Role of tumor necrosis factor-alpha in the spontaneous development ...

American Journal of Pathology, Vol. 151, No. 5, November 1997

Copyright X American Society for Investigative Pathology

Role of Tumor Necrosis Factor-a in the Spontaneous Development of Pulmonary Fibrosis in Viable Motheaten Mutant Mice

Roger S. Thrall,* Stefanie N. Vogel,t Robert Evans,f and Leonard D. Shultzt From the Pulmonary Division,* Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut; Department of Microbiology,t Uniformed Services University of the Health Sciences, Bethesda, Maryland; and The Jackson Laboratory,* Bar Harbor, Maine

The viable motheaten mutant mouse Is severely immunodeficient and dies from a naturaly occurring progressive pulmonary inflammation at approximately 10 weeks of age. The pulmonary disease is characterized by excessive macrophage accumulation in the lung and fibrosis. We correlated the development of lung injury in viable motheaten mice with tumor necrosis factor-a (TNF-a) levels in serum and lung. Significantly increased serum TNF-a levels were observed by enzyme-linked immunosorbent assay in viable motheaten mice at 4, 6, and 10 weeks of age as compared with normal control littermate mice. These ages correlated well with observed changes in lung wet weights, lung collagen content, and histological evidence of pulmonary inflammation and fibrosis. Qualitative assessment of lung tissue TNF-a levels was performed by immunohistochemical staining using immunoperoxidase techniques. These studies revealed increased levels of TNF-a in macrophage-like cells in viable motheaten mice from 5 to 10 weeks of age as compared with littermate control animals. Alveolar macrophages isolated from viable motheaten mice produced significantly greater amounts of TNF-a when stimulated with lipopolysaccharide compared with alveolar macrophages from control animals. In addition, administration of anti-TNF-a antibody to motheaten bone marrow recipient mice decreased the severity of acute lung injury. The results demonstrate a close correlation between the development of pulmonary injury and TNF-a levels in this model of spontaneously developing fibrotic lung disease. (AmJ Pathol 1997, 151:1303-1310)

Mice that are homozygous for the recessive allelic genes, motheaten or viable motheaten, are severely deficient in T and B lymphocyte and natural killer cell function, produce abnormally high levels of circulating immunoglobu-

lins, and express multiple autoantibodies.1-6 Homozygous mice die at approximately 3 weeks (motheaten) and 10 weeks (viable motheaten) of age, respectively, from a naturally occurring progressive interstitial fibrotic lung lesion similar to that observed in patients with chronic interstitial lung disease.7-10 The motheaten and viable motheaten mutations disrupt the structural gene for a protein tyrosine phosphatase termed SHP-1.11 The wild type gene symbol for SHP-1 hematopoietic cell phosphatase is Hcph. The gene symbols for the motheaten and viable motheaten mutations are Hcphme (me) and Hcphmev (mev), respectively. Both the me and mev mutations are single base mutations that result in splicing defects. The me mutation is a deletion of a cytidine residue that produces a frameshift and premature truncation resulting in a null mutation. The mev mutation is a thymidine to adenine transversion in the first SH2 domain,

which results in the destruction of a splice donor site. The activation of cryptic splice sites results in a frame insertion or deletion in the phosphatase catalytic domain, resulting in approximately 20% of normal activity' 1 (Shultz LD, Rajan TV, Greiner DL: Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. Trends Biotechnol 1997, 15:302-307). The longer survival period for the mevfmev mice makes them a more practical model to study and it has been established that the pathophysiological defects are similar in both mutant mouse populations.1 Tumor necrosis factor-a (TNF-a) is a cytokine with a wide variety of biological activities that include direct tumoricidal activity, the ability to activate various cell populations (polymorphonuclear leukocytes, endothelial cells, lymphocytes, and fibroblasts), and to stimulate the production of other mediators.12,13 TNF-a has been implicated in many diseases including cachexia,14 graft versus host disease after bone marrow transplant,15 sepsis,16 tuberculosis,17 sarcoidosis,16 pulmonary fibrosis,19 and the adult respiratory distress syndrome.20 Thus, it appears that TNF-a, with its wide biological effects, may play a critical role in several disease processes. This work was supported by NIH grants HL 39939, CA 20408, Al 18797, and 63706 NM 0095.001.9401. Accepted for publication July 31, 1997. Address reprint requests to Dr. Roger S. Thrall, Pulmonary Division, MC 1225, Department of Medicine, University of Connecticut, Health Center, Farmington, CT 06030-1225. Tel.: 860-679-4118; Fax: 860-679-1103.

