Clin. exp. Immunol. (1991) 85, 392-395
Tumour necrosis factor/cachectin plays a key role in autoimmune pulmonary inflammation in lupus-prone mice Y. DEGUCHI & S. KISHIMOTO Third Department of Internal Medicine, Osaka University School of Medicine, Osaka, Japan
(Acceptedfor publication 8 April 1991)
The role of tumour necrosis factor-alpha (TNF-ac) in the development of autoimmune pulmonary inflammation has been investigated in lupus-prone mice. An increase in TNF-ax mRNA level from whole lung preparation of lupus-prone mice was evident, from 3 weeks to 12 weeks during growing process, as shown by Northern blot analysis, but not in control mice. Furthermore, it is also found that the major source of this increase in TNF-a mRNA was attributed to infiltrating mononuclear cells found within the lung. Treatment of lupus-prone mice with rabbit anti-mouse TNF-a IgG prevented the development of pulmonary inflammation lesions such as lung fibrosis and alveolitis. These results suggest that an increased TNF-x production by infiltrating mononuclear cells in the lungs of lupus-prone mice may play a role in the development of autoimmune pulmonary inflammation and in significant changes of cytokines and the immune responses in pulmonary inflammation lesions of lupus-prone mice.
Keywords tumour necrosis factor lupus pulmonary inflammation transcription lymphoid
produce TNF (Beutler & Cerami, 1988). In the present study, the role of TNF-a in the development of pulmonary inflammation in lupus-prone mice was investigated. The evolution of lung fibrosis and alveolitis was found to be associated with an increase in the level of lung TNF-ac mRNA, suggesting an increase of local TNF production. Furthermore, injection of anti-TNF antibodies markedly prevented the development of pulmonary inflammation in lupus-prone mice. Our experiments suggest that TNF-a has a key role in at least some types of autoimmune pulmonary inflammation in lupus-prone mice. We also demonstrate that the expression of TNF-a gene in lymphoid mononuclear cells from lungs of lupus-prone mice (MRL/ lpr) is elevated in comparison to normal healthy control mice (MRL/ +). The enhancement of the expression of TNF-a could depend on the elevated transcription of the TNF-ot gene in the cells. The possible implication ofenhanced expression of TNF-a gene in the pathogenesis of pulmonary inflammation in lupusprone mice is discussed.
Pulmonary inflammation can occur as one of the manifestations of systemic autoimmune diseases (Tan et al., 1982). The manifestation of this disease is associated with increased serum levels of autoantibodies and circulating immune complexes, decreased levels of haemolytic complement activity, and occurrence of immunoglobulin deposits in tissues (Berden et al., 1983). Several mouse strains, including MRL-lpr/lpr and BXSB, develop autoimmune symptoms very similar to the human disease systemic lupus erythematosus (SLE) (Andrews et al., 1978). A characteristic of these murine strains is the development of lymphoadenopathy, splenomegaly and immune complex-mediated tissue injury involving the pulmonary system. Although the underlying mechanisms responsible for these disorders are unknown, dysregulation of cytokine production in the immune is suspected (Dixon et al., 1980). Recently there have been many reports on evidence for cytokine networks in immune system and cellular interactions. Tumour necrosis factor-alpha (TNF-ac, cachectin) is now recognized to have a wide range of biological activities in vivo and in vitro (Beutler & Cerami, 1987). It is associated with acute and chronic inflammatory states and has been implicated as the exogenous mediator of shock (Tracy et al. 1988). It was shown that activated macrophages and various macrophage cell lines
MATERIALS AND METHODS
MRL-lpr/lpr (MRL/lpr, lupus-prone) and MRL-+/+ (MRL/ +, control) mice were bred in our laboratory from breeding pairs originally obtained from The Jackson Laboratory (Bar Harbor, ME). We used MRL female mice. The ages of 50% mortality for MRL/lpr female mice are about 5 and 6 months (Andrews et al., 1978). MRL/ + mice, sex and age matched with MRL/lpr mice, were used for controls in all experiments.
Correspondence: Yasuhiro Deguchi, M.D. 10687 Weymouth Street no. 103, Bethesda, MD 20814, USA.
TNF in autoimmune pulmonary inflammation Preparation of lymphoid mononuclear cells of lungs Lungs from the mice were cut into 5-mm pieces with a razor blade and passed through a nylon sieve. The suspension was washed twice in phosphate-buffered saline (PBS). Mononuclear cells were isolated by two centrifugation steps on a Percoll gradient (Pharmacia, Uppsala, Sweden). After each centrifugation at 1000g for 20 min, cells were recovered from the interface. After centrifugation, about 90% of recovered cells were mononuclear cells and the cells displayed a high viability (> 97%), as assessed by trypan blue dye exclusion. The cells were washed twice in PBS and used for further analyses. RNA preparation and Northern blot assay Total RNA from the cells was prepared by guanidiniumthiocyanate and caesium chloride procedure (Chomcyzski & Sacchi, 1987). For Northern blot assay, 15 Mg of total RNA was applied on 0-8% formaldehyde-agarose gels. Samples were transferred to nylon membranes and baked at 80'C for 2 h. The filters were hybridized with 32P-labelled probes for murine TNFa-specific synthesized oligonucleotides (I 10-mer) or actin cDNA (Wako Pure Chemical Industries, Kyoto, Japan) (specific activity 1-5 x 108 ct/min per mg) (Deguchi et al., 1987). The final washes were done under stringent conditions (2 x SSC/ 0-1% SDS at 450C for oligonucleotide probes, and 0-1 x SSC/ 0-1% SDS at 60'C for cDNA probes) before autoradiography. Filters were exposed to Kodak AR X-Omat films at -70'C.
