Clin. exp. Immunol. (1992) 90, 68-71
Tumour necrosis factor enhances the asbestos-induced production of reactive oxygen metabolites by human polymorphonuclear leucocytes (PMN) M. KLOCKARS & H. SAVOLAINEN Institute of Occupational Health, Helsinki, Finland
(Acceptedfor publication 17 June 1992)
SUMMARY We studied the effect of recombinant tumour necrosis factor-alpha (TNF-c) on the production of reactive oxygen metabolites (ROM) by human PMN exposed in vitro to chrysotile and crocidolite asbestos fibres, quartz dusts and opsonized zymosan. TNF caused a significant increase in ROM release by PMN, and significantly and dose-dependently amplified the ROM production induced by asbestos fibres. The amplification of ROM production by TNF can be of crucial importance in the process of lung inflammation and fibrogenesis in pneumoconioses. Keywords tumour necrosis factor asbestos quartz reactive oxygen metabolites macrophages [22]. This suggests possible important interactions between TNF and ROM production by inflammatory cells. The present study was undertaken to compare the effect of TNF on the production of ROM by human PMN exposed to chrysotile and crocidolite asbestos fibres, quartz dusts and to a standard PMN-stimulating agent, opsonized zymosan, in vitro.
INTRODUCTION The pathogenic mechanisms by which asbestos dust causes fibrotic lung disease are incompletely understood. Different environmental respirable particles have varying potential to cause pulmonary disease, including chronic inflammation and cancer [1]. The PMN is a pivotal cell in the acute inflammatory response to inhaled particles. Interaction with asbestos fibres induces the inflammatory cells, including PMN and macrophages, to generate reactive oxygen metabolites (ROM) [2-4]. ROMs have been implicated as important mediators of inflammation, and many biological structures, including endothelial cells, lipoprotein membranes, enzymes and DNA, are potential targets for oxidative injury [5-9]. Tumour necrosis factor (TNF) plays a central role in the pathophysiology of immunity, and is associated with diverse inflammatory states [10,11]. TNF appears to play a critical role in acute lung injury and in the regulation of the fibroproliferative response [12,13]. Exogenous TNF triggers PMN influx into inoculated tissues and causes the activation of PMN [14-16]. TNF may augment the inflammatory response by directly stimulating the production of ROM by PMN [17-19]. In addition, the stimulation of fibroblast proliferation by TNF is relevant to the chronicity and outcome of the inflammatory response [20,21]. It has been shown that the production of endogenous cytokines during the inflammatory response results in complex antagonistic and synergistic inflammatory effects [11]. Such a regulatory role of the cytokines probably also influences the inflammatory response to mineral fibres. It has, in fact, been shown that mineral dusts such as silica particles and asbestos fibres can induce or augment TNF production by alveolar
MATERIAL AND METHODS PMN were isolated from heparinized human venous blood samples obtained from apparently healthy adults by Ficoll density centrifugation as described previously [23]. Chemiluminescence (CL) was measured with an automated microcomputer-controlled luminometer (LKB-Wallac 1251, Sweden) at 370C. The reaction mixture (final total volume 1 0 ml) consisted of 700 jil 10-4 M luminol (5-amino,2,3,-dihydro, l,4,-phthalazinedione; LKB Wallac) in Dulbecco's PBS, 50 p1 of cell suspension (stock concentration 5 x 106 cells/ml), 10-200 pi of particles suspended in PBS (stock concentration I 0 mg/ml) and cytokines at given concentrations. The CL response was followed up for 40 min after the particles had been added to the cells. Each test was performed with PMN obtained from three to five different individuals. Appropriate controls, as well as all test substances to be compared, were included in each experiment. We used IUAC (International Union Against Cancer) standard reference samples of chrysotile and crocidolite asbestos. Fyle quartz and DQ 12 quartz were gifts to our Institute; their physicochemical characteristics have been described previously [24]. Opsonized zymosan was prepared as described earlier [25]. Recombinant human TNF-a was produced by Boehringer-Ingelheim, Austria and kindly donated by Hans Sarelin from Boehringer-Ingelheim, Helsinki, Finland. TNF
Correspondence: Matti Klockars, MD, Institute of Occupational Health, Topeliuksenkatu 41 aA, SF-00250 Helsinki, Finland.
68
TNF amplifies reactive oxygen metabolite production by human PMN 20
200
20~~~~~~~~~~~~~~~~ i5
69
-
150
E 0
Time
(min)
Fig. 1. Chemiluminescence (CL) response of 2 5 x 105 polymorphonuclear leucocytes (PMN) exposed at time zero to 0 005-5 0 pg/mi of TNF. Each point represents the mean CL response of nine different mndividuals. 0, TNF 0 5 jug/mi; 0, TNF 5 0 pg/mi; 0, TNF 0 05 jug/mi; U, 0 005 pg/mi; A, cells alone.
