Immunology Letters 93 (2004) 179–187

Effect of rLcrV and rYopB fromYersinia pestison murine peritonealmacrophagesin vitro

Rajesh Kumar Sharmaa, Ajit Sodhia,∗, Harsh Vardhan Batrab, Urmil Tutejab

a School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, Indiab Division of Microbiology, DRDE, Gwalior, M.P., India

Received 29 December 2003; received in revised form 27 February 2004; accepted 16 March 2004

Available online 26 April 2004

Abstract

The interaction between macrophages and bacterial pathogens is crucial in the pathogenesis of infectious diseases. The 70 kb plasmidencodes low calcium response V (LcrV) or V antigen and a group of highly conserved yersinia outer proteins (Yops) are essential for fullvirulence. In present study, we investigated the effect of rLcrV and rYopB on macrophage functionsin vitro. It is observed that rLcrV andrYopB inhibited the LPS induced expression of TNF-�, IFN-�, KC, IP-10, and IL-12 in macrophages. rLcrV and rYopB caused increasedexpression of IL-10 and TLR2, whereas inhibited TLR4 expression in LPS treated macrophages. IL-10 and TLR2 antibodies reversed therLcrV and rYopB induced inhibition of TNF-� production by LPS treated macrophages, whereas IL-4 and TLR4 antibodies had no effect.Our data suggests a possible role of IL-10 and TLR2 in rLcrV and rYopB mediated inhibition of macrophage function.© 2004 Elsevier B.V. All rights reserved.

Keywords:Macrophages; rLcrV; rYopB; IL-10; Toll like receptors

1. Introduction

The genus Yersinia includes three human pathogenicspecies:Yersinia pestis, the etiological agent of bubonic andpneumonic plague; the enteropathogenic speciesYersiniapsudotuberculosisand Yersinia enterocolitica. Y. psudotu-berculosis, andY. enterocoliticacause adenitis, septicemia,and gastrointestinal syndromes. All pathogenic yersiniaemploy a panel of strategies to disarm macrophages andto disrupt their response to infection. For this purpose,Yersiniae engage a type III protein secretion system that ishighly conserved in the three species that are pathogenic forrodents and humans. The Yersinia type III secretion systemis activated upon host cell contact and specifically medi-ates the polarized translocation of Yersinia effector proteins(Yersinia outer proteins, Yops) inside eukaryotic cell[1].Y. pestispossesses virulence determinants encoded on eachof three plasmids and its chromosome[2]. The 70 kb con-served virulence plasmid, which is in common, enables the

∗ Corresponding author. Tel.:+91-542-2307314/2368331;fax: +91-542-2368693.

E-mail address:ajit.sodhi@lycos.com (A. Sodhi).

obligate intracellular gram-negative bacterium to resist theirhost [3]. The second plasmid of 100 kb encodes a murinetoxin and the capsular protein, F1, necessary for full vir-ulence in some animal species[4,5]. The third plasmid of10 kb encodes the bacteriocin, pesticin, and a plasminogenactivator protease (Pla) necessary in most of strains forvirulence from subcutaneous site[6,7].

Low calcium response V (LcrV) or V antigen is a secretedantihost protein with strong immunomodulatory effects andis associated with full virulence ofY. pestis[8,9]. Transloca-tion of Yops into eukaryotic cells requires LcrV[10,11]andLcrV is thought to be exposed at the bacterial surface priorto contact with host cells[10]. LcrV is a 327-residue solu-ble protein[12] whose only known homologue is PcrV ofPseudomonas aeruginosa[13,14]. Within Y. pestis, LcrV isrequired for induction of the LCR[15–19]. Recently, LcrVhas been shown to form pores in eukaryotic membranes inconjunction with YopB and YopD[20–22].

The translocator YopB, a 41.8 kDa protein encoded bysame virulence plasmid responsible for type III secretionsystem[23,24]. YopB contains putative transmembrane do-mains and is required for the translocation of Yops effec-tor proteins across the eukaryotic cell membrane, but it

0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.imlet.2004.03.010

180 R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187

is not itself translocated[24,25]. Recent reports suggestthat YopB is responsible for the formation of a channel inlipid membranes and can alone disturb artificial membranes[26].

