Thoracic Diseases Research Unit, Departments of Pulmonary, Critical Care and Internal Medicine, and Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Inflammatory cell recruitment contributes to
respiratory impairment during Pneumocystis
carinii pneumonia. We evaluated expression of
intercellular adhesion molecule-1 (ICAM-1), a key participant in
leukocyte accumulation, in rats with
P.
carinii pneumonia. Immunostaining for
ICAM-1 was most marked on bronchiolar epithelium but was also evident
on type II pneumocytes, endothelium, and macrophages. Lung from normal
and dexamethasone-treated uninfected animals exhibited markedly less
ICAM-1. We hypothesized that P. carinii promoted ICAM-1 expression in
epithelium through tumor necrosis factor-
(TNF-
) release from macrophages or that
P. carinii directly stimulated ICAM-1
expression. Alveolar macrophages were incubated with
P.
carinii, and the medium was added to
A549 epithelial cells. Treatment of macrophages with
P.
carinii enhanced A549 ICAM-1, which
was inhibited with antibody to TNF-
. To determine whether P.
carinii alone also stimulated ICAM-1,
A549 cells were cultured with P.
carinii, also augmenting ICAM-1. Of
note, A549 ICAM-1 expression from P.
carinii alone was less than with
P. carinii-exposed macrophages. Thus
ICAM-1 is enhanced in lung epithelium during
P.
carinii infection, in part, through
TNF-
-mediated mechanisms.
intercellular adhesion molecule-1; macrophage; tumor necrosis
factor-
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INTRODUCTION |
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PNEUMOCYSTIS carinii pneumonia is an opportunistic fungus causing severe pneumonia in immunocompromised hosts. Despite effective prophylaxis and treatment regimens, this infection remains a major complication of patients with human immunodeficiency virus infection or hematologic or solid malignancies or after organ transplantation (13, 22, 41). Among patients with acquired immunodeficiency syndrome (AIDS), severe P. carinii pneumonia is the most frequent cause of acute respiratory failure requiring admission to the intensive care unit (7, 39). Furthermore, the mortality of P. carinii pneumonia in all patients ranges between 15 and 40% (7, 22, 39). Prior investigations indicate that lung inflammation contributes significantly to respiratory impairment and clinical outcome during P. carinii pneumonia (22, 35). In particular, increased numbers of neutrophils in bronchoalveolar lavage predict poorer oxygenation and survival during P. carinii pneumonia (22). The contention that pulmonary inflammation potentiates lung injury during P. carinii pneumonia is further supported by clinical observations that corticosteroids prevent deterioration and improve outcome in moderate to severe P. carinii pneumonia (28).
The mechanisms regulating lung inflammation during P. carinii pneumonia remain poorly understood. Recent
investigations have focused on a family of cell surface adhesion
molecules that enable circulating leukocytes to accumulate in areas of
lung inflammation (1, 33). Of primary importance is intercellular
adhesion molecule-1 (ICAM-1), a member of the immunoglobulin (Ig)
supergene family, that, together with its ligands lymphocyte
function-associated antigen-1 and macrophage-1 antigen,
promotes the firm adherence of leukocytes onto vascular endothelium,
thereby permitting transmigration into injured tissues. In addition to
vascular endothelium, ICAM-1 is also expressed by epithelial cells,
lymphocytes, platelets, monocytes, and macrophages (6, 12, 33). The
expression of ICAM-1 is upregulated by proinflammatory cytokines, most
notably tumor necrosis factor-
(TNF-
) and
-interferon
(
-IFN; see Refs. 6, 12, and 24). ICAM-1 has previously been
implicated in the inflammatory component of several lung diseases,
including tuberculosis, asthma, and sarcoidosis (12, 36, 40). However, the expression of ICAM-1 in P. carinii
pneumonia has not yet been evaluated.
