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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Surfactant protein (SP)-A is a known opsonin for a variety of
pulmonary pathogens. SP-A enhances ingestion of these
pathogens by interaction with an SP-A receptor (SP-AR) found
on phagocytic cells such as peripheral blood monocytes
(PBMC) and alveolar macrophages. Respiratory syncytial virus
(RSV) is the most important respiratory pathogen in children.
Recent studies have indicated that SP-A levels may be decreased in RSV bronchiolitis and pneumonia. In this study we
examined the role of SP-A in uptake of RSV by both PBMC and
U937 macrophages, a human macrophage cell line known to
express SP-ARs. In addition, we studied the effect of SP-A- mediated uptake of RSV on production of tumor necrosis factor (TNF)-
and interleukin (IL)-10 by these cells because incomplete immunity to recurrent RSV infection has been partially attributed to abnormal cytokine responses by macrophages. SP-A
enhanced binding and uptake of fluorescently labeled RSV
(RSV-FITC) by PBMC in a dose-dependent manner, with a
maximal effect seen with 10 to 15 µg/ml SP-A as measured by
both percent fluorescent monocytes and linear mean fluorescence (lmf) of individual cells. SP-A also enhanced uptake of
RSV-FITC by U937 macrophages, with a maximal effect seen
with 20 µg/ml SP-A as measured by both percent fluorescent monocytes and lmf. With respect to TNF- levels, RSV alone
slightly enhanced TNF- production by PBMC and decreased
TNF- production by U937 macrophages measured at 12 h after addition of RSV. SP-A-mediated uptake of RSV significantly enhanced TNF- production by PBMC and reversed the
RSV-induced depression of TNF- by U937 macrophages. RSV
significantly enhanced IL-10 production by both cell types, which was reversed by SP-A-mediated uptake. These findings
suggest that SP-A is an important opsonin for RSV and that
SP-A-mediated uptake of RSV may alter some of the unusual
cytokine responses that are postulated to be involved in incomplete immunity to recurrent infection.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Respiratory syncytial virus (RSV) is the major respiratory pathogen in infants and young children. RSV infects all children by age 2 yr and can cause upper respiratory tract reinfection at any age (1). Most children have a relatively mild response to RSV infection, with upper respiratory symptoms predominating. However, RSV is a significant cause of morbidity and mortality associated with 90,000 pediatric hospitalizations, 4,500 deaths, and costs of $300 million annually (2).
RSV is an unusual virus in that infection does not confer lasting immunity. Despite the relative antigenic stability of RSV strains, children and most adults can be reinfected throughout life (1). The unusual cytokine response
of pulmonary macrophages in response to RSV infection
may contribute to the lack of sustained immunity (3). Peripheral blood monocytes (PBMC) and alveolar macrophages (AM) have been shown to have increased production of both inflammatory (tumor necrosis factor [TNF]-,
interleukin [IL]-1, and IL-8) and anti-inflammatory (IL-10
and IL-1 receptor antagonist) cytokines in response to RSV
infection (3). TNF- contributes to protection against RSV
infection both in vitro and in vivo by inhibiting viral replication (9). However, production of inflammatory cytokines appears delayed. Peak levels of TNF- may not occur until 48 h
after initial infection (10).
Surfactant protein (SP)-A is one of four known pulmonary surfactant-associated proteins and has recently been
found to play a very important role in host defense. SP-A
has been shown to opsonize a wide variety of pulmonary
pathogens including bacteria (11), mycobacteria (14),
and viruses (15). The role of SP-A as an opsonin for viral respiratory pathogens has not been extensively studied.
van Iwaarden and colleagues found that SP-A was twice as
effective as serum at stimulating uptake of herpes simplex virus (HSV) by rat alveolar macrophages (15). In a similar
study, the same authors studied the interaction of SP-A
with influenza A virus and found that SP-A bound to and
reduced infectivity of two influenza A virus strains (16).
Levine and associates recently reported that RSV clearance was deficient in genetically altered SP-A 
/ mice
and viral clearance was restored with addition of exogenous SP-A (17). A role for SP-A in RSV infection is further supported by the recent report that SP-A levels are
decreased in the bronchoalveolar lavage fluids (BALFs)
of infants with RSV pneumonia (18).
