proton exchangers

(Na+/H+ exchanger isoform 1, NHE1) in the membranes of keratinocytes at the stratum

corneum/stratum granulosum interface.80-84 The latter mechanism explains the decrease in pH

at the border between the stratum corneum and the stratum granulosum, as NHE1 is expressed

in the same place. Recently, a new pathway was proposed; in which epidermal ceramidase

contributes to endogenous skin pH by generating free fatty acids from ceramides.53

21

The acidic pH on the skin surface is assumed to inhibit the growth of pathogenic

microorganisms and keep the skin microflora in balance (reviewed by Fluhr et al.74). If the

skin surface pH is elevated, e.g., after usage of alkaline soaps, prolonged occlusion, or in skin

disorders like atopic dermatitis, the growth of pathogens increases.85-88 However, recent

studies reveal another important role of skin pH. The pH gradient through the epidermis

seems to be essential for several epidermal enzymes involved in the formation and function of

the skin barrier, as their activity is pH-dependent, e.g., �-glucocerebrosidase has an optimum

activity at pH 5.6, phospholipase A2 at pH 7–8, acid sphingomyelinase at pH 4.5, and

cholesterol sulfatase at pH 8 (reviewed by Redoules et al.89). Kallikrein 5 and 7 has been

shown to exhibit maximum activity at pH 8, but have a considerable activity also at pH 5.5.37

The importance of pH for activity of the epidermal enzymes, and therefore for the skin

barrier, was shown in a recent study on mice. Perturbed skin barrier recovered normally when

the skin was exposed to solutions buffered to an acidic pH, while initiation of the recovery

was delayed when the damaged skin was exposed to neutral or alkaline pH. This delay in

barrier recovery was suggested to be a consequence of a lower activity of �-

glucocerebrosidases.90

MOISTURIZERS AND THE SKIN BARRIER

The primary function of moisturizers is to smoothen the skin surface and to increase water

content in the stratum corneum, i.e., to moisture the skin. After application, water and other

volatile ingredients gradually evaporate; leaving a deposit of remaining ingredients, which

may stay on the skin surface or penetrate into the epidermis and be removed from the skin

surface by washing, friction and evaporation.

The increase in water content in the epidermis is achieved by water-binding properties of

humectants, e.g., glycerin, and by formation of a semi-occlusive layer on the skin surface,

which hampers water evaporation and increases water content in the upper epidermis.91

Moreover, an immediate increase in hydration of stratum corneum may be caused by an

uptake of water from the applied product.91 The increase in water content and the

simultaneous filling of the fractures on the skin surface, makes the skin more elastic, and

visibly and tactilely smoother, as well as decreases itch and brings relief (reviewed by

Lodén92,93)

If moisturizers are used repeatedly, as for example in the case of patients with various dry

skin disorders, who require treatment over a long period, or even over a lifetime, it may be

22

speculated what consequences this may have for the skin and its barrier function. Recurring

application of various substances of exogenous origin on the skin, followed by such

physicochemical changes as increase in water content in the stratum corneum or change in

skin surface pH, may influence epidermis, and therefore, the skin barrier function. Few

studies about long-term treatment with moisturizers on normal and diseased human skin have

shown both increases and decreases in skin barrier function, as measured by non-invasive

techniques. Two, 3 or 4-week treatments of normal or atopic skin with moisturizers

containing urea decreased transepidermal water loss (TEWL) and susceptibility to sodium

lauryl sulfate (SLS).94-98 Treatment with a moisturizer containing another humectant, glycerin,

seems to have a less pronounced impact on the skin barrier.98,99 On the other hand, in studies

by Held et al.100,101, a moisturizer containing high lipid content (70%) impaired the barrier of

normal skin after 5-day and 4-week treatments, measured as increased skin susceptibility to

SLS, although no change in TEWL of undamaged skin was found. The same cream also

increased susceptibility to nickel in nickel-allergic volunteers after 7-day treatment.102 In a

study on patients with lamellar ichthyosis, an 8-week treatment with moisturizers containing

high amount of lactic acid significantly increased TEWL, as well as dryness and scaling

decreased.103 Moisturizers have also been shown to influence skin barrier recovery after

exposure to a skin irritant.95,104 These studies demonstrate that prolonged application of

moisturizers may have a substantial impact on parameters used for evaluation of the skin

barrier. However, the factors responsible for the observed effects are unknown, especially

since the studies were performed using moisturizers containing several ingredients.

CHEMISTRY OF MOISTURIZERS

Moisturizers are formulated predominantly as oil-in-water (o/w) emulsions, where oil droplets

are dispersed in water and stabilized by emulsifiers. Reversed, water-in-oil (w/o) emulsions

are used less frequently due to their poor spreadability and the greasier feeling they leave on

the skin; however, they can offer other attributes, e.g., water resistance. Emulsions are

categorized into creams or lotions, depending on their viscosity. Moisturizers may also be gels

containing only hydrophilic material or ointments with only lipophilic ingredients. Other

forms of moisturizers exist, but they are much less common, e.g., multiple emulsions,

silicone-in-water emulsions, or suspensions. The choice of the form of a moisturizer depends

on its desired effect and the ingredients that are supposed to be incorporated.

23

Moisturizers may either have a simple composition and contain only a few ingredients, or be a

complex mixture of many substances. In the case of o/w and w/o emulsions, the simplest

possible moisturizer must contain three ingredients, namely, water, a lipid (oil), and an

emulsifier. Many ingredients used in moisturizers are the same as those found in the

epidermis or on the skin surface: fatty acids, ceramides, vitamins, urea, lactic acid, PCA, etc.

Lipids can be of vegetable, animal or mineral origin. Emulsifiers, which stabilize the lipid

droplets in an emulsion, can be either low-molecular substances, e.g., PEG-100 stearate, or

long-chained polymers of large size, such as acrylates/C10–30 alkyl acrylate crosspolymer.

However, it is rare that emulsions contain only three ingredients, and usually they are

mixtures of at least 15–20 substances. Those additional ingredients allow achieving desired

properties, efficacy, and stability of the product. Moisturizers usually contain humectants,

such as polyols: glycerin, propylene glycol, butylene glycol, sorbitol; alpha-hydroxy acids

(AHAs) and their salts e.g., sodium lactate; low-molecular substances: urea, betaine, amino

acids; and high-molecular polymers with water-binding capacity: sodium hyaluronate. To

increase stability of the emulsion and to adjust its rheology to either cream or lotion form,

viscosity-increasing agents must be added into the formulation, such as polymers: carbomer,

acrylates copolymer, xanthan gum; and high-melting waxes: glyceryl stearate, cetearyl

alcohol. Sensory properties may be modified with silicones, such as dimethicone and

cyclohexasiloxane. Additionally, moisturizers may contain several other ingredients, such as

antioxidants, vitamins, herbal extracts, salts, and UV filters. Depending on the composition of

moisturizers, their pH is adjusted to between slightly acidic and slightly alkaline and usually

ranges from 4 to 7, but in case of formulations containing stearic acid and zinc oxide, pH is

slightly alkaline.

Since the majority of moisturizers contain several ingredients, identification of the parameters

responsible for their effects on the skin barrier is difficult. Consequently, factors such as the

concentration and type of lipids, humectants, and other ingredients, as well as pH adjustment,

should be taken into account.

METHODS USED FOR EVALUATION OF THE SKIN BARRIER FUNCTION

Methods used for evaluation of the skin and its barrier function are numerous. Skin condition

may be assessed visually, e.g., for dryness, scaling and redness. There are several instruments

available for non-invasive assessments of the skin, where no skin sampling is necessary, as

they measure the functional changes on the skin surface or within a defined skin depth, e.g.,

24

TEWL, skin capacitance, skin impedance, blood flow, pH, surface topography, and elasticity.

