Department of Medical Research, SRM Medical College, Hospital and Research Centre, Kattankulathur, Tamil Nadu, India
Correspondence to: Dr D. Haripriya., PhD, Assistant Professor, Department of Medical Research, SRM Medical College Hospital and Research Centre, Kattankulathur-603203, Tamil Nadu, India. E-mail: firstname.lastname@example.org
Interleukin-1 plays a key role in inflammation and keratinocyte activation. It is an important mediator in the initiation and maintenance of psoriatic plaques and may represent an attractive therapeutic target. The aim of this study is to evaluate the effect of Methotrexate (MTX) on IL-1 α and IL-1 β levels in both plasma and skin biopsy of patients with psoriasis and to investigate their association with clinical disease activity. Forty-five control subjects and 58 patients with psoriasis were recruited for this study. The patients were treated with 7.5 mg of MTX per week for 12 weeks. Folic acid was given at 5 mg once daily except on the day of MTX for 12 weeks. Blood samples and lesional skin biopsy were taken. Disease severity was assessed by Psoriasis Area Severity Index (PASI) score. IL-1 levels in plasma and skin biopsy were analysed using ELISA. PASI score declined significantly (P <0.001) from day 0 to 12 weeks of MTX treatment. IL-1 α level in plasma and skin biopsy was reduced at day 0 sample and elevated significantly (P <0.001) after MTX treatment. IL-1β level in plasma and skin biopsy was higher at day 0 sample and reduced significantly (P <0.001) after MTX treatment. IL-1α levels and PASI score showed inverse correlation score before and after treatment with MTX. Whereas IL-1β levels showed positive correlation before and after treatment with MTX. Decreasing IL-1β levels by MTXs in psoriasis may block the Th17 differentiation. This shows the therapeutic effect of MTX in controlling the immunopathogenesis of psoriasis.
Psoriasis is a chronic immunologically based inflammatory skin disease which affects approximately 2% of the general population [1, 2]. Keratinocytes of the psoriatic skin are prematurely differentiated, as evident in the incomplete cornification of the stratum corneum, characterized by the retention of nuclei (i.e. parakeratosis) and the loss of the granular layer. The stratum corneum of psoriasiform skin is also thickened (i.e. hyperkeratosis). This heavy disruption of epidermal differentiation and skin barrier homeostasis coupled with altered levels of intercellular adhesion molecules result in the widespread scaling of psoriatic lesions .
It was proposed that in normal skin, there could be an interaction between the epidermis and circulating T cells . In psoriasis, the epidermis is primarily affected; the disease has a strong immunopathological basis, with the psoriatic skin being significantly infiltrated with immune cells and produces abnormal cytokines. Lesional skin T cells are hypothesized to contribute proinflammatory/type 1 (T1) cytokines to initiate and/or maintain the cell-mediated keratinocyte hyperplasia in inflammatory lesions. Reverse transcription–polymerase chain reaction cytokine profiles of psoriatic skin reveal the potential production of many proinflammatory cytokines such as interleukin (IL)-1 α, IL-1β, IL-2, IL-6, IL-8, tumour necrosis factor (TNF)-α, TNF-β, transforming growth factor-α and -β, and granulocyte/macrophage colony-stimulating factor [5-9] in psoriatic lesions.
IL-1 was the first cytokine detected in skin  and plays an important role in directing these responses. It is an essential proinflammatory cytokine which possesses multiple properties and affects almost all cell types [11, 12]. It is a mediator of the acute phase of inflammation by induction of local and systemic responses. Amongst others, IL-1 induces the expression of adhesion molecules on endothelial cells, which are required for the infiltration of the stressed tissue by inflammatory and immunocompetent cells . All biological effects of IL-1 are mediated by two IL-1 receptor ligands, IL-1α and IL-1β .
