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The Journal of Immunology, 2007, 179, 3287-3296
Copyright © 2007 by The American Association of Immunologists, Inc.
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Th2 Differentiation in Distinct Lymph Nodes Influences the Site of Mucosal Th2 Immune-Inflammatory Responses1

David Alvarez, Janine L. Arkinson, Jiangfeng Sun, Ramzi Fattouh, Tina Walker and Manel Jordana2

Department of Pathology and Molecular Medicine, Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Allergic individuals rarely present with concurrent multiple-organ disease but, rather, with manifestations that privilege a specific site such as the lung, skin, or gastrointestinal tract. Whether the site of allergic sensitization influences the localization of Th2 immune-inflammatory responses and, ultimately, the organ-specific expression of disease, remains to be determined. In this study, we investigated whether both the site of initial Ag exposure and concomitant Th2 differentiation in specific lymph nodes (LNs) privileges Th2 memory responses to mucosal and nonmucosal sites, and whether this restriction is associated with a differential expression in tissue-specific homing molecules. In mice exposed to Ag (OVA) via the peritoneum, lung, or skin, we examined several local and distal LNs to determine the site of Ag-specific proliferation and Th2 differentiation. Whereas respiratory and cutaneous Ag exposure led to Ag-specific proliferation and Th2 differentiation exclusively in lung- and skin-draining LNs, respectively, Ag delivery to the peritoneum evoked responses in gut-associated, as well as distal thoracic, LNs. Importantly, only mice that underwent Th2 differentiation in thoracic- or gut-associated LNs mounted Th2 immune-inflammatory responses upon respiratory or gastric Ag challenge, respectively, whereas cutaneous Th2 recall responses were evoked irrespective of the site of initial sensitization. In addition, we observed the differential expression of gut homing molecules (CCR9, {alpha}4, β7) in gut-associated LNs and, unexpectedly, a universal induction of skin-related homing molecules (CCR4, CCR10) in all LNs. These data suggest that the site of initial Th2 differentiation and differential homing molecule expression restricts Th2 immune-inflammatory responses to mucosal, but not cutaneous, tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The skin and mucosal surfaces of the respiratory and gastrointestinal (GI)3 tract are continuously exposed to environmental allergens and, thus, are likely to play an important role in allergic sensitization. In the appropriate immunological context, allergens are captured by tissue-resident dendritic cells (DCs) and transported to the local draining lymph node (LN) where they are presented to naive T cells (1, 2). Upon cognate interaction with Ag and MHC class II, naive T cells undergo activation, proliferation, and differentiation into CD4+ Th2 cells (3). In turn, these Ag-experienced CD4+ Th2 cells prime B cells for IgE class-switching (4) and, then, subsequently traffic to the target organ where they execute their immune effector program (5). Indeed, the presence of allergen-specific IgE and activated CD4+ Th2 cells is a defining feature of a number of allergic diseases (6).

Although allergic diseases share a common systemic pathogenesis, allergic individuals rarely present with concurrent multiple-organ disease but, rather, with manifestations at particular sites (7), such as the lung (allergic asthma), skin (allergic dermatitis), and GI tract (food allergy). This heterogeneity insinuates that the site of initial allergen encounter may influence the organ-specific expression of disease. In this regard, recent evidence suggests that the site of initial Ag exposure (8, 9) and, in particular, the site of concomitant T cell priming in specific draining LNs (10, 11, 12), may imprint the ensuing immune effector program with tissue-selective tropism (13, 14, 15). Indeed, it has been shown that DCs from either skin- or gut-associated lymphoid tissues (GALT) selectively induce skin- (CCR4+, CCR10+) or gut-({alpha}4β7+, CCR9+) tropic T cells that preferentially traffic to the skin or gut, respectively (16, 17, 18, 19, 20, 21, 22). In the context of allergic disease, whether the site of initial allergen exposure privileges memory Th2 immune responses to specific mucosal and nonmucosal sites remains unknown. Moreover, whether this restriction is also associated with a differential expression in tissue-specific homing molecules depending on the site (i.e., LN) of Th2 differentiation has not been determined.

We and others have previously shown that mice initially sensitized via the airways (23) or skin (24) mount Th2 immune-inflammatory responses in the lung or skin, respectively. Likewise, it has been shown that allergic responses in the GI tract can be induced in animals initially sensitized via the GALT (25). Yet, whether prior sensitization to Ag via one site is sufficient to allow for the generation of Th2 immune-inflammatory responses upon Ag re-exposure at any given site remains controversial (26, 27, 28). In this study, we investigated whether both the site of initial Ag exposure privileges Th2 immune-inflammatory responses to mucosal and nonmucosal sites, and whether this restriction is associated with a differential expression in tissue-specific homing molecules. Using murine models of Th2 sensitization targeting the peritoneum (29, 30), airways (23), or skin (24), we show that the expression of Th2 immune-inflammatory responses in mucosal, but not cutaneous, tissues is dependent on both the site of initial Ag exposure and concomitant Th2 differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female BALB/c (6–8 wk old) were purchased from Charles River Laboratories and housed in specific pathogen-free conditions with a 12-h light-dark cycle. Experiments described in this study were approved by the Animal Research Ethics Board of McMaster University.