Cells of the monocyte/macrophage lineage play an im-portant role in the host’s defense against various micro-bial infections and tumors either by direct cytotoxic activityand/or by their ability to regulate the activity of other cellsin the immune system and antigen presentation[27–29].Macrophages mediated regulation of immune response ismanifested by a variety of mechanisms involving secretionof bioactive molecules like nitric oxide (NO) and tumornecrosis factor (TNF)[30]. In the present study, we investi-gated the effect of rLcrV and rYopB on macrophage func-tions in vitro. It is observed that rLcrV and rYopB inhibitedthe expression of TNF-�, IFN-�, KC, IP-10, and IL-12. Thedata also suggests the possible role of IL-10 and TLR2 inrLcrV and rYopB induced immunomodulatory functions ofmacrophages.

2. Materials and methods

2.1. Mice

Inbred strains of Balb/c mice of either sex at 8–10 weeksof age were used for obtaining peritoneal macrophages.

2.2. Cell cultures and reagents

L929 (murine fibroblast cell line) and macrophage cul-tures were maintained in RPMI 1640 medium supple-mented with heat-inactivated fetal calf serum (10%), peni-cillin (100 U/ml), streptomycin (100 U/ml) and gentamycin(20 ug/ml) at 37◦C in humidified air containing 5% CO2.Medium RPMI 1640, LPS, proteinase K and most of theother reagents were purchased from Sigma–Aldrich, USA.Fetal calf serum was purchased from Biological Industries(Israel). Antibodies against TNF-�, TLR4, TLR2, IL-10,IL-4, iNOS, actin and HRP-conjugated anti-rabbit and -goatIgG antibodies were obtained from Santa Cruz Biotechnol-ogy Inc., CA, USA. TRIzol reagent and one-step RT–PCRkit were bought from Life Technologies Inc., GibcoBRL,USA and Qiagen, Germany. Mouse primers for TNF-�,IL-10, IFN-�, KC, IP-10, TLR2, TLR4, and GAPDH werepurchased from GENSET Singapore Biotech. Pte Ltd. (Sin-gapore). Purified recombinant proteins of Yersinia rLcrV(MW = 31 kDa), rYopB (MW = 41.8 kDa) and rYopM(MW = 40 kDa) were obtained from Dr. H.V. Batra, Di-vision of Microbiology, DRDE, Gwalior. The proteinshave been cloned and over expressed inEscherichia coliby induction with IPTG. The proteins have been purifiedusing nickel–NTA column chromatography and identi-fied by single band on SDS–PAGE. All the reagents wereendotoxin-free as determined by the limulus amoebocytelysate assay (sensitivity limit, 0.1 ng/ml).

2.3. Isolation and activation of macrophages

Macrophage monolayers were prepared as described ear-lier [31]. Peritoneal exudates cells were harvested usingchilled serum-free RPMI 1640 medium and added to wellsof 24-well tissue culture plates (Nunc, Denmark). After 2 hincubation at 37◦C in an atmosphere of 5% CO2 in airin a CO2 incubator, the non-adherent cells were removedby vigorous washing (three times) with warm serum-freemedium and the adherent cells were incubated in completemedium overnight to form macrophage monolayers. Morethan 95% of the adherent cell population was macrophagesas determined by morphology and non-specific esterasestaining.

Macrophage monolayers were incubated with rLcrV(10�g/ml) and rYopB (10�g/ml) in fresh medium for 2 h.Thereafter, macrophages were washed and further treatedwith 10�g/ml of LPS for various time intervals as indi-cated inSection 3. The cell-free culture supernatants werecollected after centrifugation at 250× g for 10 min. Thesupernatants were assayed for TNF-� production whereasthe macrophage monolayers were used for MTT assay toascertain cell viability.

2.4. Assay of TNF activity

TNF-� activity was assayed as described previously[32].The percentage cytotoxicity against L929 cells as a measureof TNF-� activity was calculated as follows:

Cytoxicity % = C − T

C× 100

whereC is the absorbance of wells containing L929 cellsincubated in medium alone, andT is the absorbance of wellscontaining L929 cells with test samples.

2.5. Preparation of cell lysates and immunoblotting

The macrophages with or without treatment were washedwith ice cold phosphate buffered saline containing 1 mMNa3VO4, then lysed in 50�l of lysis buffer [20 mMTris–HCl, pH 8, 137 mM NaCl, 10% glycerol (v/v), 1% Tri-ton X-100 (v/v), 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF,20�M leupeptin, and 0.15 units/ml aprotonin] for 20 min at4◦C. The lysates were centrifuged (13,000× g at 4◦C) for15 min and the supernatants (containing Triton X-100 solu-ble proteins) were separated on 10% SDS-polyacrylamidegels at 20 mA. The separated proteins were transferred tonitrocellulose (8 h at 125 mA) and immunoblotted withprimary antibody, incubated with second antibody con-jugated with horseradish peroxidase and visualized bythe Chemiluminescence Western Blotting Kit (Santa CruzBiotechnology, CA, USA). To monitor equal loading of pro-tein, Western blot analysis using antibody directed againstactin was done for each experiment as shown in lowerpanels.

