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BIOMOLECULAR REGULATION OF THE IgE IMMUNE RESPONSE III. CYTOKINE PROFILES IN ATOPIC DERMATITIS, INHALANT ALLERGY AND NON-ALLERGIC DONORS
BIOMOLECULAR REGULATION OF THE IgE IMMUNE RESPONSE III. CYTOKINE PROFILES IN ATOPIC DERMATITIS, INHALANT ALLERGY AND NON-ALLERGIC DONORS L.K. Poulsen,1 C. Bindslev-Jensen,2 M. Diamant,3 M.B. Hansen,3 K.F. Jepsen,1 C.M. Reimert,1 K. Bendtzen3 Cytokines—in particular interleukin 4 (IL-4), IL-5 and interferon gamma (IFN-γ)—regulate both IgE synthesis and eosinophil activation in atopic diseases. To elucidate whether distinct profiles of cytokine production were related to serum level of IgE and eosinophilia, the spontaneous and inducible in vitro cytokine secretion from peripheral blood mononuclear cells (PBMC) was investigated. PBMC were isolated and cultured from three groups of donors: (1) patients with atopic dermatitis (AD) and high levels of serum IgE (.5000 IU/ml, n 5 11), (2) patients with diagnosed inhalant allergy (IA) and serum IgE in the range of 200–2000 IU/ml (n 5 10), and (3) non-allergic individuals (NA) with serum IgE below 100 IU/ml (n 5 10). The production of cytokines was determined in cultures after 24 h [IL-1α, IL-4, IL-5, IL-6, tumour necrosis factor alpha (TNF-α), and TNF-β] or 72 h (IL-2, IFN-γ). The spontaneous production of IL-1α was increased in the AD group compared to NA (P 5 0.002), whereas for unstimulated cultures no other cytokine differed between patient groups. To identify conditions for optimal cytokine production, various combinations of phytohaemagglutinin (PHA), calcium ionophore (ION), and phorbol ester (PMA) were tested as stimuli. The combination ION 1 PMA induced the highest levels of IL-2, IL-4, IL-5, IFN-γ and TNF-α, whereas maximal production of IL-6 and TNF-β were induced by PHA and PHA 1 PMA, respectively. The AD group demonstrated a significantly lower production of TNF-α and IFN-γ compared with the two other groups, and IL-4 and IL-5 production increased in the IA group. The results suggest that in spite of the common features, i.e. raised serum IgE and eosinophilia, in IA and AD patients, the underlying aberrations in the cytokine network is different. © 1996 Academic Press Limited
The cytokines interleukin 4 (IL-4), IL-5 and interferon gamma (IFN-α) have a major impact on the synthesis of IgE and the activation of eosinophils, two important factors in the initiation and propagation of the allergic inflammation. IL-4 (which in humans may be substituted by IL-13) is mandatory for in vitro IgE synthesis, which is also counteracted by IFN-γ.1–3 Eosinophilopoiesis and subsequent activation of eosino-
From the 1Laboratory of Medical Allergology; Allergy Unit; 3 Laboratory of Medical Immunology, Institute for Inflammation Research, National University Hospital, Copenhagen, Denmark and 2Dept. of Dermatology, University Hospital of Odense, Odense, Denmark Correspondence to: Lars K. Poulsen, Laboratory of Medical Allergology, National University Hospital, TTA 7542, 20 Tagensvej, DK-2200 Copenhagen N, Denmark Received 5 October 1995; revised and accepted for publication 24 March 1996 © 1996 Academic Press Limited 1043-4666/96/08065117 $18.00/0 KEY WORDS: Allergy/atopic dermatitis/IgE/IL-1α/IL-2/IL-4/ IL-5/IL-6/IFN-γ/TNF-α/TNF-β CYTOKINE, Vol. 8, No. 8 (August), 1996: pp 651–657
phils are strongly dependent of IL-5.4 These results have mainly been derived from either in vitro systems or animal models, and the literature is more sparse regarding the clinical significance of these findings, and the cytokine network participating in the putative skewed production profile of IL-4/IL-5 vs IFN-γ. Recently reports have been published describing a defective production of IFN-γ by peripheral blood mononuclear cells (PBMC) of patients with atopic dermatitis5,6 and hyperIgE syndrome.7 An increased production of IL-4 has been found in patients being described as having a strong atopic disease6 or being allergic to several allergens.8 So far, however, atopic dermatitis and inhalation allergy have been investigated as common manifestations of atopic disease, inferring that a patient is more atopic having a higher level of IgE. The aim of the present study was to study the cytokine production profiles in patients with varying levels of IgE and an atopic disease that could be described as either inhalation allergy with moderately increased IgE or atopic dermatitis with highly increased levels of total IgE. 651
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CYTOKINE, Vol. 8, No. 8 (August 1996: 651–657)
Measurements of IL-4, IL-5, IFN-γ and other cytokines in plasma have been very difficult, and the clinical relevance of such measurements remains speculative inasmuch as inhibitory substances, e.g. soluble IL-4 receptor9 or cytokine autoantibodies (reviewed in Ref. 10) may be present at high levels. Experiments employing T-cell clones derived from peripheral blood is an interesting alternative for the study of cytokine profiles, but having the cells in culture for several weeks may by itself alter the cytokine profile.11,12 Therefore an attempt was made to establish conditions where the potential for cytokine production in cells freshly isolated from peripheral blood could be tested by different stimulators. Eight different cytokines were included in the study (Table 1).
