Document kmOmMp3zX4gN49xKNVXyaXeZE

Cttn. exp. Immunol, ( 1982) 50, 92- 98. Quantitative and functional deficit of suppressor T cells in children with atopic eczema M A R G A R E T B U TLER , D. A T H E R T O N & R. I. LEVINSK.Y Department o f Immunology. Institute o f Child Health, London, UK (Acceptedfo r publication 30 April 982) SUMMARY Helper (OKT4+) and suppressor (OKT8+) T cells were enumerated in 16 children w ith. severe atopic eczema. Compared to controls (median 1'8) the ratio of OKT4+/OKT8+ cells in the patients was significantly higher (median 2-65, P< 0 002). Functional suppressor activity in these patients was assessed by concanayalin A (Con A) activation ^ and suppression of pokewced mitogen (PWM) induced immunoglobulin production by ? plasma cells and Con A proliferation of T cells. In both assays a lack of suppression wa* shown (Con A/PWM, P < 0 02; Con A/Con A, P < 0-05). There was a significant inver: correlation between the helper/suppressor ratio and functional suppressor activit (FcO-OI). These results indicate that a defect of T cell regulation does exist in atopi eczema and if it is of primary pathogenic importance, immunotherapy to restore th balance may prove useful. IN T R O D U C T IO N It has been shown in rats that the production of IgE by plasma cells is under T celt coni (Okumura & Tada, 1971; Taniguchi & Tada, 1974). Inherently low IgE responder animals n converted into high responders by treatment which preferentially affects suppressor T cel immunosuppressive drugs (Taniguchi & Tada, 1971), adult thymectomy (Okumura 8c. Tada, I and moderate doses of whole body irradiation (Tada, Taniguchi & Okumura, 1971). heightened response can be abrogated by transfer of syngeneic thymocytes (Okumura Sc. 1971b). The evidence therefore suggests that damping o f the IgE response is by an antige non-specific T suppressor cell mechanism. Evidence for a similar mechanism in manjj circumstantial but allergy is significantly increased in immunodeficiency and very high levels orig may be associated with T cell deficiencies (Caplin et a 1977; Kikkawa et at., 1973). It has bee suggested that defective T cell regulation may play some part in the development of allergy ,,an indeed low numbers of T cells bearing Fc receptors for IgG (Ty) (these include the suppress* subpopulation) have been reported in atopic eczema (Canonico et al., 1979; Jensen, Cramcrv! Th restrup-Pederson, 1981). Recently monoclonal antisera which recognize the hclper/inducer an supprcssor/cytotoxic T cell subpopulalions more accurately have been developed (Rcmherxe/fl 1980). We have therefore studied children with atopic eczema to define their helper and suppress* T cell ratios and correlated this with two functional assays measuring T suppressor cell activity MATERIALS AND METHODS Patients. Sixteen children, who were cither inpatients at The Hospital for Sick Children < Correspondence: Dr R.J. Levinsky, Department of Immunology. Institute of Child Health, 30. Cuilfo i Street, London WCl. UK. 0009-9104/82/1000-0092*02.00 1982 Blackwell Scientific Publications Suppressor T ceil deficit in eczema 93 attending the Dermatology outpatient clinic, were studied. All of the children suffered from severe atopic eczema and had elevated serum IgE levels (> three standard deviations forage). One patient had atopic asthma in addition. None of the patients had been treated with systemic steroids. The children were aged between 3 months and 13 years with a mean age of 5-5 years. Ethical approval had been obtained from the hospital and full informed consent was received from the parents. Blood was also obtained from 15 healthy non-atopic adults (mean age 32 years). Patients and control cells were always tested at the same time. Lymphocyte separation. Ten millilitres of heparinized (preservative free) blood was diluted 1:2 with Hank's balanced salt solution (HBSS, Flow Laboratories), layered onto lymphocyte separation medium (Flow Laboratories) and centrifuged at 400 g for 20 min at 25C. The layer of mononuclear cells at the interface were collected, washed twice with HBSS and resuspended at a concentration o f 1 x IQ6 cclls/ml in bicarbonate-buffercrf RPMI 1640 (Flow