Mouse T-lymphocyte activation by Urtica dioica agglutinin

0

Res. Immunol.

INSTITUT PASTEUR/ELSEVIER

Paris 1992

1992, 143, 701-709

Mouse T-lymphocyte II. -

activation

by Urtica dioica agglutinin

Original pattern of cell activation and cytokine production induced by UDA M.A. Le Moal, J.-H. Colle, A. Galelli and P. Truffa-Bachi

(*)

Unite d’lmmunophysiologie mol~culaire, Dkpartement d’lmmunologie, Institut Pasteur, 75724 Paris Cedex 15

SUMMARY Urrica dioica agglutinin (UDAI is a T-lymphocyte-specific polyclonal activator that differs from ConA, the classical mouse T-cell mitogen, by inducing a late and limited proliferation of a distinct T-cell subset recruited among both CD4+ and CD8+ lymphocytes. We investigated the possibility that the particular kinetics may originate from UDAspecific activation processes in which the known early mandatory signals were completed only after an extended delay. We report that the time of contact required between lectin and the cell membrane to acquire the capacity to proceed into cell cycle was much longer (36-40 h) for UDA than for ConA (8-10 h). Addition of phorbol ester, which artificially induces PKC translocation, or ionomycin, which provokes Ca2+ mobilization, did not accelerate the proliferative kinetics, suggesting that these early mandatory signals are not the limiting factors in the delayed proliferation. The induction of c-myc was retarded in the UDA group, and there was a good correlation between the kinetics of c-myc induction and the kinetics of cell proliferation. The comparison of the level of transcription of the genes encoding different cytokines revealed additional differences between the two mitogens : the whole wave of cytokine gene expression was delayed with UDA. In particular, IL2, IL3 and IFNy gene expression was retarded compared to the ConA-induced single wave. An even later transcriptional wave took place at around 72 h for IL4 and IL5. Finally, this particular kinetics corresponded to an unusually high level of IL3 and IFNy and a low level of IL4 and IL5 gene transcripts. Taken together, our results indicate that UDA, by providing a particular T-cell activation pattern, is a valuable tool for the analysis of the mechanisms involved in T-cell activation and proliferation.

Key-words: terleukins,

T lymphocyte, Original pattern.

UDA,

Agglutinin,

INTRODUCTION

Antigen and cytokine binding to cell surface receptors controls T-lymphocyte proliferation.

Submitted

June 15, 1992, accepted July 18, 1992.

(*) Corresponding

author.

Cytokine;

Activation,

Mica

dioica, In-

Early events include transmembrane signalling which ends in phosphorylation-mediated activation of many proteins and of proto-oncogene transcription (reviewed in Alexander and Can-

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trell, 1989). The consecutive transcriptional events include IL2 and ~55 IL2R gene expression (Crabtree, 1989), which are the determining events leading to T-cell proliferation. In addition, a set of inducible genes encoding different cytokines are transcribed in a coordinate manner. The use of polyclonal activators or antibodies directed against different cell membrane proteins has allowed the delineation of these mechanisms. We have recently described a novel plant mitogen, Urtica dioica agglutinin, that provokes mouse T-cell proliferation with a 36-48-h delay compared to ConA, the reference polyclonal T-cell activator (Le Moal and TruffaBachi, 1988) and we have provided evidence that UDA activates a discrete murine T-cell subset (accompanying article). Since UDA triggers a subpopulation of the ConA-responding cells, the particular pattern of cell proliferation induced by this lectin might be related to the utilization of a particular activation pathway. In the present study, we investigated some features of UDAinduced T-cell activation. Our results show that UDA requires a longer time of contact with the cell membrane than ConA to ensure optimal cell proliferation ; this lag corresponds to a delay in transcription and a peculiar pattern of cytokine gene activation. These results indicate that UDA is a promising tool for outlining different patterns of T-lymphocyte activation.

