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Studies on T-cell subsets in atopic dermatitis: Human T-cell subpopulations defined by specific monoclonal antibodies
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
22, 139-146
(1982)
Studies on T-Cell Subsets in Atopic Dermatitis: Human T-Cell Subpopulations Defined by Specific Monoclonal Antibodies MICHAEL R. FAURE,* JEAN F. NICOLAS,* MARTINE A. GAUCHERAND,* AND JANUSZ *INSERM Lynn
U 209. Laboratoire de Recherche Cedex 2, France, and fDeparttnetrt
Dertnatologiyue, of Dermatology,
JEAN THIVOL.ET,*,’
M. CZERNIELEWSKI? Hbpital Edcmard Herriot, 69374 Lii& Unirsersity, Lii&. Poland
Peripheral blood T lymphocytes and T-cell subsets were explored in 40 patients with atopic dermatitis and elevated serum IgE levels. The percentages of the different T-cell subpopulations were defined by indirect immunofluorescence using monoclonal antibodies OKT3, OKT4, and OKT8 which are specific markers of, respectively, total T cells, helper T cells, and suppressor/cytotoxic T cells. Decreased peripheral blood T cells (OKT3+) and abnormal distribution between helper and suppressor T cells subsets were found as compared to controls. Results were similar in the three age-matched groups (4 months-2 years, 2- 15 years, 15-50 years) studied. Elevated helper/suppressor cells ratios with decreased percentages of suppressor (OKT8+) T cells were observed, although no correlation could be established with serum IgE levels.
INTRODUCTION
Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disorder, often seen in association with a personal or familial history of asthma, allergic rhinitis, or hay fever. Its pathogenesis remains obscure, and a variety of factors are probably involved in the initiation and evolution of the disease. Among these factors, physiologic defects, such as a blockade of /3-adrenergic receptors in tissues (1) and immunologic abnormalities (2, 3), seem to be involved in the pathogenesis, the clinical aspects, and evolution of the disease. Increased susceptibility to viral, bacterial, and fungal infection (2, 3) and association between AD and immunodeficiency diseases, such as the Wiskott-Aldrich syndrome (4) and the hyper-IgE (Buckley) syndrome with defective T lymphocytes functions, and polymorphonuclear or monocytes abnormalities (5,7) are indirect evidences for a defective cell-mediated immunity (CMI) in atopy (2). CM1 and thymus-dependent cells (T lymphocytes) functions and peripheral blood counts have been reported to be depressed in AD (8- 18), although in some cases normal (19-21). Monoclonal antibodies produced by the murine hybridome technique and directed against specific human T lymphocytes and T-cell subsets provide a new tool for the analysis of T-cell subpopulations in man (22). In the present study, we have investigated the peripheral blood T lymphocytes populations and the helper-inducer and suppressor-cytotoxic T-subsets distribution in patients with AD using monoclonal antibodies specific for surface markers of human T lympho’ Address reprint Edouard Herriot-Pav.
requests to: Monsieur le Professeur R, 69374 Lyon Cedex 2, France.
Thivolet,
Clinique
Dermatologique,
Hopital
139 0090-1229/82/010139-08$01.00/O
CopyrightQ 1982by AcademicFTCSS, Inc. All rightsof reproductionin anyformreserved
140
FAURE
ET
AI..
cytes. Our data show defective total circulating T lymphocytes counts and an abnormal distribution of the T-helper and T-suppressor subsets, with a decrease in the suppressor T-cell subset in patients with AD. MATERIALS
AND METHODS
Putients and controls. Forty patients, males and females, with AD were studied. They ranged in age from 4 months to 50 years. They were divided into three groups. Group 1 was composed of 15 patients with ages ranging from 1.5to SO years; group 2 of 12 patients, ages ranging from 2 to 15 years: group 3 of 13 patients, ages ranging from 4 months to 2 years. The diagnosis of AD was based on the following criteria, as proposed by Hanifin and Lobitz (2): a present and past history of focal or generalized lichenified, pruritic, eczematous skin rash with a predilection for the flexural areas and/or face and neck: a personal or family history of atopic. “allergic” disease: white dermographism; elevated serum IgE levels (23). All patients had typical clinical features of AD at the time of the study. Only 3 patients had a past history of infection (eczema herpeticum in one case; staphylococcal or fungal infections in three cases). Only one had a present history of skin infection when the study was performed. Serum IgE levels were high in every case of the study. Total peripheral blood (PB) lymphocytes counts were always within normal ranges (according to ages) and no decreased total PB lymphocytes counts was ever noted. The patients had not been on oral corticosteroids for at least 1 year before the study (children had never been on oral corticosteroids). They had received no antihistamines or other systemic therapy for at least 1 week, and had utilized no topical corticosteroids for at least 2 weeks before the time of the study. Controls were 30 healthy volunteers, with the same proportion of males and females, receiving no therapy. They were 20 staff members of our department and other volunteers, ages ranging from 15 to 50 years (group I) and 10 teenagers or children with ages ranging from 2 to 15 years (group 2). T-Cell studies could not be performed in controls with ages lower than 2 years, for an easily understandable ethical point of view. None had high serum IgE levels. Monoclonal antibodies. Monoclonal antibodies (Ortho Pharmaceutical Corp.. Raritan, N.J.), directed against various human T-cell antigens, were produced as already described (24, 25) by mouse hybridomas, obtained from cell fusion between mouse myeloma cells and normal spleen cells from mice immunized with human T lymphocytes. Previous studies have demonstrated that some of these antibodies recognize all peripheral T cells among PB lymphocytes, whereas others react with different PB T-cell subsets (22). Three monoclonal antibodies named OKT3, OKT4, and OKT8 were used: OKT3 reacting with all peripheral T cells, OKT4 identifying only peripheral T cells with helper-inducer function, and OKT8 directed against a T-cell subset with both suppressor and cytotoxic activity (26, 27). Erlutneration of lymphocytes subsets \r-ith tnonoclonal untihodies. Peripheral blood mononuclear cells were isolated by Ficoll-Paque (Pharmacia, Uppsala.
