activation of the cholinergic antiinflammatory pathway reduces ricin … · 2018. 9. 19. · ricin...

1 6 6 | M A B L E Y E T A L . | M O L M E D 1 5 ( 5 - 6 ) 1 6 6 - 1 7 2 , M A Y - J U N E 2 0 0 9

INTRODUCTIONRicin is a member of a family of pro-

tein toxins whose intracellular target isthe 28S rRNA of the 60S ribosomal sub-unit (1). Ricin causes the depurination of28S rRNA at a single adenine nucleotide,A4565 in humans and A4256 in mice,which results in inhibition of proteintranslation. The resulting depurinationof 28S initiates the ribotoxic stress re-sponse, which is characterized by activa-tion of the stress-activated protein ki-nases, N-terminal-c-Jun-kinases (JNK),and p38 mitogen-activated protein ki-

nase (MAPK) (2). The resulting activa-tion of these protein kinases modulatesthe expression of a variety of genes thatencode proinflammatory cytokines andchemokines. Ricin has been shown to in-crease production of proinflammatorycytokines, including tumor necrosisfactor-α (TNF-α), interleukin-1 (IL-1),IL-8, and monocyte chemotactic protein-1from both primary macrophages and celllines derived from both mice and hu-mans (3,4) via JNK and p38 activation(3). Similar results have been observedin vivo, in cases in which ricin adminis-

tration induced a systemic inflammatoryresponse and multiple organ failure(4–6).

Ricin’s wide availability and ease ofpurificaion has resulted in its use as atoxic and lethal agent by totalitarianregimes, and more recently, by terroristgroups. In humans, the estimated lethaldose of ricin is 1 to 10 μg/kg bodyweight, hence its classification by theCenters of Disease Control and Preven-tion in the USA as a level-B biothreat (6).There have been more than 750 cases ofricin intoxication, the majority of whichhave resulted from the ingestion of castorbeans, manifested by hemorrhagic diar-rhea, liver necrosis, diffuse nephritis, andsplenitis (5). Currently there is no anti-dote or vaccine available against ricin, al-though significant progress has beenmade in this area, with prospective vac-cines having been tested on human vol-unteers (7,8). With no antidote available,

Activation of the Cholinergic Antiinflammatory PathwayReduces Ricin-Induced Mortality and Organ Failure in Mice

Jon G Mabley,1 Pal Pacher,2 and Csaba Szabo3

1School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom; 2Section on Oxidative StressTissue Injury, Laboratory of Physiological Studies, National Institutes of Health, Bethesda, Maryland, United States of America; 3Department of Anesthesiology, The University of Texas Medical Branch, Galveston, Texas, United States of America

Exposure to ricin, either by accident through ingestion of castor oil plant seeds or intentionally through its use as a bioweapon,invariably leads to multiple organ damage and death. Currently there is only a vaccine in advanced development to ricin, butno other antidote. Ricin causes systemic inflammation with increased proinflammatory cytokine release and subsequent multipleorgan failure, particularly kidney and liver dysfunction. Activation of the cholinergic antiinflammatory pathway, specificallythrough the alpha7 nicotinic acetylcholine receptor (either indirectly through vagus nerve stimulation or directly through nicotinetreatment) reduces proinflammatory gene expression. This activation also increases release of proinflammatory chemokines andcytokines, and has proven effective in a variety of inflammatory diseases. The aim of this study was to investigate whether nico-tine treatment protected against ricin toxicity in mice. Male Balb/c mice exposed to ricin had increased serum levels of the in-flammatory cytokine tumor necrosis factor-α and markers of both kidney (blood urea nitrogen, creatine) and liver (alaninetranaminase) dysfunction, with a subsequent increase in mortality. Nicotine administration 2 h after ricin injection significantly de-layed and reduced ricin-induced mortality, an effect coupled with reduced serum levels of tumor necrosis factor-α and markersof kidney and liver dysfunction. Both the kidney and liver had markedly increased cellular oxidative stress following ricin exposure,an effect attenuated by nicotine administration. In conclusion, these data demonstrate that in cases of ricin poisoning, activationof the cholinergic antiinflammatory pathway may prove beneficial by reducing organ damage, delaying mortality, and allowingfor a greater chance of survival.© 2009 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2008.00105

Address correspondence and reprint requests to Jon G Mabley, School of Pharmacy and

Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton

BN2 4GJ, UK. Phone: + 44 (0) 1273 642055; Fax: + 44 (0) 1273 679333; Email: j.g.Mabley@

brighton.ac.uk.