A number of investigations indicate that TNF-
expression is augmented during P. carinii pneumonia in humans and animal models (9, 18,
19). Studies from our laboratory have demonstrated that
P. carinii stimulates release of
TNF-
from macrophages through interaction of the
cell wall of the organism with cognate macrophage receptors (29). We
hypothesized that this enhanced release of TNF-
would act locally to increase ICAM-1 expression during
P. carinii pneumonia. Accordingly,
this investigation was undertaken to address the following goals:
1) to determine the extent and cellular localization of ICAM-1 protein expression during
P. carinii pneumonia and
2) to determine potential mechanisms
by which ICAM-1 expression may be regulated during P. carinii infection, particularly whether alterations in
ICAM-1 occur through TNF-
-mediated mechanisms.
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MATERIALS AND METHODS |
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Materials.
Mouse monoclonal anti-rat ICAM-1 antibody for immunohistochemistry was
obtained from Genzyme (Cambridge, MA), and monoclonal mouse anti-human
ICAM-1 antibody used in the enzyme-linked immunosorbent assay (ELISA)
was from R & D Systems (Minneapolis, MN). Recombinant human
TNF- was purchased from Genzyme, and type I collagen
was from Vitrogen (Palo Alto, CA). Sheep anti-mouse IgG conjugated to
-galactosidase and
p-nitrophenyl-
-galactoside
were from GIBCO-BRL Laboratories (Gaithersburg, MD). A human ICAM-1
cDNA was the kind gift of Dr. Michael Dustin (Washington University,
St. Louis, MO; see Ref. 37). Ciprofloxacin was provided by Miles
Pharmaceuticals (West Haven, CT).
Preparation of P. carinii. All animal studies were approved by the appropriate institutional animal care and utilization committee. P. carinii pneumonia was induced in Harlan Sprague-Dawley rats by immunosuppression with dexamethasone and transtracheal injection with P. carinii as previously described (2, 21, 31). Specific pathogen-free rats were provided freely with drinking water containing 2 mg/l dexamethasone, 500 mg/l tetracycline, and 200,000 U/l nystatin and were fed an 8% protein diet (Teklad, Madison, WI). On a weekly basis, the animals also received 0.45 g/l oral ciprofloxacin for 2 consecutive days to further reduce the risk of bacterial infections. After 5 days of immunosuppression, rats were transtracheally inoculated with P. carinii (~500,000 cysts) prepared by homogenizing infected rat lung in a Stomacher microbiological blender (Tekmar, Cincinnati, OH). After tracheal injection, the rats were immunosuppressed for an additional 6-8 wk and were killed, and whole lung lavage was performed with 50 ml of Hanks' balanced salt solution (HBSS). P. carinii were purified from this lavage by differential centrifugation (21). Lavage fluid was centrifuged (400 g for 10 min), and associated P. carinii cysts were identified in the pellet by Diff-Quik staining. The supernatant containing predominantly suspended P. carinii organisms was recentrifuged (1,400 g for 30 min), and the pellet was resuspended in 1 ml of HBSS. Duplicate 10-µl aliquots of suspension were spotted onto glass slides and stained with Diff-Quik, and P. carinii were quantified as previously described (2, 21). If other microorganisms were noted in the lavage smear or on microbiological culture, the material was discarded. P. carinii isolates were found to contain <0.125 U/ml of soluble endotoxin using a sensitive Limulus amoebocyte lysate assay as previously described (29).
Immunolocalization of ICAM-1 in P. carinii-infected rat lung. Lung specimens were obtained from moribund rats with well-established P. carinii pneumonia 6-8 wk after inoculation, fixed with 10% phosphate-buffered Formalin at 15 cmH2O pressure, embedded in paraffin, and sectioned (20, 31). Five-micrometer sections were deparaffinized with xylene and graded alcohols and were submitted to immunohistochemical examination by an avidin-biotin-mediated immunoperoxidase method (Vectastain ABC method; Vector Laboratories). Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol for 30 min. Nonspecific staining was diminished by incubation with 1% normal goat serum. Monoclonal mouse anti-rat ICAM-1 (10 µg/ml; Genzyme) was applied to the sections overnight. After being washed, the sections were sequentially incubated with biotinylated goat anti-mouse antibody followed by an avidin-biotinylated horseradish peroxidase macromolecular complex. Bound antibodies were localized with diaminobenzidine tetrahydrochloride in the presence of H2O2 and were counterstained with 1% methyl green. To confirm the specificity of staining, parallel incubations were performed with nonimmune mouse IgG at identical concentrations.
