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Defect of CD2- and CD3-mediated activation pathways in T cells of atopic patients: Role of interleukin 2
CELLULAR
IMMUNOLOGY
139,9 1-97 (1992)
Defect of CD2- and CD&Mediated Activation Pathways in T Cells of Atopic Patients: Role of lnterleukin 2 MARIA FIAMMETTA ROMANO,*,' GIULIANA VALERIo,Jf MARIA CATERINATURCO,$GIUSEPPE SPADARO,~ SALVATOREVENLJTA,$ANDSALVATOREFORMISANO* *Dipartimento di Biologia e Patologia Cellulare e Molecolare, TDipartimento di Pediatria, SDipartimento di Biochimica e Biotecnologie Mediche, and SCattedra di Immunologia Clinica e Allergologia, II Facoltd di Medicina e Chirurgia, Napoli, Italy Received
May
1, 1991; accepted
July 25, 1991
In the present work we analyzed the proliferative response of T lymphocytes from 11 atopic patients stimulated in vitro via either the CD2 or the CD3 pathway of cell activation. In both caseswe found a significant decrease of thymidine incorporation in cell DNA in comparison with T cells from normal donors. The mechanism of this impaired proliferative response was analyzed. Atopic patients’ T cells were found to secrete low quantities of interleukin 2 (IL2) and to express low amounts of Tat antigen, measured as both a percentage of Tac-positive cells and a mean fluorescence intensity of Tat antigen per cell. Addition of recombinant IL2 to cultures completely restored both cell proliferative response and Tat antigen expression. This effect was specific of IL2 since addition of IL1 or IL4 did not significantly affect T cell proliferative response. We conclude that atopic patients’ T lymphocytes have a defect in both CD2 and CD3 pathways of cell activation relying on impairment of IL2 production, without involving IL2 responsiveness or other lymphokine defects. Q 1992 Academic PWSS, Inc.
INTRODUCTION A body of evidence suggests that atopy, the familiar allergic disorder involving an immediate type hypersensitivity to environmental allergens, is related to an impairment of T lymphocyte reactivity. Indeed, a decreased T cell proliferative response to bacterial antigens and/or an altered production of some T lymphocyte-derived cytokines in atopic patients has been reported by some authors (l-6). On the other hand, impairment of T cell reactivity in some clinical conditions (Nezelofs syndrome, WiskottAldrich syndrome, GVDH, Hodgkin’s disease) is often accompanied by developing of symptoms of atopy (7-9). T lymphocyte activation is mediated by two different pathways involving antigen receptor (Ti-CD3) or CD2 molecule. Triggering of one of these structures induces T cell transition from Go to Gr phase and production and response to interleukins 2 and 4: these in turn regulate Gr progression and entry in S phase (10). In this paper ’ To whom correspondence should be addressed at Dipartimento di Biologia e Patologia Cellulare e Molecolare (Servizio di Immunoematologia e Trasfusione), II Facolti di Medicina e Chirurgia, Via S. Pansini, S-80 13 1 Napoli, Italy. Fax: 39-8 l-209224. 91 0008-8749192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
92
ROMAN0
ET AL.
