Enumeration of T cell subsets in atopic dermatitis using monoclonal antibodies

Enumeration of T cell subsets in atopic dermatitis using monoclonal antibodies Donald Y. M. Leung, M.D., Ph.D., Arthur Raif S. Geha, M.D. Boston, Muss.

R. Rhodes,

M.D., and

Peripheral blood lymphocytes from 22 patients with atopic dermatitis, 17 age-matched healthy controls, 10 patients with other skin diseases, and 14 patients with either asthma or allergic rhiniris were characterized by reactivity with monoclonal antibodies to the sur$ace antigens of helper-inducer (T4) and suppressor-cytotoxic (T8) T cell subsets and to a common T cell antigen (T3). In contrast to healthy conrrols and controls with other skin diseases or respiratory allergic disease, patients with atopic dermatitis had a reduced percentage of T3-positive (T3+) cells (p < 0.01) and T8-positive (T8+) cells (p < 0.001) but not of T4-positive cells (T4+) (p > 0.05). A selective increase in the ratio of T4+ cells over T8+ cells was observed in I7 of 22 patients with atopic dermatitis but nor in any of the controls. Thus there is a loss of circulating suppressor-cytotoxic T cells in the majori of patients with active atopic dermatitis.

It has been suggested that a deficiency of suppressor T cells, resulting in increased IgE production, may underlie the atopic diathesis in humans. The evidence supporting this hypothesis is derived from various studies in experimental animalP5 and in human@ ’ demonstrating elevated IgE levels in disease states associated with suppressor T cell deficiency. Recently several investigators have reported that IgE synthesis in vitro by lymphocytes from atopic individuals is inhibited on coculture with T cells from normal individuals. 8, SJ Atopic dermatitis is a skin disease associated with increased serum IgE levels in up to 80% of patientslO* l1 and with impaired T cell function.“-l4 Thus it has been postulated that these patients have a suppressor cell dysfunction accounting for their elevated serum

From the Divisions of Allergy and Dermatology, Department of Medicine, Children’s Hospital Medical Center, the Department of Pediatrics of Harvard Medical School. Supported by United States Public Health Service Grant AI-05877 and grants from the Hood Foundation, the National Foundation, March of Dimes, and the Dooner/Rorer, Co. Dr. hung is the recipient of National Research Service Award lF32AIO6053-01. Received for publication Dec. 1, 1980. Accepted for publication Feb. 19, 1981. Reprint requests to: Donald Y. M. Leung, M.D., Division of Allergy, Children’s Hospital Medical Center, 300 Longwood Ave., Boston, MA 02115. Vol. 67, No. 6, pp. 460-455

IgE and possibly their skin disease. Recent studies evaluating concanavalin A-inducible suppressor cell activity in atopic dermatitis15 and in allergic rhinitis and asthma16 have concluded that suppressor cell activity is normal in such patients. However, such studies may not be sensitive enough to pick up a subtle deficiency in suppressor T cells. Recently monoclonal antibodies have become available for the identification and isolation of specific human T lymphocyte subpopulations.17 In this study we used monoclonal antibodies to the cell surface antigens of helper-inducer (T4) and suppressorcytotoxic (T8) T cell subsets and to a common T cell antigen (T3) defining all peripheral T cells.1*-20 We found that the majority of patients with atopic der-

matitis have a selective deficiency of suppressorcytotoxic T cells in their peripheral blood. In contrast patients with allergic rhinitis, asthma, and other diffuse nonatopic skin disease did not have a loss of circulating suppressor T cells. MATERIALS Subjects

AND METHODS

The sample population consisted of 22 patients with atopic dermatitis seenin either the Dermatology or the Allergy Clinic at the Children’s Hospital Medical Center, Boston. The diagnosis of atopic dermatitis was made on the following basis: (1) pruritus, (2) focal or generalized skin eruption marked by erythema, papules, or lichenification with a predilection for the flexural areasof the extremities or

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VCJLUME 67 N1lMBER 6

TABLE control -

T

I. Summary subjects

of clinical

and immunologic

findings

in patients

cell subsets in atopic dermatitis 451

with atopic

dermatitis

and in

wllctmya-et Number of @ents

Dllese

Ait. pit dermatitis Respiratory allergy &her skin disease Het&hy controls

22

14 10 17

Sex Meanage (yr)*

(%I?