RESULTS Spontaneous production of cytokines
40 000
Induction of cytokine production To enhance the production of cytokines, various combinations of stimuli were applied to the cell culIL-6
6000
pg/ml
20 000 10 000
200
40
150
30
100
3000 2000 0
IFN-γ
300
20
200 150 100
50
10
50
0
0
0
pg/ml
500
IL-4
500
400
400
300
300
200
TNF-β
250 pg/ml
50
4000
1000
0 IL-2
TNF-α
5000
30 000
pg/ml
pg/ml
250
IL-1α
pg/ml
1400 1200 1000 800 600 400 200 0
pg/ml
pg/ml
The net production of cytokines, based on the concentrations of cytokines in the supernatants, was compared between the three donor groups (Fig. 1). The cytokines predominantly produced by monocytes, i.e. IL-1α, IL-6 and TNF-α, were produced in measurable
amounts by the majority of the donors. Significantly more IL-1α (P 5 0.002) was produced by cells from the AD group (median 78 pg/ml, quartiles 45–454 pg/ml) compared to the NA group (0 pg/ml, 0–9 pg/ml). The levels in the IA group were intermediate, but not significantly different from the AD group (P 5 0.07). IL-6 was produced in considerable amounts: AD: 20.8 ng/ml (13.9–28.5), IA: 16.2 ng/ml (9.5–19.7); and NA: 17.4 ng/ml (14.0–22.3), but there were no differences between the groups. Compared to IL-6, all donors produced 10–20 times less TNF-α with no distinct pattern in any of the donor groups. Cytokines, presumably produced by Th1-like cells, i.e. IL-2, IFN-γ and TNF-β, were all produced in low amounts with no significant differences between the groups even though the median IFN-γ production in the AD group was only one third of that in the NA group. Low spontaneous cytokine production was also found for the Th2-type cytokines IL-4 and IL-5, and the majority of determinations fell below detection limit of the assays.
IL-5
200
100
100
0
0
Figure 1. Spontaneous cytokine production by atopic dermatitis patients (AD), inhalation allergic patients (IA) and non-atopic donors (NA). Cells were cultured without added stimuli and harvested at 24 h (for determination of IL-1α, IL-4, IL-5, IL-6, TNF-α and TNF-β or at 72 h (for derermination of IL-2 and IFN-γ). Cytokine concentrations were determined by ELISA or immunoradiometric assay (IL-4) as described in Materials and Methods. Atopic dermatitis patients: (d), allergic patients: (.), non-allergic individuals: (r), Medians for each donor group: (–).