we describe an impairment of Ti-CD3- and CDZmediated pathways of cell activation in T lymphocytes of atopic patients and analyze the molecular defect responsible for this impairment in these cells. MATERIAL
AND
METHODS
Patients. We studied 11 atopic patients aged between 18 and 45 years (mean age 23 years). The diagnosis was based on a history of infantile eczema, clinical features, sometimes concomitant, (active atopic eczema, rhinitis, extrinsic asthma, conjunctivitis), positive intradermal responses to common allergens, and, in some cases, raised blood titers of IgE. The control group was comprised 18 healthy blood donors aged between 18 and 55 years (mean age 22 years). Neither the controls nor the patients had received local or systemic steroid therapy at least 4 weeks before blood collection. Cells. Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood of patients and controls by Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation (400g for 30 min), washed, and resuspended at the concentration of 4 X 106/ml in medium RPM1 1640 supplemented with 5% heatinactivated fetal calf serum. Proliferative assays. These were performed in 96-well microplates by incubating 4 X lo5 PBMC in a total volume of 200 ~1 containing CD3 (30 rig/ml) or CD2 (9.1, 10 fig/ml + Bil, 10 Kg/ml) monoclonal antibodies. After 48- or 96-hr incubations of CD3or CD2-stimulated cells, respectively, at 37°C in a 5% CO2 atmosphere, 0.5 &i of [3H]thymidine (3H-dT, sp act 47 Ci/mmol, Amersham International plc., Milan, Italy) were added to each well. After 4- and 8-hr incubations at 37°C for CD3 and CD2 cultures, respectively, cells were harvested on glass fiber strips and 3H-dT incorporation was measured in a beta counter. Each point was performed in triplicate. Results were accepted when the standard deviation was lower than 15% of the mean value. Monoclonal antibodies (MoAbs) and interleukins (ILs). The anti-CD3 MoAb OKT3, an IgG2a, was purchased from Ortho Diagnostic System, Milan, Italy. The MoAbs 9.1, an IgG3, and Bil, an IgG2a, two distinct determinants of the CD2 molecule, were kind gifts of Dr. Soo Young Yang, Memorial Sloan Kettering Cancer Center (New York, NY) and Dr. Soldano Ferrone, New York Medical College (Valhalla, NY), respectively. Recombinant IL 1 and IL2 were obtained from Biogen Medical Research (Geneva, Switzerland). Recombinant IL4 was a gift of Immunex Corporation (Seattle, WA). Fluorescein isothiocyanate (FITC)-conjugated anti-IL2 receptor MoAb was purchased from Becton-Dickinson (Milan, Italy). Zmmunojluorescence (IF). Direct IF was performed by incubating 100 ~1 of a cell suspension (2 X 106/rnl) with a saturating amount of FITC-conjugated anti-IL2 receptor MoAb for 30 min at 4°C. Cells were then washed and examined with a cytofluorograph (FACS-scan, Becton-Dickinson). Results were expressed as a percentage of positive cells. Negative controls were less than 1% of stained cells. The specific cell immunofluorescence intensity was expressed as channel numbers on a log scale. The mean fluorescence intensity was calculated by integrating the fluorescence histograms for the positive cells as compared with control unstained cells. IL2 assay. IL2 biological activity of supematants was determined by the ILZdependent stimulation of PBMC activated with an anti-CD3 MoAb for 4 days (blasts) (11). Briefly, PBMC from normal donors or patients were incubated with anti-CD3 MoAb; 24 hr later, supernatants were harvested and added to the blasts (250 X 103/
T CELL PROLIFERATIVE
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ml) in 96-well microtiter plates. Recombinant IL2 (from 50 to 1.6 U/ml) was separately added to the blasts as a control. Following a 20-hr incubation at 37°C in a 5% COZ atmosphere 0.5 PCi of 3H-dT was added in each well and after an additional 6-hr incubation cells were harvested on glass fiber strips and the incorporation was measured in a beta counter. Statistical analysis of the results. It was performed using the Student t test. RESULTS Proliferative response of PBMC of atopic subjects to CD3 and CD2 A4oAbs. To analyze T cell reactivity of atopic patients to stimuli which trigger the CD3 and the CD2 pathways of lymphocyte activation, we stimulated PBMC of patients and normal donors with CD3 and CD2 MoAbs and measured 3H-dT incorporation in cell DNA. As illustrated in Fig, 1, cpm values ranged from 3000 to 45,600 and from 18,000 to 90,000 in PBMC from atopic and normal subjects, respectively, stimulated with antiCD3 MoAb. T cells of atopic patients displayed a lower response to CD3 MoAb when compared to cells of normal donors (P < 0.01). When T cell response to CD2 MoAbs was analyzed, patients’ PBMC incorporation ranged from 1300 to 18,000, while healthy donors’ PBMC incorporation ranged from 1500 to 70,000 cpm (Fig. 1). A decreased reactivity of PBMC of atopic patients could be recognized (P < 0.01). To further evaluate the defect in atopic patients’ T cell proliferative response, we analyzed whether patients and controls displayed similar dose responses to stimulants. Representative results are illustrated in Fig. 2: T lymphocytes from atopic donors, displayed a lower proliferative response than controls’ T cells to the same anti-CD3 doses, either optimal or suboptimal. Eflects of diflerent cytokines on the proliferative response of PBMC of atopic patients to CD3 MoAb. Monocyte-derived IL1 is required for Go-G1 transition of activated T lymphocytes and IL2 synthesis while T cell-produced IL2 and IL4 are involved in progression of G, phase (10). To investigate the possible role of these cytokines in the impaired proliferative response of PBMC of atopic subjects, we stimulated the cells
PATIENTS FIG. I. Normal and atopic donors’ PBMC proliferative response to CD2 and CD3 MoAbs. PBMC from normal (controls) and atopic (patients) donors were incubated with anti-CD3 (Q or anti-CD2 (a) MoAbs and 3H-dT incorporation in cell DNA was measured with a beta counter. CONTROLS
ROMAN0
0
100
ET AL.