p M

F

OKT3

OKT4

9 (1 to 30) 15 7 60 * 6$ lO(3to24) 9 5 63k6 12(2to26) 4 6 6626 17(1 to34) 11 6 66 r 5

OKT8

35 _’ 6

11 t 4f

3557 35 3- 7 37 3- 3

2124 20 2 4 22 -c 4

Meenwrm.IgE wswlP

Mslnfaement skhl imfe~meilt*

3055 (7 to28,OOO) 28 (10 to 60) 452 (36 to 1,300) 0 76 (5 to 300) 20 (10 to 40) 65 (5 to 330) 0

*N~&ers in parentheses denotethe range. tRmults areexpressedas mean + standarddeviation. SSi$ilificantdifferences(p < 0.05). face and neck in older children and adults or a predominantly facial and extensor involvement in infants, (3) tendemy toward chronic or chronically relapsing dermatitis, and 14) a personal or family history of atopic dermatitis or otha;. atopic disorders.‘-I TElreeage-matched control populations wefe studied: 17 heaiirhysubjects, 10 patients with skin rashes unrelated to atopic dermatitis but who were matched for the extent of skin involvement with the patients with atopic dermatitis, and ‘4 atopic patients with allergic rhinitis or asthma. In this idtter group five patients had a previous history of atopic derm&tis. FW this study allergic rhinitis was defined as sneezing, rhinomhea, or nasal obstruction associated with positive skin iests with inhalant antigens. Asthma was defined as recumentparoxysmal dyspnea with wheezing, responsiveto bromtiodilator treatment irrespectiveof the presenceor absence of known anergens. NW of the individuals studied had received oral steroids for at least 6 mo prior to the time of the study or topical corticjt,steroidsfor at least 2 wk prior to the study. Antihistamiqs and other systemic therapy such as bronchodilators or aeibiotics were withheld for the 72 hr preceding the study, The extent of skin disease in patients with atopic derm&itis and in the population of patients with nonatopic skin diseasewas quantitated by determining the percentage of body involvement basedon the rule of nines.*l The extent of skin diseasein these two patient populations was similar and ranged from 10% to 60% skin involvement.

n of lymphmytes Hueian peripheral blood mononuclear cells were isolated from bparinized venous blood by means of Ficoll-Hypaque gradieirt density centrifugation (PharmaciaFine Chemicals, Piscatsiway,N. J.). 22 After washing the cells three times with I&nks’ balanced salt solution (HBSS), unfractionated cells wicresuspendedin RPMI-1640 (Gibco, Grand Island, N. Y .I with 20% fetal calf serum (FCS) and incubated overnight in plastic Petri dishes (Falcon Plastics, Oxnard, Calif.) .st 37” C in 5% CO* to deplete monocytes. Nonadherent @ellswere collected by rinsing the plates with HBSS, centrifuged and resuspendedat 5 X lo6 cells/ml in RPhfI-

1640 medium supplemented with 5% he&nactivated FCS and 0.1% sodium azide. Cell suspe&ons were placed on ice prior to treating the lymphocytes with monoclorral antibody (seebelow). Preliminary experiments have shown that overnight incubation of peripheral bIood mononuclear cells did not alter the distribution of T cell subsets identified by monoclonal antibodies.

Reagents. Monoclonal antibodies anti-T3 (OKT3), antiT4 (OKT4), and anti-T8 (OKTg) ware obtained from Ortho Pharmaceutical Corp. (Raritan, N. J.). These antibodies have been previously shown to react otiy with T Jymphocytes.‘7-20OKT3 reactswith 100% of peripheml T cells. In contrast the T4 antigen is expressedon a~~~~~y 55% to 65% of peripheral T cells, and the T8 a on 20% to 30% of peripheral T cells. defines the human inducer-heiper T cell subs@a& OKT8 defines the suppressor-cytotoxicpopu&itia. ‘i&e Jr&&rsubset corresponds to the previously described TN,+ suppressor cellsz3and contains the c alin A-inducible suppressor cells. Fluoresc&n-c goat a&mouse IgG (G/M FITC) was obtained from tipple Laboratories (Cochranville, Pa.). ImmunvJluvrescence . Analysis of lymphocyte pop~lations was performed by means of inditeet i~unoiftuorescence. Lymphocytes, 1 x 106, in 0.2 ml RPMI medium supplemented with 5% FCS and 0.1% sodium atide (wash medium) were treated with 5 to 10 ~1 reconstituted monoclonal antibody solution in an ice W&W bath for 40 min. Then the cells were washed twice with wash medium suld resuspendedin 0.1 ml wash medim. One-ten& milliliter 1: 10 dilution of G/M FITC was then ad&d. After 30 min the cells were washed three times and ~~~~~d in wash medium supplemented with 3 mM e~b~~d~~~~~acetic acid (EDTA), and the fluore~ent e&s were counted with the aid of a Zeissfluorescentmic ley, Mass.). Background staining w ing the lymphocyte suspensionwith a 1: I~d~l~on of G/M FITC after incubation with mouse as&c &id. Uniformly less than 2% of the cells exhibited fluorescent staining with

