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Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, macrophage inflammatory protein-1, and monocyte
chemoattractant protein-1. In contrast, SP-A did not affect the
baseline liberation of these cytokines. The release of the
antiinflammatory cytokines IL-1 receptor antagonist and IL-6 was not
inhibited by SP-A under baseline conditions and in response to fungal
challenge. The inhibitory effect of SP-A on proinflammatory cytokine
release was retained upon reassembly of the apoprotein with natural
surfactant lipids and in the presence of serum constituents, for
mimicry of plasma leakage into the alveolar space. It was not
reproduced by the homologous proteins complement component C1q and type
IV collagen. It was independent of Candida-SP-A binding
and phagocyte C1q receptor occupancy, but apparently demanded SP-A
internalization by the mononuclear phagocytes, effecting
down-regulation of proinflammatory cytokine synthesis at the
transcriptional level. We conclude that SP-A limits excessive
proinflammatory cytokine release in AM and monocytes confronted with
fungal challenge in the alveolar compartment. These data lend further
credit to an important physiological role of SP-A in regulating
alveolar host defense and inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocytes obtained from the alveolar compartment by lavage techniques
have repeatedly been shown to be hyporesponsive to inflammatory stimuli
as compared with leukocytes isolated from peripheral blood
(14). This relative "dampening" of leukocyte activity
within the alveolar space is thought to protect the host from
persistent immune cell activation via inhaled Ags, since ongoing
inflammatory events in the alveolar compartment might result in
progressive destruction and/or fibrotic remodeling of lung tissue and
ultimately result in severe impairment of pulmonary gas-exchange
function. Surfactant has since long been implicated in this
suppression, since surfactant lipid mixtures and individual lipid
components were noted to inhibit lymphocyte proliferation and Ig
secretion (15), as well as phagocyte oxygen radical
production (16). Recently, immunosuppressive activity was
also demonstrated for SP-A, since this major surfactant protein
inhibited lymphocyte proliferation and IL-2 production
(17), and reduced TNF- generation in
endotoxin-stimulated macrophages (18). However, others
reported that SP-A per se stimulated proinflammatory cytokine
production in mononuclear cells, secretion of Igs by splenocytes, and
proliferation of lymphocytes (19, 20).
In this study, we analyzed the impact of SP-A on the Candida
albicans-induced cytokine response in alveolar macrophages (AM)
and its precursor cells, the monocytes, which rapidly expand the
alveolar mononuclear phagocyte pool under inflammatory conditions.
Isolated Candida cell wall oligosaccharides have previously
been shown to stimulate TNF- synthesis in AM (21), and
TNF- production in the alveolar compartment contributes
significantly to local inflammatory tissue injury and lethal septic
shock in disseminated candidemia (22). Further cytokines
centrally involved in lung inflammation and leukocyte recruitment
include IL-1ß, IL-8, macrophage inflammatory protein (MIP)-1 and
monocyte chemoattractant protein (MCP)-1 (23). We now show
that the Candida-elicited mononuclear phagocyte production
of all these proinflammatory cytokines, but not that of the
antiinflammatory agents IL-1 receptor antagonist (IL-1Ra) and IL-6
(24, 25), is dramatically inhibited by SP-A. Focussing on
TNF- as central proinflammatory cytokine, we further demonstrate
that the SP-A effect is robust, since it is also operative in the
presence of surfactant lipids and plasma constituents, and that it is
independent of fungal SP-A binding but is rather related to some direct
impact of the apoprotein on the phagocyte signaling events with
down-regulation of the TNF- gene expression. Interestingly, the SP-A
effect is not reproduced by the homologous proteins complement
component C1q and type IV collagen. In summary, these findings strongly
support an important role of SP-A in down-regulating AM and monocyte
inflammatory response to fungal challenge in the alveolar
compartment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stock cultures of C. albicans (kindly provided by E. Martin and S. Bakdhi, Institute of Medical Microbiology, Mainz, Germany) were maintained on Columbia agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) at 4°C. Agar cultures were restored by culturing an aliquot of frozen yeast stock for 48 h at 37°C. For each experiment, one colony was inoculated into 2 ml of tryptic soy broth (TSB; Sigma, Munich, Germany) and grown overnight at 37°C. After 12 h, 10 µl of yeast suspension was drawn into 50 ml fresh tryptic soy broth and incubated again for 12 h at 27°C on a shaker. At the end of the second culture period, yeast were centrifuged for 10 min at 1500 x g and room temperature, and resuspended in sterile, endotoxin-free saline. Under these conditions C. albicans grew as a >95% pure yeast phase population and remained >96% viable as determined by the exclusion of trypan blue dye. For the use of nonviable yeast, Candida were killed by incubation in 70% ethanol for 60 min at room temperature followed by three washing steps in sterile saline (26). The endotoxin content of saline and Candida supernatants was assayed by the Limulus amebocyte lysate assay (COATEST endotoxin; Chromogenix, Mölndal, Sweden) and always ranged <10 pg/ml. Possible bacterial contamination of the Candida suspension cultures was excluded by reculture experiments on agar plates.
