0014-2980/99/1212-4002$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

A role for lymphotoxin I receptor in host defenseagainst Mycobacterium bovis BCG infection

Rudolf Lucas1, Fabienne Tacchini-Cottier2, Reto Guler2, Dominique Vesin2, StephaneJemelin2, Maria L. Olleros2, Gilles Marchal3, Jeffrey L. Browning4, Pierre Vassalli2 andIrene Garcia2

1 Department of APSIC, University Medical Center, Geneva, Switzerland2 Department of Pathology, University Medical Center, Geneva, Switzerland3 Institut Pasteur, Paris, France4 Biogen Inc, Cambridge, USA

To investigate the role of membrane lymphotoxin (LT) § 1/ g 2 and its LT g receptor (LT g R) in theprotective immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infec-tion, we have used a soluble fusion molecule (LT g R-IgG1). LT g R-Ig treatment interferes withgranuloma formation mainly in the spleen by inhibiting macrophage activation and nitricoxide synthase activity. In addition, a large accumulation of eosinophils was observed in thespleen of LT g R-Ig-treated infected mice. Decreased blood levels of IFN- + and increased IL-4were also observed, suggesting that the LT g R pathway is important in BCG infection to favora Th1 type of immune response. The treatment of transgenic mice expressing high bloodlevels of a soluble TNFR1-IgG3 fusion protein with LT g R-Ig resulted in a still higher sensitivityto BCG infection, and extensive necrosis in the spleen. In conclusion, these results suggestthat the LT g R and the TNFR pathways are not redundant in the course of BCG infection andprotective granuloma formation: the LT g R pathway appears to be important in spleen granu-loma formation, whereas the TNFR pathway has a predominant role in other tissues.

Key words: Lymphotoxin g receptor / Bacillus Calmette-Guerin / Granuloma / Spleen

Received 10/6/99Revised 6/9/99Accepted 8/9/99

[I 19760]

Abbreviations: LT: Lymphotoxin AFB: Acid-fast bacilliiNOS: Inducible nitric oxide syntase s: Soluble

1 Introduction

Mycobacterial infections induce a cell-mediated immuneresponse, in which the contribution of macrophages andT lymphocytes is essential. This response involves theaction of at least two synergistic cytokines, TNF- § andIFN- + , which play a critical role in the development ofprotective granulomas. These granulomas contain acti-vated macrophages producing specific enzymes, suchas inducible nitric oxide synthase (iNOS), and areresponsible for the elimination of intracellular bacteria.These mechanisms form the basis for protection againstmycobacterial spreading [1–4].

The importance of TNF and IFN- + in mycobacterial infec-tions has been extensively reported. Neutralization ofTNF with anti-TNF antibodies or with soluble (s) TNFR1-Ig fusion protein during Mycobacterium bovis andM. tuberculosis infections increased sensitivity by inhib-

iting macrophage differentiation and the development ofwell-differentiated granulomas, resulting in mycobacte-rial overgrowth and rapid death of animals [5–8]. In addi-tion, the presence of TNFR1 was shown to be necessaryfor protection against M. tuberculosis infection [9]. Onthe other hand, mice incapable of using or producingIFN- + are also unable to control mycobacterial infections[10–14]. Induction of IFN- + synthesis is largely depen-dent on IL-12, and IL-18, which are important cytokinesfor the generation of a protective immunity against tuber-culosis [15, 16]. Infection of IL-12 p40–/– mice withM. tuberculosis resulted in unrestrained growth of myco-bacteria and a reduction of IFN- + mRNA [17].

