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Keywords:

  • allergy;
  • dendritic cell;
  • immune regulation;
  • specific immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

To cite this article: Zhao C-Q, Li T-L, He S-H, Chen X, An Y-F, Wu W-K, Zhou X-H, Li P, Yang P-C. Specific immunotherapy suppresses Th2 responses via modulating TIM1/TIM4 interaction on dendritic cells. Allergy 2010; 65: 986–995

Abstract

Background:  Specific immunotherapy (SIT) is the only curable remedy for allergic disorders currently; however, the underlying mechanism is not fully understood yet. This study aimed to elucidate the mechanism of SIT on suppressing TIM4 (T cell immunoglobulin mucin domain molecule 4) expression in dendritic cells (DCs) and modulating the skewed T helper 2 (Th2) responses in patients with airway allergy.

Methods:  Twenty patients with allergic rhinitis (AR) were treated with SIT for 3 months. Before and after SIT, the expression of TIM4 in peripheral DC and TIM1 in Th2 cells was examined. The role of Fc gamma receptor (FcγR) I and II in modulating the expression of TIM4 in DCs was investigated.

Results:  The interaction of TIM1/TIM4 played a critical role in sustaining the polarization status of Th2 cells in AR patients. Cross-linking FcγRI by antigen/IgG complexes increased the production of TIM4 by dendritic cells via upregulating tumor necrosis factor-alpha in DCs. Exposure to microbial products promoted the expression of FcγRI in DCs that further increased the expression of TIM4. Exposure to specific antigens alone upregulated the expression of FcγRII in DCs, that suppressed the expression of TIM4.

Conclusions:  We conclude that SIT suppresses the skewed Th2 responses via disrupting the interaction of TIM1/TIM4 in antigen-specific Th2 cells.

The prevalence of allergic diseases, such as asthma, allergic rhinitis (AR), and allergic dermatitis, keeps rising worldwide (1, 2). The main immune pathogenic features of allergic diseases include T helper 2 (Th2) polarization, elevated serum IgE levels, profound infiltration of eosinophils and mastocytosis in local tissue (1, 3, 4). In fact, allergic diseases have merged as a major health problem in the world. Although research on allergic diseases advanced rapidly in recent years, the etiology of allergic diseases remains unknown.

It is still an enigma how antigen-specific Th2 cells get skewed polarization and how they maintain a dominant status. The T-cell immunoglobulin and mucin domain molecule (TIM)1 is a newly described molecule that plays a critical role in Th2 cell development (5, 6). However, whether TIM1 expressed by Th2 cells has the capacity maintaining functional status by itself remains unclear. TIM4 is the natural ligand of TIM1 (6). It is expressed by dendritic cells (DCs) (6); the expression can be upregulated drastically upon activation (7–9). Dendritic cell-derived TIM4 can bind TIM1 on CD4+ T cells and drive them to develop into Th2 cells. Still, whether the interaction of TIM1/TIM4 plays a role in the maintenance of Th2 polarization as shown in allergic disorders is to be further understood.

Dendritic cells express Fc gamma receptor (FcγR) including FcγRI, FcγRII, and FcγRIII (10, 11). FcγRs recognize antigen/IgG complexes on the surface of DCs (12). Ligating these receptors by their ligands, IgG/Ag complexes, induces intracellular signaling and various effector responses such as antibody-dependent cellular cytotoxicity, mast cell degranulation, and phagocytosis (12–15). However, whether activation of FcγRs is involved in the expression of TIM4 in DCs is unknown.

It is proposed that production of antigen-specific IgG during specific immunotherapy (SIT) is the major effector molecule to ameliorate allergic symptoms (16, 17); however, how the IgG suppress polarized Th2 responses is less understood. Although we have known that IgG can bind FcγRs to initiate or inhibit cellular activities of FcγR-bearing cells (18), whether the interaction of IgG/FcγR is involved in therapeutic effect of SIT is not clear. As DCs are the master modulator of immune responses, DCs express FcγRI and FcγRII (10, 12), we hypothesize that SIT-induced antigen-specific IgG ligates FcγRs on DCs and modulates the skewed Th2 responses subsequently.

In this study, with isolated peripheral CD4+ T cells and DCs from patients with AR as study platforms, we were able to examine the kinetics of TIM1 expression in Th2 cells. TIM1 expression had the feature of natural decay that could be prevented in the presence of TIM4. The SIT had the capacity to suppress both TIM1 and TIM4 expression. Exposure to microbial products was involved in the regulation of FcγR expression in DCs that further modulated the expression of TIM4.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

Diagnosis of AR and SIT

Patients with AR were recruited into this study at outpatient of the Department of Otolaryngology, Head and Neck Surgery of Shanxi Medical University. Informed consent was obtained from each patient. Using human material in this study was approved by the Human Study Ethic Committee at Shanxi Medical University. Diagnosis of AR specific to Der and treatment with Der (Dermatophagoides pteronyssinus 1, the major allergen of dust mite) SIT were based on our routine procedures (SIT was performed subcutaneously). Patients using anti-allergy drugs within the previous 4 weeks and those with documented allergy to non-Der allergens were excluded. The demographic data were presented in Table S1, in supporting information.

