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*
Institute for Molecular Bioscience, and
Cooperative Research Center for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland, Australia; and
Institute for Systems Biology, Seattle, WA 98105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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were not induced in all
subclones, and there was no absolute correlation between expression of
pairs of genes. Nevertheless, the array analysis revealed clusters of
genes that were more likely to be coexpressed. RAW264 cells stably
transfected with luciferase reporter genes driven by LPS-responsive
promoters revealed the same kind of clonal heterogeneity. The results
indicate that each LPS-inducible gene has its own inherent probability
of activation in response to LPS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have used cDNA microarrays to examine the patterns of LPS-inducible genes in subclones of RAW264 cells. The results indicate a remarkable degree of heterogeneity, and also a degree of order, that has profound implications for our understanding of innate immunity and the nature of gene regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA was isolated from RAW264 cell clones using the TRIzol reagent kit (Life Technologies, Rockville, MD). Thirty micrograms of total RNA was blotted onto nylon membranes (Millipore, Bedford. MA) using a slot-blot apparatus under vacuum. The RNA was fixed by UV irradiation, and was prehybridized in Denhardt’s with 100 µg/ml of sheared salmon sperm DNA for 1 h at 55°C. Radiolabeled cDNA probes were prepared using PCR-amplified full-length cDNA as template, with the RedyPrime kit (Pharmacia, Peapack, NJ). Hybridization was conducted for 12 h at 55°C in the same buffer. The blots were washed once with 0.1% SDS, 2x SSC, twice with 0.01% SDS, 2x SSC, and then once with 2x SSC. Bound radioactivity was detected using a Bio-Rad Phosphorimager (Bio-Rad, Hercules, CA). Loading was determined by probing with a radiolabeled cDNA directed against the 16S mouse ribosomal RNA subunit. Multiple independent blots were made, and each one was used for up to four separate cDNA hybridizations plus the rRNA control.
Microarray design
We designed a 3700-element mouse microarray enriched for macrophage-expressed genes. Seventeen hundred mouse UniGenes (Research Genetics, Huntsville, AL) were sequence verified, and a panel of known macrophage-expressed and developmentally regulated control genes was added from our own laboratory resources and colleagues within the Institute for Molecular Biosciences (University of Queensland). Additionally, we produced a subtracted library from RAW264 cells that had been stimulated for 4 h with LPS. The Clontech PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA) was used exactly as per the manufacturer’s instructions, and mRNA from unstimulated RAW264 cells was used to produce cDNA for subtraction. Subtracted library clones were plated and picked individually. From an initial set of around 400, 50 were sequenced. We used a pool of multiple hit cDNAs to reprobe the library, and we selected an additional 1300 clones to array. The cDNA inserts were PCR-amplified, column purified, and cDNA arrays were printed onto polylysine-coated glass microscope slides using an Affimetrix 418 gridder at the Queensland Institute for Medical Research. The methods are described elsewhere (13).
Total RNA from RAW264 cells was harvested using Qiagen RNeasy mini-prep columns (Qiagen, Valencia, CA). The integrity of each RNA preparation was checked on a 1.2% agarose/Tris-acetate EDTA gel, and A260/A280 was measured. RNA was concentrated to 2.5 µg/µl in a microcon-30 column (Millipore). Fluorescently labeled cDNA was transcribed from each 50-µg aliquot by incorporating Cy3-dCTP or Cy5-dCTP into a modified superscript reverse transcriptase (Life Technologies) protocol. Hybridization and washing of slides was conducted as described previously (13).
Data collection, analysis, and normalization
Slides were scanned on an Affimetrix 417 scanner. Images were analyzed using ImaGene 4.1 (Biodiscovery, Los Angeles, CA) and GeneSpring V3.2.11 (Silicon Genetics, Redwood City, CA). Because the large majority of elements on the arrays were induced by LPS, the Cy3 and Cy5 fluorescence distributions were quite distinct, with the former (LPS stimulated) substantially skewed to higher values (not shown).
Promoter constructs.
The human IL-1
promoter clones into the luciferase vector pGL3 was a
gift from Dr. Matt Fenton (14). We subcloned the promoter
fragment into the corresponding sites of the Promega Renilla
luciferase vector. The IL-12 reporter was constructed by us. The murine
IL-12 p40 promoter region from -349 to +56 (15) was
generated by PCR and was cloned into the pGL2-basic
KpnI/XhoI site. The endothelial leukocyte
adhesion molecule (ELAM) luciferase reporter is as previously
described (16).
