Chronic Spontaneous Urticaria: An Etiopathogenic Study.

Importance: Exact etiopathogenesis of chronic spontaneous urticaria (CSU) remains elusive. Infections, pseudoallergens, autoimmunity, and contact sensitization are various postulated factors. Few studies are available measuring cytokine levels in CSU.
Objectives: The aim was to study various etiological factors of CSU and levels of IL-6 and IFN-ϒ in cases and controls, and correlation between various etiologies with the levels of the abovementioned interleukins in the cases. Design: Case-control study performed over 2 years with no follow-up of the participants. Setting: It was a referral-center-based study. Participants: Sixty patients of CSU and equal age and sex-matched healthy controls were recruited on the basis of convenience sampling. Exposures: Biochemical and hematological investigations with hepatitis serology, thyroid function tests, anti-thyroid antibodies, and levels IL-6 and IFN-ϒ were performed in all cases and controls. All cases were subjected to ASST. Cases with all above negative tests were patch-tested with Indian standard series. Urticaria activity score (UAS7) was calculated for all the cases and repeated in patients with positive etiological factor after 3 weeks (improvement after allergen or drug avoidance, treatment of infection). Outcomes: To study the various etiological factors (food, infection, autoimmunity, autoreactivity, and contact sensitization) and the levels of IL-6 and IFN- γ in patients of chronic spontaneous urticaria.
Results: Etiology was ascertained in 75% of patients (autoimmunity: 50%, contact sensitization: 21.67%, food and drug allergy: 1.67% each). Mean values of the interleukins and anti-thyroid antibodies were significantly higher in cases versus controls. Levels of IFN-ϒ were significantly elevated in patients with higher UAS7 scores.
Conclusion: Antithyroid antibodies, ASST, and patch testing are important tools and should be considered in patients of CSU after a thorough history and history-based workup. Elevated levels of IL-6 and IFN- ϒ in cases suggest that both Th1 and Th2 type of immune responses are implicated in pathogenesis of CSU. Copyright:

Introduction

Chronic spontaneous urticaria (CSU) is characterized by the spontaneous occurrence of wheals without an obvious stimulus that lasts for most days of the week for more than 6 weeks.[12] A point prevalence between 0.5% and 1% for CSU has been proposed.[3]

CSU is the most common subtype of chronic urticaria.[4] The long-lasting course and distressing symptoms have a great impact on the quality of life. The exact etiopathogenesis of CSU remains poorly understood. Possible causes of CSU include acute or chronic infections, pseudo allergies to food and drugs, allergic reactions to medications, insect bites and stings, and autoimmunity (including autoimmune thyroid disorders).[5] There is increasing evidence that inflammation and unbalanced immunity play critical roles in the generation and promotion of symptoms.[678910] Immunoglobulin (Ig) E-mediated allergy is probably never the cause of CSU, except in rare events that unrecognized food allergy in very young children might present with recurrent weals over a period of weeks until the cause is suspected and withdrawn.[11]

A strong link has been reported between CSU and elevated levels of IgG antithyroid antibodies,[12] with a large number of studies reporting positivity varying from 20.4% to 57.6%, of IgG against thyroid peroxidase (TPO) and 15.2%–42.5% of IgG against thyroglobulin (TG).[1314] Levels of IgG anti-thyroid antibodies are more often elevated in adults with CSU than in children and in females more than males.[15]

An autoimmune etiology characterized by the presence of histamine-releasing autoantibodies directed against the subunit of high-affinity IgE receptor (FcεRI) or IgE has been found in approximately 35%–45% of CSU patients.[16] These autoantibodies can be detected by autologous serum skin test (ASST).[17] The sensitivity and specificity of ASST for autoantibody detection in patients of CSU is reported to be 80% at best compared with the in vitro basophil histamine release assay.[18] One very important proposed association of CSU is with contact allergic sensitization. Patch test positivity in patients with CSU has been reported to vary between 42.9% and 96%.[1920] It has been hypothesized that small amounts of allergens may get absorbed through skin or gastrointestinal tract into the bloodstream over a long period of time and get delivered to Langerhan's cells in skin and other antigen-presenting cells, which provide signals for mast cell activation.[20] Thus, patch testing is a feasible and easy-to-perform procedure that has been evaluated earlier but has not been included in standard guidelines for management.