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TNF-a has been implicated in mediating pulmonary injury. It has been shown to increase collagen synthesis in isolated lung fibroblasts,21 to be elevated in bronchopulmonary secretions of patients with adult respiratory distress syndrome,22 and to cause a pulmonary inflammatory reaction when administered intravenously or directly into the lungs of animals.23 Further, depletion of circulating TNF-a by treatment with anti-TNF-a antibody inhibited the development of bleomycin-induced19 and silica-induced24 fibrotic lung disease in mice. The purpose of the current study was to investigate the role of TNF-a in the naturally occurring progressive fibrotic lung lesion of the viable motheaten mouse.

Materials and Methods Mice Motheaten and viable motheaten mice used in this study were from the C57BL/6J-mev strain propagated by +1mev brother/sister matings or from the C57BL/6-me strain propagated by +/me sibling matings. Littermate control animals (+/-) included +/mev and +/+ littermates. All mice were fed a standard diet of Wayne Lab Blocks and received chlorinated water ad libitum.

Analysis of Lung Damage Histological Evaluation Animals were killed by exsanguination under C02 anesthesia at 4, 6, and 10 weeks of age. The lungs were removed and inflated under constant pressure with Fekete's formol-acetic alcohol.25 Tissue sections were dehydrated, embedded in paraffin, and processed for light microscopic examination. Slides were routinely stained with trichrome or hematoxylin and eosin. For histochemical localization of TNF-a, lungs were fixed and paraffin embedded as described above. Sections (5 ,m) were incubated with rabbit polyclonal anti-mouse TNF-a (Genzyme, Boston, MA) or normal rabbit serum. Staining was developed with a Vectastain ABC peroxidase kit (Vector Laboratories, Burlingame, CA) and the chromogen diaminobenzidine. Lung Wet Weight

Lungs were trimmed free of major bronchi, and weights were measured on a Mettler electronic balance. Lung Hydroxyproline (Collagen) Lung collagen levels were calculated by a method described by Woessner26 for the determination of hydroxyproline in tissue. The lungs from each animal were dissected free of major bronchi, homogenized, and hydrolyzed in 6 N HCI for 18 hours at 1 10°C. The hydrolysate was neutralized with 2.5 N NaOH and assayed colorimetrically with the addition of chloramine T, perchloric acid, and dimethylaminobenzaldehyde. Samples were

read for color development in a spectrophotometer at 561 nm.

Lung Hemoglobin The lungs were trimmed to remove all major bronchi, homogenized, and brought to a volume of 5 ml with distilled water. The homogenates were centrifuged at 20,000 x g for 15 minutes and the supernatants were filtered (pore size 0.45 ,um). Hemoglobin levels were determined by the cyanmethemoglobin colorimetric assay using Drabkin's reagent (Total Hemoglobin, Sigma, St. Louis, MO).27 Briefly, 500 ,ul of sample were added to 5 ml of Drabkin's reagent, incubated at room temperature for 15 minutes, and the absorbance was read at 540 nm. The sample values were determined using a standard curve generated from human methemoglobin standards.

Quantitation of TNF-a Levels by Enzyme-Linked Immunosorbent Assay Mouse TNF-a levels in serum were quantified by enzymelinked immunosorbent assay (ELISA) (Factor-Test mTNF-a, Genzyme). This assay used a hamster monoclonal antibody specific for murine TNF-a and a goat anti-murine TNF-a antibody. A horseradish peroxidaseconjugated donkey anti-goat Ig and peroxide substrate with chromogen completed the reaction and the absorbance was read at 492 nm.

Bioassays of TNF-ca Functional Activity TNF-a functional activity was determined by both a fibroblast cell lytic assay and a fibrosarcoma cell line growth inhibition assay as previously described.28 Briefly, mouse L929 fibroblasts (American Type Culture Collection, CCL-1) were seeded overnight as monolayers in microtiter plates. Test samples were diluted twofold across the plate, UV irradiated, and then added to the prepared cell monolayers. Actinomycin D was added to each well to a final concentration of 1 gg/ml. The plates were incubated for 18 hours at 370C and scored visually under the microscope. A mouse fibrosarcoma cell line, WEHI 164 clone 13, was used to measure TNF-a activity in a growth-inhibition assay. Inhibition of the WEHI cells proliferative capacity over a 24-hour period was established using recombinant TNF-a (Genzyme) in a dose-response assay. The 50% end-point was approximately 0.02 TNF U/ml. Proliferation was measured by the incorporation of [3H]TdR for the last 6 hours of the assay period.

