IFN-γ is only partially restored by co-stimulation with IL-12, IL-2, IL-15, IL-18 or engagement of CD28

IFN-g is only partially restored by co-stimulation with IL-12,IL-2, IL-15, IL-18 or engagement of CD28

T. JUNG, K. WITZAK, K. DIECKHOFF, K. ZACHMANN, S. HEIDRICH,G. AVERSA* and C. NEUMANN

Department of Dermatology, University Go¨ttingen, Gottingen, Germany and*Novartis Research Institute, Vienna, Austria

Summary

Background Although it is well established that T cells derived from patients with atopicdiseases produce low levels of interferon-gamma (IFN-g), the mechanisms responsible forthis phenomenon are poorly understood.Objectives To elucidate whether IFN-g production may be restored by co-stimulatorymolecules known to increase IFN-g productionin vitro. Further, to investigate whetherdeficient IFN-g production is associated with disease activity.Methods Purified peripheral T cells obtained from patients with severe atopic dermatitis(AD), individuals with a history but no symptoms of AD and healthy control subjects wereactivated with anti-CD3 MoAbs in the presence or absence of anti-CD28 MoAbs,interleukin (IL-) 12, IL-2, IL-15 or IL-18. IFN-g production was determined at the singlecell level by flow cytometry, as well as by ELISA.Results Activated T cells from patients with severe AD produced less IFN-g than T cellsfrom healthy control individuals. IL-12 or engagement of CD28 enhanced IFN-g productionin both healthy and atopic T cells. However, absolute values of IFN-g were still different.IL-2, IL-15 and IL-18 did not restore IFN-g production. T cells from individuals with ahistory of AD produced more IFN-g than those from subjects with severe AD, but less thanT cells from healthy individuals. Atopic T cells expressed regular levels of CD3, CD28 andStat4, the main signal transducer and activator of transcription for IL-12. IL-4, IL-10 andTGF-b production by T cells were not different between healthy and atopic individuals.Conclusion IFN-g deficiency in atopic T cells is not due to a lack of responsiveness toCD28, IL-12, IL-2, IL-15 or IL-18. T cell-derived cytokines able to antagonize IFN-g do notcontribute to decreased IFN-g production. The extent of IFN-g deficiency seems to bedependent on disease activity.

Keywords: human, T lymphocytes, atopic dermatitis, cytokines

Clinical and Experimental Allergy, Vol. 29, pp. 207–216. Submitted 1 April 1998; revised18 June 1998; accepted 28 July 1998.

Introduction

In recent years it has become increasingly clear that T cellscontribute to the pathogenesis of atopic dermatitis (AD) [1].Activated, cytokine-producing T cells were observed in spon-taneously developed lesions [2] or provoked by epicutaneousadministration of a relevant aeroallergen [3]. When T cells wereisolated from these lesions and cloned, a substantial proportion

of the T-cell clones, especially allergen-specific clones, pro-duced interleukin-4 (IL-4) and low levels of interferon-g

(IFN-g), characteristics of a TH2-like cytokine pattern [4–6].In addition, a number of studies demonstrated that

polyclonally activated peripheral blood mononuclear cells(PBMC), or purified T cells from patients with AD,produced low amounts of IFN-g [7–12] even followingphorbol-ester stimulation circumventing the T-cell receptor(TCR) pathway [11]. Interestingly, mitogen-activated cordblood T cells from newborns at high risk of developing anatopic disorder produced less IFN-g compared with T cells

Clinical and Experimental Allergy,1999, Volume 29, pages 207–216

207q 1999 Blackwell Science Ltd

Correspondence: T. Jung, Novartis Research Institute, CMB Unit, BrunnerStr. 59, A-1235 Vienna, Austria.

from low-risk newborns [13–15]. Furthermore, deficientIFN-g production was associated with subsequent develop-ment of atopy [13,15] suggesting that an impaired IFN-g

response represents a very early immunological character-istic of atopy. However, it remains to be clarified whetherdiminished IFN-g production is due to an intrinsic defect ofT cells or whether it is merely anin vitro finding due tosuboptimal stimulation conditions.

