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The Journal of Immunology, 2004, 172: 5870-5879.
Copyright © 2004 by The American Association of Immunologists

Differential Gene Expression Patterns by Oligonucleotide Microarray of Basal versus Lipopolysaccharide-Activated Monocytes from Cord Blood versus Adult Peripheral Blood1,2

Hong Jiang*, Carmella Van de Ven*, Prakash Satwani*, Laxmi V. Baxi{dagger} and Mitchell S. Cairo3,*

Departments of * Pediatrics and {dagger} Obstetrics and Gynecology, Children’s Hospital of New York-Presbyterian, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocytes (Mo) are critically important in the generation of inflammatory mediators, cytokines/chemokines, and regulation of innate and adaptive immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated cord blood (CB) vs adult peripheral blood (APB) mononuclear cells (MNC). In this study, we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB vs CB Mo. We demonstrated a significant increase in the gene expression of several important functional groups of CB genes compared with basal levels including cytokine (IL-12p40, 5-fold), immunoregulatory (signaling lymphocytic activation molecule, 4-fold), signal transduction (Pim-2, 3-fold), and cell structure (Rho7, 4-fold) among others. Furthermore, there was significantly differentially amplified gene expression in LPS-activated APB vs LPS-activated CB Mo, including cytokine (G-CSF, 14-fold), chemokine (macrophage-inflammatory protein 1{alpha}, 5-fold), immunoregulatory (MHC DRB1, 5-fold), transcription factor (JunB, 4-fold), signal transduction (STAT4, 5-fold), apoptotic regulation (BAX, 5-fold), and cell structure (ladinin 1, 6-fold) among others. These results provide insight into the molecular basis for normal genetic regulation of Mo development and cellular function and differential inflammatory and innate and adaptive immune responses between activated CB and APB Mo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune response to microbial pathogens depends on both innate and adaptive mechanisms (1). Monocytes (Mo)4 initiate innate immune responses by internalization, phagocytosis, and killing of pathogens and concurrently produce a wide range of costimulatory molecules, inflammatory cytokines and chemokines (2, 3). The activation of innate immune response results in a sequential activation of the adaptive immune response by soluble immune mediators, costimulatory molecules, and Ags presented by MHC class I or II molecules from APC (4). We and others have previously demonstrated that hemopoietic and immune function from activated cord blood (CB) effector cells are developmentally immature when compared with their counterparts in APB (5, 6, 7, 8, 9, 10). The dysregulation and immaturity of neonatal host defense predispose newborns to an increased susceptibility to infectious morbidity and mortality from microorganisms (11).

Because of the notable immaturity of CB cellular immunity, CB has been used as an alternative source of allogeneic stem cells for repopulating bone marrow following myeloablative therapy and allogeneic transplantation. Despite the use of more HLA-disparate allogeneic CB stem cells, early clinical results have suggested that CB transplantation is associated with both a lower incidence and severity of acute graft-versus-host disease (GVHD) and chronic GVHD (5). The mechanism(s) underlying the decreased incidence and severity of acute and chronic GVHD following CB transplantation is not fully understood. Recently, Aranha et al. (12) reported that depletion of CD14-positive donor Mo suppressed donor T cell proliferation in a dose-dependent fashion and reduced the development of acute GVHD following allogeneic bone marrow transplantation.

More recently, the comparison of gene expression profiles of NFAT-dependent and -associated genes between CB and APB CD4+ T lymphocytes was demonstrated by microarray technology. Included in these genes were cytokine/receptor, chemokine/receptor, and NFAT-pathway-associated genes (13). In addition, serial analysis of gene expression has been used to examine differential gene expression in Mo/macrophages during a variety of developmental stages and in LPS-activated human Mo (14, 15, 16). We have previously demonstrated significant down-regulation of a number of cytokine genes from activated CB mononuclear cells (MNC) compared with APB MNC, including M-CSF, G-CSF, GM-CSF, TGF-{beta}, IL-8, IL-11, IL-12, IL-15, and IL-18 (6, 7, 9, 17, 18, 19). This approach, however, of examining single gene differential regulation of activated CB vs APB MNC is time-consuming. To accelerate this analysis and to more comprehensively examine differential CB vs APB Mo gene expression patterns, we used oligonucleotide microarray technology to examine entire genome-wide gene expression profiles of Mo from CB and APB in the basal (constitutive state) and following lipopolysaccharide (LPS) activation. Differential gene expression patterns between CB vs APB Mo provide insight to determining the genetic mechanism(s) underlying the differential susceptibility to infections in neonates and the decreased incidence and severity of acute and chronic GVHD following CB transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and stimulation of CB and APB Mo