200
300
CD3 MoAb: rig/ml FIG. 2. Proliferative response to CD3 MoAb in PBMC from a normal donor and an atopic patient. PBMC from a normal donor (0) and an atopic patient (0) were incubated with different doses of MoAb OKT3 and ‘H-dT incorporation in cell DNA was measured with a beta counter.
in the absence or presence of exogenous interleukins 1,2,4 and measured 3H-dT incorporation in cell DNA. IL1 and IL4 did not display any significant effect on thymidine incorporation in PBMC of atopic patients, although they slightly increased cell proliferation of PBMC of normal donors (not shown). On the other hand a marked effect of IL2 on T cell DNA synthesis was detected in both normal donors’ and atopic patients’ PBMC preparations. Indeed, in the presence of this factor, an enhanced 3HdT incorporation was observed in CD3- and especially CDZstimulated normal PBMC and comparable values were obtained with PBMC from atopic patients (Fig. 3).
CD3 CD2 IL2
+ -
FIG. 3. Effect of recombinant IL2 (10 U/ml) on ‘HdT incorporation in CD3- or CM-stimulated PBMC. PBMC from 10 controls (@) and 10 atopic subjects (0) were stimulated with the indicated reagents and ‘HdT incorporation in cell DNA was determined. Results represent mean values + standard error.
T CELL PROLIFERATIVE
IMPAIRMENT
IN ATOPIC
PATIENTS
Efect of IL2 on Tat antigen expression in PBMC of atopic patients. A
95 decreased
responsiveness to IL2 in PBMC of atopic patients had been reported by some authors (5). Our results did not appear to be in agreement with those conclusions since atopic patients’ PBMC stimulated in the presence of IL2 displayed thymidine incorporation values comparable with those of normal PBMC (Fig. 3). However, to further analyze this point, we measured the expression of IL2 receptor p55 (Tat) in PBMC of normal donors and atopic patients stimulated with CD3 MoAb. Although Tat antigen expression of atopic patients’ PBMC was lower than that of normal PBMC, the addition of exogenous IL2 resulted in comparable values of both positive cell percentages and mean Tat intensities in the two groups (Fig. 4). Production of IL2 in atopic patients. Our results indicated that atopic subjects’ PBMC might present a defect in production of IL2. To assessthis point, we measured the presence of IL2 in supematants of atopic patients’ PBMC stimulated via CD3. As illustrated in Fig. 5, following cell stimulation with MoAb OKT3, PBMC of four normal donors secreted high quantities of IL2 (X = 10 U/ml), while PBMC of five different atopic patients produced very low amounts (X = 2 U/ml) of this interleukin. DISCUSSION In the present work we have investigated the ability of atopic patients’ PBMC to proliferate when stimulated through the two pathways of T lymphocyte activation, loo-
A
In 80 = a
-
o 60+ : I- 40 0 * 20I
Ol -I
10
1
-
L.2
I L-2
I + I L.2
I
1 + I L-2
FIG. 4. Effect of recombinant IL2 (10 U/ml) on Tat antigen expression in CD3-stimulated PBMC. PBMC from normal (0) or atopic (0) donors were stimulated with anti-CD3 MoAb (100 rig/ml) in the absence or presence of recombinant IL2 for 72 hr. Then cells were washed and analyzed for Tat antigen expression in IF. (A) Percentage of positive cells; (B) mean fluorescence intensity.
