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Published ahead of print on September 11, 2003, doi:10.1165/rcmb.2003-0062OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 438-448, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0062OC

Lymphoid Tissue and Emphysema in the Lungs of Transgenic Mice Inducibly Expressing Tumor Necrosis Factor-{alpha}

Brian R. Vuillemenot, Juan F. Rodriguez and Gary W. Hoyle

Department of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana

Address correspondence to: Gary W. Hoyle, Ph.D., Section of Pulmonary Diseases, Critical Care and Environmental Medicine, SL-9, 1430 Tulane Ave., New Orleans, LA 70112. E-mail: ghoyle{at}tulane.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To develop a model in which the pathogenic effects of the proinflammatory cytokine tumor necrosis factor-{alpha} (TNF) could be investigated, transgenic mice that express TNF in the lung under the control of a doxycycline-inducible promoter were generated. TNF transgene message was expressed at a low level in the absence of doxycycline treatment and was induced in the lung by administration of the drug. Analysis of lung lavage fluid indicated increases in neutrophils and lymphocytes in doxycycline-treated transgenic mice. Histologic analysis of lungs from adult transgenic mice treated with doxycycline revealed prominent development of lymphoid tissue and increases in airspace size. Genes upregulated in TNF transgenic mice, as identified by oligonucleotide microarray analysis, included a variety of transcripts expressed in lymphoid tissues. Immunohistochemical analysis demonstrated the presence of B lymphocytes and, to a lesser extent, T lymphocytes within lymphoid aggregates in TNF transgenic mice. CD8-positive T cells were absent from lymphocytic nodules, but in the lung parenchyma were more abundant in transgenic than in nontransgenic mice. These results indicate that induction of TNF in adult lung promotes the formation of lymphoid tissue and emphysema, and provides a model in which the pathogenic effects of TNF on the lung can be investigated.

Abbreviations: doxycycline, dox • mean linear intercept, Lm • reverse tetracycline transactivator, rtTA • surfactant protein C, SPC • tumor necrosis factor-{alpha}, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} (TNF) is a proinflammatory cytokine that has been implicated in the pathogenesis of a variety of lung diseases. For example, TNF levels are upregulated in the lungs of patients with chronic obstructive pulmonary disease (1) and in patients with pulmonary disorders associated with autoimmune diseases (2) and HIV infection (3). In spite of these observations, the mechanisms by which TNF contributes to these pathologic states are not well characterized. TNF may exert pathologic effects in the lung by regulating the influx of inflammatory cells such as neutrophils and lymphocytes. Alternatively, TNF may act on resident cells in the lung to regulate gene expression and cell death. To develop a model in which the pathogenic effects of TNF could be experimentally controlled and investigated, we generated transgenic mice that express TNF in the lung under the control of a doxycycline-inducible promoter. In these mice, TNF expression is repressed in the absence of doxycycline treatment, and therefore the consequences of acutely upregulating TNF expression in the adult lung can be assessed. Results obtained from these mice in the present study indicated that induction of TNF expression resulted in focal lymphoid tissue formation in the lung. In addition, mice administered doxycycline for 1–9 mo developed significant increases in airspace size indicative of emphysema.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic Mice
The surfactant protein C (SPC)–reverse tetracycline transactivator (rtTA) construct containing the rtTA gene under control of the human SPC promoter was generated by cloning a 1.0-kb EcoRI–BamHI fragment encoding rtTA from the plasmid pTetOn (Clontech, Palo Alto, CA) downstream from the 3.7-kb human SPC promoter/enhancer (4). A 2.2-kb BamHI fragment from the human growth hormone gene (5) was cloned downstream of the rtTA fragment to provide intronic and polyadenylation sequences. The TRE–TNF construct containing the murine TNF gene under control of a doxycycline inducible promoter, tetracycline-responsive element (TRE), was generated by cloning a blunt-ended 1.1-kb EcoRI fragment encoding murine TNF (6) into pTRE-luc (Clontech) that had been cut with HinDIII and EcoRV to remove the translated portion of the luciferase fragment and treated with the Klenow fragment of DNA polymerase I to produce blunt ends. A 6.9-kb XhoI–NotI SPC-rtTA fragment and a 3.3-kb XhoI–ApaLI TRE-TNF fragment were gel-purified for microinjection. The constructs were co-injected into B6SJLF2-fertilized mouse eggs to generate transgenic mice as described (7). Transgenic lines were maintained by mating to B6SJLF1 hybrid mice. Mice were housed under specific pathogen–free conditions. For transgene induction, mice were administered 0.5 mg/ml doxycycline in the drinking water.