OVA sensitization protocols

Th2 sensitization via the peritoneum. Sensitization via the peritoneum route was achieved by conventional i.p. injections of OVA as previously described (29, 30). Briefly, mice were sensitized twice, 5 days apart, by i.p. injection of 8 µg of OVA (Sigma-Aldrich) adsorbed overnight at 4°C to 4 mg of aluminum hydroxide (Sigma-Aldrich) in a total volume of 0.5 ml of sterile PBS.

Th2 sensitization via the airways. Sensitization via the airways was achieved by exposure to aerosolized OVA in the presence of exogenous GM-CSF expressed via adenoviral (Ad) gene transfer to the airway, as previously described (23). Briefly, a replication-deficient human type 5 Ad vector encoding murine GM-CSF (Ad/GM-CSF) was delivered intranasally to isoflurane-anesthetized mice at a dose of 3 x 107 PFU 1 day before OVA aerosolization. Over the next 10 consecutive days (days 0–9), mice were placed in a Plexiglass chamber (10 cm x 15 cm x 25 cm) and exposed for 20 min daily to aerosolized OVA (grade V protein, 1% w/v in 0.9% saline; Sigma-Aldrich). The OVA aerosol was generated by a Bennet/Twin nebulizer at a flow rate of 10 L/min. We have previously shown that only the concurrent exposure of OVA with Ad/GM-CSF is able to generate Th2 sensitization, and not exposure to OVA alone, Ad/GM-CSF alone, or OVA with an empty Ad vector (23).

Th2 sensitization via the skin. Sensitization via the skin was achieved by particle bombardment of plasmid OVA-coated gold particles onto the skin using the Helios-GG (Bio-Rad), as previously described (24). In brief, mice receive three nonoverlapping deliveries of gold particles coated with OVA-expressing plasmids, totaling 1 µg of plasmid per inoculation (i.e., 0.33 µg of DNA per delivery) onto the ventral abdominal skin on three occasions at weekly intervals. The DNA-coated particles were prepared as described previously and discharged at a helium pressure of 350 lb/inch (2). The plasmids used were a modified version of pcDNA3.1+ (Invitrogen Life Technologies) containing the cDNA for OVA under control of the human CMV intermediate-early promoter. By this approach, we previously reported that delivery of plasmids expressing OVA leads to OVA-specific Th2 immunity, but not luciferase cDNA or empty control plasmids, or delivery of gold particles alone (24).

Splenocyte and LN cell culture

Spleens or LNs (axillary, inguinal, mesenteric, deep cervical, super cervical, thoracic, or Peyer’s patches) were excised at various time points following Ag sensitization via the skin, peritoneum, or respiratory mucosa and placed into sterile tubes containing sterile HBSS on ice. Spleens/LNs were triturated between the ends of sterile frosted slides and the resulting cell suspension was filtered through 40-µm nylon cell strainers (Falcon; BD Biosciences), then washed at 1200 rpm for 10 min at 4°C. RBCs were lysed from spleen suspensions by adding 1 ml of ACK lysis buffer (0.5 M NH4Cl, 10 mM KHCO3, and 0.1 nM Na2EDTA (pH 7.2–7.4)) for 1 min. Splenocytes and dispersed LN cells were washed with HBSS and then resuspended in RPMI 1640 medium supplemented with 10% FBS (Sigma-Aldrich), 1% L-glutamine, 1% penicillin/streptomycin (Invitrogen Life Technologies), and 0.1% 2-ME (Invitrogen Life Technologies). Cells were cultured in medium alone or with 40 µg of OVA/well and seeded at 8 x 105 cells/well (spleen) or 5 x 105 cells/well (LN) in a flat-bottom, 96-well plate (BD Biosciences). Following 120 h of culture incubation, supernatants were harvested and stored at –20°C for cytokine analysis.

In vitro LN cell proliferation

Ag-specific in vitro proliferation was determined by a [3H]thymidine incorporation assay. Briefly, 1 µCi/well [3H]thymidine (PerkinElmer) was added to the last 18 h of a 3-day culture, in the presence or absence of OVA (40 µg/well), and proliferative responses were measured by cell uptake of [3H]thymidine. For proliferation, splenocytes and LN cells were seeded at 5 x 105 cells/well. Cells were harvested using a Filtermate harvester (Packard Bioscience), quantified using TopCount NXT microplate scintillation and luminescence counter (Packard Bioscience), and expressed as the mean cpm ± SD of triplicate wells, where applicable.

Cytokine and Ig measurement

Cytokine/chemokine content was determined using ELISA kits purchased from R&D Systems for murine IL-4, IL-5, IL-10, IL-13, IFN-{gamma}, and eotaxin. Each of these assays has a threshold of detection between 1.5 and 5 pg/ml. Levels of OVA-specific serum IgE were measured using a previously described Ag-capture (biotinylated OVA) ELISA method (29, 30). Units of OVA-specific IgE were determined relative to in-house standardized serum, obtained from mice sensitized to OVA through a conventional i.p. sensitization model for IgE standard, and expressed in units per milliliter relative to standard mouse sera.