Following informed consent, APB was obtained as buffy coat products from normal healthy adult donors from the New York Blood Center. CB was obtained by venipuncture from umbilical cord veins immediately after delivery at Columbia Presbyterian Medical Center at New York Presbyterian Hospital. Informed consent was obtained from maternal donors. MNC from CB or APB were isolated on a standard Ficoll-Paque density gradient (Amersham Biosciences, Piscataway, NJ) and were plated onto 100-mm tissue culture dishes for the isolation of Mo-enriched population. Briefly, 50 x 106 MNC were seeded in each 100-mm tissue culture dish. Adherent CB or APB Mo were obtained by incubation of MNC at 37°C for 1 h followed by washing off nonadherent cells with warm culture medium (RPMI 1640) and PBS twice, respectively. Adherent Mo were further cultured in RPMI 1640 or activated with LPS (10 µg/ml, a nonphysiological dose; Sigma-Aldrich, St. Louis, MO) for 18 h. The purity of the monocyte population was confirmed by flow cytometry. Briefly, cells were suspended in PBS with 0.2% BSA and incubated with CD14, CD3, or CD20 fluorescent-conjugated Ab (BD Biosciences, San Diego, CA) on ice for 30 min, washed in PBS twice, and resuspended in a fixed solution (3.7% paraformaldehyde and 0.1% sodium azide; Sigma-Aldrich). Appropriate isotype controls were included in each experiment. A minimum of 10,000 events was collected for evaluation on a FACScan (BD Biosciences) and data were analyzed using CellQuest software.

Purification of total RNA, generation of cDNA, and labeling of cRNA

Approximately 50–100 x 106 adherent CB or APB Mo were mixed with 5–10 ml of TRIzol reagents (Invitrogen, Carlsbad, CA) for the isolation of total RNA. After homogenization, chloroform was added to the mixture and the aqueous phase was collected and the total RNA was precipitated by adding isopropanol. The isolated total RNA was further purified by RNeasy (Qiagen, Valencia, CA). Double-stranded cDNA was generated from 10 µg of total RNA using a poly(dT) oligonucleotide and the Superscript Choice System kit (Invitrogen). cDNA was purified by extraction with phenol-chloroform. Biotinylated labeling cRNA was conducted by in vitro transcription using the BioArray High Yield RNA Transcript Labeling System (ENZO, Farmingdale, NY). cRNA was then purified by RNeasy (Qiagen).

Oligonucleotide hybridization and microarray

Biotinylated cRNA was heated for 5 min at 95°C, allowing RNA to break down to small fragments according to Affymetrix (Affymetrix, Santa Clara, CA) protocols. Fragmented cRNA (15 µg) was subjected to oligonucleotide hybridization (Fluidit Station; Affymetrix) to the human U95Av2 gene chip (Affymetrix). After scanning (scanner from Affymetrix), the oligonucleotide hybridization data were exported for gene expression value analysis. To confirm the quality of generated cRNA, random selection of fragmented CB Mo or APB Mo cRNA samples were first hybridized to the Test Microarray chip (Test3; Affymetrix) to ensure equal hybridization to 5' and 3' oligonucleotides of housekeeping genes (GAPDH) before being subjected to the hybridization with U95Av2 chips for quality control.

RT-PCR and quantitative real-time PCR

Selected genes were first examined for their expression levels by RT-PCR using the SuperScript One-Step RT-PCR kit (Invitrogen). A total of 0.5 µg of total RNA was used to perform 40 cycles of PCR. The PCR cycles were conducted as follows: denature 94°C for 30 min; anneal 58°C for 30 min; and extension 72°C for 60 min. Quantitative real-time PCR was performed in duplicate using LightCycler-RNA amplification SYBR Green I kit (Roche Molecular Biochemicals, Indianapolis, IN) following the protocol provided by the vendor. Direct detection of PCR product was monitored in real time by measuring the increase in fluorescence by the binding of SYBR Green I dye to dsDNA.

The following primers were used to determine the levels of gene expression: IL-1{beta} forward, 5'-TGCCTTAGGGTAGTGCT-3'; IL-1{beta} reverse, 5'-GCGGTTGCTCATCAGA-3'; signaling lymphocytic activation molecule (SLAM) forward, 5'-GGAGTGAAAAGGCGGG-3'; SLAM reverse, 5'-GGCATAGATCGTAAGGCT-3'; macrophage-inflammatory protein (MIP) 1{alpha} forward, 5'-GCAACCAGTTCTCTGC-3', MIP-1{alpha} reverse, 5'-CTGGACCCACTCCTCA-3'; calmodulin 1 (CALM1) forward, 5'-TGACGATTGAGCACAGT-3'; CALM1 reverse, 5'-CCAACATACACGGGCA-3'; GAPDH forward, 5'-GGTGAAGGTCGGAGTCAACG-3'; and GAPDH reverse, 5'-CAAAGTTGTCATGGATGACC-3'. Relative quantification of IL-1{beta}, SLAM, MIP-1{alpha}, or CALM1 mRNA expression was determined by normalizing to the housekeeping gene (GAPDH). cDNA amplification efficiencies of IL-1{beta}, SLAM, MIP-1{alpha}, CALM, and GAPDH were confirmed to be equivalent and the amount of amplified dsDNA was all within the standard curve with an error of <0.1.