MATERIALS

Animals

AND

METHODS

and reagents

BALB/c female mice were obtained from the Institut Pasteur breeding facilities and used at 8-12 weeks of age. PMA and ionomycin were purchased from Sigma (St Louis, MO). a-methyl mannoside (Sigma) was kept frozen as a 1 M stock solution in “RPM1-1640”. The a-methyl-D-mannoside and the N-acetylglucosamine oligomers, N,N’-diacetylchitobiose, N,N’,N”-triacetylchitotriose and N,N’,N”, N”‘-tetraacetylchitotetraose (Sigma) were dissolved in RPM1 and stored at -20°C.

UDA PMA

= Urficu dioicu agglutinin. = phorbol my&ate

acetate.

ET AL.

Cell proliferation Cultures were made in RPMI-1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 2 mM L-glutamine, 50 pg/ml streptomycin, 50 U/ml penicillin, 5 070 heat-inactivated foetal calf serum, 5 x 10e5 M 2-mercaptoethanol, 1 mM sodium pyruvate and 10 mM Hepes in a humidified atmosphere of 5 Vo CO, in air. Proliferative responses were assessed by pulsing the cells (2 x 10’ cells/microwell) for 4 h with 0.25 $i of 3H-thymidine (2 Ci/mmol = 74GBq/mmol, Amersham, UK) as previously described (Le Moal and Truffa-Bachi, 1988). The DNA-incorporated radioactivity was measured in a scintillation counter (Kontron, Zurich, CH). RNA preparation Total cellular RNA was extracted from splenic cells (4x 106/ml, cultured in IO-cm diameter Petri dishes, 12 ml/dish) by homogenization in guanidiurn isothiocyanate (Chirgwin et al., 1979). The RNA was isolated by centrifugation with caesium chloride (Glisin et al., 1974) followed by ethanol precipitation. RNA was quantified by 260-nm absorbency. Hybridization

probes

The DNA probes used for hybridization were inserted in a “Bluescript” plasmid supplied by Stratagene (San Diego, CA). The following probes were used: the 1154-bp PstI fragment of mouse c-myc cDNA (Stanton et al., 1984), the 910-bp PstI fragment of mouse IFNy cDNA (Gray and Goeddel, 1983) (gift of G. Ward and A. Morris), the 625-bp Bg/II 3’ fragment of mouse IL2 cDNA (Kashima et al., 1985) (gift of T. Leanderson, Uppsala Biomedical Centre, Uppsala, Sweden), the 421-bp PstI fragment of mouse IL2R ~55 cDNA (Froussard et al., 1988), a 289-bp BglII-Hind111 fragment of mouse IL3 cDNA (Kindler et al., 1986), a 373-bp RsuI fragment of mouse IL4 cDNA (Noma et al., 1986) and a 445-bp PvuII-AccI fragment of mouse IL5 cDNA (Kinashi et al., 1986). Dot hybridization

analysis

Four pg of total RNA dissolved in a 30-~1 denaturation mixture composed of MOPS 20 mM pH 7, so-

PKC

= protein kinase C.

T-LYMPHOCYTE

ACTIVATION

dium acetate 5 mM, EDTA 1 mM, 1.5 % formaldehyde and 5 070deionized formamide, were incubated for 10 min at 65°C and chilled on ice for 2 min. After addition of 30 ~1of 10 x SSC, the samples were applied to Hybond N+ membranes (Amersham, Buckinghamshire, UK) using a 96-hole manifold apparatus (BioRad, Richmond, CA) under vacuum. RNA was fixed to filters by baking for 2 h at 80°C. Filters were prehybridized for 4 h at 42°C in a mixture containing 50 070formamide, 5 x SSC, 1 x Denhardt’s solution, 0.5 M sodium phosphate pH 6.5,0.1 070SDS, 100 (*g/ml salmon sperm DNA and 200 pg/ml of yeast RNA. Hybridization was carried out in the same solution at 42°C for 36 h with lo6 cpm/ml of a32PdCTP (dCTP > 800 Ci/mmol, Amersham, Btickinghamshire, UK) random priming labelled probe (Feinberg and Vogelstein, 1983). The filters were washed for 20 min once with 1 x SSC and once with 0.5 x SSC solution containing 0.1 % SDS at room temperature and, finally, with 0.2 x SSC at 55°C for 20 min. Filters were exposed to “Kodak XAR-5” film with an intensifying screen at - 70°C. RNase protection

