Could your T-shirt or Hair Dye be Altering your Immune System?

Increased expression of interleukin-9 in patients with allergic contact dermatitis caused by p-phenylenediamine

Recent studies of the effects of p-phenylenediamine (PPD) have raised awareness in public health due to its tendency to cause severe allergic reaction. The median prevalence of PPD sensitization in North America is 6.2%, and its widespread use as an ingredient in many common products has caused much concern.² PPD is found in many hair dyes, black clothing dye, henna substitute, and many rubber products.³ Allergic contact dermatitis (ACD) is a complex disease categorized as a type IV reaction caused by antigen-specific T-cells, rather than antibodies. In this type of reaction, the individual acquires lifelong memory against the allergen. This results the mounting of an immune response any time that allergen is reintroduced to the epidermis, and this causes eczema or other forms of skin irritation that can become extremely severe. In this study, the researchers aimed to analyze T cell recruitment and the cytokine production profile of patients allergic to PPD. They ultimately found here that interleukin 9 (IL-9), plays an important role in this allergic response. Interleukins (ILs) are proteins that regulate communication between immune cells. The study included 29 patients who had a positive patch test and a known allergy to PPD, and 16 control patients who had a negative patch test and no hypersensitivity to PPD.

From the skin biopsies, they observed tissue infiltration using flow cytometry, which allows the researcher to classify individual cells based on their proteins. They found that mostly CD3+ and CD4+ T cells infiltrated the dermis, indicating that T helper cells (which express CD4) were mostly responsible for the signaling that occurs during a response to PPD.

Fig. 1. Flow cytometry data on dermal cells from positive patch test biopsies. CD3+ and CD4+ cells increased the most over 48 hours.

Next, they used qRT-PCR, which measures gene expression, to evaluate the cytokines produced by the T helper cells. Here, they observed a large increase in IL-4 and IL-9 expression, suggesting that these two cytokines play important roles in activating an allergic response to PPD. IL-9 levels continued to increase even after 48 hours, suggesting a regulatory role in the response pathway. Furthermore, it was found that IL-9 expression correlated with the severity of the patch test reaction, providing more evidence for the importance of IL-9.

Fig. 2. (A) qRT-PCR data of mRNA isolated from skin biopsies at the indicated time points. IL-4 and IL-9 levels significantly increased. (C), (D) Flow cytometry data, stained for CD3 on IL-9+ cells and IL-4 positive cells. A much higher percentage of the CD3 cells are IL-9 positive.

They then turned to immune cells in the blood to evaluate their expression of cytokines. Blood samples were collected from patients both before the patch test and 48 hours after, and the cells were isolated. They found that IL-9 expression here more than doubled when allergic patients were treated with PPD. However, there was not a significant increase in IL-4 production. In PPD-tolerant patients, the cells did not secrete any IL-9.

Finally, now that it was evident that IL-9 was the most important cytokine in PPD-induced ACD, the researchers explored its role. They treated Il9r deficient mice with PPD. Il9r is the receptor IL-9 binds to to generate a response. They noticed a high level of contact hypersensitivity (CHS) compared to PPD-treated wild type mice, indicating a protective role of IL-9.

Fig. 3. Ear thickness of mice over 12 days. WT=wild type, Il9r-/-=Il9r deficient. Il9r deficient mice treated with PPD showed the most CHS development, indicated by the large increase in ear thickness.

 To conclude, this study revealed that IL-9 plays a main, protective role in the inflammatory pathway of PPD-induced ACD. This cytokine was shown to be important in the skin biopsies, immune blood cell analysis and the PPD-induced contact hypersensitivity models. Although this paper focuses on dermatologic effects, PPD can negatively impact cardiac, renal, and hepatic function as well.³ Because of the strong evidence for IL-9 presented here, it may also be significant in other types of severe allergic reactions. Since PPD is an emerging public health threat, it is important to discover the molecular mechanisms responsible for the allergic reaction to potentially develop a treatment for those who have developed lifelong allergy. Perhaps another direction this can be taken in is that of MHC-based immune phenotyping to find the differences between those who have no reaction and those who have a severe reaction to PPD. This may help the researchers find out more about the mechanism of this reaction and specifically, any genes associated with severe ACD.  However, it is essential to publicize the dangers of using henna ink, as this is the most common source of ACD. Traditional henna is a lighter brown, while substitute henna is black. Always be aware of the dye being used before you receive hair treatment or a tattoo!

If you would like to learn more about the dangers of PPD specifically in substitute henna, click here.

References:

1.      Baeck M, Herman A, Montjoye LD, et al. Increased expression of interleukin-9 in patients with allergic contact dermatitis caused by p-phenylenediamine. Contact Dermatitis. 2018. doi:10.1111/cod.13123.

2.      Thyssen JP, White JML. Epidemiological data on consumer allergy top-phenylenediamine. Contact Dermatitis. 2008;59(6):327-343. doi:10.1111/j.1600-0536.2008.01427.x.

3.      Abd-Elzaher MA, Fawzy IA, Ahmed HM, Abd-Allah AM, Gayyed MF. Some toxicological health hazards associated with subchronic dermal exposure to paraphenylene-diamine (PPD): An experimental study. Egyptian Journal of Forensic Sciences. 2012;2(3):105-111. doi:10.1016/j.ejfs.2012.06.003.

4.      Allergic Contact Dermatitis. Practice Essentials, Background, Pathophysiology. https://emedicine.medscape.com/article/1049216-overview. Published August 14, 2018. Accessed October 11, 2018.

5.      Delayed Hypersensitivity Reactions: Background, Pathophysiology, Epidemiology. Sickle Cell Anemia Differential Diagnoses. https://emedicine.medscape.com/article/136118-overview. Published May 7, 2018. Accessed October 10, 2018.

6.      Allergy skin tests. Mayo Clinic. https://www.mayoclinic.org/tests-procedures/allergy-tests/about/pac-20392895. Published August 7, 2018. Accessed October 10, 2018.

7.      Bio-Rad. The T Cell Marker, CD3 Antigen and Antibodies Mini-review. Bio-Rad. https://www.bio-rad-antibodies.com/minireview-cd3-antibody.html. Accessed October 11, 2018.

8.      Gaspari AA, Katz SI. Contact hypersensitivity. Current protocols in immunology. https://www.ncbi.nlm.nih.gov/pubmed/18432793. Published May 2001. Accessed October 11, 2018.