1 Department of Medicine, National Jewish Medical and Research Center, Denver 80206; 2 University of Colorado Health Sciences Center, Denver, Colorado 80262; and 3 The Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
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Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.
cellular activation; gene regulation; Toll-like receptor; host defense
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INTRODUCTION |
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THE INNATE IMMUNE RESPONSE is initiated by recognition of microbial surface or secreted components (1), typified by the lipopolysaccharides (LPS) of gram-negative bacteria. To contend with invading microorganisms, the innate response utilizes a number of mechanisms including: 1) generation of reactive oxygen intermediates, 2) release of antimicrobial peptides, 3) phagocytosis, and 4) production and release of cytokines and chemokines. The latter mechanism, relying on transcriptional and translational controls, acts to recruit additional immune cells to the site of infection to amplify and regulate the response.
Polymorphonuclear leukocytes (neutrophils) are important components of
innate immunity. Neutrophils alone can effectively limit infection and,
by production of chemokines, recruit and activate other immune cells
that aid in clearance of the infectious agents and mount an adaptive
immune response. An experimental model of neutrophil response to
infection involves exposure of cells to soluble LPS. Soluble LPS, found
in the circulation and at sites of infection, is recognized by the
Toll-like receptors (TLRs) present on immune cells and has been
identified as the initial signaling event in the response to LPS
(4, 6, 25, 34, 49). TLRs belong to the interleukin (IL)-1
family of receptors (4, 34, 39) and signal through the
cascade of MyD88, interleukin receptor-associated kinase (IRAK), and
tumor necrosis factor (TNF) receptor-associated factor 6 to activate
NF-B and MAP kinases (4, 34). Although neutrophils are
known to respond to LPS by production of reactive oxygen intermediates,
release of lipid mediators and cytokines, adhesion, and phagocytosis,
the genomic response of the neutrophil to infection has been believed
to be somewhat static. Recent studies indicate a robust transcriptional response (32), especially of cytokines, indicating that
the neutrophil contributes more than the release of premade mediators and formation of bactericidal agents. For example, it is well documented that neutrophils produce cytokines such as IL-1
, IL-8, and TNF-
and that this production can depend on both transcriptional and translational regulation (35). Therefore, general
mechanisms exist for regulation of gene expression in neutrophils after
exposure to LPS.
Previous studies screening for changes in gene expression in experimental situations include subtractive hybridization and sequencing, coupled with Northern blot and PCR-based analysis. Recent advances in microarray analysis have allowed the screening of a large number of changes in mRNA expression. In this way, thousands of genes can be screened in an unbiased fashion for transcript abundance and changes in expression levels. Such genomic screens in mammalian cells have involved altered expression profiles in response to agonists (11) and drug action (24) and during cell cycle progression (22). Furthermore, microarray analysis can suggest pathways important in cell function that otherwise would be difficult to unmask by traditional methods. We reasoned that neutrophil gene expression changes are not limited to cytokine production and that a more detailed examination of gene expression in stimulated neutrophils will display novel gene expression profiles with potential consequences in functional characteristics. As an initial step to test this hypothesis, we have utilized microarray technology. Furthermore, we have recognized that neutrophil life span is enhanced at sites of infection (by inhibition of spontaneous apoptosis), and, therefore, time points other than those immediately after stimulation may provide clues into neutrophil function. Thus we have examined gene expression after prolonged (4 h) stimulation with LPS.
Analysis of gene expression microarrays of naïve and
LPS-stimulated neutrophils confirmed the production of chemokine and cytokine (e.g., IL-1) mRNAs among the 28 distinct gene products found to be induced by LPS, including monocyte chemoattractant protein
(MCP)-1, described here for the first time as induced by LPS in
neutrophils. Surprisingly, a number of genes involved in distinct
cellular processes were also induced including those involved in
transcriptional regulation (e.g., NFKB) and cell signaling (e.g.,
cot). Likewise, repression of diverse genes, including those
important for cytoskeletal regulation [e.g., tubulin, Rho guanine
nucleotide dissociation inhibitor (GDI)
], was found to occur. Finally, changes in the expression levels of several expressed sequence tags were induced or repressed. The changes observed using
cDNA arrays complement our previous findings using oligonucleotide arrays and include gene expression changes not previously noted (15). Our results establish that neutrophils actively
regulate gene expression levels and suggest that, in addition to the
well-known regulation of cytokine and chemokine expression, their
exposure to infectious agents may modify several classes of genes.
