Immunology 1998 93 33–40

Differential regulation of expression of the MHC class II molecules RT1.B andRT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-c

A. ROOS,* E. J. M. SCHILDER-TOL,* M. A. CHAND,* N. CLAESSEN,* F. G. LAKKIS,† D. W. PASCUAL,‡J. J. WEENING* & J. ATEN* *Department of Pathology, Academic Medical Centre, Amsterdam, the Netherlands, †Department

of Medicine, Emory University School of Medicine, Atlanta, GA, and ‡Department of Veterinary Molecular Biology, Montana StateUniversity, Bozeman, MT, USA

SUMMARY

Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity ismultifactorial and involves genetic linkage to the major histocompatibility complex (MHC) classII haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependentsystemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediatedautoimmunity, develop immune suppression after mercuric chloride exposure. Exposure tomercuric chloride is known to enhance B-lymphocyte expression of the MHC class II moleculeRT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D wasunmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelialcells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BNrat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B(2·8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1·2-fold).B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression(2·5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells fromBN rats to interferon-c induced a moderate increase of B-cell MHC class II expression,predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observedafter stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modifyB-cell MHC class II expression; however, it induced typical morphological changes in peritonealmacrophages. These experiments demonstrate isotype-specific and strain-dependent regulation ofMHC class II expression on rat B lymphocytes, which may be of pathophysiological relevancefor the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity.

INTRODUCTION quantitatively modified, depending on the maturation, differ-entiation and activation stage of the cell. Furthermore, MHCAntigen presentation mediated by major histocompatibilityII expression can be induced on most cell types using appro-complex class II (MHC II) molecules is essential for develop-priate stimuli.1 In both types of regulation, cytokines play anment and function of the major part of CD4+ T lymphocytes.important role. Interferon-c (IFN-c) is a potent inducer ofFor efficient recognition of antigens as well as for self–MHC II expression on non-lymphoid tissue such as epitheliumnon-self discrimination, cellular expression of MHC II needsand endothelium, and on macrophages.1 Expression of MHCtight regulation, which occurs in a cell type-specific way. CellsII on B lymphocytes and macrophages can be enhanced bywhich are naturally involved in antigen presentation, such asexposure to interleukin-4 (IL-4).1−3 Furthermore, IL-13 hasdendritic cells, macrophages and B lymphocytes, do constitut-similar effects on MHC II expression in human B cells andively express MHC II on their membrane, which can bemacrophages as previously described for IL-4,4,5 but, in con-trast, mouse B cells were reported to be unresponsive toReceived 3 June 1997; revised 5 September 1997; accepted

17 September 1997. IL-13.5 Up to the present, no data have been available on theeffect of IL-13 on rat B cells.Abbreviations: BN, Brown Norway; FCM, flow cytometry; IFN,

The a- and b-chains of MHC II molecules are encoded byinterferon; IL, interleukin; LN, lymph node; mAb, monoclonala cluster of highly polymorphic genes, of which the transcrip-antibody; MFI, mean fluorescence intensity; PI, propidium iodide;

SD, standard deviation; Th, T helper. tion is regulated by a complex of protein factors binding toelements in the proximal promoters. The MHC trans-activatingCorrespondence: Dr A. Roos, Department of Pathology, Academicprotein CIITA plays an essential role in regulation of constitut-Medical Centre, PO Box 22 700, 1100 DE Amsterdam, the

Netherlands. ive as well as IFN-c- or IL-4-induced transcription of all

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A. Roos et al.34

MHC II genes, leading to co-ordinate regulation of expression antibodies (mAb) OX6 or OX17 [anti-RT1.B (a-chain) andanti-RT1.D (b-chain), respectively,17 mouse IgG1; hybridomasof MHC II molecules in most cases.6,7 However, independent

regulation of MHC II isotypes has been established in several obtained from the European Collection of Animal CellCultures (ECACC; Salisbury, UK)]. As a control mAb,systems.1,8