R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187 181

2.6. RNA isolation, reverse transcription and polymerasechain reaction

Total RNA was isolated from the murine peritonealmacrophages by TRIzol reagent (GibcoBRL) in accor-dance with the supplier’s instructions. The RNA wasreverse-transcribed using a one-step RT–PCR kit (Qiagen,Germany) and amplified by PCR using the specific murineprimers indicated inSection 3. The thermocycle conditionswere 28 cycles of 94◦C for 1 min, 55◦C for 1 min, and72◦C for 2 min, after which an additional extension step at72◦C for 5 min was included. Electrophoresis of amplifiedDNAs was carried out on a 2% agarose gel and stainedwith ethidium bromide. The sequences of primers specificfor murine genes are as follows:

TNF-� forward 5′-GGCAGGTCTACTTTGGAGTCAT-TGC-3′

TNF-� reverse 5′-ACATTCGAGGCTCCAGTGAATTC-GG-3′

IFN-� forward 5′-GGTGACATGAAAATCCTGCAGA-GC-3′

IFN-� reverse 5′-CGCTGGACCTGTGGGTTGTTGAC-C-3′

IL-10 forward 5′-GGACTTTAAGGGTTACTTGGGTT-GCC-3′

IL-10 reverse 5′-CATTTTGATCATCATGTATGCTTCT-3′

IL-12 forward 5′-CCACTCACATCTGCTGCTCAACA-AG-3′

IL-12 reverse 5′-ACTTCTCATAGTCCCTTTGGTCC-AG-3′

TLR-2 forward 5′-GGAGCGGCGGCTGCAGGACTC-3′TLR-2 reverse 5′-CCAAAGAGCTCGTAGCATCC-3′TLR-4 forward 5′-AGTGGGTCAAGGAACAGAAGCA-

3′TLR-4 reverse 5′-CTTTACCAGCTCATTTCTCACC-3′KC forward 5′-TCGCTTCTCTGTGCAGCGCT-3′KC reverse 5′-GTGGTTGACACTTAGTGGTCTC-3′IP-10 forward 5′-CCAAGTGCTGCCGTCATTTTC-3′IP-10 reverse 5′-TCGCAGGGATGATTTCAAGC-3′GAPDH forward 5′-CCTGCAGTGTCTGATATTGTTG-

3′GAPDH reverse 5′-AACACACCATTGCGATGAA-3′

The expression of house keeping gene GAPDH was checkedfor each set of RT–PCR experiment, as shown in lower panel.

2.7. Percentage viability by MTT assay

Percentage viability of macrophages were determined byMTT [3(4,5);dimethyl thiazol-2,5-diphenyl tetrazolium bro-mide] assay as described earlier[33]. The relative cell via-bility was calculated according to the formula:

Relative cell viability= Absorbance experimental

Absorbance control× 100

where ‘Absorbance control’ represents macrophages incu-bated in medium alone and ‘Absorbance experimental’ rep-resents macrophages treated with the recombinant proteinsor their vehicles. In each of the experiments, the viability ofthe peritoneal macrophages was not affected by the doses ofthe recombinant proteins used up to 48 h.

3. Results

3.1. rLcrV and rYopB inhibits TNF-α production bymacrophages in vitro

rLcrV and rYopB pretreatment significantly inhib-ited TNF-� production in LPS treated macrophages in adose-dependent manner. Whereas rYopM taken as irrele-vant control had no effect on TNF-� production (Fig. 1a).A similar inhibitory effect of these purified proteins wasobserved on TNF-� protein expression by western blot-ting and for the TNF-� gene at the transcriptional level byRT–PCR (Fig. 1b and c).

3.2. Suppression of TNF-α is abolished by boiling orpretreatment with proteinase K of rLcrV and rYopB

rLcrV and rYopB proteins boiled for 1 h did not inhibitLPS induced TNF-� production(Fig. 2a). Proteolytic de-graded rLcrV and rYopB by digestion with (20ı̀g/ml) pro-teinase K for 30 min similarly did not inhibit LPS inducedTNF-� production (Fig. 2b).

