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Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References

Background  Aqueous Cream BP is frequently prescribed for patients with eczema and is known to induce sensitivity in certain patients and also to decrease the thickness of the stratum corneum (SC). We have previously reported methodology to quantify corneocyte maturity and size, protease activity and protein content within different levels of the SC.

Objectives  The aim of the present study was to investigate changes in corneocyte size, corneocyte maturity, selected protease activities, protein content and transepidermal water loss (TEWL) in normal skin after a 28-day application of Aqueous Cream BP.

Methods  The left and right mid volar forearms of six healthy female volunteers were selected as the study sites. Aqueous Cream BP was applied twice daily to treated sites for 28 days. At the end of this period, the site was tape-stripped and corneocyte maturity, corneocyte size and protease activity of the desquamatory kallikrein proteases, KLK5 and KLK7, and the inflammatory proteases tryptase and plasmin were measured. Protein content and TEWL measurements were also recorded.

Results  Corneocyte maturity and size decreased with increasing number of tape strips, and were significantly lower in treated sites compared with untreated sites. Protease activity and TEWL values were higher (P < 0·05) for the treated sites compared with untreated sites. The amount of protein removed from deeper layers of treated sites was significantly lower than from untreated sites.

Conclusions  We report rapid minimally invasive measures of the effects of Aqueous Cream BP at the cellular and molecular level of the skin. Treatment with this formulation is associated with increased desquamatory and inflammatory protease activity. Changes in corneocyte maturity and size are also indicative of accelerated skin turnover induced by chronic application of this emollient. These findings question firmly the routine prescription of this preparation as a moisturizer in patients with atopic dermatitis.

Eczema is reported to account for up to 22·5% of cutaneous diseases treated by general practitioners in the U.K.1 For the management of eczema and dry skin the British National Formulary currently includes Aqueous Cream BP under the heading of ‘Emollients’, which are further defined as ‘preparations which soothe, smooth and hydrate the skin and are indicated for all dry or scaling disorders’.2 Aqueous Cream BP first appeared as a preparation of the British Pharmacopoeia in 1958 and its formulation, containing sodium lauryl sulphate (SLS), remains largely unchanged today.3 The continued recommendation of Aqueous Cream BP as an emollient is surprising as SLS is a known skin irritant.4–7 and it is commonly used in patch testing. The use of Aqueous Cream BP is also associated with a number of adverse skin reactions in children8 and more recently with significant thinning of the stratum corneum (SC).9 The exact mechanism by which the components of Aqueous Cream BP contribute to these effects is not understood.

We have recently reported the development of rapid minimally invasive measures of the spatial distribution of corneocyte maturity and size as well as protease activity and protein content within different levels of the SC layers.10 Quantitative indices of the mature, rigid corneocyte envelopes isolated from the outermost layer of the SC and the fragile, less mature corneocyte envelopes present in deeper layers of the SC were reported. Corneocyte size measurements were also observed to be greater for the more mature skin cells when compared with the less mature cells deeper in the SC. The activities of the key desquamatory kallikrein enzymes, tryptic-like ‘KLK5’ and chymotryptic-like ‘KLK7’, together with inflammatory-related protease ‘tryptase’ were also evaluated as a function of depth in the SC.

In the present study we examine the effects of the application of Aqueous Cream BP on these markers of epidermal turnover and skin barrier function. We have also extended the proteases studied to include plasmin as this enzyme appears to be the most responsive inflammatory-related marker for the skin.11,12 As transepidermal water loss (TEWL) is routinely used to characterize skin barrier function TEWL measurements were also conducted.13