Expression of MHC II molecules has been considered as TS2/9.1.4.3 [TS2, mAb anti-human CD58, mouse IgG1;hybridoma obtained from the American Type Culturean important issue in relation to the occurrence of autoimmun-

ity.1,9 Susceptibility to the development of autoimmune disease Collection (ATCC), Rockville, MD] was used. Endogenousperoxidase activity was blocked by PBS containing 0·1% NaN3is in many cases genetically linked to certain MHC alleles. In

addition, autoimmune disorders can be accompanied by and 0·3% H2O2. Subsequently, antibody binding was detectedusing horseradish peroxidase-conjugated goat anti-mouse IgG1aberrant or enhanced expression of MHC II. Both aspects

have been studied in an animal model for drug-induced (Southern Biotechnology Associates, Birmingham, AL)containing 1% rat serum, and the substrate 3-amino-9-ethyl-immune dysregulation, induced by mercuric chloride (HgCl2).

Exposure of Brown Norway (BN ) rats to HgCl2 induces carbazole.systemic autoimmunity, which involves T- and B-lymphocyteactivation, (auto-)antibody production and multi-organ Preparation of cells

Single cell suspensions from LN were prepared as described,18inflammation,10,11 and which is dependent on activation of Thelper-2(Th2)-type lymphocytes.12 In contrast, Lewis rats using complete Dulbecco’s modified Eagle’s medium (DMEM)

complete medium [DMEM, supplemented with 10% heat-exposed to HgCl2 develop generalized immune suppression.13Susceptibility to induction of either autoimmunity or immune inactivated fetal calf serum (FCS), 100 IU/ml penicillin,

100 mg/ml streptomycin and 2 m glutamine]. For some experi-suppression by HgCl2 is linked to the MHC II haplotype, andthe response is modified by non-MHC genes.14 Exposure of ments, B cells were purified by negative selection using the

mAb OX8 (anti-CD8a), R73 (anti-ab T-cell receptor), W3/13BN rats to HgCl2 induces expression of the rat MHC IImolecule RT1.B (HLA-DQ/H-2 I-A homologue) on epithelial (anti-CD43), and W3/25 (anti-CD4), all mouse IgG1

(hybridomas obtained from the ECACC). Unlabelled cellscells in various organs.11 Furthermore, expression of RT1.Bis transiently enhanced on B lymphocytes from BN and Lewis were purified using magnetic beads coated with rat anti-mouse

IgG1 (Dynal, Oslo, Norway). Fluorescence-activated cellrats after HgCl2 injection.15 HgCl2-induced enhancement ofH-2 I-A on mouse B cells was blocked by anti-IL-4 treatment sorter (FACS) analysis revealed that this suspension contained

<2% CD2+ cells, <0·2% CD4+ cells, <0·4% CD8+ cells,in vivo.16 However, information on B-cell expression of RT1.D(HLA-DR/H-2 I-E homologue) after HgCl2 exposure in vivo and >98% cells double-positive for MHC II and k

immunoglobulin.has not been available until now.In the present study, we demonstrate that exposure of BN Peritoneal washout cells were plated at 1×106/well in

24-well plates using DMEM complete medium. After incu-rats to HgCl2 specifically enhanced B-lymphocyte expressionof RT1.B, whereas RT1.D expression was unmodified. In bation for 2 hr at 37°, non-adherent cells were washed away

and adherent cells were used for stimulation.contrast, expression of both RT1.B and RT1.D was enhancedon epithelial cells. These observations prompted us to examinethe cytokines IL-4, IL-13 and IFN-c for their effect on B-cell Cell culture

At the start of culture, cell suspensions were at least 90%expression of RT1.B and RT1.D.viable, as assessed by trypan blue exclusion. LN or B cellswere cultured at 1×106/ml in 24-well plates (1·2 ml/well ) for

MATERIALS AND METHODS18 hr, using DMEM complete medium. The following reagentsor cytokines were used in culture: phorbol 12-myristateAnimals

