1 Divisions of Pulmonary Biology and 2 Critical Care Medicine, Cincinnati, Ohio 45229-3039; and 3 Department of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Mice lacking surfactant protein
SP-A [SP-A(/
)] and wild type SP-A(+/+) mice were infected with
influenza A virus (IAV) by intranasal instillation. Decreased clearance
of IAV was observed in SP-A(
/
) mice and was associated with
increased pulmonary inflammation. Treatment of SP-A(
/
) mice with
exogenous SP-A enhanced viral clearance and decreased lung
inflammation. Uptake of IAV by alveolar macrophages was similar in
SP-A(
/
) and SP-A(+/+) mice. Myeloperoxidase activity was reduced in
isolated bronchoalveolar lavage neutrophils from SP-A(
/
) mice. B
lymphocytes and activated T lymphocytes were increased in the lung and
spleen, whereas T helper (Th) 1 responses were increased
[interferon-
, interleukin (IL)-2, and IgG2a] and Th2
responses were decreased (IL-4, and IL-10, and IgG1) in the
lungs of SP-A(
/
) mice 7 days after IAV infection. In the absence of
SP-A, impaired viral clearance was associated with increased lung
inflammation, decreased neutrophil myeloperoxidase activity, and
increased Th1 responses. Because the airway is the usual portal of
entry for IAV and other respiratory pathogens, SP-A is likely to play a
role in innate defense and adaptive immune responses to IAV.
virus; lung; lectin; surfactant protein-A; surfactant protein-A-deficient mice
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INTRODUCTION |
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SURFACTANT PROTEIN (SP)-A is a member of the collectin family of the mammalian C-type lectins that also includes mannose-binding lectin, conglutinin, and SP-D (27, 30). The collectins are thought to be involved in innate host defense against various bacterial, fungal, and viral pathogens. Members of the collectin family of proteins share in common an NH2-terminal collagen-like domain and a COOH-terminal lectin domain that binds carbohydrates in a calcium-dependent manner (27). The C-type lectins bind carbohydrate surfaces of many microorganisms mediating phagocytosis and killing by phagocytic cells (34).
In the human lung, SP-A is expressed in alveolar type II cells, serous cells in tracheobronchial glands, and in nonciliated bronchiolar cells (17). In vitro, SP-A stimulates macrophage chemotaxis (35) and enhances the binding of bacteria and viruses to alveolar macrophages (34). SP-A enhances macrophage phagocytosis of herpes simplex virus type 1 (HSV-1; see Ref. 31) and binds to HSV-1-infected cells (32). Mannose-binding lectin, conglutinin, SP-A, and SP-D neutralize influenza virus (2, 5, 11, 13, 14), although neutralization by SP-A occurs through binding of the viral hemagglutinin (HA) to sialic acid on the SP-A molecule rather than through carbohydrate-binding activity of SP-A (5).
Influenza A virus (IAV) infection is acquired primarily by inhalation, generally causing infection of the upper respiratory tract. During infection, virus spreads to the lower respiratory tract and may result in pneumonia. Influenza infections are most frequent in children and young adults. Deaths from IAV infection occur most frequently in the very young (<1 yr), the elderly, and persons of all ages with underlying heart or lung disease (26). Prematurity has been associated with decreased SP-A levels in bronchoalveolar lavage fluid (BALF; see Ref. 8). Bronchopulmonary dysplasia and cystic fibrosis have been associated with decreased SP-A concentrations in the lung (3, 25), conditions that may increase susceptibility to infection by respiratory viruses such as IAV. In addition, viral pneumonia has been associated with decreased SP-A in BALF (23), which may further exacerbate the viral infection and increase susceptibility to bacterial superinfection.
Specific and nonspecific immune mechanisms take part in the immune response to influenza virus. IAV is a lytic infection and causes the breakdown of the blood-tissue barrier early in infection, resulting in the influx of macrophages, neutrophils, and natural killer (NK) cells into the lung. Specific immune responses to IAV are initiated by the influx of virus-specific T lymphocytes and antibody production. Cytotoxic T lymphocytes (CTL) are thought to be involved in viral clearance by direct cytolysis of virus-infected cells (37). Defects in neutrophil and monocyte chemotactic, oxidative, and bacterial killing functions have been documented in IAV infection (10, 18). In vitro, SP-A enhanced uptake of IAV by neutrophils; however, SP-A did not protect neutrophils from the inhibitory effects of IAV on the respiratory burst (15).
