Tumor necrosis factor alpha and interleukin 6 release induced by antibiotic killing of Pseudomonas aeruginosa and Staphylococcus aureus

OBJECTIVE: The purpose of this study was to determine the depth that implants may be safely placed into the distal femoral epiphysis (DFE) for the repair of distal femoral physeal fractures. STUDY DESIGN: The depth of the DFE was related to the radiographic thickness of the patella in this experimental study. ANIMALS OR SAMPLE POPULATION: Twenty immature canine cadavers. METHODS: Patella thicknesses were measured from lateral radiographs. Actual DFE depths were determined for pins driven in normograde fashion and for pins driven retrograde from the central depression between the metaphyseal pegs and from the cranial pegs. The association of DFE depth and patella thickness was evaluated using linear regression analysis. Using 95% confidence intervals, rules for estimating the safe depth of implant placement into the DFE were determined. RESULTS: DFE depth had significant correlation with patella thickness for pins placed in retrograde fashion from the central depression between the metaphyseal pegs (r2 = .83) and from the cranial pegs (r2 = .82) and for pins placed in normograde fashion (r2 = .65). CONCLUSIONS: Based on 95% confidence intervals, pins placed in retrograde fashion from the central depression between the metaphyseal pegs may be safely driven into the DFE a distance equal to 140% of patella thickness. Pins placed from the cranial metaphyseal pegs may be driven to a depth equal to 80% of patella thickness, and pins placed in normograde fashion may be driven to a depth equal to 30% of patella thickness. CLINICAL RELEVANCE: Measurement of patella thickness assists the surgeon in determining the approximate depth that pins may be driven into the DFE without penetrating the articular surface of the stifle joint.     

Effect of interferon-gamma on nitric oxide hemoglobin production in endotoxin-treated rats and its synergism with interleukin 1 or tumor necrosis factor

We studied the in vivo effect of interferon-gamma (IFN-gamma) on nitric oxide (NO) generation. ESR spectra of nitric oxide hemoglobin (HbNO) appeared after a lag time of 2h in the blood of rats treated with Escherichia coli lipopolysaccharide (LPS). IFN-gamma enhanced LPS-induced HbNO formation in rats without modifying the time lag, although IFN-gamma alone did not induce HbNO formation. The plasma nitrate concentration was approximately one order of magnitude higher than the HbNO concentration. On treatment with LPS alone, the amount of tumor necrosis factor (TNF) released decreased after 2 h. Simultaneous addition of IFN-gamma and LPS increased TNF release for at least 8 h. Interleukin 1 (IL-1) release was detected only at 2 h in both groups. We also investigated the in vivo interactions of these cytokines. TNF plus IL-1 induced the greatest HbNO generation, followed by TNF plus IFN-gamma, and then IL-1 plus IFN-gamma. These results suggest that increase of TNF release by IFN-gamma plays a key role in NO generation in LPS-treated rats.     

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.     

In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide

The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO.     

Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.     

Induction of macrophage-mediated production of tumor necrosis factor alpha by an L-form derived from Staphylococcus aureus

We investigated the capability of an L-form derived from Staphylococcus aureus to induce tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. The activity for TNF-alpha induction was found in the membrane fraction of the L-form but not in the cytoplasmal fraction purified by the sucrose step gradient centrifugation. TNF-alpha mRNA was also detected in macrophages stimulated with L-form membranes. L-form induced TNF-alpha production in macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains. Regardless of the presence of polymyxin B, the activity of TNF-alpha induction of L-form was mostly found in the phenol layer, but not in the aqueous layer, both of which were prepared by phenol extraction method. Fractions of L-form membranes representing molecular masses of approximately between 29 and 36 kDa were primarily responsible for inducing the production of TNF-alpha consistently. Moreover, this stimulatory effect was abolished by digestion with Streptomyces griseus protease. In Western blot (immunoblot) analysis with anti-lipoteichoic acid antibody, two bands (65 and 45 kDa) were observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phenol layer, whereas one band (14 kDa) was observed in either the aqueous layer or lipoteichoic acid of S. aureus. These results suggest that the component in the membrane of the L-form, distinct from cell wall components such as teichoic acid or lipopolysaccharide, possesses the capability to stimulate TNF-alpha production by macrophages.     

Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha

Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization.     