Furthermore, the gene expression profile of stimulated neutrophils
provides a "fingerprint" of their activation state and information
on intracellular signaling events. We believe these findings present a
better understanding of the role neutrophils play in inflammation and
immunity and provide evidence for molecular mechanisms of neutrophil
function after exposure to LPS.
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METHODS |
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Cell preparation and stimulation. Neutrophils were isolated from citrated blood from healthy donors and purified on a Percoll gradient, as described previously (20). Cells (25 × 106 cells/ml) were resuspended in RPMI containing 1% heat-inactivated platelet-poor plasma and 10 mM HEPES, pH 7.6, and divided into 1.5-ml tubes. Each condition for microarray analysis utilized 120 × 106 cells. Cells were stimulated with 100 ng/ml LPS (Escherichia coli 0111:B4; List Biological) and rotated continuously at 37°C for 4 h. For microarray analysis, nonstimulated and 4-h-treated cells were used from three separate donors. More detailed time courses after LPS exposure were performed for analysis by PCR and ELISA using 20 × 106 neutrophils.
Extraction of RNA and isolation of mRNA. Cell suspensions were centrifuged briefly, and cell supernatants were retained for analysis by ELISA. Cell pellets were solubilized in 0.5 ml of RNA Trizol (GIBCO) per 25 × 106 cells, and total RNA was extracted as described in the manufacturer's protocol. Briefly, 0.1 ml of chloroform was added to the Trizol suspension, shaken, incubated at room temperature for 10 min, and centrifuged for 15 min at 12,000 g. The aqueous upper phase was carefully removed, RNA was precipitated with 0.25 ml of isopropanol, and the precipitate was washed in 75% ethanol before being dried and resuspended in RNase-free water. RT-PCR was performed routinely to determine the integrity of RNA, the quiescence of neutrophils at baseline, and their responsiveness to LPS. Total RNA from each condition was pooled, and poly(A)RNA was obtained from these samples by Oligotex Mini Kit (Qiagen).
cDNA probe synthesis, hybridization to microarray, and scanning. A detailed account of hybridization of cDNA probes to microarray has been described previously in detail (24). Briefly, cDNA probes were generated by reverse transcription of mRNA (1 µg) with SuperScript II (GIBCO) in the presence of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia). Isolated single-strand cDNAs were denatured and incubated on duplicate arrayed slides in 30 ml 5× SSC, 0.1% SDS, 0.1 mg/ml salmon sperm DNA, and 50% formamide. The array was composed of 4,608 minimally redundant cDNAs from the UniGene set (24, 33). Labeled cDNA probes were allowed to hybridize overnight at 42°C in a humidified chamber. Slides were washed and dried before being scanned on the Avalanche dual-laser confocal scanner (Molecular Dynamics). Fluorescent intensities were quantified with ARRAYVISION 4.0 (Imaging Research, St. Catherine's, ON, Canada).
Data analysis. We normalized data for the individual experiments by dividing the fluorescence intensity (for both Cy3 and Cy5) of each element by the total fluorescence for each fluorophore. Ratios of the normalized values of LPS-stimulated (Cy3) to nonstimulated (Cy5) elements were calculated with Excel (Microsoft). We performed regression analysis (SigmaPlot) to determine the mean fluorescence ratio for the 4,608 elements. Significant changes in gene expression were taken as two standard deviations above the mean (induced) and less than one-half the mean (repressed). The average ratio for each element in the three experiments was calculated, and significant changes (P < 0.05) were determined by t-test. Data were deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) (accession numbers: GSM3033, GSM3034, GSM3035, GSM3038, GSM3039, GSM3040).
RT and PCR.
cDNA was prepared by reverse transcription using 2 µg of total RNA.