The role of SP-A in uptake of RSV by macrophages and
alterations in the subsequent macrophage cytokine response
has not been previously studied. In this report we demonstrate that SP-A enhances binding and uptake of RSV by
PBMC and U937 macrophages and alters the RSV-mediated effects on macrophage production of the inflammatory cytokine TNF- and the anti-inflammatory cytokine IL-10.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials
Cell tissue culture media (AIM-V, RPMI 1640, F12K) and Dulbecco's phosphate-buffered saline (PBS) were from GIBCO BRL
(Grand Island, NY). Ficoll-Paque was purchased from Pharmacia
Biotech (Uppsala, Sweden). 7--Actinomycin D was supplied by
Molecular Probes (Eugene, OR) and phycoerythrin-conjugated
CD14 (PE-CD14) was obtained from Becton Dickinson Immunochemistry (San Jose, CA). Polymixin B-agarose, chloramine-T,
and fluorescein isothiocyanate (FITC) isomer 1 were from Sigma
Chemical Co. (St. Louis, MO). Paraformaldehyde was purchased
from Mallinckrodt (St. Louis, MO). RSV-G protein was kindly
provided by Barney Graham (Department of Medicine, Vanderbilt University). Spectra/Por dialysis membrane (MWCO 3,500)
was obtained from Spectrum Medical Industries (Houston, TX).
Multi-well culture plates and cell lifters were from Costar (Cambridge, MA). Sephadex G-25 columns were from Pharmacia Biotech.
SP-A
SP-A was isolated from BALF from patients with alveolar proteinosis using a butanol extraction method as previously described (19). Alveolar proteinosis fluid was a gift from Samuel Hawgood (Department of Pediatrics, University of California-San Francisco, San Francisco, CA). Purified SP-A was treated with polymixin B-agarose to remove lipopolysaccharide (LPS). Briefly,
polymixin B-agarose was rinsed twice with PBS to remove preservatives. SP-A and polymixin B were incubated at a 1:1 (vol/
vol) ratio for 2 h at 4°C. Polymixin B was removed by centrifugation at 10,000 rpm for 60 s. The final concentration of SP-A was
determined by optical density at 280 nm (OD280). Other investigators have reported stimulatory effects of SP-A on TNF- production even in the absence of LPS. We therefore performed a
functional assay to confirm that our SP-A preparations were not
enhancing TNF- production. PBMC and U937 cells were treated with SP-A (20 µg/ml) for 1 h. Cell supernatants were collected 12 h later and assayed for TNF- production. Only SP-A
preparations that did not enhance TNF- production were used
in subsequent experiments.
Monocytes
PBMC were obtained from normal healthy volunteers without recent upper respiratory symptoms. Approximately 100 ml of blood was drawn from each volunteer and centrifuged at 1,000 × g for 20 min. The buffy coat was removed and diluted to 50 ml with 1 mM ethylenediaminetetraacetic acid in PBS, overlaid onto Ficoll-Paque, and centrifuged at 1,000 × g for 20 min. The mononuclear cell layer was aspirated and rinsed with PBS to remove the Ficoll-Paque. An aliquot of cells was stained with trypan blue to assess viability and counted using a hemocytometer. Monocytes were plated at 2 × 106 cells/ml in serum free AIM-V media in 24-well plates. The cells were allowed to attach and media were changed after 2 h. The resulting monocyte-enriched cell populations were used within 24 h of isolation in all experiments.
U937 Macrophages
U937 macrophages were obtained from American Type Culture
Collection (Rockville, MD) and maintained in RPMI 1640 with
10% inactivated fetal bovine serum (FBS). Before experiments,
U937 cells were stimulated with phorbol myristate acetate
(PMA) (10
8 M) for 12 h to promote cell attachment. Cell viability was confirmed by trypan blue exclusion before use.
RSV Labeling
RSV stocks were a gift from James Crowe (Department of Pediatrics, Vanderbilt University) and Barney Graham. HEp-2 cells
were maintained in Eagle's minimum essential medium supplemented with glutamine, amphotericin, gentamicin, penicillin G,
and 10% FBS. Working stocks of A2 and Long strains of RSV
were prepared as previously described (20) and were stored at
concentrations of 108 plaque forming units (pfu)/ml at 70°C.
RSV was labeled with FITC using a method previously described
for FITC labeling of HSV1 (15). Briefly, 1 mg of FITC was dissolved in 1 ml 1 M Na2CO3, pH 9.6, and 100 µl was added to 1 ml
of RSV stock for 45 min at 4°C. Excess FITC was removed by dialysis against PBS with two to three changes over 18 h at 4°C.
