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Expression of interleukin 4 receptors in bronchial asthma patients who underwent specific immunotherapy
Expression of interleukin 4 receptors in bronchial asthma patients who underwent specific immunotherapy Krzysztof Kowal, MD*; Joanna Osada, PhD*; Sebastian Zukowski, MD*; Milena Dabrowska, PhD*; Lawrence DuBuske, MD†; and Anna Bodzenta-Lukaszyk, MD*
Background: Interleukin (IL) 4 and IL-13 are crucial cytokines for the development of allergic reactions and have been shown to modulate the function of monocytes and macrophages. Objectives: To evaluate the expression of IL-4Rs on peripheral blood monocytes and in the serum of patients with bronchial asthma who underwent specific immunotherapy (SIT). Methods: The study was performed on 17 asthma patients with a typical clinical history and positive skin prick test results to Dermatophagoides pteronyssinus allergens. Five asthma patients who declined SIT were used as a comparator control group. Ten healthy persons served as negative controls. Flow cytometry analysis was performed on the whole blood samples using labeled monoclonal antibodies against CD14 and CD36 monocyte markers and against the CD124 ␣ chain of IL-4R. The serum levels of soluble IL-4R were evaluated using an immunoenzymatic assay. Results: Compared with controls, bronchial asthma patients before SIT had a higher mean ⫾ SD percentage of CD14-positive cells that coexpressed CD124 (3.5% ⫾ 1.8% vs 18.6% ⫾ 7.9%; P ⬍ .01). After SIT, the mean ⫾ SD percentage of CD14 cells coexpressing CD124 decreased to 8.1% ⫾ 5.1%, which was significantly lower than before SIT (P ⬍ .01) but still significantly higher than in controls (P ⫽ .01). Changes in CD124 expression were associated with up-regulation of CD14 and downregulation of CD36 expression on peripheral blood monocytes, suggesting that IL-4/IL-13–mediated signaling may be important for regulation of monocyte phenotype and function in asthma patients receiving SIT. Conclusions: Even short courses of SIT are associated with a decrease in IL-4R expression on peripheral blood monocytes, which may cause decreased IL-4/IL-13–mediated effects in patients who undergo SIT. Ann Allergy Asthma Immunol. 2004;93:68–75.
INTRODUCTION Allergic diseases, including bronchial asthma, are characterized by decreased production of interferon ␥ (IFN-␥) and increased production of interleukin (IL) 4, IL-5, and IL-13 by allergen-specific T-cell clones.1 Furthermore, on stimulation with bacterial antigens, mononuclear phagocytes derived from allergic patients release less IL-12 and IL-18 than those derived from controls.2,3 Interleukin 12 and IL-18, in turn, are strong inducers of IFN-␥ release from T cells.4 The TH2-type cytokines IL-4 and IL-13 directly inhibit IL-12 production by mononuclear phagocytes.5 Furthermore, IL-4 and IL-13 inhibit several proinflammatory functions of mononuclear phagocytes, including production of IL-1 and tumor necrosis factor ␣ (TNF-␣), release of reactive oxygen species, synthesis of nitric oxide, and surface expression of procoagulant activity.6 – 8 On the contrary, expression of CD23, major histocompatibility complex class II, some costimulatory molecules, and adhesion molecules on mononuclear phagocytes are increased by IL-4 and IL-13.9,10 * University Medical School of Bialystok, Bialystok, Poland. † Immunology Research Institute of New England, Fitchburg, Massachusetts. Received for publication April 30, 2003. Accepted for publication in revised form January 8, 2004.
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Interleukin 4 acts through a membrane receptor composed of a specific ␣ subunit (IL-4 receptor [IL-4R] ␣ or CD124) and a common ␥ chain (␥c), which also functions as a part of receptors for IL-2, IL-7, IL-9, and IL-15.11 CD124 can form a functional receptor for IL-13 and IL-4 even in the absence of ␥c by interacting with IL-13 receptor ␣ subunit (IL13R␣).11 The IL-13R␣ is not expressed by T cells, and, therefore, IL-13 does not affect T-cell function, whereas the effect of IL-4 on these cells depends on IL-4R␣ and ␥c expression.12 Mononuclear phagocytes express IL-4R␣, IL13R␣, and ␥c, and, therefore, the action of IL-4 on these cells is mimicked by the action of IL-13.8 The IL-4R is also found in serum as a soluble form (sIL-4R),13 which possibly functions to inhibit IL-4.13 The main source of sIL-4R seems to be T cells because under certain culture conditions the magnitude of sIL-4R release parallels the magnitude of IL-4 secretion by these cells.14 The importance of IL-4R–mediated effects in the pathogenesis of bronchial asthma has been emphasized by the results of experimental studies in animals and genetic studies in humans. In sensitized mice, blockade of the IL-4R inhibited development of airway hyperresponsiveness after allergen challenge, whereas in a similar experiment, blockade of endogenous IL-13 reversed airway hyperresponsiveness and pulmonary mucous cell hyperplasia.15,16 Polymorphisms in the promoter regions that affect
