Introduction

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Airway eosinophilia is a characteristic feature of asthmatic inflammation in humans, as well as in murine experimental models that recapitulate this process (1, 2). However, the accumulation of eosinophils in the airway may be viewed as a terminal step in a sequence that involves complex cellular interactions not only in the airway but also in other immunologically important sites, such as the thoracic lymph nodes. Although a critical role of T lymphocytes in the development of experimental allergic inflammation is well established (3), T cells cannot respond effectively to antigen unless it is adequately presented to them by antigen-presenting cells (APCs) (4, 5). Dendritic cells (DCs), the most potent APCs, are believed to be indispensable to the initiation of primary T-cell responses (6, 7). In addition to their ability to capture and process antigen, DCs have a considerable migratory capacity. Because both T cells and DCs display discrete migration patterns, the temporal and spatial interplay between antigen, APCs, and T cells is of central importance to our understanding of specific immunity. The objective of this study was to investigate spatial and temporal changes in the phenotype of APCs and T cells in a model of antigen-induced airways inflammation.

We used a murine model of antigen-induced airways inflammation that involves sensitization with ovalbumin (OVA) delivered intraperitoneally together with an adjuvant, followed by antigen aerosol challenge. Our model and other similar models have been characterized in a number of parameters (8, 9); these studies, however, have largely focused on events that take place in the lung after aerosol challenge. Our objective was to define, after both sensitization and challenge, cellular subsets within the APC and lymphocyte compartments at two sites, namely the lungs and the thoracic lymph nodes.

The pattern that emerged from these studies is that there is a cellular expansion, mainly of B and T cells, in the thoracic lymph nodes shortly after the second intraperitoneal (i.p.) injection. A considerable number of APCs expressed the costimulatory molecule B7.2, with no significant changes (compared with naive mice) in B7.1 expression. Although we observed increased expression of CD69 on T cells, T1/ST2 expression remained at the level detected in naive mice. Although these cells expressed GATA-3 and signal transducer and activator of transcription (STAT)-6 and were capable of cytokine production in vitro after stimulation with OVA, expression of several cytokine messenger RNA (mRNA) species in vivo was marginal. Throughout the process of antigen sensitization, the lung remained immunologically silent. However, shortly after aerosol challenge there was an expansion of APCs in the lung that was different in character from that observed in the lymph nodes. That is, whereas APC expansion in the lymph nodes was mainly due to B cells, APC expansion in the lung largely consisted of macrophages and DCs. This expansion was accompanied by enhanced expression of both B7 molecules. We also observed a marked expansion of CD69⁺ T cells in the lung after aerosol challenge. In contrast to our findings in the lymph nodes, however, we documented a remarkable increase in the number of CD4⁺ T cells expressing the T helper (Th)-2-associated marker T1/ST2. In addition, we observed a dramatic increase in effector activity, as detected by expression of interleukin (IL)-4, IL-5, and IL-13 mRNA after aerosol challenge. These Th2-associated cytokines were expressed at a considerably greater level than interferon (IFN)- and IL-15, whereas IL-2 and IL-10 were expressed at marginal levels. Our study indicates that whereas the major events, including expansion of activated T cells and APCs, occur in the lymph nodes after sensitization, the effector functions are executed within tissue presumably upon the encounter of antigen.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

Female Balb/c mice (6 to 8 wk old) were purchased from Harlan (Indianapolis, IN). Mice were maintained under a 12-h light- dark cycle in an access-restricted area. Cages, food, and bedding were autoclaved, and the handling of mice was carried out in a laminar flow hood only by gloved and masked personnel. The Animal Research Ethics Board of McMaster University approved all the experiments described here.

