Physiol. Genomics AJP: Gastrointestinal and Liver Physiology
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Physiol. Genomics 25: 203-215, 2006. First published January 10, 2006; doi:10.1152/physiolgenomics.00192.2005
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Received 29 July 2005; accepted in final form 5 January 2006.
Physiological Genomics 25:203-215 (2006)
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Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans

Shefali Talwar1, Peter J. Munson2, Jennifer Barb2, Carmen Fiuza1, Anadel Pilar Cintron1, Carolea Logun1, Margaret Tropea1, Sameena Khan1, Debra Reda1, James H. Shelhamer1, Robert L. Danner1 and Anthony F. Suffredini1

1 Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland
2 Mathematics and Statistical Computing Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To define gene expression profiles that occur during the initial activation of human innate immunity, we administered intravenous endotoxin (n = 8) or saline (n = 4) to healthy subjects and hybridized RNA from blood mononuclear cells (0, 0.5, 6, 24, 168 h) or whole blood (0, 3, 6, 24, 168 h) to oligonucleotide probe arrays. The greatest change in mononuclear cell gene expression occurred at 6 h (439 induced and 428 repressed genes, 1% false discovery rate, and 50% fold change) including increased expression of genes associated with pathogen recognition molecules and signaling cascades linked to receptors associated with cell mobility and activation. Induced defense response genes included cytokines, chemokines, and their respective receptors, acute-phase transcription factors, proteases, arachidonate metabolites, and oxidases. Repressed defense response genes included those associated with co-stimulatory molecules, T and cytotoxic lymphocytes, natural killer (NK) cells, and protein synthesis. Gene expression profiles of whole blood had similar biological themes. Over 100 genes not typically associated with acute inflammation were differentially regulated after endotoxin. By 24 h, gene expression had returned to baseline values. Thus the inflammatory response of circulating leukocytes to endotoxin in humans is characterized by a rapid amplification and subsidence of gene expression. These results indicate that a single intravascular exposure to endotoxin produces a large but temporally short perturbation of the blood transcriptome.

innate immunity; inflammation; leukocyte transcriptome


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SEPSIS IS A SYSTEMIC INFLAMMATORY disorder induced by infection and, when severe, is accompanied by shock with organ failure. It is a major cause of morbidity and death in critically ill patients (32). While the host inflammatory response is necessary to localize and resolve an infection, it has a central pathogenic role in the development and severity of sepsis syndromes (3). Therapy of septic shock is based on infection control, hemodynamic resuscitation, and attempts to modify the inflammatory response. Yet in the latter case, treatment directed at a single mediator or inflammatory pathway has met with only limited success in improving survival in sepsis (3, 4, 17, 35). Major challenges remain in understanding the mechanisms that contribute to these processes.

The pathogenesis of sepsis is directly related to innate immunity, which encompasses the immediate host inflammatory responses that result from exposure to microbial components (5). The rapid immune responses are initiated by conserved microbial structures that activate pattern recognition receptors on cell surfaces and in the circulation (e.g., endotoxin, Toll-like receptors, and lipopolysaccharide-binding protein, respectively) (6, 33). These interactions result in cell activation, the release of inflammatory molecules, and the recruitment of inflammatory cells to sites of infection (37).

Investigators have administered the gram-negative bacterial wall component endotoxin to healthy human subjects to learn more about the mechanisms associated with sepsis (48). This model results in physiological responses that are archetypal of innate immunity, cell activation and inflammatory mediator release that cause fever, leukocytosis, tachycardia, tachypnea, and decreased blood pressure, findings similar to the early signs of infection in patients (1, 41, 46). To further characterize the complex phenomena associated with this common bacterial product, we studied differential gene expression of peripheral blood from healthy subjects challenged with intravenous endotoxin using oligonucleotide gene arrays. Oligonucleotide arrays have been used to profile the complex gene networks associated with normal physiology and disease (11, 44, 45). We have previously shown (42) that some acute-phase proteins persist in the blood for 7–10 days after intravenous endotoxin challenge, and we hypothesized that alterations in the transcriptome may persist for several days mirroring this acute inflammatory response.

We show that the administration of a single dose of intravenous endotoxin to humans leads to a rich profile of gene expression changes in blood. These include the induction of genes associated with pattern recognition molecules, intracellular signaling and transcription, cell mobility, and defense function. T lymphocyte-associated genes were repressed, and many genes not previously associated with endotoxin-induced inflammation were differentially regulated during this response. Notably, these alterations in gene expression were rapidly extinguished within 24 h, and scant residua of this response were detected at 7 days.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials
Vacutainer CPT cell preparation tubes for isolation of peripheral blood mononuclear cells, mouse anti-human CD14-PE, CD45-PerCP, CD4-FITC, CD8-FITC, CD69-PE, CD25-PE, CD3-PerCP, tricolor CD3-FITC/19-PE/45-PerCP, tricolor CD3-FITC/CD16&56-PE/CD45-PerCP, tricolor CD3-PerCP/4-FITC/8-PE, IgG-PE, and IgG-FITC antibodies were obtained from Becton Dickinson (Franklin Lakes, NJ). RLT buffer, RNeasy Midi and Mini kits, and DNase 1 were purchased from Qiagen (Valencia, CA). PAXgene blood RNA tubes and PAXgene Blood RNA Kit were purchased from PreAnalytiX, Qiagen. T7-d(T)24 primer was obtained from Genset (La Jolla, CA). SuperScript Double Stranded cDNA Synthesis Kit was from Invitrogen (Rockville, MD). Phase Lock Gel was obtained from Eppendorf (Westbury, NY), and phenol-chloroform-isoamyl alcohol (25:24:1) was purchased from Invitrogen (Carlsbad, CA). HighYield RNA Transcript Labeling Kit was purchased from Enzo Diagnostic (Farmingdale, NY). GeneChip Sample Cleanup Module was obtained from Affymetrix (Santa Clara, CA). Human Multiplex Antibody Bead Kits for Luminex were purchased from BioSource International (Camarillo, CA).

