Measurement Serum Levels of IFNγ, IL-17 and IL-4 of Psoriasis patients in Al-Najaf City.

Background: Psoriasis is an inflammatory skin disorder characterized by activation of lymphocytes, systemic and local over expression of pro-inflammatory cytokines. Objectives:- The study was designed to investigate the level of cytokines in patients with psoriasis and study the correlation of the cytokines with disease severity. Patients and Methods: Blood samples were collected from seventy patients with psoriasis for measurement of (IFNγ, IL -17 and IL-4) by ELISA technique. Results: High levels of IFNγ and IL -17 were observed in patients with psoriasis. It was found that the mean levels of IFNγ and IL-17 were significantly correlated to disease severity. Moreover, the mean level of IL-4 in psoriatic patient was found to decrease significantly according to graded severity of the disease. Statistical analysis was done by using SPSS (statistical package for social sciences) in which T-test and ANOVA test were used and chi-square (X 2 ) test for categorical data. Conclusion: The study concluded that the level of IFNγ and IL -17 may play a key role as an indicator in the immunopathogenesis of psoriasis and markers of disease activity. Recommendation:- Using another cytokines and study effect of these cytokines with disease severity.


INTRODUCTION
Psoriasis is characterized by hyper-proliferation and abnormal differentiation of epidermal keratinocytes which results in the characteristic scale or flakes of the lesions 1 .T-lymphocytes, cytokines and chemokines release appear to be the principal driver of lesion development and persistence, although endothelial cells, neutrophils, and natural killer cells may play an adjunctive role along with other cytokines and selectins such as intercellular adhesion molecule (ICAM) 2 .Many studies suggest that cytokines released by activated T-lymphocytes, initiate, and maintain the psoriatic process by stimulating keratinocyte proliferation 3 .

OBJECTIVES:-
The study was designed to investigate the level of cytokines in patients with psoriasis and study the correlation of the cytokines with disease severity.

MATERIALS AND METHODS
Five ml of venous blood were collected from 70 patients with plaque psoriasis.These patients attended Al-Sadder Medical City, outpatient clinic of Dermatology in Al-Najaf city (Iraq) during the period from May 2011 to October 2011.The included patients had not received any treatment, topical or systemic for at least 3 weeks.Patients were diagnosed clinically and the disease severity was evaluated using the Psoriasis Area and Severity Index (PASI).The study also included 20 healthy individuals as a control group.Blood samples allowed for few minutes to form appropriate clot.Serum was separated by centrifugation at 1500 rpm and divided into 3 tubes (200 µl) for each aliquots and stored at freeze (-20°C) to be used for different investigations.Serum levels of IFNγ, IL-17 and IL-4 were measured by enzymelinked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay using Peprotech (USA) kit that contains the key components required for the quantitative measurement of natural and/or recombinant human IFNγ, IL-17, and IL-4 within the range of 47-1500 pg/ml, 16-2000 pg/ml, and 16-2000 pg/ml, respectively.

RESULTS
The patients comprised 36 (51.4%) females and 34 (48.6%)males and the ratio of female to male was 1.1:1.According to PASI score classification the severity of 70 patients with psoriasis was graded into mild, moderate and sever.The results were revealed that 29 (41.4%)patients had mild, 35 (50.0%) patients had moderate and 6 (8.6%) patients had severe grade (Table -1).There was no significant difference in the severity of the disease between males and females (P> 0.05).High levels of IFNγ and Il-17 were observed in patients with psoriasis (358.129± 180.504 pg/ml) and (346.026± 273.469 pg/ml), respectively compared with control group (64.984 ± 21.3571pg/ml) and (22.7±4.966pg/ml).The difference between the mean concentration of IFNγ and IL-17 level of psoriasis patients and control group was significant (p <0.05).In the present work, IL-4 mean level (45.364 ± 42.981 pg/ml) of patients with psoriasis was found to be higher than the mean level of control group (8.063 ± 3.96282 pg/ml) (Figur-1).In addition, serum levels of both IFNγ and IL-17 were found to be significantly correlated with PASI score.Serum level of IFNγ was increased in severe degree of psoriasis and the level found to be (701.1167± 91.7329 pg/ml) as compared with that in moderate (250.3960± 83.92543 pg/ml) and in mild degree (122.8759± 36.44595 pg/ml).The mean levels of IL-17 in psoriatic patients were also found to increase according to severity of disease (Table -2).Furthermore, the mean serum levels of IFNγ and IL-17 in psoriatic patient were found to be correlated to disease severity (r = 0.849, P< 0.001) and (r = 0.433, P < 0.001) respectively (Figur-2) and (Figur-3).According to IL-4 serum levels the study was found that the mean level in severe group of psoriatic patients (3.0167 ± 2.17522 pg/ml) was lower than the mean levels of mild (79.9759 ± 97.82719 pg/ml) and moderate groups (15.3784 ± 23.88903 pg/ml) (Table -2).The mean concentration of IL-4 in psoriatic patient was found to be inversely proportional with the severity of the disease.Patients with severe grade have lower mean concentration level than those with moderate and mild grades.The correlation of these cytokine mean concentration between grads severity of disease was appeared (r = 0.416, P < 0.001) (Figure -4).This table indicate that no significant difference between severity of the disease and the gender.This table indicates that serum level of IFNγ was increased in severe degree of psoriasis as compared with that in moderate and in mild degree also the mean levels of IL-17 in psoriatic patients were also found to increase according to severity of disease