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expression of both the ␣ subunit of the IL-4R and the ligand of this receptor, IL-13, are associated with an increased risk of developing allergic asthma.17 Several immunologic changes have been observed in patients undergoing specific immunotherapy (SIT). An increase in the concentration of serum-specific IgG1 and IgG4, a blunted typical seasonal rise in IgE, and, with sustained therapy, a decrease in serum-specific IgE concentration have all been reported in patients undergoing SIT.18,19 Mononuclear cells derived from patients receiving SIT have been shown to produce less IL-4 and histamine-releasing factor but increased amounts of IFN-␥ on allergen challenge.20 –22 Finally, many SIT-induced changes in the function of the effector cells of the allergic response have been described, including a decrease in both basophil sensitivity and reactivity to specific allergen challenge.21–26 In grass-sensitive hay fever patients after 4 years of SIT, increased production of IL-12 by skin macrophages has been demonstrated.27 Peripheral blood monocytes derived from grass-sensitive patients who underwent SIT are less sensitive to IL-4 –mediated suppression of IL-12 production.28 The latter effect is observed after 6 weeks of SIT, being associated with down-regulation of CD124 expression on peripheral blood monocytes.27,28 Relatively little is known about monocyte and macrophage function in patients with asthma undergoing SIT. In one study, 12 months of SIT with house dust mite allergen extract resulted in decreased in vitro production of IL-1 and TNF-␣ by Dermatophagoides pteronyssinus allergen–stimulated peripheral blood monocytes.29 The aim of this study was to evaluate the expression of IL-4R␣ on peripheral blood monocytes and sIL-4R in the serum of patients with bronchial asthma receiving SIT. Furthermore, the expression of 2 monocyte surface proteins, CD14 and CD36, which is regulated by IL-4R signaling, was also evaluated.30,31 MATERIALS AND METHODS Seventeen patients with bronchial asthma and allergic rhinitis were enrolled in this study. All patients had a history of perennial allergic rhinitis for at least 12 months and episodes of dyspnea, cough, and wheezing, especially on dust exposure. They had positive skin prick test results to D pteronyssinus. Resting forced expiratory volume in 1 second (FEV1) in all patients was greater than 75% of the predicted value, but all patients demonstrated significant bronchial reactivity to histamine and significant bronchoconstriction on bronchial
challenge with D pteronyssinus extract (Table 1). Most patients studied had already been treated with intranasal steroids before initial evaluation in our allergy clinic; therefore, we did not perform an intranasal allergen challenge. Five of the 17 patients declined to undergo SIT and served as a comparator control group. Ten healthy volunteers were used as a negative control group. They had negative skin prick test results to commonly encountered aeroallergens (house dust mites, trees, weeds, grasses, cat, Alternaria, and Cladosporium). Their lung function test results were within normal limits, and they did not demonstrate significant bronchial reactivity to histamine (provocation concentration that caused a decrease in FEV1 of 20%, ⬎32 mg/mL). No bronchial challenge with D pteronyssinus extracts was performed in the negative control group. Histamine bronchial challenge was performed according to the method of Ryan et al.32 Briefly, all patients and controls inhaled doubling concentrations of histamine starting from a concentration of 0.62 mg/mL. Aerosol was generated using a nebulizer (DeVilbis 646; DeVilbis, Somerset, PA) attached to a Rosenthal-French dosimeter. All subjects performed 5 inspiratory-capacity breaths of the given histamine concentration. Forced expiratory maneuvers were performed 90 seconds after each fifth inhalation. The procedure was continued until either at least a 20% fall in FEV1 or a histamine concentration of 32 mg/mL was reached. A bronchial provocation test with aqueous D pteronyssinus extracts (Allergopharma Joachim Ganzer KG, Reinbek, Germany) was performed according to the method of Cockcroft et al.33 Increasing doses of allergen (0.8, 4, 20, 100, 500, and 2,000 SBE) were administered using a DeVilbis 646 nebulizer attached to a Rosenthal-French dosimeter. Forced expiratory maneuvers were performed 15 minutes after inhalation of each dose of the allergen extract. Allergen inhalations were continued until either at least a 20% fall in FEV1 or a cumulative dose of 5,000 SBE was reached. Then, FEV1 was measured every 15 minutes during the first hour, every hour during the next 11 hours, and after 24 hours. The early response was evaluated as a dose of allergen causing a 20% drop in FEV1. The late response was considered positive whenever 2 consecutive records of FEV1 demonstrated at least a 15% fall observed between the third and twelfth hours of the test. Concentration of serum-specific anti-D pteronyssinus IgE was evaluated using an enzyme-linked immunosorbent assay test (Allergopharma).
Table 1. Patient Characteristics* Group
N
SIT
PC20, mg/mL
PD20, SBE
Total IgE, IU/mL
Specific anti-D pteronyssinus IgE, IU/mL
Asthma Asthma Control
12 5 10
Yes No No
2.5 ⫾ 3.6 2.9 ⫾ 2.1 ⬎32
872 ⫾ 955 965 ⫾ 723 ND
545.6 ⫾ 331 425.7 ⫾ 385 35 ⫾ 29
15.4 ⫾ 13.2 17.7 ⫾ 15.2 ND
Abbreviations: FEV1, forced expiratory volume in 1 second; ND, not determined; PD20, provocative dose that caused a 20% fall in FEV1; PC20, provocation concentration that caused a decrease in FEV1 of 20%; SIT, specific immunotherapy. * Data are given as mean ⫾ SD.
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Figure 1. Representative flow cytometer (FACS, Becton-Dickinson, Franklin Lakes, NJ) analysis of blood samples from a control person (A and D), a patient with asthma who underwent specific immunotherapy (SIT) (B and E), and a patient with asthma who did not undergo SIT (C and F). The analyses using labeled monoclonal antibodies anti-CD14 fluorescein isothiocyanate (FITC) with anti-CD124 phycoerythrine (PE) (A-C) or anti-CD36 FITC with anti-CD14 PE (D-F) were performed for the control patient on 2 separate occasions and for the patients with asthma before and after a 3-month period, which in patients who received SIT corresponded to the period necessary to reach the maintenance dose of allergen extract.