Human Subjects
Six men and four women (20–45 yr; mean ± SE, 30 ± 2 yr) in good health participated in the study. None used tobacco products, and all had normal screening blood tests, electrocardiograms, and chest radiographs. Informed written consent was obtained from each subject. Human experimental guidelines of the United States Department of Health and Human Services were followed, and the study was approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (Bethesda, MD). Eight subjects were given intravenous endotoxin [4 ng/kg, Escheria coli O:113, Clinical Center Reference Endotoxin, National Institutes of Health (NIH), Bethesda, MD], and four subjects were given intravenous saline alone. This dose of endotoxin is safe and elicits a broad variety of physiological responses that are similar to the syndrome of sepsis (41, 46, 48). Two of the four subjects given saline participated in the endotoxin portions of the study, separated by at least 6 wk. Blood was drawn from either radial arterial catheters or by venipuncture after the endotoxin or saline challenge. Temperature was monitored with tympanic membrane thermometers, heart rate by continuous electrocardiogram, and arterial pressure by arterial catheter or cuff pressure. Automated total and differential blood cell counts were determined (Coulter). The effects of endotoxin were studied in two different cell populations: peripheral blood mononuclear cells (PBMC) and whole blood.

Blood Cell Preparation and Total RNA Isolation
PBMC.
After endotoxin or saline challenge, blood was drawn at 0, 0.5, 6, 24, and 168 h. These time points were chosen to assess responses before and immediately after the lymphopenia and monocytopenia that occur with intravenous endotoxin (47). To minimize in vitro manipulations that might introduce bias into expression profiles (12, 19), blood was collected directly in cell preparation tubes containing sodium citrate and Ficoll Hypaque density fluid (Vacutainer CPT), placed on ice, and centrifuged within 30 min (2,200 g, 8°C for 30 min). The PBMC layer was removed, washed with cold PBS, and centrifuged (900 g, 8°C for 10 min). Contaminating red blood cells were lysed with ammonium chloride lysing buffer. Sample quality was confirmed by a differential blood smear, and an aliquot of cells was placed aside for surface marker analysis using flow cytometry. After a washing in PBS, the cells were lysed with RLT buffer, homogenized using a syringe and 20-gauge needle, and then stored at –80°C. The process from sample acquisition to cell lysate was performed within 90 min for each sample to minimize the affects of processing on the in vivo expression profile. After all time points per subject were acquired, total RNA was extracted as per RNeasy Midi protocol (Qiagen). The quality of the total RNA was assessed by visualization of intact 18S and 28S ribosomal RNA bands using a 1.2% formaldehyde agarose gel (Ambion, Austin, TX) stained with Sybr Green II (Molecular Probes, Eugene, OR). The 260/280 ratio of all samples was between 1.8 and 2.0. Baseline samples (0 h) revealed a negligible number of neutrophils. At 6 h, neutrophil contamination of the PBMC preparations was <10% of the total cells. Our previous work has shown that, because of the low amount of RNA present in neutrophils, this amount of contamination would contribute minimally to the gene profiles of the PBMC (28).

Whole blood cells.
In four subjects challenged with intravenous endotoxin, samples of whole blood cells were collected at 0, 3, 6, 24, and 168 h in PAXgene blood RNA tubes. These time points were chosen to sample whole blood immediately after the release of cytokines and after the onset of monocytopenia. In addition, PBMC were collected in these same subjects at 0, 6, and 24 h. Whole blood cell RNA was isolated. Total RNA was extracted and treated with DNase 1 as per PAXgene Blood RNA Kit protocol with the following modifications: after 2 h of incubation at room temperature, blood samples were kept for a minimum of 24 h at 4°C, and total RNA was precipitated overnight with 3 M sodium acetate and 100% ethanol. All samples were processed within 72 h from time of collection.

PBMC and Whole Blood Cell Total RNA Hybridization to Oligonucleotide Probe Arrays
Double-stranded cDNA was synthesized from total RNA (5–20 µg) using 7-d(T)24 primer and SuperScript Double-Stranded cDNA Synthesis Kit. cDNA was purified by Phase Lock Gel-phenol-chloroform extraction followed by ethanol precipitation. Biotin-labeled cRNA was prepared by in vitro transcription using HighYield RNA Transcript Labeling Kit followed by fragmentation with 5x fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 min. Fragmented cRNA (10 µg) was hybridized to Affymetrix Hu95Av2 oligonucleotide probe arrays for 16 h at 45°C. After removal of hybridization fluid, the arrays were washed and stained with streptavidin-phycoerythrin (SAPE), and signal was amplified by anti-streptavidin antibody using Affymetrix Fluidics Station 400. Probe set signals were measured using Agilent GeneArray Scanner (Affymetrix).

