INTISRNATIONAI. JOURNAI. OP Lr.PROSY^ Volume 67, Number 3

Printed in the U.S.A.(ISSN 0148-916X)

Serum IL-1 ra is Elevated in LepromatousLeprosy Patients 1

Mary Fafutis-Morris, Cecilia M. Guillen-Vargas, Sergio Navarro-Fierro,Roberto Morales-Ortiz, and Juan Armendariz-Borunda 2

T lymphocytes are key elements in me-diating both ccllular and humoral immuneresponses through cytokine productionagainst infectious. Two functional subsetsof CD4+ lymphocytes have been character-ized based on the production of specific cy-tokines. Thus, in animal models, Th 1 cellsproduce interleukin 2 (IL-2) and gamma in-terferon (1FN-y) prefercntially driving thecellular immune response; Th2 cells drivethe humoral immune response by secretingIL-4, IL-5 and IL-10. Likewise, feedbackamong this network of cytokines has beenobserved. Thus, IFN-y inhibits Th2 cellfunction and IL-10 and IL-4 can block Th 1action. These characteristic patterns havebeen clearly demonstrated in murine experi-mental models associated with either cellularprotection or immunopathological proc-esses. In humans, a clear-cut pattern ofthese cellular subsets remains to be estab-lished mainly due to the fact that healthy in-dividuais present a highly combined patternof cytokines. Nonetheless, studies in recentyears have shown some evidence of polar-ized subsets of CD4+ T cells in patients withinfectious diseases and allergies (' 14 . 1 ').

Immunological and clinicai manifesta-tions in leprosy patients represent a goodmodel for use in discriminating among pat-terns of response mounted by different indi-

' Received for publication on 13 August 1998. Ac-cepted for publication in revised forro on 27 April1999.

M. Fafutis-Morris, Ph.D.; C. M. Guillen-Vargas,M.Cs.; S. Navarro-Fierro, B.A.; R. Morales-Ortiz,M.D., Centro de Investigacion en Inmunologia y Der-matologia, C.U.C.S., Universidad de Guadalajara/In-stituto Dermatologico de Jalisco, Av. FederalismoNorte 3102, Guadalajara, Jalisco, Mexico 44220. J.Armendariz-Borunda, Ph.D., Instituto de BiologiaMolecular en Medicina, G.U.C.S., Universidad deGuadalajara, Guadalajara, Jalisco, Mexico.

Reprint requests to Mary Fafutis-Morris, Ph.D. atthe above address or FAX: 52-3-672-2848; e-mail:ciinde@cencar.udg.mx

viduals against the same pathogen (My-cobacteriuni leprae). Tuberculoid leprosy(TL) patients develop a strong delayed hy-persensitivity response toward M. lepraeantigens, as well as a poor antibody-medi-ated response, resembling a Th1 type re-sponse ("'. "). On the other hand, leproma-tous leprosy (LL) patients have a defectivecellular immune system; they do not mounta response against M. leprae antigens, andthey display a poor delayed hypersensitivityresponse and have abundant in vivo anti-body production. Moreover, T lymphocytesfrom LL patients do not proliferate and pro-duce low IL-2 and IFN-y leveis againstnonspecific polyclonal stimuli (PHA,ConA, anti-CD3) evoking Th2 type cellfunctions (4_9. 10-12 )

Knowledge of the active role of pro-in-flammatory and antiinflammatory cytokinesin the development of leprosy is lacking.Therefore, in this communication we havetried to elucidate the patterns of specific cy-tokines in leprosy patients. We found thatelevated serum leveis of interleukin 1 re-ceptor antagonist (IL-1 ra) is a major featurein LL patients.

MATERIALS AND METHODSStudy subjects. Eighteen patients (nine

females, nine males; ages 20 to 65) fromthe Dermatologic Institute, Guadalajara,Jalisco, Mexico, were studied. Thirteen pa-tients were classified as having polar lepro-matous leprosy (LL) and six as having tu-berculoid (TT) leprosy according to the Ri-dley-Jopling classification ( 13). The LLpatients presented positive bacilloscopy,and ali of them received treatment proposedby the World Hcalth Organization (WHO)consisting of dapsone, clofazimine and ri-fampin (WHO/MDT) ('`').

The control group consisted of 10 unre-lated healthy subjects sex- and age-rnatchedwith the patient group.

287

288^ huernational.Iournal o/ Leprosv^ 1999

Muni ,: 1. In vitro c.vtokine productionby^lvmphoc.vtc's.