Changes in the levels of IL-1 (IL-1 α, IL-1 β, and its receptor antagonist, IL-1RA) occur upon keratinocyte differentiation in vitro and are associated in vivo with abnormal differentiated and hyperproliferative states of psoriatic keratinocytes . Both pro-IL-1α and pro-IL-1β are present in normal epidermis [15, 16]. Pro-IL-1α is active, and preformed pro-IL-1α is secreted by keratinocytes under conditions of cell stress , whereas pro-IL-1β requires inflammasome-mediated processing for activity . Once secreted, these isoforms have similar functions, acting in an autocrine fashion and also on local fibroblasts, vascular endothelium and lymphocytes.
On a cellular level, IL-1 regulates the expression of numerous genes that are involved in inflammatory and immune responses. For example, IL-1 increases the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, which facilitate leucocyte entry into inflammatory sites. IL-1 also induces expression of enzymes which play a part in the synthesis of proinflammatory mediators, such as nitric oxide, prostaglandins and platelet-activating factor. IL-1 triggers T and B lymphocyte activation, leading to the generation of numerous cytokines, increased antibody production and expansion of specific T cell clones [19, 20]. Surprisingly, the apoptosis, cytoskeleton-associated and DNA repair genes are suppressed by IL-1 .
Anakinra, an interleukin-1 receptor antagonist which blocks IL-1. It has been found to be effective in treating some inflammatory conditions such as rheumatoid arthritis (RA), psoriatic arthritis. In a double-blind, placebo-controlled, multicentre study, patients with RA were treated with anakinra in combination with methotrexate (MTX) over 24 weeks. Moreover, addition of anakinra to existing MTX treatment significantly reduced signs and symptoms of active disease . Anakinra treatment could be useful in psoriatic arthritis, yet has no influence on skin lesions .
Methotrexate has been used in the treatment of psoriasis and considered as the gold standard therapy for moderate to severe psoriasis . In psoriasis, MTX appears to exert its effects by acting as both immunomodulatory agent and antimetabolite [25-29]. Immunomodulatory effect of MTX can be explained by the decreasing T cell-mediated inflammation at multiple steps . MTX inhibits the growth of keratinocytes  and is also capable of downregulating endothelial expression of the cell adhesion molecules ICAM-1 and E-selectin . In vitro study had shown that MTX decreases the markers associated with proliferation in the skin biopsies of psoriatic patients . Only few evidence based-studies have been done by treating psoriatic patients with MTX .
Thus, IL-1 is likely to be an important mediator in the initiation and maintenance of psoriatic plaques and represents an attractive therapeutic target. Therefore, the study to assess the effects of IL-1 inhibition on the clinical features of psoriasis disease is very imperative. So, the aim of this study is to assess the therapeutic effect of MTX on IL-1 α and IL-1 β levels in the plasma as well as in skin biopsy of patients with psoriasis and also to investigate their association with clinical disease activity.
Materials and methods
Patients with psoriasis vulgaris (58 in number) who visited Dermatology Department, SRM Hospital, Kattankulathur, were recruited to this study. The age of all patients ranged from 18 to 70 years (mean ± SD, 46.4 ± 14.1 years), and there were 27 men and 31 women. Patients with 18 years of age or older who had more than 20% body surface area involvement and had not received any topical or systemic therapy for at least a month were included in this study.
Exclusion criteria included children (<18 years old), pregnancy and lactating women, patients with unstable psoriasis, liver and renal impairment, infertility, anaemia, excessive alcohol intake or any other systemic diseases like diabetes, hypertension, rheumatoid arthritis, cardiovascular diseases and respiratory syndrome were excluded.
Detailed history regarding the age of disease onset, family history was recorded. Physical examination and the presence of any other dermatological condition were noted. The study protocol was approved by the institutional ethical committee. Informed consent documents were signed by all the patients.