analysis

IL3, IL4 and IL5 were detected by RNase protection. Complementary RNA probes were produced by transcription of linearized plasmid DNA with T7 or T3 polymerase as described by Pharmacia-LKB (Uppsala, Sweden). The transcription buffer contained unlabelled CTP at a final concentration of 10 PM. Fifty microcuries of u~~P-CTP (CTP > 3000 Ci/mmol) were used per 12.5 ~1of reaction. Half to one fourth of a microgram of plasmid DNA was used per labelling reaction and 50 to 70 % of the radioactive nucleotide input was usually incorporated. RNase protection was performed as described by Zinn et al. (1983) using an RNA probe (2 to 5 x 10 cpm) hybridized for 16 h at 45°C with each RNA sample (10 Kg). After RNase digestion, the reaction mixtures were denatured for 5 min at 100°C and fractionated on 6 % polyacrylamide urea sequencing gels at 42 V/cm (Maxam and Gilbert, 1980).

RESULTS

Requirement for extended lectin-membrane contact to enable UDA-induced T-cell proliferation ConA binding to the cell membrane for 6 to 12 h is mandatory to induce T-lymphocyte proliferation (Novogrodsky and Katchalski, 197 1). UDA-induced proliferation is characterized by a lag of 24-36 h with respect to ConA;

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we therefore tested whether an extended time of membrane contact was necessary to generate effective signalling. For this purpose, a mixture of N-acetylglucosamine oligomers, which compete with UDA fixation to lymphocyte membrane (Peumans et al., 1984), was added at different times to the cultures. Cell proliferation was measured at the peak of the UDA-induced proliferative response (72 h) by 3H-thymidine incorporation. For comparison, u-methyl Dmannoside (0.1 M) was added to ConAstimulated cells and proliferation was measured 48 h later. As shown in figure 1, addition of the N-acetylglucosamine oligomers (1.8 mM) at 24 or 36 h blocked T-cell proliferation by 70 %, whereas addition at 48 h no longer had any inhibitory effects. In contrast, a-methyl D-

120 -& 100

60 40 20 0

0

24

48

Sugar Addition

72 (h)

Fig. 1. Requirement for a long period of UDA-membrane contact for optimal T-cell proliferation. Spleen cells were cultured in 96-well (2x 105/well) plates in the presence of UDA or ConA. The neutralizing sugars, a mixture of N-acetylglucosamine oligomers (0.6 mM final each) for UDA or a-methyl-D-mannoside (100 mM final) for ConA, wereadded at the indicated time. Cell proliferation wasestimated at day 3 for UDA and day 2 for ConA. The results are expressedas percentage of the proliferative response (mean f SE, three cultures/group) with respect to control cell culture in the absence of the neutralizing sugar (thymidine incorporation in controls : UDA, 22 kcpm, ConA 45 kcpm).

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mannoside (100 mM), which blocks the ConAinduced proliferation by 50 % when added at 6 h, had no inhibitory effect when added at 12 h. The long signalling period required by UDA to induce cell competence suggests that the limiting step in the proliferation induced by this lectin is an early activation event.

ET AL.

therefore compared the transcription of c-myc following UDA or ConA spleen cell activation. RNA were prepared and analysed by dot blot hybridization at different times. As shown in figure 3, the two lectins induced different pro-

20 PMA and ionomycin do not accelerate the kinetics of UDA-induced T-cell proliferation An increase in phosphoinositide hydrolysis is observed after mitogen binding to the T-cell membrane. This leads to Ca*+ mobilization and to PKC translocation (Berridge and Irvine, 1984; Grove and Mastro, 1989; Rosoff et al., 1987). These second messengersare directly delivered by Ca ionophores and PMA (Mastro and Smith, 1983; Truneh et al., 1985), two chemicals which complement defective activation signals. We tested whether the addition of one or the other would complement an eventual defect in Ca mobilization or PKC translocation and thus accelerate the proliferative response to UDA. Spleen cells were cultured in the presence of the optimal mitogenic concentration of UDA (1 pg/ml), and PMA (8 nM) or ionomycin (1 PM) was added to the cultures at 0 h. As illustrated in figure 2, their addition did not accelerate the kinetics of UDA-induced T lymphocyte proliferation. The effectiveness of PMA and ionomycin was verified by their capacity to induce T-lymphocyte proliferation when added together in the absence of mitogen (fig. 2). Thus, the delayed UDA-induced proliferation cannot be ascribed to a defect in Ca*+ mobilization or PKC translocation.

