In situ hybridization of SP-A mRNA in adult human conducting airways.

Abstract

In our study, surfactant protein (SP)-A was characterized in adult human trachea and bronchi. SP-A mRNA and protein were localized to serous cells in submucosal gland by in situ hybridization and immunohistochemistry, respectively. A 2.2 kb SP-A mRNA transcript was detected in tracheal tissues by Northern blot analysis. Primer extension analysis and gene-specific reverse transcriptase polymerase chain reaction (RT-PCR) revealed the predominance of SP-A2 mRNA. However, using nested PCR, we also detected low amounts of SP-A1 mRNA in the tracheal tissues. A approximately 35 kDa SP-A immunoreactive protein was detected in the tracheal tissues by immunoblot analysis and was shown to be modified by the addition of N-linked oligosaccharides. We conclude that submucosal glands in the conducting airways produce a novel SP-A protein with a molecular weight and post-translational modification similar to the SP-A produced in the distal lung. We speculate that this SP-A2 protein, like other serous secretions from airway submucosal glands, functions in local antimicrobial host defense mechanisms in the conducting airways.

Full text

Page 1

Pediatric Pathology and Molecular Medicine 20: 349–366, 2001
Copyright # 2001 Taylor & Francis
1522-7952/01 $12.00 ‡.00
IN SITU HYBRIDIZATION OF SP-A mRNA IN ADULT HUMAN
CONDUCTING AIRWAYS
Kavita R. Khubchandani, BS, Kelli L. Goss, BA,
John F. Engelhardt, PhD, and Jeanne M. Snyder, PhD
of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa, USA
&
Department
&
mRNA and protein were localized to serous cells in submucosal glands by in situ hybridization and
immunohistochemistry, respectively. A 2.2 kb SP-A mRNA transcript was detected in tracheal tissues
by Northern blot analysis. Primer extension analysis and gene-speci®c reverse transcriptase–polymerase
chain reaction(RT–PCR)revealed the predominance of SP-A2 mRNA. However, using nested PCR, we
also detected low amounts of SP-A1 mRNA in the tracheal tissues. A ¹35 kDa SP-A immunoreactive
protein was detected in the tracheal tissues by immunoblot analysis and was shown to be modi®ed by the
addition of N-linked oligosaccharides. We conclude that submucosal glands in the conducting airways
produce a novel SP-A protein with a molecular weight and post-translational modi®cation similar to the
SP-A produced in the distal lung. We speculate that this SP-A2 protein, like other serous secretions from
airway submucosal glands, functions in local antimicrobial host defense mechanisms in the conducting
airways.
In ourstudy,surfactant protein(SP)-A was characterized in adult human trachea and bronchi. SP-A
Keywords
adult lung, SP-A1, SP-A2, submucosal glands, trachea
Pulmonary surfactant is a lipoprotein synthesized and secreted by alveolar type
II cells that reduces the surface tension at the lung alveolar air-liquid interface
[1].Surfactantis composed of phospholipids,cholesterol, and proteins;themost
abundant surfactant-associated protein is surfactant protein (SP)-A[2]. This
34–36 kDa protein facilitates the surface-tension lowering properties of
surfactant and regulates surfactant phospholipid synthesis, secretion, and recy-
cling[2]. SP-A is a member of the collectin family of C-type lectins and is
thought to play a role in innate host defense mechanisms in the lung[3].
Human SP-A is encoded by two genes, SP-A1 and SP-A2, which are 94%
identical in nucleotide sequence and 96% identical in amino acid sequence
This research was supported by grants from the National Institutes of Health, HL-50050 (JMS), DK-
47967 (JFE), and DERC DK-25295. The authors thank Dr. Michael Welsh, a Howard Hughes Medical
Institute Investigator in the Department of Internal Medicine for providing the adult human tracheal tissue
used in these studies. We also thank Philip Karp and Anubha Mittal for their help in conducting these
studies.
Address correspondence to: Jeanne M. Snyder, PhD, Department of Anatomy and Cell Biology,
University of Iowa College of Medicine, Iowa City, IA 52242, USA. E-mail: jeanne-snyder@uiowa.edu
349