Isolation of Alveolar Macrophages Animals were killed at 4, 6, and 10 weeks of age, and bronchoalveolar lavage was performed as previously described.29 Briefly, lungs were lavaged in situ by infusion of 5 ml (in 1 ml of aliquots) of sterile saline via a cannula ligated in the trachea. The recovered fluid, approximately

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4 ml, was centrifuged at 250 x g for 10 minutes and the pelleted cells resuspended in Gey's solution to lyse red blood cells. After red blood cell lysis, nucleated cells were centrifuged, washed, and resuspended at 4 x 105 cells/ml in RPMI 1640 containing gentamicin. The cells were plated out in Falcon flat-bottomed microtiter plates (100 pl/well) and were allowed to incubate and adhere at 370C, 5% C02, for 6 hours, after which nonadherent cells were washed and aspirated off. Media separately or with lipopolysaccharide (LPS) were then added to each well.

Lipopolysaccharide Treatment Animals were given an intraperitoneal injection of LPS (Escherichia coli 0127:B8, Difco, Detroit, Ml) 25 ,ug/animal, in 0.5 ml of phosphate-buffered saline, 90 minutes before serum samples were drawn. For cell culture experiments, the cells were exposed to 1 ,ug of LPS/well for 24 hours before harvesting of supernatants.

Bone Marrow Transfer As previously described,30 bone marrow cells (3 x 106) from 4-week-old me/me mice were injected intravenously into lethally irradiated (1000R), congenic, 10-week-old C57BL/6J +/+ mice. The animals were divided into two groups (seven animals/group). Mice in the experimental group were treated with rabbit anti-TNF-a antiserum (kindly provided by Drs. Steven Kunkel and Robert Streiter, Pathology Department, University of Michigan, Ann Arbor, Ml). The anti-TNF-a antibody had been shown to be effective in inhibiting serum TNF-a levels by more than 95%. The mice in the control group received normal rabbit serum. The animals received two intraperitoneal injections (0.3 ml) of anti-TNF-a or normal rabbit serum, one each on days 1 and 7 after bone marrow transfer. Animals were killed 12 days after bone marrow transfer, two animals in each group were used for histological evaluation, and the remaining five animals were used to quantify lung injury.

Statistical Analysis Student's unpaired t-test and Bonferroni's adjustment for multiple comparisons were used for data analysis.3

Results Histopathological Findings Changes in lung pathology observed in this study were similar to those previously described in both mevlmevl and me/me78 mice. Briefly, at 2 weeks there was no discernible lung injury observed at the light microscopic level. At 4 weeks, there was evidence of slight patchy areas of inflammation consisting of an interstitial infiltrate of polymorphonuclear leukocytes, lymphocytes, and mononuclear histiocyte-like cells. At 6 (Figure 1A) and 10 (Figure 1 B) weeks of age, the lesion had progressively

developed with extensive consolidation and fibrosis evident. By 10 weeks of age most of the lung was involved. Some signs of acute inflammation were still present at these later stages. Massive accumulations of mononuclear histiocyte-like cells containing eosinophilic crystals were apparent in the chronic lesions (Figure 1C). Analysis of trichrome-stained sections demonstrated the presence of increased levels of collagen that appeared to be both interstitial and intra-alveolar and evenly distributed in a linear pattern (Figure 1D). There were no histological lung abnormalities observed in any +/- control mice (data not shown).

Immunoperoxidase Localization of TNF-a in Lung Tissue Lung sections from mevlmev and +/- control mice at 2 to 10 weeks of age were stained for the presence of TNF-a. Increased positive staining was observed in mev/mev mice from 5 to 10 weeks of age. As shown in Figure 2, at 10 weeks of age, lungs from mev/mev mice contained accumulations of macrophages, many of which showed intense cytoplasmic TNF-a staining (Figure 2A). Lungs from control mice showed normal structure and were negative for TNF-a (Figure 2C). As a control for nonspecific staining, normal rabbit serum was used as the primary antibody in place of anti-TNF-a on 10-week-old mev/mev mouse lung sections (Figure 2B).

Assessment of Lung Injury Lung wet weights, lung/body weight ratio, and total lung hydroxyproline (collagen) levels of mev/mev mice were significantly (P < 0.001) elevated as compared with control +/- mice at 4, 6, and 10 weeks of age. The lung lesions increased progressively with age in the mev/mev mice (Table 1).

Serum TNF-a Levels Using the ELISA assay for TNF-a, it was determined that serum levels of immunoreactive TNF-a were significantly elevated in mev/mev mice as compared with +/- mice at 4, 6, and 10 weeks (Figure 3). Although the serum TNF-at levels of the 6-week-old mev/mev are elevated above the +/- 6-week-old animals, they are also significantly lower than both the 4- and 10-week-old mev/mev mice. To correlate immunoreactive TNF-a levels with functional TNF-a biological activity, a cell-lytic assay was used to measure TNF-a bioactivity in the serum of 6-week-old mev/mev and +/- mice. The TNF-a biological activity was elevated sixfold in the mev/mev mice.

