Changes of comedonal cytokines and sebum secretion after UV irradiation in acne patients

ARTICLE

Many causes are suggested to be involved in the pathogenesis of acne. Hormones, activity of the sebaceous gland, bacteria, genetics, drugs, occupation, and psychic stress are well-known, and many clinical and research data are available [1]. Environmental factors, including climate and ultraviolet light, are also presumed to be important in the provocation of acne [2]. In the case of climate, many researchers agree that acne is usually aggravated in hot and humid weather [2]. However, the exact role of ultraviolet light in the development and aggravation of acne lesions is uncertain and inconclusive.

Some patients believe in the suppressive effect of sunlight on acne [2]. In the cases improved by sunlight, there was no conclusion about the underlying pathophysiology. Little was known about whether it may work by direct effect on keratinocytes or other cells in the skin and pilosebaceous unit. Some people even suggest that the improvement may be caused by either the placebo effect or masking effect from sunlight tanning and erythema [2]. The controversy about the effect of sunlight on acne was further complicated by Hjorth et al. [3]. They named ''acne aestivalis'' for the acne patients who complained of the aggravation of acne lesions in summer. They contended that the main causative factor is sunlight.

Sunlight contains ultraviolet light as well as visible light. According to Sigurdsson et al. [4], it was assumed that visible light has a moderate effect on the improvement of acne lesions because it may cause photodynamic destruction of P. acnes. This study focuses on the effect of ultraviolet light. UVB causes a variety of biological reactions in the skin [5], including the evocation of inflammatory reaction and impairment of immune reaction. Though UVA is 10 to 1,000 times less efficient in producing erythema than UVB, it also has been shown to have an ability to produce cytokines from keratinocytes [6, 7]. In addition, UVA penetrates more deeply into the skin. However, the effect of ultraviolet light on comedones according to wavelength range is still unknown.

In this study, we tried to discover the effect of UVA and UVB on the changes of cytokines in comedones as well as on the sebum secretion. IL-1, which is divided into IL-1alpha and IL-1beta, is the cornerstone among cytokines because it affects many other cytokines [8]. IL-1alpha is mainly produced by keratinocytes. Its biologic action is regulated by IL-1 receptor antagonist (IL-1ra), which has 19% homology with IL-1alpha. IL-1ra is normally produced 10-100 times more than IL-1alpha by keratinocytes. IL-6 is a highly pleiotropic cytokine with multiple effects including the stimulation of proliferation of keratinocytes [9]. IL-10 is also an important cytokine because it has mainly inhibitory or regulatory effects on immune responses [10]. GM-CSF, a well-known growth factor for myeloid progenitors, is also produced by keratinocytes and enhances proliferation and differentiation of keratinocytes and melanocytes [11]. In the past literature, IL-10, IL-1ra, and GM-CSF have never been measured from comedones in acne patients.

Subjects and methods

Subjects

Thirteen acne patients (M:F = 7:6) were enrolled on a volunteer basis after informed consent. This study was permitted by IRB of the hospital. Acne lesions were distributed on their backs. The possibility of pityrosporum folliculitis was excluded by microscopic examination. Briefly, comedones were extracted and smeared on the glass slide with ten percent of KOH solution. After putting on a cover glass, it was sealed with Parker ink^® - glycerol solution (1:100). Next day, the slides were observed under a microscope to rule out the possibility of pityrosporum folliculitis.

Ultraviolet irradiation

The backs of the patients were divided into three parts. While the left side of the back was irradiated with UVA, right side was irradiated with UVB. The middle part was used as a control (no irradiation). Firstly, minimal erythema dose (MED) was checked in each patient. MED was defined as the smallest exposure dose needed to produce erythema with sharp borders 24 hrs after irradiation. UVASUN (Mutzhas Co., Germany; 330-460 nm, peak at 360 nm) was used to determine UVA MED and Waldmann UV 800 (Waldmann Co., Germany; 285-350 nm, peak at 310-315 nm) was used to measure UVB MED. Irradiance at the skin surface was measured with Waldmann UV meter (Waldmann Co., Germany). After checking MED, UVB phototherapy was performed everyday with Waldmann UV 8001K (Waldmann Co., Germany; 285-350 nm, peak at 310-315 nm) by 10% stepwise increments, starting from 2/3 MED. UVA phototherapy was also performed on the same day with UVA-003 HOUVA-II (National Biological Co., U.S.A.; 320-400 nm, 55% from 340-360 nm and 24% from 360-400 nm) by 10% increments daily, starting from 20 J/cm². UV irradiation continued for nine consecutive days. Before irradiation, we clearly marked the areas to be irradiated with a pen to avoid overlapping of the areas. Proper coverage made up of thick black fabrics was applied to other portions when the designated portion of the back was exposed to either UVA or UVB.