In spite of considerable in vitro evidence that SP-A is involved in
innate host defense, its role in vivo has only recently been
demonstrated. SP-A-deficient [SP(/
)] mice produced by targeted gene inactivation are susceptible to bacterial and respiratory syncytial virus pneumonia (20, 21). In the present study, SP-A(
/
) mice were infected intranasally with IAV. Clearance of IAV
was delayed, and lung inflammation increased in SP-A(
/
) mice in vivo.
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METHODS |
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Animal husbandry.
The murine SP-A gene locus was targeted by homologous recombination as
previously described; the lungs of SP-A(/
) mice were lacking
detectable SP-A (20). SP-A(
/
) and SP-A-sufficient [SP-A(+/+)] mice were maintained in strain 129. Animals were housed and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facility of the Children's Hospital Research Foundation (Cincinnati, OH). Male and female mice of ~20-25 g (42-56 days old) were used.
Preparation of IAV.
IAV strain H3N2 A/Philadelphia/82 (Phil/82)
(H3N1) was a gracious gift from E. M. Anders (University of Melbourne, Melbourne, Australia) and was grown in
the chorioallantoic fluid of 10-day-old embryonated hen's eggs.
Allantoic fluid was harvested after 48 h of incubation and was
clarified by centrifugation at 1,000 g for 40 min followed
by centrifugation at 135,000 g to precipitate viruses. The
virus-containing pellets were resuspended and purified on a
discontinuous sucrose density gradient, as previously described (11). Virus stocks were dialyzed against PBS, separated
into aliquots, and stored at 70°C until used. HA titers were
determined by titration of virus samples in PBS followed by the
addition of thoroughly washed human type O erythrocytes. The potency of each viral stock was measured by HA and protein assays after samples were thawed from frozen storage at
70°C. The potency of each viral
stock was also measured by the fluorescent foci assay
(25).
Fluorescent isothiocyanate labeling of IAV. Fluorescent isothiocyanate (FITC) stock was prepared at 1 mg/ml in 1 mol/l sodium carbonate, pH 9.6. FITC-labeled virus was prepared by incubating concentrated virus stocks with FITC (10:1 mixture by volume of virus in PBS with FITC stock) for 1 h, followed by dialysis of the mixture for 18 h against PBS.
Viral clearance of influenza.
Mice were lightly anesthetized with isoflurane and inoculated
intranasally with 105 fluorescent foci (ff) of IAV in 50 µl PBS. Quantitative IAV cultures of lung homogenates were performed
3 and 5 days after inoculation of the animals with IAV. The entire lung
was removed, homogenized in 2 ml of sterile PBS, weighed, and stored at
80°C. After infection (5 days), quantitative IAV cultures of spleen
homogenates were also performed. The spleen was removed, homogenized in
1 ml of sterile PBS, and stored at
80°C. Madin-Darby canine kidney
(MDCK) cell monolayers were prepared in 96-well plates. The layers were incubated with lung or spleen homogenates diluted in PBS containing 2 mM calcium for 45 min at 37°C, and the monolayers were washed three
times in virus-free DMEM containing 1% penicillin and streptomycin. The monolayers were incubated for 7 h at 37°C in DMEM and
repeatedly washed, and the cells were fixed with 80% (vol/vol) acetone
for 10 min at
20°C. The monolayers were then incubated with
monoclonal antibody directed against IAV nucleoprotein (monoclonal
antibody A-3) and then with rhodamine-labeled goat anti-mouse IgG. Ff
were counted directly under fluorescent microscopy. The resulting titer was divided by the lung or spleen weight and reported as ff per gram of
lung or spleen.
In vitro assessment of antiviral activity of SP-A. The antiviral activity of SP-A was measured by HA inhibition assay and by testing the ability of SP-A to inhibit infection of MDCK cells using the ff assay. HA inhibition was measured using human type O erythrocytes. SP-A was serially diluted in PBS in 96-well plates followed by addition of 40 HA units of Phil/82 strain of IAV and a suspension of washed erythrocytes. The ff assay was performed as described above. In this case, a dilution of purified influenza virus containing ~6,000 infectious focus U/ml was preincubated with various dilutions of SP-A followed by infection of MDCK monolayers with these samples and measurement of ff formed after 7 h.
Treatment with SP-A.
Human SP-A obtained from patients with alveolar proteinosis was
purified by the 1-butanol extraction method of Haagsman et al.
(9) and was dissolved in 2 mg/ml Na-HEPES (pH 7.2).
