Immunology 1997 90 626-631

The in vivo antibody response against exogenous antigens is not influenced by themouse Bcg (Nrampl) gene

M. SIROVA, 0. HOVORKA, I. RIHA, B. RIHOVA, M. BAUDYS,* S.-W. KIM * & E. SKAMENEt Division ofImmunologyand Gnotobiology, Institute of Microbiology, Academy ofSciences of the Czech Republic, Prague, Czech Republic, *CCCD,

University of Utah, Salt Lake City, UT, USA, and tCentrefor the Study ofHost Resistance, McGill University, Montreal, Canada

SUMMARY

The mouse Nrampl (Bcg) gene on chromosome 1 exerts pleiotropic effects on macrophagefunction. The gene is known to affect presentation of mycobacteria, and other antigens in vitro,so that macrophages carrying the resistant Bcg allele better support the proliferation of antigen-specific T cells compared with macrophages of the sensitive phenotype. To determine whether theBcg allele could affect in vivo the antibody response to antigens not related to mycobacterialinfections, we tested the primary and secondary responses to sheep red blood cells (SRBC) andglycosylated bovine insulin (G-insulin) in two pairs of Bcg congenic strains: BALB/c (Bcg?) versus

BALB/c.CD2 (Bcg?), and BlO.A (Bcg?) versus BlOAr (Bcg?), and in C57BL/lOScSn (B10; Bcg?)and A/J (Bcg?) mice. Furthermore, the antigen-specific proliferative responses of T cells primed invivo by protein antigens were also tested in Bcg congenic mice. We found no significant differencein in vivo antibody response either to SRBC or G-insulin between the Bcg' and Bcg? strains. Themagnitude of in vitro antigen-specific proliferation of lymph node cells sensitized in vivo by henegg lysozyme (HEL) or chicken ovalbumin (OVA) was also similar in Bcg? and Bcgr congenicmice. However, we have documented a higher antigen-presenting capacity of Bcg? macrophagesin in vitro antigen-specific proliferation to OVA. Since the macrophages are the only cells in whichthe Nrampl gene is expressed, we suggest that the activity of other types of antigen-presentingcells masks the effect of the Bcg? allele on antigen-presentation in vivo.

INTRODUCTION

Different individuals, and also inbred mouse strains, differ intheir ability to resist infection and challenge with antigen.Their response to such stimuli can be different both in termsof non-specific and antigen (Ag)-specific immune reactions. Inthe mouse strain C57BL/lOScSn (B10; H-2b), low levels ofIgG antibodies were found in response to a variety of antigens,including red blood cells, proteins, synthetic antigens andhaptens (reviewed in ref. 1). In comparison with the BlO mice(low responders), the inbred strain A/J (H-2a) represents ahighly responding strain in terms of IgG antibody formation.The low IgG production is dominant and it is not due to alimitation in B-cell potential per se,2 but differences/defects inantigen processing and presentation are clearly involved.Previously we have proven that antigen uptake (sheep redblood cells; SRBC)3'4 and handling, including presentation,are different in macrophages of the low responding strain B10in comparison with the high responding strain A/J. However,

Received 15 July 1996; revised 6 November 1996; accepted11 November 1996.

Correspondence: Professor B. Iihovd, Division of Immunologyand Gnotobiology, Institute of Microbiology, Academy of Sciencesof the Czech Republic, Prague, Czech Republic.

the mechanism of regulation of the IgG response is still notcompletely understood. On a genetic level, the response isregulated both by genes of the major histocompatibility com-plex (MHC; H-2 genes) and background genes.5 A dominantdown-regulatory effect of the H-2Ab allele on the response tothe allo-4-hydroxyphenylpyruvate dioxygenase (F liver pro-tein) and the allo-Thy-l was reported by Brunner et al.6 andMitchison & Simon,7 and to the mycobacterial epitope TBpl4(T68) by Ivanyi & Sharp.8

The Bcg (Lsh, Ity) locus,9 for which a candidate gene hasbeen isolated and characterized,'0 determines the natural resist-ance to infection by several unrelated intracellular pathogens.The gene was designated Nramp for 'natural resistance-associated macrophage protein', and localized to mouse chro-mosome 1° and to the syntenic region on human chromosome2." In mice, the Nramp gene is present in two allelic forms,the dominant resistance allele Bcg' and the recessivesusceptibility allele Bcgj.