Cells. To avoid phenotypic drift in cell culture (7), all experiments were conducted using RAW264 cells that had been recently obtained from the American Type Culture Collection (ATCC, Manassas, VA), expanded immediately, and then frozen in aliquots. Cells were maintained in culture for no more than 4–6 wk. Subclones of RAW264 cells were produced by random limiting dilution cloning in 96-well plates. After 3 days of culture, individual wells were inspected to identify those with a single small focus of cells, and such wells were selected and grown for further study.
Transfection.
A total of 5 x 106 RAW264 cells were
transfected by electroporation as described (6). All cell
culture, including the electroporation procedure, was conducted in RPMI
1640 medium with 10 mM HEPES and 10% FBS. The inclusion of HEPES for
transfections is a departure from previous studies (17)
and appears to increase survival and transfection efficiency. Ten
micrograms of the desired firefly luciferase reporter gene, 10 µg of
the IL-1 promoter-Renilla luciferase plasmid, and 2 µg
of the selective marker plasmid pNeoTak, which also directs expression
of the Tet repressor (18), were added to each
transfection. In this system, only the Neo resistance cassette is
relevant, but the Tet repressor could be used subsequently for studies
using inducible modifier cassettes. After transfection, the cells were
placed in a 100-mm square bacteriological petri dish (Sterilin,
Teddington, U.K.) with 
25 ml of medium. Following overnight
incubation, most viable cells adhere weakly. The medium was changed,
and geneticin (G418, 200 µg/ml) was added. The cells were left in
culture with one change of medium to remove dying cells around days 4
and 5. By days 7–10, several hundred individual foci of stably
transfected cells were evident in each dish. These were removed by
washing of the surface using a syringe with an 18-guage needle (the
cells are weakly adherent to bacteriological plastic), expanded, and
frozen in aliquots for future experiments. After around 3 wk in
culture, the stable transfectants were cloned by limiting dilution.
Time course, dose response, and agonist sensitivity of these lines will
be described in detail elsewhere (33). For this study,
stable transfectant clones were plated at 2 x
105 cells/well and cultured overnight. LPS (100
ng/ml of salmonella R595 LPS from Sigma-Aldrich, St. Louis, MO) was
added, and cells were harvested after 8 h for determination of
firefly and Renilla luciferase activity using the Promega
Dual Luciferase system. The dose of LPS and time were predetermined
maxima.
In situ hybridization. The probes were made and labeled using digoxygenin (DIG) RNA labeling mix according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany), using T7 and SP6 polymerases from Promega. Mouse 800-bp GLI3 probe in the pGEM vector was linearized with HindIII (anti-sense) and XbaI (sense). Thioglycollate-elicited peritoneal macrophages were plated at 1 x 105 cells/chamber on 16-chamber glass slides (Nuc: Lab Tek) and were cultured overnight in RPMI 1640 medium and 10% FBS. LPS (100 ng/ml) was added where required. After 4 h, cells were fixed in 4% (w/v) paraformaldehyde overnight, and were dehydrated with ethanol/glacial acetic acid (95:5). After rehydration, they were permeabilized with proteinase K (1 µg/ml) for 15 min. Cells were prehybridized for 3 h in Denhardt’s buffer at 44°C and were then hybridized overnight in Denhardt’s buffer containing 10 ng/µl of labeled probe. Posthybridization washes were also performed at 44°C as follows: two times for 15 min with 2x, one time for 15 min with 0.2x SSC, and two times for 15 min with 0.1x SSC. Washed cells were blocked using 10% sheep serum and 2% BSA in 1% blocking reagent (Boehringer Mannheim) for the last 30' and they were then incubated overnight with anti-DIG F(ab')2 conjugated to alkaline phosphatase (Boehringer Mannheim). Alkaline phosphatase activity was localized using 4-nitroblue tetrazolium chloride and 5-bromo-4-vhloro-3-indolyl-phosphate (Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine macrophage cell line RAW264 was originally obtained
from an Abelson leukemia virus-induced tumor in mice, and was cloned
(RAW264.7) and deposited in the ATCC. We obtained the line from the
ATCC, expanded it, and froze aliquots immediately so that the line used
herein is as close as possible to the original stock. Limiting dilution
subcloning of the original RAW264.7 cells gives sublines with divergent
patterns of gene expression in response to LPS. For example, individual
subclones do, or do not, express iNOS or plasminogen activator type 2
(PAI-2) in response to LPS (7, 9). To gain a greater
insight into the extent of this diversity, we selected a set of 16
known LPS-inducible genes as well as control genes that were expected
to be constitutively expressed and conducted a limited array analysis
using slot blots. Analysis was performed on 25 subclones that were
selected randomly based upon limiting dilution subcloning. The result
is shown in Fig. 1
. Only IL-6 was induced
in all of these clones, demonstrating that they are all LPS-responsive.