J. ALLERGY CLIN. IMMUNOL. JUNE 1981

452 Leung et al. TABLE

II. Analysis

of lymphocyte

populations Reactivity

Patient

2 3 4 5 6 7 8 9 10 11 I2 13 14 15 16 17 18 19 20 21 22

Age (vr)

Sex

12 2 7 4 1 2 5 16 8 14 7 12 2 30 16 2 2 11 10 21 6 4

M M M M M M M F M F M M F F M M M F F M M F

OKT3

50 55 55 65 68 65 70 62 57 64 60 59 57 55 63 59 63 50 59 54 70 54

with antibodies OKT4

in patients

OKT8

30 38 32 56 37 34 40 36 32 39 30 27 29 31 36 32 33 35 41 33 33 35

IgE determination Serum IgE levels were measured by the Phadebaspaper disk radioimmunoassay techniques (PRIST) (Pharmacia). Results are expressedas international units (NJ) per milliliter according to standards provided by the manufacturer.

methods

Statistical analysis was performed by Student’s t test. Results on lymphocyte reactivity with monoclonal antibodies are expressedas mean & 1 SD.

RESULTS The distribution of T cell subsets in peripheral blood lymphocytes from patients with atopic dermatitis, healthy controls, patients with asthma or allergic rhinitis, and patients with nonatopic skin disease is presented in Table I. The normal lymphocyte population from age-matched controls has 66% + 5% peripheral T cells as determined from the reactivity of their lymphocytes with OKT3 antiserum. In addition 37% + 3% of normal lymphocytes reacted with the OKT4

atopic

dermatitis

monoclonal (%I

the reagent control. All analyses were performed without knowledge of the patient’s clinical status.

Statistical

with

antiserum, which defines helper-

inducer T cells, and 22% + 4% of the normal lynaphocytes reacted with the OKT8 antiserum, which defines

0

7 17 10 8 11 11 13 13 13 6 7 6 13 19 14 8 8 10 12 15 10

W Wlml)

3600 30 150 28000 370 42 4700 25 400 3000 5300 7000 130 370 7000 7 3800 790 68 2000 190 250

skin

Percent involved

40 30 10 30 10 10 40 40 10 40 50 60 30 30 50 20 60 30 10 30 20 10

Associated

disease

Asthma Asthma Asthma Asthma

Asthma Asthma

Asthma

Asthma Asthma, allergic rhinitis

suppressor-cytotoxic T cells. Controls under the age of 16 yr had T cell subset profiles similar to those of controls over the age of 16 yr (data not shown). The distribution of T cell subsets among normal lymphocytes observed in this study is similar to that previously described by other investigators. l7 The clinical characteristics and the lymphocyte subset profiles of individual patients with active atopic dermatitis are shown in Table II. The lymhocyte population from these patients has 60% k 6% T3+ cells, 35% +- 6% T4+ cells, and 11% k 4% T8+ cells (Table I). Compared with healthy controls, patients with atopic dermatitis had a statistically significant reduction in circulating T3+ total T cells (p < 0.01) and T8+ suppressor-cytotoxic T cells (p < 0.001) but no significant reduction in circulating T4+ cells (p > 0.05). The percentage of T8+ cells in 18 of 22 patients with atopic dermatitis were more than 2 SD below the mean of normal controls. In contrast no statistical difference (p > 0.1) in the percentages of lymphocytes reactive with OKT3, OKT4, or OKTg was observed when patients with nonatopic skin diseases or patients with allergic rhinitis or asthma (Table I) were compared with healthy controls. The clinical characteristics and lymphocyte subset profiles of patients with nonatopic skin dis-

V:)LUME 67 NilMEER 6

T cell

T&EbLEIll. Analysis of lymphocytes

subsets

in atopic

-,

-..

