Transcript Immunolocalization of surfactant protein A and D in

SP-B Detection and Gene Expression in
Chronic Rhinosinusitis
Bradford A. Woodworth, MD
Noam A. Cohen, MD, PhD
Rachel Wood, BS
Geeta Bhargave, BS
John E. Baatz, PhD
Rodney J. Schlosser, MD
Department of Otorhinolaryngology – HNS
University of Pennsylvania Health System
& Medical University of South Carolina
Grant support

Cystic Fibrosis Foundation
Surfactant



Secreted in lungs by type II pneumocytes and
Clara cells
Phospholipids (lamellar bodies): 80-90%
– Used for premature infants
– Decrease surface tension
– Decrease viscosity of mucus
Proteins: 10-15%
– SP B and C: hydrophobic, PL processing &
trafficking, anti-microbial properties
– SP A and D: hydrophilic, immune functions
Surfactant in Airway Mucus




Coats surface of gel layer to reduce surface tension
Decreases the viscosity of mucus
Increases mucociliary clearance
SP-B facilitates properties of surfactant and is also
shown to have direct anti-microbial properties
SP-B




Extremely hydrophobic protein with multiple
post-translational modifications
SP-A and D found at mucosal and epithelial
surfaces throughout the body
SP-B originally thought to be limited to the
lungs
Recent studies show expression in the
Eustachian tube mucosa
SP-B in Pulmonary Surfactant
SP-B
LB
Tubular Myelin
What role does surfactant play in the sinuses?
Prior Studies

Phospholipid lamellar bodies in
sinonasal epithelium
Prior Studies

Hydrophilic surfactant
proteins A and D in sinus
mucosa
– Localize to epithelium and
submucosal glandular
elements
– Upregulated in cystic
fibrosis CRS mucosa
Is there a role for SP-B?
Hypothesis
SP-B is present in sinonasal mucosa and
expression is altered in several types of
CRS when compared to healthy
controls.
Methods

Sinus mucosal biopsies
– Allergic Fungal Rhinosinusitis (n=7)
– Cystic Fibrosis (n = 4)
– Non-atopic CRS with nasal polyps (n=5)
– Healthy controls (n=5)
– Quantitative RT-PCR, immunoblot,
immunohistochemistry
Methods – Cycle threshold (Ct)
Methods – Cycle threshold (Ct)

Delta Ct (∆Ct) - Ct for mRNA
subtracted from Ct of internal
control (18s rRNA).
– Eliminates effect of differences in
sample concentration on Ct.

Individual ∆Ct values of each
subtype of CRS are compared to
the healthy control tissue
SP-B Quantitative RT-PCR
*
* p = 0.004
167-fold elevation in CF patients compared to healthy controls
Detection of SP-B Proprotein
42 kDa
Detection of the proprotein and intermediate forms
confirms translated product
Where is the protein produced and secreted?
Immunofluorescence
Sinus Epithelium
Submucosal Glands
SP-B expression – green, Nuclear stain in blue
SP-B localizes to the epithelium and submucosal glands
Discussion




SP-B was significantly upregulated in CF
CRS mucosa
Pseudomonas produces proteases known to
degrade SPs (Malloy et al).
SP-B upregulated in response to degradation
of SP-B and surfactant by Pseudomonas.
Alternatively, increased submucosal glands in
CF mucosa contribute more mRNA
transcripts to sample
Conclusion

SP-B is upregulated in cystic fibrosis
CRS and is produced by the epithelium
and submucosal glands of the sinonasal
cavities.

Further studies indicated to investigate
the role that SP-B and surfactant have
in CRS.
Future directions

Anti-microbial properties of SP-B as a
potential therapy for infectious CRS
– In vivo and in vitro models

Further delineation of protein expression
and specific localization with
immunoelectron microscopy
Thank You