Validation of Gene Expression Using Real-Time PCR
Real-time PCR (RT-PCR) was used to validate the microarray results for PBMC data sets (TaqMan PCR detection; Applied Biosystems, Rockville, MD). Probes and primers for 12 target sequences were designed using Primer Express computer software (Applied Biosystems) or purchased (Applied Biosystems) (see Supplemental Table S1; available at the Physiological Genomics web site).1 Total RNA from the PBMC of four subjects was extracted as above, treated with DNase 1, and reverse transcribed to cDNA using random hexamers and High Capacity cDNA Archive Kit as per the manufacturer's instructions (Applied Biosystems). The gene of interest was then amplified and quantified using TaqMan Universal PCR master mix and TaqMan probe and primers on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). RNase P was used as internal control (Applied Biosystems). The standard curve method was used to quantify the gene of interest and then normalized to RNase P. Final results were expressed as fold change, comparing 6 with 0 h after intravenous endotoxin.

Flow Cytometry of Isolated PBMC
Cell surface markers for identifying different cell populations were analyzed at times of 0 and 6 h after endotoxin challenge. The PBMC preparations were evaluated for percentage of T cells, B cells, natural killer (NK) cells, and monocytes. T lymphocytes were analyzed for CD4 and CD8 subsets and for markers of early and late activation. Cells were resuspended in PBS, incubated with mouse anti-human antibody for 30 min at 4°C, washed with PBS, and fixed with 1% formaldehyde. Monocyte and lymphocyte differential counts were determined by staining cells with CD14-PE and CD45-PerCP. CD14-PE bright population was back-gated to light scatter to quantify monocyte percentage, and the remaining cells were considered lymphocytes. The percentage of T cells, B cells, and NK cells was determined by staining cells with tricolor CD3-FITC/19-PE/45-PerCP or tricolor CD3-FITC/CD16&56-PE/CD45-PerCP, gating on CD45-PerCP. T lymphocyte subset (CD4 and CD8) percentages were identified using tricolor CD3-PerCP/CD4-FITC/CD8-PE stain, gating on CD3-PerCP. Finally, labeling with CD3-PerCP/CD4-FITC/CD69-PE, CD3-PerCP/CD8-FITC/CD69-PE, CD3-PerCP/CD4-FITC/CD25-PE, or CD3-PerCP/CD4-FITC/CD25-PE identified early and late activation of CD4 and CD8 cells. Gated events (2,000) were collected using FACScan system and analyzed with CELLQuest software (Becton Dickinson).

Measurement of Blood Inflammatory Protein Markers
Serum samples for tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-{gamma}, regulated upon activation, normal T cell expressed, and secreted (RANTES), macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, monocyte chemotactic protein (MCP)-1, eotaxin, granulocyte macrophage colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF) were measured at 0, 3, 6, and 24 h using a multiplex bead-based assay (Human Multiplex Antibody Bead Kits for Luminex) according to the manufacturer's recommendations. The samples were measured using the antibody bead mix in duplicate with a biotinylated detection antibody followed by streptavidin-phycoerythrin. The plate was read using the Luminex XYP platform (Luminex, Austin, TX), and data were collected for 100 beads per cytokine from each well. The raw data (mean fluorescent intensity) were processed on Masterplex Quantitation software (MiraiBio, Alameda, CA) to obtain concentration values.

Statistical Analysis: Data Transformation
The average difference (AD) values (Affymetrix Microarray Suite 4.0) for the oligonucleotide arrays were stored in the NIH Laboratory Information Management System (LIMS) database and retrieved, transformed, and analyzed with the use of the Mathematical and Statistical Computing Laboratory (MSCL) Analyst's Toolbox (http://abs.cit.nih.gov/MSCLtoolbox/) written by P. J. Munson and J. Barb in the JMP scripting language (SAS Institute, Cary, NC). Values (AD) were standardized and transformed using the Symmetric Adaptive Transform, which yields quantile-normalized, homogeneous variance scale intensity values, termed S-AD (16, 28). A consistency test was applied to identify subjects with a consistent change in the S-AD across all treated subjects. The analysis specified a 1% false discovery rate for each set of detected genes found in the PBMC and whole blood (43). The list was further refined by requiring that the relative change between late time points compared with time baseline (fold change) be at least 50%. To eliminate genes that were at or below the stated detection limits for this assay, an average AD value >20 was required in at least one of the two compared groups.