BN and Lewis rats (Charles River Wiga, Someren, the 13-acetate (PMA; 10 ng/ml; Sigma), ionomycin (100 ng/ml;Calbiochem, La Jolla, CA), supernatant from a ChineseNetherlands) were used when aged 11–14 weeks. For in vivo

experiments, rats received subcutaneous injections of HgCl2 hamster ovary cell line producing recombinant rat IL-4(Serotec, Oxford, UK), supernatant from an insect cell line(1 mg/kg body weight; Sigma, St. Louis, MO) dissolved in

demineralized water at 1 mg/ml, at days 0, 2, 5, 7 and 9. (Sf9) producing recombinant rat IL-13,19 and purified recom-binant rat IFN-c (0·1–100 U/ml, containing 0·17 ng/U;Control rats received vehicle only. At day 4 or day 14, rats

were killed by aorta puncture under ether anaesthesia, and Biosource, Camarillo, CA). The specific activity of rat IL-13was measured by induction of proliferation of the humantheir submandibular and cervical superficial lymph nodes (LN )

were removed and processed for flow cytometry (FCM). erythroleukaemia cell line TF1, as described;19 1 U/ml inducedhalf-maximal proliferation. Blocking antibodies used wereSubmandibular salivary glands were snap-frozen in liquid

nitrogen. For in vitro experiments, submandibular, cervical OX81 (mAb anti-rat IL4,20 mouse IgG1, 2·4 mg/ml; kindlyprovided by Dr D. Mason, Oxford, UK), and rabbit anti-ratsuperficial, axial, mesenteric and para-aortic LN were excised

from untreated or control-treated rats. Peritoneal washout- IL-13 (unpurified antiserum,19 1/100 diluted). TS2 (2·4 mg/ml )was used as a control mAb. OX81 and TS2 were purified fromcells were obtained after injection of 10 ml cold phosphate-

buffered saline (PBS)/heparin (15 IU/ml ) into the peritoneal hybridoma culture supernatants by protein A chromatography.cavity.

Flow cytometryFCM stainings were essentially performed as describedImmunohistochemistry

Acetone-fixed frozen sections (4 mm) were pre-incubated with previously.18 Briefly, the cells were incubated with saturatingamounts of OX6 and OX17, followed by goat anti-mouse10% goat serum, followed by incubation with the monoclonal

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Differential regulation of MHC class II isotype expression 35

IgG1, conjugated to R-phycoerythrin (SBA). Fluoresceinisothiocyanate (FITC)-conjugated OX12 (mAb anti-rat kimmunoglobulin light chain, mouse IgG2a; Serotec) wasfinally added.

When FCM was performed on cultured cells, dead cellswere detected using propidium iodide (PI, 1 mg/ml; MolecularProbes, Leiden, the Netherlands); cells which were directlystained ex vivo were fixed with 1% paraformaldehyde in PBSbefore measurement. Data acquisition was performed usingFACScan or FACSCalibur flow cytometers (both from BectonDickinson, San Jose, CA). For data analysis, viable lympho-cytes were gated based on scatter parameters and negativestaining for PI. Mean fluorescence intensity (MFI) values forRT1.B and RT1.D stainings were calculated for gated OX12+lymphocytes. Specific staining was defined by comparison to

Figure 1. Immunostaining of RT1.B (a, c) and RT1.D (b, d) onnegative controls, using antibodies with non-binding specificit- submandibular salivary glands from control (a, b) and HgCl2-injectedies, as described.18 (day 14; c, d) BN rats. Incubation with a non-binding mAb resulted

in negative staining (not shown). Magnification ×53.Data analysisFor in vitro experiments, data were obtained by duplicate

previously published,15 B-cell RT1.B expression in control BNstainings on cells pooled from at least two wells. The effect of

rats was lower than in control Lewis rats (MFI ratio BN/Lewiscell stimulation on RT1.B and RT1.D expression is expressed

0·71±0·06; n=4; P<0·005); however, RT1.D expression inas the MFI ratio of stimulated and control cultures. The mean

both strains was similar (0·91±0·08; n=4; P>0·1).ratios from several experiments are presented ± standarddeviation (SD) and tested for statistical significance in a one-sample t-test. Alternatively, when indicated, one representative Differential effect of IL-4 on expression of RT1.B and RT1.Dexperiment is presented. Data from in vivo experiments were on B lymphocytes from BN and Lewis rats in vitropooled from at least four experiments, presented as the MFI