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Sweden) density centrifugation. After washing, the cells were suspended in RPM1 1640 (Eurobio, Paris, France) supplemented with 5% fetal calf serum (Gibco, Grand Island, N.Y.) and 25 m&f Hepes (Eurobio, Paris, France) at a concentration of 5 X lo6 cells/ml. Ten microliters of each reconstituted antibody (OKT3, OKT4, OKT8) was incubated with 200 ,ul of the cell suspension in hemolysis tubes and left for 30 min at 4°C in an ice bath. After washing, the pellet was added with 100 ~1 of fluoresceinconjugated goat antimouse IgG antibody (Nordic Immunological Lab., Tilburg, The Netherlands; fluorescein concentration, 10 mg’ml; F/P ratio, l-4), which has been diluted 1: 10 in RPM1 1640. Cells were left again for 30 min in an ice bath at 4”C, then washed and resuspended in medium. The percentage of fluoresceinlabeled cells was established by counting fluorescent cells on a Leitz Orthoplan microscope (E. Leitz, Wetzlar, FRG) and comparing to the total number of cells. Statistical methods. Statistical analysis was carried out using Student’s t test, with P < 0.05 indicating significant difference. RESULTS
Enumeration of PB T cells. Table 1 shows the percentages of T cells (OKT3+) in patients with AD compared to normal controls. As can be seen, in group 1 and 2, total T cell counts were lower in patients with AD (58.8 + 8.8 and 59.0 + 5.5%, respectively), than in controls (69.4 2 11.1% and 66.3 + 8%, respectively). These differences were statistically significant, with F < 0.02 in group 1, and P < 0.05 in group 2. No significant difference was observed between results in groups 1 and 2, nor between these two groups of atopic patients and group 3 patients where the OKT3+ cells accounted for 58.7 5 7.7% of PB mononuc!ear cells. Enumeration of helper and suppressor T-cell subsets. The balance between the
PERIPHERAL
BLOOD INDIRECT
TABLE 1 T-CELL ENUMERATIONS IN ATOPIC DERMATITIS IMMUNOFLUORESCENCE (IIF) WITH ANTI-HUMAN MONOCLONAL ANTIBODY (OKT3)
Patients and controls groups
Number of cases
Group 1 (15-50 years) AD” Normal subjects
I5 20
58.8 -+ W 69.4 + I 1.1 (P .: 0.02)
Group 2 (2- 15 years) AD Normal subjects
12 10
59.0 + 5.5 66.3 k 8.0
Group 3 CO- 2 years) AD
13
58.7 k 7.7
(z Atopic b Mean
dermatitis. + SE.
Percentage peripheral
(AD) ANALYZED T-CELL
OKT3 positive cells among blood mononuclear cells
(P .< 0.05)
BY
142
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ET AL.