A) Full history including:
Name, age, sex, occupation,residence and contact number
History of the disease:
Age of onset, course and duration of the disease
History of previous treatment
Family history of similar condition
History of other systemic infection
B) Clinical examination:
Site and severity of skin involvement
Distribution of the lesion, generalized or localized
Nail, scalp and joint involvement
Extent of skin involvement
Patients with psoriasis were treated with 7.5 mg of MTX per week for 12 weeks. Folic acid was given at 5 mg once daily except on the day of MTX for 12 weeks. During systemic treatment, no concomitant antipsoriatic therapy was permitted, with the exception of emollients.
Clinical evaluations were performed by the same dermatologist at four intervals (i.e. day 0, 2, 6 and 12 weeks) until completion of the study. Scoring was based on the Psoriasis Area Severity Index (PASI) scoring system .
Healthy volunteers (45 in number) consisting of 21 men and 24 women, aged from 21 to 70 years (mean ± SD, 44.6 ± 15.5 years) served as control. Blood samples of psoriatic patients were collected and analysed before (day 0) and after (6 & 12 weeks) treatment with MTX. In control subjects, blood samples were collected only once. Blood samples were collected in heparinized tube.
In each patient, 10 mm of lesional and non-lesional skin biopsies were taken after local anaesthesia, lidocaine hydrochloride and adrenaline bitartrate IP were given intradermally. Lesional biopsies were taken before (day 0) and after (6 & 12 weeks) treatment with MTX. Non-lesional skin biopsy served as a control, which was collected only once. Biopsies of psoriatic lesional skin were taken within a lesion, 1 cm from the edge of the plaque border. Biopsies of non-lesional skin were taken 2 cm beyond the plaque border. All tissue specimens were immediately immersed in protease inhibitor cocktail and stored at −20°C until further use.
Preparation of tissue for ELISA
The skin biopsy was weighed (50–100 mg) and was homogenized using a glass homogenizer with 1.5 ml extraction buffer (10 mm Tris, 150 mm Nacl, 1% Triton X-100, pH 7.4)/g of tissue. Then, the homogenate was centrifuged at 13,000 g for 10 min at 4°C and the supernatant was used ELISA .
Measurement of IL-1 beta in plasma & skin biopsy by ELISA
Freshly isolated heparinized peripheral blood samples were immediately centrifuged for 10 min at 500 g, and the plasma was separated. The concentration of IL-1β in plasma and supernatant of skin biopsy was measured using Bender MedSystem kits from Austria, Europe (catalogue no: BMS224/2 and BMS224/2TEN) according to manufacturer's instructions.
Measurement of IL-1 alpha in plasma & skin biopsy by ELISA
Freshly isolated heparinized peripheral blood samples were immediately centrifuged for 10 min at 500 g, and the plasma was separated. The concentration of IL-1α in plasma and supernatant of skin biopsy was measured using Bender MedSystem kits from Europe (catalogue no: BMS243/2 and BMS243/2TEN) according to manufacturer's instructions.
The results were expressed as mean ± SD. Control and psoriatic patients (day 0) were compared using independent sample t-test. Before (day 0) and after (6 and 12 weeks) MTX treatment, psoriatic samples were compared by repeated measures ANOVA. The Spearman's coefficient was used to identify correlations. P <0.05 was considered to be significant.
Six patients were dropped out of this study: two patients due to transient increase in alanine aminotransferase (ALT) activity and four patients were dropped out due to difficulty in follow-up.
Fig. 1(A) shows PASI scores before and after MTX treatment. MTX treatment to psoriatic patients elicited a reduction in PASI score from day 0 to 2-week treatment (26.80 ± 9.76 versus 21.19 ± 8.35, respectively); day 0 to 6-week treatment (26.80 ± 9.76 versus 13.46 ± 6.97, respectively); day 0 to 12-week treatment (26.08 ± 9.76 versus 5.77 ± 3.54, respectively) which was statistically significant (P <0.001) and showed F (1.453, 74.083) = 311.769, P <0.001) as measured by repeated measures ANOVA. The median values for PASI score at day 0, 2, 6 and 12 weeks of MTX treatment in patients with psoriasis were found to be 28.15, 20.5, 10.9 and 5.4, respectively.