was poduceby rcombiant NA tehnoloy in seherchia
ccli, and contained a specific activity of 6 x 10 U/mg, was of
0
0
8
16 24 32 40 Time (mmin) Fg .Ceiuiecne(L eos f2 O oyopo 300 Fig. 2. Chemllumlnescence (CL) reponse of 2 5 x 105 polymorE nuclear leucocytes (PMN) exposed to chrysotile (0.; 50 pg/mi), TNF (-; 5 0 pg/mi) separately, and to chrysotile (50 and TNF (5 0 p/i obnd()
pug/mi)
> 99%/ purity, and contained
Tumour necrosis factor enhances the asbestos-induced production of reactive oxygen metabolites by human polymorphonuclear leucocytes (PMN) M. KLOCKARS & H. SAVOLAINEN Institute of Occupational Health, Helsinki, Finland
(Acceptedfor publication 17 June 1992)
SUMMARY We studied the effect of recombinant tumour necrosis factor-alpha (TNF-c) on the production of reactive oxygen metabolites (ROM) by human PMN exposed in vitro to chrysotile and crocidolite asbestos fibres, quartz dusts and opsonized zymosan. TNF caused a significant increase in ROM release by PMN, and significantly and dose-dependently amplified the ROM production induced by asbestos fibres. The amplification of ROM production by TNF can be of crucial importance in the process of lung inflammation and fibrogenesis in pneumoconioses. Keywords tumour necrosis factor asbestos quartz reactive oxygen metabolites macrophages [22]. This suggests possible important interactions between TNF and ROM production by inflammatory cells. The present study was undertaken to compare the effect of TNF on the production of ROM by human PMN exposed to chrysotile and crocidolite asbestos fibres, quartz dusts and to a standard PMN-stimulating agent, opsonized zymosan, in vitro.
INTRODUCTION The pathogenic mechanisms by which asbestos dust causes fibrotic lung disease are incompletely understood. Different environmental respirable particles have varying potential to cause pulmonary disease, including chronic inflammation and cancer [1]. The PMN is a pivotal cell in the acute inflammatory response to inhaled particles. Interaction with asbestos fibres induces the inflammatory cells, including PMN and macrophages, to generate reactive oxygen metabolites (ROM) [2-4]. ROMs have been implicated as important mediators of inflammation, and many biological structures, including endothelial cells, lipoprotein membranes, enzymes and DNA, are potential targets for oxidative injury [5-9]. Tumour necrosis factor (TNF) plays a central role in the pathophysiology of immunity, and is associated with diverse inflammatory states [10,11]. TNF appears to play a critical role in acute lung injury and in the regulation of the fibroproliferative response [12,13]. Exogenous TNF triggers PMN influx into inoculated tissues and causes the activation of PMN [14-16]. TNF may augment the inflammatory response by directly stimulating the production of ROM by PMN [17-19]. In addition, the stimulation of fibroblast proliferation by TNF is relevant to the chronicity and outcome of the inflammatory response [20,21]. It has been shown that the production of endogenous cytokines during the inflammatory response results in complex antagonistic and synergistic inflammatory effects [11]. Such a regulatory role of the cytokines probably also influences the inflammatory response to mineral fibres. It has, in fact, been shown that mineral dusts such as silica particles and asbestos fibres can induce or augment TNF production by alveolar
MATERIAL AND METHODS PMN were isolated from heparinized human venous blood samples obtained from apparently healthy adults by Ficoll density centrifugation as described previously [23]. Chemiluminescence (CL) was measured with an automated microcomputer-controlled luminometer (LKB-Wallac 1251, Sweden) at 370C. The reaction mixture (final total volume 1 0 ml) consisted of 700 jil 10-4 M luminol (5-amino,2,3,-dihydro, l,4,-phthalazinedione; LKB Wallac) in Dulbecco's PBS, 50 p1 of cell suspension (stock concentration 5 x 106 cells/ml), 10-200 pi of particles suspended in PBS (stock concentration I 0 mg/ml) and cytokines at given concentrations. The CL response was followed up for 40 min after the particles had been added to the cells. Each test was performed with PMN obtained from three to five different individuals. Appropriate controls, as well as all test substances to be compared, were included in each experiment. We used IUAC (International Union Against Cancer) standard reference samples of chrysotile and crocidolite asbestos. Fyle quartz and DQ 12 quartz were gifts to our Institute; their physicochemical characteristics have been described previously [24]. Opsonized zymosan was prepared as described earlier [25]. Recombinant human TNF-a was produced by Boehringer-Ingelheim, Austria and kindly donated by Hans Sarelin from Boehringer-Ingelheim, Helsinki, Finland. TNF
Correspondence: Matti Klockars, MD, Institute of Occupational Health, Topeliuksenkatu 41 aA, SF-00250 Helsinki, Finland.
68
TNF amplifies reactive oxygen metabolite production by human PMN 20
200
20~~~~~~~~~~~~~~~~ i5
69
-
150
E 0
Time
(min)
Fig. 1. Chemiluminescence (CL) response of 2 5 x 105 polymorphonuclear leucocytes (PMN) exposed at time zero to 0 005-5 0 pg/mi of TNF. Each point represents the mean CL response of nine different mndividuals. 0, TNF 0 5 jug/mi; 0, TNF 5 0 pg/mi; 0, TNF 0 05 jug/mi; U, 0 005 pg/mi; A, cells alone.
was poduceby rcombiant NA tehnoloy in seherchia
ccli, and contained a specific activity of 6 x 10 U/mg, was of
0
0
8
16 24 32 40 Time (mmin) Fg .Ceiuiecne(L eos f2 O oyopo 300 Fig. 2. Chemllumlnescence (CL) reponse of 2 5 x 105 polymorE nuclear leucocytes (PMN) exposed to chrysotile (0.; 50 pg/mi), TNF (-; 5 0 pg/mi) separately, and to chrysotile (50 and TNF (5 0 p/i obnd()
pug/mi)
> 99%/ purity, and contained
