Pneumocystis stimulates MCP-1 production by alveolar epithelial cells
through a JNK-dependent mechanism
Jing Wang,1Francis Gigliotti,1,2Samir P. Bhagwat,1Sanjay B. Maggirwar,2and Terry W. Wright1,2
Department of1Pediatrics and2Department of Microbiology and Immunology;
University of Rochester School of Medicine and Dentistry, Rochester, New York
Submitted 16 November 2006; accepted in final form 12 February 2007
Wang J, Gigliotti F, Bhagwat SP, Maggirwar SB, Wright TW.
Pneumocystis stimulates MCP-1 production by alveolar epithelial
cells through a JNK-dependent mechanism. Am J Physiol Lung Cell
Mol Physiol 292: L1495–L1505, 2007. First published February 16,
2007; doi:10.1152/ajplung.00452.2006.—Pneumocystis carinii is an
opportunistic fungal pathogen that causes pneumonia (PCP) in immu-
nocompromised individuals. Recent studies have demonstrated that
the host’s immune response is clearly responsible for the majority of
the pathophysiological changes associated with PCP. P. carinii inter-
acts closely with alveolar epithelial cells (AECs); however, the nature
and pathological consequences of the epithelial response remain
poorly defined. Monocyte chemotactic protein-1 (MCP-1) is involved
in lung inflammation, immunity, and epithelial repair and is upregu-
lated during PCP. To determine whether AECs are an important
source of MCP-1 in the P. carinii-infected lung, in vivo and in vitro
studies were performed. In situ hybridization showed that MCP-1
mRNA was localized to cells with morphological characteristics of
AECs in the lungs of infected mice. In vitro studies demonstrated that
P. carinii stimulated a time- and dose-dependent MCP-1 response in
primary murine type II cells that was preceded by JNK activation.
Pharmacological inhibition of JNK nearly abolished P. carinii-stim-
ulated MCP-1 production, while ERK, p38 MAPK, and TNF receptor
signaling were not required. Furthermore, delivery of a JNK inhibi-
tory peptide specifically to pulmonary epithelial cells using a recom-
binant adenovirus vector blocked the early lung MCP-1 response
following intratracheal instillation of infectious P. carinii. JNK inhi-
bition did not affect P. carinii-stimulated production of macrophage
inflammatory protein-2 in vitro or in vivo, indicating that multiple
signaling pathways are activated in P. carinii-stimulated AECs. These
data demonstrate that AECs respond to P. carinii in a proinflamma-
tory manner that may contribute to the generation of immune-medi-
ated lung injury.
inflammation; epithelial; AIDS
PNEUMONIA INDUCED by Pneumocystis carinii (PCP) continues to
be the most common AIDS-defining illness as well as an
important cause of morbidity and mortality in patients with a
wide array of immunosuppressive conditions. Most recent
studies indicate that, although the presence of P. carinii is
obviously a factor in the development of lung injury, pulmo-
nary inflammation is a major determinant of the severity of
PCP (5, 27, 53, 54). In AIDS patients with profound reductions
in CD4?T cell numbers, bronchoalveolar lavage (BAL) fluid
IL-8 and neutrophil concentrations, but not organism numbers,
correlate with severity of PCP (5, 23, 28). In addition, clinical
studies of immune-reconstituted PCP patients and controlled
animal studies have both demonstrated that inflammatory me-
diators are released, and immune and inflammatory cells are
recruited to the lung in response to P. carinii (2, 25, 38, 39).
More defined studies in mice have identified specific T cell
subsets as having prominent roles in the lung injury associated
with PCP (54, 57). For example, CD8?T cells are responsible
for much of the PCP-associated lung injury that occurs in
CD4-deficient hosts (17). Therefore, recent studies have fo-
cused on the mechanisms by which T cells accumulate in the
lung during PCP.
While the close interaction of P. carinii with the alveolar
epithelium was one of the first observations offering insight
into the pathogenesis of PCP (26, 32, 58, 59), very little is
known about the epithelial response to P. carinii. In vivo
studies have most often noted the attachment of P. carinii to
the type I pneumocyte. However, this observation does not
preclude an important role for type II cells in the response to
P. carinii. Type II cells are closely positioned near the type I
cells and have been reported to interact with P. carinii in vivo
(29). These findings are particularly relevant given that the
type II cell is becoming increasingly recognized as an
immune effector cell in the alveolus (16, 46). Our prior
studies have found that immune and inflammatory cells are
recruited specifically to alveolar sites of P. carinii infection in
mice, suggesting that the interaction of P. carinii with the
alveolar epithelium targets the immune response (55, 56).