Fig. 1(B) shows the number of patients reaching PASI 75. Clinical response of patients with psoriasis was based on PASI response. In our study, 75% of patients showed PASI>75, and 25% of patients showed PASI<75.
Plasma IL-1 α and IL-1 β concentration
Table 1 shows the levels of plasma IL-1 α and IL-1 β in psoriatic patient before and after MTX treatment. Statistically significant (P <0.001) decrement of plasma IL-1 α and increment of plasma IL-1 β concentration were found at day 0 compared with control subjects. MTX treatment elicited an elevation in IL-1α concentration and reduction in IL-1 β concentration from day 0 to 6- and 12-week treatment, respectively (P <0.001), in psoriatic patients.
Table 1. Comparison of IL-1α and IL-1β levels in plasma of psoriasis vulgaris patients before treatment and after (6, 12 weeks) treatment of MTX thereafter compared with healthy controls analysed by Repeated measures ANOVA
Before treatment Day 0 (b)
After Methotrexate treatment
Greenhouse-Geisser correction F Value
6 weeks (c)
12 weeks (d)
All values were expressed as Mean (SD).
Interleukin-1 alpha (pg/ml)
F (1.867,95.201) = 737.537
Interleukin-1 Beta (pg/ml)
F (1.517,77.375) = 1159
IL-1 α concentration in skin biopsy
In lesional psoriatic skin biopsy, IL-1 α concentration was found to be lower at day 0 (2.975 ± 0.9 pg/mg) compared with non-lesional skin biopsy (7.9 ± 0.13 pg/mg; P <0.001) as measured by independent sample t-test.
Post hoc tests using the Bonferroni correction by repeated measures ANOVA revealed that MTX treatment to psoriatic patients elicited an elevation in IL-1α concentration from day 0 to 6-week treatment (2.975 ± 0.9 versus 4.39 ± 0.7 pg/mg, respectively); day 0 to 12-week treatment (2.975 ± 0.9 versus 5.83 ± 0.6 pg/mg, respectively), which was statistically significant (P <0.001). A Greenhouse-Geisser correction determined that mean IL-1α concentration differed statistically significant between time points (F (1.684, 126.280) = 697.978, P <0.001; Fig. 2).
The ratio of IL-1 α lesional/IL-1 α non-lesional in each patient was found to be 0.36 ± 0.15. After MTX treatment for 6 and 12 weeks, the ratio for all patients was found to be increased significantly (P <0.001; Table 2).
Table 2. Ratio of lesional/Nonlesional of IL-1α and IL-1β before treatment and after (6, 12 weeks) treatment of MTX analyzed by Repeated measures ANOVA
After MTX treatment
6 weeks/NL (b)
12 weeks/NL (c)
L, Lesional; NL, Nonlesional. All values were expressed as mean (SD).
Interleukin -1 alpha (pg/mg)
Interleukin -1 beta (pg/mg)
IL-1 β concentration in skin biopsy
In lesional psoriatic skin biopsy, IL-1 β concentration was found to be higher at day 0 (2.015 ± 0.29 pg/mg) compared with non-lesional skin biopsy (0.387 ± 0.4 pg/mg; P <0.001) as measured by independent sample t-test.
A repeated measures ANOVA with a Greenhouse-Geisser correction determined that mean IL-1 β concentration differed statistically significant between time points (F (1.996, 149.672) = 1263, P <0.001). Post hoc tests using the Bonferroni correction revealed that MTX treatment to psoriatic patients elicited a reduction in IL-1β concentration from day 0 to 6-week treatment (2.015 ± 0.29 versus 1.27 ± 0.3 pg/mg, respectively); day 0 to 12-week treatment (2.015 ± 0.29 versus 0.73 ± 0.04 pg/mg, respectively), which was statistically significant (P <0.001; Fig. 3).