HgCl2-exposed BN rats, but not Lewis rats, show stronglyratio±SD of rats injected with HgCl2 and H2O, and tested in

enhanced production of IL-4.21 Furthermore, IL-4 is aa one-sample t-test. Differences between BN and Lewis rats

mediator for the in vivo effects of HgCl2 on MHC II expressionwere tested in a paired t-test, using paired data from parallel

in the mouse.16 As previously described,20 exposure of aexperiments. Differences are considered statistically significant

culture of rat LN cells to rat IL-4 resulted in a dose-dependentwhen P-values are below 0·05.

increase of B-cell expression of RT1.B, which was completelyprevented by the IL-4-blocking mAb OX81,20 but not by a

RESULTS control mAb (TS2) (Fig. 3a, b). In contrast to the strongeffect of IL-4 on RT1.B expression, expression of RT1.D wasExpression of MHC class II antigens in BN and Lewis ratsonly slightly, but significantly, modified by IL-4 exposure,exposed to HgCl

2 although both MHC II molecules were expressed at a similarImmunohistochemical staining for detection of RT1.B and level on B lymphocytes in control cultures (Fig. 3a, Table 1).RT1.D expression was performed on submandibular salivaryglands from HgCl2-exposed and control BN and Lewis rats(day 14 after the first injection). As described previously,11exposure to HgCl2 induced a strong increase of RT1.Bexpression on epithelial cells, as well as increased numbers ofRT1.B+ infiltrating leucocytes, in BN rats (Fig. 1a, c). Similarresults were obtained for expression of RT1.D (Fig. 1b, d).RT1.B and RT1.D expression in salivary glands of Lewis ratswas similar to that in control BN rats, irrespective of theirexposure to HgCl2 (not shown).

B-lymphocyte expression of RT1.B and RT1.D wasexamined by FCM in LN-cell suspensions from BN and Lewisrats during the induction phase of HgCl2-induced immunedysregulation (day 4). As depicted in Fig. 2, HgCl2 exposureinduced a clearly enhanced expression of RT1.B on B lympho-cytes in the BN rat, thus confirming earlier observations.15 Incontrast, B-cell expression of RT1.D was not modified. Similarresults were obtained at day 6 [MFI ratios (HgCl2/H2O) forRT1.B and RT1.D were 1·87±0·12 and 1·01±0·06, respect-ively (n=2)]. In HgCl2-exposed Lewis rats (day 4), no alter- Figure 2. Relative expression of RT1.B and RT1.D on B lymphocytesations in B-cell expression of RT1.B (Fig. 2 and unpublished from LN from BN rats (n=6) and Lewis rats (n=4) exposed to

HgCl2 (day 4), as compared to control-treated rats. *P<0·002.data; n=10) or RT1.D (Fig. 2, n=4) were detected. As

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A. Roos et al.36

Figure 3. Dose–response of the effect of IL-4 on expression of RT1.B and RT1.D on B lymphocytes from BN rats. (a) LN cellswere exposed to different doses of IL-4 as indicated, and expression of RT1.B and RT1.D was analysed by FCM on PI− Blymphocytes after 18 hr. (b) The effect of IL-4 (5 U/ml ) on B-lymphocyte expression of RT1.B in the presence of OX81 (anti-IL-4) or TS2 (control-IgG1). Results shown are mean±SD from duplicate stainings from a representative experiment; similarresults were obtained in at least four independent experiments. (c) Representative histograms of FCM analysis of expression ofRT1.B and RT1.D. Figures are gated for PI− lymphocytes staining positive for k immunoglobulin. Peaks A represent isotypecontrols for MHC II detection, using non-binding mouse IgG1, on unstimulated B cells ( left panel ) or IL-4-exposed B cells (rightpanel ). Expression of RT1.B (left) and RT1.D (right) on unstimulated B cells (peaks B) and on IL-4-exposed B cells (peaks C)is shown. IL-4 was used at 5 U/ml.