For comparison to P. carinii-infected lung samples, parallel ICAM-1 stains were performed on rat lungs from uninfected normal rats and from animals that were identically treated with corticosteroids but that also received 160 mg/l trimethoprim and 800 mg/l sulfamethoxazole added to the drinking water to prevent development of P. carinii pneumonia. To further quantify the relative expression of ICAM-1 during P. carinii pneumonia, additional lung specimens from P. carinii-infected animals and from dexamethasone-treated control animals without P. carinii were stained, and the staining intensity was scored. The specimens were coded and reviewed in a blinded fashion. Analogous to a previously published immunohistochemical study by Limper et al. (20), each specimen was given a score from zero to four, where zero was no detectable ICAM-1 staining, one was minimally detectable staining, two was weakly positive staining, three was moderate ICAM-1 staining, and four was abundant ICAM-1 staining.P. carinii induction of ICAM-1 expression in lung epithelial cells.
P. carinii induces alveolar
macrophages (AMs) to release TNF-, a potent
stimulant of ICAM-1 expression (29). To determine whether
P. carinii-induced macrophage release
of TNF-
caused enhanced ICAM-1 expression by lung
epithelial cells, AMs were cultured with P. carinii. Subsequently, the conditioned medium was
removed and plated on A549 lung cells, and cell-surface ICAM-1 expression was determined after further overnight incubation. Specifically, uninfected Harlan Sprague-Dawley rats were killed, and
whole lung lavage was performed with 50 ml of HBSS. Such lavages contain >95% AMs (8). Macrophages were plated in 24-well plates (100,000/well) and were incubated overnight in the presence of P. carinii in Dulbecco's modified
Eagle's medium with 10% fetal calf serum, 2 mM glutamine, 10,000 U
penicillin/l, 1 µg streptomycin/l, and 25 µg
amphotericin/l at 37°C. The conditioned media were removed, clarified by centrifugation (12,000 g
for 5 min), and added to confluent monolayers of A549 lung epithelial
cells (CCL-185; American Type Culture Collection, Rockville, MD), a
cell line that has proven useful for examination of P. carinii-epithelial interactions (11, 21). The next day,
ICAM-1 expression on the A549 cells was evaluated by cell-based ELISA
(24). To study the role of TNF-
and other cytokines
in mediating epithelial ICAM-1 expression, conditioned media were
treated with TNF-
antiserum, antibody to
-IFN, or nonimmune rabbit serum or nonimmune mouse
IgG (1:40 dilution of rabbit sera or 50 µg/ml of
mouse IgG) for 30 min before and throughout the subsequent incubation
on A549 cells.
Evaluation of cell surface ICAM-1 on A549 cells.
After incubation of A549 cells with macrophage-conditioned media or
with P. carinii alone, cell surface
ICAM-1 was quantified using a cell-based ICAM-1 ELISA (24). A549 cell
monolayers were washed with tris(hydroxymethyl)aminomethane-buffered
saline containing 0.5% bovine serum albumin (BSA), 1 mM
CaCl2, and 1 mM
MgCl2 and were fixed in 1%
paraformaldehyde for 15 min at 25°C. Next, the cells were blocked
with 2% BSA for 1 h at 37°C. After being washed, the cells were
sequentially incubated with monoclonal anti-human ICAM-1 antibody (100 µl of a 10 µg/ml stock solution) at
37°C for 1 h, followed by a secondary sheep anti-mouse IgG
F(ab')2 conjugated to
-galactosidase (1:200 dilution) over an additional hour. Bound antibodies were detected with 1 mg/ml
p-ni-
trophenyl-
-D-galactoside in 50 mM phosphate buffer with 1.5 mM
MgCl2 (pH 7.2) reacting for 15 min
at 37°C, and absorbances were determined at 405 nm. Unless
otherwise stated, the amount of basal ICAM-1 expressed by unstimulated
A549 cells was subtracted from each sample to correct for background
expression of ICAM-1.