Cytokine measurements from comedones

Six open comedones per UV-irradiated and control areas were collected three times: before the study (day 1), in the middle of the study (day 5), one day after the last irradiation (day 10). At day 1 and day 5, the collection was performed just before UV irradiation. The recovery and collection procedure has been previously described [12]. Briefly, the comedone was collected aseptically without any bleeding using a comedone extractor after swabbing the skin with isopropanol. The weight of each comedone was measured, and then the homogenization of comedone was performed for one minute with a micro-tissue homogenizer. The homogenization medium was 250 mul DMEM supplemented with 2 mM L-glutamine, 0.375% (W/V) NaHCO₃, 20 mM HEPES, and 10% (V/V) FCS, penicillin (100,000 U/L) and streptomycin (100 mg/L). The homogenate was centrifuged at 10,000 g for 10 min and the supernatant was used for ELISA of cytokines. For IL-1alpha, IL-6, IL-1ra, and IL-10, Quantikine^® (R & D System, USA) ELISA kits were used. For GM-CSF, ELISA kits by Endogen, Inc. (USA) were used. Minimal detectable doses of each ELISA kit were as follows: IL-1 alpha, 0.5 pg/ml; IL-6, 0.70 pg/ml; IL-1ra, 6.5 pg/ml; IL-10, 7.8 pg/ml; GM-CSF, 2 pg/ml.

Measurement of sebum excretion

To avoid diurnal variation, sebum level was measured at 10 a.m. It was performed four times: day 1, day 4, day 7, one day after the last irradiation (day 10). At day 1, day 4 and day 7, the collection was performed just before UV irradiation. Sebumeter SM810^® (Courage + Khazaka Electronic GmbH, Germany) was used after 30 minutes rest after removing upper underwear. For all the measurements, the middle portions of the three areas (for UVA, UVB and control) were selected. Patients were required to take showers at 9 p.m. one day before each measurement. Then, additional showers as well as work or exercise which could cause perspiration, was not permitted on the days of sebum measurements. Constant temperature (20 ± 1^° C) and humidity (40 ± 2%) were maintained in the sebum measurement.

Clinical assessments

By modifying the method of Michaelsson et al. [13], objective assessment by two independent observers was undertaken regarding the change in number and size of acne lesions. The numbers of comedones, papules and pustules were recorded. Each type of lesion was given a severity index; 0.5 for comedo, 1 for papule, 2 for pustule. A total score that corresponds to the severity of the disease was obtained by multiplying the number of each type of lesion with its severity index and calculating the sum of the various lesions. If the total score was decreased by more than 50% at the end of UV irradiation, it was evaluated as "improved". If it was decreased by less than 50%, the case was rated as "stationary". In the case of increased total score after UV irradiation, it was evaluated as "aggravated". The patient's opinion of the improvement was also recorded and rated as "improved", "stationary", or "aggravated".

Statistical analysis

ANOVA, Student's t-test and Wilcoxon rank sum test were used. When p < 0.05, the difference was evaluated as being significant.

Results

Clinical assessments (Table I)

The volunteers reported that acne lesions were improved (7 persons) or stationary (6 persons). This subjective evaluation coincided with the objective assessment in each patient. No one complained of aggravation of the acne lesions.