During the study, all patients were allowed to use intranasal steroids and inhaled 2-agonists for symptom control. SIT was performed using a standardized, aqueous Dermatophagoides extract (Novo Helisen Depot, Allergopharma). The initial dose in all patients was 5 therapeutic units. The dose was doubled every week until the maintenance dose (5,000 therapeutic units) was achieved. Blood samples were taken before therapy and 1 week after administration of the first maintenance dose. In patients with asthma who did not undergo SIT, blood samples were obtained before bronchial inhalation challenge tests and after 3 months. Flow cytometry analysis was performed on whole blood samples using labeled monoclonal antibodies, including anti-CD14 fluorescein isothiocyanate and anti-CD124 phycoerythrine (PE; BD Biosciences Pharmingen, San Diego, CA). In addition, in 13 patients with asthma and 7 controls,
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flow cytometry was performed using labeled monoclonal antibodies, including anti-CD14 PE and anti-CD36 fluorescein isothiocyanate (BD Biosciences Pharmingen). Briefly, an EDTA anticoagulated blood sample was drawn from the antecubital vein and was immediately used for staining. Fiftymicroliter samples of whole blood that contained 2 to 4 ⫻ 105 leukocytes were incubated with 10 L of each monoclonal antibody solution according to manufacturer instructions. Matched labeled anti-idiotype antibodies were used as negative controls. After 30 minutes of incubation at room temperature, erythrocytes were lysed using a set of 3 wholeblood lysing reagents (ImmunoPrep Reagent System, Coulter Corp, A Beckman Coulter Co, Miami, FL). The remaining white blood cells were analyzed by flow cytometry (Coulter). Monocytes were selected based on their typical morphologic features—side scatter and forward scatter—and also based on
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the presence of the surface protein CD14. Expression of the studied surface proteins was evaluated as the percentage of monocytes staining positively with relevant labeled antibodies and also as the mean fluorescence intensity in relative fluorescence units (FU). Soluble IL-4Rs in the serum were evaluated using an enzyme-linked immunosorbent assay method (Quantikine, R&D Systems, Minneapolis, MN). The test sensitivity was less than 5 pg/mL, and the testing range was 15.6 to 1,000 pg/mL. Statistical analysis was performed using analysis of variance. The results are expressed as mean ⫾ SD. RESULTS Patients with bronchial asthma were characterized by higher total serum IgE levels and significant bronchial reactivity to histamine (Table 1). There was no significant difference in bronchial reactivity or total or specific serum IgE concentrations between asthma patients who underwent SIT and those who did not. In asthma patients, the mean number of monocytes, selected on the basis of their morphologic features, with expression of CD14 was 324 ⫾ 124 cells/L, which was not different from that found in controls (307 ⫾ 88 cells/L) (P ⫽ .71). The mean density of CD14 on individual cells evaluated as mean fluorescence intensity was lower in patients with asthma vs the negative control group, but the difference was not statistically significant (7.58 ⫾ 5.3 vs 12.5 ⫾ 8.4 FU; P ⫽ .07; Figs 1 and 2). In the negative control group, only 3.5% ⫾ 1.8% of CD14-positive cells expressed CD124 (Figs 1 and 2). In asthma patients who underwent SIT, 17.9% ⫾ 8.7% of peripheral blood monocytes expressed CD124 on their surface, which was significantly greater than that in the control group (P ⬍ .01) but not significantly different from that observed in patients with asthma who did not undergo SIT (22.7% ⫾ 21.2%; P ⫽ .51; Figs 1 and 3). One week after administration of the maintenance dose of house dust mite allergen extract, the mean percentage of CD14-positive cells coexpressing CD124 was significantly lower than before SIT (17.9% ⫾ 8.7% vs 8.3 ⫾ 5.4%; P ⬍ .01; Figs 1 and 3). Furthermore, in the 5 patients who declined SIT, no significant change in CD124 expression on peripheral blood monocytes was observed (Figs 1 and 3). In asthma patients, the density of CD124 on the cell surface, evaluated as mean fluorescence intensity, was also significantly higher than in the negative controls (2.6 ⫾ 1.4 vs 1.1 ⫾ 0.6 FU, respectively; P ⬍ .01). In patients with asthma who underwent SIT, mean fluorescence intensity of CD124 fell from 2.56 ⫾ 1.13 FU to 1.65 ⫾ 0.53 FU (P ⬍ .01; Figs 1 and 3). No change in CD124 density was detected in asthmatic patients who did not undergo SIT (Figs 1 and 3). The mean percentage of monocytes expressing CD14 after SIT (87.4% ⫾ 4.4%) was not statistically different from the baseline value (86.9 ⫾ 6.2; P ⫽ .82), but a significant increase in mean density of CD14 on monocyte cell surfaces was found after SIT (from 7.58 ⫾ 5.2 FU to 12.8 ⫾ 6.06 FU; P ⫽ .01; Figs 1 and 3).
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Figure 2. Comparison of the expression of CD14, CD36, and CD124 on peripheral blood monocytes in patients with asthma and controls shown as the mean percentage of monocytes expressing each surface protein (A) and as mean fluorescence intensity (B). Error bars represent SD.
Another monocyte surface receptor, CD36, was found on a similar number of monocytes in patients with asthma vs negative controls (85.8% ⫾ 6.8% vs 81.0% ⫾ 6.17%; P ⫽ .08; Figs 1 and 3). The mean density of CD36 on individual cells was higher in patients with asthma but not significantly different than in negative controls (12.9 ⫾ 8.13 vs 7.2 ⫾ 4.5 FU; P ⫽ .05). After 3 months of SIT, the percentage of monocytes expressing CD36 decreased from 86.4% ⫾ 6.8% to 79.8% ⫾ 5.9% (P ⫽ .02) and fluorescence intensity of CD36 decreased from 13.3 ⫾ 9.16 to 6.9 ⫾ 4.6 FU (P ⫽ .01). Neither mean percentage of CD36-expressing cells nor density of CD36 expression changed after 3 months in asthmatic patients who were not receiving SIT (Figures 1 and 3). There was no significant difference in the concentration of sIL-4R in serum samples from patients with asthma who underwent SIT vs those who did not undergo SIT (43.2 ⫾ 12.4 vs 38.1 ⫾ 14.1 pg/mL; P ⫽ .47) and vs controls (43.2 ⫾ 12.4 vs 29.1 ⫾ 14.5 pg/mL; P ⫽ .27; Fig 4). Significant changes in the concentration of the sIL-4R in serum samples were not observed in patients with asthma undergoing SIT (from 43.2 ⫾ 12.5 to 36 ⫾ 10.5 pg/mL; P ⫽ .17) or in
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Figure 3. Mean changes in the expression of CD14 (A and B), CD124 (C and D), and CD36 (E and F) on peripheral blood monocytes in patients with asthma who did and did not receive specific immunotherapy (SIT). Error bars represent SD. Both mean percentage of monocytes expressing each surface protein (A, C, and E) and mean fluorescence intensity (B, D, and F) are presented.
