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Monocyte-Derived Dendritic Cells Induce a House Dust Mite-Specific Th2 Allergic Inflammation in the Lung of Humanized SCID Mice: Involvement of CCR7

Hamida Hammad^1,*, Bart N. Lambrecht^1,, Pierre Pochard^*, Philippe Gosset^*, Philippe Marquillies^*, André-Bernard Tonnel^*, and Joël Pestel^2,*

^* Institut National de la Santé et de la Recherche Médicale, Unité 416, Institut Pasteur de Lille, Lille, France; Department of Pulmonary and Critical Care Medicine, Erasmus University, Rotterdam, The Netherlands; and Clinique des Maladies Respiratoires, Hôpital Calmette, Centre Hospitalier Régional Universitaire de Lille, Lille, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4⁺ T cells, whereas those from healthy donors induced IFN--secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ are powerful APC with a unique capacity to stimulate naive T cells in vivo (1). Airway DCs form a highly developed network of cells ideally placed to sample inhaled Ags (2, 3). In the absence of Ag exposure, lung DCs are in an immature state, which is optimal for Ag uptake and processing (4). In response to Ag encounter, immature DCs respond to a variety of inflammatory stimuli by up-regulating the CCR-7, the receptor for the chemokines secondary lymphoid tissue chemokine/CCL21 and macrophage inflammatory protein-3 (MIP-3)/CCL19, which are constitutively expressed in afferent lymph endothelium and the T cell area of lymph nodes, respectively, explaining why DCs direct their interest to the draining lymph node (5). In the T cell area of the draining lymph nodes, Ag-loaded mature DCs stably express MHC-associated antigenic peptides and attract and stimulate naive T cells to induce a primary immune response (6). This interaction between DCs and T cells occurs via a large number of noncognate interactions between cell adhesion molecule pairs (e.g., dendritic cell-specific ICAM3 grabbing nonintegrin interacting with ICAM-3) and through newly expressed costimulatory molecules such as CD80, CD86, and CD40, interacting with their ligand expressed on T cells (1, 7). By controlling the strength and duration of TCR triggering, the pattern of costimulatory molecule expression, and the production of polarizing cytokines, DCs determine the outcome of the primary T cell response of tolerance, unpolarized Th0, or polarized Th1 or Th2 (8).

Allergic asthma is a Th2-mediated disorder with three distinguishing features: IL-4-dependent production of allergen-specific IgE, chronic airway inflammation characterized by IL-5-dependent cellular tissue eosinophils and mast cell infiltration, and airway hyperresponsiveness (AHR) to specific and nonspecific stimuli (9). Although DCs may play a role in the polarization of Th immune responses in the airways, only a few studies have attempted to define their precise role in the pathogenesis of allergic reactions. We have previously shown that Ag-pulsed DCs injected intratracheally in mice induced the rapid division and activation of Ag-specific T cells after migration to the draining mediastinal lymph nodes (MLN) (6). Moreover, BALB/c and C57BL/6 mice and Brown Norway rats injected intratracheally with OVA-pulsed DCs, and subsequently challenged with OVA aerosols developed CD4⁺ Th2 cell-dependent airway eosinophilia, goblet cell hyperplasia, and bronchial hyperreactivity (7). When airway DCs were depleted from the airways of thymidine kinase transgenic OVA-sensitized mice, the secondary response to inhaled OVA, consisting of airway eosinophilia and goblet cell hyperplasia, was completely abolished, suggesting that airway DCs were necessary for the generation of effector functions in previously sensitized animals (10).

All these in vivo protocols allowed us to better understand the role of DCs in the induction and maintenance of eosinophilic airway inflammation, but were conducted exclusively in mice or rats using the OVA allergen model. The relevance of these findings to human asthma remains to be established. In an attempt to study the contribution of DCs to the induction of an allergic immune response to the relevant house dust mite (HDM) allergen Der p 1 in vivo, we have previously used the human PBMC-reconstituted SCID (hu-SCID) model. In this way we have shown that hu-SCID mice reconstituted i.p. with PBMC from HDM-allergic patients and subsequently exposed to aerosols of HDM produce human IgE (11), develop a pulmonary infiltrate composed of activated T cells and DCs (12, 13, 14), and exhibit AHR in response to bronchoconstrictor agents (15). In this study we investigated the capacity of intratracheally injected monocyte-derived DCs from allergic or nonallergic patients 1) to migrate to the draining MLN of SCID mice, 2) to induce a T cell immune response within the draining MLN, and, finally, 3) to generate Th2 effector function in reconstituted T cells leading to eosinophilic airway inflammation and allergen-specific IgE production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients

Blood was collected from HDM-allergic (n = 11) and healthy control (n = 10) donors. Allergic patients had a history of asthma, skin prick positivity toward Dermatophagoides pteronyssinus (Dpt), and serum-specific IgE Abs with an average total IgE of 486 ± 125 IU/ml. Healthy donors had negative skin prick test toward common aeroallergen and average total IgE level of 115 ± 55 IU/ml.

