Although the pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood, the identification of novel ALI biomarkers holds promise for unique insights. Expression profiling in animal models of ALI (canine and murine) and human ALI detected significant expression of pre–B-cell colony-enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. These results were validated by real-time polymerase chain reaction and immunohistochemistry studies, with PBEF protein levels significantly increased in both bronchoalveolar lavage fluid and serum of ALI models and in cytokine- or cyclic stretch–activated lung microvascular endothelium. We genotyped two PBEF single-nucleotide polymorphisms (SNPs) in a well characterized sample of white patients with sepsis-associated ALI, patients with severe sepsis, and healthy subjects and observed that carriers of the haplotype GC from SNPs T-1001G and C-1543T had a 7.7-fold higher risk of ALI (95% confidence interval 3.01–19.75, p < 0.001). The T variant from the SNP C-1543T resulted in a significant decrease in the transcription rate (1.8-fold; p < 0.01) by the reporter gene assay. Together, these results strongly indicate that PBEF is a potential novel biomarker in ALI and demonstrate the successful application of robust genomic technologies in the identification of candidate genes in complex lung disease.
Acute lung injury (ALI) is a refractory lung disease characterized by severe hypoxemia and unacceptably high mortality (30–50%) (1–3). The pathogenetic basis of ALI is incompletely understood; however, ALI survival appears to be influenced by the stress generated by mechanical ventilation (4) and by sepsis-associated factors, which initiate and amplify the inflammatory response in ALI (5). Emerging evidence also suggests that genetic factors are associated with susceptibility to ALI (6). Both a nonsynonymous single nucleotide polymorphism (SNP) in the surfactant protein B gene (7) and an intronic SNP in the angiotensin-converting enzyme gene (8) were reported to contribute to the susceptibility and outcome of patients with ALI. Associations with the susceptibility and mortality of septic shock, a frequent inciting cause of ALI, have been demonstrated with promoter polymorphisms in the tumor necrosis factor-α (TNF-α) (9) and CD14 genes (10). Additional studies are clearly needed to identify novel biochemical and genetic markers that may provide unique insights into pathogenic mechanisms and the genetic basis of ALI (11).
We used a high-throughput functional genomic approach, with extensive microarray-based lung gene expression profiling in canine, murine, and human ALI, to identify novel ALI candidate genes. In these studies, we observed significant increases in the expression of pre–B-cell colony-enhancing factor (PBEF), a relatively obscure cytokine with only 22 PubMed citations to date. PBEF was named after its effect on the maturation of B-cell precursors (12). Its expression in a human amniotic epithelial cell line in vitro was upregulated by the treatment of either mechanical force (13) or inflammatory cytokines (14). Recently, Jia and colleagues (15) reported that PBEF was significantly expressed in peripheral neutrophils of patients with sepsis, a frequent cause of ALI, and PBEF inhibited the neutrophil apoptosis. Despite these observations in nonlung tissues, the relevance of PBEF to the lung pathophysiology is unknown. The present study reports the first findings of PBEF expressed in lung tissues and overexpressed in ALI and the association of the PBEF with susceptibility to ALI. We postulated that PBEF might be a dual sensor to both the mechanical force and inflammatory stimuli to be involved in the pathogenesis of ALI. Our results from animal models and human patients with ALI and in vitro cell culture experiments strongly indicate that PBEF is a potential novel ALI biomarker and confirm the utility of genomic approaches to generate important insights into complex lung disease. A portion of the relevant results of these studies has been previously reported in abstract forms (16, 17).
All animal models were institutionally approved. Canine Model 1 used unilateral saline lavage–induced lung injury (18), with the injured left and uninjured right lungs independently mechanically ventilated for 6 hours (8 ml/kg, 0 positive end-expiratory pressure, and 10 ml/kg and positive end-expiratory pressure 5 cm H2O, respectively). Canine Model 2 used intrabronchially delivered endotoxin (LPS) with high Vt mechanical ventilation (6 hours, 17 ml/kg), as reported (19). Control animals received endobronchial saline with identical ventilation strategies. Lung tissues were processed for microarray analysis, and bronchoalveolar lavage (BAL)/serum was collected for protein analyses.
Two murine ALI models were described: (1) 24-hour spontaneous ventilation postintratracheal LPS and (2) 2-hour 17-ml/kg mechanical ventilation (19, 20). Control groups were spontaneously ventilated. Lung tissue and BAL were collected for microarray and protein analyses.
Human protocols were approved by the institutional review boards. Human BAL (n = 3 each) was obtained from patients with ALI (21, 22) and healthy control subjects.