Laboratories) containing 2mM glutamine, 5 fig/ml gentamicin and 10% fetal calf scrum (FCS, Flow Laboratories). M easurem ent o f T celt subsets. Subpopulations of T cells were estimated by indirect immunofluorescence. Monoclonal antisera recognizing all T cells (OKT3, Ortho diagnostics), hclper/inducer (OKT4) and supprcssor/cyto toxic (OKT8) subpopulations were used (Rcinherz et at.. 1980). Five microlitres of the monoclonal reagents or HBSS was added to separate aliquots of 5 x 10* lymphocytes. The cells were incubated at 4C for 30 min after which excess antibody was removed by washing three times with cold H BSS + azide (to prevent capping). Twenty microlitres of a second layer fluorosccin labelled sheep anti-mouse Ig was then added to each aliquot and left to incubate for a further 30 min at 4C. After three washes, wet slide preparations were made. Positively stained cells were counted using a Zeiss fluorescent microscope (150 cells were counted for each slide) and the results were expressed as a percentage after subtraction for non-specific staining. Functional suppressor celt assays Concanavalin A (Con A) suppression of pokeweed mitogen (PWM) stimulated immunoglobulin (Ig) synthesis (Layward, Levinsky & Butler, 1981). (a) Cell culture. Cells were cultured in triplicate (200 p\ volumes) in Linbro round bottomed microtitre plates at 37C in 5% humidified COj with or without PWM (G ibco Biocult, final dilution 1:200). Suppression of this PWM response was induced byco-culturing with 5 and 10pg Con A (Sigma) and after 7 days the amount of Ig released into the supernatant was estimated. (b) Competitive ELISA for total Ig production by cultures. An ELISA microtitre plate (Flow Laboratories) was coated overnight at 4C with 5pg/ml o f human Ig in 01 m carbonate/bicarbonate coaling buffer, pH 9-5. After extensive washing with PBS, to remove unbound Ig, the remaining binding sites were blocked by incubating with 1% FCS in coating buffer for I hr at 37UC. The plate was then washed with PBS-f0-05% Tween 20 (Sigma) (PBS/Twcen) and dried. Meanwhile SQpl of culture supernatants were incubated with 50/il o f peroxidase labelled anti-human Ig (DAKO, 1:400 final dilution) for 30 min at 37C in plates which had been coated only with 1% FCS. Eighty microlitres of this mixture containing Ig/pcroxidase labelled anti-Ig complexes were then transferred to the Ig coated ELISA plate and incubated for 1 hr at 37C during which time any uncomplexcd peroxidase labelled anti-Ig bound to the Ig coated plate. Unbound Ig was removed by extensive washing with PBS/Twcen and bound peroxidase labelled anti-Ig was estimated by the addition of lOO/il of substrate solution (40 mg O-phcnylodiaminc in cilrate/sodium dihydrogen phosphate buffer, pH 5). The enzymic reaction was carried out at 25'Cin the dark and stopped after 30 min by the addition of25/d oM n sulphuric acid. The colour which developed during the reaction was read on a Tilcrtck plate reader at the optimum wavelength of 492 nm. Sample concentrations were calculated by reference to a standard curve, range 1-50,000 ng Ig. Both samples and standards were set up n duplicate and mean values were calculated. tO Calculation o f suppression. /,, suppression = a --b ------ x 100 a 96 M argaret Butler, D. A^teuujrr. & Sjezne&y too 00 & 60 40 J 20 o 1- Contois^. Alfopr.x Fig. 3. Suppressor cell function measured by Cmjyvsajpjresxion of.ti^n`A'iiidb(Ptd"ir-j3*IigtvJ6cration. expressed as percentages. Bars indicate median'vnliw. P< 0 05). These results indicate that the finfiniRorfficcUir-id'.sui7rpts^ura^lkilyanoDoefonaIantis (OKT8+) in atopic eczema correlates with dtsr irimcltomi"! iirbuurmciltties un suppressor activi measured by the two assays. DfSaTESSDeEtK Our findings indicate that children with w e n i :atrpa:ecieinaaHmn at ddficil in the number ' function o f suppressor T cells as compared tinrtfiiJboomtat & K'mcrthiafl.ccasonf il was not possf to obtain blood from age matched childrcuoantraliPihiiUhKt&acaaimiilmcdiin-crar laboratory' testing children with suspected minor neutnritiilinmTumaiirfmceiiiHsjhasgn'en equivalent results those obtained in the adult controls. Furthermore,ihigtirflas wfhcctttf'.nrtxfflldata