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 653
TABLE 1. Culture periods and assay characteristics for the cytokines tested in this study Main Culture period Assay cellular source (h) type*
IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
Monocytes T-lymphocytes T-lymphocytes T-lymphocytes Monocytes T-lymphocytes Monocytes T-lymphocytes
24 72 24 24 24 72 24 24
Sensitivity of assay (pg/ml)
E E I E E E E E
8 8 100 100 30 15 30 15
Cytokine profiles at optimal stimulations Figure 3 shows a comparison between the three donor groups, when cultures are stimulated optimally, as described above. IL-1α is not included in this figure, because no consistent treatment could be identified, which increased the level significantly above the spontaneous production. Compared to the two other groups, the AD group showed a lower production of IL-6 and TNF-α, but this was only statistically significant in the case of TNF-α. For IFN-γ, however, there was a significantly lowered production in the AD group. The IFNγ production was somewhat heterogenous, as two subjects expressed levels comparable to the IA and NA groups, the remainder being severely depressed. These two individuals did not otherwise express any aberrant cytokine production profile compared to the rest of the AD-group. The IA-group, on the other hand, showed
*E: ELISA, I: Immunoradiometric assay.
tures. The outcome of these experiments is depicted in Figure 2, where the median changes in cytokine production of the normal donors are illustrated as bars (error bars represent quartiles). The levels of IL-2, IL5, IL-6, IFN-γ, TNF-α and TNF-β could all be significantly increased by one or more of the stimuli. For IL-1α and IL-4, the stimuli did not induce significant changes compared to the spontaneous production, whereas PMA with or without PHA, actually decreased the levels of IL-6 in the supernatant. IL-1α
200 0
20 000 0
∗∗ ∗∗
∗∗
60 000 40 000
–20 000
20 000
–400
–40 000
0
IFN-γ
10 000
3000 2000
0
∗∗ ∗∗ ∗∗ 500
∗∗
∗
IL-4
1500
100
∗∗
∗
∗∗ ∗∗
IL-5
1250 ∆ pg/ml
∆ pg/ml
200
400
0
∗∗ ∗∗ ∗∗
400 300
600
200
1000 ∗∗
TNF-β
800 ∆ pg/ml
∆ pg/ml
20 000
∗∗ ∗∗ ∗∗ ∗∗ ∗∗
1000
4000
30 000
0
IFN-γ
5000
TNF-α
80 000
–200
40 000 ∆ pg/ml
IL-6
40 000 ∆ pg/ml
∆ pg/ml
400
∆ pg/ml
Cytokine
The stimulations in the two other groups (results not shown) generally coincided with the results shown in Figure 2, and produced a parallel pattern with the NA group. The conditions producing the highest and lowest levels for each of the cytokines are summarized in Table 2.
1000 750 500 250
0
Figure 2.
0 ∗∗ Alterations in cytokine levels induced by exogenous stimuli of normal donor cell culture.
Cultures and supernatants were otherwise treated as described in legend to Figure 1. The cytokine production is expressed relatively to the spontaneous cytokine production, i.e. the levels obtained after spontaneous production has been subtracted for each individual donor. Bars represent medians, error bars represent 1st and 3rd quartile. Stimuli (from left to right): PHA (u), Ionomycin (j), PMA , PHA 1 PMA , Ionomycin 1 PMA . *P , 5%; **P , 1%.
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CYTOKINE, Vol. 8, No. 8 (August 1996: 651–657)
TABLE 2. Summary of optimal stimulus for in vitro production of cytokines Stimulus producing lowest response
Cytokine IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
Same pattern in the three donor groups?
Stimulus producing highest response
No significant differences between stimuli PMA higher in IA Spontaneous PHA 1 PMA and ION 1 PMA Yes Spontaneous ION 1 PMA Only in IA Spontaneous ION 1 PMA Yes PMA PHA Yes Spontaneous ION 1 PMA Yes Spontaneous PHA 1 PMA and ION 1 PMA Yes Spontaneous PHA 1 PMA Yes
no sign of impaired IFN-γ production, but instead IL-4 and IL-5 levels were increased. IL-4 was also slightly, but not significantly increased in the AD group. Interdependency of cytokines Table 3 shows the coefficients of correlation between the spontaneous and the maximal production of the cytokines. Only combinations producing signifi-
cant correlations are shown. For the spontaneous production the two monokines IL-6 and TNF-α correlated as did the Th1 cytokines IL-2 and TNF-β. When stimulated to maximal production, a strikingly different pattern emerged: IL-4, IL-5, TNF-α and TNF-β were closely interrelated, i.e. the production of each cytokine correlated to the three others; moreover, the production of IFN-γ was correlated to both IL-5 and IL-6.
