Recombinant SP-D carbohydrate recognition domain is a
chemoattractant for human neutrophils
Guang-Zuan
Cai1,
Gail L.
Griffin2,
Robert M.
Senior2,
William J.
Longmore1, and
Michael A.
Moxley1
1 Edward A. Doisy Department of
Biochemistry and Molecular Biology, Saint Louis University School of
Medicine, St. Louis 63104; and
2 Division of Pulmonary and
Critical Care Medicine, Department of Medicine, Washington University
School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri
63110
 |
ABSTRACT |
Human pulmonary surfactant protein D (SP-D) is a
collagenous C-type lectin with high binding specificity to
-D-glucosyl
residues. It is composed of four regions: a short
NH2-terminal noncollagen sequence,
a collagenous domain, a short linking domain ("neck" region), and
a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic
for inflammatory cells. To test which domain of SP-D might play a role
in this function, a mutant that contains only neck and CRD
regions was expressed in Escherichia
coli and purified by affinity chromatography on
maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two
antibodies (antisynthetic SP-D COOH-terminal and neck region peptides)
but not by synthetic SP-D
NH2-terminal peptide antibody. The
recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the
rest was monomeric. Analysis of the chemotactic properties of the
trimeric CRD demonstrated that the CRD was chemotactic for neutrophils
(polymorphonuclear leukocytes), with peak activity at
10
10 M equal to the
positive control [formyl-Met-Leu-Phe (fMLP) at 10
8 M]. The
chemotactic activity was abolished by 20 mM maltose, which did not
suppress the chemotactic response to fMLP. The peak chemotactic
activity of the CRD is comparable to the activity of native SP-D,
although a higher concentration is required for peak activity
(10
10 vs.
10
11 M). The chemotactic
response to CRD was largely prevented by preincubation of
polymorphonuclear leukocytes with SP-D, and the response to SP-D was
prevented by preincubation with CRD. These preincubations did not
affect chemotaxis to fMLP. These results suggest that trimeric CRD
accounts for the chemotactic activity of SP-D.
surfactant protein D; collectins; host defense proteins; chemotaxis
 |
INTRODUCTION |
PULMONARY SURFACTANT PROTEIN (SP) D is a member of a
family of collagenous C-type lectins (13). The protein is synthesized and secreted by alveolar epithelial type II cells and by nonciliated bronchiolar or Clara cells (4, 31). SP-D has been shown to be a
calcium-dependent, carbohydrate-binding protein (20) that interacts
with one of the lipid components of surfactant (19, 21) and with
microorganisms (11, 16), leukocytes (7), and macrophages (15, 18). Four
distinct regions of the protein can be identified: a short
NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" or
"hinge" region), and a COOH-terminal carbohydrate recognition
domain (CRD). Electron-microscopic studies (6) show that SP-D molecules
are assembled as tetramers of trimeric subunits (12 mers). A previous study (7) showed that SP-D is chemotactic for polymorphonuclear leukocytes (PMNs) and monocytes. This finding fits with a role for SP-D
in host defense. Because both maltose and antibodies to the
COOH-terminal domain of SP-D diminish the chemotactic
response of human PMNs to SP-D (7), this domain may be the domain
responsible for the chemotactic activity of SP-D. To test for the
chemotactic activity of this domain, a mutant that contains only the
neck and CRD regions (neck-CRD) of SP-D was expressed in
Escherichia coli and purified by
affinity chromatography on maltosyl-agarose. The chemotactic response
of human PMNs to this protein was determined.