The ratio of IL-1 β lesional/IL-1 β non-lesional in each patient was found to be 8.07 ± 0.85. After MTX treatment for 6 and 12 weeks, the ratio for all patients was found to be decreased significantly (P <0.001; (Table 2).
Correlation analysis between IL-1 and PASI
Correlation analysis of IL-1 α level in lesional skin biopsy is shown in Fig. 4. There were a significant inverse correlation between IL-1 α and PASI score before (r =−0.995; P <0.001) and after (r =−0.997; P <0.001) treatment with MTX for 12 weeks (Fig. 4A, B).
Correlation between IL-1 β level in lesional skin biopsy is shown in Fig. 5. There were a significant positive correlation between IL-1 β and PASI score before (r =0.986; P <0.001) and after (r =0.991; P <0.001) treatment with MTX for 12 weeks (Fig. 5A, B).
Correlation analysis between IL-1 levels in skin biopsy and plasma
Correlation between ratio of IL-1 α lesional/non-lesional and IL-1 α plasma was found to be positive before and after treatment with MTX for 12 weeks. Correlation between ratio of IL-1 β lesional/non-lesional and IL-1 β plasma was also found to be positive before and after treatment with MTX for 12 weeks (Table 3).
Table 3. Correlation between ratio of lesional/Nonlesional of IL-1α and IL-1β with Plasma levels of IL-1α and IL-1β before treatment and after (6, 12 weeks) treatment of MTX in psoriasis patients
Correlation of IL-1α and IL-1 β levels in lesional skin biopsy is shown in Fig. 6. There was an inverse correlation between IL-1 α and IL-1 β levels in skin biopsy before (r =−0.982; P <0.001) and after (r =−0.990; P <0.001) treatment with MTX for 12 weeks (Fig 6A, B).
Interleukins play a major role in the pathological pathway of keratinocyte proliferation  and has pleiotropic effects on epithelial cells in vitro  and in vivo . In addition, precursor IL-l α appears to be at least 10-fold more phosphorylated than the IL-I β precursor and could be phosphorylated in vitro by a cyclic CAMP-dependent protein kinase. Phosphorylation promoted the conversion of pre-IL-l α to the more mature extracellular form of IL-l α and enhanced its susceptibility to tryptic digestion, which may allow its release into the extracellular milieu .
In our study, PASI 75 was achieved in 75% of patients with psoriasis after MTX treatment. PASI 75 is defined as a reduction from baseline PASI score of >75%. PASI 75 is used as the benchmark of primary end points in assessing therapies for psoriasis. Patients reaching PASI 75 represents very meaningful changes in psoriasis severity.
In our study, we found that IL-l α levels in plasma as well as in skin biopsy of psoriatic patients were low compared with healthy subjects and non-lesional skin biopsy, respectively. Studies by Takematsu et al. [41, 42] showed that in scale extracts and suction blisters fluids of psoriatic patients IL-l α levels were decreased. Organ cultures from psoriatic patients also showed decreased IL-1α levels . Debets et al.  reported that immunostaining of skin sections with IL-1 α, located in the basal keratinocyte of normal control and non-lesional psoriatic epidermis, was decreased significantly to negligible levels in psoriatic epidermis. Contradictory to our results, study by Cohen et al.  reported that the IL-1α levels in lesional skin biopsy were high compared with non-lesional skin biopsy The major reason for this decrease could be high nerve growth factor, an IL-1 α downregulator [46, 47] and decreased cAMP levels  in psoriasis.
After MTX treatment, IL-l α levels was found to be increased in plasma and also in skin biopsy compared with healthy subjects and non-lesional skin biopsy, respectively. MTX increases the levels of cAMP through adenosine release , and this might have led to an increase in IL-1 α level as observed in the present study.