OVA recall protocols

OVA recall to the gut: allergic diarrhea. Mice initially sensitized to OVA via the skin, airways, or peritoneum, were re-exposed to OVA by the oral/GI route using a recently characterized protocol of Ag-induced allergic diarrhea (25). Briefly, 2 wk postsensitization, mice were held in the supine position and orally administered 50 mg of OVA protein in 250 µl of sterile PBS by gavage every other day up to seven times. Before each intragastric challenge, mice were deprived of food for 4 h to limit the extent of Ag degradation in the stomach. Oral challenges were performed with intragastric feeding needles (1", 1(1/4) mm ball diameter, 22G; Popper and Sons). Unsensitized naive mice served as negative controls. Clinical scoring of diarrhea was assessed by visually monitoring mice for up to 1 h following oral Ag challenge, by multiple observers. Mice demonstrating profuse liquid stool were scored as positive. Seventy-two hours following the last (i.e., seventh) OVA challenge, mice were sacrificed and the spleen and mesenteric LNs were isolated and cultured for evidence of intact OVA-specific Th2 immunity, as detailed above. In addition, segments of the small intestine (jejunum) were removed, fixed in 10% formalin, embedded in paraffin, sectioned at 3-µm thick, and stained with H&E for visualization of eosinophils.

OVA recall to the lung: allergic airways inflammation. Mice initially sensitized to OVA via the skin, airways, or peritoneum, were re-exposed to OVA via a respiratory challenge with aerosolized OVA. Approximately 3 wk postsensitization, mice were exposed to 1% OVA aerosol daily for 20 min for 5 consecutive days. Unsensitized naive mice served as negative controls. Mice were sacrificed 72 h following the last (i.e., fifth) OVA aerosolization, and the ensuing immune response was assessed in the peripheral blood (PB), bronchoalveolar lavage (BAL) fluid, and lung tissue, as described below. Moreover, thoracic LNs were removed and cultured for evidence of intact OVA-specific Th2 immunity, as detailed above.

OVA recall to the skin: late-phase cutaneous responses. Mice initially sensitized to OVA via the skin, airways, or peritoneum, were re-exposed to OVA by intradermal injection. Approximately 3 wk postsensitization, mice were injected with 10 µg of OVA in 10 µl of sterile saline into one ear, and vehicle (sterile saline) into the opposite ear. Unsensitized naive animals served as negative controls. Ear thickness was measured before and at several time points after injection using a modified low-tension thickness gauge (Dyer Company). Mice were sacrificed 48 h later, and ears fixed in 10% formalin, then sectioned and stained with H&E.

Collection and measurement of specimens

Following OVA recall to the lung, mice were sacrificed and PB was collected by retro-orbital bleeding. Total white blood cell counts determined in a blinded manner using a hemocytometer. Serum was obtained by centrifugation after incubating whole blood for 30 min at 37°C, and stored as aliquots at –20°C. Smears were prepared for differential cell analysis. In addition, BAL was performed as previously described (30). Briefly, the lungs were dissected, the trachea was cannulated with a polyethylene tube (BD Biosciences), and the lungs were lavaged twice with PBS (0.25 ml followed by 0.2 ml); ~0.3 ml of the instilled fluid was consistently recovered. Total BAL cell counts were determined in a blinded manner using a hemocytometer. Each BAL sample was centrifuged, and the supernatant was stored at –20°C for cytokine and chemokine analysis. The cell pellet was resuspended in PBS and smears were prepared by cytocentrifugation (Shandon) at 300 rpm for 2 min. PB and BAL smears were stained with the Protocol Hema 3 stain set (Fisher-Scientific). Differential cell counts of PB and BAL smears were determined in a blinded manner from at least 300–500 leukocytes using standard hemocytological criteria to classify the cells as neutrophils, eosinophils, or mononuclear cells. Lung tissue was removed and fixed in 10% formalin and embedded in paraffin. Sections, 3-µm thick, were stained with H&E (for visualization of leukocytes and histopathological features) or periodic acid-Schiff (for detection of goblet cells and mucus production).

Isolation of mRNA and preparation of cDNA samples

At various time points during skin, respiratory, or i.p. sensitization to OVA, various LNs (thoracic, mesenteric, inguinal, axillary, or cervical) or Peyer’s patches were pooled from three to four mice and stored in RNAlater (Ambion) at –20°C until processed. Total RNA was extracted from LNs using a Polytron homogenizer with TriPure isolation reagent (Roche). Genomic DNA was then removed from the samples using the DNA-free kit (Ambion). Finally, RNA was reversed transcribed to cDNA using the RETRO-script kit (Ambion) with random decamers used as primers.

Gene expression by real-time quantitative PCR

For real-time quantitative PCR, Assays-on-Demand Gene Expression Assays containing two unlabeled PCR primers and a FAM-labeled, MGB-quenched TaqMan probe for either CCR4, CCR8, CCR9, CCR10, {alpha}4 integrin chain, β7 integrin chain, or fucosyltransferase-VII were purchased from Applied Biosystems. The housekeeping gene 18S rRNA were purchased from Applied Biosystems. PCR was run using TaqMan Universal PCR mix (Applied Biosystems) on the ABI Prism (7900HT) Sequence Detection System and analyzed using the Sequence Detector software (version 2.1). Samples were run in triplicate with ~1 µg of cDNA was added per well as a single-plex PCR. Genes were expressed relative 18S rRNA and further normalized to background (i.e., naive) levels.