The Affymetrix GeneChip (Affymetrix, Inc., Santa Clara, CA) microarray system was used as described previously (23). Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot, and real-time PCR were used to validate PBEF expression in animal lung tissues and human BAL, respectively.
To evaluate the spatial localization of PBEF expression, we performed triple immunohistochemical staining in canine lung tissue (24, 25) using an anticanine PBEF polyclonal antibody (26) and antisera raised against factor VIII (to visualize vascular endothelium), neutrophils, and pro–surfactant protein C (alveolar epithelium). In addition, 4′6 diamidino-2-phenylinodole was used to visualize cell nuclei.
The total protein content in each sample was quantified using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). PBEF proteins were assessed by Western blotting with densitometric quantification.
Human lung microvascular endothelial cells (HMVEC-L) were exposed to cytokines for 4 hours or 18% cyclic stretch or static conditions for 48 hours plus or minus interleukin 1β (IL-1β) as previously described (27). PBEF content in cell lysates was analyzed as previously described.
Leukocyte DNA and serum samples (n = 8 each) from subjects with sepsis-associated ALI, subjects with sepsis alone, and healthy control subjects were obtained (Johns Hopkins University, Medical College of Wisconsin) according to consensus diagnostic criteria (21, 22) with recording of Acute Physiology and Chronic Health Evaluation (APACHE) II scores (28). SNP discovery of the human PBEF gene was performed in 36 subjects (12/group) by direct DNA sequencing. Genotyping of the PBEF SNPs (T-1001G and C-1543T) in white subjects was performed using a restriction-site polymorphism assay and an Assays-by-Design Service SNP genotyping method (Applied Biosystems, Foster City, CA), respectively.
A 272-bp fragment (either a T or G variant at the −1001 position) or a 147-bp fragment (either a C or T variant at the −1543 position) of the PBEF gene promoter was subcloned into pGL3 basic vector (Promega, Madison, WI) and transiently transfected into HMVEC-L. After 4-hour transfection, cell lysates were retrieved for luciferase activity determination.
Statistical analyses were performed using SigmaStat (version 3.1, Systat Software, Inc., Point Richmond, CA) and/or Stata (version 8.0, StataCorp LP, College Station, TX).
Evidence of the development of ALI varied among the different animal models. In the canine unilateral lavage model, lung injury was evident from the progressive increase in peak inspiratory pressure in the injured lung, eventually doubling by 6 hours, compared with minimal change in peak inspiratory pressure in the control lung. Because of the presence of the control lung and the use of 100% oxygen, blood gases did not change substantially and PaO2 remained above 400 mm Hg even after unilateral injury. In one of these animals, we performed computed tomography imaging at 5 hours, which confirmed the involvement of the entire left lung in an injury pattern of dependent flooding and collapse and increased interstitial density throughout compared with the normal appearance and density of the control lung. In the canine LPS injury model, there were dramatic increases in shunt fraction, peak inspiratory pressure, and pulmonary artery pressure, and a sharp decrease in PaO2/FiO2 ratio beginning 60 minutes after LPS instillation and sustained out to 8 hours (19). BAL protein was markedly elevated. Computed tomgraphy studies revealed dependent volume loss, increased vertical gradients of lung density, and overall increased lung tissue volumes. BAL cell counts were not performed. In the murine LPS and high-Vt models, injury was documented by quantifying alveolar protein leak with Evan's blue extravasation. The canine LPS and murine LPS and high-Vt injury model findings have recently been published (19, 20).
