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Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
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Abstract |
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, IL-10
, IL-131, IL-15, IL-17, IL-18, C1q, low-density lipoprotein, IgG Fc (CD32), and formyl peptide, Toll-like receptor 6, platelet/endothelial cell adhesion molecule-1 (CD31), P-selectin (CD62), IL-1, IL-16, and granulocyte chemoattractant protein-2 were down-regulated. Many of these genes were similarly down-regulated during Fas-mediated or camptothecin-induced apoptosis. We used flow cytometry to confirm that IL-8R (CXCR2) and IL-1 were significantly down-regulated during PMN apoptosis. We also discovered that 23 genes encoding phosphoinositide and calcium-mediated signal transduction components, which comprise complex pathways essential to the inflammatory response of host cells, were differentially regulated during PMN apoptosis. Importantly, our data demonstrate that PMNs down-regulate proinflammatory capacity at the level of gene expression during induction of apoptosis. These findings provide new insight into the molecular events that resolve inflammation following PMN activation in humans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the course of microbial infections, signal transduction events that facilitate host cell responses are relayed through PMN surface receptors, including those specific for complement receptors (CRs) and Ab (FcRs) (5), IL-8–10 (6, 7, 8), IL-13 (9), IL-15 (10, 11), and IL-18 (12), and multiple receptors for bacteria and microbial products, such as the Toll-like receptors (13). Expression of these receptors is essential for PMN functions such as chemotaxis, phagocytosis, ROS production, and release of cytokines, chemokines, and growth factors that potentiate innate immune and inflammatory responses. Importantly, recent studies from multiple laboratories indicate that mature PMNs synthesize de novo many cytokines, chemokines, and growth factors, including IL-1, IL-6, IL-8, growth-regulated gene products (GROs) and , TNF-, TGF-, oncostatin M (OSM), macrophage inflammatory protein-1, and vascular endothelial growth factor (VEGF) (14, 15, 16). Although significant progress has been made toward understanding the role of specific receptors and inflammatory mediators during the innate immune response, very little is known about their regulation during PMN apoptosis.
Phosphoinositide metabolism and calcium signal transduction mediate key PMN functions including chemotaxis, phagocytosis, ROS production, degranulation, and cell fate (17, 18, 19, 20, 21, 22, 23, 24). For example, phosphoinositide 3-kinase (PI3K)-Akt/PKB signal transduction represses programmed cell death by activating and inhibiting transcription factors that mediate cellular survival (25, 26, 27, 28) and induce apoptosis (29), respectively. Moreover, the inositol triphosphate (IP3) receptor and IP3-mediated calcium spikes control mitochondrial permeability transition pore involvement in programmed cell death (30, 31). Thus, PI3K and calcium signal transduction appear critical for modulating PMN function during the course of the inflammatory response and for termination of inflammation.
We recently proposed that programmed cell death in human PMNs is regulated by an apoptosis differentiation program, a final stage of transcriptional maturity that facilitates resolution of inflammation (32). Thus, multiple cellular processes in human PMNs are gene-regulated during apoptosis. In the present study, we used human oligonucleotide microarrays to demonstrate that the proinflammatory capacity of human PMNs is down-regulated at the level of gene expression during apoptosis. We propose that repression of PMN proinflammatory capacity is crucial for resolution of inflammation due to neutrophil activation in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sterile water and 0.9% sodium chloride (both Irrigation, U.S. Pharmacopeia) were purchased from Baxter Healthcare, Deerfield, IL. Dextran T-500 and Ficoll-Paque PLUS were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). PE-conjugated Abs specific for IL-8R, CD31, CD54, CD59, CD89, and IL-1 and corresponding isotype control Abs were purchased from BD Biosciences (Los Angeles, CA). Rabbit Ab specific for human serum albumin and human IgM was purchased from ICN Biomedicals (Costa Mesa, CA). Anti-Fas Ab (IgM clone CH-11) was purchased from Upstate Biotechnology (Lake Placid, NY). RPMI 1640 medium was from Invitrogen (Rockville, MD). Latex beads (LB; 2.0 µM) were purchased from Polysciences (Warrington, PA). Unless specified, all other reagents were from Sigma-Aldrich (St. Louis, MO).
