Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0093OC
© 2006 American Thoracic Society DOI: 10.1165/rcmb.2005-0093OC Gene Expression Profiles of Mst1r-Deficient Mice during Nickel-Induced Acute Lung InjuryDepartments of Surgery, Pediatrics, and Environmental Health and Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio Correspondence and requests for reprints should be addressed to Susan E. Waltz, Ph.D., Department of Surgery, University of Cincinnati College of Medicine, SRU/MSB G482 ML 0558, 231 Albert Sabin Way, Cincinnati, OH 45267-0558. E-mail: susan.waltz{at}uc.edu
Previous studies have shown that mice deficient in the tyrosine kinase domain (TK–/–) of the receptor Mst1r have an increased susceptibility to nickel (Ni)-induced acute lung injury (ALI). Mst1r TK–/– mice have decreased survival times, alterations in cytokine and nitric oxide regulation, and an earlier onset of pulmonary pathology compared with control mice, suggesting that Mst1r signaling, in part, may regulate the response to ALI. To examine the role of Mst1r in ALI in more detail, we compared the gene expression profiles of murine lung mRNA from control and Mst1r TK–/– mice at baseline and after 24 h of particulate Ni sulfate exposure. Microarray analyses showed a total of 343 transcripts that were significantly changed, either by Ni treatment, or between genotypes. Genes responsible for inflammation, edema, and lymphocyte function were altered in the Mst1r TK–/– mice. Interestingly, the genes for several granzymes were increased in Mst1r TK–/– mice before Ni exposure, compared with controls. In addition, the Mst1r TK–/– lungs showed clusters of cells near the vascular endothelium and airways. Immunohistochemistry indicates these clusters are composed of macrophages, T cells, and neutrophils, and that the clusters display granzyme protein production. These results suggest that Mst1r signaling may be involved in the regulation of macrophage and T-lymphocyte activation in vivo during injury. This assessment of gene expression indicates the importance of genetic factors in contributing to lung injury, and points to strategies for intervention in the progression of inflammatory diseases.
Key Words: acute lung injury • gene expression arrays • hepatocyte growth factor–like protein • Mst1r • Ron receptor
Acute lung injury (ALI) is an often fatal condition that is marked by progression through critical stages of changing endothelial and epithelial permeability, surfactant disruption, arterial hypoxemia, reduced pulmonary compliance, and, ultimately, death due to respiratory failure (1, 2). To identify genes responsible for the progression of ALI, this study used a mouse model system with nickel sulfate (NiSO4) as the pulmonary irritant. Within hours, NiSO4 induces alveolar damage that resembles the initial phase of ALI and that, with continued exposure, results in death (3, 4). Microarray analyses on mice exposed to NiSO4 have indicated that the overall pattern of gene expression during progression of ALI is consistent with increases in oxidant responses, hypoxia, extracellular matrix repair, and decreases in surfactant-associated proteins (5). One hypothesis to come out of these data is the suggestion that receptor tyrosine kinases may be important in regulating the response to Ni. One tyrosine kinase receptor, macrophage-stimulating 1 receptor (Mst1r, a c-met–related tyrosine kinase, also known as the Ron receptor), has been shown to play a critical role in Ni-induced lung injury in mice (6). Mst1r is a transmembrane receptor for macrophage stimulating protein (also known as hepatocyte growth factor–like protein [HGFL]) (7–9). Stimulation of Mst1r leads to its transphosphorylation and the ultimate activation of numerous intracellular signaling pathways, such as the classical mitogen-activated protein kinase pathway, the phosphotidylinositol (PI)3-kinase pathway, and the JNK pathway (10).