Exposure of purified B lymphocytes to IL-4 yielded similar MHC II expression with a reduced width and a higher meanfluorescence intensity.data (Table 1). The effect of IL-4 on RT1.D expression was

completely inhibitable by OX81 (not shown). As demonstrated The effects of IL-4 on B-cell expression of RT1.B andRT1.D were compared between LN cells derived from BNin Fig. 3(c), IL-4 mainly affected the cells having a relatively

low constitutive expression of MHC II, resulting in a peak of and Lewis rats. The maximal enhancement of B-cell RT1.Bexpression in response to IL-4 (Fig. 4), as well as the dose–response curves for IL-4-mediated RT1.B induction (notTable 1. Effects of cytokines on B-cell MHC II expression (BN rats)shown) were similar between both strains. However, we couldnot observe any effect of IL-4 on B-cell expression of RT1.DMFI ratio MFI ratioin cultures derived from Lewis rat LN (Fig. 4). ParallelStimulus Culture RT1.B† RT1.D† n‡experiments revealed a significant strain difference in induc-

IL-4 (5 U/ml ) LN cells 2·82±0·73* 1·20±0·15* 13 ibility of B-cell expression of RT1.D by IL-4 (n=4, P<0·02).B cells 2·58±0·15 (NT) 1·29±0·08 (NT) 2

IFN-c (10 U/ml ) LN cells 1·53±0·12* 1·21±0·11* 6Exposure to IFN-c enhances MHC class II expression on B cellsB cells 1·37±0·07 (NT) 1·14±0·09 (NT) 2

IL-13 (6 U/ml ) LN cells 0·99±0·05 (NS) 0·96±0·09 (NS) 6HgCl2-injected BN rats show enhanced mRNA expression ofB cells 0·93±0·04 (NT) 0·97±0·03 (NT) 1IFN-c, especially in a late phase of their disease.21 Additionof purified recombinant IFN-c to BN rat LN cells resulted in†Relative expression as compared to control cultures (mean±SD).increased B-cell expression of MHC II molecules, predomi-‡Number of experiments.nantly of RT1.B (Table 1). Similar results were obtained in*P<0·01; NT=statistical significance not tested; NS=not

significant. cultures of purified B lymphocytes (Table 1).

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Differential regulation of MHC class II isotype expression 37

been described many times; however, most studies do notcompare the different loci. Increased B-cell expression of bothI-A and I-E, or both RT1.B and RT1.D, in response to IL-4has been reported;2,3,23,24 the presented data in some studiessuggest a quantitative difference in inducibility of these iso-types.2,23 We now show that the effect of IL-4 on B-cell RT1.Dexpression is dependent on the genetic background.

It is believed that both constitutive and inducible expressionof the MHC II genes is regulated at the level of transcriptionalcontrol of expression of the trans-activator CIITA.6 However,promoter regions of genes encoding HLA D antigens containseveral locus-specific transcriptionally active elements.25,26Accordingly, certain cell lines show expression of HLA DQbut not DR or DP,27 or vice versa.28 Furthermore, it has beenshown that a trans-acting factor of mouse origin, whichrestores expression of HLA DR, DQ and DP in a humanMHC II-negative mutant B-cell line, is essential for expressionof I-E but not I-A in somatic cell hybrids, which indicates a

Figure 4. Relative expression of RT1.B and RT1.D on B lymphocytes species difference in isotype-specific regulation.29 As anfrom BN (n=13) and Lewis rats (n=6) after in vitro exposure to IL-4 additional level of variation, promoter regions of DQ30 and(5 U/ml ), as compared to control cultures. *P<0·002. DR genes31 were shown to be polymorphic, thus introducing

considerable allelic differences in promoter strength. Besidesdifferential transcriptional control, post-transcriptional regu-

Exposure to IL-13 does not modify MHC class II expression onlatory mechanisms may also play a role in non-co-ordinate

rat B lymphocytesexpression of MHC II molecules.32 Similar mechanisms mayvery well play a role in the observed differential and strain-IL-13 enhances MHC II expression on human but not on

mouse B cells.5 Addition of recombinant rat IL-13 to BN dependent regulation of RT1.B and RT1.D expression on ratB lymphocytes.LN-cell cultures did not modify expression of RT1.B or RT1.D

on B lymphocytes in these cultures at any concentration tested, B lymphocytes from BN rats showed a moderate increasein RT1.B expression when exposed to IFN-c. IFN-c wasin the range of 0·5–150 U/ml (Table 1). In order to test the

biological activity of the IL-13-containing supernatant on rat previously shown to be ineffective in inducing MHC IIexpression on primary mouse B lymphocytes,2,33 and toleucocytes, its effect was examined on peritoneal macrophages.

