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Previous Article | Next Article 
Infection and Immunity, August 2000, p. 4720-4724, Vol. 68, No. 8
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Down-Regulation of GATA-2 Transcription during
Pneumocystis carinii Infection
Xing
Tang,
Mark E.
Lasbury,
Darrell D.
Davidson,
Marilyn S.
Bartlett,
James W.
Smith, and
Chao-Hung
Lee*
Departments of Pathology and Laboratory
Medicine, Indiana University School of Medicine, Indianapolis,
Indiana 46202
Received 24 February 2000/Returned for modification 2 April
2000/Accepted 5 May 2000
 |
ABSTRACT |
Differences in gene expression between Pneumocystis
carinii-infected and noninfected rats were examined. Total RNA
was isolated from homogenized rat lungs and then subjected to
differential display with combinations of oligo(dT) and various
arbitrary PCR primers. Approximately 50 differentially expressed bands
were observed. Several of these DNA bands were isolated, reamplified, and cloned. The cloned DNA fragments were used as probes to perform Northern hybridization on RNA from P. carinii-infected and
noninfected rat lungs. One clone was found to react with a 3-kb mRNA
from noninfected but not from P. carinii-infected rat lung,
suggesting that the gene represented by this clone was down-regulated
during P. carinii infection. The nucleotide sequence of
this clone was determined and found to be 97% homologous to the mouse
GATA-2 transcription factor. In situ hybridization using RNA probes
derived from this clone revealed that alveolar macrophages, resident
lung monocytes, and bronchial epithelial cells express the GATA-2 gene in the lung.
| |
INTRODUCTION |
Pneumocystis carinii
causes pneumonia with a high mortality rate in immunocompromised
individuals, such as those with AIDS and organ transplants. Children
with severe malnutrition are also susceptible to P. carinii
infection. Although P. carinii is an extracellular parasite,
it must adhere to the surface of type I pneumocytes to proliferate.
However, an in vitro axenic culture of P. carinii was
recently reported (17). Adhesive proteins such as integrins
have been shown to enhance binding of P. carinii to type I pneumocytes.
During P. carinii infection, surfactant protein A and
integrins are up-regulated (1, 8, 20, 22). In contrast, the production of alveolar macrophage mannose receptor, which is
responsible for binding and engulfing P. carinii
(6), is reduced (11). The amount of total
surfactant phospholipid is also reduced during infection (2, 5,
10, 21, 23, 24, 25), with a decrease in phosphotidylcholine but
an increase in sphingomyelin (23, 25).
In order to further understand how host cells respond to P. carinii infection, we performed mRNA differential display to
detect genes that are up- or down-regulated during P. carinii infection. Three groups of two rats each were used. The
first group was immunosuppressed with dexamethasone (Dex) and then
infected with P. carinii (referred to as P. carinii infected hereafter). The second group was immunosuppressed with Dex only (referred to as Dex suppressed hereafter), and the third
group was nonimmunosuppressed and noninfected (referred to as normal
hereafter). We found that the expression of the GATA-2 transcription
factor is severely down-regulated during P. carinii infection. We also determined that ciliated bronchial epithelial cells,
alveolar macrophages, and resident lung monocytes are the cell types
that normally express the GATA-2 gene in the lung. This the first
report of GATA-2 expression in the pulmonary system and in a terminally
differentiated immune cell.
| |
MATERIALS AND METHODS |
Animals and infection of animals with P. carinii.
Three groups of two rats each were used: normal, Dex suppressed, and
P. carinii infected. The Dex-suppressed group served as a
control to detect gene expression altered by immunosuppressive treatment with Dex. The normal rats served as the negative control. P. carinii infection in immunosuppressed rats was achieved
by transtracheal inoculation of lung homogenate from P. carinii-infected rats. The lung homogenate was shown to contain
P. carinii by light microscopy performed on stained smears.
Each rat was transtracheally injected with 0.2 ml of lung homogenate
containing 106 P. carinii organisms. The rats
developed P. carinii pneumonia in 5 to 8 weeks and were then sacrificed.