Mice

C.B17 SCID mice (5–7 wk old) were maintained in isolators (La Calhène, Vélizy, Yvelines, France) with sterilized bedding at the Pasteur Institute (Lille, France). The SCID colony was regularly checked for the absence of mouse serum Igs.

PBMC preparation and purification of DCs and T cells

Platelet-rich plasma was obtained after centrifugation (120 x g, 15 min) and discarded. Blood cells were then diluted in RPMI 1640 (Life Technologies, Paisley, Scotland; v/v) and layered over a Ficoll gradient (Pharmacia, Uppsala, Sweden). After centrifugation (400 x g, 30 min), PBMC were harvested and washed.

PBMC were incubated on ice for 30 min with magnetic beads coated with CD14 Abs (Miltenyi Biotec, Paris, France), washed, and applied to a column placed in a magnetic field of a MACS separator (Miltenyi Biotec). After elution of the CD14-negative cells, the column was removed from the magnetic field, and the CD14⁺ monocytes were collected, washed twice in RPMI 1640 medium before plating (2 x 10⁶ cells/2 ml/well) into six-well flat-bottom culture plates in RPMI 1640 medium supplemented with 1% antibiotics and 10% FCS (Life Technologies). To obtain DCs, CD14⁺ cells were cultured for 7 days at 37°C in humidified 5% CO₂ in air in RPMI medium supplemented with GM-CSF (PeproTech, London, U.K.; 20 ng/ml) and IL-4 (R&D Systems, Oxon, U.K.; 200 IU/ml).

For the isolation of T cells, the eluted CD14^- cells were incubated for 10 min on ice with a mixture of hapten-conjugated CD11b, CD16, CD19, CD36, and CD56 Abs (Miltenyi Biotec), followed by a 15-min incubation with MACS microbeads coupled to an anti-hapten mAb and placement in a magnetic separator column, allowing the depletion of B cells, monocytes, NK cells, DCs, early erythroid cells, platelets, and basophils. The eluted fraction containing the T cells was collected (purity, >95%).

Allergen pulsing and intratracheal injection of DCs

Generated DCs were incubated overnight with 1 µg/ml Der p 1 (provided by G. A. Stewart, University of Western Australia, Nedlands, Australia). After allergen pulsing, DCs were washed to remove free Der p 1 and were resuspended in PBS. As a control, some DCs were not pulsed with allergen. For intratracheal injection, mice were anesthetized by i.p. injection of avertin (Sigma-Aldrich, Saint Quentin Fallevier, France; 2.5%, v/v, in PBS). Eighty microliters of DC suspension, corresponding to 1 x 10⁶ cells, was administered intratracheally under direct vision through the opening vocal cords using a 23-gauge metal catheter connected to the outlet of a micropipette as previously described (16).

Protocol I: in vivo localization of human DCs

After the intratracheal injection of 1 x 10⁶ human DCs on day 0, mice were sacrificed at 24, 36, 48, and 120 h (Fig. 1A). Bronchoalveolar lavage (BAL) fluids, draining MLN, and pooled nondraining peripheral (superficial cervical, inguinal, brachial, mesenteric, and para-aortic) lymph nodes were collected, and cells were counted and incubated for 30 min on ice with FITC-conjugated anti-human CD45, PE-conjugated anti-human CD11c, and CyChrome-conjugated anti-human HLA-DR or with an irrelevant mAb of the same isotype (all from BD Biosciences, Le Pont de Claix, France). After washing, the cells were fixed in PBS/1% paraformaldehyde and analyzed on a FACSCalibur (BD Biosciences).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Protocols of reconstitution of naive SCID mice with human cells. The in vivo migration of intratracheally injected human DCs (protocol I), their effect on the stimulation of adoptively transferred T cells (protocol II), as well as the capacity of DCs to orchestrate a pulmonary inflammatory reaction (protocol III) in humanized SCID mice were evaluated.

SCID mice were reconstituted by i.p. injection of 5 x 10⁶ T cells from HDM-allergic patients or healthy donors (Fig. 1B) on day -7. On day 0 of the experiment, mice were immunized with 1 x 10⁶ intratracheally injected Der p 1-pulsed or unpulsed DCs of the same donor. Mice were killed on days 2, 4, and 7 after the DC injection. Mediastinal and pooled nondraining peripheral lymph nodes were collected, minced, and prepared for flow cytometric analysis. Cells were incubated with different mAbs specific for human cells (FITC-conjugated CD45, PE-conjugated CD3, and CyChrome-conjugated CD4 and CD8) or with an irrelevant mAb of the same isotype (all Abs from BD Biosciences).