Although most studies have focused on the role ofTNF- § in infectious diseases, little is known about thecontribution of the lymphotoxin (LT) system in defensemechanisms. LT § is made by activated lymphocytes andsecreted as a homotrimer binding to both TNFR1 andTNFR2. Moreover, LT § can also exist as a membrane-bound heterotrimer complexed with LT g . MembraneLT § / g heterotrimers (LT § 1/ g 2 predominantly, and LT § 2/g 1) are found on activated lymphocytes and have been

reported to bind mainly to the LT g R. This receptor, which

4002 R. Lucas et al. Eur. J. Immunol. 1999. 29: 4002–4010

Figure 1. (A, B) Acid phosphatase activity of frozen tissuesections from spleens of BALB/c control (A) and LT g R-Ig-treated mouse (B) at 4 weeks of BCG infection. Magnifica-tion 320X. (C, D) Histological sections of spleen stained witheosin showing the presence of few eosinophils in BALB/ccontrol (C) and numerous eosinophils in LT g R-Ig-treatedmouse spleen section (D) at 4 weeks of BCG infection. Mag-nification 800X.

does not bind TNF, is expressed on many cell types, butnot on lymphocytes, and also interacts with the newlydefined LIGHT ligand [18–21]. Blockade of the LT g Rpathway by transgenic expression of LT g R-Ig [22], treat-ment with this protein or targeted disruption of LT § , LT gand LT g R results in alteration of splenic architecture andimpaired development of lymphoid organs [23–28]. Sig-naling through the LT g R appears therefore to be crucialfor lymph node genesis. Signaling through TNFR1 hasbeen shown to be involved mainly in Peyer’s patchdevelopment. LT g R appears to be essential for the main-tenance of lymphocytes and follicular dendritic cells inthe spleen and lymph nodes, whereas TNFR are notrequired in this process [28]. Furthermore, LT g R signalingseems to be involved in the differentiation of the splenicstromal elements driving lymphocyte segregation and inthe development of follicular dendritic cells [29–30].

To evaluate the role of the LT g R pathway in protectivemechanisms against BCG infection, we have used LT g R-Ig recombinant fusion protein to neutralize LT § / g (andLIGHT). The results presented here suggest that LT § / gcontributes to granuloma formation and iNOS activation,and that the LT g R pathway is important to favor a Th1type of immune response required for host defenseagainst BCG infection.

2 Results

2.1 LT I R-Ig treatment prevents the developmentof granulomas and the induction of NOS inthe spleen of BCG-infected mice

To explore the possible role of the LT g R pathway in gran-uloma development, a group of ten BALB/c mice wastreated with LT g R-IgG, using a protocol of weekly i.p. LT-g R-Ig injection (100 ? g/mouse), starting 1 week prior to

infection and continued until the animals were killed.Control mice were injected with the same amount ofhuman IgG. Subsequently, mice were infected i.v. with105 CFU BCG and killed at 4 weeks.

Evaluation of the granuloma number and size in thespleen was done by staining of tissue sections for acidphosphatase activity, an enzyme produced in largeamounts in differentiated macrophages, which reflectsthe macrophage activation within the granulomas [5, 8,31, 32]. Acid phosphatase is easily detectable by histo-chemistry because it stains cells in red. LT g R-Ig treat-ment resulted in an approximately fourfold decrease inthe number of observed granulomas in the spleen(Table 1). In addition, we found that granulomas fromLT g R-Ig treated mice were small and showed low acidphosphatase activity as depicted in Fig. 1. Quantification

of the acid phosphatase activity of spleen enzymeextracts from BCG-infected mice at 4 weeks post infec-tion showed 40 % reduction in LT g R-Ig-treated mice(Fig. 2 A).

Mycobactericidal mechanisms include the activation ofiNOS, which plays a role in the control of bacterialgrowth. We have analyzed nitric oxide synthase (NOS)activity from crude spleen protein extracts by monitoringthe conversion of radioactive L-arginine to L-citrulline andNO [33]. We found a 64 % decrease of NOS activity in thespleen of mice treated with LT g R-Ig at 4 weeks of BCGinfection, as compared to infected control mice(Fig. 2 B). LT g R-Ig treatment resulted in a weak but sig-nificant effect on bacterial loads in the spleen (Table 1).

