* Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Israel, and
Department of Pathology and Immunology, Washington University Medical School, St. Louis, Missouri
Correspondence: Dr. Yona Keisari, Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69778, Israel. E-mail: ykeisari{at}ccsg.tau.ac.il
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Key Words: macrophages • monocytes • mannose receptor • human • capsule • capsular polysaccharides
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When innate nonclonal surveillance and containment measures fail, bacterial proliferation may elicit a more marked or generalized inflammatory response, which may in turn promote the development of acquired immunity. Cytokines and chemokines involved in inflammatory and immunological responses can be synthesized by resident leukocytes as a result of the phagocytic process or in response to substances released by the invading bacteria. The attachment of the pathogen to receptors on phagocytic cells can trigger the production of chemokines and cytokines [1; for review, see ref. 2 ]. In addition, gram-negative lipopolysaccharides (LPSs) and a variety of other components of bacterial cell walls are capable of inducing cytokine production in monocytes and macrophages [3 4 5 6 7 8 9 10 11 12 13 ]. The resulting inflammatory or adaptive humoral and cellular immune reactions often eradicate the pathogen effectively but also have the potential to damage the host. Thus, the initial interaction of the pulmonary pathogen with the alveolar macrophages in the serum-poor environment of the lung constitutes one of the most important initial events of the host-microbial interaction.
Lung phagocytic cells recognize microorganisms either directly or indirectly. The most thoroughly studied direct mode of recognition, termed lectinophagocytosis, involves the interaction of specific surface glycoconjugates on one cell with corresponding lectins expressed on the other [14 ]. There are two mechanisms of lectinophagocytosis. One mechanism involves microbial surface lectins that bind glycoconjugates on the phagocyte; virtually nothing is known about the induction of cytokines in mononuclear phagocytic cells after this type of interaction. In the second mechanism, lectins on the phagocyte, typically a macrophage, recognize glycoconjugates expressed on the microbial surface.
Of the various macrophage lectins studied, the mannose receptor (MR), a glycoprotein carrying eight C-type lectin carbohydrate recognition domains (CRDs), is probably the most abundant on tissue macrophages, including alveolar macrophages [15 ]. It mediates the binding and phagocytosis of many microorganisms including bacteria, fungi, and protozoa [14 ].
Opsonic proteins can mediate direct recognition of invading microorganisms by mononuclear phagocytic cells in the lung. Of particular relevance for pulmonary innate immunity are the collagenous C-type lectins (collectins)-surfactant proteins A (SP-A) and D (SP-D) [16 ]. These epithelial-cell-derived proteins can increase their phagocytosis and killing by resident alveolar macrophages [17 , 18 ]. In at least some cases, this activity involves the simultaneous binding of the lung collectins to specific glycoconjugates on the bacterial surface and to collectin receptors expressed on the phagocytic-cell membrane. However, very little is known about the ability of collectin-opsonized microorganisms to trigger the biosynthesis of cytokines by phagocytic cells.
Accordingly, we examined the possible role of direct as well as
indirect collectin-mediated recognition of bacterial pathogens in
cytokine production by mononuclear phagocytes. For these studies, we
focused on Klebsiella pneumoniae as a model system. These
organisms can shift from an encapsulated phase to an unencapsulated
phenotype at a predetermined frequency, allowing the isolation of
spontaneous, unencapsulated-phase variants among a population of
capsulated cells [19
, 20
]. Previous studies
have shown that encapsulated K. pneumoniae containing
Man
2/3Man or Rha2/3Rha sequences in their capsular
polysaccharides are recognized by the MRs on macrophages, resulting in
attachment, ingestion, and killing of the Klebsiella cells
by the phagocytic cells [21
, 22
]. The same
capsular disaccharides are recognized by SP-A, which binds to the
encapsulated bacteria and mediates phagocytosis of the organisms
[23
]. Encapsulated Klebsiella strains lacking
such sequences in their capsular polysaccharides are not recognized by
either MR or SP-A.