for children & few months to 17 years has been reported,iitrnrHtiiyiihuitps^flGICirJr^Saos^ppressor (OKT8+) 1*75 + 0*3 (2 s.d.) and this is very similar totfhmtrtinuhBilimauiratdiihiaanarDls (Siegel et at., Leung, Rhodes & Gcha, 1981). There have been several studies o f siqgoxscair iKiriwG^- :iii artery* with reduced suppr reported by some(Strannegard, Strannega/i';iS'iiuti;,li!i7il;iiir^!fnarzt.Cz2rnocki & Wolff, 19 but not by others (Martinez et at., 1979). AxrubicftniiiruT:^is:lbwiintlrraumto normal numbers i successful dcsensitization has been shown in^ariiLmLewcfhaiSihmri^vtiajiiic.gard etat., 1978). Ty: include the suppressor subpopulation butiU)jn cdlii:iveiv/fSycinncir.rfr-<C, 1980) and furthermo the in vitro expression o f T cells may be ire v<nnirblj fttiiiukrt: 5>vl^G in m u n e complexes (Pic Lum & Brodcr, 1978) so that the red uctionatlMs7`v;;i)miir^J}*i.-ai*ir5:CTiuiaiyeffect of such compl Tound in allergic individuals (Brostoff et jil,. fiiTJft;. 4Iun dm: using nonoclonal antisera wLi accurately define the hciper/induccr and sitfiptaftvirAy.iiotrsii; Tcriil subpopulations indicate patients with severe atopic eczema do indeed fuiA-trs rk![(ch:n: .T.irsuqxpressor cells. These findin confirm those of Leung et at, (1978) but ri>itfdthiitm run' jiutem;/. were xhown to have functio suppressor cell deficiency. The significant IivdjrxixiirjrJiKiitniijif .Wilfior/Jiwpprcssor T cell ratio wi 94 Margaret Butler, D. Atherton & R. J. Levinsky Where a -=ng Ig produced by PWM stimulated cells b = ng Ig produced by PWM stimulated cell-f Con A (5 or lOpg/ml). Con A induced suppression of mitogen stimulation T cell proliferation (Cpn A/Con A assay) (Sho Schwartz & Good, 1976). (a) Con A pre-treatment of lymphocytes. Suppressor T cells were generated by cult lymphocytes for 24 hr at 37C in 5% COj in the presence of I0^g/ml Con A. At the end of period the oells were washed twice in HBSS and resuspended in RPMI +10% FCS. Control were treated in a similar manner but Con A was not present during the 24 hr incubation. The were given 2000 rad irradiation to prevent further cell division. . (b) Mitogen stimulation o f responder oells. The suppressor and control cells were co-cult: with allogeneic responder cells from a healthy non-atopic donor. The ratio of suppressor control cells to responder cells were kept at 1:2 (concentration of responder cells I x 10*/i T riplicate cultures of cells with or without Con A (Ipg/ml) were incubated in 5% COi for 4 dayi 37C. Two microcuries of JH-thymidine (Radiochemical Centre, Amersham) were added for.di final 24 hr of incubation. The cells were collected on thcTitertek cell harvester and 3H-thymidiig1 incorporation was measured by liquid scintillation counting (LKB ft counter). Counts were; expressed as disintegrations per minute (d.p.m.) after correction for quenching and efficiency^ the counter. The percentage of mitogen induced T cell transformation of responder cells whidi was inhibited by Con A induced suppressor cells, was calculated using the following formula^ (Dmc --Dc) % suppression = l ---------------- x 100 Where: Dmc = mean d.p.m. for suppressor cells and responder cells, mitogen added. Dc = mean d.p.m. for suppressor cells and responder cells, no mitogen added. Dm =m can d.p.m. for control cells and responder cells, mitogen added. D =m ean d.p.m. for control cells and responder cells, no mitogen added. Statistics. Statistical analysis was by Mann-Whitney U-tcst and the Spearman Rank correlate lest. RESULTS Both suppressor assays and T cell subset measurements were performed on 14 of the 16 patient Insufficient blood was obtained from the other two patients, however, and T cell subsets only ; measured in one, whilst T cell subset measurements and the Con A/Con A assay were performed] the other. Measurement o / T celt subsets There were no significant differences in absolute lymphocyte numbers between the two groups! did the eczema group differ from the control group in terms of total T cell (OKT3+) percent (control median 71%; eczema group median 65*6%). However, the eczema group had significant lower percentages of supprcssor/cytotoxic T cells (OKT8+) (median 20*5%) than the control grot (median 27-0%, P < 0-02). There was no significant difference between the two groups-;fo helper-inducer T cell (OKT4+) subpopulalion. When expressed as ratio of OKT4+/OKT8' the eczema group had a significantly higher ratio (2*65:1*8, P< 0*002) (Fig. 1). Con A{PW M suppressor assay In control subjects the addition of 10/ig/ml Con A to PWM stimulated cultures greatly reduced IS production with a median value of 88% suppression. In atopic patients this degree of suppression* was lower giving a median value of 68%. The difference between these two groups was found to!?