Therefore, we investigated the capacity of atopic T cellsto respond to IL-12, -2, -15 and -18, as well as to engage-ment of the co-stimulatory molecule CD28, all well-knownstimulators of IFN-g [16–19]. The data indicate thatalthough T cells from patients with AD responded to theseco-stimulatory signals, they produced less IFN-g thanhealthy control T cells. This deficiency was neither due todiminished expression of the receptors for the activatingsignals, nor to enhanced production of IL-4, IL-10 and TGF-which might be able to inhibit endogenous IFN-g produc-

tion in an autocrine fashion [20–22]. Therefore, the presentstudy provides evidence for an intrinsic dysregulation inT cells from atopic patients.

Materials and methods

Patients and controls

A total of 10 healthy volunteers (mean age 29 years, range25–38), seven patients having mild (mean age 30 years,range 23–40) and nine patients having severe atopic der-matitis (mean age 27.2, range 14–39) were enrolled in thisstudy. AD was diagnosed according to the criteria describedby Hanifin and Rajka [23]. Patients with mild disease had apersonal and family history of AD but nearly no symptoms.The mean SCORAD index (severity scoring of AD) [24]was 5.2 in this group. Three patients also had a history ofallergic rhinits, two had a positive Phadiatop test (Pharma-cia, Freiburg, Germany) for inhalant allergens, IgE in serumwas normal (< 100 kU/L). Patients with severe AD had apersonal and family history of atopy, mean IgE in serumwas 1115 kU/L (range 428–1820). All individuals had apositive Phadiatop test for inhalant allergens, the meanSCORAD index was 58.6 (range 31–69). At the time ofinvestigation none of the patients was treated with immu-nosuppressive drugs.

Cells and cell culture

T cells were isolated by sheep red blood cell rosetting ofPBMC obtained from peripheral blood. T cells werecultured in Iscove’s medium supplemented with 5% FCS(Biochrom, Berlin, Germany) and were stimulated withimmobilized anti-CD3 MoAbs (20mg/mL) in 96 well

flat-bottom plates (OKT-3, kindly provided by Ch. Heusser,Novartis, Basel, Switzerland). T cells were co-activatedwith anti-CD28 MoAbs (soluble 100 ng/mL, Becton Dick-inson, Heidelberg, Germany). Recombinant IL-2, IL-4, IL-12 and IL-15 were purchased from R & D Systems (Wies-baden, Germany), recombinant IL-18 from PreproTech ECLtd (London, UK) and added to the cultures at 10 ng/mL.Supernatants were harvested after 2 and 7 days of stimulation.Phorbol-ester (PMA, 10 ng/mL) and ionomycin (1mmol/L)were obtained from Sigma (Deisenhofen, Germany).

ELISA

IFN-g and IL-4 production was measured in the super-natants as described earlier [25]. Briefly, capture antibodieswere coated to 96 well round-bottom plates (Nunc, Wies-baden, Germany) at 2mg/mL 0.1 mol/L NaHCO3 pH 8.3overnight. Standard curves were generated by dilution ofrecombinant IFN-g and IL-4, respectively. Biotinylateddetection antibodies were incubated at 2mg/mL PBS/10%FCS for 1 h. Streptavidin-peroxidase (Amersham,Braunschweig, Germany) was added for 30 min and theoptical density given by the reaction with ABTS (2,20-azino-bis3-ethylbenzthiazoline-6-sulphonic acid; Sigma) wasread at 405 nm using a Dynatech spectrometer (Stuttgart,Germany). The detection limit for the IL-4 ELISA rangedbetween 4 and 8 pg/mL, while it was 20–40 pg/mL forIFN-g. The antibodies and recombinant IL-4 were kindlyprovided by Ch. Heusser. IL-10 (Pharmingen, Hamburg,Germany) and TGF-b (R & D Systems) were measuredaccording to the manufactures instructions, the detectionlimit for both ELISAs was 4 pg/mL. Recombinant cytokineswere obtained from R & D Systems.