Endotoxin contamination was not detected in SP-A preparations (<0.06
EU/ml) using the Limulus Amoebocyte Lysate assay (Sigma, St. Louis, MO) according to the manufacturer's directions. Quantitative IAV cultures of lung homogenates were performed 3 days after intranasal inoculation of mice with IAV followed immediately by intratracheal inoculation with
PBS or SP-A (100 µg). Because previous studies demonstrated that 100 µg SP-A restored resistance to bacterial pneumonia in SP-A(/
)
mice (22), this dose was used for the present study.
Bronchoalveolar lavage. Lung cells were recovered by bronchoalveolar lavage (BAL). Animals were killed as described for viral clearance, and lungs were lavaged three times with 1 ml of sterile PBS. The fluid was centrifuged at 2,000 rpm for 10 min and resuspended in 600 µl of PBS; total cells were stained with trypan blue and counted under light microscopy. Differential cell counts were performed on cytospin preparations stained with Diff-Quick (Scientific Products, McGaw Park, IN).
Cytokine production.
Lung homogenates were centrifuged at 2,000 rpm, and the supernatants
were stored at 20°C. Tumor necrosis factor-
(TNF-
), interleukin (IL)-1
, IL-6, macrophage inflammatory protein (MIP)-2, and interferon (IFN)-
were quantitated 3 and 5 days after IAV infection. IL-2, IL-4, IL-10, and IL-12 were measured 7 days after IAV
infection using murine sandwich enzyme-linked immunosorbent assay
(ELISA) kits (R&D systems, Minneapolis, MN) according to the
manufacturer's directions. All plates were read on a microplate reader
(Molecular Devices, Menlo Park, CA) and analyzed with the use of a
computer-assisted analysis program (Softmax; Molecular Devices). Only
assays with standard curves with a calculated regression line value
>0.95 were accepted for analysis.
Phagocytosis of IAV. Phagocytosis of IAV by macrophages in vivo was measured by intranasally infecting mice with FITC-labeled IAV followed by evaluation of cell-associated fluorescence by flow cytometry. After infection (2 h), macrophages from BALF were incubated in buffer (PBS, 0.2% BSA fraction V, and 0.02% sodium azide) with phycoerytherin-conjugated murine CD16/CD32 antibodies (PharMingen, San Diego, CA) for 1 h on ice and washed two times in fresh buffer. Trypan blue (0.2 mg/ml) was added to quench fluorescence of extracellular FITC. Cell-associated fluorescence was measured on a FACScan flow cytometer using CELLQuest software (Becton-Dickinson, San Jose, CA). For each sample of macrophages, 20,000 cells were counted in duplicate, and the results were expressed as the percentage of macrophages with label.
Lymphocytes in BALF. Lymphocytes in BALF were measured after intranasal IAV infection, staining of cells with fluorescent antibodies, and evaluation of cell-associated fluorescence by flow cytometry. Cells from BALF were incubated in fluorescence-activated cell sorter (FACS) buffer (PBS, 0.2% BSA fraction V, and 0.02% sodium azide) with rat anti-mouse CD16/CD32 antibodies (Fc Block), and separate aliquots were stained with FITC-conjugated mouse CD4 (T-helper lymphocytes), CD8 (CTL), CD19 (B lymphocytes), or CD56 (NK cells) antibodies (PharMingen) for 1 h on ice. Specific markers on T lymphocytes were evaluated by double staining with phycoerytherin-conjugated mouse CD3 (T cell receptor) and FITC conjugate mouse CD6 (T cell activation marker) or CD16 (Fc receptor) antibodies (PharMingen), and a lymphocyte gate was used. Cell-associated fluorescence was measured on a FACScan flow cytometer using CELLQuest software (Becton-Dickinson). For each sample, 10,000 events were analyzed, and the results were expressed as the percentage of CD4+, CD8+, CD19+, and CD56+ lymphocytes in BALF or the percentage of CD3+ cells expressing CD6 or CD16.
Isolation of spleen cells. Seven days after IAV infection, mouse spleens were removed, passed through a 70-µm nylon cell strainer (Fisher Scientific, Pittsburgh, PA) in 10 ml of fresh Hanks' balanced salt solution with 1% FCS and 10 mM HEPES, aspirated through a 21-gauge needle, and centrifuged at 800 g for 5 min at 4°C. Erythrocytes in the pellet were lysed with erythrocyte lysis buffer (Life Technologies, Rockville, MD), and spleen cells were resuspended in FACS buffer. Spleen lymphocytes were stained as described for BALF lymphocytes, and fluorescent staining was measured by flow cytometry.
Cell-mediated cytotoxicity assay.
