Agents and Actions,vol. 31, 3/4 (1990)
0065-4299/90/040275-05$1.50+ 0.20/0 9 1990 BirkhfiuserVerlag, Basel
Ambroxol inhibits interleukin 1 and tumor necrosis factor production in human mononuclear cells M. Bianchi, A. Mantovani, A. Erroi, C.A. Dinarello 1 and P. Ghezzi Laboratoryof Immunology,Istituto di RicercheFarmacologiche"Mario Negri", Via Eritrea62, 20157 Milan, Italy and 1Divisionof GeographicMedicineand InfectiousDiseases, TuftsUnversityNew England MedicalCenter, Boston,MA, USA
Abstract
We have studied the effect of Ambroxol on the production of lnterleukin-I (IL-1) and Tumor Necrosis Factor (TNF) in human mononuclear cells (MNC). For this purpose MNC were cultured for 24 h in the presence of endotoxin and different doses of Ambroxol. The results indicate that Ambroxol markedly inhibited IL-1 and TNF production at doses of 10-100 gg/ml, without any apparent toxicity.
Introduction
There is a growing literature on Interleukin 1 (IL-I) and Tumor Necrosis Factor (TNF) as inflammatory mediators [1, 2]. In particular, administration of IL-I and TNF have been shown to cause pulmonary damage and these cytokines have been suggested to play a role in silica- and Neomycin-induced lung fibrosis [3, 4]. In fact, administration to mice of anti-TNF antibodies was reported to ameliorate Neomycin-induced lung fibrosis [5]. It might therefore be important to identify compounds that inhibit the synthesis of these cytokines. Among the antiinflammatory agents, only glucocorticoids were reported to inhibit the synthesis of IL-1 and TNF by monocytes/macrophages [6, 7]. Interferon gamma was also reported to inhibit IL-1 synthesis [8] and this was suggested to be important for its pharmacological activity in rheumatoid arthritis and its inhibition of bleomycin-induced lung fibrosis [9]. In this paper we report that Ambroxol (Mucosolvan-NA 872metabolite VII1 of bromhexine), a drug used to increase surfactant production in the lungs, in-
hibits the synthesis of IL-1 and TNF by human mononuclear cells stimulated with endotoxin (lipopolysaccharide, LPS).
Materials and methods
Following informed consent, blood was obtained from healthy volunteers who had not used cyclooxygenase inhibitors during the previous 2 weeks. MNC were prepared using Ficoll-Hypaque gradients [8]. Cells were cultured at the concentration of 2.5 • 106/ml in LPS-free RPMI 1640 medium [8] in the presence and absence of 10 ng/ml LPS (E. coli 055:B5, Sigma, St Louis, USA) with the indicated concentration of Ambroxol (De Angeli, Milan, Italy). After 24 h, cultures (cells+supernatant) were subjected to three freeze-thaw cycles to allow complete cell lysis, and the lysates were assayed for IL-I beta or TNF in specific radioimmunoassay (RIA, ref. [8, 10]). In some experiments, supernatants were harvested to allow determination of the amount of IL-I in cells (cell-associated) and supernatants (secreted).
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Agents a n d Actions, vol. 31, 3/4 (1990)
For IL-1 mRNA determination, MNC were lysed in guanidine isothiocyanate and RNA was prepared by centrifugation through cesium chloride [11]. 2.Spg of RNA were analyzed by electrophoresis through 1% agarose formaldehyde gel followed by Northern blot transfer to Gene Screen Plus membranes (New England Nuclear, Boston, MA). An IL-1 beta cDNA clone [12] was labelled with alpha-/32P/dCTP (3.0 Ci/mmole, Amersham) by the random priming method (Feinberg, AB 132,6). Membranes were pretreated and hybridized in 50% formamide (Merck) with 10% dextran sulfate (Sigma) and washed twice with 2 x SSC (1 x SSC is 0.15 MNaC1 with 0.015 M N a citrate) and 1% sodium dodecyl sulfate (SDS, Sigma) at 60 ~ for 30 minutes, and then twice more with 0.1 x SSC at room temperature for 30 minutes. The membranes were exposed for 4 - 8 hours at - 8 0 ~ using intensifying screens.
Results
Figure 1 shows the effect of Ambroxol at two different concentrations (10 and 100 lag/ml) on IL-1 production by MNC from 7 different donors, cultured for 24 h in the presence of 10 ng/ml LPS.