Differences in c-myc expression between UDA and ConA stimulation The delivery by ConA of a growth stimulus to quiescent T lymphocytes is associated with the transient expression of c-myc (Kelly et al., 1983), a gene involved in the regulation of cell proliferation and differentiation (reviewed in Crabtree, 1989; Miiller et al., 1984; Studzinski, 1989). We

0

UDA

0

UDA + PMA

0

UDA + ION0

n

PMA

+ ION0

E 2

10

0

Time (d) Fig. 2. Failure of ionomycine or PMA to modify UDA-

induced T-cell proliferation. Spleencellswere cultured as describedin the legend, figure 1. lonomycin (1 FM) or PMA (8 nM) wasadded with UDA at the onsetof culture. Cellswereharvestedat the indicatedtime and thymidine incorporation wasmeasured. Results are expressedas mean&SE (three cultures/group).

Time (II)

0

1612243648

I

Fig. 3. Comparisonof c-myc transcription in UDA- or

ConA-activated spleencells. Spleencell cultures (4 x 106/ml) were stimulatedwith UDA (1 pg/ml) or ConA (2 Kg/ml). At the indicatedtime of culture, total RNA wasisolatedand 4 KgRNA/dot were blotted on Hybond+ membranes.Hybridization wascarried out with the c-myc-specific probe as specified in “Materials and Methods”. Data arerepresentativeof three independentexperiments.Autoradiography time : 6 h.

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files of c-myc accumulation. C-myc-encoded transcripts were barely detectable in UDAactivated cells at the time of their maximal expression in the ConA group (6-12 h). However, c-myc was efficiently transcribed with broad kinetics at around 36-48 h. The low c-myc RNA level can be ascribed to the lower number of cells activated by UDA. The differences in the kinetics of c-myc expression are in agreement with the long UDA-membrane contact required for this lectin to induce T-lymphocyte proliferation. Particular pattern of cytokine gene transcription induced by UDA The product of c-myc participates in the regulation of many genes involved in cell proliferation (Curran and Franza, 1988). We questioned whether its late expression in UDA-activated cells would affect the expression of different cytokine or cytokine receptors involved in lymphocyte

P55 L

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proliferation and/or differentiation. Spleen cells were cultured with UDA or ConA and the RNA prepared for analysis at selected times. The transcription of the IL2 gene and of the gene encoding the ~55 chain of IL2R was analysed by dot-blot hybridization. As shown in figure 4, the response to UDA was characterized by a broad peak of IL2 and ~55 transcripts culminating 24-48 h after cell activation, a finding which contrasts with the early expression (6-12 h) observed with ConA. As already noticed with c-myc, the level of mRNA accumulation for both genes is low in the UDA group, a finding consistent with the lower number of T cells activated by UDA. IFN-y transcripts, quantified by dot blot hybridization, also showed a 36-h lag (fig. 4). However, and in contrast with the lower level of expression of IL2 and ~55, a considerable enhancement of IFNy transcripts was found in UDA-activated cells (fig. 4).

Con A UDA

Fig. 4. Comparison of IL2, ILZR p55 and IFNy gene transcription in UDA- or Con&activated cells.

spleen

Spleen cell culture and dot hybridization were performed as described in the legend, figure 3. Specific mRNA were detected with the corresponding 32P-labelled probes. Autoradiography time: 4 h, 11 h, and 11 h for IL2, IL2R ~55 and IFNy, respectively. Data are representative of 3 independent experiments.