Most allergic and autoimmune diseases are caused by an imbalance between cytokines and T lymphocyte subgroups. This imbalance between Th1 and Th2 cytokines has long been proposed as a possible mechanism in urticaria.[2122] The majority of research works and studies on the etiology of CSU have focused on a single parameter. Contact allergic sensitization has not been studied much. Association between various etiological factors such as contact sensitization, autoreactivity, and antithyroid antibodies with cytokines such as IL-6 and IFN-γ have either not been studied previously or meagre literature is available for the same. Therefore, we planned to study various etiological factors in CSU, their association with each other, and with levels of IL-6 and IFN γ.

Materials and Methods

Study design

The study was conducted in the Department of Dermatology, Venereology, and STD and the Department of Biochemistry of a tertiary care hospital, from November 2017 to March 2019.

Patients

All patients aged above 18 years with CSU presenting to the Dermatology outpatient department (OPD) during the study period were included. Sixty patients of CSU were enrolled according to convenient sampling. Patients with dermographism or any other physical urticaria, signs of dermatitis on examination, with hives remaining fixed for more than 24 h, patients on corticosteroids or immunosuppressant drugs in 6 weeks preceding participation in the study, and pregnant and lactating females were excluded. Equal number of age and sex-matched healthy controls were enrolled in the study. Approval from ethics committee was sought and was given on 13/11/17.

Methodology

The demographic details of the enrolled patients were recorded. Detailed history and examination were carried out. Next, 6 mL of venous blood was collected in a plain vacutainer and centrifuged at 1500 rpm for 10 min after the venous blood had clotted. The sera was separated and stored in aliquot at −20°C till batch was analyzed. Serum levels of IL-6, IFN-γ, anti-TPO antibodies, and anti-TG antibodies were analyzed using ELISA-based kits, and ASST was performed.[23] If the patient was taking anti-histaminic drugs, he was advised to discontinue medication for 2 days and report to the OPD on the third day for venous blood collection and ASST. Investigations including complete blood count (CBC), erythrocyte sedimentation rate (ESR), urine routine microscopy (URM), stool routine microscopy (SRM), thyroid function test (TFT), hepatitis B and C serology, anti-streptolysin-O (ASO), and C-reactive protein (CRP) were performed in all patients. Additional investigations such as throat swabs and urine culture were carried out as suggested by the history. Patients were explained how to record their symptoms for the next 7 days, according to UAS7. Patients were called after 7 days, and investigation reports were recorded. Patients who gave history of offending food item or drug were subjected to avoidance of the same (in case of food item, avoidance of incriminating food item and low pseudoallergen diet was advised for 3 weeks, and in case of drug, patients were asked not to take the suspected drug) and were evaluated after 4 weeks for symptom severity. Table 1 summarizes the low-pseudoallergen diet that was used. In patients in whom history and above investigations did not show any abnormality, including negative ASST and no response to avoidance of drug or food items, or who had history of nickel allergy were subjected to patch test with Indian Standard Series patch test series. Patients with positive patch test were advised to avoid the allergen and to come for follow-up after 4 weeks with UAS7 as done initially. Age and sex-matched healthy controls were recruited after taking written informed consent. Detailed history and investigations were carried out to rule out any systemic disorder. IL-6, IFN-γ, and antithyroid antibodies were measured in the same way as for cases. Patients were treated as per standard treatment protocol.

Table 1

Low-pseudoallergen diet[24]

	FOOD ITEMS ALLOWED	FOOD ITEMS NOT ALLOWED
Carbohydrates:	Potatoes, rice, flour, pasta without egg, plain cereals (e.g., porridge, cornflakes)	All others (e.g., pasta with egg, noodles, popcorn, potato chips and crisps, biscuits, cakes)
Fat and oils:	Butter, cold-pressed oils (e.g., olive, sunflower)	All other (e.g., margarine, mayonnaise, butter substitute)
Dairy:	Fresh milk, cream	All others (e.g., yogurt)
Meat, fish, egg:	Fresh meat without seasoning	All others (e.g., non-organic eggs, fish, seafood, processed and smoked meat (e.g., sausages))
Vegetables:	All vegetables and salads, except those listed as not allowed	Tomatoes and tomato products, peas, spinach, sweet peppers, olives, mushroom
Fruits :	None	All fresh, dried, and sugared fruits, fruit juices
Desserts, spreads and sweets :	None except honey	All others (e.g., jams, Nutella, toppings, sweets, chewing gum)
Drinks :	Milk, water, coffee, black tea	All others (e.g., beer, wine, spirits; herbal and green teas, fruit squashes)
Herbs, spices, and nuts:	Salt, sugar, onion, all nuts (unless allergy has been shown, and avoid walnuts)	All others (e.g., chilies, garlic, coriander)

Observations and Results

The mean age group of our study population was 33.18 ± 9.59 years, while the mean age of controls was 30.7 ± 8.3 years. The difference was statistically insignificant. Women formed 73.33% of our study population and 71.67% of controls and thus had comparable sex distribution. Mean disease duration was 35.05 ± 65.50 months, with minimum being 2 month and maximum being 30 years. In our study population, 43.34% cases gave history of angioedema.