Flow cytometry

Intracellular detection of IFN-g production was performedas described [25–27]. Cells were activated with immo-bilized anti-CD3 MoAbs in the absence or presence ofanti-CD28 MoAbs or IL-12 for 24 h, Brefeldin A (Sigma)was added 8 h before harvesting. Cells were fixed with 4%paraformaldehyde, permeabilized with 0.1% saponin andstained with FITC- labelled anti-IFN-g antibodies (clone45.15, kindly provided by Ch. Heusser). Cell surface CD4was simultaneously detected with PE-labelled antibodies(Medac, Hamburg, Germany). Samples were measured on aPAS III flow cytometer (Partec, Mu¨nster, Germany)equipped with a 50 mW argon laser and analysed byWinList 3.0 software (Verity Software House, Topsham,ME, USA). Cell surface expression of CD28 and CD3 wasdetermined using dye-labelled antibodies obtained fromBecton Dickinson.

208 T. Junget al.

q 1999 Blackwell Science Ltd,Clinical and Experimental Allergy, 29, 207–216

Western blotting

Freshly isolated T cells were dissolved in 1% SDS,10 mmol/L TRIS-HCl, pH 7.4. Protein content wasmeasured using the Biorad system (Mu¨nchen, Germany).Ten micrograms protein were electrophoresed on a 8%polyacrylamide gel and transferred to PVDF membranesby semidry blotting (Biometra, Go¨ttingen, Germany). Stat4was detected using a specific polyclonal rabbit serumobtained from Santa Cruz, Heidelberg, Germany, a positivecontrol (rat testis lysate) was obtained from TransductionLaboratoires, Lexington, Kentucky. Lanes were visualizedby enhanced chemiluminescence (ECL, Amersham) andanalysed by densitography (Biometra). For some experi-ments nuclear proteins of unstimulated and anti-CD36IL-12 or IL-4-activated T cells were isolated by standardprocedures [28] and analysed for Stat4 as described above.

Statistics

The Mann WhitneyU-test (Prism 2.01, GraphPad Software,San Diego, CA, USA) was used to compare the study groupsfor significant differences.

Results

IFN-g production by atopic T cells is impaired followingTCR stimulation and engagement of CD28

IFN-g levels in supernatants of anti-CD3-activated purifiedT cells were lower in the group of patients with severe ADcompared with healthy controls (Fig. 1). Also, T cells frompatients with nearly no symptoms, but a clear history of ADproduced reduced levels of IFN-g compared with thosefrom healthy control individuals. This difference was,however, not statistically significant. Since CD28 is amajor co-stimulatory receptor involved in T-cell activation[29], IFN-g production was determined following activationwith anti-CD3 plus anti-CD28 MoAbs (Fig. 1). IFN-g wasenhanced in all three groups compared with stimulation withanti-CD3 alone. To exclude that these differences were dueto decreased receptor densities, CD3 and CD28 cell surfacefluorescence intensities were measured by flow cytometry.No major differences between the study groups weredetected (data not shown).

Diminished IFN-g production is due to a reduced frequencyof IFN-g producing cells and a diminished IFN-g synthesisat the single cell level

Intracellular IFN-g production was determined in T cellsactivated with anti-CD3þ anti-CD28 MoAbs. Simultaneousstaining of the CD4 antigen allowed an analysis of CD4þ vsCD4-IFN-g producing T cells (Fig. 2a). Since purified

T cells were stimulated, the CD4-population consistedmainly of CD8þ T cells (>80%), contaminating cells wereNK cells and fewgd-T cells (<5%). Figure 2(B) demon-strates that the frequency of IFN-g producing CD4þ T cellswas lower in both patients groups and lowest in the group ofindividuals with severe AD. The mean fluorescence inten-sity of IFN-g-producing cells as a parameter of IFN-g

content per cell was significantly reduced in activatedCD4þ T cells from patients with severe AD (Fig. 2c). Forthe CD4-T-cell population the mean frequency andSEM ofIFN-g- producing cells was 4.46 1.3 for the healthy groupand 1.46 0.5 for patients with severe (P<0.05) and 1.76 0.2 for patients with mild AD (P<0.01).