Sensitization and Antigen Challenge Protocol

The sensitization and challenge protocol has been described previously (8). In brief, mice were sensitized at Days 0 and 5 by i.p. injection of 8 µg OVA (Sigma Chemical Co., St. Louis, MO) adsorbed to 4 mg of aluminum hydroxide (Aldrich Chemicals Co., Milwaukee, WI) overnight at 4°C in a total volume of 0.5 ml of phosphate-buffered saline (PBS). At 7 d after the second sensitization, mice were placed in a Plexiglas chamber (10 × 15 × 25 cm) and exposed to aerosolized OVA (10 mg/ml in 0.9% saline) for 1 h on two occasions 4 h apart. The aerosolized OVA was produced by a Bennet nebulizer at a flow rate of 10 liters/min.

Lymph Nodes and Lung Cell Isolation

Thoracic lymph nodes, including the hilar, mediastinal, and tracheobronchial nodes, were removed and adjacent connective tissue was dissected away. The nodes were immediately placed in cold (4°C) Hanks' balanced salt solution (HBSS) (GIBCO BRL, Grand Island, NY). The nodes were ground between frosted slides and filtered through nylon mesh (BSH Thompson, Scarborough, ON, Canada). The cell suspension was centrifuged at 1,200 rpm for 10 min at 4°C and resuspended again in PBS. After this washing step, the cells were resuspended in flow cytometric analysis buffer (PBS supplemented with 0.2% bovine serum albumin or in RPMI (GIBCO BRL) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine (Sigma), and 1% penicillin/streptomycin. Cells were cultured in medium or alone with 40 µg OVA/well at 8 × 10⁵ cells/well in a 96-well flat-bottom plate (Becton Dickinson, Lincoln Park, NJ). After 5 d of culture, supernatants were harvested for cytokine measurement.

For isolation of lung cells, lungs were flushed via the right ventricle of the heart with 10 ml of warm (37^oC) HBSS (calcium- and magnesium-free) containing 5% FBS (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL). The lungs were then cut into small pieces (approximately 2 mm in diameter) and shaken at 37°C for 1 h in 15 ml of 150 U/ml collagenase III (Worthington Biochemical, Freehold, NJ) in HBSS. Using a plunger from a 5-ml syringe, the lung pieces were triturated through a metal screen into HBSS, and the resulting cell suspension was filtered through nylon mesh. After lysing red blood cells with ACK lysis buffer (0.5M NH₄Cl, 10 mM KHCO₃, and 0.1 nM Na₂-ethylenediaminetetraacetic acid at pH 7.2 to 7.4), cells were washed twice and mononuclear cells were isolated by density centrifugation in 30% Percoll (Pharmacia, Uppsala, Sweden). With this enzymatic digestion protocol approximately 60% of cells constituted mononuclear cells, and these were gated for flow cytometric analysis.

Flow Cytometric Analysis

Panels of monoclonal antibodies (mAbs) were selected to study the phenotype of APCs and T lymphocytes in lung and lymph-node cells. To minimize nonspecific binding, 10⁶ cells were preincubated with FcBlock (CD16/CD32; Pharmingen, Mississauga, ON, Canada). For each antibody combination, 10⁶ cells were incubated with mAbs at 0 to 4°C for 30 min; the cells were then washed and treated with second-stage reagents. The following antibodies were purchased from Pharmingen: anti-B7.1 (biotin-conjugated 16-10AI), anti-B7.2 (biotin-conjugated GLI), anti-CD11b (Mac1) (phycoerythrin [PE]-conjugated MI/70), anti-CD11c (PE- conjugated HL3), anti-CD3 (biotin-conjugated 145-2CII), anti-CD4 (fluorescein isothiocyanate [FITC]-conjugated L3T4), anti-CD8 (FITC-conjugated Ly-2), and anti-CD69 (PE-conjugated H1 2F3); the anti-major histocompatibility complex (MHC) class II antibody (FITC-conjugated M5/114.152) was prepared by Dr. D. P. Snider (Department of Pathology and Molecular Medicine, McMaster University). T1/ST2 (3E10) antibody was provided by Millenium Pharmaceuticals, Inc. (Cambridge, MA), and FITC-labeled in-house according to a standard protocol (10). Streptavidin PerCP (Becton Dickinson, San Jose, CA) was used as a second-step reagent for detection of biotin-labeled antibodies. Titration was used to determine the optimal concentration for each antibody. Cells were fixed in 1% paraformaldehyde and counted on a FACScan, and analyses were performed using PC-LYSIS software (Becton Dickinson, San Jose, CA). A total of 50,000 to 100,000 events was acquired.