It is clear that Ambroxol markedly inhibited IL-I synthesis in all donors at both concentrations (p
0065-4299/90/040275-05$1.50+ 0.20/0 9 1990 BirkhfiuserVerlag, Basel
Ambroxol inhibits interleukin 1 and tumor necrosis factor production in human mononuclear cells M. Bianchi, A. Mantovani, A. Erroi, C.A. Dinarello 1 and P. Ghezzi Laboratoryof Immunology,Istituto di RicercheFarmacologiche"Mario Negri", Via Eritrea62, 20157 Milan, Italy and 1Divisionof GeographicMedicineand InfectiousDiseases, TuftsUnversityNew England MedicalCenter, Boston,MA, USA
Abstract
We have studied the effect of Ambroxol on the production of lnterleukin-I (IL-1) and Tumor Necrosis Factor (TNF) in human mononuclear cells (MNC). For this purpose MNC were cultured for 24 h in the presence of endotoxin and different doses of Ambroxol. The results indicate that Ambroxol markedly inhibited IL-1 and TNF production at doses of 10-100 gg/ml, without any apparent toxicity.
Introduction
There is a growing literature on Interleukin 1 (IL-I) and Tumor Necrosis Factor (TNF) as inflammatory mediators [1, 2]. In particular, administration of IL-I and TNF have been shown to cause pulmonary damage and these cytokines have been suggested to play a role in silica- and Neomycin-induced lung fibrosis [3, 4]. In fact, administration to mice of anti-TNF antibodies was reported to ameliorate Neomycin-induced lung fibrosis [5]. It might therefore be important to identify compounds that inhibit the synthesis of these cytokines. Among the antiinflammatory agents, only glucocorticoids were reported to inhibit the synthesis of IL-1 and TNF by monocytes/macrophages [6, 7]. Interferon gamma was also reported to inhibit IL-1 synthesis [8] and this was suggested to be important for its pharmacological activity in rheumatoid arthritis and its inhibition of bleomycin-induced lung fibrosis [9]. In this paper we report that Ambroxol (Mucosolvan-NA 872metabolite VII1 of bromhexine), a drug used to increase surfactant production in the lungs, in-
hibits the synthesis of IL-1 and TNF by human mononuclear cells stimulated with endotoxin (lipopolysaccharide, LPS).
Materials and methods
Following informed consent, blood was obtained from healthy volunteers who had not used cyclooxygenase inhibitors during the previous 2 weeks. MNC were prepared using Ficoll-Hypaque gradients [8]. Cells were cultured at the concentration of 2.5 • 106/ml in LPS-free RPMI 1640 medium [8] in the presence and absence of 10 ng/ml LPS (E. coli 055:B5, Sigma, St Louis, USA) with the indicated concentration of Ambroxol (De Angeli, Milan, Italy). After 24 h, cultures (cells+supernatant) were subjected to three freeze-thaw cycles to allow complete cell lysis, and the lysates were assayed for IL-I beta or TNF in specific radioimmunoassay (RIA, ref. [8, 10]). In some experiments, supernatants were harvested to allow determination of the amount of IL-I in cells (cell-associated) and supernatants (secreted).
276
Agents a n d Actions, vol. 31, 3/4 (1990)
For IL-1 mRNA determination, MNC were lysed in guanidine isothiocyanate and RNA was prepared by centrifugation through cesium chloride [11]. 2.Spg of RNA were analyzed by electrophoresis through 1% agarose formaldehyde gel followed by Northern blot transfer to Gene Screen Plus membranes (New England Nuclear, Boston, MA). An IL-1 beta cDNA clone [12] was labelled with alpha-/32P/dCTP (3.0 Ci/mmole, Amersham) by the random priming method (Feinberg, AB 132,6). Membranes were pretreated and hybridized in 50% formamide (Merck) with 10% dextran sulfate (Sigma) and washed twice with 2 x SSC (1 x SSC is 0.15 MNaC1 with 0.015 M N a citrate) and 1% sodium dodecyl sulfate (SDS, Sigma) at 60 ~ for 30 minutes, and then twice more with 0.1 x SSC at room temperature for 30 minutes. The membranes were exposed for 4 - 8 hours at - 8 0 ~ using intensifying screens.
Results
Figure 1 shows the effect of Ambroxol at two different concentrations (10 and 100 lag/ml) on IL-1 production by MNC from 7 different donors, cultured for 24 h in the presence of 10 ng/ml LPS.
It is clear that Ambroxol markedly inhibited IL-I synthesis in all donors at both concentrations (p