Analysis of steady-state ICAM-1 mRNA content in A549 cells
stimulated with P. carinii.
Northern hybridizations were performed to additionally determine
whether P. carinii induced changes in
ICAM-1 mRNA expression in A549 cells. A549 cells were plated in
six-well plates (5 × 106
cells/well) and were grown to confluence at 37°C over 48 h.
Subsequently, the cells were incubated with P. carinii at the indicated concentrations overnight.
Total RNA was extracted from the cells with the use of a monophasic
solution of phenol and guanidine isothiocyanate followed by chloroform
extraction and isopropyl alcohol precipitation (Trizol reagent;
GIBCO-BRL). Equal amounts of total RNA from each condition (20 µg) were loaded and were separated by electrophoresis through a 1.2% agarose gel in the presence of 2.2 M formaldehyde. Equal loading of the RNA was verified by ethidium bromide staining of
18S and 28S rRNA, and the separated RNA was transferred to nitrocellulose membranes and was prehybridized (ExpressHyb; Clonetech Laboratories). A radiolabeled ICAM-1 probe was generated from human
ICAM-1 cDNA in the pAprM8 plasmid (37). This plasmid was digested with
Hind III and Pst I to
release an 800-bp fragment that was separated on agarose and was
labeled with
[-32P]dCTP
(NEN) by a random-primer method (Rediprime; Amersham). The radiolabeled
probe was added to hybridization solution (2 × 106
counts · min
1 · ml
1)
and was incubated with the membranes for 2 h at 68°C. After hydridization, the membranes were washed with 2× salt-sodium
citrate solution (SSC; 1× solution contained 150 mM NaCl and 15 mM sodium citrate at pH 7.0) with 0.05% sodium dodecyl sulfate (SDS)
at room temperature for 40 min followed by 0.1× SSC with 0.1%
SDS solution at 50°C for 40 min. The blots were visualized by
autoradiography. To further verify equal RNA loading, the membranes
were stripped in 1% SDS and were rehybridized with a
32P-labeled probe complementary to
the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene product. Scanning densitometry of the resulting
autoradiographic images was performed, and results were expressed as
ICAM-1-to-GAPDH ratios.
Statistical methods. Data are expressed as means ± SE. Differences between multiple data groups were first assessed using the one-way analysis of variance. Differences between experimental data groups were analyzed using the two-sample Student's t-test for normal parameters and the Mann-Whitney U-test for nonparametric data. P < 0.05 was defined to represent a statistically significant difference.
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RESULTS |
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Immunohistochemical localization of ICAM-1 during P. carinii pneumonia. In all rats studied, markedly enhanced ICAM-1 was detected compared with normal lung (Fig. 1). Lung tissues infected with P. carinii revealed intense staining for ICAM-1 on bronchiolar epithelial cells (Fig. 1A), proliferative type II pneumocytes (Fig. 1D), and vascular endothelial cells (Fig. 1C). Additionally, ICAM-1 was detected in smooth muscle cells surrounding airways (Fig. 1A) and blood vessels (Fig. 1C) and on AMs (Fig. 1D) in these infected animals. To confirm the specificity of the antibody staining for ICAM-1, serial sections were incubated with an identical concentration of nonimmune mouse IgG that failed to demonstrate any specific reactivity (Fig. 1B). For comparison, normal uninfected rat lungs were also stained to detect basal tissue expression of ICAM-1 protein. In normal rat lungs, ICAM-1 was detected in epithelial cells but in markedly less quantities than in infected rats (Fig. 1E). In addition, markedly less ICAM-1 staining was detected in endothelial cells and in AMs compared with tissues obtained from rats with P. carinii pneumonia. Thus ICAM-1 is present in enhanced quantities in specific lung cell populations during P. carinii pneumonia in infected rats.
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AMs infected with P. carinii induce ICAM-1 surface expression on
A549 lung epithelial cells.
