Abstract
Objectives:
First, this research was instituted to identify common allergens, and second, to test the association between IL16 gene promoter polymorphism rs4778889 T/C and allergic contact dermatitis (ACD).
Methods:
A case control study was conducted in dermatology outpatients’ clinic. Study subjects received interview-based semi-structured questionnaire, complete skin examination, IL16 gene promoter was investigated by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis, and IQ Ultra™ patch test units (Chemotechnique Diagnostics AB, Sweden) with 10 substances were used.
Results:
Most of the prevalent cases had positive patch test (93.3%). The most common clinical presentation of ACD in our patients was itching (96.7%), followed by dryness (86.75%), erythema (76.7%), and fissuring (76.7%). There was higher CC gene distribution among cases, but there was no statistically significant difference. IL16 gene distribution was nearly similar among different clinical presentations. Formaldehyde showed statistically significant higher frequency for CC. The most common allergen found was mercury chloride (76.6%), followed by potassium dichromate (26.6%) and cobalt chloride (20%).
Conclusions:
The current study found prominent metal sensitization (mercury chloride) over the previously known potassium dichromate. There was no statistically significant IL16 gene distribution among cases compared with control. However, C allele was more frequently encountered in cases. Further studies are required to test the association with IL16 genotype and ACD and highlight the new trends in metal sensitization among cement-exposed workers.
Introduction
The construction sector in Egypt is a major contributor to the country’s economy and one of its fastest growing sectors. Construction workers represent around 7% of working population in Egypt. 1
Once cement workers’ hands suffer from hand contact dermatitis, they are susceptible to recurrence, and complete recovery is very difficult which may negatively affect work efficiency 2 and allergic contact dermatitis (ACD) often carries a worse prognosis than the irritant form. 3 The common allergens affecting cement workers are as follows: epoxy resin, colophony, formaldehyde, nickel, cobalt, hexavalent chromium (Cr), and rubber gloves. 4 Reducing Cr content of cement is useful in preventing ACD, as was found in Scandinavia. 5
Allergic cement contact dermatitis (ACCD) involves the delayed-type hypersensitivity (DTH) as the result of a T-cell-mediated immune response to cement components. 3 Interleukin 16 (IL-16) is a cytokine that is strongly upregulated during DTH. Interleukin 16 is chemoattractant for T cells and other CD4-expressing cells, including monocytes, dendritic cells, and eosinophils. 6
The gene encoding IL-16 (IL16) is located on chromosome 15q26.3. One polymorphism in the promoter region of the IL16 gene (T/C single-nucleotide polymorphism [SNP] at position -295) may be associated with altered levels of gene expression and may partially account for the increased levels of IL-16 seen in inflammatory diseases including inflammatory bowel disease, bronchial asthma, atopic dermatitis, and ACD.7,8 Yet, studies in this area gave conflicting results and these studies need to be replicated.9,10 In general, the genetics of contact allergy are still only partly understood, despite decades of research. 11
Building on these conflicting results, this research was instituted first to identify common allergens among cement-exposed workers, and second, to test the association between IL16 gene promoter polymorphism rs4778889 T/C and ACCD as a form of ACD. To the best of authors’ knowledge, there is little data in literature regarding IL-16 polymorphism in ACCD in Egyptian patients.
Subject and Methods
Study design and setting
A case control study was carried out during the period of May 2016 to December 2017 in dermatology outpatient clinic, Department of Dermatology, Andrology and STDs, Mansoura University Hospital.
Study subjects
This study included 2 groups:
Cases: A convenience sample of 30 construction workers suffering from ACD mainly attributed to cement exposure;
Control: A sample of 90 age- and sex-matched healthy subjects (from service workers and laboratory workers in Mansoura Faculty of Medicine who have never been exposed to cement and did not have history of any type of allergy).
Study methods
The study methods are as follows:
1. An interview based on semi-structured questionnaire including sociodemographic data and full occupational history (job description, duration of exposure, use of personal protective equipment, short medical history, and family history of allergy).
2. Complete skin, nail, hair, and mucous membrane examination. Skin manifestations suggestive of dermatitis were assessed (eg skin lichenification, hyperkeratosis, fissuring, scaling, dryness, erythema, oozing vesiculation, and scratch marks). Sites of the lesions were determined. Photographs of the skin lesions were taken during the examination for further review.
