Macrophage Dysfunction and Susceptibility to Pulmonary
Pseudomonas aeruginosa Infection in Surfactant Protein
Stephan W. Glasser,2* Albert P. Senft,3* Jeffrey A. Whitsett,* Melissa D. Maxfield,* Gary F. Ross,*
Theresa R. Richardson,* Daniel R. Prows,†Yan Xu,* and Thomas R. Korfhagen*
To determine the role of surfactant protein C (SP-C) in host defense, SP-C-deficient (Sftpc?/?) mice were infected with the
pulmonary pathogen Pseudomonas aeruginosa by intratracheal injection. Survival of young, postnatal day 14 Sftpc?/?mice
was decreased in comparison to Sftpc?/?mice. The sensitivity to Pseudomonas bacteria was specific to the 129S6 strain of
Sftpc?/?mice, a strain that spontaneously develops interstitial lung disease-like lung pathology with age. Pulmonary bac-
terial load and leukocyte infiltration were increased in the lungs of Sftpc?/?mice 24 h after infection. Early influx of
polymorphonuclear leukocytes in the lungs of uninfected newborn Sftpc?/?mice relative to Sftpc?/?mice indicate that the
lack of SP-C promotes proinflammatory responses in the lung. Mucin expression, as indicated by Alcian blue staining, was
increased in the airways of Sftpc?/?mice following infection. Phagocytic activity of alveolar macrophages from Sftpc?/?mice
was reduced. The uptake of fluorescent beads in vitro and the number of bacteria phagocytosed by alveolar macrophages in
vivo was decreased in the Sftpc?/?mice. Alveolar macrophages from Sftpc?/?mice expressed markers of alternative acti-
vation that are associated with diminished pathogen response and advancing pulmonary fibrosis. These findings implicate
SP-C as a modifier of alveolar homeostasis. SP-C plays an important role in innate host defense of the lung, enhancing
macrophage-mediated Pseudomonas phagocytosis, clearance and limiting pulmonary inflammatory responses.
of Immunology, 2008, 181: 621–628.
Surfactant homeostasis is maintained by precise regulation of synthe-
sis, secretion, and recycling by alveolar type II epithelial cells and its
degradation by alveolar macrophages (1, 2). Conditions that compro-
include surfactant insufficiency due to premature birth or inactivation
of surfactant due to lung injury or infection. Mutation in genes es-
sential for surfactant homeostasis including Sftpb, Sftpc Gmcsf, and
Abca3 causes acute or chronic lung disease (2–5).
Pulmonary surfactant protein C (SP-C)4is synthesized in type II
cells as a 21-kDa precursor (proSP-C) that is proteolytically pro-
ulmonary surfactant is a lipoprotein-rich complex that is re-
quired for normal pulmonary function and host defense. Pul-
cessed to the active secreted peptide. The mature form of SP-C is
a 35 aa peptide that is distinguished by an extended hydrophobic
valine rich domain from residues 9–23. In a lipid environment,
SP-C forms an ? helical structure that spans lipid bilayers. The
hydrophobic nature of SP-C is further enhanced by palmitoylation
of two adjacent N-terminal cysteine residues. SP-C increases the
rate of adsorption of lipids into a surface film and alters the orga-
nization of lipid acyl side chains (2, 6). Synthetic lipid preparations
containing only SP-C restore the dynamic compliance and oxy-
genation of lungs in surfactant depleted animal models (7). Sur-
factant extracts enriched in SP-C and phospholipids are used to
treat neonatal respiratory distress syndrome in newborn infants
Surfactant proteins play important roles in innate host defense of
the lung. An elaborate host defense system suppresses infection
from inhaled microorganisms while minimizing inflammation. The
innate pulmonary defense system consists of physical, cellular, and
biochemical barriers. The surfactant layer acts as both a barrier and
a source of proteins that modulate inflammatory signaling in the
alveolus and enhance the elimination of pathogens. For example,
surfactant protein A (SP-A) and surfactant protein D (SP-D) bind
to various infectious microorganisms as well as to LPS and other
bacterial and fungal components to enhance uptake and killing by
phagocytic cells (11). SP-C binds to the lipid A component of
bacterial LPS and to CD14, a component of the cellular LPS sig-
naling complex (12, 13). SP-C inhibits LPS interactions with mac-
rophages in vitro, potentially reducing overt phagocytic cell activ-
ity in the alveolus (14). These in vitro findings imply that SP-C
neutralizes LPS to influence subsequent inflammatory stimuli in
the alveolar compartment.
Mutations in the gene encoding SP-C (Sftpc) have been
linked to hereditary forms of interstitial lung disease (ILD).
*Division of Pulmonary Biology and†Division of Human Genetics, Department of
Pediatrics, Children’s Hospital Medical Center and University of Cincinnati College
of Medicine, Cincinnati, OH 45229
Received for publication September 27, 2007. Accepted for publication April 28, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants HL50046 and HL61646 (to S.W.G., J.A.W., and
D.R.P.), HL58795 (to T.R.K.), and by the Parker B. Francis Foundation (to A.P.S.).
2Address correspondence and reprint requests to Dr. Stephan W. Glasser, Division of
Pulmonary Biology, MLC 7029, Cincinnati Children’s Hospital Medical Center, 3333
Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: email@example.com
3Current address: Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive,
SE, Albuquerque, NM 87108.