Data analysis

Data are expressed as mean ± SEM, unless otherwise noted. Statistical analysis was performed using SigmaStat software (SPSS). Results were interpreted using ANOVA followed by Fisher’s least significant difference post hoc test analysis, unless otherwise indicated. A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag-specific proliferation/Th2 differentiation in mice sensitized via the skin, lung, or peritoneum

Following OVA exposure/sensitization via the skin, lung, or peritoneal cavity (as outlined in Fig. 1), we harvested local and distant LNs to determine which secondary lymphoid organs served as the predominant site(s) of Ag presentation and concomitant T cell proliferation and Th2 differentiation. Specifically, we dissected different LNs from the head and neck region (super and deep cervical), the airways (thoracic), the GI tract (mesenteric and Peyer’s patches), and the skin (inguinal and axillary). In skin-sensitized mice we observed robust OVA-specific proliferation in the local skin-draining inguinal and axillary LNs, but not in LNs of the gut, airways, or head/neck region (Fig. 2A). In contrast to this, in mice sensitized via the lung both thoracic and deep/super cervical LNs were the predominant sites of proliferation, whereas distant gut-associated LNs and skin-draining LNs were not involved in this route of sensitization (Fig. 2B). Finally, Ag delivery to the peritoneal cavity led to marked proliferation in the gut-associated mesenteric LNs and to a lesser extent in the Peyer’s patches, as well as the thoracic LNs, but not in the LNs draining the skin or head and neck region (Fig. 2C). The magnitude of the proliferative response was comparable between the principal LNs activated in each of the different models, with an ~4- to 6-fold increase in proliferation (i.e., ratio of OVA cpm to medium cpm), with the exception of the Peyer’s patches which consistently showed a ~3-fold induction in proliferation following i.p. Ag delivery.


Figure 1
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  		FIGURE 1. Models of Th2 sensitization targeting the skin, lung, and peritoneum. A, Skin sensitization to OVA via GG delivery: mice are bombarded with gold particles coated with OVA-expressing plasmids onto the skin. B, Respiratory mucosal sensitization to OVA via aerosolization: mice are exposed to daily OVA aerosols in the context of a GM-CSF-enriched airway microenvironment. C, Conventional sensitization via i.p. injection: mice are sensitized by i.p. injection of OVA adsorbed to the adjuvant Al(OH)3. Time points examined for proliferation and Th2 differentiation, as well as homing molecule expression are indicated.

 

Figure 2
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  		FIGURE 2. Ag-specific proliferation in distinct LNs following Ag exposure to diverse sites. Individual LNs draining the skin (inguinal and axillary LNs), head and neck region (super and deep cervical LNs), airway (thoracic LNs), and gut (mesenteric LNs and Peyer’s patches) were excised from mice sensitized to OVA via the skin (A), lung (B), or peritoneum (C) and examined for Ag-specific proliferative responses. Ag-specific proliferation was assessed by [3H]Thy incorporation following in vitro stimulation of harvested LN cells with OVA or medium alone. Graphs depict peak responses for cutaneous sensitization (72 h postsensitization), respiratory mucosal sensitization (day 9 of the protocol), and peritoneal sensitization (72 h postsensitization). Data are expressed as mean ± SD of triplicate wells (were applicable) from pooled LN cells (from three to five mice) and representative of three independent experiments.

 
To determine the site(s) of Th2 differentiation, we assayed culture supernatants for the production of Th2-affiliated cytokines (IL-4, IL-5, and IL-13) by Ag-stimulated LN cells in vitro. Consistent with the proliferation data, mice sensitized via the skin, lung, or peritoneum resulted in robust Th2-cytokine production in the relevant draining LNs. Specifically, Th2 differentiation was restricted to the skin-draining LNs in skin-sensitized mice, and the thoracic and cervical LNs in respiratory mucosal-sensitized mice (Fig. 3, A and B). Moreover, in i.p.-sensitized mice Th2 differentiation occurred in the gut-associated mesenteric LNs and Peyer’s patches, as well as the thoracic LNs (Fig. 3C). Examination of culture supernatants for IL-10 levels resulted in a similar profile, with enhanced IL-10 production solely in local draining, but not distant, LNs in each of the sensitization protocols (skin sensitization: 730 pg/ml inguinal/axillary LNs; lung sensitization: 750 pg/ml deep cervical LNs, 914 pg/ml super cervical LNs, and 1459 pg/ml thoracic LNs; i.p. sensitization: 2259 pg/ml thoracic LNs and 2772 pg/ml mesenteric LNs).