3.3. Inhibition in the transcription of interferon-γ (IFN-γ)and IL-12 gene in response to rLcrV and rYopB

rLcrV and rYopB (10̀ıg/ml) treated macrophages whenincubated with LPS for 12 h showed decreased transcriptionof IFN-� and IL-12 genes, as compared to their increasedtranscription in macrophages treated with LPS alone (Fig. 3aand b).

3.4. Inhibition in the transcription of proinflammatorychemokines KC and IP-10 genes in response to rLcrV andrYopB

rLcrV and rYopB (10̀ıg/ml) treated macrophages on incu-bation with LPS for 12 h resulted in decreased transcriptionof KC and IP-10 genes, as compared to their increased tran-scription in macrophages treated with LPS alone (Fig. 4aand b).

3.5. rLcrV and rYopB elicit IL-10 expression inmacrophages in vitro

rLcrV and rYopB (10̀ıg/ml) treatment of macrophages for2 h induced expression of IL-10 as observed by immunoblot-ting and reverse transcription and polymerase chain reaction

182 R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187

Fig. 1. (a) Effect of different concentrations of rLcrV, rYopB, and rYopM on LPS induced production of TNF-�. The cell free culture supernatants werecollected and assayed for TNF-� production as described inSection 2. The above experiment is representative of three independent experiments done intriplicate. (b) TNF-� protein expression in macrophages. Lane 1, untreated; Lane 2, LPS; Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS. (c) TNF-�

gene expression in macrophages. Lane 1, untreated; Lane 2, LPS; Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS.

(RT–PCR) (Fig. 5a and b). No expression of IL-10 gene wasobserved in untreated and LPS stimulated macrophages.

3.6. Anti-IL-10 antibody abrogated the inhibitory effectof rLcrV and rYopB on the production of TNF-α inmacrophages, whereas anti-IL-4 antibody had no effect

To examine if the inhibition of LPS induced TNF-� pro-duction with rLcrV and rYopB is IL-10 or IL-4 mediated, therLcrV and rYopB treated macrophages were incubated withLPS in the presence of anti-IL-10 and anti-IL-4 antibodies

(2�g/ml). Inhibition of TNF-� production was abrogatedin macrophages treated with anti-IL-10 antibody (Fig. 6,Lane 3 and 4). Anti-IL-4 antibody did not reverse the rLcrVand rYopB induced inhibition of TNF-� production in LPStreated macrophages (Fig. 6, Lane 5 and 6).

3.7. rLcrV and rYopB upregulates the expression of TLR2and downregulates the expression of TLR4

rLcrV and rYopB induced significantly enhanced expres-sion of TLR2 protein as well as its gene expression in

R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187 183

Fig. 2. (a) Effect of boiling of rLcrV and rYopB proteins on TNF-� production in LPS stimulated macrophages. (b) Effect of proteinase K treated rLcrVand rYopB on TNF-� production in LPS stimulated macrophages.

macrophages 12 h after treatment (Fig. 7a and b). rLcrV andrYopB did not significantly induce TLR4 gene expression.However, rLcrV and rYopB inhibited TLR4 protein and geneexpression in LPS treated macrophages (Fig. 8a and b).

3.8. Anti-TLR2 antibody abolished the inhibition of TNF-α

production, whereas anti-TLR4 antibody had no effect

rLcrV and rYopB treated macrophages were incubatedwith LPS in the presence of anti-TLR2 or anti-TLR4

antibodies (2�g/ml). Inhibition in TNF-� production wasreversed in anti-TLR2 treated macrophages (Fig. 9a, Lane3 and 4), suggesting a possible role for TLR2 in inhibitionof TNF-� production. Anti-TLR4 did not reverse the rLcrVand rYopB induced inhibition of TNF-� production (Fig. 9a,Lane 5 and 6). A graph showing densitometric analysis ofFig. 9a is also provided (Fig. 9b). Addition of anti-TLR4and anti-TLR2 antibodies did not significantly abolishthe LPS mediated production of TNF-� in macrophages(Fig. 10a, Lane 3 and 4). Treatment of macrophages with

184 R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187

Fig. 3. (a) IFN-� gene expression in macrophages. Lane 1, untreated;Lane 2, LPS; Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS. (b)IL-12 gene expression in macrophages. Lane 1, untreated; Lane 2, LPS;Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS.

