© 2004 American Thoracic Society
Flies, Mice, and Surprises in Dissecting Environmental Lung InjuryDepartment of Pediatrics Harvard Medical School Boston, Massachusetts We are now at an unprecedented time in our ability to unravel the complex biology underlying many respiratory diseases. A generation ago, the main tools employed were largely based upon physiology and lung mechanics. These disciplines remain central to understanding lung biology, but here, as in virtually all of biomedical research, advances in molecular biology, genetics, and immunology are now assuming critical roles in our field. The challenge for investigators today is to be integrative, taking new approaches through old familiar terrains. Information coming from the genomes of yeast, flies, and fish are being applied toward understanding the human disease. The workhorse for analyzing new genomic information is the mouse. Genes may be added, deleted, turned on and off selectively, and the impact of these genetic manipulations may then be assessed in vivo. The study by Hollingsworth and colleagues in this issue of the Journal (pp. 126–132) is a prime example of this process (1). In 1985, Drosophila geneticists first identified a gene in fly larvae that regulated body axis development, which they named Toll (2). Subsequent research in the fly led to the discovery that after playing a critical role in development, the Toll gene was essential in adult flies for inducing innate immune responses to fly pathogens. Comparative analyses of vertebrate genomes led to the realization that the Toll gene is ancestrally related to the Interleukin-1 receptor gene family. We now know that the Toll gene family recognizes non-self products of bacteria, fungi, and foreign DNA (3). Activation of the Toll-like receptor (TLR) gene family leads to the unleashing of a powerful cascade of cytokines. In the past several years, studies based on mouse genetics has taught us that septic shock induced by endotoxin or lipopolysaccharide is the result of activation of the Toll 4 gene (4). Given this background, Hollingsworth and colleagues investigated four models of environmental lung injury using mice genetically deficient in TLR4, or their identical littermates. The four inhalational toxicants were lipopolysaccharide, residual oil fly ash, and acute and subacute ozone exposure. Each of these stimuli are known to exacerbate lung inflammation. The data clearly show, as expected, that lipopolysaccharide injury is almost entirely due to activation of TLR4. On the other hand, the complex microparticulate of residual oil fly ash appears not to interact with the TLR4, although this does not exclude other of the nine Toll-like receptors. The most interesting and surprising finding in the study concerned the role of the TLR4 receptor in ozone-induced lung injury. Two different ozone exposures were examined. An acute model challenged mice with 2 ppm ozone for 3 hours (acute model). The second exposure challenged with 0.3 ppm for 72 hours. In each case, lung lavage protein, leukocytes, and airway responsiveness to methacholine challenge were scored. In the acute model, there were no phenotypic differences between TLR4-sufficient and -deficient mice on a pure C57Black/6 background. In contrast, after subacute exposure, although there were no changes in inflammation or lavage protein levels, there was a highly significant blunting of the airways hyperresponsiveness to methacholine. There are several points to consider in analyzing these results. First, for unclear reasons, the airways responsiveness was assessed in two different ways in the acute and chronic models. The acute model used a plethysmographic technique (Buxco Electronics, Sharon, CT), which is an indirect measure of resistance and compliance. The subacute model used the more direct measurement of airways pressure/time index. Note that the method of administration and the dose response to methacholine is also different with the two techniques. Second, the dissociation between inflammation and permeability from airways responsiveness in the models is interesting. The protection from increased airways responsiveness to methacholine was recently shown in this model to be complement-dependant and neutrophil-independent (5). The real surprise in the results in the subacute model comes when the work of Hollingsworth and colleagues is compared with the work of Kleeberger and colleagues (6, 7). The latter group used a technique called quantitative trait locus analyses. In this powerful technique, two inbred lines of mice, which have a phenotypic variance in a physiologic response, are crossed, and the recombinant progeny are screened for the traits. Kleeberger and colleagues showed that in subacute ozone exposure, C57Black/6 mice are relatively resistant and C3H/HeJ mice are relatively sensitive. The resistance or susceptibility in two outcomes (neutrophil asccumulation or protein leakage) was then analyzed over sets of offspring against a background of mapped markers covering the entire mouse genome. They found that leukocyte influx and inflammation appeared to map to chromosome 11 and possibly 17, while the airway hyperpermeability (as assessed by the amount of lavage protein) was linked to a locus on chromosome 4 that contained only two candidate genes. These candidates were mouse urinary protein 1 and the TLR4 receptor. To confirm a linkage with TLR4, the authors next compared the recombinant inbred lines C3H/HeJ (TLR4 mutant) with C3H/OuJ (which has a wild-type TLR4 gene). Again, the protein leakage after ozone exposure was accounted for by the TLR4 locus. Thus the discrepancies are these: the present work clearly links airways hyperresponsiveness with TLR4 in the subacute ozone model, but does not link protein leakage. Kleeberger and colleagues clearly link protein leakage after subacute ozone exposure with TLR4 (but did not assay airway responses to methacholine). Hollingsworth and colleagues suggest that the Kleeberger and colleagues are measuring a trait linked but not identical to TLR4. While resolution of this conundrum may seem irrelevant to human disease, when both works are taken together there is strong evidence that something controlled by signaling at the TLR4 level is a critical player in some aspects of ozone lung injury and perhaps chronic obstructive pulmonary disease. FOOTNOTES Conflict of Interest Statement: C.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. REFERENCES
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
