Immunology 1993 80 45-450

Does interleukin-2 restore lymphocyte responses suppressed byTrypanosoma cruzi?

F. KIERSZENBAUM, H. MEJIA LOPEZ & M. B. SZTEIN* Department of Microbiology, Michigan State Universiti',East Lansing, Michigan and the *Center for Vaccine Development, Department of Pediatrics, University of Maryland, Baltimore,

Maryland, U.S.A.

Accepted for publication 12 July 1993

SUMMARY

There has been disagreement about the ability of exogenous interleukin-2 (IL-2) to restoreresponsiveness to lymphocytes from either Tr'panosoma cruzi-infected animals or normal indi-viduals co-cultured with this parasite. The discrepancy has been attributed to the use of differentstrains of mice or T. cruzi isolates, or to the use of lymphoid cells from different organs. As T. cruzi

inhibits the expression of IL-2 receptors by activated lymphocytes in vitro, we were able to testwhether restoration of responsiveness by exogenous IL-2 might depend on the level of suppressionpresent in the system. Human or mouse lymphocytes stimulated with phytohaemagglutinin (PHA)exhibited gradual decreases in IL-2 receptor expression, [3H]thymidine incorporation and IL-2secretion as the concentration of T. cruzi in the culture increased. Exogenous IL-2 afforded a degreeof restoration of both IL-2 receptor expression and ["H]thymidine uptake which was substantial atthe lower, but very small-if any-at the higher, parasite concentrations tested. Trypanosoma cruzicould not have competed with the lymphocytes for IL-2 because it did not bind significant amounts ofthis cytokine. These results suggested that the controversy about the corrective effects of IL-2 may bemore apparent than real, reflecting variations in the extent ofimmunosuppression present in differentmodel systems of T. cru-i-associated immunosuppression.

INTRODUCTION

Ever since the first reports of the immunosuppression thatdevelops during the acute phase of Trypanosoma cruzi infec-tion,' I investigators have tried to unravel the underlyingmechanisms. Several types of alterations occurring in lympho-cytes and macrophages from animals infected with T. cruzi havebeen described, and abnormal functions of these cells have beenpostulated to be the underlying causes.6 10 These anomalies havebeen attributed, at least in part, to an incapacity of activatedlymphocytes from T. cruzi-infected animals to produce suffi-cient amounts of interleukin-2 (IL-2).8t' 1' This notion issupported by evidence showing that exogenous IL-2 partially orcompletely restores functional capacities to lymphoid cells fromacutely infected mice both in vitro'S'o and in vito.'7 '9 Moreover,administration of IL-2 has been reported to restore immunolo-gical function in a mouse model system of chronic Chagas'disease2"2' and to reduce parasitaemia and increase survival inacutely infected mice.'179 However, it has also been reportedthat IL-2 does not restore mitogen responsiveness to lympho-cytes from T. cruzi-infected mice' 122.23 or activated humanlymphocytes co-cultured with T. cruzi.'6 This discrepancy has

Correspondence: Dr F. Kierszenbaum, Dept. of Microbiology,Michigan State University, East Lansing, Ml 48824, U.S.A.

been tentatively attributed to the use of different T. cruzi isolatesor mouse strains, and to the use of different immunologicalassays. It is also possible that reversal or prevention of T. cruzi-induced suppression by IL-2 may be contingent upon the extentof the immunosuppression afforded by the experimental condi-tions. We tested this hypothesis by using a culture system inwhich varying T. cruzi concentrations were used to increasegradually the extent of suppression of IL-2 receptor (IL-2R)expression and [3H]thymidine incorporation by mitogen-stimu-lated human and mouse lymphocytes.

MATERIALS AND METHODS

MiceFour-week-old stock CD I (ICR) Swiss (referred to as CD 1) micewere purchased from the Charles River Laboratory (Portage,MI). They were housed five animals to a cage and providedpelleted food and water ad libitum.