Fig. 4. (a) KC gene expression in macrophages. Lane 1, untreated; Lane2, LPS; Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS. (b) IP-10gene expression in macrophages. Lane 1, untreated; Lane 2, LPS; Lane3, rLcrV plus LPS; Lane 4, rYopB plus LPS.

Fig. 5. (a) IL-10 protein in macrophages. Lane 1, untreated; Lane 2, LPS;Lane 3, rLcrV; Lane 4, rYopB. (b) IL-10 gene expression in macrophages.Lane 1, untreated; Lane 2, LPS; Lane 3, rLcrV; Lane 4, rYopB.

anti-TLR4 or anti-TLR2 antibodies by themselves did notinduce TNF-� production (Fig. 10a, Lane 5 and 6). A graphshowing densitometric analysis ofFig. 10ais also provided(Fig. 10b).

Fig. 6. Effect of IL-10 and IL-4 antibodies on TNF-� protein expressionin macrophages. Lane 1, untreated; Lane 2, LPS; Lane 3, rLcrV plusIL-10 Ab plus LPS; Lane 4, rYopB plus IL-10 Ab plus LPS; Lane 5,rLcrV plus IL-4 Ab plus LPS; Lane 6, rYopB plus IL-4 Ab plus LPS;Lane 7, rLcrV plus LPS; Lane 8, rYopB plus LPS.

Fig. 7. (a) TLR2 protein expression in macrophages. Lane 1, untreated;Lane 2, rLcrV; Lane 3, rYopB; Lane 4, rLcrV plus rYopB. (b) TLR2gene expression in macrophages. Lane 1, untreated; Lane 2, rLcrV; Lane3, rYopB; Lane 4, rLcrV plus rYopB.

Fig. 8. (a) TLR4 protein expression in macrophages. Lane 1, untreated;Lane 2, LPS; Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS. (b)TLR4 gene expression in macrophages. Lane 1, untreated; Lane 2, LPS;Lane 3, rLcrV plus LPS; Lane 4, rYopB plus LPS.

Fig. 9. (a) Effect of anti-TLR2 and anti-TLR4 antibodies on TNF-�

protein expression in rLcrV and rYopB treated macrophages. Lane 1,untreated; Lane 2, LPS; Lane 3, rLcrV plus TLR2 Ab plus LPS; Lane4, rYopB plus TLR2 Ab plus LPS; Lane 5, rLcrV plus TLR4 Ab plusLPS; Lane 6, rYopB plus TLR4 Ab plus LPS. (b) Densitometric analysisof (a) showing relative intensity of the bands.

R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187 185

Fig. 10. (a) Effect of TLR2 and TLR4 antibodies on TNF-� proteinexpression in macrophages. Lane 1, untreated; Lane 2, LPS; Lane 3,TLR4 Ab plus LPS; Lane 4, TLR2 Ab plus LPS; Lane 5, TLR4 Ab; Lane6, TLR2 Ab. (b) Densitometric analysis of (a) showing relative intensityof the bands.

4. Discussion

Yersinia antigenic protein LcrV and YopB are knownas translocator of Yersinia and necessary for full virulence[34,35]. The role of rLcrV and rYopB in modulating theeffector functions of murine peritoneal macrophages hasbeen investigated. It is demonstrated that the treatment withrLcrV and rYopB inhibited production of TNF-�, IFN-�,and IL-12 as well as of IFN-� inducible proinflammatorychemokines, IP-10 and KC in LPS stimulated murine peri-toneal macrophages. It is also observed that the treatmentwith rLcrV and rYopB induced increased expression ofIL-10 and TLR2 in macrophages. The possible involvementof IL-10 and TLR2 in rYopB-mediated immunosuppressionin macrophages is a novel finding, and inhibition of otherproinflammatory cytokines like, IL-12, and IFN-� relatedchemokines IP-10 and KC are reported for the first time forrecombinant rLcrV and rYopB.

TNF-� is a proinflammatory cytokine, which is primarilyreleased by activated macrophages and plays a crucial rolein limiting the severity of the bacterial infection[36,37]. In-hibition of TNF-� production has been reported in severalbacterial infections[38]. In most cases, however, the un-derlying cytokine-suppressive mechanism has not yet beenelucidated. Although the molecular mechanism of TNF-�suppression by YopP has been assigned to the inhibitionof NF�B and of the extracellular signal regulated kinase 2,c-Jun N-terminal kinase, and p38 MAP kinase activities, theprofound suppression of proinflammatory cytokine suppres-sion duringYersinia pestisinfection was first attributed toLcrV by Nakajiama et al.[9] in 1995. Sing et al.[39] in2002 reported that LcrV inhibits zymosan-induced produc-tion of TNF-� by inducing IL-10. LPS is known to medi-ate its response through TLR4 and CD14 and it has been

