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* The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark;
Center for Biological Sequence Analysis, BioCentrum-Technical University of Denmark, Lyngby, Denmark; and
Division of Infectious Diseases, Department of Health and Environment, University of Linköping, Linköping, Sweden
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polymorphonuclear neutrophilic granulocytes (PMNs)3 are attracted to skin wounds within minutes of injury by chemotactic mediators released by thrombocytes and microorganisms. Upon migration to sites of infection such as skin wounds, PMNs get activated by microorganisms and their products, and by cytokines generated by other leukocytes (monocytes and PMNs) and the stromal environment (fibroblasts, endothelial and epidermal cells). Following activation, PMNs immediately initiate a first line of defense using a number of distinct mechanisms (2, 3). These defense mechanisms include the release of anti-microbial peptides, phagocytosis, and the generation of reactive oxygen intermediates for killing and degradation of microorganisms. De novo synthesis of chemokines and cytokines, which are essential for the regulation of the cellular immune response and the recruitment of additional effector cells to the wound, constitutes another defense mechanism of PMNs.
More recently, studies using genomic and proteomic approaches have demonstrated a significant transcriptional response of human PMNs upon in vitro activation by single agents such as bacteria, LPS, and by phagocytosis of IgG- and complement-coated latex beads (4, 5, 6, 7). However, at present, no genomic approaches have been applied to investigate how PMNs respond in vivo to inflammatory mediators in skin wounds and whether their response contributes to healing of wounds.
To gain more insight into this complex process, we applied gene array technology to compare changes of gene expression of highly purified PMNs from peripheral blood (PB) and PMNs that had transmigrated to inflammatory skin lesions in vivo. For the collection of PMNs, we used a model of skin wounding called skin chamber technique. With this model, small areas of denuded dermis, termed "skin windows", are generated and covered with skin chambers containing a medium that attracts PMNs (8).
Our study demonstrates that migration of PMNs into skin lesions is associated with an extensive change in gene expression, implicating that the cellular fate and function of PMNs attracted to skin wounds is partially regulated at the transcriptional level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PB and skin chamber samples were collected in parallel from four healthy individuals. All samples were obtained following informed consent according to the guidelines established by the Ethics Committee of the Cities of Copenhagen and Fredricksberg.
PMNs were isolated from PB by density centrifugation and subsequent immunomagnetic depletion of nongranulocytic cells. Briefly, 60–80 ml of anti-coagulated venous blood were mixed 1 + 1 with chilled saline/2% dextran (Dextran 500; Amersham Bioscience, Uppsala, Sweden) and kept on ice for 30–40 min to sediment erythrocytes. The resultant leukocyte-rich supernatant was centrifuged (200 x g, 4°C, 6 min), and the pellet was gently resuspended in chilled saline. The leukocyte suspension was then layered on 15 ml of Lymphoprep (1.077 g/ml; Nycomed, Oslo, Norway) and centrifuged (400 x g, 4°C, 30 min). The supernatant including the interphase containing mononuclear cells was discarded and the pellet highly enriched for PMNs subjected to hypotonic lysis of erythrocytes (resuspension in 5 ml of chilled H2O, gentle mixing for 30 s, and termination of lysis by addition 5 ml 1.8% NaCl). After lysis, cells were pelleted (200 x g, 4°C, 6 min) and resuspended in PBS/0.5% BSA/2 mM EDTA buffer. Subsequently, the PMN preparations were depleted of nongranulocytic cells by immunomagnetic sorting using the MACS system according to the instructions of the manufacturer (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The mAbs used for depletion were raised against Ags expressed by the following cell types: monocytes (anti-CD14), B cells (anti-CD19), T cells (anti-CD3), platelets and megakaryocytes (anti-CD61), NK cells (anti-CD56), erythroid cells (anti-glycophorin A), and eosinophils (anti-CD49d; all mAbs were provided by BD Biosciences, San Diego, CA). To minimize the activation of PMNs, all isolation procedures were performed immediately after cell collection at 
4°C, i.e., on ice, in a cold room, or a cooled centrifuge, using nonpyrogenic reagents and plasticware.