To investigate Mst1r signaling during acute injury, mice lacking the catalytic tyrosine kinase domain of Mst1r have been generated, which lack the ability to activate Mst1r-dependent downstream signaling cascades (11). Using these mice, Mst1r has been shown to be important in the response to a number of murine acute injury models (11, 12). In the Ni-induced ALI model, we demonstrated that the Mst1r tyrosine kinase–deficient mice (TK–/–) have significantly decreased survival times compared with control mice (TK+/+) (6). These Mst1r TK–/– mice displayed earlier increases in IL-6, chemokine (C-C motif) ligand 2 (CCL2; also known as MCP-1) and chemokine (CXC motif) ligand 2 (CXCL2; also known as MIP-2), and had augmented serum nitrite levels and enhanced tyrosine nitration compared with Mst1r TK+/+ mice. This increased cytokine and serum nitrate production is associated with an earlier onset of pulmonary inflammation, edema, and lethality (6). To gain insight into the potential role for Mst1r in response to NiSO4, this study used several techniques to analyze the differences between Mst1r TK+/+ and Mst1r TK–/– mice. Gene expression patterns of the lung, in response to NiSO4, were generated with oligonucleotide microarrays containing probe sets for Overall, the data obtained from our analyses suggest that genes involved in the inflammatory response may contribute to the elevation of epithelial and alveolar damage observed during Ni-induced ALI in the Mst1r TK–/– mice, possibly through enhanced lymphocyte-mediated cytotoxicity, cytokine release, and macrophage activation (3, 5). Several striking differences between the Mst1r TK+/+ and Mst1r TK–/– mice were observed. One difference was in the gene expression pattern of a family of proteins termed granzymes. Granzymes are a family of serine proteases found almost exclusively in the granules of cytotoxic T lymphocytes and natural killer (NK) cells. Another key area of difference was observed in the chemokines, cytokines, and their receptors. These changes in gene expression may result from, or be causative agents in, the accumulation of immune cells in Mst1r TK–/– mouse lungs. In either case, the functional status of the Mst1r receptor tyrosine kinase plays a significant role in regulating important immune system cells and molecules, which together lead to multiple changes in response to ALI.
Mice and Nickel Sulfate Exposure The mice used in this experiment contained a targeted disruption of the tyrosine kinase domain of the receptor Mst1r, and have been described previously (6, 11). Briefly, the experimental mice produce a truncated form of Mst1r, which contains only the extracellular and transmembrane domains of the protein plus five amino acids of the cytoplasmic domain, thus abolishing Mst1r intracellular signaling. These mice are designated Mst1r TK–/–, whereas their controls, designated as Mst1r TK+/+, express the wild-type Mst1r protein. Both genotypes are in a mixed 129/Black Swiss/CD-1 background. Mice (8–12 wk) were placed into a 0.32-m3 stainless-steel inhalation chamber and continuously exposed to filtered air containing 115 ± 7 ug/m3 NiSO4 particulate aerosol (0.2 mm mass median aerodynamic diameter, 1.85 geometric standard distribution) (1, 5). Nickel aerosol was generated from 50 mM Ni sulfate hexahydrate (Ni2SO4 · 6H2O) solution as described previously (4).
Microarray Analysis
Data Analysis
Identification of Gene Expression Patterns
RNA Analysis
Diagnostic Analysis of Granzymes D–G
Immunohistochemistry
Myeloperoxidase Assay
Due to the fact that Mst1r TK–/– mice show increased lung injury in response to NiSO4 exposure, and that this increased injury appears to be a multifactorial process, we hypothesized that changes in the expression of numerous genes may cooperate to mediate the difference between Mst1r TK–/– mice and control animals in this ALI model. To identify differentially expressed genes, RNA was isolated from lung tissue of control (Mst1r TK+/+) and Mst1r TK–/– mice at 0 and 24 h after Ni exposure. This RNA was subjected to oligonucleotide genechip analysis using the murine U74Av2 chip from Affymetrix. The hybridization profiles from technical replicate chips were combined, and differences in genes, the expression of which differed by at least 2-fold between any two groups, were considered significant (19, 20). Using this moderate level of stringency, a total of 343 transcripts were determined to be differentially expressed. Several of these genes, particularly those for granzymes, were validated by Northern analysis. The genes identified by the microarray experiments encompass a number of functions, from regulation of inflammation and the immune system, to genes involved in apoptosis and cell cycle progression. These analyses have provided two main types of information: the changes in gene expression that are related to Ni inhalation, regardless of the genotype of the animal; and the changes in gene expression that are different in animals with a deletion of Mst1r signaling compared with control mice.
