Immunological Investigations, 2014; 43(5): 504–516 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.892509
Immunoreactivity to food antigens in patients with chronic urticaria Sanvila Rasˇkovic ´ ,1,2 Ivana Z. Matic ´ ,3 Marija Ðord ic ´ ,3 Ana Damjanovic ´ ,3 Branka Kolundzˇija,3 Nada Grozdanic ´ -Stanisavljevic ´ ,3 Irina Besu,3 ˇZikica Jovic ˇ ic ´ ,1,2 Borivoj Bijelic ´ ,4 Ljiljana Jankovic ´ ,4 and Zorica Juranic ´3
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Institute of Immunology and Allergology, Clinical Center of Serbia, 11000 Belgrade, Serbia, 2 School of Medicine, University of Belgrade, 11000 Belgrade, Serbia, 3 Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia, 4 School of Dental Medicine, Clinic for Periodontology and Oral Medicine, University of Belgrade, 11000 Belgrade, Serbia The goal of study was better understanding of complex immune mechanisms that can help to evaluate patients with chronic urticaria (CU), especially those with unknown etiology. The study involved 55 patients with CU. Control group consisted of up to 90 healthy persons. The presence and intensity of serum IgG, IgA, IgM and IgE antibodies to common food antigens: cow’s milk proteins (CMP), gliadin and phytohemagglutinin were determined by ELISA. Determination of subpopulations of immunocompetent cells was performed by flow cytometry. Significantly enhanced IgE, but also IgA immunity to CMP was found in patients with CU in comparison to healthy controls: (p50.000004) and (p50.002), respectively. Notably, in 40 out of 55 CU patients, the increased levels of some type of immunoglobulin reactivity to CMP were found. Regarding gliadin, only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p50.04). Significantly enhanced percentage of CD89+ cells accompanied with significantly lower percentage of lymphocytes and significantly higher mean fluorescence intensity of CD26 expression on lymphocytes were found in patients with CU in comparison to healthy controls (p50.04), (p50.02) and (p50.003), respectively. Results of this study may help in better understanding the complex immune disturbances in patients with CU. Keywords CD16+CD56+, CD26+, CD89+, cellular immunoreactivity, chronic urticaria, food antigens, humoral immunoreactivity, serum DPPIV activity
INTRODUCTION Urticaria (hives) is characterized by recurrent, pruritic, pink-to-red edematous lesions that often have pale centers (wheals) (Kanani et al., 2011), resulting from localized edema of upper dermis. Angioedema is presented with deeper dermal or submucose swelling, while itching is less common (Altman & Chang, 2013). Swelling of the lips, tongue and cheeks are the most common oral manifestations. Temporary attacks are termed acute, while repeated episodes
Correspondence: Zorica Juranic´, Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Pasterova 14, 11000 Belgrade, Serbia. E-mail: [email protected]
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for longer than 6 weeks are termed chronic, and these conditions can affect 15–30% of the human population, at least once in their lifetime. Chronic urticaria (CU) is more common in adults, affecting mainly middle–aged women (Altman & Chang, 2013; Wardhana, 2012), so the prevalence of urticaria/ angioedema is twice as high in women as in men. The causes of CU are identifiable only in about 10% of cases (chronic physical urticaria, allergic urticaria, urticaria vasculitis, hereditary angioedema). Pathophysiological mechanisms that are implicated in the development of chronic urticaria have been studied during the past 20 years (Kaplan & Greaves, 2009). Evidence of an autoimmune etiology in approximately 30–45% of patients has been obtained. In this group, the so-called chronic autoimmune urticaria (CAU), many patients have symptomatic thyroid disease, most frequently Hashimoto’s thyroiditis (incidence of thyroid antibodies in CAU range from 5% to 90%). It is reported that autoimmune urticaria results from an IgG antibodies to the a subunit of the IgE receptor on the mast cells and basophils, or to IgE itself (Altman & Chang, 2013). The etiology of remaining, about 50% of patients, is unknown, and this group remains ‘‘idiopathic.’’ There is recent evidence that alterations in the signal transduction pathways in mast cells and basophils may be involved (Saini, 2009). Food antigens are very important for various immune functions, and the effects of dietary restriction on lymphocytes functionality were studied in animals (Meneguello-Coutinho et al., 2014). The immune reaction to some of food antigens is found in patients with urticaria; mainly IgE mediatedimmunity was considered to be connected with the existence of the symptoms of the disease (Husz et al., 1996; Martorell et al., 2006; Paranos et al., 1990; Paranos et al., 1993). Moreover, in some patients with allergy to cow’s milk proteins recurrent oral ulcerations were found (Besu et al., 2009). Recently Pleass et al. (2007) reported that crosslinking of IgA receptors, FcalphaRI (CD89), by immune complexes of antigens bound to specific IgA could induce significant not only eosinophils, but also neutrophils degranulation, and that IgA is a more potent inducer of NADPH oxidase activation and degranulation in eosinophils than IgE. Additionally, there are data that neutrophils in the reaction with microbes (but probably also with the antigens) could release reactive oxygen species (ROS), and chromatin, to liberate neutrophil extracellular traps (NET). These data provoked this investigation aiming to determine is the enhanced IgA (additionally to the known IgE) immunity to food antigens like cow’s milk proteins, or gliadin, or phytohemagglutinin at least partially connected with CU. Additionally, we examined whether the decrease in the percentage of lymphocytes observed in specific patients with CU might be associated with the enhanced percentage of CD89+ cells and enhanced levels of serum IgA, and/or IgE, and/or IgG immunity to the mentioned food antigens. Anamnestic collection of data and clinical examination of oral mucosa were performed to determine is the immunity to cow’s milk proteins found in some patients with CU related to the oral mucosal changes. To elucidate is the insufficient activity of dipeptidyl peptidase IV (serine protease that catalyzes the release of N-terminal dipeptides from proteins with proline or alanine at the penultimate position) (DPPIV), the possible reason of the enhanced immunogenicity of food proteins in CU patients, we assessed
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the serum DPPIV activity and determined the expression levels of the activation molecule CD26/DPPIV on lymphocytes.