Although SP-D has not been observed to interact with encapsulated strains of Klebsiella, it shows CRD-dependent binding to the spontaneous unencapsulated-phase variants, and can mediate their phagocytosis and killing by alveolar macrophages [17 , 18 , 24 ]. Significantly, MR and SP-A show comparatively little interaction with these unencapsulated variants [17 ]. The spontaneous emergence of unencapsulated bacteria in a population of encapsulated bacterial cells that occurs in vitro [19 , 20 ] is likely to occur in vivo as a requirement for efficient mucosal colonization. Thus, lung mononuclear phagocytes may encounter both forms of bacteria. In our attempts to better understand the mechanisms underlying innate immunity against pulmonary phatogens, we examined in this study cytokine production resulting either from the direct recognition of encapsulated Klebsiella by macrophage MRs or from the indirect SP-D dependent recognition of unencapsulated-phase variants.
We observed that cytokine release and induction of cytokine mRNA by human macrophages interacting with Klebsiella are dependent on recognition of either capsular dimannose sequences by the MRs or on coating of unencapsulated Klebsiella cells with SP-D. An intriguing finding was that the induction of cytokine mRNA via the MR occurred only in monocyte-derived macrophages, whereas that caused by SP-D-coated Klebsiella cells occurred in both monocyte-derived macrophages and peripheral-blood monocytes.
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Bacteria were grown overnight on nutrient agar, harvested by scraping the confluent growth, and resuspended at the desired density in either phosphate-buffered saline, (PBS; 0.1 M NaCl, 0.02 M PO4, pH 7.2), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered saline, pH 7.5, RPMI-1640 medium (Biological Industries, Bet-Haemek, Israel), or F-12 nutrient mixture (Biological Industries) as indicated. The latter two media were supplemented with 0.25% (w/v) NaHCO3, 1% (w/v) glutamine, 15% (v/v) heat-inactivated newborn bovine serum (Beith Sara, Bet-Haemek, Israel), 100 µg/mL of streptomycin, and 100 U/mL of penicillin G. Colony-forming-unit (CFU) counts on agar plates showed that 1 optical density (OD) at 700 nm (OD700) was equivalent to approximately 2 x 109 and 5 x 109 CFU/mL for encapsulated and unencapsulated phase variants, respectively. Stocks with a 50-fold-higher cell density were stored at -70°C with 20% (v/v) glycerol. On the day of assay, bacteria were washed three times to remove the glycerol, diluted to the desired density, and incubated on ice pending use for various assays. The thawed bacteria maintained 100% viability as assessed by CFU counts.
Preparation of SP-D
Human recombinant SP-D (RrSP-D) dodecamers were prepared as
described previously [25
]. For most of the present
studies, contamination with soluble endotoxin was not an issue because
SP-D was used to coat bacteria or beads that were washed with
endotoxin-free buffer prior to their exposure to macrophages.
Nevertheless, the level of endotoxin contamination was routinely
quantified using a sensitive, end-point chromogenic microplate assay
(Chromogenix, Sweden) with Escherichia coli O111:B4
endotoxin as standard. The endotoxin content of the purified
recombinant proteins was 0.3–5 ng/mL or approximately 50 pg/µg of
SP-D for our stock solutions. For individual assays of SP-D activity,
the stock was further diluted over 10-fold in endotoxin-free medium,
giving final endotoxin concentrations of <500 pg/mL, much less than
associated with detectable macrophage activation in our system.
Coating of Klebsiella with SP-D.
Bacterial suspensions (5x1010 CFU/mL) were prepared
in PBS alone or in PBS supplemented with 20 mM
CaCl2 or 20 mM EDTA. Equal volumes of the
bacterial suspensions and PBS or PBS containing 10 µg/mL of SP-D were
incubated for 60 min at room temperature. The bacteria were then washed
three times by centrifugation at 12,100 g to remove unbound
SP-D and resuspended in buffer. Although the bacteria were agglutinated
at the end of the incubation period, after washing off the excess of
SP-D, there were no aggregates in the bacterial suspension. The pellets
of SP-D-coated or -uncoated bacteria were resuspended to the original
density in PBS and maintained at 4°C pending use in the phagocytosis
assays. All media and buffers were assayed for the presence of
bacterial endotoxin by the gel-clot technique using the limulus
amebocyte lysate (Pyrogent, M.A., Bioproducts Inc., Walkersville, MD)
and used only when the amount of endotoxin was <75 pg/mL.