96
ROMAN0
0
CONTROLS
ET AL.
0
PATIENTS
FIG. 5. Production of IL2 by stimulated PBMC. PBMC from normal or atopic donors were stimulated with MoAb OKT3 (100 rig/ml) for 24 hr. Supematants were harvested and analyzed for the presence of IL2.
namely Ti-CD3 and CD2. In both cases PBMC proliferative response was impaired. These results indicate that the defect does not involve selectively one of the two receptorial mechanisms, i.e., the antigen receptor and the CD2 molecule, but concerns one or more steps shared by the two activation pathways. Some investigators have suggested a possible involvement of IL 1, IL2, or IL4 in the immunological defect of atopic patients (4, 12, 13). To verify the specific role of the different interleukins in the proliferative defect, we analyzed the effects of exogenous ILl, IL2, and IL4 on PBMC proliferation. Our results rule out the possibility that a defect of IL1 or IL4 is responsible for the impaired cell proliferation. Indeed, addition of these cytokines did not affect the response of the atopic patients’ PBMC, compared with that of the controls’ PBMC. On the other hand, we found that the atopic patients’ PBMC had a defect in IL2 production and their proliferative response was rescued in the presence of exogenous IL2. Therefore, this appears to be the only factor responsible for the proliferative defect of PBMC of patients with common atopy. Our results also indicate that PBMC responsiveness to IL2 is not impaired in atopic patients, in contrast with the results reported by Ohtsuki et al. (5). Two lines of evidence confirm that atopic patients’ PBMC normally respond to IL2 following cell stimulation with a mitogen: first, their proliferation in the presence of the factor was comparable with that of normal PBMC when the cells were stimulated via either CD3 or CD2; second, although stimulated PBMC of atopic donors displayed low levels of Tat antigen, they expressed higher levels of Tat and with the same intensity than normal PBMC when exogenous IL2 was added to the cultures. Therefore, the decreased expression of IL2 receptor in stimulated PBMC of atopic subjects in the absence of exogenous IL2 appeared entirely due to the lack of the upregulating effect normally exerted by the endogenous interleukin on its receptor (14). In conclusion, our results demonstrate that a deficit of IL2 is responsible for the impairment of T lymphocyte proliferation in atopic patients. Since the balance of IL2 and other interleukins (specifically IL4) regulates the secretion of specific immunoglobulin (Ig) isotypes by B cells (15, 16), it may be suggested that the diminished production of this factor is related to the altered profile of Ig isotypes produced by atopic patients during the course of in vivo antigenic challenge.
T CELL PROLIFERATIVE
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ACKNOWLEDGMENTS Authors are grateful to Ms. Cristina Maresca for her excellent secretarial help. This work is supported by funds from the Italian C.N.R., P.F. “Biotecnologie.”