Flow cytometry

Cells were stained with fluorescently labeled antibodies on ice for 30 min. The stained cells were analyzed by a FACSarray (BD Bioscience, San Jose, CA, USA). Data were analyzed by software flowjo (FlowJo flow cytometry analysis software; Tree Star, Inc., Ashland, OR, USA).

Real-time quantitative reverse transcription polymerase chain reaction (qPCR)

Total RNA was extracted from the DCs using an RNeasy Mini kit (Qiagen, Mississauga, ON, Canada). cDNA was synthesized using iScriptTMcDNA Synthesis kit (Bio-Rad, Mississauga, ON, Canada). The resulting cDNA was subjected to qPCR that was performed with a LightCycler using a SuperScript III Platinum SYBR Green Two-Step qPCR kit (Invitrogen, Burlington, ON, Canada). The amplified product was detected by the presence of an SYBR green fluorescent signal. The standard curve was designed with β-actin cDNA. The resulted amplicon was quantified with the standard curve. The primers are listed in supporting information.

Co-immunoprecipitation and Western blotting

Total proteins were extracted from cultured DCs. The immune complexes were formed with antibodies against Der, IgG, FcγRI, or FcγRII and 100 μl extracts for 12 h at 4°C. The rest procedures were the same as we reported (12). The membranes were then blotted with antibodies against Der, IgG, FcγRI, or FcγRII respectively. The proteins were detected using horseradish peroxidase conjugated second antibodies. Horseradish peroxidase enzymatic reaction was detected with enhanced chemiluminescent reagents and recorded with X-ray films.

Enzyme-linked immunosorbent assay (ELISA)

Cytokine and IgE levels were determined by ELISA from the instruction of reagent kits.

T-cell proliferation assay

Isolated CD4+ T cells were cultured in the presence or absence of specific antigen, Der with radiated DCs for 3 days. [3H] thymidine (1 μCi per well) was added for the last 16 h. The [3H] thymidine incorporation was detected by a scintillation counter.

Statistic analysis

Data are presented as the means ± SD. Differences between two groups were evaluated with the Student’s t-test; data among three or more groups were evaluated with anova. Bonferroni adjustment was applied to post hoc group comparisons when required. A P < 0.05 was accepted as a significant criterion.

The procedures of RNA interference (RNAi), LightShift chemiluminescent electrophoretic mobility shift assay, chromatin immunoprecipitation assay, symptom scoring, serum antibody measurements, and details of reagents were presented in supporting information.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

Expression of TIM1 in polarized human Th2 cells

It is reported that Th2 cells express TIM1 that has the costimulatory activity to promote Th2 cell proliferation (5–7, 19). However, whether the expression of TIM1 on Th2 cells is stable or whether the expression requires support from other molecules is not clear. With isolated peripheral CD4+ CD25+ T cells from 20 patients with AR, ratio of interleukin (IL)-4+ T cells was analyzed by flow cytometry. The results showed that IL-4+ T cells was markedly more in patients with AR (>50%) (Fig. 1Aa) than in healthy controls (20 subjects) (<20%) (Fig. 1Ba). Using flow cytometry gating technique, we further analyzed TIM1 expression in IL-4+ T cells. Significantly more TIM1+ IL-4+ T cells were detected in patients with AR (Fig. 1Ab) when compared with healthy subjects (Fig. 1Bb). CCR3 was proposed as a specific marker on Th2 cells of patients with allergic diseases (20). We thus analyzed the expression of CCR3 on IL-4+ T cells. As shown by flow cytometry data, more than 80% IL-4+ T cells was CCR3+ in patients with AR (Fig. 1Ac), whereas 5% IL-4+ T cells was also CCR3+ in healthy subjects (Fig. 1Bc).

image

Figure 1.  T-cell immunoglobulin mucin domain molecule (TIM)1 is required in the maintenance of antigen-specific T helper 2 response. (A) and (B), peripheral CD4+ CD25+ T cells were isolated from 20 patients with allergic rhinitis (AR) (A) 20 healthy subjects (B) by magnetic cell sorting. The isolated cells were stained with antibodies against interleukin (IL)-4, TIM1, and CCR3, and analyzed by flow cytometry. Panels Aa and Ba show IL-4+ T cells (the gated cells). Panels b and c in A and B show TIM1+ cells (b) and CCR3+ cells (c) in gated cells of A and B. (C) CD4+ CD25+ T cells from patients with AR were cultured for 0–5 days. Flow cytometry histograms show TIM1+ cells (the right peaks). C1, cells were from healthy subjects. C2–C6, cells were from patients with AR. C2, day 0. C3–C6, cells were analyzed on day 5 (day 5). C4, addition of exogenous TIM4 to the culture. C5, added dendritic cells (DCs) and Der. C6, siDC: DCs were transfected with TIM4siRNA in the presence of Der. (D) Bars indicate the expression of TIM1 mRNA in cultured CD4+ CD25+ T cells that was analyzed by quantitative real-time reverse transcription polymerase chain reaction. The legend numbers are the same as those in panel C. (E) Western blots show the TIM1 protein contents in cellular extracts from cultured D4+ CD25+ T cells as described in panel C. The legend numbers are the same as those in panel C. (F) CD4+ CD25+ T cells were isolated from healthy subjects and patients with AR. Bars indicate the rate of T-cell proliferation representing by [3H] thymidine incorporation. (G) Bars indicate the levels of IL-4 and IFN-γ in culture media of CD4+ CD25+ T cells. Group 1: T cells were cultured for 5 days with media alone and then exposed to Der and DCs. Group 2: T cells were exposed to Der and DCs immediately after isolation. In E, F, and G, data are presented as the means ± SD from three separate experiments. Isotype IgG was employed as a control in each experiment; no positive staining was observed (data not shown). The control siRNA did not show any effect on target genes (data not shown).