Mouse spleen cells (MSC)Normal mouse spleens were removed aseptically, under etheranaesthesia. Single-cell suspensions were prepared in RPMI-1640 medium containing penicillin (100 IU/ml), streptomycin(100 pg/ml) and 2% heat-inactivated fetal bovine serum (HyC-lone, Logan, UT) (RPMI +2%FBS) using a sterile Ten-Broeck

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F. Kierszenbaum, H. Mejia Lopez & M. B. Sztein

tissue grinder (two strokes only) refrigerated in an ice bath.Tissue debris were removed by filtration through a sterile nylongauze. The cells were then washed with the same medium bycentrifugation and resuspended in RPMI + 2%FBS. In all cases,cell viability, determined by trypan blue dye exclusion, was>98%,.

Human peripheral blood mononuclear cells (PBMC)Blood from healthy donors was drawn by venipuncture andcollected in heparin. The PBMC were purified by centrifugation(400 g, 20-`, 45 min) over Isolymph (Gallard-Schlesinger, CarlePlace, NY). The cells were washed twice with and resuspendedat the desired concentration in RPMI-1640 medium with thesame supplements as described above except for the use of 5%,FBS (RPMI+50%FBS). The cell viability of PBMC prepara-tions was consistently >99%,, as determined by trypan blueexclusion.

ParasitesTrypanosoma cruZi trypomastigotes (Tulahuen isolate) wereisolated from the blood of mice infected subcutaneously with1 x 106 organisms 10-12 days previously by centrifugation overlsolymph24 followed by chromatography through a diethylami-noethyl-cellulose column.25 The eluted flagellates were washedtwice by centrifugation with and resuspended at the desiredconcentration in RPMI+2%YFBS or RPMI+50%FBS depend-ing on whether they were to be used in experiments peformedwith mouse or human cells, respectively. The recovered organ-isms consisted of 100% trypomastigotes and were > 99%0, viable(i.e. motile).

Lymphoproliferation assaysCultures (100 pl) containing 1-25 x 105 PBMC or 2 x 105 mousespleen cells, lacking or containing I jug phytohaemagglutinin(PHA)/ml (Burroughs-Wellcome, Research Triangle Park, NC)were set up in quadruplicate in sterile 96-well flat-bottom plates.Where present, T. cruzi or recombinant IL-2 (rIL-2; specificactivity= 1-6 x I07 IU/mg; a generous gift from Dr P. Sorter,Hoffman LaRoche, Nutley, NJ) was added at time 0 (i.e.together with the mitogen), at the concentrations describedunder Results, without changing the final volume. The parasiteconcentrations and their range tested in each experimentdepended on the amount of purified trypomastigotes available.All cultures were incubated at 37' and 5% CO2 for the lengths oftime described under Results and were pulsed with I 1iCi oftritiated thymidine ([3H]thymidine; Dupont NEN Products,Boston, MA; specific activity 2 Ci/mmol) 24 hr prior totermination by automated harvesting. Incorporated radioacti-vity was measured in a liquid scintillation counter and the resultswere expressed as the mean + SD.

The percentage of suppression of either [3H]thymidineincorporation or IL-2R+ expression (see below) observedin the presence of T. cruzi was calculated by the equation:% suppression= [(value obtained in the absence of T. cruzi-value obtained in the presence of T. cruzi)/value obtained in theabsence of T. cruzi)] x 100.