Figure 3
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  		FIGURE 3. Site of initial Ag exposure leads to Th2 differentiation in distinct LNs. Individual LNs draining the skin (inguinal and axillary LNs), head and neck region (super and deep cervical LNs), airway (thoracic LNs), and gut (mesenteric LNs and Peyer’s patches) were excised from mice sensitized to OVA via cutaneous (A), respiratory mucosal (B), or peritoneal (C) routes and examined for Ag-specific Th2 cytokine (IL-4, IL-5, and IL-13) production following in vitro stimulation of harvested LN cells with OVA or medium alone. Data show pooled cytokine levels from pooled LN cells (from three to six mice) and representative of three independent experiments.

 
Systemic Th2 cytokine and IgE production

In addition to characterizing the response in individual LNs during sensitization, we also determined the contribution of the spleen compartment in each of the models of sensitization. To this end, we harvested splenocytes from skin-, lung-, or i.p.-sensitized mice and pulsed them with OVA in vitro to examine Th2 cytokine (IL-4, IL-5, and IL-13) production. As shown in Table I, we observed OVA-specific Th2 cytokine production by cultured splenocytes regardless of the site of initial Ag exposure. Of note, mice sensitized by the i.p. route produced slightly higher levels of Th2 cytokines and OVA-specific serum IgE than mice sensitized via the skin or lung. Mice sensitized to OVA via the lung also produced high levels of Th2 cytokines compared with skin-sensitized mice; however, the levels of serum IgE were lowest.


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  		Table I. OVA-specific serum IgE and splenocyte-derived Th2 cytokine production following skin, respiratory, or i.p. sensitization in micea

 
Intragastric OVA recall in mice sensitized via the skin, lung, or peritoneum

We next investigated whether both the site of initial Ag exposure and concomitant Th2 differentiation in specific LNs privileged the ensuing Th2 memory response to specific tissue compartments. To this end, mice were sensitized to OVA via the skin, lungs, or peritoneum, and then re-exposed to OVA by oral gavage using a recently characterized protocol of Ag-induced GI inflammation and acute allergic diarrhea (see Materials and Methods; Ref. 25). Despite unequivocal signs of Th2 immunity in mice previously sensitized to OVA via the skin or lung (Fig. 3, A and B), only mice that were initially sensitized to OVA via the peritoneal cavity developed OVA-induced allergic diarrhea (Fig. 4A). Indeed, i.p.-sensitized mice started developing allergic diarrhea after the third intragastric OVA challenge and, by the seventh exposure, all mice showed signs of GI dysfunction, including obvious signs of discomfort (data not shown). We also harvested mesenteric LN cells 72 h following the last (i.e., seventh) OVA challenge and stimulated them in vitro with OVA to determine whether a Th2 immune response was been developed (Fig. 4B). We found a pronounced increase in Th2 cytokine production by OVA-stimulated mesenteric LN cells from diarrhea-positive and i.p.-sensitized mice. Importantly, mesenteric LN cells harvested from diarrhea-negative and skin- or respiratory mucosal-sensitized mice also produced Th2 cytokines, but at significantly lower levels. We also examined, histopathologically, jejunum segments of the small intestine 72 h following the last oral challenge, and observed a slightly greater influx of eosinophils in diarrhea-positive/i.p.-sensitized mice compared with diarrhea-negative and skin- or respiratory mucosal-sensitized mice, or unsensitized naive controls (Fig. 4C). Despite being unresponsive to intragastric OVA challenge, splenocytes harvested from skin- and respiratory mucosal-sensitized mice produced very similar, if not higher, Th2 cytokines compared with diarrhea-positive/i.p.-sensitized mice (Fig. 4D).


Figure 4
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  		FIGURE 4. Intragastric Ag challenge elicits allergic GI responses in mice initially sensitized via the GALT. Mice were initially sensitized to OVA via the skin, airways, or peritoneum, and subsequently challenged with OVA by intragastric gavage. Mice were challenged with OVA (50 mg) seven times, every other day, and scored for the occurrence (%) of Ag-induced allergic diarrhea (A) as assessed up to 1 h postchallenge (n = 6–8). B, Mesenteric LNs were excised 72 h after the final challenge and examined for Th2 cytokine production in vitro. Data in B represent mean ± SEM (n = 3). C, Representative H & E sections of jejunum tissue following intragastric challenge with OVA in unsensitized naive (i), skin-sensitized (ii), airway-sensitized (iii), or i.p.-sensitized (iv) mice. Arrowheads depict eosinophils. Original magnification of panels, x200. Original magnification of insets, X600 oil immersion. D, Th2 cytokine production by in vitro stimulated splenocytes.

 
Respiratory OVA recall in mice sensitized via the skin, lung, or peritoneum

Next, we determined whether mice initially sensitized via the skin, lungs, or peritoneum, were equally able to mount OVA-specific Th2 immune-inflammatory responses in the lung upon re-exposure to aerosolized OVA (Fig. 5). Mice sensitized to OVA via the lung mounted a robust inflammatory response upon respiratory OVA recall including a marked increase in total cells and eosinophils in the BAL compartment (Fig. 5A). This was accompanied by an increase in PB eosinophils and BAL eotaxin levels (Fig. 5, B and C), a massive peribronchial and perivascular eosinophilic infiltrate in the lung tissue, and pronounced goblet cell hyperplasia and mucus production (Fig. 5Eiii). Likewise, mice sensitized via the peritoneum also mounted a robust Th2 immune-inflammatory response in the airways, which was both qualitatively and quantitatively similar to mice initially sensitized via the lung (Fig. 5E, iii and iv). Consistent with our previous findings (24), skin-sensitized mice failed to mount allergic responses upon OVA aerosolization. Indeed, the response elicited by skin-sensitized mice was similar to that of unsensitized naive controls (Fig. 5E, i and ii).