Isolation of human PMNs
Human PMNs were isolated from venous blood of healthy individuals as described previously (4) in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Briefly, PMNs were purified using dextran sedimentation followed by Hypaque-Ficoll gradient centrifugation (33). Cell preparations contained >99% PMNs and all reagents used contained <25.0 pg/ml endotoxin. Eosinophils accounted for 5.2 ± 2.8% of the PMNs from the three individuals used in the oligonucleotide array experiments, and comprised 5.8 ± 3.0% of the human PMNs from a random sampling of preparations (n = 13 donors). We have previously reported that contaminating lymphocytes and monocytes account for 0.71 ± 0.26% and 0.11 ± 0.07% (n = 30 PMN preparations) of the cells in our PMN preparations (4).
PMN phagocytosis and gene expression analysis
Preparation of complement (C3bi)- and Ab (IgG)-coated LB and phagocytosis experiments were performed as described by Kobayashi et al. (4). Briefly, PMNs (107) were combined on ice with or without IgG and/or C3bi-coated LB (8 x 107) in wells of a 12-well tissue culture plate (precoated with 20% normal human serum) and centrifuged at 350 x g for 8 min at 4°C to synchronize phagocytosis. Following centrifugation, plates were incubated at 37°C in a CO2 incubator for up to 24 h. Alternatively, PMNs (107) were combined with 10 µM camptothecin (CPT) or 500 ng/ml anti-Fas Ab (CH-11) and incubated at 37°C in a CO2 incubator. At the indicated times, tissue culture medium was aspirated from the plate (or cells were centrifuged in microfuge tubes before aspiration of the media) and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target (12 µg) was performed as described (4). Labeling of samples, hybridization of cRNA with Hu95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols (http://www.affymetrix.com/pdf/expression_manual.pdf) as described previously (4). Experiments were performed with three separate donors at each time point using a separate oligonucleotide array for each donor.
Gene expression data were analyzed as reported previously with GeneSpring expression analysis software version 4.04 (Silicon Genetics, Redwood City, CA) (4). Briefly, genes were defined as differentially expressed if the average expression level changed at least 2-fold in one of the treatments compared with that from time-matched unstimulated cells over three experiments, and were called "Present" in at least two experiments by GeneChip Suite (Affymetrix). Receptor-specific changes were determined as described previously (4). Graphs generated with GeneSpring expression analysis software version 4.04 (Silicon Genetics) were annotated in CorelDraw, version 10 (Corel Corporation, Ottawa, Ontario, Canada).
Relative levels of differentially expressed genes were assigned based on average difference intensity (ADI) values determined by GeneChip Suite. (A) = genes called absent; (VL) very low expression = ADI <50; (L) low expression = ADI 50–200; (M) moderate expression = ADI 200–500; (H) high expression = ADI 500-1500; (VH) very high expression = ADI >1500. ADI is an approximation of transcript abundance and not caused by oligonucleotide primer hybridization bias.
TaqMan real-time RT-PCR analysis
Phagocytosis experiments and RNA preparation for TaqMan analysis were done with conditions identical with those used for the microarray analysis. TaqMan analysis of samples from three blood donors was performed in triplicate with an ABI 7700 thermocycler (Applied Biosystems, Foster City, CA) as described (4).
Flow cytometric analysis of receptor surface expression, IL-1 production, and cell viability
PMN apoptosis following phagocytosis was measured with flow cytometry using a modified TUNEL assay (Apo-BRDU Apoptosis Detection kit; BD Biosciences) or with annexin V-FITC (Annexin V-FITC Apoptosis Detection kit II; BD Biosciences) as described by the manufacturer. For Fas- or CPT-induced PMN apoptosis experiments, PMNs (2 x 106) were combined with 10 µM CPT or 500 ng/ml anti-Fas Ab (CH-11) and incubated at 37°C in a CO2 incubator for the desired times. PMN viability was determined by propidium iodide staining with flow cytometry, and was unaffected by incubation at 37°C for at least 9 h after phagocytosis (32). PMN viability at the start of the assay after the centrifugation step (0 min) was 96.4 ± 1.3%, and was reduced only slightly to 90.0 ± 2.6% in unstimulated PMNs after incubation for 24 h at 37°C (eight separate determinations). However, PMN lysis occurred more rapidly after 24 h in culture (data not shown).