Analysis of Transgene Expression
Northern blot analysis was performed as described previously (4). RNase protection assay for mouse TNF was performed using a Riboquant assay kit (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Twenty micrograms of total lung RNA was assayed per mouse. The signal in the protected TNF band was measured by phosphorimage analysis and normalized to an internal control in the same lane (ribosomal protein L32). TNF activity in lung homogenates was measured using the L-929 cell lysis assay (8, 9). Lung extracts were prepared by homogenizing lung tissue in 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 mg/ml aprotinin, 10 µg/ml leupeptin, 40 µM bestatin, 15 µm pepstatin A, and 14 µm E-64. Homogenates were centrifuged for 10 min at 14,000 x g, and the supernatants were assayed for TNF activity. Mouse L-929 cells (kindly provided by Wayne Vedeckis, Louisiana State University Health Sciences Center) were grown in RPMI 1640 plus 10% fetal bovine serum. Cells were seeded into 96-well microtiter plates (5 x 104 cells/well) and incubated overnight at 37°C. The medium was removed and the cells were incubated overnight at 37°C with lung homogenates diluted in medium containing 1 µg/ml actinomycin D. Known concentrations of recombinant mouse TNF were assayed in parallel as standards. TNF-induced cell lysis was quantitated using the MTT cell proliferation kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. TNF levels in lung homogenates were normalized to protein content as determined by Bradford assay (BioRad, Hercules, CA) with bovine serum albumin as a standard.

Histologic and Morphometric Analyses
Lung were fixed by tracheal instillation of 10% neutral buffered formalin at a pressure of 25 cm water for 20 min followed by immersion in 10% neutral buffered formalin overnight at 4°C. Lungs were cut into individual lobes, embedded in paraffin, and cut into 5-µm sections. Analysis of lymphocytic nodules and mean linear intercept was performed on sections that had been stained with hematoxylin and eosin. Lymphocytic nodules were counted in lungs sections and normalized to section area as measured using ImagePro software (MediaCybernetics, Silver Spring, MD) on digitized images. The procedure for measuring mean linear intercept was based on that previously described (10) but was adapted for computerized measurement of the number of intercepts. For each lung, images from 4–5 fields were analyzed using public domain NIH Image software. Images were edited to remove airways, blood vessels, and lymphocytic nodules; they were then thresholded manually, made binary, and inverted. Horizontal and vertical grid lines were sequentially superimposed over the images, and the number of intercepts was measured using the "Image Math" and "Analyze Particles" function of NIH Image. Mean linear intercept (Lm) was calculated from the number of intercepts as described (10).

Lung Lavage
Lung lavage and analysis of cell differential were performed as described previously (11).

Hydroxyproline Analysis
Hydroxyproline content in whole lung was measured by the method of Woessner (12).

Microarray Analysis
Experimental procedures for GeneChip oligonucleotide microarray analysis were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Equal amounts of total RNA were pooled from three doxycycline-treated TNF transgenic mice and three doxycycline-treated nontransgenic mice, and 8 µg of RNA from each pool was used to synthesize double-stranded DNA (Superscript Choice System; GIBCO/BRL, Rockville, MD). In vitro transcription to produce biotin-labeled cRNA was performed using the BioArray High Yield RNA Transcription Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Biotinylated RNA (25 µg) was fragmented to 50–200 nucleotides and hybridized for 16 h at 45°C to Affymetrix MG-U74Av2 arrays containing ~ 12,000 mouse genes. Arrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR). Staining signal was amplified by biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA) followed by second staining with streptavidin-phycoerythrin. Arrays were scanned on a Hewlett-Packard (Palo Alto, CA) GeneArray Scanner, and expression data were analyzed using Affymetrix MicroArray Suite v5.0. Signal intensities of all probe sets were scaled to the target value of 2,500. Changes in gene expression between the samples were compared using the log signal ratio statistic, which represents the log base 2 of the ratio of the signal in the experimental group (TNF transgenic mice) to the signal in the control group (wild-type mice). A log signal ratio of > 1 (indicating a 2-fold increase in signal) or < -1 (indicating a 50% decrease in signal) was considered significant.