Mean ± S.D. pg/ml

'hAIII.L 2. Leveis o/ Thl cytokines pro-dured by PHA"-stintulated lymphocytes.

Mean ± S.D. pg/nil

Cytokine^1,L patients^

I leallhy controls^Cytokine^IA, patients

^leallhy controls

(N = 13)^

(N = 9)^

(N = 13)^

(N = 9)

11,-IO11,-4

525 ± 312'<l()

5S6 ± 350<10

224 ± 309''TNF-u^

1.35 ± 145

Culture supernatants were ohtained from monona-clear cens isolated from LL patients and healthy con-trols, and the corre.sponding cytokines were measuredby ELISA as dcscrihed in the Materiais and Mcthodssection.

''Statistical analysis was carried out using lhe Mann-Whitney U test; tu Ilerence not statistically significantcompared to controls.

Samples. Blood was obtained byvenipuncture to cstablish T lymphocyte cul-tures and to mcasurc the production in Nitroof 1FN-y, 1L-2, IL-4, IL-10 and tumornecrosis factor-alpha (TNF-a) cytokines.An aliquot of the same blood was uscd toobtain sera for furthcr evaluation of serumcytokines IL-1 í3, IL-1 ra, TNF-a and 1L-6.

Supernatants of lymphocytes stimu-lated with phytohemagglutinin (PHA).Mononucicar cells were separated overHistopaque-1077 (Sigma Chemical Co., St.Louis, Missouri, U.S.A.), washed twice inHank's balanced salt solution (HBSS), andsuspende(' in RPM' 1640 medium (Gibco,Grand Island, Ncw York, U.S.A.) supplc-mented with 2.5% heat-inactivated fetalcalf serum (FCS). Cells were adjusted to 1x 10" cens/mi and activated with 10 pgPHA/ml (' "). The cell cultues were incu-bated for 48 hr at 37°C in a humidified at-mosphere of 95% air and 5% CO,. Ccll-free supernatants were collected and appro-priatcly diluted to evaluate 1FN-y, IL-2, IL-4,1L-10 and TNF-a concentration by enzymelinked immunosorbcnt assays (ELISA) asdcscrihed by the manufacturer (R & D Sys-tems, Minneapolis, Minnesota, U.S.A.).

Serum cytokine measurements. TheQuantikinc human IL-1(3, IL-ira, TNF-aand IL-6 immunoassay (R & D Systems)were uscd for the quantitative determina-tion of human pro-inflammatory cytokinespresent in sera collected from LL patients,TL patients and controls. Appropriate stan-dard curves were uscd.

11.-2^

153 -4- 62h^

555 ± 356I FN-y^

200 ± 154'^

671 ±6.31

('ulture supernatants were obtained from mononu-clear edis isolaled from L1, patients and healthy con-trols.

Statistical analysis was carried out using lheMann-Whitney (I test; statistically signilicant differ-cnces compared to controls, p <0.05.

RN SULTSSecretion of pro- and antiinflamnnatory

cytokines by lymphocytes is an importantfeature of sevcral chronic inflammatory dis-cases. Therefore, we measured the produc-tion of IL-4, 1L-10 and TNF-a by mononu-clear cens from the LL patients and healthycontrols. Table 1 shows that control censstimulatcd with PHA did not secretc latothe cultue media different amounts of IL-10 and 1L-4 as compareci with the leproma-tous leprosy patients. lt is noteworthy thatIL-4 leveis were undetectablc in the cell-free supernatants from either group.

Table 2 shows Th 1 cytokine (IL-2, IFN-y)concentrations in the supernatants ofmononuclear cells frota LL patients andhealthy controls obtained aftcr stimulationwith PHA. LL patients produced signifi-cantly less IL-2 and 1FN-y than the healthycontrols.

Table 3 shows the in vivo leveis of IL-1(3, IL-6, TNF-a, and IL-1 ra in sera fromtuberculoid leprosy patients, lepromatousleprosy patients and healthy controls. Thecontrols showed no detectable IL-6 leveis,but slight increases were noticed in sera ofTL and LL patients. IL-1(3 serum leveis inLL patients were high as compared with thecontrol group although the difference wasnot statistically significant. On the othcrhand, TL patients showed no differences inIL-1 [3 when compared to healthy controls.

Regarding TNF-a, the serum concentra-tion in TL patients was significantly highercompared with the healthy controls. Eventhough LL patients had a tendency towardhigher leveis of TNF-a, thcre was no sta-tistically significant difference when the

67, 3^Fa¡utis-Morris, et al.: Serrim IL-ira^ 289

TABU, 3 Serunt cytokine concentrations in le/)roSV patients and controls". Data pre-sented as nu'ans ±S.1). /)g/nrl.

