Oct. 2000, p. 6069–6072
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Differential Effects of Virulent versus Avirulent Legionella
pneumophila on Chemokine Gene Expression in Murine
Alveolar Macrophages Determined by cDNA Expression
NORIYA NAKACHI, KAZUTO MATSUNAGA, THOMAS W. KLEIN, HERMAN FRIEDMAN,
Received 31 March 2000/Returned for modiﬁcation 6 June 2000/Accepted 3 July 2000
naires’ disease, a severe form of pneumonia and sometimes a
systemic infection, especially in immunocompromised individ-
uals with defective immune response mechanisms (4). L. pneu-
macrophages and other phagocytic cells. Development of cell-
mediated immunity is known to be essential in host defense to
L. pneumophila infection (6). Speciﬁc cytokines are considered
key factors in host immunity to intracellular microorganisms,
especially those produced by macrophages which help to reg-
ulate development of cellular immunity. Although numerous
studies concerning L. pneumophila infection have been con-
ducted, the interaction between this organism and alveolar
macrophages is still not well understood. The newly developed
cDNA expression array technique with membranes can differ-
entially detect more than 1,100 expressed genes at one time
and is considered an excellent method to determine gene mes-
sages which can be expressed by cells (9). Since identifying
genes that are modulated by infection may provide important
insights into the key molecular changes in the pathogenesis of
infection, this relatively new array technique to detect the
expression of different genes was used to investigate L. pneu-
mophila infection in alveolar macrophages.
For these experiments, the MH-S murine alveolar macro-
phage cell line, which was derived from BALB/c mouse alve-
olar macrophages (7), was used as the source of target cells for
ican Type Culture Collection, Manassas, Va., were maintained
in RPMI 1640 medium containing 10% heat-inactivated fetal
calf serum (Hyclone Laboratories, Logan, Utah). The MH-S
cells were adhered to a tissue culture dish (100 by 20 mm; BD
Falcon, Franklin Lakes, N.J.) at a concentration of 10
cells/dish for 2 h in 5% CO
at 37°C and then used for the
experiments. Virulent L. pneumophila M124 (3) and avirulent
L. pneumophila M124-Av, which was prepared by multiple
passage of M124 (11), were cultured on buffered-charcoal
yeast extract medium (Gibco Laboratories, Madison, Wis.) for
3 days at 37°C, as described previously (3). The virulent L.
strain A/J mice, whereas avirulent L. pneumophila strain
M124-Av was not lethal for the strain A/J mice infected intra-
peritoneally (11). The growth of the bacteria in macrophages
was also different between virulent and avirulent L. pneumo-
phila strains (11). That is, virulent L. pneumophila M124
showed a more than 100-fold increase in the number of viable
bacteria in susceptible mouse strain A/J peritoneal macro-
phages during 48 h of culture, but avirulent strain M124-Av did
not. The MH-S cells were infected with either L. pneumophila
M124 or M124-Av for 30 min at a concentration of 100 bac-
teria per cell, washed to remove noninfected bacteria with
Hank’s balanced salt solution, and then incubated in RPMI
1640 medium with 10% fetal calf serum. The number of viable
bacteria in macrophage lysates prepared with 0.1% saponin
(Sigma Chemical Co., St. Louis, Mo.) was determined by stan-
dard plate counts on buffered-charcoal yeast extract medium,
as described previously (13).
Total cellular RNA from cultured cells was extracted with
the Atlas Pure Total RNA labeling system (Clontech, Palo
Alto, Calif.) at 5 h postinfection, and quantiﬁcation of ex-
tracted RNA was performed with the RiboGreen RNA quan-
* Corresponding author. Mailing address: Department of Medical
Microbiology and Immunology, University of South Florida College of
Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Phone:
(813) 974-2332. Fax: (813) 974-4151. E-mail: email@example.com
on May 3, 2017 by guest
orometer (Molecular Probe). Since the macrophage response
to bacteria is complicated due to involvement of multiple fac-
tors in the bacteria-macrophage interaction, analysis of gene
expression at an early time point of interaction, such as 5 h
postinfection, was chosen for this study. To determine gene
expression, the membrane-based microtechnique with an Atlas
cDNA expression array (mouse 1.2 array; Clontech) was per-
formed in accordance with the manual provided. The array
included 1,176 mouse cDNAs and 9 housekeeping control
cDNAs and negative controls immobilized on a nylon mem-
brane. The cDNAs on a membrane are divided into 22 cate-
gories: (i) 25 cDNAs for cell surface antigens, (ii) 290 cDNAs
for transcription factors and DNA-binding proteins, (iii) 45
cDNAs for cell cycle regulators, (iv) 55 cDNAs for cell adhe-
sion receptors and proteins, (v) 4 cDNAs for extracellular
transporters, (vi) 81 cDNAs for oncogenes and tumor suppres-
sors, (vii) 20 cDNAs for stress response proteins, (viii) 36
cDNAs for ion channels and transport proteins, (ix) 2 cDNAs
for extracellular matrix proteins, (x) 1 cDNA for trafﬁcking and
targeting protein, (xi) 12 cDNAs for metabolic pathways, (xii)
2 cDNAs for posttranslational modiﬁcation and folding, (xiii)
one cDNA for translation, (xiv) 60 cDNAs for apoptosis-asso-
ciated proteins, (xv) 97 cDNAs for receptors, (xvi) 116 cDNAs
for extracellular cell signaling and communication, (xvii) 160
cDNAs for modulators, effectors, and intracellular transduc-
ers, (xviii) 39 cDNAs for protein turnover, (xix) 57 cDNAs for
cytoskeleton and motility proteins, (xx) 48 cDNAs for DNA
synthesis, repair, and recombination proteins, (xxi) 25 cDNAs
for other, and (xxii) 9 cDNAs for housekeeping genes.