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Leung, D. Y. M., and Geha, R. S., Clin. Rev. Allergy4, 67, 1986. Kapp, A., Gillitzer, R., Kirchner, H., and Schopf, E., Arch. Dermatol. Rex 279, S55, 1987. Schopf, E., Kapp, A., and Kim, C. W., Arch. Dermatol. Res. 262, 37, 1978. Kapp, A., Kimabauer, R., Luger, T. A., and Schopf, E., Acta Derm. Venereal. Suppl. 144,97, 1989. Ohtsuki, M., Ogata, F., Furue, M., and Ishibashi, T., Arerugi 38( IO), 1169, 1989. Chiarielli, F., Canfora, G., Verrotti, A., Amerio, P., and Morgese, G., Br. J. Dermatol. 116, 651, 1987. Buckley, R. H., and Fiscus, S. A., J. Clin. Invest. 55, 157, 1975. Ringden, O., Persson, U., and Johansson, S. G. O., Blood 61, 1190, 1983. Romagnani, S., Biagiotti, R., Amadori, A., Maggi, E., Biti, G., and Ricci, M., Znt. Arch. Allergy Appl. Immunol. 63, 64, 1980. O’Garra, A., Umland, S., De France, T., and Christiansen, J., Zmmunol. Today 9(2), 45, 1988. Cantrell, D. A., and Smith, K. A., J. Exp. Med. 158, 1895, 1985. Ma&, E., Del Prete, G., Tiri, A., Macchia, D., Parronchi, P., Ricci, M., and Romagnani, S., Clin. Exp. Immunol. 73, 57, 1988. Romagnani, S., Del Prete, G., Maggi, E., Parronchi, P., Giudizi. M. G., Almerigogna, F., and Ricci, M., Clin. Immunol. Immunopathol. SO, S13, 1989. Welte, K., Andreff, M., Platzer, E., Holloway, K., Rubin, B. Y., Moore, M. A. S., and Mertelsmann, R., J. Exp. Med. 160, 1390, 1984. Sideras, P., Takafumi, N., and Tasuky, H., immunol. Rev. 102, 189, 1988. Snapper, C. M., Finkelman, F. D., and Paul, W. E., Immunol. Rev. 102, 51, 1988.
IMMUNOLOGY
139,9 1-97 (1992)
Defect of CD2- and CD&Mediated Activation Pathways in T Cells of Atopic Patients: Role of lnterleukin 2 MARIA FIAMMETTA ROMANO,*,' GIULIANA VALERIo,Jf MARIA CATERINATURCO,$GIUSEPPE SPADARO,~ SALVATOREVENLJTA,$ANDSALVATOREFORMISANO* *Dipartimento di Biologia e Patologia Cellulare e Molecolare, TDipartimento di Pediatria, SDipartimento di Biochimica e Biotecnologie Mediche, and SCattedra di Immunologia Clinica e Allergologia, II Facoltd di Medicina e Chirurgia, Napoli, Italy Received
May
1, 1991; accepted
July 25, 1991
In the present work we analyzed the proliferative response of T lymphocytes from 11 atopic patients stimulated in vitro via either the CD2 or the CD3 pathway of cell activation. In both caseswe found a significant decrease of thymidine incorporation in cell DNA in comparison with T cells from normal donors. The mechanism of this impaired proliferative response was analyzed. Atopic patients’ T cells were found to secrete low quantities of interleukin 2 (IL2) and to express low amounts of Tat antigen, measured as both a percentage of Tac-positive cells and a mean fluorescence intensity of Tat antigen per cell. Addition of recombinant IL2 to cultures completely restored both cell proliferative response and Tat antigen expression. This effect was specific of IL2 since addition of IL1 or IL4 did not significantly affect T cell proliferative response. We conclude that atopic patients’ T lymphocytes have a defect in both CD2 and CD3 pathways of cell activation relying on impairment of IL2 production, without involving IL2 responsiveness or other lymphokine defects. Q 1992 Academic PWSS, Inc.
INTRODUCTION A body of evidence suggests that atopy, the familiar allergic disorder involving an immediate type hypersensitivity to environmental allergens, is related to an impairment of T lymphocyte reactivity. Indeed, a decreased T cell proliferative response to bacterial antigens and/or an altered production of some T lymphocyte-derived cytokines in atopic patients has been reported by some authors (l-6). On the other hand, impairment of T cell reactivity in some clinical conditions (Nezelofs syndrome, WiskottAldrich syndrome, GVDH, Hodgkin’s disease) is often accompanied by developing of symptoms of atopy (7-9). T lymphocyte activation is mediated by two different pathways involving antigen receptor (Ti-CD3) or CD2 molecule. Triggering of one of these structures induces T cell transition from Go to Gr phase and production and response to interleukins 2 and 4: these in turn regulate Gr progression and entry in S phase (10). In this paper ’ To whom correspondence should be addressed at Dipartimento di Biologia e Patologia Cellulare e Molecolare (Servizio di Immunoematologia e Trasfusione), II Facolti di Medicina e Chirurgia, Via S. Pansini, S-80 13 1 Napoli, Italy. Fax: 39-8 l-209224. 91 0008-8749192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
92
ROMAN0
ET AL.