Cytotoxicity of splenic lymphocytes was measured using the CytoTox96
cytotoxicity assay (Promega, Madison, WI), a colorimetric assay that
measures lactate dehydrogenase (LDH) release from lysed cells. EL4
cells (H-2b; ATCC, Manassas, VA) were grown in media (DMEM
with 10% horse serum), incubated with 100 HA units of IAV for 4 h, and used as target cells. Mouse spleen cells (129 mice,
H-2b) were isolated 7 days after IAV infection and were
cultured overnight in DMEM. Assays used 20,000-target cells/well with
varying effector-to-target ratios. Effectors were incubated with
targets for 4 h, and plates were read on a microplate reader at an
absorbance of 490 nm. Results are expressed as a percentage of specific
release, according to the formula
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Serum immunoglobulins.
After IAV infection (7 days), blood was collected from the inferior
vena cava of mice and centrifuged at 3,500 rpm for 5 min, and serum was
collected and stored at 20°C. Concentrations of total
immunoglubulin, IgM, and the IgG subclasses IgG1 and
IgG2a in mouse serum were measured by an ELISA using an
isotype-specific kit (Southern Biotechnology, Birmingham, AL)
with sensitivities of 2 µg/ml. Ninety-six-well plates were coated
with 10 µg of whole immunoglobulin overnight and then blocked for
1 h at room temperature with 1% BSA. Serum samples were equalized
for total protein, diluted (1:1,000 or 1:2,000) in PBS, and added to
the plate. Alkaline phosphatase-labeled isotype antibodies were used
for detection, and standard curves (31 ng to 4 µg total protein) were
generated for each isotype. All samples were run in duplicate, and the
concentrations of the samples were calculated by graphing absorbance
vs. concentrations of the standard.
Neutrophil myeloperoxidase activity.
Myeloperoxidase (MPO) activity was measured in BAL neutrophils 3 days
after intranasal infection with IAV at a concentration of
106 ff. A higher concentration of virus was used to provide
adequate neutrophils to study. BALF from three wild-type mice was
pooled to provide sufficient neutrophils, whereas a single SP-A(/
) mouse was used. Blood obtained from uninfected SP-A(
/
) and
SP-A(+/+) mice was separated on a gradient of neutrophil isolation
medium (NIM-1; Cardinal Associates, Santa Fe, NM) to isolate blood
neutrophils and was assayed after stimulation with phorbol 12-myristate
13-acetate (PMA; 100 ng/ml) or was left unstimulated. Neutrophils were
added to homogenate buffer [100 mM sodium acetate (pH 6.0), 20 mM EDTA (pH 7.0), and 1% hexadecyltrimethylammonium bromide (HETAB)] in a
96-well microtiter plate in a final volume of 50 µl. The neutrophil mixtures were incubated at 37°C for 1 h to lyse the neutrophils and allow release of MPO from the granules. Assay buffer (100 µl)
containing 1 mM H2O2, 1% HETAB, and 3.2 mM
3,3'5,5'-tetramethylbenzidine was added to each well, and readings were
taken at 650 nm using a THERMOMAX microplate reader for a period of 4 min. Readings were the average of at least three individual wells, and
MPO activity was reported as maximum MPO activity per 4 min per 3 × 103 neutrophils.
SP-D concentrations.
Concentrations of SP-D in BALF were determined with an ELISA. After IAV
infection (5 days), lungs from infected and uninfected SP-A(+/+) and
SP-A(/
) mice were lavaged with 2 ml of sterile saline. SP-D
concentrations were measured in a double-antibody ELISA using rabbit
and guinea pig anti-SP-D sera. Each assay plate included a standard
curve generated with purified mouse SP-D. All samples were run in
duplicate, and the concentrations of the samples were calculated by
graphing absorbance vs. concentrations of the standard.
Statistical methods. Lung viral titers, total cell counts, cytokines, immunoglubulins, and MPO activity were compared using ANOVA and Student's t-test. Findings were considered statistically significant at probability levels <0.05.
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RESULTS |
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Pulmonary pathology after IAV administration.
Intranasal administration of IAV (105 ff) was well
tolerated, and all animals survived the study period. Mice infected
with IAV had weight loss over 4 days postinfection, with 1.5 ± 1.0 and 3.6 ± 1.5% weight loss in the SP-A(+/+) and SP-A(/
)
mice, respectively (means ± SE). SP-A(
/
) mice had increased
total cell counts in BALF 3 and 5 days after IAV infection (Fig.
1). Baseline total cell counts in BALF
from controls inoculated with PBS were 9.6 ± 1.1 × 104 and 8.8 ± 0.4 × 104 for the
SP-A(+/+) and SP-A(
/
) mice, respectively (means ± SE). A
significantly greater percentage of polymorphonuclear neutrophils was
detected in BALF from SP-A(
/
) compared with SP-A(+/+) mice 3 and 5 days postinfection (Fig. 1). Pulmonary inflammation was not
observed in wild-type mice inoculated with sterile PBS
(n = 5; data not shown).