PMNs that had transmigrated from the blood circulation to skin lesions generated by epidermal detachment and blister formation were collected by the skin chamber technique as described previously (8, 9). Briefly, a cylindrical acrylic suction device containing 3 holes of 5 mm in diameter, was placed on the volar surface of the nondominant forearm and negative pressure of 200 mm Hg was applied for 2 h by a vacuum pump resulting in the formation of suction blisters. After detachment of the vacuum pump, the blister roofs were removed with sterile tweezers and scissors, resulting in 3 uniform skin lesions, termed "skin windows", of 
0.2 cm2. A collection chamber with 3 collection compartments of 7 mm in diameter, was placed on top of the skin window, filled with 0.5 ml of autologous serum, and sealed with tightly fitting lids. The collection chamber was then fixed to the forearm by tape and an elastic bandage. After 18 h, the collection compartments were emptied, washed, and refilled with autologous plasma. After an additional 6 h, exudated cells were collected from skin chambers and depleted of nongranulocytic cells by immunomagnetic sorting as described above. Notably, serum contains biological active components of the coagulation and complement system. When used in skin chambers, serum is a documented strong chemoattractant, induces exocytosis of gelatinase granules and secretory vesicles by PMNs, and up-regulates Mac-1/CD11b on exudated PMNs, whereas plasma has no such effects (9). Thus, serum itself might activate PMNs. With the applied skin chamber protocol, PMNs were collected in plasma rather than serum to minimize the influence of skin chamber fluid on PMN activation. However, plasma alone is a poor chemoattractant and does not allow the collection of sufficient PMNs for gene expression analysis. Moreover, with plasma, it is technically not possible to extend the exudation period beyond 6 h to obtain more cells, since this will activate coagulation and trap exudated cells in a clot (P. Follin, unpublished observations). Based on these findings the exudation process was first initiated with serum for 18 h, followed by washing and refilling the skin chambers with plasma, before collecting freshly exudated PMNs after an additional 6 h (9). Since the applied skin chamber protocol is reproducible and uses an aseptic inflammation to attract cells to skin lesions, it is in our opinion currently one of the most suitable techniques that mimics the in vivo activation and resultant transcriptional response of PMNs in skin lesions. However, the protocol did not discriminate to what extent the migration process or the various stimuli (cytokines etc.) in the skin window exudates contributed to the observed transcriptional response of PMNs.
The purity of PMN preparations was assessed by microscopy of Wright-Giemsa-stained cytospins before and after immunomagnetic sorting. Cells were enumerated using a Neubauer hemocytometer.
Total RNA and proteins for gene expression analysis and Western blot analysis, respectively, were isolated from purified PMN preparations using TRIzol (Invitrogen, Paisley, U.K.) according to the guidelines of the manufacturer.
Gene expression analysis
For gene expression analysis, total RNA was biotinylated and hybridized to Hu95A GeneChips (Affymetrix, Santa Clara, CA) according to instructions of the manufacturer (www.affymetrix.com/pdf/expression_manual.pdf/). Briefly, first-strand cDNA was generated by reverse transcription of 2–5 µg of total RNA at 42°C for 1 h using a T7-oligo(dT)24 primer and Superscript II (Invitrogen). DNA second-strand synthesis was accomplished using DNA polymerase I and RNase H (Invitrogen) at 16°C for 2 h. Biotinylated cRNA was subsequently generated by in vitro transcription of dsDNA using T7 RNA polymerase at 37°C for 6 h in the presence of biotinylated nucleotides (BioArray High Yield RNA transcript labeling kit; Enzo Diagnostics, Farmingdale, NY). Finally, biotinylated cRNA was fragmented and the quality was confirmed on a test GeneChip before hybridization to Hu95A GeneChips (Affymetrix).
The expression index for each gene was calculated using the Li-Wong weighted average difference (10). Array signals on individual gene arrays were normalized by the Qspline method developed by Workman et al. (11). Qspline is a robust nonlinear method for normalization using array signal distribution analysis and cubic splines. Qspline fits cubic splines to the quantiles of the array signal distribution, and uses those splines to normalize signals dependent on their intensity.
