Expression of Surfactant Protein D in the Human Gastric Mucosa and during Helicobacter pylori Infection

Abstract

Helicobacter pylori establishes persistent infection of gastric mucosa with diverse clinical outcomes. The innate immune molecule surfactant
protein D (SP-D) binds selectively to microorganisms, inducing aggregation and phagocytosis. In this study, we demonstrated
the expression of SP-D in gastric mucosa by reverse transcription-PCR and immuohistochemical analysis. SP-D is present at
the luminal surface and within the gastric pits, with maximal expression at the surface. Levels of expression are significantly
increased in H. pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used to demonstrate binding
and agglutination of H. pylori by SP-D in a lectin-specific manner. These activities resulted in a 50% reduction in the motility of H. pylori, as judged on the basis of curvilinear velocity measured by using a Hobson BacTracker. Lipopolysaccharides extracted from
three H. pylori strains were shown to bind SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding
among the strains. SP-D may therefore play a significant role in the innate immune response to H. pylori infection.

Full text

INFECTION AND IMMUNITY, Mar. 2002, p. 1481–1487 Vol. 70, No. 3
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.3.1481–1487.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Expression of Surfactant Protein D in the Human Gastric Mucosa and
during Helicobacter pylori Infection
Emma Murray,
1
Wafa Khamri,
2
Marjorie M. Walker,
2
Paul Eggleton,
1
Anthony P. Moran,
3
John A. Ferris,
3
Susanne Knapp,
3
Q. Najma Karim,
3
Mulegata Worku,
3
Peter Strong,
1
Kenneth B. M. Reid,
1
and Mark R. Thursz
3
*
MRC Immunochemistry Unit, Oxford,
1
and Faculty of Medicine, Imperial College of Science, Technology and
Medicine, St. Mary’s Hospital Campus, London,
2
United Kingdom, and Department of Microbiology,
National University of Ireland, Galway, Ireland
3
Received 18 January 2001/Returned for modification 11 April 2001/Accepted 15 October 2001
Helicobacter pylori establishes persistent infection of gastric mucosa with diverse clinical outcomes. The
innate immune molecule surfactant protein D (SP-D) binds selectively to microorganisms, inducing aggrega-
tion and phagocytosis. In this study, we demonstrated the expression of SP-D in gastric mucosa by reverse
transcription-PCR and immuohistochemical analysis. SP-D is present at the luminal surface and within the
gastric pits, with maximal expression at the surface. Levels of expression are significantly increased in H.
pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used
to demonstrate binding and agglutination of H. pylori by SP-D in a lectin-specific manner. These activities
resulted in a 50% reduction in the motility of H. pylori, as judged on the basis of curvilinear velocity measured
by using a Hobson BacTracker. Lipopolysaccharides extracted from three H. pylori strains were shown to bind
SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding among
the strains. SP-D may therefore play a significant role in the innate immune response to H. pylori infection.
Helicobacter pylori is a gram-negative bacterium which col-
onizes gastric mucosa. It is one of the most common patho-
gens, with a prevalence of up to 90% in developing countries
(17). Once infection is established, gastric mucosal inflamma-
tion develops, and although infection persists for life, only 30%
of those infected become symptomatic. The outcome of infec-
tion is diverse and includes duodenal ulcer, gastric ulcer, and
gastric malignancy—both carcinoma and lymphoma. Such het-
erogeneous consequences are dependent on the time span and
topography of mucosal inflammation (28). The route of infec-
tion is not proven, but the most likely event is direct ingestion
of gastric contents. Gastric mucosa is markedly adverse to
bacterial colonization, as the physical and chemical barriers
encountered (mucus, enzymes, and acid) inhibit colonization
by common bacteria.
H.pylori-related gastritis is characterized by both lympho-
cytic and neutrophil infiltrates. In addition, there is a strong
humoral immune response with specific antibodies of both
immunoglobulin G (IgG) and IgA classes. Despite evidence of
an immune response, H.pylori infection is frequently persis-
tent, suggesting that the organism may evade conventional
innate and adaptive immune responses. A number of factors
appear to contribute to the virulence of H.pylori, including the
possession of flagella, which confer motility (6).