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To clarify whether the expression of TIM1 in polarized Th2 cells was stable, another batch of CD4+ CD25+ T cells from the same group of patients with AR was cultured for 1–5 days. As shown by flow cytometry data, the rate of TIM1+ cells in total CD4+ CD25+ T cells decreased gradually that dropped down to control level on day 5 (Fig. 1C). The results were further confirmed by qPCR (Fig. 1D) and immunoblotting assay (Fig. 1E). The total cell number on day 0 and 5 was counted. The results showed that the total cell number increased 1.56 ± 0.66 times on day 5. The fact indicates that the reduction of TIM1 in CD4+ CD25+ T cells was not caused by natural dying of TIM1+ T cells, but caused by natural decay of TIM1 expression during the 5-day culture.

We next sought to determine whether the decay of TIM1 expression on Th2 cells was accompanied by suppression of Th2 response. CD4+ CD25+ T cells from the same group of patients with AR were divided to group 1 and group 2. Group 1 was cultured with media alone for 5 days and then exposed to specific mite antigen (Der) of these patients, and freshly isolated peripheral DCs (irradiated) for another 3 days. Group 2 was directly exposed to Der and DCs for 3 days. As shown by [3H] thymidine incorporation assay, T-cell proliferation was significantly higher in group 2 than in group 1 (Fig. 1F). Meanwhile, we also examined Th1 and Th2 cytokines in culture media. As expected, the levels of IL-4, but not IFN-γ, were markedly higher in group 2 when compared with group 1 (Fig. 1G).

DC-derived TIM4 contributes to the maintenance of skewed Th2 response

It is reported that TIM-1 is critical in Th2 response (21). The present data show a positive correlation between the expression of TIM1 and the maintenance of Th2 response (Fig. 1). Therefore, it is possible that interaction of TIM1/TIM4 is one of the approaches to sustain TIM1 activities in Th2 cells. Thus, we added exogenous TIM4 to the culture of another batch of CD4+ CD25+ T cells from patients with AR at a dose of 5 μg/ml (the media and reagents were changed on day 3). The results showed that TIM1 expression in CD4+ IL-4+ T cells kept high levels throughout the 5-day culture (Fig. 1C–E, item 4). The fact indicates that TIM4 has the capacity to sustain Th2 activities in the presence of TIM1.

Dendritic cells express TIM4 in response to various stimuli (7–9) that can be the endogenous source of TIM4. We then examined the effect of DC-derived TIM4 in the maintenance of TIM1 expression in Th2 cells. CD4+ CD25+ T cells from patients with AR were cocultured with DCs from the same patients in the presence of Der for 5 days. As expected, the expression of TIM1 in T cells as well as the Th2 responses was maintained at high levels (Fig. 1C–E, item 5). However, when the TIM4 gene was knocked down in DCs by RNAi, the expression of TIM1 could not be maintained in cocultured polarized Th2 cells (Fig. 1C–E, item 6).

Specific immunotherapy suppresses TIM4 expression in DCs of patients with AR

Currently, SIT is the only specific therapy for the treatment of allergic diseases (1, 2). The underlying mechanisms of SIT are not fully understood yet. From the data we got from interaction of TIM1/TIM4 (Fig. 1), we reasoned that the interaction of TIM1/TIM4 might be one of the targets in SIT. To test the hypothesis, 20 patients with AR with Der sensitization were treated with the Der desensitization therapy for 3 months. The results of ELISA showed that levels of serum IL-4 (Fig. 2A) and IL-4-secretion by CD4+ CD25+ T cells in culture (Fig. 2B) were significantly inhibited by SIT. The expressions of TIM4 in peripheral DCs and TIM1 in peripheral CD4+ CD25+ T cells were examined by flow cytometry before and after SIT. TIM4 expression in gated CD11c+ cells (Fig. 2C,D) and TIM1 expression in gated IL-4+ T cells (Fig. 2E,F) were also significantly decreased after the 3-month therapy (Fig. 2). The downregulation of TIM1 and TIM4 had a correlation with decreased Th2 responses. Levels of IFN-γ in culture media were not significantly changed.