The transformed data matrix [samples (columns) by genes (rows)] was subjected to principal components analysis to visualize the relative location of chips in a low-dimensional space allowing for detection of outliers or other relevant patterns. Genes were annotated using the Gene Ontology (GO) database (http://www.geneontology.org) and the GO-Significant Collection of Annotations (SCAN) program (http://abs.cit.nih.gov/goscan/) for assessing significantly overrepresented annotation terms. GO-SCAN presents an interactive table of terms and permits drill-down into the supporting expression data. Overrepresentation of the annotation category was determined using a one-sided Fishers exact test. The annotation terms were then analyzed using the GO-MAP program (http://abs.cit.nih.gov/goscan/), which performs a 2-D hierarchical clustering of terms and genes to group possibly redundant annotations and corresponding genes into blocks that can be presented graphically. Groups of similarly annotated genes may then be studied together in relation to their expression results. One hundred twelve genes of the final gene list have been reported in part as a comparison population describing gene expression in patients with stable sickle cell anemia (28). The entire data set of the above investigation has been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO; http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi), accession number GSE3026.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Systemic Responses to Endotoxin
All subjects given endotoxin developed mild to moderate degrees of malaise, headache, and fever (0 h, 37.1 ± 0.1°C, and 3 h, 39.03 ± 0.2°C) tachycardia (0 h, 62 ± 5 beats/min, and 3 h, 96 ± 4 beats/min) and a decrease in mean arterial pressure (0 h, 90 ± 3 mmHg, and 5 h, 74 ± 3 mmHg) (maximum change from baseline, all P < 0.01, paired t-test). Saline-challenged subjects had similar baseline vital signs with no significant changes during the study period. Within 1 h, total leukocyte counts reached their nadir due to margination of cells to activated vascular endothelium (31, 39, 47, 51). This was followed by a rapid rise in total leukocyte counts composed primarily of neutrophils, while mononuclear cells were decreased in number compared with baseline. At 6 h, the number of mononuclear cells began to rise and returned toward normal at 24 h (Table 1). Blood cytokines (TNF-{alpha}, IL-1ß, IL-6, IL-8, IL-10, MCP-1, MIP-1{alpha} and -ß) rose at 3 h and returned to baseline values by 6–24 h (Table 2). These eight cytokines and chemokines have been described in normal subjects given endotoxin compared with saline in previous publications (reviewed in Ref. 48). In these previous studies, no changes occur from baseline in the saline-challenged subjects.


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  		Table 1. Changes in peripheral blood leukocytes after intravenous endotoxin administration

 

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  		Table 2. Acute-phase cytokines in serum after intravenous endotoxin challenge

 
Cell Components of Mononuclear Cell Preparations
The proportion of lymphocytes and monocytes present after density centrifugation separation at 0 and 6 h is summarized in Fig. 1. Compared with baseline, endotoxin administration resulted in the percentage of lymphocytes falling by almost 50% and the percentage of monocytes increasing by threefold at 6 h. The percentage of T lymphocytes and NK cells remained the same, while the proportion of B cells increased at 6 h (Fig. 1, inset). The percentage of CD3+ lymphocytes at 6 h expressing CD4 and CD8 surface markers did not change from baseline (0 h: CD4 58.0 ± 2.8%; CD8, 35.0 ± 3.2%). CD4+ and CD8+ cells expressing an early activation marker (CD69) increased at 6 h (CD4+69+ 0 h, 0.8 ± 0.3%, and 6 h, 5.0 ± 1.7%, P = 0.035; and CD8+69+ 0 h, 1.9 ± 0.7%, and 6 h, 7.6 ± 3.1%, P = 0.07, paired t-test). No changes in CD4+69+ or CD8+69+ cells occurred in the control subjects, suggesting that cell processing was not associated with induction of this early marker of lymphocyte activation. The percentage of CD4+ or CD8+ cells expressing a late marker of activation (CD25) was unchanged at 6 h (0 h: CD4+25+, 18.1 ± 4.4%; CD8+CD25+, 1.4 ± 0.4%).


Figure 1
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  		Fig. 1. Proportion of lymphocytes and monocytes present after density centrifugation separation at 0 and 6 h. Compared with baseline, endotoxin administration resulted in the percentage of lymphocytes falling by almost 50% and the percentage of monocytes increasing by 3-fold at 6 h. Inset: percentage of T lymphocytes and natural killer (NK) cells remained the same while the proportion of B cells increased at 6 h.

 
Differential Gene Expression in PBMC After Endotoxin
Endotoxin is cleared from the blood within 30 min after intravenous administration, suggesting that many of its downstream effects are due to secondary mediators that amplify the initial host response to this bacterial component (50). A major source of TNF, an important secondary mediator that enhances the host response to endotoxin, is from the liver (23, 34). The resultant inflammatory milieu in blood has profound effects on genes associated with both the afferent and efferent arms of innate immunity. At 6 h, >800 genes were differentially regulated; 439 genes were induced, and 428 genes were repressed compared with baseline (Fig. 2A and Supplemental Table S2A). GO categories that were significantly (P < 0.01) represented among these genes are depicted by GO-MAPs found in Supplemental Table S2, B (induced genes) and C (repressed genes). Twelve genes were differentially regulated at other time points: 30 min (4 induced), 24 h (5 induced and 2 repressed), and 168 h (1 induced). Eight genes were altered in control subjects, of which only two genes (Hgb-ß and AP1G2) overlapped with the endotoxin-associated gene list and were removed from further analysis.


