Smoke and Mirrors - ATS Journals

Editorials References 1. Fischereder M, Kretzler M. New immunosuppressive strategies in renal transplant recipients. J Nephrol 2004;17:9–18. 2. Konishi K, Inobe M, Yamada A, Murakami M, Todo S, Uede T. Combination treatment with FTY720 and CTLA4IgG preserves the respiratory epithelium and prevents obliterative disease in a murine airway model. J Heart Lung Transplant 2002;21:692–700. 3. Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk M, Volpi M, Sha’afi RI, Hla T. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 1999;99:301–312. 4. Quadri S, Bhattacharjee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelial barrier strengthening by activation of focal adhesion kinase. J Biol Chem 2003;278:13342–13349. 5. Safdar Z, Wang P, Ichimura H, Issekutz AC, Quadri S, Bhattacharya J. Hyperosmolarity enhances the lung capillary barrier. J Clin Invest 2003; 112:1541–1549. 6. Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, English D. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J Clin Invest 2001;108:689–701. 7. Dudek SM, Jacobson JR, Chiang ET, Birukov KG, Wang P, Zhan X, Garcia JG. Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase. J Biol Chem 2004;279:24692–24700.

929 8. 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. 9. Hla T, Lee MJ, Ancellin N, Paik JH, Kluk MJ. Lysophospholipids– receptor revelations. Science 2001;294:1875–1878. 10. Morales-Ruiz M, Lee MJ, Zollner S, Gratton JP, Scotland R, Shiojima I, Walsh K, Hla T, Sessa WC. Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells. J Biol Chem 2001;276: 19672–19677. 11. Cummings RJ, Parinandi NL, Zaiman A, Wang L, Usatyuk PV, Garcia JG, Natarajan V. Phospholipase D activation by sphingosine 1-phosphate regulates interleukin-8 secretion in human bronchial epithelial cells. J Biol Chem 2002;277:30227–30235. 12. Ryan AJ, McCoy DM, McGowan SE, Salome RG, Mallampalli RK. Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity. J Appl Physiol 2003;94:253–258. 13. Jolly PS, Rosenfeldt HM, Milstien S, Spiegel S. The roles of sphingosine1-phosphate in asthma. Mol Immunol 2002;38:1239–1245. 14. Ammit AJ, Hastie AT, Edsall LC, Hoffman RK, Amrani Y, Krymskaya VP, Kane SA, Peters SP, Penn RB, Spiegel S, et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001;15:1212–1214. DOI: 10.1164/rccm.2408008

Smoke and Mirrors Mouse Models as a Reflection of Human Chronic Obstructive Pulmonary Disease In this issue of the Journal (pp. 974–980), Guerassimov and colleagues exposed five strains of mice to cigarette smoke and found impressive variability in the development of emphysema between strains (1). Questions that some readers of the Journal might be asking are: Why are mice “smoking,” and why am I reading about mice in the Blue journal? Why the mouse? The mouse has been an invaluable tool to dissect disease pathways in humans. Mice are becoming the animal of choice for many types of research because: (1 ) like us, they are mammals; (2 ) we have great understanding of mouse biology and many molecular probes to study the mouse; (3 ) breeding is rapid; (4 ) genetic homology maps are available to allow translation of genetic findings in laboratory mice to the human genome; and (5 ) we can manipulate the murine genome eliminating (“knock-outs”) or enhancing (transgenics) the expression of individual gene products or introducing specific variants within a gene of interest (“knock-ins”). Thus, controlled genetic experiments in mammals can be performed. Disadvantages of the mouse include their small size, which precludes some surgical models. Their small size also makes physiologic assessment more difficult, although much progress has been made in this regard. In fact, the phenotyping in the Guerassimov manuscript, which includes quantitative measurements of morphometry and physiology, is a major strength. The biggest criticism of the mouse, of course, is that mice are not (wo)men (note the whiskers). Mouse biologists would argue that basic biological processes are usually conserved in mammals. Overall, information derived from studies in mice is best used to guide studies in humans, many of which have been incredibly informative, including important insights into obesity and cancer (2, 3). The marked strain-to-strain variability in mice exposed to the same environmental conditions strongly suggests that genetic differences between the strains influence the differential susceptibility to develop emphysema. These results provide further evidence that genetic factors can predispose to emphysema, and