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The activation of IL3, IL4 and IL5 genes was analysed by RNase protection using IL3, IL4 or IL5 probes protecting 289 bp, 373 bp and 445 bp of IL3, IL4 or IL5 mRNA, respectively. The autoradiography presented in figure 5 shows that the peak of IL3 gene transcription was shifted from 12 h (ConA group) to 48 h (UDA group). Remarkably, as with IFNy, specific transcripts were more abundant in the UDA than in the ConA group. Figure 5 also shows that the peak of IL4 and IL5 gene transcription was shifted from 12 h (Con4 group) to 60 h (UDA group). In addition, the amount of mRNA accumulation for both cytokines was extremely low in the UDA group.

ET AL. DISCUSSION

The studies detailed in the present report have addressed some characteristics of UDA-induced T-lymphocyte activation. With respect to ConA, the reference T-lymphocyte mitogen in the mouse, UDA requires a prolonged mandatory membrane-occupancy period, induces a late transcription of c-myc proto-oncogene and displays a particular pattern of expression of various cytokine-encoding genes. The cell membrane and cytoplasmic events associated with ConA binding, Ca2+ mobilization and PKC translocation are completed in minutes, yet the lectin has to interact with the

21/36

Time (h) Con A

ND NI: 1

IL-3

-

UDA

ND -

Con A -

IL-4 .__

UDA _.

-w-

IL-5

Fig.

5. Comparison of IL3, IL4 and IL5 gene transcription in UDA- or ConA-activated spleen cells.

The spleen cell culture was performed as described in the legend, figure 3. RNase protection was performed as described in “Materials and Methods” (10 pg RNA/lane). Autoradiography time: 5 days. ND = not done. Data are representative of 2 independent experiments. The arrows indicate the size of the protected radioactive IL3, IL4 and IL5 probes.

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ACTIVATION

cell membrane for hours before DNA replication begins (Novogrodsky and Katchalski, 1971). This minimal time of contact with the cell membrane is defined as the commitment period (Milner, 1977). One of the distinctive features of UDA-induced T-cell proliferation is that the commitment time is in the order of 36-40 h. It has been suggested that prolonged membrane occupancy is required to stabilize a labile second messenger (Crabtree, 1989; Gardner, 1989). The Ca*+ concentration, which increases and then fluctuates with time and the cell activation state (Goldsmith and Weiss, 1988; Kimball et al., 1988), is thought to be the labile messenger (Crabtree, 1989). Ca ionophores such as ionomytin augment the Ca intracellular concentration ; in association with PMA, a PKC activator, ionomycin provokes T-cell proliferation (Mastro and Smith, 1983 ; Truneh, 1985). These two chemicals have been extensively used as costimulators, and are complementary for ineffective or defective signals given by weak T-cell activators. The addition of each of these costimulators to UDA-activated cells did not change the kinetics of proliferation, suggesting that Ca*+ mobilization and/or PKC translocation are not the limiting messengers in the delayed proliferation provoked by UDA. Mitogen binding to the T-cell membrane induces the rapid and transient accumulation of mRNA for the proto-oncogene encoding c-Myc, one of the essential regulatory proteins controlling cell proliferation (Kelly et al., 1983). Compared to ConA, T-cell activation by UDA is characterized by a 24-36 h delayed c-myc transcription. The lag in c-myc induction is somehow linked to the late proliferation induced by UDA. The reason(s) why c-myc is not induced in the few hours following UDA binding to the cell membrane, as it is with ConA, are currently being analysed and should allow for the delineation of the mechanisms underlying the UDA-induced uncommon T-lymphocyte activation. One of the remarkable features of T-lymphocyte activation is the ordered expression of the inducible genes, indicating that their transcription is controlled by common transactivating factors (Crabtree, 1989; Ullman et al., 1990). As

