CED
Clinical dermatology • Original article
Clinical and Experimental Dermatology
Effect of psoriasis activity on epidermal growth factor (EGF) and the concentration of soluble EGF receptor in serum and plaque scales I. Flisiak,1 M. Szterling-Jaworowska,1 A. Baran1 and M. Rogalska-Taranta2 1
Department of Dermatology and Venereology, and 2Department of Infectious Diseases and Hepatology, Medical University of Białystok, Białystok, Poland
doi:10.1111/ced.12356
Summary
Background. Epidermal growth factor receptors (EGFRs) are overexpressed in psoriatic keratinocytes, and regulate cell growth, proliferation and differentiation through binding to epidermal growth factor (EGF). The role of EGF and EGFRs in the pathogenesis of psoriasis and the contribution of their measurement to psoriasis management are still unknown. Aim. To evaluate serum concentrations of EGF, soluble (s)EGFRs and EGF content in psoriatic scales of patients with severe psoriasis, and to analyse their association with the clinical activity of the disease. Methods. Serum samples and plaque scales were collected from 51 patients with plaque-type psoriasis. Concentrations of EGF and sEGFR in serum and of EGF in scales were measured using enzyme immunoassay. Data were analysed with respect to baseline Psoriasis Area and Severity Index (PASI). Results. Mean serum EGF concentration in patients was higher than in controls (701 72 vs. 586 63 pg/mL), but the difference was not significant. Mean serum concentration of sEGFR was significantly lower than controls (40.8 1.4 vs. 86.4 11.3 ng/mL, P < 0.001). Serum levels of EGF showed a significant positive correlation and EGFR showed a significant negative correlation with PASI (P < 0.05). No correlation was seen between PASI and EGF content in scales or between EGF and sEGFR levels. Serum EGF concentrations reached the highest mean level (914 138 pg/mL) in patients with PASI > 20, and this was significantly higher than the mean of 414 82 pg/mL in the group with PASI < 10. Mean sEGFR serum concentrations remained significantly lower than those of controls, irrespective of disease severity. Conclusions. Compared with controls, patients with psoriasis had increased EGF and decreased sEGFR levels in serum. EGF and sEGFR levels correlated with disease severity.
Introduction Psoriasis is an inflammatory skin disease characterized by keratinocyte hyperproliferation, vascular expansion, Correspondence: Professor Iwona Flisiak, Department of Dermatology and _ Venereology, Medical University of Białystok, ul. Zurawia 14, 15-540, Białystok, Poland E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 14 December 2013
ª 2014 British Association of Dermatologists
fibroblast activation and immune cell infiltration resulting in changes to cytokine production.1 Hyperplasia of keratinocytes can be explained by an imbalance between the growth factors responsible for epidermal proliferation and the activity of their receptors in the cells of affected skin.2 The most important factors involved in this process are excessive proliferation of keratinocytes in the basal layer, disturbances in the cycle of keratinocye maturation and increases in angiogenesis. As we reported previously, some inflammatory cytokines and their soluble receptors
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
[transforming growth factor (TGF)-b1 and vascular endothelial growth factor] are involved in the pathogenesis of psoriasis.3,4 The factor that appears to play a crucial role in the pathogenesis of psoriasis is epidermal growth factor (EGF).5 EGF receptors (EGFRs) are overexpressed in epidermal keratinocytes in psoriatic lesions, and regulate cell growth, proliferation and differentiation through binding to EGF.6,7 Serum EGF was previously reported to be increased in patients with psoriasis, but these studies were not able to demonstrate any association with disease activity because of the small numbers of patients (mainly with mild psoriasis) who were enrolled.8–10 In active psoriatic lesions, an increase in EGFR content in all layers of epidermis is regularly observed.11,12 Binding of the EGFR receptor to monoclonal antibodies or inhibitors can ameliorate proliferation of keratinocytes.13,14 These substances are capable of binding EGF, and thereby preventing proliferative signal transduction, which can be a clinically relevant strategy for the inhibition of keratinocyte hyperplasia.15 By contrast, activation of EGFR by EGF and transforming growth factor (TGF)-a upregulates production of Decoy receptor (Dcr)3, which has a pleiotropic role in many inflammatory and autoimmune disorders and malignant diseases.16 However, there are limited data currently available on the role of EGF and sEGFR in the pathogenesis of psoriasis and the possible application of their measurement in psoriasis management. The aim of this study was to evaluate concentrations of EGF and soluble (s)EGFR in serum as well as EGF content in scales of patients with severe psoriasis. We analyzed their association with clinical activity of the disease, for possible follow-up of the disease.
Methods The study was approved by the Bioethical Committee of the Medical University of Bialystok, and informed consent was obtained from all participants.