more importantly, that these genetic factors are amenable to genetic dissection by careful study of the histologic and physiologic quantitative phenotypes examined by Guerassimov and colleagues. This type of detailed phenotyping is crucial in human chronic obstructive pulmonary disease (COPD) studies as well, because phenotypic heterogeneity is a potential contributor to the inconsistent results of previously reported human COPD candidate gene association studies (4). Although lung histology is not usually readily available from patients with emphysema, high-resolution chest CT scans may provide a useful surrogate. It is unclear how many genes influence the development of emphysema in these mouse strains, where they are located, and what their individual effect size is. The next steps will likely involve localization of broad genomic regions that influence these quantitative phenotypes; this can be performed using crosses between susceptible and nonsusceptible strains and assessment of the relationship in subsequent generations between the phenotype and genetic variants across the genome that differ between the parental strains—traditional quantitative trait locus (QTL) mapping. However, the investigation of complex traits in mice has been accelerated with the development of new analytic methods and computer software, and in silico gene mapping using comparative SNP maps between inbred mouse strains is now an important addition to QTL mapping (5). Using a bioinformatic approach to relate known genetic variation between strains to phenotypic variation may speed the discovery of disease genes in COPD, as it eliminates some of the time and cost involved in classical QTL approaches. In either case, the size of the genomic region identified will depend on the magnitude of the experiment. In classical QTL analysis, phenotyping and genotyping a larger number of mice in crosses between strains will increase the number of recombination events and lead to a narrower genomic region of interest. Including a larger number of strains for bioinformatic comparisons will similarly lead to a more refined estimate of QTL localization.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 170 2004

TABLE 1. COMPARISON OF COMPLEX DISEASE GENE IDENTIFICATION IN HUMANS AND MICE Human

Mouse

Evidence that genetic factors influence trait

Disease-related phenotypes aggregate in families

Localization to broad genomic region

Genome scan linkage analysis

Fine mapping and susceptibility gene identification

(1 ) Systematic approach: testing SNPs for association across linked region (2 ) Positional candidate gene studies (1 ) Gene level: in vitro cell gene interference assays (e.g., siRNA) (2 ) Specific variant: in vitro cell transfection assays for promoter variants; protein structure assessment of amino acid changes

Assessment of function of the gene and/or specific variant

The next step gets really hard. The process of fine mapping and gene identification for a QTL, in mouse or in man, remains quite challenging. Direct positional cloning efforts, by successively narrowing the key region using repeated crosses in mice, are time-consuming and expensive. Searching for reasonable pathophysiologic candidate genes within the regions of linkage or assessing expression differences in RNA from relevant tissues to select candidate genes are additional approaches to identify the key susceptibility variant or set of variants within a linked region. The recent development of novel approaches, such as chromosome substitution strains in mice, in which strains of mice are created that contain a single chromosome of one strain and the remainder of their chromosomes of a second strain, will likely make it easier to identify QTL signals and to localize them. Research continues to move rapidly in this area (6). During the process of fine mapping a QTL, the ability to compare murine genetic results to human genetic association analysis and linkage analysis results can be very useful. As shown in Table 1, analogous processes in human and murine studies are involved in demonstrating that genes likely influence a trait, using linkage approaches to achieve broad genomic localization of the susceptibility gene, and performing fine mapping and gene identification. If a potentially functional variant is identified, murine studies are especially critical, although investigation of function using in vitro methods can also be considered. Ideally, the human and mouse studies should be synergistic, rather than isolated pathways of investigation. Human studies of candidate gene variants within regions of murine linkage can provide strong evidence that a positional candidate gene is the QTL, whereas human linkage studies that show evidence for linkage in chromosomal regions syntenic to murine linkage signals suggest that the murine linkages are relevant to human disease. To date, this intersection of mouse and human results has not been applied in COPD, but efforts to link mouse QTL studies to human genetic association studies are ongoing in asthma (7, 8). COPD is a complex disease, influenced by genetic factors, environmental determinants (mainly cigarette smoking), and gene-by-environment interactions. Few nonsmokers in the United States develop COPD, but only a minority of smokers ever develop the disease, suggesting variation between humans for COPD susceptibility similar to the variable susceptibility observed between different mouse strains. Thus, exposure to a noxious agent, invariably cigarette smoke in developed countries, combined with likely multiple predisposing genes, results in COPD. Several genomic regions have shown suggestive or significant linkage to pulmonary function phenotypes in families with severe, early-onset COPD, and a region of significant linkage on chromosome 2 to FEV1/FVC in these families with early-