Preparation of AM
Healthy volunteers underwent single bronchoscopy and bronchoalveolar lavage (BAL). All participants were nonsmokers, had no smoking history and no history of cardiac or pulmonary disease, were free of respiratory symptoms, were not taking any medication, and had normal lung function testing. The study was approved by the local ethics committee, and written, informed consent was obtained from all participants. Bronchoscopy and BAL were performed by means of a fiberoptic bronchoscope, with 10 aliquots of 20 ml warm sterile saline being infused into one segment of the middle lobe or lingula and removed by gentle suction with a total recovered volume of 150–180 ml. The lavage fluid was centrifuged for 10 min at 200 x g and room temperature, and the cell pellet was washed twice in endotoxin-free HBSS without Ca2+ and Mg2+ (HBSS; Life Technologies, Eggenstein, Germany). Cells were counted with a hemocytometer, viability was assessed by trypan blue exclusion, and differential cell counting was performed in Pappenheim-stained cytocentrifuge preparations. The BAL cells were composed of 93–96% macrophages, 3–6% lymphocytes, and 0–1% neutrophil granulocytes. Viability of the cells consistently ranged >95%.
Monocyte isolation
Human monocytes were isolated using a combination of ficoll density gradient centrifugation (Ficoll Hypaque, Pharmacia, Freiburg, Germany) and counterflow centrifugal elutriation (Beckmannn J2–21 M/E centrifuge with JE-B6 Elutriator Rotor, standard 5-ml Elutriation Chamber, Beckman Instruments, Palo Alto, CA). Cell counts and viability were determined by hemocytometer counts of trypan blue-stained aliquots. Cytocentrifuge preparations were examined after Pappenheim staining for differential cell counting. The monocyte fraction consisted of 88–93% monocytes, 7–12% lymphocytes, and 0–1% granulocytes; cell viability always ranged >96%. Buffers and reagents were tested for endotoxin content, which always ranged <10 pg/ml. The elutriation chamber was rinsed with Detoxaclean (Sigma) to eliminate possible endotoxin contamination and was sterilized by the use of 70% ethanol. The endotoxin content of chamber effluents ranged always <10 pg/ml.
Induction of cytokine synthesis in mononuclear phagocytes by C. albicans
Monocytes and AM were cultured at a concentration of 5 x 105/ml in MEM (Life Technologies) with 0.1% human serum albumin (MEM) in 48-well tissue culture plates (Costar, Cambridge, MA) in absence or presence of 5% human AB serum (Life Technologies). After incubation for prescribed periods at 37°C and 5% CO2, and stimulation with viable or nonviable C. albicans, supernatants were collected, centrifuged, and stored at -80°C until cytokine measurements. The endotoxin content of MEM, human serum albumin, and human serum was assayed by the Limulus lysate assay and always ranged below the detection limit of 10 pg/ml.
Influence of SP-A on mononuclear phagocyte cytokine secretion
Human recombinant SP-A (generously provided from K. Schäfer, Byk Gulden Pharmazeutica, Konstanz, Germany) or canine SP-A from silica-treated dogs (a gift from U. Pison, Department of Anesthesiology and Intensive Care Medicine, Humboldt University, Berlin, Germany) were used in concentrations of 0.01, 0.1, 1, 10 and 100 µg/ml during the coincubation of mononuclear phagocytes with C. albicans. In selected experiments, leukocytes or yeast were preincubated with 10 µg/ml SP-A and washed twice in warm MEM to remove nonbound SP-A before starting the coincubation period.