Having observed markedly enhanced quantities of ICAM-1 on lung
epithelium during P. carinii
infection, we next sought to determine potential mechanisms by which
ICAM-1 expression might be mediated. Prior studies indicate that AMs
bind and phagocytize P. carinii and
are stimulated by the organism to release oxidants, eicosanoids, and
TNF-, a potent stimulant of ICAM-1 expression in the
epithelium (8, 15, 29). To investigate whether macrophage interaction with P. carinii causes cytokine
release, thereby promoting ICAM-1 expression in epithelial cells, AMs
were preincubated with P. carinii, and
the resulting conditioned medium was removed and recultured on A549
alveolar epithelial cells (Fig. 2).
Conditioned media obtained from cultures of control macrophages
incubated in the absence of P. carinii
mildly stimulated basal ICAM-1 expression in A549 cells. However,
culturing AMs with increasing numbers of P. carinii resulted in significant enhancement in
immunoreactive ICAM-1 expression in the A549 epithelial cells.
Incubating P. carinii with AMs at a
P.
carinii-to-AM ratio of 1:1 resulted in 292 ± 107% enhanced expression of ICAM-1 in A549 epithelial cells compared with control macrophage-conditioned media incubated without organisms (P = 0.009). Incubations of
P. carinii with macrophages at a
P.
carinii-to-AM ratio of 5:1 resulted in
199 ± 41% enhanced ICAM-1 expression
(P = 0.006 compared with control).
These data indicate that the interaction of P. carinii with AMs enhances ICAM-1 expression on cultured
lung epithelial cells.
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P. carinii directly promotes enhanced ICAM-1 surface expression on A549 cells. During the life cycle of the organism, P. carinii trophozoites attach to alveolar epithelial cells by intimate approximation and interdigitation of their surfaces with the cell membranes of host epithelium (21). We postulated that the interaction of P. carinii with A549 lung epithelial cells might directly stimulate surface expression of ICAM-1. To examine this, freshly purified P. carinii were incubated overnight on A549 cell monolayers. The next day, the monolayers were washed, and A549 surface ICAM-1 expression was determined by ELISA with a monoclonal antibody specific for human ICAM-1 (Fig. 3). Increased ICAM-1 expression occurred between a P. carinii-to-A549 ratio of 0:1 compared with 1:1, 2.5:1, 5:1, and 10:1. A P. carinii-to-A549 ratio of 1:1 resulted in significantly increased A549 ICAM-1 surface expression (P = 0.022), whereas a P. carinii-to-A549 ratio of 5:1 yielded maximal enhancement of expression (P = 0.0217 compared with A549 cells incubated without P. carinii). Limitations in P. carinii enumeration and in assay sensitivity made it difficult to reliably assess ICAM-1 differences using ratios of infectivity between 0:1 and 1:1. At the higher ratio of 10:1 P. carinii to A549 cells, a minor reduction in maximal A549 ICAM-1 expression was observed compared with the 5:1 concentration. However, the ratio of P. carinii to A549 cells of 10:1 reliably induced A549 ICAM-1 expression, and this level of ICAM-1 was not statistically different from that induced by the 5:1 concentration of P. carinii. These data indicate that the interaction of P. carinii organisms with A549 lung epithelial cells also exerts a direct stimulation of ICAM-1 expression. It should be noted, however, that the maximal absolute level of ICAM-1 surface expression induced by P. carinii alone was approximately one-third of that induced by P. carinii-exposed AMs in parallel experiments. Therefore, P. carinii directly stimulate cultured A549 lung epithelial cells to express ICAM-1, however, at an apparently lower level than that induced by AM products after P. carinii challenge.
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P. carinii increase steady-state ICAM-1 mRNA in cultured A549 lung
cells.
It was conceivable that the P. carinii
preparations might contain small amounts of contaminating rat ICAM-1
that, if transferred into the A549 cell cultures, might be detected
with the cell-based ELISA assay. This was considered rather unlikely
because the A549 cell monolayers were thoroughly washed before analysis
and were examined using a monoclonal antibody specific for the human
ICAM-1 expressed by A549 cells. However, to further confirm that
P. carinii stimulates the expression
of ICAM-1 in lung epithelial cells, we examined steady-state ICAM-1
mRNA expression in A549 cells cultured with increasing numbers of
P. carinii using a cDNA probe specific
for human ICAM-1 (Fig. 4). The human cDNA
probe was hybridized with a 1.3-kb RNA species from A549 cells.