Immunostaining
Immunohistochemistry was performed with fluorescent-conjugated monoclonal anti-mouse antibodies (BD Pharmingen, San Diego, CA). Acetone-fixed frozen sections of TNF transgenic and wild-type mice were stained with phycoerythrin-conjugated anti-B220 (clone RA3–6B2, 4 µg/ml), phycoerythrin-conjugated anti-CD3 (clone 17A2, 4 µg/ml), phycoerythrin-conjugated anti-CD11c (clone HL3, 4 µg/ml), FITC-conjugated anti-CD4 (clone H129.19, 10 µg/ml), and FITC-conjugated anti-CD8a (clone 53–6.7, 10 µg/ml).

Data Analysis
Quantitative data are presented as group means ± SEM. Group means were compared by Student's t test or ANOVA. The criterion for significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF Transgene Expression
The constructs used to produce transgenic mice inducibly expressing TNF in the lung are shown in Figure 1. Transcription of the mouse TNF cDNA is under control of a TRE consisting of tet operator sequences upstream of a minimal promoter (13). The rtTA (13) is expressed from the lung-specific SPC promoter (4, 14). The SPC–rtTA and TRE–TNF constructs were co-injected into fertilized mouse eggs to generate transgenic mice in which the two constructs were integrated at the same chromosomal location. Fifteen founder transgenic mice carrying the SPC–rtTA and TRE–TNF transgenes were generated. Ten of these founder mice gave rise to transgenic progeny that were assessed for transgene expression in the lung by Northern blot analysis. In six lines, TNF transgene message was detected in lung RNA after doxycycline treatment (not shown). Following doxycycline treatment, animals from all six of these lines developed lung pathology (described in detail below) characterized by the development of lymphocytic nodules, and mice from three of the lines developed enlarged airspaces. The line in which these effects were most pronounced, designated 320–1, was investigated more extensively.



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  		Figure 1. DNA constructs. (A) SPC-rtTA construct. The construct contains the 3.7-kb human SPC promoter (hatched), the a 1.0-kb fragment encoding rtTA (white), and a 2.2-kb fragment from the human growth hormone gene that contains intronic and polyadenylation sequences (black). (B) TRE-TNF construct. The construct contains the 0.7-kb doxycycline-responsive promoter (hatched), a 1.1-kb fragment encoding murine TNF (white), and fragments derived from luciferase (black) and SV40 (shaded) that are not translated in this construct.

 
Analysis of TNF mRNA expression in the 320–1 line is shown in Figure 2A. TNF transgene RNA was present at a low level in the lungs of mice that had not been treated with doxycycline (lane 3), and was induced by doxycycline treatment (lane 4). TNF RNA levels in transgenic mice with or without doxycycline treatment were somewhat variable; typical levels are shown. Mice in which TNF transgene expression had been induced also exhibited an upregulation of the endogenous TNF message. Analysis by RNase protection assay indicated that TNF mRNA levels were significantly increased in transgenic mice treated with doxycycline (Figure 2B). Tissue specificity of expression was assessed by performing Northern blot analysis on RNA from other tissues from doxycycline-treated mice. No TNF transgene message was observed in heart, liver, kidney, muscle, spleen, or thymus; however, a weak band was observed in brain (not shown).



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  		Figure 2. Transgene expression. (A) Northern blot analysis of lung RNA. Lung RNA was hybridized with a murine TNF probe (top) or with an 18S ribosomal probe as a loading control (bottom). Lane 1, nontransgenic; lane 2, nontransgenic + doxycycline; lane 3, transgenic; lane 4, transgenic + doxycycline. Mice were treated with doxycycline for 7 d. The sizes of the TNF transgene and endogenous TNF transcripts are indicated at the right. (B) RNase protection assay. TNF message levels were quantitated by RNase protection assay in transgenic (Tg) and wild-type (WT) mice with and without doxycycline (dox) treatment. Mice were treated with doxycycline for 1 mo. *P < 0.02 versus all other groups (n = 4 mice/group). (C) TNF bioassay. TNF polypeptide levels were quantitated by cell lysis assay in lung homogenates from transgenic (Tg) and wild-type (WT) mice with and without doxycycline (dox) treatment. Mice were treated with doxycycline for 1 wk. *P < 0.02 versus all other groups (n = 4 mice/group).

