|Year : 2018 | Volume
| Issue : 2 | Page : 52-58
Evaluation of macrophage migration inhibitory factor in patients with alopecia areata before and after the treatment
Shereen Farouk Gheida1, Ghada Abdel-Moemen Soliman2
1 Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||15-Jan-2018|
|Date of Acceptance||09-Jun-2018|
|Date of Web Publication||17-Aug-2018|
Shereen Farouk Gheida
Assistant Professor of Dermatology & Venereology Department, Tanta University, Tanta
Source of Support: None, Conflict of Interest: None
Background Alopecia areata (AA) is a common disease affecting the anagen phase of the hair follicles. It is considered as an autoimmune disorder. Many cytokines play a role in its pathogenesis; one of these cytokines is macrophage migration inhibitory factor (MIF).
Aim The aim of this study was to evaluate the MIF level to elucidate its role in the pathogenesis of AA.
Patients and methods This study included 50 patients with AA and 30 normal healthy individuals who served as the control group. Serum MIF levels were evaluated by enzyme-linked immunosorbent assay in patients (before and after the treatment) and in the controls. The clinical severity of the disease was done using severity of alopecia tool scoring system and it was correlated with the serum MIF levels.
Results There was a statistically significant increase in the serum MIF level in patients before the treatment, compared to the controls. In addition to this, a significant elevation in its level was found before than found after the treatment. There was a positive significant correlation between the MIF level and both duration of the disease and its clinical severity, whereas there was a negative significant correlation between MIF level and the age of the patients.
Conclusion Macrophage MIF plays an important role in the etiopathogenesis of AA as it is increased in patients when compared to the controls and in patients before than after the treatment. So, it could be considered as a prognostic factor for disease severity and might be a potential new target as a future therapy.
Keywords: alopecia areata, macrophage migration inhibitory factor, pathogenesis
|How to cite this article:|
Gheida SF, Soliman GA. Evaluation of macrophage migration inhibitory factor in patients with alopecia areata before and after the treatment. Egypt J Dermatol Venerol 2018;38:52-8
|How to cite this URL:|
Gheida SF, Soliman GA. Evaluation of macrophage migration inhibitory factor in patients with alopecia areata before and after the treatment. Egypt J Dermatol Venerol [serial online] 2018 [cited 2020 Jun 2];38:52-8. Available from: http://www.ejdv.eg.net/text.asp?2018/38/2/52/235982
| Introduction|| |
Alopecia areata (AA) is a chronic, relapsing immune-mediated inflammatory disorder affecting the hair follicles, resulting in nonscarring hair loss. It represents around 0.7–0.8% of all diseases encountered in dermatology clinics ,. The disease affects males and females equally . AA can be classified into patchy AA (partial loss of scalp hair), alopecia totalis (100% of scalp hair is lost), and alopecia universalis (100% loss of scalp and body hair) . It may be associated with other autoimmune disorders such as vitiligo and thyroid autoimmune diseases (Hashimoto’s thyroiditis). These associations may give strong evidence that there is relation between AA and autoimmunity .
Many cytokines are involved in the pathogenesis of AA. Tumor necrosis factor (TNF) , interleukin (IL), and macrophage migration inhibitory factor (MIF) might be critical inducers of losing hair in this disease . MIF is a pleotropic cytokine that functions to promote inflammation, drives cellular proliferation, inhibits apoptosis, and regulates the migration and activation state of immune cells . MIF was found to have an important role in many autoimmune and inflammatory skin disorders such as psoriasis, vitiligo, and pemphigus .
Therefore, a similar mechanism may be involved in AA. It has been demonstrated that a potential source of serum MIF might be the activated T-cells. When MIF is released, it stimulates the production of other proinflammatory cytokines by macrophages such as IL-1 and TNF-α, and vice versa. It is believed that there is a positive feedback mechanism which might be the cause of interaction between the three cytokines (IL-1, TNF-α, and MIF) in this disease . Therefore, the aim of this study was to evaluate the MIF level to elucidate its role in the pathogenesis of AA.
| Patients and methods|| |
The study was approved by the research ethics committee (approval code 2755/09/14). All participants provided signed informed consent. This was a case-control study of 50 patients having AA (group A) and 30 normal individuals served as the control group (group B). Newly diagnosed cases of AA, old cases who did not take the treatment for at least 6 weeks before the start of the study, and patients who do not have other dermatological or systemic diseases were included in this study. However, patients with any other autoimmune diseases such as thyroid diseases, systemic lupus erythematosus, diabetes mellitus, rheumatoid arthritis, ulcerative colitis, hypertension, ultra violet exposure, malignancy, and dermatological diseases, other than AA, such as vitiligo and psoriasis were excluded from the study.