Incubation of A549 cells in the presence of P. carinii augmented the levels of steady-state ICAM-1
mRNA at P.
carinii-to-A549 ratios of 5:1 and
10:1. This was comparable with the pattern of hybridization observed
using RNA obtained from A549 cells incubated with
TNF-, a potent inducer of epithelial ICAM-1
expression. No appreciable ICAM-1 mRNA induction was observed in A549
cells incubated in the absence of P. carinii. Equality of RNA loading under the different
conditions was initially confirmed by ethidium bromide stains of the
18S and 28S rRNA bands. However, to further verify equivalency of
loading, the membranes were rehybridized with GAPDH cDNA, a
constitutively expressed target RNA (Fig. 4). Scanning densitometry of
these blots (Fig. 5) demonstrated that the
ICAM-1-to-GAPDH ratio increased from 0.07 ± 0.02 in A549 cells
incubated in the absence of P. carinii to 0.42 ± 0.17 in A549 cells incubated with a
P.
carinii-to-A549 cell ratio of 5:1 and
to 0.86 ± 0.28 in A549 cells cultured with a ratio of 10:1
(P = 0.025 each compared with A549
cells incubated without P. carinii).
Taken together, these data confirm that P. carinii significantly augments ICAM-1 gene expression
in A549 lung epithelial cells. In addition, the above findings further confirm that the enhancement of surface ICAM-1 expression on A549 cells
infected with P. carinii was due to
direct stimulation of the epithelial cells by the organisms and does
not likely represent ICAM-1 contamination carried over with
P. carinii inoculae.
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P. carinii enhance ICAM-1 expression in A549 lung cells through
TNF--mediated mechanisms.
To evaluate potential mechanisms by which P. carinii induce ICAM-1 expression in lung epithelial
cells, we next studied the role of macrophage-derived
TNF-
in our model system of P. carinii infection and ICAM-1 expression. Accordingly,
conditioned media derived from AMs cultured with P. carinii (P.
carinii-to-AM ratio of 10:1) were
clarified by centrifugation and were treated with a neutralizing
TNF-
antiserum (Genzyme; 1:40 dilution) for 30 min
before and throughout the subsequent incubation on A549 cells (Fig.
6). Treatment of conditioned media with
anti-TNF-
resulted in 80.7 ± 12.3% inhibition
of the maximal ICAM-1 expression induced by untreated conditioned media
(P = 0.002). Nonimmune antiserum at
identical concentrations had no significant effect on ICAM-1 expression
by A549 cells. Furthermore, treatment of macrophage-conditioned media
with antibody to
-IFN, a cytokine that is known to
induce epithelial ICAM-1 expression but that is not released from AMs in substantial quantities, had no significant effect on A549 ICAM-1 expression (P = 0.09, not
significantly different from control).
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DISCUSSION |
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This study demonstrates that ICAM-1, a potent leukocyte cell adhesion
molecule, is present in enhanced quantities in the lung during
P. carinii pneumonia in a rat model.
Increased ICAM-1 protein expression was chiefly observed on bronchial
and type II epithelial cells, endothelial cells, smooth muscle cells,
and AMs in animals with established P. carinii infection. We have further demonstrated that
P. carinii induces enhanced ICAM-1
expression in cultured A549 lung epithelial cells through interaction
of the organisms with AMs and through the subsequent release of
TNF-. In addition, direct contact of
P. carinii with cultured lung
epithelial cells also significantly augments ICAM-1 expression through
alternate mechanisms.
