Lung response to Bordetella pertussis infection in mice identified by gene-expression profiling

Host genetics determines the course of Bordetella pertussis infection in mice. Previously, we found four loci, Tlr4 and three novel loci, designated Bps 1–3, that are involved in the control of B. pertussis infection. The purpose of the present study was to identify candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. We, therefore, studied the course of mRNA expression in the lungs after B. pertussis infection. Of the 22,000 genes investigated, 1,841 were significantly differentially expressed with 1,182 genes upregulated and 659 genes downregulated. Upregulated genes were involved in immune-related processes, such as the acute-phase response, antigen presentation, cytokine production, inflammation, and apoptosis, while downregulated genes were mainly involved in nonimmune processes, such as development and muscle contraction. Pathway analysis revealed the involvement of granulocyte function, toll-like receptor signaling pathway, and apoptosis. Nine of the differentially expressed genes were located in Bps-1, 13 were located in Bps-2, and 62 were located in Bps-3. We conclude that B. pertussis infection induces a wide and complex response, which appears to be partly specific for B. pertussis and partly nonspecific. We envisage that these data will be helpful in identifying polymorphic genes that affect the susceptibility and course of B. pertussis infection in humans.
Introduction
The airway pathogen Bordetella pertussis can cause the disease known as whooping cough. Despite high vaccination coverage, pertussis is still an endemic disease with peaks in incidence every 2–3 years during the last decade (Crowcroft and Pebody 2006; de Greeff et al. 2003, 2005). Susceptibility to and severity of B. pertussis infection in infants and children vary widely (Kimman 2001). The spectrum of clinical symptoms ranges from subclinical infection to mild disease, severe whooping cough, and death. In previous studies, we examined genetic susceptibility of mice to B. pertussis and identified loci in the mouse genome that are involved in restriction of colonization and/or clearance of B. pertussis. We and others have provided indications for the role of several polymorphic host genes in the course of B. pertussis infection, including the interferon gamma receptor (Mahon et al. 1997), toll-like receptor 4 (Tlr4; Higgins et al. 2003; Mann et al. 2004, 2005), and three putative disease loci, B. pertussis susceptibility locus 1, 2, and 3 (Bps-1, 2, and 3; Banus et al. 2005). Tlr4 is a major host factor involved in the course of B. pertussis infection. C3H/HeJ mice, which carry a nonfunctional Tlr4 gene, exhibit an aberrant course of infection. Functional Tlr4 is essential for an efficient IL1-β, TNF-α, and IFN-γ response, efficient clearance of bacteria from the lung, and therewith reduced lung pathology (Banus et al. 2006). The Bps-1 locus is located on chromosome 12, spanning a region of 185 genes and has a dominant positive effect on the clearance of B. pertussis from the lung. The function of most genes in this locus is unknown. Two other loci, Bps-2 and Bps-3, showed genetic interaction and are located on chromosomes 5 and 11, respectively (Banus et al. 2005).
The highest linkage between the number of colony-forming units (CFU) and the Bps-1 locus that we observed showed a logarithm of the odds (LOD) score of 4.6. This LOD score and the corresponding P value (P = 0.000025) are statistically significant also after correction for multiple comparisons (P = 0.0019). We interpreted these data as suggestive for linkage warranting further investigation (Banus et al. 2005). In the present study, we aimed to enhance the insight in the host response and pathogenesis of B. pertussis infection by identifying genes that are differentially expressed upon infection. In particular, we were interested in identifying candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. For this purpose, we infected mice and analyzed gene-expression profiles in the lungs on days 1, 3, and 5 after inoculation using microarrays. We were especially interested if genes, located in the Bps-1, 2, and 3 loci, were differentially expressed after B. pertussis infection or that the susceptibility of these loci was regulated alternately.
Materials and methods
Experimental design
Female C3H/DISnA mice (eight mice per group, total of 48) were intranasally inoculated with 2 × 107 CFU of the streptomycin-resistant Tohama strain of B. pertussis (B213) in 40 μl of Verwey medium (The Netherlands Vaccine institute, Bilthoven, The Netherlands) or as a control with Verwey Medium only. One, 3, and 5 days after inoculation, mice were euthanized. To remove blood cells from the lungs, mice were perfused with phosphate-buffered saline, and the lungs and trachea were collected (King et al. 2001; Willems et al. 1998). For the extraction of RNA, the right lung was collected in the RNA stabilization reagent (RNAlater, Qiagen, Venlo, The Netherlands). The left lung was fixed intratracheally using 4% formalin for histological examination.
The number of viable B. pertussis bacteria was determined in the trachea to confirm a proper infection (Verwey et al. 1949). Therefore, approximately 1 cm of the trachea was collected in 500 μl of Verwey medium. Bacterial suspensions were diluted in Verwey medium, and the number of CFU was determined by plating on Bordet Gengou agar supplemented with 15% sheep blood and 30 μg/ml streptomycin (Tritium Microbiology, Veldhoven, The Netherlands). Plates were incubated for 4 days at 35°C before counting the number of CFUs using a ProtoCOL Colony counter (Synbiosis, Cambridge, UK).
Animals
Female C3H/DISnA mice were kindly supplied by Dr. Peter Demant (Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY). Mice were bred to the appropriate numbers at our animal testing facility in Bilthoven, The Netherlands. Mice were acclimatized for at least 1 week before the start of the experiments. Mice received standard laboratory food (SRM-A, Hope Farms, Woerden, The Netherlands) and tap water ad libitum. All animal experiments were approved by the Institute’s Animal Ethics Committee and were performed according to the National Institutes of Health (1996) guidelines.
Clinical and pathological examinations
Mice were weighed before inoculation and subsequently every day after inoculation to determine the relative change in weight. Lung weights were determined postmortem as a parameter for lung inflammation. Formalin-fixed lungs were embedded in paraplast (Monoject, St Louis, MO). Sections (5 μm) were stained with hematoxylin eosin. Lung lesions were examined for infiltration of inflammatory cells in the peribronchiolar space (peribronchiolitis), infiltration of inflammatory cells in the alveoli (alveolitis), infiltration of inflammatory cells in the perivascular space (perivasculitis), hypertrophy of mucus-producing glands, free protein (exudate), and eosinophilia. Lung lesions were scored semiquantitatively as absent, minimal, slight, moderate, marked, or strong as previously described (Barends et al. 2004).
Transcription profiling
Lungs were incubated in RNAlater (Qiagen) at 4°C for 1 day, after which, the tissues were transferred to a fresh tube and stored at −80°C. RNA was extracted by using the midi-RNA isolation kit (Qiagen). RNA concentrations were measured using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and was expressed as the RNA Integrity Number (RIN) as defined by the manufacturer. Only samples with a RIN score of at least eight (on a 1–10 scale) were used for expression profiling.
Microarray slides were spotted at the Microarray Department of the University of Amsterdam. The slides contain 21,997 65-mer oligo nucleotides from the Sigma-Compugen Mouse oligonucleotide library, 192 additional 65-mer oligo nucleotides of genes, of which 185 were located in the Bps-1 region spotted in duplicate (Isogen Life science, IJsselstein, The Netherlands), as well as appropriate controls and blank spots.