Consistent phenotypic differences across strains in same environment QTL Analysis (1 ) Intercrosses/backcrosses (2 ) Cross-strain bioinformatic comparisons (1 ) Systematic approach: isolation of QTL by repeated crosses and/or bioinformatic approaches (2 ) Positional candidate gene studies (1 ) Gene level: knock-out or transgenic mice (2 ) Specific variant: knock-in mice

onset COPD has been shown to influence FEV1/FVC in families from the general population (9, 10). The intersection of these human linkage results with mouse strain QTL analysis will be essential. In this regard, the genetic loci in cigarette smoke– susceptible mice may mirror the susceptibility loci in people who develop COPD. Additional linkage studies of COPD in humans are required, and QTL mapping in mice together with fine mapping of linked regions in both humans and mice has the potential to identify novel COPD susceptibility genes. Improved understanding of COPD pathophysiology would result, potentially with new opportunities for treatment. Conflict of Interest Statement : S.D.S. has participated on the Advisory Board for Wyeth and receives a fixed stipend from the American Thoracic Society for editorial responsibilities for the American Journal of Respiratory Cell and Molecular Biology; D.L.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.K.S. has received grant support and honoraria from GlaxoSmithKline for a study of COPD genetics.

Steven D. Shapiro, M.D. Dawn L. DeMeo, M.D., M.P.H. Edwin K. Silverman, M.D., Ph.D. Brigham and Women’s Hospital, Harvard Medical School Boston, Massachusetts References 1. Guerassimov A, Hoshino Y, Takubo Y, Turcotte A, Yamamoto M, Ghezzo H, Triantafillopoulos A, Whittaker K, Hoidal JR, Cosio MJ. The development of emphysema in cigarette smoke–exposed mice is strain dependent. Am J Respir Crit Care Med 2004;170:974–980. 2. Brockmann GA, Bevova MR. Using mouse models to dissect the genetics of obesity. Trends Genet 2002;18:367–376. 3. Demant P. Cancer susceptibility in the mouse: genetics, biology and implications for human cancer. Nat Rev Genet 2003;4:721–734. 4. Silverman EK. Genetics. In Calverley PMA, MacNee W, Pride NB, Rennard SI, editors. Chronic obstructive pulmonary disease, 2nd ed. London: Arnold; 2003. 5. Grupe A, Germer S, Usuka J, Aud D, Belknap JK, Klein RF, Ahluwalia MK, Higuchi R, Peltz G. In silico mapping of complex disease-related traits in mice. Science 2001;292:1915–1918. 6. Singer JB, Hill AE, Burrage LC, Olszens KR, Song J, Justice M, O’Brien WE, Conti DV, Witte JS, Lander ES, et al. Genetic dissection of complex traits with chromosome substitution strains of mice. Science 2004;304:445–448. 7. Barnes KC, Caraballo L, Munoz M, Zambelli-Weiner A, Ehrlich E, Burki M, Jimenez S, Mathias RA, Stockton ML, Deindl P, et al. A novel promoter polymorphism in the gene encoding complement component 5 receptor 1 on chromosome 19q13.3 is not associated with asthma and atopy in three independent populations. Clin Exp Allergy 2004; 34:736–744. 8. Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Kohl J, Wahl L, Kuperman D, Germer S, et al. Identification of comple-

Editorials ment factor 5 as a susceptibility locus for experimental allergic asthma. Nat Immunol 2000;1:221–226. 9. Silverman EK, Palmer LJ, Mosley JD, Barth M, Senter JM, Brown A, Drazen JM, Kwiatkowski DJ, Chapman HA, Campbell EJ, et al. Genomewide linkage analysis of quantitative spirometric phenotypes in severe early-onset chronic obstructive pulmonary disease. Am J Hum Genet 2002;70:1229–1239.

931 10. Malhotra A, Peiffer AP, Ryujin DT, Elsner T, Kanner RE, Leppert MF, Hasstedt SJ. Further evidence for the role of genes on chromosome 2 and chromosome 5 in the inheritance of pulmonary function. Am J Respir Crit Care Med 2003;168:556–561.