Page 2

[4–6]. Native SP-A is made up of six SP-A trimers that form a ¯ower-bouquet
structure[7]. The human SP-A trimers are thought to be composed of two SP-
A1 and one SP-A2 molecules[7]. Post-translational modi®cations of SP-A
include glycosylation with N-linked oligosaccharides[2]. SP-A mRNA and
protein have been detected in human fetal alveolar type II cells[8]and in
human fetal tracheal and bronchial epithelium and submucosal glands[9, 10].
Our laboratory has demonstrated that human fetal lung airways only express
SP-A2 mRNA[11]. SP-A protein and mRNA also have been detected in
human adult lung alveolar type II cells and bronchiolar epithelium[12, 13].
Recently, SP-A protein was reported in adult human airway submucosal
glands, and SP-A2, but not SP-A1, mRNA is present in the airway submucosal
glands, as determined by Southern blot analysis of RT–PCR products[14].
In our study, we localized SP-A mRNA in serous cells of conducting air-
way submucosal glands in adult human proximal conducting airway tissues,
speci®cally trachea and bronchi, by in situ hybridization. We also character-
ized the submucosal gland SP-A protein by gel electrophoresis, enzymatic
digestion, and immunoblot analysis, and we showed that it has molecular
weight and post-translational modi®cations similar to the SP-A produced in
fetal distal lung alveolar type II cell. Consistent with previous reports, we
found that the submucosal glands express primarily the SP-A2 gene
products. In addition, the existence of small amounts of SP-A1 mRNA in
adult human tracheal submucosal tissues was also shown by RT-PCR.
These new observations contribute to our understanding of the role of SP-A
in the human conducting airways.
MATERIALS AND METHODS
Tissue
The experimental protocol for this study was approved by the Human
Subjects Review Committee at the University of Iowa, Iowa City, IA.
Segments of adult human trachea were obtained from the Iowa State Organ
Procurement Organization,IowaCity,IA,and fromtheInternationalInstitute
forthe Advancementof Medicine,Exton,PA.Twoadditionalhumanbronchial
samples were obtained from Dr. John Engelhardt. Tracheal submucosal tissue
was isolated using sterile technique. First, the trachea was pinned on a wax
surface with the lumenal surface up. The epithelium between the cartilages
was then peeled from the surface with forceps. Submucosal tissues between the
cartilages were dissected free, placed in sterile microfuge tubes, and frozen in
liquid nitrogen. Table 1 summarizes patient information for the 11 tracheal
and 2 bronchial samples included in this study. Fetal lung explants (prepared
from distal lung tissue) that had been maintained in vitro for 6 days and an
adult lung adenocarcinoma cell line (NCI-H441 cells) were used as positive
350
K. R. Khubchandani et al.