Surfactant protein D (SP-D) is a collagenous glycoprotein
which contains trimeric arrays of C-type (calcium-dependent)
lectin domains and which belongs to a family of proteins im-
plicated in innate immunity, termed the collectins (5, 25, 27).
The protein and molecular structures for SP-D are well char-
acterized, and the gene has been localized to human chromo-
some 10q22.2-23.1 (8, 13, 18). SP-D has been shown to recog-
nize and bind selectively to the surfaces of viruses, bacteria,
protozoa, and fungi and is currently being considered for use as
a therapeutic agent for the treatment of both bacterial infec-
tions and allergies in humans (4, 19, 23, 24, 26). SP-D is
believed to bind directly to the lipopolysaccharide (LPS) on
the surface of gram-negative bacteria via the carbohydrate
recognition domain (16). This process may result in the aggre-
gation of microorganisms followed by enhanced phagocytosis
by neutrophils and macrophages (11). Although phagocytosis
of microorganisms appears to be enhanced by interaction with
SP-D or SP-A, it is unclear whether such interactions promote
further immunological or inflammatory responses (2, 9, 11, 30).
In humans, SP-D has been shown to be synthesized in the
lungs, specifically alveolar type II and Clara cells, and to be
present in the fluid phase of the airways. SP-D has also been
detected at other sites, including tears, amniotic fluid, and fetal
membranes. In the rat, SP-D has been found in mucus-secret-
ing cells of the gastric mucosa but not in the duodenum or
remaining intestine, and a speculative role in mucus barrier
assembly and possibly host defense has been proposed (7).
In order to establish whether SP-D may play a role in de-
fense against H.pylori infection or in the pathogenesis of es-
tablished H.pylori infection, the aim of this study was to de-
termine whether SP-D is present in the human gastric mucosa
and whether SP-D interacts with H.pylori. Further studies were
performed to determine if the motility of H.pylori is impaired
by SP-D. In addition, SP-D binding studies were performed to
establish whether SP-D binds to LPS from bacterial cell walls.
* Corresponding author. Mailing address: Faculty of Medicine, Im-
perial College of Science, Technology and Medicine, St. Mary’s Hos-
pital Campus, Norfolk Place, London W2 1NY, United Kingdom.
Phone: 44 207 594 3851. Fax: 44 207 706 9161. E-mail: m.thursz
@ic.ac.uk.
1481

MATERIALS AND METHODS
Preparation of SP-D and antisera to SP-D. SP-D was purified from lung lavage
fluid from patients with alveolar proteinosis by the method of Strong et al. (29).
Briefly, lavage fluid was centrifuged at 10,000 ⫻gfor 40 min. The supernatant
was applied to a maltosyl-agarose column, and bound SP-D was specifically
eluted by using MnCl
2
. SP-D was further purified by gel filtration on Superose-6.
A recombinant preparation of human SP-D head and neck regions expressed
in Escherichia coli was used to raise polyclonal antisera in rabbits. The IgG
fraction was purified from rabbit serum by sodium sulfate precipitation followed
by protein A chromatography as previously described (20). The specificity of the
antisera for native human SP-D was confirmed by an enzyme-linked immunosor-
bent assay (ELISA) and Western blotting.
Detection of SP-D mRNA. mRNA for SP-D was detected by reverse transcrip-
tion (RT)-PCR. Twenty-eight patients with nonulcer dyspepsia were selected at
the time of endoscopy and gave informed consent. Thirteen patients had normal
histological findings, and 7 had H.pylori-associated antral gastritis. RNA was
extracted from three gastric antral biopsy specimens taken from each patient.