C1q is homologous to SP-A with respect to its macromolecular organization, and both proteins are composed of a collagen-like triple helical domain that binds to the C1qR on monocytes and macrophages and show immunologic homology with type IV collagen (27). Against this background, human recombinant C1q and bovine type IV collagen (Sigma) were employed in concentrations of 0.01, 0.1, 1, and 10 µg/ml to examine whether C1q substituted for SP-A in its impact on cytokine production by mononuclear phagocytes and whether the collagenous domain was involved. To assess the role of C1qR in the modulation of cytokine synthesis, the receptor ligands C1q and SP-A were coated to 48-well tissue culture plates (10 µg/well in 100 µl of 0.1 M carbonate buffer for 3 h at 37°C). Coating of culture plates was verified by ELISA technique using a monoclonal mouse anti-human SP-A (PE10; generously provided by T. Akino, Department of Biochemistry, Sapporo Medical College, Sapporo, Japan) or a polyclonal goat anti-human C1q Ab (Sigma), HRP-coupled goat anti-mouse or rat anti-goat Ig Abs (Dianova, Hamburg, Germany) and the substrate ABTS (Dianova). Monocytes and AM were allowed to adhere to coated wells for 30 min at 37°C to cluster C1qR at the basal surface of the cells (10). C1qR clustering was assumed since a C1q-binding assay, using biotinylated human C1q, streptavidin-coupled HRP (Dianova), and the substrate ABTS demonstrated a decrease of C1q binding to phagocytes that previously adhered to C1q- or SP-A-coated wells in comparison to cells adhered to native tissue culture plates or albumin-coated wells (data not presented). After the adherence step, mononuclear phagocytes were stimulated with C. albicans in absence or presence of 10 µg/ml soluble SP-A for 24 h at 37°C, and supernatants were collected and processed as described above.
The effect of surfactant lipids as well as the impact of SP-A-assembly with these lipids on the profile of mononuclear phagocyte cytokine synthesis was investigated by use of the natural bovine surfactant Alveofact, which is composed of phospholipids, cholesterin, glycerides, the hydrophobic surfactant proteins SP-B and SP-C, and free fatty acids (Dr. Karl Thomae GmbH, Biberach, Germany). Reassembly of SP-A-lipid complexes was generated by incubation of 10 µg SP-A with 200 µg Alveofact for 1 h at 37°C under continuous rotation as described (28). The endotoxin content of buffers and MEM was routinely assayed by the Limulus amebocyte lysate assay and always ranged below the detection limit of 10 pg/ml. SP-A, C1q, and type IV collagen were routinely treated with polymyxin B (Pierce, Rockford, Illinois) to reduce significant endotoxin contamination (>1 µg/mg protein). The endotoxin level in the SP-A, C1q, and type IV collagen preparations after polymyxin B treatment always ranged below 10 pg/mg protein.
Cytokine ELISA
TNF-, IL-1ß, IL-6, IL-8, MCP-1, and IL-1Ra in cell culture
supernatants were measured by ELISA technique. Maxisorp microtiter
plates (Nunc, Wiesbaden, Germany) were coated overnight at 4°C with
polyclonal goat Abs to human TNF-, IL-1ß, IL-6, IL-8, MIP-1,
MCP-1, or IL-1Ra (R&D, Abingdon, U.K.) followed by three washing steps
with PBS containing 0.05% Tween 20 (Sigma). Fifty-microliter samples
of culture supernatant were dispensed into the wells and incubated for
2 h at room temperature. After washing, application of a
monoclonal mouse Ab directed against TNF-, IL-1ß, IL-6, IL-8,
MIP-1, MCP-1, or IL-1Ra (R&D) was followed by sequential incubation
with a biotinylated donkey anti-mouse Ig Ab, avidin, and
biotinylated HRP, and the substrate ABTS (Dianova). Serial dilutions of
human recombinant cytokines (R&D) provided a standard curve for each
individual ELISA. Plates were read at 405 nm with an ELISA photometer.
Quantification of each cytokine was performed in triplicate with
detection ranges of 10–1000 pg/ml.