PCR was performed using specific primers for TNF-
(5'-AGCCCATGTTGTAGCAAACC-3', 5'-TTTGGGAAGGTTGGATGTTC-3'), MCP-1
(5'-TCTGTGCCTGCTGCTCATAGC-3', 5'-GGGTAGAACTGTGGTTCAAGAGG-3'),
A1 (5'-GATGACTGACTGTGAATTTGG-3', 5'-TGGAGTGTCCTTTCTGGTCA-3'), Mx-1
(5'-TGTGCAGCCAGTATGAGGAG-3', 5'-CTCAGCTGGTCCTGGATCTC-3'),
S100A4 (5'-TTCTTTCTTGGTTTGATCCT-3', 5'-CATCAGAGGAGTTTTCATTTC-3'), and
GAPDH (5'-TCATCCATGACAACTTTGGTATCG-3', 5'-TGGCAGGTTTTTCTAGACGGC-3').
ELISA.
Secretion of IL-1, MCP-1, macrophage inflammatory protein
(MIP)-1
, and MIP-1
was quantified by ELISA kits (R&D), using undiluted supernatant, as directed by the manufacturer.
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RESULTS AND DISCUSSION |
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Human neutrophils were left untreated or incubated in the presence
of 100 ng/ml LPS for 4 h. We used the 4-h time point in appreciation of the ability of LPS to inhibit spontaneous
apoptosis and to explore the possibility that long-term changes
in gene expression were important in neutrophil activation. Initially, to confirm that the neutrophils were not activated and that LPS resulted in normal stimulation, we performed PCR for TNF-. Little TNF-
expression was seen in nonstimulated cells, whereas LPS treatment led to a significant increase in expression in each of the
donors subsequently used for microarray analysis (data not shown; see
Fig. 3A).
Microarrays were hybridized simultaneously with fluorescent cDNA probes
derived from mRNAs of nonstimulated (Cy5-labeled) and LPS-stimulated
(Cy3-labeled) neutrophils. A sample grid from one donor is shown in
Fig. 1A. Predominately red
spots indicate induction, green spots indicate repression, and yellow
indicates no change in gene expression; areas of undetectable
fluorescence indicate that the gene is not expressed or is expressed
below the level of detection. The fluorescence intensities at each
element were normalized to the total fluorescence for each fluorophore, and the ratio of stimulated to nonstimulated fluorescence was determined. The ratio for each element was subjected to regression analysis, and the mean fluorescence ratio for the entire set was determined. Furthermore, we searched cDNA arrays for monocyte markers
to determine whether monocyte contamination would complicate the
analysis. RNA for the macrophage-colony stimulatory factor receptor was
not expressed in nonstimulated neutrophils, and CD14, which is
expressed at higher levels in monocytes compared with neutrophils, was
barely detectable. These observations are consistent with the low
contamination (<5%) of monocytes when neutrophils are isolated
(20).
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Reproducibility of genes induced by LPS in neutrophils.
Because of the inherent variability of primary human cells, we wished
to determine the reproducibility of changes detected on gene expression
microarrays. Analysis of microarray data for each donor indicates that
36-48 nonredundant genes (average 40.0) were induced by greater
than two standard deviations from the mean fluorescence ratio for the
entire gene set as determined by regression analysis (Fig.
2A). We wished to compare the
reproducibility of gene induction between any two experiments by
comparing the number of common genes induced. When those genes
upregulated by any two donors were assessed, 22-25 genes (average
23.7) were induced, corresponding to ~60% of the genes induced in
each experiment. All three donors shared 18 upregulated genes
(~45%). Therefore, microarray analysis conveys both the complexity
of human genetic response and the potential to establish common events
in primary human cells, since all three donors shared almost 50% of
the induced changes. Although neutrophil preparations provide a highly
purified cell population, the variability of the response may be
explained by donor variability, previous activation state of the
neutrophils, cell maturity, and differences in the time course of the
response.