All patients were subjected to: complete history-taking including age, duration, precipitating factors (e.g. psychic stress), family history of same disease, past history of autoimmune diseases, and previous treatments. The severity degree of AA lesions was assessed according to the severity of alopecia tool (SALT) score.
Severity of alopecia tool score
Scalp was divided into four areas: Vertex: 40% (0.4) of scalp surface area; right profile of the scalp: 18% (0.18) of scalp surface area; left profile of the scalp: 18% (0.18) of scalp surface area; posterior aspect of scalp: 24% (0.24) of the scalp surface area ([Figure 1]). Percentage of hair loss in any of these areas is the percentage hair loss multiplied by the percent surface area of the scalp in that area. SALT score is the sum of percentages of hair loss in all the above mentioned areas. Further subgrouping of percent scalp hair loss into the following (SALT subclasses): S0=no hair loss, S1<25% hair loss (mild), S2=25–49% hair loss (moderate), S3=50–74% hair loss, S4=75–99% hair loss, and S5=100% hair loss (S3–S5 were considered as sever or extensive AA) .
|Figure 1 Visual aid (Olsen/Canfield) for estimating the percentage scalp hair loss.|
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All patients were subjected to treatment with topical and intralesional steroids and 40 patients (80%) were completely cured and participated in the study as group C for evaluation of MIF level after the treatment.
A total of 3 ml of venous blood were collected from each patient and the control in plain tubes. The tubes were placed in a water bath for about 30 min and then centrifuged for 10 min. The sera from the patients (before and after the treatment) and controls were isolated in different tubes and stored at −20°C till the time of analysis of MIF, which was done by enzyme-linked immunosorbent assay kit provided by Chongqing Biopsies Co. Ltd (Shanghai, China).
This kit was based on the standard sandwich enzyme-linked immunosorbent assay technology. The purified anti-MIF antibody was precoated onto 96-well plates. The horseradish-peroxidase (HRP)-conjugated anti-MIF antibody was used as a detection antibody. The standards, test samples, and HRP-conjugated detection antibody were added to the wells subsequently, mixed, and incubated, and then the unbound conjugates were washed away with wash buffer. Tetramethyl benzidine substrates (A and B) were used to visualize HRP enzymatic reaction. Tetramethyl benzidine was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the MIF amount of sample captured in the plate. The optical density (OD) absorbance can be detected at 450 nm in a microplate reader, and then the concentration of MIF can be calculated: the relative OD450=the OD450 of each well−the OD450 of zero well. The standard curve was plotted as the relative OD450 of each standard solution (y) versus the respective concentration of the standard solution (x). Human MIF concentration in the samples was interpolated from the standard curve  ([Figure 2]).
|Figure 2 Standard curve of macrophage migration inhibitory factor level.|
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Data were fed to the computer and analyzed using IBM SPSS software package, version 20.0 (version 16; SPSS Inc., Chicago, Illinois, USA).
| Results|| |
This study included 50 patients, 34 (68%) females and 16 (32%) males, with female : male ratio of 2.1 : 1. Their age ranged from 13 to 45 years, with the mean age of 24.92±8.60 years. In addition, 30 healthy persons who served as the control group, in this, 18 (60%) patients were females and 12 (40%) were males, and their age ranged from 15 to 50 years, with the mean age of 29.07±10.39 years. The comparison between the two study groups according to the demographic data showed no statistically significant difference between the two groups (P>0.05).
The duration of AA lesions ranged from 1 to 6 months, with a mean of 2.80±1.44 months. As for the family history of AA in this study, it was found that 18 (36%) patients had positive family history of AA, whereas 32 (64%) patients had negative family history. As for the past history of similar condition, it was found that 20 (40%) patients had a past history of similar condition.
Clinically, according to the SALT score, 18 patients had mild alopecia (SALT=S1) ([Figure 3]), 20 patients had moderate alopecia (SALT=S2) ([Figure 4]), and 12 patients had severe alopecia (SALT=S3).
|Figure 3 Patient with mild alopecia areata (severity of alopecia SALT 1).|
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|Figure 4 Patient with moderate alopecia areata (severity of alopecia SALT 2).|
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The serum level of MIF in the patients (group A) ranged from 5.90 to 25.20 ng/ml, with a mean of 12.46±3.85 ng/ml. Whereas in the controls (group B), it ranged from 3.20 to 6.70 ng/ml, with a mean of 4.65±1.21 ng/ml. There was a statistically significant increase in the MIF level among patients when compared to the controls (P<0.001).