 
TNF polypeptide levels were measured in lavage fluid by ELISA. The amount of TNF in lavage fluid from TNF transgenic mice was not elevated compared with wild-type mice. In both it was near the level of detection of the assay, suggesting that TNF was produced at relatively low levels and was bound locally so it did not appear in the lavage fluid. TNF activity in lung homogenates was measured by cell killing in the TNF-sensitive L-929 mouse fibroblast line. TNF was significantly increased ~ 6-fold in the lungs of doxycycline-treated transgenic mice compared with wild-type mice (Figure 2C). Uninduced transgenic mice exhibited an intermediate level of TNF that was not statistically elevated over wild-type mice.

Lung Lavage
To examine inflammatory cell influx that occurred as a result of TNF production, TNF transgenic mice were administered doxycycline for 7 d and were then subjected to lung lavage (Figure 3). Compared with nontransgenic mice, doxycycline-treated transgenic mice exhibited increases in the number of lymphocytes and neutrophils that were recovered in lavage fluid. However, these increases were fairly modest, with lymphocytes and neutrophils comprising 2% and 6% of total cells respectively in doxycycline-treated transgenic mice.



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  		Figure 3. Lavage fluid inflammatory cells. Transgenic (Tg) and wild-type (WT) mice with or without doxycycline (dox) treatment for 1 wk were subjected to lung lavage, and the recovered cells were analyzed. *P < 0.02 versus both groups of nontransgenic mice; **P < 0.01 versus all other groups; n = 5–7 mice/group. Open bars, WT; shaded bars, Tg; striped bars, WT dox; filled bars, Tg dox.

 
Lung Pathology
TNF transgenic mice and age-matched wild-type litter mates starting at 2–3 mo of age were administered doxycycline for 1, 3, 6, or 9 mo, after which lungs were collected for histologic analysis. Doxycycline treatment in wild-type mice had no effect on lung histology (Figure 4A). Transgenic mice that received doxycycline exhibited lung inflammation characterized by cells that had the histologic appearance of lymphocytes (Figures 4B–4G). Inflammatory cells were generally not observed within the airways or airspaces but were clustered within lymphocytic nodules adjacent to airways, within the parenchyma, and adjacent to the pleural surface (Figures 4B and 4C). Some nodules contained loose collections of lymphocytes with uniformly small nuclei (Figure 4B), whereas others appeared to be more organized lymphoid tissue that was similar to bronchus-associated lymphoid tissue (BALT; Figures 4C and 4F). This organized lymphoid tissue exhibited histologic features suggestive of lymphoid follicles, e.g., a lighter stained germinal center (Figure 4F, black asterisk) surrounded by a darker lymphocyte corona (Figure 4F, white asterisk). However, these features were not as clearly defined morphologically as in follicles of spleen or lymph nodes. The lymphoid nodules contained lymphocytes at various stages of activation, apoptotic cell debris, and tingible body macrophages (Figure 4G), all of which are characteristic of germinal centers. In TNF transgenic mice that were not treated with doxycycline, varying degrees of inflammation were noted, but overall the inflammation was much less pronounced than in doxycycline-treated transgenic mice. Some animals contained multiple small to medium sized lymphocytic nodules, some contained a few small nodules (Figure 4E), and some had lungs that were indistinguishable from wild-type. This observation is consistent with the Northern blot analysis, which indicated a low yet variable level of transgene expression in the absence of induction. The number of lymphocytic nodules in histologic sections of TNF transgenic mice was counted and normalized to section area (Figure 5). The number of these clusters of inflammatory cells was significantly higher in doxycycline-treated transgenic mice than in untreated transgenic mice. In doxycycline-treated transgenic mice, the number of nodules was similar between 1 and 6 mo of treatment, but decreased between 6 and 9 mo. No inflammatory nodules were observed in nontransgenic mice (not shown).