FITC-labeled virus was used immediately after dialysis in all experiments. Viral viability was confirmed by plaque assay as previously described (20). A 100-fold loss of viral viability was caused
by the labeling process, and the multiplicity of infection (MOI)
was adjusted in subsequent experiments to account for this loss.
Fluorometric Uptake Assay
The uptake assay was adapted from a method previously described by van Iwaarden and colleagues (15). Briefly, fluorescently labeled RSV (RSV-FITC) (1 × 106 pfu) was preincubated
with SP-A at varying concentrations (0 to 20 µg) for 30 min at
37°C. The RSV/SP-A mixture was added to either PBMC or
U937 cells (106 cells/well) in triplicate at an MOI of 1.0 and an
SP-A concentration of 0 to 20 µg/ml. After 45 min, cells were
rinsed three times with media and stained with PE-CD14 (monocytes only) and 7--actinomycin D. The cells were collected using
a cell lifter, centrifuged at 500 × g for 5 min, and fixed with 500 µl
of 1% paraformaldehyde in PBS. Cell fluorescence was analyzed
using a FACSCalibur flow cytometer (Becton Dickinson) using
an argon-ion laser at 15 mW and 488 nm. Filters included FL1-green fluorescence (530 +/ 30 nm bandpass filter), FL2-orange
(585 +/ 42 nm bandpass filter), and FL3-red (650 nm longpass
filter). A total of 10,000 events were collected per sample. Both
percentage of fluorescent cells and linear mean fluorescence
(lmf) data were collected.
Fluorescence Quenching Technique
To determine whether cell-associated fluorescence was extracellular or intracellular, extracellular quenching of fluorochrome emission was performed with trypan blue as previously described (15). Briefly, in initial experiments, an aliquot of each sample was resuspended in PBS containing 0.2 mg/ml trypan blue. After 1 min the cells were washed twice and fixed with 1% paraformaldehyde as described earlier, followed by cell fluorescence analysis.
Confocal Fluorescent Microscopy
In addition to flow cytometry, cell-associated fluorescence was also analyzed by fluorescent microscopy using a Zeiss LSM 410 confocal laser scanning inverted microscope. Cells were prepared in a manner similar to preparation for flow cytometry. Briefly, RSV-FITC (1 × 106 pfu) was preincubated with SP-A at varying concentrations (0 to 20 µg) for 30 min at 37°C. The RSV/SP-A mixture was added to either PBMC or U937 cells (106 cells/well) in triplicate. After 45 min, cells were rinsed three times with media, fixed with 500 µl of 1% paraformaldehyde in PBS, and mounted on 35-mm dishes (Mat-Tek Co., Ashland, MA) using Aqua-Poly-Mount (PolySciences, Warrington, PA). Laser wavelengths used were 488 nm for green (FITC) fluorescence and 543 nm for red fluorescence.
RSV-G Purification
RSV-G protein was a gift from Barney Graham and was purified as previously described (21). Briefly, BSC40 cells were infected with a vaccinia virus expressing a truncated form of RSV-G that is secreted from infected cells. Culture supernatant was collected and overlaid on a cushion of 1.46 M sucrose/1 mM NaH2PO4, pH 7.2, and centrifuged at 62,000 × g for 90 min. The supernatant was dialyzed overnight against 20 mM Tris-HCL/0.5 M NaCl, pH 7.4, and then applied to a Concanavalin A-Sepharose lectin column (Pharmacia Biotech, Piscataway, NJ). Bound proteins were eluted with a continous gradient of 0 to 0.5 M methyl-a-D-mannopyranoside in 20 mM Tris-HCl/ 0.5 M NaCl, pH 7.4. Fractions were screened by capture enzyme-linked immunosorbent assay (ELISA) and G-containing fractions were pooled. Purity of isolated G protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and compared with commercially available RSV-G (Figure 1).
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SP-A Iodination
SP-A was labeled with Na125I using a chloramine-T protocol as previously described (22). Iodinated SP-A has previously been shown to bind to SP-A receptors and mediate phagocytosis of pathogens in a fashion similar to unlabeled SP-A (14, 23). Briefly, SP-A (100 µg) was mixed at 4°C with 0.2 M K2PO4 to a volume of 200 µl, followed by addition of 10 uCi 125I and 10 µl fresh chloramine-T (0.33 mg/ml). After 15 min the reaction was terminated with 100 µl sodium metabisulfite (5 mg/ml) and 100 µl potassium iodide (20 mg/ml). Labeled SP-A was separated from free 125I using a Sephadex G-25 column pre-equilibrated with 5 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 150 mM NaCl, and 0.1% bovine serum albumin (BSA), pH 7.4. Iodinated SP-A was used within 24 h of labeling.