The first experiments in which PBEF was identified assessed lung gene expression in the canine unilateral lavage model of ALI (three animals, from which 22 lung tissue samples, including 11 injured and 11 uninjured, were obtained) with canine RNA cross-hybridized to individual human HG-U133A chips. Uniformly, the gene exhibiting the highest level of expression (5.79-fold increase; n = 3, p < 0.01) was PBEF (Table 1)
Control | ALI | ||||
---|---|---|---|---|---|
Type | N | (Mean ± SE)† | (Mean ± SE)† | RF‡ | p Value |
Canine | 3* | 312 ± 22 | 1807 ± 505 | 5.79 | < 0.01 |
Murine | 4 | 186 ± 14 | 396 ± 74 | 2.13 | < 0.05 |
Human | 3 | 597 ± 91 | 2190 ± 716 | 3.67 | 0.05 |
To validate canine lung PBEF gene expression, we cloned the full-length canine PBEF cDNA (1,476-bp open reading frame encoding 491 amino acids) from canine lung tissues using RT-PCR based on the human PBEF sequence (26). On the basis of the cloned canine and published murine and human PBEF cDNA sequences, specific primers were next designed for either semiquantitative RT-PCR or real-time PCR. Consistent with the microarray results, significantly higher lung PBEF expression was observed in the surfactant-depleted canine ALI model (3.32-fold increase; n = 3, p < 0.05), in the LPS-murine ALI model (2.23-fold increase; n = 6, p < 0.01; Figure 1)
, and in BAL cells from human patients with ALI (4.83-fold increase; n = 3, p < 0.001) relative to controls (Figure 2). We further examined changes in PBEF gene expression using RT-PCR in lung tissue samples from the intratracheal LPS-treated canine ALI model versus intratracheal saline controls. There was a significant increase in PBEF expression in the LPS-injured canine lungs (2.46-fold increase; n = 3, p < 0.01; Figure 1). Western blot results also confirmed the increased PBEF protein expression in Canine Model 1 ALI lung tissues (2.02-fold increase; n = 3, p < 0.05; Figure 3). The expression of constitutively expressed genes, such as ribosomal protein S18 (RPS18) or β-actin, was unchanged. These results firmly corroborate the increased PBEF expression in both human and animal models of ALI detected by microarray profiling.To evaluate spatial localization of PBEF expression, we performed triple immunohistochemical staining in canine lung tissue samples from the unilateral lavage injury model and colocalized PBEF expression in vascular endothelial cells, neutrophils, and type II alveolar epithelial cells. Antibodies to factor VIII (an endothelial marker), neutrophils, and ProSPC (a type II alveolar epithelial cell marker) were obtained commercially. Figure 4
depicts strong canine PBEF expression in the vascular endothelium (Figure 4C) within infiltrating leukocytes in the surfactant-depleted, injured canine lung (Figure 4F) and type II alveolar epithelial cells (Figure 4I), whereas PBEF immunoreactivity was minimally detectable in the uninjured but ventilated control canine lung (Figures 4B, 4E, and 4H, respectively). Figures 4A, 4D, and 4G demonstrated background staining of injured/ventilated lungs with only secondary antibodies. Preimmune serum did not show any significant staining of PBEF in an injured/ventilated canine lung (data not shown). These results indicate that lung vascular endothelial cells, type II alveolar epithelial cells, and infiltrating neutrophils overexpress PBEF in the injured lung.To examine the potential utility of PBEF as an ALI biomarker, we measured BAL and serum levels of PBEF protein in animal models of ALI and humans with ALI using Western blots. PBEF protein levels were significantly increased in canine LPS ALI BAL fluid (2.23-fold increase; n = 3, p < 0.01) and serum (2.01-fold increase; n = 3, p < 0.01; Figure 5)
. PBEF protein levels were increased in BAL fluid obtained from the two murine models: that induced by high Vt ventilation without LPS (2.62-fold increase; n = 7, p < 0.01) and LPS-mediated lung injury (1.67-fold increase; n = 3, p < 0.05; Figure 5). Finally, PBEF protein levels in human patients with ALI were significantly increased in BAL (4.96-fold increase; n = 3, p < 0.01) and serum (2.25-fold increase; n = 8, p < 0.01) relative to healthy control subjects (Figure 6). These results support PBEF as a potential biomarker in ALI and further validate the microarray-based enhanced PBEF expression in animal and human ALI.To examine effects of inflammatory stimuli (LPS, TNF-α, and IL-1β) and mechanical stress (18% cyclic stretch) on PBEF protein expression and to confirm our immunohistochemical localization of PBEF immunoreactivity, we next challenged human lung microvascular endothelium with LPS (50 ng/ml), TNF-α (10 ng/ml), or IL-1β (10 ng/ml), and 18% cyclic stretch in the absence or presence of IL-1β (10 ng/ml), and evaluated PBEF protein content in cellular lysates by Western blotting and densitometric quantification. Each cytokine produced significantly increased PBEF protein expression (2.2- to 4.2-fold increases; Figure 7)