Such equipment is often portable and easy to use, and consequently, non-invasive

measurements are a common tool in dermatological research. Today, assessment of TEWL is

the most common method for evaluation of the skin barrier function. TEWL is increased

when the skin barrier is impaired, e.g., in dry skin disorders or after damage with an irritant,

but also when the skin is hydrated.

However, in order to investigate processes in the skin in greater detail, at the molecular and

cellular level, skin samples are required. They may be punch or shave biopsies or samples

obtained by tape stripping. Studies utilizing such invasive methods are more complicated to

perform, require more resources and assessment from ethical perspective. Although they may

be common in basic dermatological research, they have rarely been used in research about

moisturizers and their effect on the skin barrier. It is also possible to perform studies in vivo

on mice or in vitro on keratinocyte cultures or skin equivalents. However, results obtained

from experiments performed using animal models or in vitro systems do not always correlate

to the in vivo situation in humans. Analyses of human skin biopsies may give a lot of

information about changes in epidermal structure, and gene and protein expression, as well as

allowing for staining with antibodies against various proteins.

AIM OF THE RESEARCH

The objective of the present work was to increase the understanding of the mechanism by

which long-term treatment with moisturizers influences the skin and its barrier function. The

impact of formulation variables such as pH, lipid type and humectants was assessed in vivo in

healthy human volunteers. Functional changes in the skin were explored using non-invasive

techniques. Moreover, the impact of moisturizers on the skin barrier function was assessed

also at molecular and cellular level, investigating gene and protein expression as well as

histology of epidermis.

25

MATERIALS AND METHODS

VOLUNTEERS

All studies were performed on healthy adult human volunteers. Exclusion criteria were skin

diseases, pregnancy, and allergy to ingredients used in the test preparations. Informed consent

and health declarations were obtained from volunteers before commencement of the studies.

The studies were approved by the Regional Ethical Review Board at Uppsala University,

Uppsala, Sweden. The number and age of the volunteers participating in each study are given

in Table 2.

Table 2 � Number and age of volunteers participating in each study.

Study Number of volunteers Women Men Age

(years)

Paper I 18 15 3 21–54

Paper II Treatment with test moisturizers Irritancy patch test

78 11

58 8

20 3

25–60 27–46

Papers III and IV 20 15 5 23–59

TEST MOISTURIZERS

Altogether, five test preparations were used in the investigations: one ordinary cream,

hereafter called “Complex cream”; three simplified creams emulsified with a long-chained

polymer, with the hydrophobic lipid phase consisting of either the hydrocarbons

isohexadecane and paraffin (“Hydrocarbon cream”) or a vegetable triglyceride oil, canola oil

(“Canola cream” and “Canola/urea cream”); and one lipid-free gel consisting of the polymer

used in simplified creams (“Polymer gel”). All test moisturizers, except for the Polymer gel,

were oil-in-water (o/w) emulsions. Table 3 shows the detailed compositions of the test

moisturizers, their pH, the number of volunteers testing each preparation, and the papers in

which the results are presented.

Tabl

e 3 �

Com

posit

ion

of th

e te

st m

oist

uriz

ers,

thei

r in

gred

ient

s and

pH

, and

the

pape

rs in

whi

ch th

e re

sults

are

pre

sent

ed.

Num

ber

of v

olun

teer

s T

est

prep

arat

ion

Lip

ids

Em

ulsi

fiers

O

ther

ingr

edie

nts

Ure

a pH

Pa

per

I Pa

per

II

Pape

rs II

I an

d IV

4.0

7.5

18

Com

plex

cr

eam

a

20%

ca

pric

/cap

rylic

tri

glyc

erid

e, c

anol

a oi

l, ce

tear

yl

alco

hol,

para

ffin

, gl

ycer

yl st

eara

te

1.3%

PE

G-1

00 st

eara

te,

carb

omer

, po

lyso

rbat

e 60

wat

er, p

ropy

lene

gl

ycol

, gly

cery

l po

lym

etha

cryl

ate,

di

met

hico

ne,

sodi

um la

ctat

e,

met

hylp

arab

en,

prop

ylpa

rabe

n,

lact

ic a

cid,

citr

ic

acid

5%

5

15

10

Hyd

roca

rbon

cr

eam

40%

is

ohex

adec

aneb

(20%

), pa

raffi

nc (2

0%)

0.4%

ac

ryla

tes/

C10

–30

alky

l acr

ylat

e cr

ossp

olym

erd

wat

er

0%

5

16

10

Can

ola

crea

m

40%

ca

nola

oile

0.4%

ac

ryla

tes/

C10

–30

alky

l acr

ylat

e cr

ossp

olym

erd

wat

er

0%

5

15

Can

ola/

urea

cr

eam

40

%

cano

la o

ile

0.4%

ac

ryla

tes/

C10

–30

alky

l acr

ylat

e cr

ossp

olym

erd

wat

er

5%

5

16

Poly

mer

gel

0%

0.4%

ac

ryla

tes/

C10

–30

alky

l acr

ylat

e cr

ossp

olym

erd

wat

er,

met

hylp

arab

enf

0%

5

16

a Can

oder

m

krä

m 5

%, A

CO

HU

D N

OR

DIC

AB

, Sto

ckho

lm, S

wed

en; b A

rlam

ol H

D, U

niqe

ma,

Gou

da, T

he N

ethe

rland

s; c M

erku

r W

hite

Oil

Phar

ma,

Mer

kur

Vas

elin

e,

Ham

burg

, Ger

man

y; d Pe

mul

en T

R-2

, Nov

eon

Inc.

, Cle

vela

nd, O

H, U

SA; e A

kore

x L,

Kar

lsha

mns

AB

, Kar

lsha

mn,

Sw

eden

; f Nip

agin

M, C

laria

nt In

tern

atio

nal,

Pont

yprid

d,

Uni

ted

Kin

gdom

.

27

EXPERIMENTAL DESIGN

The studies were double-blinded and randomized. Test moisturizers were distributed to

volunteers in white coded tubes. Volunteers were allowed to wash normally, but not to use

any skin care products on the test areas (forearms and gluteal skin) at least three days before,

and during, the test period.

Paper I

Volunteers treated each volar forearm with one of two test preparations, twice daily for 8

days. The test preparations were identical creams, except for pH, which was adjusted to either

4.0 or 7.5 (Table 3; Complex cream). Before the first application of the creams, one area of

each forearm was exposed to the skin irritant SLS for 24 hours, which induced irritancy.

Assessments of TEWL, blood flow, and skin capacitance, as well as visual scoring of

irritancy, were performed repeatedly during the subsequent days. On day 8, the examined

areas were exposed once again to SLS for 7 hours and evaluated finally on day 9.

Paper II

This study consisted of two parts: a long-term treatment study with the test preparations, and a

test of their irritancy potential. In the long-term treatment study, volunteers treated one volar

forearm twice daily for 7 weeks with one test preparation (Table 3), leaving the other forearm

to serve as the untreated control. After 7 weeks, on day -1, both volar forearms, treated and

control, were exposed to SLS for 24 hours. TEWL and blood flow were assessed on SLS-

exposed and undamaged skin on each forearm on day 1. Skin capacitance was also measured

on undamaged skin. Moreover, test preparations were assessed for their acute irritancy

potential using a 24-hour patch test, evaluated by visual scoring and TEWL measurements.