two major T-cell subsets, OKT4+ and OKT8-t cells, was strongly altered in patients with AD, as seen in Table 2. Lower percentages of OKT4 and OKTS+ cells were observed in groups 1 and 2, as compared to control subjects of the same age groups. The decrease in the percentages of OKT8+ cells was highly significant (27.4 k 7.7%, controls: 41.9 + 12.7%, P < 0.002 in group 1: 22.3 k 4.7%, controls: 30.0 t 9.7%, P < 0.02 in group 2) and higher than what was observed with OKT4+ cells. These changes appeared still more clearly when the OKT4-OKT8 balance was expressed by the ratio between the percentages of OKT4+ and OKT8+ cells (helper/suppressor ratio). Such calculation eliminated the fluctuation of OKT4+ and OKT8+ cells percentages due to variable content of PB mononuclear cells in monocytes (28, 29). Furthermore, some overlap between the two OKT4+ and OKT8+ subpopulations (as we have previously reported with control studies when the cells were reacted in the same experiment with the two antibodies OKT4 and OKT8 (29)), is also eliminated in the determination of helper/suppressor balance when expressing it by the OKT4/OKT8 ratio. As also seen in Table 2, these OKT4/OKT8 ratios were increased in each group of patients with AD, as compared to controls: 1.82 t 0.52, controls: 1.38 ? 0.37 in group 1; 2.01 t 0.37, controls: 1.59 k 0.26 in group 2. with p r 0.02; in group 3, the OKT4/OKT8 ratio was similar (2.02 t 0.67). with a decrease in the OKT8+ cell subset similar to the results in the two other groups. These results indicate a T helper/suppressor imbalance with a decreased T TABLE 2 PERIPHERAL B~oou HELPER AND SUPPRF~SOR T-CFI.I. tOKT4t %NL) OKTg+ CEI tsi ENUMF.RATIONS AND BALANCES IN AD ANALYZED BY IIF WITH AN I‘I-HUMAN T-SUBSET MONOCLONAL ANTIBOIX~S tOKT4 ANI) OKTS) Antibodies t’J positive cells among peripheral blood mononuclear cells) Patients and controls groups
Number of cases
Group I (15-W years) AD” Normal subjects
I5 20
46.5 -t 7.1” 54.4 -+ 10.8 (P < 0.03
‘7.4 T 7.7 41.9 -t 12.7 (P c- 0.002)
I .82
Group 2 (2~ 15 years) AD Normal subjects
12 IO
43.6 i- 8.0 46.4 i- 10.3 NS”
22.3 -* 4.7 30.0 2 9.7 (P -: 0.02)
2.01 f 0.37 I .5Y -c 0.26 (P -= 0.02)
Group 3 (O-2 years) AD
13
49.5 f 9.x
26.5 -t 8.1
2.02 2 0.67
‘I Atopic dermatitis. * Mean -r SE. ” Nonsignificant
OKT4
OKTX
Ratio ‘;OKT4+/VOKT8+
z 0.52h
1.38 i 0.37 (P i 0.02)
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suppressor-cytotoxic subpopulation in peripheral blood of patients with atopic dermatitis. No significant difference was observed in the enumeration of OKT3 + , OKT4+, and OKT8+ cells or in the OKT4/OKTS balance between males and females or according to the clinical history (intensity and severity of skin lesions, presence or absence of infection, etc). Because a decreased suppressor cell population may allow increased IgE production (30-32) in atopy, we tried to correlate this helper/suppressor imbalance with serum IgE levels in our atopic patients. As seen in Fig. 1, no positive correlation was observed between serum IgE levels and the values of OKT4+/OKT8+ cell ratios. Furthermore, no inverse correlation was observed between the percentages of OKT8+ suppressor-cytotoxic cells and serum IgE levels. DISCUSSION
We have studied in patients with atopic dermatitis and in normal controls T-cell markers as recognized in indirect immunofluorescence on cell suspensions by monoclonal antibodies specific for T lymphocytes, helper-inducer T cells, and rh Ratio
%OKT4+/%0KT8+
. .
.
. .
.
5--
.
.
. l
. .
’
.
l*
.
l *
.
. .
,5-, .
.
.
.
.