Cluster Analysis of Gene Expression in Mst1r TK+/+ and TK–/– Mice
The largest number of differentially expressed genes occurred in the two clusters that are modulated during NiSO4-induced ALI, independent of genotype (Set 2 and Set 4). In Set 2, there are 114 genes with decreased transcript levels in lung tissue after NiSO4 exposure to approximately the same extent in Mst1r TK–/– mice as in TK+/+ mice. The decreased expression of many genes in this group is likely related to the epithelial and endothelial cell injury and decreased differentiated function seen in this model. Genes whose expression is increased to the same extent after Ni exposure in both groups of mice form the next largest set, Set 4, with 96 genes. Included in this group are genes for heat shock proteins (Hspa1b, Hsp70–3), serum amyloid proteins, signaling molecules (JunB), and IL (IL-1b, ILr-2). Another broad grouping of genes is those that increased in both genotypes after NiSO4 inhalation, but to a greater extent in one genotype than the other. Set 3 shows 26 genes that increased slightly more in Mst1r TK+/+ mice than in Mst1r TK –/– mice after Ni treatment. Similar to Set 3 is Set 6, consisting of 11 genes that were very strongly increased in wild-type mice, but which barely changed in TK–/– mice after treatment. This group has a number of contractile proteins, including actin, troponin T, and several myosin genes (Myl2, Myh1). Two groups of genes increased more in Mst1r TK–/– animals than in control animals. Set 1 is comprised of five genes that were more strongly increased in Mst1r TK–/– mice after NiSO4 treatment than in control mice, including IL-6 (Il6), and the IL-1 receptor antagonist (Il1rn). The genes of Set 7 were similarly, though not quite as strongly increased. Several important chemokines (CXCL5, CCL9, CCL22) are in this group. Two groups of genes were decreased in a genotype-dependent manner after Ni exposure. The 20 genes in Set 9 were decreased in Mst1r TK+/+ mice more than they decreased in Mst1r TK–/– mice after NiSO4 treatment. This group includes IFN regulatory factor 4 (Irf4) and at least 2 Ig-related genes. The genes of Set 11 also decreased more in Mst1r TK+/+ mice than in Mst1r TK–/– mice, but these genes started out with a higher baseline expression level before Ni treatment, as well. There are five genes in this group, including procollagen (Col15a1), TNF receptor–associated factor (Traf1), and the PI3-kinase regulatory subunit (Pik3r2). This is especially noteworthy, as the PI3 kinase pathway is one of the cascades used in Mst1r intracellular signaling. The genes of Set 10 show an interesting regulation; they were highly expressed in untreated Mst1r TK–/– mice, but not in wild-type animals. After NiSO4 treatment, the expression of these transcripts in Mst1r TK–/– mice was decreased to the baseline levels seen in control mice. Four of the five genes in this group are members of the granzyme (Gzm) family (granzymes D, E, F, and G). There were two groups of genes that were initially more highly expressed in one genotype than the other, and although there was some regulation of those genes by NiSO4 exposure, these genes always remained higher in the select genotype. Of these genes, Set 5 was higher in Mst1r TK–/– mice throughout the experiment, compared with Mst1r TK+/+ controls. This set includes several Ig genes (for both the light and heavy chains) as well as the B-cell antigen receptor gene (Igh-VJ558). Finally, Set 8 is composed of 13 genes that remained higher in Mst1r TK+/+ mice than in the Mst1r TK–/– mice, regardless of treatment.
Functional Classification of Genes
Genes controlling cytokine and chemokine production also show regulation in this model (Table 1 and Figure 2). Most of these genes were increased in both genotypes to a similar extent. However, IL-6 and three chemokine ligand genes, CCL9, CCL22, and CXCL5, were increased more in Mst1r TK–/– mice than they were in control mice (Figure 2).
Confirmation of Gene Expression Data To provide an additional confirmation of the results obtained from the microarray hybridization experiments, mRNA levels for seven selected genes were assayed by Northern analysis. These genes showed a significant change after 24-h exposure in at least one genotype. This group includes amphiregulin, IFN-  , serum amyloid, and troponin T (Figure 3), as well as granzymes D, F, and G (Figures 4, 5, and 6).
