Mice expressing the transgene for the DO11.10 TCR-α/β, which recognizes residues 323–339 of chicken OVA in association with I-Ad, were provided by Dr. D. Loh (Washington University, St. Louis, MO 15
). Transfer recipients were 6–8-wk-old female BALB/cJ mice (The Jackson Laboratory).
In Vitro Polarization of T Cells.
OVA-specific TCR-transgenic CD4+ T cells were isolated from the spleen and cultured in complete RPMI 1640 medium with OVA323–339 (1 μg/ml) and mitomycin C–treated splenocytes. For Th1 phenotype development, recombinant murine IL-12 (40 ng/ml; Endogen) and neutralizing anti–IL-4 Ab (11B11, 20 μg/ml; R&D Systems) were added; for Th2 phenotype development, recombinant murine IL-4 (50 ng/ml) and anti–IL-12 (TOSH-2, 10 μg/ml; Endogen) were used. Cells were cultured for three rounds of antigenic stimulation under polarizing conditions. At this point, the cells were divided into two portions, with the majority being used to induce pulmonary inflammation as described below. A small sample (2 × 105 cells) from each culture was activated on immobilized anti-CD3 mAb (2C11, 10 μg/ml; PharMingen) in the presence of human (h)IL-2 (10 U/ml; Endogen) for 48 h to determine the integrity of the polarization. Culture supernatants were collected for measurement of IL-4, IL-5, and IFN-γ levels by ELISA (Endogen), and cell pellets were collected for RNA extraction and PCR analysis. Th2 cells produced high IL-4 and IL-5 levels but little IFN-γ, whereas Th1 cells produced high IFN-γ levels but little IL-4 and IL-5 (Th2 cells: 100–300 ng/ml IL-4, 50–150 ng/ml IL-5, and <20 pg/ml IFN-γ; Th1 cells: 7,000–15,000 ng/ml IFN-γ). Similarly, RNA expression analysis revealed that Th2 cells expressed predominantly IL-4 and IL-5 but little if any IFN-γ, whereas the reverse was true of Th1 cells.
Induction of Pulmonary Inflammation.
In preparation for induction of allergic inflammation, Th1 or Th2 cells produced as described above were rested in hIL-2 (10 U/ml; Endogen) for 48 h before being washed in tissue culture medium. Recipient BALB/c mice were given 2 × 106 cells intravenously. 24 h later, mice were exposed to an aerosol of OVA (50 mg/ml, Grade V; Sigma Chemical Co.) for 20 min. Thereafter, mice were challenged daily and were killed 24 h after the last aeroallergen challenge on day 4, 7, or 14. Control mice received cells but were challenged with aerosolized PBS. After the mice were killed, bronchoalveolar lavage (BAL) was collected by cannulation of the trachea and lavage with 1 ml of PBS. Lungs were then inflated with optimum cutting temperature (OCT) compound and removed, and the right lobes of the lung were snap-frozen in liquid nitrogen while the left were fixed in 10% buffered formalin.
For blocking studies, mice were injected daily with 100 μg per mouse polyclonal rabbit antieotaxin Abs 7
or polyclonal rabbit anti–murine MDC 13
30 min before OVA challenge. Mice were then killed at day 4 after T cell transfer (after three antigen challenges) or at day 7 (after six antigen challenges), as shown in .
Figure 1 Adoptive transfer of Th effector cells leads to AAD. BALB/c mice were injected with 2 × 106 cells followed by daily aerosolized OVA. Mice were killed on day 4 or 7 for analysis. Results shown are from two different experiments with five mice per (more ...)
Determination of BHR.
Airway responsiveness was measured in Th2 recipient mice 24 h after the last aerosol challenge by recording respiratory pressure curves using whole body plethysmography (Buxco; EMKA Technologies) in response to inhaled methacholine (Sigma Chemical Co.) at concentrations ranging from 2.5 to 25 mg/ml for 1 min. Airway responsiveness was expressed in enhanced pause (Penh), a calculated value that correlates with measurement of airway resistance, impedance, and intrapleural pressure in the same mouse: Penh = (te/tr1) × Pef/Pif (te, expiration time; tr, relaxation time; Pef, peak expiratory flow; Pif, peak inspiratory flow).