Sex, F/M Age, years Family history of psoriasis, n (%) Effect of sun on exacerbation, n (%) Nail involvement, n (%) Disease duration, years* Duration of relapse, months*
Patients
We enrolled 51 patients (15 women, 36 men: mean SE age 48.7 2.2, range 19–84) with exacerbated chronic plaque-type psoriasis. As shown in Table 1, disease duration ranged from 1 to 60 years (mean SD 17.7 1.9 years), and duration of present relapse ranged from 1 week to 24 months (mean 3.9 0.7 months). Patients with a history of any other inflammatory chronic disease including other forms of psoriasis were excluded from the study. All patients were treated initially with a topical application of salicyl 5% ointment for desquamation, and then with 0.3% dithranol ointment. Blood samples were collected before the start of any treatment for psoriasis and then 14 days later. Serum levels of EGF and sEGFR were analysed with respect to the Psoriasis Area and Severity Index (PASI), and to normal serum values determined in 15 healthy controls (9 women, 6 men; mean SD age 44.5 3.7, range 22–78). EGF concentration in scales was also measured; this was expressed per protein content of the scales, and analysed with respect to PASI. For PASIrelated analysis, patients were divided into three groups with PASI ranges of < 10 (mild), 10–20 (moderate) and > 20 (severe). Analysis also included association with the disease duration (from the first relapse) and duration of present relapse (from the most recent appearance of papular lesions) as well as basic laboratory indices of inflammation. Mean age of controls (43.7 2.7 years) did not differ significantly from the patients (Table 1). PASI calculation
PASI was calculated in all patients as described by Fredriksson and Pettersson.17 Each region of the body (the head, the trunk, and the upper and lower limbs) were assessed separately for erythema, infiltration and desquamation, then summed to obtain the final PASI, as described previously.3,4
Patients
Controls
15/36 48.7 2.2 (19–84) 20 (40.8) 31 (63.3) 34 (69.4) 17.7 1.9 (1–60) 3.9 0.7 (1–24)
6/9 44.5 3.7 (22–68) 0 – – – –
Table 1 Characteristics of patients with psoriasis and controls.
*Mean SE (range).
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Clinical and Experimental Dermatology (2014) 39, pp461–467
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
Measurement of epidermal growth factor and soluble epidermal growth factor receptor
Fasting venous blood samples were collected into vacutainer tubes and allowed to clot for 30 min, then centrifuged for 15 min at 1000 g. Scales were collected from lesions with the most intensive inflammation and desquamation, and then homogenized in buffer (50 mmol/ L Tris–HCl, 75 nmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl fluoride), and centrifuged at 1000 g. Samples were divided into small tubes and stored at 20 °C until measurement of EGF (serum and scales), protein content (scales) and sEGFR (serum). Samples were assayed in duplicate with quantitative solid-phase sandwich ELISA techniques according to the relevant manufacturer’s instructions. The kit used to measure EGF concentrations in serum and scale samples (Quantikine Human EGF Immunoassay; R&D Systems Inc. Minneapolis, MN, USA) has a sensitivity of 0.7 pg/mL, while that used to measure sEGFR (Quantikine Human Soluble EGFR Immunoassay; R&D Systems Inc.) had a sensitivity of 0.014 ng/mL. Optical density was read at 450 nm, using a microtitre plate photometer, and concentration was determined by interpolation from a curve prepared with standard samples supplied by the manufacturer. Serum concentrations were expressed as pg/mL for EGF
P = 0.401
and ng/mL for sEGFR, and the EGF concentration in scales was calculated as ng/g of protein measured by a bicinchoninic acid (BCA) assay (QuantiProTM BCA Assay; Sigma-Aldrich GmbH, Steinheim, Germany). Statistical methods
Values are expressed as mean SE. Statistical comparison of group means was calculated by two-tailed Student t-test. For correlation analysis, Pearson product moment correlation was used, and linear regression performed. P < 0.05 was considered significant.
Results Mean concentration of EGF was higher in sera of patients with psoriasis (701 72 pg/mL) than in sera of healthy controls (586 63 pg/mL), but this difference was not significant. Treatment with salicyl 5% ointment caused a decrease in serum concentration of EGF to 683 46 pg/mL. Mean serum concentration of sEGFR was less than half that observed in controls (40.8 1.4 vs. 86.4 11.3 ng/mL, respectively) and this difference was significant (P < 0.001 (Fig. 1). After treatment with salicyl 5% ointment, there was a significant
P = 2x10–9
Figure 1 Concentrations of epidermal growth factor (EGF) and soluble EGF receptor (sEGFR) in the sera of patients with psoriasis and
controls, and concentration of EGF in scales collected from psoriatic lesions.