RSV-G Binding Assay
RSV-G (100 ng/100 µl 0.5 M Na2CO3, pH 9.6) was bound to a 96-well ELISA plate in triplicate for 1 h at room temperature. The plate was washed four times with PBS-0.05% Tween, then blocked for 60 min with 1% BSA. 125I-SP-A (1 µg/100 µl PBS) was added to each well for 60 min, then rinsed as described earlier. Triton X-100 (0.1%, 100 µl/well) was added for 15 min. The solubilized RSV/SP-A was collected and radioactivity counted.
Statistical Analysis
All experiments were repeated four to six times with data expressed as means +/ standard error. Statistical analysis of differences between doses of SP-A was performed by two-tailed t
test with significance detected at P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Uptake of RSV by PBMC
PBMC were used for these experiments because they express a high level of the 210-kD SP-A receptor (23). SP-A enhanced uptake of RSV by PBMC in terms of both the percentage of fluoresecent monocytes and the lmf (Figure 2). The percentage of fluorescent monocytes denotes the percentage of cells that have ingested labeled virus. Without SP-A, 30% of the PBMC were fluorescent. Addition of SP-A enhanced uptake in a dose-dependent manner, with the maximum response seen with 15 µg/ml SP-A (57% fluorescent PBMC, P < 0.05). The lmf denotes the fluorescence intensity of a population of cells and is proportional to the number of fluorescent viral particles that each cell ingests. Compared with addition of RSV alone, SP-A enhaced the lmf at all doses, with a maximal response seen with 10 µg/ml (425% of RSV alone, P < 0.05). Confocal fluorescent microscopy of monocytes yielded similar results (Figure 3). Quenching of cell-surface fluorescence with trypan blue in initial experiments resulted in a 15% reduction in the lmf signal that was uniform across all SP-A samples and RSV controls. Therefore, cell-associated fluorescence was primarily intracellular with a small amount of extracellular binding.
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Uptake of RSV by U937 Macrophages
U937 cells are known to express the 210-kD SP-A receptor (23) and have been used as a model system to study
RSV infection (24). We therefore examined the effect of
SP-A on RSV uptake by U937 cells. U937 cells were pretreated with PMA (108 M) before experiments to promote cell attachment. Treatment of macrophages with
PMA also further enhances expression of SP-A receptors
(25). SP-A enhanced uptake of RSV by U937 macrophages in a dose-response fashion in terms of both percentage of fluorescent cells (34% RSV plus SP-A versus
20% RSV alone, P < 0.05) and lmf (137% of RSV control,
P < 0.05) (Figure 4). The maximal effect was seen with an
SP-A concentration of 20 µg/ml. Confocal fluorescent microscopy of U937 cells yielded similar results (Figure 5).
Quenching of cell-surface fluorescence with trypan blue in
initial experiments resulted in only a 5% reduction in the
lmf signal that was uniform across all SP-A samples and
RSV controls, indicating that cell-associated fluorescence
was predominately intracellular.
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Effect of SP-A-Mediated Uptake of RSV on TNF- and
IL-10 Production by PBMC and U937 Cells
RSV preincubated with SP-A was added to PBMC or
PMA-treated U937 cells. Control cells without addition of
RSV or SP-A were treated in a similar fashion. Media were
collected at various time points between 1 and 48 h after
addition of RSV and SP-A. The maximal effect on TNF-
levels was noted at 12 h and IL-10 at 24 h. Therefore, in
subsequent experiments cell supernatants were collected at
12 and 24 h for cytokine analysis. TNF- and IL-10 levels
are expressed as picograms corrected for micrograms of cellular protein (pg/µg protein). Control monocytes expressed minimal TNF- at 12 h (0.5 +/ 0.2 pg TNF-/mg protein).
Uptake of RSV in the absence of SP-A caused a significant
enhancement of TNF- production by PBMC (12.3 +/ 1.2 pg TNF-/mg protein). SP-A-mediated uptake further
stimulated TNF- compared with RSV alone, with the maximal effect seen at 20 µg/ml (29.2 +/ 1.8 pg TNF-/mg
protein, P < 0.05) (Figure 6A). Addition of SP-A alone at
20 µg/ml without RSV caused no enhancement of TNF-
compared with controls (data not shown).