Impact of SP-A on TNF- gene expression
AM (2 x 106) were cocultured with
5 x 107 C. albicans in absence or
presence of 0.1, 1, and 10 µg/ml SP-A in six-well tissue culture
plates in 2 ml MEM at 37°C. After 2 h of coincubation,
supernatants were removed, and total cellular RNA was isolated using
the acid guanidinium thiocyanate-phenol-chloroform method as previously
described (29). The constituent mRNA was reverse
transcribed according to the instructions of the manufacturer
(StrataScript RT-PCR kit; Stratagene, Heidelberg, Germany) in a final
volume of 25 µl. The synthesis of complementary DNA was conducted in
a GeneAmp PCR System 2400 (Perkin-Elmer, Norwalk, CA) for 50 min at
37°C, and enzyme inactivation was achieved by heating the reaction to
94°C for 7 min. Subsequently, the reaction mixture was diluted with
RNase-free water to 60 µl and stored at -85°C until use. The PCR
was performed in 1 x PCR buffer (Perkin-Elmer), 1 mM of each dNTP
(dATP, dCTP, dGTP, and dTTP), 1 µM of intron-spanning specific
primers (ß-actin: 5'-AAAGAACCTGTACGCCAACACAGTGCTGTCT-3',
5'-CGTCATACTCCTGCTTGCTGATCCACATCTG-3'; TNF-:
5'-CGGGACGTGGAGCTGGCCGAGGAG-3', 5'-CACCAGCTGGTTATCTCAGCTC-3';
Stratagene), 0.75 U AmpliTaq DNA Polymerase (Perkin-Elmer), and 2 µl
of first strand cDNA in a total volume of 25 µl. PCR profiles
consisted of initial denaturation at 94°C (1.5 min), followed by 25
(ß-actin) or 35 (TNF-) cycles of denaturation (94°C, 50 s),
primer annealing (60°C, 60 s), and primer extension (72°C,
60 s) in a GeneAmp PCR System 2400. The final extension was
performed at 72°C for 7 min. Aliquots of PCR products were
electrophoresed through 1.8% (w/v) Nusieve/agarose gels stained with
ethidium bromide for 
2 h at 75 V. Negative controls were routinely
performed by running PCR without cDNA-template to exclude false
positive amplification products. Positive controls were performed using
cDNA preparations obtained from LPS-stimulated AM. To verify the
specificity of PCR amplifications obtained from the above mentioned
procedure, automated DNA sequencing was conducted on the purified cDNA
samples according to the instructions of the manufacturer (model 373 A;
Applied Biosystems, Darmstadt, Germany). By comparing the resulting DNA
sequences with the corresponding published sequences, we identified PCR
products as expected segments of spliced TNF- or ß-actin mRNA
species. With PCR conditions optimized for primer and magnesium
concentrations and cycle numbers, amplification of cDNA samples was
verified to be in the exponential phase of PCR by comparing the amount
of input RNA equivalents with the yield of the TNF- and ß-actin
PCR products.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from monocytes and AM
(Fig. 1
).
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production in monocytes and AM was not
affected by LPS-decontaminated human recombinant (Fig. 2) or natural canine SP-A, the augmented
TNF- secretion in response to C. albicans-challenge was
dose-dependently suppressed by human recombinant and natural canine
SP-A (Fig. 3). Maximal reduction of
Candida-stimulated TNF- release was achieved by 10
µg/ml human recombinant SP-A, which decreased AM TNF- secretion by
60% and nearly completely inhibited monocyte TNF- production
(Fig. 3). The natural canine SP-A isolated from silica-treated dogs by
butanol extraction was less active. A total of 100 µg/ml of the
canine SPA preparation reduced the AM and monocyte TNF- release to
approximately the same levels achieved with 10 µg/ml human SP-A
(Fig. 3).
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, we analyzed the impact of SP-A on the
production of the proinflammatory cytokines IL-1ß, IL-8, MIP-1,
and MCP-1, as well as IL-1Ra and IL-6, all known to be produced by AM
during the acute inflammatory response in the alveolar space (Ref.
30 and unpublished data). SP-A did not influence the
baseline production of these cytokines, but nearly totally inhibited
the Candida-elicited secretion of IL-1ß and MCP-1, and
significantly decreased the release of IL-8 and MIP-1 in response to
the fungal agent. In contrast, no significant inhibition of the
Candida-induced secretion of the antiinflammatory cytokines
IL-6 and IL-1Ra was noted (Fig. 4).
