Inflammatory response to orthopaedic cobalt chromium alloy wear debris in a rodent air pouch model
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Abstracts / Toxicology Letters 196S (2010) S37–S351 S199 P205-022 Allergen-stimulated expression of interleukin (IL)-17 isoforms in mice I. Kimber, M. Hayes, R. Dearman University of Manchester, United Kingdom The development of immune and allergic responses is orches- trated by T helper (Th) subsets that are characterized by selective cytokine secretion profiles. Topical exposure of BALB/c strain mice to different classes of chemical allergen has been shown to result in preferential activation of divergent Th cell subsets. Thus, Th1 and Th2 cells are induced by the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizer trimellitic anhydride (TMA), respectively. A further subset of inflammatory Th cells that secrete IL-17 may play an important role in some autoimmune and inflammatory conditions. In the current investigations, the ability of chemical allergens to provoke changes in expression of the expression of IL-17 isoforms has been exam- ined. BALB/c strain mice were exposed to a single topical dose of either 1% DNCB or 25% TMA, or to vehicle alone, for 30 min to 72 h. At various times thereafter, cytokine production by skin explants (prepared from dorsal halves of ear tissue) or by draining auric- ular lymph node cells (LNC) was measured by cytokine-specific enzyme-linked immunosorbant assay (ELISA). Topical treatment with DNCB, but not to TMA or vehicle alone, provoked a rapid increase in IL-17A and IL-17F isoforms, peaking at 2–3 h. Levels of IL-17F were generally some 5-fold higher than those observed for IL-17A. DNCB-activated LNC also produced high levels of both IL-17A and IL-17F, reaching maximal levels after 6 h of exposure. The heterodimer (IL-17A/F) was also detected, but at considerably lower levels. Treatment with TMA induced the same pattern of cytokines in the draining lymph node, but with slightly delayed kinetics, peaking at 48–72 h. These data demonstrate that chemical allergens provoke the rapid production of IL-17 isoforms in the skin and draining lymph node which may play a role in the acquisition of sensitization. doi:10.1016/j.toxlet.2010.03.673 P205-023 Toxic effects of Senecio brasiliensis on rat phagocytes F. Elias 1 , I. Hueza 2 , A. Latorre 1 , S. Gorniak 1 1 University of São Paulo/Research Center of Veterinary Toxicology (CEPTOX), Brazil, 2 Federal University Of São Paulo/School of Pharmacy and Biochestry, Brazil Senecio brasiliensis is one of the most toxic poisonous plant species of Senecio’s genus. It contains pyrrolizidine alkaloids (PAs) that are associated with disease in livestock and human beings after be metabolized into the liver in a high reactive electrophiles (pyrrol compound) capable to link with cellular macromolecules, mainly DNA, forming adducts which may initiate acute or chronic toxi- city. The development of oxidative stress with the generation of superoxides and lipid-peroxy radicals in PAs toxicity in cattle and cytotoxicity to macrophages mediated by reactive oxygen species were recently documented. Therefore, the aim of this study was to verify if S. brasiliensis induce toxicity in innate immune system cells (macrophages and neutrophils) during phagocytosis activ- ity. PAs was extracted and concentrated into a butanolic residue (BR) and rats were then treated with BR daily, by gavage, dur- ing 28 days. Two experiments were performed, one to evaluate peritoneal macrophages and the other to determine peripheral neutrophils activity. For each experiment 40 adult rats were sepa- rated into four groups treated with 0.0 (control), 3.0 (A), 6.0 (B), and 9.0 (C) mg/kg of BR. The results obtained showed a signifi- cant enhancement in basal (A- and C-group) and PMA induced (C-group) oxidative burst of peritoneal macrophages and also an increase percentage of macrophage phagocytosis (A-, B-, and C- group). However, in relation to neutrophils activities, it was only observed an increase in basal oxidative burst (A- and B-group). The intracellular redox potential is maintained in a reduced state by constant elimination of superoxides, peroxides and cellular H 2 O 2 by glutathione (GSH). The pyrrolic metabolites produced from PAs are capable to bind with GSH to form GSH conjugates. Thus, it is pos- sible to hypothesized that, one of the mechanism that S. brasiliensis exerts its immunotoxicity on macrophage and neutrophil activities is by depleting