IL-6 600 000 400 000 200 000 80 000
TNF-α
//
60 000
pg/ml
pg/ml
80 000
40 000
60 000 40 000
20 000
20 000 0
0
P = 0.03
40 000
4000
30 000
3000
20 000
IFN-γ
TNF-β
pg/ml
5000
pg/ml
pg/ml
50 000
IL-2
2000
10 000
1000
0
0
1400 1200 1000 800 600 400 200 0
P = 0.01 IL-4
IL-5 3000
800
2500 pg/ml
pg/ml
600 400 200
1500 1000 500
0
0 P = 0.009
Figure 3.
2000
P = 0.02
Optimal cytokine production by the groups of donors.
Cultures were cultured with the optimal stimulus (as described in Table 2) and harvested for cytokine determination as described in Fig. 1. Stimulation with PHA was used for IL-6, stimulation with PHA 1 PMA for TNF-β and ION 1 PMA for TNF-α, IL-2, IFN-γ, IL-4 and IL-5. Atopic dermatitis patients: (d), allergic patients: (.), non-allergic individuals: (r), Medians for each donor groups: (–).
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 655
TABLE 3. Spearman’s coefficients of correlation between spontaneous and maximal production of cytokines Spontaneous
Maximal
IL-1α IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
IL-2
IL-4
IL-5
IL-6
IFN-γ
TNF-α
TNF-β
— —
0.37* — 0.49*
—
0.43* 0.44*
0.49* 0.63** 0.59**
— 0.45*
0.39* — — 0.53**
—
*P , 0.05; **P , 0.01. All donors combined.
Correlation of IL-4, IL-5 and IFN-γ with IgE levels and eosinophil levels A negative correlation (r 5 20.42, P 5 0.022) was found between the level of serum IgE and the maximal IFN-γ production, but this correlation mainly reflected the differences between serum IgE and IFN-γ production for the AD group compared to the two other groups. No significant correlations were found between serum IgE and the maximal production of IL-4 and IL-5. A clear correlation was found between the spontaneous production of IL-1α and eosinophil number (r 5 0.63, P 5 0.00013). Moreover, correlations were found between IL-1α production and the levels of the eosinophilic proteins, eosinophilic cationic protein (ECP), (r 5 0.75, P , 1025) and eosinophilic protein X (EPX) (r 5 0.64, P 5 0.00012), released to serum during a clotting time of 2 h. Eosinophil numbers were not correlated to the spontaneous production of other cytokines.
DISCUSSION Atopic patients have been reported to have an aberrant cytokine production profile.5–8,13,14 Looking at the spontaneous production of cytokines, similar and low levels were found of the cytokines that are primarily T-cell derived. Monokines, however, were produced in considerable amounts, and increased levels of IL-1α were noted in the AD group. Because cytokine production is highly inducible, different forms of non-specific in vitro stimulations of PBMC were examined. It is interesting that the optimal conditions for cytokine production varied even between cytokines that are believed to be produced by the same cells. This was especially prominent for IL-1α, IL-6 and TNF-α. Thus, IL-1α production was not increased by any stimuli; the IL-6 response was optimally potentiated by PHA, and TNF-α was optimally induced by PHA or ionomycin in combination with PMA. IL-6 was the only cytokine
exhibiting a reduced response when PMA was added. It should be emphasized that the concentrations of cytokines in culture supernatants are dependent not only on production but also on degradation and consumption. It is likely, for example, that the different stimuli induce different ratios between a cytokine and its receptor. The kinetics in the system may also be important. Thus, in accordance with these results, Tang et al. found optimal IL-4 and IFN-γ production at 24 h and 72 h, respectively,6 while Reinhold et al. found optimal IFNγ production at 24 h.5 Takahashi et al. found optimal concentrations of the cytokines IL-4, IL-2, IFN-γ and TNF-α at 24 h.13 Looking at the cytokine profile under optimal conditions, it is clear that two patient groups express different abnormalities. While the IA group has increased expression of the Th2-type cytokines IL-4 and IL-5, the AD group demonstrates an impaired IFN-γ production, i.e. a Th1 defect. It is worth noticing, however, that the latter impairment is not accompanied by a reduction in IL-2 or TNF-β production. Several investigators have focused on the ratio between IL-4 and IFN-γ,6 and from this study it is clear that both patient groups will have an increased IL-4: IFN-γ ratio. It is important to recognize, however, that the two groups differ in their IgE response. While the IA group has a relatively large fraction of their total IgE directed to distinct inhalation allergens,15,16 the AD patients are probably subject to a polyclonal activation. This could be interpreted in the way that IA patients have relatively few, but allergenspecific Th2-type cells, which will help production of large amounts of allergen-specific IgE. AD patients, however, have a reduced level of Th1-type cells, and a larger panel of antigen-specificities may escape the otherwise very tight control of the IgE synthesis exerted by a large number of inhibiting T-cells. Because T-cell development is believed to be controlled by professional antigen presenting cells, it was of interest to try to identify differences between the three groups for the monokine production profiles.