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) and monocytes. Preincubation of AM or
monocytes with SP-A for 30–60 min and removing unbound SP-A before
admixing Candida did not suffice to inhibit TNF- release,
but preincubation for 120–240 min significantly inhibited cytokine
secretion (Fig. 5), which demonstrated that SP-A directly acted on the
mononuclear phagocytes. Culturing AM or monocytes for another 24 h
in MEM after preincubation with SP-A for 240 min and removing unbound
protein, however, completely restored the cytokine response to C.
albicans (Fig. 5).
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release (Fig. 6). This time course did
suggest a transcriptional modulation of TNF- synthesis in the
presence of SP-A. Indeed, SP-A significantly and dose-dependently
down-regulated TNF- gene expression in Candida-stimulated
AM (Fig. 6).
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in mononuclear phagocytes (Ref. 21 and Fig. 1
). This
is important in vivo, since blastoconidia disseminate into the lungs
during Candida septicemia and undergo hyphal transition in
the alveolar space coincident with an overwhelming, compartmentalized
TNF- production (22). We could show that SP-A not only
diminished mononuclear phagocyte TNF- release induced by nonviable
Candida yeast (Figs. 3 and 7),
but also inhibited cytokine secretion stimulated by viable hyphae (Fig. 7), which developed from viable blastoconidia during the 24 h
coculture period of fungi and leukocytes.
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); SP-A, however, also abrogated this serum-enhanced
Candida-elicited TNF- response in both cell types
(Fig. 7).
The structural homologous proteins C1q and type IV collagen share the
N-terminal collagenous region with SP-A but lack the C-terminal
carbohydrate recognition domain. Like SP-A, both proteins bind to C1qR
with their collagenous domain, but, in contrast to the surfactant
protein, they failed to inhibit the Candida-induced cytokine
response in monocytes and AM. LPS-decontaminated type IV collagen
itself stimulated TNF- secretion in both cell types and
dose-dependently augmented the Candida-induced cytokine
release (Fig. 8). Polymyxin B-treated C1q
did not influence the baseline secretion of TNF-, but slightly
increased the Candida-stimulated cytokine production at a
concentration of 10 µg/ml (Fig. 8).
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secretion (Fig. 9). Soluble SP-A, in contrast, maintained
its inhibitory capacity even under conditions of AM adherence to SP-A-
or C1q-coated tissue culture plates (Fig. 9), which clustered C1qR at
the basal surface of adherent cells. Altogether, these data suggested
that binding of the collagenous domain of SP-A to C1qR was not involved
in reducing TNF- release from Candida-stimulated
monocytes or AM.
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). Despite SP-A assembly with
surfactant lipids, the surfactant protein displayed a strong inhibitory
capacity, surpassing that of surfactant lipids and even slightly (not
significantly) surpassing that of SP-A alone, rather suggesting that
surfactant lipids and SP-A act in an additive manner (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis in
response to endotoxin challenge (18). Interestingly, the
SP-A homologue MBP also decreased the production of TNF- from
oligosaccharide-activated monocytes (31). Various cell
types might respond differentially to SP-A to explain these different
findings; however, the origin and chemical features of the SP-A batches
employed in these studies must also be taken into consideration. Human
alveolar proteinosis material, a common source of SP-A, differs from
physiological surfactant (43). SP-A might be susceptible
to inactivation or denaturation during a commonly employed butanol
extraction procedure (44), and significant contamination
with endotoxin might have additional impact. To address this issue, we
currently employed two different SP-A preparations, one recombinant
human protein, the functional integrity of which has been characterized
in detail (43, 45), and one extracted from natural
material (canine SP-A; Ref. 40). Corresponding results
were obtained with these two SP-A preparations, with the only
difference of lower spec. act. (related to weight) of the canine SP-A
in the human cell types currently investigated. It has to be kept in
mind, however, that we presently employed a LPS-decontamination step to
minimize the endotoxin load of the SP-A preparations used, since
considerable LPS-contamination of these SP-A preparations was noted in
pilot experiments, alongside with marked induction of mononuclear
phagocyte cytokine production under baseline conditions (data not
shown).