Based on RNA quality and yield, RNA isolated from six mice per group was selected for amplification and labeling. RNA amplification and labeling were carried out using the Amino Allyl MessageAmp II aRNA kit (Ambion, Austin, TX) using 1 μg of total RNA as starting material. RNA samples from individual mice were labeled with Cy3. A common reference containing a RNA pool of all samples isolated was labeled with Cy5. After hybridization and washing, the arrays were scanned using a ScanArray 4000XL microarray scanner (Perkin-Elmer, Wellesley, MA).
Median Cy3 and Cy5 spot signals were determined using ArrayVision (Imaging Research, St. Catharines, ON, Canada). Quality control was performed by means of visual inspection of the scanned images, raw data scatter, and MA plots (M is a mnemonic for the log-ratios of expression, A is a mnemonic for the log-intensity of each spot; Smyth and Speed 2003), as well as a normal probability plot to assess signal distribution (Pennings and Heisterkamp 2004). If more than 10% of the spots were flagged as missing data, slides were excluded from data analysis. At least five slides per group were used for further analysis.
Raw microarray signal data were normalized in the R statistical software environment (R Development Core Team 2005) using a four-step approach of (1) natural log transformation, (2) quantile normalization of all scans, (3) correcting for differences in the reference signal, and (4) averaging replicate spot data.
Significance of differences in gene expression between the experimental groups was calculated in R with analysis of variance. The false discovery rate (FDR) was calculated according to Benjamini and Hochberg (1995). The FDR is the expected proportion of false positives among the genes considered significantly differentially expressed (Benjamini et al. 2001; Benjamini and Hochberg 1995). Gene-expression data were visualized by hierarchical clustering (using Euclidian distance and Ward linkage) using GeneMaths (Applied Maths, St-Martens-Latem, Belgium). For this purpose, gene-expression values were corrected for the overall average to allow a neutral comparison between all B. pertussis- and mock-inoculated groups. Gene categories were defined by Gene Ontology (GO, http://www.geneontology.org). Classification and enrichment according to GO categories were determined using DAVID/EASE (Dennis et al. 2003; Hosack et al. 2003), using EASE scores as P values for enrichment. Additional pathway analysis was performed using MetaCore™ (GeneGo, http://www.genego.com).
Results
Bacteriologic and pathologic findings
All B. pertussis-inoculated mice had between 102 and 105 CFUs in the trachea during the first 5 days after inoculation thereby confirming a proper infection. A statistically significant decrease in the number of bacteria in the trachea was observed over time during the first 5 days postinoculation (P = 0.022, linear regression, SPSS, data not shown).
Infected mice lost up to 4% of their body weight during the first 5 days postinoculation, while all mock-inoculated mice gained weight up to 4% during these 5 days. This difference in relative gain of weight between mock- and B. pertussis-inoculated mice is significant from day 1 to 5 postinoculation (Supplementary data Figure 2).
We determined lung weights in relation to body weight as a marker for inflammation. All mock-inoculated mice had relative lung weights of approximately 1% during the first 5 days after inoculation, while B. pertussis-infected mice had relative lung weights of up to 3% of their total body weight. This difference in relative lung weights between mock- and B. pertussis-inoculated mice is significant from day 1 to 5 postinoculation (Supplementary data Figure 3).
A summary of histopathological findings is presented in Table 1. Intranasal inoculation of B. pertussis causes an acute inflammatory response that is characterized by influx of polymorphonuclear leukocytes (PMNs) and macrophages starting in perivascular and peribronchiolar areas on day 1 and extending to alveolar walls and lumina on days 3 and 5.
Table 1Histological evaluation of lung changes after B. pertussis inoculationParameter and gradeNo. of animalsaMockB. pertussisDay 1Day 3Day 5Day 1Day 3Day 5PeribrochiolitisbMinimal23422Slight125Moderate3Totalc(25%)(0%)(38%)(63%)(88%)(88%)PerivasculitisMinimal22221Slight433Moderate35Marked1Total(25%)(25%)(25%)(75%)(100%)(100%)HypertrophyMinimal1112Slight1Moderate31Marked24Strong1Total(13%)(0%)(13%)(13%)(88%)(88%)AlveolitisMinimal21Slight42Moderate31Marked13Strong31Total(0%)(0%)(0%)(75%)(100%)(88%)ExudateMinimal22Slight22Moderate11Marked1Strong1Total(0%)(0%)(0%)(0%)(100%)(63%)aEight mice per group were inoculated with medium (mock) or B. pertussis.bHematoxylin/eosin-stained slides were examined for peribronchiolitis, alveolitis, perivasculitis, hypertrophy of mucus-producing glands, and free protein (exudate). Lung lesions were scored semiquantitatively as absent (not included in table), minimal, slight, moderate, marked, or strong.cTotal percentage of animals with at least minimal scoring in the specified category
Expression profiling
Gene-expression profiles were determined in at least five animals per group. The three mock-inoculated groups, euthanized at days 1, 3, and 5, were statistically indistinguishable (i.e., there were no genes with FDR < 0.6). Of the approximately 22,000 genes tested, 1,841 genes were significantly differentially expressed in B. pertussis-inoculated lungs (FDR < 0.05, corresponding to P < 0.0042). Expression levels of all 1,841 genes as well as comparisons to other studies are presented in the supplementary data Table 5.
Hierarchical clustering showed time-dependent changes in gene expression with 1,182 upregulated genes and 659 downregulated genes. Of these genes, 439 were more than twofold upregulated, and 134 were more than twofold repressed. All genes followed a similar trend in time. One day postinoculation differential gene expression was already observed, but the effect was larger on days 3 and 5 postinoculation (Fig. 1). There was no identifiable subset of genes that displayed a different kinetic trend in gene expression.
Fig. 1Presentation of microarray data from mock- and B. pertussis-inoculated mice. Analysis was performed on 1,841 genes whose expression was found to be significant at an FDR < 0.05. a Hierarchical clustering shows the segmentation of the genes clustered by gene expression. Relatively increased gene expression is presented in red, while relatively decreased gene expression is presented in green. The color scale shows log(2) ratios of gene expression levels compared to the global average. b Course of B. pertussis-regulated gene expression; diamonds represent the average of 1,182 upregulated genes, and squares represent the average of 659 downregulated genes. Error bars show the standard deviation (SD) of the average gene expression
To assign functions to differentially expressed genes, we classified them according to GO categories. In addition, we determined enrichment for GO categories to identify pathophysiological processes involved in response to B. pertussis. The most important GO categories, i.e., with the highest percentage differentially expressed genes, are summarized in Table 2. Most upregulated genes are involved in immune response-related processes, such as the acute phase response, antigen presentation, cytokine activity, inflammation, and apoptosis; whereas downregulated genes are involved in nonimmune processes, such as development, muscle contraction, cytoskeleton, and general processes like metabolism.
Table 2Classification of genes induced upon B. pertussis infectiona UpDownbTotal on array P valuecAcute-phase responsed12(55%)0(0%)222.4 × 10−7 (up)Antigen presentation21(66%)0(0%)327.7 × 10−15 (up)Apoptosis47(15%)6(2%)3248.0 × 10−5 (up)Cell cycle43(8%)22(4%)5104.3 × 10−1 (up)Chemokine activity21(58%)0(0%)361.2 × 10−13 (up)Complement activation8(26%)1(3%)319.7 × 10−3 (up)Cytokine activity48(25%)6(3%)1927.8 × 10−13 (up)Cytoskeleton31(6%)40(8%)5312.0 × 10−3 (down)Development96(7%)94(7%)1,4339.8 × 10−1 (down)Immune response162(40%)3(1%)4086.3 × 10−75 (up)Inflammatory response46(38%)2(2%)1211.1 × 10−19 (up)Metabolism353(7%)220(5%)4,7711.8 × 10−1 (down)Muscle contraction0(0%)12(24%)496.7 × 10−6 (down)Listed in any of the above576(9%)318(5%)6,574Other328(6%)204(4%)5,639Unannotated278(3%)137(1%)9,744Total1,182(5%)659(3%)21,957aClassification according to gene-ontology (GO) categoriesbAnalysis was performed on 1,841 regulated genes.cEase score for GO category enrichment in the specified set of genesdSome genes are classified into multiple categories.