suggested that LcrV also uses CD14[39]. Brubaker[40]in 2003 also supported the role of LcrV and other Yops ininhibition of innate immunity. Recently Sing et al.[41] in2003 further suggested a crucial role of LcrV in evasion ofY. enterocolitica. In the present investigation, it is suggestedthat IL-10 possibly play a role in the rLcrV and rYopBinduced inhibition of LPS mediated production of TNF-�and other proinflammatary cytokines and chemokines inmacrophages. Anti-IL-10 antibody completely reversed theinhibitory effect of rLcrV and rYopB on LPS induced pro-duction of TNF-�, whereas anti-IL-4 antibody had no effect.LcrV-induced production of IL-10 has been proposed by Ne-dialkov et al.[42] in 1997 to explain this immunosuppres-sion duringY. pestisinfection. Schmidt et al.[43] in 1999reported that activated T cells are required for LcrV medi-ated TNF-� suppression upon LPS-IFN-� stimulation in aperitoneal exudates model. However, in the present investi-gations it is observed that rLcrV directly inhibits the TNF-�production in LPS stimulated macrophages. The cells ofthe monocyte-macrophages lineage are the main or the onlypossible source of both IL-10 and TNF-�. It can be, there-fore, suggested that rLcrV induced IL-10 leads to TNF-�suppression by “silencing” macrophages in a paracrine orautocrine loop.

To show that inhibition in TNF-� production and theIL-10 induction by macrophages are specific effects of rLcrVand rYopB and not due to endotoxin contamination, stud-ies with denatured rLcrV or rYopB were undertaken. Bothboiling and proteinase K degradation of rLcrV and rYopBcompletely reversed the inhibition of TNF-� production inmacrophages. Beuscher et al. in 1995 reported that YopBpurified from the culture supernatant ofY. enterocoliticain-hibits synthesis of tumor necrosis factor (TNF-�) by murinemacrophages and is required for bacterial colonization ofintestinal lymphoid tissue of mice[44]. However, in presentinvestigation recombinant YopB is used excluding any pos-sibility of LcrV contamination in it (as LcrV have affinityfor YopB). Further, the data suggests that the inhibitory ef-fect of rYopB on macrophages is mediated through TLR2and IL-10.

The observation that rLcrV and rYopB treatment inhibitsthe production of IL-12, IFN-� as well as that of IFN-� in-ducible proinflammatory chemokines, IP-10 and KC is in-teresting in view of the role of IFN-� in modulating theNK cells and T-cell-mediated responses[45]. The ability ofchemokines IP-10 and KC to attract activated T-cell dur-ing inflammatory reactions[46], and the role of IL-12 inthe initiation of antigen-specific T-cell response is well rec-ognized[47]. A role of V antigen in IFN-� suppression inmixed macrophage T cell cultures has been reported previ-ously [43]. However, the role of YopB in modulating thesechemokines production is not well documented.

The Toll like receptors are PRRs that have a unique andessential function in animal immunity. It is reported thatthese receptors have a vital role in microbial recognition,induction of the antimicrobial genes and the control of the

186 R.K. Sharma et al. / Immunology Letters 93 (2004) 179–187

adaptive immune responses[48,49]. It is observed that rL-crV and rYopB induced increased expression of TLR2, butnot TLR4 in macrophages. However, both rLcrV and rYopBdown regulated the expression of TLR4 in LPS stimulatedmacrophages. It is, therefore, suggestive of a possible rolefor TLR2 in rLcrV and rYopB induced TNF-� suppression.It is further supported by that the anti-TLR2 antibodies sig-nificantly reversed the inhibitory effect of rLcrV and rY-opB in LPS induced TNF-� production, whereas, anti-TLR4antibodies had no effect. Pretreatment of LPS stimulatedmacrophages with anti-TLR4 antibodies caused slight inhi-bition in TNF-� production, thereby, suggesting that TNF-�suppression is primarily due to inhibitory effect of rLcrVand rYopB and not due to anti-TLR4 antibodies. Treatmentof macrophages only with anti-TLR2 or anti-TLR4 antibod-ies did not induce TNF-� production.

Acknowledgements

This work was supported by grants from DRDO, NewDelhi to professor A. Sodhi. Rajesh Kumar Sharma is arecipient of CSIR-JRF.

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