Isolation of RNA from animals for mRNA differential display.
The lungs of P. carinii-infected rats were lavaged with
normal saline, and the lavage fluids were assayed for the presence of
P. carinii by PCR, using the mitochondrial rRNA gene primers (29). The lavage fluids from both the Dex-suppressed and
normal rats were negative in the mitochondrial rRNA gene PCR,
indicating that P. carinii infection did not develop in
these rats. The lavage fluids from P. carinii-infected rats
were positive in the PCR, indicating that the rats were indeed infected
with P. carinii. The lungs were perfused from the pulmonary
artery with Hanks balanced salt solution to remove blood in order to
avoid interference with RNA isolation. Total RNA was isolated from
homogenized lung tissue. The isolated RNAs were treated with RNase-free
DNase I and then reverse transcribed using various oligo(dT) primers.
The cDNAs thus generated were used for mRNA differential display.
mRNA differential display.
The RNAmap Kit B, obtained from
GenHunter Co. (Nashville, Tenn.), was used to perform mRNA differential
display. Four different sets of oligo(dT) primers
(5'-T12MN-3') with the following sequences were used for
reverse transcription (RT): 5'-T12MG-3',
5'-T12MA-3, 5'-T12MT-3', and
5'-T12MC-3'. These primers are composed of 12 T residues
followed by a degenerate base M, which includes G, A, and C, and then
either G, A, T, or C at the extreme 3' end. Two hundred nanograms of
lung RNA from each animal group was used in each 20-µl RT mixture.
The RT mixture contained 25 mM Tris-HCl (pH 8.3), 37.6 mM KCl, 1.5 mM
MgCl2, 5 mM dithiothreitol, 50 µM deoxynucleoside
triphosphate, 1 µM T12MN primer, and 100 U of Moloney
murine leukemia virus reverse transcriptase. The reaction was carried
out in a thermocycler at 65°C for 5 min, 37°C for 60 min, and
95°C for 5 min. After 10 min at 37°C, the reverse transcriptase was
added to each reaction tube. The reverse-transcribed cDNA species were
then amplified by PCR with various combinations of a
5'-T12MN-3' primer used in RT and one of the five 10-bp
random primers (AP1 to -5). The PCR was run in a mixture containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.001%
gelatin, 2 µM deoxynucleoside triphosphate, 200 nM AP primer, 1 µM
T12MN primer, 2 µl of RT product, 10 µCi of
[
-33P]dATP, and 1 U of AmpliTaq (Perkin-Elmer, Foster
City, Calif.) at 94°C for 30 s, 42°C for 2 min, and 72°C for
30 s for 40 cycles. The labeled PCR products were electrophoresed
on 6% DNA sequencing gels and detected by autoradiography of the gels.
Isolation and reamplification of differentially expressed
bands.
The DNA in the differentially expressed bands was eluted by
boiling the gel slice containing the band for 15 min in 100 µl of
water. The eluted DNA was precipitated with ethanol, dried, and
resuspended in water. To obtain a sufficient amount of DNA for
subsequent studies, the eluted DNA of each band was reamplified with
the same primer set which produced the isolated band. The reamplified
products were electrophoresed on agarose gels, and the band of the
correct size was isolated, purified with a Qiaex II kit (Qiagen,
Valencia, Calif.), and then cloned into the TA cloning vector pCRII.
PCR was then performed on colonies containing recombinant plasmids
using primers which anneal to the vector flanking the cloning site
(EcoRI) to determine the sizes of the inserts.
In situ hybridization.
Rat lungs were cut into small pieces
and then immersed for 24 h in a fixative composed of 4%
formaldehyde and 1% glutaraldehyde. The tissues were washed with
phosphate-buffered saline (PBS) (pH 7.4) three times for 30 min each
and then dehydrated by being passed through a series of increasing
concentrations of ethanol. After replacement of the ethanol with
xylene, the tissue was embedded in paraffin. Five-micrometer sections
were cut with a microtome (Leitz, Rockleith, N.J.) and mounted on
ProbeOn Plus microscope slides (FisherBiotech, Pittsburgh, Pa.). Each
slide contained both immunosuppressed and P. carinii-infected rat lung sections.