Amphiregulin is a ligand for the epidermal growth factor receptor, and the binding of amphiregulin to epidermal growth factor receptor stimulates proliferation of lung epithelial cells (21). The microarray data revealed 20- and 10-fold increases in expression of this gene in Mst1r TK+/+ and Mst1r TK–/– mice after 24 h of Ni exposure, respectively. The Northern analysis illustrates a similar pattern of expression, with amphiregulin RNA levels increasing after Ni exposure in both genotypes, but increasing to a much greater extent in Mst1r TK+/+ lungs compared with the Mst1r TK–/– lungs (Figure 3). Expression of genes related to serum amyloids were also identified in the microarray analysis. Serum amyloid A (Saa) 3 is an acute-phase apoprotein of high-density lipoproteins found in mammals. This protein is produced in response to inflammation. The transcript level of Saa3 in both genotypes was increased after 24-h exposure to NiSO4, with the Northern blot showing slightly higher expression in Mst1r-deficient animals (Figure 3). A similar expression pattern was observed with troponin-T, which is part of the contractile apparatus and binds other troponin subunits to tropomyosin (22, 23). Very little troponin T was detected at baseline in either genotype. By 24 h after NiSO4 treatment, troponin levels had increased to a similar extent in both TK+/+ and TK–/– mice (Figure 3).
IFN-
Expression Analysis of Granzymes To confirm the increased granzyme gene expression seen on the microarray, a Northern analysis was performed using RNA isolated from the lung, liver, thymus, and spleen of unchallenged Mst1r TK+/+ and Mst1r TK–/– mice (Figure 4). The thymus and spleen were chosen because they are expected to contain a high percentage of T cells and NK cells (two cell types that are known to express high granzyme activity), whereas the liver is a representative tissue that should have relatively few of these cells. Northern analysis for lung tissue confirms the microarray finding that granzymes D, F, and G are elevated in untreated mice that are deficient in Mst1r TK signaling compared with untreated control mice (Figure 4). In addition, the Northern blot identifies granzyme C as being increased in untreated Mst1r TK–/– mice compared with untreated control mice. These four granzymes (C, D, F, and G) are also elevated in the spleen of Mst1r TK–/– animals and, except for granzyme C, in the thymus as well. In contrast, liver tissue shows similar, baseline granzyme levels in Mst1r TK+/+ and Mst1r TK –/– animals (Figure 4). The genes that code for granzymes D through G have a very high degree of sequence homology (ranging from 84 to 94%) (25, 26). Due to this high degree of homology, a probe for any one of these granzymes genes may hybridize to the other genes in this group (18). To determine if all four of these transcripts are simultaneously expressed in the Mst1r TK–/– mice, RT-PCR was performed on lung RNA, and the amplification products were then subjected to diagnostic restriction enzyme analysis (18). One primer set was generated that would specifically amplify RNA products from granzymes D–G, but not the rest of the granzyme family members (18). After RT-PCR with these primers, restriction analysis with BstXI, BglII, or HindIII/PstI was used to distinguish between the mRNAs encoding granzymes D–G (Figure 5). The results of the RT-PCR analysis show the expected restriction digest fragments for these four granzymes, confirming that granzymes D, E, F, and G are all expressed in the same sample. To determine the status of the granzyme genes as a family, cluster analyses were performed (Figure 6). Although a number of members of this family were upregulated in the TK–/– mice at baseline (granzymes D, E, F, and G), other members of this family increased after NiSO4 treatment. These Ni-responsive genes include granzymes M and B. The striking Mst1r-dependent impact on this gene family suggests Mst1r as an important coordinate regulator of this gene cluster in vivo.
Histology of Mst1r TK+/+ and TK–/– Mouse Lungs
To access overall lung histology in greater detail, tissues were taken from untreated Mst1r TK+/+ or Mst1r TK–/– mice and processed for histologic analyses by hematoxylin and eosin staining (Figure 7). Microscopic analysis of these sections revealed cellular clusters in the lungs of Mst1r TK–/– mice, but not in the lungs of wild-type mice (Figure 7B). These clusters appear near larger vessels and airways in the lung. To determine if there is a potential for Mst1r signaling to influence this cellular clustering, immunohistochemistry was performed on sections taken from lungs of wild-type mice, using an anti-Mst1r antibody. Mst1r-positive staining was principally limited to airway epithelial cells (Figure 7A).