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Reproducibility of genes repressed by LPS in neutrophils. The number and reproducibility of genes repressed by LPS treatment were also analyzed. The criterion for genes negatively regulated by LPS was a fluorescence ratio less than one-half of the average fluorescence ratio (Fig. 1B, green circles). The concordance for genes repressed among the individual donors, and between the individual and averaged experiment, was less than that for the induced genes. The three donors downregulated 49-68 genes (average 56.0), but any pair of individuals shared only 23, 24, and 25 (average 24.0) repressed genes (representing ~43% of the total in the individual experiments), and there were eight genes (~27%) shared by all three donors (Fig. 2B). When the average data were analyzed there were 18 genes determined to be repressed.1 Therefore, a greater variability in response was found with repressed genes, although several common changes were reproducibly observed.
The averages of the fluorescence ratios from the three experiments were determined. By this method, a fuller picture of changes in gene expression could be formed. However, the use of three donors may mask significant changes in some genes. Thus, the data presented here are conservative in their appraisal of LPS-regulated genes, and the list of genes provided likely includes only those genes whose regulation is most profound. Those genes whose ratios were, on average, greater than the threshold of two standard deviations of the average mean fluorescence (1.00 ± 0.51 SD) were further analyzed. A scatter plot of the average ratios for each of the 4,608 genes is shown in Fig. 1B. In all, 32 different gene products2 were found to be induced (Fig. 1B, red circles); redundant appearance of some genes on the array accounts for the 50 elements induced in Fig. 1B. Of the 32 genes upregulated, 24-27 genes (average 25.3, or 79%) were upregulated by each donor. Although some variability in array data was observed, we believe this is due largely to donor differences and is not inherent in the array analysis itself. In fact, the data described here are likely to represent robust changes in gene expression. Thus microarray analysis is a reproducible means of determining changes in gene expression in primary human neutrophils. Individual genes induced in LPS-stimulated neutrophils will be described below.Genes differentially expressed in LPS-stimulated neutrophils:
upregulated genes.
Genes whose expression changed after LPS treatment were verified by
sequencing, PCR, ELISA, or additional array experiments and were
required to be significantly changed. As expected, the genes for
several cytokines and chemokines were upregulated by LPS treatment of
neutrophils. These include IL-1, IL-8, MIP-1
, and MIP-1
(Table
1). Upregulation of these inflammatory
mediators is well documented in neutrophils exposed to LPS and in
animal models of LPS-induced sepsis syndrome and acute respiratory
distress syndrome, a neutrophil-mediated illness (23, 44).
PCR was performed to confirm and substantiate the results from the
microarray analysis (Fig. 3A).
Analysis of selected genes indicates that the time course for changes
can be rapid or delayed but parallels the changes found in the array at
the 4-h time point (Fig. 3A), thus confirming expression
changes found using cDNA arrays. However, the possibility arises that
transient changes in gene expression were missed because only the later
time point was used. Secretion of IL-1
, MIP-1
, and MIP-1
were
confirmed by ELISA (data not shown). Finally, mRNAs for IL-1
receptor antagonist (RA) and TSG-6, a known TNF-
-induced gene
(28), were also induced in LPS-treated neutrophils.
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Genes differentially expressed in LPS-stimulated neutrophils:
downregulated genes.
Genes downregulated by LPS again provide enticing information on
neutrophil function in the face of infection. There were several genes
whose products are structural in nature (e.g., 2-actin, coronin, tubulin) (Table 2) and suggest
that neutrophil motile response is repressed in the face of infection
(45). The downregulation of Rho GDI
, involved in the
regulation of the actin cytoskeleton, is also consistent with this
suggestion, as are our data indicating upregulation of the adhesion
molecule CD44 (Table 1). The calcium-binding protein S100A4,
downregulated in LPS-treated neutrophils (Table 2), has been implicated
in cell motility and metastasis (3). Furthermore, LPS is
known to increase adhesion and cell rigidity and decrease chemotaxis
(45). Thus gene expression array analysis has identified
genes involved in cell motility and suggests a genetic basis for
structural and motile changes induced by LPS. These findings complement
the cell survival signals suggested previously to prolong neutrophil
residency at sites of infection.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. C. Malcolm, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: malcolmk{at}njc.org).
1 We were unable to verify the sequence of one of these genes.
2 We were unable to verify the sequence of five of these genes.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 20, 2002;10.1152/ajplung.00094.2002
Received 29 March 2002; accepted in final form 11 December 2002.
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