Distribution of etiology on basis of history

Out of 60 patients, based on history, we could ascertain the plausible cause of CSU in 39 (65%) patients [Figure 1].

Figure 1

Distribution of etiology on basis of history

Distribution of associated illnesses based on history

In our study population, history of thyroid disorders and jaundice was present in 10% each. History of chikungunya was found in 6.67%, pulmonary tuberculosis in 3.34% patients, hypertension in 3.34% cases, and diabetes mellitus in only 1.67% (n = 1) patients [Figure 2].

Figure 2

Distribution of associated illnesses based on history

Distribution of thyroid disorders based on history and investigations

Six patients had a history of hypothyroidism, with all of them on medication. On investigation, only one was found to be hypothyroid; all others were euthyroid. Without a prior history of thyroid disorders, 11 patients were found to have hypothyroidism, and seven were found to have hyperthyroidism. Thyroid dysfunction (abnormal test results or normal test results on medication) was thus seen in 24 (40%) patients.

Distribution of cases with dietary allergy

Four patients gave a history of intolerance to certain food items, with two of them incriminating eggs and non-vegetarian food, one incriminating sour food items, and the fourth incriminating cow's milk. Relevance of giving a low-pseudoallergen diet was proven only in one patient who had a history of symptom aggravation on eating eggs or non-vegetarian food.

Distribution of ASST results

Approximately 22% of patients were ASST positive [Figure 3].

Figure 3

Distribution of ASST results

Distribution of patch test results

Patch test was performed in 40 (66.67%) patients in whom either history of nickel allergy was present or no other etiological cause for urticaria could be ascertained. Out of these, 20 (33.34%) patients showed positive patch test results, 18 showed negative results, and two were doubtful. None of the results were irritant. Of the 20 positive results, 13 (21.67%) were etiologically relevant, that is, there was improvement in symptoms on avoidance of incriminating allergen, while seven were irrelevant [Table 2].

Table 2

Distribution of positive patch test results

ALLERGENS	TEST RESULTS					TOTAL POSITIVES	RELEVANCE (%)
	+	++	+++	+/-	IR
MERCAPTOBENZATHIAZOLE	0	0	0	2	0	0	0 (0)
NICKEL SULFATE	4	2	0	0	0	6	5 (83.34)
COLOPHONY	1	0	0	0	0	1	0 (0)
EPOXY RESIN	1	0	0	0	0	1	0 (0)
PARAPHENYLENE DIAMINE	5	0	0	1	0	5	0 (0)
FRAGRANCE MIX	7	2	0	0	0	9	8 (88.89)
THIURAM MIX	1	0	0	0	0	1	0 (0)

IR: irritant

Distribution of thyroid autoantibodies among cases and controls

Thyroid autoantibodies were seen in 10 (16.67%) cases (nine cases had anti-TPO and one had anti-TG) and one control (both anti-TPO and anti-TG). This difference in autoantibodies was statistically significant in cases compared to controls (P = 0.004). Six of these cases, that is, 60% of patients with elevated antibodies had thyroid dysfunction (thyroid dysfunction + euthyroid on medication).

Proportion of various etiological factors after evaluations and investigations

Upon performance of all the investigations and tests such as ASST and patch test, etiology could be found in 45 (75%) patients. Autoimmunity was judged based on the findings of either positive ASST, positive thyroid autoantibodies, or both. Thus, autoimmunity was found in 30 patients, wherein 20 (33.34%) had positive ASST, eight (13.34%) had positive thyroid autoantibodies (13.34%), and two (3.34%) patients had both. Approximately 21.67% (n = 13) patients had contact sensitization, and one patient each had incriminating drug and dietary allergy. However, there was some overlap of etiological factors [Figure 4].