Determination of IL-2R expressionTo determine IL-2R expression, cultures were set up asdescribed above except that the final culture volume was I ml.All cultures were harvested at 24 hr. The cells were washed in ice-cold Dulbecco's phosphate-buffered saline (PBS) pH 71,resuspended in the same buffer containing 1% bovine serumalbumin (BSA; Sigma, St Louis, MO) and 0-1 /o sodium azide.Human PBMC and MSC were stained with fluorescein-labelledanti-human p55IL-2R (FITC-anti-human CD25; Becton Dick-inson, San Jose, CA) and phycoerythrin-labelled rat anti-mousep55IL-2R (PE-anti-mouse CD25; Boerhinger-Mannheim,Indianapolis, IN) monoclonal antibodies (mAb), respectively.To establish background fluorescence we stained aliquots of thecell suspensions with FITC- or PE-labelled mAb of irrelevantspecificity and the same isotypes as the anti-IL-2R antibodies(Becton Dickinson and Caltag, South San Francisco, CA).Trjypanosoma cruzi did not stain with either the FITC-anti-human or PE-anti-mouse IL-2R antibody. An EPICS ELITEflow cytometer/cell sorter (Coulter Cytometry, Hialeah, FL)was used for all flow cytometric determinations; the data wereprocessed by using the Elite software package. Parasites,platelets, non-viable cells, debris and cells other than lympho-cytes were excluded from analysis by setting an appropriate gateon the forward versus 90 light scatter parameters. Twentythousand cells were individually examined in each sample; thestatistical dispersion of data resulting from the analysis of thisnumber ofcells falls within 0 05% of the results presented in thispaper.

IL-2 bioassayThe supernatants of cultures were assayed for IL-2 levels usingthe IL-2-dependent CTLL-2 cell line as previously described.26Because murine interleukin-4 (IL-4) stimulates proliferation ofCTLL-2 cells,'27r8 the supernatants from MSC cultures weretested for IL-2 in the presence of an anti-IL-4 mAb [1 lB 1;American Type Culture Collection (ATCC), Rockville, MD] toblock IL-4 activity.29 This antibody was added to the CTLL-2cell cultures at their onset at a concentration that completelyblocked proliferation of these cells induced by 600 U/ml ofrecombinant murine IL-4 (Genzyme, Boston, MA). Resultswere expressed as units IL-2/ml relative to a standard IL-2preparation [a supernatant of a concanavalin (Con A)-stimu-lated rat splenocyte culture], which was arbitrarily assigned avalue of 1000 U/ml.

RESULTS

Effects of increasing T. cruzi concentrations on lymphocyteactivation parameters

The extent of suppression of IL-2R expression by PHA-stimulated PBMC or MSC co-cultured with T. cruzi increasedwith T. cruzi concentration (Table 1). A similar correlation wasobserved when [3H]thymidine incorporation by activatedPBMC or MSC was monitored instead of IL-2R expression(Table 2). It should be noted that, although the actual levels ofsuppression of IL-2R expression or [3H]thymidine uptake

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IL-2 and T. cruzi-induced immunosuppression

Table 1. Correlation between Trspanosoma cruzi concentration and thelevel of suppression of IL-2R expression by PHA-stimulated PBMC or

MSC*

T. cruziconcentration

(million ' IL-2R+ "Cells parasites/ml) PHA cells suppression

PBMC 0 Absent 3-30 Present 68-12-5 Present 50 0 26-65 Present 41 2 39.510 Present 36-3 46-720 Present 30-1 55-8

MSC 0 Absent 5-40 Present 40-03 Present 26 1 34 86 Present 19 0 52 512 Present 9-5 76 3

* Cultures of PBMC or MSC lacking or containing PHA and theindicated concentrations of T. cruzi were incubated at 37 and 5¾% CO2for 24 hr. The cells were then harvested, stained with FITC-anti-humanIL-2R or PE-anti-mouse-IL-2R, as appropriate and analysed by flowcytometry. The percentage of suppression caused by the presence of T.cruli was calculated as described under Materials and Methods. Thesesets of results are representative of four and three repeat experimentsperformed with cells from different human blood donors and mice,respectively.

Table 2. Correlation between T. cruzi concentration and the level ofreduction of [3H]thymidine incorporation by PHA-stimulated PBMC or

MSC*

T. cruziconcentration

(million 0/Cells parasites/ml) PHA C.p.m. + SD suppression

PBMC 0 Absent 468+ 1480 Present 52,088 + 18075 Present 41,515+2172 20 310 Present 21,894+ 1614 58 020 Present 2700+ 353 94-8

MSC 0 Absent 948 + 360 Present 46,264+ 20273 Present 34,572+ 1954 25 36 Present 14,574+819 68-512 Present 1331 +28 97-1

* Cultures of PBMC or MSC lacking or containing PHA and theindicated concentrations of T. cruzi were incubated at 37T and 50/o CO2for 72 hr. The cultures were pulsed with [3H]thymidine during the last 24hr. All reductions caused by the presence of T. cruzi were statisticallysignificant (P < 0-05; Student's t-test). Each set of results is representa-tive of at least two repeat experiments performed with cells fromdifferent individuals.