4Abbreviations used in this paper: SP-C, surfactant protein C; SP-A, surfactant pro-
tein A; SP-D surfactant protein D; ILD, interstitial lung disease; BALF, bronchoal-
veolar lavage fluid; MMP, matrix metalloproteinase; PND14, postnatal day 14; AAM,
alternatively activated macrophage.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
Mutations that alter the structure of the proSP-C precursor pre-
dominate, whereas a smaller number of patients lack a primary
mutation yet still have a selective deficiency of mature SP-C
(15, 16). Clinical findings in SP-C-dependent ILD are highly
variable even among affected individuals from the same family
(17). Morphological changes due to SP-C deficiency include
cellular infiltrates, alveolar remodeling, airspace loss, and fi-
brosis. Episodes of respiratory insufficiency in SP-C-deficient
individuals are often preceded by pulmonary infection from a
variety of viral or bacterial sources (16, 17). These findings
suggest that the inability to process the aberrant proSP-C or loss
of SP-C in the alveolus predisposes the lungs to infection. The
SP-C gene-targeted mice (Sftpc?/?) on a 129S6 background
develop a progressive lung injury with features similar to those
of familial ILD in humans (18, 19). To determine whether SP-C
plays a role in innate host defense of the lung, Sftpc?/?mice
were challenged with the pulmonary pathogen Pseudomonas
aeruginosa. P. aeruginosa is a common pathogen associated
with pulmonary infection in cystic fibrosis and chronic diseases
requiring ventilator support (20). In the present study we dem-
onstrate that Sftpc?/?mice are susceptible to pulmonary infec-
tion by P. aeruginosa.
Materials and Methods
Sftpc?/?mice were generated by targeted gene inactivation as previously
described (18, 19). Sftpc?/?mice were previously bred onto 129S6/
SvEvTac (129S6) and FVB/N backgrounds to generate congenic 129S6
and FVB/N Sftpc?/?lines. Mice were maintained in a barrier facility and
housed in sterilized cages with access to sterilized food, water, and filtered
air. Lung homogenates were negative for culture of bacteria and fungus.
Sentinel mice in the colony were not infected by bacterial or viral patho-
gens. All animals were handled under aseptic conditions. Animal studies
were performed under protocols approved by the Institutional Animal Care
and Use Committee of the Children’s Hospital Research Foundation
A clinical isolate of mucoid P. aeruginosa from a single freezer stock was
used to minimize variation in virulence throughout the study. Bacteria were
grown overnight in tryptic soy broth at 37°C with continuous shaking.
Broth cultures were centrifuged, and the pelleted bacteria washed and re-
suspended in 1 ml of sterile PBS. The concentration of the inoculum was
determined by quantitative culture on tryptic soy blood agar. Administra-
tion of P. aeruginosa was performed by intratracheal inoculation of bac-
teria diluted in PBS. Mice were anesthetized with isofluorane and the tra-
chea exposed by a small anterior midline incision. A 30-gauge needle
attached to a tuberculin syringe was inserted into the trachea, and 50 or 100
?l of inoculum dispensed into the lung of postnatal day 14 (PND14) pups
or adult mice, respectively. Sterile PBS was injected as a control. The
incision was closed with one drop of Nexaband. For in vivo macrophage
phagocytic assays, mice were instilled with 8 ? 105P. aeruginosa that
constitutively express bacterial GFP, a gift of Dr. T. Machem (University
of California, Berkeley, CA).
Survival and bacterial clearance
To determine an effective bacterial dose 14-day-old Sftpc?/?and
Sftpc?/?mice were given 105–107bacteria intratracheally. Mortality
occurred at the 1 ? 107dose. Therefore, PND14 mice were intratra-
cheally injected with 1–4 ? 107bacteria and placed in warmed incu-
bators. Cages were checked at 1-h intervals for the first 4–6 h and then
monitored continuously for changes in survival. For clearance studies,
PND14 mice were administered 2–4 ? 106bacteria. Quantitative cul-
tures of lung homogenates were obtained 4 and 24 h postinoculation at
which time mice were given a lethal i.p. injection of ketamine/xylazine/
acepromazine. The abdomen was opened and the animal exsanguinated
by transection of the inferior vena cava to minimize pulmonary hem-
orrhage. Lungs were removed, weighed, and homogenized in 1 ml of
sterile PBS. Aliquots of the homogenates were serially diluted and
plated on tryptic soy-blood agar plates to quantitate the surviving bac-
teria. Bacterial colony counts were normalized to milligrams of lung
wet weight per milliliter of recovered homogenate.
Lungs were inflated via a tracheal cannula at 20 cm of pressure with 10%
buffered formalin and removed en bloc from the thorax. Each lobe was
bisected, dehydrated, and embedded in paraffin. The 5-micron tissue sec-
tions were cut and stained with H&E or Alcian blue (Poly Scientific).
For adult mice, lungs were lavaged three times with 1 ml of sterile PBS.
For PND14 mice, the lungs were lavaged three times with 350-?l aliquots.
bronchoalveolar lavage fluid (BALF) was centrifuged at 1250 rpm for 5
min, and the cell pellet resuspended in 1 ml of PBS. Total cell counts were
obtained using a hemocytometer and differential cell counts were made on
cytospin preparations using Diff-Quik stain (Scientific Products).
Macrophage phagocytic assays
Phagocytic activity of alveolar macrophages isolated from BALF was as-
sessed by fluorescent bead uptake in vitro. Lungs from PND14 mice were
lavaged with cold Hanks’ buffer and placed on ice. Lavage from six mice
of each genotype was pooled, and cells were pelleted and resuspended in
1 ml of DMEM of 5% FBS with antibiotics. Cell counts were determined
and 5 ? 105cells plated in 6-well dishes. Cells were allowed to adhere for
15 min, the medium and nonadherent cells removed, and the medium re-
placed. Examination of Diff-Quik-stained adherent cells indicated that the
cultures were macrophages and free of polymorphonuclear leukocytes or
lymphocytes. Fluorescent beads were added at a ratio of 100 beads per cell.
The wells were gently washed after 1.5 h to remove nonadherent cells. The
remaining cells were dislodged by scraping and collected by low speed
challenge with P. aeruginosa. The survival of 2-wk-old mice was moni-
tored after intratracheal instillation of 1 ? 107bacteria. Bacteria were
poorly tolerated for the Sftpc?/?mice (n ? 12 mice of each genotype). p ?
0.017 determined by Kaplan-Meier survival.
Decreased survival of Sftpc?/?mice following intratracheal
Concentrations of P. aeruginosa were determined 24 h after intratracheal
administration of 1–4 ? 107bacteria to mice at 2 wk of age. Bacterial
counts were significantly increased in the PND14 Sftpc?/?mice (n ? 5 or
6 mice per genotype per experiment). ?, p ? 0.001, by t test.