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Control monocytes expressed IL-10 (7.4 +/ 0.2 pg IL-10/mg protein) which was significantly increased by addition of RSV (9.7 +/ 0.2 pg IL-10/mg protein). SP-A-
mediated uptake of RSV suppressed the RSV-induced
enhancement of IL-10, with a maximal effect seen at 10 µg/
ml (4.8 +/ 0.3 pg IL-10/mg protein, P < 0.05) (Figure 6B).
Addition of SP-A alone at 20 µg/ml without RSV caused no difference in IL-10 levels compared with controls.
In experiments with U937 cells, RSV significantly decreased the production of TNF- compared with controls
(2.1 +/ 0.2 RSV versus 3.6 +/ 0.1 control pg TNF-/mg
protein, P < 0.05). SP-A-mediated uptake of RSV reversed the effect of RSV on TNF-, with a maximal response seen at 20 µg/ml (9.6 +/ 1.6 pg TNF-/mg protein,
P < 0.05) (Figure 7A). SP-A alone had no effect on TNF-
levels compared with controls. RSV significantly enhanced
IL-10 production by U937 cells (73.8 +/ 13.4 RSV versus
1.3 +/ 1.0 Control pg IL-10/mg protein, P < 0.05). SP-A-
mediated uptake reversed the RSV-induced increase, with
a maximal effect seen at 5 µg/ml (31.8 +/ 14.6 pg IL-10/
mg protein, P < 0.05) (Figure 7B). SP-A alone had no effect on IL-10 levels compared with controls.
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Binding of SP-A to RSV-G Protein
The RSV G-attachment protein is heavily glycosylated and a potential binding site for SP-A (26, 27). Binding of SP-A to RSV-G was analyzed after immobilization of the G protein to ELISA plates. SP-A bound to RSV-G in a calcium-dependent fashion (Figure 8).
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Discussion |
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Materials and Methods
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SP-A has long been recognized as an important protein in pulmonary host defense. SP-A has been shown to opsonize a wide variety of bacterial, mycobacterial, fungal, and viral pathogens (11, 28). SP-A acts as an opsonin by binding to carbohydrate groups on the pathogen surface via a carbohydrate recognition domain formed as SP-A folds into its quaternary structure. The SP-A-pathogen complex then interacts with a 210-kD SP-A receptor found on the surface of phagocytic cells such as peripheral blood monocytes, AM, and bone marrow-derived macrophages (23). Although the mechanisms of internalization of the SP-A- pathogen complex remain unclear, pathogen killing appears to be enhanced by this pathway (14).
In the current report we describe the role of SP-A in opsonization and uptake of RSV. RSV is an ideal candidate for opsonization by SP-A because it has several mannose-containing proteins on the viral capsid, including the F (fusion) and G (attachment) proteins (26, 27). In our studies, SP-A enhanced uptake of RSV by both PBMC and U937 macrophages in two specific ways. First, SP-A enhanced the percentage of cells in a given population that were able to phagocytose RSV. Second, individual cells appeared able to internalize more viral particles as expressed by the lmf of individual cells in the presence of SP-A.
Both PBMC and U937 cells were selected for these studies due to their known expression of fairly high quantities of SP-A receptor (SP-AR) (25). PBMC express high levels of SP-AR when first isolated but over a period of 4 to 5 d in cell tissue culture will lose their SP-AR expression as they differentiate into monocyte-derived macrophages. U937 cells express fairly high levels of SP-AR under normal culture conditions, but both cell types can increase SP-AR expression after stimulation with inflammatory mediators such as LPS or PMA (25). Alternatively, SP-A could be interacting with other cell-surface receptors for C-type lectins, most notably the C1q and C1qRP receptors, which can also be upregulated by inflammatory mediators (29). However, C1q receptors are downregulated by PMA in other macrophage cell lines (30).