image

Figure 2.  Specific immunotherapy (SIT) suppresses T-cell immunoglobulin mucin domain molecule (TIM)1/TIM4 and modulates T helper 2 response. Twenty patients with allergic rhinitis (AR) were treated with SIT for 3 months. Serum levels of interleukin (IL)-4 and IFN-γ were determined by enzyme-linked immunosorbent assay (ELISA) (A). Peripheral CD4+ CD25+ T cells were isolated and cultured with or without specific antigen Der for 3 days in the presence of dendritic cells (DCs) from the same patients. Levels of IL-4 and IFN-γ in culture media were determined by ELISA (B). Data are presented as the means ± SD. *P < 0.05, compared with healthy group. (C) and (D), peripheral mononuclear cells were isolated from the patients with AR and healthy subjects. Cells were stained with anti-CD11c and anti-TIM4 and analyzed by flow cytometry. The flow cytometry dot plots show DCs (the gated cells). (D) The histograms show TIM4+ DCs within the gated DCs (pointed by arrows). (E) and (F), peripheral CD4+ CD25+ T cells were isolated from the patients with AR and healthy subjects. Cells were stained with anti-IL-4 and anti-TIM1 and analyzed by flow cytometry. The flow cytometry dot plots show IL-4+ T cells (the gated cells). (F) the histograms show TIM1+ T cells within the gated DCs (pointed by arrows). AR: samples were taken from patients with AR. Before (or after): before (or after) SIT. (C–F), the digital numbers: 1, healthy controls; 2, patients with AR before SIT; 3, patients with AR after SIT. Mean ± SD was presented in each flow cytometry graph.

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In addition, we also examined the serum levels of Der-specific IgE and IgG4 before and after SIT. The AR clinical symptom scores were documented. The results indicated that SIT significantly suppressed the Der-specific IgE and increased Der-specific IgG4 levels in the sera (Fig. S1) as well as the clinical score (Fig. S2). Correlation assay was performed between the expression of TIM4 in peripheral DCs and serum Der-specific IgE (TIM4 and IgE), TIM4 and IgG4, TIM4 and symptom score respectively. The results showed significant correlation between TIM4 and IgE (r = 0.58, P < 0.05), TIM4 and IgG4 (r = −0.71, P < 0.01, TIM4 and symptom score (r = 0.53, P < 0.05).

Interaction of antigen-specific IgG and FcγRII downregulates TIM4 production by DCs

As shown by Figs 1 and 2, SIT reduced the expression of TIM4 on DCs and TIM1 on Th2 cells of patients with AR. We further explored the underlying mechanism. It is proposed that increase in antigen-specific IgG produced in SIT plays a major role in the amelioration of allergic symptoms (21, 22). However, the targets of antigen-specific IgG still remain obscure. As DCs express FcγRs that recognize IgG to form complexes on the surface (23), we hypothesized that SIT produced antigen-specific IgG bound FcγRs to prime DCs. Re-exposure to specific antigens, such as those using in SIT, activated DCs to suppress the production of TIM4 by DCs. To test the hypothesis, we prepared DCs from the peripheral mononuclear cells of patients with AR and healthy controls with our established protocols (the purity of DCs was over 96.5% as examined by flow cytometry). The first attempt was to identify the complexes of Der/IgG/FcγR on DCs. DC cellular extracts were analyzed by coimmunoprecipitation assay with antibodies against Der, IgG, and FcγRs (including FcγRI, FcγRII and FcγRIII) respectively. The separated proteins on membranes were blotted by antibodies against Der, IgG, FcγRI, or FcγRII. As shown in Fig. 3, before SIT, the abundance of Der/IgG/FcγRI was significantly higher in AR group than healthy group; less Der/IgG/FcγRII was found, whereas FcγRIII was under detectable level (data not shown). The examination was carried out again after the 3-month SIT with DCs obtained from the same group of patients. The Der/IgG/FcγRII complex was increased significantly, whereas the complex of Der/IgG/FcγRI was decreased on DCs. Using as a comparison, another group of six patients with AR with Der sensitization were treated with Veramyst (a nasal steroid spray) alone for 3 months. Complexes of Der/IgG/FcγRI and Der/IgG/FcγRII were also detected in isolated peripheral DCs from these patients, which were similar to SIT group (before SIT treatment); but no detectable changes in the immune complexes were identified after treatment with Veramyst (data not shown).

image

Figure 3.  Specific immunotherapy modulates expression of Fc gamma receptors (FcγRs) on dendritic cells (DCs). Isolated peripheral mononuclear cells were cultured in the presence of granulocyte colony-stimulating factor (20 ng/ml) for 3 days. DCs were isolated and cultured in the presence of Der (10 μg/ml) for 6 h. The cellular protein extracts of DCs were subjected to coimmunoprecipitation assay to identify the Der-specific IgG/FcγR complex. Immune blots show FcγRI or FcγRII in DC cellular extracts precipitated by antibodies against IgG, FcγRI, or FcγRII.

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To understand whether the complex of antigen/IgG/FcγR plays any roles in TIM4 expression in DCs, TIM4 protein in isolated peripheral DCs were analyzed by Western blotting with the same set of antibodies in Fig. 3 and anti-TIM4. Indeed, we detected TIM4 in DC cellular extracts that was positively correlated with the amount of FcγRI and negatively correlated with the amount of FcγRII in samples before and after SIT (Fig. 4).

image

Figure 4.  Specific immunotherapy (SIT) suppresses T-cell immunoglobulin mucin domain molecule 4 (TIM4) in dendritic cells (DCs). Peripheral DCs were isolated from healthy subjects or patients with allergic rhinitis (AR) before and after SIT. Cellular protein extracts of DCs were analyzed by Western blotting to evaluate the expression of Fc gamma receptor (FcγR)I, FcγRII, and TIM4. A. The immune blots show proteins of FcγRI, FcγRII, and TIM4 in DC cellular extracts. B. Bars indicate the data of densitometry of immune blots. Data were normalized to the percentage of β-actin blots (the internal control) and presented as mean ± SD from 12 patients with AR and eight healthy controls. *P < 0.05, compared with healthy control.