SP-C is required for normal clearance of P. aeruginosa.
622SP-C-DEFICIENT MICE AND P. aeruginosa INFECTION
centrifugation (2000 ? g for 5 min). Cells were resuspended in 1 ml of
PBS with 0.004% trypan blue to quench background fluorescence. FACS
analysis was used to identify fluorescent-positive cells. The phagocytic
index was calculated as the mean fluorescence multiplied by the number of
gated events. Incremental peaks in fluorescence intensity corresponded to
the number of beads internalized per cell.
For determination of phagocytic activity in vivo, mice were instilled
with 8 ? 105fluorescent bacteria, P. aeruginosa that constitutively express
bacterial GFP, and bronchoalveolar lavage performed 4 h postinfection.
BALF was analyzed from individual mice or pooled from four mice for
each genotype. BALF cells were pelleted, washed, and resuspended in 500
?l of PBS for FACS analysis. Event recording gates were set to exclude
nonmacrophage cells based upon size and complexity. BALF from mice
instilled with nonfluorescent P. aeruginosa was used in FACS determina-
tions as a control for nonspecific autofluorescence.
Identification of protein differences in Sftpc?/?and Sftpc?/?
Macrophages were collected from BALF of adult mice by selective adher-
ence to tissue culture dishes, washed, and lysed in situ with 100 ?l of SDS
lysis buffer (50 mM Tris-HCl (pH 7.4)) supplemented with protease in-
hibitors (Sigma-Aldrich) and protein determine by bicinchoninic acid assay
for cell lysate and cell-free BALF. BALF protein (5 ?g) was dried, resus-
pended in Laemmli buffer, boiled 5 min, and loaded on 8–16% polyacryl-
amide gels (Invitrogen). Gels were silver stained, and select bands that
tion in the lungs of Sftpc?/?mice af-
ter infection with P. aeruginosa.
Lungs of 2-wk-old Sftpc?/?
Sftpc?/?mice were assessed before
(A and B), and 24 h after (C and D)
administration of P. aeruginosa. Ar-
eas of subtle interstitial thickening
and inflammatory cells were infre-
quently seen in Sftpc?/?lungs before
challenge (B). Extensive granulocytic
infiltrates were observed in the lungs
of infected Sftpc?/?mice (D) com-
pared with infected control mice (C).
Arrows indicate bronchiolar epithe-
lium. Final magnification is shown at
sia in lungs of Sftpc?/?mice in re-
sponse to P. aeruginosa challenge.
Lung sections were stained with Al-
cian blue before (A and B) and after
(C and D) infection. Alcian blue stain-
ing was not detected in lungs of
Sftpc?/?mice (A). Alcian blue-posi-
tive staining was present in cells of
the conducting airways of 2-wk-old
Sftpc?/?mice (B). Alcian blue stain-
ing was detected in small clusters of
goblet cells along the bronchiolar ep-
ithelium of Sftpc?/?lungs following
infection (C). In contrast, the number
and intensity of Alcian blue staining
cells were increased in the lungs of
Sftpc?/?mice after infection (D). Fi-
nal magnification is shown at ?77 (A
and B) and at ?280 (C and D).
Goblet cell hyperpla-
623The Journal of Immunology
differed in abundance were extracted and submitted for trypsin digest.
Fragments were analyzed using a Bruker MALDI-TOF mass spectrometer
(University of Cincinnati Proteomics and Mass Spectrometry Core Facil-
ity). Unique tryptic fragments were searched against the Profound
Establishment of Pseudomonas infection model in Sftpc?/?
On the 129S6 background, Sftpc?/?mice have normal lung mor-
phology at birth but develop lung pathology consistent with ILD
associated with hereditary SP-C deficiency. PND14 mice were
chosen to assess pathogen susceptibility before development of
overt pathological changes in the lungs of Sftpc?/?mice. PND14
also represented the earliest age at which intratracheal inoculation
of bacteria and recovery of lavage fluid was reproducible.
Survival and bacterial clearance in Sftpc?/?mice
To determine an appropriate bacterial dose, 129S6 Sftpc?/?and
Sftpc?/?mice were inoculated by intratracheal injection with con-
centrations of P. aeruginosa ranging from 106to 108CFU and
monitored for 24 h. Bacteria were poorly tolerated by the Sftpc?/?
mice. Six hours after bacterial instillation, the Sftpc?/?mice were
inactive and physically unresponsive to mild stimulation in com-
parison to the wild-type mice that remained active. The survival of
PND14 Sftpc?/?mice was decreased when challenged with 107P.
aeruginosa (Fig. 1). In contrast, there was no difference in survival
of adult 129S6 Sftpc?/?and Sftpc?/?mice infected with P.
aeruginosa. To determine whether SP-C deficiency altered bacte-
rial clearance, PND14 mice were inoculated with a smaller dose of
bacteria (2–4 ? 106). Twenty-four hours after instillation, the
lungs were removed and homogenized, and bacteria quantified by
plating serial dilutions of the lung homogenates on tryptic soy-
blood agar plates. By inspection of the excised lungs, injury was
more extensive in Sftpc?/?than in Sftpc?/?mice noted by exten-
sive discoloration. Bacterial colony counts were 24-fold higher in
the lung homogenates from PND14 129S6 Sftpc?/?mice (Fig. 2).
When age-matched congenic FVB/N Sftpc?/?and Sftpc?/?mice
were similarly infected, there was no difference in the colony
counts from lung homogenates. Based upon the reduced survival
of the 129S6 Sftpc?/?mice, 2–4 ? 106bacteria were used for
subsequent clearance and phagocytic assays and all studies com-
pleted in the 129S6 strain of Sftpc?/?mice.
Increased inflammation following P. aeruginosa challenge
Increased inflammation was observed in histological sections of
lungs from PND14 Sftpc?/?and Sftpc?/?mice 24 h postinfection.
