Clin. exp. Immunol. (1990) 81, 311-314
Enhanced expression of the tumour necrosis factor/cachectin gene in peripheral blood mononuclear cells from patients with systemic vasculitis Y. DEGUCHI, N. SHIBATA* & S. KISHIMOTO Third Department of Internal Medicine, Osaka University School of Medicine, Osaka; and *Division of Internal Medicine, Centre for Adult Diseases, Osaka, Japan (Acceptedfor publication 8 March 1990)
SUMMARY The expression of tumour necrosis factor-alpha (TNF-a) gene in peripheral blood mononuclear cells from patients with systemic vasculitis, periarteritis nodosa and Wegener's granulomatosis, has been studied by RNA dot blot and Northern blot assays. We further examined the transcriptional level of TNF-a gene in peripheral blood mononuclear cells from these patients by performing nuclear run on transcription assay. We demonstrate enhanced TNF-a gene expression in mononuclear cells from these patients compared with healthy subjects and patients with bronchial asthma. These findings suggest that the enhanced transcription of TNF-a gene in peripheral blood mononuclear cells from patients with systemic vasculitis may be involved in the pathophysiology/pathogenesis of these diseases by cytokine dysregulation. Keywords tumour necrosis factor
transcription systemic vasculitis
Systemic vasculitis is a group of clinical disorders characterized by damage and inflammation of blood vessels. Several immunological and pathological studies of systemic vasculitis (Fauci, Haynes & Katz, 1978) have shown the importance of circulating and tissue-bound immune complexes in the pathogenesis of systemic vasculitis was demonstrated (Cupps & Fauci, 1981). This deposition is enhanced by cytokines derived from peripheral blood cells (Kniker & Cochrane, 1976). Causative antigens have been implicated in systemic vasculitis only rarely. Cellmediated immunological reactions may also contribute to the pathogenesis of vasculitis associated with granulomatous inflammation (Epstein, 1967). While the pathogenesis of systemic vasculitis is not completely understood, there is considerable evidence for the importance of cytokines in the immune system.
Tumour necrosis factor (TNF, 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
that the expression of TNF-a gene in peripheral blood mononuclear cells from patients with systemic vasculitis such as periarteritis nodosa and Wegener's granulomatosis is elevated, compared with healthy subjects and patients with bronchial asthma. This enhanced expression of the TNF-a gene depended upon the elevated levels of the gene transcription in the cells. The possible implication of this enhancement of TNF-a gene expression in pathophysiology/pathogenesis of systemic vasculitis is discussed.
MATERIALS AND METHODS Human subjects We examined five patients with periarteritis nodosa and five patients with Wegener's granulomatosis. The diagnoses of these diseases were made clinically in addition to the biopsy findings in some cases (Mowrey & Lundberg, 1967). Clinical findings of these cases are shown in Table 1. We also examined five patients with bronchial asthma and five healthy volunteers who were laboratory or hospital personnel and had no history of use of any medication known to affect immune functions.
Preparation of peripheral blood mononuclear cells Heparinized blood was obtained from the subjects. Peripheral blood mononuclear cells were obtained with Ficoll-Paque method. The cells were suspended in RPMI 1640 medium (Bioproducts), layered on Ficoll-Paque, and centrifuged at 1800 rev/min for 20 min. Peripheral blood mononuclear cells were
mediator of shock (Tracey et al., 1986). Activated macrophages and various macrophage cell lines have been shown to produce TNF (Beutler & Cerami, 1988). In this study, we demonstrate Correspondence: Yasuhiro Deguchi, MD, Ph.D., NIAID, NIH, 10687 Weymouth Street, no. 103, Bethesda, MD 20814, USA. 311
Y. Deguchi, N. Shibata & S. Kishimoto
Table 1. Clinical findings of periarteritis nodosa (PN) and Wegener's granulomatosis (WG) cases in this study
PN 1 2 3 4 5 WG
M M M M F
45 41 37 40 35
+ + + + +
K,M,G,F K,H,F K,M,C K,M,F K,G,F
M M M M F
35 44 47 37 38
+ + + + +
L,M,N L,K,N M,N K,M,E L,K,E
1 2 3 4 5