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Collectively, the results indicate that antigen-specific IgG can bind both FcγRI and FcγRII to form complexes. IgG/FcγRI complexes recognizing specific antigens increase the expression of TIM4, whereas IgG/FcγRII complexes recognizing specific antigens decrease the expression of TIM4 in DCs.

Microbial products modulate FcγRs on DCs

Dendritic cells express both FcγRI and FcγRII. Antigen-specific IgG has equal opportunity to bind either FcγRI or FcγRII on DCs. The mechanism by which antigen-specific IgG choose to bind to FcγRI or FcγRII is unclear. Prompted by our previous studies that microbial products are involved in the initiation of Th2 responses (8, 19), we postulated that microbial products promotes FcγRI activities in DCs that further promotes the expression of TIM4 in DCs. Thus, we next exposed naïve human DCs (generated from peripheral mononuclear cells) or exposed DCs from patients with AR to Der or microbial products [lipopolysaccharide (LPS), staphylococcal enterotoxin B, or cholera toxin] in culture for 48 h. As shown by qPCR and Western blotting, microbial products markedly increased the expression of FcγRI and TIM4 in DCs, while Der increased FcγRII but not TIM4 in DCs (Fig. 5A,B). Because exposure to microbial products induces DCs to produce IL-12 and TNF-α that play important roles in immune inflammation (24), the autocrine IL-12 and TNF-α in DCs might be involved in the expression of FcγRs and TIM4. Thus, in further experiments, DCs were transfected with IL-12 small interference RNA (siRNA) or TNF-α siRNA prior to exposure to microbial products in separate experiments. The results showed that TNF-α-deficient DCs did not express TIM4 in response to stimulation of microbial products, whereas those IL-12-deficient DCs still had the capacity to produce TIM4 upon exposure to microbial products (Fig. 5C). To further confirm the role of TNF-α on TIM4 expression in DCs, a batch of DCs was exposed to TNF-α at graded doses for 48 h. The results showed that TIM4 was expressed by DCs in a TNF-α dose-dependent manner (Fig. 5D).

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Figure 5.  Microbial products modulate T-cell immunoglobulin mucin domain molecule 4 (TIM4) expression in dendritic cells (DCs). Isolated DCs were exposed to lipopolysaccharide (LPS, 1 μg/ml), staphylococcal enterotoxin B (SEB, 5 μg/ml), or cholera toxin (CT, 5 μg/ml) for 24 h. (A) Bars indicate TIM4 mRNA level in DCs that was determined by quantitative real-time reverse transcription polymerase chain reaction. Data were presented as the percentage of β-actin (mean ± SD from three separate experiments). Iso: isotype IgG. (B) Immune blots show proteins of Fc gamma receptor (FcγR)I, FcγRII, and TIM4 in isolated DCs after exposure to microbial products for 24 h (the presented data were from CT-stimulated DCs; data from LPS- or SEB-stimulated DCs were not shown). (C) Immune blots show TIM4 protein in DCs after exposure to microbial products (CT). Interleukin (IL)-12 (or TNFα): IL-12- (or TNFα-)deficient DCs [genes were knocked down by RNA interference (RNAi)]. (D) Immune blots show TIM4 protein in DCs after exposure to exogenous TNF-α. (E) Immune blots show TIM4 protein in DCs after cross-linking by IgG. FcγRII (or FcγRI): FcγRII- (or FcγRI-)deficient DCs (genes were knocked down by RNAi).

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Alternatively, a batch of peripheral DCs was cultured in IgG (100 μg/ml)-coated plates for 48 h to cross-link the FcγRs on DCs. Unexpectedly, no increase in TIM4 was detected in these DCs. Considering both FcγRI and FcγRII might be cross-linked simultaneously in response to the exposure to nonspecific IgG, DCs with FcγRI- or FcγRII-knocked down were cultured in IgG (100 μg/ml)-coated plates for 48 h. The results showed that abundant TIM4-expression was detected in FcγRII-deficient DCs, but not in those FcγRI-deficient DCs (Fig. 5E). The fact further confirmed that the FcγRI activities were involved in TIM4 expression in DCs, whereas the FcγRII activities inhibit TIM4 expression in DCs.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

The present study reports several novel findings from a study with human specimens in which we found that the expression of TIM1 in polarized Th2 cells from patients with AR decayed quickly when cultured in vitro. The decay of TIM1 expression in Th2 cells correlated with the decrease in Th2 responses; addition of TIM4 stabilized the TIM1 expression. Dendritic cell-derived TIM4 could be one of the endogenous sources of TIM4 in the body. We also found that SIT increased the activities of FcγRII, whereas microbial products increased FcγRI on DCs; interaction between specific antigen, specific IgG and FcγR modulated the expression of TIM4 in DCs.