Apart from analyzing the spleen, we also examined liverand lung granulomas on hematoxylin/eosin-stained tis-sue sections. The patterns of hepatic granulomas was

Eur. J. Immunol. 1999. 29: 4002–4010 LT g R in BCG infection 4003

Figure 2. LT g R treatment in BCG-infected BALB/c micedecreased the acid phosphatase and NOS activity andincreased the number of eosinophils in the spleen. (A) Acidphosphatase activity of spleen enzyme extracts from humanIg- or LT g R-Ig-treated mice, collected at 4 weeks of BCGinfection. Results are shown as means of the absorbance(570 nm) values after subtraction of basal levels from unin-fected mice (0.136 ± 0.43) from four different samples pergroup ± SEM, and are representative of two independentexperiments. (B) NOS activity was determined in spleenenzyme extracts from control and LT g R-Ig-treated mice (fourmice per group). Data are represented as means (after sub-traction of the value from uninfected mice: 1172 ± 215) ±SEM of the cpm/mg of tissue. These results are representa-tive of two independent experiments. (C) Evaluation of thenumber of eosinophils in histological sections after eosinstaining of the spleen sections from mice injected withhuman Ig (n = 4), and mice treated with LT g R-Ig (n = 4). Val-ues represent means of the number of eosinophils per 1000spleen cells ± SEM. A minimum of 3000 cells were countedper section. Differences between control and LT g R-Ig arestatistically significant (p X 0.001). Data are representativeof two independent experiments.

Table 1. Granuloma formation and bacterial load in controland LT g R-Ig-treated mice

Control mice LT g R-Ig-treatedmicea)

Spleen

Granulomasb)

(number/mm2)57.64 ± 19.72 15.65 ± 9.52**

CFU/mg tissuec) 373.34 ± 83.27 1013.36 ± 140.48*

AFB/mm2 d) 56.75 ± 22.73 230.05 ± 49.04*

Liver

Granulomas(number/mm2)

43.50 ± 5.46 33.60 ± 7.50*

CFU/mg tissue 566.08 ± 140.35 1147.20 ± 290.41

AFB/mm2 69.34 ± 23.61 217.58 ± 52.56*

Lung

CFU/mg tissue 554.15 ± 69.40 1678.25 ± 258.05*

a) BALB/c mice were treated with human Ig (control) or withLT g R-Ig (100 ? g/mouse/week). Liver, lung and spleenwere isolated from BCG-infected mice after 4 weeks.

b) The number of BCG granulomas in spleens was countedon acid phosphatase-stained tissue sections and thenumber of liver granulomas was counted on hematoxylin/eosin-stained tissues sections as described [8]. Resultsare represented as average number ± SD per mm2 oftissue section (n = 5).

c) CFU were determined in four mice per group.d) Histological sections were stained with Ziehl-Neelsen

stain and the number of AFB counted per mm2 of tissuesection (n = 5). Differences between groups arestatistically significant, * p p 0.05; ** p p 0.001.

similar in both experimental groups, but we observed amoderate reduction in their number in LT g R-Ig treatedmice compared to control mice (Table 1). To verify theefficiency of the injection of soluble receptors in BCGinfection, a group of mice (n = 5) were treated weeklywith (100 ? g/mouse) sTNFR1-IgG1 recombinant protein.From our previous work on transgenic mice expressingsTNFR1-IgG3 fusion protein, we know that this protein,when expressed at permanently high blood levels, has aprofound effect on BCG granuloma formation in the liverof infected mice [8]. We observed that administration ofsTNFR1-Ig reduced the number of granulomas in theliver at 4 weeks of BCG infection only twofold whereasunder the same conditions, well-formed granulomaswere absent in transgenic mice.

Histological analysis of lung lesions did not showmarked differences between control and LT g R-Ig treated

4004 R. Lucas et al. Eur. J. Immunol. 1999. 29: 4002–4010

Figure 3. Histological appearance of liver (A–D) and lungsections (E–H) from control (A, C, E, G) and LT g R-Ig-treatedmice (B, D, F, H), 4 weeks after BCG infection. Hematoxylin/eosin staining is shown in (A, B, E, F). Ziehl-Neelsen stainingis shown in (C, D, G, H). Magnification 100x (A, B), 250x (E,F) and 1000x (C, D, G, H). Results are representative of threeindependent experiments (five mice per group).

mice, although we observed a higher number of acid-fast bacilli (AFB) in infected alveolar macrophages asshown in Fig. 3. Moreover, CFU determined in the lung ofLT g R-Ig-treated mice were increased (Table 1).