Submitted January 31, 2009; Accepted for publication February 4, 2009; Epub

(www.molmed.org) ahead of print February 5, 2009.

R E S E A R C H A R T I C L E

M O L M E D 1 5 ( 5 - 6 ) 1 6 6 - 1 7 2 , M A Y - J U N E 2 0 0 9 | M A B L E Y E T A L . | 1 6 7

the treatment of ricin intoxication consistsof removing the toxin from the body asquickly as possible and the use of sup-portive measures to maintain organfunction.

The cholinergic antiinflammatorypathway has been identified as a majorneuromodulator of immune-cell func-tion (9,10). Stimulation of the vagusnerve has been demonstrated to protectagainst endotoxic shock by directly af-fecting immune cells (11,12). Nicotinehas been shown to be antiinflammatoryin a variety of animal models of diseasestates, including colitis (13,14), arthritis(15), and type I diabetes (16), an effectobserved in humans (17,18). Nicotinehas also proven effective in reducinglung inflammation in asthma, pneu-monitis, chronic obstructive pulmonarydisease, and acid-induced injury(19,20). The nicotine-mediated reduc-tion in the proinflammatory cytokineprofile is common to all disease statesagainst which nicotine has proven pro-tective, including colitis (13,21), arthri-tis (15), diabetes (16), lung inflamma-tion (19,20), and endotoxic shock(10,12).

With the proinflammatory effects ofricin exposure proposed to be the majoreffector mechanism of toxicity, the aim ofthis study was to determine whethernicotine administration protected micefrom ricin-induced mortality and organfailure.

MATERIALS AND METHODSReagents were obtained from the fol-

lowing sources: ricin was from Vector lab-oratories (Bulingame, CA, USA); nicotine,thiobarbituric acid, and sodium dodecylsulfate were from Sigma (St. Louis, MI,USA.); BALB/c mice were from Taconicfarms (Germantown, NY, USA); and spe-cific cytokine ELISA kits were from R&Dsystems (Minneapolis, MN, USA).

Animal StudiesIn vivo studies were performed in ac-

cordance with National Institutes ofHealth guidelines and with the approvalof the local institutional animal care and

use committee. Male BALB/c were in-jected with ricin (50 or 100 μg/kg intra-peritoneally) ± either nicotine (0.4 μg/kg,subcutaneously) or vehicle administeredat 2 h and 26 h after the ricin injection.The mice were then monitored for mor-tality over a 48-h period. Control miceand those receiving nicotine alone hadthe appropriate volumes of saline admin-istered at the required time pointsthroughout the experimental period.Mortality measurements were obtainedfrom two separate experiments, with 10animals per group with a final n of 20 foreach treatment protocol. In a second se-ries of studies male BALB/c mice wereexposed to ricin (50 μg/kg) ± either nico-tine (0.4 μg/kg, subcutaneously) or vehi-cle administered 2 h after the ricin injec-tion for 14 h prior to being killed. Serumwas then taken for cytokine and bio-chemical analysis and the liver and kid-ney were taken for oxidative stress deter-mination; results were obtained fromtwo separate experiments with 5 animalsper group, with a final n of 10 for eachtreatment protocol.

Serum TNF-α LevelsThe concentration of the proinflam-

matory cytokine TNF-α was deter-mined in the serum by use of a com-mercially available ELISA, followingthe protocol provided by the manufac-turer (R&D Systems).

Serum Levels of Alanine Tranaminase,Blood Urea Nitrogen, and Creatine

The serum levels of alanine tranami-nase (ALT), blood urea nitrogen (BUN),and creatine were determined enzymati-cally using an automated Vetscan chem-istry analyzer (Abaxis, Union City, CA,USA).