ICAM-1 likely provides a number of key host defense functions during P. carinii pneumonia. Endothelial expression of ICAM-1 promotes the adherence, migration, activation, and subsequent recruitment of mononuclear cells, neutrophils, and T lymphocytes into tissues (1). Consistent with this, perivascular monocytoid infiltrates are typically observed during development of P. carinii pneumonia. Furthermore, patients with severe P. carinii pneumonia document a marked accumulation of neutrophils in the lung (22). Interestingly, T lymphocyte recruitment is also prominent during P. carinii pneumonia. Although CD4 lymphocytes are present in deficient numbers in the lower respiratory tract of patients with P. carinii pneumonia associated with AIDS or malignancy, recent investigations indicate that CD8 lymphocytes are recruited into the lungs during P. carinii infection (5, 27). These CD8 cells, although less effective than CD4 cells in mediating host defense, participate in a weaker fashion in suppressing the establishment of P. carinii pneumonia (5).
We observed augmented ICAM-1 expression on lung epithelial cells during P. carinii pneumonia. It is noteworthy that epithelial surfaces represent key sites controlling the life cycle of P. carinii. Adherence of P. carinii trophozoites to respiratory epithelial cells promotes proliferation of the organism and is a central event in the establishment of lung infection (21, 23). ICAM-1 expression on these epithelial cells concomitantly promotes adherence, migration, and activation of recruited macrophages and neutrophils that interact with the organisms and reduce its viability (1, 33). Thus enhanced ICAM-1 with augmentation of host defense cells at the organism-epithelium interface may critically interfere with this essential phase of the P. carinii life cycle.
Our investigations also evaluated potential mechanisms through which
epithelial ICAM-1 expression may be augmented during P. carinii infection. AMs release
TNF- when challenged with P. carinii by interaction of
-glucan on
the organisms with macrophage glucan receptors, a process that is
augmented by vitronectin, fibronectin, and specific
anti-Pneumocystis antibodies (29). TNF-
expression is upregulated during
P. carinii pneumonia in both animals
and humans and is necessary for optimal clearance of infection (9, 18,
19). TNF-
additionally promotes endothelial and
epithelial cell ICAM-1 expression (1, 14). As anticipated, in this
model system we observed that interaction of P. carinii with AMs promoted increased ICAM-1 expression
by A549 lung epithelial cells through release of
TNF-
from P. carinii-activated macrophages.
Cultured bronchial, tracheal, and alveolar epithelial cells have all
been reported to express ICAM-1 (10, 12, 18). Look and colleagues (24)
demonstrated selective -IFN responsiveness of
bronchial and tracheal epithelial cell lines. In our current study,
antibody neutralization of
-IFN in conditioned media
from P. carinii-stimulated macrophages
had minimal effect on ICAM-1 expression by A549 lung epithelial cells.
However,
-IFN is predominately produced by large T
lymphocytes, and minimal amounts of
-IFN are
released from AMs (17). The current findings do not preclude a role for
-IFN-mediated expression of ICAM-1 in vivo but
likely reflect the small amount of
-IFN released by
macrophages during P. carinii
infection in the system employed. Indeed, prior investigations suggest
that
-IFN promotes optimal clearance of
P. carinii in vitro
(9). It has been proposed that lymphocytic
destruction in patients with AIDS results in a relative deficiency of
-IFN during P. carinii pneumonia (4, 27). Such relative deficiency of
-IFN during P. carinii pneumonia may reduce ICAM-1 expression in the
lung, thus hindering complete clearance of infection in these hosts.
Interestingly, our study revealed that purified P. carinii also directly stimulate ICAM-1 by A549 lung
epithelial cells. The mechanisms of this direct ICAM-1 stimulation are
not yet clear but may involve either surface antigens of the organism
or host proteins bound to the microbe that stimulate the epithelial
cells. The interaction of P. carinii
with alveolar epithelial cells is a complex process involving
glycoprotein A (gpA), a mannose-rich surface protein of
P. carinii, as well as host alveolar
proteins, including fibronectin, vitronectin, surfactant components,
and other host-derived molecules that coat the organisms and promote adherence to the lung epithelium (28, 31, 34).