Page 3

controls for some experiments[15, 16]. Both the cultured fetal lung explants
and the H441 cells produce both human SP-A proteins (SP-A1 and SP-A2)
and SP-B[11, 17]. The fetal lung explants also produce SP-C[17].
RNA Isolation
Frozen tissue was homogenized in 1 ml of RNA extraction mixture and
total RNA was isolated as described previously[18]. The precipitated RNA
pellet was washed with 75% ethanol, air-dried, and resuspended in sterile
RNase-free, DNase-free water. RNA was quanti®ed by absorbance at 260 nm
and purity determined by the ratio of absorbance at 260 to 280 nm; 50mg of
RNA were electrophoresed on 1.2% agarose/5% formaldehyde gels. A
Polaroid photograph of the ethidium bromide-stained gel was obtained
under ultraviolet light (UV). The RNA was transferred by capillary action
to a positively charged nylon membrane (Nytran plus, Schleicher and Schuell,
Kenne, NH) and cross-linked to the membrane by UV irradiation in a GS
Gene Linker (Bio-Rad, Hercules, CA).
Northern Blot Analysis
A human cDNA probe for SP-A (gift of Dr. J. Whitsett, University of
Cincinnati, Cincinnati, OH) was radiolabeled with[¬-32P]deoxycytosine
triphosphate (¹3,000 Ci/mmol; Amersham, Bedford, MA) using a random-
primer kit (Roche Molecular Biochemicals, Indianapolis, IN). Prehybridi-
zation of the membranes was carried out for 6 h, then the labeled cDNA
probe was added (1 £ 106cpm/ml) and allowed to hybridize overnight.
SP-A2 mRNA in Human Conducting Airways
351
TABLE 1 Summary of Donor Information
Donor #
Age Sex*Cause of Death Smoking History
1
2
3
4
5
6
7
8
9
84
51
52
33
20
46
51
53
74
42
35
58
31
M
M
F
M
M
M
M
M
M
M
M
M
M
Pneumonia
Intracranial hemorrhage
Intracranial hemorrhage
Gunshot wound to the head
Head trauma
Head trauma
Intracranial hemorrhage
Cardiovascular disease
Cardiovascular stroke
Intracranial hemorrhage
Gunshot wound to the head
Unknown
Unknown
Quit in 1950
No
No
Heavy smoker**
No
Quit 1.5 years ago
Quit 20 years ago
No
Quit 50 years ago
No
Heavy smoker**
Unknown
No
10
11
12
13
* M ˆ male; F ˆ female, **1–2 packs per day.

Page 4

Membranes were washed and exposed to x-ray ®lm (Eastman Kodak,
Rochester, NY) with an intensifying screen at ¡708C as described[11].
RT±PCR and Restriction Digests
RT–PCR was performed on 1mg of RNA as described[19]. A description
of the oligonucleotides used for the PCR ampli®cations is given in Table 2.
Oligonucleotide (oligo) number 3, which hybridized to common SP-A1 and
SP-A2 cDNA sequences, was used in combination with either oligo number 4
or oligo number 5 to speci®cally amplify SP-A1 or SP-A2, respectively. In
samples in which the PCR ampli®cation was not adequate to detect either SP-
A1 (using oligos 3 and 4) or SP-A2 (using oligos 3 and 5) cDNA, nested PCR
was performed using another set of oligos that hybridized to sequences com-
mon to both SP-A1 and SP-A2 cDNAs (oligos 1 and 2). For the nested SP-A
PCR, 5ml of the ®rst PCR was used as a template for the second round of
PCR. SP-B and SP-C cDNAs were ampli®ed using oligos 6 and 7, 8 and 9,
respectively. Ten ml of each PCR reaction were electrophoresed on a 1%
agarose gel. A Polaroid photograph of the ethidium bromide-stained gel was
obtained under UV light. The speci®city of the SP-A1 and SP-A2 PCR was
con®rmed by digesting the PCR products with Apa I (New England Biolabs,
Beverly, MA), a restriction enzyme that speci®cally cleaves the SP-A1 PCR
product and does not cleave the SP-A2 PCR product, and with SbfI (New
England Biolabs), a restriction enzyme that speci®cally cleaves the SP-A2
PCR product and does not cleave the SP-A1 PCR product. The speci®city
of the nested SP-A1 and SP-A2 PCR ampli®cations was con®rmed by digest-
ing the nested PCR products with SbfI, a restriction enzyme that speci®cally
cleaves the SP-A2 nested PCR product and does not cleave the SP-A1 nested
PCR product.
352
K. R. Khubchandani et al.
TABLE 2 PCR Oligonucleotides
Oligonucleotide
Number
Gene
Speci®city
5’to3’Sequence OrientationNucleotide Position*
1SP-A1, SP-A2 CTAGACGAGGAGCTCCAAGCCACASense2418–2441 of SP-A1 gene and
2439-2463 of SP-A2 gene
3256–3279 of SP-A1 gene and
3268–3291 of SP-A2 gene
182–197 of SP-A1 gene and
178–193 of SP-A2 gene
3374–3393
3372–3393
18–38
6696–6717
1383–1403
2786–2806
2SP-A1, SP-A2 CCCATCTGTGTACATCTCCACACAAntisense
3 SP-A1, SP-A2TGGAGGCTCTGTGTGTSense
4
5
6
7
8
9
SP-A1
SP-A2
SP-B
SP-B
SP-C
SP-C
AGCCTCAGGATGGAGGCCGA
AACTGAAGGCCAGACAGGATCC
GCTGAGTCACACCTGCTGCAG
AAGGTCGGGGCTGTGGATACA
TTTGGCATTCCCTGCTGCCCA
GATGTAGTAGAGCGGCACCTC
Antisense
Antisense
Sense
Antisense
Sense
Antisense
*bp number as given in ref[6]for SP-A1, ref[5]for SP-A2, ref[29]for SP-B and ref[30]for SP-C.