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PATIENTS AND METHODS Patients The study involved 55 patients with chronic urticaria (age range 18–81 years). The control group consisted of up to 90 healthy persons (age range 24–62 years). Oral status (anamnesis and clinical examination) was evaluated in 25 hospitalized CU patients. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia and by the Ethics Committee of the Clinical Center of Serbia. Written informed consent was obtained from each patient. ELISA tests The presence and intensity of humoral immunoreactivity to food antigens that are commonly used in everyday nutrition—such as cow’s milk proteins (CMP), gliadin from wheat and phytohemagglutinin (PHA) from red beans—were determined by ELISA test, as described previously (Besu et al., 2009; Juranic´ et al., 2009). The levels of serum IgG, IgA, IgM and IgE antibodies were measured for each of the examined antigens, and were presented in arbitrary units AU/ml. Human sera with the high anti-CMP, anti-gliadin or anti-PHA immunoreactivity were used for the calibration. Cut-off values for each of the assessed immunoglobulins to examined food antigens were Xav + 2SD. Flow cytometry analysis Determination of the specific subpopulations of immunocompetent cells: CD16+, CD16+CD56+, CD26+ and CD89+ was performed by flow cytometry. Monoclonal antibody specific for CD56 was FITC-stained, while monoclonal antibodies specific for CD16, CD26 and CD89 were PE-stained (Becton Dickinson Immunocytometry Systems, CA, USA). Expression of mentioned antigens on white blood cells was determined using a FACSCalibur flow cytometer (BD Biosciences Franklin Lakes, NJ, USA). Acquired data were analyzed using CELLQuest software (BD Biosciences). It is noteworthy that the reference cut-off values (Xav ± SD) for the investigated parameters are established in our laboratory by analyzing the blood samples of 41 (for CD16, CD16CD56 and CD89) or 40 healthy control persons (for CD26); these data are already published (Ðordic´ et al., 2012; Matic´ et al., 2012). Determination of in vitro PBMC stimulation Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of patients or healthy control persons by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in the nutrient medium (RPMI 1640, pH 7.2, supplemented with 10% autologous plasma, 3 mM L-glutamine, 100 mg ml1 streptomycin, 100 IU ml1 penicillin and 25 mM Hepes). Target PBMC
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(150 000/well) were seeded into 96-well microtiter plates and were incubated in 150 ml of nutrient medium, in the presence of cow’s milk proteins, gliadin, PHA or in the mixture of PHA and CMP or gliadin. Control PBMC were incubated in nutrient medium only. All experiments were done in triplicate. PBMC were incubated in the presence of investigated food antigens for 72 h. Cell survival was determined by the MTT test according to the method of Mosmann (1983) and modified by Ohno and Abe (Ohno & Abe, 1991). Briefly, after 72 h of treatment, 10 ml of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 ml of 10% SDS. Absorbance at 570 nm was measured the next day. In vitro stimulation of PBMC to proliferate was determined as the increase in the number of cells in the presence of antigen(s) in comparison to the control PBMC incubated in nutrient medium only. DPPIV activity in serum The activity of DPPIV in serum was determined by the direct photometric method adapted to 96-well microtiter plates and according to the procedure described by Jarmolowska et al. (Jarmolowska et al., 2007) but modified to some extent, as we reported previously (Matic´ et al., 2012). Serum DPPIV enzymatic activity was calculated according to the formula: A serum sample A blank of serum sample Enzyme activity ¼ 100 A standard A blank of standard Enzyme activity : IU=L ¼ 1 mM= min =L of serum It is important to mention that the reference cut-off values (Xav ± SD) for the DPPIV activity are established in our laboratory by analyzing the sera samples of 40 healthy control persons; these data are already published (Matic´ et al., 2012). Statistical analysis Two-tailed Student’s t-test was used for statistical analysis of experimental data. p values below 0.05 were considered as significant.
RESULTS Humoral immunity to food antigens Data presented in Figure 1 showed that there was a significantly enhanced IgE immunity to CMP in patients with CU in comparison to healthy controls (p50.000004). Additionally, IgA immunity to CMP in patients with CU was also significantly enhanced (p50.002). Higher than cut-off levels of anti-CMP IgA antibodies were found in 30 out of 55 patients, of IgE antibodies were found in 27 out of 55 patients, of IgG antibodies in 6 out of 55, and of IgM antibodies in 7 out of 55 analyzed patients’ sera. Shortly, in 40 out of 55 analyzed CU patients, the increased levels of some type of immunoglobulins reactivity to CMP were found (Table 1). Only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p50.04), as seen in Figure 2. It could be seen
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Figure 1. (A) Serum IgG immunoreactivity to cow’s milk proteins (CMP), (B) serum IgA immunoreactivity to cow’s milk proteins, (C) serum IgM immunoreactivity to cow’s milk proteins, (D) serum IgE immunoreactivity to cow’s milk proteins in healthy controls and in patients with chronic urticaria.