ª 2014 British Association of Dermatologists
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(P = 0.02) increase in EGR-R levels (50.8 3.9 ng/mL) compared with baseline. A significant positive correlation was found between EGF serum levels and individual PASI results (Fig. 2a), while a significant negative correlation was found between sEGFR and individual PASI results (Fig. 2b). By contrast, there was no correlation between disease severity and EGF content in plaque scales. There was also no association between EGF or sEGFR levels and patient age, disease duration, duration of present relapse, or a number of laboratory measures of inflammation activity, such as erythrocyte sedimentation rate (ESR) and platelet count (Table 2). Mean PASI was 17.5 1.1, and varied from 5.0 to 34.2. Of the 51 studied patients, 12 (23.5%) had mild (PASI < 10), 17 (33.3%) moderate (PASI 10–20) and 22 (43.1%) severe (PASI > 20) psoriasis. Despite the lack of significance compared with normal values, serum EGF concentrations in patients with psoriasis increased with disease activity as measured by PASI (Table 3), and reached the highest level of 914 138 pg/mL in patients with severe psoriasis, which was significantly higher than that in patients with mild disease (414 82 pg/mL). Although the differences between particular groups were not significant, mean sEGFR concentrations remained significantly lower than normal values irrespective of disease severity (Table 3), and the significant negative correlation with PASI values was confirmed (Fig. 2b). There was no significant difference in EGF concentrations in plaque scales related to disease activity.
(a) 3000
R = 0.317 P < 0.05
2500
EGF [pg/mL]
2000 1500 1000 500 0 0
10
20
30
40
30
40
PASI (b)
100 90
R = –0.322 P < 0.05
80
EGF-R [ng/mL]
70 60 50 40 30 20 10 0 0
10
20
PASI Figure 2 Correlation between Psoriasis Area and Severity Index
Discussion
(PASI) and (a) epidermal growth factor (EGF) or (b) soluble EGF receptor (EGFR) concentrations.
Mean serum EGF concentration in the study group was higher than in the control group, although the
Correlation (r) Serum Mean SE Age, years Duration of present relapse, months Disease duration, years PASI ESR, mm/h Platelets, 9 103/mL
48.7 3.9 17.7 17.5 24.6 219.8
2.2 0.7 1.9 1.1 2.8 9.7
EGF 0.31* 0.19 0.09 0.32* 0.12 0.22
Scales sEGFR 0.25 0.07 0.17 0.32* 0.16 0.21
Table 2 Age, disease characteristics, PASI and laboratory values, and correlations between these and EGF or sEGFR concentrations.
EGF 0.02 0.049 0.11 < 0.01 0.13 0.22
EGF, epidermal growth factor; ESR, erythrocyte sedimentation rate; PASI, Psoriasis Area and Severity Index; sEGFR, soluble epidermal growth factor receptor. *Significant (P < 0.05).
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
Table 3 Concentrations of EGF and EGFR in serum as well as EGF in plaque scales in patients depending on PASI range.
Patients with PASI of: Measurement
Controls (n = 15)
< 10 (n = 12)
10–20 (n = 17)
> 20 (n = 22)
EGF in serum, pg/mL sEGFR in serum, ng/mL EGF in scales, ng/g
586 63 86.4 11.3 –
414 82 40.5 2.2* 46.1 10.8
629 70 46.5 2.9* 53.6 17.4
914 138† 36.6 1.4* 53.0 17.7
EGF, epidermal growth factor; ESR, erythrocyte sedimentation rate; PASI, Psoriasis Area and Severity Index; sEGFR, soluble epidermal growth factor receptor. Significant at P < 0.05 vs. *control and †PASI < 10.