GATA-2 riboprobes were used for the in situ hybridization and were
produced by in vitro transcription using the TA vector containing the
insert (pGATA-2a or pGATA-2s) as the template. Both pGATA-2a and
pGATA-2s were linearized with BamHI and transcribed with T7
RNA polymerase to produce antisense (from pGATA-2a) and sense (from
pGATA-2s) probes. The BamHI-digested plasmids were purified
by the Qiaex II gel elution method. Riboprobes were labeled with biotin
using a biotin RNA labeling kit (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.). In vitro transcription was performed in a 20-µl
reaction mixture containing 1 µg of linearized vector DNA, 2 µl of
biotin RNA labeling mix (10 mM ATP, 10 mM CTP, 10 mM GTP, and 6.5 mM
UTP), 3.5 mM biotin-UTP, 2 µl of 10× transcription buffer (pH 7.5),
and 2 µl (20 U) of T7 RNA polymerase. The reaction mixture was
incubated at 37°C for 2 h. Two microliters of RNase-free DNase I
(20 U) was then added to the reaction mixture and incubated for 15 min
at 37°C to digest the template DNA. Two microliters of 0.2 M EDTA
solution (pH 8.0) was then added to stop the reaction. The RNA probes
were precipitated by adding 2.5 µl of 4 M LiCl and 75 µl of cold
ethanol. After cooling at 
70°C for 1 h, the samples were
centrifuged at 16,000 × g for 15 min at 4°C. The
pellets were washed with 200 µl of cold 75% ethanol, dried, and
resuspended in 100 µl of diethyl pyrocarbonate-treated water
containing 1 µl of RNase inhibitor (RNasin) (20 U). The concentration
of RNA thus produced was determined by spectrophotometry, measuring the
absorbance at 260 nm.
The paraffin sections on the ProbeOn Plus microscope slides were
deparaffinized, rehydrated, and treated with
H2O2 in PBS (pH 7.4) for 15 min at room
temperature to inactivate the native peroxidase activity. The sections
were digested with 50 µg of proteinase K per ml in PBS at 42°C for
20 min, rinsed with diethyl pyrocarbonate-treated H2O for 2 min, and then treated with 0.25% (vol/vol) acetic anhydride in 0.1 M
triethanolamine HCl (pH 8.0) for 10 min at room temperature, followed
by washing with neutralizing solution (0.1 M Tris-HCl [pH 7.5], 0.2 M
NaCl, 5 mM MgCl2) for 10 min. The sections were then
incubated with the hybridization cocktail, which is composed of 1×
Denhardt's solution, 50% deionized formamide, 10% dextran sulfate,
4× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50 mM
dithiothreitol, 0.1 mg of tRNA per ml, and 400 ng of biotin-labeled
riboprobes per ml, in a humidified chamber at 50°C overnight. After
hybridization, the sections were washed two times for 15 min each with
solution I (2× SSC, 0.1% sodium dodecyl sulfate [SDS], and 0.05%
Brij) and then two times for 15 min each with solution II (0.5× SSC,
0.1% SDS, and 0.05% Brij) at room temperature. The final wash was
done two times for 5 min each with solution III (0.1× SSC, 0.1% SDS, and 0.05% Brij) at 42°C.