Cytokine secretion in lymph node CD4⁺ and CD8⁺ T cells was detected using surface affinity matrix technology (17). Lymph node cells were restimulated in vitro with anti-CD3 and anti-CD28 Abs for 5 h, washed, and incubated for 5 min on ice with a bispecific Ab directed against human CD45 and the human cytokines IL-4 and IFN- (Miltenyi Biotec). Cells were washed in PBS/BSA/EDTA and incubated in RPMI 1640/human serum 10% at 37°C for 45 min. After washing, cells were incubated for 15 min with different Abs: PE-conjugated anti-human IL-4 or IFN-, or CyChrome-conjugated anti-human CD4 or CD8. Cells were washed and analyzed by flow cytometry.

Protocol III: capacity of DCs to prime for Th2 effector function and allergen-induced airway inflammation

On day -7, SCID mice were reconstituted by i.p. injection of 10 x 10⁶ cells previously depleted in monocytes from allergic patients or healthy donors (Fig. 1C). On day 0, mice were immunized with 1 x 10⁶ intratracheally injected Der p 1-pulsed or unpulsed DCs from the same donor (i.e., syngeneic set-up). From days 8–12 mice were exposed to one aerosol of 100 index of reactivity of the HDM Dpt/day for 5 consecutive days. Mice were killed 48 h after the last allergen exposure (day 14), BAL was performed, serum IgE was measured, and tissue samples were taken.

BAL was performed with 1 ml PBS. The BAL fluid was centrifuged (7 min, 4°C, 700 x g), and the supernatant was collected and stored at -80°C until analysis of cytokine content. The pellet was resuspended in PBS, and cell number was determined. Differential cell counts were performed on cytospins stained with May-Grünwald Giemsa by classification of 300 cells using standard morphologic criteria. The presence of the human cytokines IL-4, IL-5, and IFN- in BAL fluids was measured by specific ELISA using Eli-pairs (Biotest, Buc, France). The sensitivity of detection of IL-4, IL-5, and IFN- was 2 pg/ml.

For immunohistochemical analysis, 6-mm-thick serial frozen or paraffin tissue sections of the lungs were performed. After a 20-min permeabilization in TBS/Triton 0.3%, the slides were saturated for 1 h at room temperature with TBS/20% normal human serum. Sections were incubated for 1 h with the following primary Abs: anti-human CD45 (BD Biosciences), anti-human CD3 and murine major basic protein (MBP; provided by G. Gleich), or an isotype-matched mouse IgG (DAKO, Trappes, France) as a negative control. After saturation with 10% normal human serum diluted in TBS, sections were stained using an alkaline phosphatase-anti-alkaline phosphatase technique (DAKO). All slides were lightly counterstained with hematoxylin (Sigma-Aldrich). The Abs used in this study did not cross-react with murine tissues.

Total human IgE in murine serum was investigated by using a UniCAP system method (Pharmacia, St. Quentin, France) as previously described (12). The sensitivity of detection was 0.1 IU/ml.

Protocol IV: effect of neutralizing anti-murine secondary lymphoid tissue chemokine (SLC) Abs on DC migration, T cell stimulation, and Th2 effector generation

To test the functional importance of the chemokine SLC on the migration potential of DCs and their potential to stimulate T cells, SCID mice previously reconstituted with 5 x 10⁶ T cells from allergic patients were administered intratracheally with 25 µg goat anti-mouse SLC polyclonal Ab (R&D Systems) or the same dose of goat IgG (R&D Systems) as a control simultaneously with autologous Der p 1-pulsed DCs. Two or 4 days later BAL fluids and MLN were collected. The presence of T cells in lymph nodes and that of DCs in both BAL and lymph nodes were determined by flow cytometry (see protocols I and II)

To test the functional importance of SLC in priming for Th2 effector function and airway inflammation (protocol III), SCID mice were reconstituted by i.p. injection of PBMC from allergic patients. On day 0 mice were injected intratracheally with 1 x 10⁶ Der p 1-pulsed DCs simultaneously with 25 µg goat anti-mouse SLC Abs or with the same dose of goat IgG. From days 8–12 mice were exposed to one aerosol of 100 index of reactivity Dpt/day for 5 consecutive days. Mice were analyzed as described in protocol III.