The binding of SP-D, was determined by enzyme-linked immunosorbent assay (ELISA). SP-D coated bacteria (107 bacteria in 50 µL) were allowed to dry overnight at 37°C in wells of microtiter plates. After the wells were washed, the amount of SP-D bound to the immobilized bacteria was estimated by ELISA using anti SP-D diluted to 1:1,000 as the first antibody. The anti SP-D was prepared as previously described [26 ]. ELISA OD values (±SD) of the SP-D bound to Klebsiella were 1.1 ± 0.08 and 0.4 ± 0.03 (P>0.001) for bacteria precoated with SP-D in buffer and EDTA (10 mM)-supplemented buffer, respectively.
Harvest of human monocytes and monocyte-derived macrophages
Peripheral blood human monocytes (HuMo) were obtained from the
buffy coat of normal blood bank donors. The mononuclear fraction was
separated on Ficoll-Hypaque [27
, 28
], and
adherent monocytes were separated as previously described
[28
]. Mononuclear cells (2x106/well in
96-well tissue culture plates or 2.5x107 in
75-cm2-surface-area tissue culture flasks) were incubated
for 1 h, and the resulting monolayers were reconstituted with
RPMI-1640 supplemented with 100 µg/mL of streptomycin, 100 U/mL of
penicillin, 300 µg/mL of glutamine and 10% newborn bovine serum.
Cultured cells were further incubated without changing the medium for
10–14 days in the presence of 100 U/mL of granulocyte-macrophage
colony-stimulating factor (Behringwerke, Marburg, Germany) to obtain
HuMo-derived macrophages (HuMoDM) and promote MR expression
[29
, 30
].
All the media and buffers used in this study were assayed for the presence of bacterial endotoxin by the gel-clot technique using the Limulus Amebocyte Lysate reagent (Pyrogent; BioWhittaker Inc., Walkersville, MD). Reagents were used only if no detectable LPS was found (sensitivity, 0.06 endotoxin U/mL).
Determination of MR-positive cells by immunofluorescence flow
cytometry
Monocytes and MoDM grown in suspension for 4 days in
polypropylene round-bottom tubes (106 cells/tube) were
incubated for 30 min with mouse antihuman MR (PAM-1) monoclonal
antibodies in 50 µL of PBS containing 2% fetal calf serum and 0.1%
sodium azide at 4°C. After three washings the cells were incubated
with fluorescein-isothiocyanate-labeled, affinity-purified
F(ab')2 fragment of goat anti-mouse immunoglobulin G
(Jackson Immunoresearch Laboratories, West Grove, PA). Ten-thousand
gated events were collected in a flow cytometer (FACSsort; Beckton
Dickenson, San Jose, CA), and the number of positively stained cells
was calculated after subtraction of controls, that received the second
antibody only.
Anti-human MR PAM-1 [31 ] was kindly provided by Alberto Mantovani (Mario Negri Institute, Milan, Italy). This antibody (immunoglobulin G1) was obtained by immunizing mice with human alveolar macrophages [32 ].
Production of interleukin-6 by human monocytes and HuMoDM
HuMo and HuMoDM monolayers (2x105 cells per well)
were treated with 2 x 104 CFU of K.
pneumoniae (K2, K21a, K10, and K50) per well or with (LPS, 1
µg/mL) in 96-well tissue culture plates. After 24 h at 37°C
and 7.5% CO2, the supernatants were collected and
interleukin (IL)-6 levels were determined using ELISA with a kit
(Endogen, MA) according to manufacturers instructions.
Induction of cytokine mRNA expression by attachment of K.
pneumoniae to HuMo and MoDM
HuMo and HuMoDM monolayers (107 cells per flask)
were incubated with medium containing 1 µg/mL of LPS or medium
containing 109 CFU/mL of either uncoated
Klebsiella or Klebsiella precoated with SP-D in
the absence or presence of maltose, lactose (50 mM), or mannan (10
mg/mL from Saccharomyces cerevisiae; Sigma). After 30 min of
incubation at 37°C and 7.5% CO2, nonadherent bacteria
were washed away with warm Hank’s balanced salt solution, and the
monolayers were supplemented with RPMI-1640 containing 10% newborn
bovine serum and further incubated for 4 h at 37°C and 7.5°
CO2. The supernatants were removed after incubation, and
total RNA from the cell monolayers was extracted by the single-step
method using guanidinium thiocyanate [33
]. Total
cellular RNA (1 µg) was reverse-transcribed in a 20-µL reaction
mixture containing 1 x PCR buffer [0.1% (v/v) Triton X-100, 10
mM Tris HCl , pH 8.8], 5 mM MgCl2, 1 mM deoxynucleoside
triphosphate, 1 U of ribonuclease inhibitor, 2.5 µM
oligo(dT)15 (Promega, Madison, WI), and 15 U of avian
myeloblastosis virus reverse transcriptase. The reaction mixture was
sequentially incubated for 1 h at 42°C, 5 min at 99°C, and 5
min at 4°C to yield the cDNA from the total mRNA in the original
sample. The mixtures were stored at -20°C pending PCR amplification.