Total BAL cells were counted, and aliquots (5 × 105 cells per slide) were pelleted onto glass slides by cytocentrifugation. A differential cell count was then performed after Wright-Giemsa staining (Fisher Diagnostics). Percentages of eosinophils, lymphocytes, neutrophils, and macrophages were determined by counting in eight randomly selected high-power fields (hpf; magnification: ×40; total area: 0.5 mm2) and dividing this number by the total number of cells per hpf. To obtain the absolute number of each leukocyte subtype in the lavage, these percentages were multiplied by the total number of cells recovered from the BAL fluid.
In Vivo Measurement of Cytokine Production.
Levels of the cytokines IL-4, IL-5, IFN-γ, IL-6, and IL-10 were determined in the lavage fluid of mice using ELISA kits (Endogen).
Measurement of Chemokine Ligand and Receptor Expression by PCR Analysis.
PCR was performed using the Advantage® KlenTaq polymerase (Clontech Laboratories) according to the manufacturer's instructions. cDNA derived from 25 ng of total RNA was used for each 30-μl reaction containing 0.5 μM primers, 0.2 mM dNTP mix, 1× PCR reaction buffer, and 0.5 μl polymerase. Samples were amplified at 94°C for 30 s, 52–60°C for 1 min, and 68°C for 1 min for 20, 25, or 32 cycles. 10 μl of each reaction was loaded per well on 1.5% agarose gels. Primer sequences were as follows: for CCR3, 5′-TCTGTGGAATGAGTGGGGTTTTG and 5′-GTAATACGACTCACTATAGGGACTTCTGGATAGCGAGGACTG; for CCR4, 5′-ATCGTGCACGCGGTATTCTCC and 5′-GACGGGGTTAAGGCAGCAGTGA; for MDC, 5′-GGTGAAGAAGCTACTCCATAAACT and 5′-GTAATACGACTCACTATAGGGAGAAGGGATAGAGGGGAGGTA; and for eotaxin, 5′-TCTCCCTCCACCATGCAGAG and 5′-CAGATCTCTTTGCCCAACCT. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were purchased from Clontech Laboratories.
Lung Histology and Immunohistochemistry.
The left lobe of the lungs was fixed in 10% neutral buffered formalin (NBF; J.T. Baker) and paraffin embedded. Sections (4 μm) were stained for cyanide-resistant peroxidase according to standard protocols 16
, then counterstained with hematoxylin to depict eosinophils or with chloroacetate esterase to show neutrophils. The composition of infiltrates was then determined by counting the total number of infiltrating cells in five peribronchiolar fields and determining the percentage of eosinophils, neutrophils, and mononuclear cells. General morphology was assessed on hematoxylin and eosin–stained sections.
For determination of antigen-specific T cells within lung tissue, serial frozen sections (4 μm) were stained with either anti-CD4 (PharMingen) or an Ab specific for the transgenic TCR, KJ126 17
. Both of these Abs were biotinylated, and positive staining was detected using streptavidin-peroxidase (DAKO Corp.) followed by diaminobenzidine (DAB; Vector) before counterstaining in hematoxylin. Eosinophils were stained for cyanide-resistant peroxidase as described above.
The number of cells per hpf was obtained by counting positively stained cells (CD4 cells or eosinophils) in five fields per section at a magnification of 400. To calculate the percentage of KJ126+ CD4 cells, CD4 cells were counted; the same field was located on the KJ126-stained serial section, and positive cells were enumerated. At least 250 CD4 cells were counted for each section, and the percentage of KJ126+ CD4 cells was then calculated.
CCR3+ or CCR4+ Th2 cells were detected in lung sections by immunohistochemical staining using polyclonal Abs specific for the COOH terminus of CCR3 or CCR4 (Santa Cruz Biotechnology). Staining of primary Abs was visualized with a biotinylated donkey anti–goat Ig Ab (Jackson Immunochemicals) followed by streptavidin-peroxidase as described above. Positively stained Th2 cells were enumerated by locating an infiltrate in the serial KJ126-stained section and counting the percentage of KJ-stained cells that were either CCR3+ or CCR4+. At least 50 KJ126-stained cells were counted in each section from lungs obtained at days 4 (n = 6), 7 (n = 4), and 14 (n = 4), with between two and three lobes stained per mouse.