Malondialdehyde Assay Malondialdehyde (MDA) formation

was utilized to quantify the lipid peroxi-dation in the liver and kidney, measuredas thiobarbituric acid-reactive material.Tissues were homogenized (100 mg/mL)in 1.15% KCl buffer; 200 μL of the ho-mogenate was then added to a reaction

mixture consisting of 1.5 mL 0.8% thio-barbituric acid, 200 μL 8.1% sodium do-decyl sulfate, 1.5 mL 20% acetic acid (pH3.5), and 600 μL distilled H2O. The mix-ture was then heated at 90°C for 45 min.After cooling to room temperature, thesamples were cleared by centrifugation(10,000g, 10 min), and their absorbancewas measured at 532 nm, with 1,1,3,3-tetramethoxypropane as an external stan-dard. The level of lipid peroxides wasexpressed as nanomoles MDA per mil-ligram protein, which was determinedusing the Bradford assay (22).

Statistical AnalysisThe results are presented as mean ±

SEM; statistical analysis was performedusing either one-way analysis of variancefollowed by Student–Newman–Keulsmultiple comparisons post-hoc analysis orKaplan–Meier survival analysis as ap-propriate, with a P value of less than 0.05considered significant.

RESULTS

Nicotine Treatment Increases Survivalfollowing Ricin Administration

Because there is currently no antidoteto ricin and the only treatment option isto remove the toxin from the body andprovide support to failing organs, anystrategy that can extend the survivaltime to allow for these clinical interven-tions to become effective would be thera-peutically useful. Mice exposed to ricinthrough a variety of administrationroutes, including nasal, oral, and in-traperitoneal, develop severe inflamma-tory reactions and organ damage result-ing in increased mortality (4,6,23).Nicotine has been identified as antiin-flammatory in a variety of disease states,an effect linked to its activation of thecholinergic antiinflammatory pathwaythrough specific nicotinic receptors lo-cated on inflammatory cells (24). Activa-tion of the cholinergic antiinflammatorypathway either via nicotine administra-tion or vagal stimulation has proven par-ticularly effective in endotoxic shock in-duced by injection of lipopolysaccharide,


N I C O T I N E P R O T E C T S A G A I N S T R I C I N - I N D U C E D M O R T A L I T Y

reducing inflammation and, most impor-tantly, improving survival (25). There-fore, we investigated whether nicotinetreatment following ricin exposure coulddelay mortality and increase survivalrates. Nicotine treatment was started 2 hafter ricin exposure to mimic what islikely to be observed clinically with ricin-exposed patients presenting many hoursafter the initial contact.

Ricin dose-dependently induced mor-tality during a 48-h postexposure period,with 50% mortality seen at 24 h with50 μg/kg and 18 h with 100 μg/kg (Fig-ure 1). Nicotine treatment (0.4 μg/kg)administered at 2 h and 26 h after ricinexposure significantly delayed mortalityand improved survival in both the 50and 100 μg/kg ricin groups (Figure 1),with 45% and 20% of the nicotine-treatedmice surviving at 48 h compared with15% and 0% in the vehicle-treated 50 and100 μg/kg ricin-treated groups, respec-tively (Figure 1).

Nicotine Reduces Serum TNF-α Levelsfollowing Ricin Exposure

Exposure to ricin has been linked to amarked increase in levels of serum proin-flammatory cytokines, including TNF-α,IL-1β, and IL-6 (5). This effect has beenproposed to be the major mechanism ofricin-induced organ dysfunction andmortality. Because nicotine delayed mor-tality and improved survival of mice ex-posed to ricin, we wanted to determinewhether this result was mediatedthrough the antiinflammatory effects ofnicotine. Using serum TNF-α as a markerof overall inflammation following ricinexposure, we determined whether nico-tine treatment reduced the proinflamma-tory effects of ricin.

Treatment with ricin (50 μg/kg) for14 h increased serum TNF-α levels ten-fold compared with vehicle treatment(Figure 2). Treatment with nicotine(0.4 μg/kg) 2 h after ricin exposure signif-icantly reduced the serum TNF-α levels

by 50% (Figure 2). Nicotine alone had noeffect on serum TNF-α levels (Figure 2).