The examination was combined with symptom questionnaire “Nordic Occupational Skin Questionnaire” (NOSQ-2002/SHORT translation master). The NOSQ was developed by a group of Nordic occupational dermatology researchers to survey work-related skin dermatoses and exposures to environmental factors. The questionnaire was created to provide a standardized method by which results across countries can be compared for epidemiological studies. The 14-question shorter form (NOSQ-2002/SHORT) is a 4-page questionnaire. The questionnaire covers demographics, occupational history, atopic symptoms, self-reported hand or forearm eczema, exacerbating factors, consequences and life impact of dermatoses, self-reported contact urticaria on hands or forearms, skin symptoms, skin tests, and exposures. 12
3. Skin patch test: The IQ Ultra™ patch test units (Chemotechnique Diagnostics AB, Sweden) with 10 substances were used. The tested allergens were potassium dichromate, epoxy resin, mercapto mix, mercury chloride, paraben, cobalt chloride, formaldehyde, nickel sulfate, neomycin, and normal saline (0.9%). Most of the tested allergens were reported as components in the cement. The tested allergens were prepared in Ain Shams University Hospital, Faculty of Medicine, Ain Shams University, Immunology and Allergy Lab according to standard series of patch test allergens (European standard series). The test was done for cases only.
At the time of testing, the dermatitis was in a quiescent phase and patients were off systemic corticosteroids. The procedure required 3 visits at intervals of 48 hours and is therefore most commonly performed on Saturday (Day 0), Monday (Day 2), and Wednesday (Day 4). On Day 0, the allergens were applied to rows of plastic chambers mounted on hypoallergenic porous tape and then fixed to the patients’ upper back and secured by 3M tape. Patients were instructed to keep their back dry for the whole week and to avoid exercise and sweating. At Day 2, the location of the panels was marked on the patients’ back and the strips were removed. Patch test results were evaluated 30 minutes after removal of test material to allow for irrelative effects from adhesive material. The reactions were noted at this time and again when the patients return at Day 4 by the same dermatologist. Later readings may be necessary. The reading at Day 4 was considered as potassium dichromate; nickel and cobalt may also present at this time.4-13 Patients showing doubtful reactions were excluded. Patch test reactions were graded according to the guide of the manufacturer as follows:
4. IL16 promoter polymorphism rs4778889 T/C: Genomic DNA was extracted from 3 cm blood on EDTA, and the T-to-C transition at position -295 of the IL16 gene promoter was investigated using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis with AhdI as a restriction enzyme. Primer sequence was as follows:
Forward: 5′-CTCCACACTCAAAGCCTTTTGTTCCTATGA-3′; Reverse: 5′-CCATGTCAAAAC GGTAGCCTCAAGC-3′.
Polymerase chain reaction steps were as follows: denaturation at 95°C for 3 minutes, followed by 30 cycles at 94°C for 1 minute, at 59°C for 1 minute, at 72°C for 1 minute, and then a final extension for 5 minutes at 72°C. DNA products were visualized on 2% agarose gels. 14
The amplified 280-bp PCR fragment was then digested by addition of 1 unit of AhdI (Fermentas, Germany) and incubated for 3 hours at 37°C. AhdI recognizes the restriction GACNNN ↓ NNGTC, present T: 280 & C: 246+34 (Figure 1).
Agarose gel with the PCR product and different genotypes of IL16 SNP rs 4778889 in studied groups, from left to right: Lanes 1&2 PCR product; Lane 3 ladder 50 bp; lanes 7&9&12&13&16&18 CT genotype; lanes 6 CC genotype; lanes 4&5&8&10&14&15&17 for TT genotype.
Ethical consideration
All study subjects gave oral consent to participate and accepted to attend in required follow-up visits. Also, they received follow-up card after diagnosis of their condition and received necessary treatment. Approval was obtained from the Institutional Review Board (IRB) of Mansoura Faculty of Medicine (No. R/16.04.46)
Statistical analysis
Data entry and analysis were done by SPSS (Version 16) and Epi Info statistical program. Quantitative data were summarized using mean and standard deviation, and qualitative data were described by frequencies and percentages. Chi-square test—“linear-by-linear association”—was used to compare qualitative data. Fisher’s exact test was used when 50% of cell values were less than 5. Odds ratios (95% CI) were calculated at confidence interval 95%. A P-value less than .05 was considered statistically significant.
Results
The mean age of the studied cases was 35.3 (11.13) years compared with 33.05 (9.14) years in controls. All of them were males. No statistically significant differences were found between cases and controls regarding age, residence, marital status, education, and duration of employment. Smoking and work hours were significantly higher among cases than controls (P < .05) (Table 1).
Sociodemographic characteristics of studied groups.
P < .05 is statistically significant.
The genotypes for IL16 were 22 TT (73.3%), 7 CT (23.3%), and 1 CC (3.4%). A higher proportion was found for the CC/CT IL16 gene polymorphism in cases compared with controls (26.7 vs 12.2%) and also with C allele (15% vs 6.7%), but the difference was not statistically significant (P > .05) (Table 2).
Genotype and alleles distribution in studied groups.
Abbreviations: ACD, allergic contact dermatitis; CI, confidence interval; OR, odds ratio; r, reference groups.