Collection, Extraction, Separation, and Isolation of mRNA from Tissues

Thoracic lymph nodes, spleens, and lungs (typically the left lobe and one right lobe) were collected and placed in 1 ml TriPure Isolation Reagent, a monophasic solution of phenol and guanidine thiocyanate (Boehringer Mannheim Canada, Laval, PQ, Canada). Tissues were then homogenized with a Polytron 7-mm power homogenizer (Kinematica, Lucerne, Switzerland) and RNA was isolated according to the TriPure Isolation Reagent protocol. The RNA pellet was resuspended in 20 µl of diethypyrocarbonate-treated ribonuclease (RNase)-free water. To determine the concentration of total RNA collected, the optical density was calculated using an Ultraspec 1000 UV/Visible spectrophotometer (Pharmacia Biotech [Biochrom] Ltd., Cambridge, UK). The RNA was stored in a 70°C freezer until analysis.

RNase Protection Assay

The RiboQuant Multi-Probe RNase Protection Assay (Pharmingen) was used to detect and quantify mRNA species from lungs and lymph-node tissues. Briefly, on Day 1 of the assay, an [-³²P]uridine triphosphate (30,00 Ci/mmol, 10 mCi/ml)-labeled antisense RNA probe set was synthesized using the mCK-1 Multi-Probe Template Sets. The probe (~ 1 × 10⁶ counts per min [cpm]/µl) was then hybridized with the desired amount of target RNA, typically 15 µg, overnight at 56°C. PharMingen control RNA (2 µg) and yeast transfer RNA (2 µg) were used as positive and negative controls, respectively. On Day 2, the unhybridized RNA and protein were digested and the protected probes were purified, precipitated, and resuspended in loading buffer according to the manufacturer's protocol. The samples were loaded at approximately 2,000 cpm/lane and electrophoresed at 60 W constant power on a denaturing 5% polyacrylamide gel to resolve the RNase-protected probes. The gel was dried for ~ 1 h at 80°C under a vacuum gel drier (Bio-Rad Laboratories Canada Ltd., Mississauga, ON, Canada) and placed on a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) overnight. The gel was visualized and analyzed using ImageQuant software (Molecular Dynamics). To quantify, each test and housekeeping band, as well as the background of each lane, was captured by first drawing a rectangle around it and then integrating the volume of intensity inside the rectangle. Background volumes were subtracted, and the ratio of the test band volume to the average of the housekeeping band volume was generated and expressed as RNase protection assay (RPA) units (× 10³).

Real-Time Polymerase Chain Reaction (Taqman)

STAT-6 and GATA-3 expression was evaluated using real-time quantitative polymerase chain reaction (PCR) analysis. In brief, an oligonucleotide probe was designed to anneal to the STAT-6/ GATA-3 genes between two PCR primers. The probe was then fluorescently labeled with 6-carboxyl-fluorescein (reporter gene) on the 5' end and with 6-carboxyl-tetramethyl rhodamine (quencher dye) on the 3' end. A similar probe and PCR primers were purchased for rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The probe for this gene incorporated VIC as the reporter dye. PCR reactions were run that included the primers and probes for these two genes as well as complementary DNA (cDNA) made from cells isolated from lymph nodes. As the polymerase moved across the gene during the reaction, it cleaved the dye from one end of each probe, which caused a fluorescent emission that was measured by the Sequence Detector 7700. The emissions recorded for each cDNA were then converted to determine the level of expression for STAT-6/GATA-3 normalized to the expression of mGAPDH. Expression of STAT-6 and GATA-3 was determined on cells isolated from lymph nodes after a second i.p. sensitization.