Papers III and IV

The volunteers applied one of the test preparations (Table 3; Complex cream or Hydrocarbon

cream) on one volar forearm and one buttock twice daily for 7 weeks, leaving the other

forearm and buttock untreated to serve as control sites. The side of treatment was randomized.

After 7 weeks, one shave and one punch biopsy were taken from each buttock, preceded by

TEWL measurements of the biopsy area. The shave biopsies were used for gene expression

analysis and the punch biopsies for histological and other molecular evaluations. Moreover,

the skin of the forearms was patch-tested with SLS for 24 hours. Non-invasive evaluations

were performed on undamaged and SLS-exposed skin.

28

EVALUATIONS

A list of evaluation techniques used in all presented studies is given in Table 4. The

techniques are described in detail in the “Materials and methods” section of each paper.

Table 4 – Summary of analyses performed in Papers I–IV

Analyses performed Paper I Paper II Paper III Paper IV

Non-invasive in vivo evaluations of the skin

TEWL x x x

Blood flow x x x

Skin capacitance x x x

Visual scoring x x

Molecular analyses of skin biopsies RNA isolation and cDNA synthesis x x

QRT-PCR x x

Histological evaluations x

Immunofluorescence x

Immunohistochemistry x

Lipid staining x TEWL = transepidermal water loss; RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid;

QRT-PCR = quantitative real-time polymerase chain reaction

CALCULATIONS AND STATISTICS

The results are expressed either as percentage ratio to the corresponding values obtained from

control (untreated) skin areas, which are given as 100%, or as absolute values. They are

presented in graphs as box plots with the median value as a line across the box and the first

quartile value at the bottom and the third at the top. The whiskers are lines that extend from

the top and bottom of the box to the lowest and the highest observation within a defined

region, with outliers plotted as asterisks outside this region. For results presented in a table or

in the text, the median value is given, followed by the lower (Q1) and upper (Q3) quartiles in

brackets.

To analyze differences between results of non-invasive measurements and molecular

analyses, a Wilcoxon signed rank test on paired data was used. Possible linear relationships

between two variables were analyzed using the Pearson product moment correlation

29

coefficient. A Kruskal-Wallis test of equality of medians was used to investigate the

differences between various moisturizers.

Minitab® statistical software (Minitab Inc., State College, PA, USA), was used for

calculations and plots. The level of significance was set at p < 0.05.

30

RESULTS

PAPER I

Treatment of surfactant-damaged skin in humans with creams of different pH values

After the initial exposure to SLS, there was no difference in the skin barrier recovery between

skin treated with the pH 4.0 cream and skin treated with the pH 7.5 cream, evaluated as

TEWL, blood flow, and skin capacitance, and by visual scoring. After 8 days of cream

application, there was no difference between treatment groups in susceptibility to SLS at the

second SLS exposure.

PAPER II

Changes in skin barrier function following long-term treatment with moisturizers, a

randomized controlled trial

Treatment of normal skin for 7 weeks with Hydrocarbon cream, Canola cream, Canola/urea

cream, and Polymer gel increased TEWL in comparison to the untreated areas. The opposite

effect was found for Complex cream, where TEWL was reduced. Skin capacitance decreased

following treatment with Hydrocarbon cream, but no difference was found for the other test

preparations. Treatment with all test moisturizers did not change blood flow in comparison to

control areas.

After SLS-exposure, the TEWL of skin treated with Hydrocarbon cream, Canola cream,

Canola/urea cream, and Polymer gel was increased in comparison with the corresponding

SLS-exposed skin on the untreated forearm, while after treatment with Complex cream, it was

reduced. However, only Canola/urea cream resulted in a higher relative increase in TEWL

compared with the untreated control skin after SLS exposure.

There were no differences between the simplified creams, Hydrocarbon cream, Canola cream,

and Canola/urea cream, in their effects on TEWL of undamaged skin, TEWL of SLS-exposed

skin, or blood flow of SLS-exposed skin. However, there was a difference in impact on skin

capacitance.

The irritancy patch test showed that the five test preparations were not more irritant than

water.

A summary of the effects of the test preparations on the skin barrier is presented in Table 5.

31

Table 5 – Summary of the effects of test moisturizers on the skin barrier

after 7 weeks of exposure.

Undamaged skin SLS-exposed skin Moisturizer

TEWL Capaci-tance

Blood flow TEWL Blood

flow

Complex cream – –

Hydrocarbon cream � – � �

Canola cream � – – � �

Canola/urea cream � – – � �

Polymer gel � – – � – � = increased in comparison with untreated skin; = decreased; – = no difference;

SLS = sodium lauryl sulfate; TEWL = transepidermal water loss

PAPER III

Long-term treatment with moisturizers affects the mRNA levels of genes involved in

keratinocyte differentiation and desquamation

After 7 weeks of twice-daily treatment of buttock and forearm skin, TEWL was increased on

the Hydrocarbon cream-treated sites, but decreased on the Complex cream-treated sites, as

compared with untreated skin. TEWL measured on untreated areas of gluteal skin was

significantly higher than on untreated forearms: 10.8 (8.7–14.3) vs 7.3 (6.5–12.3) gm-2h-1,

respectively (p<0.001). None of the creams induced changes in superficial skin blood flow or

influenced the messenger ribonucleic acid (mRNA) expression of interleukin-1� (IL1A),

indicating absence of inflammation.

After SLS-exposure of forearms, a higher degree of irritation was found in skin treated with

Hydrocarbon cream compared with its corresponding untreated control skin site, while skin

treated with Complex cream showed less irritation than control skin. Skin capacitance

decreased after exposure to Hydrocarbon cream, while no difference was found for Complex

cream.

Histological analysis of sectioned skin biopsies showed no differences in the thickness of

epidermis or stratum corneum after treatment with either of the creams compared with

controls. In addition, the corneocyte size was not significantly influenced by any of the

treatments.

32

Treatment with Complex cream decreased the expression of cyclin-dependent kinase inhibitor

1A (CDKN1A), while the remaining genes were unaffected. Hydrocarbon cream significantly

increased the gene expression of involucrin (IVL), transglutaminase 1 (TGM1), kallikrein 7

(KLK7), and kallikrein 5 (KLK5) compared with untreated controls, while no changes were

found in the levels of profilaggrin (FLG) and cyclin-dependent kinase inhibitor 1A

(CDKN1A). In an attempt to identify genes important for the normal barrier function we

examined possible correlations between TEWL and mRNA expressions, thickness of the

stratum corneum, and size of corneocytes of untreated buttock skin. However, no linear

correlation was found between TEWL and gene expressions of analyzed genes, thickness of

stratum corneum, and size of corneocytes. A summary of gene expressions analyses presented

in Papers III and IV is given in Table 6.

The immunofluorescence staining did not reveal significant changes at the protein level of

involucrin, transglutaminase 1, and filaggrin (Figure 3). An example of photographs used for

semi-quantitative analyses of protein expression is given in Figure 4.

Perc

enta

ge ra

tio [%

]

Filaggrin Transglutaminase 1 Involucrin

350

300

250

200

150

100

50

p=0.

415

p=0.

083

p=0.

683

p=0.

083

p=0.

067

p=0.

919

Complex creamHydrocarbon cream

Figure 3 – Protein expression of involucrin, transglutaminase 1 and filaggrin in skin treated

with test moisturizers (n=10), assessed using the semi-quantitative method. For an

explanation of the boxplots, see the “Calculations and statistics” section. The untreated

control site is given as 100%. P-values relate to differences between treated and control areas.