Statistical analysis

Parametric statistical analysis of the data obtained from allergic patients and healthy donors was performed using Student’s t test. Values of p < 0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo localization of injected DCs

The presence of human DCs was investigated in BAL and lymph nodes of mice at different time points following intratracheal administration of DCs. Human DCs were characterized as cells positive for the human markers CD45, CD11c, and HLA-DR (Fig. 2, A and B). DCs from allergic patients pulsed with Der p 1 were detectable in the BAL of mice 24 h after they were injected. The number of DCs rapidly decreased until day 5 following intratracheal immunization. In parallel, the number of CD45⁺ CD11c⁺ HLA-DR⁺ cells in the MLN rapidly increased and appeared maximal by 36 h after injection (Fig. 2C). When the cells were not previously pulsed with Der p 1, <100 cells could be detected in the lymph nodes of the mice (data not shown). No DCs were detected in pooled non-draining lymph nodes (data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Localization of human DCs in SCID mice. DCs from allergic patients or from healthy donors were injected into the trachea of SCID mice. Human DCs were identified as CD45+ cells expressing CD11c and HLA-DR (A and B). DCs from allergic patients (C) or from healthy donors (D) were detected in BAL fluids and in MLN of mice. Results are expressed as the mean ± SEM from six to eight mice per group.

Interaction of Der p 1-pulsed DCs with T cells in the draining lymph nodes

As human DCs instilled in the trachea of mice were able to migrate into the MLN, we next investigated whether these cells were able to stimulate syngeneic T cells, injected 7 days previously.

In mice reconstituted with T cells from allergic patients, the number of CD45⁺ CD3⁺ human T cells appeared to be maximal on day 4 after injection of pulsed DCs, as shown in (Fig. 3B), and was higher than that following injection of unpulsed DCs (Fig. 3A). T cells were still detectable in the MLN 7 days after DC administration The increase in the number of T cells following immunization of mice with Der p 1-pulsed DCs was related to T cell proliferation. This was confirmed by labeling of T lymphocytes with CFSE (Interchim, Montluçon, France) (18) before injection into SCID mice (Fig. 3E). Interestingly, in mice reconstituted with pulsed DCs from healthy donors, the number of CD3⁺ cells was also maximal on day 4 (Fig. 3D) and was higher than in mice receiving unpulsed DCs (Fig. 3C). However, the number of T cells was much lower than that in mice reconstituted with cells from allergic patients.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Stimulation of T cells by DCs in MLN. Animals received T cells 7 days before intratracheal administration of 1 x 106 unpulsed (A) or Der p 1-pulsed (B) autologous DCs from allergic patients. Similar experiments were performed with cells from healthy donors (C and D). MLN were collected 2, 4, and 7 days after immunization with DCs. CD3+, CD4+, and CD8+ cells were enumerated. The proliferation of T cells from allergic patients and from healthy donors stimulated with Der p 1-pulsed autologous DCs was evaluated by staining of lymphocytes with CFSE (E and F, respectively). Results are expressed as the mean ± SEM from 10–12 mice/group.

To investigate whether the intratracheal administration of Der p 1-pulsed DCs led to a selective accumulation of lymphocytes producing either Th1- or Th2-type cytokines in the draining lymph nodes, MLN cells were analyzed for production of human IFN- and human IL-4 by surface affinity matrix technology and flow cytometry. Fig. 4 shows representative dot plots of IFN- or IL-4 expression in either CD4⁺ or CD8⁺ lymphocytes in SCID mice reconstituted with cells from allergic patients and healthy donors, respectively. As shown in Table I, after administration of Der p 1-pulsed DCs from allergic patients, only the percentage of IL-4-producing cells, and not that of IFN--producing cells, was increased. In contrast, in mice injected with Der p 1-pulsed DCs from healthy donors, the numbers of both IL-4- and IFN--producing cells were increased. However, the percentage of IFN--producing cells was higher than the percentage of IL-4-producing cells.

View larger version (61K): [in this window] [in a new window]   FIGURE 4. IL-4 and IFN- secretion by T cells from MLN of mice injected with Der p 1-pulsed DCs from allergic patients (A) or from healthy donors (B). Data shown are obtained by flow cytometry on MLN cells after stimulation with anti-human CD3 and anti-human CD28 Abs. Cells were stained for IL-4, IFN-, and isotype control. The dot plots shown were gated on CD45+ cells and are representative of six to eight mice in the group.