Other groups have found that disruption of alveolar epithelial
cell (AEC) signaling in vivo modifies pulmonary immune and
inflammatory responses (21, 46). Evidence has also accumu-
lated that transformed human lung epithelial-like cell lines can
produce inflammatory mediators in response to P. carinii
stimulation (4, 41, 60). In addition, a murine AEC line and
primary rat and mouse type II cells undergo NF-?B-dependent
macrophage inflammatory protein-2 (MIP-2) production when
stimulated with P. carinii or purified P. carinii glucan (15, 52).
However, recent evidence indicates that neutrophils are not
critical to the development of lung injury during PCP (47).
Thus, focus has shifted to CC chemokines, including monocyte
chemotactic protein-1 (MCP-1), which have a role in the tissue
recruitment of T cells. Pulmonary epithelial cells are capable of
secreting MCP-1 in response to infectious stimulation, and
epithelial MCP-1 mediates the pulmonary recruitment of
CD8?T cells (42, 62). In addition, a role for MCP-1/CC
chemokine receptor 2 (MCP-1/CCR2) signaling in the repair of
damaged pulmonary epithelium has been suggested (10).
These findings, combined with the fact that lung MCP-1 levels
are dramatically elevated in mice with PCP, make this chemo-
Address for reprint requests and other correspondence: T. W. Wright, Dept.
of Pediatrics, P.O. Box 850, Univ. of Rochester School of Medicine and
Dentistry, 601 Elmwood Ave., Rochester, NY 14642 (e-mail: Terry_Wright
The costs of publication of this article were defrayed in part by the payment
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Am J Physiol Lung Cell Mol Physiol 292: L1495–L1505, 2007.
First published February 16, 2007; doi:10.1152/ajplung.00452.2006.
1040-0605/07 $8.00 Copyright © 2007 the American Physiological Society http://www.ajplung.org L1495
kine of interest for its contribution to the pathological T cell
response that is critical to the progression of PCP.
Identification of epithelial-specific responses to P. carinii,
and the signaling cascades leading to these responses, will aid
in understanding the role of AECs as immune effector cells in
the generation of pulmonary immune and inflammatory re-
sponses. The hypothesis of the current study is that the inter-
action of P. carinii with type II AECs induces MCP-1 produc-
tion through NF-?B and mitogen-activated protein kinase
(MAPK)-dependent mechanisms. To test this hypothesis, pri-
mary murine type II cell cultures, in vivo mouse models, and
specific pharmacological and protein inhibitors of NF-?B, p38,
JNK, and ERK signaling were utilized. This study will help
determine the mechanism by which P. carinii-epithelial inter-
action promotes and targets the pathological immune/inflam-
matory response and also determine whether this interaction
might be exploited as a therapeutic intervention.
MATERIALS AND METHODS
Animals. CB.17 wild-type and severe combined immunodeficient
(SCID) mice were purchased from Taconic. C57BL/6 mice were
purchased from the Jackson Laboratory. B6.129-Tnfrsf1atm1Mak/J and
B6.129S2-Tnfrsf1btm1Mwm/J mice on a C57BL/6 background were
also purchased from the Jackson Laboratory. These mice were then
crossed to produce mice deficient in both TNFR1 and TNFR2
(“TNFR-deficient”) on a C57BL/6 background.
All animal protocols were preapproved by University Committee
for Animal Research (UCAR) at the University of Rochester Medical
Isolation and culture of primary murine type II cells. Primary type
II pneumocytes were isolated from mouse lungs using a modification
of the method of Corti et al. (11). Briefly, the lung was perfused with
saline. Two milliliters of dispase solution (BD Biosciences) was
instilled by tracheal catheter, followed immediately by slow insertion
of 0.45 ml of low-melting-point agarose (GIBCO-BRL) at 45°C. The
lungs were cooled briefly on ice and then incubated at room temper-
ature in dispase for 45 min. The lung tissue was microdissected,
incubated briefly in DMEM with 0.01% DNase at room temperature,
filtered through nylon monofilament screens (100, 40, and 25 ?m; BD
Falcon), and centrifuged at 150 g at 4°C. Type II cells were purified
from inflammatory cells by incubation with biotin-conjugated anti-
bodies against CD32 and CD45 followed by recovery with streptavi-
din-conjugated magnetic beads in a magnetic separator. With this
procedure, type II cell yield was ?1 ? 106cells/mouse. Typically the
type II cells were ?95% viable and ?92% pure as assessed by
papanicolaou staining. In addition, ?95% of the isolated cells were
typically positive for surfactant protein C (SP-C) expression as as-
sessed by intracellular staining and fluorescence-activated cell sorting
The isolated type II cells were cultured under conditions previously
demonstrated to maintain a type II phenotype as described by Rice
et al. (43). The cells were cultured on Matrigel/rat tail collagen-coated
plates (ratio 70/30, vol/vol) (BD Biosciences). Cells were maintained
at 37°C with 6% CO2 in bronchial epithelial cell growth medium
(BEGM) without hydrocortisone (Cambrex), supplemented with 5%
charcoal-stripped FBS (Hyclone, Logan, UT) and 10 ng/ml keratino-
cyte growth factor (KGF) (Calbiochem) to promote maintenance of a
type II cell phenotype.