 |
METHODS |
Construction and expression. A human
lung cDNA of SP-D, which was kindly supplied by E. C. Crouch
(Washington University, St. Louis, MO) was used as a template for a
PCR. Two oligonucleotides were synthesized on the basis of the
published cDNA sequence (8), engineering
Sal I and
Nde I restriction
sites at the 5'-end of the neck region oligonucleotide
(5'-GACAGTCGACCATATGGATGTTGCTTCTCTGAGG-3') and
BamH I and a stop codon at the 5'-end of the CRD
oligonucleotide (5'-CCAAGGATCCCTATCAGAACTCGCAGACCAC-3'). The PCR
product was purified with a Gene Clean kit
(Bio101, Vista, CA) after electrophoresis on a 1.5%
SeaPlaque agarose gel (FMC Bioproducts, Rockland, ME). The DNA fragment was digested with Sal
I and BamH I and ligated into the
Sal I and
BamH I side of pBluescript IIKS with T4 ligase. The
ligation mixture was transformed into competent cells of
E. coli strain DH5 for color
selection. The correct clone was digested with BamH I
and Nde I and ligated into pET/3xa
vector under the control of the T7 promoter. The target plasmid was
transformed into E. coli strain
BL21(DE3) for expression.
E. coli was grown in Luria-Bertani
broth (Difco, Detroit, MI) containing 200 mg/l of
ampicillin at 37°C. Expression of the CRD was achieved by adding 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO) at an optical density of 0.9 at 600 nm. Cells were harvested 2 h after IPTG induction by
centrifugation at 1,500 g for 5 min.
Sequencing. Plasmids isolated from
single colonies were subjected to double-stranded sequencing with the
T7 sequencing kit (Amersham Life Science, Arlington Heights, IL).
Purification of neck-CRD. The
purification protocol was adapted from Weis et al. (33). Briefly, the
pellet of the induced cells was washed with 10 mM Tris, pH 7.8, sonicated (30 s, 6×), and centrifuged at 10,000 g for 15 min at 4°C. The pellet
was extracted with 6 M guanidine HCl (GuHCl) in 100 mM Tris containing 0.01% 2-mercaptoethanol for 2 h on ice. The mixture was centrifuged at
100,000 g for 60 min at 4°C. The
supernatant was dialyzed extensively against loading buffer (1.25 M
NaCl, 25 mM CaCl2, and 25 mM Tris, pH 7.8) and again centrifuged at 100,000 g for 60 min at 4°C. The
supernatant of the resulting solution was further purified by
maltosyl-agarose affinity chromatography (20). Briefly, the supernatant
was loaded onto the maltosyl-agarose column, and the column was washed
with the loading buffer. The protein was then eluted from the column
with 1.24 M NaCl, 25 mM EDTA, and 25 mM Tris, pH 7.8. After the protein
content of each fraction was determined (1.5 ml/fraction), 1 µg of
each fraction was loaded onto an SDS-tricine polyacrylamide gel to
identify CRD-containing fractions.
The trimeric neck-CRD was isolated by HPLC isocratically on a Progel
TSK-GEL G3000SW (TSK; 30 cm × 7.5-mm ID)
size-exclusion column (Supelco, Bellefonte, PA) with a mobile phase of
buffer A (100 mM
NaH2PO4,
100 mM
Na2SO4,
and 0.05% NaN3, pH 6.8).
Thyroglobulin, BSA, ovalbumin, and myoglobin (Sigma) were used to
calibrate the TSK column.
Antibody preparations and
characterization. Three polyclonal antibodies to
different regions of human SP-D were prepared in rabbits with synthetic
peptides conjugated to thyroglobulin. The three synthetic peptides used
for immunization were the COOH terminus of human SP-D (amino acids
281-293), the NH2 terminus of
human SP-D (amino acids 37-50), and the neck or hinge region of
SP-D (amino acids 169-183). The antisera were purified on a
protein A-Sepharose 4B column (Sigma). The antibodies were
characterized by Western blot with both specific peptide-thyroglobulin
conjugates and human SP-D as antigens. Human SP-D was isolated from
human alveolar proteinosis lavage fluid (5). Each antibody detected its
specific antigen, and each was capable of detecting human SP-D (data
not shown). For Western blot analysis of the CRD, the three antibodies
were used in a 1:250 dilution. A 1:3,000 dilution of goat anti-rabbit
IgG alkaline phosphate conjugate was used as the secondary antibody.
Color was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium as substrate.