The biopsy specimens were snap-frozen in liquid nitrogen and stored at ⫺70°C
until processed. When required, biopsy specimens were homogenized in saline
and RNA was isolated by using a total RNA isolation kit according to the
manufacturer’s protocol (Ambion, AMS Biotechnology, Europe Ltd.). cDNA
synthesis and PCR amplification were carried out as a single step by using a
one-step RT-PCR kit according the manufacturer’s protocol (ABgene, Surrey,
United Kingdom). Primers from exons 3 and 4 of the SP-D gene were designed
to give product sizes of 475 bp from genomic DNA and 155 bp from cDNA. The
primer sequences were as follows: exon 3, 5⬘-GAACATAGGACCTCAGGGC
A-3⬘, and exon 4, 5⬘-TGTGTTTCCAGGGACTCCAC-3⬘. Parallel RT-PCRs
were performed with primers for the human ␤-actin gene to serve as a reference.
First-strand synthesis was performed with a single cycle at 47°C for 30 min. The
reverse transcriptase was then inactivated by denaturation at 94°C for 2 min.
PCR amplification was performed with 43 cycles of annealing at 63°C for 30 s,
extension at 72°C for 1 min, and denaturation at 94°C for 30 s. There was a final
extension step at 72°C for 5 min. Negative and positive control amplifications
were performed in every experiment. RT-PCR products for SP-D and ␤-actin
were run on a 2% (wt/vol) agarose gel, stained with ethidium bromide, and
visualized under UV light. Digital images were obtained, and densitometry was
performed by using a Syngene gel documentation system (Synoptics Ltd., Cam-
bridge, United Kingdom) and Genetools v3.0 software (Synoptics). Semiquanti-
tative measurement of SP-D mRNA was achieved by comparing the area under
the peak of the SP-D band with the area under the peak of the ␤-actin band and
expressing the result as a simple ratio.
Immunohistochemical analysis. Immunohistochemical analysis was per-
formed with the anti-SP-D polyclonal antisera described above and with mono-
clonal antibody 245.2 (a gift from U. Holmskov, Odense, Denmark). The spec-
ificity of this antibody has been confirmed by Western blotting and ELISA
analyses (21).
Formalin-fixed, paraffin-embedded gastric biopsy specimens were randomly
selected from the pathology archives for immunocytochemical staining, and 24
specimens were classified according to histological appearances. Of the 24 cases
selected, 11 were H.pylori-associated antral gastritis, 4 were H.pylori-negative
chemical antral gastritis, and 9 were H.pylori-negative normal antral mucosa.
Two 5-mm sections were cut from each biopsy specimen; one of these was
stained with the rabbit anti-SP-D polyclonal antisera, and the other was stained
with the SP-D-specific monoclonal antibody. Staining was demonstrated by using
a routine avidin-biotin-horseradish peroxidase system. Negative controls were
stained with the monoclonal antibody which had been preincubated with purified
SP-D, with an irrelevant antibody, and with preimmune sera from the same
rabbits.
SP-D expression was evaluated by a quantitative method with a standard grid
(10). Positive immunostaining was assessed by site in the gastric mucosa (lumen,
foveola, and pit) by counting cells expressing SP-D in a given area delineated by
the grid.
Binding and agglutination. H.pylori strain J178 (cagA positive) was cultured in
brain heart infusion broth at 37°C under microaerophilic conditions to a con-
centration of 10
8
to 10
9
bacteria ml
⫺1
. The organisms were centrifuged at 3,000
⫻gand resuspended in Tris-buffered saline (pH 7.4) (TBS).
Binding of SP-D to H.pylori was demonstrated by immunofluorescence. H.
pylori was incubated with SP-D (10 ␮g/ml) in TBS containing 10 mM CaCl
2
or 10
mM EDTA to demonstrate calcium-dependent binding. Controls were incubated
in calcium buffer alone. To assess lectin specificity, 100 mM maltose was added
to bacteria treated with SP-D and 10 mM CaCl
2
. Finally, SP-D binding to
bacteria was detected by probing with biotinylated anti-human SP-D antisera
(1:200 dilution of a 1-mg/ml IgG stock solution) followed by streptavidin-fluo-
rescein isothiocyanate (1:200; Sigma Chemical Co., Dorset, United Kingdom).