Nicotine Protects against Ricin-Mediated Organ Dysfunction

Ricin exposure has previously beenshown to cause significant organ dam-age, particularly in the kidney (5,6) andthe liver (26). This ricin-mediated dam-age has been linked to its proinflamma-tory actions and cytokine release lead-ing to a proinflammatory transcriptionalresponse in the target organs, increasedoxidative stress, and subsequent cellulardysfunction. Because nicotine signifi-cantly attenuated the increase in serumproinflammatory cytokines as assessedby TNF-α levels following ricin expo-sure, we hypothesized that nicotine mayalso protect against ricin-induced organdamage. Following ricin exposure,using serum markers of organ function,we focused on kidney (BUN and crea-tine) and liver (ALT), the two primary

Figure 1. Increased survival of ricin-treated mice following nicotine treatment. Male BALBc mice received an intraperitoneal injection ofricin (50 or 100 μg/kg) followed by a single dose of nicotine (0.4 μg/kg) administered subcutaneously 2 h after the ricin injection. Mousesurvival was monitored over a 48-h period. Results are expressed as percentage of surviving mice from 20 animals per group; **P < 0.01versus ricin (50 μg/kg); ††P < 0.01 versus ricin (100 μg/kg).



organs affected by ricin, to determinewhether nicotine was able to maintaintheir function following exposure. Addi-tionally, following ricin exposure oxida-tive stress in the liver and kidney, asmeasured by tissue MDA levels, was de-termined and again any protective effectof nicotine assessed.

Exposure to ricin (50 μg/kg) for 14 hresulted in multiple organ failure withsignificantly increased serum levels ofALT (Figure 3A), BUN (Figure 3B), andcreatine (Figure 3C), indicating liver andkidney damage. Nicotine (0.4 μg/kg)administration 2 h after ricin exposureattenuated the rise in serum levels ofALT, BUN, and creatine following ricintreatment (Figure 3). Liver and kidneylevels of MDA, a marker of oxidativestress, were increased following 14-h ex-posure to ricin (50 μg/kg) (Figure 4).The rise in liver MDA levels followingricin treatment was attenuated by nico-tine (0.4 μg/kg) treatment (Figure 4),and the rise in kidney MDA levels wascompletely prevented by nicotine treat-ment (Figure 4).

DISCUSSIONOur results have demonstrated that

nicotine reduces organ failure and im-proves mouse survival following ricinexposure. The protective effect of nico-tine appears to be associated with its an-tiinflammatory effect, suggesting a possi-ble therapeutic strategy of activating thecholinergic antiinflammatory pathwayfollowing ricin exposure to protectagainst multiple organ failure. Nicotinetreatment reduced levels of the inflam-matory cytokine TNF-α and improvedboth liver and kidney function while re-ducing the oxidative stress observed inthese organs following ricin exposure.The overall effect of nicotine on main-taining liver and kidney function whilereducing systemic inflammation may ac-count for the reduced mortality observedwith ricin exposure.

Ricin has been shown to induce a se-vere inflammatory response that hasbeen linked to development of acuterenal failure (5,6). Ricin exposure also

Figure 3. Ricin-mediated liver (A) and kidney (B and C) damage is reduced by nicotinetreatment. Exposure of mice to ricin (50 μg/kg) for 14 h resulted in increased serum levelsof ALT (A), BUN (B), and creatine (Cre) (C), demonstrating significant liver and kidney dys-function, effects attenuated by nicotine treatment. Nicotine alone had no effect onserum levels of ALT, BUN or Cre. Results are mean ± SEM (n = 10), *P < 0.05, **P < 0.01 versusvehicle-treated mice, †P < 0.05, ††P < 0.01 versus ricin-treated mice.

Figure 2. Nicotine attenuates ricin-mediated increase in serum TNF-α levels. Exposure ofmice to ricin (50 μg/kg) for 14 h increased serum TNF-α levels, an effect reduced by nico-tine (0.4 μg/kg) treatment. Nicotine alone had no effect on serum cytokine levels. Resultsare mean ± SEM (n = 10), **P < 0.01 versus vehicle treated mice, ††P < 0.01 versus ricintreated mice.



results in hypoglycemia and hepatic in-jury, effects linked to an increased neu-trophil and monocyte infiltration with asubsequent increase in inflammatory cy-tokine levels (26). Ricin may increasethe multiple organ damage induced byinflammatory cytokines by affecting cel-lular antioxidant enzyme levels; ricinexposure reduces the activities of super-oxide dismutase and glutathione peroxi-dase while increasing xanthine oxidaseactivity in both kidney and liver, result-ing in increased lipid peroxidationproducts (27–30), an effect observed inthis study. Human airway cells exposedto ricin show a marked inflammatoryresponse with activation of nuclear fac-tor (NF)-κB and increased expression ofcytokines such as TNF-α (31). The re-sulting organ failure, which has beenobserved in humans following exposureto ricin (32) eventually leads to death(26,33).