Measurement of Cytokines

Enzyme-linked immunosorbent assay (ELISA) kits for murine IL-4 and IL-5 were purchased from R&D Systems (Minneapolis, MN) and Amersham (Buckinghamshire, UK), respectively; each of these systems has a threshold of detection of 1.5 to 5 pg/mL.

Data Analysis

Data are expressed as means ± standard error of the mean (SEM). Whenever suitable, results were interpreted using analysis of variance (ANOVA) test with Tukey's post hoc test or Student's t test. Differences were considered statistically significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Phenotype of APCs in the Thoracic Lymph Nodes and Lungs

Mice were killed at Days 0 (naive mice), 6 (24 h after sensitization), and 13 (24 h after challenge). We observed an enlargement of mediastinal, bronchial, and tracheobronchial lymph nodes after the second i.p. injection with OVA/alum. Numerically, this translated into a significant cellular increase from 1.2 ± 0.4 × 10⁶/mouse at Day 0 to 6.4 ± 0.7 × 10⁶/mouse at Day 6, as assessed by counting with hemocytometer; this enlargement was also observed at Day 13 after challenge (4.9 ± 0.8 × 10⁶/mouse). In the lungs, the total cell numbers at Days 0 and 6 were not significantly different from one another. However, they increased more than 3-fold from 2.2 ± 0.4 × 10⁶/mouse at Day 0 to 6.9 ± 1.6 × 10⁶/mouse at Day 13.

Having documented an expansion in total cell number at Day 6 in the lymph nodes and at Day 13 in the lungs, we then examined the types of APCs contributing to this expansion. To this end, B cells, macrophages, and DCs were identified on the basis of B220⁺ (11), MHC II⁺/Mac-1⁺ (12), and MHC II⁺/CD11c⁺ (13) expression, respectively. As shown in Figure 1, it is apparent that the increase in APCs in the lymph nodes at Day 6 was due largely to B cells (40.9% compared with 18.8% in naive mice). The same distribution of APCs in the lymph nodes was observed at Day 13 (data not shown). In contrast, the increase in APCs observed in the lungs at Day 13 was due to increases in macrophages (16.6 ± 2.3 versus 3.8 ± 0.2) and DCs (9.1 ± 1.8 versus 2.2 ± 0.2).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Proportion of B cells, macrophages, and DCs in the lung and lymph nodes at different time points after sensitization and challenge with OVA. Totals of 50,000 and 100,000 events were collected for lungs and lymph-node cells, respectively, and analyzed for MHC/CD11c (DCs), MHC/Mac-1 (macrophages), and B220 (B cells). Inserts indicate changes in the number of APCs (MHC II+) at the time of major expansion. Lymph nodes and lungs from five to 10 mice were pooled for each group. Values represent means ± SEM of three to five experiments. Statistical analysis was performed by t test; *P < 0.05.

Expression of B7.1 and B7.2 on APCs

Next, we examined the expression of the costimulatory molecules B7.1 and B7.2 on APCs in lymph nodes and lung. As shown in Figure 2, MHC II⁺/B7.2⁺ cells increased considerably at Day 6, from 14.7% to 38.4%, and remained at a similar level at Day 13 (45.5%) in the lymph nodes. In contrast, the percentage of MHC II⁺ cells expressing B7.1 did not increase at any time point tested. Further, Table 1 shows that a large proportion of the increase in B7.2⁺ cells was contributed by B cells (from 12.3 ± 3.4% to 28.1 ± 7.1%). This preferential expression of B7.2 on B cells was also detected at Day 13 after challenge (40.3 ± 6.3%) (data not shown). We also observed a trend for increased B7.2 expression on DCs from 3.4 ± 1.6% in naive to 8.3 ± 2.1% at Day 6 and 14.8 ± 4.1% at Day 13. The proportion of B cells, macrophages, and DCs expressing B7.1 did not change at any time point.