33

Figure 4– Examples of photographs used for semi-quantitative analyses of protein expression

of transglutaminase 1: untreated skin of a volunteer using Complex cream (a); treated skin of

a volunteer using Complex cream (b); untreated skin of a volunteer using Hydrocarbon cream

(c); and, treated skin of a volunteer using Hydrocarbon cream (d). The dashed line represents

the dermal–epidermal border. Bar = 50 �m.

PAPER IV

Moisturizers change the mRNA expression of enzymes synthesizing skin barrier

lipids

After a 7-week exposure of the skin to Hydrocarbon cream, an increased mRNA expression of

the ceramide synthesizing enzymes �-glucocerebrosidase (GBA), serine palmitoyltransferase

2, (SPTLC2) and acid sphingomyelinase (SMPD1), but not of UDP-glucose ceramide

glucosyltransferase (UGCG) was observed. Identical treatment with Complex cream did not

affect the expression of any of these enzymes. Regarding the two analyzed enzymes involved

in cholesterol synthesis, treatment with Hydrocarbon cream increased the expression of

HMG-CoA synthase 1 (HMGCS1), but not HMG-CoA reductase (HMGCR), while Complex

cream had no effect on either. None of test moisturizers significantly influenced the mRNA

expression of enzymes involved in free fatty acid metabolism: acetyl-CoA carboxylase beta

(ACACB), fatty acid synthase (FASN) and acyl-CoA synthetase long-chain family member 1

34

(ACSL1). Treatment with Complex cream increased the mRNA expression of one nuclear

receptor, PPAR-� (PPARG), while exposure to Hydrocarbon cream decreased it. There was

no effect of any of the treatments on the expression of PPAR-� (PPARA), PPAR-� (PPARB)

and RXR-� (RXRA). Moreover, treatment with Hydrocarbon cream increased expression of

both analyzed lipoxygenases arachidonate 12-lipoxygenase 12R type (ALOX12B) and

epidermal arachidonate lipoxygenase 3 (ALOXE3), while Complex cream did not (a

summary of gene expression analyzed in Papers III and IV, is presented in Table 6).

In biopsies from the untreated skin, we examined whether there was a correlation between the

mRNA expression of any of the analyzed genes and TEWL (TEWL results were reported in

Paper III). The mRNA expression of two of the 15 examined genes, PPARG and ACACB,

exhibited an inversed linear correlation to TEWL, and high expression of these genes was

associated with low TEWL.

The amount and organization of non-polar lipids, examined by Nile Red in situ staining,

revealed no changes induced by either of the two treatments, in comparison with control

areas.

Since treatment with the two test moisturizers altered mRNA expression of PPARG in

opposite directions, the protein expression of PPAR-� was examined in the biopsies. Two

patterns of nuclear staining were observed: one was the staining of the entire viable epidermis

and the other was more restricted to the rete ridges. These patterns were equally distributed

between the volunteers, irrespective of the type of moisturizer used. In most cases, every

volunteer exhibited the same pattern, both in treated and in control sites, and when performing

semi-quantitative analysis of the staining intensity, the cream-exposed areas were no different

from control areas. An example of photographs used for semi-quantitative analysis of PPAR-

��is presented in Figure 5.

35

Figure 5 – Examples of photographs used for semi-quantitative analyses of protein expression

of PPAR-�: untreated skin of a volunteer using Complex cream (a); treated skin of a volunteer

using Complex cream (b); untreated skin of a volunteer using Hydrocarbon cream (c); and,

treated skin of a volunteer using Hydrocarbon cream (d). Bar = 50 �m.

36

Table 6 - Summary of the gene expression analysis presented in Papers III and IV.

Function Gene Complex cream

Hydrocarbon cream

IVL – �

TGM1 – � FLG – –

Proteins involved in keratinocyte differentiation

CDKN1A –

KLK5 – � Enzymes involved in the process of desquamation KLK7 – �

GBA – �

SPTLC2 – �

SMPD1 – � Enzymes involved in ceramide synthesis

UGCG – –

HMGCS1 – � Enzymes involved in cholesterol synthesis HMGCR – –

ACACB – –

FASN – – Enzymes involved in fatty acid metabolism

ACSL1 – –

PPARA – –

PPARB – –

PPARG � Nuclear hormone receptors

RXRA – –

ALOX12B – � Lipoxygenases

ALOXE3 – � Interleukin IL1A – –

� = increased messenger ribonucleic acid (mRNA) expression in comparison with untreated skin; = decreased mRNA expression; – = no difference; ACACB = acetyl-CoA carboxylase beta; ACSL1 = acyl-CoA synthetase long-chain family member 1; ALOX12B = arachidonate 12-lipoxygenase, 12R type; ALOXE3 = epidermal arachidonate lipoxygenase 3; CDKN1A = cyclin-dependent kinase inhibitor 1A; FASN = fatty acid synthase; FLG = profilaggrin; GBA = �-glucocerebrosidase; HMGCR = HMG-CoA reductase; HMGCS1 = HMG-CoA synthase 1; IL1A = interleukin-1�; IVL = involucrin; KLK5 = kallikrein 5; KLK7 = kallikrein 7; PPARA = PPAR-�; PPARB = PPAR-�; PPARG = PPAR-�; RXRA = RXR-�; SMPD1 = acid sphingomyelinase; SPTLC2 = serine palmitoyltransferase 2; TGM1 = transglutaminase 1; UGCG = UDP-glucose ceramide glucosyltransferase.

37

DISCUSSION AND CONCLUSIONS

Moisturizers are often used as supplements to topical and/or systemic anti-inflammatory drugs

in various types of skin conditions and disorders, such as contact dermatitis, atopic dermatitis,

psoriasis, and ichthyosis, in order to bring relief and break a dry skin cycle (reviewed by

Lodén93,105 and Proksch et al.106). Such skin conditions usually require long-lasting treatment

with moisturizers, and in the case of atopic dermatitis, their use is recommended even when

the eczema is cleared.107 Vehicles of many topical drugs are moisturizes as well. Use of

moisturizer is also widespread among people that self-perceive their skin as dry or rough, e.g.,

in elderly, due to a dry climate or frequent contact with cleaning agents, and they use

moisturizers to obtain relief and for smoothening of the skin. Moreover, skin protection

creams (also called “barrier creams”) are widespread at various workplaces to minimize the

percutaneous penetration of chemicals. Use of moisturizers is therefore common and is

practiced by a significant percentage of the population. Although the importance of using

moisturizers often is overlooked, and they are not perceived as an “active” treatment, they

have been shown to influence skin properties and the barrier function in both healthy and

diseased skin.94-98,100-103,108

Studies on the impact of moisturizers on the skin barrier have mostly focused on short-term

effects, showing that moisturizers are able to increase skin hydration, decrease roughness and

scaling, and improve the condition of dry skin (reviewed by Lodén et al.92,93,105,109). However,

little is known about the effects of their long-term use, lasting weeks, months, or even years,

which better reflects the real-life situation. The few studies that have looked into such effects,

have shown that the moisturizers studied influenced the skin barrier function and recovery, as

measured by non-invasive techniques, such as TEWL, skin capacitance and susceptibility to

an irritant, e.g., SLS and nickel salts.94-98,100-103,108 Therefore, the aim of present research was

to gain further understanding of the mechanism by which long-term treatment with

moisturizers influences the skin in vivo in healthy human volunteers, using not only non-

invasive techniques, but also other tools, such as quantitative real-time polymerase chain

reaction (QRT-PCR), immunofluorescence, immunohistochemistry, and histological

evaluations, allowing investigating the epidermis at molecular and cellular level.