difference was not significant, which was probably due to an insufficient number of controls. Further analysis revealed an association between serum EGF and psoriasis activity, which was confirmed both by the significant correlation with PASI (r = 0.32) and by the significantly higher serum EGF levels in patients having PASI > 20 compared with those having PASI < 10. To our knowledge, EGF levels in psoriatic scales have not been analysed with respect to psoriasis activity, and our data clearly demonstrate lack of any association. Serum EGF concentrations in our study were higher than those in the study of Anderson et al., who reported a mean of 323 pg/mL.10 This is not unexpected, as the patients in the Andersen et al. study had milder disease (mean PASI 8.5; range 2.0–25.3) than our population (mean PASI 17.5; range 5.0– 34.2). Another study by Ma et al.9 showed even lower EGF serum concentrations (175.4 179.9 pg/mL), but these patients also had mild disease, with mean PASI being 9.7 5.8. In contrast to our study, linear regression analysis showed no correlation between serum EGF level and PASI in these two studies, probably due to the smaller numbers of patients included (14 and 31, respectively, vs. 51 in our study) and the small number of patients with high PASI. Pietrzak et al.8 also found a lower mean EGF serum level (221 170 pg/mL) in 17 patients with chronic exacerbated psoriasis, but unfortunately, they did not provide details on disease severity. Mean serum concentration of sEGFR in patients with psoriasis was only 47% of that in controls, and this difference was significant irrespective of disease activity. In contrast to serum EGF, there was no significant difference between serum EGFR concentrations in patients with mild vs. severe psoriasis, which was probably due to the small number of patients with mild psoriasis. However, there was a trend towards lower sEGFR levels with higher disease activity, which
ª 2014 British Association of Dermatologists
was confirmed by the significant negative correlation between serum sEGFR levels and PASI. As there were no associations between EGF or EGFR and ESR or platelet count, a relationship with inflammatory activity seems to be unlikely. EGFRs are known to be strongly expressed in psoriatic lesions.11,12 However, there are still limited data on the role of EGFR in development of psoriatic lesions, and there are no data on the possible role of sEGFR in management of patients with psoriasis. Binding of EGF to sEGFR in serum could explain our finding of a continual decrease in serum EGFR associated with psoriasis activity, which is responsible for EGF overproduction. Because of the binding of circulating EGF to sEGFR, serum EGF concentration is not able to reach a significantly raised level compared with controls, but does show a significant correlation with psoriasis activity as reflected by PASI. The abnormal histological features of psoriatic tissue can be ameliorated with antibodies to EGFR.14 As shown recently by Hsieh et al.,15 activation of EGFRs in psoriatic lesions can be inhibited by indirubin and thus ameliorate epidermal keratinocyte proliferation. Recently described cases of successful treatment with EGFR inhibition resulting in complete resolution of psoriatic lesions has encouraged research into new EGFR inhibitors designed exclusively for the treatment of psoriasis.18–20
Conclusion In this study, we found that levels of EGF were increased and those of sEGFR decreased in serum of patients with psoriasis compared with controls, and both had a correlation with disease severity. Measurement of both EGF and sEGFR in serum could be useful for assessment of psoriasis severity, which is an important step in the possible application of EGFR inhibitors for the treatment of psoriasis.
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What’s already known about this topic? Serum EGF was previously shown to be raised
in patients with psoriasis, but those studies were not able to demonstrate association related to the disease activity because the patient population was too small and had only mild psoriasis. In active psoriatic lesions, an increase in EGFR content in all layers of epidermis is regularly observed, but there are very limited data on the role of sEGFR in the pathogenesis of the disease. There have been no studies on the possible association between psoriasis severity and EGF content in plaque scales.
What does this study add? Serum concentrations of EGF showed a positive
and EGFR a negative correlation with disease severity, and both were statistically significant. EGF concentrations in plaque scales did not demonstrate statistically significant differences related to the disease activity.
Acknowledgements The study was supported by a grant (no. 11349732L) from the Medical University of Białystok.
References 1 Green L. An overview and update of psoriasis. Nurs Stand 2011; 25: 47–55. 2 Krueger JG, Krane JF, Carter DM, Gottlieb AB. Role of growth factors, cytokines, and their receptors in the pathogenesis of psoriasis. J Invest Dermatol 1990; 94 (Suppl 6): 135–40. 3 Flisiak I, Zaniewski P, Rogalska M et al. Effect of psoriasis activity on VEGF and its soluble receptors concentrations in serum and plaque scales. Cytokine 2010; 52: 225–9. 4 Flisiak I, Zaniewski P, Rogalska-Taranta M, Chodynicka B. Effect of psoriasis therapy on VEGF and its soluble receptors serum concentrations. J Eur Acad Dermatol Venereol 2012; 26: 302–7. 5 Wieduwilt MJ, Moasser MM. The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci 2008; 65: 1566–84.