To reveal intergene relationships, we performed MetaCore™ analysis on the 1,841 regulated genes in addition to the GO-based analysis presented above. The most significant pathways are presented in Table 3. This analysis revealed the significance of several pathways, such as regulation of granulocyte function, the toll-like receptor (TLR) signaling pathway, the classic complement pathway, and apoptosis.
Table 3Pathway analysis of genes induced upon B. pertussis infectionName of Pathway involvedaMain processP valueNo. of genes in pathwayExpressedTotalTranscription regulation of granulocyte developmentImmune response0.00000192517(38%)45TLR ligands and common TLR signalling pathwayImmune response0.0000181416(34%)47ECM remodelingMetabolism0.0000390618(30%)60Role of IAP proteins in apoptosisApoptosis0.0000545813(36%)36HETE and HPETE diosynthesis and metabolismMetabolism0.000324913(31%)42TPO signaling via JAK-STAT pathwayCell cycle0.00038939(39%)23Leukotriene 4 biosynthesis and metabolismMetabolism0.000465516(27%)60RhoA regulation pathway (extension, GEFs/GAPs)Immune response0.00241210(29%)34MIF in innate immunity responseImmune response0.00243514(25%)57Retinol metabolismMetabolism0.00284212(26%)46Classic complement pathwayComplement activation0.00284212(26%)46Putative erythropoietin signaling pathway (part 1)Immune response0.00290513(25%)52MAPK cascadeCell cycle0.00497512(24%)49Ceramide-dependent NO antiapoptotic actionApoptosis0.00673813(23%)57IL9 signaling pathwayCytokine activity0.0068099(27%)33Ephrins signalingOther0.00784713(22%)58Polyamine metabolismMetabolism0.0087317(30%)23Integrin outside-in signalingCell cycle0.00944416(20%)79Chemokines and adhesionChemokine activity0.00965734(16%)209Analysis was performed on 1,841 genes whose expression was found to be significant at an FDR < 0.05. The list is presented by descending P values. P values represent the probability of particular mapping arising by chance given the numbers of genes in all Metacore pathways, genes involved in the specified pathway, and genes regulated upon B. pertussis inoculation.ECM Extracellular matrix, GAP GTPase-accelerating protein, GEF guanine nucleotide exchange factor, HPETE hydroperoxyeicosatetraenoic acid, HETE hydroxyeicosatetraenoic acid, IAP inhibitor of apoptosis, IL9 interleukin 9, JAK Janus tyrosine kinase, MIF macrophage migration inhibitory factor, MAPK mitogen-activated protein kinase, NO nitric oxide, RhoA ras homolog gene family member A, STAT signal transducers and activators of transcriptionaClassification according to main gene-ontology (GO) categories
Genes located in Bps-1 that were differentially regulated upon B. pertussis inoculation and their function according to the GO database are presented in Table 4. Bps-1 contains 185 genes, of which nine were differentially expressed. The strongest upregulated genes in this locus were tryptophanyl-tRNA synthetase (Wars, fourfold) and tumor necrosis factor alpha-induced protein 2 (Tnfaip2, 5.4-fold). Zinc finger FYVE domain containing 21 (Zfyve21, 1.5-fold) and kinesin family member 26A (Kif26a, 1.7-fold) were the strongest downregulated genes.
Table 4Genes located in Bps-1 that are differentially regulated upon B. pertussis inoculationGenBank accessionGene symbolLocationaFold ratio changeP valueDescriptionPathwaybDay 1Day 3Day 5NM_011705Vrk11061.291.521.410.00003Vaccinia-related kinase 1Protein kinase activityNM_011710Wars1091.953.584.010.00000Tryptophanyl-tRNA synthetaseAminoacyl-tRNA ligase activityNM_010480Hspca111−1.141.401.270.00064Heat shock protein 1, alphaProtein foldingNM_009396Tnfaip21121.434.485.400.00000Tumor necrosis factor, alpha-induced protein 2AngiogenesisXM_138272Gm266112−1.08−1.25−1.350.00110Gene model 266, (NCBI)G-protein coupled receptor activityNM_026752Zfyve21112−1.33−1.41−1.500.00001Zinc finger, FYVE domain containing 21Zinc ion bindingXM_138275Kif26a113−1.32−1.68−1.730.00030Kinesin family member 26AMicrotubule motor activityNM_178911Pld41131.211.822.520.00000Phospholipase D family, member 4Catalytic activityXM_619046LOC4341661131.131.421.420.00141Similar to hypothetical protein MGC37588UnknownaBps-1 is located on chromosome 12; location is presented as distance from centromere (Mb).bClassification according to gene-ontology (GO) categories
Genes located in Bps-2 and -3 that were differentially regulated upon B. pertussis inoculation are presented in Supplementary data Table 6. Bps-2 is located on chromosome 5 and contains 262 genes, of which 13 were differentially expressed. Interleukin-6 (IL-6) was the strongest upregulated gene (4.7-fold) in this locus, and general transcription factor IIIC, polypeptide 2, beta was the strongest downregulated gene (twofold) in this locus. Bps-3 is located on chromosome 11 and contains 922 genes, of which 62 were differentially expressed. Chemokine (C–C motif) ligand 8 was the strongest upregulated gene (15.3-fold) in this locus, and RIKEN cDNA 9530033F24 gene was the strongest downregulated gene (2.3-fold) in this locus.
Discussion
We have recently shown that genetic differences of the host play an important role in the restriction of colonization and/or clearance of B. pertussis from the lungs of mice (Banus et al. 2005, 2006). In the present study, we wanted to extend the understanding of the host response to B. pertussis and identify genes and pathways that play a role in the pathophysiological response to B. pertussis infection. In particular, we were interested in identifying candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. For these purposes, we studied the time course of mRNA-expression in the lungs of mice after a B. pertussis infection by microarray analysis. We were especially interested if genes, located in the Bps-1, 2, and 3 loci, were differentially expressed after B. pertussis infection or that the susceptibility conferred by these loci was regulated alternately.
Of the approximately 22,000 genes investigated, we found 1,841 (8.4%) genes to be significantly differentially expressed. Differential gene expression was already observed on day 1 postinoculation, but the maximum effect was observed on day 3 postinoculation. This effect remained constant till at least day 5 postinoculation. The observed course in gene expression after B. pertussis inoculation was similar to that induced by another gram-negative lung pathogen, Francisella tularensis (Andersson et al. 2006).In a study of expression profiles in the lungs of F. tularensis-infected C57BL/6 mice, 424 (2.3%) genes out of the 18,500 genes investigated were upregulated. Genes involved in immune responses were activated strongly on day 4 postinoculation but hardly or not at all on days 1 and 2 (Andersson et al. 2006).