2.2 LT I R-Ig treatment down-regulates IFN- q andup-regulates IL-4 in blood, and increases therecruitment of eosinophils in spleens ofBCG-infected mice

Mycobacterial infections induce a Th1 type of immuneresponse, characterized by the release of IFN- + and IL-12, which are required for host protection [10–15, 34,35]. To investigate whether the partial neutralization ofmembrane LT affects the pattern of cytokines induced by

BCG infection, we evaluated the amount of IFN- + andIL-4 in the serum of BCG-infected mice. We observeddecreased levels of IFN- + , but increased levels of IL-4following LT g R-Ig treatment (Fig. 4). Weekly treatment ofthe infected mice with 100 ? g/mouse sTNFR1-IgG1recombinant protein had a similar effect as LT g R-Ig treat-ment, i.e. a reduction of the blood level of IFN- + and anincrease of IL-4 (Fig. 4).

We also observed a reduction of IFN- + in spleen proteinextracts from LT g R-Ig-treated mice (spleens from fourmice, average in pg IFN- + /250 ? g tissue: 1772.73 ±195.46 in control mice and 1396.33 ± 222.66 in LT g R-Ig-treated mice; p X 0.05).

An enhancement of eosinophil recruitment may reflect alocal deviation of the Th1 to the Th2 type of immuneresponse. To explore if LT g R-Ig treatment may affect thelocal accumulation of eosinophils in the spleen, we eval-uated their number in spleens from BCG-infected miceat 4 weeks of infection. A fivefold increase in the numberof eosinophils in the spleen was observed in LT g R-Ig-treated mice, as compared to control mice (p p 0.001).Fig. 1 illustrates a stronger accumulation of eosinophilsin a spleen section of BCG-infected mice treated withLT g R-Ig, as compared to infected control mice.

2.3 LT I R-Ig treatment of sTNFR1-transgenicmice results in a still increased susceptibilityto BCG infection, enhancement of bacterialgrowth and spleen necrosis

As mentioned above, we have previously shown thattransgenic expression of sTNFR1 results in increasedsensitivity to M. tuberculosis and M .bovis infections,with bacterial overgrowth, marked inhibition of macro-phage differentiation within granulomas and a significantreduction in the number of liver granulomas [8]. Toexplore the respective roles of TNF- § and LT § / g duringBCG infection, transgenic mice expressing largeamounts of a sTNFR1-IgG3 fusion protein were treatedwith LT g R-IgG1 fusion protein to interfere with both theTNF and LT § /LT g pathways, and compared to untreatedtransgenic mice. These mice showed earlier cachexiaand two out of seven died at day 22 and 25 post infec-tion, while the untreated transgenic mice all surviveduntil they were killed at day 28 after BCG infection. His-tological sections of spleens of the untreated transgenicmice showed small scattered granulomas distributed inthe red pulp as well as in the lymphoid follicles. Theycontained many AFB and occasionally displayed somenecrosis. In contrast, the spleens of LT g R-Ig-treatedtransgenic mice showed extensive areas of necrotizingtissue corresponding to enlarged granulomas, as

Eur. J. Immunol. 1999. 29: 4002–4010 LT g R in BCG infection 4005

Figure 4. Effect of LT g R-Ig on IFN- + and IL-4 release following BCG infection of BALB/c mice. (A) IFN- + was evaluated by ELISAin the serum of BALB/c mice infected with BCG (after 4 weeks) and injected either with human Ig (n = 7), LT g R-Ig (n = 5) orsTNFR1-Ig (n = 5). Data are shown as means ± SEM and are representative of two experiments. (B) IL-4 was determined byELISA in the serum of control mice and those treated either with LT g R-Ig or sTNFR1-Ig (n = 5/group). Results are shown as meanpg protein/ml of serum ± SEM and are representative of two experiments. Serum levels of IFN- + and IL-4 were undetectable inuninfected mice.