The proinflammatory effects of ricinhave been linked to increased stimula-tion of the secondary messenger systemsERK (extracellular signal-regulated pro-tein kinase), JNK, and p38 MAPK, withresulting activation of transcription fac-tor NF-κB, which leads to inflammatory

gene expression (2,3,31,34). These path-ways are activated by ricin not only inmacrophages and inflammatory cells(3,35,36) but also other cell types such ashepatocytes and lung cells (6,23,26,31).The resulting increased expression ofproinflammatory chemokines and cyto-kines can enhance immune cell infiltra-tion and increase the damage to specificorgans, including the lung, kidney, andliver (33). Various routes of administra-tion of ricin: intrapulmonary (6,37),intraperitoneal (26,30), oral (4, 37), andintravenous (5), have all resulted in asystemic inflammatory response andmultiple organ damage, demonstratingthat ricin can achieve a systemic distribu-tion and inflammatory response.

Activation of the antiinflammatorycholinergic pathway has been demon-strated to have an antiinflammatory ef-fect in a wide variety of disease states(38), including reducing systemic inflam-mation. This effect can be observed ei-ther with vagal activation (25,39) or di-rect administration of nicotine (16,19,20).More recently the cholinergic antiinflam-matory pathway has been shown to beactivated following pharmacologiccholinesterase inhibition (40,41) or appli-

cation of the acetylcholine precursorcholine (42). Specific receptors for nico-tine have been shown to be present onlymphocytes from humans, rats, andmice. Nicotine has been shown to haveT-cell dependent and independent ef-fects, and the antiinflammatory effects ofnicotine-receptor activation have beenidentified as mediated through activationof the alpha7 nicotinic acetylcholine re-ceptor (alpha7nAChR) on immune cells(42,43), with subsequent activation ofjak2 and STAT 3 (44) and a resultant de-crease in proinflammatory cytokine pro-duction, particularly TNF-α (42,45).Nicotine suppresses production of in-flammatory mediators such as IL-1, IL-8,and prostaglandin-E2 from humanmacrophages (46) and IL-2 and TNF-αfrom human mononuclear cells (42), aneffect mediated through modulation ofNF-κß activation (46). Recently, adminis-tration of the cholinesterase inhibitorphysostigmine in an experimental modelof sepsis was shown to reduce activationof NF-κB and suppress circulating proin-flammatory cytokines (TNF-α, IL-1, andIL-6) and neutrophil infiltration (41).

The protection against ricin-mediatedinflammation and systemic organ failureby nicotine is likely due to the antiin-flammatory effect of nicotine-blockingNF-κB activation and inflammatory geneexpression. The antiinflammatory effectof nicotine requires signaling through thealpha7nAChR, with the subsequent inhi-bition of NF-κB activation and suppres-sion of the release of proinflammatorycytokines, including TNF-α, the cytokinethat appears to be the major ricin-in-duced proinflammatory mediator. Morerecently nicotine has been shown to haveprotective effects against both the kidneyand liver (47,48), organs severely dam-aged by ricin exposure. Nicotine pre-treatment protected the kidney from is-chemia-reperfusion injury, reducingglomeruli damage, neutrophil infiltra-tion, and expression of inflammatorychemokines and cytokines includingTNF-α, effects again mediated throughthe alpha7nAChR (47). Activation of thevagus nerve or administration of nicotine

Figure 4. Ricin-induced increases of oxidative stress in liver and kidney and protection bynicotine. Exposure of mice to ricin (50 μg/kg) for 14 h resulted in significantly increasedliver and kidney MDA levels indicative of oxidative stress. Nicotine partially prevented theincreased oxidative stress in the liver while completely protecting the kidney from ricin-mediated increases in oxidative stress. Nicotine alone had no effect on oxidative stress ineither organ. Results are mean ± SEM (n = 10), *P < 0.05, **P < 0.01 versus vehicle-treatedmice, †P < 0.05, ††P < 0.01 versus ricin-treated mice.



prevents Fas-induced apoptosis in mouseliver, again through activation of thealpha7nAChR (48). The mechanism ofactivation in this case is proposed to bethrough reduced hepatocyte productionof reactive oxygen species and subse-quent reduction in cellular oxidativestress (48), mechanisms of cell damagethat may be activated by ricin.