0 FIG. 1. Correlation (%OKT4+/%OKT8+ 1 and 2).
10
‘ 20I
t 50
I 100
I
I * 500 1000
I ’
, 5oob 1o’oon
between serum IgE levels and circulating cells ratios) in patients with atopic dermatitis:
I a
I 50 600’
I@
helper/suppressor no correlation
(
&‘I,
T-subset is observed
balance (Groups
144
FAURE
ET
AL
suppressor-cytotoxic T cells in man. Similar studies with the same monoclonal antibodies have previously shown significantly decreased percentages of suppressor T cells in patients with lepromatous and borderline leprosy (28) and, in the Sezary syndrome, a large increase in helper T cells with a parallel decrease in suppressor T lymphocytes (29, 33, 34). Our present data clearly document in patients with atopic eczema: (1) a decrease in the PB OKT3+-T lymphocyte population, and (2) an abnormal PB T-cell distribution between OKT4+ -helper and OKT8+ -suppressor T cells. with decreased percentages of suppressor cells. These results are in agreement with several other reports of a defective cellmediated immunity in atopy. Patients may have a decreased delayed cutaneous hypersensitivity to natural antigens (3.5) and a decreased rate of sensitization to experimentally applied antigens (36, 37). In vitro evaluations for T lymphocytes functions, and determinations of PB T cells counts have documented significally decreased rosette-forming cells levels compared to normals ( I I). and decreased lymphocytes transformation to T-cell mitogens (8, IO, 12. 14- 18, 38) and lymphokine production by T cells after T-cell-mitogen stimulation (39). Furthermore, our finding of a decreased proportion of PB OKT8+ suppressor-cytotoxic T cells correlates other observations of PB suppressor T cell defect in atopic disease (30, 38, 40, 41). Low suppressor cell counts have been documented on the basis of reduced relative levels of T cells bearing a receptor for the Fc fragment of IgG (T y subset) in patients with atopy (40). Abnormal irr \‘itro suppressor cell activity was recently observed, with a defective concanavalin A-inducible suppressor cell function as measured by the proliferative responses to pokeweed mitogen (41). These findings and the present data with monoclonal antibodies as markers of T-cell subsets, raise the question of the exact meaning of the T-cell subsets imbalance in atopy. Several studies have shown that depressed T-cell-functions balance and relative counts may be correlated with serum IgE levels (12, 14, 15. 18. 20). It has been postulated that excessive IgE antibody responses found in atopic diseases may be due to impaired suppressor cell function (30-32). On the other hand, it has been suggested that IgE may modulate immune reactions such as T-cell activity (42). In contrast with previous studies of defective suppressor T-cell functions (41). we failed to observe a correlation between supressor cell levels (determined as OKT8+ cells) or OKT4+/OKT8+ cell ratios, and serum IgE levels in our patients with atopic dermatitis. This difference in results may be due to the difference in the methods used for the evaluation of the T-cell suppressor subset, or, more likely, to the fact that all our patients with AD had elevated serum IgE levels prior to, and at the time of the study. In summary, patients with AD and high serum IgE levels appear to have decreased percentages of peripheral blood T lymphocytes, and a helper/suppressor T-cell imbalance, with a decreased suppressor-cytotoxic T-cell subset, as established using monoclonal antibodies specific for T cells and T-cell subsets. These monoclonal antibodies represent a new, easy, and useful tool for the determination of T-cell subsets balance and functions in various disorders.
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ACKNOWLEDGMENT The authors CILAG-FRANCE,
wish
to thank Mrs. J. Bielsky for statistical analysis. This work affiliate of ORTHO P.C., and in part by UER GB Bl 1981.
was supported
by
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
Szentivanyi, A., .I. Allergy C/in. Immunol. 42, 203, 1968. Hanifin, J. M., and Lobitz, W. C., Arch. Dermatol. 113, 663, 1977. Maize, J. C., Int. J. Dermatol. 15, 555, 1976. Mackie, R. M., Alcorn, M. J., Stevenson, R. D., Cochran, T., and McSween, R. N., &it. J. Dermatol. 98, 567, 1908. Buckley, R. H., Wray, B. C., and Belmaker, E. Z., Pediatrics 49, 59, 1972. Hill, H. R., and Quie, P. G., Lancer 1, 183. 1974. Jeune, R., Faure, M., Leung-Tack, J., Schmitt, D., and Thivolet, J.. Arch. l)ermato/. 114, 1372, 1978. Andersen, E., and Hjort, N., Acta Dermatol. Venereal. 55, 345, 1975. Capareto, F. J., Winkelmann, R. K., and Jordan, R. E., Arch. Dermatol. 112, 1095, 1976. Fjelde. A., and Kopecka, B., Acta Dermutol. Venereal. 46, 168, 1967. Gottlieb, B. R., and Hanifin, J. M., C/in. Res. 22, 159 A, 1974. Lobitz, W. C.. Honeyman, J. F., and Winkler, M. W., Brit. J. Dermatol. 86, 317, 1972. Luckasen, J. R.. Sabad, A., Goltz, R. W.. and Kersey, J. H., Arch. Dermatol. 110, 375, 1974. McGeady, S. J.. and Buckley, R. H., J. Allergy C/in. Immunol. 56, 393, 1975. Rachelefsky, G. S., Opelz, G., Mickey, M. R., Kiudchi, M., Terasaki, P. I., Siegel, S. C., and Stiehm, E. R.. J. Allergy C/in. Immunol. 57, 569, 1976. Rogge. J. L., and Hanifin, J. M., Arch. Dermatol. 112, 1391, 1976. Strannegard, I. L., Lindholm, L., and Strannegard, O., Arch. A//erg. Appl. fmmttd. 50, 684, 1976. Thestrup-Pedersen, K., Ellegaard, J., Thulin, H., and Zachariae, H., C/;,I. Ex,D. Immunol. 27, 118, 1977. Dupree, E., Friedman, J. M., Land, R. N., and Goldman, A. S., J. Allergy C/in. Immunol. 55, 102 A, 1975. Grove, D. J., Reid, J. G., and Forbes, J. J., Brit. J. Dermutol. 92, 611, 1974. Howmark, A., Acta Dermatol. Venereal. 55, 181, 1975. Reinherz, E. L., and Schlossman, S. F., Cell 19, 821, 1980. Juhlin. L., Johansson. S. G., Bennich. H., Hogman, C.. and Thyresson, N.,Arch. Derrnutol. 100, 12, 1969. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., J. Immrrd. 123, 1312, 1979. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., Proc. Nat. Acad. Sci. USA 76, 4061, 1979. Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H., and Schlossman, S. F., Proc. Nat. Acad. Sci. USA 77, 1588, 1980. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., J. Immunol. 124, 1301, 1980. Bach, M. A., Chatenoud, L., Wallach. D., Phan Dinh Tuy, F., and Cottenot, F., C/in. Exp. Immunol. 44, 491, 1981. Fulton. R., Thivolet, J.. Garcier, F.. and Gaucherand, M., Ann. Dermatol. Venereal. 108, 243, 1981. Buckley, R. H., and Becker, W. G., Immunol. Rev. 41, 288, 1978. Okumura, K., and Tada, T., J. fmmunol. 107, 1682, 1971. Tada. T., Prog. A//erg. 19, 122, 1975. Laroche, L., and Bach, J. F., C/in. Immunol. Immunopathol. 20, 278, 1981. Schmitt, D., Thivolet, J., Souteyrand, P., Fulton, R., and Germain, D., Arch. Dermatol. Res. 270, 487, 1981. Sultzberger, M. B., and Goodman, J., J. Amer. Med. SOL.. 106, 1000, 1936. Jones, H. E., Lewis, C. W., and McMarlin. S. L., Arch. Dermatol. 107, 217, 1973.