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the UDA-activated T-cell subset is included in the cells triggered by ConA, a similar pattern of induction was expected, although with delayed kinetics; a lower level of transcription of these genes was also predicted due to the lower number of UDA-activated T lymphocytes. While this prediction is fulfilled for IL2 and the ~55 chain of IL2R genes, which exhibit the expected delay and lower expression, the other cytokine genes analysed displayed a peculiar pattern of transcription. In particular, IL3 and IFNy mRNA accumulation is much higher in the UDA group. With these high transcription levels coincides an increase in the biological activity of the corresponding cytokines in the supernatants (data not shown). In contrast, very low amounts of IL4 and IL5 transcripts were found in UDAactivated cells. In addition to the particular pattern of gene induction, the kinetics of expression are also different. UDA provides two sequential waves of induction, the first occurring at around 36 h for IL2, IL3 and IFNy, and the second at around 60-72 h for IL4 and IL5. These data clearly demonstrate that UDA activation does not conform to the classical pathway of T-cell activation and that the regulatory elements controlling cytokine gene expression utilized by classical mitogens do not work, or else operate differently, during UDA activation. UDA thus appears to be an appropriate tool for outlining different patterns of T lymphocyte activation and regulatory element expression. Although every T cell binds UDA to an equal extent (accompanying article), this lectin functionally delineates responding and nonresponding T-cell subpopulations. The existence of several T-cell subsets is well documented. Mosmann and collaborators (for review, see Mosmann and Coffman, 1989) have described two classesof CD4+ cells, the Th-1 subset that produces IL2, IFNy and most of IL3, and the Th-2 that secretes IL4 and IL5 (Gajewski et al., 1989). Another subdivision relies on the naive versus primed status of T cells associated with modulation of CD45R and Pgp-1 membrane markers (Budd et al., 1987; Pilarski and Deans, 1989; Sanders et al., 1988). Superantigens also delineate T-cell subsets by their capacity to activate all T cells sharing a TCR belonging to a

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unique or restricted family of TCR genes. The functional definition of UDA-sensitive and UDA-insensitive T-cell subsets and their relevance to these categories is currently under way.

Acknowledgements This investigationwassupportedin part by the Institut Pasteur-CNRS(URA 040 359)and the Associationpour la Recherchesur le Cancer (ARC 6244). The authorsgratefully acknowledgeDr. W. Peumans for the generousgift of UDA. We thank Ms. L. Perrayon for the pilot experimentson activation with PMA and ionomycin. We are indebted to Drs. G. Langsley and A. Dautry for helpful discussion,andto Drs. P. Cracker, J. MacDougalI and I. Motta for critically reading the manuscript.We thank Mr. R. Perret for technical help.

Activation

II. -

des lymphocytes T par I’agglutinine de la grande ortie:

Particularit&

de I’activation

et de la production de cytokines L’agglutinine de la grande ortie (UDA) est un acti-

vateur polyclonal des lymphocytes T murins. L’UDA se diffkrencie de la concanavaline A (ConA), le mitogtne classique de lymphocytes T, par sa propriM d’activer une sous-population des lymphocytes T CD4+ et CD8+. Nous avons analysC si le retard dans la cinktique de prolifkration induite par I’UDA &ait da ti la mise en place retardte des mCcanismes

d’activation. Le temps de contact entre UDA et membrane lymphocytaire nkcessaire pour obtenir la prolifkration optimale est de 36-40 heures, tandis que 8-10 heuresde contact sont suffisantes pour la ConA. L’addition d’ester de phorbol ou d’ionomycine ne modifie pas le profil de prolifkration. Cela suggkre que ni la translocation de la prottine kinase C, induite par le phorbol ester, ni I’augmentation de Ca intracellulaire, provoquke par l’ionomycine, ne sont les facteurs limitants dans l’activation produite par I’UDA. Par contre, l’induction de c-myc est retardCe. L’Ctude de l’expression des genes codants pour diffkrentes cytokines rCv&le d’autres diffkrences entre les deux lectines. La transcription des g&es codants pour l’IL2, l’IL3 et I’interfkon y est plus tardive; et ce dkalage est encore plus important pour l’IL4 et I’ILS. A ces cinktiques d’expression particulieres fait pendant un niveau d’expression ileve des g&es codant pour l’IL3 et I’interfkron y. L’ensemble de ces don&es indique que I’UDA provoque une activation trb diffkrente de la ConA et qu’elle est done un outil prometteur dans I’ttude des mkanismes d’activation des lymphocytes T.

ET AL.

Mot.+cl&: Lymphocyte T, UDA, Agglutinine, Cytokine; Activation, Interleukines, Modele original.

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