To identify the cell types that compose these clusters, additional immunohistochemistry was performed on lung tissue sections from Mst1r TK–/– mice (Figure 8). An anti-CD3e antibody, which recognizes the T-cell receptor antigen, showed strong staining of cells within the clusters, consistent with the presence of a potentially broad population of T-cell subtypes. Antibodies against CD 4 and CD 8 both showed positive staining in some cells of the clusters, indicating the presence of CD4- and CD8-positive lymphocytes. Staining for the F4/80 antigen, which is present on macrophages, was also seen in some cells of the clusters, whereas neutrophils were identified in the clusters using an antineutrophil antibody. No staining was observed in Mst1r TK+/+ lungs for any of the antibodies utilized in these analyses (Figure 8). In addition, no background staining was observed in serial sections of the Mst1r TK–/– lungs stained with isotypic control antibodies (data not shown). The staining pattern in Figure 8 indicates that the clusters of cells in lungs of untreated Mst1r TK–/– mice include cells critical to innate immunity, which are not seen in the lungs of Mst1r TK +/+ mice. Whereas the composition of cells within individual clusters varied, the clusters were made up of mainly T cells, followed by macrophages and neutrophils. To determine if the cell clusters contributed to the differential expression of granzyme proteins observed in the Mst1R TK–/– mice, immunohistochemistry was performed. As is apparent in Figure 9, granzyme D protein expression is present in the cellular clusters of the lungs of the TK–/– mice, whereas no expression is detected in control lungs.
To determine if the cell clusters seen in lungs from Mst1r TK–/– mice correspond to an increased number of neutrophils, the level of MPO activity was measured in homogenates from untreated wild-type and Mst1r TK–/– lungs. MPO, a prominent enzyme contained in azurophilic granules, is an index of neutrophil infiltration. The Mst1r TK–/– lungs had about a 4.5-fold greater MPO activity than that in the Mst1r TK–/– lungs (Figure 10), a finding consistent with an increase in neutrophil numbers in the Mst1r TK–/– lungs noted with the antineutrophil staining (Figure 8).
The Mst1r receptor TK is the receptor for macrophage-stimulating protein (also known as HGFL). Signaling through this ligand-receptor complex influences numerous physiologic functions, including cell adhesion and motility, carcinogenesis, inflammation, and modulation of the immune system (10–12, 27). It is known that Mst1r signaling occurs through several intracellular pathways, with the potential to activate transcription of many different genes (10, 28). ALI evokes many changes in gene expression, including changes in genes involved in innate immunity. Previous work has shown that mice lacking the ability to signal through Mst1r (Mst1r TK–/–) are more susceptible to Ni-induced ALI and have a significantly decreased survival time after Ni exposure than control mice (6). This increased lethality of NiSO4 inhalation in Mst1r TK–/– mice may indicate a protective role for Mst1r signaling, whereby activity of Mst1r serves to modulate the tissue-damaging effects that can be present during acute immune system activation. Due to the potential for Mst1r to influence expression of a wide variety of genes, this study was undertaken to evaluate the contribution of Mst1r signaling to gene expression in the NiSO4-induced ALI model, and to correlate the genetic changes to differences in the biological phenotype of these mice. Changes in gene expression after Ni-induced ALI were monitored using an Affymetrix murine microarray, which surveys more than 12,000 different DNA sequences. A comparison of the gene expression profile from control (Mst1r TK+/+) mice and Mst1r TK–/– mice, both before and after 24 h of continuous exposure to aerosolized Ni particles, revealed 343 probe sets, the level of which was changed at least two-fold in one or more pair-wise comparisons. Some of these changes may have resulted from the alveolar cellular defense against damage, whereas others may have potentiated the damage when repair became impossible. Among this set of 343 differentially expressed transcripts, 8 were further characterized by Northern analyses. The results of the microarray and Northern analyses showed similar levels of gene activation, indicating that the changes seen on the gene array reflect actual differences in mRNA expression. Earlier work exploring the changes in gene expression profile after NiSO4 treatment in C57BL/6 mice showed both increasing and decreasing gene expression over a time course of 3–96 h (6). Many of the genes identified in that study also showed changes in expression here. Included in this group is metallothionein-1 and galectin-3, which increased