The increase/decrease of gene expression in PMNs from skin lesions relative to PMNs from PB was calculated as a log2-fold change to obtain a symmetric distribution around zero (up-regulated genes have positive log2 values and down-regulated genes have negative log2 values). For functional clustering, genes were annotated with Gene Ontologies (www.geneontology.org/), which provides a unique identifier for genes having a characterized biological function.
Western blot analysis
TRIzol (Invitrogen) purified cell lysates corresponding to 5 x 105 PB-PMNs (pb-PMNs) and skin lesion-PMNs (sl-PMNs) were electrophoresed on 10–14% SDS polyacrylamide gels (BDH Laboratory Supplies, Poole, U.K.) and transferred to nitrocellulose membranes (Amersham Bioscience) by electroblotting. Subsequently, the membranes were incubated with primary Abs raised against IL-8, macrophage inflammatory protein (MIP)-1
(both from R&D Systems, Minneapolis, MN), and urokinase plasminogen activator (uPA; a gift from Dr. J. Pass, The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark) followed by a secondary HRP-conjugated swine anti-rabbit Ab or rabbit anti-mouse Ab (DAKO, Glostrup, Denmark). Binding of Abs was visualized by ECL (Amersham Bioscience, Uppsala, Sweden). Loading of equal amounts of protein was assessed by probing membranes with a monoclonal primary anti-
-actin Ab (12).
Statistics
The statistical analysis was performed using the R statistics program environment available from www.r-project.org/. The variability between subjects was low as estimated by the correlation coefficient, which ranged from 0.96 to 0.97 for pb-PMN genechips and 0.91 to 0.98 for sl-PMN genechips. In contrast, the correlation coefficient between pb-PMN genechips and sl-PMN genechips ranged from 0.78 to 0.80.
A Student t test was applied to identify differentially expressed genes in pb-PMNs and sl-PMNs. In the t test, the variance among individuals in the two categories of pb-PMNs and sl-PMNs was calculated for each gene, and differences between the two categories were only considered significant if they far exceeded the variance. The p values calculated by the t test were corrected for multiple testing (Benjamini-Hochberg, 13) to estimate the false discovery rate for differentially regulated genes. The false discovery rate for differentially regulated genes in the present study was 0.032.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most critical for the comparison of gene expression in cell populations collected in vivo is the application of highly purified cell preparations to minimize the false positive rate of differentially regulated genes due to contamination with other cell types. Thus, we reasoned to use a purification strategy based on density centrifugation and immunomagnetic depletion of nongranulocytic cells to obtain highly purified PMN preparations from PB and skin lesions for array analysis. In alignment with previous studies, density centrifugation of PB cells resulted in PMN preparations containing 95–97% PMNs, 2–4% eosinophils, and <1% mononuclear cells (5, 7). Additional lineage-depletion increased the purity of PMN preparations from PB to >99.5% (n = 4, mean purity 99.7%). Cells collected from skin chambers contained 85–95% PMNs and 5–15% contaminating monocytes/macrophages. After depletion of nongranulocytic cells, the purity of PMN preparations increased to >99% (n = 4, mean purity 99.4%). Subsequent array analysis revealed no detectable levels of lineage-specific transcripts for other relevant cell types such as eosinophils, basophils, monocytes, T cells, endothelial cells, fibroblasts, and epidermal cells in any of the purified PMN preparations (Table I; Affymetrix absent call). These findings demonstrate that the applied purification protocol resulted in highly purified PMN preparations from PB and skin lesions, and thus, minimized the false positive rate of differentially expressed genes due to contamination of nongranulocytic cells.
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Microarray analysis was applied to compare differentially the expression of 
12,500 genes in highly purified PMNs from PB and PMNs, which had transmigrated to skin lesions. Genes were defined as differentially expressed if they were among the 1000 most significant differentially expressed genes (range of p values: 2.6 x 10–3–3.6 x 10–9, estimated false discovery rate 0.032 (Benjamini-Hochberg)), and if they changed gene expression by 
0.5 log2-fold. By these criteria, 314 differentially expressed genes assigned to various functional gene categories of the Gene Ontology database were detected in PMNs upon migration to skin lesions (Fig. 1). Almost no up- or down-regulated genes were detected in categories critical for defense, cellular movement/transport or cell structure indicating that functions such as the migration to sites of infection, changes of cellular structure, and immediate anti-microbial defense are not regulated at the transcriptional level. In contrast, the high numbers of differentially expressed genes in categories such as apoptosis regulators, signal transducers, and enzymes, indicated that PMN activity in skin lesions is partially regulated at the transcriptional level.