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Figure 4. Mean changes in serum concentrations of soluble interleukin 4 receptor (sIL-4R) in patients with asthma who did and did not undergo specific immunotherapy (SIT). NS indicates not significant. Error bars represent SD.
asthmatic patients who did not receive SIT (from 38.1 ⫾ 14.1 to 39.7 ⫾ 15.2 pg/mL; P ⫽ .87; Fig 4). DISCUSSION In patients with bronchial asthma, elevated production of IL-4 and IL-13 by T cells has been documented.1 Concomitant increased expression of IL-4R␣ on peripheral blood monocytes in patients with asthma may be responsible for the increased effect of IL-4 and IL-13 on these cells. In T and B cells, IL-4 has been shown to increase the expression of its own receptor.34 If this is also true for mononuclear phagocytes, increased expression of IL-4R␣ subunit might be a result of increased IL-4 or IL-13 production by T cells and possibly mast cells and basophils. One of the results of this interaction could be decreased production of IL-12 by mononuclear phagocytes. Decreased production of IL-12 by mononuclear phagocytes has been described in patients with asthma.2 Decreased production of IL-12 results in decreased production of IFN-␥ and promotion of TH2-type immune responses. In allergic patients, increased expression of CD124 has been demonstrated on peripheral blood monocytes and on granulocytes, including basophils.35,36 In grasspollen allergic patients, increased expression of CD124 on peripheral blood monocytes was associated with decreased production of IL-12 and IFN-␥ by phytohemagglutinin-stimulated mononuclear cells.28,35 Furthermore, exogenous IL-4 more efficiently suppressed IL-12 production in patients with grass-pollen allergy, indicating that increased CD124 expression is associated with enhanced function of the IL-4 –IL-4R system in allergic patients.28 Up to now, little information has been available concerning the effect of SIT on CD124 expression on circulating leukocytes. In patients allergic to Hymenoptera venoms who underwent venom immunotherapy, a significant decrease in the expression of CD124 on
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peripheral blood basophils was observed 1 week after the end of rush immunotherapy.36 In patients with grass-sensitive hay fever who received SIT, the expression of CD124 on peripheral blood monocytes decreased already after 6 weeks of SIT.28 Furthermore, monocytes derived from the patients who underwent SIT were less sensitive to IL-4 –mediated suppression of IL-12 production.28 Relatively little is known about the exact mechanisms responsible for the IL-4/IL-13–mediated inhibition of IL-12 production by mononuclear phagocytes in allergic patients and the possible effect of SIT on this cytokine network. As observed in this study, up-regulation of CD14 during SIT may represent a possible mechanism to increase the susceptibility of monocytes to release proinflammatory cytokines, including IL-12. Lipopolysaccharide, which is a ligand of CD14, has been shown to be a very potent stimulus of IL-12 release by peripheral blood monocytes.2,5 Changes in IL-4/ IL-13 receptor signaling may be directly involved in the regulation of CD14 and CD36 expression on peripheral blood monocytes in patients undergoing SIT because IL-4 and IL-13 have been shown to up-regulate CD36 expression and down-regulate CD14 expression on these cells.30,31 Several intracellular molecules are involved in IL-4/IL-13 receptormediated signaling. One of the best characterized is the signal transducer and activator of transcription 6, which, when phosphorylated, represses the biological actions of IFN-␥–activated signal transducer and activator of transcription 1 and TNF-␣–activated nuclear factor–B.37 Furthermore, it has recently been demonstrated that the IL-4/IL-13 receptor mediates up-regulation of peroxisome proliferator activator receptor gamma (PPAR-␥) and induces synthesis of its natural ligands.31 Activation of PPAR-␥, in turn, inhibits the proinflammatory effects of nuclear factor–B and inhibits IL-12 production by stimulated monocytes.38 These 2 mechanisms may be potentially involved in IL-4R–mediated suppression of IL-12 production. During SIT there is down-regulation of CD36, the expression of which is PPAR-␥ dependent, supporting the hypothesis that this intracellular molecule may also be involved in the IL-4R–mediated effects seen in patients with asthma.31,38 Although changes in cell surface CD124 expression have been demonstrated, there is little information on how the potential activation of this receptor occurs. The presence of sIL-4Rs at significant concentrations in the peripheral blood of both negative controls and patients with asthma argues against an endocrine action of IL-4. A potential paracrine action of IL-4 released from T cells during the T cell– monocyte interaction cannot be excluded. Furthermore, IL13, the action of which mimics the action of IL-4 on monocytes, also uses CD124 as a part of the IL-13 receptor yet is not inhibited by sIL-4R. Further studies are needed to clarify whether the change in CD124 expression reflects changes in monocyte function and whether this phenomenon is transient or persists throughout SIT, perhaps constituting an important mechanism responsible for the beneficial clinical effects of SIT.
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34. Renz H, Domenico J, Gelfand EW. IL-4 dependent upregulation of IL-4 receptor expression in murine T and B cells. J Immunol. 1991;146:3049 –3055. 35. Grzela K, Grzela T, Lazarczyk D, et al. CD124 on monocytes in grass pollen allergy. Allergy. 2001;57:254 –255. 36. Siegmund R, Vogelsang H, Machnik A, Hermann D. Surface membrane antigen alteration on blood basophils in patients with Hymenoptera venom allergy under immunotherapy. J Allergy Clin Immunol. 2000;106:1190 –1195. 37. Ohmori Y, Hamilton TA. Interleukin-4/STAT6 represses STAT1 and NF-B– dependent transcription through distinct mechanisms. J Biol Chem. 2000;275:38095–38103.