Data analysis and statistics

Gene expression values were analyzed by the Microarray Suite 5.0 (Affymetrix) software package. Pairwise analysis was performed with the APB Mo, LPS-activated CB Mo, or LPS-activated APB Mo data as the experimental chip file and CB Mo or LPS-activated CB Mo data as the baseline file. All comparisons were conducted using five sets of APB or five sets of LPS-activated CB Mo vs three sets of basal CB Mo (15 comparisons) and five sets of LPS-activated APB Mo vs five sets of LPS-activated CB Mo (25 comparisons). The results from these comparisons provided a difference call of "I" for increased, "D" for decreased, and "NC" for no change based on the average intensity change and background noise in the microarray. In addition, a fold change value (expressed in log2) was also provided for each gene.

To generate a list of up-regulated or down-regulated genes in APB Mo vs CB Mo in response to LPS that would be significant, genes with increased or decreased calls in 15 of 25 comparisons were considered for further analyses. From this group, all genes demonstrating smaller than 2-fold change were eliminated from further consideration. Furthermore, the genes that showed absence in LPS-activated APB or LPS-activated CB were removed from the up-regulated or down-regulated gene lists, respectively. The signal intensities of these genes were imported into GeneSpring software 5.0 (Silicon Genetics, Redwood City, CA) for further analysis to present relative expression for a given gene. The signal intensity of each probe was normalized to the medium value of all intensities measured in all samples. To determine whether the difference in gene expression in CB vs APB was biologically significant, a Mann-Whitney test was used to perform statistical analysis based on the intensity values. Values of p < 0.05 were considered to be significant.

Bioinformatics

The identified gene information and accession numbers were first obtained through NetAffx provided by Affymetrix, and their identities were further confirmed by searches of LocusLink (www.ncbi.nlm.nih.gov/LocusLink). These genes were grouped based on their known molecular and/or biological function from LocusLink. Gene symbols were obtained from NetAffx and are the currently defined official symbols described in the National Center for Biotechnology Information LocusLink database.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Flow cytometry results demonstrated that 60–70% of the adherent mononuclear cell populations expressed CD14. These populations were also found to be CD3 and CD20 negative, indicating no contamination of T and B cells. We analyzed the differential gene expression in these enriched MNC populations of Mo for the basal states of APB vs CB Mo, LPS-activated CB vs basal CB, and LPS-activated APB vs LPS-activated CB. We focused on genes with three or greater fold differential expression and separated them into different categories based on molecular function as determined by public databases. These molecular families included cytokine, cytokine receptor, chemokine, Ag/immunoregulatory molecule, transcription factor, signaling molecule, apoptosis/cell growth regulator, and cell structure regulatory genes.

Comparison of differential gene expression of basal APB vs CB Mo

Among the genes whose basal expression were significantly higher in APB compared with CB Mo were the cytokines G-CSF (3.92-fold), IL-15 (3.03-fold), and IL-24 (MDA-7; 16.39-fold), cytokine receptors IL-2R{alpha} (4.75-fold), IL-2R{beta} (4.26-fold), and chemokines RANTES (5.30-fold), GCP-2 (3.78-fold), and MIP-3{beta} (12.7-fold). Furthermore, Ags or surface molecules found to be expressed at basal levels greater than 3-fold in APB vs CB Mo were SLAM (5.88-fold), IL-1R antagonist (IL-1RN) (3.68-fold), and oncostatin M (21.11-fold). Genes encoding transcription factors NF-{kappa}B p65 were expressed 3.73-fold higher, while the kinases and phosphatases MAK2K4, PAC-1, and DUSP-4 were also found to be expressed 3.32-, 4.59-, and 3.82-fold higher in APB vs CB Mo, respectively. In addition, an inhibitor of STAT3 (SSI-3) was amplified 3.96-fold. Cell structure regulatory genes ladinin and PLD1 were expressed 3.7- and 3.03-fold higher in APB vs CB Mo, respectively. Lastly, human cyclooxygenase 2 (17.15-fold) and PAI-2 (3.10-fold) were all increased in basal APB Mo compared with CB Mo (Table I). In contrast, our analysis also identified genes whose expression was higher in CB compared with APB Mo, including adhesion molecules CD9 (3.1-fold), integrin-{alpha}M (13-fold), and cyclin-dependent kinase inhibitor 1C (3-fold). Because of the difficulty of obtaining enough volume of CB for Mo microarray studies, we performed three independent CB microarray experiments for basal levels and the data were reproducible.