To measure surface expression of IL-8R, CD31, CD54, and annexin V-FITC, phagocytosis assays were performed as described above, but with the following modifications. PMNs (106) were mixed on ice with IgG and/or C3bi-coated LB (8 x 106) in 96-well microtiter plates precoated with 20% normal human serum and centrifuged at 350 x g for 8 min at 4°C to synchronize phagocytosis. Following centrifugation, plates were incubated at 37°C in a CO2 incubator for up to 9 h as indicated. Alternatively, PMNs were treated with 500 ng/ml anti-Fas Ab (CH-11) as described above. Cells were stained with the indicated Abs and with annexin V-FITC as recommended by the manufacturer (BD Biosciences). Staining for intracellular IL-1 was performed 6, 9, and 16 h following phagocytosis in the presence of GolgiPlug (brefeldin A) as suggested by the manufacturer (BD Biosciences). The percentage of unstimulated PMNs staining positive for intracellular IL-1 was routinely 6–13% (individual variability) at 9 h, and this percentage increased significantly over time to 21–28% after 16 h due to accumulating cytokine in the endoplasmic reticulum (ER) after treatment with brefeldin A. Increasing the time of brefeldin A treatment increased the level of detectable IL-1. Thus, it is likely that all PMNs constitutively produce IL-1, albeit not to detectable levels. Despite individual variability in IL-1 staining, there was always less intracellular IL-1 in FcR/CR-stimulated PMNs compared with unstimulated PMNs. All samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and a single gate was used to eliminate debris.
Statistics
One-way ANOVA with Dunnett’s correction for multiple comparisons, Kruskal-Wallis ANOVA on ranks, and Student’s t test were performed in Sigma Stat for Windows, version 2.03 (SPSS, Chicago, IL) where indicated.
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Results |
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Unstimulated human PMNs undergo spontaneous or constitutive apoptosis, which presumably facilitates normal cell turnover in vivo (Ref. 2 ; Fig. 1). PMN apoptosis is accelerated significantly following receptor-mediated phagocytosis (phagocytosis) (4, 32), Fas ligation (34), and treatment with the topoisomerase inhibitor CPT (Ref. 35 ; Fig. 1, upper panel). The initial stages of phagocytosis-induced apoptosis occurred 3–6 h after PMN activation (Fig. 1). Therefore, analysis of gene regulation within this time period is critical for understanding molecular processes related to cell fate in PMNs, and ultimately, resolution of inflammation due to PMN activation.
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We screened 
12,500 human genes for changes in expression 3–6 h following phagocytosis, and compared expression of the up- and down-regulated genes in cells treated with anti-Fas Ab or CPT (Figs. 2 and 3, and Table I4). A total of 212 differentially regulated genes encoded proteins that mediate inflammatory response-related processes, signal transduction, adhesion, or cytoskeletal/structural function. A total of 133 genes (62.7%) were down-regulated and 79 genes (37.2%) were up-regulated.
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, GRO
, VEGF, OSM, and IL-6 were up-regulated, as were those encoding acute-phase reactant proteins, pentraxin 3 and orosomucoid 1 (Fig. 2). Several of these genes were correspondingly up-regulated during Fas- and/or CPT-induced PMN apoptosis (Fig. 2A). We reported previously that genes encoding several of these cytokines were induced between 30 and 90 min after phagocytosis, and that the gene encoding TNF- was up-regulated between 3 and 6 h after phagocytosis (4).
During the induction of phagocytosis-induced apoptosis, 32 genes encoding key surface molecules including receptors for IL-8, IL-10, IL-131, IL-15, IL-17, IL-18, C1q, and formyl peptide, Toll-like receptor 6, P-selectin (CD62), carcinoembryonic Ag-related cell adhesion molecule-4, platelet/endothelial cell adhesion molecule-1 (CD31), syndecan 1, FcRIIB (CD32), two Ig-like receptors (LILRB2 and LILRA1), and three purinergic receptors were down-regulated (Fig. 2). Consistent with the idea that there is general down-regulation of proinflammatory capacity during programmed cell death, 24 of these genes were down-regulated during Fas- and/or CPT-induced apoptosis (Fig. 2A). Down-regulation of these key PMN effector molecules, especially the IL-8R, would alter host-pathogen interactions normally mediated by PMNs (37). Genes encoding IL-1 and IL-16, key proinflammatory cytokines, and granulocyte chemoattractant protein 2 were significantly down-regulated (Fig. 2). Moreover, genes encoding sialic acid binding lectin 7 (SIGLEC7), peptidoglycan recognition protein (PGLYRP), cytotoxic granule-associated RNA binding protein (TIAL1), and calgranulin C, all involved directly in host defense, were down-regulated (Fig. 2). Taken together, these data suggest that key components of PMN proinflammatory and bactericidal capacities are regulated at the level of gene expression during the early stages of apoptosis.