we describe an impairment of Ti-CD3- and CDZmediated pathways of cell activation in T lymphocytes of atopic patients and analyze the molecular defect responsible for this impairment in these cells. MATERIAL
AND
METHODS
Patients. We studied 11 atopic patients aged between 18 and 45 years (mean age 23 years). The diagnosis was based on a history of infantile eczema, clinical features, sometimes concomitant, (active atopic eczema, rhinitis, extrinsic asthma, conjunctivitis), positive intradermal responses to common allergens, and, in some cases, raised blood titers of IgE. The control group was comprised 18 healthy blood donors aged between 18 and 55 years (mean age 22 years). Neither the controls nor the patients had received local or systemic steroid therapy at least 4 weeks before blood collection. Cells. Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood of patients and controls by Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation (400g for 30 min), washed, and resuspended at the concentration of 4 X 106/ml in medium RPM1 1640 supplemented with 5% heatinactivated fetal calf serum. Proliferative assays. These were performed in 96-well microplates by incubating 4 X lo5 PBMC in a total volume of 200 ~1 containing CD3 (30 rig/ml) or CD2 (9.1, 10 fig/ml + Bil, 10 Kg/ml) monoclonal antibodies. After 48- or 96-hr incubations of CD3or CD2-stimulated cells, respectively, at 37°C in a 5% CO2 atmosphere, 0.5 &i of [3H]thymidine (3H-dT, sp act 47 Ci/mmol, Amersham International plc., Milan, Italy) were added to each well. After 4- and 8-hr incubations at 37°C for CD3 and CD2 cultures, respectively, cells were harvested on glass fiber strips and 3H-dT incorporation was measured in a beta counter. Each point was performed in triplicate. Results were accepted when the standard deviation was lower than 15% of the mean value. Monoclonal antibodies (MoAbs) and interleukins (ILs). The anti-CD3 MoAb OKT3, an IgG2a, was purchased from Ortho Diagnostic System, Milan, Italy. The MoAbs 9.1, an IgG3, and Bil, an IgG2a, two distinct determinants of the CD2 molecule, were kind gifts of Dr. Soo Young Yang, Memorial Sloan Kettering Cancer Center (New York, NY) and Dr. Soldano Ferrone, New York Medical College (Valhalla, NY), respectively. Recombinant IL 1 and IL2 were obtained from Biogen Medical Research (Geneva, Switzerland). Recombinant IL4 was a gift of Immunex Corporation (Seattle, WA). Fluorescein isothiocyanate (FITC)-conjugated anti-IL2 receptor MoAb was purchased from Becton-Dickinson (Milan, Italy). Zmmunojluorescence (IF). Direct IF was performed by incubating 100 ~1 of a cell suspension (2 X 106/rnl) with a saturating amount of FITC-conjugated anti-IL2 receptor MoAb for 30 min at 4°C. Cells were then washed and examined with a cytofluorograph (FACS-scan, Becton-Dickinson). Results were expressed as a percentage of positive cells. Negative controls were less than 1% of stained cells. The specific cell immunofluorescence intensity was expressed as channel numbers on a log scale. The mean fluorescence intensity was calculated by integrating the fluorescence histograms for the positive cells as compared with control unstained cells. IL2 assay. IL2 biological activity of supematants was determined by the ILZdependent stimulation of PBMC activated with an anti-CD3 MoAb for 4 days (blasts) (11). Briefly, PBMC from normal donors or patients were incubated with anti-CD3 MoAb; 24 hr later, supernatants were harvested and added to the blasts (250 X 103/
T CELL PROLIFERATIVE