Nicotine’s effect on antioxidant systemsis complex, with reports of decreased ac-tivities of antioxidant enzymes such asglutathione peroxidase and catalase, butnicotine has also been shown to upregu-late expression of heme oxygenase-1, astress-inducible protein that functions asan antioxidant enzyme (49), as well assuperoxide dismutase levels in the kid-ney (50). With ricin having significantprooxidant effects (26,30,51), any protec-tive antioxidant enzymes that may be in-duced by nicotine may prove effective inmaintaining organ function followingricin exposure. However, it is likely thatnicotine’s main protective mechanism isthrough the cholinergic antiinflamma-tory pathway.

Currently there is no antidote to ricin,and the treatment options following ex-posure consist of providing supportivemeasures to maintain organ function andremoving the toxin from the body. There-fore, the development of new therapiesthat maintain organ function and delaymortality following ricin poisoningwould be invaluable both for accidentaland deliberate exposure of the humanpopulation. The data presented here sug-gest activation of the cholinergic antiin-flammatory pathway may prove to be aneffective therapeutic strategy to improvesurvival following ricin exposure. Thiseffect may be induced centrally, by stim-ulating the vagus nerve or inhibitingcholinesterases, or peripherally, usingnicotine or a specific alpha7nACh-receptor agonist.

DISCLOSUREWe declare that the authors have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-

sults and discussion reported in thispaper.

REFERENCES1. Barbieri L, Battelli MG, Stirpe F. (1993) Ribo-

some-inactivating proteins from plants. Biochim.Biophys. Acta 1154:237–282.

2. Iordanov MS, et al. (1997) Ribotoxic stress re-sponse: activation of the stress-activated proteinkinase JNK1 by inhibitors of the peptidyl trans-ferase reaction and by sequence-specific RNAdamage to the alpha-sarcin/ricin loop in the 28SrRNA. Mol. Cell. Biol. 17:3373–81.

3. Gonzalez TV, Farrant SA, Mantis NJ. (2006) Ricininduces IL-8 secretion from humanmonocyte/macrophages by activating the p38MAP kinase pathway. Mol. Immunol. 43:1920–3.

4. Yoder JM, Aslam RU, Mantis NJ. (2007) Evidencefor widespread epithelial damage and coincidentproduction of monocyte chemotactic protein 1 ina murine model of intestinal ricin intoxication.Infect. Immun. 75:1745–50.

5. Korcheva V, Wong J, Corless C, Iordanov M,Magun B. (2005) Administration of ricin inducesa severe inflammatory response via nonredun-dant stimulation of ERK, JNK, and P38 MAPKand provides a mouse model of hemolytic ure-mic syndrome. Am. J. Pathol. 166:323–39.

6. Wong J, Korcheva V, Jacoby DB, Magun B. (2007)Intrapulmonary delivery of ricin at high dosagetriggers a systemic inflammatory response andglomerular damage. Am. J. Pathol. 170:1497–510.

7. Smallshaw JE, Richardson JA, Pincus S, SchindlerJ, Vitetta ES. (2005) Preclinical toxicity and effi-cacy testing of RiVax, a recombinant protein vac-cine against ricin. Vaccine 23:4775–84.

8. Vitetta ES, et al. (2006) A pilot clinical trial of a re-combinant ricin vaccine in normal humans. Proc.Natl. Acad. Sci. U. S. A. 103:2268–73.

9. Ulloa L. (2005) The vagus nerve and the nicotinicanti-inflammatory pathway. Nat. Rev. Drug. Dis-cov. 4:673–84.

10. Tracey KJ. (2007) Physiology and immunology ofthe cholinergic antiinflammatory pathway. J. Clin.Invest. 117:289–96.

11. Wang H, et al. (2004) Cholinergic agonists inhibitHMGB1 release and improve survival in experi-mental sepsis. Nat. Med. 10:1216–21.

12. Wittebole X, et al. (2007) Nicotine exposure altersin vivo human responses to endotoxin. Clin. Exp.Immunol. 147:28–34.