TaqMan confirmation of microarray data
We selected several up- and down-regulated genes (n = 7) representative of the microarray data set for confirmation by TaqMan real-time PCR (Fig. 2B). There was almost absolute correlation (r = 0.99) of gene expression levels between the two methods at either 3 or 6 h following phagocytosis, including those for CD59 and CD54 (Fig. 2B). Microarray data for the other genes confirmed by TaqMan in Fig. 2B are presented below. These results are consistent with the strong positive correlation we reported between TaqMan and microarray data previously (4, 32). Importantly, these results confirm that regulation of key signal transduction mediators occurs at the level of gene expression during the initial stages of phagocytosis-induced apoptosis.
Comparison of gene expression during phagocytosis-, Fas-, and CPT-induced PMN apoptosis
To gain insight into common components of PMN apoptosis, we compared expression of selected apoptosis-related genes shown previously to be up- or down-regulated after phagocytosis (4) with gene expression in cells after Fas ligation or CPT treatment (Fig. 3A). Genes encoding glycoprotein NMB, BTG3, early growth response 2, TR3 orphan receptor, TNF-, and optineurin (FIP2) were up-regulated by all of the apoptosis-inducing stimuli (Fig. 3A). Furthermore, 13 genes encoding apoptosis-related proteins such as apoptosis inhibitor 5, DRAKs 1 and 2, and DEFCAP were down-regulated in all treatments (Fig. 3A) These findings provide compelling evidence that regulation of these genes is important for PMN apoptosis per se (rather than specific to phagocytosis).
In contrast, 24 genes encoding mediators of programmed cell death were differentially regulated during Fas- and/or CPT-induced apoptosis, but not following phagocytosis (Fig. 3B). Genes encoding UBL1 and IER3, inhibitors of Fas- and TNFR-mediated apoptosis, were down-regulated after Fas-ligation, consistent with concomitant induction of apoptosis (Fig. 3B). In contrast, the gene encoding TRAILR4 decoy receptor (apoptosis inhibitor) was up-regulated, and genes encoding CD95 (Fas), TRAILR2, BID, and BNIP3L (each promotes apoptosis) were down-regulated (Fig. 3B). Although these findings seem at variance with induction of apoptosis (see Fig. 1), several of these genes function in the initial stages of apoptosis (e.g., Fas and TRAILR2) rather than being required for downstream effector processes.
Down-regulation of proinflammatory mediators during PMN apoptosis
Although there were nearly equivalent numbers of up- and down-regulated receptors, surface molecules, and proinflammatory mediators during the onset of PMN apoptosis (Fig. 2), we hypothesized that expression of up-regulated proinflammatory molecules would diminish as apoptosis progressed. To test that hypothesis, we extended the time-course for analysis of up-regulated genes to 24 h after PMN stimulation (Fig. 4). In 20 of 28 up-regulated genes examined, expression decreased after 3 or 6 h of phagocytosis; albeit, expression was still generally greater than that in unstimulated PMNs (Fig. 4). For example, increased expression of genes encoding CCR1 and CD48 peaked at 3 and 6 h, respectively, following phagocytosis and then decreased over the next 21 h (Fig. 4). Diminishing expression levels for up-regulated cytokines, chemokines, and growth factors was even more striking at this time period, as 9 of 11 up-regulated cytokines and growth factors decreased markedly (Fig. 4). Decreasing expression of CCR1, CD48, CD59, CD71, TNF-, and GRO was especially striking after 3 or 6 h (Fig. 4). By 24 h, expression of all but Galectin 3, CD22, and Integrin,7 had returned to levels of expression comparable if not identical with that in unstimulated PMNs (Fig. 4). We note that expression of genes encoding proinflammatory molecules in unstimulated PMNs routinely decreased after 6 h in culture, concomitant with induction of spontaneous apoptosis in those cells (compare Figs. 1 and 4). These results suggest that PMNs terminate production of immune response mediators during apoptosis. Importantly, the implication of this discovery is that a significant portion of PMN proinflammatory capacity is down-regulated at the level of gene expression during the initial stages of apoptosis.