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  		Figure 4. Lung histology. (A) Wild-type mouse treated with doxycycline for 3 mo showing normal lung histology. (B) TNF transgenic mouse treated with doxycycline for 1 mo showing lymphocytic nodules and enlarged airspaces. (C) Low power of TNF transgenic mouse treated with doxycycline for 3 mo showing lymphoid nodules. (D) Transgenic mouse treated with doxycycline for 3 mo showing the edge of a lymphoid nodule at top left and the emphysematous phenotype in the lung parenchyma (compare with A and E). (E) Untreated transgenic mouse of same age as mouse in D (5 mo) showing small lymphocytic nodule but normal airspaces. (F) Lymphoid nodule from a transgenic mouse treated with doxycycline for 3 mo showing light (black asterisk) and dark (white asterisk) zones characteristic of follicles with germinal centers. (G) High-power view of a lymphoid nodule from a transgenic mouse treated with doxycycline for 3 mo. The white arrow indicates a cell with a small dark nucleus characteristic of an unactivated lymphocyte. The white arrowhead indicates a cell with a large nucleus and diffuse chromatin characteristic of an activated lymphocyte. The black arrow indicates a tingible body macrophage. The black arrowhead indicates apoptotic debris. (H) Transgenic mouse treated with doxycycline for 6 mo showing a fibrotic lesion adjacent to an airway. Bar in A equals 100 µm in A, B, D, and E; 250 µm in C; 50 µm in F and H; and 25 µm in G.

 


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  		Figure 5. Lymphocytic nodules in TNF transgenic mice. Lymphocytic nodules were counted in lung sections from TNF transgenic mice with (solid bars) or without (shaded bars) doxycycline treatment and normalized to section area. *P < 0.05 versus uninduced; **P < 0.01 versus uninduced; n = 4–9 mice/group.

 
Airspace enlargement was observed in TNF transgenic mice treated with doxycycline (Figures 4B and 4D). In affected animals, airspace enlargement was observed throughout the lung, but tended to be more pronounced adjacent to lymphoid aggregates. Airspace size was quantitated by measuring mean linear intercept (Lm; Figure 6). Significant increases in Lm were observed in TNF transgenic mice treated with doxycycline for 1–9 mo compared with wild-type mice. No change in the extent of airspace enlargement was observed over the time course of doxycycline treatment, as Lm values in doxycycline-treated transgenic mice were not significantly different among the 1, 3, 6, and 9 mo treatment groups. Lm was not significantly different between uninduced transgenic mice and wild-type mice through 8–9 mo of age (control mice for 6 mo of doxycycline treatment). Untreated transgenic mice eventually exhibited significant airspace enlargement at 11–12 mo of age (control mice for 9 mo of doxycycline treatment), indicating that the low amount of transgene expression in uninduced transgenic mice can, over time, produce emphysema. Treatment of nontransgenic mice with doxycycline had no effect on airspace size. These results confirmed the histologic findings of enlarged air spaces and demonstrated that induction of the TNF transgene induces emphysema in these mice.



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  		Figure 6. Emphysema in TNF transgenic mice. Mean linear intercept (Lm) was measured as an indicator of airspace size. (A) One month of doxycycline treatment. (B) Three months of doxycycline treatment. (C) Six months of doxycycline treatment. (D) Nine months of doxycycline treatment. *P < 0.05 versus wild-type mice; **P <= 0.01 versus wild-type mice; {dagger}{dagger}P < 0.001 versus uninduced transgenic mice; n = 4–6 mice/group.

 
Some fibrotic lesions were observed in TNF transgenic mice. These were focal in nature and were most commonly observed adjacent to airways (Figure 4H). Hydroxyproline content in transgenic and wild-type lungs after 6 mo of doxycycline treatment was not significantly different (wild-type, 207 ± 7 µg/lung; wild-type dox, 208 ± 12 µg/lung; transgenic, 202 ± 23 µg/lung; transgenic dox, 207 ± 9 µg/lung; n = 4–5 per group).