656 / Poulsen et al.
The production of TNF-α was significantly lowered in the AD group, whereas IL-6 was reduced although not significantly. Kapp et al. have likewise reported a reduced production of TNF-α in AD patients—a reduction that was not seen for psoriasis patients;14 the findings in AD patients were confirmed by Takashi et al.13 A close correlation between the production of TNF-α and the Th2-type cytokines, IL-4 and IL-5 was observed. This correlation only became significant, however, if all three groups were combined. The additional finding that the spontaneous production of IL-1α was increased in the AD group could suggest that an increased IL-1α:TNF-α ratio might be of importance for the relative deficiency in IFN-γ production. In considering a possible role for IL-1α in atopic diseases, it is interesting that allergic patients express a much lower frequency of autoantibodies to IL-1α compared to normals.10 There was also a close correlation between the spontaneous IL-1α production and the number of eosinophil counts or the levels of eosinophil counts and the serum levels of the eosino-phil proteins, ECP and EPX/Eosinophil-derived neurotoxin (EDN), high coefficients of correlation were achieved (r 5 0.63; 0.75; and 0.64; P , 0.0001, respectively). It is tempting to speculate that IL-1α plays an important role in the development of Th2-like cells, but a more direct association between the proinflammatory cytokine and eosinophils should also be kept in mind. In conclusion, a marked difference in cytokine production profiles between patients with atopic dermatitis and inhalation allergies has been demonstrated. Whereas a deficiency in the ability to produce TNF-α and IFN-γ upon stimulation is an important feature in the AD group, the group of inhalation allergic patients suffers from an excessive production of “atopic” cytokines like IL-4 and IL-5.
MATERIALS AND METHODS Allergic and non-allergic donors Three groups of donors were admitted to the study: (1) Patients with atopic dermatitis with hyper serum-IgE (AD): 11 individuals with serum IgE . 5000 IU/ml. All patients in this group demonstrated atopic dermatitis according to the criteria of Hanifin and Rajka;18 (2) Allergic patients (IA): 10 individuals with total IgE in the range of 200–2000 IU/ml, and inhalant allergy to pollen, house dust mites or animal dander. The diagnosis was established by case history, positive skin prick test and presence of allergen-specific IgE; (3) Non-allergic donors (NA): 10 individuals with total IgE , 100 IU/ml and no history of allergy. None of the donors had received systemic glucocorticoid treatment or cytostatic treatment within 8 weeks of the study, and no patients were undergoing allergen specific immunotherapy. The age profiles were similar in the three groups (mean 6 SD: 36 6 11, 39 6 11 and 35 6 6 years) as were the distri-
CYTOKINE, Vol. 8, No. 8 (August 1996: 651–657)
bution between the two sexes (F/M): 7/4, 3/7 and 6/4. Total serum IgE in the three groups were: 12 000 6 9200, 485 6 416 and 32 6 30 IU/ml.9 Apart from the eosinophilic counts (0.51 3 106/ml, 0.18 3 106/ml and 0.13 3 106/ml) no abnormal or varying haematological parameters were observed.17
Blood sampling, isolation of cells and culture conditions Blood was drawn in Vacutainers (Labco Inc., Bucks, UK) with heparin for isolation of mononuclear cells. PBMC were isolated from heparinized blood using LymphoPrep™ (Specific density: 1.077) (Nycomed, Oslo, Norway). Cells were grown in 96-well plates (200 µl at 2 3 106/ml) in the medium described by Yssel19 at 37°C, in a humidified atmosphere containing 5% CO2. Cytokine levels were determined in supernatants of cultures with or without addition of various combinations of phytohaemagglutinin (Difco, Detroit, MI) (PHA, 1%), calcium ionophore (Ionomycin, Calbiochem, La Jolla, CA) (ION 1 µM), and phorbol myristate acetate (Sigma, St Louis, MO) (PMA, 50 ng/ml). Preliminary experiments had revealed maximum levels of IL-4 and IL-5 in 24 h cultures vs higher levels of IL-2 and IFN-γ in 72 h cultures, and these two culture periods were subsequently used. Based on kinetic studies, IL-1α, IL-6, TNF-α and TNF-β were determined in the 24 h cultures. Determination of cytokines IL-1α, IL-2, IL-4, IL-5, IL-6, TNF-α, TNF-β and IFN-γ Analytical sensitivities and references to detailed descriptions of the cytokine assays are given in Table 1.