, indicating that enhanced PBEF expression occurs in response to cytokines implicated in the pathogenesis of ALI. To further confirm the increased PBEF expression observed in mechanical ventilation models of murine and canine ALI (Table 1; Figures 1–5), we applied 18% cyclic stretch to human lung microvascular endothelium in vitro for 48 hours and observed significantly augmented PBEF protein expression (3.1-fold increase; n = 6, p < 0.05) relative to static controls (Figure 8). The combination of both 18% cyclic stretch and IL-1β seems to further increase PBEF expression in HMVEC-L (3.7-fold increase; n = 6, p < 0.01; Figure 8). Thus, increases in mechanical stress appear to contribute to increased PBEF expression in lung tissues and in HMVEC-L with inflammatory stimuli exerting a possible additive effect.Because our findings strongly implicated PBEF as a novel candidate gene in ALI, we next examined whether common variants in the human PBEF gene might be associated with susceptibility to sepsis-associated ALI. Direct DNA sequencing in 36 subjects with ALI, subjects with sepsis, and healthy control subjects identified 11 PBEF SNPs (see Table E2 in the online supplement) with a T-1001G transversion in the human PBEF gene immediate promoter (−1 to –3000 bp) having the highest degree of representation in 12 subjects with ALI (40% minor allelic frequency). The PBEF (T-1001G) SNP was genotyped in a case-control population of white subjects with sepsis-associated ALI (n = 87), subjects with sepsis alone (n = 100), and healthy control subjects (n = 84); relevant characteristics of the study population are presented in Table 2
Control | Sepsis | ALI | |
---|---|---|---|
Variable | (n = 84) | (n = 100) | (n = 87) |
Age, mean ± SD | 32.9 ± 10.5 | 59.7‡ ± 19.2 | 52.0‡ ± 16.9 |
Sex, M/F | 40/44 | 48/52 | 51/36 |
APACHE II, mean ± SD | NA | 21.3 ± 8.7 | 23.4 ± 6.9§ |
60-d survival,*L/D (%)† | 84/0 (100%) | 71/23 (75.5%)‡ | 55/26 (67.9%)‡ |
Cancer, % | NA | 22 | 17 |
Immunosuppression, % | NA | 16.5 | 29.9 |
Liver disease, % | NA | 10.9 | 11.5 |
ESRF, % | NA | 9.9 | 8 |
COPD, % | NA | 12.1 | 4.5 |
Alcohol abuse, % | NA | 8.8 | 12.6 |
Diabetes, % | NA | 19.8 | 19.5 |
CHF, % | NA | 9.8 | 6.9 |
HIV-positive, % | NA | 1.1 | 4.6 |
Anemia, % | NA | 26.4 | 17.2 |
ARF, % | NA | 8.8 | 25.3 |
Use of vasopressors, % | NA | 47.1 | 35.2 |
Insult | |||
Lung, % | NA | 37.5 | 70.1 |
UTI, % | NA | 15.4 | 2.3 |
Other, % | NA | 37.4 | 28.7 |
Factor | Control | Sepsis | p Value† | ALI | p Value‡ | p Value§ |
---|---|---|---|---|---|---|
(% frequency) | (% frequency) | (% frequency) | ||||
T-1001G | ||||||
N | 82 | 75 | 69 | |||
TT genotype | 64 (78) | 44 (55) | 29 (42) | |||
GT genotype | 16 (20) | 33 (44) | 0.004 | 39 (57) | < 0.001 | NS |
GG genotype | 2 (2) | 1 (1) | 1 (1) | |||
T allele | 144 (88) | 115 (77) | 97 (70) | |||
G allele | 20 (12) | 35 (23) | 0.01 | 41 (30) | < 0.001 | NS |
C-1543T | ||||||
N* | 83 | 98 | 75 | |||
CC genotype | 42 (51) | 56 (57) | 47 (63) | |||
CT genotype | 31 (37) | 37 (38) | 0.226 | 26 (35) | 0.059 | NS |
TT genotype | 10 (12) | 5 (5) | 2 (2) | |||
C allele | 115 (69) | 149 (76) | 120 (80) | |||
T allele | 51 (31) | 47 (24) | 0.136 | 30 (20) | 0.026 | NS |
A significant association was observed between the PBEF (T-1001G) genotype and subjects with ALI and subjects with sepsis compared with healthy control subjects (p < 0.001 and p = 0.004, respectively). However, the difference in the genotype frequency between ALI and sepsis was not statistically significant. Because a dominant-G model fit the data best, the GT and GG genotypes were included as a single risk group. In a univariate analysis, carriers of the G allele had a 2.75-fold increased risk of ALI compared with control subjects (p = 0.002). Multiple logistic regression analysis using relevant clinical risk factors revealed that, after controlling for age and sex and other comorbidity factors (cancer, immunosuppression, liver disease, end-stage renal failure, chronic obstructive pulmonary disease, alcohol abuse, diabetes, congestive heart failure, anemia, acute renal failure), the G-mutant allele remained an independent risk factor for ALI susceptibility (odds ratio 2.16, 95% confidence interval [CI] 1.01–4.62) but not for sepsis without ALI. The G allele was not associated with mortality among patients with sepsis-associated ALI or sepsis only after controlling for age (odds ratio 1.26, 95% CI 0.47–3.38) and sex (odds ratio 1.67, 95% CI 0.59–4.73). For both sepsis and sepsis-associated ALI, the APACHE II score was the single best predictor of mortality in this population (data not shown). A borderline association was observed between the PBEF (C-1543T) genotype and ALI (p = 0.059), but no association was observed between the PBEF (C-1543T) genotype and sepsis (p = 0.226).