The inhibitory effect on proinflammatory cytokine release from
mononuclear phagocytes was specific for SP-A and was not reproduced by
C1q and type IV collagen, which share the N-terminal collagenous region
with SP-A, but lack the C-terminal carbohydrate recognition domain
(6, 8, 9). These findings and the previous observation of
calcium-dependent SP-A-binding to C. albicans
(26) initially favored the hypothesis that the lectin
domain of SP-A might "shield" fungal mannan and glucan moieties,
thereby blocking their interaction with corresponding receptors on AM
and monocytes (2, 31). Fungal cell wall polysaccharides
have indeed been reported to provoke TNF- synthesis in mononuclear
phagocytes (21). However, precoating of yeast with SP-A in
the absence of soluble SP-A completely failed to inhibit the cytokine
release in response to fungal challenge. This finding is in line with
the previous observation that SP-A binds only to Lipid A of rough LPS,
but interferes with macrophage TNF- secretion in response to smooth
LPS (18, 46, 47). Altogether, these data strongly suggest
that SP-A blocked the cytokine response of macrophages and monocytes to
Candida challenge not by its binding to the fungal surface,
but by some direct impact on the mononuclear phagocytes. This is
supported by the finding that preincubation of AM with SP-A and removal
of unbound SP-A before starting the AM-Candida coincubation
sufficed to suppress the cytokine response to fungal challenge.
Moreover, internalization of SP-A by macrophages seemed apparently necessary for the interference with the cytokine response to Candida exposure, since SP-A immobilized on tissue culture plates was entirely ineffective. Multiple SP-A membrane receptors on monocytes and AM have been suggested, all of which might be operative for achieving SP-A internalization into these cells (48, 49). One of these is C1qR, which recognizes the collagenous region of SP-A and its homologues (49, 50). Employment of immobilized C1q for clustering C1qR at the basal surface of adhering mononuclear phagocytes and blocking C1qR by this technique (10, 48), did, however, not abrogate the inhibitory activity of soluble SP-A. Though further detailed analysis of the mechanisms of SP-A binding and internalization, underlying the suppressive effect on proinflammatory cytokine synthesis, was not undertaken in the current study, this finding supports the notion that the carbohydrate recognition domain of SP-A rather than its collagenous region is centrally involved in this effect.
Analysis of the time-dependency of the SP-A effect on the phagocyte
TNF- response, as well as direct assessment of the TNF- gene
expression in absence and presence of SP-A, indicated that the
down-regulation of TNF- synthesis by SP-A occurred at the
transcriptional level. This observation is reminiscent of the
previously reported effect of surfactant lipid mixtures, which were
noted to reduce mononuclear cytokine mRNA via inhibition of the
transcription factor NF-
B (36). Accordingly, the
suppressive effect of SP-A was not exclusive for TNF-, but was also
demonstrated for the proinflammatory cytokines IL-1ß, IL-8, MCP-1,
and MIP-1. In contrast, the Candida-elicited synthesis of
the antiinflammatory cytokines IL-1Ra and IL-6 was not suppressed by
SP-A, and SP-A did not affect the baseline levels of cytokine
generation in AM and monocytes. These data indicate a distinct
immunomodulatory rather than a general inhibitory effect of SP-A on
mononuclear phagocyte cytokine responsiveness. The intracellular
signaling events underlying this profile of SP-A efficacy in
macrophages and monocytes clearly demand further elucidation.
The robustness of the SP-A effect on mononuclear phagocyte cytokine
generation is demonstrated by the fact that it was noted for both
viable and nonviable Candida as well as AM and monocytes,
and that it was also operative in the presence of surfactant lipids and
plasma proteins. The vast majority of SP-A recovered from lung lavage
is associated with the lipid fraction of surfactant (51),
and the lipids have been noted to counteract several effects
demonstrated for purified SP-A (11, 19, 20). We could show
that the capacity of SP-A to suppress the proinflammatory cytokine
response of AM was retained in the presence of the natural surfactant
material, including surfactant lipids and the hydrophobic surfactant
apoproteins SP-B and SP-C, and this was true after a preceding maneuver
to establish reassembly of SP-A-lipid complexes. Most impressively,
SP-A also retained its inhibitory capacity under conditions mimicking
plasma protein leakage into the alveolar compartment, a prominent
feature of acute inflammatory lung disease. In the presence of serum
constituents, the TNF- secretory response to both viable and
nonviable Candida was manyfold increased, possibly related
to an opsonization effect, and this strong TNF- response was
markedly reduced in the presence of SP-A, to a level approaching that
in the absence of serum. This finding is reminiscent of the previous
observation that SP-A counteracts the inhibitory effect of plasma
constituents on alveolar surfactant function (32).