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To determine whether differential expression of proinflammatory genes resulted in concomitant regulation of protein levels, we measured surface expression of IL-8R (CXCR2; Ref. 6), CD31, and CD54 during phagocytosis- and Fas-induced PMN apoptosis (Fig. 5). Freshly isolated human PMNs expressed all three proteins (Fig. 5, 0 h). Compared with unstimulated cells, expression of IL-8R, a receptor critical to the inflammatory response, was significantly reduced at 1.5, 3, and 6 h following phagocytosis, and 3 and 6 h after Fas ligation (p < 0.006 vs unstimulated PMNs for each stimulus; Fig. 5A). Importantly, decreased surface expression of IL-8R correlated with increased staining for annexin V following phagocytosis and Fas ligation, confirming that the receptor is down-regulated during PMN apoptosis (Fig. 5B). Surface expression of CD31 was also significantly reduced 1.5 h following phagocytosis (p < 0.006 vs unstimulated PMNs; Fig. 5A). However, CD31 was surface-expressed on neither stimulated nor unstimulated cells by 3 or 6 h (Fig. 5A). Although plasma membrane-associated CD54 decreased slightly over time, surface expression was increased significantly compared with unstimulated cells at 6 h (p < 0.004 vs unstimulated PMNs; Fig. 5, A and B). We note that an overall decrease in expression of genes encoding CD31 and CD54 in unstimulated PMNs after 6 h correlated with the initial stages of spontaneous apoptosis (compare Figs. 1 and 5). Compared with unstimulated cells, surface expression of CD59 and CD89 was also up-regulated during the early stages of activation-induced apoptosis (+13.7% and +7.7% for CD59, and +34.0% and +27.3% for CD89 compared with unstimulated cells at 3 and 6 h, respectively, n = 3; p = 0.01 for surface expression of CD89 in stimulated vs unstimulated PMNs at 6 h), consistent with gene expression (Fig. 2A). Taken together, there was general correlation of gene transcription and expression of receptors at the cell surface (compare panels in Fig. 5).
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IL-1 is a critical mediator of inflammatory processes, especially those mediated by PMNs, and its down-regulation during the induction of apoptosis would likely be important in the resolution of inflammation. To determine whether down-regulation of the gene encoding IL-1 resulted in a concomitant decrease in protein levels, we measured intracellular IL-1 at 6 and 9 h following phagocytosis in human PMNs (Fig. 6A). There was a significant decrease in PMN-associated IL-1 at both 6 and 9 h after phagocytosis (during PMN apoptosis) compared with unstimulated cells (reduced by 44.8 ± 9.3% and 45.8 ± 24.5%, respectively; *, p = 0.03 and 0.009 vs unstimulated PMNs, respectively; Fig. 6B). Down-regulation of the gene encoding IL-1 correlated with decreased expression of IL-1 protein at those times, indicating that IL-1 is down-regulated at the level of gene expression (compare A and C in Fig. 6).
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Although a focus of these studies was to identify differentially expressed inflammatory mediators during apoptosis, we hypothesized that many genes encoding pro- and anti-inflammatory molecules are expressed in human PMNs, but not differentially regulated. Therefore, we examined expression of 114 genes that encode a significant number of the known inflammatory mediators, but were not up- or down-regulated following phagocytosis by our criteria (Fig. 7). Of these genes, there were a limited number (38) identified as present in human PMNs, and most encoded receptors rather than cytokines (Fig. 7). Importantly, the gene encoding IL-1R antagonist (IL1RN) was very highly expressed, consistent with down-regulation of IL-1 and proinflammatory capacity during this period of time (Fig. 7). We also note that genes encoding receptors which mediate apoptosis, such as TRAILR2 and CD120a, although not differentially regulated following phagocytosis, were highly expressed in human PMNs (Fig. 7).
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Inasmuch as signal transduction is coupled to regulation of PMN function following receptor ligation, we identified 80 signal transduction-related genes differentially expressed between 3 and 6 h after phagocytosis (Fig. 8). Sixty-three of these genes were down-regulated and 17 were up-regulated (Fig. 8). Thirty of 34 genes encoding kinases and associated proteins were down-regulated, including key signal transduction regulators such as class I PI3K, p110 subunits , 
, and , and calcium/calmodulin-dependent kinases II and (Fig. 8). Moreover, genes encoding 12 phosphatases and 14 of 24 G-protein-related molecules were down-regulated during induction of phagocytosis-induced apoptosis (Fig. 8). Genes encoding phospholipases D2 and C
2, critical regulators of receptor-mediated signal transduction, were down-regulated (Fig. 8). We also identified 27 differentially regulated genes that encode structural proteins or those comprising and/or involved in modifying the actin cytoskeleton (Fig. 8). For example, genes encoding 2-actin, myosin 5A, and isoforms 1 and 4 of tropomyosin were down-regulated during the induction of PMN apoptosis (Fig. 8).