Table 2. LT g R-Ig treatment of sTNFR1-transgenic mice

Treatmenta) Mouse no. sTRNFR1-IgG( ? g/ml)b)

Spleennecrosisc)

Ig 409 6.5 +456 222.3 +457 207.1 +458 14.7 +459 10.7 +

LT g R-Ig 405 0.7 +++439 312.4 ++++440 277.2 ++441 289.0 ++

442 233.2 +++

a) Transgenic BALB/c mice expressing sTNFR1-IgG3 weretreated with LT g R-Ig or with human Ig (100 ? g/mouse/week). Mice were infected i.v. with BCG and killed at 4weeks of infection.

b) Before infection, serum levels of the transgene wereevaluated by ELISA as previously described [38].

c) Spleen necrosis was estimated by microscopicobservation of the number of cells and the extent ofnecrosis on histological sections stained withhematoxylin/eosin. +: granulomas with a few necroticcells; ++: granulomas with more than 20–30 necroticcells; +++: granulomas presenting more than 100necrotic cells as in Fig. 5 B; ++++: extensive areas ofnecrosis encompassing more than 20 % of spleensections.

depicted in Fig. 5 (Table 2). Ziehl-Neelsen stainingshowed numerous AFB in the spleens, with a very highaccumulation in the areas of necrosis (Fig. 5). In histologi-cal sections of livers, the untreated transgenic mice hadwell-delimited small granulomas which contained numer-ous AFB and occasionally areas of necrosis as previouslyreported [8]. In the LT g R-Ig-treated transgenic mice,granulomas were not well demarcated and were largersize, and also showed areas of necrosis (data not shown).

3 Discussion

The present study is an investigation of the involvementof LT § / g (and possibly of LIGHT) in the development ofprotective granulomas during BCG infection, using ansLT g R-Ig fusion molecule. The main impact of LT, amembrane molecule expressed on activated T lympho-cytes, but also on activated B cells and NK cells, is onvarious cells of the monocytic lineage and on follicularstromal cells in lymphoid organs, i.e. on all sites wheredirect cellular interactions between T cells and cellsbearing LT g R take place. Treatment of adult BALB/cmice with LT g R-Ig causes disruption of spleen architec-ture and follicular dendritic cell networks [27, 29]. Herewe demonstrate that this treatment results in an impair-ment of BCG granuloma formation in the spleen, as indi-cated by a reduced number of granulomas and theirincomplete differentiation. Under normal conditions, themacrophages forming granulomas become differenti-

4006 R. Lucas et al. Eur. J. Immunol. 1999. 29: 4002–4010

Figure 5. Histological sections stained with hematoxylin/eosin (A, B) or with Ziehl-Neelsen stain (C, D) of spleen ofTNFR1-transgenic mice (BALB/c) after 4 weeks of BCGinfection. In (B) and (D), transgenic mice treated with LT g R-Ig. These data are representative of two independent experi-ments (five mice analyzed per group).

ated into epithelioid cells, capable of limiting the expan-sion of intracellular bacilli. This differentiation is accom-panied by an accumulation of acid phosphatase andiNOS, leading to the production of NO and reactive nitro-gen intermediates, which represents an important bac-tericidal pathway [36]. LT g R-Ig treatment markedlyreduced both the total acid phosphatase activity (by40 %) and the NOS activity (by 60 %) of spleen extracts.The induction of macrophage differentiation and themycobactericidal potency of granulomas has so far beenattributed to the synergistic effect of soluble cytokines,namely IFN- + (and IL-12) and TNF. Our data show thatdirect contact between cells bearing the LT g R and thoseexpressing its membrane ligand LT § 1/ g 2 is involved incell-mediated immune response against BCG in thespleen. We cannot exclude a role of the new ligandLIGHT in this process.