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Requests for reprints should be addressed to: Lawrence DuBuske, MD 358 Elm St Gardner, MA 01440 E-mail: [email protected]
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Background: Interleukin (IL) 4 and IL-13 are crucial cytokines for the development of allergic reactions and have been shown to modulate the function of monocytes and macrophages. Objectives: To evaluate the expression of IL-4Rs on peripheral blood monocytes and in the serum of patients with bronchial asthma who underwent specific immunotherapy (SIT). Methods: The study was performed on 17 asthma patients with a typical clinical history and positive skin prick test results to Dermatophagoides pteronyssinus allergens. Five asthma patients who declined SIT were used as a comparator control group. Ten healthy persons served as negative controls. Flow cytometry analysis was performed on the whole blood samples using labeled monoclonal antibodies against CD14 and CD36 monocyte markers and against the CD124 ␣ chain of IL-4R. The serum levels of soluble IL-4R were evaluated using an immunoenzymatic assay. Results: Compared with controls, bronchial asthma patients before SIT had a higher mean ⫾ SD percentage of CD14-positive cells that coexpressed CD124 (3.5% ⫾ 1.8% vs 18.6% ⫾ 7.9%; P ⬍ .01). After SIT, the mean ⫾ SD percentage of CD14 cells coexpressing CD124 decreased to 8.1% ⫾ 5.1%, which was significantly lower than before SIT (P ⬍ .01) but still significantly higher than in controls (P ⫽ .01). Changes in CD124 expression were associated with up-regulation of CD14 and downregulation of CD36 expression on peripheral blood monocytes, suggesting that IL-4/IL-13–mediated signaling may be important for regulation of monocyte phenotype and function in asthma patients receiving SIT. Conclusions: Even short courses of SIT are associated with a decrease in IL-4R expression on peripheral blood monocytes, which may cause decreased IL-4/IL-13–mediated effects in patients who undergo SIT. Ann Allergy Asthma Immunol. 2004;93:68–75.
INTRODUCTION Allergic diseases, including bronchial asthma, are characterized by decreased production of interferon ␥ (IFN-␥) and increased production of interleukin (IL) 4, IL-5, and IL-13 by allergen-specific T-cell clones.1 Furthermore, on stimulation with bacterial antigens, mononuclear phagocytes derived from allergic patients release less IL-12 and IL-18 than those derived from controls.2,3 Interleukin 12 and IL-18, in turn, are strong inducers of IFN-␥ release from T cells.4 The TH2-type cytokines IL-4 and IL-13 directly inhibit IL-12 production by mononuclear phagocytes.5 Furthermore, IL-4 and IL-13 inhibit several proinflammatory functions of mononuclear phagocytes, including production of IL-1 and tumor necrosis factor ␣ (TNF-␣), release of reactive oxygen species, synthesis of nitric oxide, and surface expression of procoagulant activity.6 – 8 On the contrary, expression of CD23, major histocompatibility complex class II, some costimulatory molecules, and adhesion molecules on mononuclear phagocytes are increased by IL-4 and IL-13.9,10 * University Medical School of Bialystok, Bialystok, Poland. † Immunology Research Institute of New England, Fitchburg, Massachusetts. Received for publication April 30, 2003. Accepted for publication in revised form January 8, 2004.
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Interleukin 4 acts through a membrane receptor composed of a specific ␣ subunit (IL-4 receptor [IL-4R] ␣ or CD124) and a common ␥ chain (␥c), which also functions as a part of receptors for IL-2, IL-7, IL-9, and IL-15.11 CD124 can form a functional receptor for IL-13 and IL-4 even in the absence of ␥c by interacting with IL-13 receptor ␣ subunit (IL13R␣).11 The IL-13R␣ is not expressed by T cells, and, therefore, IL-13 does not affect T-cell function, whereas the effect of IL-4 on these cells depends on IL-4R␣ and ␥c expression.12 Mononuclear phagocytes express IL-4R␣, IL13R␣, and ␥c, and, therefore, the action of IL-4 on these cells is mimicked by the action of IL-13.8 The IL-4R is also found in serum as a soluble form (sIL-4R),13 which possibly functions to inhibit IL-4.13 The main source of sIL-4R seems to be T cells because under certain culture conditions the magnitude of sIL-4R release parallels the magnitude of IL-4 secretion by these cells.14 The importance of IL-4R–mediated effects in the pathogenesis of bronchial asthma has been emphasized by the results of experimental studies in animals and genetic studies in humans. In sensitized mice, blockade of the IL-4R inhibited development of airway hyperresponsiveness after allergen challenge, whereas in a similar experiment, blockade of endogenous IL-13 reversed airway hyperresponsiveness and pulmonary mucous cell hyperplasia.15,16 Polymorphisms in the promoter regions that affect