Figure 5 illustrates that IL-13 strongly stimulated cell spread- decrease IL-4-mediated effects in these cultures.33 In ourexperiments, IFN-c was always far less potent than IL-4 ining and formation of large cellular processes, as has been

shown previously for human and mouse macrophages.4,22 inducing B-cell RT1.B expression, and, similar to IL-4, hadonly a slight effect on RT1.D. However, exposure of the LNThese morphological changes were blocked by a rabbit anti-

serum raised against rat IL-13 (Fig. 5f ). Similar effects were cell culture to PMA and ionomycin resulted in strong andearly induction of RT1.D, thus suggesting a qualitative differ-observed upon addition of rat IL-4 (Fig. 5b), which could be

inhibited by OX81 (not shown). Moreover, both IL-4 and ence in regulation of expression of RT1.D, as compared toRT1.B. Furthermore, this experiment excludes the possibilityIL-13 were able to enhance RT1.B expression on rat peritoneal

macrophages (day 4; not shown). of methodological incapability in detection of RT1.Dexpression as an explanation for its observed minormodification by IL-4 and IFN-c.

B-lymphocyte expression of RT1.D is rapidly increased afterThe in vitro evidence for differential regulation of B-cell

addition of PMA plus ionomycinRT1.B and RT1.D expression is strongly supported by theeffect of HgCl2 on B-cell MHC II expression in BN rats inRegulation of B-cell expression of RT1.B and RT1.D was

compared upon stimulation of BN LN cells either by IL-4 or vivo. HgCl2-exposed BN rats show strong and early productionof IL-4,21 which is a likely candidate to mediate the observedby a combination of PMA and ionomycin, at different time-

points (Fig. 6). As described above, IL-4 enhanced predomi- effects on B-cell MHC II expression.34 However, other cyto-kines produced in response to HgCl2 may also play a role,nantly RT1.B expression. In contrast, addition of PMA and

ionomycin strongly enhanced RT1.D expression, and the such as IFN-c and IL-10;21 unfortunately, the latter cytokinewas not available for use in our study. Induction of B-cellkinetics of up-regulation were more rapid than up-regulation

of RT1.B expression by PMA and ionomycin, and than the MHC II expression by HgCl2 in vivo may in addition involvemodification of cell–cell interactions.18 Furthermore, theeffects of IL-4.B lymphocyte may be a direct target of HgCl2. We haveobserved that HgCl2 increases B-cell expression of both RT1.B

DISCUSSIONand RT1.D in BN and Lewis rat LN-cell cultures;35 similarresults were recently obtained in cultures of purified BNThe present study demonstrates that IL-4-mediated regulation

of cell surface expression of MHC class II molecules on rat B lymphocytes (A. Roos et al., unpublished data).IL-13 did not modify MHC II expression on rat B cells,B lymphocytes is isotype-specific. The enhancing effect of IL-4

on B-cell MHC II expression, both in vitro and in vivo, has although the cytokine was shown to be biologically effective.

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A. Roos et al.38

Figure 5. The effects of IL-4 and IL-13 on the morphology of rat macrophages. Peritoneal macrophages were cultured during 2days in the presence of culture medium only (a); IL-4, 5 U/ml (b); culture supernatant from normal Sf9 cells (c, e, g); supernatantfrom Sf9 cells producing recombinant rat IL-13, 6 U/ml (d, f, h). Specificity of the effect of IL-13 was controlled by addition of arabbit antiserum raised against IL-13 (e, f ) or normal rabbit serum (g, h). Magnification ×220. Similar effects of IL-4 and IL-13were observed in four independent experiments.

Possibly, rat B lymphocytes are unresponsive to IL-13, as hasbeen reported for mouse B lymphocytes;5 this has to beconfirmed by using other readout-systems: human B lympho-cytes do also show increased CD23 expression and IgEproduction upon IL-13 exposure.4,5 Until now, biochemicalevidence for the presence or absence of IL-13 receptors onmouse or rat B lymphocytes has been lacking. In any case,the observed species difference in the B-cell response to IL-13will strongly limit the applicability of these animals for extra-polation of the function of IL-13 to the human situation.