Table 3. Correlation between T. cruzi concentration and IL-2 produc-tion by PHA-stimulated PBMC or MSC*

T. crudlconcentration

(millionCells parasites/ml) PHA IL-2 (U/ml) IL-2R' cells

PBMC 0 Absent 19 5 60 Present 368 70 510 Present 348 (5 4)t 42-1 (40 3)20 Present 96 (73 9) 16 9 (76-0)

MSC 0 Absent 9 5-40 Present 443 40-05 Present 479 (-8l1) 32 5 (18 8)10 Present 395 (10-8) 9-5 (76-3)20 Present 184 (58-5) 7 6 (81-0)

* Cultures of PBMC or MSC lacking or containing PHA and theindicated concentrations of T. cruzi were incubated at 37 and 5'S, CO2for 24 hr. The supernatants of the cultures were then assayed for IL-2activity. The sets of results obtained with PBMC and MSC arerepresentative of two and three repeat experiments, respectively,performed with cells from different individuals.

t The percentages of suppression relative to the correspondingcontrol values obtained with PHA in the absence of T. cruoi are given inparentheses.

caused by any given parasite concentration varied among blooddonors or individual mice, the suppressive effects and thecorrelation between parasite concentration and the degree ofsuppression was always demonstrable.

Whereas suppression of IL-2R expression graduallyincreased with T. cruIi concentration, reductions in IL-2 activitywere observed only in the supernatants of co-cultures contain-ing the highest parasite concentration tested (Table 3).

Effects of exogenous IL-2 on lymphocyte responsiveness by T.cruzi-suppressed lymphocytes

We examined whether exogenous IL-2 would reverse thesesuppressive effects of T. cruzi and, if it did, if the reversal wasinfluenced by the parasite concentration, i.e. by the level ofsuppression exerted on the responder cells. To these ends,experiments were performed in which a constant amount of rIL-2 was added to cultures of PHA-stimulated cells containingincreasing T. cruzi concentrations. As shown in Table 4, theaddition of rIL-2 enhanced the IL-2R expressing capacity ofactivated PBMC or MSC in the cultures containing the lower T.cruzi concentration(s) tested. A smaller degree of recovery or nosignificant recovery was observed as the parasite concentrationincreased. Similar observations were made in experiments inwhich [3H]thymidine incorporation was measured instead of IL-2R expression (Table 5). The doses of rIL-2 used in theseexperiments were selected on the basis of their ability tostimulate from half to maximal proliferative responses incultures of the IL-2-dependent CTLL-2 cell line (data notshown). Solutions containing these doses of rlL-2 were incu-bated with 2 x 107 T. cruzi/ml to find out if the organism wouldremove some or all of their activity but no significant reductionwas observed (data not shown).

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F. Kierszenbaum, H. Mejia Lopez & M. B. Sztein

Table 4. Effects of exogenous rlL-2 on the level of IL-2R expression by PHA-activated PBMC or MSC co-cultured withincreasing concentrations of T. cruci*

/, IL-2R1 cells

Cells+PHA+ T. cruzi

Cells rlL-2 Cells alone Cells+ PHA 5 million parasites/ml 10 million parasites/ml 20 million parasites/ml

PBMC Absent 5-6 70 5 57 1 (19 0)+ 42.1 (40 3)t 16 9 (76 0)Present 9-3 75 5 69 1 (8 5) 609 (19 3) 19 5 (74-2)

MSC Absent 7 1 28-9 ND 19 0 (34-3)§ 16-1 (44 3)Present 7-1 28 4 ND 27 6 (2 8) 15 1 (46 8)