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References

Materials

Standard D-Squame® tape (2·2 cm in diameter, 3·8 cm2) was obtained from CuDerm Corporation (Dallas, TX, U.S.A). Sodium lauryl sulphate, ethylenediaminetetraacetic acid, Triton X-100, acetic acid and Nile red were obtained from Sigma-Aldrich (Gillingham, U.K.). HPLC analytical grade water and methanol were obtained from Fisher Scientific (Loughborough, U.K.). dl-Dithiothreitol and Tris–HCl buffer (pH 8·0) were obtained from Fluka Analytical (Gillingham, U.K). Dimethyl sulphoxide was obtained from VWR International Ltd (Lutterworth, U.K.) and phosphate-buffered saline tablets were obtained from Oxoid (Cambridge, U.K.). TEWL was measured using an Aquaflux AF103 (Biox Systems Ltd, London, U.K.) and protein absorbance was measured at 850 nm using a SquameScan A850 infrared densitometer (Heiland Electronic, Wetzlar, Germany). The primary monoclonal antibody, antihuman involucrin (clone SY5), was purchased from Cambridge Scientific, Monosan (Cambridge, U.K.) and the rabbit polyclonal antibody to mouse fluorescent IgG H & L (whole molecule) fluorescein–isothiocyanate antibody was obtained from Abcam (Cambridge, U.K.). Aminomethyl coumarin (AMC) and all fluorogenic peptide substrates were generous gifts from Pentapharm Ltd (now part of DSM Nutritional Products Ltd, Basel, Switzerland). Aqueous Cream BP was obtained from Boots The Chemist (The Boots Company PLC, Nottingham, U.K.).

Methods

Volunteer recruitment

Six healthy female Caucasian volunteers aged 23–29 years were recruited in November 2010 and the study was performed from mid November 2010 to mid December 2010. None of the volunteers had any history of skin disease. The research protocol was approved by the South West London Research Ethics Committee (Reference 10/H0801/69). A participant information leaflet was supplied to the volunteers prior to the study. Subjects were asked, apart from daily washing, not to apply any moisturizer or cosmetic product to the region of interest during the study.

Application of Aqueous Cream BP

Aqueous Cream BP was applied according to a previously published protocol.9 Briefly, the left and right mid volar forearm were delineated to provide well-separated treatment and control sites. Cream (2 mL) was applied using a needleless syringe for 10 min to the treatment sites (approximately 40 cm2) twice a day for 28 days. Excess cream was gently removed by soft facial tissue away from the control sites. A 1-day washout period was allowed before any experimental measurements, during which participants refrained from the use of any moisturizing or other skin care treatment on their forearms. Intra individual and regional differences were minimized by conducting both control and treated experiments on the same arm.

Tape stripping

Standard D-Squame® tape was applied to the forearm sites at a constant pressure as described previously.10 Four areas (two within the treated and two within the untreated sites) within each mid volar forearm were selected. Twenty consecutive tape strippings were performed for all the selected areas within the treated and untreated sites.

Transepidermal water loss measurement

Volunteers were allowed to acclimatize for 15 min in a controlled atmosphere (20 ± 1 °C and 45 ± 5% relative humidity) prior to the tests, in order to equilibrate to room conditions and to minimize insensible perspiration. TEWL was then measured (using an Aquaflux AF102 instrument; Biox Systems Ltd). TEWL measurements were performed initially and after each series of four tape strips were removed.

Protein content

Protein content (Cprotein) was measured directly from the tape strips. Protein absorption was determined at 850 nm using an infrared densitometer SquameScan 850A (Heiland Electronic) as previously reported.11 The amount of protein was subsequently quantified as follows:11

  • image
Corneocyte maturity

Corneocyte maturity was measured by immunofluorescence and Nile Red staining of tape strip numbers 1, 5, 9, 13 and 17 and full details of the methodology have been reported elsewhere.10 Fluorescence was monitored with a fluorescence microscope equipped with a Fuji S2 Pro 6.2 megapixel camera (Fuji, Tokyo, Japan). The object was magnified × 10, giving 0·8 pixels μm−1 with a resolution of 1440 × 960 pixels. ImageJ® image analysis software (NIH-Image, Bethesda, MD, U.S.A.) was used to analyse the red pixels obtained from Nile Red stained cells and the green pixels from the immunostained cells.

Measurement of corneocyte surface area

ImageJ® imaging software was calibrated to measure the corrected number of pixels based on the images acquired (0·8 pixels μm−1 with a resolution of 1440 × 960 pixels).10 Corneocyte surface areas were measured for the same tape strip numbers as those measured for corneocyte maturity, i.e. tape numbers 1, 5, 9, 13 and 17. A minimum of 10 images, randomly selected, was taken from each slide.