Other investigators have studied SP-A as an opsonin for
viral pathogens. Van Iwaarden and coworkers found that
SP-A enhanced uptake of HSV (15) and influenza A virus
(16). In these studies interaction of SP-A with the virus was
inhibited by removal of the complex carbohydrate moiety
from the SP-A molecule, indicating a viral and SP-A glycoconjugate interaction. SP-A has been implicated to be important in clinical RSV infections, inasmuch as BALF SP-A levels have been found to be decreased in infants with RSV
bronchiolitis (18). Recent studies by Levine and associates in
genetically altered SP-A / mice further demonstrated that
in the absence of SP-A, RSV clearance is deficient and can be
restored by the addition of exogenous SP-A (17). They also
found that cytokine levels in lung homogenates from SP-A
/ mice were significantly altered compared with wild-type mice.
The effect of RSV infection on monocytes and macrophages has been intensely studied. Compared with epithelial cells, monocytes and macrophages are not as susceptible
to RSV infection and virus replication, and spread of virion
particles appears to be limited (31). Monocytes are more
susceptible to RSV infection than bronchoalveolar macrophages (32) but again virus replication appears limited. Despite limited viral replication, the effects of RSV infection on the subsequent cytokine response of monocytes and
macrophages may have a more significant impact on clinical
disease. RSV infection of monocytes has been shown to induce TNF- secretion, which could have a protective effect
by limiting further viral replication (5). RSV infection of
AM causes a more dramatic enhancement of TNF- compared with monocytes (4). However, the balance of inflammatory (TNF-) and anti-inflammatory (IL-10) cytokines may be important in determining the ultimate immunoregulatory response. RSV has been shown to enhance IL-10 production dramatically and subsequently decrease TNF- by
LPS-activated AM, and this cytokine imbalance may contribute to the incomplete immune response to RSV (3).
We found that SP-A-mediated uptake of RSV reversed
the effects of RSV on TNF- and IL-10 production by
both monocytes and U937 cells. In monocytes, SP-A-mediated uptake of RSV enhanced TNF- and decreased IL-10 production. The effects of SP-A-mediated uptake of
RSV by U937 cells on cytokine production was even more striking. RSV alone actually decreased TNF- and enhanced IL-10 production compared with control cells. SP-A-mediated uptake of RSV reversed these effects, with
enhanced TNF- and suppressed IL-10.
As mentioned previously, RSV is an ideal candidate for opsonization by SP-A because it has several heavily glycosylated proteins on the viral envelope, including the F (fusion) and G (attachment) proteins (26, 27). We studied the binding of SP-A to the G (attachment) protein and found that it was calcium-dependent. Calcium is required for binding of the carbohydrate recognition domain of SP-A to carbohydrate moieties. The RSV-G protein is known to be heavily mannosylated with extensive post-translational modification (26, 27).
In the clinical setting of RSV pneumonia, SP-A recovered in BALF has been shown to be decreased in critically
ill infants compared with normal controls (18). However, in
these infants the clinical severity of disease and length of
illness appeared to have an inverse correlation with SP-A
levels. SP-A levels have previously been shown to be decreased in other models of lung injury (33, 34). These findings would suggest that RSV clearance from the human
lung may be enhanced by SP-A. Our findings that SP-A-
mediated uptake of RSV enhances TNF- production by
monocytes and U937 cells would support this finding, inasmuch as TNF- has been shown to decrease viral replication in animal models of RSV pneumonia (9).
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Footnotes |
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Address correspondence to: Frederick E. Barr, M.D., Pediatric Critical Care and Anesthesia, Vanderbilt Children's Hospital, 714 Medical Arts Bldg., Vanderbilt University Medical Center, Nashville, TN 37232-1565. E-mail: rick.barr{at}mcmail.vanderbilt.edu
(Received in original form April 20, 1999 and in revised form June 14, 2000).
Abbreviations: alveolar macrophages, AM; bronchoalveolar lavage fluid, BALF; enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC; interleukin, IL; linear mean fluorescence, lmf; lipopolysaccharide, LPS; peripheral blood monocytes, PBMC; phosphate-buffered saline, PBS; phorbol myristate acetate, PMA; respiratory syncytial virus, RSV; fluorescently labeled RSV, RSV-FITC; surfactant protein, SP; SP-A receptor, SP-AR; tumor necrosis factor, TNF.Acknowledgments: The authors are grateful to Sheryl Vick for technical support, David McFarland for his expertise with flow cytometry, Jonathan Sheehan for assistance with confocal fluorescent microscopy, and Barney Graham and James Crowe for manuscript review and supply of RSV and RSV reagents. This work was supported by a Research Grant from the American Lung Association and a Founder's Grant from the Society of Critical Care Medicine to one author (F.E.B.).
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References |
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Discussion
References
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