View larger version (32K): [in this window] [in a new window]   Figure 2.   B7.1 and B7.2 expression on MHC II+ cells isolated from lymph nodes and lung tissue at the indicated time points after sensitization and challenge. Cells were gated on MHC II+ cells and then evaluated for the distribution of B7 markers on the cell surface. Representative histograms from one experiment are shown; unfilled histograms represent cells stained with isotype-matched control mAbs. Lymph nodes and lungs from five to 10 mice were pooled for each group. One of four representative experiments is shown.

In the lung, we observed an increase in the proportion of APCs expressing either B7.1 or B7.2 (Figure 2). Table 1 shows that there was a large increase in the proportion of macrophages expressing both B7 molecules. Interestingly, although the proportion of DCs expressing B7.1 did not change significantly, we observed an increase in the proportion of DCs expressing B7.2 at Day 13.

Activation Status of T Cells in the Thoracic Lymph Nodes and Lungs

Figure 3 depicts the content and distribution of activated (CD69⁺) T-cell subsets. In the lymph nodes, the absolute number of activated CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells significantly increased after sensitization (Day 6). Our findings show that 5 to 6% of CD4⁺ or CD8⁺ cells expressed CD69 in the lymph nodes of naive mice. Intraperitoneal sensitization resulted in a doubling of the proportion of activated cells, which remained at a similar level at Day 13 of the protocol.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Activated T-lymphocyte subsets in lung and lymph nodes at different time points after sensitization and challenge with OVA. Values represent means ± SEM of three to five experiments. For each time point, cells were pooled from five to 10 mice. Statistical analysis was performed by t test (lymph nodes) or ANOVA with Tukey's post hoc test (lung); *P < 0.05.

In the lung, there were no statistically significant differences in the numbers of activated CD3⁺/CD4⁺ and CD3⁺/ CD8⁺ cells at Days 6 and 13 compared with naive mice. Interestingly, at Day 17 (i.e., 5 d after challenge) the number of CD3⁺/CD4⁺ T cells that were CD69⁺ increased significantly, whereas the increase in CD8⁺/CD69⁺ cells was comparatively marginal. In terms of percentages, the proportion of CD3⁺/CD4⁺ cells expressing CD69⁺ reached 19 ± 5.4%.

Cytokine Expression

Cytokines are a defining component of an effector immune response. To examine expression of a broad range of cytokines in both the thoracic lymph nodes and the lung in vivo we chose to evaluate mRNA expression using an RPA. The mCK-1 template, which includes cytokines that are particularly relevant to allergic airways inflammation, was used. mRNA was obtained from lung and lymph nodes at 3, 6, 12, and 18 h after sensitization and after challenge. The kinetics of mRNA expression for all cytokines in both compartments followed a similar pattern: peak expression at 3 h after either sensitization or challenge, with a return to baseline levels between 12 and 18 h (data not shown). Consequently, only the data at 3 h are shown in Figure 4. Cytokine mRNA expression in naive lungs and spleens was minimal for the eight cytokines examined in this template. Likewise, very low mRNA expression was detected in the lung and lymph nodes at Day 5. In contrast, in the challenged lung (Day 12) we observed robust expression of mRNA for IL-4, IL-13, IL-5, and IL-6; comparatively modest expression of IFN- and IL-15; and minimal expression of IL-2 and IL-10. Given the unaltered expression of cytokine mRNA in the lymph nodes at the time of major cellular expansion (Day 6), we sought evidence for Th2 polarization by examining the expression of STAT-6 and GATA-3 in the thoracic lymph nodes. Figure 5 shows that, compared with naive lymph nodes, expression of both genes was upregulated after i.p. sensitization (Day 5). Hence, we next examined the ability of lymph-node cells to produce Th2 cytokines upon antigen recall. To this end, cells were cultured with OVA or medium alone for 5 d, and IL-5 and IL-4 production in culture supernatants was measured by ELISA. IL-5 (5,050 ± 2,730 pg/ml) and IL-4 (628 ± 230 pg/ml) were detected only in supernatants from cells stimulated with OVA (Figure 6).