Immunoradiometric assay for IL-4 An antiserum towards IL-4 was produced by immunizing rabbits with recombinant human IL-4 (a kind gift from Dr Jan de Vries, DNAX, Palo Alto, CA).9 IL-4 was determined in culture supernatants by an immunoradiometric assay using the IgG fraction of the rabbit anti-IL-4 antiserum which were coated onto “Maxisorp” tubes. Samples or standard (Genzyme, Cambridge, MA) were diluted in RPMI 1640 1 10% fetal calf serum, and the detecting antibody was radioiodinated rabbit anti IL-4 IgG. There was no cross-reactivity between the seven other natural cytokines or recombinant IL2, IL-5 and IFN-γ. The inter-assay coefficient of variation was 15%.
ELISA for IL-5 IL-5 was measured by a sandwich enzyme-linked immunoabsorbent assay (ELISA) using monoclonal rat antibodies. Briefly, Maxisorp plates (NUNC, Roskilde, Denmark) were coated with mAb rat anti-mouse/human IL-5 (clone TRFK5) (Pharmingen, San Diego, CA) and non-attached sites were blocked with 1% human serum albumin (Behringwerke, Marburg, Germany) in phosphate-buffered (10 mM, pH 7.4) saline (150 mM) (PBS). Samples and standard (recombinant human IL-5, Pharmingen) were diluted in RPMI 1640 1 10% fetal calf serum. Biotinylated monoclonal rat anti-human IL-5 (clone JES-A1 Pharmingen, cat no 18522
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 657
D) and the streptavidin system were used to detect IL-5. Interassay variation was 10%. The ELISA did not show any crossreactivity with recombinant IL-2, IL-4 or IFN-γ.
ELISAs for IL-1α, IL-2, IL-6, TNF-α, TNF-β and IFN-γ IL-1α, IL-2, IL-6, TNF-α, TNF-β and IFN-γ was measured by a double sandwich ELISA using monospecific, polyclonal rabbit antibody.20 Immuno-Maxisorp plates (NUNC) were coated with protein A affinity-purified IgG. Nonattached sites were blocked with 5% human serum albumin in PBS. Samples were diluted in phosphate buffered saline (PBS) supplemented with 2% normal rabbit serum (DAKO, Glostrup, Denmark), EDTA 10 mM, aprotinin 2000 KIE/ml. The assays were calibrated with international standards of the respective cytokines (National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK). Biotinylated rabbit antibodies were used as reported along with streptavidin peroxidase (Kirkegaard and Perry La., Gaithersburg, MD). Development was caried out with 1,2phenylendiamine dihydrochloride and measured at 492 nm. The inter- and intra-assay coefficients of variation were less than 15%. The detection limits of these ELISAs are shown in Table 1.
Determination of Eosinophilic Cationic Protein (ECP) and Eosinophilic Protein X (EPX) ECP and EPX were determined in plasma and serum (clotting time: 2.0 h) as previously described.17
Statistics The Mann–Whitney test for unpaired and the Wilcoxon test for paired observations were applied for testing differences between groups. Coefficient of correlation was calcuated as Spearman’s ρ.
Acknowledgements This study was supported by the Danish Biotechnology Program. CM Reimert is a senior research fellow of the Danish Medical Research Council. The excellent technical assistance of Lena Baron, Ulla Minuva and Marianna Thomsen are gratefully acknowledged.