Page 5

Primer Extension Analysis
An oligonucleotide, 5’-GGGGATACCAGGGCTTCCAACACAAACG-
3’, that is complementary to nucleotides 1117–1144 of the human SP-A1
gene[6]and nucleotides 1144–1171 of human SP-A2 gene[5]was labeled
at its 5’end with ®32P-ATP (5000 Ci/mmol; Amersham) using T4 polynucleo-
tide kinase (New England Biolabs). The unincorporated nucleotides were
removed using a Sephadex G-25 quick spin column (Roche). Primer extension
analysis was performed on 10mg of tracheal submucosal tissue total RNA and
0.2 pmol of labeled primer. The protocols for primer extension and extraction
of radiolabeled DNA transcripts have been described[11]. Transcripts were
analyzed on a 6% polyacrylamide/7M urea sequencing gel at a constant
power of 58 W. The gel was transferred to ®lter paper and dried using a gel
drier (Bio-Rad) for 2 h. The gel was exposed to x-ray ®lm at ¡708C using an
intensifying screen (Lightning Plus; Dupont, Wilmington, DE).
In Situ Hybridization
In situ hybridization was carried out as described[20]. First, 7mm-thick
frozen sections were cut at ¡208C and mounted on glass slides (Superfrost
Plus; Fisher, Chicago, IL). Sense or antisense[3H]cRNA probe (¹ 3 £ 105
cpm) was applied to each slide. Coverslips (HybriWell Chambers; Lab Vision;
Fremont, CA) were placed atop the sections and sealed, and the slides were
incubated at 608C overnight in a humid chamber. After removal of coverslips,
the slides were rinsed as described[20]. The sections were dehydrated through
an ethanol series, air-dried and coated with NTB-2 emulsion (Eastman
Kodak), dried in a slide oven, and exposed in a light-proof box at 48C.
Autoradiograms were developed in a D-19 developer, rinsed in distilled
water, ®xed in rapid ®xer, rinsed in distilled water, stained with hematoxylin,
then dehydrated and mounted with glass coverslips.
Immunostaining
Tracheal submucosal tissue was cut into 7-mm frozen sections at ¡208C
and mounted on glass slides (Superfrost Plus, Fisher). Sections were ®xed and
stained as described previously[19]using a rabbit antihuman SP-A antibody
(Chemicon International, Temecula, CA, 1:1000 in phosphate-buÚered
saline[PBS]) followed by a biotinylated secondary antibody for 30 min,
then rinsed and incubated in avidin-peroxidase reagent for 45 sec. After
rinsing, the sections were then incubated in substrate, 0.7 mg/ml diaminoben-
zidine (Sigma, St. Louis, MO) in PBS. Sections were rinsed, counterstained
with hematoxylin, dehydrated, and mounted on coverslips. For preabsorption
of the SP-A antibody, 2mg of antibody and 2mg of SP-A, puri®ed from
SP-A2 mRNA in Human Conducting Airways
353