Tissue and airspace infiltrates contained neutrophils and enlarged
macrophages. The cellular infiltration was more extensive in the
lungs of Sftpc?/?mice and included areas of complete consolidation
(Fig. 3). Airway inflammation occurred in response to P. aeruginosa
infection and included distinct goblet cell hyperplasia. Mucin produc-
tion was assessed by Alcian blue histochemistry (Fig. 4). Alcian blue
Total cell number was significantly higher (?, p ? 0.035) in the BALF of adult Sftpc?/?mice (A) or (?, p ? 0.03) the BALF of PND14 Sftpc?/?mice
(C), compared with with Sftpc?/?mice. Neutrophils comprise the majority of BALF cells after P. aeruginosa infection in adult Sftpc?/?mice (B) and more
significantly (?, p ? 0.01) in the young PND14 Sftpc?/?mice (D), compared with Sftpc?/?mice. Although the percentage of neutrophils were similar,
total neutrophils were increased in lungs of uninfected Sftpc?/?mice compared with Sftpc?/?mice.
Increased inflammatory cells in BALF from Sftpc?/?mice after infection. Total cell counts were elevated 4 h after challenge with bacteria.
624SP-C-DEFICIENT MICE AND P. aeruginosa INFECTION
staining cells were present in the airways of Sftpc?/?mice be-
fore exposure to bacteria. Following infection, the number of
Alcian blue-positive cells and the intensity of staining increased
dramatically in the airway epithelia of Sftpc?/?mice relative to the
Sftpc?/?airways (Fig. 4, B and D).
Total cell counts were increased in BALF from adult and
PND14 mice 4 h postinfection (Fig. 5). Cell counts from BALF of
infected adult Sftpc?/?mice were 2-fold higher than cell counts in
Sftpc?/?mice (Fig. 5A). Cell counts from BALF of infected
PND14 Sftpc?/?mice were increased 4-fold over PND14 Sftpc?/?
mice (Fig. 5C). Neutrophils accounted for the majority of the cells
in BALF after infection (Fig. 5, B and D). Large, foamy macro-
phages were identified in cytospin preparations from the BALF of
Sftpc?/?mice. Neutrophil counts were also elevated in the
BALF of uninfected adult and PND14 Sftpc?/?mice relative to
BALF from Sftpc?/?mice, consistent with a pre-established
chronic low level inflammation due to SP-C deficiency (Fig. 5,
B and D). In both Sftpc?/?and Sftpc?/?mice, total protein
levels in BALF increased 3 fold following infection consistent
with increased injury. The BALF from Sftpc?/?or Sftpc?/?
mice (n ? 6) was collected 4 h postinfection with Pseudomo-
nas, pooled, and screened using specific ELISA kits (R&D Sys-
tems) for IL-1?, IL-4, IL-6, IL-13, and with an Ab array to 20
inflammatory cytokines (Ray Biotech) to profile changes in in-
flammatory mediators. Surprisingly there were no observable
differences in cytokine levels between the two groups of mice
(data not shown).
teria were mixed with a synthetic phospholipid mixture or purified SP-C with
phospholipids before infection of 2-wk-old Sftpc?/?mice. Bacterial counts were
determined 24 h after infection. SP-C did not enhance bacterial clearance.
SP-C does not restore phagocytic killing of P. aeruginosa. Bac-
mice and incubated with fluorescent beads. The internalization was determined by FACS analysis. Peaks of descending magnitude indicate smaller
populations of cells containing an increased number of beads. The phagocytic index of Sftpc?/?and Sftpc?/?macrophages is shown (right) of the
FACS profile (n ? 3 mice). p ? 0.04. B, In vivo phagocytosis. Phagocytosis by alveolar macrophages was determined by FACS analysis after
instillation of P. aeruginosa that constitutively express bacterial GFP. The open peak corresponds to specific GFP fluorescence generated by
ingestion of GFP expressing P. aeruginosa. The filled peak is nonspecific fluorescence produced by wild-type P. aeruginosa instilled into separate
control mice. Phagocytosis of bacteria by Sftpc?/?macrophages was reduced (n ? 3 mice). Mean fluorescence intensity is summarized (right) for
macrophage-associated fluorescence in cells. p ? 0.01. C and D, Sftpc?/?macrophages express markers of alternative activation. Arginase I gene
(Arg I) expression in Sftpc?/?and Sftpc?/?macrophages was assessed by RT-PCR of cDNA. Relative expression was normalized to ?-actin
expression (C). Gel electrophoresis identified a 40-kDa protein that was increased in macrophages and BALF from Sftpc?/?mice (D). The band was
collected for sequence analysis by mass spectroscopy. The sequence of peptide fragments that match the protein Ym1 from both the macrophages
and the BALF samples is shown below the gel image.
Phagocytic activity of macrophages from Sftpc?/?mice. A, In vitro phagocytosis. Alveolar macrophages were collected from PND14
625The Journal of Immunology
SP-C does not opsonize P. aeruginosa
SP-C preparations were mixed with bacteria before infection to
determine whether direct interaction of SP-C with bacteria en-
hances phagocytosis or clearance. Bacterial colony counts from
lung homogenates 24 h postinfection were not altered by pretreat-
ment of the bacteria with a phospholipid preparation containing
2.5% purified human SP-C (Fig. 6). Thus, SP-C does not directly
kill or enhance the uptake of bacteria to Sftpc?/?mice.
Phagocytic activity is reduced in alveolar macrophages from
Changes in alveolar macrophage morphology were observed in the
lungs of Sftpc?/?mice. A subpopulation of the enlarged macro-
phages with numerous cytoplasmic inclusions is characteristic of
Sftpc?/?mice. Multinucleated giant cells were observed in the
alveolar spaces. Increased cell size and granularity of Sftpc?/?
macrophages was verified by flow cytometry. To assess phagocytic
activity, macrophages were isolated from PND14 mice and incu-
bated with fluorescent-labeled beads in vitro. After 1.5 h, macro-
phages were recovered, and the uptake of beads determined by
FACS analysis. The phagocytic index of Sftpc?/?macrophages
was 64% of the index calculated for Sftpc?/?macrophages (Fig.