A tyramide signal amplification kit (NEN Science Products, Boston,
Mass.) was used to amplify the hybridization signals in tissue
sections. The sections were rinsed with solution I and then soaked in
100 µl of TNB blocking buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M
NaCl, and 0.15% blocking reagent supplied in the kit) for 30 min at
room temperature to block the nonspecific biotin binding sites. After
blocking, the sections were incubated for 30 min at room temperature in
100 µl of horseradish peroxidase-conjugated streptavidin (SA-HRP)
diluted 1:100 in TNB buffer. The excess SA-HRP was removed by washing
three times for 5 min each in TNT buffer (0.1 M Tris-HCl [pH 7.5],
0.15 M NaCl, 0.05% Tween 20) at room temperature. The sections were
then incubated in 300 µl of biotin-tyramide working solution (1:50
dilution of the biotin-tyramide stock solution with the amplification
diluent supplied in the kit) for 10 min at room temperature. After
being washed three times for 5 min each in TNT buffer, the sections
were again incubated in 100 µl of diluted SA-HRP for 30 min at room
temperature. The sections were washed three times for 5 min each in TNT
buffer to remove the excess SA-HRP. The hybridization signals were
developed by incubating the sections with 1×
3,3'-diaminobenzidine-CoCl2 (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.) for 15 min. After the reaction was
stopped by rinsing the sections in Tris-EDTA buffer, the hybridization
signal was further enhanced by incubating the sections with 100 µl of
1% osmium tetraoxide at room temperature for 5 to 30 s. The
sections were washed with water to stop the reaction, counterstained
with hematoxylin for 30 s, dehydrated in ethanol gradients,
cleared with xylene, and sealed with Permount under a coverslip. The
sections were then examined under a light microscope.
| |
RESULTS |
Approximately 50 differentially expressed bands were observed.
Some bands were present only in the samples of P. carinii-infected rat lungs; these may represent expressed P. carinii genes or host genes that are turned on or up-regulated by
P. carinii infection. There were also bands that were
present in samples of Dex-suppressed and normal rats but not in
P. carinii-infected rats; these bands may represent host
genes that are turned off or down-regulated by P. carinii
infection. Seven of the down-regulated bands were isolated,
reamplified, and cloned from the differential display gel.
To confirm that these clones represent genes that are differentially
expressed, a Northern hybridization was performed. The inserts of these
clones were isolated, labeled with 32P, and used as probes
to react with RNA samples from Dex-suppressed and P. carinii-infected rat lungs. These two RNA samples (10 µg each)
were run side by side on an agarose gel in eight replicates. Each
replicate was separately transferred to a Nytran membrane. One of the
membranes was reacted with the 
-actin gene probe to demonstrate that
the same amounts of RNA samples were loaded into each well. The other
membranes were each reacted with different probes. The -actin gene
probe gave approximately the same intensity of hybridization signal
with both Dex-suppressed and P. carinii-infected RNA samples
(Fig. 1), indicating that approximately
equal amounts of RNA were loaded. A separate membrane was probed with
the -actin gene probe because it was unknown whether there would be
interference from hybridization signals of other probes to mRNA of the
same length as -actin.
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FIG. 1.
Confirmation of differential expression by Northern blot
hybridization. Inserts containing a portion of potential differentially
expressed genes were labeled with 32P and used as probes to
react with electrophoresed RNA samples from lungs of Dex-suppressed
(lanes D) and P. carinii-infected (lanes I) rats.
|
|
All of these probes gave a more intense hybridization signal with the
RNA sample from Dex-suppressed rat lung cells than with that from
P. carinii-infected rat lung cells (Fig. 1). This result indicates that the genes represented by these probes are down-regulated during P. carinii infection. The gene represented by A8II
has the highest level of expression, followed by those represented by
the C6I and T6II clones and then by A8I, A7IV, A8IV, and A8III. Probe
A8II generated two bands that are very close in size (~1.8 and 1.5 kb). Probes C6I and T6II produced the same hybridization patterns, and
the intensities of hybridized bands of P. carinii-infected samples are approximately one-fourth of those of noninfected cells. Probes A8IV and A8III also produced the same hybridization patterns; a
weak band was seen in Dex-suppressed RNA samples and no band was seen
in P. carinii-infected samples, suggesting that these two
genes are expressed at a very low level in Dex-suppressed rats and are
severely down-regulated in P. carinii-infected rats.