TNF-ca Levels in Alveolar Macrophage Culture Supernatants Tissue culture media harvested from LPS-stimulated alveolar macrophages from 4-, 6-, and 10-week-old mevl mev mice had significantly (P < 0.001) higher levels of

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A

B

C Figure 1. Histopathological changes observed in 6- and 10-week-old me"lme" mice. A: Lung section from a 6-week-old me/me" mouse shows a relatively early inflammation of the lung primarily in the interstitium and composed of lymphocytes, neutrophils, and mononuclear histiocyte-like cells. The alveolar spaces are focally infiltrated with histioctye-like cells containing brightly eosinophilic material. Large areas of open alveolar space is preserved. H&E; magnification, X 100. B: Lung section from a 10-week-old mev/me" mouse shows a more advanced stage of the disease characterized by marked increased intra-alveolar infiltration of histiocyte-like cells containing eosinophilic crystals; notice that they occupy the entire alveolar space. The interstitial inflammatory infiltrate has remained the same. H&E; magnification, X 100. C: Lung section from a 10-week-old melme" mouse demonstrates the characteristic features of the degenerated histioctye-like cells containing the needle-like eosinophilic crystals. These cells are primarily located in the alveolar space. The interstitial infiltrate is composed of lymphocytes, neutrophils, and occasional larger round cells (histioctye-like) that are seen in various stages of degeneration, ie, accumulation of the crystals. H&E; magnification, x400. D: Lung section of a 10-week-old me"lme" mouse demonstrates the deposition of collagen around the individual histiocyte-like cells as well as surrounding the clusters of them. It appears that some of these cells do not contain significant amounts of the crystalline material and are not fully degenerated. Trichrome; magnification, X400.

TNF-a activity as determined by the WEHI growth inhibition assay, as compared with LPS-stimulated alveolar macrophages from age-matched control +/- mice. There was no detectable TNF-a activity in the culture media of unstimulated macrophages from +/- mice, however, with LPS stimulation, low (< 4 ng/ml) measurable TNF-a activity was demonstrated for these control mice at 4, 6, and 10 weeks of age. All unstimulated alveolar macrophages from mev/mev mice had low but detectable levels of TNF-a in their culture media (Table 2). Consistent with the progressive increase in injury with age in the mev/mev mice, the alveolar macrophages from these animals demonstrated a progressive increase in TNF-a levels with age.

Effect of Anti-TNF-a Treatment in me/me Bone Marrow Chimeras The transfer of me/me bone marrow cells to normal recipients resulted in severe lung injury as previously described.30 Briefly, me/me bone marrow recipients exhibited abnormal lung histology and significantly increased lung weight, lung weight/body weight ratio, and lung hemoglobin. Treatment of me/me bone marrow chimeras with anti-TNF-a serum significantly reduced both the lung weight/body weight ratio (P < 0.04) and the lung hemoglobin levels (P < 0.003), which indicates reduced hemorrhage into alveoli as compared with similar animals treated with normal rabbit serum (Table 3).

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A~~

Figure 2. Lung sections from 9-week-old mev/mev and +/-control mice stained for the presence of TNF-a by immunoperoxidase technique. Magnification, X500. A: A mel/meV lung showing a severe inflammatory response predominated by macrophages. Many of the macrophages show intense cytoplasmic staining positive for TNF-a. The primary antibody used was rabbit anti-TNF-a. B: A mev/me7 lung showing a similar severe inflammatory response as above. Normal rabbit serum was used as the primary antibody in place of the specific anti-TNF-a. Note the absence of any positive staining for TNF-a. C: A control +/- mouse lung section showing normal lung structure. Rabbit anti-TNF-a was the primary antibody used. There was no positive staining for TNF-a.