Few available studies demonstrate that moisturizers may have an impact on epidermis at the

molecular level, when used on normal skin. Short et al.110 showed that a moisturizer

containing high amounts of glycerin and silicones increased maximum epidermal thickness,

38

decreased epidermal melanin content, and altered protein expressions of keratins 6, 10, and

16, as assessed with immunohistochemistry after a 4-week treatment. Moreover, all these

changes were accompanied by decreased TEWL. Another study revealed that a 6-week

treatment with a moisturizer containing glycerin and erythritol increased the number of

keratinocytes with a well-matured cornified envelope; also, the interleukin-1 receptor

agonist/interleukin-1� (IL-1ra/IL-1�) ratio in the epidermis was altered.111 However, it may

well be individual ingredients that affect the skin at a molecular level, i.e., not the moisturizer

as a whole. Linoleic acid, found in certain vegetable oils commonly used in topical

preparations, is a ligand of peroxisome proliferator activated receptors (PPAR) which has

been found to have effects comparable to a potent topical glucocorticoid in animal models.112

Another substance, nicotinamide, was shown to increase levels of ceramides and free fatty

acids and to decrease TEWL, as compared with placebo-treated skin.113

CHOICE OF TEST MOISTURIZERS

Three test moisturizers used in the present studies were formulated in such way as to

minimize their complexity, in order to diminish the number of possible confounding factors.

Therefore, they contained only few ingredients, in contrast to the majority of commercially

available topical preparations that contain over a dozen substances. In addition, one

moisturizer, Complex cream, containing several ingredients and previously shown to

influence the skin barrier in normal and atopic skin, was chosen as a reference for this

investigation.96,108 The following factors were examined: the impact of a humectant (urea);

difference between creams of minimum complexity (Hydrocarbon, Canola and Canola/urea

creams) and a cream containing more ingredients (Complex cream); and different types of

lipids, which were either pure hydrophobic hydrocarbons derived from mineral oil (paraffin

and isohexadecane) or a more polar vegetable oil consisting of triglycerides and sterols

(canola oil). The simple creams were stabilized with a polymeric emulsifier, which was

expected not penetrate and influence skin barrier function due to its large molecular size. A

gel, consisting of water and this polymer, was also investigated (Polymer gel). By contrast,

Complex cream contained a mixture of lipids of various origins and was emulsified with a

combination of polymers and low-molecular emulsifiers. To evaluate the effect of pH on the

skin barrier, Complex cream was adjusted to pH 4.0 or 7.5, as certain epidermal enzymes

have optimum activity in either acidic or alkaline pH. To investigate the effect of long-term

39

use of moisturizers, treatment time was chosen to be 7 weeks, as the average turnover time of

epidermis is reported to be 40–45 days.114,115

EFFECT OF 7-WEEK USE OF TEST PREPARATIONS ON THE SKIN

BARRIER

Twice-daily treatment of normal skin for 7 weeks with all test preparations examined in this

research, induced changes in the skin barrier function, as evaluated with non-invasive

methods. Hydrocarbon cream, Canola cream and Canola/urea cream appear to have a negative

effect on the skin barrier, as their application resulted in elevated TEWL and increased skin

susceptibility to SLS. Polymer gel, consisting of polymer and water, had similar effects.

Moreover, Hydrocarbon cream decreased skin capacitance. By contrast, treatment with

Complex cream decreased TEWL and susceptibility to SLS. Molecular analyses of skin

biopsies after use of Hydrocarbon cream and Complex cream on normal skin for 7 weeks

revealed that these two creams induced different effects on the mRNA expression of genes

involved in the keratinocyte differentiation, corneocyte formation and desquamation as well

as lipid metabolism. Treatment with Hydrocarbon cream changed the expression of 11 out of

22 analyzed genes, while exposure to Complex cream affected expression of only two of

them. At the same time, the moisturizers had no effect on protein expression of four analyzed

proteins. Stratum corneum thickness, epidermal thickness, the size of corneocytes, and non-

polar lipid staining, were also unaffected by the treatments.

The studies therefore revealed that moisturizers had different effects on the barrier function of

normal skin and that these changes were dependent on the composition of the moisturizer.

The test preparations had an impact on the normal function and/or structure of the skin. The

observed changes may be caused either by the ingredients of the test preparations (e.g., lipids,

humectants, emulsifiers, or water), which have a direct or indirect effect on skin barrier

components, or by the physical effects of moisturizers on the skin, such as occlusion. The

impact of pH of moisturizers was also investigated, and it was assessed to have no

significance for their effects on the skin barrier.

POSSIBLE EXPLANATIONS OF OBSERVED EFFECTS

Despite the apparent relationship between gene expression and the skin barrier function, it

was not possible to ascertain whether the observed functional changes, such as

40

increased/decreased TEWL, susceptibility to SLS or skin capacitance, were the effect of

molecular changes in the epidermis, or vice versa (“hen-and-egg” situation). The first

possibility is that functional changes in the skin barrier, induced by moisturizers, could trigger

epidermal keratinocytes to alter their gene expression. An example of such functional change

may be the delivery of exogenous lipids from the moisturizers into the intercellular lipids of

the stratum corneum, resulting in altered barrier function, or the impact of emulsifiers,

humectants or exposure to water.

Lipids

Lipids in moisturizers may remain on the skin surface or enter the skin116, and more

physiological lipids may penetrate into the epidermis and affect skin barrier structure and

recovery.117-119 Changes in lateral packing of stratum corneum lipids were observed in

patients with atopic dermatitis and psoriasis after 3-week treatment with a moisturizer

containing 10% petrolatum.20 Therefore, in our study, the possible penetration of lipids from

test moisturizers could alter skin barrier properties.

The three simplified creams investigated in our study, all of which had a negative effect on

the skin barrier, contained high proportions of lipids, 40%, but the type of lipid (hydrocarbons

or triglycerides) was of no importance for the effect. At the same time, Complex cream,

containing 20% lipids, did not deteriorate the skin barrier. Similar effects as for simplified

creams have been obtained also with a moisturizer containing 70% lipids, which made the

skin more susceptible to SLS.100,101 This suggests that differences in the lipid content or

uptake rate may be important for the effect of moisturizers on the skin barrier function. As

exogenous lipids may change the highly organized structure of intercellular lipid layers,

TEWL or the ion flux may also be altered. Such changes may be recognized as barrier

impairment by epidermal keratinocytes, initiating a repair process including altered gene

expression.

Water

Penetration of lipids, however, cannot explain the impairment of the skin barrier obtained

after treatment with Polymer gel, as it contains no lipids but much more water compared with

other moisturizers, 99%. After application of a moisturizer, water, which is one of the main

ingredients, evaporates within a short time.120,121 The effect on the skin barrier of twice-daily

exposure to water for a few weeks is unknown. It has been shown that prolonged contact with

water disrupts the intercellular lipid lamellar structure in the stratum corneum, and may

41

contribute to dryness and increased TEWL.122 Moreover, changes in gene expression of

epidermal enzymes and non-enzymatic proteins were found after exposure of skin only to

water under occlusion.123 It has also been suggested that higher TEWL of the dorsal surface of

hands, compared with the forearm and back, may be due to more frequent contact with

water.124 Increased TEWL and dryness of the skin could also be caused by contact with

irritants, but the test preparations, including Polymer gel, were shown not to be irritant, by an

acute irritancy test and by blood flow measurements. Moreover, treatment with Hydrocarbon

cream and Complex cream did not alter expression of IL1A, indicating lack of inflammation.