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Detailed analysis of apoptosis regulators demonstrated the up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes (Tables II and III). Up-regulated anti-apoptotic genes included IEX1 (immediate early response (IER)3; protects cells from Fas-induced apoptosis) (14, 15), BCL2-related protein A1 (BCL2A1; blocks mitochondrial release of cytochrome c) (16), and FLIP (cFLAR; inhibits Fas-associated death domain protein-mediated activation of CASP8) (17). Among the down-regulated proapoptotic genes were Fas-associated death domain protein (activates CASP8), CASP8 (activates downstream caspases affecting apoptosis), APAF1 (activates CASP9 in a complex with cytochrome c), death-associated protein kinase 2 (18), and TNFR (activates apoptosis pathway by ligand binding). This change of gene expression among members of the apoptotic pathway suggests a transient anti-apoptotic priming of PMNs immediately after migration to skin lesions, regulated at the transcriptional level.
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The complex process of wound healing is orchestrated by signal transduction through chemokines, cytokines, and their respective receptors. Upon migration to skin lesions, PMNs up-regulated 26 and down-regulated 68 mediators of signal transduction (Tables II and III). Up-regulated chemokines and cytokines were critical for the recruitment of additional macrophages, T cells, and PMNs, and for the modulation of inflammatory responses (IL-8, monocyte chemoattractant protein (MCP)-1 (CC chemokine ligand 2), MIP-1 (CC chemokine ligand 3), growth-related oncogene (GRO)- (CXCL2), GRO-
(CXCL3), IL-1 (IL1B), and TNF- (TNF)) (19). Of interest, receptors mediating chemotaxis and cellular activation (IL-8RA (CXCR1), IL-8RB (CXCR2), G-CSFR, Toll-like receptor (TLR)1, and TLR6) (19, 20) were down-regulated concomitant with the up-regulation of other receptors modulating inflammatory responses (IL-1R1, TGF-BR1, G protein-coupled receptors (GPR) 65, GPR18, and HM74). Hence, PMNs might change their responsiveness to chemotactic and immunoregulatory mediators once activated in skin wounds.
Other effects of up-regulated chemokines/cytokines included the promotion of angiogenesis (vascular endothelial growth factor (VEGF), IL-8, GRO-, and MCP-1) (21), proliferation of keratinocytes and fibroblasts (IL-8, IL-1, and MCP-1) (19), and the induction of anti-microbial gene expression in keratinocytes (IL-1 and TNF-) (22). Additional up-regulated genes potentially involved in wound healing were laminin 5 3 (LAMB3) (23), which promotes adhesion of keratinocytes to the dermal layer, and uPA (PLAU), which supports tissue remodelling by breakdown of fibrin clots and degradation of extracellular matrix (24, 25). Other wound healing activities that are stimulated by uPA include the proliferation, migration, and adhesion of keratinocytes, fibroblasts, and endothelial cells in skin wounds (25).
The up-regulation of tripeptidyl-peptidase I/ceroid-lipofuscinosis, neuronal 2, legumain/asparaginyl endopeptidase, and the mannose-6-phosphate receptor suggested a priming of lysosomal activity once PMNs have migrated to skin lesions (26, 27, 28, 29).