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expression of both the ␣ subunit of the IL-4R and the ligand of this receptor, IL-13, are associated with an increased risk of developing allergic asthma.17 Several immunologic changes have been observed in patients undergoing specific immunotherapy (SIT). An increase in the concentration of serum-specific IgG1 and IgG4, a blunted typical seasonal rise in IgE, and, with sustained therapy, a decrease in serum-specific IgE concentration have all been reported in patients undergoing SIT.18,19 Mononuclear cells derived from patients receiving SIT have been shown to produce less IL-4 and histamine-releasing factor but increased amounts of IFN-␥ on allergen challenge.20 –22 Finally, many SIT-induced changes in the function of the effector cells of the allergic response have been described, including a decrease in both basophil sensitivity and reactivity to specific allergen challenge.21–26 In grass-sensitive hay fever patients after 4 years of SIT, increased production of IL-12 by skin macrophages has been demonstrated.27 Peripheral blood monocytes derived from grass-sensitive patients who underwent SIT are less sensitive to IL-4 –mediated suppression of IL-12 production.28 The latter effect is observed after 6 weeks of SIT, being associated with down-regulation of CD124 expression on peripheral blood monocytes.27,28 Relatively little is known about monocyte and macrophage function in patients with asthma undergoing SIT. In one study, 12 months of SIT with house dust mite allergen extract resulted in decreased in vitro production of IL-1 and TNF-␣ by Dermatophagoides pteronyssinus allergen–stimulated peripheral blood monocytes.29 The aim of this study was to evaluate the expression of IL-4R␣ on peripheral blood monocytes and sIL-4R in the serum of patients with bronchial asthma receiving SIT. Furthermore, the expression of 2 monocyte surface proteins, CD14 and CD36, which is regulated by IL-4R signaling, was also evaluated.30,31 MATERIALS AND METHODS Seventeen patients with bronchial asthma and allergic rhinitis were enrolled in this study. All patients had a history of perennial allergic rhinitis for at least 12 months and episodes of dyspnea, cough, and wheezing, especially on dust exposure. They had positive skin prick test results to D pteronyssinus. Resting forced expiratory volume in 1 second (FEV1) in all patients was greater than 75% of the predicted value, but all patients demonstrated significant bronchial reactivity to histamine and significant bronchoconstriction on bronchial
challenge with D pteronyssinus extract (Table 1). Most patients studied had already been treated with intranasal steroids before initial evaluation in our allergy clinic; therefore, we did not perform an intranasal allergen challenge. Five of the 17 patients declined to undergo SIT and served as a comparator control group. Ten healthy volunteers were used as a negative control group. They had negative skin prick test results to commonly encountered aeroallergens (house dust mites, trees, weeds, grasses, cat, Alternaria, and Cladosporium). Their lung function test results were within normal limits, and they did not demonstrate significant bronchial reactivity to histamine (provocation concentration that caused a decrease in FEV1 of 20%, ⬎32 mg/mL). No bronchial challenge with D pteronyssinus extracts was performed in the negative control group. Histamine bronchial challenge was performed according to the method of Ryan et al.32 Briefly, all patients and controls inhaled doubling concentrations of histamine starting from a concentration of 0.62 mg/mL. Aerosol was generated using a nebulizer (DeVilbis 646; DeVilbis, Somerset, PA) attached to a Rosenthal-French dosimeter. All subjects performed 5 inspiratory-capacity breaths of the given histamine concentration. Forced expiratory maneuvers were performed 90 seconds after each fifth inhalation. The procedure was continued until either at least a 20% fall in FEV1 or a histamine concentration of 32 mg/mL was reached. A bronchial provocation test with aqueous D pteronyssinus extracts (Allergopharma Joachim Ganzer KG, Reinbek, Germany) was performed according to the method of Cockcroft et al.33 Increasing doses of allergen (0.8, 4, 20, 100, 500, and 2,000 SBE) were administered using a DeVilbis 646 nebulizer attached to a Rosenthal-French dosimeter. Forced expiratory maneuvers were performed 15 minutes after inhalation of each dose of the allergen extract. Allergen inhalations were continued until either at least a 20% fall in FEV1 or a cumulative dose of 5,000 SBE was reached. Then, FEV1 was measured every 15 minutes during the first hour, every hour during the next 11 hours, and after 24 hours. The early response was evaluated as a dose of allergen causing a 20% drop in FEV1. The late response was considered positive whenever 2 consecutive records of FEV1 demonstrated at least a 15% fall observed between the third and twelfth hours of the test. Concentration of serum-specific anti-D pteronyssinus IgE was evaluated using an enzyme-linked immunosorbent assay test (Allergopharma).
Table 1. Patient Characteristics* Group
N
SIT
PC20, mg/mL
PD20, SBE
Total IgE, IU/mL
Specific anti-D pteronyssinus IgE, IU/mL
Asthma Asthma Control
12 5 10
Yes No No
2.5 ⫾ 3.6 2.9 ⫾ 2.1 ⬎32
872 ⫾ 955 965 ⫾ 723 ND
545.6 ⫾ 331 425.7 ⫾ 385 35 ⫾ 29
15.4 ⫾ 13.2 17.7 ⫾ 15.2 ND
Abbreviations: FEV1, forced expiratory volume in 1 second; ND, not determined; PD20, provocative dose that caused a 20% fall in FEV1; PC20, provocation concentration that caused a decrease in FEV1 of 20%; SIT, specific immunotherapy. * Data are given as mean ⫾ SD.
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Figure 1. Representative flow cytometer (FACS, Becton-Dickinson, Franklin Lakes, NJ) analysis of blood samples from a control person (A and D), a patient with asthma who underwent specific immunotherapy (SIT) (B and E), and a patient with asthma who did not undergo SIT (C and F). The analyses using labeled monoclonal antibodies anti-CD14 fluorescein isothiocyanate (FITC) with anti-CD124 phycoerythrine (PE) (A-C) or anti-CD36 FITC with anti-CD14 PE (D-F) were performed for the control patient on 2 separate occasions and for the patients with asthma before and after a 3-month period, which in patients who received SIT corresponded to the period necessary to reach the maintenance dose of allergen extract.