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6 Mascia F, Cataisson C, Lee TC et al. EGFR regulates the expression of keratinocyte-derived granulocyte/ macrophage colony-stimulating factor in vitro and in vivo. J Invest Dermatol 2010; 130: 682–93. 7 Schneider MR, Werner S, Paus R, Wolf E. Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am J Pathol 2008; 173: 14–24. 8 Pietrzak A, Miturski R, Krasowska D et al. Concentration of an epidermal growth factor in blood serum of males during topical treatment of psoriasis. J Eur Acad Dermatol Venereol 1999; 12: 1–5. 9 Ma LL, Chen PF, Luo F. Impact of serum epidermal growth factor on progressive psoriasis vulgaris and regulation of Chinese herbal medicine for cleaving heat, cooling blood, and detoxicating on it. Zhongguo Zhong Xi Yi Jie He Za Zhi 2008; 28: 733–5. 10 Anderson KS, Petersson S, Wong J et al. Elevation of serum epidermal growth factor and interleukin 1 receptor antagonist in active psoriasis vulgaris. Br J Dermatol 2010; 163: 1085–9. 11 Nanney LB, Stoscheck CM, King LE. Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J Invest Dermatol 1984; 83: 385–93. 12 Green MR, Couchman JR. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition. J Invest Dermatol 1985; 85: 239– 45. 13 Stoll SW, Elder JT. Retinoid regulation of heparinbinding EGF-like growth factor gene expression in human keratinocytes and skin. Exp Dermatol 1998; 7: 391–7. 14 Varani J, Kang S, Stoll S, Elder JT. Human psoriatic skin in organ culture: comparison with normal skin exposed to exogenous growth factors and effects of an antibody to the EGF receptor. Pathobiology 1998; 66: 253–9. 15 Hsieh WL, Lin YK, Tsai CN et al. Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes. J Dermatol Sci 2012; 67: 140–6. 16 Wu NL, Huang DY, Hsieh SL et al. EGFR-driven up-regulationof decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis. Biochim Biophys Acta 2013; 1832: 1538–48. 17 Fredriksson T, Pettersson U. Severe psoriasis – oral therapy with a new retinoid. Dermatologica 1978; 157: 238–44. 18 Wierzbicka E, Tourani JM, Guillet G. Improvement of psoriasis and cutaneous side-effects during tyrosine kinase inhibitor therapy for renal metastatic adenocarcinoma. A role for epidermal growth factor receptor (EGFR) inhibitors in psoriasis? Br J Dermatol 2006; 155: 213–14.
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19 Overbeck TR, Griesinger F. Two cases of psoriasis responding to erlotinib: time to revisiting inhibition of epidermal growth factor receptor in psoriasis therapy? Dermatology 2012; 225: 179–82.
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20 Petch D, Anderson RJ, Cunningham A et al. Design and synthesis of EGFR dimerization inhibitors and evaluation of their potential in the treatment of psoriasis. Bioorg Med Chem 2012; 20: 5901–14.
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Clinical dermatology • Original article
Clinical and Experimental Dermatology
Effect of psoriasis activity on epidermal growth factor (EGF) and the concentration of soluble EGF receptor in serum and plaque scales I. Flisiak,1 M. Szterling-Jaworowska,1 A. Baran1 and M. Rogalska-Taranta2 1
Department of Dermatology and Venereology, and 2Department of Infectious Diseases and Hepatology, Medical University of Białystok, Białystok, Poland
doi:10.1111/ced.12356
Summary
Background. Epidermal growth factor receptors (EGFRs) are overexpressed in psoriatic keratinocytes, and regulate cell growth, proliferation and differentiation through binding to epidermal growth factor (EGF). The role of EGF and EGFRs in the pathogenesis of psoriasis and the contribution of their measurement to psoriasis management are still unknown. Aim. To evaluate serum concentrations of EGF, soluble (s)EGFRs and EGF content in psoriatic scales of patients with severe psoriasis, and to analyse their association with the clinical activity of the disease. Methods. Serum samples and plaque scales were collected from 51 patients with plaque-type psoriasis. Concentrations of EGF and sEGFR in serum and of EGF in scales were measured using enzyme immunoassay. Data were analysed with respect to baseline Psoriasis Area and Severity Index (PASI). Results. Mean serum EGF concentration in patients was higher than in controls (701 72 vs. 586 63 pg/mL), but the difference was not significant. Mean serum concentration of sEGFR was significantly lower than controls (40.8 1.4 vs. 86.4 11.3 ng/mL, P < 0.001). Serum levels of EGF showed a significant positive correlation and EGFR showed a significant negative correlation with PASI (P < 0.05). No correlation was seen between PASI and EGF content in scales or between EGF and sEGFR levels. Serum EGF concentrations reached the highest mean level (914 138 pg/mL) in patients with PASI > 20, and this was significantly higher than the mean of 414 82 pg/mL in the group with PASI < 10. Mean sEGFR serum concentrations remained significantly lower than those of controls, irrespective of disease severity. Conclusions. Compared with controls, patients with psoriasis had increased EGF and decreased sEGFR levels in serum. EGF and sEGFR levels correlated with disease severity.