The product of the Nramp gene is expressed exclusively inthe macrophage lineage, 10,12,13 depending on the state of celldifferentiation. It has been shown that macrophages fromresistant animals (Bcg?) are superior to macrophages fromsensitive animals (Bcg?) in supporting antigen-specific and non-specific T-cell proliferation.'4-'7 This led us to a question

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whether the Bcg allele could exert any effect on the in vivoantibody response to exogenous antigens. Speculatively, thesuperior ability of Bcg' macrophages to be activated byantigens and mitogens'5" 6 could be the underlying mechanism.To test such a possibility, we studied the in vivo mouse antibodyresponse to SRBC, glycosylated insulin (G-insulin), hen egglysozyme (HEL) and chicken ovalbumin (OVA), using twopairs of Bcg congenic strains: BALB/c (Bcg3) versusBALB/c.CD2 (Bcg?), and BlO.A (Bcg') versus BlO.Ar (Bcg?).B10 and A/J strains were used for comparison of antibodyresponses. The A/J (Bcg?, H-2a) mice are high IgG respondersto a number of T-dependent antigens including SRBC," 5 butare low responders to insulin and its G-derivatives.'8 Incontrast, B10 (Bcg', H-2b) mice are low IgG responders toSRBC and T-dependent antigens, but are high insulinresponders."15" 8 BALB/c mice (Bcg", H-2d) are high IgGproducers, but their IgG antibody levels are lower whencompared with A/J mice (Bcg?, H-2a). In contrast, the BlO.A(Bcg', H-2a) strain produces low levels of IgG in comparisonwith A/J and BALB/c strains, but higher levels than the B10strain (Bcg").' Thus, the question was whether the comparisonof in vivo antibody production in these mouse strains couldreveal a potential effect of the Nrampl (Bcg) gene.

Since we did not find any significant difference in in vivoantibody formation depending on the Bcg phenotype, the invitro response of Ag-sensitized T cells was also studied in Bcgcongenic strains. The in vitro results are in keeping with thefinding of Denis et al. 15.16 that the Bcg? macrophages are

superior to the Bcgs macrophages in supporting the prolifer-ation of Ag-sensitized T cells. In vivo, several cell populationshave been shown to act as antigen-presenting cells (APC). Inaddition to macrophages/monocytes, these include dendriticcells, B lymphocytes and enterocytes. Dendritic cells are con-sidered to be the principal cells responsible for presentingantigen during the primary response, both in vitro and invivo.19'20 Thus, we conclude that the superiority of the Bcg?macrophages over the Bcgs ones to present antigens to T cellsis not apparent in vivo when other types of APC are active.

MATERIALS AND METHODS

MiceThe congenic strains BALB/c (Begs, H-2d), BALB/c.CD2(Bcgr, H-2d)2l and B10.A (Bcg', H-2a), B10.Ar (Bcg?, H-2a)lSl6were used. Breeding pairs of BlO.Ar, BALB/c andBALB/c.CD2 strains were kindly donated to our laboratoryby the Centre for the Study of Host Resistance, McGillUniversity (Montreal, Canada), and mice were bred in our

animal facility. B10.A mice were obtained from the Instituteof Molecular Genetics, Academy of Sciences of the CzechRepublic (Prague, Czech Republic), and A/J (Bcg?, H-23) andC57BL/lOScSn (B10; Bcg', H-2b) mice from the Institute ofPhysiology, Academy of Sciences of the Czech Republic. Allmice were kept under standard conditions and given food andwater ad libitum. For the experiments, 8-12-week-old micewere used, both sex and age matched. Expression of therespective Bcg allele was checked in adult animals randomlyselected from either Bcg congenic strain throughout the experi-ments. DNA samples were analysed by polymerase reaction(PCR) using two microsatellite markers (DlMcg2 andDlMit7), which are located on either side of BegNramPl. 10,22

AntigensSRBC and a glycosylated derivative of bovine insulin(G-insulin; digalactosyl Gly Al, Lys B29 bovine insulin)'8were employed for antibody responses in vivo. HEL (Serva,Heidelberg, Germany) and chicken OVA (Sigma, St Louis,MO) were used both for the antigen-specific proliferation ofT cells in vitro and antibody responses in vivo.