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  		Table I. Differential gene expression between basal CB and APB Moa

 
Induction of gene expression in CB Mo following LPS stimulation

Since gene expression profiling in APB Mo after LPS stimulation has been previously documented (16, 20), we compared the gene expression levels in basal CB Mo vs LPS stimulation. Our analysis revealed that there was some overlap and also some distinct gene expression patterns in LPS-activated CB Mo when compared with basal CB Mo (Table II). The highest induction of LPS-activated compared with basal CB cytokine gene expression included IL-7, IL-10, and IL-12p40 with increases of 3.69-, 3.48-, and 4.98-fold, respectively. Chemokine genes CXCL6 (GCP-2) and CCL8 (monocyte chemoattractant protein (MCP) 2) were induced 3.78- and 5.72-fold in CB Mo following LPS stimulation, and the Ag and immune regulatory genes TNF-inducible TSG-14 gene (21.86-fold) and SLAM (4.22-fold) were both increased in LPS-activated vs basal CB Mo. Additionally, NK cell stimulation factor (NKSF) IL-12p40 (4.98-fold), IL-1RN (3.01-fold), CD83 (3.1-fold), CD79 (5.66-fold), and oncostatin M (4.92-fold) were induced in CB Mo in response to LPS as well as the apoptotic genes JNKK1 and DUSP which were increased by 8.0- and 3.78-fold, respectively. Plectin, a structure regulatory gene, was also elevated by 8.98-fold. Several kinase and phosphatase signaling molecules were increased, including mitogen-activated protein kinase kinase (MAPKK; 5.22-fold), Pim-2 (3.03-fold), PAC-1 (5.66-fold), SH-PTP3 (3.65-fold), and SSI-3 (6.96-fold) and the transcription factors/regulator NF-KB2 (5.34-fold) was also elevated in CB Mo following LPS stimulation. Furthermore, a group of genes encoding GTP-binding proteins were highly elevated in response to LPS in CB Mo including Rab5 (4.65-fold), Rho7 (4.34-fold), GTP-binding protein G{alpha} subunit (3.86-fold), and Cdc42-interacting protein 4 (3.21-fold; Table II). Lastly, we also found that gene expression in CD14 Ag, insulin receptor substrate 1, and myogenic factor 3 was down-regulated upon LPS activation in CB Mo (6.96-, 4.34-, 4.29-fold, respectively).


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  		Table II. Significant induction of gene expression in LPS-activated vs basal CB Moa

 
Differential gene expression between CB and APB Mo in response to LPS

Our analysis revealed that 82 genes have expression profiles in LPS-activated APB Mo vs LPS-activated CB Mo that were significantly amplified in APB Mo by >3-fold. These differentially expressed genes can be separated into various molecular categories based on their function as determined by public databases. Cytokine and cytokine receptor genes found to have increased expression in LPS-activated APB Mo vs LPS-activated CB included IL-1{beta} (5.95-fold), IL-15 (3.88-fold), IL-15R (3.74-fold), IL-17R (4.35-fold), IL-2R{alpha} (5.81-fold), IL-2R{beta} (3.44-fold), IL-4R (3.26-fold), IL-6 (3.29-fold), IL-8 (8.32-fold), IL-7R (3.39-fold), G-CSF (13.39-fold), and GM-CSF (3.1-fold) (Table III and Fig. 1A). A cluster of CC family chemokines, CCL2 (4.40-fold), CCL3 (5.29-fold), CCL4 (8.64-fold), CCL5 (5.34-fold), CCL19 (10.84-fold), and CCL22 (7.89-fold) was found to be significantly increased in the LPS-activated APB vs CB Mo. The expression of chemokine genes CXCL2, CXCL6, CXCL8, and macrophage inhibiting factor (MIF) was also significantly amplified in LPS-activated APB vs CB Mo by 3.40-, 14.71-, 8.32-, and 16.6-fold, respectively (Table III and Fig. 1B).