Transcriptionally highly induced genes are up-regulated at the protein level
To investigate whether the transcriptional up-regulation of genes in sl-PMNs detected by array analysis correlated with increased protein levels, protein lysates were extracted from the same samples used for array analysis and subjected to Western blot analysis. These analyses demonstrated that transcriptionally highly induced genes in sl-PMNs including IL-8, MIP-1, and uPA were up-regulated at the protein level (Fig. 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, uPA, IL-8, VEGF, and IL-1) only IL-8 and IL-1 were induced by bacteria and LPS (4, 5), only MIP-1 and VEGF were induced by phagocytosis of IgG- and complement-coated latex beads (7), and uPA was not induced by any of these agents. These findings demonstrate that PMNs generate distinct transcriptional responses depending on the type of stimuli and activated signaling pathway. The different responses of PMNs stimulated in vitro and in vivo further demonstrate that multiple, and not individual, stimuli and signaling pathways contribute to the transcriptional response of PMNs in skin lesions. To define how migration of PMNs to skin lesions affects biological functions on a global level, differentially regulated genes were assigned to categories according to their biological functions using the Gene Ontology database. Functional clustering revealed that genes critical for migration, cellular structure, and immediate host defense were not activated, but were partially down-regulated, whereas genes involved in apoptosis, wound healing, and other distinct cellular functions were highly differentially regulated. Physiologically, these findings are meaningful, as basic functions such as migration and immediate host defense are inherent to circulating PMNs and do not require a prolonged phase of transcriptional activation. In contrast, the data demonstrated that PMNs were capable to transcriptionally activate specific functions such as the promotion of wound healing once they have migrated to sites of infection.
The cellular fate of PMNs at sites of infection includes necrotic death, immediate apoptosis, or the prolongation of life span by inhibition of apoptosis. Upon necrotic death, PMNs release toxic granule proteins resulting in tissue damage, whereas the phagocytosis of apoptotic PMNs by macrophages protects against such damage (30, 31). When cultured in vitro, PMNs rapidly undergo apoptosis, a process, which is delayed by addition of G-CSF and a variety of inflammatory mediators to the medium (32, 33). Hence, cytokines and inflammatory mediators present at sites of infection might augment the inflammatory response of PMNs by prolongation of cellular life span. This statement is supported by the present study showing that PMNs in skin wounds up-regulate anti-apoptotic genes and down-regulate proapoptotic genes, and thus, might acquire a transient "anti-apoptotic state". Notably, two of the up-regulated anti-apoptotic genes, i.e., IEX1 (IER3) and BCL2A1, have been defined as target genes of NF-
B, a transcription factor that was found to be up-regulated in sl-PMNs and, which is activated through IL-1 and TNF- signaling and binding of pathogens to TLRs (34). Because macrophages and PMNs both produce IL-1 and TNF- at sites of infection (35, 36), the "anti-apoptotic state" of PMNs in skin lesions might partially be regulated at the transcriptional level through NF-B activation pathways.
The present study supports the notion that PMNs are a rich source of cytokines and chemokines in skin wounds (36). Moreover, PMNs clearly outnumbered macrophages at 24 h in our skin-wounding model, suggesting that PMNs are the major source of inflammatory mediators during the initial phase of wound healing. Some of the factors found to be up-regulated in the present study including IL-1, TNF-, and IL-8, have been described as up-regulated in PMNs 1 day after incisional wounding (36, 37). However, to the best of our knowledge, the up-regulation of VEGF, MCP-1, MIP-1, and GRO- has not been reported earlier in PMNs upon migration to skin wounds. These findings demonstrate that up-regulated chemokines not only recruit more PMNs (IL-8), but also specifically attract macrophages (MCP-1 and MIP-1) (38) and T cells (MCP-1), which have been reported to be the most abundant leukocyte populations 2 days after incisional wounding (37).
The cellular response to cytokines and chemokines highly depends on the profile of receptors expressed on the cell membrane. The up-regulation of the IL-1R1 concomitant with its own ligand, IL-1 in sl-PMNs, suggests an autoregulatory enforcement of their inflammatory response through the NF-B activation pathway. Up-regulation of the TGF-R indicates an increased responsiveness to TGF-, a cytokine that is secreted by activated macrophages and perhaps is the most potent endogenous negative regulator of hemopoietic cells (39, 40). Hence, one might speculate that once PMN-chemokines have attracted sufficient macrophages, the macrophages will decrease the activity of PMNs through TGF- signaling, and thus, take over and initiate the next step in wound healing. Indeed, this hypothesis is supported by in vivo experiments showing that leukocyte infiltrates in skin wounds are initially dominated by PMNs, which decline in numbers concomitant with the increase of macrophage numbers 2 days after injury (37).