Salivary gland epithelium of HgCl2-exposed BN ratsexpressed high levels of RT1.B as well as RT1.D, and no evid-ence for isotype-specific regulation was obtained. Induction ofI-A on renal epithelium from HgCl2-exposed mice can beinhibited by anti-IFN-c treatment in vivo.36 It was previouslyreported that Lewis rats, which are susceptible to Th1-mediatedautoimmune diseases such as experimental autoimmune ence-phalomyelitis, have a strongly enhanced tissue-specific induc-ibility of RT1.B expression by IFN-c, in comparison toresistant strains such as BN rats.37 We could not detect anyup-regulation of MHC II on B lymphocytes (day 4) or salivarygland epithelium (day 14) from HgCl2-exposed Lewis rats;however, HgCl2-injected Lewis rats show clear signs of T-cell

Figure 6. Kinetics of induction of B lymphocyte expression of RT1.B activation at day 4 (A. Roos et al., manuscript in preparation).and RT1.D by IL-4 (5 U/ml ) and by PMA plus ionomycin. LN cells

In contrast, Dubey et al. have shown an increase of B-cellfrom a BN rat were cultured in the presence or absence of theRT1.B expression in HgCl2-injected Lewis rats at day 3 andindicated stimuli. At various time-points, as indicated, the relative6,15 which may be due to unidentified genetic differencesexpression of RT1.B and RT1.D, as compared to the control culture,between sublines of inbred Lewis rats used. LN from HgCl2-was measured by FCM on PI− B lymphocytes. The results representexposed Lewis rats show enhanced mRNA levels of IL-10, butmean±SD from duplicate stainings from one out of two similar

experiments. not of IL-4 or IFN-c.21

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Differential regulation of MHC class II isotype expression 39

P.J. & W J.J. (1988) Mercuric chloride-induced auto-The relevance of enhanced expression of MHC II forimmunity in the Brown Norway rat. Cellular kinetics and majorantigen-dependent T-cell activation has been established for ahistocompatibility complex antigen expression. Am J Pathol 133,long time.38 Signal transduction after ligation of MHC II is127.known to also activate B cells.39 Furthermore, the density of

12. G M., D P. & G E. (1991) TH2 cells inthe MHC–peptide complex modifies the type of T-cell responsesystemic autoimmunity: insights from allogeneic diseases and

which develops, i.e. either Th1 or Th2.40 Differential regulationchemically induced autoimmunity. Immunol Today 12, 223.

of MHC II isotype expression during T–B-cell contacts may 13. P L., P R., R J. & D P. (1987) HgCl2play an important role in the susceptibility to Th1- or induces nonspecific immunosuppression in Lewis rats. EurTh2-mediated autoimmune diseases. Based on our findings, J Immunol 17, 49.IL-4 will differentially alter the antigen presentation for RT1.B- 14. A J., V A., D H E. et al. (1991) Susceptibility torestricted and RT1.D-restricted T cells. T-cell lines which are the induction of either autoimmunity or immunosuppression by

mercuric chloride is related to the major histocompatibility com-able to transfer metal ion-induced immune dysregulation wereplex class II haplotype. Eur J Immunol 21, 611.RT1.B restricted.41,42 In the mouse, expression of I-E was

15. D C., B B., H F. et al. (1991) Increased expressionfrequently associated with decreased immune responsiveness,of class II major histocompatibility complex molecules on B cellsin particular in models of autoimmunity;43,44 expression ofin rats susceptible or resistant to HgCl2-induced autoimmunity.I-E also conferred resistance to HgCl2-induced autoimmun-Clin Exp Immunol 86, 118.ity.45 The mechanism of this I-E-linked protection remains

16. V V E., U M., S C. & G E. (1993)unclear, but at least it indicates distinct functions for the

MHC control of IL-4-dependent enhancement of B cell Iadifferent MHC molecules. Differential regulation of expression expression and Ig class switching in mice treated with mercuricof these molecules, in a stimulus- and cell type-specific way, chloride. Int Arch Allergy Immunol 101, 392.may thus function as a mechanism for regulation of the 17. F T., MM W.R. & W A.F. (1982) Mouseimmune response. monoclonal antibodies against rat major histocompatibility anti-

gens. Two Ia antigens and expression of Ia and class I antigensin rat thymus. Eur J Immunol 12, 237.ACKNOWLEDGMENTS

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