that 12 out of 55 analyzed CU patients had the enhanced IgE immunity to gliadin. Although some patients with CU had the enhanced IgA and IgM reaction to PHA, (10/49 and 8/49, respectively), the IgE immunity to PHA in the examined group of patients was significantly lower when compared to healthy controls, (p50.02). This could be seen in Figure 3. Flow cytometry analysis Results presented in Figure 4 showed that although the percentage of CD16+CD56+ cells (NK cells), and CD16+ lymphocytes was not changed compared to controls, the percentage of CD16+CD56+ overall white blood cells was significantly lower (p50.04). The percentage of CD89+ overall immunocompetent cells (granulocytes) was significantly higher in patients with CU in relation to values obtained for healthy controls (p50.04). Data presented in Figure 5 showed that the percentage of CD26+ lymphocytes, as well as the percentage of CD26+ overall white blood cells in patients with CU did not differ from healthy controls. But, the mean fluorescence intensity (MFI) of CD26 expression on lymphocytes was significantly higher in patients with CU in comparison to healthy controls (p50.003). Percentage of lymphocytes in patients with CU The significant decrease in the percentage of lymphocytes in patients with CU (p50.02) could be seen too, and this decrease could be the reason for the
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Table 1. Frequencies of Patients with Chronic Urticaria with Disturbed Parameters. Lower % of lymphocytes Lower % of lymphocytes and enhanced IgA antibodies to food antigens Lower % of lymphocytes and enhanced IgA and/or IgE antibodies to food antigens Lower % of lymphocytes and enhanced IgA, IgG and/or IgE antibodies to food antigens Enhanced IgA anti-CMP antibodies Enhanced IgG anti-CMP antibodies Enhanced IgM anti-CMP antibodies Enhanced IgE anti-CMP antibodies Enhanced both: IgA and IgG anti-CMP antibodies Enhanced both: IgA and IgM anti-CMP antibodies Enhanced both: IgA and IgE anti-CMP antibodies Enhanced both: IgM and IgG anti-CMP antibodies Enhanced both: IgG and IgE anti-CMP antibodies Enhanced both: IgM and IgE anti-CMP antibodies Enhanced IgA anti-gliadin antibodies Enhanced IgG anti-gliadin antibodies Enhanced IgM anti-gliadin antibodies Enhanced IgE anti-gliadin antibodies Enhanced both: IgA and IgG anti-gliadin antibodies Enhanced both: IgA and IgM anti-gliadin antibodies Enhanced both: IgA and IgE anti-gliadin antibodies Enhanced both: IgM and IgG anti-gliadin antibodies Enhanced both: IgG and IgE anti-gliadin antibodies Enhanced both: IgM and IgE anti-gliadin antibodies Enhanced IgA anti-PHA antibodies Enhanced IgG anti-PHA antibodies Enhanced IgM anti-PHA antibodies Enhanced IgE anti-PHA antibodies Enhanced both: IgA and IgG anti-PHA antibodies Enhanced both: IgA and IgM anti-PHA antibodies Enhanced both: IgM and IgG anti-PHA antibodies Enhanced IgG antibodies to food antigens Enhanced IgA antibodies to food antigens Enhanced IgM antibodies to food antigens Enhanced IgE antibodies to food antigens
15 (43) 9 (15) 13 (15) 13 (15) 30 6 7 27 6 5 19 1 6 3 4 3 1 12 0 0 1 0 3 0 10 3 8 0 1 3 1 12 37 13 36
(55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (49) (49) (49) (49) (49) (49) (49) (55) (55) (55) (55)
mentioned lower percentage of CD16+CD56+ overall immunocompetent cells. In 15 out of 43 analyzed patients the decrease in the percentage of lymphocytes was found. The enhancement of only IgA immunity to food antigens was found in 9, while enhanced serum IgA and/or IgE immunity to food antigens was found in 13 out of these 15 patients, respectively. Regarding associated health disorders observed in patients with CU who had a decreased percentage of lymphocytes, 4 out of 15 patients had hypertension (two of those patients had only hypertension, one had gastritis as well, while one patient had gastritis, diabetes mellitus type 2 and rhinitis in addition to hypertension). Furthermore, one patient with decreased percentage of lymphocytes had diabetes mellitus and hypothyroidism, and two patients had Hashimoto’s thyroiditis.
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Figure 2. (A) Serum IgG immunoreactivity to gliadin, (B) serum IgA immunoreactivity to gliadin, (C) serum IgM immunoreactivity to gliadin, (D) serum IgE immunoreactivity to gliadin in healthy controls and in patients with chronic urticaria.
Figure 3. (A) Serum IgG immunoreactivity to phytohemagglutinin (PHA), (B) serum IgA immunoreactivity to phytohemagglutinin, (C) serum IgM immunoreactivity to phytohemagglutinin, (D) serum IgE immunoreactivity to phytohemagglutinin in healthy controls and in patients with chronic urticaria.
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Figure 4. (A) Percentage of CD16+CD56+ lymphocytes, (B) percentage of CD16+CD56+ overall white blood cells, (C) percentage of CD16+ overall white blood cells and (D) percentage of CD89+ overall white blood cells in healthy controls and in patients with chronic urticaria.
Figure 5. (A) Percentage of CD26+ overall white blood cells, (B) percentage of CD26+ lymphocytes, (C) mean fluorescence intensity (MFI) of CD26 expression on lymphocytes and (D) percentage of lymphocytes in healthy controls (Matic ´ et al., 2012) and in patients with chronic urticaria.
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Figure 6. In vitro stimulation of proliferation of peripheral blood mononuclear cells (PBMC) isolated from healthy controls and patients with chronic urticaria by cow’s milk proteins (CMP) (A), mixture of CMP and PHA (B), gliadin (C), mixture of gliadin and PHA (D) or PHA (E).
Figure 7. DPPIV serum activity in healthy controls (Matic ´ et al., 2012) and in patients with chronic urticaria.
