Our results demonstrate that IIP, regardless of type, involves disordered homeostatic control of genes that regulate the extracellular matrix and chemokine activity in the lung. Moreover, our findings demonstrate that although familial and nonfamilial forms of IIP appear to be transcriptionally distinct, the histopathologic categories of UIP and NSIP appear to be remarkably similar. In aggregate, our findings have identified several categories of genes, as well as specific genes, that appear to be important in the development and progression of IIP.
Our findings suggest that familial IIP is simply a more extreme biological variant of IIP. Genes that distinguish the inherited form of the disease from sporadic cases of IIP belong to the same functional categories as genes that are differentially expressed among all patients with the disease relative to normal control subjects (28
). In fact, our results suggest that the expression changes are simply more extreme among familial cases of IIP when compared with sporadic cases. These findings suggest that identifying susceptibility genes in families with a propensity for this disease will also be informative in understanding the pathogenesis of sporadic IIP.
Although IPF/UIP and NSIP are thought to be clinically distinct disorders of the interstitium, our results indicate that these diseases are transcriptionally similar. Thus, despite the differences in clinical and morphologic features of IPF/UIP and NSIP, gene expression profiles suggest that these are similar disease processes. It is tempting to speculate that NSIP occurring earlier in life and having a better prognosis has the potential to progress to IPF/UIP, and that IPF/UIP simply represents that late presentation of untreated (or poorly responsive) NSIP. This speculation is supported by independent observations. First, it is well known that a substantial number of patients have histologic evidence of both UIP and NSIP in the same lung (6
). Moreover, we have found that a substantial portion of the families with familial IIP had several radiographic or histologic patterns of IIP (most often including UIP and NSIP), suggesting that the different histologic types of IIP may be related etiologically and even pathogenically (7
). Although more research is needed, multiple lines of evidence suggest that UIP and NSIP do not always represent distinct forms of IIP. However, different subtypes of NSIP were identified on the basis of gene expression profiles (similar to IPF, similar to HP, or not similar to either) (29
). Our study identified only a few differences between transcription profiles of whole lungs despite differences in cell types (i.e., the presence of lymphocytes in NSIP) and biological processes (formation of fibroblastic foci, and aberrant epithelial and vascular remodeling in UIP) present in the two disease subtypes. One limitation of both the previous and present studies is the small sample size for NSIP, suggesting that future studies with more cases will be needed to validate our initial findings. In addition, gene expression profiling of different cell types isolated from NSIP and UIP lungs may provide further insight into biological processes underlying these two disease subtypes.
Our results indicate that chemokines of the CXCL family play a role in the fibroproliferative response of the lung. Although this has been suggested by others (34
), our findings are specifically supported by a study (37
) demonstrating that circulating fibrocytes are recruited to bleomycin-treated lung in response to CXCL12. Phillips and coworkers also found that fibrocyte trafficking to the lung could be attenuated by using a specific neutralizing anti-CXCL12 antibody. Hashimoto and coworkers (35
) used chimeric mice whose bone marrow–derived cells were labeled with the green fluorescent protein to show that lung fibroblasts in pulmonary fibrosis can be derived from bone marrow progenitor cells and that CXCL12 and CXCR4 are overexpressed in the lungs of bleomycin-treated chimeric mice. Although the role of the circulating fibrocytes once in the lung is at present uncertain, it is becoming clear that the CXCL chemokines play an important role in fibroblast recruitment and fibroproliferation.
Although the finding that mice lacking one copy of the CXCR4 gene develop substantially less lung fibrosis than do wild-type control mice is supported by other published studies (35
), the increased infiltration of lymphocytes to the lungs of C57BL/6CXCR4+/−
animals compared with C57BL/6CXCR4+/+
control mice 2 wk after bleomycin administration is a novel observation. Inflammatory response generally precedes the development of fibrosis in the bleomycin model of lung injury and mostly resolves by 14 d after bleomycin treatment (33
). The fact that more inflammation persists in the CXCR4 heterozygous animals than in homozygous control animals on Day 14 is a result that requires further investigation. It is possible that the CXCR4–CXCL12 axis is involved in the development of pulmonary fibrosis via mechanism(s) other than fibrocyte homing, especially considering the fact that CXCR4 is expressed on a number of different hematopoietic and nonhematopoietic cell types. Another possibility is that there is a compensatory increase in expression of other chemokines/receptors in response to bleomycin in the absence of one copy of CXCR4.
Expression of CXCR4 and its ligands was examined at the RNA and protein levels in lung biopsies from patients with UIP or NSIP, and in normal margins from tumor cases (40
). Choi and coworkers showed that no significant differences in expression of CXCR4 exist between UIP and normal tissue nor between UIP and NSIP, which is in agreement with our expression data. However, the finding that CXCL12 is not differentially expressed in IIP compared with normal control subjects is different from the results of our study and quite surprising, considering both our findings and those by Phillips and coworkers (37
). One possible explanation for this difference is the choice of normal control subjects; gene expression in normal tissue from tumor margins can be altered due to the close proximity to tumor cells. Clearly more studies will be needed to address the expression of chemokines and their receptors in IIP.
Aberrant activation of the Wnt/β-catenin signaling pathway has been proposed as a potential molecular event leading to dysregulated repair in the fibrotic lung (41
). Our findings indicate that several components of the Wnt signaling pathway are differentially expressed in the lungs of patients with IIP; for example, SFRP2 is almost fourfold upregulated in the diseased tissue. Interestingly, SFRP1 was identified as important in the susceptibility to bleomycin-induced lung injury of mice, using a combination of quantitative trait locus mapping and gene expression analysis (42
). Defects in Wnt signal transduction lead to initiation of various tumors, but the role of the pathway in the context of fibroproliferation and fibrogenesis is still being defined. One possible mechanism might involve the metalloproteinase matrilysin/MMP-7, a target of Wnt/β-catenin trans
-activation and a molecule found to be involved in IPF (28
). There is also evidence for a role of Wnt signaling in the induction of epithelial–mesenchymal transition (43
), an important process that occurs during fibroproliferative repair after lung injury. Finally, studies have implicated a role for the Wnt pathway in self-renewal of hematopoietic stem cells and at several stages of lymphocyte development by providing proliferative and/or maintenance signals to these cell populations (44
). Thus, the role of the Wnt/ β-catenin signaling pathway in lung fibrosis is in need of further investigation.
In summary, we have identified several functional categories of differentially expressed genes in the fibrotic lung compared with normal lung tissue that are associated with a familial history of IIP. We have also shown that there are considerable gene expression changes between familial and sporadic IIPs but that UIP and NSIP are transcriptionally similar. Finally, our findings highlight the importance of CXCL12/CXCR4 in the pathogenesis of IIP. In aggregate, our study has identified several categories of genes that are involved in fibroproliferation and suggests that transcriptionally regulated genes may more precisely define the type and activity of the interstitial fibroproliferative disease process.