13. Eliakim R, Fan QX, Babyatsky MW. (2002)Chronic nicotine administration differentially al-ters jejunal and colonic inflammation in inter-leukin-10 deficient mice. Eur. J. Gastroenterol.Hepatol. 14:607–14.

14. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H,Verdu EF, Collins SM. (2006) The vagus nerve: atonic inhibitory influence associated with in-flammatory bowel disease in a murine model.Gastroenterology 131:1122–30.

15. van Maanen MA, et al. (2009) Stimulation of nico-

tinic acetylcholine receptors attenuates collagen-induced arthritis in mice. Arthritis Rheum.60:114–22.

16. Mabley JG, Pacher P, Southan GJ, Salzman AL,Szabo C. (2002) Nicotine reduces the incidence oftype I diabetes in mice. J. Pharmacol. Exp. Ther.300:876–81.

17. Ingram JR, Rhodes J, Evans BK, Thomas GA.(2005) Preliminary observations of oral nicotinetherapy for inflammatory bowel disease: anopen-label phase I-II study of tolerance. Inflamm.Bowel Dis. 11:1092–6.

18. Carlsson S, Midthjell K, Grill V. (2004) Smoking isassociated with an increased risk of type 2 dia-betes but a decreased risk of autoimmune diabetesin adults: an 11-year follow-up of incidence of dia-betes in the Nord-Trondelag study. Diabetologia47:1953–6.

19. Gwilt CR, Donnelly LE, Rogers DF. (2007) Thenon-neuronal cholinergic system in the airways:An unappreciated regulatory role in pulmonaryinflammation? Pharmacol. Ther. 115:208–22.

20. Su X, et al. (2007) Activation of the alpha7nAChR Reduces Acid-Induced Acute Lung In-jury in Mice and Rats. Am. J. Respir. Cell Mol.Biol. 37:186–92.

21. Wu WK, Cho CH. (2004) The pharmacologicalactions of nicotine on the gastrointestinal tract.J. Pharmacol. Sci. 94:348–58.

22. Bradford MM. (1976) A rapid and sensitivemethod for the quantification of microgramquantities of protein utilizing the principle ofprotein-dye binding. Anal. Biochem. 72:248–54.

23. DaSilva L, et al. (2003) Pulmonary gene expres-sion profiling of inhaled ricin. Toxicon 41:813–22.

24. Pavlov VA, Tracey KJ. (2006) Controlling inflam-mation: the cholinergic anti-inflammatory path-way. Biochem. Soc. Trans. 34:1037–40.

25. Borovikova LV, et al. (2000) Vagus nerve stimula-tion attenuates the systemic inflammatory re-sponse to endotoxin. Nature 405:458–62.

26. Roche JK, et al. (2008) Post-exposure targeting ofspecific epitopes on ricin toxin abrogates toxin-induced hypoglycemia, hepatic injury, and lethal-ity in a mouse model. Lab. Invest. 88:1178–91.

27. Muldoon DF, Hassoun EA, Stohs SJ. (1992) Ricin-induced hepatic lipid peroxidation, glutathionedepletion, and DNA single-strand breaks inmice. Toxicon 30:977–84.

28. Sadani GR, Soman CS, Deodhar KK, NadkarniGD. (1997) Reactive oxygen species involvementin ricin-induced thyroid toxicity in rat. Hum. Exp.Toxicol. 16:254–6.

29. Battelli MG, Buonamici L, Polito L, Bolognesi A,Stirpe F. (1996) Hepatoxicity of ricin, saporin or asaporin immunotoxin: xanthine oxidase activityin rat liver and blood serum. Virchows Arch.427:529–35.

30. Kumar O, et al. (2007) Dose dependent effect ofricin on DNA damage and antioxidant enzymesin mice. Cell. Mol. Biol. (Noisy-le-grand) 53:92–102.

31. Wong J, Korcheva V, Jacoby DB, Magun BE.(2007) Proinflammatory responses of human air-



way cells to ricin involve stress-activated proteinkinases and NF-kappaB. Am. J. Physiol. Lung CellMol. Physiol. 293:L1385–94.

32. Doan LG. (2004) Ricin: mechanism of toxicity,clinical manifestations, and vaccine develop-ment: a review. J. Toxicol. Clin. Toxicol. 42:201–8.