Microarray Analysis
To identify genes that are regulated by TNF and may mediate lung pathology in this model, relative transcript levels in transgenic and nontransgenic mice were measured by oligonucleotide microarray analysis. RNA from doxycycline-treated TNF transgenic and wild-type mice was hybridized with Affymetrix MG-U74Av2 GeneChip expression arrays containing sequences from ~ 12,000 mouse genes. This analysis identified 136 probe sets for which the corresponding RNA levels were considered to be significantly upregulated in TNF transgenic compared with nontransgenic mice. Among the probe sets corresponding to upregulated genes, 69 were derived from immunoglobulin sequences, including µ and {gamma} heavy chains and {kappa} and {lambda} light chains. These were among the most highly induced genes detected (up to 30-fold increase in signal), indicating that immunoglobulin production is a feature of the lung phenotype in this model. Elimination of the immunoglobulin sequences resulted in 67 unique sequences, of which 64 were derived from identified genes (Table 1). The majority of the 64 upregulated genes could be grouped into 9 functional categories: signal transduction molecules (11 genes), receptors (10 genes), cytokines (9 genes), histocompatibility antigens (6 genes), complement components (5 genes), transcription factors (4 genes), metabolic enzymes (4 genes), proteases (2 genes), and growth factors (2 genes). Consistent with the observation of lymphoid follicles in the lungs of TNF transgenic mice, many of the upregulated genes are characteristically expressed on lymphoid cells, particularly B cells (mb-1, B29, CD 19, CD 20, CD 37, CD 72, complement receptor 2, BLNK, Fig-1, and Ly-6D) and antigen presenting cells (MHC class II antigens and CD 80). In addition, this analysis indicated increased expression of chemokines that act on lymphocytes (CXC chemokine ligand 13 [CXCL13; also known as B lymphocyte chemoattractant {BLC} or B cell attracting chemokine {BCA-1}], CXCL12 [also known as stromal cell-derived factor-1 {SDF-1}], CC chemokine ligand 7 [CCL7], CCL8, CCL17, and CCL19). The upregulation of two protease genes, macrophage metalloelastase (MMP-12) and cathepsin K, is also of interest in light of the development of emphysema in TNF transgenic mice. The upregulation of MMP-12 RNA was confirmed by RNase protection assay, which revealed that MMP-12 message was upregulated 2.6-fold in the lungs of TNF transgenic mice (not shown), which was similar to the 3-fold increase in signal detected in the microarray analysis. The microarray analysis also identified 40 known genes (Table 2) and 13 ESTs whose expression was downregulated in TNF transgenic mice compared with wild-type mice. These included genes that have previously been established to be downregulated by TNF (adipsin, adipoQ, and thrombomodulin), as well as a gene required for collagen synthesis (lysyl oxidase), which may be potentially relevant to the emphysema observed in TNF transgenic mice.


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  		TABLE 1 Nonimmunoglobulin genes upregulated in TNF transgenic mice

 

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  		TABLE 2 Genes downregulated in TNF transgenic mice

 
Immunohistochemical Analysis of Lymphoid Nodules
Because the histologic and microarray analyses indicated the presence of lymphoid tissue and the expression of lymphoid-specific genes in the lungs of TNF transgenic mice, immunostaining was performed to identify lymphoid cells in lung sections. Staining for the B cell marker B220 revealed the presence of large numbers of B lymphocytes within lymphoid follicles in TNF transgenic mice (Figures 7A and 7B). Many lymphoid nodules contained zones in which a higher intensity of B220 staining was observed (Figure 7B), suggesting a mixture of mature and immature B cells and the presence of germinal centers (15, 16). CD11c immunostaining was used to localize dendritic cells, which tended to be localized on the periphery of lymphoid nodules (Figure 7C). Some nodules contained T cell–rich zones that were identified by CD3 immunostaining (Figure 7D). CD4-positive cells were observed in some nodules but CD8-positive cells were not (not shown). These results suggested that the CD3-positive cells observed within lymphoid nodules were helper T lymphocytes. CD8-positive cells were observed within the lung parenchyma of both TNF transgenic and wild-type mice, but were present in greater numbers in the transgenic mice (Figures 7E and 7F).



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  		Figure 7. Immunohistochemistry for lymphoid markers. TNF transgenic mice were administered doxycycline for 1–2 mo and were used to prepare frozen sections for immunostaining. (A) Staining of small lymphoid nodules with the B cell marker B220. (B) B220 staining in a large lymphoid nodule adjacent to an airway. The asterisk indicates the location of the airway. (C) Staining of a lymphoid nodule for the dendritic cell marker CD11c. Dendritic cells are located around the periphery of the nodule. (D) Staining of a lymphoid nodule for the T cell marker CD3. (E) Staining for CD8 in the lung parenchyma of a transgenic mouse. (F) Staining for CD8 in the lung parenchyma of a wild-type mouse. Bar in A equals 100 µm for A and 50 µm for B–F.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF expression has been documented in a variety of disease states associated with chronic inflammation. As a means to investigate mechanisms of TNF-induced lung pathology, we developed transgenic mice that express TNF in the lung under control of a doxycycline-inducible promoter. TNF transgenic mice had an influx of inflammatory cells into the lung in the absence of exposure to any inflammatory agent. A modest increase in the numbers of neutrophils and lymphocytes in lavage fluid was observed. This observation is consistent with the known ability of TNF to promote neutrophilic and lymphocytic inflammation. Histologically, lymphoid nodules adjacent to airways and within the lung parenchyma were the dominant inflammatory phenotype. These aggregates contained primarily B cells, with lesser numbers of CD4-positive lymphocytes and antigen-presenting dendritic cells. This phenotype resembles the human pulmonary pathologies of lymphocytic interstitial pneumonia and follicular bronchiolitis, which are often associated with either viral infections, such as HIV (17, 18), or autoimmune diseases, such as systemic lupus erythematosus (19), Sjogren's syndrome (20), and rheumatoid arthritis (21). Because TNF is known to be produced by HIV-infected cells and in autoimmune conditions, this cytokine may function in promoting the chronic pulmonary inflammatory reactions that often occur in these diseases.