Agglutination was demonstrated by direct observation of the bacteria with phase-
contrast microscopy. H.pylori organisms resuspended in TBS containing 10 mM
CaCl
2
at 10
8
organisms/ml were incubated for 30 min with buffer alone, with 10
␮g of SP-D/ml, with 10 ␮g of SP-D/ml and 10 mM EDTA, or with 10 ␮gof
SP-D/ml and 100 mM maltose. Phase-contrast microscopy and fluorescence
microscopy were performed by using a Zeiss Axioskop transmitted-light micro-
scope. H.pylori was photographed with a Zeiss MC 100 camera (final magnifi-
cation, ⫻400) by using TX 400-ASA black and white film for phase contrast and
Provia 1600 color slide film for fluorescence.
Quantitative estimates of SP-D-mediated agglutination were obtained for clin-
ical isolates of H.pylori. Bacteria were placed in optical cuvettes suspended in
TBS plus 10 mM CaCl
2
. Two aliquots of each isolate were adjusted to an optical
density at 700 nm (OD
700
) of 0.7, and SP-D at a final concentration of 2.5 ␮g/ml
was added to one aliquot. OD
700
values were measured at 15-min intervals up to
60 min and again at 90 min. Agglutination was expressed as the difference in
OD
700
units between control and SP-D-containing aliquots at 60 min.
Inhibition of motility. Assessment of H.pylori motility was made by using a
Hobson BacTracker as previously described (15). The technique gives a quanti-
tative measure of motility by using an image-processing computer combined with
a phase-contrast microscope and video equipment. Motility was assessed in the
presence and absence of 5 ␮g of SP-D/ml in calcium buffer with and without the
addition of 100 mM maltose. Motility is expressed as curvilinear velocity, which
is the length of a track (total path length) divided by the time taken to travel it
in micrometers per second.
LPS binding studies. Binding of SP-D to LPS was demonstrated in a compet-
itive inhibition ELISA. LPS was extracted from three strains of H.pylori: clinical
strain J178, cagA-positive reference strain 007, and mouse-adapted strain SS1.
After pretreatment of bacterial biomass with pronase (Calbiochem, Los Angeles,
Calif.), LPS was extracted by the hot phenol-water technique. The resulting
crude LPS preparations, recovered from the water phase of extracts, were puri-
fied by treatment with RNase, DNase II, and proteinase K (Sigma) and by
ultracentrifugation as described previously (22).
Mannan (10 ␮g/ml) in carbonate buffer (pH 9.6) was used to coat a Maxisorb
Immunoplate (Life Technologies, Paisley, United Kingdom) overnight at 4°C.
The plate was washed three times in TBS and then blocked with TBS plus 3%
(wt/vol) bovine serum albumin for1hat37°C. Between each step, the plate was
washed three times in TBS plus 0.05% Tween 20 (TBST). LPS (200 to 1 ␮g/ml)
from H.pylori (J178, 007, or SS1) was incubated with a fixed concentration of
SP-D (1 ␮g/ml) for1hatroom temperature before addition to the mannan-
coated, blocked plate. The LPS–SP-D mixtures and SP-D alone (1,000 to 0
ng/ml) were incubated on the plate for3hat37°C. An aliquot (100 ␮l) of
biotinylated rabbit anti-SP-D polyclonal antisera (1:1,000 dilution of a 1-mg/ml
stock) in TBST was added to each well and incubated for2hat37°C. After
washing, 100 ␮l of ExtrAvidin-peroxidase conjugate (Sigma) diluted 1:10,000 in
TBST was added and incubated for 30 min at 37°C. The plate was developed with
tetramethylbenzidine substrate (Bio-Rad, Hemel Hempstead, United Kingdom),
the reaction was stopped with 100 ␮lof1NH
2
SO
4
, and readings were carried
out at 450 nm (Titertek Multiscan PLUS MKII). The resulting graphs (see Fig.
5) were generated by using Prism software (GraphPad Software Inc., San Diego,
Calif.), and the data were used to calculate the concentration of LPS required for
50% inhibition of SP-D binding.