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2. Pene J, Rousset F, Briere F, Chretien I, Bonnefoy JY, Spits H, Yokota T, Arai N, Arai K, Banchereau J, de Vries JE (1988) IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferon γ and α and prostaglandin E2. Proc Natl Acad Sci USA 85:6880–6884. 3. Punnonen J, de Vries JE (1994) IL-13 induces proliferation, Ig isotype switching, and Ig synthesis by immature human fetal B cells. J Immunol 152:1094–1102. 4. Owen WF (1991) Cytokine regulation of eosinophil inflammatory disease. ACI News 3:85–89. 5. Reinhold U, Wehrmann W, Kukel S, Kreysel HW (1990) Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities. Clin Exp Immunol 79:374–379. 6. Tang M, Kemp A, Varigos G (1993) IL-4 and interferongamma production in children with atopic disease. Clin Exp Immunol 92:120–124. 7. Del Prete G, Tiri A, Maggi E, De Carli M, Macchia D, Parronchi P, Rossi ME, Pietrogrande MC, Ricci M, Romagnani S (1989) Defective in vitro production of gamma-interferon and tumor necrosis factor-alpha by circulating T cells from patients with hyperimmunoglobulin E syndrome. J Clin Invest 84:1830–1835. 8. Pene J, Rivier A, Lagier B, Becker WM, Michel FB, Bousquet J (1994) Differences in IL-4 release by PBMC are related with heterogeneity of atopy. Immunology 81:58–64. 9. Poulsen LK, Reimert CM, Bindslev-Jensen C, Hansen MB, Bendtzen K (1995) Biomolecular regulation of the IgE immune response. II. In vitro IgE synthesis and spontaneous production of cytokines. Int Arch Allergy Immunol 106:55–61. 10. Bendtzen K, Hansen MB, Ross C, Poulsen LK, Svenson M (1995) Cytokines and autoantibodies to cytokines. Stem Cells 13: 206–222. 11. Marshall JD, Wen Y, Abrams JS, Umetsu DT (1993) In vitro synthesis of IL-4 by human CD4 1 T cells requires repeated antigenic stimulation. Cell Immunol 152:18–34. 12. Kelso A (1995) Th1 and Th2 subsets: paradigms lost? Immunol Today 16:374–379. 13. Takahashi T, Sasaki Y, Hama K, Furue M, Ishibashi Y (1992) Production of IL-4, IL-2, IFN-gamma and TNF-alpha by peripheral blood mononuclear cells of patients with atopic dermatitis. J Dermatol Sci 3:172–180. 14. Kapp A, Textor A, Krutmann J, Moller A (1990) Immunomodulating cytokines in atopic dermatitis and psoriasis: production of tumour necrosis factor and lymphotoxin by mononuclear cells in vitro. Br J Dermatol 122:587–592. 15. Gleich GJ, Jacob GL (1975) Immunoglobulin E antibodies to pollen allergens account for high percentages of total immunoglobulin E protein. Science 190:1106–1108. 16. Poulsen LK, Pedersen MF, Malling H-J, Søndergaard I, Weeke B (1989) Maxisorp RAST. A sensitive method for detection of absolute quantities of antigen-specific IgE. Allergy 44:178–189. 17. Poulsen LK, Reimert CM, Bindslev-Jensen C (1994) In vitro release of eosinophil proteins in allergic and atopic dermatitis patients. Med Inflam 3:223–227. 18. Hanifin JM, Rajka G (1980) Diagnostic features of atopic dermatitis. Acta Derm Venereol (Stockh) 92:44–47. 19. Yssel H, de Vries JE, Koken M, van Blitterswijk W, Spits H (1984) Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J Immunol Methods 72:219–227. 20. Hansen MB, Svenson M, Bendtzen K (1991) Human antiinterleukin-1α antibodies. Immunol Lett 30:133–140.