We observed that IL-1 β concentration in plasma and skin biopsies of psoriatic patients at day 0 was elevated compared with healthy subjects and non-lesional skin biopsy, respectively. Studies have showed that immunoreactive IL-1 β was found only in lesional skin and not in non-lesional skin from psoriatic patients [43, 50]. Debets et al.  clearly showed that IL-1 β RNA expression was increased in psoriatic epidermal cells, which may be related to the inflammatory response in psoriasis. Normal keratinocytes do not contain a biologically active form of IL-I β-converting enzyme (ICE) (caspases-1), which convert IL-I β precursor to active form , thereby the level of Il-1 β was low in respective controls. Johansen et al.  observed an increase in active caspases-1 in psoriatic epidermis, which leads to increase in IL-1 β level in psoriasis.
After MTX treatment, IL-I β levels were found to be reduced in plasma and also in skin biopsy compared with their respective controls. Mizutani et al.  found that IL-1 β levels in PBMC of psoriatic patients were decreased after 2 weeks of MTX treatment, due to the suppression of keratinocyte paracrine system by MTX. The decrease in IL-1 β levels by MTX can also be attributed to the inhibitory effect of the drug on 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase (ATIC), which results in accumulation of AICAR and increased adenosine release into the circulation. Extracellular adenosine increases cAMP that inhibits the production of proinflammatory cytokine IL-1 β . MTX decreases IL-1 β levels by decreasing the infiltration of lymphocytes and monocytes [54, 55]. Apart from decreasing the circulating and systemic levels of IL-1 β, MTX was also shown to block the binding of interleukin 1 beta to the interleukin 1 receptor on monocytes, lymphocytes and granulocytes using a recombinant human cytokine probe . Studies have shown that IL-l α downregulates IL-1 β via prostaglandin E2 synthesis . Therefore, increase in IL-1 α levels by MTX definitely downregulates interleukin-1 β by above said mechanisms.
Hu et al.  showed that blocking of IL-17 pathway, an important cytokine in pathogenesis of psoriasis, can be achieved by decreasing or inhibiting upstream targets such as IL-6, IL-23, IL-1β and TGF-β that control Th17 differentiation.
Our results showed that the ratio of IL-1 α lesional/non-lesional was increased compared with after-treatment ratio levels, and ratio of IL-1 β lesional/non-lesional was decreased compared with after-treatment ratio levels. This indicates the effective use of the non-lesional control and also indicates the therapeutic action of MTX on IL-1 levels.
We observed that there is an inverse correlation between IL-1 α levels in lesional skin biopsy and PASI score before and after MTX treatment. One of the study shows that the serum levels of IL-1 α correlated negatively with clinical disease severity expressed as PASI score and with duration of psoriasis .
Our results showed a positive correlation between IL-1 β levels in lesional skin biopsy and PASI score before treatment, suggesting that IL-1 β could be regarded as an indicator of psoriasis activity. We also observed a positive correlation between IL-1 β levels in lesional skin biopsy and PASI score after treatment. These results seem to point out IL-1 β is an indicator of treatment response.
Additionally, we correlate the IL-1 α and IL-1 β levels in the skin biopsy of before and after MTX treatment This negative correlation further added that IL-1 β act as a activation signal for keratinocyte in psoriatic plaques.
In conclusion, the decrease in IL-1α and increase in IL-1β in psoriatic patients is reversed by MTX by more than one mechanism. IL-1 acts as an upstream molecule for Th17 differentiation and is considered as a main pathway for pathogenesis of psoriasis. Decreasing or inhibiting IL-1 β levels in psoriasis may lead to block the Th17 differentiation, thereby reducing psoriatic plaques. This shows the therapeutic effect of MTX in controlling the immunopathogenesis in patients with psoriasis.
Conflicts of interest
All authors have declared: no support from any organization for the submitted; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.