Immunity in Chronic Urticaria
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PBMC stimulation in vitro Results presented in Figure 6 showed that there was a significant decrease (p50.04) in the PBMC (lymphocytes) stimulation only by the combined action of both gliadin and PHA in patients with CU in comparison to healthy controls. Anamnestic data Anamnestic data of patients’ oral status during episodes of CU or angioedema, revealed that 18 out of 25 evaluated patients reported swelling of the lips and/or tongue, while 13 of them reported breathing difficulties, heavy swallowing and sore throats. Six patients reported burning of oral mucosa, the other four reported mucosal redness, while five patients said that they sometimes had oral ulcerations. Clinical examination Clinical examination revealed changes of oral mucosa: 8/25 had hairy tongue, atrophy of the papillae of tongue 4/25, while one patient had lingua nigra. Enanthema or pigmentations of oral mucosa were present in 3 patients, while the recurrent oral ulcers (which were the focus of this study) also occurred 3/25. One of them had palate herpes simplex recidivans. Chelitis angularis was noticed in 8/25 patients. In all CU patients with ROU and/or enanthema or pigmentations the enhanced immunity to CMP was found. Serum DPPIV activity Determination of serum DPPIV activity in CU patients was done to assess is the insufficient activity of this enzyme present in these patients, especially in patients with enhanced immunity to food antigens. Results presented in Figure 7 indicate that there was no significant difference in the activity of DPPIV between patients with CU and healthy controls. It needs to be mentioned that in eight patients with CU decreased DPPIV activity was found; five these patients had IgE, (three out of these five had additionally IgA) imunity to CMP. Only one out of these eight patients had IgA anti-gliadin immunity. These data point out that decreased serum DPPIV activity is not the determining factor for the immunogenicity of some of the food antigens as the majority of patients with CU without decreased DPPIV activity had immunity to food antigens. One of these eight patients also had angioedema, and regarding the associated health disorders, one out of these eight patients had hypothyroidism, and two patients had diabetes mellitus type II, while four patients were without associated health disorders.
DISCUSSION Results from this study showing the enhanced immunity to cow’s milk proteins in patients with chronic urticaria (and ROU) are in accordance with previous reports (Besu et al., 2009; Husz et al., 1996; Martorell et al., 2006; Paranos et al., 1990; Paranos et al., 1993; Paranos & Nikolic, 1996). The higher level of total IgE is increased in patients with urticaria (Huilan et al., 2010), and the analysis of their damaging role is intriguing. Moreover, it is found that additionally to IgE, the IgA immunity to food antigens is very frequently present in these patients (Table 1).
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The enhancement of only IgA immunity to food antigens, found in 9 out of 15 patients who had decreased percentage of lymphocytes, points that only IgA (even without IgE immunity) could be involved in the lymphocytes’ destruction. It could be that some IgA and/or IgE autoantibodies to some others (non-tested) antigens are also present in circulation in the remaining 2 of 15 patients with decreased percentage of lymphocytes. Data on the significantly enhanced percentage of FcalphaRI positive (CD89+) cells (and significantly decreased percentage of lymphocytes) found in patients with CU in our study are also new in the scientific literature, and are in accordance with proposed possibility of their damaging role (through generation of ROS), after their reaction with IgA, or IgE complexed with their antigens, to normal tissue, but also to autologous lymphocytes, by mechanisms published earlier (Pleass et al., 2007; van der Steen et al., 2012). According to Pleass et al., the liberation of ROS from neutrophils is induced by crosslinking of receptors for IgA (of only those IgA bound to their specific antigens). As food antigens are mostly present in circulation it is to be expected that the specific interaction of these food antigens with their corresponding IgA could be of general importance for the start of the complex reaction of liberation of ROS and the damage of neighboring immune cells present in circulation. The significant decrease obtained in stimulation of lymphocytes in the presence of autologous plasma, induced by the combined action of both: gliadin and PHA in patients with CU (Figure 6), indicates that in the presence of a negligible percentage of granulocytes (which are present in PBMC isolates), there could be some decrease in stimulation of lymphocytes by these combined antigens. The reason for this could be in different (immunologically more suppressive) presentation of gliadin and PHA immunogenic epitopes when they are exposed in combination, in relation to when they are exposed alone. Although it is already reported that decreased DPPIV activity could be associated with angioedema (Brown et al., 2009; Byrd et al., 2010; Byrd et al., 2011), results of this work showing that there is no significant difference in the activity of this enzyme between patients with CU and healthy controls, and that only one out of eight patients with decreased DPPIV activity also had angioedema, indicate that decrease in DPPIV enzymatic activity is not always connected with angioedema. Results that the MFI of CD26 expression on lymphocytes was significantly higher in patients with CU compared to healthy control subjects are first reported in this work, and are in accordance with the previously known co-stimulatory function of CD26. Perhaps this phenomenon is the basis for the observed hyperreactivity of the immune system to food antigens in CU. They are in accordance with reports that CD26 expression in patients with acute T-lymphoblastic leukemias could be related to a worse prognosis, in comparison with patients with CD26-negative tumors (Carbone et al., 1995), and with the findings that the increase in CD26 cell surface expression was reported in B-CLL too (Bauvois et al., 1999; Cro et al., 2009). The opposite finding that the MFI of CD26 expression on lymphocytes in breast cancer patients was significantly decreased in comparison to healthy control group (Eric´-Nikolic´ et al., 2011) points to totally changed possibility of the functions of lymphocytes in patients
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with CU (or with some hematological diseases), compared to that determined in patients with malignant breast tumors. Findings of this research reveal a few new questions: Should the therapeutically decreased percentage of granulocytes (applied with appropriate restriction diet) decrease the ROS generation and subsequent inflammatory reaction? What about the consideration of the therapeutical use of MoAb to FcalphaRI on granulocytes in order to block their interaction with IgA antibodies bound in immune complexes to food antigens to prevent healthy tissue or healthy immune cells damage induced by subsequent ROS release reaction (van der Steen et al., 2012)? Should the use of lower immunogenic milk like goat (Rodrı´guez del Rı´o et al., 2012) or camel (Ehlayel et al., 2011) after the results on the absence of immunity to mentioned milk proteins obtained with the appropriate ELISA tests help in resolving the symptoms of CU in specific patients who originally had IgE or IgA immunoreactivity to CMP? In conclusion, better understanding of these complex immune mechanisms can help us to evaluate our patients with CU, especially those with unknown etiopathogenesis. In the future we might include new therapeutical possibilities for those patients who have enhanced immunoreactivity to food antigens.