Cytokine measurements (Table II)

IL-1alpha production was strongly elevated on day 5 (251 pg/ml by UVA and 290 pg/ml by UVB), and then on day 10, it showed approximately the same level as day 1 (Fig. 1). UVB as well as UVA showed similar results, although UVB showed a little more stimulatory effect. When the patients were divided into "improved" and "stationary" groups (Fig. 2), the improved groups showed a different pattern in IL-1 alpha production. Compared with clinically improved groups, it was significant (p < 0.05) that the stationary groups showed a dramatic increase of IL-1alpha production. These tendencies were shown on both UVA (16.6 fold increase) and UVB (18.1 fold increase) sites. IL-1ra production had the same tendency as IL-1alpha, in that IL-1ra showed a high increase in production on day 5 (14,033 pg/ml by UVA and 16,050 pg/ml by UVB), followed by a decrease on day 10 (Fig. 3a). When divided into "improved" and "stationary" groups according to the response to UV irradiation, the "improved" groups had initial IL-1ra value of 10,765 (pg/ml/mg of comedone) on average, and they did not show great changes in the amount throughout the study (Fig. 3b). This tendency was shown in both UVA- and UVB-treated sites. On the contrary, "stationary" groups had an initial IL-1ra value of 3,639 (pg/ml/mg of comedone) on average, and the level was steeply increased on day 5, followed by a drop in the level on day 10 (Fig. 3c). UVB as well as UVA showed the similar tendency in this sense, although UVB (22,963 pg/ml) induced somewhat higher increases in IL-1ra production than UVA (17,556 pg/ml).

IL-10 productions were also significantly higher in "stationary" groups (p < 0.05) (Fig. 4). UVB (2.8 fold) was a stronger stimulant than UVA (1.9 fold). However, in the improved groups, the IL-10 level showed values which were not significantly different from the initial values (p > 0.05). IL-6 in comedones was significantly decreased by both UVB and UVA (p < 0.05) (Fig. 5). UVA showed a stronger depressing effect than UVB at day 10. GM-CSF was not detected in any samples.

Sebum measurements (Fig. 6)

At day 1, the sebum levels were 18.2 (mug/cm²) on the UVA irradiation site and 19.1 on the UVB irradiation site. There was no significant difference between them (p > 0.1). On these sites, the amount of sebum showed a peak at day 4 (37.6 mug/cm² by UVA and 40.0 mug/cm² by UVB), and progressively normalized thereafter. The control sites had an average sebum amount of 48.4 (mug/cm²), and revealed no significant change (p > 0.1) during the study.

Discussion

In a study of the inflammatory cytokine content of comedones [12], bioactive IL-1 alpha-like material was found to be present. Eady et al. [14] suggested that IL-1alpha in comedones was produced by ductal keratinocytes. They also suggested that the enhancement of IL-1alpha production in comedones by therapy may be related to the resolution of lesions, because IL-1alpha is recognized to play a role in wound healing.

In our study, IL-1alpha was detected by ELISA and its content in comedones was increased on day 5 by UVA and UVB. If the source of cytokine in comedones is follicular keratinocytes, this increase may be well expected because IL-1alpha is enhanced by UV irradiation [15]. IL-1alpha was almost normalized at day 9, despite the continuous UV irradiation. At this point, we should consider the negative control of IL-1alpha production. T_H2-derived cytokines IL-4, IL-10 and IL-13 are known to suppress IL-1 expression [8]. In our study, IL-10 in comedones was elevated, and its pattern of increase was very similar to that of IL-1alpha, in that the "stationary" groups had a greater ratio of elevation (vs initial value). Therefore, there is a possibility that UV irradiation stimulated T_H2-derived cytokines including IL-10, so that it might down-regulate IL-1alpha production. These relationships between cytokines in comedones may be understood in a diagram (Fig. 7).

IL-10 is one of the anti-inflammatory cytokines [16]. It is also connected with immunosuppressive activity [17]. It is already known that UVB causes increased production of IL-10, and this is strongly associated with the inhibition of Ag presentation of Langerhans cells and the immunosuppression caused by ultraviolet light [18, 19]. Interestingly, in our study, IL-10 production was more pronounced in clinically stationary patients. It suggests that there may be a disturbance in immunoregulation in these patients, so that the beneficial effect of UV, such as the healing of acne lesions induced by the increase of IL-1alpha, may be counteracted by the greater, simultaneous production of IL-10. IL-10 production was well enhanced after UVA as well as UVB irradiation in the clinically stationary patients. It is true that there is still debate as to whether UVA causes an increase in epidermal IL-10 expression [6, 20]. However, it must be remembered that the UVA dosage we used in this study was much higher compared with those studies, and that this is a human in vivo study in which cytokines were measured directly from comedones. In the clinically improved patients, it seemed that UVA was rather stronger than UVB in IL-10 production although the difference was not significant.