Even the TNF- gene, an archetypal LPS responsive gene that is
implicated in LPS-induced shock in vivo (2), was induced
in only 72% of the clones, whereas most other genes were inducible in
significantly fewer. One surprise was that the GAPDH gene, an intended
"control," was induced in the majority of clones. The GAPDH gene
was used as a normalization control in the previous study on an RAW264
population (4). Fig. 1 shows the mean and variation of
induction in the clones in which the gene was induced, from which it is
clear that each gene was either induced greatly or not at all. Pairwise
analysis revealed no absolute correlation between the induction of this
limited set of genes in individual clones (not shown).
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It seemed almost inconceivable that there would be no coregulation
of sets of genes given that some must share transcriptional regulatory
elements. To analyze the regulatory diversity in greater detail, we
decided to use cDNA microarrays. For this purpose, we used a 1.7-K
mouse Unigene set. To expand the representation of LPS-inducible genes,
we prepared a subtracted, normalized cDNA library from RAW264 cells
stimulated for 4 h with LPS (see Materials and
Methods). After selecting 400 clones, we sequenced 50. Among
these, macrophage-inflammatory protein (MIP)-2, RANTES, virus-like
30S retrotransposon, PGH synthase (cyclooxygenase-2),
and glucocorticoid-attenuated response gene-39, all known
LPS-inducible genes, were represented more than once. We probed the
library to identify and remove from consideration these abundant
clones, then selected an additional 1300 cDNAs. Additional to these
sets, we included 400 known macrophage-expressed cDNAs and other
developmentally regulated genes available in our laboratory. The total
set of 3700 cDNAs was arrayed on glass slides. We selected the five
most divergent RAW264 clones from the original analysis to assay in
detail, using the 4-h time point (the same time as used in the library
construction).
To normalize the data for relative labeling efficiency with Cy3/Cy5 and between clones, we selected a pool of 30 relatively abundant genes that were expressed consistently relative to each other in the induced and uninduced states over all the clones and that include several commonly used loading controls (e.g. cyclophilin). The Cy3/Cy5 ratio for this set of genes was relatively consistent in different clones regardless of LPS stimulation.
Fig. 2 shows a global comparison of the
induction or repression by LPS of the individual elements of the array
in the five clonal lines. There are several features that warrant
comment in the following paragraphs..
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. The heterogeneous gene expression
observed using the slot blots in Fig. 1
for genes such as MIP-1,
RANTES, and TNF- and previously reported for PAI-2 and iNOS was
confirmed and validated using the microarrays (Fig. 3).
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, the lower four panels
show four genes that were not used in normalization; mitochondrial
aspartate-amino transferase, ubiquitin conjugating enzyme, elongation
factor 1A, and propidium iodide-3-kinase. Each was induced marginally
if at all by LPS, but uniformly among the five clones. These controls
validate the normalization. By contrast, again validating the data in
Fig. 1, -actin and GAPDH, previously used as normalization controls
in uncloned RAW264 cells (4) were clearly inducible in
some clones.
The five lines tested can also be clustered hierarchically into three
groups (Fig. 2) based upon the number of inducible genes that they
share. Table I lists some of the known
genes that are coregulated in the three different regulatory
"patterns." This observation suggests that certain sets of genes
are, indeed, more likely to be coexpressed. Each clone is still unique
and can be distinguished from even the most closely related clone by
the lack of coexpression of many individual genes. To assess the
probability of coexpression of any two genes, a much larger set of
individual subclones would need to be assessed.