Analysis of BAL fluid cells revealed that repeated allergen exposure induced a significant increase in the number of lymphocytes and murine eosinophils in reconstituted SCID mice immunized with Der p 1-pulsed DCs of allergic patients compared with mice immunized with unpulsed DCs (Fig. 5A). In contrast, SCID mice reconstituted with PBMC of healthy donors and receiving Der p 1-pulsed DCs did not show any difference in the number of lymphocytes or eosinophils compared with mice receiving unpulsed DCs (Fig. 5B).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Effect of Dpt exposure on the cellular composition of BAL fluids. Mice were injected with both B and T cells 7 days before intratracheal administration of 1 x 106 Der p 1-pulsed or unpulsed autologous DCs. On days 8–12 mice were exposed to 30-min daily Dpt aerosols. At 48 h after the last aerosol (day 14), BAL was performed. Differential cell counts based on Giemsa staining were performed on BAL fluid cells, and IL-4, IL-5, and IFN- levels were measured by ELISA in unconcentrated BAL fluids of mice immunized with DCs from allergic patients (A and C, respectively) or from healthy donors (B and D, respectively). Results are expressed as the mean ± SEM from 8–10 mice/group.

Effect of aerosol exposure on the cell recruitment into the lungs of hu-SCID mice injected with DCs

As shown in Fig. 6 and Table II, mice reconstituted with PBMC from allergic patients and subsequently immunized with Der p 1-pulsed DCs developed a pulmonary inflammatory reaction characterized by an infiltrate of human CD45⁺ cells (Fig. 6A). Lung sections also revealed an increase in the number of MBP⁺ murine eosinophils and human CD3⁺ T cells (Table II). When mice were immunized with unpulsed DCs, only a few CD45⁺ cells were detectable (Fig. 6B), and the number of eosinophils was very low (Table II) compared with that in mice immunized with pulsed DCs. In mice reconstituted with PBMC of healthy donors and immunized with Der p 1-pulsed or unpulsed DCs, no CD45⁺ cells were detectable in the lungs (data not shown).

View larger version (53K): [in this window] [in a new window]   FIGURE 6. Effect of Dpt exposure on cellular recruitment into the lung of mice injected with DCs. Mice were treated as described in Fig. 5. At 48 h after the last aerosol, lungs were fixed and processed for histologic analysis (magnification, x400). CD45+ human cells are detected in the lungs of mice injected intratracheally with pulsed DCs from allergic patients (A). Control mice injected with unpulsed DCs show a reduced number of CD45+ human cells (B). Br, bronchia; v, vessel.

Following allergen aerosol exposure, human IgE production was significantly increased in hu-SCID mice immunized with Der p 1-pulsed DCs from allergic donors (30.3 ± 2.7 IU/ml) compared with that in mice immunized with unpulsed DCs (98.2 ± 3.6 IU/ml; data not shown). In contrast, no modulation of IgE production was observed in mice immunized with allergen-pulsed DCs from healthy donors (26.7 ± 12.5 IU/ml) compared with that in mice immunized with unpulsed DCs (28.6 ± 17.8 IU/ml).

CCR7 expression on Der p 1-pulsed DCs and effect of anti-murine SLC (anti-mSLC) Abs on DC migration and induced airway responses

To determine whether the murine CC-chemokine SLC was involved in the migration of human DCs from the lung to the MLN, attempts to block SLC were conducted. First, the expression of CCR7, the receptor for SLC, was evaluated on DCs by flow cytometry. Unpulsed DCs from allergic patients showed a low expression of CCR7. Following overnight incubation with Der p 1 or LPS, CCR7 expression was increased in DCs from allergic patients. The increase in CCR7 expression following Der p 1 exposure was higher in DCs from allergic patients than in DCs from healthy donors (Fig. 7). A similar effect was observed at the mRNA level using real-time PCR. A 6- to 8-fold increase in the up-regulation of the mRNA encoding CCR7 was detected with DCs from allergic patients, but only a 2-fold increase was found with DCs from healthy donors (data not shown). The results were confirmed by testing the functionality of CCR7 in response to Der p 1. A chemotaxis assay using a Boyden’s chamber with 5-µm pore polycarbonate filters (Nucleopore, Pleasanton, CA) performed with DCs from healthy donors and from allergic patients showed that DCs from allergic patients and those from healthy donors could migrate in a dose-dependent manner in response to murine SLC. The migration was more important with DCs from allergic patients than with those from healthy donors (Fig. 8).

View larger version (29K): [in this window] [in a new window]   FIGURE 7. CCR7 expression by DCs. After 7 days of culture in the presence of GM-CSF and IL-4, DCs generated from monocytes of healthy donors (left panels) or allergic patients (right panels) were pulsed overnight with 1 µg/ml Der p 1 or LPS or were not pulsed. Cells were then collected and analyzed by flow cytometry for CCR7 expression (grey line). Black line, reactivity of fluorochrome-matched isotype control mAbs.