Figure 5
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  		FIGURE 5. Respiratory Ag challenge elicits allergic airways inflammation in mice initially sensitized via the thoracic LNs. Mice were initially sensitized to OVA via the skin, airways, or peritoneum, and subsequently challenged with OVA by aerosolization. Mice were challenged daily with 1% OVA aerosol for 20 min for 5 consecutive days then sacrificed 72 h later. A, The number of total cells, mononuclear cells and eosinophils as assessed in the BAL. B, Total circulating cells and eosinophils in the PB. C, Eotaxin levels in BAL fluid. D, Total LN cells and Th2-cytokine production (IL-4) following aerosolized OVA challenge. Thoracic LNs were excised 72 h postchallenge, enumerated, and stimulated in vitro for IL-4 production. Data are pooled from three to four mice per group. E, Light photomicrographs of lung tissue stained with H & E or periodic acid-Schiff (insets) following respiratory challenge with OVA in unsensitized naive (i), skin-sensitized (ii), airway-sensitized (iii), or i.p.-sensitized (iv) mice. Original magnification of panels, x50. Original magnification of insets, x400.

 
We also examined whether respiratory OVA recall provoked Th2 immune activity in local thoracic LNs. Indeed, mice initially sensitized via the lung or peritoneum experienced a massive expansion in total thoracic LN cells upon respiratory OVA recall, unlike skin-sensitized mice or naive controls (Fig. 5D). Upon OVA stimulation in vitro, thoracic LN cells from mice initially sensitized via the lung or peritoneum produced enhanced levels of Th2-associated cytokines (IL-4) compared with skin-sensitized or naive mice (Fig. 5D).

Cutaneous OVA recall in mice sensitized via the skin, lung, or peritoneum

Next, we investigated whether OVA recall to the skin could lead to cutaneous Th2 immune-inflammatory responses irrespective of the site of initial Ag exposure or sensitization. To this end, mice were initially sensitized to OVA via the skin, lung, or peritoneum (or unsensitized), and 3 wk later exposed to OVA by dermal injection into the ear. In skin-sensitized mice, we observed a prominent late-phase cutaneous response as determined by a significant increase in ear thickness following OVA, but not vehicle, injection (Fig. 6A). Moreover, this was accompanied by a marked influx of eosinophils into the ear tissue (Fig. 6Bii). Likewise, mice initially sensitized to OVA via the lung or peritoneum were able to mount late-phase cutaneous responses upon dermal challenge with OVA, as indicated by an increase in ear thickness and eosinophil infiltration. Histopathologically we also observed a marked influx of eosinophils into ear tissue challenged with OVA (but not saline or unsensitized naive mice challenged with OVA) in respiratory or i.p.-sensitized mice (Fig. 6B, iii and iv).


Figure 6
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  		FIGURE 6. Skin Ag challenge elicits late-phase cutaneous Th2 immune-inflammatory responses. Mice were initially sensitized to OVA via the skin, airways, or peritoneum, or unsensitized (naive) and subsequently challenged with OVA (or saline) by intradermal injection. A, Ear thickness measurements following OVA (filled symbols) or saline (open symbols) injection at the indicated time points. B, Light photomicrographs of H & E-stained cross-sections of ear tissue 48 h postchallenge. All panels represent sections obtained following dermal OVA challenge in naive (i), skin-sensitized (ii), respiratory-sensitized (iii), or i.p.-sensitized (iv) mice. Arrows depict eosinophils. Original magnification of panels, x50. Original magnification of insets, x400.

 
Differential expression of tissue-specific homing molecules in distinct LNs

The differential expression of unique homing molecules on various T cell subsets is a defining feature in the establishment of tissue-selective trafficking patterns. To determine whether sensitization via diverse sites (skin, lung, or peritoneum) led to a differential induction of tissue-specific homing molecules in vivo, we examined the expression of several putative skin-tropic (CCR4, CCR8, and CCR10) and gut-tropic (CCR9, {alpha}4 integrin, β7 integrin) molecules. To this end, the principal draining LNs from each sensitization protocol (see Fig. 1) were excised at various time points and examined for the differential expression of tissue-homing molecules. In mice sensitized via the skin, we excised axillary LNs at several time points post the third gene-gun (GG) delivery, and observed a selective induction of CCR4 and CCR10, but not the gut-tropic molecules (CCR9, {alpha}4 integrin, or β7 integrin; Fig. 7A). We did not observe an up-regulation in CCR8. Moreover, in mice sensitized to OVA via the lung, we also observed an induction of skin-tropic CCR4 and CCR10, but not gut-tropic molecules, in thoracic LNs (Fig. 7B). In contrast, in mesenteric LNs of mice sensitized to OVA via the peritoneum, we observed the induction of gut-tropic molecules (both {alpha}4 and β7 integrins, as well as CCR9; Fig. 7C). Again, we also observed an increase in the expression of the skin-tropic homing molecules (CCR4, CCR8, CCR10; Fig. 7C).