Protease activity

Stratum corneum samples were obtained by tape stripping as described above. Protease activity was measured for tape strip numbers 2–4, 6–8, 10–12, 14–16 and 18–20 as previously described.10

Statistical analysis

SPSS version 18 (SPSS, IBM, Somers, NY, U.S.A.) was used to analyse the data. Parametric statistical tests (one-way between-group anova and paired t-test to compare means) were used to investigate statistical differences between treated and untreated sites. A probability of P < 0·05 was considered statistically significant. All results are presented as mean ± SEM.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References

TEWL measurements for control and treated sites are shown in Figure 1. TEWL measurements were conducted before tape stripping and after removal of 4, 8, 12, 16 and 20 tape strips.

image

Figure 1.  Transepidermal water loss (TEWL) measurement of the mid volar forearm with increasing number of tape strips for control and treated sites (n = 24, mean ± SEM).

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The difference between control and treated sites was statistically significant up to removal of 16 tape strips (P < 0·05), i.e. with increasing amounts of stratum corneum removed, water loss was always higher for sites exposed to Aqueous Cream BP compared with untreated sites. After removal of 17–20 tape strips, the mean value of TEWL was not significantly lower for treated sites vs. control sites (P = 0·09). The initial values of TEWL were of a similar magnitude to those reported by Tsang and Guy9 for control sites and sites treated with Aqueous Cream BP over the same time-frame and using the same instrumentation.

Protein content

Figure 2 shows the protein content, which was measured for each individual tape strip. As noted previously10 the amount of protein removed decreased was more SC was removed, reflecting increased cohesion of the deeper layers.14–16

image

Figure 2.  Stratum corneum protein content removed from mid volar forearm for each tape strip for control and treated sites (n = 24, mean ± SEM).

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Significant differences (P < 0·05) in protein content between control and treated sites were evident after removal of 12 tape strips. A correlation between amount of protein removed and weight of tape strips also allowed an estimation of the depth of SC probed for both control and treated sites (data not shown). For the same number of tape strips, the depth to which the SC was tape stripped may be estimated as 8·26 ± 1·50 μm for control sites and 7·58 ± 1·09 μm for treated sites (P < 0·05).

Corneocyte maturity

Representative micrographs showing corneocytes stained with Nile Red and immunostained for native involucrin to assess corneocyte maturity are shown in Figure 3. More mature corneocytes can be seen in control-site micrographs when compared with those for the treated site.

image

Figure 3.  Micrographs showing corneocyte maturity from tape 1 of the volar forearm of a subject. (a, c) are control and treated, respectively, stained with Nile Red. (b, d) are control and treated, respectively, and immunostained for native involucrin; 1440 × 960 pixels, 0·8 pixels μm−1.

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Figure 4 illustrates the maturity of corneocytes for both control and treated sites (calculated as a ratio of red/green pixels, described previously10). For the control site, more mature cells were found in the outer layers of the SC with progressively fewer mature corneocytes found with increasing depth into the SC.

image

Figure 4.  Corneocyte maturity of skin samples removed from the mid volar forearm by tape stripping (n = 24, mean ± SEM).

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There were significant differences (P < 0·05) in the maturity of corneocytes from control sites when compared with treated sites. For all series of tape strips analysed, corneocytes were always more mature in control sites than in treated sites.

Corneocyte surface area

Corneocyte surface area decreased with each tape strip series for both groups (Fig. 5). However, the cells from control sites were significantly larger (P < 0·05) than those from the treated sites, consistent with the maturity data above (Fig. 4).

image

Figure 5.  Mean corneocyte surface area of skin samples removed from the mid volar forearm by tape stripping (n = 24, mean ± SEM).

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When the correlation between corneocyte maturity and surface area was probed using simple regression analysis for both treated and control groups, high correlation coefficients were obtained (i.e. more mature corneocytes were larger, in line with our previous findings10), providing further confidence in the robustness of the methods used to determine these properties (Fig. 6).

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Figure 6.  Correlation of corneocyte maturity with corneocyte size for control and treated sites. Control (inline image) y = 55·7x + 761·4, r2 = 0·91; treated (inline image) y = 84·2x + 693·6, r2 = 0·83 (n = 24; mean ± SEM).

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Protease activity

For the proteases studied (KLK5, KLK7, tryptase and plasmin), activity was found to decrease with increasing depth (Fig. 7). For both control and treated sites, elevated activities of the desquamatory and inflammatory enzymes were observed in the outermost layers of the SC compared with the deeper layers, in line with the findings of Voegeli et al.11 and with our own work.10

image

Figure 7.  Stratum corneum protease activity of the mid volar forearm with depth; (a) KLK5; (b) KLK7; (c) tryptase; (d) plasmin (n = 24, mean ± SEM).