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Decreased viral clearance in SP-A(/
) mice.
Quantitative IAV cultures of lung homogenates were performed 3 and 5 days after inoculation of the animals with IAV. SP-A(
/
) mice had
significantly increased viral titers of IAV in the lung 3 and 5 days
after infection compared with SP-A(+/+) mice (Fig. 1). Systemic
dissemination of IAV was assessed by quantitative culture of the
spleen, and no IAV was isolated from the spleens of either group 5 days
after IAV infection.
SP-A inhibits hemagglutination activity and virus infectivity in vitro. The hemagglutination inhibition assay was used to test the ability of the human SP-A used for treatment in vivo to inhibit HA activity of the Phil/82 strain of IAV in vitro. SP-A at a concentration of 812 ± 187 ng/ml completely inhibited HA activity of 40 HA U/ml of the Phil/82 strain of IAV. The ff assay was used to determine the effect of SP-A on the ability of IAV to infect monolayers of MDCK. SP-A at a concentration of 7.2 µg/ml reduced the number of foci to 36 ± 14% of control. These results are comparable to those previously reported using SP-A in vitro (5, 15). Data represent mean ± SE for n = 3 preparations.
Cytokine concentrations in lung homogenates.
After IAV infection (3 and 5 days), the proinflammatory cytokines
TNF-, IL-1
, and IL-6 were significantly increased in lung homogenates from SP-A(
/
) compared with SP-A(+/+) mice (Fig. 2). IFN-
was also increased in the
lungs of SP-A(
/
) mice after viral infection. Concentrations of
IFN-
5 days after IAV infection were 91 ± 20 and 696 ± 82 pg/ml for SP-A(+/+) and SP-A(
/
) mice, respectively
(P < 0.05). MIP-2, a neutrophil chemoattractant, was
significantly increased in lung homogenates from SP-A(
/
) mice after
viral infection (Fig. 2).
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Exogenous SP-A increased viral clearance and decreased lung
inflammation in SP-A(/
) mice.
After infection (3 days), the clearance of IAV in the SP-A(
/
) mice
was significantly enhanced when IAV was coadministered with SP-A (100 µg). Cytokine levels in lung homogenates (TNF-
, IL-6, and IFN-
)
were significantly reduced in lungs of SP-A(
/
) mice treated with
SP-A (Fig. 3).
|
Macrophage phagocytosis of IAV.
Phagocytosis of FITC-labeled IAV by alveolar macrophages was similar in
SP-A(+/+) and SP-A(/
) mice. The percentage of macrophages with
fluorescent IAV were 11.1 ± 1.9 and 9.6 ± 2.8% in
SP-A(+/+) and SP-A(
/
) mice, respectively, 2 h after IAV
infection, suggesting that macrophage phagocytosis of IAV is not a
major factor in the decreased clearance of IAV observed in the
SP-A(
/
) mice.
CD4+ and CD8+ cells.
After IAV infection, CD4+ (helper T lymphocytes, Th) and CD8+ (CTL)
cells were measured in BALF and from the spleen. CD4+ cells in BALF
were similar in SP-A(/
) and SP-A(+/+) mice 3 and 5 days after IAV
infection; however, 7 days after infection, significantly less CD4+
cells were present in BALF from SP-A(
/
) mice (Fig. 4). The greatest increase in BALF CD8+
cells was observed 7 days after IAV infection for both groups. After
infection (3 days), CD8+ cells were increased in BALF from SP-A(
/
)
compared with SP-A(+/+) mice. CD4+ and CD8+ cells in BALF were similar
from uninfected SP-A(+/+) and SP-A(
/
) mice (Fig. 4). After IAV
infection (7 days), less CD4+ (33.0 ± 1.5 and 38.5 ± 1.4)
and CD8+ (9.3 ± 0.3 and 18.0 ± 1.5) cells were found in the
spleens of SP-A(
/
) compared with SP-A(+/+) mice, respectively
(mean ± SE, P < 0.05).
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Lymphocytes in BALF and spleen.