*Erythrocyte sedimentation rate; +, ESR> 10 mm/h. K, kidney; M, musculocutaneous; G, gastrointestinal tract; H, hypertension; F, fever; C, central nervous sytem; L, lung; N, nose or nasopharynx; E, eye disorder. collected from the interface and washed twice with RPMI 1640. The cell population was over 97% viable (trypan blue exclusion). RNA preparation and RNA dot blot and Northern blot assay Total RNA from the cells was prepared by guanidiumthiocyanate and caesium chloride procedure (Chomcyzski & Sacchi, 1987). For RNA dot blot assay, 0 5 pg of total RNA was dotted on the nylon membranes (Biodyne, Pall). For Northern blot assay, 15 pg of total RNA were applied on 0 8% formaldehyde-agarose gels. Samples were transferred to nylon membranes and baked under vacuum at 80'C for 2 h. The filters were hybridized (Deguchi et al., 1987) in a solution including 32P-labelled probes for human TNF-a cDNA or anti-sense oligonucleotides (Amgen, Los Angeles, CA) at first or actin cDNA (Wako Pure Chemical Industries, Japan) (specific activity, 15 x 108 ct/min per pg) after autoradiography and removing the TNF probe from the same filters. The final washes were done under conditions with 6 x SSC and 0 1% SDS for oligonucleotide probe and 0 2 x SSC and 0 1% SDS for cDNA probe at 45°C for 1 h, before autoradiography. Filters were exposed to Kodak AR X-Omat films at - 70°C.
solution to a final concentration of 1 % and 5 mm, respectively, followed by treatment with proteinase 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 NaCl, 0-5% SDS, 2 mm EDTA, 50 mm HEPES (pH 7 0), Denhardt's solution and denatured salmon sperm DNA (500 pg/ml) (Marzluff& Huang, 1984). Finally, the RNA preparation was hybridized to the nitrocellulose filter onto which the human TNF-a cDNA probe or beta-actin cDNA probe (Wako Pure Chemical Industries, Japan) had been dotted in appropriate conditions. After a 24-h incubation, the filter was washed/dried and exposed to X-ray film with intensifying screen at 70°C. In all experiments, the hybridized dot was excised from the filter and directly counted by beta-counter (Reed et al., 1986). -
RESULTS As shown in Fig. 1, the amount of TNF-a mRNA was increased in peripheral blood mononuclear cells from patients with periarteritis nodosa and Wegener's granulomatosis compared with healthy subjects and patients with bronchial asthma by RNA dot blot assay. We applied equal amount of RNA in each dot on the filters. We also found that the increased TNF-o mRNA isolated from the peripheral blood mononuclear cells from these patients with systemic vasculitis was of normal length (1 9 kbp) as assessed by Northern blot assay (Fig. 2). We detect no significant changes in expression of the actin gene in peripheral blood mononuclear cells from the patients and controls. Finally, Fig. 3 shows the representative date obtained by nuclear run on transcription assay. We found the enhanced TNF-ac gene transcription in peripheral blood mononuclear cells from these patients with systemic vasculitis. In contrast, using a probe for beta-actin gene, we showed no remarkable changes in the transcription of actin gene in peripheral blood mononuclear cells from patients with systemic vasculitis and control subjects (healthy subjects and patients with bronchial asthma).
Nuclear run on transcription assay Nuclei were prepared from the cells by lysing the cells in the 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. Twelve-million nuclei were suspended into 100 p1 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 MgC12, 5 mM DTT, 1 mm of ATP, CTP, GTP, and 200 units of RNasin (ribonuclease inhibitor, 500 U, Amersham International, Amersham, UK). The preparation was then incubated at 28°C for 20 min after addition with 50 pCi 32P-radiolabelled UTP (3000 mCi/ml, Amersham). SDS and EDTA were added to the
8 TNF RNA
Fig. 1. Comparative expression of TNF-a gene by peripheral blood mononuclear cells from patients with systemic vasculitis and control subjects. The amount of TNF-a-specific mRNA was evaluated by RNA dot blot assay. Purified RNA from peripheral blood mononuclear cells from patients with periarteritis nodosa (PN) and Wegener's granulomatosis (WG), normal healthy subjects and patients with bronchial asthma were dotted on the same filters and hybridized using a 32P-labelled TNFa probe. Each subject was evaluated in duplicate. Autoradiographic images of the hybridization were quantified by densitometry and expressed in U/105 cells. Mean+s.d.