The most common clinical presentation of ACD in our patients was itching (96.7%), followed by dryness (86.75%), erythema (76.7%), and fissuring (76.7%), and there was nearly similar IL16 genotype distribution among different clinical presentations. The differences were statistically non-significant (P > .05) (Table 3).
Clinical presentation of studied cement dermatitis cases.
Abbreviation: ACCD, allergic cement contact dermatitis.
Fisher’s exact test.
Regarding the results of patch test in our patients, the most common allergen found was mercury chloride (76.6%), followed by potassium dichromate (26.6%); most of the patients were sensitized to 1 or 2 substances (89.3%) and 10.7% were sensitized to 3 or more substances (Table 4).
Patch test results among allergic cement contact dermatitis cases.
n = 28 (2 cases with doubtful reaction).
Patch test for 20 control subjects revealed that 12 subjects (60%) were negative by patch test and 6 (30%) were positive (2+mercury cl, 2+K dichromate, 1+formaldehyde, and 1+nickel sulfate). Two cases had doubtful reaction (results are not tabulated).
Regarding IL16 gene distribution, there was no statistically significant difference according to the results of patch test (P > .05). Also, there was no statistically significant difference between gene distribution and type of tested allergens except for formaldehyde which showed statistically significant higher frequency for CC/CT (P < .05) (Table 5).
Association between genotype frequency and type of allergens.
Abbreviations: CI, confidence interval; OR, odds ratio.
Chi-square test: linear-by-linear association.
P value less than 0.05 is considered statistically significant.
All cases had hand lesions either alone or combined with other sites. The most common sites were hands and foot (31.0%), followed by hands alone (27.6%), and then hands with forearm (23.3%). Hands with elbow and/or abdomen were least common (10%; results are not tabulated).
Discussion
Immune/inflammatory responses are under control of cytokines such as TNF-α, IL-1β, IL-6, IL-10, and IL-16. Several studies explored IL-16 and its important role in DTH. 10 Regarding the role of cytokine gene polymorphisms in the development of ACD, it was noted that TNF-α and IL-16 likely represent markers for increased skin immune activity in general. 15
Exposure in the workplace is responsible for a wide range of cutaneous problems. Contact dermatitis accounts for 90% of all cases of occupational dermatoses, and ACD is responsible for 20% of cases of occupational contact dermatitis. 13 A study found that about 65.7% of 350 workers in the cement sector in Egypt, selected randomly, showed skin disorders. 16
The current study showed that, cigarette smoking and working hours in ACCD patients were significantly higher than controls. It was noticed that there was a direct relationship between occurrence of ACCD and severity of involvement, and duration of cement contact. In addition, 100% of cement workers had contact dermatitis after 10 or less years of work. 17
Moreover, a review article in 2015 reported that statistically significant positive association between tobacco smoking and hand eczema prevalence rate was found in 20 articles. The association was stronger for studies in occupational settings than for population-based studies. Out of 5 studies, 2 found a positive association between smoking and hand eczema severity. 18
In the current work, the most common clinical presentation of ACCD was itching, followed by dryness, erythema, and fissuring. In agreement with a study, occupational contact dermatitis presents as eczema in 90% of cases. Acute lesions begin as pruritic erythematous and edematous urticarial-looking plaques studded with vesicles and sometimes tense bullae. Erythema and edema are still present in the subacute stages, but vesiculation becomes less visible, replaced by erosions, oozing, crusting, and desquamation. In chronic cases, the skin appears dry and rough, fissured, grayish, and thickened with increased skin lines, a process called lichenification. 13
In addition, a study in Taiwan reported nearly similar finding in cement workers but with different percentages. Thickened skin/lichenification was the commonest (64.9%), followed by hyperkeratosis (59.8%), scaling (35.1%), dryness (33.0%), erythema (25.8%), fissure (20.6%), pigmentation (14.4%), vesicles/papules (13.4%), itching (5.2%), scratch (3.1%), edema (2.1%), erosion (2.1%), and finally ulceration (1%). There was thickening of the skin over the dorsal surface of the hand, especially near the metacarpophalangeal joint, and hyperkeratosis over the palm. 19
Furthermore, a retrospective analysis of patch tests among 2 groups of bricklayers and non- bricklayers in Brazil found that in patients with ACCD, the hands were affected in 61% of patients, the feet were affected in 42%, and the forearms were affected in 21% of patients. In addition, more than 1 area was affected in some patients. 20 Similarly, in the current study, all cases had hand lesions. The most common sites were hands and foot (31.0%), followed by hands alone (27.6%), and then hands and forearm (23.3%). Hands with elbow and/or abdomen were least common (10 %).