strongly in both genotypes after treatment. In addition, the present study also identified metallothionein-2 as strongly increased in both genotypes after treatment. Metallothioneins are small, cysteine-rich proteins that bind a number of metals, including zinc and cadmium. Evidence indicates that they are produced by the body in response to stress, and thus may play a role in immune function. Another gene that was identified by McDowell and colleagues as being increased after Ni exposure was elastin (6). Elastin is an extracellular matrix protein that is responsible for the elastic properties of many tissues. This macromolecule is a critical component of the lung interstitium and helps to provide the elastic recoil necessary for proper ventilation. Our study also identified elastin as being increased after Ni exposure, but only in the Mst1r TK+/+ animals, not in the Mst1r TK–/–. This lack of elastin induction in Mst1r TK–/– mice indicates that Mst1r signaling is likely needed for expression of this gene after severe lung insult. The inability of Mst1r TK–/– mice to increase production of this elastic macromolecule after lung injury may lead to decreased ventilation, which could contribute to the decreased survival seen in Mst1r TK–/– mice. One particularly interesting grouping of genes to emerge from this study was the genes in Set 6 of Figure 1. These genes were increased in control animals after 24 h of Ni exposure, but were not increased in Mst1r TK–/– mice. Included in this grouping were three genes for myosin, one for troponin, and one for actin; all are important for muscle function (23). If the changes in gene expression seen in these samples reflects the changes seen in airway or vascular smooth muscle, this finding may point to a functional mechanism for the previously observed decrease in survival seen in Mst1r TK–/– mice after Ni-injury (6). The inability of Mst1r TK–/– mice to increase these muscle protein mRNAs may correlate with decreased survival of Mst1r TK–/– mice after NiSO4 exposure. This suggests a novel role for Mst1r during an acute challenge. Another novel finding of this study is that granzymes C, D, E, F, and G were dramatically overexpressed in Mst1r TK–/– mice before injury. After 24-h exposure to NiSO4, transcript levels for granzymes D–G were significantly decreased in Mst1r TK–/– mice (to the levels observed in control animals). Termed "Orphans" (24), the function of granzymes D–G in mice is unknown, although their related family members, granzymes A–C are important in cytolytic cell killing. Grossman and colleagues have suggested that granzymes C–H may be a "fail-safe" mechanism for murine innate immune function (24). Such redundancy would allow cytotoxic T and NK cells (where granzymes are produced) to clear a multitude of infectious agents. However, our data suggest that granzymes C, D, E, F, and G, at least, may also have a functional role in noninfectious models of ALI. One possible role of NK cells may be recognition and removal of apoptotic and necrotic epithelial cells, which may be a critical event in wound healing. On this note, the increase in Amphiregulin may have worked in consort to improve healing in the wild-type mice, because it was increased more in these mice than in Mst1r TK–/– mice. The coordinated regulation of granzymes D–G is potentially significant, as these genes cluster together on mouse chromosome 14 (24). Expression of these four genes in unchallenged mice that are devoid of Mst1r signaling may indicate that Mst1r serves to downregulate expression of this group of granzymes under normal conditions. In addition, because differences were noted more in the lung, thymus, and spleen than in the liver, these changes in granzymes may reflect a shifting in T cell and NK cell lineage under control of Msp/HGFL and Mst1r. The present study also provides information on Saa, which is increased in the lung during ALI. A large increase in the level of Saa3 was observed in both mouse genotypes after NiSO4 exposure, and may possibly be linked to the pathophysiology of acute inflammatory diseases. Serum amyloids have a role in the maintenance of the epithelial cells, similar to other epithelium-specific genes, such as keratins or junction proteins (29). Because epithelium-specific gene expression is fundamental to the maintenance and function of lung tissue, the induction of serum amyloid family members in mice after the lung airway epithelia was exposed to NiSO4 may be part of the host defense mechanism that protects these tissues from potential injuries. Earlier studies indicated that an important feature of this ALI model is the increase of endothelial nitric oxide synthase activity and nitric oxide production after Ni treatment, which, in part, contributes to the pathogenesis of ALI (1, 4–6). In the context of the