METHODS Penh In addition to measurement of APTI, airway hyperresponsiveness to methacholine was also evaluated within 24 hours of the last allergen challenge by means of measurement of Penh (Buxco Electronics, Inc, Sharon, Conn) responses to 6 mg/ml nebulized methacholine, as we have done before. E1 These measurements were performed in free-moving unanesthetized mice in whole-body plethysmographic chambers. Measurements and ribonuclease protection assay BAL of the right lung and fix inflation of the left lung with 10% neutral buffered formalin to an inflation pressure of 25 cm H 2 O were performed, as previously described. E1 In some studies lungs and TLNs were harvested for either collagenase digestion to harvest leukocytes for flow cytometry or for RNA extraction, as we have done before, E1 according to the method of Chomczynski and Sacchi. E2 Messenger RNA expression was evaluated by using the ribonuclease protection assay and riboprobe templates (PharMingen, San Diego, Calif), as we have previously described. E1 RT-PCR Real-time PCR was performed as previously described E3 by using 24-mers designed with Primer 3 and published GenBank sequences for murine MCP-1/CCL2 (NM_011333), MCP-2/CCL8 (NM_ 021443), and leucine-slc/ccl21 (MN_011335). Lung morphometry The area of cuffing around the pulmonary arterioles was measured with ImagePro 4.5 software analysis of digitized photomicrographs of hematoxylin and eosin stained lung sections calibrated with a micrometer. In separately stained lung sections, periodic acid Schiff positive cells are expressed per 100 mm of the mucosal perimeter of a bronchial cross-section, as measured by using Image- Pro software, to evaluate goblet cell hyperplasia. Flow cytometry Lungs were harvested from 4 mice in each of the 4 treatment groups, and harvested cells were pooled. Lymph nodes were pooled from 4 to 7 mice in each group. The lung vasculature was perfused with 1 ml of 0.9% NaCl containing 100 units of heparin injected through a right ventricular injection, after which the lungs were removed. The lungs were inflated with 2 ml of RPMI containing 5% heat-inactivated FCS, 400 U/mL collagenase, 1000 U/mL hyaluronidase, and 30 mg/ml Dnase I. TLNs were placed in identical media. After mincing of the lungs with scalpel blades and mechanical dispersion of lymph node cells, the tissue was incubated for 2 hours at 378Cin5%CO 2. The cells were further dispersed, filtered over cotton plugs, and counted. The dendritic cell population was then separated by using positive selection with anti-cd11c and anti-pdca-1 magnetic microbeads (Pan DC microbeads; Miltenyi Biotec, Auburn, Calif). The flowthrough was used to analyze lymphocyte expression of CCR7. Four-color flow cytometry was performed with a FACSalibur instrument (BD Biosciences, San Diego, Calif). For all leukocyte subsets, initial gating was performed by means of forward and side scatter, followed by selection of live cells by means of 7-amino-actinomycin D exclusion. Total lung dendritic cells (CD11c 1 I-A 1 ) were then identified (FL4 vs FL1), and CCR7 expression was identified by gating on FL2 (CCR7) versus FL1 (I-A). The CD103 1 epithelial dendritic cell population was identified by gating on a subset of cells that were CD11c 1 (FL4) and CD103 1 (FL1). CD103 dendritic cell expression of CCR7 was then identified by using a graph displaying CCR7 (FL2) versus CD103 (FL1). Lung plasmacytoid dendritic cells (I-A 1, PDCA 1, B220 1 ) were identified by an initial gate on PDCA 1 cells (FL4), followed by a subsequent gate on B220 (FL2) versus I-A (FL1). To estimate CCR7 expression by plasmacytoid dendritic cells, parallel staining was performed in which CCR7 staining was substituted for B220 but with identical gating to the gate used for the B220 1 cells in FL2. Lastly, for lymphocytes, after initial gating on CD3 1 cells (FL4) versus forward scatter, CCR7 expression (FL2) was evaluated in either CD4 1 or CD8 1 cells (FL1). In all studies the threshold for positive CCR7 staining was set by staining with control antibody for anti-ccr7. Intracellular cytokine staining Lymphocytes were isolated from TLNs and stimulated for 6 hours, as previously described by Nishikomori et al, E4 with 20 ng/ml phorbol 12-myristate 13-acetate, 1 mmol/l ionomycin, and 2 mmol/l monensin. Cells were then fixed, permeabilized, and stained with fluorescein isothiocyanate or phycoerythrin-conjugated anti-cd4, anti-cd8, anti-il-4, anti-il-5, and anti-ifn-g antibodies. ELISAs Cytokines and chemokines. Proteins were assayed by means of ELISA with antibody pairs for IL-4, IL-5, IFN-g, MCP-1/CCL2, SLC/ CCL21, and MDC/CCL22 (R&D Systems, Inc, Minneapolis, Minn). IgE and IgG1. OVA-specific serum IgE and IgG1 levels were measured as previously described. E5 Briefly, OVA was absorbed overnight on Immunolon 1 plates (Dynatech, Chantilly, Va), followed by the addition of appropriate dilutions of serum samples or standards. After incubation for 2 hours at room temperature, the wells were washed with PBS, after which either horseradish peroxidase (HRP) labeled antibodies to mouse IgG1 or biotinylated antibody to mouse IgE was added. After an additional wash, avidin-labeled HRP was added to wells intended for measurement of IgE. After washing, wells were incubated with o-phenylenediamine, with detection of plate-bound HRP with an ELISA plate reader at 490 nm. Standard curves were generated by using pooled mouse sera containing OVA-specific antibodies, with maximally detected antibody arbitrarily defined at 1000 units. Lymphocyte antigen stimulation Lymphocytes from TLNs and splenic antigen-processing cells were isolated by means of collagenase digestion. Cocultures of the lymphocytes and irradiated splenocytes in the absence or presence of 250 mg/ml OVA were performed as previously described. E1 RESULTS The Penh responses were comparable between 1/1 and plt-sal/sal control animals (Table E1). Penh increased in 1/1OVA/OVA mice compared with that seen in the 1/1SAL/SAL control group. Penh responses in plt-ova/ova mice were not only increased compared with those in plt-sal/sal mice, but responses were also enhanced to greater than those in 1/1OVA/OVA mice. Lung IL-4 and MDC/CCL22 levels increased significantly in plt-ova/ova mice compared with 1/1OVA/ OVA mice (Table E2). Lung MCP-1/CCL2, IL-5, IFN-g, and SLC/CCL21 levels were comparable in all groups. TLN SLC/CCL21 protein levels were significantly decreased in plt mutants compared with in 1/1 mice (Table E2).
In the draining TLNs the numbers of myeloid (I-A 1 CD11b 1 ), epithelial (CD11c 1 CD103 1 ), and plasmacytoid (I-A 1 PDCA 1 B220 1 ) dendritic cells were comparable in 1/1SAL/SAL and plt-sal/sal mice and increased with OVA/OVA immunization in both genotypes (Table E3). However, there appeared to be increased numbers of myeloid and epithelial (CD103 1 ) dendritic cells and CD4 and CD8 cells in the TLNs of plt-ova/ova mice. In addition, the numbers of myeloid, epithelial, and plasmacytoid dendritic cells and CD4 and CD8 cells expressing CCR7 appeared to be greater in plt-ova/ova mice compared with in all other groups (Table E3, brackets). There was an increase in the number of TLN CD4 cells that stained for IL-4, IL-5, or IFN-g with OVA treatment compared with saline controls for both genotypes (Table E4). However, there appeared to be more than 7-fold more CD4 cells in TLNs from plt-ova/ova mutants that stained positively for IL-4 and IL-5 compared with that seen in 1/1OVA/OVA mice (Table E4). In addition, there appeared to be 4-fold more TLN CD4 cells that stained for IFN-g from plt-ova/ova mice than from 1/1OVA/OVA mice. DISCUSSION The dissociation between Penh and APTI responses in plt-ova/ova mice supports increasing concern regarding the reliability of Penh in the assessment of enhanced bronchial provocation responses. E6 Although APTI responses to methacholine were enhanced in OVA-challenged plt mutants, the response was comparable with that seen in OVA-challenged WT mice. From this, it appears that airway hyperactivity, although increased with OVA challenge in both WT and plt mice over that seen in saline control animals, is not enhanced in plt mutants over 1/1 mice. The cause for the discrepancy between Penh and APTI responses is unclear. Possible explanations include an increase in nasal airway resistance or increased sensitivity to changes in plethysmographic box humidity or temperature in plt mice. Nevertheless, these results add to the concern regarding the utility of Penh in the evaluation of airway hyperresponsiveness. REFERENCES E1. Kim YB, Sung SJ, Kuziel WA, Feldman S, Fu SM, Rose CE Jr. Enhanced airway Th2 response after allergen challenge in mice deficient in CC chemokine receptor-2 (CCR2). J Immunol 2001;166:5183-92. E2. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. E3. Sung SJ, Fu SM, Rose CE Jr, Gaskin F, Ju S-T, Beaty SR. A major lung CD103 (ae)-beta7 integrin-positive epithelial dendritic cell population expressing langerin and tight junction proteins. J Immunol 2006;176:2161-72. E4. Nishikomori R, Ehrhardt RO, Strober W. T helper type 2 cell differentiation occurs in the presence of interleukin 12 receptor beta2 chain expression and signaling. J Exp Med 2000;191:847-58. E5. Sung SJ, Rose CE Jr, Fu SM. Intratracheal priming with ovalbumin- and ovalbumin 323-339 peptide-pulsed dendritic cells induces airway hyperresponsiveness, lung eosinophilia, goblet cell hyperplasia, and inflammation. J Immunol 2001;166:1261-71. E6. Bates J, Irvin C, Brusasco V, Drazen J, Fredberg J, Loring S, et al. The use and misuse of Penh in animal models of lung disease. Am J Respir Cell Mol Biol 2004;31:373-4.