TNF and lymphotoxin, the soluble form of which signals through TNF receptors, have been demonstrated to be important factors controlling the development of secondary lymphoid tissues. Mice with a targeted mutation in the lymphotoxin gene were devoid of lymph nodes and had abnormal splenic architecture (22). TNF-deficient mice developed lymph nodes, but lacked B cell follicles and germinal centers in the spleen (23). Overexpression of lymphotoxin in murine pancreas and kidney resulted in the formation of lymphoid tissue that appears similar to that observed in the lungs of inducible TNF transgenic mice (24). The formation of secondary lymphoid organs and the homeostatic trafficking of lymphocytes are dependent on lymphocyte homing chemokines that are constitutively expressed by stromal cells (25). CXCL13 is a critical chemokine for the formation of B cell follicles within the spleen and lymph nodes (26, 27). Expression of CXCL13 was reduced in TNF, lymphotoxin, and TNF receptor I knockout mice (28). These results indicate that TNF family ligands signaling through TNF receptor I control the homing of B cells to follicles via regulation of CXCL13. These same events appear to occur during ectopic formation of lymphoid tissue in inflammatory conditions (29, 30). In our TNF transgenic mice, we detected upregulation of CXCL13 message, so this chemokine may also be instrumental in the homing of B lymphocytes to the lungs in this model.

The nature of the inflammation in the inducible TNF transgenic mice was distinct from that observed in other models of TNF overexpression in the lung. Mice constitutively expressing TNF from the SPC promoter exhibited extensive lymphocytic inflammation in younger mice that was diffuse throughout the lung parenchyma rather than restricted to distinct clusters as in the inducible TNF transgenic mice (31, 32). The inflammation consisted primarily of CD4-positive T lymphocytes, and follicles of B lymphocytes were not reported. In older mice the inflammatory process subsided and pulmonary fibrosis or enlarged air spaces were the predominant phenotypes. A similar phenomenon appears to occur in mice inducibly expressing TNF, as the number of lymphoid nodules decreased between 6 and 9 mo of doxycycline administration. Diffuse inflammation of the lung parenchyma was also observed in rat lung transduced with a TNF-expressing adenoviral vector (33). These animals exhibited neutrophils within air spaces and lymphocytes and neutrophils within the lung parenchyma. One possible explanation for the distinct nature of the inflammation in the present model is the inducible expression of the transgene. Exposure of the lung to high levels of TNF during development, as occurs when expressed from the SPC promoter, may blunt the ability of cells to maintain the upregulation of some TNF-responsive genes in the adult. This could account for differences between the constitutive versus inducible transgenic expression in mice. In addition, the amount of TNF produced in the lungs is likely to affect the observed phenotype. In constitutively expressing SPC-TNF mice and TNF adenovirus-treated rats, very high levels (in excess of 60 ng/ml) of TNF could be measured in lavage fluid (32, 33). In contrast, TNF could not be detected in lavage fluid from inducible TNF transgenic mice and was elevated 6-fold in lung homogenates over nontransgenic mice. These observations indicate that TNF in the present model is produced at much lower and more physiologically relevant levels and was bound locally at the sites of synthesis. TNF acting locally may induce the formation of lymphocytic follicles rather than diffuse alveolitis.

TNF transgenic mice treated with doxycycline for 1–9 mo developed enlarged airspaces. Enlarged airspaces were not noted in uninduced transgenic mice until they were ~ 11 mo old. This effect is therefore a result of tissue destruction in adult lungs, i.e., emphysema. Emphysema was observed after 1 mo of doxycycline treatment and did not appear to progress significantly during an additional 8 mo of treatment. Thus, similar to the attenuation of inflammation over time, other chronic effects of TNF appear to be self-limiting.