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ml) in 96-well microtiter plates. Recombinant IL2 (from 50 to 1.6 U/ml) was separately added to the blasts as a control. Following a 20-hr incubation at 37°C in a 5% COZ atmosphere 0.5 PCi of 3H-dT was added in each well and after an additional 6-hr incubation cells were harvested on glass fiber strips and the incorporation was measured in a beta counter. Statistical analysis of the results. It was performed using the Student t test. RESULTS Proliferative response of PBMC of atopic subjects to CD3 and CD2 A4oAbs. To analyze T cell reactivity of atopic patients to stimuli which trigger the CD3 and the CD2 pathways of lymphocyte activation, we stimulated PBMC of patients and normal donors with CD3 and CD2 MoAbs and measured 3H-dT incorporation in cell DNA. As illustrated in Fig, 1, cpm values ranged from 3000 to 45,600 and from 18,000 to 90,000 in PBMC from atopic and normal subjects, respectively, stimulated with antiCD3 MoAb. T cells of atopic patients displayed a lower response to CD3 MoAb when compared to cells of normal donors (P < 0.01). When T cell response to CD2 MoAbs was analyzed, patients’ PBMC incorporation ranged from 1300 to 18,000, while healthy donors’ PBMC incorporation ranged from 1500 to 70,000 cpm (Fig. 1). A decreased reactivity of PBMC of atopic patients could be recognized (P < 0.01). To further evaluate the defect in atopic patients’ T cell proliferative response, we analyzed whether patients and controls displayed similar dose responses to stimulants. Representative results are illustrated in Fig. 2: T lymphocytes from atopic donors, displayed a lower proliferative response than controls’ T cells to the same anti-CD3 doses, either optimal or suboptimal. Eflects of diflerent cytokines on the proliferative response of PBMC of atopic patients to CD3 MoAb. Monocyte-derived IL1 is required for Go-G1 transition of activated T lymphocytes and IL2 synthesis while T cell-produced IL2 and IL4 are involved in progression of G, phase (10). To investigate the possible role of these cytokines in the impaired proliferative response of PBMC of atopic subjects, we stimulated the cells
PATIENTS FIG. I. Normal and atopic donors’ PBMC proliferative response to CD2 and CD3 MoAbs. PBMC from normal (controls) and atopic (patients) donors were incubated with anti-CD3 (Q or anti-CD2 (a) MoAbs and 3H-dT incorporation in cell DNA was measured with a beta counter. CONTROLS
ROMAN0
0
100
ET AL.
200
300
CD3 MoAb: rig/ml FIG. 2. Proliferative response to CD3 MoAb in PBMC from a normal donor and an atopic patient. PBMC from a normal donor (0) and an atopic patient (0) were incubated with different doses of MoAb OKT3 and ‘H-dT incorporation in cell DNA was measured with a beta counter.
in the absence or presence of exogenous interleukins 1,2,4 and measured 3H-dT incorporation in cell DNA. IL1 and IL4 did not display any significant effect on thymidine incorporation in PBMC of atopic patients, although they slightly increased cell proliferation of PBMC of normal donors (not shown). On the other hand a marked effect of IL2 on T cell DNA synthesis was detected in both normal donors’ and atopic patients’ PBMC preparations. Indeed, in the presence of this factor, an enhanced 3HdT incorporation was observed in CD3- and especially CDZstimulated normal PBMC and comparable values were obtained with PBMC from atopic patients (Fig. 3).
CD3 CD2 IL2
+ -
FIG. 3. Effect of recombinant IL2 (10 U/ml) on ‘HdT incorporation in CD3- or CM-stimulated PBMC. PBMC from 10 controls (@) and 10 atopic subjects (0) were stimulated with the indicated reagents and ‘HdT incorporation in cell DNA was determined. Results represent mean values + standard error.