ACKNOWLEDGEMENTS The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for the financial support (Project 175011). Also the authors would like to thank Tatjana Petrovic´ for her excellent technical assistance.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Immunoreactivity to food antigens in patients with chronic urticaria Sanvila Rasˇkovic ´ ,1,2 Ivana Z. Matic ´ ,3 Marija Ðord ic ´ ,3 Ana Damjanovic ´ ,3 Branka Kolundzˇija,3 Nada Grozdanic ´ -Stanisavljevic ´ ,3 Irina Besu,3 ˇZikica Jovic ˇ ic ´ ,1,2 Borivoj Bijelic ´ ,4 Ljiljana Jankovic ´ ,4 and Zorica Juranic ´3
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Institute of Immunology and Allergology, Clinical Center of Serbia, 11000 Belgrade, Serbia, 2 School of Medicine, University of Belgrade, 11000 Belgrade, Serbia, 3 Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia, 4 School of Dental Medicine, Clinic for Periodontology and Oral Medicine, University of Belgrade, 11000 Belgrade, Serbia The goal of study was better understanding of complex immune mechanisms that can help to evaluate patients with chronic urticaria (CU), especially those with unknown etiology. The study involved 55 patients with CU. Control group consisted of up to 90 healthy persons. The presence and intensity of serum IgG, IgA, IgM and IgE antibodies to common food antigens: cow’s milk proteins (CMP), gliadin and phytohemagglutinin were determined by ELISA. Determination of subpopulations of immunocompetent cells was performed by flow cytometry. Significantly enhanced IgE, but also IgA immunity to CMP was found in patients with CU in comparison to healthy controls: (p50.000004) and (p50.002), respectively. Notably, in 40 out of 55 CU patients, the increased levels of some type of immunoglobulin reactivity to CMP were found. Regarding gliadin, only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p50.04). Significantly enhanced percentage of CD89+ cells accompanied with significantly lower percentage of lymphocytes and significantly higher mean fluorescence intensity of CD26 expression on lymphocytes were found in patients with CU in comparison to healthy controls (p50.04), (p50.02) and (p50.003), respectively. Results of this study may help in better understanding the complex immune disturbances in patients with CU. Keywords CD16+CD56+, CD26+, CD89+, cellular immunoreactivity, chronic urticaria, food antigens, humoral immunoreactivity, serum DPPIV activity
INTRODUCTION Urticaria (hives) is characterized by recurrent, pruritic, pink-to-red edematous lesions that often have pale centers (wheals) (Kanani et al., 2011), resulting from localized edema of upper dermis. Angioedema is presented with deeper dermal or submucose swelling, while itching is less common (Altman & Chang, 2013). Swelling of the lips, tongue and cheeks are the most common oral manifestations. Temporary attacks are termed acute, while repeated episodes
Correspondence: Zorica Juranic´, Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Pasterova 14, 11000 Belgrade, Serbia. E-mail: [email protected]
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for longer than 6 weeks are termed chronic, and these conditions can affect 15–30% of the human population, at least once in their lifetime. Chronic urticaria (CU) is more common in adults, affecting mainly middle–aged women (Altman & Chang, 2013; Wardhana, 2012), so the prevalence of urticaria/ angioedema is twice as high in women as in men. The causes of CU are identifiable only in about 10% of cases (chronic physical urticaria, allergic urticaria, urticaria vasculitis, hereditary angioedema). Pathophysiological mechanisms that are implicated in the development of chronic urticaria have been studied during the past 20 years (Kaplan & Greaves, 2009). Evidence of an autoimmune etiology in approximately 30–45% of patients has been obtained. In this group, the so-called chronic autoimmune urticaria (CAU), many patients have symptomatic thyroid disease, most frequently Hashimoto’s thyroiditis (incidence of thyroid antibodies in CAU range from 5% to 90%). It is reported that autoimmune urticaria results from an IgG antibodies to the a subunit of the IgE receptor on the mast cells and basophils, or to IgE itself (Altman & Chang, 2013). The etiology of remaining, about 50% of patients, is unknown, and this group remains ‘‘idiopathic.’’ There is recent evidence that alterations in the signal transduction pathways in mast cells and basophils may be involved (Saini, 2009). Food antigens are very important for various immune functions, and the effects of dietary restriction on lymphocytes functionality were studied in animals (Meneguello-Coutinho et al., 2014). The immune reaction to some of food antigens is found in patients with urticaria; mainly IgE mediatedimmunity was considered to be connected with the existence of the symptoms of the disease (Husz et al., 1996; Martorell et al., 2006; Paranos et al., 1990; Paranos et al., 1993). Moreover, in some patients with allergy to cow’s milk proteins recurrent oral ulcerations were found (Besu et al., 2009). Recently Pleass et al. (2007) reported that crosslinking of IgA receptors, FcalphaRI (CD89), by immune complexes of antigens bound to specific IgA could induce significant not only eosinophils, but also neutrophils degranulation, and that IgA is a more potent inducer of NADPH oxidase activation and degranulation in eosinophils than IgE. Additionally, there are data that neutrophils in the reaction with microbes (but probably also with the antigens) could release reactive oxygen species (ROS), and chromatin, to liberate neutrophil extracellular traps (NET). These data provoked this investigation aiming to determine is the enhanced IgA (additionally to the known IgE) immunity to food antigens like cow’s milk proteins, or gliadin, or phytohemagglutinin at least partially connected with CU. Additionally, we examined whether the decrease in the percentage of lymphocytes observed in specific patients with CU might be associated with the enhanced percentage of CD89+ cells and enhanced levels of serum IgA, and/or IgE, and/or IgG immunity to the mentioned food antigens. Anamnestic collection of data and clinical examination of oral mucosa were performed to determine is the immunity to cow’s milk proteins found in some patients with CU related to the oral mucosal changes. To elucidate is the insufficient activity of dipeptidyl peptidase IV (serine protease that catalyzes the release of N-terminal dipeptides from proteins with proline or alanine at the penultimate position) (DPPIV), the possible reason of the enhanced immunogenicity of food proteins in CU patients, we assessed
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the serum DPPIV activity and determined the expression levels of the activation molecule CD26/DPPIV on lymphocytes.