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  		Table III. Up-regulated cytokine, cytokine receptor, chemokine, and immunoregulatory gene expression in LPS-stimulated APB Mo vs CB Moa

 


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  		FIGURE 1. Differential expression of immunoregulatory genes in LPS-activated CB vs APB Mo. mRNA expression of each gene was determined from LPS-activated CB and APB Mo by oligonucleotide microarray. The experiments were performed by using Mo populations isolated from LPS-activated CB (n = 5) and LPS-activated APB (n = 5). The data analysis was performed by using Affymetrix MAS 5.0 and GeneSpring 5.0. The differential expressed genes following LPS stimulation are separated into cytokine and cytokine receptor (A), chemokine (B), and immunoregulation (C), transcription factor (D), signal transduction (E), apoptosis (F), and cell structure/cytoskeleton (G) based on molecular function. Each row represents a gene as labeled and columns represent samples. Color squares represent the relative expression of each gene after normalization to the medium expression value. Red indicates higher expression and green indicates lower expression of genes, while black represents expression at the medium value.

 
Furthermore, a group of Ags, surface molecules, and/or immunoregulatory genes demonstrated differential gene expression between APB and CB Mo following LPS stimulation. The ferritin H chain (4.82-fold) and a cluster of leukocyte-IgG-like receptors A and B (3.73- and 5.49-fold), MHC classes I–F (4.16-fold), and MHC class II molecules HLA-DPB1 (4.81-fold) and DRB1 (4.78-fold) were amplified in LPS-activated APB Mo compared with CB Mo (Table III and Fig. 1C). In addition, the expression of a scavenger receptor gene, CD163, was elevated 3.75-fold higher in APB vs CB Mo in response to LPS. Other immunoregulatory gene expressions were also increased in LPS-activated APB vs CB Mo, including TNFR family members 6 (3.03-fold), 1B (3.48-fold), SLAM (7.49-fold), and IL-1RN (4.41-fold) (Table III and Fig. 1C). In addition, the TGF-{beta}-activated gene TSC22 and IFN {gamma}-inducible gene (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) were both amplified in LPS-APB vs CB Mo by 5.19- and 3.87-fold, respectively (Table III and Fig. 1C). Conversely, the gene expression of a number of Ags/adhesion molecules was found to be increased in LPS-activated CB vs LPS-activated APB Mo, including CD209L (4.4-fold), CDW52 (3.9-fold), and integrin {beta}6 (5.1-fold) among these genes.

The transcription factor genes JunB (3.79-fold), JunD (4.31-fold), Egr3 (3.60-fold), Etr101 (3.91-fold), and RUNX3 (3.08-fold) were also significantly elevated in APB vs CB Mo after LPS stimulation (Table IV and Fig. 1D). Furthermore, a group of signaling molecule genes were also induced in response to LPS, including STAT3 (3.28-fold), STAT4 (5.49-fold), SOCS3 (SSI-3) (3.08-fold), Pim-1 (3.05-fold), Pim-2 (3.51-fold), protein kinase C{eta} (3.54-fold), hemopoietic cell kinase (HCK; 3.07-fold), Lyn substrate 1 (3.74-fold), LIM domain kinase 2 (LIMK2; 4.27-fold), PLC{beta}3 (3.72-fold), CALM1 (5.15-fold), and guanine nucleotide binding protein (G protein) 10 (3.08-fold), and Gq (4.59-fold) (Table IV and Fig. 1E). Conversely, the gene expression of zinc finger proteins (–6 (11.5-fold), –197 (6.27-fold), and –202 (4.75-fold)) was elevated in LPS-activated CB vs LPS-activated APB Mo.


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  		Table IV. Up-regulated transcription factor and signaling molecule gene expression in LPS-stimulated APB Mo vs CB Moa

 
There was also a significantly increased expression of apoptotic genes in LPS-activated APB vs LPS-activated CB Mo including BAX (4.50-fold), DAPK1 (3.51-fold), DUSP4 (3.71-fold), and TNFRSF-21 (3.88-fold) in LPS-activated APB compared with LPS-activated CB Mo (Table V and Fig. 1F). However, several genes regulating apoptotic process were expressed higher in LPS-activated CB vs LPS-activated APB Mo, including apoptotic protease activating factor 1 (5.88-fold), CASP2 (3.3-fold), BID (4.06-fold), and TRAIL (3.31-fold) (Table V and Fig. 1F). The growth-regulating genes casein kinase 1 (3.12-fold) and MCL1 (3.12-fold) were significantly amplified between LPS-activated APB and LPS-activated CB (Fig. 1F and Table V). Furthermore, growth factor genes including VEGF (8.90-fold), ECGF1 (5.77-fold), and GRN (7.49-fold) had higher expression in LPS-activated APB vs CB Mo (Table V). Lastly, cytoskeleton/cell structure regulatory genes were significantly amplified in LPS-activated APB Mo compared with CB Mo, including the genes actinin 1 (3.18-fold), ladinin 1 (6.31-fold), tubulin 1 (3.23-fold), profilin (4.41-fold), plectin 1 (4.32-fold), Rho GDI (3.03-fold), IQGAP1 (4.91-fold), RabR 1 (6.23-fold), Rab4B (3.14-fold), and Rab13 (3.14-fold) (Fig. 1G and Table V).