Introduction Psoriasis is an inflammatory skin disease characterized by keratinocyte hyperproliferation, vascular expansion, Correspondence: Professor Iwona Flisiak, Department of Dermatology and _ Venereology, Medical University of Białystok, ul. Zurawia 14, 15-540, Białystok, Poland E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 14 December 2013
ª 2014 British Association of Dermatologists
fibroblast activation and immune cell infiltration resulting in changes to cytokine production.1 Hyperplasia of keratinocytes can be explained by an imbalance between the growth factors responsible for epidermal proliferation and the activity of their receptors in the cells of affected skin.2 The most important factors involved in this process are excessive proliferation of keratinocytes in the basal layer, disturbances in the cycle of keratinocye maturation and increases in angiogenesis. As we reported previously, some inflammatory cytokines and their soluble receptors
Clinical and Experimental Dermatology (2014) 39, pp461–467
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
[transforming growth factor (TGF)-b1 and vascular endothelial growth factor] are involved in the pathogenesis of psoriasis.3,4 The factor that appears to play a crucial role in the pathogenesis of psoriasis is epidermal growth factor (EGF).5 EGF receptors (EGFRs) are overexpressed in epidermal keratinocytes in psoriatic lesions, and regulate cell growth, proliferation and differentiation through binding to EGF.6,7 Serum EGF was previously reported to be increased in patients with psoriasis, but these studies were not able to demonstrate any association with disease activity because of the small numbers of patients (mainly with mild psoriasis) who were enrolled.8–10 In active psoriatic lesions, an increase in EGFR content in all layers of epidermis is regularly observed.11,12 Binding of the EGFR receptor to monoclonal antibodies or inhibitors can ameliorate proliferation of keratinocytes.13,14 These substances are capable of binding EGF, and thereby preventing proliferative signal transduction, which can be a clinically relevant strategy for the inhibition of keratinocyte hyperplasia.15 By contrast, activation of EGFR by EGF and transforming growth factor (TGF)-a upregulates production of Decoy receptor (Dcr)3, which has a pleiotropic role in many inflammatory and autoimmune disorders and malignant diseases.16 However, there are limited data currently available on the role of EGF and sEGFR in the pathogenesis of psoriasis and the possible application of their measurement in psoriasis management. The aim of this study was to evaluate concentrations of EGF and soluble (s)EGFR in serum as well as EGF content in scales of patients with severe psoriasis. We analyzed their association with clinical activity of the disease, for possible follow-up of the disease.
Methods The study was approved by the Bioethical Committee of the Medical University of Bialystok, and informed consent was obtained from all participants.
Sex, F/M Age, years Family history of psoriasis, n (%) Effect of sun on exacerbation, n (%) Nail involvement, n (%) Disease duration, years* Duration of relapse, months*
Patients
We enrolled 51 patients (15 women, 36 men: mean SE age 48.7 2.2, range 19–84) with exacerbated chronic plaque-type psoriasis. As shown in Table 1, disease duration ranged from 1 to 60 years (mean SD 17.7 1.9 years), and duration of present relapse ranged from 1 week to 24 months (mean 3.9 0.7 months). Patients with a history of any other inflammatory chronic disease including other forms of psoriasis were excluded from the study. All patients were treated initially with a topical application of salicyl 5% ointment for desquamation, and then with 0.3% dithranol ointment. Blood samples were collected before the start of any treatment for psoriasis and then 14 days later. Serum levels of EGF and sEGFR were analysed with respect to the Psoriasis Area and Severity Index (PASI), and to normal serum values determined in 15 healthy controls (9 women, 6 men; mean SD age 44.5 3.7, range 22–78). EGF concentration in scales was also measured; this was expressed per protein content of the scales, and analysed with respect to PASI. For PASIrelated analysis, patients were divided into three groups with PASI ranges of < 10 (mild), 10–20 (moderate) and > 20 (severe). Analysis also included association with the disease duration (from the first relapse) and duration of present relapse (from the most recent appearance of papular lesions) as well as basic laboratory indices of inflammation. Mean age of controls (43.7 2.7 years) did not differ significantly from the patients (Table 1). PASI calculation
PASI was calculated in all patients as described by Fredriksson and Pettersson.17 Each region of the body (the head, the trunk, and the upper and lower limbs) were assessed separately for erythema, infiltration and desquamation, then summed to obtain the final PASI, as described previously.3,4
Patients
Controls
15/36 48.7 2.2 (19–84) 20 (40.8) 31 (63.3) 34 (69.4) 17.7 1.9 (1–60) 3.9 0.7 (1–24)
6/9 44.5 3.7 (22–68) 0 – – – –
Table 1 Characteristics of patients with psoriasis and controls.
*Mean SE (range).