GSH. doi:10.1016/j.toxlet.2010.03.674 P205-024 Inflammatory response to orthopaedic cobalt chromium alloy wear debris in a rodent air pouch model M. Akbar 1 , M.H. Grant 1 , J. Brewer 2 1 University of Strathclyde, United Kingdom, 2 University of Glasgow, United Kingdom Cobalt chromium (Co–Cr) alloy metal-on-metal (MOM) hip resur- facing has become increasingly common in younger, more active patients. However, there is growing concern regarding levels of Co–Cr wear debris and ions released. Reports have described immunological reactions in some MOM hip arthroplasty patients. These patients have shown symptoms of aseptic lymphocytic vas- culitis associated lesions (ALVAL) as well as an aseptic soft-tissue groin mass (pseudotumour) composed largely of necrotic connec- tive tissue around the prostheses. Using the rodent air pouch model, described as a facsimile synovium, we assessed the inflammatory response to artificially generated Co–Cr wear debris. Air pouches were raised by two subcutaneous injections of sterile air in BALB/c mice. The air- pouch cavity was then injected with either 500 l sterile phosphate buffered saline (PBS), 1 g lipopolysaccharide (LPS) or 2.5 mg Co–Cr wear debris. The in situ inflammatory response was monitored 4, 24 and 48 h later when the pouches were lavaged and excised. B and T-lymphocytes, monocytes/macrophages and neutrophils in the lavage fluid were identified using flow cytometry. Histologi- cal analysis was also carried out following hematoxylin and eosin staining. Exposure to Co–Cr wear debris resulted in recruitment of mono- cytes/macrophages and neutrophils into the air-pouch cavity by 4 h. Monocyte/macrophage accumulation peaked at 24 h whereas neutrophil number decreased. In contrast, LPS initially induced neutrophil accumulation (4 h) in the pouch cavity followed by monocyte/macrophage recruitment (24 h). Histological analysis also showed metallic debris accumulated in the pouch wall and this was accompanied by a vast cellular infiltration around the debris. These data demonstrate that metallic debris triggers an inflammatory response that leads to monocyte/macrophage accu- mulation, a process different to LPS induced inflammation. The air pouch model is therefore useful in identifying the cells and molecules involved in the inflammatory response following expo- sure to Co–Cr wear debris. Understanding this process will suggest new rationales for therapeutic intervention. doi:10.1016/j.toxlet.2010.03.675
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Abstracts / Toxicology L
205-022llergen-stimulated expression of interleukin (IL)-17 isoforms
n mice
. Kimber, M. Hayes, R. Dearman
University of Manchester, United Kingdom
he development of immune and allergic responses is orches-rated by T helper (Th) subsets that are characterized by selectiveytokine secretion profiles. Topical exposure of BALB/c strainice to different classes of chemical allergen has been shown
o result in preferential activation of divergent Th cell subsets.hus, Th1 and Th2 cells are induced by the contact allergen,4-dinitrochlorobenzene (DNCB) and the respiratory sensitizerrimellitic anhydride (TMA), respectively. A further subset ofnflammatory Th cells that secrete IL-17 may play an important rolen some autoimmune and inflammatory conditions. In the currentnvestigations, the ability of chemical allergens to provoke changesn expression of the expression of IL-17 isoforms has been exam-ned. BALB/c strain mice were exposed to a single topical dose ofither 1% DNCB or 25% TMA, or to vehicle alone, for 30 min to 72 h.t various times thereafter, cytokine production by skin explants
prepared from dorsal halves of ear tissue) or by draining auric-lar lymph node cells (LNC) was measured by cytokine-specificnzyme-linked immunosorbant assay (ELISA). Topical treatmentith DNCB, but not to TMA or vehicle alone, provoked a rapid
ncrease in IL-17A and IL-17F isoforms, peaking at 2–3 h. Levelsf IL-17F were generally some 5-fold higher than those observedor IL-17A. DNCB-activated LNC also produced high levels of bothL-17A and IL-17F, reaching maximal levels after 6 h of exposure.he heterodimer (IL-17A/F) was also detected, but at considerablyower levels. Treatment with TMA induced the same pattern ofytokines in the draining lymph node, but with slightly delayedinetics, peaking at 48–72 h. These data demonstrate that chemicalllergens provoke the rapid production of IL-17 isoforms in the skinnd draining lymph node which may play a role in the acquisitionf sensitization.