- highly significant ( P<0 02) (Fig. 2). At 5 rg/ml Con A the degree of suppression was less in both; groups and controls gave less consistent results with a median value of 80%. Nevertheless, tKj patients still had a significantly reduced degree of suppression and gave a median value of 5T (P < 0*02). 4 Con AfCon A suppressor assay We have previously established the optimal conditions for this assay and found that at a 1:2 Mtippressor/rcsponder cell ratio consistent results are obtained. The control subjects had a median value of 80% suppression whereas the atopic eczema group had a significantly lower level of suppression with a median value of 50% (F<0*05) (Fig. 3). Correlation between the data Results from the PWM assay (using 10 pg Con A) and the Con A/Con A assay were found to correlate significantly (r, = 0'5t /* <0-01 ). There was a significant inverse correlation between the ratio o f OKT4 to OKT8 positive cells and the results from the PWM assay (r, = 0-49, P<0-01) and the Con A/Con A assay (r, = 0-3, 100 r 4 ss I 40"<QS.. uco 20- # ><0-02 q (_ Control! Atopies 2. Suppressor cell function measured by Con A suppression of PWM induced immunoglobulin production plasma cells. Results expressed as percentages. Bars indicate median values. 96 Margaret Butler, IX A^txnrujrr. & 3L.X Lemnsky 100 eo so 40 I 20- -7*r>03: o 1- Contoi AWopmc Fig. 3. Suppressor cell function measured by Cmijfeuigjrecsidn of.tTjn'A'iiiilLiiEeii'ir.ecKpnjJlnfiration. R expressed as percentages. Bars indicate median ^vnhws. P < 0-05). These results indicate that the fircfii rcUuvidsiirprptJiViuTiltebymoDocfona I anti*" (OKT8+) in atopic eczema correlates with ;thr ffunnliundi ubirurmriRti >insuppressor activi measured by the two assays. CrSUTIL*i?KXiKiV Our findings indicate that children with w tr tipjiceczam sa haucai defidt in the number>: function o f suppressor T cells as comparedrtn.RdiihaanitritiE.ff;ipe3thiail.seasoi h was nqt possi ; vf- to obtain blood from age matched childrcioonttalLmutdht&acajuTTuilincd in -crur laboratory' testing children with suspoctcd minor ncutorjihHmmumniidEeiunssihfisigN'cn equivalent results those obtained in the adult controls. Furthexmoc^.invfitudiEZ'WhcE^itantiuEtldaSa for children ag few months to 17 years has been reported,thtmnrtbxriflhdtp3r(iEKff4*2)la5xprcssor (OKT8 +)_ 1*75+0*3 (2 s.d.) and this is very similar touted irtiTtHmciiin)(jiiiraibihima*nols (Siegel et al.t T Leung, Rhodes & Geha, 1981). There have been several studies of siqipzamn- -.mriMihy tin aitqpy with reduced suppress! reported by some (Strannegard, Slranncgay#Jluti;.iiK7il;:dhYiltnHzzLCz2rneclci & Wolff, 198 but not by others (Martinez el at., 1979). Axwlncfirmihi'H>yiaihi^Hnilrriunnto normal numbers i successful desensitization has been shown iu^imomLTwtfh2iKinmr'iEira.naejtard era/., 1978).Tyi include the suppressor subpopulation but null i^liLiwdy,-(Sieinne:ir.rfC<C, 1980) and furthermo the in vitro expression of T cells may be in3vnirblyhjbit&fd:iJy IfeG uenurunc complexes (Pick! Lum & Brodcr, 1978) so that the red uciionnUMar-viiiJmiii^ *anKs:rnidBny effect of such complex found in allergic individuals (Brostoff et jil,, HlT/ft;. tilun dt:b using zzonoclonal antisera whi accurately define the hclper/induccr and sipiprswviv/'.yiUatnsti: Trarill.