Peti#m 1 2 3 4 5 6 7 8 9

16 15 13 26 14 6 4 8 2

10

15

sex

Percent skin invdwed

Di-

F F F F F M F M M M

Dyshidrotic eczema Pyoderma Contact dermatitis Psoriasis Psoriasis Pyoderma Nummular eczema Tinea corporis Urticaria pigmentosa Acne vulgaris

TA%I,E IV. Analysis of lymphocyte -.

populations

wftll Frkawetonel

~~~

(%I

,iJFEl,

DKT3

#CT4

DKT8

IO

29

40 20 20

105

52 68 69 71 6.5 65 61 73 71 65

38 29 30 36 2s 33 31 41 48 41

22 19 16 17 I6 27 22 21 16 25

105 46 5 82 37 9 46 300

30 20 20

10 15

10

in patients with respiratory allergic d R

Pleimt 1 2 3 4 5 6 7 8 9 10 11 12 13 P..

14

t!z 9 3 15 6 24 29 2 4 7 5 4 17 7 6

453

in patients with other skin diseases R&utty

Age (Yr)

dermatitis

Q#

Sex

Disease

M M F F F M M M M M M F F M

Allergic rhinitis Allergic rhinitis Allergic rhinitis Allergic rhinitis Allergic rhinitis Allergic rhinitis Asthma Asthma Asthma, allergic rhinitis Asthma, S/P AD Asthma, S/P AD Asthma, S/P AD Allergic rhinitis, S/P AD Allergic rhinitis, S/P AD

Wlmif

el

OK73

Qlm

740 330 74 58 68 60 36 430 300 1300 300 280

57 54 74 67 60 70 60 69 60 53 69 65

loo0 1350

61

25 30 43 41 39 47 28 42 36 24 29 38 39 32

64

OK-f% 20 21 24 24 23 21 12 23 17 20 27 18 21

21

S/P Ad = pastmedicalhistoryof atopicdermatitis.

eases and patients with asthma or allergic rhinitis are shown in Tables III and IV. Among both groups of s, in only one patient with asthma (Table IV, patient 7) did the percentage of circulating T8+ cells (lZ%,b fall between 2 and 3 SD below the mean of healthy controls. It is of interest that five patients with allergic rhinitis or asthma (Table IV, patients 10 to 14) hqd a documented past medical history of atopic tis but had no skin disease at the time of the study ;d9ndwere found to have normal T3-t and T8+ lymp&zyre distributions. A selective decrease in T8+ cells in patients with atopic dermatitis was further apparent from analysis of ratios of T4+ to T8+ cells: in normal controls the ratio ok T4+ to T8+ cells was 1.75 rtl 0.3; As shown in Fig, I, in patients with atopic dermatitis the ratio was 3 6 it 1.1; and in 17 of 22 patients the ratio

exceeded 2.5 : 1, or greater than 2 SD above the mean

T4+ to T8 + ratios found in healthy controls. These abnormalities were seen in only one of tbe EOpatients with other skin diseases and not in any of the 14 patients with asthma or allergic rhinitis or in any of 17 normal controls. No relationship could be established between the degree of T8+ depression, the serum IgE Ievels, or the extent or type (exudative vs li~~~i~) of skin involvement in patients with atopic dermatitis (Tables II and III). D

N To date the status of suppressor T cells in atopic dermatitis has been controversial. The ma&B obtained in our study clearly indicate that active atopie dermatitis is associated with a selective reduction in

454

J. ALLERGY CLIN. IMMIJNOL. JUNE 1981

Leung et al.

6:

J $5P P : i3-

-

: : :. ; .:.

0

!

I= p-

l

:

. l-

I

N=22

ATOPIC DERMATITIS

N=lO

OTHER SKIN DISEASES

N-14

‘RESPIRATORY ALLERGIC DISEASES

I I I 1 I / 1 I I 1

. N=l7

HEALTHY CONTROLS

FIG. 1. Ratio of T4+ to T8+ cells in control subjects and patients with atopic dermatitis. The mean + 1 SD is shown for each group.