Denaturation and renaturation of
neck-CRD. The purified neck-CRD was subjected to
denaturation by GuHCl to demonstrate possible effects of the extraction
procedure on the conformation of the CRD. Two samples of purified
trimeric neck-CRD (final concentrations of 1 and 0.5 µM in
buffer A) were mixed with varying
amounts of GuHCl to give final GuHCl concentrations ranging from 0 to 2 M. Samples were equilibrated at 20°C for 1 h. The denaturation was monitored by fluorescence at 20°C. The emission spectra were
recorded from 300 to 450 nm in 0.5-nm wavelength increments, with an
excitation wavelength of 295 nm (2). The renaturation of the neck-CRD was accomplished by removing GuHCl from the samples by extensively dialyzing the denaturation mixture against buffer
A at 4°C.
Chemotaxis assay. Neutrophil
(PMN) chemotaxis to recombinant neck-CRD and native
SP-D was examined in modified Boyden chamber assays at 37°C with a
membrane sandwich technique (25). PMNs were isolated from human
peripheral venous blood by density gradient centrifugation with
Histopaque (1) and resuspended in DMEM with 0.5 mg/ml of human serum
albumin. PMN migration was expressed as cells per high-power grid by
light microscopy (×400). In each experiment, the data points
represent the means of cell counts of five high-power fields on each of
three membrane pairs from the Boyden chambers
(n = 15 cell counts). The
lower compartment of each chamber was filled with 240 µl of test
solution or basal medium, and the upper compartment contained 360 µl
of cell suspension. Negative controls consisting of medium only in the
lower compartment, and positive controls of medium containing fMLP
(10
8 M) in the lower
compartment were included in each experiment. All cell counts were
corrected for medium blanks. In experiments involving maltose, 20 mM
maltose was added to the upper and lower compartments. To determine
further whether CRD accounts for the chemotactic activity of SP-D for
PMNs, we preincubated the PMNs with CRD (15 min, room temperature) and
then tested for residual chemotactic responsiveness to SP-D (24). In
other desensitization experiments, cells were preincubated with SP-D
and then exposed to CRD.
Other procedures. Protein
concentrations were measured by the modified method of
Lowry (22) with BSA as a standard. SDS-PAGE was
performed according to the method of Laemmli (17). Immunoblotting of
proteins was performed by the method of Towbin et al. (26). Digital
images of the gels and blots were captured and notated with a
Macintosh-based imaging system utilizing a charge-coupled device camera
(Digital Imagers, Elburn, IL) and National Institutes of Health Image
1.60 software.
 |
RESULTS |
Expression and purification. Two
colonies from the BL21(DE3)/pET-3xa-CRD plate were incubated at
37°C in Luria-Bertani broth containing 200 mg/l of ampicillin until
the optical density of the medium reached 0.9 at 600 nm. After 1 ml of
culture medium was taken as a negative control, 1 mM IPTG was added to
the medium and the incubation continued for another 2 h. Cells were
harvested and analyzed by SDS-PAGE and Western blot. Figure
1 shows the results of silver staining and
Western blotting of a 16% SDS-PAGE, which indicated a dominant band 17 kDa in size and was recognized by COOH-terminal peptide antibody.
Noninduced cell extracts were negative. The higher molecular-weight
species apparent on the gel represent undenatured trimer as determined
by molecular-weight analysis with National Center for Supercomputer
Applications GelReader 2.0. The
NH2-terminal amino acid sequencing
confirmed that the 17-kDa protein starts with the neck region
(Asp-Val-Ala-Ser-Leu-Arg).

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Fig. 1.
Expression of carbohydrate recognition domain (CRD).
Two colonies were incubated at 37°C until optical density at
600 nm equaled 0.9. Colonies were then induced by
adding 1 mM
isopropyl- -D-thiogalactopyranoside
(IPTG). (In each case, 1 ml of cells was saved as a negative control.)
Lanes 1, 2, 7, and
8, negative controls that were
noninduced cell extracts; lanes
3, 4,
5, and
6, induced cell extracts.
Lanes
1-4
were stained with silver. Lanes
5-8
were blotted with antibody raised against COOH-terminal peptide of
surfactant protein (SP) D. +, With; , without. Nos. at
left, molecular mass.