Surface markers on lymphocytes from BALF and spleen were measured by
flow cytometry. CD6, a receptor for T cell activation, and CD16, a
receptor important for signaling for antibody production, were assessed
on CD3+ T lymphocytes. CD6 expression on T lymphocytes from BALF and
spleen was significantly greater in SP-A(/
) mice. CD16 expression
was similar for BALF T lymphocytes and increased on T lymphocytes from
the spleen of SP-A(
/
) mice. The percentage of CD19+ (B lymphocytes)
and CD56+ (NK) cells was measured in BALF and spleen. B lymphocytes
were increased in BALF and spleen of SP-A(
/
) compared with
SP-A(+/+) mice. The percentage of NK cells in BALF and spleen was
similar in both groups (Table 1).
|
Cytotoxic activity of spleen lymphocytes.
Cytolytic potential of spleen lymphocytes was assessed in an
LDH release assay with IAV-infected EL4 cells as targets. Lymphocytes from the spleen of IAV-infected SP-A(/
) and SP-A(+/+) mice
exhibited a comparable degree of specific cytolytic activity on
virus-infected target cells in vitro (Fig.
5). These results suggest that SP-A is
not a critical determinant of the specific cytotoxic T lymphocyte response in the spleen.
|
Increased immunoglobulins in serum of SP-A(/
) mice.
Total immunoglubulins, IgM, IgG, and its subclasses were measured in
serum. After IAV infection (7 days), total immunoglubulins, IgM, and
the IgG subclass IgG2a were significantly increased in serum from SP-A(
/
) compared with SP-A(+/+) mice. IgG subclass IgG1 was significantly less in the serum of SP-A(
/
)
mice, and no difference was observed for IgG3 (Fig.
6). Serum immunoglubulins were increased
after IAV infection in both SP-A(
/
) and SP-A(+/+) mice, and total
immunoglobulins, IgG1, and IgG3 were lower in serum of uninfected SP-A(
/
) compared with SP-A(+/+) mice (data not
shown).
|
Increased Th1 and decreased Th2 cytokines in the lung of
SP-A(/
) mice.
CD4+ Th cells have been categorized into at least two distinct subsets
based on their profiles of cytokine secretion. Th1 cells produce IL-2
and IFN-
, whereas Th2 cells secrete IL-4, IL-5, and IL-10
(7). IL-12, produced by macrophages, promotes the
development of Th1 cells (28). IL-2 and IL-12 levels were increased, whereas IL-4 and IL-10 levels were deceased in the lungs of
SP-A(
/
) compared with SP-A(+/+) mice 7 days after IAV infection
(Fig. 6). The production of IgG2a is also a Th1-driven response, and IgG2a was increased in the serum of
SP-A(
/
) mice. In contrast, IgG1 is selectively induced
by the Th2 cytokine IL-4. Both IgG1 and IL-4 were decreased
in SP-A(
/
) mice; therefore, in the absence of SP-A, Th1 responses
to IAV predominated in the lung.
Decreased neutrophil MPO activity in SP-A(/
) mice.
After IAV infection, MPO activity from isolated BAL neutrophils was
significantly decreased in SP-A(
/
) compared with SP-A(+/+) mice
(Fig. 7). Control neutrophils isolated
from the blood of uninfected SP-A(+/+) and SP-A(
/
) mice had similar
MPO activity and greater MPO activity compared with BAL neutrophils
from IAV-infected SP-A(
/
) mice. MPO activity from PMA-stimulated
blood neutrophils was similar for SP-A(
/
) and SP-A(+/+) mice (data
not shown).
|
IAV infection enhances SP-D accumulation in the lung.
Concentrations of SP-D in BALF increased ~12-fold in SP-A(+/+) mice
and 7-fold in SP-A(/
) mice 5 days after IAV infection (Fig.
8). The results are consistent with
previous studies demonstrating increased SP-D levels in the lung of
wild-type mice early after IAV infection (24).
|
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DISCUSSION |
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Pulmonary clearance of intranasally administered IAV was reduced
in SP-A(/
) mice compared with SP-A(+/+) mice. Pulmonary inflammation was increased in SP-A(
/
) mice compared with wild-type controls, as indicated by increased total cell counts and
proinflammatory cytokines in the lung after IAV infection. Treatment
with exogenous SP-A enhanced viral clearance and decreased lung
inflammation. In the absence of SP-A, association of IAV with alveolar
macrophages was similar to wild-type levels. Activated T lymphocytes
and B lymphocytes were increased in the lung and spleen of SP-A(
/
) mice and were associated with increased serum immunoglubulins. Th1
cytokines were increased and Th2 cytokines decreased in the lung in the
absence of SP-A. Neutrophil MPO activity was decreased in SP-A(
/
)
mice, suggesting that neutrophil clearance of IAV may be impaired.
These findings support the concept that SP-A plays an important role in
the initial pulmonary host defense against IAV and modulates innate and
adaptive immune responses to the virus.
