for eotaxin, CXCL1, and TNF-a was 3.0, 2.0, and 8.0 pg/ml, respectively. MMP-2 and MMP-9 enzyme activity in nasal lavage fluids Gelatin zymography was performed for MMP-2 and MMP-9 detection. Gelatin was co-polymerized into a 10% polyacrylamide resolving gel. NAL fluids from mice were then subjected to separation by gelatin-SDSPAGE gel. Following electrophoresis, the gel was washed with buffer containing 0.1% Triton X-100, 10 mM Tris/HCl, 10 mM NaCl, 10 mM CaCl2, 100 mM ZnSO4, pH 7.4 at room temperature for 15 min three times and then incubated with the same buffer for 12 hrs to allow enzymatic gelatin substrate hydrolysis. The gel was stained in 0.1% Coomassie blue in a mixture of methanol:acetic acid:water for 1 hr and destained with a mixture of ethanol:acetic acid:water for 12 hrs. Densitometric analyses of triplicate data were performed using the NIS-Elements AR 3.0 software. Nasal lavage and cytology Nasal airway lavage on mice and analysis of infiltrating inflammatory cells were performed using the trans-pharyngeal nasal lavaging technique developed by our laboratory. Different from previously used trans-tracheal technique, this technique minimizes cells loss and gives consistent cytology results. Briefly, the choana was cannulated with 24G catheter through the pharyngeal opening above the vocal cord by transpharyngeal approach. The nasal cavities were gently lavaged with 350 mL of cold PBS twice and the fluid from the nostrils was collected. NAL fluids were centrifuged, and supernatants were stored at 280uC until assayed. Cell pellet was resuspended with 100 mL PBS and the total cells were counted using hemocytometer. Cytospin slides were prepared and stained with Diff-Quick stain, followed by differential cell count of at least 200 cells per slide. Statistical analysis The total and differential cell counts data were collected from multiple groups that showed normal distribution. Data were analyzed and compared with two-way ANOVA. All values are shown as Mean6Standard deviation of the mean. The mRNA expression, protein concentration of cytokines and Histological evaluation of nasal 221244-14-0 tissues H&E and Alcian blue stains were performed on 4 mm thick, paraffin-embedded nasal tissue sections after overnight decalcification with TBD-2 and fixation with formalin solution 10% Spontaneous Rhinitis in SHP-1 Deficient Mice chemokines, and densitometric results of MMPs were compared using one-way ANOVA. Difference with P,0.05 was considered statistically significant. Results were analyzed with statistical software Prism 4. Regulation of inflammatory cytokines and chemokines by Th2/Th1 cytokines To further understand the nasal inflammatory responses at the molecular level, we examined the chemokine expression in the nasal tissues of WT and mev mice using RT-PCR and ELISA. The mRNA levels of CCL2, CCL5, and GM-CSF did not increase in mev mice as compared with those in WT mice. Expression of GM-CSF decreased in mev mice on IL13 KO background. CCL2, CCL5 and GM-CSF all significantly increased in the mev mice with IFN-c KO when compared with mev mice. However, deficiency of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657886 IL-4 did not affect the expression levels of these chemokines. Similar results 
were seen for CCL2 and CCL5 in IL-13 deficient mev mice but GM-CSF was significantly reduced in the absence of IL-13. We next measured the protein concentrations of eotaxin, CXCL1 and TNF-a using ELISA. The eotaxin concentration in the NAL fluids was significantly increased in the mev mice compared
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