Amplification of human cytokine cDNA was performed by using the
following sense and antisense PCR primers (Bio-Technology General Ltd,
Rehovot, Israel), respectively: human (h)IL-1ß,
5'-atggcagaagtacctaagctcgc-3', 5'-acacaaattgcatggtgaagtcagtt-3; hIL-6,
5'-atgaactccttctccacaagcgc-3', 5'-gaagagccctcaggctggactg-3'; hIL-10,
5'-atgccccaagctgagaaccaagaccca-3', 5'-aagtctcaaggggctgggtcagctatccca-3;
hß -actin, 5'-atggatgatgatatcgccgcg-3',
5'-ctagaagcatttgcggtggacgatggaggggcc-3; hIL-12 (p40),
5'-gcttcttcatcagggacatca-3', 5'-gctgaggtcttgtcggtgaa-3'; and human
tumor necrosis factor (hTNF)-, 5'-atgagcactgaaagcatgatccgg-3',
5'-gcaatgatcccaaagtagacctgccc-3'. The cDNA samples were amplified in a
total volume of 25 µL containing 2.5 mM MgCl2, 50 mM KCl,
1x PCR buffer (10 mM Tris-HCl, pH 8.8), 0.5 U Taq
polymerase (Takara, Tokyo, Japan), and 0.4 mM deoxynucleoside
triphosphate. Reactions were performed in a PCR minicycler (MJ
Research, Boston, MA) for 28 cycles of 94°C for 2 min, 60°C for 2
min, and 72°C for 3 min.
PCR products were then analyzed by agarose (0.2% w/v) gel electrophoresis (Ultrapure agarose, BRL life technologies Inc., Gaithersburg, MD) in 1x Tris-boric acid-EDTA (TBE) buffer (0.045 M Tris, 0.1 M boric acid, and 0.001 EDTA, pH 8.5) supplemented with ethidium bromide (0.005% w/v) for DNA staining. Each PCR product (20 µL) was mixed with 5 µL of loading solution [0.1 M EDTA, 40% sucrose (w/v), and 0.05% (w/v) bromophenol blue and 0.05% sodium lauryl sulfate] and applied to an agar-gel well. Gels were run in 1x TBE buffer for 60 min at 100 V. The DNA bands of the PCR products were visualized on a UV transilluminator, and their density was determined by imaging densitometry using a Kodak DC-120 digital camera and Kodak DS-10 software (Kodak, New Haven, CT).
The densitometry ratios of IL-6 to ß-actin transcripts derived from the macrophages stimulated with indicated Klebsiella strains were expressed as percentages of the ratio obtained with LPS (1 µg/mL). LPS as standard stimulant was included in all experiments and gave an IL-6/ß-actin ratio (±SD) of 0.62 ± 0.11.
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 View larger version (49K): [in a new window] |
Figure 1. Determination of MR-positive cells by Immunofluorescence flow
cytometry. Monocytes (A, B) and MoDMs (C, D) grown in suspension for 4
days in polypropylene round-bottom tubes (106 cells/tube)
were incubated for 30 min with mouse anti-human MR monoclonal
antibodies (PAM-1) at 4°C (A, C). After three washings, the cells
were incubated with fluorescein-isothiocyanate-labeled
affinity-purified F(ab')2 fragment of goat anti-mouse
immunoglobulin G. Ten-thousand gated events were collected in a flow
cytometer.