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  		Table V. Differential expression of apoptotic, cell cycle regulatory, growth factor, and cytoskeleton genes in LPS-stimulated APB Mo vs CB Moa

 
Selected gene expression by quantitative real-time PCR

To confirm the levels of gene expression generated from microarray profiling, we selected several genes to compare their expression by quantitative real-time PCR. The expression level of each transcript was compared with microarray data. Four genes, IL-1{beta}, SLAM, MIP-1{alpha}, and CALM1, were selected to examine for the basal gene expression levels in CB and APB Mo and in LPS-activated CB and APB Mo (Fig. 2A). The expression of each of these genes was induced in response to LPS in both CB and APB Mo. The quantitative comparison of fold change between CB and APB in the basal and LPS-activated states was also determined (Fig. 2B). The fold changes by quantitative real-time PCR in the basal levels of APB vs CB Mo for IL-1{beta}, SLAM, MIP-1{alpha}, and CALM1 were 1.52-, 4.78-, 2.15-, and 1.82-fold, respectively, and 6.59-, 5.74-, 7.11-, and 7.16-fold, respectively, in the LPS-activated state. These results of gene expression by quantitative real-time PCR were comparable to those derived from the oligonucleotide microarray studies.



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  		FIGURE 2. Real-time PCR examination of genes differentially expressed in LPS-activated CB vs APB Mo. A, RT-PCR of genes for IL-1{beta}, SLAM, MIP-1{alpha}, and CALM were performed using total RNA isolated from basal and LPS-activated APB or CB Mo. The PCR products were run on 1.2% agarose gel with DNA markers and the size of PCR products was labeled for each gene. One representative experiment for each gene is shown. B, The quantitative expression of IL-1{beta}, SLAM, MIP-1{alpha}, and CALM gene fragments was determined and the fold change for basal (APB vs CB) and LPS-activated (LPS-APB vs LPS-CB) Mo was calculated. The data (±SEM) are generated from three independent experiments. The fold changes generated from quantitative RT-PCR were compared with that from the microarray analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present study demonstrates that the expression of several cytokines and their respective receptor genes is significantly enhanced in CB Mo following stimulation with LPS compared with basal levels and in LPS-activated APB vs LPS-activated CB Mo. These results are supported by recent data demonstrating up-regulation of SLAM in macrophages following activation with LPS and suggesting that decreased SLAM gene expression in CB vs APB may in part contribute to the immaturity of CB T cell function (21, 22).

In this study, we also demonstrate amplified gene expression of G-CSF in APB vs CB Mo at both the basal and LPS-activated level and enhanced expression of GM-CSF only in LPS-activated APB vs LPS-activated CB Mo. Kaminski et al. (13) also demonstrated increased GM-CSF expression in APB vs CB CD4 T cells by oligonucleotide microarray. However, no significant increase for either G-CSF or GM-CSF gene expression was demonstrated at either basal or activated levels in CB Mo. These data are consistent with the previous reports from our laboratory that demonstrated negligible basal expression of G-CSF mRNA and a significant reduction in G-CSF mRNA gene expression and G-CSF production by activated CB Mo vs APB Mo (23). Furthermore, we reported a 7-fold decrease of GM-CSF protein production in activated CB Mo vs APB Mo (23, 24). In the present study, we observed no significant difference in the gene expression of IL-12 (p40) in APB Mo vs CB Mo either at basal levels or after LPS activation; however, a significant increase was seen in LPS-activated CB Mo. These data are consistent with our previous studies where we reported IL-12 mRNA expression was induced within 6 h following LPS stimulation and reached peak levels at 12 h in CB MNC (7, 25).

Our study also demonstrates enhanced gene expression of IL-15 in APB compared with CB Mo at both the basal and activated levels. Kaminski et al. (13) reported that IL-15 gene expression differs in CB vs APB CD4+ T lymphocytes in both the basal and activated states. Additionally, we observed no significant increase in IL-15 gene expression in LPS-activated CB Mo compared with basal levels. These results are consistent with our previous reports that both IL-15 mRNA expression and protein production are significantly lower in activated CB compared with APB (6). In addition, our analysis showing an increase in IL-10 gene expression in activated CB but not APB is consistent with a recent report demonstrating that during primary TCR stimulation, neonatal naive T cells secrete high levels of IL-10 while adult T cells began to secrete IL-10 only after repeated stimulation (26).