146
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37. 38. 39. 40.
ET
AL..
Palacios. J.. Fuller, E. W., and Blaylock. W. K.. J. IIII~c,.s~. I)er-/,rrrrr>/. 47, 484. 1966. Ogden, B. E.. Krueger. G. G.. and Hill, H. R.. c’li~. Lvp. I~~~rtr~~o/. 35. 269. 1979. Horsmanheimo, M.. and Horsmanheimo, A.. J. I//~Y~.sI. fIrr/t~/r~/. 72, 128. 1979. Schuster. D. L.. Bongiovanni. B. A.. Pierson. D. L.. Barbaro. J. F.. Wong. D. T. 0.. and Levi son. A. 1.. J. I~?/~nr~ro/. 124, 1662. 1980. 41. Rola-Plesrcynski. M.. and Blanchard. R., ./. IuI~c,.\I. /Ie~/r~tr/~~/ 26. 279. I981 42. Hanifin. J. M.. and Gottlieb. B. R.. C‘lirr. Rc\. 22. 328 A. 1 74. Received
July 9. 1981: accepted
August
X. 1981.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
22, 139-146
(1982)
Studies on T-Cell Subsets in Atopic Dermatitis: Human T-Cell Subpopulations Defined by Specific Monoclonal Antibodies MICHAEL R. FAURE,* JEAN F. NICOLAS,* MARTINE A. GAUCHERAND,* AND JANUSZ *INSERM Lynn
U 209. Laboratoire de Recherche Cedex 2, France, and fDeparttnetrt
Dertnatologiyue, of Dermatology,
JEAN THIVOL.ET,*,’
M. CZERNIELEWSKI? Hbpital Edcmard Herriot, 69374 Lii& Unirsersity, Lii&. Poland
Peripheral blood T lymphocytes and T-cell subsets were explored in 40 patients with atopic dermatitis and elevated serum IgE levels. The percentages of the different T-cell subpopulations were defined by indirect immunofluorescence using monoclonal antibodies OKT3, OKT4, and OKT8 which are specific markers of, respectively, total T cells, helper T cells, and suppressor/cytotoxic T cells. Decreased peripheral blood T cells (OKT3+) and abnormal distribution between helper and suppressor T cells subsets were found as compared to controls. Results were similar in the three age-matched groups (4 months-2 years, 2- 15 years, 15-50 years) studied. Elevated helper/suppressor cells ratios with decreased percentages of suppressor (OKT8+) T cells were observed, although no correlation could be established with serum IgE levels.
INTRODUCTION
Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disorder, often seen in association with a personal or familial history of asthma, allergic rhinitis, or hay fever. Its pathogenesis remains obscure, and a variety of factors are probably involved in the initiation and evolution of the disease. Among these factors, physiologic defects, such as a blockade of /3-adrenergic receptors in tissues (1) and immunologic abnormalities (2, 3), seem to be involved in the pathogenesis, the clinical aspects, and evolution of the disease. Increased susceptibility to viral, bacterial, and fungal infection (2, 3) and association between AD and immunodeficiency diseases, such as the Wiskott-Aldrich syndrome (4) and the hyper-IgE (Buckley) syndrome with defective T lymphocytes functions, and polymorphonuclear or monocytes abnormalities (5,7) are indirect evidences for a defective cell-mediated immunity (CMI) in atopy (2). CM1 and thymus-dependent cells (T lymphocytes) functions and peripheral blood counts have been reported to be depressed in AD (8- 18), although in some cases normal (19-21). Monoclonal antibodies produced by the murine hybridome technique and directed against specific human T lymphocytes and T-cell subsets provide a new tool for the analysis of T-cell subpopulations in man (22). In the present study, we have investigated the peripheral blood T lymphocytes populations and the helper-inducer and suppressor-cytotoxic T-subsets distribution in patients with AD using monoclonal antibodies specific for surface markers of human T lympho’ Address reprint Edouard Herriot-Pav.
requests to: Monsieur le Professeur R, 69374 Lyon Cedex 2, France.