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The genes that were induced reproducibly by LPS in some subclones
include some very unexpected genes. Among the Unigene set they include
the muscle-specific transcription factor MyoD (19), the
key regulator of cardiac development Nkx2.5 (20, 21), and
the patched hedgehog pathway target gene, Gli-3 (22). We
have confirmed that each of these genes is also induced in primary bone
marrow-derived macrophages stimulated with LPS (see below). The array
results formed the basis for prioritizing additional sequencing of the
clones from the subtracted library. Some of the most highly inducible
sequenced genes, and the numbers of clones in which they were detected,
are shown in Table II. Several are
uncharacterized expressed sequence tags.
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The data in Fig. 1 and the array data suggest that each inducible
gene in macrophages has its own intrinsic probability of being
activated in response to LPS challenge in any RAW264 subclone,
presumably reflecting a stable difference in chromatin structure at the
level of individual alleles. If the probability of being in an
inducible state is allele autonomous, we might expect the same
stochastic pattern to be manifest with reporter genes. To address this
issue in a novel way, we created stably transfected RAW264 lines in
which different LPS-responsive promoters driving either
Renilla luciferase or firefly luciferase were introduced
into the same line. A pool of stable transfectants was produced in
which either the ELAM promoter (a commonly used indicator of
NF-
B-dependent transcription) or the IL-12 promoter were coupled to
firefly luciferase, and the IL-1 promoter was coupled to
Renilla luciferase. The IL-12 and IL-1 genes did not
cosegregate in apparent clusters in RAW264 clones (not shown). The
pools were subcloned by limiting dilution cloning to generate 24
individual lines. Reporter gene activities in the subclones are shown
in Table III. The ELAM promoter, which is
a relatively short fragment, produced quite divergent basal activity in
subclones, ranging over at least 3 orders of magnitude. Despite the
baseline variation, activity was induced at least 20-fold in 23 of 24
lines by LPS. By contrast, basal activity of the IL-1 promoter was
remarkably consistent between subclones, suggesting that is not subject
to effects of position of integration. Despite the consistent baseline,
only 8 of 24 clones exhibited induction by LPS. In the combination of
the IL-12 and IL-1 promoters, approximately the same frequency of
clones showed inducible IL-1 promoter activity. The IL-12 promoter
was also expressed quite consistently in the basal state, but like the
IL-1 promoter, it was activated by LPS in only a subset of clones (5
of 24). As would be expected if the inducibility of the integrated IL-1
and IL-12 promoters segregates independently and randomly, only two of
these clones also exhibited LPS-activated IL-1 promoter activity.
These findings show unequivocally that all clones are LPS responsive.
In addition, the activation of the transcription factor NF-B, which
is strongly implicated in many LPS actions and underlies induction of
the ELAM promoter (16), is not sufficient to ensure
induction of all LPS target genes (although it may well contribute to
an increased probability). For the purpose of this study, the results
reinforce the concept that each gene has an intrinsic probability of
being activated in individual subclones of RAW264 cells and demonstrate
that the determinant of probability lies within the
cis-acting elements of the individual gene promoter.
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A systematic validation of the expression of all of the
LPS-inducible genes discovered in this study is beyond the scope of the
current paper. As noted in the introduction, single cell heterogeneity
in primary macrophages has already been demonstrated for the iNOS,
PAI-2 IL-6, and IL-12 genes (7, 8, 9). To extend this
evidence, and to cross-validate array data from RAW264 cells to primary
macrophages, we focused on a newly discovered LPS-inducible gene (see
above), the transcription factor, Gli-3 (22). Fig. 4 shows detection of Gli-3 in primary
thioglycollate-elicited peritoneal macrophages. In the unstimulated
state in adherent macrophages, Gli-3 mRNA was detected at low levels in
a subset of cells (around 50%). Upon LPS-stimulation, Gli-3 mRNA was
clearly induced. Both the proportion of expressing cells and the level
of expression per cell was increased, but even in the LPS-stimulated
state, not all cells expressed the gene.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, we would suggest that the increased level of expression of
Gli-3 mRNA individual macrophages in LPS-stimulated compared with
control cells reflects a transition from monoallelic to biallelic
expression.