View larger version (16K): [in this window] [in a new window]   Figure 4.   RPA. Detection of mRNA expression for selected cytokines in lung and lymph nodes after sensitization and challenge. Mice were sensitized or sensitized and challenged and total RNA was extracted from lymph nodes and lungs at the indicated times after the second sensitization (Day 5; 3 h after second i.p. sensitization) or challenge (Day 12; 3 h after challenge). For naive mice, RNA was extracted from spleen. Data are representative of two independent experiments.

View larger version (10K): [in this window] [in a new window]   Figure 5.   Effect of sensitization on the levels of GATA-3 and STAT-6 in lymph nodes. Mice were sensitized twice by i.p injection of OVA/aluminum hydroxide, the lymph nodes were removed, and total RNA was extracted. Real-time quantitative PCR (TaqMan) was run. Data are representative of two experiments.

View larger version (9K): [in this window] [in a new window]   Figure 6.   Th2 cytokine production from lymph nodes cultured in vitro with or without OVA. At the indicated time points, lymph nodes were removed, pooled, and placed in culture for 5 d in either medium or OVA. Cytokines were measured by ELISA (n = 3; means ± SEM). Three independent experiments yielded similar results.

T1/ST2 Expression in the Lymph Nodes and Lungs

Table 2 shows the percentage of CD4⁺ T cells that expressed T1/ST2. We did not observe statistically significant differences at Days 6 or 13 in either the lung or the lymph nodes compared with naive mice. However, there was a significant increase in the proportion of CD4⁺ T cells expressing T1/ST2⁺ in the lung at Day 17 of the protocol (18.5 ± 1.5% compared with 3.6 ± 1.2% in naive animals).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The primary objective of this study was to define, spatially and temporally, immune activity in a conventional murine model of allergic airways inflammation: one that involves the introduction of antigen (OVA) together with adjuvant (aluminum hydroxide) into the peritoneal cavity followed by respiratory exposure to aerosolized antigen. We have previously documented cell and cytokine changes in the bronchoalveolar lavage fluid (BALF), peripheral blood, and bone marrow of the model described in this study (8). Here, we focused our analysis on mononuclear cells, particularly APCs and T cells, because together with antigen they constitute the tripartite interaction that determines the nature of the immune-inflammatory response.

Our data document a remarkable expansion of cells in the lymph nodes after sensitization. The increase in the APC compartment was largely due to B cells (Figure 1) which translates into the establishment of humoral responses (immunoglobulin [Ig] E production; ref. 8) and may implicate B cells as major APCs. However, recent studies have demonstrated that T cells can still be primed, and airways inflammation established, in B cell-deficient mice (14). This indicates the involvement of different cell types in the process of antigen presentation, possibly DCs, which among all APCs represent the subset with greater antigen-presenting capacity crucial for the activation of naive T cells both in vitro and in vivo (15, 16). Thus, the functional significance of the comparatively small changes in DCs that we observed in the lymph nodes after sensitization may be much greater than what the quantitative changes would imply.

In the lung, robust APC accumulation was observed at Day 13 (24 h after aerosol challenge). In sharp contrast to our findings in the lymph nodes, DC and macrophage expansion predominated in the lung, with no significant changes in B cells (Figure 1). These findings are consistent with the notion that an increase in DCs is a universal feature of the response after mucosal exposure to bacteria and to viral and soluble protein antigens (17, 18). The importance of lung DCs as APCs and, specifically, in the induction of secondary responses to surrogate allergens has recently been demonstrated (19). With respect to macrophages, the functional significance of the rather remarkable increase that we observed is unclear. This expanded macrophage population expressed the costimulatory molecules B7.1 and B7.2. Hence, antigen challenge might alter the functional phenotype of lung macrophages from poor APCs (20, 21), as has been described previously, to more effective APCs. We cannot exclude the possibilty that our observed increases in macrophages are due to DC precursors in the lung vasculature, particularly because DCs have been shown to share certain phenotypic characteristics with monocytes/macrophages (22). Alternatively, the major role of macrophages may be, as proposed by Gong and colleagues (23), to degrade soluble proteins into antigenic peptides that are then captured by DCs to be presented to T cells. Our current understanding of the interactions between the innate and adaptive immune systems in general and, particularly, among macrophages, DCs, and T cells is incomplete, especially as it relates to responses to aeroallergens.