This study also demonstrates a significant increase in the gene expression of the MHC class II molecules HLA-DPB1 and DRB1 in LPS-activated APB vs CB Mo. Our data are consistent with reports showing the reduced expression of class II HLA Ags in CB Mo and dendritic cells (27, 28). Furthermore, the degree of histocompatibility between donor and recipient is predictive of engraftment and potential development of GVHD following allogeneic stem cell transplantation. Thus, the decreased expression of these MHC class II molecules in CB Mo in basal and LPS-activated states may in part be responsible for the reduced severity of acute GVHD after CB stem cell transplantation even following one or two HLA-disparate Ag umbilical CB transplantation (29, 30, 31).

Many transcription factors and signaling pathways leading to activation of these transcription factors that regulate gene expression of LPS-induced proinflammatory cytokines have been reported (32). Our studies have shown several transcription factors, immediate early genes JunB and JunD, Egr3, Etr101, and Runx3, whose expression was increased in APB compared with CB Mo following LPS stimulation. Although these transcription factor genes are expressed at similar levels in APB and CB Mo and the induction of these genes in response to LPS in CB was low (1- to 3-fold), the significantly elevated expression of these genes in LPS-induced APB Mo suggests that these transcription factor genes may in part play a role in regulating transcription of their targeted genes involved in inflammatory responses.

LPS induces the activation of a number of signaling pathways including three mitogen-activated protein (MAP) kinase pathways, MEK-extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 (33). Our results have demonstrated a group of signaling molecule genes whose expression was amplified higher in APB vs CB following LPS stimulation. Among these genes were STAT3, STAT4, and SOCS3 (SSI-3). Previous studies have demonstrated that the production of STAT4 was correlated with IL-12 and IFN-{gamma} production in mice, suggesting the functional importance of STAT4 in the regulation of IFN-{gamma} and IL-12 production and further playing a key role in Th1 cell differentiation for adaptive immunity (34, 35). Thus, elevated expression of the IFN-{gamma} gene might be secondary to the higher expression of STAT4 in Mo in LPS-activated APB vs LPS-activated CB. STAT3 is activated by both IL-6 and IL-10 and mediates IL-6- and IL-10-dependent gene expression, and SOCS3 selectively inhibits IL-6-activated STAT3, but not IL-10 (36). STAT3 has also been reported to play a crucial role in controlling inflammatory response in myeloid cells and G-CSF-induced cell proliferation and differentiation (37, 38). Thus, the enhancement of expression of STAT3, STAT4, and SOCS3 genes following LPS stimulation of APB vs CB Mo may in part explain the differences in the inflammatory responses between APB vs CB immune function.

The gene expression of nonreceptor tyrosine kinase Src family members HCK and LIMK, known to regulate cell shape change and migration (39, 40, 41, 42), was significantly amplified in response to LPS in APB vs CB Mo. Enhancement of HCK and LIMK expression by LPS in APB vs CB may in part contribute to enhanced APB vs CB Mo migration and structure change following activation. Similarly, we have also found an array of increased expression of cytoskeleton regulatory signaling genes, phospholipase C{beta}3, phospholipase D2, protein kinase C{eta}, and CALM1 in LPS-activated APB vs LPS-activated CB Mo. These signaling proteins were previously reported as downstream effectors for receptor-mediated phagocytosis (43). Thus, these signaling molecules, if presenting in the LPS-induced cellular signaling network, may participate in specific signaling pathways, exerting their functional roles during endocytosis by monocyte/macrophage following the challenge of pathogens, and their involvement appears to be more profound in APB vs CB.

Interestingly, we observed an increase in gene expression of apoptotic regulating genes, including death-associated protein kinase 1 (DAP kinase 1), BAX, and TNFR family member 21 and dual specificity phosphatase 4. Dual specificity phosphatase 4 has been shown to play a role in dephosphorylation of MAP kinase, causing the attenuation of MAPK-mediated proliferation pathways (44). DAP kinase 1 was recently reported to induce apoptosis via the TGF-{beta} pathway (45). Indeed, we have observed significantly elevated expression of the TGF-{beta}-dependent gene TSC-22 (TGF-{beta}-activated protein 22) in LPS-activated APB vs LPS-activated CB Mo. These results suggest a potential TGF-{beta}-regulated apoptotic pathway involved in LPS-induced apoptosis in APB Mo. Furthermore, our observation of significantly higher expression of TNF-{alpha}-induced gene 2, gene 3, as well as several members of the TNFR family in APB vs CB Mo further provides evidence to support the suggestion that autocrine production of TNF-{alpha} may be a mechanism for inducing apoptosis of Mo/macrophages (46). In addition, our analysis has revealed increased gene expression of BAX in APB vs CB in response to LPS stimulation. This observation appears to be in agreement with other reports in which LPS activation results in overproduction of NO, which in turn induces BAX-regulated cell apoptosis (46, 47). Taken together, our results have added further evidence to support the previous observation that LPS-induced apoptosis of macrophages/Mo is mediated through autocrine production of TNF-{alpha} and NO. Furthermore, our results also postulate a TGF-{beta} and DAP kinase-mediated apoptotic pathway, which might be another mechanism involved in the apoptotic process of macrophage/Mo following LPS administration.