Although the IL-12 and IL-1 promoters, and the cytokine and
chemokine genes in Fig. 1, showed no absolute coregulation, the data in
Fig. 2 suggest that there are sets of genes that are commonly
coexpressed in macrophages. Because of the probabilistic basis of the
variation, such order only becomes evident if one examines many
different genes or many different cells. There is a clear analogy with
studies of the Th1/Th2 dichotomy in stimulated T helper cells where
sets of lymphokines tend to be expressed together in a cell population,
but at a single cell level, the archetypal Th1 (IFN-
) and Th2 (IL-4)
can clearly be expressed in the same cell (see Ref. 23).
Clusters of genes that tend to be induced together can define a
population phenotype even if individual cells cannot be classified
based upon analysis with any one marker. The phenotypes that emerge
among RAW264 cell clones are consistent with the proposed existence of
alternative activation phenotypes in macrophages (25, 26, 27)
in which the balance between pro-inflammatory and anti-inflammatory
secretory products is altered. These proposed phenotypes, like the
corresponding Th1 and Th2 T cell phenotypes, were based upon
differences in the patterns of inducible cytokines between inbred mouse
strains, and indeed the term M-1/M-2 has been proposed
(10). In this study, we have show that distinguishable
macrophage activation phenotypes can arise on a single mouse genetic
background in a cell line.
Coexistence of macrophages with different phenotypes in a single
inflammatory site may be an important part of host defense. For
example, the data in Table I shows that the natural
resistance-associated macrophage protein genes (NRAMP1 and NRAMP2) may
tend to be coinduced in the related lines Clone 14 and Clone 30 along
with high levels of TNF-, MIP-1, MIP-1, and IL-6. The NRAMP 1
gene is mutated in mouse strains that display wide-ranging sensitivity
to intracellular pathogens such as salmonella and mycobacteria
(28). The observed induction of nRAMP2 confirms a recent
report (29). We may speculate that in wild-type
(NRAMP1+) mice the nRAMP1/2 genes are coinduced
by pathogen in only a subset of macrophages that provide a
nonpermissive environment as a consequence. Those macrophages that fail
to induce NRAMP1 and NRAMP2 to help resist the pathogen will eventually
be killed, but will also provide a reservoir of Ag and much higher
amounts of inducible cytokines (30, 31) to permit priming
of the acquired immune system. Given that NRAMP1 and NRAMP2 have
distinct functions in moving iron and other divalent cations across the
endosomal and plasma membranes (32), the intracellular
environment may be different again in cells that induce one gene but
not the other.
Where it has been examined, primary macrophages display the same
heterogeneity in inducible gene expression as RAW264 cells
(7, 8, 9). We have extended this evidence to include the
newly discovered LPS-inducible transcription factor, Gli-3.
Heterogeneous expression of transcription factors like Gli-3 clearly
indicates that both trans-acting and cis-acting
mechanisms might determine transcription probability. Heterogeneity in
primary macrophages was shown most clearly by Witsell and Schook
(12) who reported that individual macrophage colonies
derived by cultivation of mouse bone marrow cells in CSF-1 are
heterogeneous in terms of their ability to express IL-1 and several
other inducible cytokines in response to LPS. These authors favored a
hierarchical determinist model in which the ability to produce
particular cytokines arises as different subsets of macrophages develop
and mature. If the underlying mechanism is actually probabilistic, as
is demonstrated clearly in this work, the progeny of each committed
macrophage progenitor are unique and the innate immune system must
present an infinitely complex team of foes to resist an invading
pathogen.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Hume, Institute for Molecular Bioscience, University of Queensland, Molecular Biosciences Building, Brisbane, QLD 4072, Australia. E-mail address: d.hume{at}imb.uq.edu.au
3 Abbreviations used in this paper: iNOS, inducible NO synthase; DIG, digoxygenin; PAI-2, plasminogen activator type 2; MIP, macrophage-inflammatory protein; ELAM, endothelial leukocyte adhesion molecule; NRAMP, natural-resistance associated macrophage protein gene.
Received for publication June 7, 2001. Accepted for publication October 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transforming growth factor: a quantitative analysis at the single-cell level. Eur. J. Immunol. 19:2037.[Medline]
A-binding element, not an overlapping NF-IL-6-binding element, is required for maximal IL-1 gene expression. J. Immunol. 152:4444.[Abstract]
B half-site. Mol. Cell. Biol. 15:5258.B binding sites in the human E-selectin gene required for maximal tumor necrosis factor -induced expression. Mol. Cell. Biol. 14:5820. and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophages. Eur. J. Biochem. 267:6586.[Medline]
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