Thivolet,
Clinique
Dermatologique,
Hopital
139 0090-1229/82/010139-08$01.00/O
CopyrightQ 1982by AcademicFTCSS, Inc. All rightsof reproductionin anyformreserved
140
FAURE
ET
AI..
cytes. Our data show defective total circulating T lymphocytes counts and an abnormal distribution of the T-helper and T-suppressor subsets, with a decrease in the suppressor T-cell subset in patients with AD. MATERIALS
AND METHODS
Putients and controls. Forty patients, males and females, with AD were studied. They ranged in age from 4 months to 50 years. They were divided into three groups. Group 1 was composed of 15 patients with ages ranging from 1.5to SO years; group 2 of 12 patients, ages ranging from 2 to 15 years: group 3 of 13 patients, ages ranging from 4 months to 2 years. The diagnosis of AD was based on the following criteria, as proposed by Hanifin and Lobitz (2): a present and past history of focal or generalized lichenified, pruritic, eczematous skin rash with a predilection for the flexural areas and/or face and neck: a personal or family history of atopic. “allergic” disease: white dermographism; elevated serum IgE levels (23). All patients had typical clinical features of AD at the time of the study. Only 3 patients had a past history of infection (eczema herpeticum in one case; staphylococcal or fungal infections in three cases). Only one had a present history of skin infection when the study was performed. Serum IgE levels were high in every case of the study. Total peripheral blood (PB) lymphocytes counts were always within normal ranges (according to ages) and no decreased total PB lymphocytes counts was ever noted. The patients had not been on oral corticosteroids for at least 1 year before the study (children had never been on oral corticosteroids). They had received no antihistamines or other systemic therapy for at least 1 week, and had utilized no topical corticosteroids for at least 2 weeks before the time of the study. Controls were 30 healthy volunteers, with the same proportion of males and females, receiving no therapy. They were 20 staff members of our department and other volunteers, ages ranging from 15 to 50 years (group I) and 10 teenagers or children with ages ranging from 2 to 15 years (group 2). T-Cell studies could not be performed in controls with ages lower than 2 years, for an easily understandable ethical point of view. None had high serum IgE levels. Monoclonal antibodies. Monoclonal antibodies (Ortho Pharmaceutical Corp.. Raritan, N.J.), directed against various human T-cell antigens, were produced as already described (24, 25) by mouse hybridomas, obtained from cell fusion between mouse myeloma cells and normal spleen cells from mice immunized with human T lymphocytes. Previous studies have demonstrated that some of these antibodies recognize all peripheral T cells among PB lymphocytes, whereas others react with different PB T-cell subsets (22). Three monoclonal antibodies named OKT3, OKT4, and OKT8 were used: OKT3 reacting with all peripheral T cells, OKT4 identifying only peripheral T cells with helper-inducer function, and OKT8 directed against a T-cell subset with both suppressor and cytotoxic activity (26, 27). Erlutneration of lymphocytes subsets \r-ith tnonoclonal untihodies. Peripheral blood mononuclear cells were isolated by Ficoll-Paque (Pharmacia, Uppsala.