In vivo antibody response to SRBCSRBC were used as an antigen for the determination of thenumber of plaque-forming cells (PFC) in vivo. The mice wereimmunized either with a single intraperitoneal (i.p.) dose of1 x 109 SRBC in phosphate-buffered saline (PBS) for a primaryresponse, or, for a secondary response, with two doses of1 x 109 SRBC injected i.p. with an interval of 2 weeks. Spleensof the immunized mice were removed and the plaque-formingassay conducted 5 days after the primary SRBC dose and 4days after the secondary dose, respectively.

The number of PFC was estimated in individual mice (fivemice per one group) by a direct23 or indirect plaque tech-nique.24'25 A simple statistical analysis of the data was per-formed by conventional Student's t-test.

In vivo antibody response to glycosylated insulinThe first and second doses of G-insulin (50 pg per dose/animal) were given in incomplete Freund's adjuvant (IFA;Institute of Sera and Vaccines, Prague, Czech Republic) subcu-taneously (s.c.) with a 2-week interval. The third dose (10 Yug)was given i.p. in alum precipitate. Seven days after the lastimmunization, the mice were killed and the serum tested foranti-G-insulin antibody by enzyme-linked immunosorbentassay (ELISA), as described previously.'8 Anti-G-insulin anti-body was detected by swine anti-mouse immunoglobulin anti-body labelled by horseradish peroxidase (SwAM/Px; Instituteof Sera and Vaccines).

In vivo antibody response to OVA andHELA/J, B10, BALB/c, BALB/c.CD2, B1O.A and B1O.Ar micewere immunized with OVA or HEL. OVA or HEL, 50 pg,were applied twice i.p. as alum precipitate. The time intervalbetween the first and the second dose was 14 days. Ten daysafter the last immunization the serum was collected and storedfrozen. Detection of anti-OVA or-anti HEL antibodies was

performed using ELISA.

Ag-specific proliferation oflymph node cellsFifty micrograms of the protein (HEL, OVA) in incompleteFreund' s adjuvant (IFA; Difco, Detroit, MI) was injecteds.c. in two sites at the base of the tail, and lymph node (LN)cells were used for in vitro restimulation 7 days after theimmunization. LN cells, 4 x 105, were incubated in 250 p1 ofcomplete RPMI-1640 medium (Institute of Sera and Vaccines)supplemented with 2 mM L-glutamine, non-essential aminoacids, 40 pg/ml gentamicin, 5 x 10-5 M 2-mercaptoethanol, andcontaining 10% of a selected batch of fetal calf serum (FCS;Biocom, Brno, Czech Republic), in 96-well flat-bottomedplates (Linbro, London, UK). Cells were stimulated by differ-ent concentrations of the antigen used for the sensitization aswell as by an irrelevant antigen to prove the specificity of thereaction. Cells cultivated without any stimuli were consideredas a negative control. After 68 hr of incubation, 0-5 pCi of

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[3H]thymidine was added to the cultures for an additional6 hr and DNA-incorporated activity was measured by liquidscintilation.

Preparation ofT cells and macrophagesA T-cell-enriched population was prepared from the LNof mice immunized as specified above, by non-adherenceto nylon.26 Flow cytometry analysis (FACSort; Becton-Dickinson, Mountain View, CA) of the resulting populationrevealed 85% of cells within the lymphocytic gate, 88% ofwhich were CD3+ [flourescein isothiocyanate (FITC)-labelledanti-CD3 antibody; Pharmingen, San Diego, CA].Macrophages from the spleens of non-immunized mice wereisolated by adherence to a plastic surface. 4-5 x 107 spleencells were incubated in tissue culture Petri dishes (100 x 90 mm;Nunc, Roskilde, Denmark) in complete RPMI containing 1%FCS for 2 hr at 370, in an atmosphere of 7% CO2. Non-adherent cells were removed by thorough washing by PBSprewarmed to 250. Adherent cells were obtained using a cellscraper, washed, and cell counts and viability estimated. 2 x 105Tcells were co-cultured with different numbers of splenicmacrophages in the presence of the respective Ag and/or anirrelevant Ag as a control of specificity. Proliferation of thecells and cytokine levels in supernatants were determinedas above.

RESULTS

In vivo antibody response to SRBC

The primary anti-SRBC antibody reaction following a singlei.p. dose of antigen (SRBC) was determined on day 5. TheIgM-PFC assay revealed no significant difference between theBceg and Bcgr mice in both Bcg congenic systems (BALB/cversus BALB/c.CD2, and BlO.A versus BlO.Ar) (Fig. 1). Twoadditional mouse strains, A/J (Bcg') and BlO (Bcg'), wereincluded in the experiment for comparison of the numberof PFC.