Tumor necrosis factor alpha regulates nitric oxide synthase expression in portal hypertensive gastric mucosa of rats

While the clinical features of sclerosing cholangitis secondary to opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosporidium parvum associated with this syndrome actually cause disease are obscure. We established an in vitro model of biliary cryptosporidiosis employing a human biliary epithelial cell line. Using morphological and biochemical techniques, we examined the interaction of C. parvum with cultured human cholangiocytes. When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C. parvum oocysts that had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The infectious process was both plasma membrane domain- and cell-specific, because no attachment or invasion occurred when the basolateral membrane of cholangiocytes was exposed to the parasite, or when a human hepatocyte cell line (HepG2) was used. Time-lapse video microscopy and scanning electron microscopy (SEM) showed that sporozoite attachment was rapid, involved extensive cholangiocyte membrane ruffling, and culminated in parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane similar to those found in naturally occurring infection in vivo. Transmission electron microscopy (TEM) showed that C. parvum organisms formed parasitophorus vacuoles and were able to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects were noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morphologically and biochemically beginning within hours after exposure to the organism. The novel finding of specific cytopathic invasion of biliary epithelia by C. parvum may be relevant to the pathogenesis and possible therapy of the secondary sclerosing cholangitis seen in AIDS patients with biliary cryptosporidiosis.     

Differential induction of tumor necrosis factor alpha in murine and human leukocytes by Mycoplasma arthritidis-derived superantigen

Mycoplasma arthritidis-derived superantigen (MAS) is exclusively produced by M. arthritidis, which is the only known mycoplasma to produce a superantigen. As a superantigen, MAS shows properties similar to those of the staphylococcal enterotoxins and related substances, such as binding to major histocompatibility complex (MHC) class II and V beta-specific stimulation of T cells. In this series of experiments, we demonstrate some differences between MAS and other superantigens. MAS induced the production of tumor necrosis factor alpha (TNF-alpha) mRNA in human as well as in murine leukocytes. However, only in murine leukocytes was the mRNA adequately translated into the protein. In human peripheral blood mononuclear cells, we found only small amounts of TNF, whereas in murine spleen cells we detected levels more than three times higher. The proliferative response to MAS has been shown to be restricted to I-E alpha in the murine MHC. Furthermore, TNF was induced in I-E alpha+ bone marrow-derived macrophages by MAS. In these cells, MAS rapidly induced very high levels of TNF and the amounts of mRNA detected correlated to the amount of protein produced. In comparison with other superantigens, including the staphylococcal enterotoxins, toxic shock syndrome toxin 1, and exfoliative toxin A, the failure of MAS to induce TNF-alpha in human peripheral blood mononuclear cells is specific for MAS and not common to all superantigens. The direct activation of bone marrow-derived macrophages also seems to be specific for MAS. These data suggest that the induction of TNF-alpha by MAS is dependent on the strength of binding to the MHC class II molecule.     

Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent

Tumor necrosis factor (TNF) alpha has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNFalpha antibody clinical trials. Although signaling pathways leading to TNFalpha expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNFalpha synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system in vitro we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IkappaB alpha, the natural inhibitor of NF-kappaB, that induction of TNFalpha in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNFalpha from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-kappaB pathway is an essential step for TNFalpha synthesis in synovial macrophages and demonstrating that NF-kappaB should be an effective therapeutic target in this disease.     

Effects of interferons on tumour necrosis factor alpha production from human keratinocytes

Interferons (IFNs) have been reported to have pleiotrophic effects including the ability to induce the production of other cytokines in several cell types. Tumour necrosis factor alpha (TNF-alpha) is pro-inflammatory cytokine a known to be produced by a variety of cells including human keratinocytes. In the present study, we sought to determine the effects of IFNs on TNF-alpha production from human keratinocytes. IFN-gamma (50-100 ng/ml) induced TNF-alpha production dose dependently, but no induction of TNF-alpha was observed with IFN-alpha or IFN-beta. Since in the epidermis cytokines often work with in a cascade fashion and keratinocytes are a source of primary cytokine, IL-1 alpha, whether combined treatment with IFN-gamma and IL-1 alpha had a synergistic effect on TNF-alpha production was examined. Combined treatment with IFN-gamma (100 ng/ml) and IL-1 alpha (10 ng/ml) induced 2-3-fold higher level of TNF-alpha than IL-1 alpha alone. These results suggest that IFN-gamma is a positive regulator for the production of TNF-alpha from human keratinocytes and likely to increase skin inflammation.     

Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.     