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Gene-regulated phosphoinositide and calcium-mediated signal transduction
Twenty-three of the signal transduction-related genes identified as differentially regulated during the initial stages of PMN apoptosis (21 down-regulated and 2 up-regulated) comprised complex pathways for phosphoinositide- and calcium-mediated signal transduction (Fig. 8). The observation that genes encoding all three known class I PI3Ks (PIK3CA, PIK3CD, and PIK3CG), Akt1 (AKT1) and Akt2 (AKT2), and anti-apoptotic transcription factors CREB1 (CREB1) and Forkhead box O1A (FOXO1A) were down-regulated is consistent with the parallel finding that PMNs are undergoing the initial stages of apoptosis (Ref. 4 , Fig. 8, and Table I4). We previously described down-regulation of AKT1 and FOXO1A during phagocytosis-induced PMN apoptosis (4). Although CREB1 encodes a transcription factor, it was included in this study because of its critical importance as a downstream effector for PI3K activation. PI3Ks are also critical for the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), and repression of these genes is a potential mechanism to regulate IP3 synthesis, and thus, calcium signal transduction (Fig. 8). Consistent with those findings, genes encoding proteins directly involved in calcium-mediated signal transduction, such as protein kinase C
and calcium/calmodulin-dependent protein kinases II and II, were down-regulated during phagocytosis-, Fas-, and CPT-induced apoptosis (Fig. 8).
Of the differentially expressed genes comprising PI3K/calcium signal transduction, only genes encoding the ITPR1 and PI3K,C2 (PIK3C2B) were up-regulated (Figs. 2A and 8A). This finding is intriguing because class II PI3Ks phosphorylate phosphatidylinositol and phosphatidylinositol 4-phosphate rather than PIP2, and their role in leukocyte signal transduction remains unclear (38). Up-regulation of ITPR1 is at variance with the observation that other critical regulators of calcium signal transduction are down-regulated (Fig. 8). However, ITPR1 has been implicated in T lymphocyte programmed cell death (39), and it is probable that it has a similar role in PMN apoptosis.
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Discussion |
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, VEGF, OSM, IL-6, and GROs and (Fig. 4). These results indicate that proinflammatory signals up-regulated because of phagocytosis/PMN activation are subsequently repressed at the onset of apoptosis.
Repression of genes encoding CD31 and IL-8R correlated with down-regulation of the surface expression of these proteins. Down-regulation of CD31, a molecule required for transendothelial migration of PMNs (41), has been shown to accompany spontaneous PMN apoptosis, although no link was provided between gene and protein expression (42). Although it could be argued that decreased surface expression of IL-8R and CD31 in stimulated cells is caused by internalization mediated by endocytosis (43), we found no significant difference in the surface expression of IL-8R (between unstimulated and stimulated PMNs) until there was concomitant change in gene expression (Fig. 5). Of note, little evidence has been provided for the modulation of IL-8R during PMN apoptosis. The gradual decrease in IL-8R on the PMN surface in our studies (over several hours) contrasts with very rapid (within 10 min) down-regulation observed during endocytotic IL-8R internalization (43). Loyd and coworkers (44) previously demonstrated a link between IL-8R gene transcription and expression of protein on the PMN surface, which occurred on the order of hours rather than minutes, just as we found in our studies. Moreover, down-regulation of IL-8R surface expression occurred at a point in time in which PMN secretion of IL-8 and other CXCR2 ligands was likely diminished compared with earlier time points (Fig. 2). Because IL-8 is a potent chemokine that modulates key leukocyte responses such as chemotaxis (45), we propose that down-regulation of IL-8R during apoptosis is critical for the resolution of inflammation following PMN activation. Of note, the gene encoding 2 arrestin (ARRB2), a protein that modulates IL-8R internalization, was down-regulated (Fig. 7).
Phagocytosis-induced PMN apoptosis was accompanied by significant decrease in the gene encoding IL-1 and importantly, the corresponding level of IL-1 protein was diminished significantly. Although a seemingly small population of PMNs expressed detectable IL-1 in our assays (Fig. 6A), we found that the percentage of PMNs with detectable IL-1 increased significantly over time because of accumulating cytokine in the ER after treatment with brefeldin A. As a result, when the time of brefeldin A treatment was extended, the level of detectable IL-1 increased (from 1.78 ± 0.38% IL-1-positive PMNs at 6 h, to 8.1 ± 3.0% and 24.2 ± 4.9% at 9 and 16 h, respectively). Most significantly, there was always less intracellular IL-1 in stimulated PMNs compared with unstimulated cells (Fig. 6). Production of IL-1 by PMNs has not been characterized, but the cytokine has a critical role in the innate immune and inflammatory responses in humans (46). Pro-IL-1, the fully active IL-1 precursor, is released upon cell death (47), and would occur during necrotic PMN lysis. Because IL-1 is an important inflammation-inducing cytokine, its down-regulation by human PMNs during apoptosis has important implications for termination of the inflammatory response. In support of these findings, the gene encoding IL-1R accessory protein, a molecule critical for IL-1 signal transduction (48), was down-regulated (Fig. 2).