Figure 2
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  		Fig. 2. A: heat map of changes in gene expression over time (0.5 h, n = 6; 6 h, n = 8; 24 h, n = 7; and 168 h, n = 6) of 982 differentially expressed probe sets in peripheral blood mononuclear cells (PBMC) after intravenous endotoxin challenge. Each row represents a gene probe set and each column a subject at the respective time point. Change from baseline is determined by the average difference in the probe set (standardized and transformed using the symmetric adaptive transform as per METHODS), with increased gene expression depicted by red and decreased gene expression depicted by green. B: heat map of changes in gene expression over time (3 h, n = 3; 6 h, n = 4; 24 h, n = 4; and 168 h, n = 4) of 224 differentially expressed probe sets in whole blood cells after intravenous endotoxin challenge. Each row represents a gene probe set and each column a subject at the respective time point. Change from baseline is determined by the average difference in the probe set, as above.

 
Afferent Limb of Innate Immunity
The afferent limb of innate immunity encompasses those responses necessary for the host to recognize and to activate inflammatory responses to a pathogen (Table 3). Endotoxin administration was associated with the induction of cell surface receptor genes whose products enhance cell recognition of microbial signals [TLR1 and -2, CD14, FPRL1, and scavenger receptors (MARCO)]. Soluble pathogen-recognition molecules and their respective receptors or receptor components were induced [i.e., bactericidal permeability-increasing protein, properdin P factor, C1q receptor 1, C3a receptor 1, and C (3b/4b) receptor 1]. The mRNA of immune complex receptors (Fc-{gamma} receptor I and II) and leukocyte immunoglobulin-like receptors (LILRA3, LILRB2, LILRB3, and LILRB4) was upregulated. The latter are associated with major histocompatibility complex (MHC) class I antigens and may result in a stimulatory (LILRA3) or inhibitory cascade (LILRB2, -3, -4). These responses suggest that the mononuclear cells are primed to respond to further microbial component exposure.


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  		Table 3. Defense response genes altered in peripheral blood mononuclear cells 6 h after intravenous endotoxin challenge

 
Simultaneously, major changes occur in the induction of genes associated with intracellular signaling cascades: G protein-linked receptors (protein kinase C or PRKCD), MAPKs (MAPK14 or p38), p21 activated kinase 2 (PAK2), receptor tyrosine kinases [phosphatidylinositol 3-kinase (PI3K), GTPase], and receptor tyrosine kinase-associated genes (Src, JAK, Ras, Rho). These pathways are linked to receptor families that affect cell mobility, proliferation, activation, and effector function.

Efferent Limb of Innate Immunity
The activation of host inflammatory cells initiates the efferent limb of innate immunity (Table 3). This results in the release of inflammatory molecules and changes in cell structural components to enhance association with activated endothelium and migration to a nidus of infection. The activation of the mononuclear cells results in changes in cytoskeleton (MARCKS, actin), membrane and ion channel genes (aquaporin 9), and genes that enhance cell motility (uPAR, L-selectin or CD63, CD11b or complement receptor type 3, {alpha}-subunit). Anti-proteases (SERPINA1 and -B1) and disintegrins with domains that have potential for adhesion and metalloprotease activity are upregulated (ADAM8 and -9). Immediate anti-pathogen responses include the induction of microbicidal oxidases [CYBA (p22-phox), CYBB (gp91-phox)] and their regulators [superoxide dismutase (SOD)]. These efferent responses are modulated by the induction of cytokines (IL-1Ra), cytokine and chemokine receptors (INFGR2, IL-10R-ß, IL-17R, CCR1, CCR2), growth factors (VEGF, ECGF1), and growth factor receptors (CSF2RA and -B, BST1 and -2). Arachidonate metabolism is potentiated by the upregulation of platelet-activating factor acetylhydrolase (PLA2G7) and leukotriene B4 receptor and the induction of arachidonate 5-lipoxygenase-activating protein (ALOX5AP), which, with 5-lipoxygenase (ALOX5), is required for leukotriene synthesis. Genes associated with cell stress are upregulated [heat shock (HS) 90-kDa protein 1ß; HS 70-kDa protein 1A, 1B, and 5; HS 27-kDa protein 1; HS-binding protein 1, a negative regulator of HS response].

Transcriptional activator genes responsive to cytokine induction increase in expression including STAT3 (acute-phase response factor-binding IL-6 response elements), STAT1 (transcription factor that binds IFN-stimulated response elements), and enhancer of STAT responses (NMI or N-myc). Genes encoding proteins that bind to regulatory regions involved in inflammatory responses (especially the IL-1 response element of IL-6 gene), CCAAT/enhancer-binding protein (CEBP)B and CEBPD, as well as members of the Fos and JunB family [which dimerize to regulate AP-1/activating transcription factor (ATF) transcription sites] are induced. Negative regulators of transcription appeared as well: BCL6; calreticulin (CALR), which modulates nuclear hormone receptor activity; BATF, a regulator of AP-1/ATF transcriptional events; and HSBP1, a regulator of the HS response.

Repression of Genes After Endotoxin Administration
Signals initiated by the induction of innate immunity (i.e., cytokine, chemokine, and co-stimulatory molecules) control the activation of adaptive immune responses (Table 3). Activated antigen-presenting cells process proteins from microorganisms into antigenic peptides that are complexed with MHC class II molecules on the cell surface and, in conjunction with co-stimulatory molecules (CD80, CD86), activate T cells. Within hours of the sterile inflammation induced by endotoxin, we observed suppression of genes associated with antigen presenting cell function (CD86, MHC complex class II antigens) and T and B lymphocyte genes (adenosine deaminase; T cell receptor complex components; CD2-, CD3-, and CD4-related genes; IL-2 receptor). Genes associated with T and B cell receptor activation (TNFRSF7) and one of its binding proteins [CD27-binding (siva) protein] as well as CD160, which activates NK, and cytolytic T cells are repressed.