Atopic T cells are responsive to IL-12

Since IL-12 is well known to stimulate IFN-g production byactivated T cells, IFN-g secreted by anti-CD3- activated Tcells in the presence of IL-12 was determined. While IFN-g

increased 2.4-fold in the healthy group, a 3.4- and 5-foldenhancement was found in the group with mild AD andsevere AD, respectively (Fig. 3a). Compared with activationwith anti-CD3 alone these differences were significant in allthree groups (P< 0.05). Absolute values of IFN-g stilldiffered between the groups. Additional engagement ofCD28 moderately enhanced IFN-g production (Fig. 3a),but again levels of IFN-g differed between healthy andsevere AD T cells. Therefore, the results indicate that T cellsfrom atopic patients are responsive to IL-12. To furtherunderline this finding, Stat4, the major signal transducer and

Mechanisms of deficient interferon-g production in atopic diseases 209


Fig. 1. IFN-g production by atopic T cells is decreased even byengagement of CD28. Freshly isolated T cells from healthy donors(n¼ 9), patients with mild (n¼ 7) and severe atopic dermatitis(n¼ 8) were stimulated with anti-CD3 or anti-CD3þ anti-CD28MoAbs. Supernatants were harvested after 48 h and IFN-g wasmeasured by ELISA. Data are presented as mean andSEM.

210 T. Junget al.


Fig. 2. IFN-g production by atopic T cellsis diminished at the single cell level.Peripheral T cells from five healthy donors,five patients with mild and five with severeatopic dermatitis were activated with anti-CD3þ anti-CD28 MoAbs. Cells wereharvested 24 h later, fixed and stained asdescribed. Two-colour flow cytometry wasperformed with isotype control antibodies(examplified in a1) and anti-CD4þ anti-IFN-g MoAbs (examplified in a2). IFN-g-producing cells were calculated asproportion of gated CD4þ T cells (b). Themean fluorescence intensity (MFI) ofIFN-g-producing cells was calculated asmean channel of gated CD4þ T cells (c).Horizontal lines represent the median.

activator of transcription for IL-12, was detected in freshlyisolated T cells. As shown in Fig. 3b, Stat4 was observed inall total cell lysates without any major difference betweenhealthy and atopic T cells. Furthermore, IL-12-driventranslocation of Stat4 was demonstrated by western blottingof nuclear extracts of unstimulated and stimulated T cells asexamplified in Fig. 3c. Even in unstimulated T cells a weakband was detectable, while anti-CD3 activation enhancedStat4 translocation, which was further enhanced by co-activation with IL-12, but not IL-4. This type of response

did not differ between T cells from four atopic and fourhealthy donors, indicating a regular nuclear translocation inatopic T cells following IL-12 stimulation (data not shown).

IL-2 and IL-15 enhanced IFN-g production by activatedT cells, while IL-18 only moderately increased IL-12-drivenIFN-g production

Since IFN-g production by T cells is increased in thepresence of the T-cell growth factors IL-2 and IL-15, both



Fig. 3. Atopic T cells respond to IL-12 andexpress normal levels of Stat4. (a) Freshlyisolated T cells from healthy donors(n¼ 9), patients with mild (n¼ 7) andsevere atopic dermatitis (n¼ 8) werestimulated with IL-12 in addition to anti-CD3 or anti-CD3þ anti-CD28 MoAbs.Supernatants were harvested after 48 h andIFN-g was measured by ELISA. Data arepresented as mean andSEM. (b) Westernblot analysis of Stat4 in lysates of freshlyisolated T cells from healthy controlindividuals, patients with mild and severeatopic dermatitis. ECL-marker (M),positive control (P). (c) Western blotanalysis of Stat4 in nuclear extracts of Tcells from a healthy donor. Lane 1unstimulated, lane 2 anti-CD3-stimulatedfor 2 h, lane 3 anti-CD3þ IL-4 (10 ng/mL),lane 4 anti-CD3þ IL-12.

cytokines were tested for their capacity to restore IFN-g

production in atopic T cells. As shown in Fig. 4a, IL-2 morethan IL-15 moderately enhanced IFN-g synthesis by T cellsfrom all individuals ranging from a 1.6- to 2.4-fold increase(statistically not significant). IL-18 itself did not increaseIFN-g production by activated T cells, but moderatelyenhanced IL-12-driven IFN-g production in both healthyand atopic T cells (Fig. 4b).