the subset of circulating suppressor-cytotoxic T cells as defined by the recently developed mouse monoclonal antibody OKTS. It has been previously shown by Reinherz et al. 17,*O that the subset of T cells reacting with OKT8 includes the conconavalin-inducible suppressor T cells as well as the precursors of cytotoxic T cells such as those that arise in response to allogeneic stimulation in vitro. At the present time there are no readily available markers that distinguish between precursors of suppressor cells and cytotoxic cells. Thus it is not known whether these precursors are indeed different. It is also not known whether the observed deficiency in T8+ cells in patients with atopic dermatitis reflects a deficiency of suppressor as well as cytotoxic cells. To resolve this issue we are currently using a monoclonal antibody to human cytotoxic T cells developed by Dr. Steven Burakoff (Sidney Farber Cancer Institute) to enumerate the number of cytotoxic T cells generated from the circulating T cells of patients with atopic dermatitis following stimulation with alloantigens. The observed deficiency of T8+ cells in patients with atopic dermatitis did not appear to be related to nonspecific skin inflammation, inasmuch as this finding was not observed in patients who suffered from nonatopic skin diseases matched for the extent of skin involvement. It was also found that the deficiency of T8+ cells observed in atopic dermatitis did not correlate with the elevation of serum IgE levels in individual patients with atopic dermatitis

(Table II). This lack of correlation does not negate a role for T8+ cells in the regulation of IgE synthesis. Indeed we have recently shown that T8+ cells can suppress IgE synthesis in vitro.24 It only suggests that the T8+ cells involved in IgE regulation either constitute a small proportion of the T8+ subset or that in some cases they may be present in the circulation but are functionally deficient. The number of circulating T8 + cells was decreased in most patients with active atopic dermatitis (18 of 22 patients) and were normal in five patients who gave a past medical history of atopic dermatitis but exhibited no active skin disease at the time of the study (Table IV). This observation suggests an association between active atopic dermatitis and deficiency of circulating T8+ cells, but further studies are required to substantiate this hypothesis. The exact relationship between a deficiency of suppressor T cells as demonstrated in this study and atopic dermatitis is not clear. In vitro experiments in humans and laboratory animals suggest that under normal circumstances suppressor T cells play an important role in supressing the cytotoxic activity of lymphocytes against autologous skin fibroblasts.25, 26 Our laboratory is currently investigating the possibility that a loss of suppressor cells in atopic dermatitis results in an immune-mediated skin injury by naturally occurring autocytotoxic lymphocytes. The observation that the skin lesion in atopic dermatitis is infiltrated by mononuclear cells2’* 28 and preliminary results from our laboratory showing cell-mediated autocytotoxicity against skin fibroblasts29 tend to support this hypothesis. More studies are needed, however, to determine whether the immunologic abnormalities we found in atopic dermatitis are primary or secondary events in the course of this disease. We wish to thank Thomas Gesnerfor technical assistance and Melissa Smith for secretarial assistance. REFERENCES 1. Tada T: Regulation of reaginic antibody formation in animals. Rag Allergy 19122, 1975. 2. Tada T, Taniguchi M, Okumura K: Regulation of homocytotropic antibody formation in the rat. II. Effect of X-irradiation. J Immunol 106:1012, 1971. 3. Okumura K, Tada T: Regulation of homocytotropic antibody formation in the rat. IV. Inhibitory ef&ect of thymocytes on homocytottopic antibody response. J Immunol 187~1682, 1971. 4. Ishizaka K: Cellular events in the IgE antibody response. Adv Immunol 23~1, 1976. 5. Katz DH, Hamaoka T, Neuberger PE, Benacerraf B: Haptenspecific IgE antibody responses in mice. IV. Evidence for distinctive sensitivities of IgE and IgG B lymphocytes to the regulatory influences of T cells. J Immunol 1132974, 1974. 6. Geha RS, Rappaport JM, Twarog FJ, Parkman R, Rosen FS:

WI&ME 67 I-WblBEfi 6

Increased serum IgE levels following allogeneic bone marrow transplantation. J ALLERGY CLIN IYMUNOL 66:78, 1980. I. Waldmann TA, Pohnan SV, Balestra ST, Jost MC, Bruce RM, Tetry WD: Immurmglobulin E in immunologic deficiency diseases. J Immunol lti304, 1972. +. Fiier PM, Buckley RH: Human IgE biosynthesis in vitro: Studies with atopic and normal blood mononuclear cells and subpopulations. J Immunol 123:1788, 1979. 1:. Saxon A, Morrow C, Stevens RH: Subpopulations of circulating B cells and regulatory T cells involved in in vifro immunoglobulin E production in atopic patients with elevated serum immunoglobulin E. J Clin Invest 65:1457, 1980. I(; Juhlin L, Johansson SGG, Bennich H, Hogman C, Thyresson N: Immunoglobulin E in dermatoses: Levels in atopic dermatitis and urticaria. Arch Dermatol 100~12, 1969. 1;. Byrom NA, Timlin DM: Immune status in atopic eczema: A survey. Br J Dermatol lOOr491, 1979. 1:: Lobitz WC Jr, Honeyman JF, Winkler NW: Suppressed cell mediated immunity in two adults with atopic dermatitis. Br J Dermatol g&317, 1972. 13. McGeady SJ, Buckley RH: Depression of cell-mediated immunity in atopic dermatitis. J ALLERGY CLIN IMMUNOL 96:393, 1975. 14 Hanifin JM, Lobitz WC Newer concepts of atopic dermatitis. Arch Dermatol 113~663, 1977. 15. Schuster DL, Pierson D, Bongiovanni B, L-evinson AI: Suppressor cell function in atopic dermatitis associated with elevatedimmunoglobu1inE.J ALLERGY CLIN~MMUNOL~~Z~~~, 1979. 16. Martinez JD, Santos J, Stechschulte DJ, Abdon NI: Nonspecific suppressor cell function in atopic subjects. I ALLERGY CLIN ,MMUNOL64:485, 1979. 17. Reinherz EL, Schlossman SF: Regulation of the immune reponse: Inducer and suppressor T-lymphocyte subsets in human beings. N Engl J Med 303:370, 1980. 18. i
T cell

subsets

in atopic

dermatitis

455

19. Reinherz EL, Kung PC, Goldstein G, Schlossman SF: Further characterization of the human inducer T cell subser defined by monoclonal antibody. J Immunol 123~2894, 1979. 20. Reinberz EL, Kung PC, Goldstein 6, Sclliossman SF: A monoclonal antibody reactive with tbe human cytotoxic/ sup pressor T cell subset previously defined by a heteroantiserum termed THr. J Immunol 124~1301, 1980. 2 1, Evans EI: Fluids and electrolytes requirements in severe bums. Am J Surg 13;5:804, 1952. 22. Thor&y E, Bra&e A: A rapid method for meparation of pure lymphocyte suppressions, in Terasaki, P, editor: Histocompatibility testing. Baltimore, 1970, The Williams & Wilkins Co., p. 655. 23. Evans RL, Lazarus H, Penta AC, Schlossman SF: Two functionally distinct subpopulations of human T cells that collaborate in the generation of cytotoxic cells mspon5ibie for cell mediated lympholysis. J Immunol 120:1423, 1978. 24. Geha R, Reiuberz E, Leung D, Schlossman S, Rosen F: Suppressor T cell deficiency in the hyper IgE syndrome. Clin Res (In press.) 25. Parkman R, Rosen FS: Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts. J Exp Med 144~1520, 1976. 26. O&and M, Parkman R: Lack of automactivity in murine spleens is due to concomitant presence of suppressor and autocytotoxic lymphocytes. J Immunol 121: 179, 1978. 27. Mihm MC, Soter NA, Dvorak HF, Austen KF: The structure of normal skin and the morphology of atopic eczema. J Invest Dermatol67:305, 1976. 28. Braathen LR, Form 0, Natvig JB, &f-Lament T: Predominance of T lymphocytes in the derma infiltrate of atopic dermatitis. Br J Dermatol lMk511, 1979. 29. Leung DYM, Parkman R, Feller J, Geha RS: Cell mediated autocytotoxicity against skin tibroblasts in atopic dermatitis. (In preparation. )