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 View larger version (65K): [in a new window] |
Figure 2. IL-6 production by HuMoDMs stimulated with encapsulated and
unencapsulated variants of K. pneumoniae. Cultured
macrophages were incubated with 105 CFU/mL of each
encapsulated parent strain of indicated capsular serotypes or the
corresponding unencapsulated variants as described in the text. Values
are means plus or minus standard deviations of at least three
experiments. IL-6 release by nonstimulated macrophages was 19 ± 5
µg/mL.
|
and IL-10
mRNA and moderate levels of IL-1 mRNA. Both LPS and the K21a
Klebsiella strain triggered in the differentiated
macrophages high mRNA expression for all the cytokines. Exposure of the
differentiated macrophages to K2 bacteria resulted in the expression of
low levels of IL-6, IL-10, and IL-12 and moderate levels of IL-1 and
TNF-. Cytokine mRNA expression triggered by K2 was above the level
exhibited by nonstimulated macrophages but lower than levels observed
for K21a- or LPS-triggered cells (Fig. 3)
.
 View larger version (68K): [in a new window] |
Figure 3. PCR-assisted mRNA amplification of cytokines in monocytes and HuMoDMs
stimulated with K2 and K21a encapsulated Klebsiella strains.
Representative agarose gel electrophoresis of reverse
transcriptase-PCR-amplified samples of cDNA corresponding to ß-actin
and indicated cytokines produced by monocytes (left gel) and MoDMs
(right gel). Lane 1, mRNA expressed in unstimulated macrophages; lane
2, macrophages stimulated with LPS (1 µg/mL); lanes 3 and 4,
stimulated with K2 and K21a, respectively.
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Figure 4. mRNA cytokine expression in monocytes (A) and macrophages (B)
stimulated by encapsulated and unencapsulated K50 strains and the
effect of SP-D coating. Values shown are the means and standard
deviations of triplicates for indicated cytokine mRNA stimulation by
K50-encapsulated, K50-3OF-unencapsulated, and SP-D-coated
unencapsulated strains of K. pneumoniae as percent of
cytokine/actin ratios obtained in cells stimulated by LPS (1 µg/mL).
Cytokine/actin ratios after LPS activation of macrophages were:
0.53 ± 0.08, 0.35 ± 0.06, and 0.45 ± 0.1 for IL-6,
IL-10, and IL-12, respectively. LPS activation of monocytes yielded
cytokine/actin ratios of 2.6 ± 0.8, 1.7 ± 0.2, and 3.2 ± 0.4 for IL-6, IL-10, and TNF-, respectively. IL-6, IL-10, and
TNF- mRNA expression by nonstimulated monocytes was 21%, 18%, and
22% of LPS, respectively. IL-6, IL-10, and IL-12 mRNA expression by
nonstimulated macrophages was 21%, 20%, and 27% of LPS,
respectively.
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It is interesting that the SP-D-opsonized, unencapsulated Klebsiella also stimulated cytokine mRNA expression in fresh monocytes to a level comparable to that obtained with LPS (Fig. 4A) . However, the magnitude of stimulation of monocytes with the encapsulated parent strain preincubated with SP-D was not significantly different (P<0.1) from that of unstimulated macrophages (data not shown).
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The encapsulated K21a, K10, and K50 strains, all of which are recognized by MR, stimulated IL secretion by macrophages, whereas their corresponding unencapsulated-phase variants did not. In addition, the encapsulated K2 strain, which lacks the dimannose residues in its capsule, did not stimulate secretion. Similarly, the encapsulated K21a strain stimulated transcription of cytokine mRNA by macrophages. Significantly, the stimulation was inhibited by mannan, a ligand for MR. By contrast, the encapsulated K2 strain or its unencapsulated variant showed little effect on macrophage-cytokine transcription, consistent with the cytokine secretion data. Blood monocytes, which do not express detectable MR, were poorly stimulated by the encapsulated K21a strain.
These data taken together suggest that encapsulated K. pneumoniae can stimulate human macrophages if recognized by the MR via corresponding glycoconjugates in their capsular polysaccharides. These results are consistent with studies showing that the fungi Candida albicans, Candida neoformans, and Pneumocystis carinii can interact with macrophage MR and trigger the production of cytokines. [1 , 13 , 35 , 36 ]. The MR may also mediate cytokine production by dendritic cells triggered with enveloped viruses [37 ] or by spleen cells phagocytosing chitin [38 ].