Antigen-specific Th2 polarization plays a critical role in the clinical attacks and the recurrence of allergic disorders (25). One of the characteristics of antigen-specific Th2 cells is that once developed, this cell population sustains in the body. Currently, we do not have any recognized remedies to eliminate these cells (25). The discovery of TIM1 expression in Th2 cells shed a new light in the study of the pathogenic mechanism of allergic diseases (5, 6). The present study reveals that TIM1 not only plays a critical role in the development of Th2 responses (5, 6) but also is important in the sustaining of antigen-specific Th2 responses as manifested that the natural decay of TIM1 expression correlates with the decreases in Th2 responses. Previous studies also showed that using blocking antibodies of TIM1 could suppress allergic responses in animal models (8, 19, 26, 27).

A correlation between the TIM1 expression in Th2 cells and allergic disorders was noted by several clinical studies (8, 28–34), so did the present study. The data show that over 90% CD4+ IL-4+ T cells in patients with AR express TIM1. TIM1 is also recognized in atopic dermatitis (28), AR (35), asthma (36), rheumatoid arthritis (37), and systemic lupus erythematosus (30). These findings indicate that TIM1 expression in Th2 cells of patients with immune diseases is relatively common; the fact implicates that there must have been one or more specific factors (34) in the body playing roles in the maintenance of TIM1 expression in Th2 cells under allergic environment. TIM4 is the natural ligand of TIM1 (6). It is possible that TIM4 is one of the factors to maintain TIM1’s function. This notion is supported by our previous study (8) in which we recognized that TIM4 favors the expression of TIM1 in CD4+ T cells. The present data further confirm that TIM4 is capable of maintaining the TIM1 expression in Th2 cells.

The endogenous source of TIM4 is gradually elucidated. Meyers et al. (6) found that DCs express TIM4. In response to microbial stimulation, the expression of TIM4 is highly strengthened, as we reported recently (8). A recent study indicates that activated DCs show high expression of TIM4 in DCs (7). Although it is not exclusive, at least we have known that DCs have the capacity to produce TIM4. DCs have the tendency to migrate to draining lymph nodes where the DCs have opportunity to interact with T cells. Thus, it is feasible that DCs can transfer the TIM4 to T cells when necessary. In other words, polarized Th2 cells can get TIM4 from activated DCs to maintain the polarization status.

The clinical effect of SIT on amelioration of allergic diseases has been recognized via several administration routes including subcutaneous injection, epicutaneous route, inhalation and sublingual route (16, 17); but the mechanism is not completely understood yet. The present data add novel information to the mechanism of SIT. The fact that after 3-month SIT, levels of TIM1 in Th2 cells and TIM4 in DCs of patients with AR markedly reduced indicates that SIT modulates the allergic status, at least partially, via suppressing the expression of TIM1 and TIM4. The fact of the maintenance of TIM1 on Th2 cells relies on the activities of TIM4 implicates that the downregulation of TIM1 by SIT is a consequence of TIM4 suppression. The data provide supportive evidence to this event. As shown by present study, as well as reported by others (10, 11), human DCs express both FcγRI and FcγRII. FcγRs have the capacity to bind IgG or complexes of antigen/IgG. Specific immunotherapy produces antigen-specific IgG in the body to bind to FcγRI or FcγRII to form complexes on DCs to make the DCs primed as shown by the present data. Re-exposure to specific antigen forms triple complexes, the antigen/IgG/FcγRI or antigen/IgG/FcγRII. The present data and our recent publication (12) also show these complexes on DCs. It is suggested that activation of FcγRII generates negative signal, and activation of FcγRI conducts positive signal (11). The present data are in agreement with these studies by showing that more Der/IgG/FcγRI was detected in DCs from patients with AR before SIT, while high amount of Der/IgG/FcγRII was detected in DCs after SIT.

This study collected a group of patients with AR sensitized to mite antigen, Der. We also treated these patients with Der during SIT. In fact, naturally exposed Der and therapeutic Der that we used in SIT have the same properties. Then, what makes the consequences different that the natural exposure to Der evokes attacks of AR, while the injection with Der in SIT inhibits AR attacks remains to be elucidated. As an extension of our recent studies (8, 19) that microbial products promote the expression of TIM4 in DCs, the present study further revealed that exposure to microbial products favors the expression of FcγRI in DCs, which in turn promotes the expression of TNF-α, the latter is involved in the expression of TIM4 as shown by the present study. Meanwhile, we also found that exposure to antigen (such as Der) alone promote the expression of FcγRII in DCs. Thus, it is conceivable that natural antigens (such as Der) are inevitable to be contaminated by microbial products such as LPS that distributes in natural environment ubiquitously. The present study suggests that such contaminated antigens have the capacity to activate DCs to express FcγRI, to produce TIM4 and to process antigens that favor the antigen-specific Th2 polarization. During SIT, however, injection with pure antigen induces FcγRII expression in DCs that inhibits TIM4 expression resulting in the TIM1 decaying subsequently in antigen-specific Th2 cells and stops AR attacks eventually.