In conclusion, the major surfactant protein SP-A was found to strongly suppress the proinflammatory cytokine response of AM and monocytes to both viable and nonviable C. albicans. In contrast, the baseline cytokine generation and the release of antiinflammatory cytokines upon fungal challenge were not affected. This SP-A effect was retained upon reassembly of the protein with natural surfactant lipids and in the presence of serum constituents, as mimicry of inflammatory conditions with plasma leakage into the alveolar space. Direct impact of the apoprotein on phagocyte regulatory events rather than its binding to fungal surfaces is suggested as the underlying mechanism. These data lend further credit to a physiological function of SP-A in regulating alveolar host defense and inflammation, by suggesting a fundamental role of this apoprotein in limiting excessive proinflammatory cytokine release in AM and invading monocytes confronted with microbial challenge in the alveolar compartment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Simone Rosseau, Department of Internal Medicine, Justus-Liebig-University, Klinikstrasse 36, 35385 Giessen, Germany. E-mail address:
3 Abbreviations used in this paper: SP-A, surfactant protein A; MBP, mannose-binding protein; AM, alveolar macrophage; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; IL-lRa, IL-1R antagonist; BAL, bronchoalveolar lavage.
Received for publication March 4, 1999. Accepted for publication August 2, 1999.
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S. Piboonpocanun, H. Chiba, H. Mitsuzawa, W. Martin, R. C. Murphy, R. J. Harbeck, and D. R. Voelker Surfactant Protein A Binds Mycoplasma pneumoniae with High Affinity and Attenuates Its Growth by Recognition of Disaturated Phosphatidylglycerols J. Biol. Chem., January 7, 2005; 280(1): 9 - 17. [Abstract] [Full Text] [PDF]
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 Y. Wu, S. Adam, L. Hamann, H. Heine, A. J. Ulmer, U. Buwitt-Beckmann, and C. Stamme Accumulation of Inhibitory {kappa}B-{alpha} as a Mechanism Contributing to the Anti-Inflammatory Effects of Surfactant Protein-A Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 587 - 594. [Abstract] [Full Text] [PDF]
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J. M. Hickman-Davis, J. Gibbs-Erwin, J. R. Lindsey, and S. Matalon Role of Surfactant Protein-A in Nitric Oxide Production and Mycoplasma Killing in Congenic C57BL/6 Mice Am. J. Respir. Cell Mol. Biol., March 1, 2004; 30(3): 319 - 325. [Abstract] [Full Text] [PDF]
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 J. A. Gold, Y. Hoshino, N. Tanaka, W. N. Rom, B. Raju, R. Condos, and M. D. Weiden Surfactant Protein A Modulates the Inflammatory Response in Macrophages during Tuberculosis Infect. Immun., February 1, 2004; 72(2): 645 - 650. [Abstract] [Full Text] [PDF]
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J. F. Alcorn and J. R. Wright Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L129 - L136. [Abstract] [Full Text] [PDF]
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M. F. Reidy and J. R. Wright Surfactant protein A enhances apoptotic cell uptake and TGF-{beta}1 release by inflammatory alveolar macrophages Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L854 - L861. [Abstract] [Full Text] [PDF]
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M. Sato, H. Sano, D. Iwaki, K. Kudo, M. Konishi, H. Takahashi, T. Takahashi, H. Imaizumi, Y. Asai, and Y. Kuroki Direct Binding of Toll-Like Receptor 2 to Zymosan, and Zymosan-Induced NF-{kappa}B Activation and TNF-{alpha} Secretion Are Down-Regulated by Lung Collectin Surfactant Protein A J. Immunol., July 1, 2003; 171(1): 417 - 425. [Abstract] [Full Text] [PDF]
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S. Hussain, J. R. Wright, and W. J. Martin II Surfactant Protein A Decreases Nitric Oxide Production by Macrophages in a Tumor Necrosis Factor-{alpha}-Dependent Mechanism Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 520 - 527. [Abstract] [Full Text] [PDF]
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C. Stamme, M. Muller, L. Hamann, T. Gutsmann, and U. Seydel Surfactant Protein A Inhibits Lipopolysaccharide-Induced Immune Cell Activation by Preventing the Interaction of Lipopolysaccharide with Lipopolysaccharide-Binding Protein Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 353 - 360. [Abstract] [Full Text] [PDF]