7A). Phagocytic activity was evaluated in vivo after administration
of fluorescent P. aeruginosa. Macrophage-associated fluorescence
was determined in cells isolated from BALF 4 h after administra-
tion. Nonfluorescent P. aeruginosa were instilled in separate mice
to control for nonspecific fluorescence. The phagocytic index of
macrophages recovered from Sftpc?/?mice was reduced (Fig.
7B), supporting the concept that the lack of SP-C causes macro-
phage dysfunction leading to impaired bacterial uptake.
Sftpc?/?macrophages express markers associated with
Alveolar macrophages from Sftpc?/?and Sftpc?/?mice were an-
alyzed for changes in gene expression or protein levels of mole-
cules associated with an alternatively activated phenotype. By RT-
PCR analysis, arginase I expression was increased 4-fold in
Sftpc?/?macrophages (Fig. 7C). Increased arginase activity in
macrophages contributes to matrix remodeling by promoting poly-
amine and subsequent collagen synthesis. The protein composition
of Sftpc?/?and Sftpc?/?macrophages was compared by gel elec-
trophoresis of either whole cell lysates or BALF supernatant fol-
lowing cell isolation. Several abundant proteins were detected in
Sftpc?/?macrophages after silver staining of the gel. Selected
bands were excised from the gel, and the sequence determined for
peptides generated by trypsin digestion. The peptide sequence gen-
erated from a prominent band of ?40 kDa had identity with the
murine chitinase protein Ym1 (Fig. 7D). Sequence of 12 peptides
from the macrophage sample matched with Ym1, covering 35% of
the primary sequence. Sequence of eight peptides from the BALF
sample matched Ym1 covering 28% of the primary sequence. The
sequence of five tryptic fragments common to the whole macro-
phage lysate and supernatant bands are shown below the gel in Fig.
7D. Increased chitinase expression is associated with asthma and
epithelial inflammation, and Ym1 is thought to contribute to innate
The increased susceptibility of Sftpc?/?mice to infection by the
pulmonary pathogen P. aeruginosa was demonstrated in vivo.
Bacterial clearance and survival was decreased and pulmonary in-
flammation was increased in Sftpc?/?mice compared with
Sftpc?/?mice after intratracheal delivery of P. aeruginosa. SP-C
deficiency was associated with changes in macrophage activation
state, supporting the critical role of SP-C in the maintenance and
innate defense in the lung. These findings indicate that SP-C plays
an important role in innate defense of the lung during Pseudomo-
nas-induced pneumonia. The present study was designed to deter-
mine whether the susceptibility to P. aeruginosa was related to
direct effects of SP-C on opsonization, to factors related to the
inflammatory state of the lung, or to changes in macrophage func-
tion in Sftpc?/?mice.
The progressive pulmonary disease seen in 129S6 Sftpc?/?
mice shares features with the familial SP-C-associated ILD found
in humans (2, 19). Although a severe pulmonary disorder develops
in 129S6 Sftpc?/?mice, there is virtually no pathology in the
lungs of Sftpc?/?mice of the FVB/N background unless lung in-
jury is induced in these mice. This strain-specific effect in mice
suggests that other genes modify the severity of the SP-C-deficient
phenotype. A role for modifier genes is also suggested in human
disease because phenotypic heterogeneity is seen in SP-C-associ-
ated ILD among affected family members for whom injury can
range from mild pulmonary insufficiency to severe fibrosis (17).
The reports of bacterial and viral infections associated with exac-
erbations in SP-C-deficient patients supports the hypothesis that
SP-C deficiency increases the effect of pathogens on lung inflam-
mation and repair. For the current study the young mice were
chosen for infection before the onset of observed inflammation or
histopathologic changes to minimize the inflammatory effects seen in
Sftpc?/?mice in pathogen clearance. PND14 129S6 Sftpc?/?mice
had reduced survival and clearance of P. aeruginosa when compared
with infected Sftpc?/?mice. There was no difference in survival or
clearance between PND14 FVB/N Sftpc?/?and Sftpc?/?mice (data
not shown). The impaired response of Sftpc?/?to Pseudomonas chal-
lenge was specific to the 129S6 Sftpc?/?strain that developed spon-
taneous lung injury with age. In contrast, there was no difference in
survival between infected adult 129S6 Sftpc?/?and Sftpc?/?mice.
The selective susceptibility of the PND14 129S6 mice may be related
to total lung macrophage content in which the less mature lungs have
a smaller total resident macrophage population to respond to the
Pseudomonas challenge. Alternatively, there may age-dependent dif-
clearance of Pseudomonas in adult mouse lung and rapid injury when
large bacteria inocula are used is problematic in other models of
Pseudomonas-related lung disease. An outcome of the current study
may be to improve Pseudomonas lung interactions by using young
mice. The strain-specific response to P. aeruginosa challenge indi-
cates that genetic factors modify pathogen susceptibility as well as the
development of SP-C-deficient ILD.
The genetic factors affecting the host response to microbial in-
fection among different inbred strains of mice are largely unde-
fined. Comparison of susceptibility between the 129 and FVB
mouse strains is even more limited. The 129 mice have been re-
ported to be one of the most highly susceptible inbred strains to
viral pulmonary pathogens (20). In a survey of the immediate re-
sponse to P. aeruginosa in infection in 11 strains of adult inbred
mice, 129 and FVB mice strains had similar clearance and inflam-
mation at 6 h postinfection (21). The lack of strain sensitivity in
that study is similar to the response of adult 129S6 and FVB/N
mice in the current study. Effects of strain background in younger
mice were not reported.
The genetic influence of the 129S6 background on susceptibility
in young Sftpc?/?mice was unanticipated but reflects the variable
disease progression that occurs among Sftpc?/?family members
and among family members that carry the same cystic fibrosis
mutation (22). Susceptibility to infection may be influenced by
early activation of inflammatory signaling in the lungs of 129S6
Sftpc?/?mice. Microarray comparison of gene expression in the
626SP-C-DEFICIENT MICE AND P. aeruginosa INFECTION
lungs of day 1 newborn 129S6 Sftpc?/?and Sftpc?/?mice iden-
tified increased expression of host defense-related genes (?1.6-
fold increase, p ? 0.05 on triplicate samples), including chitinase
3-like 1 that is a family member with Ym1 as identified in this
study, Ecsit a TLR accessory protein, Rhamm that is induced dur-
ing oxidant-induced lung inflammation (23), and two antiproteases
(data not shown). These initial findings suggest that there is acti-
vation of inflammation-related genes in the unchallenged 129S6
Sftpc?/?lung at birth. Lung microarray comparison between
129S6 and FVB/N strains of Sftpc?/?mice may identify sets of
genes that contribute to SP-C-deficient disease.