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There were significant differences in the activities of all proteases for control vs. treated sites (P < 0·05). In all cases, treatment with Aqueous Cream BP resulted in significantly increased activity of the desquamatory kallikreins as well as the inflammatory enzymes, plasmin and tryptase at all SC depths assayed. The findings are also consistent with those of Suzuki et al.,17,18 where treatment with SLS resulted in an increase in trypsin-like and chymotrypsin-like protease activity in the SC.

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References

For effective turnover of the skin, proteolysis of the cohesive corneodesmosomes which bind the corneocytes in the SC must occur. This process is mediated by proteases which catalyse peptide bond hydrolysis.19 The human tissue kallikreins (KLKs) are found in a variety of tissues20 and have been suggested to function as an enzymatic cascade pathway.21–23 Corneodesmosomes are known to be broken down by several of the desquamatory-related serine proteases, such as KLK5, KLK6, KLK7, KLK8, KLK10, KLK11 and KLK13, present within the SC.17,24 As well as having a vital role in the proteolysis of the SC corneodesmosomes, KLKs are also reported to be involved in degradation of lipid-processing enzymes, such as β-glucocerebrosidase.25

The expression of tryptic (e.g. KLK5) and chymotryptic (e.g. KLK7) enzymes has been reported to be reduced in the outer layers of the SC in dry skin.26–28 However, increased expression of KLK7 has been reported in two major chronic inflammatory diseases: psoriasis and atopic dermatitis.24,29 Changes in the activity of serine proteases KLK5 and KLK7 also appear to be associated with skin barrier disturbances.30–32 This may partly be due to uncontrolled corneodesmolysis leading to weakening of SC integrity and cohesion.25,27 Other enzymes such as plasmin, tryptase and urokinase are also reported to be present in the SC but are not necessarily involved in the desquamatory process. However, these enzymes may have an important role in the inflammatory process in the skin.11,29

Because chronic use of Aqueous Cream BP has previously been associated with skin irritation and thinning of the skin, we hypothesized that these effects might be linked to the influence of this formulation on the molecular markers of epidermal turnover such as protease activity, corneocyte maturity, size and skin protein content. The application of Aqueous Cream BP appears to disrupt the normal maturation process, i.e. the corneocytes do not mature fully because of exposure to the preparation. The findings of our study also confirm increased protease activity for both the desquamatory enzymes KLK5 and KLK7, and the inflammatory enzymes plasmin and tryptase with repeated application of Aqueous Cream BP. Elevated levels for the latter two enzymes also suggest an inflammatory response in sites treated with this preparation and, indeed, the reduction in corneocyte surface area and maturity is entirely consistent with increased epidermal turnover. The depth to which skin could be probed for treated sites was approximately 0·7 μm less than for control sites. This compares well with the reported decrease in SC thickness of 1·1 μm, following the same treatment protocol by Tsang and Guy.9 Finally, increased TEWL values are consistent with this disturbance in corneocyte maturation during the treatment period. The results provide a mechanistic understanding for the observed thinning of the SC associated with use of Aqueous Cream BP and for the irritant effects reported following the use of this preparation in patients with atopic dermatitis.

In conclusion, robust, minimally invasive and sensitive methods have, for the first time, been developed and used to delineate the effects of Aqueous Cream BP on healthy skin at the cellular and molecular level. It is reasonable to hypothesize that, in patients with atopic dermatitis, further deterioration in these markers of skin health would be manifested with continual use of Aqueous Cream BP. Future studies will extend these findings to evaluate the effects in a cohort of patients with a history of atopic dermatitis and to examine the effects of other dermatological preparations (and their constituents) on the SC biomarkers described in this publication.

What’s already known about this topic?

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References
  • • 
    The repeated application of Aqueous Cream BP is known to induce skin irritation and has been associated with a decrease in skin thickness.
  • • 
    The exact mechanism by which the components of Aqueous Cream BP contribute to these effects is not known.

What does this study add?

  • • 
    Treatment with Aqueous Cream BP increases desquamatory and inflammatory protease activity.
  • • 
    Accelerated skin turnover is also induced by chronic application of this emollient.
  • • 
    Aqueous Cream BP should not be prescribed as a moisturizer for patients with atopic dermatitis.

Acknowledgment

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References

Diar Mohammed wishes to acknowledge Proctor & Gamble for studentship funding.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. What’s already known about this topic?
  7. Acknowledgment
  8. References