According to the relations between the serum level of MIF and clinical parameters, it was found that there were no statistically significant relations between serum level of MIF and either sex, family history, or a history of previous episode ([Table 1]).
|Table 1 Relationship between the serum level of migration inhibitory factor and different clinical parameters in the patient group|
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According to the relationship between serum MIF level and severity of AA, the mean MIF level was 8.66±1.45 ng/ml in mild cases (S1), 13.27±0.56 ng/ml in moderate cases (S2), and 16.83±4.10 ng/ml in severe cases (S3). This means that there was a significant relation between the severity of AA and serum level of MIF (P<0.001). MIF level was higher in severe cases than in moderate and mild cases ([Table 1]). A statistically significant negative correlation was found between serum level of MIF and age of the patients, while, there was a statistically positive correlation between serum level of MIF and duration of the disease. In addition, the correlation between serum levels of MIF and severity of AA was statistically significant positive correlation ([Table 2]).
|Table 2 Correlations between the serum level of migration inhibitory factor and clinical parameters in the patient group|
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The MIF level in patients after the treatment (group C) ranged from 5.82 to 9.75 ng/ml, with a mean level of 6.78±1.32 ng/ml. There was a statistically significant decrease in the MIF level in group C, when compared to patients before the treatment (group A) (P≤0.05). The MIF level in group C was higher than group B (controls), but there was no statistically significant difference (P>0.05).
| Discussion|| |
The etiopathogenesis of AA is still unclear and many factors are involved in its pathogenesis. The genetic background of the patients, the atopic condition, and stress, either emotional or environmental, are claimed to be involved in its development .
Macrophage MIF was one of the first cytokines that was identified after being isolated from the supernatants of T-lymphocytes. It was initially described as a soluble factor that inhibits macrophage migration. Later, it has been demonstrated that MIF is produced by a number of other cells such as macrophages, monocytes, granulocytes, epithelial cells, and endothelial cells .
MIF is a pleiotropic upstream proinflammatory integral mediator of the innate and adaptive immune systems. It stimulates the release of multiple cytokines including IL-1 and TNF-α. MIF has been implicated in the pathogenesis of sepsis, cardiovascular diseases, and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus . The aim of this study was to evaluate the role of MIF in the pathogenesis of AA.
In this study, positive family history was found in 36% of patients and 60% of patients were with negative family history, this reflected the genetic background of the pathogenesis of AA. This was in agreement with the study conducted by Blaumeiser et al.  that found that 21.8% of patients had a positive family history of AA. The frequency of AA was greatest in parents (7.4%), then in siblings (5.5%), and lastly in children (2.3%). A positive family history was noted in 34% of patients when information from second-degree relatives was included. In addition, Akhyani et al.  found that 18.8% of AA patients had a positive family history of AA, whereas Tan et al.  found that only 4.6% of AA patients had a positive family history.
In the current study among the 50 patients, only 40% suffered from previous episode of AA and 60% were with no previous episode of AA. Akhyani et al.  found that 40.2% of AA patients experienced only one episode, which was similar to the results of this study, and 10.5% of AA patients had more than four episodes of alopecia. Fakkar et al.  disagreed with the current results as they found that only 20% of AA patients suffered from previous episode of AA.
The results of the present study demonstrated that the mean serum level of MIF was significantly higher in patients (group A) than in the controls (group B), which may suggest a role of MIF in the etiopathogenesis of AA. This finding was in accordance with that of Shimizu et al.  who performed immunohistochemical studies using anti-MIF antibodies, and was positive in perifollicular-infiltrated lymphocytes of telogen hair follicles in patients with extensive AA. It was also in accordance with Shimizu et al.  who studied patients with AA with variable degrees of severity and found that the MIF serum level was significantly higher in patients than in healthy controls. Younan et al.  and Salem et al.  found significantly elevated level of MIF in both serum and skin biopsies of patients with AA. They were in agreement with our results.
As for the relation between the serum level of MIF and sex of the patients, there was no statistically significant relation. This comes in agreement with the last two studies , which showed that MIF level was unaffected by sex in either AA patients or controls. On the contrary, Aloisi et al.  performed their research on the plasma levels of MIF and several hormones (cortisol, estradiol, and testosterone). They evaluated their mutual behavior in the controls and in chronic pain patients. They concluded that MIF levels were significantly higher in males when compared to females.