The pulmonary inflammatory response to Pseudomonas infec-
tion in PND14 129S6 Sftpc?/?mice was vigorous and included
increased cell infiltration and goblet cell hyperplasia. Pulmonary
mucins are heterogeneous glycoproteins; increased mucin produc-
tion is associated with colonization by P. aeruginosa in cystic
fibrosis and chronic obstructive pulmonary disease patients (24–
26). P. aeruginosa has been shown to adhere to respiratory mucins
(27) and directly stimulate increased mucin synthesis by airway
epithelial cells (28). Mucin production by airway epithelial cells is
activated by MMP9 activity (29). Macrophages from the lungs of
adult 129S6 Sftpc?/?mice were previously shown to have in-
creased MMP9 activity (19). Therefore, the increased MMP9 lev-
els produced by Sftpc?/?alveolar macrophages could influence
mucin production and bacterial retention.
The surfactant proteins SP-A and SP-D play important roles in
innate defense of a variety of pulmonary pathogens including P.
aeruginosa. SP-A and SP-D null mice were found to have im-
paired clearance of mucoid P. aeruginosa (30, 31). SP-A was
shown to opsonize and enhance phagocytic clearance of mucoid P.
aeruginosa (32). SP-D also binds directly to P. aeruginosa and
enhances phagocytosis (33). These findings demonstrated that
SP-A and SP-D directly contribute to limiting a Pseudomonas in-
fection. P. aeruginosa secretes proteases that degrade both SP-A
and SP-D (34, 35). Thus proteolytic degradation of SP-A or SP-D
might contribute to the increased mortality of Sftpc?/?mice in the
current study. By Western blot analysis SP-A and SP-D levels
were unchanged in the BALF of Sftpc?/?and Sftpc?/?mice be-
fore or after infection (data not shown). When a purified human
SP-C/phospholipid mixture was incubated with the P. aeruginosa
before instillation, there was no increase in bacterial clearance by
Sftpc?/?mice. Thus, SP-C does not appear to augment P. aerugi-
nosa clearance by opsonization as do SP-A and SP-D. It is unlikely
that the observed impaired clearance of P. aeruginosa is an opso-
nization defect. Our finding that SP-C does not enhance host de-
fense by opsonization is consistent with reports suggesting that
SP-C may confer alveolar protection by neutralizing inhaled in-
flammatory compounds or microorganisms. SP-C was shown to
bind to bacterial LPS and reduce macrophage release of cytokines
in vitro (14). It is thus conceivable that the lack of SP-C alters the
alveolar microenvironment by increasing the exposure of the al-
veolar epithelium or sentinal alveolar phagocytes to inflammatory
stimuli. The pulmonary inflammation and ILD-like injury that de-
velops with age in 129S6 Sftpc?/?mice may arise from chronic
alveolar inflammation when SP-C does not sequester inhaled
A prominent feature of familial SP-C ILD is extensive alveolar
infiltration and accumulation of macrophages. The SP-C patient
index cases were initially classified as desquamative interstitial
pneumonitis and chronic pneumonitis of infancy to reflect the mac-
rophage injury (15). The pneumonitis-like histopathology of both
the affected human and adult 129S6 Sftpc?/?lungs suggest an
impaired macrophage response as a common link. The alveolar
macrophages from 129S6 Sftpc?/?mice had altered morphology
and reduced phagocytosis in vitro and in vivo and were previously
shown to have increased metalloproteinase activity (19). The im-
paired phagocytosis and increased MMP9 activity suggested that
the status of the Sftpc?/?macrophage had shifted from the classic
protective function to a repair and remodeling function termed the
alternatively activated macrophage (AAM) (36). The alternative
activation state is associated with markers affecting cell-cell and
cell-matrix interactions and other markers with only partially de-
fined function. Murine AAM produce and secrete Ym1, a 42-kDa
protein with chitinase sequence homology. Ym1 is thought to con-
tribute to innate defense through lectin-like binding to glu-
cosamine oligosaccharides and to heparan sulfate components of
the extracellular matrix (37, 38). Ym1 protein was abundant in
isolated alveolar macrophages and in the BALF collected from
129S6 Sftpc?/?mice. AAM have increased expression of the ar-
ginase I gene (ArgI) that alters arginine metabolism and macro-
phage function (38). ArgI mRNA expression was increased in the
129S6 Sftpc?/?macrophages. ArgI converts arginine to precursors
of polyamines and collagen used in extracellular matrix produc-
tion, tissue remodeling, and eventual fibrosis. ArgI activity de-
pletes arginine from the NO synthetic pathway thus reducing mac-
rophage bacteriocidal activity (38, 39). The increased Ym1 protein
and ArgI expression of Sftpc?/?macrophages is consistent with an
AAM phenotype and a reduced ability to suppress infection. The
presence of herpesvirus has been linked to familial pulmonary
fibrosis and idiopathic pulmonary fibrosis (40). Pulmonary mac-
rophages acquire an AAM phenotype in herpesvirus-positive pa-
tients with pulmonary fibrosis and in mice with either herpesvirus-
induced lung fibrosis or bleomycin-induced lung fibrosis (41, 42).
Macrophages positive for ArgI and Ym1 localized to fibrotic lung
tissue of herpes virus-infected mice and ArgI-positive macro-
phages associated with fibrotic regions of lungs from bleomycin-
treated mice (42). Similarly ArgI-positive macrophages were as-
sociated with fibroblastic foci of idiopathic pulmonary fibrosis
patients (41). Collectively these data implicate AAM as a shared
feature between experimentally induced fibrotic lung disease, clin-
ical idiopathic pulmonary fibrosis, and the ILD of Sftpc?/?mice,
wherein fibrosis develops with age. Characteristics of macro-
phages from affected SP-C patients have not been reported.
