ORIGINAL ARTICLE Differential Expression of Surfactant Protein A in the Nasal Mucosa of Patients With Allergy Symptoms Christopher T. Wootten, MD; Robert F. Labadie, MD, PhD; Anton Chen, MD; Kirk F. Lane, PhD Objective: To characterize surfactant protein A (SP-A) cosa, polymerase chain reaction amplification of SP-A mes- expression in human nasal tissue and correlate differen- senger RNA, and rhinitis symptom scores. tial expression of SP-A with symptoms suggestive of al- lergic rhinitis. Results: Immunostaining localized SP-A to the mucosa Design: Allergic rhinitis symptom data were prospectively and submucosal glands in specimens. Quantitative collected in the form of the Rhinitis Symptom Utility In- polymerase chain reaction demonstrated correlation dex, the Rhinoconjunctivitis Quality of Life Questionnaire, between SP-A messenger RNA concentration and the and a Visual Analog Scale. Immunohistochemical stain- total Rhinitis Symptom Utility Index score (0.51, ing for SP-A was performed on resected nasal tissue. Quan- P=.009) as well as “sneezing over the previous week” titative polymerase chain reaction amplification of the SP-A (0.40, P=.049), “runny nose over the previous week” gene referenced to ␤-actin was performed on complemen- (0.55, P=.005), and “sneezing today” (0.47, P=.02). tary DNA samples synthesized from total RNA isolates. Conclusions: To our knowledge, this is the first Setting: Academic tertiary referral center, department report of SP-A expression in human nasal tissue. Fur- of otolaryngology laboratories. thermore, the degree of expression correlated with severity of disease as measured by the Rhinitis Symp- Patients: Twenty-five consecutive patients undergo- ing nasal surgery. tom Utility Index in patients with allergic rhinitis symptoms. Main Outcome Measures: Immunohistochemical staining of SP-A in human nasal mucosa and submu- Arch Otolaryngol Head Neck Surg. 2006;132:1001-1007 URFACTANT PROTEIN A (SP-A) tory regulatory protein-␣ on the surface of is a surfactant-related pro- tissue macrophages. Alternatively, associa- tein thought to have a role be- tion of its collagenous tail region with the yond phosphatidylcholine calreticulin/CD 91 complex on tissue mac- cycling, including immune rophages stimulates cytokine production, Smodulation along the respiratory tract.1 P38 phosphorylation, and nuclear factor ␬B A member of the collectin (calcium-de- activation, promoting local inflamma- pendent lectin) family, SP-A is related to tion.6 In such a role, SP-A also acts as an surfactant protein D, mannose-binding pro- opsonin and stimulates phagocytosis. tein, and C1q.2-4 Based on differential post- Given its varied role in the innate im- translational modifications, SP-A exists as munity of the respiratory tract, SP-A ex- a 28 to 36 kDa protein, but in vivo it ag- pression has been characterized in sev- gregates into octadecameric oligomers with eral disease states. Surfactant protein A a flower bouquet shape.5 down-regulation in allergy was demon- Author Affiliations: Its bipolar anatomy, with collagenous strated in a mouse asthma model in which Departments of Otolaryngology tails opposing carbohydrate-binding globu- SP-A expression decreased after allergen (Drs Wootten, Labadie, and 7 Chen) and Medicine, Division lar heads, underpins its biphasic immune challenge in sensitized animals. This of Pulmonary and Critical Care function. For example, SP-A is able to down-regulation phenomenon has also Medicine (Dr Lane), Vanderbilt down-regulate local inflammation by bind- been observed in eustachian tube mu- University, Nashville, Tenn. ing with its globular heads to signal inhibi- cosa after middle ear perfusion of aller- (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 132, SEP 2006 WWW.ARCHOTO.COM 1001 ©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 gen in sensitized rats (A.C., C.T.W., K.F.L., and R.L.L., (VAS) of their symptoms. Allergic symptoms over the past 1 week unpublished data, 2006). Consistent with its biphasic role were measured via a modified Rhinoconjunctivitis Quality of Life in inflammation, in the human respiratory tract, SP-A ex- Questionnaire (RQLQ),15 a 28-item, self-administered assess- ment, and symptoms over the past 2 weeks were assessed by the pression may also be elevated in the setting of allergy or 14 infection.8-10 Bronchoalveolar lavage specimens from pa- 5-item RSUI. In the operating room and under sterile conditions, re- tients with chronic bronchial asthma have demon- 11 12 sected specimens were dissected free of gross bone, parti- strated increased SP-A, as have reports of differential tioned, and snap-frozen in liquid nitrogen and stored in la- SP-A expression in patients with pneumocystis pneu- beled nuclease-free