The secondary anti-SRBC response was evaluated on day4 after the second dose of SRBC. For BALB/c andBALB/c.CD2 mice, no significant differences were found forboth IgM and IgG PFC (Fig. 2a, b). A slightly higher levelof IgM-PFC was detected in the BlO.A (Bcgs) strain comparedwith its congenic partner BlO.Ar (Bcg') (Fig. 2a). AlthoughB1O.A (Bcgs) mice had lower numbers of IgG-producing cells

8o T l T F T

CD2 B1O.A B1O.A' B10

I

ANJ

Figure 1. Primary anti-SRBC response, detected by a direct PFC assay

5 days after the immunization of mice with 1 x 109 SRBC i.p. A/J andB10 strains were included in the experiment for comparison of thenumber of PFC. The BALB/c.CD2 strain is referred to as CD2. Themean PFC number of 10 mice+ SD is shown.

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Figure 2. Secondary anti-SRBC response in mice immunized by aprimary dose of 1 x 109 SRBC and a secondary dose of 1 x 109 SRBC2 weeks later. The antibody production was evaluated 4 days afterthe secondary immunization. IgM anti-SRBC Antibody was detectedby a direct PFC assay (a), and IgG anti-SRBc antibody by an indirectassay (b). The BALB/c.CD2 strain is referred to as CD2. The meanPFC number of 10 mice+ SD is shown.

than BIO.Ar (Bcg7) mice (Fig. 2b), these differences were notsignificant at the level of P = 0-05. There was a significantdifference in IgG-PFC numbers between the B10 (H-2b) miceand the H-2 congenic (H-2a) strains BlO.A and BlO.Ar(Fig. 2b).

Antibody response to glycosylated insulin

The anti-G-insulin antibody levels were expressed as log2 titresof mouse sera by ELISA testing (Fig. 3). The log2 titres ofanti-G-insulin antibody were not substantially differentbetween BALB/c (Bcg') and BALB/c.CD2 (Bcg') mice. In theBlO.A congenic system, the levels of anti-G-insulin antibodywere lower than those in the BALB/c and BALB/c.CD2strains. However, the anti-G-insulin antibody levels in B1O.A

BALB/c CD2 B1O.A B1O.A' B10 AJ

Figure 3. Log2 titres of anti-G-insulin antibody in mouse sera, detectedby ELISA. The mice were immunized by two doses of 50 pg G-insulinin IFA and a third dose of 10 pg in alum precipitate. The mice wereinjected at 2-week intervals and killed for serum 7 days after thelast injection.

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(Bcg') and the BlO.Ar (BcgF) mice were equivalent (Fig. 3).Compared with the BlO (H-2b) strain, the B1O.A and BlO.Ar(H-2') mice had significantly lower titres of anti-G-insulinantibody.

Antigen-specific proliferation ofLN cells

We determined the Ag-specific proliferation ofLN cells primedby a single dose of antigen (OVA or HEL) in vivo andrestimulated by the same antigen in vitro. The sensitized T cellsfrom Bcg' and Bcg' strains did not differ in ability to respondby Ag-specific proliferation, since we could not detect anysubstantial difference either in BALB/c versus BALB/c.CD2mice (Fig. 4a) or in B1O.A versus BIO.Ar mice (Fig. 4b). Theproliferation response of BALB/c.CD2 (Bcg) LN cells was

slightly higher than that of BALB/c (Bcg') cells in the wholerange of antigen concentrations tested except the highest one

(500 Mg/ml) (Fig. 4a). However, the proliferation of unstimu-lated LN cells was also higher in BALB/c.CD2 (Bcgr) mice.As a consequence, the response expressed as a stimulatoryindex (SI =c.p.m. of stimulated cells/c.p.m. of control) didnot differ between the BALB/c and BALB/c.CD2 strains (datanot shown). Comparison of BIO.A and BIO.Ar strains alsorevealed uniform levels of LN proliferation in these congenicpartners (Fig.4b). In BlO.A congenic strains, the specificresponse was only twofold higher than the background pro-liferation, resembling that of the BlO strain (data not shown).