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The GuHCl extraction yielded 10 mg of the recombinant neck-CRD per
liter of culture. The neck-CRD was eluted in a single peak by
maltosyl-agarose affinity chromatography (Fig.
2). The eluted fraction was subjected to
Western blot analysis with antibodies generated against the COOH
terminus, the NH2 terminus, and
the neck or hinge region of SP-D. The CRD was recognized
by the neck region and COOH-terminal antisera but not by
NH2-terminal and nonimmune sera
(Fig. 3).

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Fig. 2.
Purification of CRD by guanidine HCl (GuHCl)
extraction and maltosyl-Sepharose chromatography (silver-stained
polyacrylamide gel). Protein was extracted from a sonicated cell pellet
as described in METHODS. Dialyzed
extract was loaded on a maltosyl-agarose 4B column and eluted as
described in METHODS.
Lane
1, molecular-mass markers;
lane
2, extract dialyzed against loading
buffer before loading on column; lane
3, proteins that did not bind to
maltose column; lane
4, column wash;
lanes
5-7,
CRD eluted from column. Nos. at left,
molecular mass.
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Fig. 3.
Western blot of CRD with SP-D antibodies.
Lane
1, molecular-mass markers stained with
amido black; lanes
2-7,
1 µg of CRD loaded in each and run on SDS-PAGE under
reducing conditions. Lanes
2, 4,
and 6 were reacted with nonimmune
serum; lane
3 was reacted with antibody raised
against COOH terminus of SP-D; lane
5 was reacted with antibody against
neck region; and lane
7 was reacted with antibody against
NH2 terminus of SP-D. Nos. at
left, molecular mass.
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Separation of trimeric neck-CRD. To
determine whether the recombinant CRD was isolated as a monomer or as a
trimer, we subjected the protein to size-exclusion chromatography. Five
micrograms of the CRD purified from a maltosyl-agarose column were
injected onto the TSK column that was calibrated by thyroglobulin, BSA, ovalbumin, and myoglobin (Fig.
4A).
Analysis of two preparations showed that an average of 84% of the
recombinant neck-CRD was found to assemble into the trimeric form (Fig.
4, B and
C). These results demonstrate that
our isolation procedure allows us to isolate the CRD in a conformation
similar to its conformation in native SP-D.

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Fig. 4.
Recorder output of size-exclusion chromatography of
maltose affinity-purified CRD. A:
Progel TSK-GEL G3000SW column was calibrated with thyroglobulin
(1), BSA
(2), ovalbumin
(3), and myoglobin
(4).
B: 76% of CRD is present as trimer
(T). M, monomer. C: 93% is present as
trimer. B
and C represent 2 separate preparations.
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Denaturation and renaturation. To
determine whether the extraction procedure had any effect on the
conformation of the CRD, denaturation-renaturation experiments were
carried out. Fluorescence emission spectra were used to monitor the
denaturation of neck-CRD at 20°C. Figure
5 shows that the trimeric neck-CRD was
completely dissociated by GuHCl at 1 M. Renaturation was accomplished
by extensively dialyzing the neck-CRD denatured by 2 M GuHCl to remove GuHCl at 4°C. Again monitored by fluorescence emission spectra, the
intensity of the fluorescence returned to normal. As shown in Fig. 5,
the intensity of the renatured CRD was 98% of that of the starting
material.

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Fig. 5.
Denaturation of CRD by GuHCl. CRD was exposed to
increasing concentrations of GuHCl. Fluorescence intensity decreased as
denaturation increased. At 1 M GuHCl, CRD was completely denatured.
After extensive dialysis to remove GuHCl, fluorescence intensity
returned to control values (data not shown).
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Chemotactic activity. Purified
recombinant neck-CRD showed dose-dependent effects on human PMN
migration, with a maximal response at a concentration of 2 ng/ml
(~10
10 M; Fig.
6). At this concentration, neck-CRD was as
active as the optimal concentration of fMLP, a potent leukocyte
chemoattractant.

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Fig. 6.
CRD is chemotactic for human polymorphonuclear
leukocytes and chemotactic activity is abolished by maltose. Peak
chemotactic activity is at
10 10 M and approximated
activity of formyl-Met-Leu-Phe (fMLP) at
10 8 M. Chemotaxis was
determined as described in METHODS.