Genes associated with NK cells were likewise suppressed (killer cell lectin-like receptors, or KLRC), as were killer inhibitory receptors (KIR2DL3, KIR3DL1). The latter bind human leukocyte antigen (HLA) class I molecules and are associated with inhibitory cell signals. Genes associated with cell products from cytolytic T cells and NK cells that have a direct lytic potential for target cells were repressed (granulysin; perforin; granzyme A, M, and K; cathepsin).

Cytokines, chemokines, and chemokine receptors that regulate lymphocyte activation (lymphotoxin LTB) and trafficking (CCL5-RANTES, XCL1 lymphotactin) are downregulated, as are G protein-coupled chemokine receptors (CCR7 and CXCR3). Suppression of multiple genes associated with protein synthesis (ribosomal genes), translation, and processing was observed.

Forty-eight genes whose absence or mutation has been associated with overt immunodeficiency (i.e., JAK3, NCF1, CYBA, LRBA), metabolic defects (i.e., CTNS, PYGL, GM2A), neurological syndromes (i.e., TBL2, HIP1, FACL4), tumorgenesis (i.e., RB1, NBS1, CD63), and eye diseases (i.e., VMD2, RP2) were differentially regulated by endotoxin. Many of these genes have not typically been associated with the acute-phase response or innate immunity (Table 4).


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  		Table 4. Atypical acute-phase genes differentially regulated after intravenous endotoxin challenge

 
Gene Expression in Whole Blood Samples
To assess changes in a complementary tissue, we measured gene expression in whole blood from four subjects (Fig. 2B and Supplemental Table S3A). Whole blood gene expression was altered at 3 h (40 up- and 9 downregulated genes), 6 h (100 up- and 92 downregulated genes), and 24 h (1 upregulated gene). No genes in whole blood were differentially regulated at 168 h. At 6 h, we measured simultaneous changes in gene expression in the PBMC of the same four subjects; 134 genes were induced and 129 genes were repressed compared with baseline. Twenty-nine induced and 32 repressed genes were common between the PBMC and whole blood samples. GO categories that were significantly (P < 0.01) represented among these genes are depicted by GO-MAPs found in Supplemental Table S3, B (induced genes) and C (repressed genes).

The defense response was an overrepresented gene category that included genes from the afferent arm of innate immunity (peptidoglycan recognition protein, TLR5, CD14, C3aR1, LILRA3), intracellular signaling proteins (MAPK14, MAP4K4, FGR, JUNB), transcription factors (NFkB2, BATF, ETV6), cell mobility (VASP, LIMK2, MARCKS, aquaporin 9), and cytokine and cytokine receptors (IL-1ß, IL-1RAP, IL-1RN, IL-1RII, IL-4R, IL-18R, IL-18RAP) (Supplemental Table S3B).

Downregulated defense response included T cell- and B cell-associated genes [CD2 antigen (p50); CD3D antigen; T cell receptor-{alpha}, -ß, and -{gamma}; CD79A; CD79B], cytolytic and NK cell-associated genes (perforin 1, granulysin, granzyme M), MHC molecules (MHC II DP-{alpha}1 and -ß1, MHC II DQ-{alpha}1), and cytokine receptors (IL-2Rß, IL-7R) as well as genes for multiple ribosomal proteins (Supplemental Table S3C).

Thus similar biological themes emerge from analysis of whole blood gene expression compared with mononuclear cells: increased gene expression associated with the defense response and response to pathogens, and decreased expression of genes associated with lymphocytes and ribosomal proteins (Supplemental Table S4).

Quantitative PCR and Array Expression
Confirmatory assays of relative gene expression across a range of array expression were performed on 12 genes using real-time quantitative PCR (TaqMan). Results are represented in fold change, comparing 6 with 0 h. TaqMan fold changes were comparable with the array results (Fig. 3).


Figure 3
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  		Fig. 3. Real-time PCR (RT-PCR) was used to validate the microarray results for PBMC data sets (TaqMan PCR detection, Applied Biosystems). Probes and primers for 12 target sequences were designed using Primer Express computer software (Applied Biosystems). Data are presented as median values [±95% confidence limit (CL)]; n = 4. CD8A, CD8 antigen, {alpha}-polypeptide (p32); CD3G, CD3G antigen, {gamma}-polypeptide (TiT3 complex); SH2D1A, SH2 domain protein 1A; TRB, T cell receptor-ß locus; IL7R, interleukin (IL)-7 receptor; LAT, linker for activation of T cells; CSFR2, colony-stimulating factor 2 receptor-ß, low affinity; FAS, tumor necrosis factor receptor superfamily, member 6; SOD2, superoxide dismutase 2, mitochondrial; ADM, adrenomedullin; S100A12, S100 calcium-binding protein A12; MMP9, matrix metalloproteinase 9.