Ni-induced ALI, the pathogenesis of this disease may involve multiple signaling pathways; one that directs an insult on epithelial and alveolar cells, and others that involve activation of alveolar macrophages in the lung. Leukocyte recruitment requires intercellular signaling between infiltrating leukocytes and the endothelial cells. These events are mediated via the generation of molecules such as chemokines. For instance, the present study showed a number of chemokines and cytokines, such as IL-1b, CXCL1, and CCL7, CCL9, CCL11, CCL17, and CCL22, that all increased after NiSO4 challenge. The changes seen in these chemokines and cytokines may, in fact, be causative agents in the increased PMN infiltration seen in Mst1r TK–/– mice after 36 h of Ni treatment. Before any treatment, clusters of cells were clearly seen in the lungs of all of the Mst1r TK–/– mice examined, but were not found in wild-type mice. These clusters form near larger blood vessels and around the airway epithelium of the lungs, where Mst1r immunostaining is normally found. An analysis of the clusters shows cells with markers for several immune system subtypes, including T cells, neutrophils, and macrophages. The presence of clusters of immune cells in mice that are deficient in Mst1r signaling may indicate that this TK receptor is responsible for regulating immune cell infiltration, possibly through changes in cytokine production. With these cells already accumulating in Mst1r TK–/– mice, the potential exists for them to cause cytotoxic damage as soon as they are activated after insult, such as occurs in this model of ALI. The various consequences of increased cytotoxic damage during ALI may be a factor in the decreased survival time of Mst1r TK–/– mice during NiSO4 inhalation. The data presented here indicate a role for the Mst1r signaling pathway in the regulation of macrophage and lymphocyte activation and provide an in vivo model with which to examine the biochemical mechanism beyond the action of cytokines and chemokines. Current data point to a broad role for the Mst1r pathway in regulating macrophage and lymphocyte activation and the resulting proinflammatory signals. The mice described here are a valuable tool with which to define the Mst1r signaling pathway and its regulation of cytokine/chemokine signaling in a biologically relevant system. Future work will be required to identify the mechanisms by which Mst1r mediates suppression of regulators of cytokine-inducible gene expression, and to determine whether the Mst1r signaling pathway affects other signals implicated in macrophage activation. In summary, this study revealed the expression patterns of genes associated with ALI in relation to the lack of the TK domain of the receptor Mst1r. It also points to relationships initiated from the temporal and functional analyses of differential gene expression during Ni-induced ALI. The most obvious genetic changes were increases in the expression of genes involved in inflammation. In addition, untreated Mst1r TK–/– mice display perivascular and peribronchial clustering of cells within the lung. These cells stain positive for markers found on a variety of lymphocytes, as well as for granzyme D, which is potentially important in cytotoxic T-cell function. The important suggestions that arise from these data are that a variety of lymphocytes may contribute to alveolar injury in mice lacking the TK domain of Mst1r. Activation of these lymphocytes may contribute to the alterations observed in these animals after Ni exposure, and may provide insights into a novel pathway that can influence ALI.
The authors acknowledge Kenya Toney for expert technical support.
* These authors contributed equally to this work.  This work was supported by Public Health Services grants DK073552 (S.E.W.), ES10562 (G.D.L.), HL-65612 (G.D.L.), ES06096 (G.D.L.), National Institutes of Health Training Grant T32 GM08478–10 (L.W.K.), by Cincinnati Shriner's Hospital for Children Project No. 8950 (S.E.W.). S.A.M. was a recipient of the U.S. Environmental Protection Agency Science to Achieve Results Graduate Fellowship and the Albert J. Ryan Fellowship. Originally Published in Press as DOI: 10.1165/rcmb.2005-0093OC on September 15, 2005 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 3, 2005 Accepted in final form September 2, 2005
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Abstract
Introduction
12,000 murine genes. Changes in gene expression patterns were monitored at baseline and after 24 h NiSO4 exposure in control mice and Mst1r tyrosine kinase–deficient mice. Histologic analysis was performed on lung tissue from both control and Mst1r-deficient mice before Ni exposure. These studies revealed an accumulation of immune cells in the lungs of Mst1r TK–/– mice, which was not seen in wild-type mice. 
-V8, and Ig