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Sweden) density centrifugation. After washing, the cells were suspended in RPM1 1640 (Eurobio, Paris, France) supplemented with 5% fetal calf serum (Gibco, Grand Island, N.Y.) and 25 m&f Hepes (Eurobio, Paris, France) at a concentration of 5 X lo6 cells/ml. Ten microliters of each reconstituted antibody (OKT3, OKT4, OKT8) was incubated with 200 ,ul of the cell suspension in hemolysis tubes and left for 30 min at 4°C in an ice bath. After washing, the pellet was added with 100 ~1 of fluoresceinconjugated goat antimouse IgG antibody (Nordic Immunological Lab., Tilburg, The Netherlands; fluorescein concentration, 10 mg’ml; F/P ratio, l-4), which has been diluted 1: 10 in RPM1 1640. Cells were left again for 30 min in an ice bath at 4”C, then washed and resuspended in medium. The percentage of fluoresceinlabeled cells was established by counting fluorescent cells on a Leitz Orthoplan microscope (E. Leitz, Wetzlar, FRG) and comparing to the total number of cells. Statistical methods. Statistical analysis was carried out using Student’s t test, with P < 0.05 indicating significant difference. RESULTS
Enumeration of PB T cells. Table 1 shows the percentages of T cells (OKT3+) in patients with AD compared to normal controls. As can be seen, in group 1 and 2, total T cell counts were lower in patients with AD (58.8 + 8.8 and 59.0 + 5.5%, respectively), than in controls (69.4 2 11.1% and 66.3 + 8%, respectively). These differences were statistically significant, with F < 0.02 in group 1, and P < 0.05 in group 2. No significant difference was observed between results in groups 1 and 2, nor between these two groups of atopic patients and group 3 patients where the OKT3+ cells accounted for 58.7 5 7.7% of PB mononuc!ear cells. Enumeration of helper and suppressor T-cell subsets. The balance between the
PERIPHERAL
BLOOD INDIRECT
TABLE 1 T-CELL ENUMERATIONS IN ATOPIC DERMATITIS IMMUNOFLUORESCENCE (IIF) WITH ANTI-HUMAN MONOCLONAL ANTIBODY (OKT3)
Patients and controls groups
Number of cases
Group 1 (15-50 years) AD” Normal subjects
I5 20
58.8 -+ W 69.4 + I 1.1 (P .: 0.02)
Group 2 (2- 15 years) AD Normal subjects
12 10
59.0 + 5.5 66.3 k 8.0
Group 3 CO- 2 years) AD
13
58.7 k 7.7
(z Atopic b Mean
dermatitis. + SE.
Percentage peripheral
(AD) ANALYZED T-CELL
OKT3 positive cells among blood mononuclear cells
(P .< 0.05)
BY
142
FAURE
ET AL.
two major T-cell subsets, OKT4+ and OKT8-t cells, was strongly altered in patients with AD, as seen in Table 2. Lower percentages of OKT4 and OKTS+ cells were observed in groups 1 and 2, as compared to control subjects of the same age groups. The decrease in the percentages of OKT8+ cells was highly significant (27.4 k 7.7%, controls: 41.9 + 12.7%, P < 0.002 in group 1: 22.3 k 4.7%, controls: 30.0 t 9.7%, P < 0.02 in group 2) and higher than what was observed with OKT4+ cells. These changes appeared still more clearly when the OKT4-OKT8 balance was expressed by the ratio between the percentages of OKT4+ and OKT8+ cells (helper/suppressor ratio). Such calculation eliminated the fluctuation of OKT4+ and OKT8+ cells percentages due to variable content of PB mononuclear cells in monocytes (28, 29). Furthermore, some overlap between the two OKT4+ and OKT8+ subpopulations (as we have previously reported with control studies when the cells were reacted in the same experiment with the two antibodies OKT4 and OKT8 (29)), is also eliminated in the determination of helper/suppressor balance when expressing it by the OKT4/OKT8 ratio. As also seen in Table 2, these OKT4/OKT8 ratios were increased in each group of patients with AD, as compared to controls: 1.82 t 0.52, controls: 1.38 ? 0.37 in group 1; 2.01 t 0.37, controls: 1.59 k 0.26 in group 2. with p r 0.02; in group 3, the OKT4/OKT8 ratio was similar (2.02 t 0.67). with a decrease in the OKT8+ cell subset similar to the results in the two other groups. These results indicate a T helper/suppressor imbalance with a decreased T TABLE 2 PERIPHERAL B~oou HELPER AND SUPPRF~SOR T-CFI.I. tOKT4t %NL) OKTg+ CEI tsi ENUMF.RATIONS AND BALANCES IN AD ANALYZED BY IIF WITH AN I‘I-HUMAN T-SUBSET MONOCLONAL ANTIBOIX~S tOKT4 ANI) OKTS) Antibodies t’J positive cells among peripheral blood mononuclear cells) Patients and controls groups
Number of cases
Group I (15-W years) AD” Normal subjects
I5 20
46.5 -t 7.1” 54.4 -+ 10.8 (P < 0.03
‘7.4 T 7.7 41.9 -t 12.7 (P c- 0.002)
I .82
Group 2 (2~ 15 years) AD Normal subjects
12 IO
43.6 i- 8.0 46.4 i- 10.3 NS”
22.3 -* 4.7 30.0 2 9.7 (P -: 0.02)
2.01 f 0.37 I .5Y -c 0.26 (P -= 0.02)
Group 3 (O-2 years) AD
13
49.5 f 9.x
26.5 -t 8.1
2.02 2 0.67
‘I Atopic dermatitis. * Mean -r SE. ” Nonsignificant
OKT4
OKTX
Ratio ‘;OKT4+/VOKT8+
z 0.52h
1.38 i 0.37 (P i 0.02)
STUDIES
ON T-CELL
SUBSETS
IN ATOPIC
143
DERMATITIS
suppressor-cytotoxic subpopulation in peripheral blood of patients with atopic dermatitis. No significant difference was observed in the enumeration of OKT3 + , OKT4+, and OKT8+ cells or in the OKT4/OKTS balance between males and females or according to the clinical history (intensity and severity of skin lesions, presence or absence of infection, etc). Because a decreased suppressor cell population may allow increased IgE production (30-32) in atopy, we tried to correlate this helper/suppressor imbalance with serum IgE levels in our atopic patients. As seen in Fig. 1, no positive correlation was observed between serum IgE levels and the values of OKT4+/OKT8+ cell ratios. Furthermore, no inverse correlation was observed between the percentages of OKT8+ suppressor-cytotoxic cells and serum IgE levels. DISCUSSION
We have studied in patients with atopic dermatitis and in normal controls T-cell markers as recognized in indirect immunofluorescence on cell suspensions by monoclonal antibodies specific for T lymphocytes, helper-inducer T cells, and rh Ratio
%OKT4+/%0KT8+
. .