Multiple PMN functions are regulated by phosphoinositide metabolism and calcium-mediated signal transduction (reviewed in Refs. 20, 21, 22, 23, 24). Ligation of cell surface receptors, including IL-8R, activates PI3Ks, which in turn moderate a balance between PIP2 and PIP3, important second messengers for downstream signal transduction events and subsequent cell activation. PIP3 concentrations increase dramatically upon PMN stimulation because of the conversion of PIP2 to PIP3 by class I PI3Ks (49). By comparison, PIP2 is present at high constitutive levels in human PMNs (49) and is hydrolyzed to IP3 and diacylglycerol by phospholipase C. It is PIP2-derived IP3 that binds to the ITPR1 and elicits efflux of calcium from the ER into cytosol, thus facilitating subsequent calcium-regulated cellular processes. Of note, PI3K signal transduction has been directly linked to ROS generation in PMNs (17, 18, 19). Therefore, regulating PI3K and calcium-mediated signal transduction is important in maintaining a balance between pro- and anti-inflammatory signals in the cell. The observation that at least 21 genes encoding PI3K and calcium signal transduction pathways were down-regulated suggests that the ability of the cell to respond to proinflammatory stimuli, such as IL-8, was limited. Thus, proinflammatory mediators and the signal transduction pathways linking them to cellular responses are down-regulated at the level of gene expression during PMN apoptosis. Importantly, these studies define precisely the regulation of specific components of complex signal transduction pathways essential for PMN function.
In conclusion, we discovered that proinflammatory capacity is 1) down-regulated during the induction of PMN apoptosis, and 2) regulated at the level of gene expression. We recently proposed that an apoptosis differentiation program regulates, in part, cell-fate, energy metabolism, and responses to oxidative stress, and represents a final stage of transcriptionally regulated PMN maturation (32). Based on the findings reported herein, the apoptosis differentiation program down-regulates proinflammatory capacity as well. We propose that these processes are critical for the resolution of inflammation and bacterial infections in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840. E-mail address: fdeleo{at}niaid.nih.gov
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; CR, complement receptor; LB, latex bead; CPT, camptothecin; ROS, reactive oxygen species; ADI, average difference intensity; ER, endoplasmic reticulum; PIP2, phosphatidylinositol 4,5-biphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; VEGF, vascular endothelial growth factor; OSM, oncostatin M; PI3K, phosphoinositide 3-kinase; GRO, growth-regulated gene product; IP3, inositol triphosphate; ITPR1, IP3 receptor 1.
4 The online version of this article contains supplemental material.
Received for publication August 14, 2002. Accepted for publication January 13, 2003.
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 C. Burlak, A. R. Whitney, D. J. Mead, T. Hackstadt, and F. R. DeLeo Maturation of Human Neutrophil Phagosomes Includes Incorporation of Molecular Chaperones and Endoplasmic Reticulum Quality Control Machinery Mol. Cell. Proteomics, April 1, 2006; 5(4): 620 - 634. [Abstract] [Full Text] [PDF]
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B. Puthengady Thomas, C. X. Sun, E. Bajenova, R. P. Ellen, and M. Glogauer Modulation of Human Neutrophil Functions In Vitro by Treponema denticola Major Outer Sheath Protein Infect. Immun., March 1, 2006; 74(3): 1954 - 1957. [Abstract] [Full Text] [PDF]
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 S. D. Kobayashi, J. M. Voyich, A. R. Whitney, and F. R. DeLeo Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor J. Leukoc. Biol., December 1, 2005; 78(6): 1408 - 1418. [Abstract] [Full Text] [PDF]
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 B. J. Stephens, H. Han, V. Gokhale, and D. D. Von Hoff PRL phosphatases as potential molecular targets in cancer Mol. Cancer Ther., November 1, 2005; 4(11): 1653 - 1661. [Abstract] [Full Text] [PDF]
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J. M. Voyich, K. R. Braughton, D. E. Sturdevant, A. R. Whitney, B. Said-Salim, S. F. Porcella, R. D. Long, D. W. Dorward, D. J. Gardner, B. N. Kreiswirth, et al. Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils J. Immunol., September 15, 2005; 175(6): 3907 - 3919. [Abstract] [Full Text] [PDF]
