Sensitization of Mice with Latex Allergens
Latex allergy in BALB/c mice was induced according to a protocol described previously [20-22]. In brief, 8–10 week old BALB/c mice were divided into three groups. The first group (Group 1) was challenged with latex allergens, the second group (Group 2) was challenged with latex and treated with curcumin (Sigma Chemicals), and the third group (Group 3) consisted of controls treated with curcumin only. 100 μg of a Malaysian non-ammoniated (MNA) latex extract, isolated from sap collected from the rubber plant Hevea brasiliensis, was injected intraperitoneally into the mice, once a week for two weeks. Remaining challenges were done intranasally twice a week for four weeks (50 μg of latex in 30 μl of PBS per challenge) (Groups 1 and 2). Intranasal inoculations with latex antigen and intragastric administration of curcumin (250 μg in 250 μl PBS) (Group 2) were carried out after anesthetizing the mice with xylazine. Control animals (Group 3) received PBS intranasally and curcumin intragastrically. The levels of total serum IgE and latex specific IgG1 were measured by ELISA as described previously . When a significant antibody response was detected, the animals were challenged with a final dose of latex allergens, and euthanized 48 hours later. Blood, lung tissue, and spleens were collected and evaluated as described below. The animal studies were approved by the Veterans Affairs Animal Care Committee.
Total Serum IgE
Total serum IgE levels were determined in all mice before sensitization and after euthanization as previously reported [22,23]. Serum IgE levels was expressed as ng/ml after comparing the optical density (O.D. at 480nm) values to mouse IgE standards.
Latex Specific IgG1 and IgG2a in the Sera of Mice
Levels of latex antigen-specific IgG1 and IgG2a in collected serum samples were studied by ELISA as previously described . In brief, micro titer plate wells (Immulon II, Fisher Scientific, Itasca, IL) were coated with 5 μg/ml of latex proteins in PBS (pH 7.4). The plates were incubated at room temperature for 3 hours, followed by overnight incubation at 4°C. The plates were then washed with PBS and after blocking the wells with 0.5% Bovine serum albumin in PBS, 100 μl of serum diluted in PBS containing 0.05% Tween 20 (PBS-T) was added to the wells, and the plates incubated at room temperature for three hours. The wells were washed with PBS-T and isotype specific biotinylated anti-mouse antibody was added for an additional hour. The plates were washed again and streptavidin conjugated horse radish peroxidase was added to the wells for one hour. After washing the plates, the substrate was added and the color developed with O-phenylene diamine (OPD). The color development was stopped by adding 2N H2S04, and the optical density (O.D.) read at 490nm using an ELISA reader. The O.D. values of several serum dilutions were used to calculate log10 titer, and the different groups were compared.
Peripheral blood eosinophils were plated on slides stained with Eosin-Y and enumerated using a hemacytometer . Eosinophil numbers were assessed before and during sensitization and at the end of the experiment.
Immediately after sacrifice, the lungs were inflated with 10% neutral buffered formalin to prevent atelectasis. The specimens then were fixed in formalin and processed. Sections were cut at 5 μm thickness and stained with hematoxylin-eosin and PAS. Lung inflammation was scored with special reference to the infiltrating cell types and the severity of lesions as described previously [22-24].
Lung sections were examined for cells producing IFN-γ, IL-4, IL-5, IL-10, and IL-13 by immunohistochemistry as previously described . Lungs were frozen in liquid nitrogen and frozen sections from different groups of mice were fixed in 4% paraformaldehyde. The sections were incubated in 0.3% H2O2 in PBS for 15 minutes. After washing three times with PBS for five minutes each, the sections were blocked with PBS containing 5% bovine serum albumin (BSA) for three hours. The sections were then incubated for two hours at room temperature with 1:20 diluted biotinylated anticytokine antibodies (Pierce, R&D) in PBS containing 3% BSA. The slides were then incubated for 30 minutes at room temperature with streptavidin peroxidase (1:50 diluted). The color was developed with 3,3-diaminobenzidine tetra chloride (DAB) (Sigma). Numbers of cytokine positive cells were determined by counting five different microscopic fields.