To identify these genes, the inserts of these clones were sequenced.
The sequences thus obtained were compared with all of the sequences in
GenBank using the BLAST-n search tool. The sequences of clones C6I and
T6II were found to be identical to each other and are 90% homologous
to that of the MM-1 gene (GenBank accession no. D89667). The sequences
of clones A8IV and A8III were also identical to each other and are 97%
homologous to that of the mouse GATA-2 transcription factor gene at the
3' noncoding region (Fig. 2). The
sequence of clone A8I was found to be 83% homologous to that of an
unidentified gene, KIA0026 (GenBank accession no. D14812). The
sequences of clones A8II and A7IV were found to be novel. These two
sequences have no significant homology with those of any of the genes
in the nucleotide sequence banks.
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FIG. 2.
Sequence comparison between A8IV and GATA-2. The
nucleotide sequence of the clone A8IV was compared with all sequences
stored in GenBank using the BLAST program. The A8IV sequence (positions
85 to 236) was found to be homologous to the mouse GATA-2 gene at
nucleotides 2957 to 3108.
|
|
Among the three known genes identified to be down-regulated during
P. carinii infection, the GATA-2 gene is the most well characterized. The function of KIA0026 is unknown. MM1 was recently identified to be a c-Myc binding protein. GATA-2 is a transcription factor and regulates the development of hematopoietic cells. Therefore, a decision was made to further study the function of the GATA-2 gene in
the lung. In situ hybridization using biotin-labeled sense and
antisense GATA-2 riboprobes was performed to identify cells expressing
the GATA-2 gene in the lung.
No hybridization signal was seen in any sections that reacted with the
sense riboprobe, indicating that no nonspecific hybridization occurred
in the reaction (Fig. 3). In sections of
Dex-suppressed rat lung, cells that showed positive hybridization
signals with the antisense GATA-2 probes were located in the
epithelium of bronchioles, interstitium, alveolar walls, and
alveoli (Fig. 3A). Ciliated bronchial epithelial cells were most
prominently stained, indicating high expression of the GATA-2
gene. The cells in the interstitium and alveolar walls that react with
the probe had the typical appearance of monocytes, with horseshoe or
uniform nuclei and a moderate amount of vacuolated cytoplasm. These
resident lung monocytes are commonly referred to as histiocytes. There was no hybridization signal seen in interstitial fibrocytes or endothelial cells in these sections. Cells in the alveoli that reacted
with the probe were large and had an amoeboid shape with shaggy cell
margins and phagocytic vacuoles in the cytoplasm, characteristic of
alveolar macrophages.
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FIG. 3.
In situ hybridization. Sections of various rat lungs
were reacted with different probes (A) Dex-suppressed rat lung probed
with antisense GATA-2 riboprobes. (B) P. carinii-infected
rat lung probed with GATA-2 antisense riboprobes. (C) Dex-suppressed
rat lung probed with sense GATA-2 riboprobes. (D) P. carinii-infected rat lung probed with GATA-2 sense riboprobes.
Three types of cells, i.e., ciliated bronchial epithelial cells (E),
monocytes (M), and alveolar macrophages (AM), reacted with antisense
GATA-2 riboprobes. Representatives of these cells are indicated by
arrows.
|
|
In sections of P. carinii-infected lung, the alveolar spaces
were filled with an amphophilic foamy amorphous exudate composed of
cell debris and P. carinii organisms. The organisms were
also seen in the lumen of some small bronchioles. The alveolar septa were wider than those of noninfected rats and contained more
inflammatory cells such as neutrophils and lymphocytes. The numbers of
bronchial epithelial cells that showed positive hybridization with the
antisense GATA-2 probes were much fewer than those seen in noninfected
rat lungs (Fig. 3B). The intensity of the brown color in
hybridization-positive cells, including ciliated bronchial epithelial
cells, monocytes, and alveolar macrophages, was reduced. The numbers of
monocytes that reacted with the probes were also reduced.
| |
DISCUSSION |
In this study, we have investigated differences in gene expression
between noninfected and P. carinii-infected rat lungs. The
expression of the GATA-2 gene was found to be down-regulated during
P. carinii infection. This GATA-2 down regulation was first detected by mRNA differential display experiments. Identification of
the gene was achieved by comparing the nucleotide sequence of the
differential displayed product with sequences stored in GenBank (Fig.