Other up-regulated receptors in sl-PMNs that might modulate cellular activity in skin lesions included the GPR18/65 and HM74. Whereas induction of GPR18/65 in PMNs has not been described so far, the induction of HM74 has been reported upon stimulation of PMNs by LPS and bacteria in vitro (4, 5). HM74 has been defined as a receptor for nicotinic acid, which mediates decrease in cAMP levels in adipose tissue when binding to its ligand (41). Down-regulated receptor transcripts included IL-8R, IL8-R, G-CSFR, and the TLRs 1 and 6, which mediate chemotaxis and cellular activation (19, 20). Overall, the altered expression of receptor transcripts suggests a change in responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated into skin lesions and have been activated.
The healing of skin wounds is a multistep process where cytokines and chemokines orchestrate the collaboration of various cell types. VEGF, probably the most important angiogenic cytokine, stimulates both proliferation and migration of endothelial cells (42). The present study demonstrates for the first time the up-regulation of VEGF by PMNs in a human skin wounding model. Moreover, sl-PMNs up-regulated the chemokines IL-8, GRO-, and MCP-1, which have been reported to stimulate growth of endothelial cells, keratinocytes, and fibroblasts (19, 21). Other genes that affect wound healing and were up-regulated by sl-PMNs included uPA and LAMB3. This was somewhat surprising as uPA is not up-regulated by PMNs when activated in vitro by various stimuli (4, 5, 7). However, incubation of plasma with PMNs has been shown to generate thrombolytic uPA activity (24). Hence, activated PMNs in skin wounds might generate uPA resulting in plasminogen activation and subsequent breakdown of fibrin clots and extracellular matrix (25). Moreover, uPA has been reported to promote the proliferation, migration, and adhesion of keratinocytes, fibroblasts, and endothelial cells in skin wounds (25). LAMB3 is an important adhesion molecule of the basal membrane and the deficiency of LAMB3 results in a blistering skin disease (junctional epidermolysis bullosa) due to the disruption of epidermal-dermal coadhesion (23). The up-regulation of LAMB3 by PMNs in skin lesions might therefore support adhesion of keratinocytes at wound margins, and thus, promote epitheliazation.
An important function of PMNs at sites of infection is the release of antimicrobial proteins as well as the phagocytosis and subsequent lysosomal degradation of microorganisms and cellular debris. Importantly, PMNs in skin lesions demonstrated no transcriptional regulation of antimicrobial peptides and lysosomal enzymes. However, the up-regulation of tripeptidyl-peptidase I/ceroid-lipofuscinosis, neuronal 2 and legumain/asparaginyl endopeptidase, which activate lysosomal enzymes by cleavage (26, 27, 28), and the up-regulation of the mannose-6-phosphate receptor (29), which target proteins to the lysosome, suggested a priming of PMNs for degradation of phagocytosed material upon migration to skin lesions.
We conclude that PMNs generate a substantial transcriptional response upon migration to skin lesions. This response fits with a model where PMNs change from a state primed for chemotaxis and activation by immunoregulatory mediators and microorganisms toward a state promoting wound healing, and the recruitment and activation of additional inflammatory effector cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kim Theilgaard-Mönch, The Granulocyte Research Laboratory, Department of Hematology-9322, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK 2100 Copenhagen, Denmark. E-mail address: kimthei{at}rh.dk
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophilic granulocyte; PB, peripheral blood; pb-PMN, PB PMN; sl-PMN, skin lesions; MIP, macrophage-inflammatory protein; uPA, urokinase plasminogen activator; MCP, monocyte chemoacttractant protein; GRO, growth-related oncogene; GPR, G protein-coupled receptor; IER3, immediate early response 3; BCL2A1, BCL2-related protein A1; CASP8, caspase 8, apoptosis-related cystein protease 8; CXCL, CXC chemokine ligand; TLR, Toll-like receptor; LAMB3, laminin 5 3; VEGF, vascular endothelial growth factor.
Received for publication February 3, 2004. Accepted for publication April 9, 2004.
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