Nuclear run on transcription assay Nuclei were prepared from the cells by lysing the cells in a solution containing 10 mm Tris (pH 7-5), 2 mm MgCl2, 3 mM CaC12, 5 mm DTT and 0-02% of NP40, with subsequent centrifugation through 2 M sucrose solution. Three-million nuclei were suspended into 100 ul of 50% glycerol solution with 50 mM Tris (pH 7 5), 5 mM MgCl2 and 0-1 mM EDTA. The suspension of nuclei was immediately mixed with an equal volume of buffer containing 0-2 M KCI, 5 mM MgCl2, 5 mM DTT, 1 mm of ATP, CTP, GTP, and 200 U of RNAsin (ribonuclease inhibitor, 500 U, Amersham International, Amersham, UK). The preparation was then incubated at 28°C for 20 min after addition of 50 pCi of 32P-radiolabelled UTP (3000 mCi/ml, Amersham). To the preparation were added SDS and EDTA solution to a final concentration of 1 % and 5 mm, respectively, followed by treatment with protein K (1 mg/ml) at 42°C for 30 min. RNA was extracted with phenol and chloroform from the preparation, and precipitated with ethanol. The pellet was resuspended into 3 ml of hybridization buffer which contained 50% formamide, 0-75 M NaCI, 0-5% SDS, 2 mm EDTA, 50 mM HEPES (pH 7-0), 1/10 dilution of Denhardt's solution, and denatured salmon sperm DNA (500 pg/ml) (Marzluff& Huang, 1984). Finally, the preparation was applied to the nitrocellulose filter onto which the murine TNF-a probe or beta-actin probe (Wako Pure Chemical Industries) had been dotted. After 24 h of incubation, the filter was washed at stringent conditions, dried and exposed to an X-ray film with intensifying screen at - 700C. In all experiments, the hybridized dot was excised from the filter and directly counted in a beta counter (Reed, Tsujimoto & Alpers, 1987). Treatment of MRL/lpr mice with rabbit anti-mouse TNF anti-
body The preparation of rabbit anti-TNF antibody using synthetic
(a ) 2
( b) 2
Fig. 1. Expression of TNF-a and beta-actin gene in lungs from lupusprone mice (MRL/lpr) and control mice (MRL/+) during growing process. (a) Representative northern blot assay with 32P-labelled TNF-cr probe. For each lane, 15 pg of total RNA were used. Lane I and 2, MRL/+ mice aged 2 weeks and 4 months, respectively; lanes 3-13, MRL/lpr mice, lane 3, 2 weeks old, lane 4, 3 weeks, lane 5, 4 weeks, lane 6, 6 weeks, lane 7, 8 weeks, lane 8, 10 weeks, lane 9, 1 1 weeks, lane 10, 3 months, lane 1 1, 4 months, lane 12, 5 months, and lane 13, 6 months; (b) Representative northern blot assay with 32P-labelled beta-actin probe. For each lane, 15 pg of total RNA were used. Lanes 1 and 2; MRL/ + mice, aged 2 weeks and 4 months, respectively; lanes 3 and 4, MRL/lpr mice, aged 2 weeks and 4 months, respectively.
peptides of TNF-ca as the immunizing agent was performed as described (Grau et al., 1988). Normal control rabbit sera were
obtained before immunization. Different bleeds from the same immunized animal as well as from separate immunized rabbits were tested with comparable results. IgG fraction from immunized and pre-immunized serum (control) were deaggregated by ultracentrifugation at 150 000g for 180 min, and I mg of control or anti-TNF IgG antibody was injected intravenously into lupus-prone and control mice at age 7 and 8 weeks (twice). Pulmonary tissues were examined for evidence of pulmonary
Y. Deguchi & S. Kishimoto 9
Fig. 2. Expression of TNF-a gene (top) and beta-actin gene (bottom) in pulmonary lymphoid mononuclear cells from lupus-prone mice during growing process. Representative results of Northern blot assay for TNF-a and beta-actin RNA. For each lane, 5 yg of total RNA were used. Lane 1, 2 weeks old; lane 2, 3 weeks old; lane 3, 8 weeks old; lane 4, 3 months old.
(a) Fig. 4. Representative histopathological sections from the lungs of lupus-prone (MRL/lpr) mice (10 weeks old, 3 weeks after the final treatment) after treatment with anti-TNF IgG (a) or control (b) serum. Magnification x 26.