No.^IL-6^IL-1(3^IL- Ira^TNFa

Controls 9 <10 28.6 ± 7.7 217 ± 52 58.6 ± 6.1TI. patients 6 13.2 ± 11.8 21.8 ± 2 . 4h. 210±60.5. 70.5 ±1.1. patients 13 21.5±24.1 258.0 ± 374.8" 3755 ± 60` 217.3±491.8' ,

Blood was obtaincd from patients and matched healthy controls as indicated, and serum concentrations ofcytokines were determined by ELISA according to manufacturer's instructions. Statistical analysis wasperfürmcd usine the Mann-Whitney U test.

Statistically no significant differences compared to controls.Statistically significant differenees compared to controls, p <0.05.

' Statistically significant differences compared to controls, p <0.01.

TNF-a serum leveis were compared to thehcalthy controls.

Lastly, our most remarkablc finding wasthe 1 5-fold increase of IL- Ira in sera fromLL patients as compared with the healthycontrols. The iL- 1 ra serum concentration inTL patients was no diffcrent than that of thecontrol group.

DISCUSSIONThe clinicai and imnutnological manifes-

tations in leprosy represent a well-defincdsystcm of variable cellular immune re-sponses against the same antigen. On onehand, TL patients develop a good, delayedimmune response against M. leprae, effec-tively controlling the bacterial growth andspreading of the disease. On the other hand,LL patients do not respond specifcallyagainst M. leprae antigens. The immune re-sponse is poor, thus presenting a multibacil-lary disease with spreading. The Thl censresponse plays a major role in the develop-ing and the control of severa! infections,while Th2 cytokines (IL-4, IL-5 and IL-10)have been associated with progression ofdisease C. ",).

Based on results obtained by severa!groups of researchers, the tuberculoid poleof the leprosy spectrum has bccn associatcdwith a Th 1 -type response, while the lepro-matous pole clearly correlates with Th2 re-sponse. This was due to the fact that LL pa-tients produce very low concentrations ofIL-2 and IFN-y and high concentrations ofIL-4 and IL-10 when compared to healthyindividuais (5.7' 5. "). Neverthcicss, other in-vestigators point out that the pattern of cy-tokines seems not to correlate with the clin-icai stage of leprosy patients ( 4 ).

In this work we have evaluated super-natant concentrations of Th 1 and Th2 cy-tokines in cultures of LL paticnts' mononu-ciear cens, and we found no difference inthe Th2 responses between LL patients andhealthy individuais. Regarding Th 1 cy-tokine leveis, our data presented here con-firm and extend previous observations con-cerning IL-2 and IFN-y production by lym-phocytes (`'). Thus, lymphocytes from LLpatients produced far less amounts of IL-2and IFN-y as compared to healthy subjects( 5 . "• '') On the other hand, data obtaincdfrom the measurement of pro-inflammatorycytokines demonstrated that both TL andLL patients had higher IL-6 serum concen-trations compared with controls was ex-pected given the characteristics of this dis-ease.

Our results concerning IL-1 ra serumconcentrations are important. IL- 1 ra is anatural inhibitor of cytokine IL- 1 ( 15 ). Aclear-cut relationship among upregulationof IL-1 ra and inflammatory events has beendemonstrated in HIV infection, rheumatoidarthritis, glomerulonephritis and septicshock ('). In keeping with this, murine ex-perimental models induced by infectionwith M. at'innr have shed light on the role ofIL- 1 ra. Thus, IL- 1 ra promotes increased M.ai'irnn growth in the infected animais (`).High IL- ira leveis in granulomatous lesionsof patients with tuberculosis and sarcoido-sis have also been demonstrated ( 2). Consis-tent with these data, TNF-a is also an im-portant inducer of IL- 1 ra production bycells in granulomatous diseases (" ). In ad-dition, Apostopulos, et al. (') have shownthat where IL-Ira is exogenously added, itinhibits proliferation of PHA-stimulatedthymocytes.

290^ Intcrnationcrl Journal of Leprosy^ 1999

Taken together, there tindings are consis-tem with our previous results in which wefound that lymphocytes from LL patientsclearly displayed an inability to proliferateiu t'itro before stimulus given by Al. lepra'antigens and PI-IA. In this study we havefound a dramatic increase of iL-1 ra in thesera of LL patients. The interrclationshipamong thesc facts deserves further consider-ation, and the mechanisms involved are cur-rently being investigated in our laboratory.