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T-cell response to antigens presented by splenic macrophages

We performed experiments in which macrophages from thespleens of Bcg' and Bcg mice presented antigens to T cellsand/or macrophage-depleted LN cells. First, LN cells ofimmunized (BALB/c x BALB/c.CD2)Fl mice were depletedof adherent cells and co-cultivated with the adherent cellfraction of spleen cells from both parent strains. We foundthat splenic adherent cells from the BALB/c.CD2 (Bcg) parentstrain were superior in presenting antigen to non-adherent LNcells (Fig. 5). Although the BALB/c.CD2 adherent cellsinduced higher background proliferation of F1 cells (i.e. with-out antigen), the response in the presence of antigen washigher than that induced by BALB/c adherent cells (SI3-4 forBALB/c.CD2 adherent cells versus 2-5 for BALB/c). In asubsequent experiment, T-cell-enriched cells from the LN ofimmunized mice were cultivated with macrophages isolatedfrom the spleens of non-immunized mice in the presence ofseveral concentrations of the antigen. In both pairs of congenicstrains, we detected higher levels of Ag-specific T-cell prolifer-ation induced by macrophages expressing the Bcg' allele(Fig. 6). The results were further confirmed by the estimationof interleukin-2 (IL-2) and IL-3 levels in supernatants. In all

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(a)0 BALB/c + OVA

CD2 + OVAo--0*---- BALB/c+ HEL..*CD2+HEL

0 0-1 l10 10o 500

(b)B1O.A

* B1l0.A'----O-- B10.A + HSA

.-- ---- B10.A' + HSA

0 0.1 1 10 100

Figure 4. Ag-specific proliferative response of LN cells sensitized bya single injection of antigen 7 days prior to in vitro restimulation.Proliferation was detected on day 3 of cultivation. (a) Proliferation ofBALB/c (Bcg') and BALB/c.CD2 (Bcg') LN cells. The mice were

immunized by 50 pg of OVA in IFA s.c. HEL was used as a controlof specificity in vitro. (b) Proliferation of Bl0.A (Bcg') and BlO.Ar(Bcg') LN cells. In this experiment, the mice were immunized by 50 pgof HEL in IFA s.c., and OVA was used as a control of specificity forthe in vitro restimulation.

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0

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* OVA 10

M HEL 100

BALB/c BALB/c.CD2 0

Figure 5. Proliferation of a non-adherent fraction of LN cells of(BALB/c x BALB/c.CD2)Fl mice, co-cultivated with macrophagesisolated from spleens of non-immunized mice of the parent strainsBALB/c (Bcg') and BALB/c.CD2 (Bcgr). The Fl mice were immunizedwith 50 pg of OVA in IFA s.c., and cells from their LN were isolated7 days later by non-adherence to a plastic surface. In vitro, the cellswere stimulated by the same antigen (OVA; 100 or 10 pg per ml), andby an irrelevant antigen (HEL; 100 pg per ml) as a control ofspecificity. Proliferation was evaluated on day 3 of cultivation.

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-0--- B10.A 1 x 1050 B1O.A'1 x 105

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Figure 6. Ag-specific proliferation of isolated T cells from BlO.A mice,cultivated with splenic macrophages either of BlO.A (Bcg') or ofBlOAr (Bcg') origin. The T cells were isolated from LN 7 days afterthe mice were immunized by 50 pg of OVA in IFA s.c. 2 x l05 T cellswere cultivated with x l05 or 5 x 104 macrophages isolated fromspleens of non-immunized mice by adherence to plastic, in the presenceof the antigen. Proliferation was detected on day 3 of cultivation.

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cases, the cytokine levels correlated with the proliferativeresponses (data not shown).

DISCUSSION

In the present paper we studied a possible effect of the Bcggene on the antibody response against antigens unrelated tomycobacterial infection. The Nrampl protein, encoded by theBcg gene, has been shown to affect many biological functionsof macrophages.10'27"28 Numerous studies have shown that theability of the Bcgr macrophages is superior over the Bcgs inmany aspects of immune activation,10,56 including antigenpresentation.15'17 Macrophages process and present antigensas professional APC.29 However, in our experiments weobserved no significant difference in the in vivo antibodyresponse against SRBC, G-insulin, HEL and OVA betweenmouse strains carrying the resistant or the sensitive allele ofthe Bcg gene.