( bI Fig. 3. Representative results of nuclear run on transcription assay of TNF-a (a) and beta-actin (b) gene expression. C, pulmonary lymphoid mononuclear cells from a control mouse (MRL/+, no. 5); L, from a lupus-prone mice (MRL/lpr, no. 8). Both mice were 3 months old. We examined 10 lupus-prone and 11 control mice in each age-point in Figs 1 and 2 with similar results.
inflammation from the mice treated with anti-TNF antibody or with control serum at 10 weeks of age (2 weeks after the final treatment). RESULTS Increased TNF-cx mRNA accumulation in lungs of lupus-prone mice RNAs from lungs of lupus-prone and control mice were extracted and analysed by Northern blot hybridization at various time-points during the growing process. As shown in Fig. 1, a significant increase of TNF mRNA level was detected in MRL/lpr mice, compared with the control mice. Additionally, the TNF-a mRNA level was found to increase during the
growing process (Fig. la). The difference between lupus-prone and control mice was minimal at 2 weeks of age, but was much more accentuated after 4 weeks of age. In contrast, the level of
beta-actin mRNA was not significantly changed between lupusand normal control mice (Fig. I b), nor during the growing process (Fig. b, Fig. 2).
Increased TNF-L by lung lymphoid cells of lupus-prone mice To study the source of TNF-cx production in lung, we used RNA from the mononuclear cells isolated from lungs for Northern blot assay. Each lane in Northern blots was loaded with the same amount of each 5 /,g of total RNA. As shown in Fig. 2, the strong signal of TNF-a transcripts was only detected in lupusprone mice. Furthermore, the transcript level of TNF-a gene in lung lymphoid mononuclear cells from lupus-prone mice increased during the growing process. We used the same mice as in the study of total lung RNA in Fig. 1. The increased levels of TNF-ax expression in total lung from lupus-prone mice were comparable with those in lung lymphoid cells.
TNF mRNA accumulation by enhanced transcription in lung lymphoid cells of lupus-prone mice We examined transcriptional level of the TNF-a gene by nuclear run on transcription assay. As shown in Fig. 3, enhanced transcription of the TNF-cx gene was found in 3-month-old MRL/lpr mice (female), as compared with age-matched MRL/n control mice (female). By densitometrical analysis, this
TNF in autoimmune pulmonary inflammation
increased level of transcription found in lupus-prone mice is more than 12 times that found in control mice. There was no significant change in transcriptional level of beta-actin gene between lupus-prone and control mice.
ary inflammation in lupus-prone mice has been investigated. Molecular mechanisms of enhanced transcription of TNF-x gene by infiltrated lymphoid mononuclear cells in lungs of lupus-prone mice are now in progress.
Effect of anti- TNF antibody on pulmonary legions of lupus-prone
Figure 4 is representative of the pulmonary histopathology of lupus-prone mice after treatment of anti-TNF antibody and control serum (10 weeks old). The pathological changes of pulmonary inflammation were significantly reduced in lupusprone mice treated with anti-TNF IgG, in comparison to control serum. No significant change of proteinuria level between these lupus-prone mice treated with anti-TNF antibody and control serum was detected in this study (no improvement or deterioration) (data not shown). DISCUSSION These studies have demonstrated that lungs from lupus-prone mice, as compared with normal healthy mice, have enhanced transcription of TNF-La gene and that the major sources of TNFa transcripts in lungs are infiltrated lymphoid mononuclear cells. Our data suggest that TNF-a is an important factor in the development of pulmonary inflammation in lupus-prone mice. The enhanced production of TNF-a can suggest that pathological lesions in lungs of lupus-prone mice are mediated by excessive action of TNF-a (TNF-mediated immunopathological reactions). Infiltrated lymphoid mononuclear cells are, in quantitative terms, the most important procedures of TNF-a. We found no detectable granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in lungs of lupus-prone mice (data not shown). Recently, it was shown that continuous in vivo infusion of TNF-cx induced a diffuse alveolar damage, notably with necrosis of alveolar epithelial and endothelial cells (Tracy et al., 1988). Pulmonary vasculitis induces pneumopathy in lupus-prone mice. Reports have also indicated that MRL/lpr and BXSB mice develop vasculitic cell infiltrates in various organs, including lungs (Andrews et al., 1978; Berden et al., 1983; Alexander et al., 1985). The susceptibility of the lesions to the anti-TNF antibody strongly suggests that TNF-a, microenvironmentally produced by infiltrated lymphocytes, may have an important role in the development of pulmonary inflammation in lupus-prone mice. We detected no significant changes of proteinuria level between the lupus-prone mice treated with anti-TNF antibody and control serum (no improvement or deterioration). Various other cytokines (e.g. IL-1) have been shown to influence immunological reactions in lungs in vivo and in vitro. A further element of complexity results from the possibility that these mediators may induce each other, as the induction of IL- 1 by TNF-ot has been shown (Ruff & Gifford, 1981). In this context, the results of anti-TNF antibody might indicate that other mediators are not produced in significant quantities and/ or that involvements by other mediators could be dependent on TNF-a. The key role of TNF-a in the development of pulmon-
This study was supported in part by grants from the Ministry of Culture, Education and Science and the Ministry of Health and Welfare of Japan.
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