Our study also revealed the direct mechanism by which cross-linking FcγRs modulates the expression of TIM4 in DCs. TNF-α is the critical molecule that mediates the expression of TIM4 in DCs induced by FcγRs; its production by DCs can be upregulated by cross-linking FcγRI and inhibited by cross-linking FcγRII. Our finding is in line with previous reports (38) that TNF-α is a critical molecule in the development of optimal antigen-specific immune responses (39). The results implicate that modulating TNF-α expression in immune cells has potential to develop a novel approach in the therapeutics of allergic diseases.

In summary, the present study reports that TIM1 is required in the maintenance of antigen-specific Th2 responses. DC-derived TIM4 maintains TIM1 in Th2 cells in a stable status that plays a critical role in sustaining the Th2 polarization. FcγRI recognizes antigen/IgG complexes to increase, while FcγRII recognizes antigen/IgG complexes to decrease the expression of TIM4 in DCs that was confirmed in a group of patients with AR after treatment with SIT. Finally, we found that exposure to microbial products increases the expression of FcγRI, while exposure to pure antigens increases the expression of FcγRII in DCs. Activation of FcγRI favors the expression of TIM4 in DCs.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

This study was supported by grants from the Natural Science Foundation of China, the Canadian Institute of Health Research (CIHR), and the Natural Science Engineering Research Council of Canada. Dr P. Yang holds a New Investigator Award of CIHR.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