* Cultures of PBMC or MSC lacking or containing PHA and the indicated concentrations of T. cruzi were incubated at37 and 5%, CO, for 24 hr in the absence and presence of rlL-2. The cells were harvested, stained with FITC-anti-human IL-2R or PE-anti-human IL-2R, as appropriate, and analysed by flow cytometry. The concentrations of rIL-2 used in theexperiments with PBMC and MSC were 2000 and 500 U/ml, respectively. Each set of results is representative of at least tworepeat experiments performed with cells from different individuals.

t The percentages of suppression relative to the corresponding control values obtained with PHA in the absence of T.cruci are given in parentheses.

+ ND, not determined.

Table 5. Effects of exogenous rIL-2 on the level of [3H]thymidine incorporated by PHA-activated PBMC or MSC co-culturedwith increasing concentrations of T. crui*

C.p.m.

Cells+ PHA +T. cruci

Cells rlL-2 Cells alone Cells+ PHA 5 million parasiteslml 10 million parasites 'ml 20 million parasites/ml

PBMC Absent 246+24 66,862+4212 42.682+2154 (36 2)t 30,659+ 1377 (54 1) 5700+ 1068 (91 5)Present 18,389 + 635 68,796 + 4656 61.670 + 931 (10 4) 41 914 + 1463 (39 1) 6169 + 562 (91 0)

MSC Absent 590+ 119 26,907+ 1971 19.409+ 1276 (27 9) 9956+365 (63 0) ND+Present 1446 + 179 24.083 + 1679 23.032 + 1070 (4 4) 15,771 + 626 (34-5) ND

* Cultures ofPBMC or MSC lacking or containing PHA and the indicated concentrations of T. cruoi were incubated at 37and 5 "%, CO, for 72 hr in the presence or absence of rlL-2 and pulsed with [3H]thymidine during the last 24 hr. The concentrationof rIL-2 used in these experiments was 2000 U/ml. Each set of results is representative of at least two repeat experimentsperformed with cells from different individuals.

t The percentages of suppression relative to the corresponding control values obtained with PHA in the absence of T. crociare given in parentheses.

+ ND, not determined.

Interestingly, the PBMC from one of the blood donors were

found to be much more sensitive to T. cruoci-induced suppressionthan the cells from the other donors. The cells from thisindividual were comparably suppressed in the presence T. cruciconcentrations ranging from 2 5 x 106 to I x 107 organisms/ml(Table 6). Although 1000 U rlL-2/ml restored responsiveness inall cases, the level of recovery decreased as the parasiteconcentration increased.

DISCUSSION

It is generally thought that the genetic background of the hostand the virulence and infective doses of different T. cruci isolatesare important factors in determining the degree of immunosup-pression that ensues. Other variables are the time post-infectionat which lymphoid cells are obtained for study and the

conditions used in the various assay systems. Because of thesevariables, it has been difficult to regulate the extent of immuno-suppression that develops in T. cruci-infected animals or reachunanimity about the ability of IL-2 to reverse this suppression.The co-culture system we used makes it possible to vary the levelof suppression by adjusting the parasite concentration. Ourresults showed that, in this system, restoration of T-lymphocyteresponsiveness by exogenous IL-2 was a function of the degreeof suppression, whether monitored by measuring levels of IL-2R expression or [3H]thymidine incorporation.

It is known that IL-2 binding to specific receptors on

activated lymphocytes up-regulates IL-2R expression."0 Thiseffect was readily demonstrable in cultures containing the lowand intermediate T. cruci concentrations tested but was notobserved at the highest parasite concentration, i.e. when IL-2Rexpression was maximally suppressed. Conceivably, in the latter

448

IL-2 and T. cruzi-induced immunosuppression 449

Table 6. Effects of rlL-2 on the level of [3H]thymidine incorporated by PHA-activated PBMC from a donor highlysensitive to T. cru i-induced suppression*

C.p.m.