Costimulatory molecule expression is clearly a central requirement for the generation of a productive immune response (24). Specifically, the importance of the CD28/ CTLA4/B7 pathway in the elicitation of immune responses in models of allergic airways inflammation has recently been demonstrated (25). Our data document an increase in B7.2⁺ APCs, particularly B cells, in the thoracic lymph nodes 24 h after sensitization (Day 6 of the protocol) (Figure 2 and Table 1). In contrast to B7.2, we did not detect significant changes in the number of APCs expressing B7.1. Thus, our data demonstrate that the dominant B7 costimulatory molecule expressed in the thoracic lymph nodes at the time of sensitization is B7.2, a finding that is consistent with the notion that expression of B7.1 or B7.2 will privilege CD4 T-cell differentiation toward the Th1 or Th2 phenotypes, respectively (30, 31).

In the lung, however, our data demonstrate that expression of both B7.1 and B7.2 molecules increases considerably after challenge. Although B7.2 is considered the dominant B7 costimulatory molecule in this model on the basis of evidence that treatment with anti-B7.2 antibodies prevents pulmonary eosinophilia, secretion of Th2-type cytokines, IgE production, and bronchial hyperreactivity, it is likely that B7.1 also plays a meaningful, perhaps somewhat unappreciated, role in this process. In this regard, several studies have shown that administration of anti-B7.1 antibodies significantly decrease airways eosinophilia (29, 32). Particularly informative is the finding by Harris and associates (32), who showed that although treatment with anti-B7.1 antibodies significantly decreased airways eosinophilia, it did not decrease peripheral blood eosinophilia. Moreover, Masten and coworkers (33) demonstrated that B7.1 signaling by lung DCs plays an important role in costimulation. The concept that B7.1. and B7.2 likely play complementary roles has recently been reinforced by studies using B7.1 and/or B7.2 knockout mice (34). Our data support the hypothesis that whereas B7.2 has a predominant role in the sensitization event that takes place in the lymph nodes, B7.1 plays an important role in the lung after secondary antigen exposure. For example, B7.2 may be essential in the process leading to the generation of peripheral blood eosinophilia, whereas the influx of eosinophils into the tissue may require additional signals mediated by B7.1 in the lung/airway. Thus, both B7.1 and B7.2 are probably required for the full expression of the allergic phenotype, with discrete requirements for both molecules depending on the time, site, and context of their expression.

The APC increase in the lymph nodes after the second i.p. sensitization is concomitant with the expansion of activated (CD69⁺) T lymphocytes (Figure 3), a phenomenon that is sustained after OVA challenge. There is very limited information with respect to events in the thoracic lymph nodes in this type of experimental model. Krinzman and colleagues (35) reported an increase of CD4⁺ T cells in the thoracic lymph nodes of sensitized mice after challenge. Our findings extend this observation because we demonstrate that such changes are already established after sensitization. This pattern reflects the general model in which primary T-cell activation and expansion, facilitated by APCs, occur in lymphoid tissues.

In the lung, we observed an initial increase in activated T cells 24 h after challenge. However, the major expansion of CD4⁺/CD69⁺ cells took place at Day 17 (5 d after challenge), in accordance with our earlier studies describing massive influx of lymphocytes into BALF (8). It is of interest to note that whereas the ratio of CD4⁺/CD69⁺ to CD8⁺/CD69⁺ cells in the thoracic lymph nodes at Day 6 (24 h after sensitization) was 2.9, this ratio was 7.5 in the lung 5 d after challenge, indicating preferential participation of CD4⁺ T cells in airways inflammation. Indeed, the involvement of CD4⁺ T cells in the development of airway inflammatory responses to allergens is well established (36, 37). In fact, the role of CD4⁺ T cells is pivotal: CD4 knockout mice cannot be sensitized, and depletion of CD4 cells in wild-type animals prevents antigen-induced airway hyperreactivity and airways eosinophilia (38).