RESULTS
Detection of SP-D mRNA. SP-D expression at the mRNA
level was assessed in gastric mucosa by use of RT-PCR. mRNA
for SP-D could be detected in 12 (100%) of 12 gastric tissue
samples where H.pylori infection was present and in 10
(62.5%) of 16 samples from individuals who did not have the
infection (Fig. 1A). The levels of gastric SP-D mRNA expres-
sion in individuals with H.pylori infection were significantly
higher than those in individuals without the infection. The
median SP-D/␤-actin ratios were 0.34 (range, 0.01 to 0.79) in
individuals with H.pylori infection and 0.08 (range, 0 to 0.45)
in individuals without the infection (Fig. 1B) (P⫽0.004)
(Mann-Whitney U test).
Immunohistochemical analysis. In order to assess, at the
cellular level, the site of SP-D expression, immunocytochemi-
1482 MURRAY ET AL. INFECT.IMMUN.

cal analysis was performed on endoscopic gastric mucosal bi-
opsy specimens taken routinely from 24 patients with dyspep-
sia. Immunohistochemical analysis was performed with rabbit
polyclonal antisera and the SP-D-specific monoclonal anti-
body. Negative controls, performed with the monoclonal anti-
body preabsorbed with SP-D, an irrelevant antibody, and pre-
immune sera from the same rabbits, revealed no
immunohistochemical staining. Staining with the anti-SP-D
monoclonal antibody gave results similar to those seen with the
polyclonal antisera.
The staining demonstrated SP-D protein expression in epi-
thelial cells, and SP-D coating of H.pylori was observed in situ
FIG. 1. SP-D mRNA is expressed in gastric mucosa. (A) RT-PCR results for RNA extracted from gastric mucosal tissue samples taken from
patients with dyspepsia. Bands specific for actin and SP-D are seen at 325 and 155 bp, respectively. “Helicobacter”indicates the presence (⫹)or
absence (⫺)ofH.pylori infection, as determined by histological examination; s, standards. (B) Level of SP-D mRNA expression, assessed by
densitometry of the RT-PCR bands and presented as plots (mean and standard error of the mean) of the SP-D/␤-actin ratio. H.pylori-positive and
H.pylori-negative gastric samples were compared.
VOL. 70, 2002 SP-D AND H.PYLORI 1483

(Fig. 2). The level of SP-D expression was observed to vary at
different epithelial levels in the mucosa, with maximal intensity
in the luminal mucosa compared to the foveolar region and the
gastric pits. No staining was observed in sections stained with
preimmune sera.
Semiquantitative analysis of cellular expression at different
levels in the mucosa was performed by comparing the intensi-
ties of expression in patients with H.pylori-associated gastritis,
reactive (chemical) gastritis, and normal histological findings.
SP-D was expressed at equal intensities in the basal region of
epithelial cells at each of the anatomical sites examined in all
three patient groups. However, in H.pylori-associated gastritis,
there was a significant increase in cell surface expression at all
sites (P⬍0.05) (Mann-Whitney U test) (Fig. 3). Increased cell
surface expression was not seen in reactive gastritis.
Binding and agglutination. Binding of SP-D to heat-fixed H.
pylori was clearly seen with immunofluorescence staining. The
calcium dependence and lectin-specific nature of this binding
FIG. 2. SP-D expression demonstrated by immunohistochemical analysis. Immunohistochemical analysis was performed with an anti-SP-D
monoclonal antibody and antral gastric mucosa by using a standard avidin-biotin technique as described in Materials and Methods. (A) Negative
control after the antibody was preabsorbed with native SP-D. (B) Low-level expression of SP-D (as judged by the brown staining) in the basal
regions of epithelial cells on the luminal surface of the mucosa. (C) Luminal epithelial cells of H.pylori-positive antral gastric mucosa, with strong
SP-D expression on the surface and in the basal regions of the cells. (D) Coating of H.pylori with SP-D (arrowheads). Original magnifications,
⫻1,000 (A, B, and C) and ⫻3,000 (D).