 

   DISCUSSION
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 INTRODUCTION
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To model gene profiles in the early phases of a systemic infection, we administered endotoxin to normal volunteers and measured serial changes in gene expression in PBMC and whole blood. The greatest changes occurred by 6 h and resolved by 24 h. Exposure to a single microbial component resulted in a rich variety of changes in gene expression including the induction of genes associated with pattern recognition molecules, signal transduction, transcription factors, cell mobility, cell proliferation, and microbial killing, the downregulation of lymphocyte-associated genes, and expression of genes not previously associated with endotoxemia or acute inflammation. The endotoxin phenotype did not persist in the blood transcriptome at 24 h. This broad and rapid on-off response of gene expression is in stark contrast to the sustained inflammatory phenotype that occurs during clinical sepsis, due in part to continued exposure to microbial components with recruitment of pathways that promote a sustained inflammatory response (3, 25). Several different counterregulatory mechanisms may have contributed to the extinction of the endotoxin phenotype: anti-inflammatory cytokines (i.e., IL-1 receptor antagonist, IL-10), inflammatory mediator clearance, removal of circulating inflammatory cells by adherence to endothelium or by apoptosis, and marrow replenishment of cells to the circulation.

Human immune cell responses to pathogens and their components have a shared core pattern of gene expression as well as pathogen-specific responses (26, 38). Monocyte-derived macrophages activate genes encoding receptors, signal transduction molecules, and transcription factors that prime the macrophage for subsequent environmental interactions (38). Gene profiles induced by endotoxin stimulation of monocyte-derived dendritic cells mimic almost the entire core gene expression induced by intact bacteria in vitro (26). While transcript levels of genes associated with phagocytosis and pathogen recognition decline immediately after contact with pathogens, a wide variety of genes associated with immunity are activated (26). Gene levels of cytokines, chemokines and their receptors, and cytoskeleton, signaling, and transcription factors are increased. Antigen processing and presentation genes as well as genes involved in reactive oxygen species are induced in a sustained fashion (26). In contrast to these studies of in vitro monocyte-derived cells, our expression profiles of PBMC and whole blood in vivo show a strong upregulation of genes associated with pathogen recognition and phagocytosis at 6 h.

Other investigators have evaluated gene expression changes in PBMC in vitro after exposure to endotoxin or killed bacteria using amplified polyA RNA and a cDNA microarray format (7). A stereotypic program of gene expression was found with the preponderance of induced genes associated with cell-cell signaling pathways and proinflammatory mediators and repressed genes that included chemokine and chemokine receptors, pathogen recognition and adhesion molecules, components of the respiratory burst, and genes encoding MHC class II molecules (7). Only a modest overlap was found between our 439 induced genes at 6 h and those described by Boldrick et al. (7) in PBMC stimulated with endotoxin; 35 (7.8%) of our induced genes were found in their gene database. Eleven genes were concordant whereas 24 were discordant with the direction of change. Of our 428 repressed genes observed at 6 h after endotoxin, 50 (12%) were found in their database: 30 were concordant and 20 were discordant with our results. Our gene list differs from these in vitro observations with an enhanced role for genes associated with pathogen recognition, chemokines and their receptors, adhesion molecules, and respiratory burst but is similar regarding the suppression of MHC class II genes.

The lack of similarity of our observations of gene expression in PBMC after endotoxin challenge with in vitro data may be due to culture conditions and different array targets (i.e., arrays enriched for detection of lymphocyte-associated genes) (7). Gene expression profiles obtained from in vitro experiments provide important information regarding gene expression potential, yet several factors must be considered when extrapolating these results to clinical states. Cell isolation and culture conditions, including in vitro differentiation (i.e., monocyte-derived macrophages or dendritic cells), may affect gene expression profiles (14, 30). Cultured cells stimulated with endotoxin are further altered by the accumulation of secreted mediators. The lack of primary and secondary mediator clearance limits the ability to assess physiological levels of inflammatory molecules on gene expression. Furthermore, expression profiling of isolated cells precludes an assessment of cell interactions on differential gene regulation. Thus fundamentally different information is obtained from assessment of gene expression in vivo.

Mononuclear cells undergo changes in number and proportion after endotoxin challenge, and this will affect the relative contribution of RNA associated with a specific cell type (i.e., T lymphocyte) in the array results. Activated T cells contain almost twice as much total RNA as resting cells (49), and analyses based on the proportion of cells present in a mixed cell sample may not account for differences in RNA content per cell. In our study, the percentage of T lymphocytes remained constant within the PBMC preparation (Fig. 1). When a fixed proportion of blood cells (whole blood) are stimulated in vitro with endotoxin, genes for ribosomal proteins, cytotoxic T lymphocyte factors, and T cell receptor and nuclear activation factors are downregulated, similar to our in vivo observations (22). A functional consequence of ribosomal gene downregulation in vitro and in vivo is suggested by a decrease in T lymphocyte protein synthesis (~58%) immediately after endotoxin challenge in humans (27).