IL-1ra interferes with IL-1 activity by competitive binding to IL-1 R1, preventing IL-1 from reacting excessively [21]. IL-1ra production is increased by stimulants like UV, which elevates IL-1 level [22]. IL-10 and GM-CSF also enhance LPS-induced expression of IL-1ra in both monocytes and PMN [23]. In our study, IL-1ra level was rather constant during the study in clinically improved patients. On the other hand, in the clinically stationary patients, the initial level of IL-1ra was low compared with those of the improved groups and production was highly increased after UV irradiation. Such an abrupt increase at day 5 was very similar to the results of IL-1alpha and IL-10 in clinically stationary patients.

Therefore, it may be suggested that the clinically stationary patients are less prepared for stimulants like UV irradiation because their initial IL-1ra level is lower compared with improved groups. In response to UV irradiation, IL-1ra production will be steeply increased in the stationary groups to cope with the inflammation caused by UV irradiation, because IL-1alpha is steeply increased in these stationary patients as shown in Fig. 2. In a future study, it may be necessary to study many acne patients to confirm whether or not the initial low level of IL-1ra in comedones is correlated with a poor response to UV therapy.

IL-6 production is increased by IL-1alpha [24]. It is inhibited by IL-10 administration in mice and rats [21]. The expression and production of GM-CSF are increased by UVA and UVB [11]. IL-1alpha also enhances GM-CSF production. On the other hand, IL-10 prevents the release of GM-CSF in mouse and rat models [23]. In our study, IL-6 level was gradually decreased and GM-CSF was not detected by ELISA. This may be further evidence that IL-10 is the key regulator in the cytokine network in comedones (Fig. 7) because the effects of IL-10 are believed to overcome those of IL-1alpha and to suppress the production of IL-6 and GM-CSF.

The reason why the control site showed high sebum levels is that the location of control site is mid-back, which has intrinsically high sebum excretion. Sebum levels of UVA and UVB irradiation sites were elevated in the first three days, then it gradually normalized. Although there is no direct evidence that sebum excretion is controlled by ultraviolet light, UV irradiation might cause the elevation of sebum excretion. After several days, the sebaceous gland might be adapted to UV stimulation, so that it returned to the original functional activity.

In this study, we did not perform the culture and enumeration of skin microorganisms in comedones. It may be thought that the improvement of acne by UV may be due to its anti-bacterial effect because the ultraviolet light affects bacteria in vitro. However, it was not necessary to work on the bacteria because it was known that cutaneous microflora are not able to stimulate IL-1alpha production by keratinocytes [25]. Therefore, it is less likely that the cytokine changes shown in this study were caused mainly by microorganisms.

CONCLUSION

This work was presented as a poster at the 8th EADV Congress.

Acknowledgements

We thank Hyo Sook Kim for excellent advice. This work was supported by a grant (04-97-025) from Seoul National University Hospital Research Fund.

REFERENCES

2. Cunliffe WJ. Acne. London: Martin Dunitz, 1989.

3. Hjorth N, Sjolin KE, Sylvest B, Thomsen K. Acne aestivalis-Mallorca acne. Acta Derm Venereol 1972; 52: 61-3.

4. Sigurdsson V, Knulst AC, Van Weelden H. Phototherapy of acne vulgaris with visible light. Dermatology 1997; 194: 256-60.

5. Pathak MA, Nghiem P, Fitzpatrick TB. Acute and chronic effects of the sun. In: Dermatology in general medicine. Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, Fitzpatrick TB, eds. New York: McGraw-Hill. 1999: 1598-607.

6. Grewe M, Gyufko K, Krutmann J. Interleukin-10 production by cultured human keratinocytes: Regulation by ultraviolet B and ultraviolet A1 radiation. J Invest Dermatol 1995; 104: 3-6.