Spleens were processed into single cell suspensions and antigen dependent proliferation studied by tritiated thymidine uptake . Briefly, spleen cells (1 × 105/well) were cultured for seven days in 96 well plates in 200 μl of RPMI 1640 medium supplemented with glutamine, sodium pyruvate, 10% heat inactivated fetal bovine serum (FBS), and penicillin and streptomycin (complete RPMI). Latex antigen (5 μg/ml) or Concanavalin A (5 μg/ml) was added to experimental wells . One μCi of 3 [H] thymidine was added for the final 18 hours of culture. The incorporated 3H thymidine was measured by liquid scintillation counting, and the stimulation indices (SI) were calculated as described before .
Cytokine Production by Spleen Cells
Spleen cells (1 × 107) were placed in complete RPMI and cultured in 24 well plates for 60 hours. Latex antigen (5 μg/ml) was added to experimental wells at the beginning of culture. After incubation, cell free supernatants were collected and analyzed for cytokines by ELISA, including IL-4, IL-5, IL-10, IL-13, and IFN-γ .
Flow Cytometric Analysis of Lung Cells
Lungs were removed aseptically and cut into small pieces of about 2 to 3 mm in size. The pieces were digested enzymatically by treating with 120 μg/ml Dispase (Invitrogen) and Collagenase (Sigma) for one hour at 37°C. After incubation, the tissue was homogenized gently in a tissue grinder and the cells were collected. These cells were washed three times, and the lung cells for each group were pooled (equal numbers from each mouse) and suspended in complete RPMI. The cells were stained with combinations of FITC and PE conjugated antibodies specific for CD4, CD8, CD25, CD28, CD80, CD86, CD152, OX40, B220, or Mac-1 (26). The stained cells were run through a FACS Calibur flow cytometer (Becton-Dickinson, Mountain View, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA).
Total RNA was isolated using RNeasy mini kits (Qiagen, Valencia, CA) . Briefly, lung tissues were homogenized using disposable pestles and tubes (Kontes Glass Company, Vineland, NJ) in the presence of lysis buffer. Lysates were transferred to QIAshredder spin columns (Qiagen), spun for 2 minutes, the eluates collected, and RNA isolated as per the manufacturer's protocol. The RNA was quantified by measuring OD (Nanodrop ND-1000, Wilmington, DE).
One Step RT-PCR
One-step RT-PCR reactions were performed in triplicate using TaqMan one-step RT PCR (Applied Biosystems, Branchburg, NJ) . Briefly, the sequence specific FAM-labeled Taqman primer-probe pairs (Applied Biosystems, Foster City, CA) and 10 ng total RNA were mixed with reaction buffer supplied by the manufacturer in a 20 μl reaction volume. The sequential one-tube reverse transcription and real time PCR were performed in an Opticon 2 thermal cycler (MJ Research/Bio Rad Laboratories, Hercules, CA). The temperature conditions included an initial 48°C incubation for 30 minutes, followed by AmpliTaq Gold activation at 95°C for 10 min, 40 cycles of amplification at 95°C for 30 sec and 60°C for 1 min cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; assay id: Mm99999915_g1) was used as an internal control. Applied Biosystem Taqman gene expression primer-probe pairs specific to lymphocyte antigen 75 (Ly5, Assay id: Mm00522144_m1), matrix metallopeptidase 9 (MMP9; Assay id: Mm00600163_m1), ornithine amino transferace (OAT; Assay id: Mm00497544_m1) and thymic stromal lymphopoietin (TSLP; Assay id: Mm00498739_m1) were used.
Total serum IgE levels, latex specific IgG1 and IgG2a, peripheral blood eosinophils, cytokine production and stimulation of spleen cells in response to latex antigens in vitro were compared among different groups of mice. The data were analyzed and compared using student 't' test with unequal variance and the results expressed as means ± SEM. 'P values'