To investigate whether expansion in the lymph nodes and lungs reflects the establishment of effector activity, we evaluated cytokine mRNA expression. As shown in Figure 4, expression of the prototypic Th2 cytokines IL-4, IL-5, and IL-13 was minimal and virtually identical to that observed in naive mice (in the thoracic lymph nodes) at all time points examined. We think that it is very unlikely that we missed cytokine upregulation because mRNA was obtained at 3, 6, 12, and 18 h after the second i.p. injection. We find it informative that despite clear evidence of T-cell expansion and preferential activation, as assessed by CD69 expression especially on CD4⁺ cells, actual effector activity, as assessed by cytokine mRNA expression, was not apparent. Interestingly, OVA sensitization led to increased levels of STAT-6 and GATA-3, which are implicated in the differentiation of naive T cells into Th2 cells (Figure 5) (39, 40). In our view, these findings convey a functional logic: there is no immunologic advantage of producing effector molecules in the regional lymph nodes, whereas the production of effector molecules in the target organ (in our case, the lung) is essential. Indeed, in vitro stimulation of lymph-node cells with OVA induced the production of typical Th2 cytokines such as IL-5 and IL-4 (Figure 6). The ability of lymph-node cells to respond to antigen in vitro can be explained within the model proposed by Sallusto and associates in which immunologic memory is displayed by distinct T-cell subsets: lymph node-homing cells lacking inflammatory functions and tissue-homing cells endowed with various effector functions such as cytokine production (41). Because in vitro conditions simulate the in vivo tissue environment, we observed the transition from nonproducers to cytokine producers.

The findings discussed earlier argue that for Th cells to execute their effector program they need to encounter antigen in the right environment in the tissue. Indeed, in agreement with observations by Krzesicki and coworkers (42), we detected substantial effector activity in the lung after challenge as indicated by considerable upregulation of IL-4, IL-13, IL-5, and IL-6, but not IFN-, mRNA. However, we also found that mRNA expression for the cytokines IL-2 and IL-15 was minimal and, in fact, very similar to that observed in naive mice. It is interesting that, in the face of a major expansion of mononuclear cells, expression of cytokines with well-established proliferative activity remains so modest. This suggests that recruitment/ relocalization, rather than in situ proliferation, may play a prominent role in the mononuclear cell expansion that we observed in the lung.

Further, and in sharp contrast to our findings in the thoracic lymph nodes, the proportion of CD3⁺/CD4⁺ cells expressing T1/ST2 increased as early as 72 h after challenge, with a considerable increase 5 d after challenge. T1/ST2 has significant homology to the IL-1 receptor, but does not bind IL-1 or IL-1 (43). More recently, T1/ST2 has been identified as a marker of advanced differentiation to the Th2 phenotype in Th cells polarized in vitro toward Th2 but not Th1 (44, 45). Interestingly, expression of T1/ST2 in the lymph nodes during sensitization remained at the level detected in naive mice (approximately 5%). Together, these data might argue for preferential recruitment of activated T1/ST2 CD4⁺ T cells to the lung after antigen aerosol challenge. Alternatively, T cells may acquire this phenotype in the lung as a consequence of the Th2-polarized immune response. The geography of T1/ST2 expression (i.e., in the lung) is consistent with this logic and suggests that T1/ST2 may best be characterized as a marker of effector Th2 cells rather than simply of Th2 cells.

In summary, our studies provide an elaborate description of immunologic activity in the thoracic lymph nodes and lung during a response to the soluble antigen OVA. Our data identify the main sites of the primary and secondary immune responses, elucidate distribution and phenotype of APCs and T cells, and provide evidence that may prompt the revisiting of accepted concepts of activation and effector activity, particularly in allergic inflammation.