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PATIENTS AND METHODS Patients The study involved 55 patients with chronic urticaria (age range 18–81 years). The control group consisted of up to 90 healthy persons (age range 24–62 years). Oral status (anamnesis and clinical examination) was evaluated in 25 hospitalized CU patients. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia and by the Ethics Committee of the Clinical Center of Serbia. Written informed consent was obtained from each patient. ELISA tests The presence and intensity of humoral immunoreactivity to food antigens that are commonly used in everyday nutrition—such as cow’s milk proteins (CMP), gliadin from wheat and phytohemagglutinin (PHA) from red beans—were determined by ELISA test, as described previously (Besu et al., 2009; Juranic´ et al., 2009). The levels of serum IgG, IgA, IgM and IgE antibodies were measured for each of the examined antigens, and were presented in arbitrary units AU/ml. Human sera with the high anti-CMP, anti-gliadin or anti-PHA immunoreactivity were used for the calibration. Cut-off values for each of the assessed immunoglobulins to examined food antigens were Xav + 2SD. Flow cytometry analysis Determination of the specific subpopulations of immunocompetent cells: CD16+, CD16+CD56+, CD26+ and CD89+ was performed by flow cytometry. Monoclonal antibody specific for CD56 was FITC-stained, while monoclonal antibodies specific for CD16, CD26 and CD89 were PE-stained (Becton Dickinson Immunocytometry Systems, CA, USA). Expression of mentioned antigens on white blood cells was determined using a FACSCalibur flow cytometer (BD Biosciences Franklin Lakes, NJ, USA). Acquired data were analyzed using CELLQuest software (BD Biosciences). It is noteworthy that the reference cut-off values (Xav ± SD) for the investigated parameters are established in our laboratory by analyzing the blood samples of 41 (for CD16, CD16CD56 and CD89) or 40 healthy control persons (for CD26); these data are already published (Ðordic´ et al., 2012; Matic´ et al., 2012). Determination of in vitro PBMC stimulation Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of patients or healthy control persons by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in the nutrient medium (RPMI 1640, pH 7.2, supplemented with 10% autologous plasma, 3 mM L-glutamine, 100 mg ml1 streptomycin, 100 IU ml1 penicillin and 25 mM Hepes). Target PBMC
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(150 000/well) were seeded into 96-well microtiter plates and were incubated in 150 ml of nutrient medium, in the presence of cow’s milk proteins, gliadin, PHA or in the mixture of PHA and CMP or gliadin. Control PBMC were incubated in nutrient medium only. All experiments were done in triplicate. PBMC were incubated in the presence of investigated food antigens for 72 h. Cell survival was determined by the MTT test according to the method of Mosmann (1983) and modified by Ohno and Abe (Ohno & Abe, 1991). Briefly, after 72 h of treatment, 10 ml of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 ml of 10% SDS. Absorbance at 570 nm was measured the next day. In vitro stimulation of PBMC to proliferate was determined as the increase in the number of cells in the presence of antigen(s) in comparison to the control PBMC incubated in nutrient medium only. DPPIV activity in serum The activity of DPPIV in serum was determined by the direct photometric method adapted to 96-well microtiter plates and according to the procedure described by Jarmolowska et al. (Jarmolowska et al., 2007) but modified to some extent, as we reported previously (Matic´ et al., 2012). Serum DPPIV enzymatic activity was calculated according to the formula: A serum sample A blank of serum sample Enzyme activity ¼ 100 A standard A blank of standard Enzyme activity : IU=L ¼ 1 mM= min =L of serum It is important to mention that the reference cut-off values (Xav ± SD) for the DPPIV activity are established in our laboratory by analyzing the sera samples of 40 healthy control persons; these data are already published (Matic´ et al., 2012). Statistical analysis Two-tailed Student’s t-test was used for statistical analysis of experimental data. p values below 0.05 were considered as significant.
RESULTS Humoral immunity to food antigens Data presented in Figure 1 showed that there was a significantly enhanced IgE immunity to CMP in patients with CU in comparison to healthy controls (p50.000004). Additionally, IgA immunity to CMP in patients with CU was also significantly enhanced (p50.002). Higher than cut-off levels of anti-CMP IgA antibodies were found in 30 out of 55 patients, of IgE antibodies were found in 27 out of 55 patients, of IgG antibodies in 6 out of 55, and of IgM antibodies in 7 out of 55 analyzed patients’ sera. Shortly, in 40 out of 55 analyzed CU patients, the increased levels of some type of immunoglobulins reactivity to CMP were found (Table 1). Only the levels of serum IgE anti-gliadin antibodies were significantly enhanced in patients with CU (p50.04), as seen in Figure 2. It could be seen
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Figure 1. (A) Serum IgG immunoreactivity to cow’s milk proteins (CMP), (B) serum IgA immunoreactivity to cow’s milk proteins, (C) serum IgM immunoreactivity to cow’s milk proteins, (D) serum IgE immunoreactivity to cow’s milk proteins in healthy controls and in patients with chronic urticaria.