Furthermore, though all simple creams and Complex cream contain similar amounts of water,

around 60%, they induced different changes in the skin barrier.

Emulsifiers and polymers

Emulsifiers are essential in moisturizers, as they stabilize the emulsion. Emulsifiers

commonly used in o/w emulsions have been shown to influence the skin barrier function in

normal and SLS-exposed skin.125 Although the emulsifier used in the simple creams and

Polymer gel, acrylates/C10–30 alkyl acrylate crosspolymer, was expected not to penetrate into

the epidermis due to its large molecular size, its negative effect could have been caused by

monomers, which may be present at low concentrations. However, the Complex cream also

contained a polymeric emulsifier, carbomer, which is similar in structure to acrylates/C10–30

alkyl acrylate crosspolymer, lacking a negative impact on the skin barrier. Interestingly, it has

been suggested that polymers themselves may have an effect on the skin barrier, as tested on

mice, accelerating or delaying the barrier recovery.126 Although this phenomenon is not fully

understood, it is possible that polymers together with their counter-ions form an electric

double layer on the skin surface, influencing the skin barrier.126

Humectants

Humectants that are added to moisturizers, such as urea, glycerin, and propylene glycol, may

penetrate into the skin.127,128 It has been suggested that the decreased TEWL and lower

response to SLS after treatment with Complex cream was due to its urea content.96,97,108

Moreover, it could be expected that the addition of urea to a moisturizer would be beneficial,

as it has been reported that urea replaces water in the skin, leaving the physical properties of

the stratum corneum intact.129 However, not only did the addition of urea to Canola/urea

cream not improve the skin barrier, but it also made the skin more susceptible to SLS in

comparison to Canola cream without urea. Urea may have favoured the absorption of

42

potentially damaging ingredients in this formulation, or allowed for increased penetration of

SLS during the 24-hour patch test exposure. It might be that in Canola/urea cream,

penetration of urea was different than in case of Complex cream, due to another emulsion

composition.

Urea has been reported to act as a keratolytic agent;130 however, absence of a measurable

thinning of the stratum corneum by the 5% urea in Complex cream in the present

investigation, does not support keratolytic properties at a concentration commonly found in

moisturizing creams.

Although treatment with Complex cream containing 5% urea significantly decreased TEWL

and susceptibility to SLS, it had only minor effects on the mRNA expression of the analyzed

genes. Therefore, the effect of urea on the barrier function may depend on the whole

composition of the moisturizers, including the lipid content. Urea has also been found to

diminish epidermal proliferation in psoriasis, measured as a decreased expression of

involucrin and an increased expression of cytokeratins.131 However, no difference in

expression of proteins or the genes involved in keratinocyte differentiation and desquamation,

apart from CDKN1A, was detected in the present study. Decreased expression of CDKN1A

suggests influences in cell cycle progression after treatment with the urea-containing Complex

cream, which could be interpreted as decrease in cell differentiation,34,132 though not

detectable by histological evaluations, since the thickness of the epidermis and the stratum

corneum remained unchanged, as did corneocyte size.

Occlusion

As an alternative explanation, gene expression may be influenced by altered activity of

various signaling pathways, resulting in a changed skin barrier function. However, it is still

not clear what type of signal triggers changes in gene expression: the changes could be due to

ingredients included in the moisturizers, or could have been indirectly induced by, e.g.,

changes in ion or water flux. One possible signal may be a reduction in TEWL due to

occlusion by the topically applied moisturizer, hypothetically resulting in water flux changes.

We have previously shown that occlusion of the skin with a semi-permeable membrane,

mimicking the occlusion effect by a moisturizer, decreased TEWL and susceptibility to an

irritant, in a group wearing the membrane 23 hours a day for 3 weeks, suggesting changes in

skin barrier function.133 However, there is no evidence of a correlation between occlusive

properties of creams and their effects on the skin barrier, determined as degree of irritation

43

after SLS-exposure, since Complex cream and a lipid-rich cream show similar occlusive

capacity133, but opposite effects on the skin barrier function.96,97,100,101,108 Therefore, the

detected difference in the effect on the skin barrier is more likely to have causes other than

difference in occlusion between the creams. It is still not known whether a prolonged

occlusion per se influences mRNA expression, since no investigation of this aspect has yet

been performed. It is also worth noting that the semi-occlusive layer formed by a moisturizer

is usually removed from the skin within a few hours.

pH

The impact of the pH of topical preparations on the skin barrier is still not fully understood.

The results presented in Paper I show that the pH of moisturizers seems not to be of major

importance for their effects on the skin barrier. We found no difference in the impact on skin

barrier recovery or susceptibility to an irritant between moisturizers of pH 4 and 7.5. The lack

of difference in effect on skin barrier recovery of the moisturizers of acidic or alkaline pH

values in our study disagrees with a previous study in mice, where barrier recovery was

delayed after exposure to slightly alkaline pH.90 However, the endogenous mechanisms

involved in the formation of the pH gradient in stratum corneum,80-84 as well as continuous

exogenous excretion of sweat and sebum and NMF, could be expected to counteract the

change in the skin surface pH induced by a topical application. It has been shown that after

use of an alkaline soap, initially elevated skin surface pH decreases back towards acidic

values,87 and therefore the same effect can be expected after a moisturizer.

Moisturizers usually contain only small quantities of buffering ingredients, which make them

unable to produce persistent changes in the skin surface pH, while the mentioned skin barrier

recovery study on mice was performed using strong buffer systems.90 Moreover, the majority

of moisturizers have pH in a range of 4 to 6, which is in the range of the skin surface pH.

However, it cannot be excluded that some ingredients of moisturizers may penetrate into the

epidermis, and influence the pH gradient there, which would have an effect on the skin barrier

function.

44

MOLECULAR CHANGES INDUCED BY HYDROCARBON AND

COMPLEX CREAMS

Regardless of the mechanisms involved, Hydrocarbon cream and Complex cream influenced

the skin barrier in a way detectable by non-invasive measurements and molecular analyses of

mRNA expression: the latter, however, were not confirmed at the protein level.

Effect of Hydrocarbon cream on the skin barrier

Hydrocarbon cream appears to have a negative effect on the skin barrier, since it elevates the

TEWL and makes the skin more susceptible to SLS. In the present study, Hydrocarbon cream

increased the mRNA expression of genes responsible for the synthesis of ceramides and

cholesterol: GBA, SPTLC2, SMPD1, and HMGCS1, but not the expression of free fatty acid

metabolizing enzymes. These results can be interpreted as an epidermal response to barrier

damage. Our results support such a hypothesis, since Hydrocarbon cream increased also

mRNA expression of IVL and TGM1, both being key proteins in formation of cornified

envelope. Other studies of skin barrier damage induced in mice by acetone, surfactant, tape

stripping, or an essential fatty acid-deficient diet, also describe increased mRNA expressions

or activities of lipid-processing enzymes.54,55,134,135

However, in a recent study on healthy human volunteers, two creams containing 5% urea, the

same amount as in Complex cream, increased mRNA expression of IVL, FLG, loricrin, and

TGM1 after a 2-week treatment, which was also accompanied by a significant decrease in

TEWL.136 This gene expression profile is more similar to the effect of Hydrocarbon cream,

although the effect on TEWL is like that of Complex cream. This suggests that the

relationship between mRNA expression and TEWL is not so straightforward, and also that the

gene expression profile may change during treatment time.