2). Down-regulation of the GATA-2 gene was confirmed by Northern blot
analysis. The GATA-2 gene in normal rat lung was found to be expressed
at a very low level based on the intensity of the RNA band that
hybridized with the probe. Its expression in P. carinii-infected lung is even lower, as no RNA band was found to
react with the probe (Fig. 1). With in situ hybridization, alveolar
macrophages, resident lung monocytes, and ciliated bronchial epithelial
cells are found to express the GATA-2 gene in the lung (Fig. 3). The
expression of the GATA-2 gene in these three types of cells in P. carinii-infected lungs is much lower than that in normal rat lung,
confirming the results of Northern blot analyses.
GATA-2 is one of the six transcription factors of the GATA family that
have been identified. All members of the GATA family contain two copies
of zinc finger DNA binding motifs (14). The consensus
binding sequence of GATA transcription factors is (A/T)GATA(A/G) (19), which is present in the promoters or enhancers of many genes. GATA-2 plays a crucial role in the development of hematopoietic cells (27). It has been found to be expressed in erythroid
and early myeloid cells and regulates the expression of -globin and erythropoietin genes (9, 16, 18). It has also been shown to
control the expression of the endothelin-1 gene (12), the endothelial nitric oxide synthase gene (30), and the gene
encoding the platelet and endothelial cell adhesion molecule-1
(12).
Down-regulation of GATA-2 in alveolar macrophages may have an impact on
host defenses against P. carinii infection. In vitro, alveolar macrophages have been shown to be activated by the whole organism or the major surface glycoprotein of P. carinii to
release inflammatory substances such as tumor necrosis factor alpha,
prostaglandin E2, and leukotriene B4 (3,
8, 26). This activation is enhanced by vitronectin or
fibronectin, which accumulates in the lung during P. carinii
infection. Furthermore, alveolar macrophages from normal rat lung are
able to bind, phagocytize, and degrade P. carinii (6,
13, 15, 28). However, alveolar macrophages appear to have
decreased functional abilities during P. carinii infection.
Using the SCID mouse model, Chen et al. (4) demonstrated that phagocytosis of P. carinii is not common. In addition,
Hanano et al. (7) showed that activated alveolar macrophages
are insufficient to resolve P. carinii infection. The
mannose receptors of alveolar macrophages are found to be defective in
AIDS patients with P. carinii pneumonia (11). It
is possible that down-regulation of GATA-2 in alveolar macrophages
renders them unable to phagocytose P. carinii. This
hypothesis is consistent with the finding that phagocytosis of
P. carinii is uncommon in heavily infected lungs.
We also found that GATA-2 is expressed in resident lung monocytes and
that this expression is also decreased during P. carinii infection. Resident lung monocytes are interstitial
monocytes in the lung with the potential of becoming alveolar
macrophages. Since GATA-2 plays a key role in the development of many
types of tissues, down-regulation of the GATA-2 gene in resident lung monocytes may prevent them from becoming alveolar macrophages. Down-regulation of GATA-2 in resident lung monocytes and macrophages may be one of the mechanisms by which P. carinii ensures its
own survival. Studies are being conducted to test these hypotheses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology and Laboratory Medicine, Indiana University School of
Medicine, 1120 South Dr., FH 419, Indianapolis, IN 46202-5113. Phone:
(317) 274-2596. Fax: (317) 278-0643. E-mail:
chlee{at}iupui.edu.
Editor:
W. A. Petri Jr.
| |
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Infection and Immunity, August 2000, p. 4720-4724, Vol. 68, No. 8
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