Tumour necrosis factor in vasculitis 3
Fig. 2. Representative result of Northern blot assay for TNF-a RNA expression. Lane 1, peripheral blood mononuclear cells from a subject with periarteritis nodosa (subject no. 2); lane 2, subject with bronchial asthma (subject no 3); lane 3, with Wegener's granulomatosis (subject no. 5); lane 4, a healthy subject (subject no. 5). We examined all patients and control subjects in Fig. 1, and results were similar to these representative ones. A
result of nuclear
and beta-actin gene
nuclear cells from
periarteritis nodosa (no. 2);
Wegener's granulomatosis (subject
healthy subject (no. 5); B, from
bronchial asthma; C, from and D, with
expression. A, peripheral
I and results
5). We examined all
similar to these
systemic vasculitis, such
periarteritis nodosa variety of
symptoms including general fatigue, fever, myalgia and abdo-
minal, head and extremity pains. The pathogenesis/pathophy-
siology of the diseases is unknown. TNF is a multi-meric protein (Beutler & Cerami, 1987) and a cytokine which has many
mediator of shock,
we examined the expression of TNF-a gene in peripheral blood mononuclear cells from patients with systemic vasculitis. In RNA dot blot and Northern blot assays with probes for human TNF-a transcript, we first demonstrated that the amount of TNF-a mRNA was of normal size in peripheral blood mononuclear cells from these patients and increased in comparison to healthy subjects and patients with bronchial asthma as controls (Figs 1 and 2). To examine the transcriptional level of the TNF-a gene in peripheral blood mononuclear cells, we further used the nuclear run on transcription assay (Fig. 3). As shown in Fig. 3, we found the enhanced transcription of the TNF-a gene in peripheral blood mononuclear cells from patients with systemic vasculitis, compared with controls. This suggests that the increased amount of TNF-Lx mRNA in the cells is due to enchanced transcription of the TNF-a gene in the cells. We also analysed DNA amplification and gross translocation of TNF gene by Southern blot assay. We found neither gene amplification nor gross translocation of TNF gene in this study (data not shown). Within the limited number of cases in this study, it seemed that there was a positive correlation between the enhanced transcription of the TNF-a gene and clinical disease activity. We need more cases to clarify this possible correlation and this study is in progress. We also examined plasma TNF levels in these patients and controls. Plasma TNF levels were determined by ELISA using recombinant human TNF and purified monoclonal antibody to human TNF. We found increased TNF level in these patients in mean value (normal < 38 pg/ml, patients 59 pg/ml), but in some cases TNF levels were increased in controls. The range of the values of TNF level is very wide in systemic vasculitis. There was no clear positive correlation between plasma TNF-a levels and disease activity by measuring plasma TNF levels only. TNF has many biological activities in vivo and in vitro (Beutler & Cerami, 1988). TNF-cx has been reported to inhibit endothelial cell growth, contributing to vascular damage (Kahaleh et al., 1988). TNF administration in vivo is associated with severe endothelial cell damage and development of a vascular leak syndrome (Tracey et al., 1987). Our observation suggests that the enhanced TNF-c gene expression in peripheral blood mononuclear cells may be involved in cytokine dysregulations on vascular-endothelial cells in systemic vasculitis and may contribute to an understanding of the pathophysiology/pathogenesis of chronic inflammatory disorders in systemic vasculitis. We are now investigating the mechanisms of enhancement of TNF-oe gene expression in peripheral blood mononuclear cells from patients with systemic vasculitis, and its interaction with other cytokines. The major source of TNF-a transcription in peripheral blood mononuclear cells from patients with systemic vasculitis is of monocytic origin (data not shown). The major target cells and biological effects of TNF-a in patients with systemic vasculitis are unknown, but the marked increase of transcription of TNF-a gene in peripheral blood mononuclear cells from patients with systemic vasculitis could indicate that TNF-ax plays a major role in the pathophysiology/pathogenesis of systemic vasculitis.
activity and activity as a mesenchymal cell growth (Ruff & Gifford, 198 1). To examine the cytokine systems
and cellular activation in
peripheral blood mononuclear cells
vasculitis and its
of systemic vasculitis,
ACKNOWLEDGMENTS 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. We thank Ms Kyoto Sugita for excellent editorial assistance.
Y. Deguchi, N. Shibata & S. Kishimoto
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