The puriﬁed RNA, which was analyzed for genomic DNA
contamination by PCR with primers speciﬁc for
processed with the gene-speciﬁc CDS primer mix (Clontech),
deoxynucleoside triphosphate, [
P]dATP, and reverse tran-
P-labeled cDNA was
labeled cDNA in a solution of ExpressHyb (Clontech) with
heat-denatured, sheared-salmon-testes DNA was then hybrid-
ized overnight to the Atlas array membrane at 68°C. The mem-
brane was washed in 2
ϫ SSC (1ϫ SSC is 0.15 M NaCl plus
0.015 M sodium citrate) with 1% sodium dodecyl sulfate, 0.1
SSC with 0.5% sodium dodecyl sulfate, and 2
tially, and then exposed to a PhosphorImager (Storm 860;
Molecular Dynamics, Sunnyvale, Calif.). Results of the gene
expression were analyzed by computer with Atlas image soft-
L. pneumophila readily infected the MH-S cells, as shown in
Table 1, and 5 h after infection, the bacteria numbers mini-
mally increased in both virulent- and avirulent-L. pneumo-
phila-infected cultures. However, by 24 h after infection, it was
obvious that the virulent L. pneumophila strain grew remark-
ably, but the avirulent strain did not, similar to previous results
concerning growth of virulent versus avirulent L. pneumophila
in primary peritoneal macrophages from genetically suscepti-
ble strain A/J mice (13).
The MH-S cells infected with either L. pneumophila M124
FIG. 1. Phosphorimages of cDNA expression array membranes for MH-S
cells infected with or not infected with L. pneumophila. The total RNA was
extracted from cells infected with either virulent (B) or avirulent (C) L. pneu-
postinfection and subjected to cDNA expression array assay. Arrowheads, dou-
ble arrowheads, and arrows indicate
␤-actin, MIP-1␤, and MCP-3 cDNA, re-
TABLE 1. L. pneumophila growth in MH-S alveolar macrophage
Time after infection
Ϯ SD of L. pneumophila strain
Ϯ 0.5 ϫ 10
Ϯ 0.4 ϫ 10
Ϯ 0.6 ϫ 10
Ϯ 0.8 ϫ 10
Ϯ 0.9 ϫ 10
Ϯ 0.2 ϫ 10
Number of viable bacteria (CFU) in MH-S cell lysates measured at indicated
time after infection by plate count method. Data are from triplicate cultures and
represent three experiments.
array technique at an early-infection time point, such as 5 h
postinfection, as compared to gene expression in noninfected
control cells. Comparison of phosphorimages of DNA from
the control cultures not infected with L. pneumophila as com-
pared to cultures infected with virulent L. pneumophila M124
showed that only select genes were signiﬁcantly modulated
(Fig. 1). Figure 2 shows a semiquantitative analysis of select
genes as the ratio of target gene expression versus housekeep-
infected cells. Since there were many genes affected by infec-
tion in terms of expression level, stable up-regulated genes
were speciﬁcally assessed between experiments after infection.
As was apparent by analysis of speciﬁc gene expression levels
in comparison to
␤-actin gene expression, there were several
genes which were markedly induced by the virulent but not by
the avirulent bacteria (Fig. 2). It should be noted that the lower
expression levels of genes varied between experiments. How-
ever, the expression of several genes related to inﬂammation
was signiﬁcantly modulated by infection with L. pneumophila.