SP-A, SP-D, and GM-CSF null mice have macrophages with
altered morphology and impaired phagocytosis of P. aeruginosa
yet apparently do not develop populations of AAM or fibrosis (30,
31, 43). Thus the loss of SP-C appears to elicit a distinct alveolar
injury that modifies both macrophage function and airway cell re-
sponse to challenge. The susceptibility of Sftpc?/?mice to
Pseudomonas challenge supports the emerging concept that
chronic Ag stimulation may underlie rare familial and idiopathic
pulmonary fibrosis. The current findings indicate that SP-C defi-
ciency perturbs the activation state of alveolar macrophages re-
sulting in decreased phagocytosis and clearance of P. aeruginosa.
SP-C deficiency per se renders mice susceptible to pulmonary
Pseudomonas infection as well as replicating the progressive ILD
and fibrosis seen in human SP-C deficiency.
We thank Dusti Folger, Teah Witt, and William Hull for technical assis-
tance with experiments, Ann Maher for manuscript preparation, and
Chenxia Duan for purification of human SP-C.
The authors have no financial conflict of interest.
1. Goerke, J. 1998. Pulmonary surfactant: functions and molecular composition.
Biochim. Biophys. Acta 1408: 79–89.
627 The Journal of Immunology
2. Whitsett, J. A., and T. E. Weaver. 2002. Hydrophobic surfactant proteins in lung Download full-text
function and disease. N. Engl. J. Med. 347: 2141–2148.
3. Nogee, L. M. 2006. Genetics of pediatric interstitial lung disease. Curr. Opin.
Pediatr. 18: 287–292.
4. Whitsett, J. A., S. E. Wert, and B. C. Trapnell. 2004. Genetic disorders influ-
encing lung formation and function at birth. Hum. Mol. Genet. 13: R207–R215.
5. Whitsett, J. A. Genetic disorders of surfactant homeostasis. 2006. Paediatr. Re-
spir. Rev. 7(Suppl. 1): S240–S242.
6. Beers, M. F., and S. Mulugeta. 2005. Surfactant protein C biosynthesis and its
emerging role in conformational lung disease. Annu. Rev. Physiol. 67: 663–696.
7. Davis, A. J., A. H. Jobe, D. Hafner, and M. Ikegami. 1998. Lung function in
premature lambs and rabbits treated with a recombinant SP-C surfactant.
Am. J. Respir. Crit. Care Med. 157: 553–559.
8. Jobe, A. H. 1993. Pulmonary surfactant therapy. N. Engl. J. Med. 328: 861–868.
9. Jobe, A. H. 2006. Mechanisms to explain surfactant responses. Biol. Neonate 89:
10. Jobe, A. H. 2000. Which surfactant for treatment of respiratory-distress syn-
drome. Lancet 355: 1380–1381.
11. Wright, J. R. 2005. Immunoregulatory functions of surfactant proteins. Nat. Rev.
Immunol. 5: 58–68.
12. Augusto, L. A., J. Li, M. Synguelakis, J. Johansson, and R. Chaby. 2002. Struc-
tural basis for interactions between lung surfactant protein C and bacterial lipo-
polysaccharide. J. Biol. Chem. 277: 23484–23492.
13. Augusto, L. A., M. Synguelakis, J. Johansson, T. Pedron, R. Girard, and
R. Chaby. 2003. Interaction of pulmonary surfactant protein C with CD14 and
lipopolysaccharide. Infect. Immun. 71: 61–67.
14. Augusto, L. A., M. Synguelakis, Q. Espinassous, M. Lepoivre, J. Johansson, and
R. Chaby. 2003. Cellular antiendotoxin activities of lung surfactant protein C in
lipid vesicles. Am. J. Respir. Crit. Care Med. 168: 335–341.
15. Nogee, L. M., A. E. Dunbar, III, S. E. Wert, F. Askin, A. Hamvas, and
J. A. Whitsett. 2001. A mutation in the surfactant protein C gene associated with
familial interstitial lung disease. N. Engl. J. Med. 344: 573–579.
16. Amin, R. S., S. E. Wert, R. P. Baughman, J. F. Tomashefski, Jr., L. M. Nogee,
A. S. Brody, W. M. Hull, and J. A. Whitsett. 2001. Surfactant protein deficiency
in familial interstitial lung disease. J. Pediatr. 139: 85–92.
17. Thomas, A. Q., K. Lane, J. Phillips, III, M. Prince, C. Markin, M. Speer,
D. A. Schwartz, R. Gaddipati, A. Marney, J. Johnson, et al. 2002. Heterozygosity
for a surfactant protein C gene mutation associated with usual interstitial pneu-
monitis and cellular nonspecific interstitial pneumonitis in one kindred.
Am. J. Respir. Crit. Care Med. 165: 1322–1328.
18. Glasser, S. W., M. S. Burhans, T. R. Korfhagen, C. L. Na, P. D. Sly, G. F. Ross,
M. Ikegami, and J. A. Whitsett. 2001. Altered stability of pulmonary surfactant
in SP-C-deficient mice. Proc. Natl. Acad. Sci. USA 98: 6366–6371.
19. Glasser, S. W., E. A. Detmer, M. Ikegami, C. L. Na, M. T. Stahlman, and
J. A. Whitsett. 2003. Pneumonitis and emphysema in SP-C gene targeted mice.
J. Biol. Chem. 278: 14291–14298.
20. Anh, D. B., P. Faisca, and D. J. Desmecht. 2006. Differential resistance/suscep-
tibility patterns to pneumovirus infection among inbred mouse strains.
Am. J. Physiol. 291: L426–L435.
21. Wilson, K. R., J. M. Napper, J., Denvir, V. E. Sollars, and H. D. Yu. 2007. Defect
in early lung defence against Pseudomonas aeruginosa in DBA/2 mice is asso-
ciated with acute inflammatory lung injury and reduced bactericidal activity in
naı ¨ve macrophages. Microbiology 153: 968–979.