Figure 4

Proportion of various etiological factors after evaluations and investigations

Mean levels of IL-6 in cases as compared to controls

On comparing the mean values of IL-6 for cases (14.68 ± 35.92 pg/ml) and controls (30.0 ± 24.37 pg/ml), the difference was statistically significant, that is, levels of IL-6 were statistically higher in cases of CSU compared to controls (P = 0.003) [Figure 5].

Figure 5

Mean levels of IL-6 in cases as compared to controls

Mean levels of IFN-ϒ in cases as compared to controls

On comparing the mean values of IFN-ϒ for cases (154.9 ± 98.93 pg/ml) and controls (97.7 ± 155.22 pg/ml), the difference was statistically significant, that is, levels of IFN-ϒ were statistically higher in cases of CSU compared to controls (P = 0.006) [Figure 6].

Figure 6

Mean levels of IFN-ϒ in cases as compared to controls

Various correlations

The correlation of thyroid autoimmunity, ASST, and patch test results with levels of IL-6 and IFN-ϒ was statistically insignificant. Similarly, the correlation between thyroid autoimmunity and ASST was also statistically insignificant. It was found that urticaria severity did not correlate with ASST positivity, thyroid autoimmunity, patch test positivity, and relevance. There was also no correlation of female sex with thyroid autoimmunity and ASST positivity. Urticaria severity correlated statistically significantly with levels of IFN-ϒ but not with levels of IL-6.

Discussion

CSU is a distressing condition with a severe impact on quality of life. Establishing causality is difficult, and more often than not, treatment is needed for prolonged periods. Various studies have tried to elucidate the cause of CSU, but to our knowledge, none have tried to establish the correlation between various suspected etiological factors, not just amongst themselves but also with levels of cytokines suspected to be involved in pathogenesis. Thus, we undertook this prospective evaluation in 60 patients to ascertain various causative factors, their proportions, their correlation with levels of IFN-ϒ (Th1 immune response) and IL-6 (Th2 immune response), and comparison of these cytokine levels between cases and an equal number of healthy age and sex-matched controls.

In our study population, the maximum number of patients fell in the age group of 20–30 years, that is, 36.67%. However, in an epidemiological study of urticaria in the Korean population, it was found that the maximum number of chronic urticaria patients fell in the age group of 40–59 years,[25] while in an Italian study, it was found that the highest prevalence of CSU was in the age group of below 20 years.[26] In our study, women outnumbered men (44 vs. 16), similar to studies in Korean, Italian, and Taiwanese populations, where females were predominantly affected.[252627]

Based on the history, 21 of our patients had associated illnesses as outlined in the observations.

While infective foci is a common trigger for acute urticaria, it is uncommonly associated with CSU. In our study group, history suggestive of infective foci was present in 19 patients in the form of sore throat, vaginal discharge, dental complaints, burning micturition, and loose stools. On detailed investigation, the infection could not be established etiologically in any of them. However, in a retrospective analysis of 100 chronic urticaria patients referred for dental evaluation, the authors found that 76 had dental infection.[28]

Pseudoallergy to food and drugs has been postulated to be an important incriminating factor that is often overlooked and improperly investigated. However, a history of aggravation of symptoms with dietary components was present in only four of our patients, one of whom reported improvement of symptoms while on a low-pseudoallergen diet and one patient reported resolution of symptoms after withdrawal of nitrofurantoin. Similar results were found by Rajan[29] who recruited 100 patients of CSU to study the prevalence of sensitivity to drug and food additives in patients of CSU, of whom 43 gave positive history, but on double blinded provocative testing, all had negative test. Contrarily, out of 81 children enrolled by Cavkaytor[30] to ascertain implication of aspirin hypersensitivity, it was found that 24% of children with chronic persistent urticaria had positive provocation test.

Autoimmunity accounts for 30%–50% of cases of CSU. While the standard test for its evaluation is basophil histamine release assay, it is available only for research purposes. On the other hand, ASST is 65%–71% sensitive and 78%–81% specific.[31] Thus, all patients in our study population were subjected to ASST, and 22 (36.67%) patients showed positive ASST. Higher ASST positivity was reported by Kumar et al.[32] in 111 CSU patients. Approximately 43.62% of patients had ASST positivity. Similarly, Al-Hamamy et al.[33] performed ASST in 54 patients of CSU, of which 22 (40.7%) were positive.