View larger version (11K): [in this window] [in a new window]   FIGURE 8. Cross-reactivity between murine SLC and human DCs. After 7 days of culture in the presence of GM-CSF and IL-4, DCs generated from monocytes of allergic patients or from healthy donors were pulsed overnight with 1 µg/ml Der p 1. Human DC migration capacities in response to different doses of murine SLC (0, 10, 100, and 1000 ng/ml) were evaluated in a Boyden’s chamber with 5-µm pore size polycarbonate filters (Nucleopore). After 2 h at 37°C in 5% CO2, cells located on the filter were enumerated under a light microscope with a magnification of 500-fold using a hemocytometer. Each condition was performed in triplicate, and at least four fields were counted for each well. Results are expressed as the mean number of DC per field ± SEM from three donors per group.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Effect of anti-SLC Abs on DC migration and T cell proliferation. T cells from allergic patients were injected into SCID mice 7 days before intratracheal administration of Der p 1-pulsed DCs simultaneously either with goat anti-SLC Abs or with goat IgG as a control. The effect of anti-SLC Abs or goat IgG was evaluated on the number of migrating DCs in the MLN (A) and non-migrating DCs in the BAL (B). The number of T cells was also evaluated in the MLN of mice that received anti-SLC Abs (C) or goat IgG (D). Results are expressed as the mean ± SEM from six to eight mice per group.

View larger version (23K): [in this window] [in a new window]   FIGURE 10. Effect of anti-SLC Abs on induced airway responses. Mice were injected with B and T cells from allergic patients 7 days before intratracheal administration of 1 x 106 Der p 1-pulsed autologous DCs simultaneously with either goat anti-murine SLC or goat IgG. On days 8–12 mice were exposed to daily Dpt aerosols. At 48 h after the last aerosol (day 14), BAL was performed, and serum IgE were measured. Differential cell counts based on Giemsa staining (A), cytokine levels (B), and IgE production (C) were evaluated in mice treated, or not, with anti-mSLC Abs. Results are expressed as the mean ± SEM from 8–10 mice/group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

DCs that have taken up Ag in the lung migrate to the draining lymph nodes where they interact with T cells, and the rapidity of the ensuing T cell response reflects the kinetics of migration of DCs into the T cell area (6, 19). To confirm this in our model, T cells from either allergic patients or healthy donors were injected i.p. into SCID mice. One week later mice were instilled with autologous Der p 1-pulsed or unpulsed DCs. Four days after the injection of Der p 1-pulsed DCs from allergic patients the number of T cells in the MLN was increased 3 times compared with that in control mice injected with unpulsed DCs. Moreover, T cells located in the MLN of mice reconstituted with cells from allergic patients predominantly expressed the marker CD4. In these mice the CD4 to CD8 ratio was 2:1. In mice injected with cells from healthy donors the response to Der p 1 pulsed DCs remained very low, and the CD4 to CD8 ratio was 1:1. The T cell response induced by Der p 1-pulsed DCs from allergic patients might be due to recruitment and/or local proliferation of allergen-specific cells in the MLN. The latter was confirmed by the adoptive transfer of CFSE-labeled T cells, allowing the detection of cell divisions on a cell basis. Together, these findings underline the capacity of DCs to specifically select and activate only the Ag-specific T cells and are in agreement with results obtained in mice in which intratracheally injected OVA- or moth cytochrome c-pulsed DCs were shown to induce the recruitment and division of naive Ag-specific T cells only (6). However, in the current experiments using allergic donors as a source of T cells, the majority of allergen-specific T cells were expected to be of memory phenotype, as the precursor frequency of HDM-specific naive T cells would be low. When MLN lymphocytes were restimulated in vitro with anti-CD3 and anti-CD28 Abs, significant cytokine production, as measured on an individual cell basis, was observed only in mice immunized with Der p 1-pulsed DCs, but not with unpulsed DCs. This finding by itself suggests that circulating T cells, even if they were previously primed by Ag in vivo, were not able to produce cytokines in response to polyclonal TCR and CD28 activation in vitro. In mice injected with cells from allergic patients, Der p 1-pulsed DCs mainly induced an increase in the number of IL-4- and CD4-producing cells, while the number of IFN--producing cells remained low, suggesting a Th2 profile. In contrast, in mice reconstituted with cells from healthy donors, the administration of Der p 1-pulsed DCs induced a mixed cytokine response, with T cells producing both IL-4 and IFN-. However, the percentage of IFN--producing cells was higher than that of IL-4-producing cells, suggesting a Th1 profile. It was previously shown in humans that grass pollen allergen- or Der p 1-pulsed DCs from allergic patients enhanced the production of the type 2 cytokines IL-4 and IL-5 by autologous CD4⁺ T cells in vitro (27, 28). Interestingly, this Th2 cytokine profile induced by DCs from allergic patients was not modified even in the presence of exogenous IL-12, a Th1-polarizing cytokine. Both these in vitro data and our in vivo data suggest that DCs induce the preferential secretion of Th2 cytokines in previously primed CD4 T cells when allergic patients are used.