containers at −70°C. monia. Surfactant protein A expression has also been as- sessed in the sinonasal mucosa. In a semiquantitative IMMUNOHISTOCHEMICAL ANALYSIS study, Dutton et al13 found SP-A to be elevated in the mu- cosa of rabbits with intercurrent sinusitis or antibiotic- After thawing to −20°C and overnight decalcification, followed treated sinusitis compared with pathogen-free animals. by fixation in 4% paraformaldehyde, 5-µm sections of paraffin- Given the important role of nasal inflammation in hu- embedded human turbinate samples were placed on positively man disease, our study sought to correlate SP-A expres- charged slides and allowed to dry overnight. The paraffin was re- sion with rhinitis symptomatic traits in the nasal mu- moved from the slides, and heat-induced epitope retrieval was cosa of human subjects. Using immunohistochemical performed with EDTA buffer (Richard-Allan Scientific, Kalama- staining and real-time, quantitative polymerase chain re- zoo, Mich) for 20 minutes. Endogenous peroxidase activity was squelched with 0.03% hydrogen peroxide and rinsed. The sec- action (PCR), we demonstrate differential expression of tions were treated with casein for 20 minutes to block nonspe- SP-A that varies with the severity of allergic rhinitis symp- cific binding of the primary antibody. Rabbit anti-human SP-A toms as measured by the Rhinitis Symptom Utility In- (Santa Cruz Biotechnology Inc, Santa Cruz, Calif) diluted in an- 14 dex (RSUI). itbody diluent (Dako Corp, Carpinteria, Calif) by 1:200 was ap- plied to the tissues and stored in a humidity chamber overnight METHODS at 4°C. Sections without primary antibody served as negative con- trols. We produced localized, visible staining (DakoCytomation Envisionϩ System, DAB/Peroxidase; DakoCytomation, Carpin- A prospective, blinded analysis of SP-A expression in the na- Ն teria, Calif) after secondary antibody treatment with polyclonal sal mucosa of 25 consecutive adult patients (age, 18 years) anti-rabbit IgG and horseradish peroxidase. The slides were fi- undergoing turbinate (middle or inferior) reduction surgery nally counterstained with Mayer hematoxylin. was performed using immunohistochemical and quantitative PCR technologies. Institutional review board approval was obtained. All participants had preoperative nasal congestion RNA PURIFICATION AND COMPLEMENTARY and/or chronic sinusitis listed as a presenting complaint in DNA CONSTRUCTION their initial clinical encounter. Preoperative computed tomo- graphic imaging was obtained for each patient, demonstrating Total RNA purification was performed on the 25 specimens (us- anatomic disease persisting despite appropriate nonsurgical ing an Aurum Total RNA Mini Prep-Kit; Bio-Rad, Hercules, treatment. The indication for inferior turbinectomy was turbi- Calif), including a DNase 1 digest, according to the manufac- nate hypertrophy contributing to airflow obstruction. Middle turer’s instructions. Reverse transcriptase–PCR was per- turbinates were partially resected if involved with obstructive formed on a portion of the purified RNA for each specimen to concha bullosa changes or for improved postoperative sur- create complementary DNA (cDNA) libraries using the iScript veillance and debridement in difficult revision cases. At cDNA Synthesis Kit (Bio-Rad) under the following conditions resection, none of the study patients had gross mucosal dis- in a conventional thermocycler: 5 minutes at 25°C, 30 min- ease suggesting atrophy, suppuration, invasive fungal, or utes at 42°C, and 5 minutes at 85°C neoplastic lesions. Patients who were not undergoing tissue resective surgery QUANTITATIVE PCR and those unwilling or unable to supply information on their rhinitis symptoms or their histories were excluded from the Intron-exon spanning SP-A primers were used in the quanti- study. Allergy testing (skin testing or radioallergosorbent test tative PCR amplification reactions with the following design: assay) was not mandated prior to entry into the study, and only 5Ј-AAGCCACACTCCACGACTTTAGA-3Јand 5Ј-GCCCATT- 6 patients had allergy testing data in their medical records. These GCTGGAGAAGACCT-3Ј (Integrated DNA Technology Inc, results were excluded from analysis. Thus, the clinical instru- Coralville, Iowa). Commercially available ␤-actin primers for ments employed in this study assess the presence of symp- the amplification of the housekeeping gene were used: 5Ј- toms commonly attributed to nasal allergy, but they are not a TCATGAAGTGTGACGTTGACATCCGT-3 and 5Ј- substitute for objective testing. In addition, institutional re- CTTAGAAGCATTTGCGGTGCACGATG-3 (Promega, Madi- view board approval for this study did not allow us to control son, Wis). In a quantitative PCR detection system (My iQ Thermal