Haplotype-weighted analysis of T-1001G and C-1543T SNPs revealed four haplotypes: GC, GT, TC, and TT (Table 4)
Haplotype Frequencies | ||||||||
---|---|---|---|---|---|---|---|---|
Haplotype* | ALI | Sepsis | Control | Total | ||||
GC | 34.635 | 27.635 | 14.28 | 76.55 | ||||
GT | 67.365 | 79.365 | 93.72 | 240.45 | ||||
TC | 2.365 | 3.365 | 2.72 | 8.45 | ||||
TT | 21.635 | 27.635 | 47.28 | 96.55 | ||||
Total | 126 | 138 | 158 | 422 | ||||
χ2 test: p < 0.01 | ||||||||
Logistic Regression Analysis, OR (95% CI) | ||||||||
Haplotype* | ALI | Sepsis | p Value‡ | |||||
GC | 7.71† (3.01–9.75) | 4.84† (1.97–11.90) | NS | |||||
GT | 2.09 (0.74–5.87) | 2.63 (0.74–9.37) | NS | |||||
TC | 2.46 (0.69–8.75) | 2.73 (0.86–8.67) | NS | |||||
TT | 0.84 (0.33–2.10) | 1.04 (0.45–2.39) | NS |
To examine whether the T-1001G or T-1543C polymorphisms directly alter gene transcription, we performed a transient luciferase reporter gene assay by transfecting T-1001 variant or G-1001 variant-pGL3 basic vector as well as C-1543 variant or T-1543 variant-pGL3 basic vector into HMVEC-L for 4 hours. No significant difference in luciferase activities between T variant– 1001–containing and G variant– 1001–containing pGL3 basic vector constructs was observed. However, the T variant in the PBEF gene promoter SNP C-1543T resulted in nearly a twofold decrease in the reporter gene expression (Figure 9)
.Candidate gene identification in a complex lung disorder such as ALI poses a serious challenge because of the heterogeneity in inciting stimuli and the lack of available linkage studies. We applied emerging functional genomic technologies, specifically DNA microarray profiling and genotyping, to the study of the ALI pathogenesis in hope of providing mechanistic insights and identifying novel biomarkers and therapeutic targets. Gene expression profiling in lung tissue from animal models of ALI identified PBEF as a highly upregulated gene in ALI, and results were reinforced and validated by several complementary approaches (molecular cloning of canine PBEF, RT-PCR, immunohistochemical analysis). Furthermore, PBEF protein levels were significantly increased in BAL, serum, and lung tissues from canine, murine, and human ALI models, suggesting its potential as a biomarker.
The published literature on PBEF is sparse, and our studies provide the first observation that PBEF is significantly upregulated in the lung and in other models of lung injury (16, 17, 26). PBEF was first isolated from an activated peripheral blood lymphocyte cDNA library and found to be involved in B-cell precursor maturation (12). Subsequently, dysregulated PBEF gene expression was described in human fetal membranes of severe chorioamnionitis (14), with increased expression in an amniotic epithelial cell line following challenge with inflammatory cytokines (LPS, IL-1β, TNF-α, and IL-6) (14) and during IFN-γ–induced maturation of pre–B cells (29) in a B lymphoma cell line (30) and in IFN-induced preterm labor gestational membrane (31). Recombinant PBEF protein significantly increased expression of IL-6 and IL-8 in amniotic epithelium (32, 33). Despite the findings of PBEF expression in nonlung tissues, the molecular physiologic and pathophysiologic relevance of PBEF to lung pathophysiology is unknown. The robust expression of PBEF in murine and canine models of ALI in our study suggests that PBEF may be an inflammatory signal transducer in the pathogenesis of ALI. Immunohistochemical colocalization studies revealed increased PBEF expression in lung endothelium, type II alveolar epithelial cells, and infiltrating neutrophils, and upregulation of PBEF expression in inflammatory cytokine-stimulated HMVEC-L in vitro. These results strongly support a potentially important role for PBEF in the inflammatory lung processes observed in ALI. Several clinical studies implicate a complex network of inflammatory cytokines and chemokines in mediating, amplifying, and perpetuating the lung injury process (34). The immunohistochemical colocalization of increased PBEF expression in infiltrating neutrophils and lung endothelium suggests a novel role for PBEF as a signal transducer during lung inflammation. This notion is supported by a recent report (15) that PBEF expression is significantly increased in circulating peripheral blood neutrophils derived from patients with sepsis, including data that convincingly demonstrated that PBEF inhibits neutrophil apoptosis. Because the rate of clearance of apoptotic neutrophils is associated with resolution of neutrophilic lung inflammation (35), prolonging neutrophil survival via PBEF inhibition of apoptosis may sustain neutrophilic inflammation and contribute to the pathogenesis of ALI and other neutrophil-mediated disorders.