LT g R-Ig injections had but a very limited effect on granu-loma formation in the lung and liver. When we used a

comparable pattern of injection with an sTNFR1-Igfusion protein, we only observed a small effect on livergranulomas whereas, in contrast, a very markeddecrease in granuloma formation in liver, accompaniedby fatal mycobacterial outgrowth, was observed in trans-genic mice overexpressing the construct [8]. Thus, thecontribution of membrane LT § / g in BCG granulomas isprobably underevaluated by the protocol used in the pre-sent study. We cannot exclude that different tissue distri-bution of the injected material occurred, leading to differ-ential tissue susceptibilities. Even taking into account thelimited efficiency of few injections of receptor fusion pro-tein, it remains that the contribution of LT § / g in granu-loma formation is more marked in the spleen. This can beunderstood from the fact that, in the spleen, all the cellsrequired for granuloma formation are already present insitu at the time of infection. Moreover, their organizationand interplay is very rapidly altered by the injection of thefusion protein. In contrast, cells necessary for the forma-tion of granulomas in the liver and the lung have to berecruited from the blood. This process, being slower, willnot be interfered with by sLT g R-Ig as efficiently and rap-idly as in the spleen.

When LT g R-Ig treatment was applied to transgenic miceexpressing sTNFR1, the contribution of membrane LT toefficient mycobactericidal granuloma formation couldagain be observed in the spleen, since extensive necro-sis with overgrowth of bacilli occurred, which was notobserved in non-treated transgenic mice. This leads tothe conclusion that, while sTNF is of major importancefor the constitution of granulomas occurring in non-lymphoid organs [8], membrane LT § / g may be the cyto-kine of the TNF family which plays a major role in lym-phoid organs. Interestingly, infection of TNFR1 gene-deficient mice with a low virulent mycobacterial strainM. avium, resulted in lethal necrotizing liver granulomaswithout both exacerbated mycobacterial growth andspleen necrosis [37].

LT g R-Ig treatment caused systemic changes such as areduction of IFN- + levels and an increase of IL-4 levels inthe blood. Similar changes were found following treat-ment with sTNFR1-Ig, which has been shown to exacer-bate murine tuberculosis in several studies [6–8]. Onestriking effect of LT g R-Ig treatment was the dramaticaccumulation of eosinophils in the spleen of BCG-infected mice. The presence of numerous eosinophils inthe spleen together with the increased blood levels ofIL-4 suggested that LT g R-Ig treatment deviated theimmune response from Th1 to Th2, leading to a distinctpattern of effector functions.

In summary, we have shown that the LT g R pathway isinvolved in host defense mechanisms against BCGinfection. Its role is related to granuloma formation, mac-

Eur. J. Immunol. 1999. 29: 4002–4010 LT g R in BCG infection 4007

rophage differentiation and the maintenance of theappropriate Th1/Th2 cytokine balance. Our data suggestthat the TNFR1 and LT g R pathways act synergistically toobtain differentiation of BCG bactericidal granulomas inthe spleen.

4 Materials and methods

4.1 Mice

BALB/c mice were obtained from our breeding facility at theUniversity Medical Center, housed under conventional bar-rier protection and handled in accordance with veterinarianauthorization. Transgenic BALB/c mice expressing sTNFR1-IgG3 proteins were obtained as previously described [38]and handled under the same conditions.

4.2 Reagents and treatment protocols

Fusion proteins comprised of the extracellular domain ofeither murine LT g R, and murine TNF-R55 fused to hinge,CH2 and CH3 domains of human IgG1 were prepared asdescribed [39]. Purified human IgG used as control was pur-chased from Sandoz (Basel, Switzerland). Mice received100 ? g human IgG1, LT g R-IgG1 or TNF-R55-IgG1 per injec-tion. Mice were treated 1 week prior to BCG infection andthen received one injection per week for 4 weeks.

4.3 BCG infections

BALB/c and sTNFR55 transgenic mice on BALB/c geneticbackground (8 to 10 weeks old) were infected i.v. with 105

CFU BCG strain 1173 P2 (Pasteur Institute) and killed4 weeks post infection.

4.4 Determination of bacterial numbers

One third of the organs from infected mice were homoge-nized in PBS; the homogenates were diluted, plated on agarMiddlebrook 7H10 medium and incubated at 37 °C for 21days. Four mice were used for CFU determination, andduplicates of three dilutions were done per organ.