As expected, most upregulated genes are involved in immune and inflammation-related processes, such as antigen presentation, complement, cytokine, and chemokine activity. Other processes involved include the acute phase response and metabolism. Most downregulated genes are involved in nonimmune processes, such as metabolism, development, cell cycle, or muscle contraction. We speculate that by downregulating these genes, the energy balance is shifted in favor of the immune response. Evidently, cellular influx in the lungs is likely to influence the gene-expression profile.
Our data provide more detailed information on the central role of PMNs in the pathogenesis of B. pertussis infection. Pathogenesis of B. pertussis infection is characterized by colonization and proliferation of the ciliated mucosal cells resulting in damage of the respiratory epithelium and an acute increase in the levels of inflammatory cytokines resulting in cellular infiltrate in the alveolar spaces (Mattoo and Cherry 2005; Mills 2001; Preston 2005). Circulating PMNs are rapidly recruited to the lungs to bind and ingest B. pertussis, subsequently killing the bacteria by a combination of reactive oxygen and granule components. Finally, the PMNs undergo apoptosis (Kobayashi et al. 2005a). TLR ligands such as lipopolysaccharides (LPS) are critical components for the recruitment and priming of PMNs. Serum antibody-mediated clearance of B. pertussis also requires a TLR-induced early recruitment of PMNs. However, pertussis toxin limits this rapid serum antibody-mediated clearance by inhibiting PMN recruitment (Kirimanjeswara et al. 2005). As determined by pathway analysis in this study, the most significant pathways involved in the response to B. pertussis infection are regulation of PMN function, TLR signaling pathway, extracellular matrix remodeling, and apoptosis. It has been described that apoptosis of PMNs, in which TLR2 plays an important role (Jablonska et al. 2006), is accelerated after phagocytosis of bacteria (Kobayashi et al. 2005a, b). Therefore, the involvement of three out of four pathways may, at least partly, be explained by the influx of PMNs. Our data thus further illustrate the central role of PMNs in the course of B. pertussis infection and the involved mechanisms in PMN function.
We found the response to B. pertussis to be wide and complex. Some remarkable findings were the involvement of mucin genes, intracellular pathogen resistance gene, and the excessive upregulation of serum amyloid A (SAA) genes. We found the mucin (Muc) genes Muc4 and Muc5b to be significantly upregulated. Expression of mucin in the respiratory tract is one important innate host defense mechanism that respiratory pathogens must overcome (Belcher et al. 2000). Muc4, which encodes a respiratory tract mucin glycoprotein, was expressed 3.3-fold at day 5 upon B. pertussis inoculation. Muc5b, which encodes mucin 5, subtype B protein, in tracheobronchial epithelial cells, was expressed 1.8-fold 3 days upon inoculation. Belcher et al. (2000) described that B. pertussis induces mucin gene transcription by BEAS-2B cells. Evidently, gene expression studies do not allow to define the functional role of regulated genes. Further studies should define whether the mucin response is favorable to the host, the pathogen, or both.
In our study, we found a threefold upregulation of the intracellular pathogen resistance gene 1 (Ipr1). Ipr1 mediates innate immunity to mycobacterium tuberculosis (Pan et al. 2005). Pan et al. (2005) speculated that the Ipr1 gene product might function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death, and pathogenesis. Our data therefore suggest that Ipr1 may be involved in intracellular presence of B. pertussis. Interestingly, B. pertussis was shown to survive intracellularly in macrophages, PMNs and bronchoalveolar lavage (BAL) cells of mice (Hellwig et al. 1999; Mills 2001).
Among the upregulated genes upon B. pertussis inoculation, SAA genes were upregulated to a remarkably high extent. SAA is an acute phase protein whose expression is markedly upregulated during inflammation and infection (Uhlar and Whitehead 1999). Among other functions, this protein plays an important role in high density lipoprotein cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways (van der Westhuyzen et al. 2005). In our study, SAA1, 2, and 3 were induced 14-, 54-, and 44-fold, respectively, after B. pertussis infection, again underlining the important role for the acute phase response after B. pertussis infection. Although strongly upregulated, it is clear that the acute-phase response is not specific for B. pertussis infection.
In a previous study, we observed a 1.5-fold upregulation of the membrane-receptor Tlr4 and a fivefold upregulation of the downstream cytokine gene Tnf-α 3 days post-B. pertussis inoculation by means of quantitative real-time polymerase chain reaction (PCR). Thus, a slight upregulation (1.5-fold) of this receptor induces a more pronounced (fivefold) induction of this downstream proinflammatory cytokine (Banus et al. 2006). In the present study, we obtained similar results by using microarray analysis. Tlr4 expression was increased 1.6-fold, while the expression of Tnfaip was increased 4.5-fold 3 days postinoculation. The results of the present study hereby confirm our previous observations. In a recent study, a high correlation between gene-expression values determined by quantitative real-time PCR and microarray was observed (Canales et al. 2006). Given these results and our observations, we conclude that the results, at least for the two given genes, are similar for both techniques.
It is very likely that the expression profile to B. pertussis is partly unique to B. pertussis and partly nonspecific. To examine the specificity of B. pertussis-regulated gene expression, we compared our data to other studies. In a recent review by Jenner and Young (2005), a comparison was made between published transcriptional profiling data from 32 studies that involved 77 different host–pathogen interactions. A set of 417 genes that comprise a common host transcriptional response was defined. We found that 122 genes affected by B. pertussis infection were listed in this common host–pathogen response, further illustrating that many genes regulated by B. pertussis are indeed not specific for B. pertussis infection but are involved in a common response to pathogens. One explanation for the broad response in expression profiles is the complexity of the bacterium. It is, for instance, known that LPS is a very potent immune stimulus, which induces a strong inflammatory response (Kawai et al. 2001; Qin et al. 2005; Wells et al. 2003). Transcriptional profiling of LPS-induced acute lung injury in mice resulted in 71 immune-related upregulated genes (Jeyaseelan et al. 2004). Of these 71 genes, 43 were also regulated upon B. pertussis inoculation in this study.
Previously, we have demonstrated that the Bps-1 locus has a dominant positive effect on the clearance of B. pertussis from the lung. Of the 185 genes in this locus, nine were differentially expressed. Some of these genes could explain the linkage of this locus with susceptibility to B. pertussis infection. Tumor necrosis factor, alpha-induced protein 2 (Tnfaip2), has been described as a tumor necrosis factor-alpha-inducible primary response gene in endothelial cells (Sarma et al. 1992). Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA with their cognate amino acid. Tryptophanyl-tRNA synthetase (Wars) catalyzes the aminoacylation of tRNA(trp) with tryptophan and is induced by interferon (Ibba and Soll 2000).The role of the nine genes in the B. pertussis infection process is unknown and will be investigated in future experiments. Thus, by using a microarray approach, we selected nine possible candidate genes from the 185 present in Bps-1. However, by this approach, we focused on transcriptionally regulated genes, and the possibility remains that susceptibility to B. pertussis is regulated in a different way, such as phosphorylation or protein binding.
In comparison to the Bps-1 region, the Bps-2 and -3 regions contain considerably more genes, namely, 262 and 922 genes, respectively, of which 13 and 62 were differentially expressed post-B. pertussis inoculation. Among these differentially expressed genes are several interesting candidate genes that may explain the linkage of these loci with susceptibility to B. pertussis infection. IL-6 was the most strongly induced gene in the Bps-2 region (4.7-fold). IL-6 is a proinflammatory cytokine that can be secreted by macrophages after Tlr4 activation (Akira and Takeda 2004). Among the genes differentially expressed in the Bps-3 region was a large group of chemokines, of which Chemokine (C–C motif) ligand 8 was the strongest upregulated gene (15.3-fold). Upon B. pertussis infection, this large group of chemokines is induced to promote immune cells to migrate to the site of infection. The role of the other B. pertussis-induced genes in these loci is still unknown and will be investigated in future experiments.