In addition to inflammatory cytokines, another clinically relevant stimulus for PBEF expression is increased mechanical stress, a major contributing factor to both ALI mortality and ventilator-associated ALI (3, 36). The application of 18% cyclic stretch to HMVEC-L in vitro for 48 hours resulted in significant augmentation in PBEF protein expression (3.1-fold), which is consistent with the increased PBEF gene expression observed in distended human fetal membranes in vitro (13). The PBEF promoter contains two nuclear factor κB binding elements that may potentially participate in conferring mechanical stretch responsiveness (14). Besides demonstrating the survival benefit of a lung-protective ventilatory strategy, the landmark Acute Respiratory Distress Syndrome Network study also highlighted a marked reduction in the number of neutrophils and the concentration of proinflammatory cytokines released into the airspaces of the injured lung (34). Studies are ongoing to establish the potential contribution of PBEF to the pathogenesis of ALI. At a minimum, however, increased PBEF protein expression, either in BAL fluid or serum, has promise as a novel and useful biomarker to assist in the clinical diagnosis of inflammatory lung disease (37–39).
Given that our genomic studies identified PBEF as a viable candidate gene and potential biomarker in ALI, we selected two PBEF promoter variants (T-1001G and C-1543T) and conducted genetic studies to test for an association between these PBEF SNPs and sepsis-associated ALI. Both T-1001G and C-1543T SNPs conform to the HWE.
The HWE implies maintenance of allele and genotype frequencies in a steady state from generation to generation. Departures from HWE can be used as an indication of population genetic features in a sample and also can be used to judge potential genotyping errors. Both allele and genotype frequencies of T-1001G and C-1543T SNPs were in HWE (p = 0.50 and p = 0.46, respectively), suggesting that genes of subjects used in this study were picked independently from the gene pool, which is equivalent to picking patient samples at random from the population. It also supports that the genotyping work was correctly performed in this study using two well established techniques (restriction-site polymorphism and 5′ nuclease assays). Only after HWE was verified for each SNP did we initiate the individual SNP or haplotype association tests. For the T-1001G SNP, a significantly higher frequency of the minor G allele and TG/GG genotypes and lower frequencies of the minor T-allele and CT/TT genotypes in the C-1543T SNP were associated with ALI. The former also was associated with sepsis without ALI. Multiple logistic regression analysis revealed that, after controlling for 12 other risk factors, the G-variant allele from T-1001G remains an independent risk factor for ALI susceptibility. Haplotype analysis revealed four possible haplotypes (GC, GT, TC, TT; Table 4) from two SNPs. Among them, carriers of the GC haplotype had a 7.71-fold higher risk of ALI and a 4.84-fold higher risk of sepsis (both p < 0.01). Trends of differences in the minor allele, genotype, and haplotype frequencies comparing subjects with sepsis-associated ALI and subjects with sepsis were evident but not statistically significant. This finding may reflect, in part, a limited sample size, which prevented the detection of a difference between sepsis and ALI groups. An alternative postulate is that this locus is more strongly associated with the sepsis severity rather than ALI susceptibility. For example, we observed a slightly higher APACHE II score (a measure of severity) in patients with sepsis-associated ALI compared with patients with sepsis only, although the difference didn't achieve significance. Our ongoing recruitment of additional patients will allow us to test this hypothesis in the future. In addition, further analysis of DNA from patients with ALI from causes other than sepsis may be necessary to distinguish whether the haplotype GC is a risk factor or the haplotype TT a protective factor specific for ALI, rather than for severe sepsis, which frequently leads to ALI.