Interestingly, TNF- also binds to a soluble cell
wall fraction of P. carinii and is
present on freshly isolated organisms (30, 32). The mechanisms of
TNF-
binding to P. carinii are not fully known, but recent investigations
demonstrate that TNF-
interacts with fungal cell
wall
-glucans through lectin-mediated binding (30). Lectin interactions utilize a region of the TNF-
molecule distinct from that which recognizes mammalian
TNF-
receptors (30). Although we postulated that
P. carinii-associated
TNF-
might mediate enhanced ICAM-1 expression after
organism binding to epithelial cells, ICAM-1 expression by A549 cells
directly stimulated with freshly isolated P. carinii was not significantly inhibited by
TNF-
antiserum, suggesting that alternate mechanisms
confer this effect. In additional experiments, antibody to
-IFN also did not reduce ICAM-1 expression in A549
epithelial cells cultured with P. carinii (data not shown). Further studies are necessary
to determine the roles of other native and host proteins in mediating
this direct stimulation of epithelial ICAM-1 by P. carinii.
Although the absolute level of ICAM-1 induced in epithelial cells
directly by P. carinii was less than
that evoked by P. carinii-exposed macrophages, our findings do suggest that both mechanisms may occur
during infection. For comparison of the response induced by
P. carinii alone to a maximal
stimulus, we further included TNF- in selected
experiments in which A549 cells were directly stimulated with
P. carinii. The maximal surface ICAM-1
expression resulting from direct P. carinii stimulation ranged between 30 and 50% of that
observed with TNF-
stimulation alone (1,000 U/ml). Similarly, we observed that P. carinii
stimulated roughly one-third of the ICAM-1 mRNA compared with a maximal
stimulation of A549 cells with TNF-
(Fig. 5). Our
investigation further indicates that P. carinii directly influence epithelial activation of
ICAM-1 expression through mechanisms independent of macrophage-derived TNF-
. Additional studies are required to determine
the relative extent to which these mechanisms contribute to lung
inflammation in vivo during P. carinii
pneumonia.
Although ICAM-1 promotes inflammatory cell recruitment necessary for
lung defense, exuberant ICAM-1 expression may also be detrimental to
the host. Excessive neutrophils in bronchoalveolar lavage from patients
with P. carinii pneumonia correlate
with enhanced morbidity, increased gas-exchange abnormalities,
and poorer survival during infection (22, 35). Marked ICAM-1
expression in P. carinii infection may
exacerbate this deleterious neutrophil recruitment. Furthermore,
modulation of pulmonary inflammation with corticosteroids improves
survival and reduces the occurrence of respiratory failure in patients
with moderate to severe P. carinii
pneumonia (28). Corticosteroids are known to reduce TNF- release from stimulated AMs and to directly
suppress ICAM-1 expression (16, 25, 38). Recent investigations further
indicate that excess pulmonary neutrophil influx may be selectively
inhibited with monoclonal antibodies that recognize ICAM-1 (3, 26, 40).
Accordingly, selective modulation of ICAM-1 may also have a potential
therapeutic benefit during severe P. carinii pneumonia; however, additional animal and
preclinical studies are warranted to evaluate the merits of such a
strategy.
The mechanisms of lung inflammation that accompany P. carinii infection are only beginning to be defined.
This study provides the first evidence that ICAM-1 is upregulated
during P. carinii pneumonia. Our data
further support a role for macrophage-derived TNF-
in the mediation of increased ICAM-1 expression in cultured lung
epithelial cells. In addition, P. carinii organisms also appear to directly augment
ICAM-1 protein and mRNA expression by cultured lung cells. Further
understanding of lung inflammation during this serious infection should
provide additional insights and potential therapeutic strategies for
patients with P. carinii pneumonia.
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ACKNOWLEDGEMENTS |
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We appreciate the many helpful discussions with Drs. Zvezdana Vuk-Pavlovic, Ulrich Specks, and Rolf Hubmayr. In addition, we thank Kathy Stanke for assistance in the final preparation of this manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants R29 AI-34336-04, R01 HL-55934-02, and R01 HL-57125-01 and by funds from the Mayo Foundation (to A. H. Limper). These studies were performed during the tenure of an American Heart Association Clinician-Scientist Award (to A. H. Limper).
Address for reprint requests: A. H. Limper, Thoracic Diseases Research Unit, 601A Guggenheim Bldg., Mayo Clinic and Foundation, Rochester, MN 55905.
Received 19 November 1996; accepted in final form 25 August 1997.
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