Tumor necrosis factor alpha augments nitric oxide-dependent macrophage cytotoxicity against Entamoeba histolytica by enhanced expression of the nitric oxide synthase gene

Nitric oxide (NO measured as nitrite, NO2-) is the major effector molecule produced by activated macrophages for in vitro cytotoxicity against Entamoeba histolytica trophozoites. In this study, we determine whether tumor necrosis factor alpha (TNF-alpha) produced by activated bone marrow-derived macrophages (BMM) is involved in the induction of the inducible NO synthase gene (mac-NOS) for NO-dependent amebicidal activity. TNF-alpha alone did not directly induce macrophage NO2- production to kill amebae; however, in combination with increasing concentrations of TNF-alpha and gamma interferon (IFN-gamma), BMM amebicidal activity and NO2- production progressively increased and showed a significant linear correlation. Antiserum to TNF-alpha and the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) inhibited the synergistic effects of TNF-alpha and IFN-gamma. BMM activated with increasing concentrations of lipopolysaccharide (LPS) and IFN-gamma showed a significant linear correlation between TNF-alpha release and NO2- production. Antiserum to TNF-alpha suppressed TNF-alpha release, NO2- production, and amebicidal activity by 93, 53, and 86%, respectively. L-NMMA diminished NO2- production by 74% and macrophage amebicidal activity by 83% but had no effect on TNF-alpha release. Quantification by Northern (RNA) blot analyses demonstrated that IFN-gamma in combination with TNF-alpha or LPS increased markedly the accumulation of mac-NOS and TNF-alpha mRNAs in a time-dependent manner with a concomitant increase in NO and TNF-alpha production. Peak induction of mac-NOS occurred after 24 h, whereas TNF-alpha mRNA was rapidly expressed after 4 h and remained stable for 48 h. Taken together, these data argue that TNF-alpha augments NO-dependent macrophage cytotoxicity against E. histolytica via elevated levels of mac-NOS mRNA expression which may be associated with the accumulation of TNF-alpha mRNA.     

Structural significance of the benzoyl group at the C-3'-N position of paclitaxel for nitric oxide and tumor necrosis factor production by murine macrophages

The antitumor agent paclitaxel (Taxol) mimics the actions of lipopolysaccharide (LPS) on murine macrophages (M phi). Recently, we have shown that the benzoyl group at the C-3' position of paclitaxel is the most important site to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by C3H/HeN M phi (Biochem. Biophys. Res. Commun. 210, 678-686, 1996). In the present study, synthetic analogs of paclitaxel with replacement of the C-3'-N position were examined for their potencies to induce NO and TNF production by peritoneal M phi of LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice, by human blood cells and human M phi. In this structure-activity relationship study, we found that (i) the p-substitution of the benzoyl group definitely affects the activity to activate C3H/HeN M phi, (ii) the analogs having a methyl or chloro group at the p-position exhibit stronger activity than that of paclitaxel, (iii) there is good correlation between NO and TNF production by the M phi in response to compounds, (iv) the compounds tested do not induce either NO or TNF production by C3H/HeJ M phi or TNF production by human cells, (v) a previous treatment of C3H/HeN M phi with the inactive compounds can hardly affect either paclitaxel- or LPS-induced TNF production by the M phi, (vi) paclitaxel and its analogs marginally affect LPS-induced TNF production by human blood cells, and (vii) there is no correlation between the NO/TNF inducibility to C3H/HeN M phi and growth inhibitory activity against M phi-like J774.1 and J7.DEF3 cells.     

Prevention of Jarisch-Herxheimer reactions by treatment with antibodies against tumor necrosis factor alpha

BACKGROUND: In patients with louse-borne relapsing fever (Borrelia recurrentis infection), antimicrobial treatment is often followed by sudden fever, rigors, and persistent hypotension (Jarisch-Herxheimer reactions) that are associated with increases in plasma concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6, and interleukin-8. We attempted to determine whether sheep polyclonal Fab antibody fragments against TNF-alpha (anti-TNF-alpha Fab) could suppress the Jarisch-Herxheimer reaction. METHODS: We conducted a randomized, double-blind, placebo-controlled trial in 49 patients with proven louse-borne relapsing fever. Immediately before the intramuscular injection of penicillin, the patients received an intravenous infusion of either anti-TNF-alpha Fab or a control solution. RESULTS: Ten of the 20 patients given anti-TNF-alpha Fab had Jarisch-Herxheimer reactions with rigors, as compared with 26 of the 29 control patients (P = 0.006). The controls had significantly greater mean maximal increases in temperature (1.5 vs. 0.8 degrees C, P < 0.001), pulse rate (31 vs. 13 per minute, P < 0.001), and systolic blood pressure (25 vs. 15 mm Hg, P < 0.003), as well as higher mean peak plasma concentrations of interleukin-6 (50 vs. 17 micrograms per liter) and interleukin-8 (2000 vs 205 ng per liter) (P < 0.001 for both comparisons). Levels of TNF-alpha were undetectable after treatment with anti-TNF-alpha Fab. CONCLUSIONS: Pretreatment with sheep anti-TNF-alpha Fab suppresses Jarisch-Herxheimer reactions that occur after penicillin treatment for louse-borne relapsing fever, reduces the associated increases in plasma concentrations of interleukin-6 and interleukin-8, and may be useful in other forms of sepsis.     