During the study, all patients were allowed to use intranasal steroids and inhaled 2-agonists for symptom control. SIT was performed using a standardized, aqueous Dermatophagoides extract (Novo Helisen Depot, Allergopharma). The initial dose in all patients was 5 therapeutic units. The dose was doubled every week until the maintenance dose (5,000 therapeutic units) was achieved. Blood samples were taken before therapy and 1 week after administration of the first maintenance dose. In patients with asthma who did not undergo SIT, blood samples were obtained before bronchial inhalation challenge tests and after 3 months. Flow cytometry analysis was performed on whole blood samples using labeled monoclonal antibodies, including anti-CD14 fluorescein isothiocyanate and anti-CD124 phycoerythrine (PE; BD Biosciences Pharmingen, San Diego, CA). In addition, in 13 patients with asthma and 7 controls,
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flow cytometry was performed using labeled monoclonal antibodies, including anti-CD14 PE and anti-CD36 fluorescein isothiocyanate (BD Biosciences Pharmingen). Briefly, an EDTA anticoagulated blood sample was drawn from the antecubital vein and was immediately used for staining. Fiftymicroliter samples of whole blood that contained 2 to 4 ⫻ 105 leukocytes were incubated with 10 L of each monoclonal antibody solution according to manufacturer instructions. Matched labeled anti-idiotype antibodies were used as negative controls. After 30 minutes of incubation at room temperature, erythrocytes were lysed using a set of 3 wholeblood lysing reagents (ImmunoPrep Reagent System, Coulter Corp, A Beckman Coulter Co, Miami, FL). The remaining white blood cells were analyzed by flow cytometry (Coulter). Monocytes were selected based on their typical morphologic features—side scatter and forward scatter—and also based on
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the presence of the surface protein CD14. Expression of the studied surface proteins was evaluated as the percentage of monocytes staining positively with relevant labeled antibodies and also as the mean fluorescence intensity in relative fluorescence units (FU). Soluble IL-4Rs in the serum were evaluated using an enzyme-linked immunosorbent assay method (Quantikine, R&D Systems, Minneapolis, MN). The test sensitivity was less than 5 pg/mL, and the testing range was 15.6 to 1,000 pg/mL. Statistical analysis was performed using analysis of variance. The results are expressed as mean ⫾ SD. RESULTS Patients with bronchial asthma were characterized by higher total serum IgE levels and significant bronchial reactivity to histamine (Table 1). There was no significant difference in bronchial reactivity or total or specific serum IgE concentrations between asthma patients who underwent SIT and those who did not. In asthma patients, the mean number of monocytes, selected on the basis of their morphologic features, with expression of CD14 was 324 ⫾ 124 cells/L, which was not different from that found in controls (307 ⫾ 88 cells/L) (P ⫽ .71). The mean density of CD14 on individual cells evaluated as mean fluorescence intensity was lower in patients with asthma vs the negative control group, but the difference was not statistically significant (7.58 ⫾ 5.3 vs 12.5 ⫾ 8.4 FU; P ⫽ .07; Figs 1 and 2). In the negative control group, only 3.5% ⫾ 1.8% of CD14-positive cells expressed CD124 (Figs 1 and 2). In asthma patients who underwent SIT, 17.9% ⫾ 8.7% of peripheral blood monocytes expressed CD124 on their surface, which was significantly greater than that in the control group (P ⬍ .01) but not significantly different from that observed in patients with asthma who did not undergo SIT (22.7% ⫾ 21.2%; P ⫽ .51; Figs 1 and 3). One week after administration of the maintenance dose of house dust mite allergen extract, the mean percentage of CD14-positive cells coexpressing CD124 was significantly lower than before SIT (17.9% ⫾ 8.7% vs 8.3 ⫾ 5.4%; P ⬍ .01; Figs 1 and 3). Furthermore, in the 5 patients who declined SIT, no significant change in CD124 expression on peripheral blood monocytes was observed (Figs 1 and 3). In asthma patients, the density of CD124 on the cell surface, evaluated as mean fluorescence intensity, was also significantly higher than in the negative controls (2.6 ⫾ 1.4 vs 1.1 ⫾ 0.6 FU, respectively; P ⬍ .01). In patients with asthma who underwent SIT, mean fluorescence intensity of CD124 fell from 2.56 ⫾ 1.13 FU to 1.65 ⫾ 0.53 FU (P ⬍ .01; Figs 1 and 3). No change in CD124 density was detected in asthmatic patients who did not undergo SIT (Figs 1 and 3). The mean percentage of monocytes expressing CD14 after SIT (87.4% ⫾ 4.4%) was not statistically different from the baseline value (86.9 ⫾ 6.2; P ⫽ .82), but a significant increase in mean density of CD14 on monocyte cell surfaces was found after SIT (from 7.58 ⫾ 5.2 FU to 12.8 ⫾ 6.06 FU; P ⫽ .01; Figs 1 and 3).
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Figure 2. Comparison of the expression of CD14, CD36, and CD124 on peripheral blood monocytes in patients with asthma and controls shown as the mean percentage of monocytes expressing each surface protein (A) and as mean fluorescence intensity (B). Error bars represent SD.
Another monocyte surface receptor, CD36, was found on a similar number of monocytes in patients with asthma vs negative controls (85.8% ⫾ 6.8% vs 81.0% ⫾ 6.17%; P ⫽ .08; Figs 1 and 3). The mean density of CD36 on individual cells was higher in patients with asthma but not significantly different than in negative controls (12.9 ⫾ 8.13 vs 7.2 ⫾ 4.5 FU; P ⫽ .05). After 3 months of SIT, the percentage of monocytes expressing CD36 decreased from 86.4% ⫾ 6.8% to 79.8% ⫾ 5.9% (P ⫽ .02) and fluorescence intensity of CD36 decreased from 13.3 ⫾ 9.16 to 6.9 ⫾ 4.6 FU (P ⫽ .01). Neither mean percentage of CD36-expressing cells nor density of CD36 expression changed after 3 months in asthmatic patients who were not receiving SIT (Figures 1 and 3). There was no significant difference in the concentration of sIL-4R in serum samples from patients with asthma who underwent SIT vs those who did not undergo SIT (43.2 ⫾ 12.4 vs 38.1 ⫾ 14.1 pg/mL; P ⫽ .47) and vs controls (43.2 ⫾ 12.4 vs 29.1 ⫾ 14.5 pg/mL; P ⫽ .27; Fig 4). Significant changes in the concentration of the sIL-4R in serum samples were not observed in patients with asthma undergoing SIT (from 43.2 ⫾ 12.5 to 36 ⫾ 10.5 pg/mL; P ⫽ .17) or in
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Figure 3. Mean changes in the expression of CD14 (A and B), CD124 (C and D), and CD36 (E and F) on peripheral blood monocytes in patients with asthma who did and did not receive specific immunotherapy (SIT). Error bars represent SD. Both mean percentage of monocytes expressing each surface protein (A, C, and E) and mean fluorescence intensity (B, D, and F) are presented.