In conclusion, we found 1,841 genes in the lungs of mice that are differentially expressed after B. pertussis inoculation. Most upregulated genes are involved in immune and inflammation-related processes or in nonspecific processes. Most downregulated genes are involved in nonimmune processes. A substantial number of genes and pathways provide more detailed information on the central role of PMNs in the pathogenesis of B. pertussis. In particular, the transcriptional profiles indicate the significance of genes involved in TLR activation, the recruitment and activation of PMNs, and apoptosis.
Electronic supplementary material
Raw data as well as the detailed description of the experiment will be uploaded to the freely accessible online database ArrayExpress (
http://www.ebi.ac.uk/arrayexpress/
) upon acceptance of the manuscript. Expression levels of all 1,841 genes as well as comparisons to other studies are presented in the supplementary data Table
5
.
Supplementary data Fig. 2
Gain of body weight after infection. Relative gain of body weight at different days postinoculation with medium (mock) or B. pertussis. Boxes represent the mean of the relative weights of B. pertussis infected mice; diamonds represent the mean of the relative weights of mock inoculated mice (GIF 10 kb).
High resolution image file (EPS 493 kb)
Supplementary data Fig. 3
Lung weight after infection. Relative lung weight of mice after infection with B. pertussis at several days postinoculation. Solid bars represent the mean relative weight of B. pertussis infected mice; grey bars represent the mean relative lung weight of mock inoculated mice (GIF 13 kb).
High resolution image file (EPS 680 kb).
Supplementary data Table 5
All B. pertussis induced genes and overlapped with other studies. a1,841 genes induced upon B. pertussis inoculation whose expression was found to be significant at an FDR < 0.05, of which 1,395 had an FR higher then 1.5; bJenner and Young (2005); cPalmer et al. (2006) BMC Genomics; dB cells and following columns are subsets described by Palmer et al. (2006) (XLS 665 kb)
Supplementary data Table 6
Genes located in Bps-1, -2, and -3 that are differentially regulated upon B. pertussis inoculation (XLS 25 kb)

bordetella pertussis
expression profiles
immunity
pathway analysis
gene expression


Functional analysis of a novel potassium channel (KCNA1) mutation in hereditary myokymia

Myokymia is characterized by spontaneous, involuntary muscle fiber group contraction visible as vermiform movement of the overlying skin. Myokymia with episodic ataxia is a rare, autosomal dominant trait caused by mutations in KCNA1, encoding a voltage-gated potassium channel. In the present study, we report a family with four members affected with myokymia. Additional clinical features included motor delay initially diagnosed as cerebral palsy, worsening with febrile illness, persistent extensor plantar reflex, and absence of epilepsy or episodic ataxia. Mutation analysis revealed a novel c.676C>A substitution in the potassium channel gene KCNA1, resulting in a T226K nonconservative missense mutation in the Kv1.1 subunit in all affected individuals. Electrophysiological studies of the mutant channel expressed in Xenopus oocytes indicated a loss of function. Co-expression of WT and mutant cRNAs significantly reduced whole-oocyte current compared to expression of WT Kv1.1 alone.
Introduction
Myokymia is defined as spontaneous involuntary muscle fiber group contraction, is visible as vermiform movement of the overlying skin, and may be hereditary or acquired. Electromyography (EMG) shows continuous motor unit activity with spontaneous discharges of high intraburst frequency. Myokymia occurs in autoimmune disease with antibodies to voltage-gated potassium channel subunits in Isaacs–Merton syndrome and Morvan fibrillary chorea [1]. Hereditary myokymia with episodic ataxia type 1 (OMIM #160120) arises from mutations in the voltage-gated potassium channel KCNA1 [2]. The ataxia is characterized by profound attacks of cerebellar ataxia lasting seconds to minutes. Partial epilepsy has been reported in some families with KCNA1-associated disease [3, 4]. Another rare cause of myokymia with neonatal epilepsy but lacking episodic ataxia results from a KCNQ2 potassium channel mutation [5]. Reduced frequency and severity of myokymia and episodic ataxia have been reported after treatment with acetazolamide or carbamazepine [3, 6].
Approximately a dozen missense mutations and one truncating mutation in KCNA1 have been reported [3, 4, 7–10]. The functional consequences of human KCNA1 mutations causing typical myokymia and EA1 have been reported [11], much less is known about the functional consequences of mutations causing unusual KCNA1-associated phenotypes.
In the present study, we describe the clinical and genetic features of a new family with hereditary myokymia lacking epilepsy or episodic ataxia. Molecular studies of KCNA1 in the proband revealed a c.676C>A transversion resulting in the substitution of Lys for Thr at codon 226 (T226K) and a c.1355A>C transversion resulting in Y452S. Further genetic studies showed that all affected family members carried the T226K mutation, which was absent in unaffected family members and ethnically matched normal controls, whereas the Y452S substitution was present in both affected and unaffected family members and normal controls. In transfected mammalian cells, electrophysiologic studies demonstrated functional effects of the c.676C>A transversion with no currents above background levels recorded from oocytes injected with the mutant subunit. Co-injection of the wild-type and mutant cRNAs produced significantly smaller whole-oocyte currents than injection with the WT cRNA alone.
Materials and methods
Clinical data
The family pedigree is shown in Fig. 1. Six family members (two unaffected and four affected) were available for clinical and genetic studies approved by the Yale Human Investigations Committee.
Fig. 1Family pedigree. Squares indicate males, circles indicate females. Blackened symbols denote individuals with myokymia. Alleles at codon 226 are indicated (Thr = wild-type, Lys = mutation)
Subject II-1
The proband, a 13-year-old boy (II-1), was the product of an unremarkable pregnancy and delivery. He was diagnosed with cerebral palsy because of leg stiffness and delayed walking at 18 months old. Cognitive development was normal; his medical history was significant for esotropia. At 4 years old, he was hospitalized with increasing leg pain, stiffness, and inability to walk during the course of a flu-like illness. Creatine kinase (CK) was elevated at 520 U/l acutely (normal <195 U/l), but was normal when repeated interictally. Examination showed periorbital myokymia, mild abdominal and leg muscle hypertrophy, leg stiffness, spastic gait, hyperreflexia, and bilateral Babinski sign. Magnetic resonance imaging (MRI) of the brain and spine were normal. Metabolic studies including electrolytes, amino and organic acids, carnitine, ammonia, lactate, and thyroid functions were normal. Routine studies of cerebral spinal fluid and an electroencephalogram were normal. Needle EMG of deltoid and iliopsoas muscles showed irregular, polyphasic continuous motor unit discharges with normal interference pattern.