Effect of esculentoside H on release of tumor necrosis factor from mouse peritoneal macrophages

Effect of esculentoside H (EH) on release of tumor necrosis factor (TNF) from murine peritoneal macrophage (Mphi) in vitro was studied. The results showed that EH (12.5-200 micrograms.ml-1) induced the thioglycolate-broth elicited peritoneal Mphi to release TNF into supernatants in a dose-dependent manner, and higher levels of TNF activity were detected in the supernatants from EH-stimulated calcimycin-primed M? culture. EH-induced TNF release had a different type of kinetics compared with that of lipopolysaccharides (LPS). LPS-induced release of TNF increased rapidly until 6 h after LPS stimulation, then declined gradually, while EH-induced TNF release increased gradually after EH stimulation and reached its peak at approximately 24 h later. These results suggested that the anti-tumor mechanisms of Phytolaccaceae may be related to the capacity of EH for TNF release.     

Effects of ginkgolides on interleukin-1, tumor necrosis factor-alpha and nitric oxide production by rat microglia stimulated with lipopolysaccharides in vitro

The effects of ginkgolide A (CAS 15291-75-5, BN52020, GA) and B (CAS 15291-77-7, BN52021, GB) on interleukin (IL)-1, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production in resting and lipopolysaccharide (LPS)-stimulated neonatal rat microglia were studied. Apafant (CAS 105219-56-5), a platelet activating factor (PAF) antagonist of triazolobenzodiazepine type was used as control. The biological activities of IL-1 and TNF-alpha were tested by mouse thymocyte proliferation and L929 cytotoxicity assay, respectively. NO concentration was represented by nitrite and determined by Griess reaction. GA 1 nmol/1-10 mumol/l inhibited IL-1 production, and 100 nmol/l-10 mumol/l decreased TNF-alpha and NO production in dose-dependent manner. GB inhibited IL-1, TNF-alpha and NO production at the concentrations 10 nmol/l-10 mumol/l, 100 nmol/l-10 mumol/l and 10 nmol/l-10 mumol/l, respectively. Apafant inhibited IL-1, but not TNF-alpha and NO production. GB plus apafant (50 mumol/l) showed IL-1 and NO inhibitory effects, but not on TNF-alpha. The manner was different from that of GB or apafant alone. The results suggested that GA and GB inhibited proinflammatory cytokines and NO production from LPS-stimulated rat microglia, however, apafant inhibited IL-1 production only. The effects of GA and GB on proinflammatory cytokines and NO production from rat microglia do not seem to be based on PAF receptor antagonism. In addition, GA and GB are regarded as promising agents for the treatment of some neurodegenerative diseases in the central nervous system.     

Amiodarone-induced pulmonary toxicity in Fischer rats: release of tumor necrosis factor alpha and transforming growth factor beta by pulmonary alveolar macrophages

Amiodarone is an antiarrhythmic drug with numerous side effects, the most serious being the development of pulmonary toxicity. We have previously reported that a single intratracheal instillation of amiodarone to Fischer 344 rats results in pulmonary fibrosis within 6 wk of treatment. Presently, the mechanism of amiodarone-induced pulmonary toxicity is unknown. Cytokines that stimulate fibroblast proliferation and/or collagen production may play a role in amiodarone-induced pulmonary toxicity. To investigate this possibility, female rats were given a single intratracheal instillation of amiodarone (6.25 mg/kg), its metabolite desethylamiodarone (5 mg/kg), or vehicle (sterile water). At 1, 2, 3, or 6 wk after treatment the lungs were lavaged and the recovered cells were counted and identified. The alveolar macrophages were isolated by attachment to plastic petri dishes, cultured overnight, and the spent media collected for tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) analyses. Desethylamiodarone treatment resulted in a neutrophilic alveolitis, but the levels of TNF-alpha and TGF-beta were not significantly different from control animals. In contrast, amiodarone treatment resulted in a lymphocytic alveolitis and significantly higher amounts of TNF-alpha were observed at 3 and 6 wk after treatment. A trend toward higher levels of TGF-beta was also noted in the amiodarone-treated group at wk 1-3 but the values were not significantly different from those of controls. In conclusion, the release of TNF-alpha may play a role in the development of amiodarone-induced pulmonary toxicity.