Maltose at 20 mM does not affect chemotactic response to fMLP. Data are
means ± SD from a representative experiment in triplicate, with 5 high-power fields counted for each membrane pair
(n = 15 cell counts). HPG, high-power
grid.
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Maltose (20 mM), an inhibitor of SP-D binding to various saccharide
ligands (16, 20), reduced the chemotactic activity of neck-CRD by 98%
at all concentrations of the CRD (Fig. 6). In contrast, as observed
previously (7), maltose had no effect on the chemotactic activity of fMLP.
In two separate experiments, each done in triplicate, preincubation of
PMNs with CRD reduced the chemotactic activity of SP-D at its optimal
chemotactic activity of
10
11 M by 75 and 91% (Fig.
7B),
without inhibiting the response of the cells to fMLP. In two other
experiments, also in triplicate, cells preincubated with SP-D displayed
83 and 68% reductions in chemotaxis to CRD (Fig. 7A). These
findings suggest that the neck-CRD contains the chemotactically active
domain of SP-D.

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Fig. 7.
Effect of preincubations with SP-D or CRD on
neutrophil chemotaxis to CRD and SP-D. See
METHODS for details.
A: CRD chemotactic activity after
cells were preincubated with SP-D. B:
SP-D chemotactic activity after cells were preincubated with CRD. Data
are means ± SD from a representative experiment as noted in Fig. 6.
In each case, preincubations reduced chemotactic activities of both CRD
and SP-D without having an effect on chemotactic activity of fMLP.
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 |
DISCUSSION |
Pulmonary surfactant is a complex mixture of lipids and proteins lining
the alveolar surface. The major function of the complex is to prevent
the alveoli from collapsing at end expiration. The major active
component of surfactant is disaturated phosphatidylcholine (3, 23), but
both hydrophilic and hydrophobic proteins are required (12). The two
hydrophilic proteins SP-A and SP-D are produced by alveolar type II
cells and nonciliated bronchiolar or Clara cells (4, 31). These two
proteins are members of the family of
Ca2+-dependent, collagenous
carbohydrate-binding proteins known as C-type lectins or collectins
(13). These pulmonary proteins have been suggested to have a role in
nonimmune host defense (13, 27). SP-A interacts with specific
saccharide ligands (10) and with various microorganisms and stimulates
the uptake of bacteria and viruses by macrophages (29, 30). SP-D also
interacts with specific carbohydrate ligands (20) and binds to
carbohydrates on the surface of a variety of microorganisms (11, 16) as well as macrophages (15) and leukocytes (7). SP-A has been shown to be
chemotactic for macrophages (34), and SP-D has been shown to elicit a
chemotactic response in human neutrophils (7). The chemotactic response
of macrophages to SP-A appears to be at least partially dependent on
the collagenous nature of SP-A (34). However, the response of human
PMNs to SP-D may be dependent on the lectin nature of the protein
because it is inhibited by an antibody to the COOH-terminal domain and
by maltose (7). This study was undertaken to further assess directly
the lectin domain-dependent nature of the chemotactic response to SP-D.
The recombinant CRD was constructed and expressed as a peptide
containing both the CRD and the neck or hinge region of SP-D. This was
done because it was considered that such a structure would be more
likely to trimerize and thus be analogous to the native structure of
SP-D. Wang et al. (32) demonstrated that a recombinant conglutinin
polypeptide composed of the CRD and neck region of that molecule
self-associated into homotrimers, and Kishore et al. (14) recently
demonstrated that the neck region is required for homotrimer formation
of a recombinant SP-D CRD derived from a fusion protein. Our method of
preparation differs somewhat from that described by Kishore et al. in
that the peptide is not prepared as a fusion protein, thus eliminating
the requirement for a cleavage step followed by further purification.
In addition, lysis of the cells and extraction of the peptide utilized
the same medium, eliminating the denaturation and refolding step
described by Wang et al. (32). It also differs from the preparation
described by Eda et al. (9) in that it does not contain any portion of the collagen-like domain of SP-D. Figure 1 shows that the peptide was
successfully expressed in E. coli and
was recognized by an antibody to the COOH terminus of SP-D.