4.5 Acid phosphatase activity

Acid phosphatase activity was determined on frozen tissuesections as previously reported [5, 8]. The method used ontissue sections was modified and adapted for detection ofacid phosphatase activity on organ extracts, as describedhere: spleens were homogenized in 25 mM Tris-HCl(pH 7.4), 10 mM EDTA and 10 mM EGTA (250 mg tissue/ml).Crude supernatant was obtained by centrifuging thehomogenate at 10 000 × g for 15 min. Subsequently, super-

natants were collected and stored at − 80 °C until use. Acidphosphatase activity was determined using naphthol AS-BIphosphate as substrate. In brief, spleen extracts (10 ? l) wasmixed with 490 ? l reaction buffer (Michaelis buffer contain-ing 0.04 % pararosaniline and 0.04 % sodium nitrate at pH 5and 0.03 % naphthol AS-BI phosphate). Enzyme activitywas determined at different time points (570 nm).

4.6 Histological analyses

Livers, lungs and spleens were fixed in 4 % buffered formal-dehyde, embedded in paraffin and stained with hematoxy-lin/eosin or with light eosin (for eosinophil detection). Ziehl-Neelsen acid-fast staining was done on tissue sectionscounterstained with hematoxylin. AFB were counted induplicated sections from four to five infected mice pergroup.

4.7 Determination of NOS activity of spleen extracts

Evaluation of the induction of NOS activity was done using acrude enzyme preparation from frozen spleen samples.Spleens were homogenized in 25 mM Tris-HCl (pH 7.4),1 mM EDTA and 1 mM EGTA (250 mg tissue/ml of buffer).Crude supernatant was obtained by centrifugation of thehomogenate at 10 000 × g for 5 min. NOS activity was mea-sured by the ability of the supernatant to convert radioactiveL-[14C] arginine (Amersham Life Science) to L-[14C] citrullineby using a NOSdetect assay kit (Stratagene). Supernatant(5 ? l) was incubated in the presence of 1 ? Ci L-[14C] arginine,1 mM NADPH, 3 ? M tetrahydrobiopterine, 1 ? M flavin ade-nine dinucleotide, 1 ? M flavin mononucleotide and 25 mMTris-HCl, pH 7.4, at room temperature for 30 min. The reac-tion was stopped by adding the stop buffer (50 mM HepespH 5.5 and 5 mM EDTA) and applied to a Dowex 50W col-umn following the recommended protocol (Stratagene).Samples were assayed in the absence and in the presenceof NG-nitro-L-arginine methyl ester (a selective NOS inhi-bitor).

4.8 Cytokine determination in blood and frozen tissues

Blood samples were obtained from retroorbital sinuses at 2and 4 weeks after infection. Proteins were extracted fromfrozen spleens (stored at − 80 °C) by homogenization inRPMI 1640 containing 1 % CHAPS [40]. IFN- + was evalu-ated by ELISA using the Innobasics10 mIFN + kit (Innogenet-ics, Ghent, Belgium) with a sensitivity of X 20 pg/ml. IL-4levels in sera were determined using an ELISA kit with a sen-sitivity of 2 pg/ml (Ready&System).

4.9 Statistical analysis

The differences between experimental groups were evalu-ated using Student’s t-test.

4008 R. Lucas et al. Eur. J. Immunol. 1999. 29: 4002–4010

Acknowledgements: The authors wish to thank Ms. Chen-Da Kan, Mr. C. Vesin and P. Chavarot for excellent technicalassistance, Ms. G. Levraz, J. Stalder, Mr. T. Le Minh andM. Redard for histological work, and the protein chemistrygroup at Biogen for the LT g R fusion protein. We thank Dr.S. Izui and Dr. J. Louis for reading the manuscript. This workwas supported by Grants 31-42275.94, 32-41729.94 and3200-054401.98 (to I.G.) from the Swiss National Fund, andfrom the Societe Academique de Geneve.

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Correspondence: Irene Garcia, Department of Pathology,CMU, 1, rue Michel-Servet, CH-1211 Geneva 4, SwitzerlandFax: +41-22 7025746e-mail: Garcia — cmu.unige.ch

F. Tacchini-Cottier’s present address: WHO-IRTC, Instituteof Biochemistry, Lausanne, Switzerland

4010 R. Lucas et al. Eur. J. Immunol. 1999. 29: 4002–4010