The production of T cell-derived cytokines able to down-regulate IFN-g production is not different in healthy andatopic T cells

To rule out whether the observed IFN-g deficiency was dueto enhanced production of cytokines able to decrease IFN-g

production, supernatants of activated T cells were analysedfor IL-4, IL-10 and TGF-b. Regardless of the stimulus used,no major differences between healthy and atopic T cellswere observed (Fig. 5).

Discussion

In the present study we demonstrated that impaired IFN-g

production by T cells from patients with AD is neither simplydue to unresponsiveness following engagement of CD28 orIL-12R nor to over expression of IFN-g antagonizingcytokines in these T cells.

TCR-stimulated T cells from individuals with severe ADproduced lower levels of IFN-g than T cells from healthyindividuals. Co-stimulation with anti-CD28 MoAbsenhanced IFN-g production, but did not restore IFN-g tonormal levels. Downregulation of CD3 and CD28 receptors,which might explain an impaired response [30,31], was notfound in atopic T cells. We evaluated differences in IFN-g

responses between immobilized and soluble anti-CD3MoAbs, respectively. Activation with soluble anti-CD3plus anti-CD28 MoAbs induced approximately half of theIFN-g levels observed following stimulation with immo-bilized antibodies. Again, T cells from patients with severeAD produced lower levels of IFN-g than T cells fromhealthy individuals (data not shown) suggesting that it wasindependent from the method used for TCR stimulation.Furthermore, although values of IFN-g were higher insupernatants harvested after 7 days of culture, again atopicT cells produced lower levels of IFN-g (data not shown).These data were confirmed at the single cell level showingthat in atopic individuals not only the frequencies of IFN-g-producing cells were diminished, but also total IFN-g

protein content per cell, confirming and extending previousreports [11,12]. It is interesting to note that followingphorbol-ester stimulation the authors [11] and Nakagawaet al. [32] did not detect significant differences in CD4-IFN-g producing cells, while the present study and that of

Nakazawaet al. [12] demonstrated reduced frequenciesfollowing TCR stimulation. This indicates that the type ofstimulus seems to influence the results predominantly inCD8þ T cells.

The reasons for impaired IFN-g production in AD remainunknown so far. While our studies and those by others[7,33] suggest that the T-cell itself has an intrinsic defect toproduce sufficient amounts of IFN-g, others have shownthat decreased IFN-g production is dependent on antigen-presenting cells and their soluble mediators, e.g. prostaglan-din E2 [34]. IL-12 is produced by antigen-presenting cellsand, in addition with functional IL-12 receptors and Stat4, isrequired to generate a TH1-immune response [35–37]. It istherefore possible that T cells from atopic patients insuffi-ciently respond to IL-12 or that IL-12 production is dimin-ished in leucocytes from atopic patients. However, thepresent study demonstrates that atopic T cells are highlysusceptible to IL-12. Moreover, regular expression of Stat4as the major signal transducer and activator of transcription(Stat) for IL-12 [36,38,39] and its translocation to thenucleus following IL-12 stimulation further underlines thisfinding. Our observation is in accordance with regularexpression of the IL-12Rb-chain and production of IL-12by activated PBMC from AD patients [33]. Furthermore,IL-12 mRNA was also found in skin biopsies taken fromallergen patch tests [40] and in lesional skin [41] of patientswith AD. Thus, it may be concluded that neither IL-12production nor signalling plays a crucial role for impairedIFN-g production in patients with AD. On the other hand,IL-12 production was found to be reduced in whole bloodcultures from patients with asthma [42] and AD [43] there-fore at present the exact role of IL-12 for impaired IFN-g

production in atopy remains to be defined. Co-activationwith IL-2 or IL-15 moderately enhanced IFN-g productionin T cells from all donors. This indicates that activation ofthegc-chain-JAK3 pathway [44] is normal in atopic T cells,which is further supported by regular levels of JAK3 inT cell lysates from atopic individuals (data not shown).Although IL-18 was described as a potent inducer of IFN-g

production in TH1-clones [19], the presented data here showthat IL-18 itself did not induce IFN-g production in acti-vated T cells, but synergized moderately with IL-12 toenhance IFN-g production. This finding is in accordancewith the observation that IL-18 did not induce IFN-g

production in murine T cells, but enhanced IL-12-drivenIFN-g production [45]. This is further supported by the veryrecent description of a human immunodeficiency caused bymutations in the IL-12R1 gene where IFN-g production wasseverely impaired, suggesting that IL-18 did not signifi-cantly contribute to IFN-g production in the absence ofIL-12 signalling [37]. Taken together, our results suggestthat although atopic T cells are equally susceptible topotent inducers of IFN-g gene expression, absolute values

212 T. Junget al.


of IFN-g differed between healthy and severe atopicpatients.