Additional family members
Six family members were evaluated. Four affected members (mother and three sons) had myokymia on clinical examination, EMG examination, or both. Nerve conduction velocities were normal. There was no history of seizures or episodic ataxia in any of them. The mother (I-2, Fig. 1) is a 40-year-old woman who had had muscle twitching since childhood, hyperreflexia, bilateral Babinski sign, and tendency to run on her toes. Her husband is unaffected and had a normal neurological exam. A 12-year-old son (II-2) also had delayed walking, leg stiffness, and was diagnosed with cerebral palsy. At age 3 years, he had an episode of prolonged generalized paralysis with no muscle response to tetanic stimulation after general anesthesia for esotropia surgery. He recovered within 1 day. MRI of the brain and spine were normal. An 8-year-old daughter (II-3) is unaffected. A 5-year-old son (II-4) had symptoms identical to his brothers and was hospitalized twice for episodes of increased muscle stiffness with presumed viral gastroenteritis, elevated CK during both episodes (606 U/l and 997 U/l) but had normal CKs measured while well. Carbamazepine treatment resulted in marked improvement of muscle symptoms in three of the brothers who remain asymptomatic. The mother, with milder symptoms, declined treatment. Three family members with myokymia had esotropia; one patient required three eye surgeries.
Molecular studies
Genomic DNA was extracted from blood samples or buccal swabs. The coding region of the single-exon 1,448-bp gene KCNA1 (GenBank sequence NM_000217) was amplified. Polymerase chain reactions (PCRs) were performed in a 25-μl reaction volume containing 50 ng genomic DNA, 50 ng of each primer, 1.5 mM MgCl2, 200 μM dNTPs, 1× PCR buffer (Finnzyme), and 2.5 U of Finnzyme using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). Cycling parameters consisted of a modified touchdown (stepdown) protocol as follows: an initial hot start denaturation at 94°C for 5 min (hot start); 94°C for 45 s, annealing temperature (three cycles each at 70, 67, 64, 61,and 58°C) for 45 s, and extension at 72°C for 2 min for a total of 15 cycles followed by 25 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min; a final extension step of 72°C for 10 min followed by a 4°C hold. PCR products were separated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. PCR products were purified by spin column (Qiagen, Valencia, CA) or by enzymatic method (ExoSAP-IT, USB, Cleveland, OH) and analyzed for mutations by automated sequencing (Applied Biosystems, Foster City, CA). The c.1355A>C transversion resulted in the creation of a DdeI restriction site, so the presence of the sequence variant was tested by sequencing and confirmed with restriction digest in all subjects. Restriction digests were performed with DdeI (New England Biolabs, Beverly, MA) at 37°C for 2 h.
Oocyte expression and electrophysiological studies
Human Kv1.1 cDNA was subcloned into Kpn1 and HindIII sites of psGEM. The Kv1.1-T226K mutation was introduced by site-directed mutagenesis and confirmed by automated sequencing. cRNAs were synthesized using an mMessage mMachine kit (Ambion, Austin, TX) and quantified by spectroscopy.
Oocytes were isolated from Xenopus laevis and defolliculated by collagenase treatment. Each oocyte was injected with either 5 ng of human Kv1.1-WT, 5 ng Kv1.1-T226K, or 2.5 ng WT plus 2.5 ng T226K cRNA. Whole-oocyte currents were measured with two electrode voltage clamp techniques (Oocyte Clamp, Warner Instruments, Hamden, CT) with constant perfusion (1 ml/min, solution exchange\3 s) after 16 h. Data were sampled at 1 kHz and filtered at 0.25 kHz. Standard bath solution was ND-96 (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES/NaOH, pH 7.5.
Results
Mutation analysis of KCNA1
Mutation analysis of KCNA1 identified two sequence variants: a c.676C>A transversion resulting in the substitution of Lys for Thr at codon 226 (T226K) and a c.1355A>C transversion resulting in Y452S and creating a DdeI restriction site. Either a Tyr (NM_000217) or a Ser (AAI01734) had been reported at codon 452, but the population frequency and association with disease were unknown. Therefore, we tested all six family members (four affected, two unaffected) and 10 normal controls for the SNP and found that all 16 subjects were homozygous for the Y452S substitution, compatible with a common polymorphism. In contrast, all (4/4) affected subjects were heterozygous for the previously unreported T226K missense mutation, which was absent in both unaffected family members and absent in 190 chromosomes from ethnically matched normal controls (Fig. 2).
Fig. 2Sequence chromatograms demonstrating c.676C>A resulting in T226K substitution in KCNA1. A DNA sequence from an affected patient (I-2) heterozygous for the mutation (top) and from a normal control (bottom) are presented
Electrophysiological studies of the human Kv1.1 mutant The genetic studies of the segregation of the T226K mutation favored it as a pathological mutation, but given that two missense mutations were identified, we sought additional supporting evidence. We tested whether the missense mutation alters human Kv1.1 channel function by expressing Kv1.1-T226K in Xenopus oocytes. No currents above background levels were recorded from Kv1.1-T226K cRNA-injected oocytes, whereas Kv1.1 wild-type channels produced typical delayed rectifier currents (Fig. 3). We then mimicked the heterozygous situation by co-injecting equal amounts of Kv1.1 WT and T226K mutant cRNAs. Co-injection produced significantly smaller whole-oocyte currents than injection with Kv1.1 WT cRNA alone (Fig. 3c and d). These results indicate that the T226K mutation significantly reduced efflux of potassium ions during depolarization and is therefore likely to result in increased muscle cell excitability.
Fig. 3Missense mutation T226K induces loss of function of human Kv1.1 channels expressed in Xenopus oocytes. a–c Representative family of whole-oocyte currents recorded from oocytes expressing human Kv1.1-WT (a), Kv1.1-T226K (b), and both Kv1.1 WT and T226K (c). Currents were evoked by a series of 350-ms test pulses from −80 to +60 mV in 10-mV increments from a hold potential of −90 mV. Tail currents were recoded at −40 mV. Scale bar, 100 ms and 1 μA. d I–V curves for three experimental conditions in a–c. Data were collected for 10–15 oocytes in each group. The peak currents in a and c are significantly different at test voltages between 0 and +60 mV (P < 0.05)
Discussion
Mutations in KCNA1 are typically associated with episodic ataxia type 1 (EA1), characterized by profound cerebellar ataxia lasting seconds to minutes and interictal myokymia [6]. The expanded phenotypic spectrum associated with proven KCNA1 mutation includes a family with prolonged episodes of episodic ataxia and epilepsy, but without myokymia [12]. Other families have been reported with myokymia and adducted thumbs, congenital contractures, kyphoscoliosis, or epilepsy with myokymia [4, 7–10]. We report a family with autosomal dominant myokymia lacking either ataxia or epilepsy. The mother had self-described lifelong “muscle twitchiness” but never sought medical attention for her condition until the hospitalizations of her sons. Myokymia in the periorbital region and on EMG were the features that led to genetic studies of KCNA1.
Although the stiffness, toe walking, and esotropia could be caused by continuous muscle contraction, the extensor plantar reflex [observed on multiple exams by experienced two neurologists (LRM and FMH)] is a new finding for KCNA1-associated disease and suggests involvement of the corticospinal tract. The KCNA1 knockout mouse has been reported to exhibit altered inhibition of layer V pyramidal neocortical neurons [13].
KCNA1 on chromosome 12p13 encodes Kv1.1, a potassium channel subunit. Delayed-rectifier voltage-gated-type potassium channels are important in the rapid neuronal repolarization after an action potential and are multimeric proteins containing four subunits around a central pore. The Kv1.1 potassium channel subunit is expressed during development in neuronal and glial cells in the central and peripheral nervous systems [14]. Kv1.1 plays an important role in regulating neuronal excitability in the central nervous system, as shown by studies of the KCNA1 knockout mouse with an increased susceptibility to seizures [15].