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J. F. Hedges, K. J. Lubick, and M. A. Jutila {gamma}{delta} T Cells Respond Directly to Pathogen-Associated Molecular Patterns J. Immunol., May 15, 2005; 174(10): 6045 - 6053. [Abstract] [Full Text] [PDF]
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D. L. Borjesson, S. D. Kobayashi, A. R. Whitney, J. M. Voyich, C. M. Argue, and F. R. DeLeo Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils J. Immunol., May 15, 2005; 174(10): 6364 - 6372. [Abstract] [Full Text] [PDF]
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L. Allen, D. H. Dockrell, T. Pattery, D. G. Lee, P. Cornelis, P. G. Hellewell, and M. K. B. Whyte Pyocyanin Production by Pseudomonas aeruginosa Induces Neutrophil Apoptosis and Impairs Neutrophil-Mediated Host Defenses In Vivo J. Immunol., March 15, 2005; 174(6): 3643 - 3649. [Abstract] [Full Text] [PDF]
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F. Shen, M. J. Ruddy, P. Plamondon, and S. L. Gaffen Cytokines link osteoblasts and inflammation: microarray analysis of interleukin-17- and TNF-{alpha}-induced genes in bone cells J. Leukoc. Biol., March 1, 2005; 77(3): 388 - 399. [Abstract] [Full Text] [PDF]
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 R. Guo, Y. Wang, A. W. Minto, R. J. Quigg, and P. N. Cunningham Acute Renal Failure in Endotoxemia is Dependent on Caspase Activation J. Am. Soc. Nephrol., December 1, 2004; 15(12): 3093 - 3102. [Abstract] [Full Text] [PDF]
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 A. Strassburg, D. Droemann, G. van Zandbergen, H. Kothe, and K. Dalhoff Enhanced PMN response in chronic bronchitis and community-acquired pneumonia Eur. Respir. J., November 1, 2004; 24(5): 772 - 778. [Abstract] [Full Text] [PDF]
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M. Aleman, P. Schierloh, S. S. de la Barrera, R. M. Musella, M. A. Saab, M. Baldini, E. Abbate, and M. C. Sasiain Mycobacterium tuberculosis Triggers Apoptosis in Peripheral Neutrophils Involving Toll-Like Receptor 2 and p38 Mitogen Protein Kinase in Tuberculosis Patients Infect. Immun., September 1, 2004; 72(9): 5150 - 5158. [Abstract] [Full Text] [PDF]
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X. Zhang, Y. Kluger, Y. Nakayama, R. Poddar, C. Whitney, A. DeTora, S. M. Weissman, and P. E. Newburger Gene expression in mature neutrophils: early responses to inflammatory stimuli J. Leukoc. Biol., February 1, 2004; 75(2): 358 - 372. [Abstract] [Full Text] [PDF]
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S. D. Kobayashi, J. M. Voyich, K. R. Braughton, A. R. Whitney, W. M. Nauseef, H. L. Malech, and F. R. DeLeo Gene Expression Profiling Provides Insight into the Pathophysiology of Chronic Granulomatous Disease J. Immunol., January 1, 2004; 172(1): 636 - 643. [Abstract] [Full Text] [PDF]
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 S. D. Kobayashi, K. R. Braughton, A. R. Whitney, J. M. Voyich, T. G. Schwan, J. M. Musser, and F. R. DeLeo From the Cover: Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils PNAS, September 16, 2003; 100(19): 10948 - 10953. [Abstract] [Full Text] [PDF]
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), following FcR/CR phagocytosis (red circle), after incubation with anti-Fas Ab (green circle), or CPT (•, inset) as indicated. Results for annexin V-FITC are the mean ± SD of two to eight experiments. *, p < 0.001 vs CTL for each stimulus; **, p < 0.03 vs CTL for each stimulus (ANOVA). Results for TUNEL are from a representative experiment performed with each the three individuals used in the oligonucleotide array experiments.
) were selected from the overall dataset for confirmation by TaqMan real-time PCR (
) following FcR/CR-mediated phagocytosis. Inset, The correlation (r = 0.99) between real-time PCR and microarray analyses. Data represent the mean ± SD of fold changes in gene expression from the three donors used in the microarray experiments. Microarray data are presented as the mean fold change of the three individuals.
0.05 vs gene expression in unstimulated PMNs at either 3 or 6 h. Results are illustrated as the mean ± SD of relative gene expression from three separate individuals.