Clearance of IAV from the lungs of SP-A(/
) mice was impaired,
supporting the importance of SP-A in viral host defense of the lung.
SP-A is a member of the C-type lectin family of polypeptides that
includes mannose-binding lectin, conglutinin, and SP-D. C-type lectins
share structural features, including collagenous
NH2-terminal and "globular" COOH-terminal domains, the
latter serving as a carbohydrate recognition domain that functions in
opsonization. Influenza virus has two membrane glycoproteins, the HA
and neuraminidase. Collectins bind to oligosaccharides on influenza
virus glycoproteins, neutralizing the virus, with heavily glycosylated
strains of viruses being the most sensitive to SP-A (15).
Binding of SP-A to IAV likely enhances viral clearance by binding to
the virus, perhaps blocking access of cell surface receptors used by
the virus, thus interfering with internalization. In addition, SP-A
agglutinated IAV (15), which may also enhance viral
removal from the lung through mucociliary and phagocytic clearance.
Phagocytosis of IAV by alveolar macrophages was similar in SP-A(
/
)
and SP-A(+/+) mice in vivo, a finding that contrasts with in vitro
studies demonstrating that SP-A enhanced the association of IAV with
alveolar macrophages (4). The reasons for this discrepancy
are unclear; however, we chose early time points to assess macrophage
phagocytosis. Because large quantities of ingested FITC-labeled virus
were necessary to detect macrophage fluorescence, we were unable to
assess uptake at a lower inoculum or later time point. Nevertheless,
the finding that phagocytosis of IAV was similar in SP-A(
/
) and
wild-type mice suggests that SP-A is not a critical determinant for
macrophage clearance of IAV in vivo under our experimental conditions.
After IAV infection, inflammatory cells and proinflammatory cytokine
concentrations were increased in the lungs of SP-A(/
) mice.
SP-A(
/
) mice were able to mount an immune response to IAV
infection; however, the inflammatory response was increased compared
with wild-type controls. Increased cytokine production may lead to
increased numbers of cells in BALF after viral infection. Increased
cytokines, TNF-
, IL-1
, IL-6, and IFN-
have been demonstrated in a mouse model of IAV infection in association with lymphocytic and
mononuclear infiltrates in the lung (16). In the absence of SP-A, cytokine responses were similar to that observed in
previous mouse models of IAV infection; however, cytokine
production and inflammation were increased in SP-A(
/
) compared with
wild-type controls. The increased numbers of neutrophils found in lungs from SP-A(
/
) mice after infection may also contribute to the increase in inflammatory cytokines.
During lung injury, concentrations of SPs may be influenced by changes
in SP-A synthesis or degradation. SP-A levels were reduced in BALF from
children with viral pneumonia (23). In the present study,
IAV clearance was enhanced and proinflammatory cytokines decreased in
SP-A(/
) mice receiving 100 µg SP-A. In a murine model of
idiopathic pneumonia syndrome after bone marrow transplant, exogenous
SP-A (100 µg) administered to wild-type mouse lungs suppressed lung
inflammation and decreased pulmonary edema (36). Thus
enhancement of alveolar levels of SP-A during IAV infection may augment
viral clearance and limit the tissue-damaging production of
inflammatory mediators.
SP-D, another collectin family member, bound, agglutinated, and
enhanced the association of neutrophils with IAV (15). In previous studies, SP-D concentrations in the lungs of wild-type mice
increased after IAV infection (24). Similarly, in the
current study, SP-D levels increased in the lungs of wild-type and
SP-A(/
) mice after IAV infection, but the increase in SP-D was less
in the lungs of SP-A(
/
) mice. Clearance of IAV from the lungs of SP-A(
/
) mice was impaired, and enhanced production of SP-D in the
lung was not sufficient to compensate for the absence of SP-A. These
findings suggest that SP-D may play a role that differs from SP-A in
IAV clearance, and both proteins may be necessary for optimal viral
clearance from the lung.
Activated T lymphocytes were increased in the lung and spleen in
SP-A(/
) mice. Recent in vitro studies support a role of SP-A in
modulating the adaptive immune responses (6). In vitro, SP-A inhibited proliferation of human peripheral blood and tonsillar mononuclear cells after stimulation with either phytohemagglutinin or
anti-CD3 (6) and inhibited allergen-stimulated lymphocyte proliferation (33). In a murine model of idiopathic
pneumonia syndrome, SP-A suppressed T cell immune responses and T
cell-dependent macrophage activation (36). In the current
study, decreased Th cells were observed in BALF from SP-A(
/
) mice.