Intracellular SP-C staining. Intracellular SP-C expression was
measured to determine the purity of type II cells. Briefly, primary
AECs were gently dislodged from wells and washed with PBS ? 1%
FBS staining buffer, spun at 250 g for 10 min, and resuspended in
staining buffer. The cells were incubated with Cytofix/Cytoperm (BD
Biosciences) solution on ice for 20 min. The cells were stained with
rabbit-anti-human SP-C (Chemicon International), which cross-reacts
with mouse SP-C, followed by goat anti-rabbit conjugated with
allophycocyanin or primary antibody alone. Unstained cells were used
as an additional negative control. SP-C expression was measured by
a FACS caliber cytofluorometer and analyzed by CellQuest software
(Becton-Dickinson, San Jose, CA).
Isolation and enumeration of mouse P. carinii. P. carinii was
isolated from the lungs of heavily infected SCID mice and enumerated
by the Gomori methenamine silver staining as described before (52).
Antibody-mediated depletion of P. carinii. Since there is no reliable
in vitro culture system for P. carinii, the organisms used in these
experiments were purified from the lungs of infected SCID mice.
Therefore, to ensure that MCP-1 secretion by AECs was a response to
P. carinii and not to potentially copurified contaminants or mouse
lung proteins, an antibody/magnetic bead-based technique for the
specific removal of P. carinii from the partially purified P. carinii
preparation was employed. The purified preparation was depleted of
P. carinii using magnetic beads coated with a pool of anti-P. carinii
antibodies. Enumeration of cysts before and after P. carinii deple-
tion, as well as real-time PCR analysis, demonstrated that this
method removed ?96% of the P. carinii organisms as previously
In vitro AEC treatments. Primary murine type II cells were grown
to ?90% confluence and then incubated in BEGM without KGF for
6 h prior to experimental treatments. In some experiments cells were
pretreated with sulfasalazine (SSA, 2 mM; Calbiochem), SB203580
(0.6 ?M), SP600125 (10 ?M), or PD20359 (50 ?M; Calbiochem), to
inhibit IKK, p38 MAPK, JNK, and ERK, respectively. Cells were
pretreated with inhibitors in serum-free media for 2 h prior to
P. carinii stimulation. In addition, JNK inhibitor 1, L-stereoisomer
(L-JNKI1) (Alexis Biochemicals), was used as a more specific inhib-
itor of JNK than SP600125. A protein named JNK interacting protein
1/IB1 (JIP-1/IB1) has been described that competitively blocks the
interaction between JNK and c-Jun, thereby inhibiting the signaling
events downstream of JNK (1, 7). To convert JIP-1/IB1 into a
cell-permeable inhibitor of JNK (L-JNKI peptides), the minimal 20-
amino-acid inhibitory sequence of JIP-1/IB1 was covalently linked to
the 10-amino-acid HIV Tat transporter sequence recognized by TAT
transporter (8, 50). L-JNKI1 is a potent and specific inhibitor of JNK.
The control peptide (L-TAT), which lacks JNK inhibitory activity,
was also used in the culture experiments. L-JNKI1 and L-TAT (Alexis
Biochemicals) were used at a concentration of 20 ?M. Monolayers of
type II cells were pretreated with inhibitors or control in serum-free
DMEM media without KGF for 1 h and then stimulated with freshly
isolated mouse P. carinii organisms suspended in serum-free DMEM.
In some experiments, cells were treated with ?-glucan (Sigma) as a
control for the whole freshly isolated P. carinii. Experiments were
terminated at 6, 12, or 24 h. Cell supernatants were recovered
for MCP-1 ELISA, and cells were used to either isolate total RNA for
ribonuclease protection assay (RPA) or produce cell lysates for
Cytokine enzyme-linked immunosorbent assay (ELISA). Culture
supernatants were collected, centrifuged at 12,000 g for 5 min to
remove debris, and then stored at ?80°C. MCP-1 and MIP-2 concen-
trations were measured using a commercially available ELISA kit ac-
cording to the manufacturer’s instructions (R&D, Minneapolis, MN).
In situ hybridization. In situ hybridization was performed as pre-
viously described (54, 56). Murine clones for MCP-1 were subcloned
into the plasmid vector, pBluescript II SK?(Stratagene, La Jolla,
CA), for the in vitro transcription of RNA. Sense and antisense
orientations were confirmed by DNA sequencing. MCP-1 antisense
RNAs were transcribed from 1 ?g of linearized plasmid template
according to the procedure. After ethanol precipitation, the riboprobes
were dissolved in diethylpyrocarbonate-treated water. Full-length
transcripts were ?0.7 kb for MCP-1. Prior to hybridization, limited
alkaline hydrolysis was performed to create riboprobes ranging in
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