Preliminary studies addressing the functionality of the T-1001G variant using the luciferase reporter gene assay did not demonstrate a significant role for this variant in gene transcription regulation (data not shown). However, the T variant in the C-1543T SNP, 542 bp upstream from T-1001G in the PBEF promoter region, resulted in nearly a twofold decrease in the reporter gene expression (Figure 9). The frequency of the T allele was significantly lower in subjects with ALI (20%, n = 75) than that in normal control subjects (31%; n = 83, p = 0.026). This result is consistent with our observations from animal models of ALI, human subjects with ALI, and in vitro cell culture experiments, and suggests that higher expression of PBEF is implicated in the pathogenesis of sepsis-associated ALI. These results further suggest that genetically determined increased PBEF expression contributes to susceptibility to ALI.
In summary, using a candidate gene approach and a series of diverse cellular, animal, and human studies, we identified PBEF as a potential novel biomarker and candidate gene in sepsis- and mechanical stress–induced inflammatory lung disease, such as ALI. Although further studies are required to both define the pathophysiologic role of altered PBEF expression in ALI and to more clearly link PBEF variants to ALI susceptibility, our results strongly support that PBEF may be a potential novel biomarker in ALI. Finally, this study underscores the powerful potential of using genomic approaches to deciphering the genetic basis of complex lung disorders.
The authors thank Tera Lavoie, James R. McGlothin, Maria Portella, Saad Sammani, Perry Iannaconi, and Lakshmi Natarajan for their excellent technical assistance.
1. | Matthay MA, Zimmerman GA, Esmon C, Bhattacharya J, Coller B, Doerschuk CM, Floros J, Gimbrone MA Jr, Hoffman E, Hubmayr RD, et al. Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group. Am J Respir Crit Care Med 2003;167:1027–1035. |
2. | Goss CH, Brower RG, Hudson LD, Rubenfeld GD. ARDS network. Incidence of acute lung injury in the United States. Crit Care Med 2003;31:1607–1611. |
3. | Bersten AD, Edibam C, Hunt T, Moran J. Australian and New Zealand Intensive Care Society clinical trials group. Incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three Australian States. Am J Respir Crit Care Med 2002;165:443–448. |
4. | Brower RG. Mechanical ventilation in acute lung injury and ARDS. Tidal volume reduction. Crit Care Clin 2002;18:1–13. |
5. | Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342:1334–1349. |
6. | Villar J, Flores C, Mendez-Alvarez S. Genetic susceptibility to acute lung injury. Crit Care Med 2003;31(4 Suppl):S272–S275. |
7. | Lin Z, Pearson C, Chinchilli V, Pietschmann SM, Luo J, Pison U, Floros J. Polymorphisms of human SP-A, SP-B, and SP-D genes: association of SP-B Thr131Ile with ARDS. Clin Genet 2000;58:181–191. |
8. | Marshall RP, Webb S, Bellingan GJ, Montgomery HE, Chaudhari B, McAnulty RJ, Humphries SE, Hill MR, Laurent GJ. Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome. Am J Respir Crit Care Med 2002;166:646–650. |
9. | Mira JP, Cariou A, Grall F, Delclaux C, Losser MR, Heshmati F, Cheval C, Monchi M, Teboul JL, Riche F, et al. Association of TNF2, a TNF-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study. JAMA 1999;282:561–568. |
10. | Heesen M, Bloemeke B, Schade U, Obertacke U, Majetschak M. The -260 C→T promoter polymorphism of the lipopolysaccharide receptor CD14 and severe sepsis in trauma patients. Intensive Care Med 2002;28:1161–1163. |
11. | Jacobson JR, Garcia JGN. Genomics made “functional” in ventilator-associated lung injury. Am J Respir Crit Care Med 2003;168:1023–1025. |
12. | Samal B, Sun Y, Stearns G, Xie C, Suggs S, McNiece I. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol 1994;14:1431–1437. |
13. | Nemeth E, Millar LK, Bryant-Greenwood G. Fetal membrane distention: II. Differentially expressed genes regulated by acute distention in vitro. Am J Obstet Gynecol 2000;182:60–67. |
14. | Ognjanovic S, Bao S, Yamamoto SY, Garibay-Tupas J, Samal B, Bryant-Greenwood GD. Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes. J Mol Endocrinol 2001;26:107–117. |
15. | Jia SH, Li Y, Parodo J, Kapus A, Fan L, Rotstein OD, Marshall JC. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J Clin Invest 2004;113:1318–1327. |