1484 MURRAY ET AL. INFECT.IMMUN.

were confirmed by the lack of binding in the absence of cal-
cium and competitive inhibition by maltose (Fig. 4A to D).
SP-D caused agglutination of live H.pylori into large clumps
that was inhibited by both EDTA and maltose (Fig. 4E to H).
Agglutination of clinical isolates of H.pylori was measured
as the difference in the OD
700
after 60 min of incubation with
2.5 ␮g of SP-D/ml (Fig. 5). There was a wide range of values,
from 0.100 to 0.475; the mean was 0.218. After 60 min of
incubation, the difference in the OD
700
reached a plateau in
some isolates.
Motility. The motility studies demonstrated that SP-D re-
duced the curvilinear velocity of H.pylori by approximately
50%. H.pylori in TBS plus 10 mM CaCl
2
had a curvilinear
velocity of 17.1 ␮m/s; this value fell to 9.65 ␮m/s in the pres-
ence of 5 ␮g of SP-D/ml. H.pylori motility was not reduced in
the presence of maltose or in the absence of calcium. Direct
observation of binding showed that the bacteria formed encir-
cling clusters. Free movement was impeded by agglutination
but was not halted.
LPS binding. SP-D bound to mannan in a concentration-
dependent fashion with a linear relationship to the absorbance
at 450 nm at between 50 and 1,000 ng/ml. LPS inhibited SP-D
binding in a dose-dependent manner. There was clear variation
in the ability of each LPS preparation to inhibit SP-D binding,
indicating variability in the avidity of SP-D for LPS binding.
The 50% inhibitory concentration for H.pylori J178 LPS was
5.3 ␮g/ml, that for SS1 LPS was 13.4 ␮g/ml, and that for 007
LPS was 91.5 ␮g/ml. The 50% inhibitory concentration ob-
tained with E.coli LPS was 73.7 ␮g/ml (Fig. 6).
DISCUSSION
This study has demonstrated the expression of SP-D in hu-
man gastric antral mucosa at both the mRNA and the protein
levels. Expression at this site suggests that SP-D may play a
role either in the structure of the gastric mucus layer or in host
defense. Low-level SP-D expression in the stomach was re-
cently reported by Madsen et al. (21). However, this study did
not take into account the H.pylori status or the anatomical site
of tissue collection. In contrast, our data suggest that the gas-
tric epithelium is capable of high levels of SP-D expression in
the context of H.pylori infection.
Recent studies with SP-D gene knockout mice have indi-
cated that SP-D is not an essential component of the mucus
layer in respiratory epithelium, as these mice develop without
significant respiratory pathology (3). However, these experi-
ments do not completely rule out a structural role for SP-D in
the gastric mucus layer. SP-D is related to the collectin family
of proteins, which are components of the innate immune sys-
tem. Evidence suggests that the major role of SP-D is the
recognition of foreign carbohydrate structures expressed on
the cell walls of microorganisms (26). SP-D may therefore be
one of the first lines of defense in the gastric mucosa.
H. pylori is a common human pathogen which is capable of
establishing a chronic infection of gastric mucosa. The binding,
agglutination, and motility studies presented here indicate that
SP-D recognizes and interacts with H.pylori, causing aggluti-
nation of the organism and inhibition of bacterial motility.
Agglutination is thought to facilitate phagocytosis by polymor-
FIG. 3. Site-specific expression of SP-D in gastric mucosa. (Left) Schematic representation of the sites used for immunocytochemical assess-
ment. (Right) Semiquantitative comparison of SP-D expression in H.pylori-associated gastritis, reflux-type gastritis, and samples with normal
histological findings at the epithelial surface (top), the foveolar region (middle), and the gastric pit region (bottom).
VOL. 70, 2002 SP-D AND H.PYLORI 1485

phonuclear cells or macrophages (9). While some reports sug-
gest that there may be a specific SP-D receptor on neutrophils
and macrophages, this idea is controversial, and other studies
suggest that SP-D does not act as an opsonin (1, 12).