Except for the Bcg congenic strains, A/J (Bcg?) and B10(Bcg?) strains were used for comparison of antibody responses.A/J mice are high producers of IgG antibody in response toa variety of T-dependent antigens including SRBC, whereasB10 mice are low responders in terms of IgG antibodyformation.1'5 A suppressive effect of the H-2Ab allele has alsobeen found in immune responses to the allo-HPPD,6 allo-Thy-17 and a mycobacterial epitope TBpl4.8 Brunner et al.6suggest that in the presence of the H-2Ab allele the T-helpertype 1 (Thl ) response is enhanced and that of the Th2 cells issuppressed. However, due to the promoter polymorphism onlysome H-2Ab alleles favour Thl activation while other allelesexpressed in other types of APC activate the Th2 subpopul-ation. In our present study, we found similar numbers of IgMPFC in all mouse strains after the primary immunization bySRBC. In the secondary response to SRBC, the A/J mice(Bcgr) responded with high levels of IgG while B10 mice (Bcg?)produced very low levels of IgG antibody. However, the IgGresponse of the two pairs of Bcg congenic strains, BALB/c(Beg?), BALB/c.CD2 (Bcg?) and B1O.A (Bcg?), BlO.Ar (Bcg),was comparable (Fig. 2b). The same was true for HEL andOVA antigens. In contrast to most T-dependent antigens, theimmune response against insulin and its G-derivatives doesnot depend on its intracellular processing, but is more probablytriggered by a reduction of Cys residues of the insulin A-chainwithout proteolytic degradation.30 In response to insulin andG-insulins, the B10 strain produces high antibody levels whilethe A/J strain is a low responder both in MHC classI-restricted31 and MHC class II-restricted responses.18 Again,no difference linked to the Bcg resistant or sensitive alleleswas observed in Bcg congenic strains (Fig. 3).

As we did not observe any difference between Bcg? andBcgs strains in antibody formation in vivo, we sought to verifywhether a potential difference could be found at the levelof T-cell activation by an antigen. In vivo, naive T cells arepredominantly activated by dendritic cells in an antigen-specific manner while other APC present antigens mainly toprimed T cells.32 It is not clear whether macrophages caninitiate the responses of naive T cells. The macrophage is themain cell type able to phagocytose particulate antigens and somight be expected to present such antigens to naive T cells. Ithas been shown that a toxic treatment of macrophages via thephagocytosis of an encapsulated toxic drug enhanced rather

than suppressed a T-cell-dependent response to a solubleprotein antigen, but had an opposite effect with a particulateantigen, almost shutting off the response.33 This suggests thatmacrophages are obligatorily involved in the initiation ofprimary responses to complex antigens such as SRBC.However, it has also been shown that macrophages transferthe antigen to dendritic cells before the presentation occurs.33'34Therefore, the dendritic cells predominate as the cell typeresponsible for the antigen presentation to T cells in vivo.

Less information is available on APC for memory T cells.Ag-specific memory T cells are also stimulated by dendriticcells, although activated B cells can be equally as efficient. Incontrast to naive cells, both resting B cells and macrophagescan induce proliferation and cytokine secretion from memoryT cells, but 5-20 times less efficiently than dendritic cells.35

It has been proven that Nrampl is expressed exclusively inmacrophages.'0 Previously, Denis et al.' "6 showed that thereis no difference in antigen-presenting capacity of dendritic cellsbetwen Bcg' and Bcgs congenic mice. Thus, the predominantparticipation of cell populations other than macrophages(probably dendritic cells)19'20 on antigen presentation canexplain the discrepancy between our results in vivo and theresults of others, who observed superiority of Bcgr splenicmacrophages in supporting the growth of antigen- andmitogen-activated T cells in vitro. 15'16 In accordance with theobservation of Denis et al. 15"16 we also demonstrated a signifi-cant difference between Bcgr and Bcgs mice in those in vitroexperiments where the antigen-presenting population consistedmainly of splenic macrophages expressing the Nrampl. Weconclude that the in vivo immune response of Bcgr and Bcgshosts is comparable due to the antigen-presenting capacity ofcells other than macrophages, which are the only cells wherethe Nrampl gene operates.

ACKNOWLEDGMENTS

The authors thank the Centre for the Study of Host Resistance,McGill University, Montreal, Canada, represented by Professor E.Skamene, for the kind donation of breeding pairs of mice, and DanielleMalo (Centre for the Study of Host Resistance) for performing theBcg allele determination. We thank E. Buschman (Centre for theStudy of Host Resistance) for critical reading of the manuscript. Wealso thank Mrs H. Misurcova and Mrs H. Semoradovi for technicalassistance and D. Putnam for reading the manuscript. This work wassupported by grant no. 310/95/0634 of the Grant Agency of theCzech Republic.

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