In the current study, there was a statistically nonsignificant relation between serum level of MIF and positive family history of AA. This had the same results found by Younan et al.  and Salem et al. . In addition to this, there was no significant relation in serum MIF levels between patients with and without a history of previous episode. This was same as the results of the study conducted by Salem et al. .
Regarding the serum MIF level and its comparison with the clinical severity of AA in this study, a significantly higher serum level of MIF was found in severe AA (S3) than in other clinical presentation of this disease (i.e. mild and moderate). This means that the serum MIF level increases with increasing severity of AA, which may suggest that MIF was implicated in the severity of the disease. This was in agreement with results of the other studies ,, which concluded that the mean level of serum MIF was significantly higher in extensive forms of AA than in those with moderate and mild forms of the disease. In addition to this, a statistically significant positive correlation was found between serum MIF level and severity of AA. This is also supported by the results conducted by Shimizu et al. ,.
A statistically significant negative correlation between serum MIF level and age of the patients was demonstrated. This was in accordance with Shimizu et al.  who found that MIF gene promoter polymorphisms contribute to the risk of early-onset forms of AA (<20 years). Also, this was in accordance with Younan et al.  and Salem et al.  who found the higher MIF levels were in the younger patients. This might explain the risk of development of AA in younger age groups than in older patients.
In the present study, there was a positive significant correlation between MIF level and duration of AA. This came in accordance with the previous studies ,,and it supports the potential detrimental influence of MIF in the pathogenesis of the disease, and perhaps contributes to its chronicity.
AA is considered as one of the autoimmune diseases, the results of this study came in agreement with similar studies performed on other autoimmune diseases such as psoriasis , pemphigus vulgaris , vitiligo , systemic lupus erythematosus , and systemic sclerosis . The MIF level was significantly higher in patients when compared to the control in all of the previously mentioned studies.
In the previous mentioned studies done on AA ,,, the MIF level was not evaluated in patients after the treatment. Therefore, we evaluated its level in 40 completely cured patients after the therapy. A statistically significant decrease in MIF level was found in patients after the treatment (group C) when compared to its level before the treatment (group A).These results confirm that MIF level has an important role in the pathogenesis of the disease. When comparing the MIF level in group C and control group B, the MIF level was still higher in group C than in group B, but with no statistical significant difference (it did not return to the normal level). This might give an idea about the tendency of this disease to be recurrent and chronic in patients with AA.
The results presented here demonstrated that the mean levels of MIF in the sera were significantly elevated in patients with extensive AA. AA is considered to be a T-cell-mediated autoimmune disease involving the hair follicle in the anagen phase. It is characterized by infiltration of activated T-cells in the peribulbar area of the hair follicle. Although the function of these T-cells in pathogenesis is still unclear, cytokines released from the T-cells are considered as important mediators which lead to hair loss in AA. These released proinflammatory cytokines lead to a decrease in growth factor secretion by fibroblasts in the dermal papilla and result in premature development of catagen stage. This stage is characterized by an abnormal pattern of apoptosis, degeneration, and death of hair cells .The definite role of MIF in AA remains unclear. It was demonstrated that MIF may be produced by activated T-lymphocytes, macrophages, and monocytes. It is involved in many inflammatory and autoimmune diseases. It was reported that MIF induces growth-inhibiting effect on the fibroblasts. It was speculated that imbalance between proinflammatory cytokines and cytokine antagonists is one of the factors that may predispose patients to the initiation, activation, or perpetuation of autoimmune disorders including AA. It was known that proinflammatory mediators (IL-1 and TNF-α) are potent inhibitors of hair follicle cell proliferation, with a concomitant inhibition of hair growth leading to hair loss. There is a positive feedback mechanism between MIF and TNF-α. MIF is upregulated by TNF-α and, in turn, it stimulates the secretion of TNF-α. Therefore, these inflammatory cytokines may be involved in the induction or continuation of damage to hair follicles. MIF may play an important role in the pathophysiology of inflammatory hair loss as in AA and that the control of MIF production may have important therapeutic implications .
From this study, it could be concluded that elevation of total serum MIF is associated with AA and this may reflect the autoimmunity background in the pathogenesis and severity of the disease. Further studies are recommended especially in severe cases, chronic cases, and cases resistant to the treatment. Larger studies are needed to consider MIF as one of the predictors of AA especially in young age. Anti-MIF therapy should be further studied as one of the new biological treatment for AA.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]