oi:10.1016/j.toxlet.2010.03.673
205-023oxic effects of Senecio brasiliensis on rat phagocytes
. Elias 1, I. Hueza 2, A. Latorre 1, S. Gorniak 1
University of São Paulo/Research Center of Veterinary ToxicologyCEPTOX), Brazil, 2 Federal University Of São Paulo/School ofharmacy and Biochestry, Brazil
enecio brasiliensis is one of the most toxic poisonous plant speciesf Senecio’s genus. It contains pyrrolizidine alkaloids (PAs) thatre associated with disease in livestock and human beings after beetabolized into the liver in a high reactive electrophiles (pyrrol
ompound) capable to link with cellular macromolecules, mainlyNA, forming adducts which may initiate acute or chronic toxi-ity. The development of oxidative stress with the generation ofuperoxides and lipid-peroxy radicals in PAs toxicity in cattle andytotoxicity to macrophages mediated by reactive oxygen speciesere recently documented. Therefore, the aim of this study was
o verify if S. brasiliensis induce toxicity in innate immune system
ells (macrophages and neutrophils) during phagocytosis activ-ty. PAs was extracted and concentrated into a butanolic residueBR) and rats were then treated with BR daily, by gavage, dur-ng 28 days. Two experiments were performed, one to evaluate196S (2010) S37–S351 S199
peritoneal macrophages and the other to determine peripheralneutrophils activity. For each experiment 40 adult rats were sepa-rated into four groups treated with 0.0 (control), 3.0 (A), 6.0 (B),and 9.0 (C) mg/kg of BR. The results obtained showed a signifi-cant enhancement in basal (A- and C-group) and PMA induced(C-group) oxidative burst of peritoneal macrophages and also anincrease percentage of macrophage phagocytosis (A-, B-, and C-group). However, in relation to neutrophils activities, it was onlyobserved an increase in basal oxidative burst (A- and B-group). Theintracellular redox potential is maintained in a reduced state byconstant elimination of superoxides, peroxides and cellular H2O2by glutathione (GSH). The pyrrolic metabolites produced from PAsare capable to bind with GSH to form GSH conjugates. Thus, it is pos-sible to hypothesized that, one of the mechanism that S. brasiliensisexerts its immunotoxicity on macrophage and neutrophil activitiesis by depleting GSH.
doi:10.1016/j.toxlet.2010.03.674
P205-024Inflammatory response to orthopaedic cobalt chromium alloywear debris in a rodent air pouch model
M. Akbar 1, M.H. Grant 1, J. Brewer 2
1 University of Strathclyde, United Kingdom, 2 University of Glasgow,United Kingdom
Cobalt chromium (Co–Cr) alloy metal-on-metal (MOM) hip resur-facing has become increasingly common in younger, more activepatients. However, there is growing concern regarding levels ofCo–Cr wear debris and ions released. Reports have describedimmunological reactions in some MOM hip arthroplasty patients.These patients have shown symptoms of aseptic lymphocytic vas-culitis associated lesions (ALVAL) as well as an aseptic soft-tissuegroin mass (pseudotumour) composed largely of necrotic connec-tive tissue around the prostheses.
Using the rodent air pouch model, described as a facsimilesynovium, we assessed the inflammatory response to artificiallygenerated Co–Cr wear debris. Air pouches were raised by twosubcutaneous injections of sterile air in BALB/c mice. The air-pouch cavity was then injected with either 500 �l sterile phosphatebuffered saline (PBS), 1 �g lipopolysaccharide (LPS) or 2.5 mg Co–Crwear debris. The in situ inflammatory response was monitored 4,24 and 48 h later when the pouches were lavaged and excised. Band T-lymphocytes, monocytes/macrophages and neutrophils inthe lavage fluid were identified using flow cytometry. Histologi-cal analysis was also carried out following hematoxylin and eosinstaining.
Exposure to Co–Cr wear debris resulted in recruitment of mono-cytes/macrophages and neutrophils into the air-pouch cavity by4 h. Monocyte/macrophage accumulation peaked at 24 h whereasneutrophil number decreased. In contrast, LPS initially inducedneutrophil accumulation (4 h) in the pouch cavity followed bymonocyte/macrophage recruitment (24 h). Histological analysisalso showed metallic debris accumulated in the pouch wall and thiswas accompanied by a vast cellular infiltration around the debris.
These data demonstrate that metallic debris triggers aninflammatory response that leads to monocyte/macrophage accu-mulation, a process different to LPS induced inflammation. Theair pouch model is therefore useful in identifying the cells and
molecules involved in the inflammatory response following expo-sure to Co–Cr wear debris. Understanding this process will suggestnew rationales for therapeutic intervention.doi:10.1016/j.toxlet.2010.03.675