Discussion We have characterized the development of lung injury in mevlmev mice and have shown significant progressive increases in lung wet weight, lung to body weight ratio, and lung collagen in mice from 4 to 10 weeks of age. These changes were associated with severe pneumonitis. No detectable differences were observed at earlier time points and most mev/mev mice die by 10 weeks of age. Elevated TNF-a levels in serum and lung tissue correlated with the progressive development of the fibrotic lung lesion in mev/mev mice. Increased serum TNF-a levels measured by ELISA were confirmed by functional activity determined by a cytolytic assay. Qualitative assessment of lung tissue TNF-a levels was performed by immunohistochemical staining that revealed high levels of immunoreactive TNF-a in macrophage-like cells. Alveolar macrophages isolated from mev/mev mice produced significantly higher levels of TNF-a on LPS stimulation than alveolar macrophages from littermate control animals. In addition, we have shown that treatTable 1.

ment of irradiated recipients of me/me bone marrow with anti-TNF-a inhibits the development of acute lung injury. Along these lines, others have shown that treatment of motheaten mice with TNF-binding protein improved survival and decreased lung inflammation.32 The close correlation of elevated TNF-a levels to lung injury in mev/mev mice and the fact that antibodies to TNF-a can reduce the severity of the lesion in me/me bone marrow chimeras are strong evidence for a major role for TNF-a in the progression of this fibrotic process. In addition to being capable of directly stimulating lung fibroblast proliferation and collagen synthesis,21 TNF-a has been implicated in the pathogenesis of several animal models of fibrotic lung disease.1924'33 TNF-a or TNF-a mRNA levels have been shown to be elevated in the silica,24'33 bleomycin,19 and asbestos33 animal models. Asbestos and silica have been shown to directly stimulate TNF-a production from alveolar macrophages.33 Treatment of animals with anti-TNF-a1924 or recombinant soluble TNF-a receptor34 reduced the de-

Development of Lung Injury in mel/mev Mice

Animals

Body Weight (g)*

Lung Weight (mg)

Weight Ratio

Lung Hydroxyproline (,ug/lung)

13.4 ± 0.8 11.8 ± 0.4

145 ± 10 280 ± 34t

10.8 23.4

126 ± 12 197 ± 24t

19.6 ± 0.8 9.4 ± 0.4t

141 ± 6 371 ± 37t

7.2 39.5

105 ± 9 249 ± 16t

22.1 ± 0.4 12.2 ± 0.8t

150 ± 7 727 ± 10t

6.8 59.6

114 ± 6 576 ± 26t

Lung/Body

4 weeks

+/mevlmev 6 weeks

+/mevlmev 10 weeks

+/mevlmev

*Data represent the mean ± SEM of at least 6 animals in each group. control animals.

tSignificant difference (P < 0.001) from the age-matched +/-

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* Im

60-

Table 3.

Effect of Anti-TNF-a Antibody on Lung Damage in Irradiated C57BL/6j +/+ Recipients of Motheaten Mouse Bone Marrow

Treatment Groups Rabbit AntiNormal TNF-a Rabbit Serum

50*

Body weight (g)t Lung weight (mg) Lung weight/body weight ratio Lung hemoglobin (mg/lung)

40+ LL

z

E 30. c

w Co)

20-

1

04

01 4 WK

4 10

W-

a6

WK WK

WWK

10 WK

meV/meV

1-I-

CONTROLS Figure 3. Immunoreactive serum TNF-a levels in memelev and +/- littermate control mice examined at 4 to 10 weeks of age. Values represent the means +/- SEM of 5 to 10 mice per group. * All melme' serum TNF-a levels were significantly elevated from their respective littermate controls (P < 0.01). *' Also, the 4- and 10-week-old me'lme' serum TNF-a levels were significantly elevated fromn the 6-week time point (P < 0.03).

velopment of lung damage in both the bleomycin and silica animal models. Furthermore, pulmonary fibrosis has been shown to develop in transgenic mice in which the TNF-a transgene is expressed in the lung.35 These results are consistent with our findings in the progression of lung injury in mev/mev mice. The significance of TNF-a in each of these diverse fibrotic models would favor a broad general role of TNF-a in the fibrotic process. The stimulus for increased TNF-a production in the silica and asbestos models seems to be the inciting agent directly; however, this may not be the case in either the bleomycin-treated or mev/mev animals. Other inflammatory mediators may stimulate elevated TNF-a levels in these anTable 2.

TNF-a Levels in Culture Supernatants from in Vitro Stimulated Lung Macrophages Isolated from me'lme' and Control Mice

Source of Cells

LPS

+/- control +/- control 4 weeks mev/mev 4 weeks mev/mev 6 weeks mev/mev

+

6 weeks mev/mev 10 weeks mev/mev 10 weeks mev/mev

+

TNF-a (ng/ml)* Not detectable 3.3 + 0.1 3.2 + 0.2 11.4 + 1.0t 3.4 + 0.1 19.4 + 2.Ot 3.5 ± 0.1 28.8 + 1.0t t

*Data represent the mean + SEM from at least five different culture supernates for each group. tSignificant difference (P < 0.001) from LPS stimulated cells from

+/- control animals. tSignificant difference (P