that 12 out of 55 analyzed CU patients had the enhanced IgE immunity to gliadin. Although some patients with CU had the enhanced IgA and IgM reaction to PHA, (10/49 and 8/49, respectively), the IgE immunity to PHA in the examined group of patients was significantly lower when compared to healthy controls, (p50.02). This could be seen in Figure 3. Flow cytometry analysis Results presented in Figure 4 showed that although the percentage of CD16+CD56+ cells (NK cells), and CD16+ lymphocytes was not changed compared to controls, the percentage of CD16+CD56+ overall white blood cells was significantly lower (p50.04). The percentage of CD89+ overall immunocompetent cells (granulocytes) was significantly higher in patients with CU in relation to values obtained for healthy controls (p50.04). Data presented in Figure 5 showed that the percentage of CD26+ lymphocytes, as well as the percentage of CD26+ overall white blood cells in patients with CU did not differ from healthy controls. But, the mean fluorescence intensity (MFI) of CD26 expression on lymphocytes was significantly higher in patients with CU in comparison to healthy controls (p50.003). Percentage of lymphocytes in patients with CU The significant decrease in the percentage of lymphocytes in patients with CU (p50.02) could be seen too, and this decrease could be the reason for the
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Table 1. Frequencies of Patients with Chronic Urticaria with Disturbed Parameters. Lower % of lymphocytes Lower % of lymphocytes and enhanced IgA antibodies to food antigens Lower % of lymphocytes and enhanced IgA and/or IgE antibodies to food antigens Lower % of lymphocytes and enhanced IgA, IgG and/or IgE antibodies to food antigens Enhanced IgA anti-CMP antibodies Enhanced IgG anti-CMP antibodies Enhanced IgM anti-CMP antibodies Enhanced IgE anti-CMP antibodies Enhanced both: IgA and IgG anti-CMP antibodies Enhanced both: IgA and IgM anti-CMP antibodies Enhanced both: IgA and IgE anti-CMP antibodies Enhanced both: IgM and IgG anti-CMP antibodies Enhanced both: IgG and IgE anti-CMP antibodies Enhanced both: IgM and IgE anti-CMP antibodies Enhanced IgA anti-gliadin antibodies Enhanced IgG anti-gliadin antibodies Enhanced IgM anti-gliadin antibodies Enhanced IgE anti-gliadin antibodies Enhanced both: IgA and IgG anti-gliadin antibodies Enhanced both: IgA and IgM anti-gliadin antibodies Enhanced both: IgA and IgE anti-gliadin antibodies Enhanced both: IgM and IgG anti-gliadin antibodies Enhanced both: IgG and IgE anti-gliadin antibodies Enhanced both: IgM and IgE anti-gliadin antibodies Enhanced IgA anti-PHA antibodies Enhanced IgG anti-PHA antibodies Enhanced IgM anti-PHA antibodies Enhanced IgE anti-PHA antibodies Enhanced both: IgA and IgG anti-PHA antibodies Enhanced both: IgA and IgM anti-PHA antibodies Enhanced both: IgM and IgG anti-PHA antibodies Enhanced IgG antibodies to food antigens Enhanced IgA antibodies to food antigens Enhanced IgM antibodies to food antigens Enhanced IgE antibodies to food antigens
15 (43) 9 (15) 13 (15) 13 (15) 30 6 7 27 6 5 19 1 6 3 4 3 1 12 0 0 1 0 3 0 10 3 8 0 1 3 1 12 37 13 36
(55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (55) (49) (49) (49) (49) (49) (49) (49) (55) (55) (55) (55)
mentioned lower percentage of CD16+CD56+ overall immunocompetent cells. In 15 out of 43 analyzed patients the decrease in the percentage of lymphocytes was found. The enhancement of only IgA immunity to food antigens was found in 9, while enhanced serum IgA and/or IgE immunity to food antigens was found in 13 out of these 15 patients, respectively. Regarding associated health disorders observed in patients with CU who had a decreased percentage of lymphocytes, 4 out of 15 patients had hypertension (two of those patients had only hypertension, one had gastritis as well, while one patient had gastritis, diabetes mellitus type 2 and rhinitis in addition to hypertension). Furthermore, one patient with decreased percentage of lymphocytes had diabetes mellitus and hypothyroidism, and two patients had Hashimoto’s thyroiditis.
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Figure 2. (A) Serum IgG immunoreactivity to gliadin, (B) serum IgA immunoreactivity to gliadin, (C) serum IgM immunoreactivity to gliadin, (D) serum IgE immunoreactivity to gliadin in healthy controls and in patients with chronic urticaria.
Figure 3. (A) Serum IgG immunoreactivity to phytohemagglutinin (PHA), (B) serum IgA immunoreactivity to phytohemagglutinin, (C) serum IgM immunoreactivity to phytohemagglutinin, (D) serum IgE immunoreactivity to phytohemagglutinin in healthy controls and in patients with chronic urticaria.
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Immunity in Chronic Urticaria
Figure 4. (A) Percentage of CD16+CD56+ lymphocytes, (B) percentage of CD16+CD56+ overall white blood cells, (C) percentage of CD16+ overall white blood cells and (D) percentage of CD89+ overall white blood cells in healthy controls and in patients with chronic urticaria.
Figure 5. (A) Percentage of CD26+ overall white blood cells, (B) percentage of CD26+ lymphocytes, (C) mean fluorescence intensity (MFI) of CD26 expression on lymphocytes and (D) percentage of lymphocytes in healthy controls (Matic ´ et al., 2012) and in patients with chronic urticaria.
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Figure 6. In vitro stimulation of proliferation of peripheral blood mononuclear cells (PBMC) isolated from healthy controls and patients with chronic urticaria by cow’s milk proteins (CMP) (A), mixture of CMP and PHA (B), gliadin (C), mixture of gliadin and PHA (D) or PHA (E).
Figure 7. DPPIV serum activity in healthy controls (Matic ´ et al., 2012) and in patients with chronic urticaria.