The present family has generalized myokymia due to a T226K mutation in the voltage-gated potassium channel KCNA1. Unusual clinical features include pyramidal dysfunction and worsening with febrile illness or anaesthesia. One previously reported 3-year-old boy presented in a similar fashion to our patients with a 12-h episode of inability to walk, increased tone, and elevated CK during a febrile illness [3]. No anesthesia exposure was reported. That child had a P244H mutation in the intracellular loop between transmembrane segments 2 and 3. The whole-cell current amplitudes elicited by injection of P244H did not differ from wild-type amplitudes. Co-injection of P244H plus an equal amount of wild-type cRNA yielded a peak current amplitude that was 200% of wild-type alone.
We identified a novel, nonconservative T226K missense mutation that is located in the second transmembrane domain of Kv1.1 and is invariant from Drosophila melanogaster to mice and humans [6]. In contrast to the functional effects reported for the P244H mutation, heterologous expression of the T226K mutation identified in the present family showed that the mutant potassium channel expressed in oocytes demonstrated no current above background levels, and its co-expression with the wild-type channel significantly reduced whole-oocyte current, compatible with a severe effect of the mutation. The T226K mutation is likely to reduce efflux of potassium ions during depolarization, thereby increasing cellular excitability. The phenotypic similarity between the P244H and the T226K mutations contrasts with their biophysical effects. Therefore, additional factors must contribute to the functional effects of these mutations. Kv1.1 forms heteromeric channels with other subunits and interacts with other accessory subunits [16]. In addition to their well-known role as ion channels that regulate neuronal firing, potassium channels have also been shown to activate enzymes linked to intracellular signaling pathways [16, 17]. One of these novel interactions may contribute to differences in the manifestation of disease-causing mutations.


Bench-to-bedside review: Ethical challenges for those in directing roles in critical care units

Though much attention in the medical literature has focused on the ethics of critical care, it seems to be disproportionately weighted toward clinical issues. On the presumption that the operational management of an intensive care unit (ICU) also requires ethical considerations, it would be useful to know what these are. This review undertook to identify what literature exists with regard to the non-clinical issues of ethical importance in the ICU as encountered by clinician–managers. We found that in addition to issues of resource allocation, there exist many areas of ethical importance to clinician–managers in the ICU that have been described only superficially. We argue that a renewed focus on ICU ethics is merited to shed light on these other, non-clinical, issues.
Introduction
As a specialized field of philosophy, ethics has demanded that more institutions self-assess their actions so as to implement and maintain ethical practice (see the Additional file for definitions of 'ethical practice' and 'ethics'). In healthcare, technological and bureaucratic complexities have created dilemmas never before encountered, at least on the scale in which they now occur. Nowhere are these two issues, a push to self-analyze critically and an increase in novel dilemmas, more present than in the intensive care unit (ICU). The ICU is a place both where patients are exposed to modern advances in health technology and where some of the most challenging questions for bioethicists occur. Because of the pervasiveness of ethical considerations, it is logical to assume that the ethics knowledge base would be well documented in the ICU. In fact, a cursory search of the phrase 'critical care ethics' in PubMed between 1966 and 2004 cited an impressive 1090 articles. Because a more focused search on 'end of life ethics' returned 986 articles, it seems that much of the published literature has a particular focus.
The assumption that end-of-life issues represent the only ethical issue in the ICU was challenged by DeVita and colleagues' review [1] of all of the ethics manuscripts published in Critical Care Medicine. Although they identified a spectrum of ICU ethical issues in addition to end-of-life care such as futility, research, resource distribution, informed consent, and resuscitation, three issues are apparent to us. First, the number of manuscripts that they classified as 'end-of-life' far exceeded all other ethical topics (45 more than the second most common topic). Second, most of the additional ethical topics identified are issues that occur in the context of patient–physician or patient–nurse interactions. Third, on the presumption that the process of managing an ICU requires difficult moral decisions to be made, there seems to be a gap in the published literature with regard to the process of non-clinical decisions of ethical importance in the ICU.
ICU directors and nurse managers are required to make difficult decisions with respect to protocols, staffing, and administration of the ICU. Arguably, these have equal, maybe even greater, ethical importance than decisions made at the bedside, because management decisions can affect multiple patients, in a less direct, and transparent, manner. Perhaps the bedside can be described as a simpler ethical environment in that it involves fewer external factors and agents to consider, and the consequences of actions are immediately apparent to all.
If the dual roles that ICU physician directors and nurse managers occupy create unique ethical challenges that cannot be adequately captured by either the traditional principalism of medical bioethics because bioethics does not take into account the fundamentals of business, or the existing models of business ethics because these fail to account for the values of medicine, it is possible they should be addressed as a separate entity. The aim of this paper is to review briefly what ethical issues faced by ICU 'clinician–managers' have been described, and to understand the context in which they are addressed.
Method
To identify publications that focused on ethical issues faced by professionals who occupy both clinical and administrative roles in ICUs, abstract and title searches were performed in Medline/PubMed and CINAHL databases using combinations of the following keywords: Ethics, Clinician-Manager, Critical Care, Intensive Care Unit (ICU), Management, Leadership, Decision-making, Roles, Administration, Medical Directors, and Policy. Our search included primary literature, review, and opinion articles and the inclusion criteria were: 1966 to July 2004, English language, mention of critical or intensive care, direct mention of ethics OR discussed an ethical issue. An article was considered to have discussed an ethical issue if there was recognition of uncertainty about the correct choice of action in a given situation. Thus, any article that asserted one practice to be superior to another, whether anecdotally or as demonstrated by some research, was not considered to recognize an ethical concern. Articles were excluded if managed care was the source of the ethical dilemma and/or the ethical issue was strictly clinical in nature, not including the use of treatment policies because clinician–managers were considered to have a special interest in policy; that is, any ICU physician dealing with the issue was qualified to decide on the appropriate course of action.
In addition, references from captured articles were searched to identify additional literature that might not have been captured in the initial search. We also hand-searched the following journals: Bioethics (1997 to present), Critical Care, Critical Care Clinics, Critical Care Medicine (1985 to present), American Journal of Respiratory and Critical Care Medicine (1994 to present), and Intensive Care Medicine (1993 to present). Finally, the following key authors, identified by their previous ethics-related literature, were contacted to identify any articles that our search did not capture: Martin Strosberg, Kurt Darr, Dr G Rubenfeld, Dr C Sprung, and Dr J Luce.
Results
Of the roughly 1500 articles identified in our search, only 55 met the screening criteria that identified them as concerning an ethical challenge for clinician–managers in critical care. A distribution across time of these articles can be seen in Fig. 1. After an initial review of all the articles that met the screening criteria, broad categories were arbitrarily chosen to sort the results as follows: resource allocation; organizational ethics (namely, how intensive care ought to be organized); policies and protocols (formulation and implementation); professional roles (namely, what the role of 'directors' should be); ethics and law; general ICU ethics (ICU ethics in the broadest sense); and other (Table 1). PubMed categorized 23 (42%) of the 55 articles that met all screening criteria as either review articles or editorials; these review articles and editorials were evenly distributed between our categories.
Articles classified as 'resource allocation' papers mostly included discussions on which principles (such as justice, reasonableness as fairness) ought to apply when distributing scarce resources in the ICU. Some articles were more detailed, and described dilemmas such as age-based rationing [2] while still framing the question in terms of specific ethical principles and how they each affect the decision. One article sought to use cases to identify the ICU physicians' ethical role in distributing scarce resources [3].