Cells +PHA + T. cruzi

rIL-2 Cells alone Cells+ PHA 5 million parasites/ml 7 5 million parasites/ml 10 million parasites/ml

Absent 347 + 16 10,442+495 2332 + 114 (77-7)t 1920+ 336 (81-6) 2171 + 175 (79-2)

Present 495+91 10,475+ 142 8602+770 (17 9)t 5603±+ 110 (46-5) 4109+417 (60 8)

* Co-cultures ofPBMC lacking or containing PHA and the indicated concentrations of T. cruzi were incubatedat 37,248 and 50% CO2 for 72 hr in the presence or absence of rlL-2 and pulsed with [3H]thymidine during the last 24hr. The concentration of rIL-2 used in the experiments was 1000 U/ml.

t The percentages ofsuppression relative to the corresponding control values obtained with PHA in the absenceof T. cruzi are given in parentheses.

case, rIL-2 may not have encountered a sufficient number of IL-2R to bind and induce significant up-regulation. This notion isin line with our previous finding that, in cultures containingI x 107 T. cruzi/ml, between 70 and 90% of PHA-activatedPBMC remain arrested at the Go/Gia phase while progressionthrough the cell cycle continues unimpeded in the absence ofparasites.3'

Co-culture of PHA-stimulated PBMC with 5 x 106 T. (cruzitrypomastigotes/ml was previously reported not to inhibit IL-2secretion. 16 Here, we confirm this observation but show also thatat a much higher parasite concentration IL-2 secretion isimpaired.

It is known that the suppressive effects of T. cruzi do notresult from consumption of essential medium nutrients ormitogen absorption by the parasite,'6-32 and that the numbers ofPBMC and their viability in cultures containing or lacking T.cruzi, in the presence or absence of mitogen, are comparableover a 96-hr period.'6 Therefore, T. cruzi does not causesuppression by killing the responder or accessory cells. Theseconclusions are reinforced by the present results, since exoge-nous IL-2 could not have substituted for missing mediumcomponents or mitogen molecules, and restored responsivenesswould not have been possible had the responder or accessorycells been dead. The recent report that cytoplasmic levels of IL-2R in PHA-activated PBMC co-cultured with T. cruzi are lowerthan those found in cells from parasite-free cultures,33 arguesagainst the possibility that reduced IL-2R expression mighthave resulted from cleavage by a parasite-released enzyme(s), asextracellular enzymes could not possibly digest intracellularmolecules. Moreover, the expression of EA-l molecules by Tlymphocytes34 and that of either CD 19 or CD20 molecules by Blymphocytes35 is not reduced by T. cruzi, denoting the selectiveimpairment of the expression of certain surface molecules. Wealso know that, at suppressive concentrations, filtrates of T.cruzi suspensions do not affect the level of ['251]IL-2 binding toPHA-activated, normal PBMC,33 indicating that once IL-2Rare expressed on the surface their number is not reduced by T.cruzi products. We have also ruled out nutrient depletion by theparasite,36 or the infection of macrophages/monocytes as poss-ible causes of T. cruzi-induced immunosuppression in vitro.32.37

We also showed that IL-2 secretion decreased gradually asthe T. cruzi concentration increased. Yet, whereas exogenous

rIL-2 afforded a significant recovery of responsiveness incultures containing the lower T. cruz-i concentrations tested,very little, if any, restoration occurred at higher parasiteconcentrations, i.e. when cells expressed minimal levels of IL-2R. Tri'panosoma cruzi did not remove rIL-2 activity fromsolutions of this cytokine and, therefore, could not haveprevented restoration of lymphocyte responsiveness by seques-tering the cytokine. If a similar correlation existed in infectedhosts, it might help explain the apparently contradictory resultsreported from several laboratories, which would then beattributable to variable degrees of immunosuppression presentin different model systems.

ACKNOWLEDGMENTS

The authors wish to thank Mr M. K. Tanner and Miss B. Dyas for theirskilful technical assistance. This work was supported by grant Al-26542from the United States Public Health Service.

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