Nuclear receptors and lipoxygenases

Hydrocarbon cream decreased the mRNA expression of PPARG, as well as influenced

mRNA expression of the two lipoxygenases, ALOX12B and ALOXE3. PPAR-� and other

PPARs are involved in regulation of keratinocyte differentiation and expression of several of

the lipid-processing enzymes. In skin equivalent models, activation of PPAR-� receptors by

synthetic ligands resulted in an increase in the mRNA expression of lipid-metabolizing

enzymes, three of which were the same as in our present study (GBA, SPTLC2 and

HMGCS1).137 Although Hydrocarbon cream does not contain any known PPAR�agonists, the

45

elevated expressions of ALOX12B and ALOXE3, genes of lipoxygenases that produce

derivatives acting as PPAR-� agonists,62 may suggest an endogenous formation of PPAR-�

agonists by the Hydrocarbon cream treatment.

While Hydrocarbon cream decreased the mRNA expression of PPARG, Complex cream

increased it. However, no difference in protein expression of PPAR-� was found after use of

any of the test preparations. Nevertheless, the opposite effect on the expression of PPARG

and TEWL induced by Hydrocarbon and Complex cream, as well as a linear correlation

between these two parameters in untreated skin, suggests an importance of PPAR-��for the

skin barrier function. Usually, PPAR-��activation affects cell proliferation, cell

differentiation, immune responses, and apoptosis in the skin. PPAR�� signaling is triggered by

various types of ligands, including linoleic acid (reviewed by Feingold,3 Schmuth et al.,58 and

Sertznig et al.59). Although Complex cream contains a vegetable oil, which may contain a

small fraction of free fatty acids, e.g., linoleic acid, our present results do not suggest any

activation of PPARs.

A linear correlation between TEWL and mRNA expression in untreated skin was also found

for ACACB. Enzyme encoded by this gene is involved in synthesis of fatty acids and its

importance for the skin barrier has been shown after barrier disruption in mice, which

increased mRNA expression of this enzyme.54 The regulation of ACACB in keratinocytes is

unknown, but in mouse hepatocytes, it has been shown that the PPAR-� agonist WY-16,643

increases the expression of ACACB.138

Enzymes and proteins involved in keratinocyte differentiation and

desquamation

The treatment with Hydrocarbon cream increased the mRNA level of INV and TGM1, but it

was not accompanied by a corresponding increase in protein expression. This may indicate

that the epidermis was preparing itself for possible repair of the impaired skin barrier, or that

the repair phase had already been completed, but there was still ongoing transcription.

Another reason for the lack of correlation between mRNA and protein expression after

treatment may be that the mRNA had not been translated into protein, or that the protein

turnover had increased for unknown reasons. The inhibition of mRNA translation has been

shown to be caused by micro RNA. For example, certain genes in the epidermis of psoriasis

patients exhibit diminished translation due to the presence of certain micro RNAs.139

Furthermore, since protein and mRNA expressions were analyzed on a single occasion, after 7

46

weeks of treatment, it is possible that changes in protein levels occurred at another time,

perhaps after a few days, but became “normalized” after a few weeks due to some adaptation

mechanism. As already mentioned before, a recent study with two creams containing 5%

urea, showed another mRNA expression profile of IVL, FLG and TGM1 than in our study,

but analyzed after a 2-week treatment.136

It is also possible that the analytical method used was not sufficiently sensitive to detect

differences in protein expression. The mRNA expression increased by about 55% in the case

of IVL and 120% in the case of TGM1, which may not be enough to show in detectable

differences using an immunofluorescence technique. Other techniques, e.g. western blot, may

have been more powerful, but would have necessitated obtaining additional biopsies from the

volunteers, which was not possible for ethical reasons. However, another study describes

changes in mRNA expression of a magnitude similar to that seen in the present study, resulted

in visible increased protein levels after 1–7 days after exposure to SLS.123 It suggests also that

the effect of moisturizers at the protein level may also have occurred earlier.

Exposure to Hydrocarbon cream also increased the gene expression of KLK5 and KLK7,

which may suggest an excessive desquamation, since these two proteases are involved in this

process.36-39 However, since the thickness of the stratum corneum remained unchanged, it

could be possible that treatment with Hydrocarbon cream initially induced thickening of

stratum corneum, but increased performance of kallikreins counteracted it. However, the

altered expression of kallikreins may also be due to other, not well known, functions in the

skin barrier, since it has been shown that kallikrein 7 degrade some lipid-processing

enzymes.140

The altered expression of kallikreins may also be linked in some way to decreased skin

capacitance after use of Hydrocarbon cream, indicating low skin hydration levels,141 since

these enzymes have been found to have increased protein expression in and psoriasis41 and

atopic dermatitis.42 However, the protein expression of kallikreins was not assessed in the

present study. Interestingly, decreased skin capacitance was not accompanied by any change

in the mRNA expression of FLG or the protein expression of filaggrin, which degrades into

free amino acids and PCA, the main components of NMF (reviewed by Rawlings et al.27,28).

47

CONCLUSIONS

In the present investigation, moisturizers were examined for their effect on the skin barrier

after long-term treatment, with regard to such factors as pH, lipid type, and presence of a

humectant, as well as complexity of the product. In conclusion, moisturizers are able to

modify the skin barrier function, detected as changes in TEWL, skin capacitance, and

susceptibility to an irritant, and also to change the mRNA expression of certain genes

involved in the assembly, differentiation and desquamation of the stratum corneum, as well as

lipid metabolism. Therefore, moisturizers should not be perceived simply as inert topical

preparations, even if they do not contain any ingredients commonly perceived as “active”.

However, the mechanisms behind the observed effects are still not fully understood. The

observed outcome is most likely the combination of interplay and effects of several factors,

rather than only one, which makes it more difficult to draw any firm conclusions. More

research of this kind is needed to understand better the mechanism of action of moisturizers.

Such research would aid in the treatment of various skin disorders through improved design

of moisturizers, which can target specific skin problems and conditions both more efficiently

and safely.

48

FUTURE PERSPECTIVES

In the present thesis, we used traditional, non-invasive methods to investigate the skin barrier

function, combined with invasive techniques allowing us to study changes in the epidermis

after use of moisturizers at the molecular and cellular level. Analysis of gene and protein

expressions and histological evaluations lead to new hypotheses and answer more questions

than non-invasive methods alone.

Our investigation showed that treatment with moisturizers might change gene expression of a

number of epidermal proteins, including structural proteins and enzymes. The study was

performed on selected genes known to be important for the skin barrier, and since their

number was not very high, QRT-PCR was used for analyses. However, further investigations

could include also analyses using cDNA microarrays, which allow for screening several

thousands of genes at the same time. This would give a broader perspective over changes in

gene expression in epidermis and help to identify additional genes and proteins that should be

investigated more in details.

Since our research investigated the effects of moisturizers after 7 weeks of treatment, one may

consider exploring also their impact on the skin barrier at earlier time points. The course of

gene and protein expression and histological changes may vary between shortly after

application to after few-week use. It would also be interesting to continue the investigation for

some time after use of a moisturizer is terminated, following the return of the skin barrier to

the state from before the treatment.

Although molecular analyses provide plenty of information about the skin barrier, non-

invasive evaluations should be utilized as well, as they do not require skin sampling and

provide a different type of data, i.e. skin barrier function. The new types of non-invasive

methods developed during recent years help us acquire detailed facts about the skin structure

and function in vivo and may be used in studies investigating the effects of moisturizers. For

example, Raman spectroscopy helps to evaluate the moisturizing effect by giving a detailed

water profile in the epidermis and assess stratum corneum and epidermal thickness,69,70,142 as

well as to follow drug penetration (reviewed by Wartewig et al.143). Multiphoton laser

tomography144 and confocal laser scanning microscopy (reviewed by Branzan et al.145), allow

us to “look” inside the skin and may be exploited in various ways.