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 I. Shalit, L. Horev-Azaria, I. Fabian, H. Blau, N. Kariv, I. Shechtman, H. Alteraz, and Y. Kletter Immunomodulatory and Protective Effects of Moxifloxacin against Candida albicans-Induced Bronchopneumonia in Mice Injected with Cyclophosphamide Antimicrob. Agents Chemother., August 1, 2002; 46(8): 2442 - 2449. [Abstract] [Full Text] [PDF]
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 F. Meloni, A. Alberti, A. Bulgheroni, A. Lupi, E. Paschetto, A. Marone Bianco, G. Rodi, A. Fietta, M. Luisetti, and A. Baritussio Surfactant apoprotein A modulates interleukin-8 and monocyte chemotactic peptide-1 production Eur. Respir. J., June 1, 2002; 19(6): 1128 - 1135. [Abstract] [Full Text] [PDF]
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K. J. Haley, A. Ciota, J. P. Contreras, M. R. Boothby, D. L. Perkins, and P. W. Finn Alterations in lung collectins in an adaptive allergic immune response Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L573 - L584. [Abstract] [Full Text] [PDF]
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S. Murakami, D. Iwaki, H. Mitsuzawa, H. Sano, H. Takahashi, D. R. Voelker, T. Akino, and Y. Kuroki Surfactant Protein A Inhibits Peptidoglycan-induced Tumor Necrosis Factor-alpha Secretion in U937 Cells and Alveolar Macrophages by Direct Interaction with Toll-like Receptor 2 J. Biol. Chem., February 22, 2002; 277(9): 6830 - 6837. [Abstract] [Full Text] [PDF]
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O. A. Quintero and J. R. Wright Clearance of surfactant lipids by neutrophils and macrophages isolated from the acutely inflamed lung Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L330 - L339. [Abstract] [Full Text] [PDF]
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K. R. Khubchandani, R. E. Oberley, and J. M. Snyder Effects of Surfactant Protein A and NaCl Concentration on the Uptake of Pseudomonas aeruginosa by THP-1 Cells Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 699 - 706. [Abstract] [Full Text] [PDF]
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J. M. Hickman-Davis, F. C. Fang, C. Nathan, V. L. Shepherd, D. R. Voelker, and J. R. Wright Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L517 - L523. [Abstract] [Full Text] [PDF]
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J. R. Wright, P. Borron, K. G. Brinker, and R. J. Folz Surfactant Protein A . Regulation of Innate and Adaptive Immune Responses in Lung Inflammation Am. J. Respir. Cell Mol. Biol., May 1, 2001; 24(5): 513 - 517. [Full Text]
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S. Yang, C. Milla, A. Panoskaltsis-Mortari, D. H. Ingbar, B. R. Blazar, and I. Y. Haddad Human Surfactant Protein A Suppresses T Cell-Dependent Inflammation and Attenuates the Manifestations of Idiopathic Pneumonia Syndrome in Mice Am. J. Respir. Cell Mol. Biol., May 1, 2001; 24(5): 527 - 536. [Abstract] [Full Text]
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 B. W. KRAMER, A. H. JOBE, C. J. BACHURSKI, and M. IKEGAMI Surfactant Protein A Recruits Neutrophils into the Lungs of Ventilated Preterm Lambs Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 158 - 165. [Abstract] [Full Text]
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C. Stamme, E. Walsh, and J. R. Wright Surfactant Protein A Differentially Regulates IFN-gamma - and LPS-Induced Nitrite Production by Rat Alveolar Macrophages Am. J. Respir. Cell Mol. Biol., December 1, 2000; 23(6): 772 - 779. [Abstract] [Full Text]
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S. YANG, A. PANOSKALTSIS-MORTARI, D. H. INGBAR, S. MATALON, S. ZHU, E. R. RESNIK, C. L. FARRELL, D. L. LACEY, B. R. BLAZAR, and I. Y. HADDAD Cyclophosphamide Prevents Systemic Keratinocyte Growth Factor-induced Up-Regulation of Surfactant Protein A after Allogeneic Transplant in Mice Am. J. Respir. Crit. Care Med., November 1, 2000; 162(5): 1884 - 1890. [Abstract] [Full Text]
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P. Borron, J. C. McIntosh, T. R. Korfhagen, J. A. Whitsett, J. Taylor, and J. R. Wright Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo Am J Physiol Lung Cell Mol Physiol, April 1, 2000; 278(4): L840 - L847. [Abstract] [Full Text] [PDF]
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