-Bubpopulalions indicate patients with severe atopic eczema do indeed j.hiher.in: rrtirsuqiprcssor cells. These findin confirm those of Leung et ai. (1978) but iniadthtiirn/urr^icttirmf- were shown to have functio suppressor cell deficiency. The significant iivcmax-jnjTiJiiiiiini'.jti Avllferr,iiopprcssor T cell ratio wi Suppressor T cell deficit in eczema 97 functional suppressor assays further emphasizes this point. The functional assays were antigen non-specific and suppressor cell activity was induced by activation with the mitogen Con A. In the niouse it has been shown that IgE helper T cells are more sensitive to non-specific suppressor T cell activity than IgG helper T cells (Karz el a t, 1974; Takatsu Sl Ishizaka, 1976). If a similar situation exists in man, the moderately depressed non-specific suppression seen in our study is likely to lead to preferential enhancement of IgE helper T cell activity and increased levels of IgE. We do not know if our findings are of primary pathogenic importance in the development of uiopic eczema or secondary to the disease. Prospective studies on I month old babies who later developed atopic disease, showed significantly lower total T cell numbers than control children, suggesting a primary defect in these children (Juto Sc. Strannegard, 1979). Similarly, the improvement in antigen specific suppressor activity observed following successful desensitization for ragweed allergy suggests that this response can be modulated (Rocklin, 1981). Descnsitization 10 the provoking antigens is not feasible in atopic eczema; improvement in clinical symptoms can be obtained by allergen avoidance especially offending foods, but in the majority of severe cases these are not known. We have recently shown that a synthetic pentapeptide thymic hormone (TP5, Ortho Pharmaceuticals) used for the treatment of immunodeficiency corrects imbalances o f T cell subpopulations (Levinsky, Davies Si Goldstein, unpublished observations). It seems reasonable to suggest that such immunotherapy may be of clinical benefit in patients with severe atopic eczema where trials of allergen avoidance have not shown improvement. Wtf gratefully acknowledge (he National Eczema Society for grant support and Ortho Pharmaceuticals Tor supplying monoclonal reagents. REFERENCES Hrostoff. J.. Carini. C.. Wraith. D.G.. Paganelli, lymphoblastic leukaemia. B r.J. Haematol. 49,251. R. & Levinsicy. R.J. (1979) Immune complexes in Leung. D.Y.M., R hodes, A.R, &. G eha, R.S. (1981) atopy. In The Mast Cell; its role in health anddisease Enumeration o f T cell subsets in atopic dermatitis (ed. by J. Pepys & A.M. Edwards), p. 360--393. using monoclonal antibodies. J. Allerg. clin. Im Proceedings of the International Symposium. munol. 67, 450. Davos, Switzerland. Pitman Medical, London. Martinez, J.D .. Santos, /., Stechschute. D J . & Canonico. S.W., Mingari, M.C., Meltolt, G., A bdou, N.I. (1979) Non-spedfic suppressor cell Colon batti, M. 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Immunol. 45, 1[8. tors on human T lymphocytes. I. Transition of Tp Jutu. R. & Strannegard, O. (1979) T lymphocytes to Ty cells. J. Immunol. 121, 1540, and blood eosinophils in early infancy in relation to Reinherz, E.L., M oretta, L., Roper, M., Breard, hereditary for allergy and type of feeding. / . Allerg. din. Immunol, 64, 38. Kak?,, O.H., Hanaoka, T., Neuberger. P.E. & J.M ., Mingari, M .C., Gooper, M.D. & Schldssman.S.F. (1980) Human T lymphocyte subpopula tions defined by Fc receptors and monoclonal HenacerraF. B. (1974) Haptcn-specific IgE ami- antibodies. J. exp. Med. 151, 969. btxiy responses in mice. IV. Evidence for distinctive sensitisation o f IgE and IgG lymphocytes to the # fi'gulatory influence o f T oclls. J. Immunol. 113,974. Kuckawa. Y.. Kanimura. T.. Sekiguchi. T.. Hama,uv<a. T.. Kawai, T.. T akenaka. M. & T aoa. T. (IV7.1) Thymic alymphoplasia with hyper tg-g(o- Rockun, R.E. (1981) Antigen specific suppressor cells in human disease. In Suppressor cells in human disease (ed. by J.S. Goodwin) p. 229-246. Marcel Dckkcr Inc. New York and Basel. Shou, L.. Schwartz. S.A. & Good, R.A. (1976) Suppressor cell activity after concanavalin A treat ^ulinacmia. Paediatrics. 51. 690. Lavward. i-- Levinsky. R.J. & Butler. M. (1981) Longicrm abnormalities in T and B lymphocyte function in children following treatment for acute ment of lymphocytes from normal donors. J. exp. Med. 143, 1100. Siegel. R.L., Issekutz, T .. Schwaber. J.. Rosen. F.S. & G eha. R.S. (1981) Deficiency o fT helper cells in