Overproduction of cytokines able to downregulate IFN-g

may be a possible cause for impaired IFN-g production.Either directly or via antigen-presenting cells, IL-4 [20],IL-10 [21] and TGF-b [22] are well known to inhibit IFN-gproduction. The data presented here clearly show normallevels of IL-4, IL-10 and TGF-b in supernatants of atopicT cells activatedin vitro. In contrast, IL-4 production wasreported to be enhanced by activated T cells from atopicpatients [9,12,46–49], however, a number of reports demon-strate normal IL-4 production [11,32,50,51]. These differentresults may be related to the source of investigated cells, the

stimulus and the patients included in the studies. Pre-incubation of T cells with IL-10 and IL-4 decreases IFN-g

production during subsequent TCR activation (data notshown). Therefore, it seems possible that a local micro-environment enriched for IFN-g antagonizing cytokinescontributes to impaired IFN-g production in atopy.However, since IL-10 production by T cells, as shownhere, monocytes [43] and dendritic cells (unpublishedobservation) from patients with AD is not enhanced, itremains open whether this is a pathogenetically importantmechanism.

To find out whether the differences in IFN-g productionbetween healthy and atopic donors’ T cells were related to



Fig. 4. IFN-g production by atopic T cellsis not restored by IL-2, IL-15 or IL-18. (a)T cells from healthy donors (n¼ 7),patients with mild (n¼ 6) and severe atopicdermatitis (n¼ 8; n¼ 4 in case of IL-15stimulation) were activated with anti-CD3MoAbs in the absence or presence of IL-2or IL-15. Supernatants were harvested 48 hlater and IFN-g was measured by ELISA.Data are presented as mean andSEM. (b)T cells from three healthy donors and threepatients with severe AD were activatedwith anti-CD3 MoAbs in the absence orpresence of IL-18, IL-12 or both.Supernatants were harvested after 48 h andIFN-g was measured by ELISA. Data arepresented as mean andSEM.

disease activity, a group of patients with nearly no symp-toms of dermatitis, but a clear personal and family history ofAD was investigated. Compared with patients with severeAD, their T cells exhibited a stronger IFN-g response undermost experimental conditions, indicating that disease

activity indeed plays a role for IFN-g production as hasbeen observed also for allergic asthma [51]. Furthermore,successful treatment of patients with AD-increased levels ofIFN-g produced by activated T cellsin vitro [52,53]suggesting that the inflammatory response contributes todeficient IFN-g production. However, mean values of IFN-g

production usually were lower than those observed in thehealthy control group as shown by ELISA and flow cyto-metry. This observation suggests that the atopic status alonein the absence of clinical symptoms may be associated witha reduced capacity to produce IFN-g. This conclusionconfirms those studies which show that cord blood cellsfrom newborns at high risk of developing atopy, but withoutclinical symptoms, produce low levels of IFN-g followingpolyclonal activation [13–15] and this may point to animpaired IFN-g production as a hallmark of the atopicgenotype.

In summary, impaired IFN-g production by T cells frompatients with AD could only partially be restored byengagement of CD28- or IFN-g-enhancing cytokines.Since also T cells from patients with very mild disease,although less pronounced, demonstrated this defect, it issuggested that defective IFN-g production is part of thegenetic background of atopic dermatitis. Elucidation of themolecular basis for this phenomenon might possibly behelpful in defining strategies to reorganize an imbalancedcytokine production.

Acknowledgements

This work was supported by Deutsche Forschungsge-meinschaft grant Ju 351/1–1.

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IFN-γ is only partially restored by co-stimulation with IL-12, IL-2, IL-15, IL-18 or engagement of CD28