49

The results presented in this thesis as well as other studies demonstrate that the composition

of moisturizers plays an essential role in their effects on the skin and its barrier function.

Therefore, it would seem reasonable at first to systematically test various ingredients,

combinations of ingredients and complete moisturizers for their effects, in order to find those

giving the desired efficacy, both in healthy and in diseased skin. However, taking into

consideration that over 500 ingredients classified as humectants and about 1,500 emollients

are available,146 the number of possible combinations is endless, even if the number of

ingredients were limited to only those used more frequently. Therefore, it is more realistic to

continue the research about these few ingredients, which are common in many moisturizers

and for which some data is already available, regarding the effect of glycerin, urea, vegetable

oils, and hydrocarbons.

As the knowledge about the effect of various ingredients and their combinations on the skin

barrier is scarce, testing of complete moisturizers is currently the best way to assure benefits

for the consumers. However, although clinical trials are mandatory for pharmaceuticals (over-

the-counter or prescription drugs) to prove their efficacy, moisturizers available as cosmetic

products are rarely evaluated for their effect on the skin barrier. Therefore, in theory, some

commercially available moisturizers may have a negative impact on the skin, and their use

could worsen the skin condition, facilitate penetration of irritants, and even lead to eczema.

Consequently, the composition of a moisturizer should be an important issue when

recommending it to a patient with skin problems, since this choice may have an impact on the

skin status and therefore on quality of life. Treatment with corticosteroids and other drugs

improves the condition of the skin with eczema, but a relapse may only be a question of time.

However, while the use of a moisturizer with the skin barrier-improving effect after

corticosteroid therapy may increase an eczema-free period in comparison with no

treatment147, it is not known if use of a moisturizer with barrier-impairing properties causes an

earlier relapse of eczema. This issue should be investigated further, as it may have a major

impact on the approach to the treatment of dry skin disorders.

Improving or maintaining the skin status and quality of life may also involve more than

choosing a proper moisturizer, since compliance to treatment is also required. If a moisturizer

is not attractive to a patient from a cosmetic perspective, e.g., because it is too greasy and

tacky, difficult to apply, has an unpleasant odour, or its package is impractical, there is a high

probability that the patient will not use it according to guidelines, and as a result will require

more medical attention. The same problem may exist in the case of barrier creams, which are

50

supposed to inhibit penetration of irritant substances. Therefore, the cosmetic properties of

moisturizers must be taken into consideration during the early stages of the development,

which is a challenge for researchers and formulating chemists, as various ingredients added to

make a product more stable and attractive may weaken its performance or cause adverse

reactions.

In conclusion, it is important to remember that moisturizers differ in their composition, which

determines their impact on the skin barrier. A better understanding of the influence of

moisturizers on the skin barrier, obtained by combination of non-invasive methods and

molecular analyses, would facilitate designing skin care products adjusted to specific skin

problems. It would also provide more efficient management of dry skin disorders, and,

hopefully, help to influence the development of moisturizers so that fewer products will have

negative effects on the skin.

51

SVENSK SAMMANFATTNING (SUMMARY IN SWEDISH)

Mjukgörande krämer (s.k. mjukgörare) används vid behandling av olika hudsjukdomar, men

också av personer med frisk hud. Det är inte ovanligt att mjukgörare används kontinuerligt i

veckor, månader eller till och med under flera år. Studier har visat att vissa mjukgörare

förstärker hudens barriärfunktion medan andra har en negativ påverkan, av orsaker som inte

är helt klarlagda. En bättre förståelse kring mekanismer hur långtidsbehandling med

mjukgörande krämer påverkar hudbarriären är av stor klinisk betydelse. Krämer som

försvagar hudens barriärfunktion kan leda till ökad penetration av allergener eller irriterande

ämnen vilket i sin tur kan ge upphov till torr hud och eksem. Mjukgörare som stärker

hudbarriären skulle dock kunna motverka många av dessa problem.

I den här avhandlingen har icke-invasiva tekniker kombinerats med analyser av hudbiopsier

vilket möjliggjort studier av epidermis på cellulär och molekylär nivå. Olika mjukgörande

formuleringar och deras effekt på hudbarriären har studerats på friska försökspersoner.

Parametrar såsom mjukgörarens pH, lipidtyp, innehåll av fuktbindande ämne samt

komplexiteten av formuleringar har studerats.

Efter sju veckors behandling med de olika mjukgörarna detekterades förändringar i hudens

vattenavgivande förmåga, hudens kapacitans samt dess känslighet för irritation, vilket

indikerar förändringar i hudens barriärfunktion. Dessutom, mRNA-uttryck av flera gener som

är involverade i differentieringen och avfjällningen av stratum corneum, liksom

lipidmetabolismen, var förändrad i hud behandlad med en av mjukgörarna medan en annan

inducerade färre förändringar.

Långtidsanvändning med mjukgörare kan förstärka hudens barriärfunktion eller försämra den

och ge upphov till torrhet. Mjukgörare har en signifikant påverkan på hudens biokemi, vilket

kan mätas på molekylär nivå. Genom att noggrant val av innehållsämnen skulle det vara

möjligt att styra mjukgörarnas effekt på huden till en önskad klinisk effekt.

52

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to:

Hans Törmä, my supervisor, for taking me under your wings and being incredibly patient and

understanding, in all situations. Your guidance helped me to look at the skin from a broader

perspective and ignited my enthusiasm about investigating the skin barrier at molecular level.

Thank you for our vigorous discussions.

Marie Lodén, my co-supervisor, for introducing me into a field of the skin barrier and giving

the opportunity to perform research in this area. Your vision and knowledge about

moisturizers, countless ideas and passion for research are sincerely inspiring. I am deeply

grateful for your guidance and persuading me to challenge the barriers, no matter what they

are.

Berit Berne, my co-supervisor, for excellent discussions, creative brainstormings, and being

very thorough in checking the manuscripts, as well as for all “clinical” help. Your inner

calmness was a great counterbalance to my nervousness.

Magnus Lindberg, my co-supervisor, for sharing your knowledge and experience, teaching

me to be cautious about interpretation of obtained results and to express my ideas as concise

as possible (I still need to master that). I am grateful for making me to understand that stratum

corneum should be treated as a ‘black box’.

Hans, Marie, Berit and Magnus–it was an honour to work with you.

Annika Grindborg, managing director of ACO HUD, for believing in me and making this

research possible.

Anders Vahlquist, head of Department of Dermatology, for providing excellent work

conditions.

Tove Agner, Marie Virtanen and Lars Norlén, for valuable comments and interesting

discussion during my “half-time control”.

Jacek Arct, the president of the Polish Society of Cosmetic Chemists, who significantly

influenced my life and showed me that cosmetics are fascinating from all perspectives.

All volunteers, for sacrificing their skin for science, although sometimes it was painful.

My parents, Janina and Krzysztof, and my brother Adam, for their love and support.

53

My dear Tomas, for patience and being by my side.

Ragna Lindeke-Strömqvist, Emil Schwan and Jouni Frid, for moral support, encouragement

in time of despair and discussions about meaning of life.

All my colleagues at ACO HUD, for help and being enthusiastic about my research.

My colleagues at Uppsala, for all support, scientific and not only (otherwise I would have

gone crazy in my dark room), and especially to Inger Pihl-Lundin, for skilful technical

assistance.

Proper English AB, for help with language corrections.

This research was financially supported by ACO HUD NORDIC AB, Uppsala University and

the Welander and Finsen Foundation.

54

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