Figure 7
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  		FIGURE 7. Differential expression of tissue-specific homing molecules in skin, lung, and gut-draining LNs during sensitization. Mice were exposed to OVA via the skin, lung, or peritoneum and at various time points, RNA was isolated from the relevant skin, lung, or gut-draining LNs and examined for the expression of gut- (CCR9, {alpha}4β7 integrins) or skin- (CCR4, CCR8, CCR10) tropic homing molecules by quantitative PCR. Relative expression of CCR4, CCR8, CCR9, CCR10, {alpha}4 integrin, and β7 integrin in skin (axillary)- (A), lung (thoracic)- (B), or gut (mesenteric)- (C), draining LNs. Data are expressed as fold increase over naive, and represent pooled means of triplicate measurements relative to housekeeping gene (18S rRNA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we investigated whether the localization of memory Th2 immune responses to specific mucosal and nonmucosal sites was dependent on both the site of initial allergen exposure and concomitant Th2 differentiation in specific draining LNs. We show that cutaneous and respiratory Ag delivery evoked responses exclusively in the skin and cervical/thoracic LNs, respectively, whereas i.p. Ag exposure induced responses both in the gut-draining LNs and, importantly, also in the thoracic LNs. This latter response is consistent with several reports showing that the i.p. route also drains to the thoracic LNs (31). Despite differences in the Ag delivery systems (i.e., aerosol, injection, and GG) the ensuing immune response was distinctly Th2 in nature. The differences in the levels of Th2 cytokine production between protocols may be attributable to kinetics, a greater number of OVA-specific T cell clones induced in one model vs the other, or whether there are differences in the phenotype of the Th2 cells induced. It is known, however, that the generation of Th2 differentiation can be differentially programmed by specific cytokines (IL-4, IL-10, and IL-13) depending on the site of sensitization (32, 33, 34).

Next, we determined whether the site of initial Th2 differentiation in specific LNs influenced the localization of memory Th2 immune-inflammatory responses. We found, that allergic GI responses were clearly induced only in mice initially sensitized via the peritoneum. In fact, despite evidence of systemic Th2 immunity in cutaneous- and respiratory mucosal-sensitized mice, these mice were unable to develop GI manifestations upon intragastric Ag challenge. Previous studies have demonstrated that oral Ag-induced diarrhea and allergic GI responses in mice are dependent on the induction of Th2 immunity including GI inflammation mediated by IgE-primed mast cells, eosinophils, and CD4+ Th2 cells (25, 35, 36). In those studies, disease-positive animals had also been sensitized systemically although the precise involvement of specific LNs during sensitization was not examined (25). In our study, the ability to mount allergic GI responses correlated not with the existence of Th2 immunity per se, but rather with Th2 differentiation having occurred initially in the gut-draining LNs which we observed only in mice sensitized via the peritoneum. Notably, mice initially sensitized via the lung or skin, albeit unresponsive to oral Ag challenge, also showed signs of Th2 immune responsiveness in the mesenteric LNs. However, the levels were markedly lower and, perhaps, of insufficient strength (or below a certain threshold) to establish effector Th2 immunity in the GI mucosa. That the levels of serum OVA-specific IgE were not significantly different in mice sensitized via peritoneum compared with skin- or respiratory-sensitized mice argues against diminutive sensitization in the latter groups. Moreover, while mesenteric LN cytokine responses (post-oral Ag challenge) were highest in the i.p.-sensitized and disease-positive mice (Fig. 4B), markers of systemic Th2 immunity (i.e., spleen-derived Th2 cytokine production) during intragastric Ag challenge were on par between all mice (Fig. 4D).

Similar to the induction of GI manifestations, airway inflammation only developed in mice where Th2 differentiation had occurred in the thoracic, but not skin, draining LNs. Although it is not surprising that i.p.-sensitized mice mounted allergic airways responses because, as previously mentioned, thoracic LNs are involved during priming, it is unexpected, however, that priming via the respiratory mucosa can prime for effector responses in the airway but cannot lead to effector responses at the GI mucosa. These data are at variance with the view of a common mucosal system (37, 38, 39), but consistent with a growing number of reports showing that immunization at one mucosal site does automatically lead to protective immunity in all other mucosal organs (40, 41). In contrast to the selective expression of mucosal Th2 responses in the GI tract or lung, late-phase cutaneous responses were similarly provoked in mice regardless of the site of initial Ag exposure, despite the complete absence of involvement of skin-draining LNs during i.p. or respiratory sensitization.