33. Audi J, Belson M, Patel M, Schier J, Osterloh J.(2005) Ricin poisoning: a comprehensive review.JAMA 294:2342–51.

34. Fu XJ, et al. (2004) Role of p38 MAP kinase path-way in a toxin-induced model of hemolytic ure-mic syndrome. Pediatr. Nephrol. 19:844–52.

35. Hassoun E, Wang X. (2000) Ricin-induced toxic-ity in the macrophage J744A.1 cells: the role ofTNF-alpha and the modulation effects of TNF-alpha polyclonal antibody. J. Biochem. Mol.Toxicol. 14:95–101.

36. Gray JS, Bae HK, James CB, Lau AS, Pestka JJ.(2008) Double-stranded RNA-activated proteinkinase (PKR) mediates induction of IL-8 expres-sion by deoxynivalenol, Shiga toxin 1 and ricinin monocytes. Toxicol. Sci. 105:322–30.

37. Smallshaw JE, Richardson JA, Vitetta ES. (2007)RiVax, a recombinant ricin subunit vaccine, pro-tects mice against ricin delivered by gavage oraerosol. Vaccine 25:7459–69.

38. Oke SL, Tracey KJ. (2008) From CNI-1493 to theimmunological homunculus: physiology of theinflammatory reflex. J. Leukoc. Biol. 83:512–7.

39. Rosas-Ballina M, et al. (2008) Splenic nerve is re-quired for cholinergic antiinflammatory pathwaycontrol of TNF in endotoxemia. Proc. Natl. Acad.Sci. U. S. A. 105:11008–13.

40. Pavlov VA, et al. (2009) Brain acetylcholinesteraseactivity controls systemic cytokine levels throughthe cholinergic anti-inflammatory pathway. BrainBehav. Immun. 23:41–5.

41. Hofer S, et al. (2008) Pharmacologiccholinesterase inhibition improves survival in ex-perimental sepsis. Crit. Care Med. 36:404–8.

42. Parrish WR, et al. (2008) Modulation of TNF re-lease by choline requires alpha7 subunit nicotinicacetylcholine receptor-mediated signaling. Mol.Med. 14:567–74.

43. Wang H, et al. (2003) Nicotinic acetylcholine re-ceptor alpha7 subunit is an essential regulator ofinflammation. Nature 421:384–8.

44. de Jonge WJ, et al. (2005) Stimulation of thevagus nerve attenuates macrophage activationby activating the Jak2-STAT3 signaling pathway.Nat. Immunol. 6:844–51.

45. Yoshikawa H, et al. (2006) Nicotine inhibits theproduction of proinflammatory mediators inhuman monocytes by suppression of I-kappaBphosphorylation and nuclear factor-kappaB tran-scriptional activity through nicotinic acetyl-choline receptor alpha7. Clin. Exp. Immunol.146:116–23.

46. Sugano N, Shimada K, Ito K, Murai S. (1998)Nicotine inhibits the production of inflammatorymediators in U937 cells through modulation ofnuclear factor-kappaB activation. Biochem. Bio-phys. Res. Commun. 252:25–8.

47. Sadis C, et al. (2007) Nicotine protects kidneyfrom renal ischemia/reperfusion injury throughthe cholinergic anti-inflammatory pathway. PLoSONE 2:e469.

48. Hiramoto T, et al. (2008) The hepatic vagus nerveattenuates Fas-induced apoptosis in the mouseliver via alpha7 nicotinic acetylcholine receptor.Gastroenterology 134:2122–31.

49. Chang YC, Lai CC, Lin LF, Ni WF, Tsai CH.(2005) The up-regulation of heme oxygenase-1expression in human gingival fibroblasts stimu-lated with nicotine. J. Periodontal Res. 40:252–7.

50. Husain K, Scott BR, Reddy SK, Somani SM.(2001) Chronic ethanol and nicotine interactionon rat tissue antioxidant defense system. Alcohol25:89–97.

51. Kumar O, Sugendran K, Vijayaraghavan R.(2003) Oxidative stress associated hepatic andrenal toxicity induced by ricin in mice. Toxicon41:333–8.

activation of the cholinergic antiinflammatory pathway reduces ricin … · 2018. 9. 19. · ricin...

Documents