In particular, infection with the virulent L. pneumophila M124
strain signiﬁcantly up-regulated gene expression for monocyte
chemotactic protein 3 (MCP-3) and macrophage inﬂammatory
␣ (MIP-1␣), and p38-2G4 (the gene speciﬁcally for
cell cycle-modulated nuclear protein ). Other genes, such as
those for MIP-1
␤ and CD40 and the L-myc gene, were induced
by the virulent L. pneumophila strain in some experiments;
however, there was no signiﬁcant difference between infected
and noninfected groups due to a high variation in gene expres-
sion levels between experiments. It is important to note that
modulation of the gene for MCM5 DNA replication licensing
factor (the CDC46 homolog), which is involved in the initiation
of cell-cycle-speciﬁc DNA replication and expression at the
late G1 to S phase (5), seemed to occur readily in the alveolar
macrophage cells infected with avirulent L. pneumophila, but
not in cells infected with the virulent bacteria. However, the
pathophysiological role of this gene in L. pneumophila infec-
tion is not known.
Since an effective host defense against bacterial invasion is
characterized by the vigorous recruitment and activation of
inﬂammatory cells, chemokine production is considered a crit-
ical event during infection (10). In this regard, chemokine
MCP-3 and MIP-1
␤ messages in L. pneumophila-infected cells
were further investigated by reverse transcription-PCR (RT-
PCR). RNA extraction and RT-PCR were performed as de-
scribed previously (12). The PCR primers for
(housekeeping gene), MIP-1
␤, and MCP-3 were designed from
GenBank cDNA sequences using a website program Primer 3
(http://www.path.cam.ac.uk/cgi-bin/primer3.cgi). The PCR was
performed in a Minicycler (MJ Research, Watertown, Miss.)
for either 25 cycles (
␤) or 30 cycles
(MCP-3) and at a 60°C annealing temperature. PCR products
FIG. 2. Gene expression levels for selected genes in MH-S cells infected with L. pneumophila at 5 h postinfection or not infected. Results represented are means
plus standard deviations from three independent experiments.
ء, P Ͻ 0.05 compared to noninfected control analyzed by Student’s t test. Open column, noninfected
control; closed column, infected with virulent L. pneumophila (M124); gray column, infected with avirulent L. pneumophila (M124-Av).
semiquantitated, and normalized to
Both MCP-3 and MIP-1
␤ belong to the CC subfamily of
chemokines and are involved in early inﬂammatory responses,
including infection (1). In previous studies, we observed induc-
tion of MIP-1
␤ and other chemokines, such as MIP-2 and KC,
by L. pneumophila infection of macrophages (12, 14). How-
ever, chemokine induction by virulent versus avirulent L. pneu-
mophila infection has not yet been reported. The cDNA ex-
pression array experiments revealed that the virulent L.
bacteria did not at 5 h postinfection. As shown in Fig. 3, the
results of the cDNA expression array experiments were con-
ﬁrmed by RT-PCR. That is, the virulent L. pneumophila M124
strain markedly induced MCP-3 messages, and this was evident
even at 24 h after infection. In contrast, the avirulent L. pneu-
MCP-3 messages during infection (P
Ͻ 0.05 compared with
noninfected control group or virulent-bacteria-infected group).
␤ induction also was stimulated at a sim-
ilar level by both virulent and avirulent L. pneumophila strains.
Thus, the virulent L. pneumophila strain induced both MCP-3
␤, but the avirulent bacteria induced only MIP-1␤.
The mechanism of selective chemokine induction by the
virulent bacteria is not clear. MCPs are known to down-regu-
late interleukin 12 induction induced by bacteria such as Staph-
ylococcus aureus (2). Other current studies also showed inter-
leukin 12 down-regulation by virulent L. pneumophila, but not
by avirulent organisms (K. Matsunaga, T. W. Klein, H. Fried-
man, and Y. Yamamoto, unpublished data). Therefore, it can
be speculated that MCP induced by virulent bacteria plays an
immunoregulatory role in infection. Nevertheless, the results
obtained showed that alveolar macrophages respond to viru-
lent L. pneumophila infection differently than to infection with
avirulent bacteria. Particularly, the differential regulation of
chemokine MCP-3 induction by L. pneumophila was revealed
in this study. Thus, it is apparent that the cDNA expression
array technique is a powerful tool for analysis of host cell
responses to infection by L. pneumophila, and the results ob-
tained conﬁrm that important modulations of gene expression
following exposure to infectious agents can be readily screened
by this technique.
This work was supported by grant AI45169 from the National Insti-
tute of Allergy and Infectious Diseases.
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Editor: J. D. Clements
FIG. 3. Levels of MIP-1
␤ (A) and MCP-3 (B) mRNA in MH-S cells infected
with virulent (strain M124) or avirulent (strain M124-Av) L. pneumophila. RNA
was extracted from the cells at 5 h (open column) or 24 h (closed column)
postinfection. The mRNA expression for chemokines was determined by RT-
PCR and normalized to
are presented as the ratio (mean plus standard deviation) of chemokines to
-microglobulin densities from three independent experiments. —, noninfected
ء, P Ͻ 0.05 compared to noninfected control.