By focusing attention on the physicians' role and not the ethical principles themselves, a whole new series of questions arose. It is noteworthy that the American Thoracic Society addresses the issue in a consensus statement outlining ethical guidelines for fair resource allocation [4], and the American College of Chest Physicians and the Society of Critical Care Medicine similarly discuss the ethics of resource allocation in their moral guidelines regarding the withdrawal of intensive care [5]. Articles classified as 'organizational' had a scope that ranged from what makes an ICU safe, to what should be taught to students in the ICU, to the ethical issues surrounding the use of restraints. Nelson describes an organization issue by illustrating how ethics is an everyday concern, with regard to issues like collaboration, staff conflict, and moral burnout [6]. This view is in contrast to the assumption that bioethics matters only when there is a specific dilemma. Additional articles on the use of restraints were classified as 'protocols and policies' because their focus was primarily on the policies themselves. Also in 'protocols and policies' was a discussion on the value of having a family presence protocol for life-saving procedures [7]; that is, is allowing families to witness resuscitation the right thing to do? The two articles under the heading 'professional roles' discussed the changing roles of ICU physicians, especially with regard to a transition to managerial duties; this issue is discussed below. 'General ethics' articles included all papers that discussed ICU ethics in the broadest sense with less attention to the details; many of these articles were brief reviews of the numerous ethical considerations in an ICU.
Discussion
Ethical issues are usually expressed as a conflict of ideas, values, and/or norms that are often role dependent. It should therefore be expected that ICU physician directors and nurse managers, who have both clinical and non-clinical duties, should face some of the more difficult moral conflicts in the ICU. In one respect, these professionals follow a patient-centered code of conduct, either the Hippocratic oath or the Nursing Professional Code, which in part defines them. At the same time, they are also agents of the hospital as 'a business', and implicitly society as a whole. Although some clinicians completely relinquish their clinical duties on transition to management, most do not; the professional nature of medicine therefore gives the clinician a patient-centered outlook that is not as easily set aside: once a doctor or nurse, always a doctor or nurse. In summary, the physician director and nurse manager will always be in the unique position of having two separate professional standpoints from which to assess situations, which can therefore lead to unique ethical challenges.
We have begun to characterize the scope and uniqueness of ethical issues that are raised by the dual roles of clinician–managers in the ICU. In reviewing the health literature, we found that almost half of the articles identified that discussed ethical concerns for ICU clinician–managers were concerned with resource allocation. This is probably not surprising given that ICU clinicians are increasingly adopting the role of economic rationalist [8]. Perhaps the real surprise of these results is how many articles concerned issues other than resource allocation. Although DeVita and colleagues pointed out that 'end-of-life' was not the only ethical concern in the ICU [1], which they took to be a common assumption, we argue that there is more to the ethics of directing roles in the ICU than issues of resource allocation.
In this initial survey of the ethical issues experienced by those in dual management–clinician roles in the ICU, important to our conclusion was the development of a categorization scheme. In the absence of any unique approach, we were arbitrary in our definition of categories. Although some of the articles we identified could have been placed in more than one category, or in categories not used, we believe our approach to be valid for the modest purposes of this survey. Additionally, some articles that discussed issues of clinical ethics in the ICU might have contained less prominent opinions or notes relevant to clinician–managers and therefore might have been missed by our search. In spite of these potential issues, the articles selected were distributed to give both a clear and defined picture of what currently exists with regard to ICU ethics for those in both clinical and directing roles at the same time. We also believe it is likely that there were many articles that discussed ethical issues relevant to our review, yet failed to recognize the issues as being 'ethical' in nature. For example, many articles described the nuances of resource allocation in the ICU (see the Introduction), yet neither mentioned ethics or recognized any uncertainty with regard to the 'right' thing to do. The fact that many articles fail to address their ethical components might indicate a lack of awareness of what constitutes an ethical dilemma, but even if this is not so, the goal of better recognition and acknowledgement of the ethical issues that suffuse the operational management of the ICU is desirable.
Resource allocation is a well-defined topic of ethical interest that has stimulated much discussion. However, it is important not to perceive resource allocation as the beginning and end of the ethics discussion for clinician–managers in critical care. Perhaps it is also time to move beyond the commentary on resource allocation and devote more research initiatives toward this topic (for example by studying the different approaches to resource allocation).
The term 'organizational ethics' is used to denote how a business or institution ought to be organized in any number of ways, including management functions, working environments, and its infrastructure. It should not come as a surprise that organizational ethics should constitute a concern for either an ICU director or a nurse manager, yet over the past 20 years only a handful of articles have been written about the organizational ethics of intensive care and have recognized them as such. Although policies and protocols for an ICU could also fall under the heading of 'organizational ethics', we believe that determining and implementing policies might require ethical concerns that merit special attention. The use of any policy that deals with either patients or staff is to apply one rule to many different people, and necessarily ignores factors that make individual cases unique. Because policies tend to generalize in this way, they create unique ethical challenges. Little reflection is required to determine that both of these issues, organizational ethics and the ethics of policy, constitute ethical concerns for directors in the ICU in which further study is merited.
The role of the ICU physician director, or nurse director/ manager, is an ethical issue itself. Although two articles were identified that addressed this issue, we believe that the paucity of articles found indicate a need for greater consciousness of the ethical factors that influence and are influenced by clinical leaders in the dual roles of 'clinician' and 'manager'. Although healthcare leaders are familiar with the importance of ethics, they may be unaccustomed to thinking of their own role in terms of ethics [9]. Because the ICU director, or nurse manager, may engage in the decision making process from multiple professional standpoints (as both clinician and manager), the likelihood of conflicting rational and justifiable solutions, leading to ethical dilemmas, increases. The clinician–manager role, then, may require a higher level of ethical proficiency, or perhaps expertise.
Conclusion
In summary, we believe it is important that future study be directed toward understanding ethical issues surrounding the dual roles of clinician–managers in the ICU. Although it has been acknowledged that hospitals should pay as much attention to managerial ethics as to clinical ethics [10], it is not yet clear that this in fact occurs. One step in a research agenda would be to undertake a survey to determine if the published literature, identified by this review, is in fact an accurate representation of what is experienced. Understanding the scope of ethical issues experienced by clinician–managers in the ICU will foster a more complete dialogue. Further, although there are studies that describe the process of ethical reasoning in nurses and other clinicians [11-13], similar studies have not been performed with clinician–directors, be they nurses or physicians. These investigative steps toward a better understanding of the issues, and how they are understood and dealt with, ought to include direct input from ICU clinician–managers. Input from ICU clinician–managers with a background in, or special knowledge of, ethics would be particularly beneficial, because they would possess the language to describe what most feel by intuition. This experiential knowledge would best be conveyed in an open-interview or narrative format in which actual dilemmas can be discussed. Finally, the resulting data will require detailed qualitative analysis and might benefit from the support of a medical sociologist.
For all critical care leaders, there is now an opportunity to promote a better understanding of the complexity of the ICU environment and to prompt further learning.
Abbreviations
ICU, intensive care unit.
Competing interests
The author(s) declare that they have no competing interests.
Supplementary Material
Additional File 1
A complete list of categorized references for all articles captured in this review can be found here.
click here for file

ethics
critical care
management
clinician–manager