In this respect, macrophage MR may be responsible for immediate recognition and elimination of pathogens expressing the corresponding sugar residues and for initiating inflammatory or immunological reactions that can prevent the spread and colonization of the pathogens. SP-A appears to fulfill similar functions because it recognizes dimannose-containing strains and acts as an opsonin to enhance their phagocytosis via SP-A receptors of macrophages [23 ]. The apparent overlapping functions of SP-A and the MR are not compatible with the observations showing that otherwise healthy transgenic mice lacking a functional SP-A gene exhibit increased susceptibility to strains of group B streptococci, Staphylococcus aureus and Pseudomonas aeruginosa [reviewed in ref. 39 ]. Since the sugar specificity of the interaction of these strains with SP-A versus MR was not defined, it is difficult to assess the relative role of each receptor in the defense against these bacteria. Moreover, the relative susceptibility of the SP-A knockout mice to dimannose-expressing and -nonexpressing bacteria was not assayed. It is too early, therefore, to speculate whether the MR alone would be sufficient to provide innate immunity against infections caused by dimannose-expressing bacteria.
SP-D, which serves as an LPS-mediated opsonin in vivo [40 ], may bind to macrophage membranes via receptors such as the glycoprotein receptor gp-340 [41 ] and serve as an opsonin for Klebsiella [18 , 24 ]. In this study we report for the first time that SP-D-coated unencapsulated Klebsiella strain K50-3OF potently stimulated cytokine transcription by both macrophages and blood monocytes, whereas unencapsulated Klebsiella strains alone did not alter cytokine production. The SP-D-dependent stimulation was not observed when the coating with SP-D was performed in the presence of maltose, a specific inhibitor of SP-D-bacteria interactions [16 ]. The magnitude of cytokine expression triggered by dimannose-carrying encapsulated bacteria with SP-D was not significantly different from that triggered by uncoated bacteria alone, suggesting that there is no overlap in function between the MR and SP-D.
Our findings cannot be attributed to contaminating endotoxin because
the measured levels of endotoxin in the preparations and solutions used
in this study are considerably below those needed to stimulate human
monocytes and macrophages. We found that the minimal LPS concentrations
required to stimulate IL-1ß and TNF- production by monocytes and
MoDM were in the range of 1–10 ng/mL and 10-100 ng/mL, respectively
(unpublished data).
The concentration of SP-D used in this study (10 µg/mL) may fall in the range of physiological lung concentrations. There is considerable uncertainty regarding the physiological concentration of SP-D at potential sites of microbial interaction in the lung. It is secreted by more than one epithelial-cell type, and the fraction secreted by bronchiolar cells is probably subject to regulation. Concentrations could conceivably be very high in the local vicinity of a Clara cell secreting its granules. The best estimates are based on recovery by bronchoalveolar lavage normalized for estimated alveolar surface area. These estimates range from 3 µg/mL in rats to estimates as high as 60 µg/mL in humans [reviewed in ref. 42 ].
Opportunistic pathogens such as K. pneumoniae primarily attack immunocompromised individuals who are hospitalized and have severe underlying diseases [43 ]. Colonization of the upper respiratory tract by gram-negative bacteria precedes entry of the organisms into the lung [44 , 45 ]. Because a capsule interferes with the expression of adhesins required for colonization of epithelial cells by the organisms, it is likely that most of the bacteria colonizing the upper respiratory tract (or other mucosal surfaces) are in the unencapsulated phase [19 , 46 ]. MR-equipped macrophages might provide partial protection by eliminating specific encapsulated Klebsiella strains through recognition of the dimannose ligand. Klebsiella opsonization mediated by SP-D can serve as a complementary defense mechanism against unencapsulated phenotypes, because SP-D interacts with the conserved core region of bacterial LPS [16 ].
The present study showed that the induction of inflammatory cytokines could be mediated by the macrophage MR as well as by SP-D-coated bacteria, and both mechanisms are implicated in the protection of the lung against Klebsiella infections. An interesting feature of the SP-D-dependent stimulation of phagocytic cells is that, unlike MR-dependent stimulation, it involves also blood monocytes. Although SP-D may protect the lung from unencapsulated phase variants and prevent their proliferation in the lower respiratory tract, the presence of large numbers of unencapsulated SP-D-coated bacteria could trigger or amplify an inflammatory response that might be further exacerbated by the infiltration of blood monocytes.
Received April 25, 2000; revised July 8, 2000; accepted February 7, 2001.
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