Many factors could be responsible for this obvious difference in Th cytokine balance between allergic and non-allergic donors. One obvious reason could be the presence of stably differentiated memory Th2 cells among the T cell inoculum derived from allergic donors, as opposed to Th1 cells in healthy controls. It has indeed been shown that T cell reactivity to HDM can be found in the blood of almost all exposed individuals, with atopics demonstrating a Th2 profile, and non-atopics demonstrating a Th1 profile of cytokine secretion following exposure to HDM in serum-free conditions in vitro (29). Alternatively, in our in vivo experiments Th2 responses might be preferentially unmasked in mice receiving Der p 1-pulsed DCs from allergic donors, because more DCs reached the draining lymph nodes continuously compared with pulsed DCs from non-atopic donors, leading to a stronger boosting in the former group. It has been shown that sustained triggering of the TCR preferentially stimulates Th2 effector cells from naive T cells (30).

During the effector phase of the pulmonary immune response, effector T cells are recruited into the lung, release cytokines, and orchestrate airway inflammation. We have previously shown in a humanized SCID mouse model that DCs might play a role in the generation of an allergic effector response developing after a challenge with the HDM allergen. Increased numbers of DCs were detected in the lung of animals reconstituted i.p. with mononuclear cells from allergic patients. Moreover, the injection of DCs from allergic patients in the peritoneal cavity of mice further exposed to HDM aerosols led to an increase in the IgE production (14). In the current study we also determined whether injection of Der p 1-pulsed DCs in the trachea of reconstituted mice also induced effector cells with a capacity to orchestrate IgE production and eosinophilic airway inflammation after challenge with HDM aerosol. No significant levels of HDM-specific IgE were detectable in the serum of the animals administered Der p 1-pulsed DCs from allergic patients in the absence of secondary allergen challenge (data not shown). Other protocols in which protein-specific Th2-dependent Igs were detected after injection of DCs included the injection of a booster of soluble protein 1 wk after DC injection (31). Similarly in our experiments following allergen challenge to Der p 1-pulsed DC-immunized mice, there was production of human IgE, illustrating the generation of Th2 effector cells regulating IgE. Another strong argument for the induction of a Th2 effector response by DCs was the accumulation of human T cells and murine eosinophils in the lungs of mice immunized with Der p 1-pulsed DCs from only allergic patients. A very similar pulmonary infiltrate rich in T cells and eosinophils is a feature of the inflammatory reaction in human allergic asthma (32, 33). The development of a cellular infiltrate following exposure of mice to the relevant allergen was observed, for example, by Duez et al. (12) in SCID mice reconstituted i.p. with mononuclear cells from allergic patients. As eosinophilic airway inflammation is dependent on the production of IL-5 by CD4⁺ T cells, and IgE secretion is dependent on IL-4 production, not surprisingly we observed increased levels of IL-4 and IL-5, but no IFN-, in BAL fluids of mice instilled with Der p 1-pulsed DCs from allergic patients. These results obtained in hu-SCID mice are in agreement with the data obtained in human BAL fluids, where an increased number of Th2 cells expressing IL-3, IL-4, and IL-5 mRNA was observed (9, 32).