The authors do not have any conflict of interest on this paper.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information
  • 1
    Kay AB. Allergy and allergic diseases. First of two parts. N Engl J Med 2001;344:3037.
  • 2
    Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature 2008;454:445454.
  • 3
    Munitz A, Seidu L, Cole ET, Ahrens R, Hogan SP, Rothenberg ME. Resistin-like molecule alpha decreases glucose tolerance during intestinal inflammation. J Immunol 2009;182:23572363.
  • 4
    Burke SM, Issekutz TB, Mohan K, Lee PW, Shmulevitz M, Marshall JS. Human mast cell activation with virus-associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism. Blood 2008;111:54675476.
  • 5
    Umetsu SE, Lee WL, McIntire JJ, Downey L, Sanjanwala B, Akbari O et al. TIM-1 induces T cell activation and inhibits the development of peripheral tolerance. Nat Immunol 2005;6:447454.
  • 6
    Meyers JH, Chakravarti S, Schlesinger D, Illes Z, Waldner H, Umetsu SE et al. TIM-4 is the ligand for TIM-1, and the TIM-1–TIM-4 interaction regulates T cell proliferation. Nat Immunol 2005;6:455464.
  • 7
    Rodriguez-Manzanet R, Meyers JH, Balasubramanian S, Slavik J, Kassam N, Dardalhon V et al. TIM-4 expressed on APCs induces T cell expansion and survival. J Immunol 2008;180:47064713.
  • 8
    Yang PC, Xing Z, Berin CM, Soderholm JD, Feng BS, Wu L et al. TIM-4 expressed by mucosal dendritic cells plays a critical role in food antigen-specific Th2 differentiation and intestinal allergy. Gastroenterology 2007;133:15221533.
  • 9
    Liu T, He SH, Zheng PY, Zhang TY, Wang BQ, Yang PC. Staphylococcal enterotoxin B increases TIM4 expression in human dendritic cells that drives naïve CD4 T cells to differentiate into Th2 cells. Mol Immunol 2007;44:35803587.
  • 10
    Hulse KE, Reefer AJ, Engelhard VH, Satinover SM, Patrie JT, Chapman MD et al. Targeting Fel d 1 to FcgammaRI induces a novel variation of the T(H)2 response in subjects with cat allergy. J Allergy Clin Immunol 2008;121:756762.
  • 11
    Laborde EA, Vanzulli S, Beigier-Bompadre M, Isturiz MA, Ruggiero RA, Fourcade MG et al. Immune complexes inhibit differentiation, maturation, and function of human monocyte-derived dendritic cells. J Immunol 2007;179:673681.
  • 12
    Chen X, Feng BS, Zheng PY, Liao XQ, Chong J, Tang SG et al. Fc gamma receptor signaling in mast cells links microbial stimulation to mucosal immune inflammation in the intestine. Am J Pathol 2008;173:16471656.
  • 13
    Desai DD, Harbers SO, Flores M, Colonna L, Downie MP, Bergtold A et al. Fc gamma receptor IIB on dendritic cells enforces peripheral tolerance by inhibiting effector T cell responses. J Immunol 2007;178:62176226.
  • 14
    Ravetch JV, Clynes RA. Divergent roles for Fc receptors and complement in vivo. Annu Rev Immunol 1998;16:421432.
  • 15
    Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol 1998;76:231248.
  • 16
    Rodríguez-Pérez N, Penagos M, Portnoy JM. New types of immunotherapy in children. Curr Allergy Asthma Rep 2008;8:484492.
  • 17
    Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol 2007;119:780791.
  • 18
    Heyman B. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu Rev Immunol 2000;18:709737.
  • 19
    Feng BS, Chen X, He SH, Zheng PY, Foster J, Xing Z et al. Disruption of T-cell immunoglobulin and mucin domain molecule (TIM)-1/TIM4 interaction as a therapeutic strategy in a dendritic cell-induced peanut allergy model. J Allergy Clin Immunol 2008;122:5561.
  • 20
    Plewako H, Holmberg K, Oancea I, Gotlib T, Samoliński B, Rak S. A follow-up study of immunotherapy-treated birch-allergic patients: effect on the expression of chemokines in the nasal mucosa. Clin Exp Allergy 2008;38:11241131.
  • 21
    Nathan RA. Management of patients with allergic rhinitis and asthma: literature review. South Med J 2009;102:935941.
  • 22
    Wachholz PA, Durham SR. Mechanisms of immunotherapy: IgG revisited. Curr Opin Allergy Clin Immunol 2004;4:313318.
  • 23
    Benitez-Ribas D, Tacken P, Punt CJ, De Vries IJ, Figdor CG. Activation of human plasmacytoid dendritic cells by TLR9 impairs Fc gammaRII-mediated uptake of immune complexes and presentation by MHC class II. J Immunol 2008;181:52195224.
  • 24
    Hirata N, Yanagawa Y, Ebihara T, Seya T, Uematsu S, Akira S et al. Selective synergy in anti-inflammatory cytokine production upon cooperated signaling via TLR4 and TLR2 in murine conventional dendritic cells. Mol Immunol 2008;45:27342742.
  • 25
    Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2 polarization in mucosal immunity. Nat Med 2002;8:567573.
  • 26
    Encinas JA, Janssen EM, Weiner DB, Calarota SA, Nieto D, Moll T et al. Anti-T-cell Ig and mucin domain-containing protein 1 antibody decreases TH2 airway inflammation in a mouse model of asthma. J Allergy Clin Immunol 2005;116:13431349.
  • 27
    Sizing ID, Bailly V, McCoon P, Chang W, Rao S, Pablo L et al. Epitope-dependent effect of anti-murine TIM-1 monoclonal antibodies on T cell activity and lung immune responses. J Immunol 2007;178:22492261.
  • 28
    Graves PE, Siroux V, Guerra S, Klimecki WT, Martinez FD. Association of atopy and eczema with polymorphisms in T-cell immunoglobulin domain and mucin domain-IL-2-inducible T-cell kinase gene cluster in chromosome 5 q 33. J Allergy Clin Immunol 2005;116:650656.
  • 29
    Wu WK, An YF, Zhao CQ. Immunoglobulin domain and mucin domain-1 in helper T lymphocytes in allergic rhinitis. Zhonghua Yi Xue Za Zhi 2008;88:33923396.
  • 30
    Wang Y, Meng J, Wang X, Liu S, Shu Q, Gao L et al. Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients. Scand J Immunol 2008;67:6370.
  • 31
    Liu Q, Shang L, Li J, Wang P, Li H, Wei C et al. A functional polymorphism in the TIM-1 gene is associated with asthma in a Chinese Han population. Int Arch Allergy Immunol 2007;144:197202.
  • 32
    McIntire JJ, Umetsu SE, Macaubas C, Hoyte EG, Cinnioglu C, Cavalli-Sforza LL et al. Immunology: hepatitis A virus link to atopic disease. Nature 2003;425:576.
  • 33
    Marsh DG, Neely JD, Breazeale DR, Ghosh B, Freidhoff LR, Ehrlich-Kautzky E et al. Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 1994;264:11521156.
  • 34
    McIntire JJ, Umetsu SE, Akbari O, Potter M, Kuchroo VK, Barsh GS et al. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol 2001;2:11091116.
  • 35
    Xu G, Cheng L, Lu L, Zhu Y, Xu R, Yao X et al. Expression of T-cell immunoglobulin- and mucin-domain-containing molecule-1 (TIM-1) is increased in a mouse model of asthma and relationship to GATA-3. Life Sci 2008;82:663669.
  • 36
    Chae SC, Song JH, Heo JC, Lee YC, Kim JW, Chung HT. Molecular variations in the promoter and coding regions of human Tim-1 gene and their association in Koreans with asthma. Hum Immunol 2003;64:11771182.
  • 37
    Chae SC, Park YR, Song JH, Shim SC, Yoon KS, Chung HT. The polymorphisms of Tim-1 promoter region are associated with rheumatoid arthritis in a Korean population. Immunogenetics 2005;56:696701.
  • 38
    Skarica M, Wang T, McCadden E, Kardian D, Calabresi PA, Small D et al. Signal transduction inhibition of APCs diminishes th17 and Th1 responses in experimental autoimmune encephalomyelitis. J Immunol 2009;182:41924199.
  • 39
    McLachlan JB, Shelburne CP, Hart JP, Pizzo SV, Goyal R, Brooking-Dixon R et al. Mast cell activators: a new class of highly effective vaccine adjuvants. Nat Med 2008;14:536541.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Conflict of interest
  8. References
  9. Supporting Information

Methods.

Figure S1. Specific immunotherapy modulates the serum levels of IgE and IgG4 in allergic rhinitis (AR) patients.

Figure S2. Specific immunotherapy (SIT) reduces the allergic rhinitis (AR) clinical symptom score.

Table S1. Demographic data.

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