16. | Ye SQ, Lavoie T, Gao L, Easley RB, Ma S-F, Simon BA, Garcia JGN. Molecular cloning of canine pre-B cell colony enhancing factor (PBEF) cDNA [abstract]. Am J Respir Crit Care Med 2003;167:A779. |
17. | Simon BA, Ye SQ, Easley RB, Lavoie T, Gregovey D, Garcia JGN. Microarray analysis of regional cellular responses to mechanical stress in canine ventilator-associated lung injury (VALI) [abstract]. Am J Respir Crit Care Med 2003;167:A777. |
18. | Downie JM, Nam AJ, Simon BA. Pressure-volume curve does not predict steady-state lung volume in canine lavage lung injury. Am J Respir Crit Care Med 2004;169:957–962. |
19. | McVerry BJ, Peng X, Hassoun PM, Sammani S, Simon BA, Garcia JG. Sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury. Am J Respir Crit Care Med 2004;170:987–993. |
20. | Peng X, Hassoun PM, Sammani S, McVerry BJ, Burne MJ, Rabb H, Pearse D, Tuder RM, Garcia JG. Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 2004;169:1245–1251. |
21. | Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, Lamy M, Legall JR, Morris A, Spragg R. The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994;149:818–824. |
22. | American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992;20:864–874. |
23. | Cappola TP, Cope L, Cernetich A, Barouch LA, Minhas K, Irizarry RA, Parmigiani G, Durrani S, Lavoie T, Hoffman EP, et al. Deficiency of different nitric oxide synthase isoforms activates divergent transcriptional programs in cardiac hypertrophy. Physiol Genomics 2003;14:25–34. |
24. | Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L, Kasahara Y, Salvemini D, Voelkel NF, Flores SC. Oxidative stress and apoptosis interact and cause emphysema due to VEGF receptor blockade. Am J Respir Cell Mol Biol 2003;29:88–97. |
25. | Nemeth E, Tashima LS, Yu Z, Bryant-Greenwood GD. Fetal membrane distention: I. Differentially expressed genes regulated by acute distention in amniotic epithelial (WISH) cells. Am J Obstet Gynecol 2000;182:50–59. |
26. | McGlothlin JR, Gao L, Lavoie T, Simon B, Easley RB, Ma S-F, Rumala BB, Garcia JGN, Ye SQ. Molecular cloning and characterization of canine pre-B-cell colony-enhancing factor cDNA. Biochem Genet (In press) |
27. | Birukov KG, Jacobson JR, Flores AA, Ye SQ, Birukova AA, Verin AD, Garcia JG. Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. Am J Physiol Lung Cell Mol Physiol 2003;285:L785–L797. |
28. | Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit Care Med 1985;13:818–829. |
29. | Patrone L, Damore MA, Lee MB, Malone CS, Wall R. Genes expressed during the IFN gamma-induced maturation of pre-B cells. Mol Immunol 2002;38:597–606. |
30. | Xu LG, Wu M, Hu J, Zhai Z, Shu HB. Identification of downstream genes up-regulated by the tumor necrosis factor family member TALL-1. J Leukoc Biol 2002;72:410–416. |
31. | Marvin KW, Keelan JA, Eykholt RL, Sato TA, Mitchell MD. Use of cDNA arrays to generate differential expression profiles for inflammatory genes in human gestational membranes delivered at term and preterm. Mol Hum Reprod 2002;8:399–408. |
32. | Ognjanovic S, Bryant-Greenwood GD. Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes. Am J Obstet Gynecol 2002;187:1051–1058. |
33. | Ognjanovic S, Tashima LS, Bryant-Greenwood GD. The effects of pre-B-cell colony-enhancing factor on the human fetal membranes by microarray analysis. Am J Obstet Gynecol 2003;189:1187–1195. |
34. | Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokine-mediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003;14:523–535. |
35. | Matute-Bello G, Martin TR. Science review: apoptosis in acute lung injury. Crit Care 2003;7:355–358. |
36. | Frank JA, Gutierrez JA, Jones KD, Allen L, Dobbs L, Matthay MA. Low tidal volume reduces epithelial and endothelial injury in acid-injured rat lungs. Am J Respir Crit Care Med 2002;165:242–249. |
37. | Noel-Georis I, Bernard A, Falmagne P, Wattiez R. Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage. Dis Markers 2001;17:271–284. |
38. | Ma PC, Blaszkowsky L, Bharti A, Ladanyi A, Kraeft SK, Bruno A, Skarin AT, Chen LB, Salgia R. Circulating tumor cells and serum tumor biomarkers in small cell lung cancer. Anticancer Res 2003;23:49–62. |
39. | Pan T, Nielsen LD, Allen MJ, Shannon KM, Shannon JM, Selman M, Mason RJ. Serum SP-D is a marker of lung injury in rats. Am J Physiol Lung Cell Mol Physiol 2002;282:824–832. |