TNF production is associated with emphysema in certain human conditions such as chronic obstructive pulmonary disease (COPD) and HIV infection. Cigarette smoke induces upregulation of TNF expression and this molecule may therefore be involved in the development of emphysema induced by cigarette smoke. It should be noted that the airspace enlargement observed in TNF transgenic mice was not as severe as that observed in patients with COPD and with emphysema. However, in the TNF transgenic mice, the airspace enlargement occurred rapidly (within 1–3 mo) but did not continue to progress. In patients with COPD and with emphysema, the lung tissue destruction occurs over a period of decades. Thus the rate of TNF-mediated airspace enlargement in the mouse model is likely to be sufficient to generate severe emphysema if it were to continue unabated for a longer period of time. HIV-infected individuals are at risk for developing pulmonary disease characterized by decreased diffusing capacity (34). Examination of such individuals revealed a high prevalence of emphysematous lesions observed by high-resolution computed tomography (35, 36). The fact that TNF production is increased in the lungs by HIV infection raises the possibility that TNF may mediate the development of emphysema in patients with HIV-associated pulmonary disease.

The mechanisms by which TNF promotes airspace enlargement remain undefined. One potential mechanism is the stimulation of neutrophil influx by TNF that would in turn induce lung tissue destruction by neutrophil proteases. Support for this mechanism comes from studies in which cigarette smoke–induced neutrophil influx and acute extracellular matrix breakdown were inhibited in TNF receptor knockout mice (37). A second potential mechanism is the upregulation of matrix metalloproteinase (MMP) expression by TNF. TNF is known to positively regulate MMPs, which have the capability to degrade lung extracellular matrix components. MMP-12, macrophage metalloelastase, mediates cigarette smoke–induced emphysema in mice (38). This molecule is a candidate for mediating TNF-induced emphysema as well, because in the present study, MMP-12 message was upregulated in TNF transgenic mice. MMP-12 has also been implicated as being required for the release of membrane-bound TNF from murine alveolar macrophages, so it has the potential to act in multiple ways to promote the development of emphysema (9). A third possible mechanism by which TNF may cause airspace enlargement is the action of CD8-positive T lymphocytes, which have been proposed to contribute to the pathology of COPD because of their presence in increased numbers in small airways and alveolar walls of smokers with COPD (39–41). CD8-positive cytotoxic T cells may induce lung tissue destruction by apoptotic mechanisms or by the release of cytokines such as interferon-{gamma} (41, 42). In the present study, abundant CD8-positive cells were observed in the lung parenchyma of TNF transgenic mice, raising the possibility that cytotoxic T cells may mediate TNF-induced pathology in mouse and human lungs.

The use of microarray technology allowed the identification of genes whose expression was altered in TNF transgenic mice. Because of the inflammatory process in the transgenic mice, the microarray analysis detects not only genes upregulated by TNF in resident lung cells, but also genes expressed in the inflammatory cells that migrate into the lung. Many of the upregulated genes that we identified were transcripts known to be expressed in lymphoid cells, particularly B cells, which was consistent with the histologic findings. Also upregulated were a number of chemokine genes, which may be instrumental in the initial development of lymphoid follicles in the lung. Surprisingly, TNF was not identified as an upregulated gene by microarray analysis, although its increased expression in the samples used for this analysis was verified by RNase protection assay (not shown). The Affymetrix Microarray Suite software uses data analysis algorithms to assess the significance of signal changes between test samples. These are designed to assure that all identified changes are robust, but apparently they may fail to identify some genes whose expression is different between the samples.

In summary, we have generated mice in which a TNF transgene is inducibly expressed in the lung, which resulted in the development of lymphoid follicles and emphysema. These mice displayed pulmonary pathologies similar to those in human inflammatory conditions in which TNF is known to be upregulated. The TNF transgenic mice provide a model in which the mechanisms underlying the pathogenic effects of TNF expression can be investigated.


   Acknowledgments
 
The authors thank Carol Gadwaw for excellent technical assistance, Dr. Mansour Zadeh for assistance with immunostaining, Dr. Cesar Fermin and the Centralized Tulane Imaging Center for help with fluorescence microscopy, and the Tulane University Center for Gene Therapy for microarray analysis. This work was supported by NIH grant HL58610, the Louisiana Millennium Trust Health Excellence Fund, and the Tulane/Xavier Center for Bioenvironmental Research.

Received in original form February 26, 2003

Received in final form August 28, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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