22. Cutting, G. R. 2005. Modifier genetics: cystic fibrosis. Annu. Rev. Genomics
Hum. Genet. 6: 237–260.
23. Zaman, A., Z. Cui, J. P. Foley, H. Zhao, P. C. Grimm, H. M. Delisser, and
R. C. Savani. 2005. Expression and role of the hyaluronan receptor RHAMM in
inflammation after bleomycin injury. Am. J. Respir. Cell Mol. Biol. 33: 447–454.
24. Sadikot, R. T., T. S. Blackwell, J. W. Christman, and A. S. Prince. 2005. Patho-
gen-host interactions in Pseudomonas aeruginosa pneumonia. Am. J. Respir.
Crit. Care Med. 171: 1209–1223.
25. Randell, S. H., and R. C. Boucher, University of North Carolina Virtual Lung
Group. 2006. Effective mucus clearance is essential for respiratory health.
Am. J. Respir. Cell Mol. Biol. 35: 20–28.
26. Livraghi, A., and S. H. Randell. 2007. Cystic fibrosis and other respiratory dis-
eases of impaired mucus clearance. Toxicol. Pathol. 35: 116–129.
27. Lillehoj, E. P., S. W. Hyun, B. T. Kim, X. G. Zhang, D. I. Lee, S. Rowland, and
K. C. Kim. 2001. Muc1 mucins on the cell surface are adhesion sites for Pseudo-
monas aeruginosa. Am. J. Physiol. 280: L181–L187.
28. Kohri, K., I. F. Ueki, J. J. Shim, P. R. Burgel, Y. M. Oh, D. C. Tam, T. Dao-Pick,
and J. A. Nadel. 2002. Pseudomonas aeruginosa induces MUC5AC production
via epidermal growth factor receptor. Eur. Respir. J. 20: 1263–1270.
29. Deshmukh, H. S., L. M. Case, S. C. Wesselkamper, M. T. Borchers, L. D. Martin,
H. G. Shertzer, J. A. Nadel, and G. D. Leikauf. 2005. Metalloproteinases mediate
mucin 5AC expression by epidermal growth factor receptor activation.
Am. J. Respir. Crit. Care Med. 171: 305–314.
30. LeVine, A. M., K. E. Kurak, M. D. Bruno, J. M. Stark, J. A. Whitsett, and
T. R. Korfhagen. 1998. Surfactant protein-A-deficient mice are susceptible to
Pseudomonas aeruginosa infection. Am. J. Respir. Cell. Mol. Biol. 19: 700–708.
31. Giannoni, E., T. Sawa, L. Allen, J. Wiener-Kronish, and S. Hawgood. 2006.
Surfactant Proteins A and D enhance pulmonary clearance of Pseudomonas
aeruginosa. Am. J. Respir. Cell Mol. Biol. 34: 704–710.
32. Mariencheck, W. I., J. Savov, Q. Dong, M. J. Tino, and J. R. Wright. 1999.
Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mu-
coid strain of P. aeruginosa. Am. J. Physiol. 277: L777–L786.
33. Restrepo, C. I., Q. Dong, J. Savov, W. I. Mariencheck, and J. R. Wright. 1999.
Surfactant protein D stimulates phagocytosis of Pseudomonas aeruginosa by
alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 21: 576–585.
34. Mariencheck, W. I., J. F. Alcorn, S. M. Palmer, and J. R. Wright. 2003. Pseudo-
monas aeruginosa elastase degrades surfactant proteins A and D. Am. J. Respir.
Cell Mol. Biol. 28: 528–537.
35. Beatty, A. L., J. L. Malloy, and J. R. Wright. 2005. Pseudomonas aeruginosa
degrades pulmonary surfactant and increases conversion in vitro. Am. J. Respir.
Cell Mol. Biol. 32: 128–134.
36. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3:
37. Chang, N.-C., S.-I. Hung, K.-Y. Hwa, I. Kato, J.-E. Chen, C.-H. Liu, and
A. C. Chang. 2001. A macrophage protein, Ym1, transiently expressed during
inflammation is a novel mammalian lectin. J. Biol. Chem. 276: 17497–17506.
38. Nair, M. G., K. J. Guild, and D. Artis. 2006. Novel effector molecules in type 2
inflammation: lessons drawn from helminth infection and allergy. J. Immunol.
39. Noe ¨l, W., G. Raes, G. Hassanzadeh Ghassabeh, P. De Baetselier, and A. Beschin.
2004. Alternatively activated macrophages during parasite infections. Trends
Parasitol. 20: 126–133.
40. Tang, Y.-W., J. E. Johnson, P. J. Browning, R. A. Cruz-Gervis, A. Davis,
B. S. Graham, K. L. Brigham, J. A. Oates, Jr., J. E. Loyd, and A. A. Stecenko.
2003. Herpesvirus DNA is consistently detected in lungs of patients with idio-
pathic pulmonary fibrosis. J. Clin. Microbiol. 41: 2633–2640.
41. Mora, A. L., E. Torres-Gonza ´lez, M. Rojas, C. Corredor, J. Ritzenthaler, J. Xu,
J. Roman, K. Brigham, and A. Stecenko. 2006. Activation of alveolar macro-
phages via the alternative pathway in herpesvirus-induced lung fibrosis.
Am. J. Respir. Cell Mol. Biol. 35: 466–473.
42. Endo, M., S. Oyadomari, Y. Terasaki, M. Takeya, M. Suga, M. Mori, and
T. Gotoh. 2003. Induction of arginase I and II in bleomycin-induced fibrosis of
mouse lung. Am. J. Physiol. 285: L313–L321.
43. Ballinger, M. N., R. Paine, III, C. H. C. Serezani, D. M. Aronoff, E. S. Choi,
T. J. Standiford, G. B. Toews, and B. B. Moore. 2006. Role of granulocyte
macrophage colony-stimulating factor during Gram-negative lung infection with
Pseudomonas aeruginosa. Am. J. Respir. Cell Mol. Biol. 34: 766–774.
628SP-C-DEFICIENT MICE AND P. aeruginosa INFECTION