Figure-2: The correlation between mean serum levels of IFNγ and the severity of psoriasis patients
This figure shows the significant difference in increase level of IFNγ in sever stage compare with mild, moderate, and the correlation the level of this cytokine with disease severity.

Figure-3: The correlation between mean serum levels of IL-17 and the severity of psoriasis patients
This figure indicates the significant increase level of IL-17 with disease severity of psoriasis patients.

Figure-4: The correlation between mean serum levels of IL-4 and the severity of psoriasis patients
This figure indicates that level of IL-4 not effect with severity of psoriasis patients.

DISCUSSION
Several researchers reported that psoriasis occurs in any gender or race with equal male to female ratio 7 .The results of this study showed a slightly more female predominance (approaching 1:1) which yet again may reflect a sex-related preference in psoriatic patients.Until recently, T cells were classified as Th1 or Th2 cells by production of defining cytokines, IFNγ and IL-4, respectively.However, a new type of T cell, Th17, has been linked to autoimmune inflammation 8,9 .Stimulated T cells play an important role in the expansion of psoriatic plaques 10 .However, the present study showed that the mean concentration of IFNγ was significantly higher in patients with psoriasis compared with control groups.Similar result was shown by other studies 11,12,13,14 who found that IFNγ and other some cytokines were elevated in Severity of disease Concentration of IL-4 (pg/ml) Severity of disease Concentration of IL-17 (pg/ml) psoriatic patients.It was agreed with the hypothesis that psoriasis considered as a Tcell-mediated inflammatory skin disease.Th1-lymphocytes produce IFNγ and induce cellular reactions, resulting in marked increases of keratinocyte proliferation, abnormal patterns of keratinocyte differentiation, concomitant inflammation, and dermal proliferation of small vessels 3 .
The study reveals that the level of IL-17 was higher in patients with psoriatic than that of control group.This observation supports the hypothesis that the high level of IL-17 may be critical mediators of the persistently altered epidermal growth and differentiation and local inflammation that was characteristic of psoriasis and Th17 cells may be proximal regulators of psoriatic skin inflammation 18.However, many other studies were support this result, they found that the IL-17 level was increased in lesional tissue and serum of psoriatic patients 15,16,17 .The mean level of IL-4 in psoriatic patients was higher than control group in our work.This result was agreed with another study that showed the higher percentage of mean concentration of IL-4 in patients with psoriasis as compared with volunteers 18 .On the other hand, other study reported that serum levels of IL-4 in psoriatic patients were comparable with normal control 17 .The present study revealed that serum levels of the studied cytokines (IFNγ, IL-4 and IL-17) of psoriatic patients were significantly elevated in comparison with control groups.In addition, the mean serum levels of both IFNγ and IL-17 in patients found to be significantly correlated to disease severity index PASI of psoriasis.This result was supported by similar study that found a significant correlation between the level of IFN-γ and psoriatic disease severity 18 .However, the mechanisms by which T cells, keratinocytes, and macrophages achieve and maintain their activated state in psoriatic skin lesions were poorly understood.

Figure- 1 :
Figure-1: Mean distribution of IFNγ, IL-17 and IL-4 levels (pg/ml) among psoriatic patients and healthy control groupThis figure indicates that the levels of cytokines increase significantly in serum of psoriasis patients compare with control group.Groups of study