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
Measurement of epidermal growth factor and soluble epidermal growth factor receptor
Fasting venous blood samples were collected into vacutainer tubes and allowed to clot for 30 min, then centrifuged for 15 min at 1000 g. Scales were collected from lesions with the most intensive inflammation and desquamation, and then homogenized in buffer (50 mmol/ L Tris–HCl, 75 nmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl fluoride), and centrifuged at 1000 g. Samples were divided into small tubes and stored at 20 °C until measurement of EGF (serum and scales), protein content (scales) and sEGFR (serum). Samples were assayed in duplicate with quantitative solid-phase sandwich ELISA techniques according to the relevant manufacturer’s instructions. The kit used to measure EGF concentrations in serum and scale samples (Quantikine Human EGF Immunoassay; R&D Systems Inc. Minneapolis, MN, USA) has a sensitivity of 0.7 pg/mL, while that used to measure sEGFR (Quantikine Human Soluble EGFR Immunoassay; R&D Systems Inc.) had a sensitivity of 0.014 ng/mL. Optical density was read at 450 nm, using a microtitre plate photometer, and concentration was determined by interpolation from a curve prepared with standard samples supplied by the manufacturer. Serum concentrations were expressed as pg/mL for EGF
P = 0.401
and ng/mL for sEGFR, and the EGF concentration in scales was calculated as ng/g of protein measured by a bicinchoninic acid (BCA) assay (QuantiProTM BCA Assay; Sigma-Aldrich GmbH, Steinheim, Germany). Statistical methods
Values are expressed as mean SE. Statistical comparison of group means was calculated by two-tailed Student t-test. For correlation analysis, Pearson product moment correlation was used, and linear regression performed. P < 0.05 was considered significant.
Results Mean concentration of EGF was higher in sera of patients with psoriasis (701 72 pg/mL) than in sera of healthy controls (586 63 pg/mL), but this difference was not significant. Treatment with salicyl 5% ointment caused a decrease in serum concentration of EGF to 683 46 pg/mL. Mean serum concentration of sEGFR was less than half that observed in controls (40.8 1.4 vs. 86.4 11.3 ng/mL, respectively) and this difference was significant (P < 0.001 (Fig. 1). After treatment with salicyl 5% ointment, there was a significant
P = 2x10–9
Figure 1 Concentrations of epidermal growth factor (EGF) and soluble EGF receptor (sEGFR) in the sera of patients with psoriasis and
controls, and concentration of EGF in scales collected from psoriatic lesions.
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
(P = 0.02) increase in EGR-R levels (50.8 3.9 ng/mL) compared with baseline. A significant positive correlation was found between EGF serum levels and individual PASI results (Fig. 2a), while a significant negative correlation was found between sEGFR and individual PASI results (Fig. 2b). By contrast, there was no correlation between disease severity and EGF content in plaque scales. There was also no association between EGF or sEGFR levels and patient age, disease duration, duration of present relapse, or a number of laboratory measures of inflammation activity, such as erythrocyte sedimentation rate (ESR) and platelet count (Table 2). Mean PASI was 17.5 1.1, and varied from 5.0 to 34.2. Of the 51 studied patients, 12 (23.5%) had mild (PASI < 10), 17 (33.3%) moderate (PASI 10–20) and 22 (43.1%) severe (PASI > 20) psoriasis. Despite the lack of significance compared with normal values, serum EGF concentrations in patients with psoriasis increased with disease activity as measured by PASI (Table 3), and reached the highest level of 914 138 pg/mL in patients with severe psoriasis, which was significantly higher than that in patients with mild disease (414 82 pg/mL). Although the differences between particular groups were not significant, mean sEGFR concentrations remained significantly lower than normal values irrespective of disease severity (Table 3), and the significant negative correlation with PASI values was confirmed (Fig. 2b). There was no significant difference in EGF concentrations in plaque scales related to disease activity.
(a) 3000
R = 0.317 P < 0.05
2500
EGF [pg/mL]
2000 1500 1000 500 0 0
10
20
30
40
30
40
PASI (b)
100 90
R = –0.322 P < 0.05
80
EGF-R [ng/mL]
70 60 50 40 30 20 10 0 0
10
20
PASI Figure 2 Correlation between Psoriasis Area and Severity Index
Discussion
(PASI) and (a) epidermal growth factor (EGF) or (b) soluble EGF receptor (EGFR) concentrations.
Mean serum EGF concentration in the study group was higher than in the control group, although the
Correlation (r) Serum Mean SE Age, years Duration of present relapse, months Disease duration, years PASI ESR, mm/h Platelets, 9 103/mL
48.7 3.9 17.7 17.5 24.6 219.8
2.2 0.7 1.9 1.1 2.8 9.7
EGF 0.31* 0.19 0.09 0.32* 0.12 0.22
Scales sEGFR 0.25 0.07 0.17 0.32* 0.16 0.21
Table 2 Age, disease characteristics, PASI and laboratory values, and correlations between these and EGF or sEGFR concentrations.
EGF 0.02 0.049 0.11 < 0.01 0.13 0.22
EGF, epidermal growth factor; ESR, erythrocyte sedimentation rate; PASI, Psoriasis Area and Severity Index; sEGFR, soluble epidermal growth factor receptor. *Significant (P < 0.05).
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
Table 3 Concentrations of EGF and EGFR in serum as well as EGF in plaque scales in patients depending on PASI range.