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Figure 4. Mean changes in serum concentrations of soluble interleukin 4 receptor (sIL-4R) in patients with asthma who did and did not undergo specific immunotherapy (SIT). NS indicates not significant. Error bars represent SD.
asthmatic patients who did not receive SIT (from 38.1 ⫾ 14.1 to 39.7 ⫾ 15.2 pg/mL; P ⫽ .87; Fig 4). DISCUSSION In patients with bronchial asthma, elevated production of IL-4 and IL-13 by T cells has been documented.1 Concomitant increased expression of IL-4R␣ on peripheral blood monocytes in patients with asthma may be responsible for the increased effect of IL-4 and IL-13 on these cells. In T and B cells, IL-4 has been shown to increase the expression of its own receptor.34 If this is also true for mononuclear phagocytes, increased expression of IL-4R␣ subunit might be a result of increased IL-4 or IL-13 production by T cells and possibly mast cells and basophils. One of the results of this interaction could be decreased production of IL-12 by mononuclear phagocytes. Decreased production of IL-12 by mononuclear phagocytes has been described in patients with asthma.2 Decreased production of IL-12 results in decreased production of IFN-␥ and promotion of TH2-type immune responses. In allergic patients, increased expression of CD124 has been demonstrated on peripheral blood monocytes and on granulocytes, including basophils.35,36 In grasspollen allergic patients, increased expression of CD124 on peripheral blood monocytes was associated with decreased production of IL-12 and IFN-␥ by phytohemagglutinin-stimulated mononuclear cells.28,35 Furthermore, exogenous IL-4 more efficiently suppressed IL-12 production in patients with grass-pollen allergy, indicating that increased CD124 expression is associated with enhanced function of the IL-4 –IL-4R system in allergic patients.28 Up to now, little information has been available concerning the effect of SIT on CD124 expression on circulating leukocytes. In patients allergic to Hymenoptera venoms who underwent venom immunotherapy, a significant decrease in the expression of CD124 on
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peripheral blood basophils was observed 1 week after the end of rush immunotherapy.36 In patients with grass-sensitive hay fever who received SIT, the expression of CD124 on peripheral blood monocytes decreased already after 6 weeks of SIT.28 Furthermore, monocytes derived from the patients who underwent SIT were less sensitive to IL-4 –mediated suppression of IL-12 production.28 Relatively little is known about the exact mechanisms responsible for the IL-4/IL-13–mediated inhibition of IL-12 production by mononuclear phagocytes in allergic patients and the possible effect of SIT on this cytokine network. As observed in this study, up-regulation of CD14 during SIT may represent a possible mechanism to increase the susceptibility of monocytes to release proinflammatory cytokines, including IL-12. Lipopolysaccharide, which is a ligand of CD14, has been shown to be a very potent stimulus of IL-12 release by peripheral blood monocytes.2,5 Changes in IL-4/ IL-13 receptor signaling may be directly involved in the regulation of CD14 and CD36 expression on peripheral blood monocytes in patients undergoing SIT because IL-4 and IL-13 have been shown to up-regulate CD36 expression and down-regulate CD14 expression on these cells.30,31 Several intracellular molecules are involved in IL-4/IL-13 receptormediated signaling. One of the best characterized is the signal transducer and activator of transcription 6, which, when phosphorylated, represses the biological actions of IFN-␥–activated signal transducer and activator of transcription 1 and TNF-␣–activated nuclear factor–B.37 Furthermore, it has recently been demonstrated that the IL-4/IL-13 receptor mediates up-regulation of peroxisome proliferator activator receptor gamma (PPAR-␥) and induces synthesis of its natural ligands.31 Activation of PPAR-␥, in turn, inhibits the proinflammatory effects of nuclear factor–B and inhibits IL-12 production by stimulated monocytes.38 These 2 mechanisms may be potentially involved in IL-4R–mediated suppression of IL-12 production. During SIT there is down-regulation of CD36, the expression of which is PPAR-␥ dependent, supporting the hypothesis that this intracellular molecule may also be involved in the IL-4R–mediated effects seen in patients with asthma.31,38 Although changes in cell surface CD124 expression have been demonstrated, there is little information on how the potential activation of this receptor occurs. The presence of sIL-4Rs at significant concentrations in the peripheral blood of both negative controls and patients with asthma argues against an endocrine action of IL-4. A potential paracrine action of IL-4 released from T cells during the T cell– monocyte interaction cannot be excluded. Furthermore, IL13, the action of which mimics the action of IL-4 on monocytes, also uses CD124 as a part of the IL-13 receptor yet is not inhibited by sIL-4R. Further studies are needed to clarify whether the change in CD124 expression reflects changes in monocyte function and whether this phenomenon is transient or persists throughout SIT, perhaps constituting an important mechanism responsible for the beneficial clinical effects of SIT.
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34. Renz H, Domenico J, Gelfand EW. IL-4 dependent upregulation of IL-4 receptor expression in murine T and B cells. J Immunol. 1991;146:3049 –3055. 35. Grzela K, Grzela T, Lazarczyk D, et al. CD124 on monocytes in grass pollen allergy. Allergy. 2001;57:254 –255. 36. Siegmund R, Vogelsang H, Machnik A, Hermann D. Surface membrane antigen alteration on blood basophils in patients with Hymenoptera venom allergy under immunotherapy. J Allergy Clin Immunol. 2000;106:1190 –1195. 37. Ohmori Y, Hamilton TA. Interleukin-4/STAT6 represses STAT1 and NF-B– dependent transcription through distinct mechanisms. J Biol Chem. 2000;275:38095–38103.
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38. Azuma Y, Shinohara M, Wang PL, Ohura K. 15-Deoxy␦(12,14)-prostaglandin J2 inhibits IL-10 and IL-12 production by macrophages. Biochem Biophys Res Commun. 2001;283: 344 –346.
Requests for reprints should be addressed to: Lawrence DuBuske, MD 358 Elm St Gardner, MA 01440 E-mail: [email protected]
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