Additionally, we found a distinct group of apoptotic regulating genes that expressed at higher levels in the LPS-activated CB vs LPS-activated APB, including apoptotic protease-activating factor, TNFR-associated factor-interacting protein, TNF ligand family members 8, 9, 10 (TRAIL), and BID (48). The expression of cell death and apoptotic regulating genes that may be potentially activated in apoptotic pathways of CB was reported (49). A role of apoptosis in controlling CB CD34+ cell numbers was suggested previously (50). Since TRAIL is an anti-inflammatory ligand (51), a potential role for TRAIL in CB may be the inhibition of T cell proliferation which could in part play a role in reducing the severity and incidence of acute GVHD following CB transplantation.

It is noteworthy to mention that we observed the up-regulated gene expression of several signaling molecules that are reported to be involved in the G13-protein-mediated signaling pathway, including RhoA, RhoGDI, IQGAP1, and profilin in APB Mo vs CB Mo following LPS stimulation. This pathway has been shown to be responsible for regulating actin reorganization and cell-cell adhesion (52). The involvement of small G protein family members Rho, Rac, and Rap1 has been shown in LPS-activated pathways to regulate cellular morphologic and functional changes (53). In particular, Rho GTPase plays a pivotal role in mediating alternation of morphology of Mo-derived dendritic cells (54). LPS-induced expression of IL-8 and PAI genes is also dependent on Rho protein (55, 56). Furthermore, Rho also mediates G13-induced activation of capping protein profilin (57). Coincidentally, we found increased gene expression of Rho GTPase-activating factor RhoGDI, which may augment Rho-mediated signal events. IQGAP1 has been well documented as a scaffolding protein modulating cross-talk among diverse pathways including actin cytoskeleton signaling by Rho GTPase and calmodulin (52). All together, these observations may provide information in characterizing the differential activities of the G13 pathway in the LPS-activated APB vs LPS-activated CB Mo, which may in part explain the differential cell structure remodeling required during inflammatory responses.

In summary, we have demonstrated differential gene expression patterns between CB and APB Mo in the basal state and in the LPS-activated state. Although our results have confirmed previous observations of LPS-activated APB Mo (16, 20), this study has also discovered additional LPS-activated genes previously unidentified. Moreover, we have identified an array of functional genes whose expression is significantly different in CB and APB in the basal and LPS-activated states. These differential levels of gene expression in APB Mo and CB Mo may explain the differential regulation of intracellular events in APB Mo and CB Mo after activation by LPS. These results provide additional information for further characterizing differential CB vs APB Mo in vitro functional activity and clinical observations following CB vs APB allogeneic stem cell transplantation. Further studies are warranted to determine which genes and/or signaling pathways are required for optimal Mo function and translational opportunities for the exploitation of these differences in gene expression patterns between activated CB and APB Mo.


   Acknowledgments
 
We thank Linda Rahl for the excellent editorial assistance in the preparation of this manuscript. We also thank Vladam Miljkovic for his performance of gene chip hybridization and data processing.


   Footnotes
 
1 This work was supported in part by grants from the Pediatric Cancer Research Foundation, Swim Across America, and National Institute of Arthritis and Musculoskeletal and Skin Diseases (R21AR49330 to M.S.C.). Back

2 Presented in part at the American Society of Hematology, December 2002, Philadelphia, PA. Back

3 Address correspondence and reprint requests to Dr. Mitchell S. Cairo, Pediatrics, Medicine and Pathology, Director, Leukemia, Lymphoma, Myeloma Program, Herbert Irving Comprehensive Cancer Center, Columbia University, 161 Fort Washington Avenue, Irving 7, New York, NY 10032. E-mail address: mc1310{at}columbia.edu Back

4 Abbreviations used in this paper: CB, cord blood; APB, adult peripheral blood; GVHD, graft-versus-host disease; Mo, monocyte; MNC, mononuclear cell; SLAM, signaling lymphocytic activation molecule; MIP, macrophage-inflammatory protein; CALM1, calmodulin 1; NKSF, NK cell stimulation factor; MIF, macrophage inhibiting factor; MCP, monocyte chemoattractant protein; LIMK2, LIM domain kinase 2; MAP, mitogen-activated protein; MAPKK, MAP kinase kinase; HCK, hemopoietic cell kinase; DAP, death-associated protein. Back

Received for publication October 10, 2003. Accepted for publication February 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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