These data are particularly intriguing in light of recent evidence that the tissue origin of DCs (8) and the specific LN microenvironment (10, 11, 12) imprint T and B cells with tissue-specific homing molecules (16, 19, 42, 48). In this regard, we found that gut-tropic homing molecules (CCR9 and {alpha}4β7 integrins) were differentially up-regulated in mesenteric LNs of i.p.-sensitized mice, but not in the major draining LNs of skin- or respiratory-sensitized mice. As yet, chemokine receptors/integrins specific for T cells homing to the lung are not well defined (43). Of particular significance, we found that regardless of site of initial Th2 differentiation, skin-tropic molecules (CCR4 and CCR10) were uniformly up-regulated in all LNs. This is consistent with recent reports suggesting that activated T cells acquire skin tropism by default unless they encounter signals from distinct DC subsets, at which point they alter their expression pattern of homing molecules (12). The pervasive ability to mount cutaneous responses observed in our studies, despite no involvement of cutaneous LNs in two of the models studied supports the concept of skin tropism by default. Alternatively, higher numbers of Ag-specific T cells evoked in some models could make available more Ag-specific clones to respond distally; a notion consistent with a "selection" view of T cell homing (49). Moreover, the use of different adjuvants during Ag delivery may promote DC migration to distal LNs or spleen, thereby inducing diverse homing phenotypes. Indeed, restricting Ag delivery and DC traffic to local LNs may ultimately limit pervasive responses as discussed below.

In our study, there are a number of variables, including the formulation of Ag (DNA vs protein), dose, and adjuvant (alum, GM-CSF, and gold particles) that differ depending on the protocol. The type of adjuvant used during Ag delivery has dramatic effects on the migratory capacity of DCs from the site of Ag exposure to local and even distal LNs (44). Indeed, transcutaneous immunization strategies incorporating bacterial adjuvants like cholera toxin has been shown to enhance migration of skin-derived DCs to immune-inductive sites of the gut mucosa, like the Peyer’s patches, to elicit robust mucosal CTL responses (44). It remains to be determined whether the different adjuvants used in our study stimulate DC migration beyond local LNs. A number of reports have shown that Ag sensitization via the skin primes for subsequent Th2 immune responses in the lung (26, 27, 34) and the systemic compartment (45, 46, 47). It is unknown whether the Ag delivery modes in those studies exclusively confined the Ag to specific tissues and, more importantly, LNs. For the most part, these studies did not specifically set out to investigate the compartmentalization of Th2 immunity and, consequently, a comprehensive examination of the sites of Th2 differentiation in local and distal LNs is lacking. In our study, we have incorporated two models of Th2 sensitization that target Ag to the respiratory tract or skin. Importantly, these models also generated responses in the spleen compartment, either by Ag leakage into the circulation or via DC transport. Although, blood-borne Ags captured in the marginal zone of the spleen can be eventually presented to naive T cells, it is unknown whether T cells primed in that compartment would be imprinted with any tissue-selective homing program. It is our view that the GG Ag-delivery system, in particular, is better suited, but by no means perfect, to study the compartmentalization of immune responses, compared with alternatives like aerosolization, skin painting, or i.p. priming. Vast improvements in targeting and controlling the expression and dose of Ag in specific peripheral and, ideally, lymphoid tissues through the use of tissue-specific and inducible promoters will provide us with the necessary tools to begin to uncover the mechanisms underlying immune compartmentalization in vivo.

In summary, our data argue for the compartmentalization of Th2 immune responses at mucosal sites, and the pervasive or default ability to mount cutaneous responses. What are the physiological implications of these findings? That the immune system is designed to compartmentalize the effector response to the site of Ag entry hinges on the imprinting of particular sets of tissue-selective homing molecules in the local LN microenvironment. Indeed, the precise manner by which lymphocytes selectively traffic to specific tissues may be overridden under circumstances where Ags permeate beyond regional immune control and drift systemically, as would be the case for live pathogens. Such adaptation of default immune surveillance patterns not only provides for a more tailored immune effector response, it may also be, in itself, reflective of the danger associated with the Ag. In the context of allergic disease, perhaps the prerequisite for developing multiple organ manifestations arises not through allergen exposure per se, but rather as allergic sensitization becomes increasingly systemic. It is under these conditions where severity overwhelms the need for finesse and places allergic GI responses at the far end of the spectrum of allergic disease.


   Acknowledgments
 
We extend our thanks to Susanna Goncharova, Katherine Arias, and Bobae Kim for technical assistance. We also gratefully acknowledge the secretarial assistance of Mary Kiriakopoulos.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Canadian Institutes for Health Research (CIHR). D.A. and R.F. were supported by CIHR Doctoral Research Awards. M.J. holds a CIHR-Canada Research Chair. Back

2 Address correspondence and reprint requests to Dr. Manel Jordana, Department of Pathology and Molecular Medicine, Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics, McMaster University, Michael G. DeGroote Centre for Learning and Discovery, Room 4013, 1200 Main Street, West, Hamilton, Ontario, L8N 3Z5, Canada. E-mail address: jordanam{at}mcmaster.ca Back

3 Abbreviations used in this paper: GI, gastrointestinal; DC, dendritic cell; LN, lymph node; GALT, gut-associated lymphoid tissue; Ad, adenoviral; PB, peripheral blood; BAL, bronchoalveolar lavage; GG, gene gun. Back

Received for publication May 4, 2006. Accepted for publication June 26, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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