.
. .
.
5--
.
.
. l
. .
’
.
l*
.
l *
.
. .
,5-, .
.
.
.
.
0 FIG. 1. Correlation (%OKT4+/%OKT8+ 1 and 2).
10
‘ 20I
t 50
I 100
I
I * 500 1000
I ’
, 5oob 1o’oon
between serum IgE levels and circulating cells ratios) in patients with atopic dermatitis:
I a
I 50 600’
I@
helper/suppressor no correlation
(
&‘I,
T-subset is observed
balance (Groups
144
FAURE
ET
AL
suppressor-cytotoxic T cells in man. Similar studies with the same monoclonal antibodies have previously shown significantly decreased percentages of suppressor T cells in patients with lepromatous and borderline leprosy (28) and, in the Sezary syndrome, a large increase in helper T cells with a parallel decrease in suppressor T lymphocytes (29, 33, 34). Our present data clearly document in patients with atopic eczema: (1) a decrease in the PB OKT3+-T lymphocyte population, and (2) an abnormal PB T-cell distribution between OKT4+ -helper and OKT8+ -suppressor T cells. with decreased percentages of suppressor cells. These results are in agreement with several other reports of a defective cellmediated immunity in atopy. Patients may have a decreased delayed cutaneous hypersensitivity to natural antigens (3.5) and a decreased rate of sensitization to experimentally applied antigens (36, 37). In vitro evaluations for T lymphocytes functions, and determinations of PB T cells counts have documented significally decreased rosette-forming cells levels compared to normals ( I I). and decreased lymphocytes transformation to T-cell mitogens (8, IO, 12. 14- 18, 38) and lymphokine production by T cells after T-cell-mitogen stimulation (39). Furthermore, our finding of a decreased proportion of PB OKT8+ suppressor-cytotoxic T cells correlates other observations of PB suppressor T cell defect in atopic disease (30, 38, 40, 41). Low suppressor cell counts have been documented on the basis of reduced relative levels of T cells bearing a receptor for the Fc fragment of IgG (T y subset) in patients with atopy (40). Abnormal irr \‘itro suppressor cell activity was recently observed, with a defective concanavalin A-inducible suppressor cell function as measured by the proliferative responses to pokeweed mitogen (41). These findings and the present data with monoclonal antibodies as markers of T-cell subsets, raise the question of the exact meaning of the T-cell subsets imbalance in atopy. Several studies have shown that depressed T-cell-functions balance and relative counts may be correlated with serum IgE levels (12, 14, 15. 18. 20). It has been postulated that excessive IgE antibody responses found in atopic diseases may be due to impaired suppressor cell function (30-32). On the other hand, it has been suggested that IgE may modulate immune reactions such as T-cell activity (42). In contrast with previous studies of defective suppressor T-cell functions (41). we failed to observe a correlation between supressor cell levels (determined as OKT8+ cells) or OKT4+/OKT8+ cell ratios, and serum IgE levels in our patients with atopic dermatitis. This difference in results may be due to the difference in the methods used for the evaluation of the T-cell suppressor subset, or, more likely, to the fact that all our patients with AD had elevated serum IgE levels prior to, and at the time of the study. In summary, patients with AD and high serum IgE levels appear to have decreased percentages of peripheral blood T lymphocytes, and a helper/suppressor T-cell imbalance, with a decreased suppressor-cytotoxic T-cell subset, as established using monoclonal antibodies specific for T cells and T-cell subsets. These monoclonal antibodies represent a new, easy, and useful tool for the determination of T-cell subsets balance and functions in various disorders.
STUDIES
ON
T-CELL
SUBSETS
IN ATOPIC
145
DERMATITIS
ACKNOWLEDGMENT The authors CILAG-FRANCE,
wish
to thank Mrs. J. Bielsky for statistical analysis. This work affiliate of ORTHO P.C., and in part by UER GB Bl 1981.
was supported
by
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FAURE
37. 38. 39. 40.
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July 9. 1981: accepted
August
X. 1981.