DOI: 10.1164/rccm.2408007

Obliterative Bronchiolitis after Lung Transplantation A Repetitive Multiple Injury Airway Disease The success of lung transplantation has followed in the wake of other solid organ transplant populations. Regrettably, the survival of lung transplant recipients lag behind that of other solid organ transplant recipients (1, 2). However, emerging data suggest that graft survival after lung transplantation is set to improve. Unique characteristics may influence survival after lung transplantation. Unlike other solid organ transplants, the lung allograft is exposed to the environment. Approximately 11,000 liters of air, including particulates and microorganisms, move through the lung each day. A mucociliary escalator system normally maintains a sterile environment. This mechanism is disrupted in lung transplant recipients who have both abnormal ciliary function and an impaired cough reflex resulting from denervation of the lung. Therefore, the lung allograft is potentially exposed to two major sources of injury: first, the expected phenomenon of rejection leading to alloimmune injury, and second, nonalloimmune injury such as infection. Obliterative bronchiolitis results in graft failure late after lung transplantation. This is a patchy process that limits the ability of transbronchial biopsy to secure a diagnosis. This problem of sampling error has lead to the emergence of a clinical classification as a surrogate for obliterative bronchiolitis. This clinical correlate is referred to as bronchiolitis obliterans syndrome (BOS) and is primarily based on estimates of FEV1 and FEF25–75 (3). Alloimmune injury, identified histologically as acute cellular rejection, is a significant risk factor for the development of obliterative bronchiolitis. Both the frequency and the grade of severity are important. Three or more episodes of acute rejection within 12 months of transplantation results in a three- to fourfold increase in risk of BOS (4). At least one episode of mild (A2) rejection is associated with BOS (5). New insights into the natural history of acute cellular rejection and the impact of recurrent minimal (A1) rejection on graft survival are provided by Hopkins and coworkers in this issue of the Journal (pp. 1022–1026) (6). Their study challenges the conventional belief that minimal A1 rejection does not have significant consequences. In this clinical series, 42% of patients experienced multiple A1 rejection episodes in the first 12 months after transplantation. In 34% of patients, surveillance biopsies showed progression from minimal A1 to high-grade rejection. BOS developed in 68% of patients with multiple minimal A1 rejection biopsies, at a mean of 599 days. These data indicate that repetitive low-grade acute cellular rejection episodes lead to clinically relevant alloimmune-mediated injury. Broad interpretation of this detailed study suggests that all lung transplant patients should be subjected to histologic surveillance for rejection and also that augmented treatment for rejection prevents the onset of BOS. However, neither assumption can currently be corroborated. Valentine and colleagues have shown that the outcome of patients who are not subjected to a routine surveillance biopsies for the identification of acute rejection is similar to that of the

international registry (7). They used a strict care pathway that triggered a biopsy only in specific clinical circumstances. Using this approach, 55% of patients were managed with only a single biopsy procedure. These results imply that the elective identification and treatment of acute rejection may not influence longterm graft function. These contrasting approaches to the elective histologic diagnosis of rejection emphasize the need for noninvasive estimates of rejection after lung transplantation. Recent data describe a possible role for serum concentrations of hepatocyte growth factor (HGF) as a surrogate marker for rejection (8). In a prospective blinded study of 106 lung transplant recipients, elevated levels of HGF discriminated between infection and rejection. Patients with rejection had significantly greater HGF levels, 3,972 ng/L compared with 1,559 ng/L for those with infection. Receiver–operator curve characteristics were predictive of rejection at 0.99 (95% confidence interval, 0.997–1.001). Perhaps a surprising observation was the rapid escalation of HGF, within 1 to 2 days, before the diagnosis of rejection. Biologically, one might expect a delayed lead in time for the evolution of rejection in patients receiving immunosuppression. If the observations pertaining to HGF are validated, its measurement may provide further insight into the natural history of alloimmune injury and BOS. Hopkins and coworkers imply that the treatment of minimal A1 rejection may lead to reduced rates of BOS. Although treatment largely leads to histologic resolution of rejection, there is no compelling evidence that augmented treatment of acute rejection prevents BOS. In a large international study, 32% of patients demonstrated progression from recurrent acute rejection to BOS despite optimization of immunosuppression (9). Alternative sources of injury may therefore be important. Data provided by Hopkins and colleagues support this concept, as BOS occurred in 43% of patients with less than one episode of minimal A1 rejection. Nonalloimune injury represents an increasingly relevant form of injury in which preventative interventions may impact on graft survival. Gastroesophageal reflux disease (GERD) is recognized as a common event after lung transplantation, believed to result from vagal injury at the time of surgery. GERD has been documented in as many as 73% of patients using esophageal pH probe monitoring (10). Those with normal pH studies had significantly better survival, 82% at 5 years compared with 48% for patients with GERD. On the basis of a high index of suspicion for aspiration, patients were subjected to laparoscopic fundoplication. There was a 24% increase in FEV1 after fundoplication in a cohort of 43 patients. This intervention was most successful when undertaken at the early stages of BOS; only 17% of patients with advanced Grade 3 BOS improved after fundoplication. Infection, the most frequent cause of death after lung transplantation, is another potential source of nonalloimmune injury. Bronchial dilatation, characteristic of bronchiectasis, on HRCT is a typical finding in patients with BOS. Bacterial infection is