In contrast, the percentage of CTL and NK cells was similar to the wild
type after IAV infection, suggesting that SP-A regulation of CTL and NK
cells is not a critical determinant for pulmonary clearance of IAV. The
production of the potent T cell mitogen IL-2 was inhibited by SP-A in
vitro (6). The present observation that IL-2 levels are
increased in the lungs of SP-A(
/
) mice supports these in vitro
findings. IL-12, which stimulates IL-2 production, was also increased
in the lungs of SP-A(
/
) mice after IAV infection. Mononuclear
phagocytes and dendritic cells produce IL-12, which is a key inducer of
cell-mediated immune responses that play a critical role in
lung defense against viral infection but can also cause tissue
damage (28). SP-A inhibited activation of macrophages in
vivo (36), which may decrease IL-12 production and serve
an anti-inflammatory role to control the inflammatory response and
limit tissue damage during viral infection.
In the absence of SP-A, Th1 responses were increased (IFN-, IL-2,
and IgG2a) and Th2 responses were decreased (IL-4, IL-10, and IgG1; see Refs. 7 and 29). After IAV
infection, IL-12, which promotes Th1 responses, was increased in
SP-A(
/
) mice. Because SP-A has an important role in the initial
innate host defense response, impaired early viral clearance may
stimulate an exaggerated adaptive Th1 immune response. Alternatively,
SP-A may suppress lymphocyte proliferation and macrophage activation, promoting Th2 responses to reduce inflammation and tissue damage in the
lung during viral infection.
B lymphocytes were increased in the lung and spleen of SP-A(/
) mice
in association with increased levels of serum immunoglubulins. Expression of CD16, a receptor for antibody signaling, was also increased on splenic T lymphocytes. Adaptive immunity against viral
infections is mediated by antibodies that block virus binding and entry
into host cells and by CTL that eliminate the infection by killing
infected cells. Although in vitro studies suggest that SP-A may
stimulate the production of IgA, IgG, and IgM from splenocytes (19), total immunoglubulin levels were increased in the
serum of SP-A(
/
) mice. Distinct subclasses of immunoglubulins were altered in SP-A(
/
) mice after IAV infection. IgM, an antibody produced in the early primary immune response to viral antigens, was
increased in the absence of SP-A. Interestingly, IgG1,
induced by the Th2 cytokine IL-4, was decreased, and IgG2a,
induced by the Th1 cytokine IFN-
, was increased in the absence of
SP-A. These findings suggest that SP-A may potentiate the capacity of specific cytokines to promote production of particular immunoglubulin isotypes or SP-A may act directly on the B lymphocyte or Th cell to
stimulate production of a particular immunoglubulin isotype. Alternatively, decreased viral clearance in the absence of SP-A may
favor specific cytokines that promote production of a particular immunoglubulin isotype.
Neutrophil accumulation was greater in the lungs of the SP-A(/
)
than in SP-A(+/+) mice after infection. However, MPO activity of these
neutrophils was decreased in the absence of SP-A. Defects in neutrophil
chemotactic, oxidative, and bacterial killing functions have been
documented after pulmonary IAV infection (12), which may
underlie a predisposition to bacterial superinfections
(1). SP-D but not SP-A inhibits the effects of IAV on the
neutrophil respiratory burst responses in vitro (15). In
the current study, it is unclear whether neutrophil MPO activity was
decreased because of the absence of SP-A or because of impaired
clearance and increased IAV titers in the lung.
In summary, in the absence of SP-A, IAV clearance from the lung was
impaired. Lung inflammation was more severe in SP-A(/
) mice, suggesting that SP-A plays a role in modulating cytokine production and inflammatory responses during viral infection. Th1
responses were increased, whereas Th2 responses were decreased in
SP-A(
/
) mice. Exogenous SP-A restored viral clearance in the
SP-A(
/
) mice. Because the airway is the usual portal of entry for
influenza virus and other respiratory pathogens, the local production
of SP-A is likely to play a role in innate defense responses to inhaled viruses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Gary Ross for isolation and purification of surfactant protein-A, Jaymi Semona for assistance with animal husbandry, and Drs. Mitchell White and Tirsit Mogues for assistance with viral titers.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-03905 (A. M. LeVine), HL-58795 (T. Korfhagen), HL-61646, HL-56387 (J. Whitsett), and HL-5891 (K. Hartshorn).
Address for reprint requests and other correspondence: A. M. LeVine, Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: levia0{at}chmcc.org).
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. Section 1734 solely to indicate this fact.
10.1152/ajplung.00280.2001
Received 23 July 2001; accepted in final form 30 October 2001.
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