Immunity in Chronic Urticaria
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PBMC stimulation in vitro Results presented in Figure 6 showed that there was a significant decrease (p50.04) in the PBMC (lymphocytes) stimulation only by the combined action of both gliadin and PHA in patients with CU in comparison to healthy controls. Anamnestic data Anamnestic data of patients’ oral status during episodes of CU or angioedema, revealed that 18 out of 25 evaluated patients reported swelling of the lips and/or tongue, while 13 of them reported breathing difficulties, heavy swallowing and sore throats. Six patients reported burning of oral mucosa, the other four reported mucosal redness, while five patients said that they sometimes had oral ulcerations. Clinical examination Clinical examination revealed changes of oral mucosa: 8/25 had hairy tongue, atrophy of the papillae of tongue 4/25, while one patient had lingua nigra. Enanthema or pigmentations of oral mucosa were present in 3 patients, while the recurrent oral ulcers (which were the focus of this study) also occurred 3/25. One of them had palate herpes simplex recidivans. Chelitis angularis was noticed in 8/25 patients. In all CU patients with ROU and/or enanthema or pigmentations the enhanced immunity to CMP was found. Serum DPPIV activity Determination of serum DPPIV activity in CU patients was done to assess is the insufficient activity of this enzyme present in these patients, especially in patients with enhanced immunity to food antigens. Results presented in Figure 7 indicate that there was no significant difference in the activity of DPPIV between patients with CU and healthy controls. It needs to be mentioned that in eight patients with CU decreased DPPIV activity was found; five these patients had IgE, (three out of these five had additionally IgA) imunity to CMP. Only one out of these eight patients had IgA anti-gliadin immunity. These data point out that decreased serum DPPIV activity is not the determining factor for the immunogenicity of some of the food antigens as the majority of patients with CU without decreased DPPIV activity had immunity to food antigens. One of these eight patients also had angioedema, and regarding the associated health disorders, one out of these eight patients had hypothyroidism, and two patients had diabetes mellitus type II, while four patients were without associated health disorders.
DISCUSSION Results from this study showing the enhanced immunity to cow’s milk proteins in patients with chronic urticaria (and ROU) are in accordance with previous reports (Besu et al., 2009; Husz et al., 1996; Martorell et al., 2006; Paranos et al., 1990; Paranos et al., 1993; Paranos & Nikolic, 1996). The higher level of total IgE is increased in patients with urticaria (Huilan et al., 2010), and the analysis of their damaging role is intriguing. Moreover, it is found that additionally to IgE, the IgA immunity to food antigens is very frequently present in these patients (Table 1).
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The enhancement of only IgA immunity to food antigens, found in 9 out of 15 patients who had decreased percentage of lymphocytes, points that only IgA (even without IgE immunity) could be involved in the lymphocytes’ destruction. It could be that some IgA and/or IgE autoantibodies to some others (non-tested) antigens are also present in circulation in the remaining 2 of 15 patients with decreased percentage of lymphocytes. Data on the significantly enhanced percentage of FcalphaRI positive (CD89+) cells (and significantly decreased percentage of lymphocytes) found in patients with CU in our study are also new in the scientific literature, and are in accordance with proposed possibility of their damaging role (through generation of ROS), after their reaction with IgA, or IgE complexed with their antigens, to normal tissue, but also to autologous lymphocytes, by mechanisms published earlier (Pleass et al., 2007; van der Steen et al., 2012). According to Pleass et al., the liberation of ROS from neutrophils is induced by crosslinking of receptors for IgA (of only those IgA bound to their specific antigens). As food antigens are mostly present in circulation it is to be expected that the specific interaction of these food antigens with their corresponding IgA could be of general importance for the start of the complex reaction of liberation of ROS and the damage of neighboring immune cells present in circulation. The significant decrease obtained in stimulation of lymphocytes in the presence of autologous plasma, induced by the combined action of both: gliadin and PHA in patients with CU (Figure 6), indicates that in the presence of a negligible percentage of granulocytes (which are present in PBMC isolates), there could be some decrease in stimulation of lymphocytes by these combined antigens. The reason for this could be in different (immunologically more suppressive) presentation of gliadin and PHA immunogenic epitopes when they are exposed in combination, in relation to when they are exposed alone. Although it is already reported that decreased DPPIV activity could be associated with angioedema (Brown et al., 2009; Byrd et al., 2010; Byrd et al., 2011), results of this work showing that there is no significant difference in the activity of this enzyme between patients with CU and healthy controls, and that only one out of eight patients with decreased DPPIV activity also had angioedema, indicate that decrease in DPPIV enzymatic activity is not always connected with angioedema. Results that the MFI of CD26 expression on lymphocytes was significantly higher in patients with CU compared to healthy control subjects are first reported in this work, and are in accordance with the previously known co-stimulatory function of CD26. Perhaps this phenomenon is the basis for the observed hyperreactivity of the immune system to food antigens in CU. They are in accordance with reports that CD26 expression in patients with acute T-lymphoblastic leukemias could be related to a worse prognosis, in comparison with patients with CD26-negative tumors (Carbone et al., 1995), and with the findings that the increase in CD26 cell surface expression was reported in B-CLL too (Bauvois et al., 1999; Cro et al., 2009). The opposite finding that the MFI of CD26 expression on lymphocytes in breast cancer patients was significantly decreased in comparison to healthy control group (Eric´-Nikolic´ et al., 2011) points to totally changed possibility of the functions of lymphocytes in patients
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with CU (or with some hematological diseases), compared to that determined in patients with malignant breast tumors. Findings of this research reveal a few new questions: Should the therapeutically decreased percentage of granulocytes (applied with appropriate restriction diet) decrease the ROS generation and subsequent inflammatory reaction? What about the consideration of the therapeutical use of MoAb to FcalphaRI on granulocytes in order to block their interaction with IgA antibodies bound in immune complexes to food antigens to prevent healthy tissue or healthy immune cells damage induced by subsequent ROS release reaction (van der Steen et al., 2012)? Should the use of lower immunogenic milk like goat (Rodrı´guez del Rı´o et al., 2012) or camel (Ehlayel et al., 2011) after the results on the absence of immunity to mentioned milk proteins obtained with the appropriate ELISA tests help in resolving the symptoms of CU in specific patients who originally had IgE or IgA immunoreactivity to CMP? In conclusion, better understanding of these complex immune mechanisms can help us to evaluate our patients with CU, especially those with unknown etiopathogenesis. In the future we might include new therapeutical possibilities for those patients who have enhanced immunoreactivity to food antigens.
ACKNOWLEDGEMENTS The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for the financial support (Project 175011). Also the authors would like to thank Tatjana Petrovic´ for her excellent technical assistance.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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