Motility is thought to be an important virulence determinant
in H.pylori, allowing the organism to migrate to suitable niches
within the gastric mucosa; inhibition of motility prevents col-
onization (6, 31). In these experiments, motility was signifi-
cantly inhibited even in the absence of agglutination.
An attempt was made to measure the levels of SP-D in
gastric juice and to correlate these measurements with the
histological and immunocytochemical findings. Although it was
possible to detect low concentrations of SP-D in gastric juice
(results not shown), SP-D appeared to be rapidly digested by
proteases. It has been suggested that SP-D secreted from sal-
ivary glands may also interact with H.pylori. However, our
observations suggest that SP-D from salivary secretions would
be rapidly digested in the gastric lumen, whereas SP-D ex-
pressed by gastric epithelial cells would be effectively protected
under the mucus-bicarbonate barrier.
SP-D is believed to bind to LPS, which is the major compo-
nent of the cell wall in gram-negative bacteria. LPS was there-
fore considered to be the most likely target for SP-D binding to
H.pylori, and the data from the competitive inhibition assays
appear to confirm this notion. Our data do not rule out the
possibility that other components of the H.pylori cell wall are
also involved in SP-D binding. Significant interstrain variations
in the terminal O-chain structures of H.pylori LPS and in the
core oligosaccharide have been described (14). Wide variations
in agglutination measurements in clinical isolates and varia-
tions in binding affinities between LPSs extracted from differ-
ent H.pylori strains and SP-D were observed, potentially re-
flecting these structural differences. In the inhibition studies,
LPS of H.pylori J178 was the most inhibitory, with H.pylori SS1
LPS being next and H.pylori 007 LPS being the least inhibitory.
The structure of J178 LPS is presently under investigation, and
serological analysis indicates that LPS of H.pylori SS1 ex-
presses Le
y
(A. P. Moran et al., unpublished results); however,
the O chain of H.pylori 007 LPS consists of a polymeric Le
x
chain, lacking terminal Le
y
, thus indicating variations in the
FIG. 4. Binding of SP-D to H.pylori and agglutination. (A) Immu-
nofluorescence detection of SP-D binding to H.pylori by biotin-labeled
anti-human SP-D antibody (1:200 dilution of a 1-mg/ml stock).
(B) Phase-contrast image of the same bacteria as in panel A. (C) Effect
of 100 ␮M maltose on SP-D binding to H.pylori, as detected by
immunofluorescence. (D) Phase-contrast image of the same bacteria
as in panel C. (E to H) Agglutination of H.pylori after incubation with
10 ␮g of SP-D/ml and 10 mM CaCl
2
buffer (E), 10 mM EDTA (F), or
100 ␮M maltose (G) or without treatment (H). Immunofluorescence
microscopy and phase-contrast microscopy were performed with a
Zeiss Axioskop transmitted-light microscope with a Zeiss MC 100
camera. Final magnifications, ⫻400.
FIG. 5. Quantitative assessment of SP-D-mediated agglutination of
H.pylori clinical isolates. Agglutination was measured as the difference
in OD
700
between isolates incubated with or without SP-D at 2.5
␮g/ml.
FIG. 6. Inhibition of SP-D binding to mannan by H.pylori LPS.
Dose-dependent competitive inhibition of SP-D–mannan binding by
LPSs prepared from three H.pylori strains (J178, 007, and SS1) and
one E.coli strain (O128:B8) is shown. Curve fitting was performed by
using GraphPad Prism software.
1486 MURRAY ET AL. INFECT.IMMUN.

O-chain structures of these strains. However, these later stud-
ies also have indicated variations in the core oligosaccharide of
these strains. Therefore, at present, no clear conclusions can
be made as to the structures within H.pylori LPS involved in
SP-D binding.
The expression of SP-D in human gastric mucosa and a
functional interaction between SP-D and H.pylori have been
demonstrated. It therefore remains to be explained how per-
sistent infection with this organism is established and how the
organism can evade this component of innate immunity.
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Editor: B. B. Finlay
VOL. 70, 2002 SP-D AND H.PYLORI 1487