All these data indicate that human DCs from allergic patients might play a key role in the development of a pulmonary allergic reaction by activating primed Th2 cells to become effector cells. Consistent with this, our previous experiments in mice have shown that restimulation of primed CD4 T cells to become effector cells that have the potential to regulate eosinophilic airway inflammation is critically dependent upon their stimulation by endogenous airway DCs (10). It has been suggested that the generation of effector function in previously primed T cells could occur locally within the airways, as primed T cells are biased to migrate to the periphery (34). To test the requirement of DC migration to the draining lymph nodes in generating effector function in adoptively transferred T cells, experiments were conducted to block DC migration. It is now well established that DCs that have recognized and taken up Ags in tissues migrate into the draining lymph nodes to interact with T cells. This migration from non-lymphoid tissues to the nodes is a specificity of mature DCs expressing CCR7 and CXCR4 (5, 23). A ligand for the CCR7 is the CC chemokine SLC. This chemokine is constitutively expressed at high levels on the endothelium of the afferent lymphatics and high endothelial venules of lymph nodes and has been implicated in the entry of DCs into afferent lymphatics (35, 36). Indeed, treatment of mice with anti-SLC Abs inhibits DC migration to lymphoid organs (36, 37). In addition, blocking of the CC chemokine MIP-3, another CCR7 ligand, using Abs in vivo leads to inhibition of DC migration to the draining nodes, suggesting that both ligands induce essential and non-overlapping signals via the CCR7 to direct DCs into the T cell area of lymph nodes (38). The importance of both CCR7 ligands is furthermore demonstrated in vivo in spontaneously SLC and MIP-3 deficient (plt, paucity of lymph node T cell) mice, in which their absence was associated with the inability of DCs to migrate from peripheral to lymphoid tissues (25). In our work using human DCs injected in vivo into mice, we showed that DCs exposed in vitro to Der p 1 had increased CCR7 expression, indicating a maturation of the cells in response to the allergen. A potential major issue was represented by the fact that the SCID mouse model is a chimeric model where both human and murine T cells have to interact. We had to determine a possible cross-reactivity of recombinant murine SLC with human DCs, and we showed that only Der p 1-induced mature DCs could migrate in vitro in response to the murine chemokine (not shown). More importantly, the in vivo blockade of murine SLC with neutralizing Abs led to a dramatic decrease in the number of DCs reaching the MLN and in the number of T cells in the MLN and to an accumulation of DCs in the BAL of the mice, illustrating that SLC is essential to get human DCs into the draining lymph nodes. However, as CCR7 is also expressed by naive T cells, the fact that anti-SLC treatment could also affect T cell migration into the draining lymph nodes, as previously described in plt mice (36, 37), cannot be completely excluded. Our results furthermore suggest that T cells and DCs have to interact physically within the draining lymph node to generate the human T cell response, which excludes the possibility that the passive transfer of antigenic material from transferred DCs to endogenous mouse APCs would be the only reason why we see a T cell response. Moreover, the neutralization of mSLC induced a significant decrease in the Th2 effector response induced by DCs, as assessed by the decrease in the number of eosinophils, in the amounts of the cytokines IL-4 and IL-5, and in the production of IgE. One caveat, however, is the recent observation that anti-SLC-treated or plt mice are not truly deficient in T cell activation, but, rather, show delayed kinetics of T cell activation, with T cell responses being dependent on SLC on day 2 following antigenic stimulation, but not on day 6. At the latter time point, T cell responses were even enhanced in plt mice (37, 39). However, in our experiments, there was a 14-day delay (see protocols III and IV) before we actually measured T cell effector responses in terms of IgE production, Th2 cytokines, and airway eosinophilia, largely excluding any effects of delayed immune responses in anti-SLC-treated mice.

It remains to be shown whether migration of endogenous airway DCs to the draining lymph nodes is similarly implied in the generation of effector function in primed T cells, leading to airway inflammation. In this regard, two types of human memory T cells have recently been described; one subset expresses the CCR7 chemokine receptor. These so-called central memory T cells are long-lived cells that are biased to recirculate via central lymphoid organs rather then via peripheral tissues (40). It is our hypothesis that migratory DCs will be needed to activate these recirculating resting T cells to generate effector function (10). Taken together, these results suggest that DCs from allergic patients might play a key role in the development of pulmonary allergic responses mainly through interactions with allergen-specific CD4⁺ cells in the draining lymph nodes, leading to the efficient generation of effector Th2 cells that have the capacity to regulate IgE production and airway eosinophilia. This hu-SCID mouse model will allow us to test new therapeutic strategies interfering with airway DCs in allergic patients with the hope of reducing airway inflammation.

	Acknowledgments

 
We thank Dr. A. Tsicopoulos and the personnel of the Calmette Hospital for the selection of patients and the blood collection involved in this study, Dr. G. A. Stewart for providing Der p 1 allergen, and Dr. G. Gleich for providing anti-murine MBP Abs.

	Footnotes

 
¹ H.H. and B.N.L. contributed equally to this manuscript.

² Address correspondence and reprint requests to Dr. Joël Pestel, Institut National de la Santé et de la Recherche Médicale, Unité 416, Institut Pasteur de Lille 1, rue du Prof. Calmette, BP 24559019, Lille Cedex, France. E-mail address: jpestel{at}voila.fr

³ Abbreviations used in this paper: DC, dendritic cell; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; Dpt, Dermatophagoides pteronyssinus; HDM, house dust mite; hu-SCID, human PBMC-reconstituted SCID; MBP, major basic protein; MIP-3, macrophage inflammatory protein-3; MLN, mediastinal lymph nodes; mSLC, murine secondary lymphoid tissue chemokine; plt, paucity of lymph node T cell; SLC, secondary lymphoid tissue chemokine.

Received for publication July 6, 2001. Accepted for publication May 8, 2002.

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