T CELL PROLIFERATIVE
IMPAIRMENT
IN ATOPIC
PATIENTS
Efect of IL2 on Tat antigen expression in PBMC of atopic patients. A
95 decreased
responsiveness to IL2 in PBMC of atopic patients had been reported by some authors (5). Our results did not appear to be in agreement with those conclusions since atopic patients’ PBMC stimulated in the presence of IL2 displayed thymidine incorporation values comparable with those of normal PBMC (Fig. 3). However, to further analyze this point, we measured the expression of IL2 receptor p55 (Tat) in PBMC of normal donors and atopic patients stimulated with CD3 MoAb. Although Tat antigen expression of atopic patients’ PBMC was lower than that of normal PBMC, the addition of exogenous IL2 resulted in comparable values of both positive cell percentages and mean Tat intensities in the two groups (Fig. 4). Production of IL2 in atopic patients. Our results indicated that atopic subjects’ PBMC might present a defect in production of IL2. To assessthis point, we measured the presence of IL2 in supematants of atopic patients’ PBMC stimulated via CD3. As illustrated in Fig. 5, following cell stimulation with MoAb OKT3, PBMC of four normal donors secreted high quantities of IL2 (X = 10 U/ml), while PBMC of five different atopic patients produced very low amounts (X = 2 U/ml) of this interleukin. DISCUSSION In the present work we have investigated the ability of atopic patients’ PBMC to proliferate when stimulated through the two pathways of T lymphocyte activation, loo-
A
In 80 = a
-
o 60+ : I- 40 0 * 20I
Ol -I
10
1
-
L.2
I L-2
I + I L.2
I
1 + I L-2
FIG. 4. Effect of recombinant IL2 (10 U/ml) on Tat antigen expression in CD3-stimulated PBMC. PBMC from normal (0) or atopic (0) donors were stimulated with anti-CD3 MoAb (100 rig/ml) in the absence or presence of recombinant IL2 for 72 hr. Then cells were washed and analyzed for Tat antigen expression in IF. (A) Percentage of positive cells; (B) mean fluorescence intensity.
96
ROMAN0
0
CONTROLS
ET AL.
0
PATIENTS
FIG. 5. Production of IL2 by stimulated PBMC. PBMC from normal or atopic donors were stimulated with MoAb OKT3 (100 rig/ml) for 24 hr. Supematants were harvested and analyzed for the presence of IL2.
namely Ti-CD3 and CD2. In both cases PBMC proliferative response was impaired. These results indicate that the defect does not involve selectively one of the two receptorial mechanisms, i.e., the antigen receptor and the CD2 molecule, but concerns one or more steps shared by the two activation pathways. Some investigators have suggested a possible involvement of IL 1, IL2, or IL4 in the immunological defect of atopic patients (4, 12, 13). To verify the specific role of the different interleukins in the proliferative defect, we analyzed the effects of exogenous ILl, IL2, and IL4 on PBMC proliferation. Our results rule out the possibility that a defect of IL1 or IL4 is responsible for the impaired cell proliferation. Indeed, addition of these cytokines did not affect the response of the atopic patients’ PBMC, compared with that of the controls’ PBMC. On the other hand, we found that the atopic patients’ PBMC had a defect in IL2 production and their proliferative response was rescued in the presence of exogenous IL2. Therefore, this appears to be the only factor responsible for the proliferative defect of PBMC of patients with common atopy. Our results also indicate that PBMC responsiveness to IL2 is not impaired in atopic patients, in contrast with the results reported by Ohtsuki et al. (5). Two lines of evidence confirm that atopic patients’ PBMC normally respond to IL2 following cell stimulation with a mitogen: first, their proliferation in the presence of the factor was comparable with that of normal PBMC when the cells were stimulated via either CD3 or CD2; second, although stimulated PBMC of atopic donors displayed low levels of Tat antigen, they expressed higher levels of Tat and with the same intensity than normal PBMC when exogenous IL2 was added to the cultures. Therefore, the decreased expression of IL2 receptor in stimulated PBMC of atopic subjects in the absence of exogenous IL2 appeared entirely due to the lack of the upregulating effect normally exerted by the endogenous interleukin on its receptor (14). In conclusion, our results demonstrate that a deficit of IL2 is responsible for the impairment of T lymphocyte proliferation in atopic patients. Since the balance of IL2 and other interleukins (specifically IL4) regulates the secretion of specific immunoglobulin (Ig) isotypes by B cells (15, 16), it may be suggested that the diminished production of this factor is related to the altered profile of Ig isotypes produced by atopic patients during the course of in vivo antigenic challenge.
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ACKNOWLEDGMENTS Authors are grateful to Ms. Cristina Maresca for her excellent secretarial help. This work is supported by funds from the Italian C.N.R., P.F. “Biotecnologie.”
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