SUMMARYPatterns of production of specific cy-

tokines are accepted as standards for T-lym-phocyte subsets in diseases caused by intra-cellular parasites. These lymphocyte sub-sets (Th 1 and Th2) have been associatedwith the different potes of the leprosy spec-trum. Lepromatous leprosy (LL) onset cor-relates with cytokines produced by Th2cells on the grounds of the patient's poorcellular immune response, i.e., interleukin 2(IL-2) and gamma interferon (IFN-y) defi-ciency. On the other hand, tuberculoid lep-rosy (TL) has been associated with a Thlresponse. Moreover, pro-in(]ammatory cy-tokines like IL-1(3 and tumor necrosis fac-tor-alpha (TNF-a) play a major role inchronic intlammatory pathologies being IL-1 ra and TNF-a soluble receptors, naturalcounterbalancing inhibitors.

In light of this background, we decidedto measure serum leveis of IL-1(3, IL-1 ra,TNF-a and IL-6 in LL and TL patients, andwe also studied the production in Nitro ofTh 1 (IFN-y, IL-2), Th2 (IL-4, IL-10) andTNF-a cytokines. Our data showed that IL-Ira is highly elevated in sera from LL pa-tients; there were no differences in Th2 cy-tokine leveis and there were diminishedleveis in Thl cytokines.

RESUMENSe reconocen patrones específicos de producción

de citocinas por las subpoblaciones de linfocitos T enenfermedades causadas pro parásitos intracelulares.Estas subpoblaciones de linfocitos (Thl y Th2) se hanencontrado asociadas con los diferentes poios dei es-pectro de la lepra. La lepra lepromatosa correlacionacon las citocinas producidas por las células Th2 (inter-leucina 2 e interferón gamma) y con una pobre res-puesta immune celular. Por el contrario, la lepra tuber-culoide se ha asociado con una respuesta Thl,Además, las citocinas pro-inflamatorias corno la IL-1(3y el factor de necrosis tumoral alfa (TNFa) juegan un

papel importante en las patologias inflamatorias cróni-cas, siendo cl receptor soluble 1L-Ira y el receptor paraTNFu, las moléculas reguladoras naturales. Sobre estabase decidimos medir los niveles en cl suem de iL-113,1L-Ira, TNFu e iL-6 en pacientes LL y Ti., y la pro-ducción in litro de las citocinas Th I (iFNy, 1L 2), Th2(li.-4, iL-IO) y TNFu. Nuestros ditos mostraron que1L-1ra está muy elevado en el suem de los pacientesLL; no hubieron diferencias en los niveles de citocinasTh2 amigue los niveles de citocinas Th 1 estuvierondisminuidos.

RÉSUMÉLes profils de production dy cytokines spécifiques

sont des mesures maintenant acceptées d'évaluationdes sous-populations de lymphocytes T présentes dansles maladies causécs par des parasites intracellulaires.Ces sous-populations de lymphocytes T (Thl et Th2)ont été associées aux différents piles du spectre de lalèpre. La laepre lépromateuse (LL) est associée à lasécrétion de cytokines par des lymphocytes de typeTh2 et ets caractérisée par une faible réponse immuni-taire à médiation cellulaire du patient, c'est-à-dire unedéficience de production d'interleukine 2 (IL-2) etd'intcrféron garnma (IFN-y). La larpre tuberculoïde(TL), en revanche, a ;aaté associée a une réponse detype Thl. De plus, les cytokines pro-inflammatoirescomine l'iL-1[3 et le facteur de nécrose des tumeurs-alpha (TNF-u) jouent un rôle majeur dans les étatspathologiqucs intlammatories chroniques avec,comme inhibiteurs et rétrocontrôles négatifs, l'IL-Iraet les récepteurs solubles de TNF-u. Au vu de ces con-naissances, nous avons décidé de mesurer les niveauxsériques de l'1L-1[3, de l'IL- ira, du TNF-u et de l'iL-6 chez des patients LL et TL, ainsi que la productionin vilro des cytokines de type Thl (IFN-y, iL-2), detype Th2 (IL-4, IL-10) et de TNF-a. Nos résultats in-diquent que IL- ira est présente en grande quantitédans le sérum des patients LL. II n'y avait pas de dif-férence de niveaux de sécrétion des cytokines de typeTh2 et les niveaux de sécrétion des cytokines de typeTh l étaient diminués chez les patients LL>

Acknowledgment. We are indehted to ing. Roge-lio Troyo for his invaluable help in statistical analyses.This study was supported by a grant froni Conacyt1485P-M.

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