Patients with PASI of: Measurement
Controls (n = 15)
< 10 (n = 12)
10–20 (n = 17)
> 20 (n = 22)
EGF in serum, pg/mL sEGFR in serum, ng/mL EGF in scales, ng/g
586 63 86.4 11.3 –
414 82 40.5 2.2* 46.1 10.8
629 70 46.5 2.9* 53.6 17.4
914 138† 36.6 1.4* 53.0 17.7
EGF, epidermal growth factor; ESR, erythrocyte sedimentation rate; PASI, Psoriasis Area and Severity Index; sEGFR, soluble epidermal growth factor receptor. Significant at P < 0.05 vs. *control and †PASI < 10.
difference was not significant, which was probably due to an insufficient number of controls. Further analysis revealed an association between serum EGF and psoriasis activity, which was confirmed both by the significant correlation with PASI (r = 0.32) and by the significantly higher serum EGF levels in patients having PASI > 20 compared with those having PASI < 10. To our knowledge, EGF levels in psoriatic scales have not been analysed with respect to psoriasis activity, and our data clearly demonstrate lack of any association. Serum EGF concentrations in our study were higher than those in the study of Anderson et al., who reported a mean of 323 pg/mL.10 This is not unexpected, as the patients in the Andersen et al. study had milder disease (mean PASI 8.5; range 2.0–25.3) than our population (mean PASI 17.5; range 5.0– 34.2). Another study by Ma et al.9 showed even lower EGF serum concentrations (175.4 179.9 pg/mL), but these patients also had mild disease, with mean PASI being 9.7 5.8. In contrast to our study, linear regression analysis showed no correlation between serum EGF level and PASI in these two studies, probably due to the smaller numbers of patients included (14 and 31, respectively, vs. 51 in our study) and the small number of patients with high PASI. Pietrzak et al.8 also found a lower mean EGF serum level (221 170 pg/mL) in 17 patients with chronic exacerbated psoriasis, but unfortunately, they did not provide details on disease severity. Mean serum concentration of sEGFR in patients with psoriasis was only 47% of that in controls, and this difference was significant irrespective of disease activity. In contrast to serum EGF, there was no significant difference between serum EGFR concentrations in patients with mild vs. severe psoriasis, which was probably due to the small number of patients with mild psoriasis. However, there was a trend towards lower sEGFR levels with higher disease activity, which
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was confirmed by the significant negative correlation between serum sEGFR levels and PASI. As there were no associations between EGF or EGFR and ESR or platelet count, a relationship with inflammatory activity seems to be unlikely. EGFRs are known to be strongly expressed in psoriatic lesions.11,12 However, there are still limited data on the role of EGFR in development of psoriatic lesions, and there are no data on the possible role of sEGFR in management of patients with psoriasis. Binding of EGF to sEGFR in serum could explain our finding of a continual decrease in serum EGFR associated with psoriasis activity, which is responsible for EGF overproduction. Because of the binding of circulating EGF to sEGFR, serum EGF concentration is not able to reach a significantly raised level compared with controls, but does show a significant correlation with psoriasis activity as reflected by PASI. The abnormal histological features of psoriatic tissue can be ameliorated with antibodies to EGFR.14 As shown recently by Hsieh et al.,15 activation of EGFRs in psoriatic lesions can be inhibited by indirubin and thus ameliorate epidermal keratinocyte proliferation. Recently described cases of successful treatment with EGFR inhibition resulting in complete resolution of psoriatic lesions has encouraged research into new EGFR inhibitors designed exclusively for the treatment of psoriasis.18–20
Conclusion In this study, we found that levels of EGF were increased and those of sEGFR decreased in serum of patients with psoriasis compared with controls, and both had a correlation with disease severity. Measurement of both EGF and sEGFR in serum could be useful for assessment of psoriasis severity, which is an important step in the possible application of EGFR inhibitors for the treatment of psoriasis.
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EGF and its soluble receptor in psoriasis I. Flisiak et al.
What’s already known about this topic? Serum EGF was previously shown to be raised
in patients with psoriasis, but those studies were not able to demonstrate association related to the disease activity because the patient population was too small and had only mild psoriasis. In active psoriatic lesions, an increase in EGFR content in all layers of epidermis is regularly observed, but there are very limited data on the role of sEGFR in the pathogenesis of the disease. There have been no studies on the possible association between psoriasis severity and EGF content in plaque scales.
What does this study add? Serum concentrations of EGF showed a positive
and EGFR a negative correlation with disease severity, and both were statistically significant. EGF concentrations in plaque scales did not demonstrate statistically significant differences related to the disease activity.
Acknowledgements The study was supported by a grant (no. 11349732L) from the Medical University of Białystok.
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