TABLE E1. Airway hyperreactivity Penh 0.5 6 0.1 (29) 1.0 6 0.1* (29) 0.4 6 0.1 (10) 1.6 6 0.3* (13) APTI (cm H 2 O sec) 577 6 55 (15) 1027 6 158* (12) 482 6 86 (8) 1179 6 196* (8) Numbers in parentheses indicate number of observations in each group. *Significantly different from respective genotype saline control: P<.05. Significantly different from comparably treated 1/1 group: P<.05.
TABLE E2. Lung cytokine and chemokine concentrations Lung MCP-1/CCL2 (pg/ml) 951 6 62 (13) 828 6 54 (16) 982 6 110 (4) 768 6 111 (7) IL-4 (pg/ml) 37 6 3 (11) 53 6 4* (14) 31 6 2 (3) 89 6 3* (4) IL-5 (pg/ml) 147 6 22 (12) 188 6 28 (14) 113 6 11 (3) 140 6 12 (4) IFN-g (pg/ml) 104 6 22 (8) 100 6 11 (11) 56 6 4 (3) 60 6 3 (4) MDC/CCL22 (pg/ml) 351 6 26 (9) 427 6 64 (10) 421 6 50 (3) 669 6 29 (4) SLC/CCL21 (pg/ml 3 10 3 ) 4.2 6 1.1 (16) 3.0 6 0.5 (19) 1.8 6 0.6 (4) 2.2 6 0.8 (7) TLNs SLC/CCL21 (pg/ml 3 10 3 ) 32.8 6 4.5 (14) 29.7 6 3.4 (16) 5.1 6 1.7 (4) 2.9 6 1.1 (7) Numbers in parentheses indicate number of observations in each group. *Significantly different from respective genotype saline control: P<.05. Significantly different compared with comparably treated 1/1 group: P<.05.
TABLE E3. TLN dendritic cell and T-cell subsets (310 3 /per mouse) I-A 1 CD11b 1 48 6 6 (2) [2] 148 6 41 (2) [3] 52 6 10 (2) [3] 285 6 131 (2) [24] CD11c 1 CD103 1 4 6 3 (2) [2] 9 6 5 (2) [4] 4 6 3 (2) [2] 14 6 7 (2) [8] I-A 1 PDCA 1 B220 1 12 6 1 (2) [0.03]* 27 6 16 (2) [0.03]* 16 6 10 (2) [0]* 26 6 2 (2) [0.6]* CD4 522 6 11 (2) [171] 742 6 251 (2) [174] 194 6 26 (2) [92] 871 6 189 (2) [338] CD8 188 6 38 (2) [15] 253 6 103 (2) [20] 154 6 36 (2) [25] 565 6 193 (2) [47] Numbers in parentheses indicate number of observations, and numbers in brackets indicate number of cells positive for CCR7. *I-A 1 PDCA 1 CCR7 1 cells.
TABLE E4. Cytokine production by TLN CD4 cells (310 3 per mouse) CD4 533 491 220 681 No. IL-4 1 * 0 1.3 0.8 9.9 No. IL-5 1 * 0.5 1.2 1.5 9.8 No. IFN-g 1 * 2.5 4.5 2.7 18.9 *Number of cells expressing the cytokine, as determined by means of flow cytometry.