NH2-terminal amino acid sequencing
confirmed that the 17-kDa product started with the neck region (data
not shown). To purify the protein, we took advantage of its lectin
nature and specificity for
-D-glucosyl residues (20).
The peptide was successfully purified by maltosyl-Sepharose affinity
chromatography (Fig. 2) and is recognized by antibodies to the neck
region and the COOH terminus but not by an antibody to the
NH2-terminal domain (Fig. 3).
These results were predicted because the recombinant protein was
constructed to lack the
NH2-terminal region of the lectin.
Size-exclusion chromatography indicated that the majority of the
isolated peptide was present in the trimeric form. Denaturation with
GuHCl followed by renaturation indicated that the isolation procedure
did not permanently affect the conformation of the isolated peptide.
The recombinant CRD was shown to elicit a chemotactic response in human
PMNs. Significant chemotaxis was demonstrated at
10
12 M CRD, with a maximum
response at 10
10 M. This is
an order of magnitude higher than the maximally effective concentration
of native SP-D. The differing chemotactic response to native SP-D and
the CRD may indicate that the tetrameric conformation of native SP-D
with four available CRDs elicits a greater response than a single
recombinant consisting of a single trimeric subunit of the tetramer.
However, it must be acknowledged that these studies do not completely
eliminate the possible contribution of the
NH2-terminal and collagen domain
regions to the chemotactic activity of SP-D. The maximum response is
comparable to that elicited by fMLP at 10
8 M. The dependence of
the response on the lectin nature of the peptide was demonstrated by
the response of the cells to the peptide in the presence of a known
carbohydrate ligand for SP-D. Maltose at 20 mM inhibited the
chemotactic response at all concentrations of the CRD up to
10
7 M (Fig. 6). This is in
agreement with the demonstrated effect of maltose on the chemotactic
activity of SP-D for PMNs (7).
Desensitization studies (Fig. 7) supported the probable role of the CRD
in the chemotactic response to SP-D. Cells preincubated with SP-D
(10
8 M) were much less
responsive to 10
10 M CRD,
and cells preincubated with CRD were likewise much less responsive to
SP-D. Preincubation with either protein had no effect on the response
of PMNs to fMLP. These results suggest that the chemotactic response of
the cells to SP-D is largely dependent on the CRD domain of the
protein. The precise sequence or conformation of the recombinant
neck-CRD was not determined; however, trimeric conformation is required
for high-affinity binding to carbohydrates (14). Whether this
high-affinity binding is required for chemotaxis or whether the
monomeric form of the CRD can function as a chemoattractant remains to
be determined. The fact that native SP-D and the recombinant neck-CRD
show similar chemoattractant activity suggests that the trimeric form
may be required.
These results and others (7, 15, 16) indicate that the
proposed role of SP-D as a host defense protein is multifaceted. As
noted above, the protein has been shown to interact with a variety of
microorganisms, and, at higher concentrations than in the present
study, it has been shown to stimulate oxygen radical production in rat
macrophages (28). The demonstrated ability of the protein to function
as a chemoattractant suggests that it could play a role in the
accumulation of inflammatory cells in lung injury. It is of interest to
note that the chemoattractant activity of SP-D (7) or its CRD
(present study) is demonstrable at concentrations several orders of
magnitude lower than that required to show chemoattractant activity of
SP-A for macrophages, suggesting that SP-D is a more potent
chemoattractant in vivo.
 |
ACKNOWLEDGEMENTS |
We thank Thomas Broekelmann and Robert Mecham for sequencing the
recombinant peptide.
 |
FOOTNOTES |
This investigation was supported by National Heart, Lung, and Blood
Institute Grants HL-13405 and HL-29594.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. A. Moxley, 1402 South Grand Blvd.,
Saint Louis Univ. School of Medicine, Edward A. Doisy Dept. of
Biochemistry and Molecular Biology, St. Louis, MO 63104-1079.
Received 6 July 1998; accepted in final form 7 October 1998.
 |
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