Moreover, a study in Taiwan on 153 current cement workers who had regular contact with cement in the past 12 months found that a high percentage (39%) of cement workers was noted to have hand dermatitis. Forearms, legs, and back were the other sites that showed dermatitis. 21
The current study showed nearly similar distribution of IL16 gene among different clinical presentations. Among the patients with ACCD, the types of IL16 gene were TT (73.3%), CT (23.3%), and CC (3.4%). The higher proportions of cases with C allele for the IL16 gene were statistically non-significant. In agreement with our results, some authors found non-significant association between IL16 gene polymorphism and some allergic conditions. One study has reported that the T-295 C promoter polymorphism was not associated with asthma, disease severity, or atopy in Australian population. 22 Also, another research found that despite increased IL16 level, there was no association between IL16 rs4778889 T/C polymorphism and asthma or atopy in Iranian patients. 23
However, the distribution of IL16-295 genotypes, in particular the IL16-295CC, differed between ACD patients and healthy controls and might increase susceptibility to ACD. 14 Also, in the current study, the most common genotype in ACCD was IL16-295 TT. Furthermore, the IL16-295 genotype polymorphism was associated with allergic asthma in white population in United Kingdom. 24
Regarding gene distribution and polysensitization, IL16-295CC genotype was over-represented among polysensitized individuals and not in monosensitized persons or in atopic dermatitis patients as compared with controls. Polysensitization means sensitization to 3 or more unrelated allergens, and monosensitization means patients sensitized only to para-acrylic compounds. 25
Regarding our study, according to patch test, 25 cases (89.3%) were monosensitized (15 sensitive to 1 substance and 10 sensitive to 2 substances) and 3 cases (10.7%) were polysensitized. These results can be explained by differences in ethnicity; environmental exposures between populations and different phenotype definitions might have contributed to these conflicting findings. It is believed that the genetic control of DTH reactions is antigen-specific, at least in some instances, as was shown for nickel, 26 mercury, 27 Cr, 28 and organic haptens. 29
Finally, some authors stated that the genetics of contact allergy are still only partly understood, despite decades of research. The polymorphisms of NAT1, NAT2, GSTM, GSTT, ACE, TNF, and IL-16 were shown to be associated with an increased risk of contact allergy. In one of their studies, there was increased risk conferred by the TNF and IL-16 polymorphisms which were confined to polysensitized individuals. 9
Many studies asked for a replication of these studies and further investigations to solve these conflicts in results.9,11,30 Most of the studied cases were sensitive to mercury chloride, followed by potassium dichromate (26.6%), cobalt chloride (20%), formaldehyde (16.6%), epoxy resin (16.6%), and nickel sulfate (6.6%), respectively. Most of the cases (65.2%) sensitive to mercury chloride showed extreme positive reaction (+++). It was reported that the common allergens affecting cement workers are epoxy resin, colophony, formaldehyde, nickel, cobalt, Cr, and rubber gloves. 4 Many previous studies regarding ACCD showed that most of the patients were sensitive to potassium dichromate.5,19-21
However, recent study found that contact sensitization to chromate decreased from 43.1% to 29.0%, and sensitization to epoxy resin increased from 8.4% to 12.4%. Logistic regression analysis revealed a significantly decreased risk of chromate sensitization (OR = 0.42) and a significantly increased risk of sensitization to epoxy resin (OR = 2.79) among patients who started work in building trade after 1999. Also, mercury chloride was not continuously tested as part of the German Contact Dermatitis Research Group standard series in studies of ACCD till 2006 and then became regularly tested and its sensitivity increased by time. 5
In the study in Southern Taiwan, it is concluded that among workers frequently exposed to cement, a high proportion of dichromate sensitization (12.4%) was seen in cement workers, followed by mercuric ammonium chloride (5.9%), nickel, benzalkonium, cobalt, fragrance mix, and phenyl mercuric acetate. 21 In addition, another study reported ACD and mercury exanthema due to mercury chloride in plastic boots. 31
This study highlights a new important possible and toxic sensitizing substance in cement workers in our locality, which is mercury chloride. This allergen needs further studies to evaluate the magnitude of problem and how to solve.
Regarding IL16 gene, CC/CT genotype was nearly similar to TT among cases with positive patch test. There was no statistically significant difference between gene distribution and type of tested allergens except for formaldehyde which showed statistically significant higher frequency for CC.
Limitations of the study
Few studies explored IL-16 and its important role in delayed hypersensitivity reaction. Further studies are needed with larger sample size in different ethnic groups to confirm the role of IL-16 SNP and pathogenesis of ACD. Patch test was completed for subset of healthy control group due to early removal of patches before the required time of follow-up visits and fear of side-effects. It is recommended for future study design to add serum IL16 level using ELISA in addition to genotype study and test the difference in serum level of IL16 among different genotypes and relation to severity of ACD.
Footnotes
Funding:
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests:
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Author Contributions
MAG & AFS: data collection and share in writing revised manuscript. ME: lab work and share in writing revised manuscript. EOK: concept and design of research, share in writing manuscript, statistical analysis and publication.