Animal and human studies of systemic endotoxin challenge further support our observations. Whole blood arrays of rats challenged with endotoxin show that ribosomal and MHC class II genes are repressed as observed in the current study (20). With the use of a cDNA array, qualitative changes in 23 downregulated (i.e., ribosomal genes and HLA antigens) and 31 upregulated genes (i.e., transcription factors, cytokines, CD14, and mitochondrial proteins) were described in humans challenged with either 2 or 3 ng/kg endotoxin (40). The relationship of mRNA and protein expression for cytokines, cytokine receptors, and adhesion molecules was variable, suggesting either non-PBMC sources of protein or the effects of posttranscriptional regulatory events (40).

Recently, other investigators (8) have studied the effects of endotoxin (2 ng/kg) in four healthy subjects and, using a higher-density oligonucleotide array, described differential regulation of 3,714 genes in whole blood leukocytes. Their analysis revealed dysregulation of leukocyte bioenergetics and modulation of translational machinery. Genes for cytokines and chemokines and their receptors as well as members of the NF-{kappa}/relA family of transcription factors were activated within the first 2–4 h (8). These data complement our observations by providing data from whole blood leukocytes (buffy coat) with similar differentially regulated genes and biological themes. We compared our 6-h mononuclear cell data (HU95Av2 microarray, Affymetrix) with the 6-h whole blood data (HU133A/B, Affymetrix) from Calvano et al. (8). Using a >50% increased or decreased relative change from baseline, we evaluated probe sets in common and different between the two studies (Supplemental Table S5). We found 162 induced and 299 repressed probe sets that were common between the two studies and noted 264 upregulated and 143 downregulated probe sets unique to PBMC and distinct from the 281 (upregulated) and 856 (downregulated) probe sets discovered by Calvano et al. in whole blood (Supplemental Table S5). The unique PBMC probe sets included genes associated with heat shock proteins and chemokine and cytokine receptors (all induced) and an enrichment of T lymphocyte-associated genes (repressed). These data demonstrate that, while a common set of genes are expressed in blood leukocytes after endotoxin challenge, unique gene expression profiles emerge when enriching the RNA pool with specific cell types such as PBMC.

The repression of genes associated with lymphocytes in PBMC and whole blood preparations is consistent with previous observations of a state of immunosuppression that occurs after acute inflammation (3, 25, 36). We found that, while an early marker of lymphocyte activation (CD69 surface marker expression) was increased on lymphocytes after endotoxin challenge, a broad category of genes associated with T and B cell surface receptors, NK cell-related cytolytic granules, and MHC antigens were downregulated. Th1 cytokine (IL-2 and IFN-{gamma}) blood levels did not increase. IL-10 levels increased in blood, whereas other Th2 cytokines (IL-4 and IL-5) were unchanged. Mice stimulated with endotoxin activate T cells, as reflected by increased CD69 and CD25 expression, but have a decreased capacity to produce Th1 cytokines (9). Whole blood obtained from volunteers after intravenous endotoxin challenge respond to T cell stimuli with reduced IFN-{gamma} and IL-2 production, and this tolerant state was due in part to serum factors (29). Similarly, mononuclear cells from patients with sepsis or traumatic injury or in the postoperative state have decreased responsiveness to secondary stimuli (i.e., endotoxin, anti-CD3/CD28) with impaired Th1 cytokine secretion and T cell proliferative responses and decreased expression of HLA-DR molecules (2, 13, 15, 18, 24). This tolerance phenomenon has been postulated to be an adaptive response to limit tissue damage (10). However, tolerance is not characterized by a global hyporesponsiveness to all bacterial components (2). The latter case is consistent with our observation of increased expression of genes associated with pathogen recognition molecules.

We analyzed whole blood cells with the PAXgene blood collection system, which has the advantage of rapid stabilization of nucleic acids on collection. This ease of sample acquisition facilitates its use in clinical studies. However, this method has limitations because of an abundant globin and reticulocyte message that contributes to increased signal noise and decreased sensitivity compared with gene expression profiles of blood cells isolated by other methods (12, 19, 21). Despite these limitations, we show that similar biological themes of whole blood compared with PBMC emerge from analysis of the activated cells at 6 h.

Thus the interrogation of the transcriptome associated with endotoxin-induced inflammation integrates a broad variety of biological themes that collectively reflect the activation of innate immunity. We conclude that, while some of the observed changes in gene expression on endotoxin administration are due to gene regulation at a single cell level (i.e., changes in the transcriptome), others may be caused by changes in the proportion of each blood cell type (e.g., different subtypes of mononuclear cells) and are not due to gene regulation. Further experiments are needed to determine which of the differentially expressed genes are regulated at the mRNA level. Nevertheless, it is likely that a significant fraction of the observed changes are due to gene regulation, and that the group of regulated genes reveals a broad variety of biological themes that reflect the activation of innate immunity. The pathway redundancy and parallelism of the innate immunity support the usefulness of broadly acting anti-inflammatory agents during severe infections (35). These observations provide a blueprint of expected changes as well as new pathways and molecules that are engaged during the early response to this common bacterial component.


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 ABSTRACT
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This work was supported by NIH intramural funds.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. F. Suffredini, Bldg. 10, Rm. 7D-43, CCMD/CC/NIH, 10 Center Dr., Bethesda, MD 20892-1662 (e-mail: asuffredini{at}mail.cc.nih.gov).

1 The Supplemental Material for this article (Supplemental Tables S1–S5) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00192.2005/DC1. Back


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