7. Kondo S. The roles of keratinocyte-derived cytokines in the epidermis and their possible responses to UVA-irradiation. J Invest Dermatol Symp Proc 1999; 4: 177-83.

8. Mantovani A. The interleukin-1 system. In: The cytokine network and immune functions. Thèze J, ed. New York: Oxford University Press. 1999: 85-96.

9. Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT, Kupper TS, Sehgal PB, Gottlieb AB. Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 1989; 86: 6367-71.

10. Garssen J, Vandebriel RJ, De Gruijl FR, Wolvers DAW, Van Dijk M, Fluitman A, Van Loveren H. UVB exposure-induced systemic modulation of Th1- and Th2-mediated immune responses. Immunology 1999; 97: 506-14.

11. Park KC, Jung HC, Hwang JH, Youn SW, Ahn JS, Park SB, Kim KH, Chung JH, Youn JI. GM-CSF production by epithelial cell line: upregulation by ultraviolet A. Photodermatol Photoimmunol Photomed 1997; 13: 133-8.

12. Ingham E, Eady EA, Goodwin CE, Cove JH, Cunliffe WJ. Pro-inflammatory levels of interleukin-1 alpha-like bioactivity are present in the majority of open comedones in acne vulgaris. J Invest Dermatol 1992; 98: 895-901.

13. Michalsson G, Juhlin L, Vahlquist A. Effects of oral zinc and vitamin A in acne. Arch Dermatol 1977; 113: 31-6.

14. Eady EA, Ingham E, Walters CE, Cove JH, Cunliffe WJ. Modulation of comedonal levels of interleukin-1 in acne patients treated with tetracyclines. J Invest Dermatol 1993; 101: 86-91.

15. Kupper TS, Chua AO, Flood P, McGuire J, Gubler U. Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. J Clin Invest 1987; 80: 430-6.

16. Enk CD, Sredni D, Blauvelt A, Katz SI. Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro. J Immunol 1995; 154: 4851-6.

17. Katiyar SK, Challa A, McCormick TS, Cooper KD, Mukhtar H. Prevention of UVB-induced immunosuppression in mice by the green tea polyphenol (-)-epigallocatechin-3-gallate may be associated with alterations in IL-10 and IL-12 production. Carcinogenesis 1999; 20: 2117-24.

18. Hochberg M, Enk CD. Partial protection against epidermal IL-10 transcription and Langerhans cell depletion by sunscreens after exposure of human skin to UVB. Photochem Photobiol 1999; 70: 766-72.

19. Kurimoto I, Kitazawa T, Streilein JW. Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity, 2. Evidence that interleukin-10 from UVR-treated epidermis is the critical mediator. Immunology 2000; 99: 134-40.

20. Shen J, Bao S, Reeve VE. Modulation of IL-10, IL-12, and IFN-r in the epidermis of hairless mice by UVA (320-400 nm) and UVB (280-320 nm) radiation. J Invest Dermatol 1999; 113: 1059-64.

21. Schwarz T, Urbanska A, Gschnait F, Luger TA. UV-irradiated epidermal cells produce a specific inhibitor of interleukin 1 activity. J Immunol 1987; 138: 1457-63.

22. Bigler CF, Norris DA, Weston WL, Arend WP. Interleukin-1 receptor antagonist production by human keratinocytes. J Invest Dermatol 1992; 98: 38-44.

23. Mire-Sluis AR, Thorpe R. Cytokines. San Diego: Academic Press, 1998.

24. Chung JH, Youn SH, Koh WS, Eun HC, Cho KH, Park KC, Youn JI. Ultraviolet B irradiation-enhanced interleukin (IL)-6 production and mRNA expression are mediated by IL-1 alpha in cultured human keratinocytes. J Invest Dermatol 1996; 106: 715-20.

25. Ingham E, Walters CE, Eady EA, Cove JH, Kearney JN, Cunliffe WJ. Inflammation in acne vulgaris: Failure of skin micro-organisms to modulate keratinocyte interleukin 1 alpha production in vitro. Dermatology 1998; 196: 86-8.