Thyroid autoimmunity may also be causative in patients with CSU with thyroid autoantibodies. All our cases and controls were subjected to measurement of levels of anti-TPO and anti-TG antibodies. Nine cases had elevated levels of anti-TPO, of which six (10%) patients had thyroid dysfunction (hypothyroidism: 5; hyperthyroidism: 1). Two of these patients had positive ASST. A case-control study performed in Karachi reported similar but higher positivity. They recruited 90 subjects that were divided into three groups: group 1 had 30 patients with diagnosed CSU, group 2 had 30 patients diagnosed with hypothyroidism, and group 3 had 30 age and sex-matched healthy controls. Out of a total of 47 patients with chronic urticaria (30 from group 1 and 17 from group 2 who were also found to have chronic urticaria), elevated titers of anti-TG antibodies and anti-TPO antibodies were found to be positive in 42.5% and 57.6% of patients, respectively.[14] Higher prevalence of thyroid autoimmunity was also reported in other studies.[343536]

Contact allergy has been proven to be causative in CSU in various studies. Stem cell growth factors, neuropeptides, and histamine release factors (HRFs) are implicated in non-IgE-mediated mast cell degranulation. It has been theorized that in some patients, after cutaneous or systemic exposure to some allergens, T-cells are stimulated to produce these specific HRFs, which lead to mast cell degranulation and thus lead to type IV, T-cell-mediated urticaria.[19] In our study, 15 patients gave a history of nickel allergy and were subjected to patch testing with ISS, along with all those patients in whom no other etiological cause could be ascertained. Thus, in total, 40 patients were patch tested. Out of these, 20 patients showed positive patch test results and 13 were etiologically relevant (fragrance mix in eight and nickel sulfate in five patients). Higher positivity was seen in various studies ranging from 42.9% to 96%.[192037] Of the 23 patients enrolled in a study by Hession et al.,[19] 22 (96%) had at least one positive patch test reaction of 1+ or stronger, whereas nine patients (39%) had at least one positive reaction of 2+ or stronger. In another study conducted by Chen et al.,[20] 543 CSU patients were patch-tested with European baseline series with 42.9% testing positive to the contact allergens. Similarly, Alghamdi et al.[37] evaluated patch test reactivities in 93 patients with CSU. Patients were also subjected to SPT. Patients with negative SPT were patch tested. Of the 58 patients of CIU who were patch-tested, 31 (53.4%) had positive results.

Elevated immune mediators in chronic urticaria indicate the presence of pro-inflammatory and autoimmune basis for the disease. While Th2 mediators predominate in acute urticaria, Th1 mediators predominate in chronic urticaria. However, it has been postulated that patients with generalized Th1/Th2 along with Th17 immune response activation are implicated in chronic urticaria.[38] In our study, it was found that while mean levels of IFN-ϒ in cases were 154.9 ± 98.93 pg/ml, it was 97.7 ± 155.22 pg/ml in controls. On statistical analysis, it was found that levels in cases were significantly elevated compared to controls (P = 0.0006). Similarly, levels of IL-6 were statistically significantly elevated in cases compared to controls with respective means being 14.68 ± 35.92 pg/ml and 30.0 ± 24.37 pg/ml (P = 0.003).

To reiterate the same, Alasandagutti et al.[39] recruited 100 CSU patients and 200 healthy controls and measured the levels of IL-6 and IFN-ϒ. A significant increase was observed in mean serum levels of IFN-Y (80.762–62.056) and IL-6 (39.37–11.06) in patients compared to healthy controls (24.79–21.84 and 7.175–4.81), respectively, at P < 0.0001. In another study, IL-6 trans-signaling was studied in 58 CSU patients. Plasma concentration of IL-6 was significantly higher in CSU patients as compared with the healthy subjects.[20]

Elevated levels of both the cytokines in our patients point toward an imbalance of both Th1 and Th2 immune response. Whether this finding can be helpful in predicting which patients of acute urticaria can progress to chronicity needs to be explored further.

Limitations of the study

There were a few limitations in our study. The sample size was small, and drug and food provocation tests were not performed. Due to lack of resources, skin prick testing, serum IgE levels and basophil histamine release assay could not be performed.

Conclusion

In conclusion, a significant proportion of our patients were found to have autoimmune urticaria and patch test positivity with only a few cases attributing infection, dietary allergens, and drugs as triggers. There were some limitations in our study such as small sample size and inability to perform certain tests such as basophil histamine release assay, skin prick test, and IgE level measurement. Based on the findings of our study, we recommend larger multicentric studies to explore etiopathogenesis of chronic urticaria with focus on autoimmune causation (including thyroid autoimmunity), patch testing, and cytokine levels (both Th1 and Th2) for better management of patients.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.