The cytokines interleukin 4 (IL-4), IL-5 and interferon gamma (IFN-α) have a major impact on the synthesis of IgE and the activation of eosinophils, two important factors in the initiation and propagation of the allergic inflammation. IL-4 (which in humans may be substituted by IL-13) is mandatory for in vitro IgE synthesis, which is also counteracted by IFN-γ.1–3 Eosinophilopoiesis and subsequent activation of eosino-
From the 1Laboratory of Medical Allergology; Allergy Unit; 3 Laboratory of Medical Immunology, Institute for Inflammation Research, National University Hospital, Copenhagen, Denmark and 2Dept. of Dermatology, University Hospital of Odense, Odense, Denmark Correspondence to: Lars K. Poulsen, Laboratory of Medical Allergology, National University Hospital, TTA 7542, 20 Tagensvej, DK-2200 Copenhagen N, Denmark Received 5 October 1995; revised and accepted for publication 24 March 1996 © 1996 Academic Press Limited 1043-4666/96/08065117 $18.00/0 KEY WORDS: Allergy/atopic dermatitis/IgE/IL-1α/IL-2/IL-4/ IL-5/IL-6/IFN-γ/TNF-α/TNF-β CYTOKINE, Vol. 8, No. 8 (August), 1996: pp 651–657
phils are strongly dependent of IL-5.4 These results have mainly been derived from either in vitro systems or animal models, and the literature is more sparse regarding the clinical significance of these findings, and the cytokine network participating in the putative skewed production profile of IL-4/IL-5 vs IFN-γ. Recently reports have been published describing a defective production of IFN-γ by peripheral blood mononuclear cells (PBMC) of patients with atopic dermatitis5,6 and hyperIgE syndrome.7 An increased production of IL-4 has been found in patients being described as having a strong atopic disease6 or being allergic to several allergens.8 So far, however, atopic dermatitis and inhalation allergy have been investigated as common manifestations of atopic disease, inferring that a patient is more atopic having a higher level of IgE. The aim of the present study was to study the cytokine production profiles in patients with varying levels of IgE and an atopic disease that could be described as either inhalation allergy with moderately increased IgE or atopic dermatitis with highly increased levels of total IgE. 651
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Measurements of IL-4, IL-5, IFN-γ and other cytokines in plasma have been very difficult, and the clinical relevance of such measurements remains speculative inasmuch as inhibitory substances, e.g. soluble IL-4 receptor9 or cytokine autoantibodies (reviewed in Ref. 10) may be present at high levels. Experiments employing T-cell clones derived from peripheral blood is an interesting alternative for the study of cytokine profiles, but having the cells in culture for several weeks may by itself alter the cytokine profile.11,12 Therefore an attempt was made to establish conditions where the potential for cytokine production in cells freshly isolated from peripheral blood could be tested by different stimulators. Eight different cytokines were included in the study (Table 1).
RESULTS Spontaneous production of cytokines
40 000
Induction of cytokine production To enhance the production of cytokines, various combinations of stimuli were applied to the cell culIL-6
6000
pg/ml
20 000 10 000
200
40
150
30
100
3000 2000 0
IFN-γ
300
20
200 150 100
50
10
50
0
0
0
pg/ml
500
IL-4
500
400
400
300
300
200
TNF-β
250 pg/ml
50
4000
1000
0 IL-2
TNF-α
5000
30 000
pg/ml
pg/ml
250
IL-1α
pg/ml
1400 1200 1000 800 600 400 200 0
pg/ml
pg/ml
The net production of cytokines, based on the concentrations of cytokines in the supernatants, was compared between the three donor groups (Fig. 1). The cytokines predominantly produced by monocytes, i.e. IL-1α, IL-6 and TNF-α, were produced in measurable
amounts by the majority of the donors. Significantly more IL-1α (P 5 0.002) was produced by cells from the AD group (median 78 pg/ml, quartiles 45–454 pg/ml) compared to the NA group (0 pg/ml, 0–9 pg/ml). The levels in the IA group were intermediate, but not significantly different from the AD group (P 5 0.07). IL-6 was produced in considerable amounts: AD: 20.8 ng/ml (13.9–28.5), IA: 16.2 ng/ml (9.5–19.7); and NA: 17.4 ng/ml (14.0–22.3), but there were no differences between the groups. Compared to IL-6, all donors produced 10–20 times less TNF-α with no distinct pattern in any of the donor groups. Cytokines, presumably produced by Th1-like cells, i.e. IL-2, IFN-γ and TNF-β, were all produced in low amounts with no significant differences between the groups even though the median IFN-γ production in the AD group was only one third of that in the NA group. Low spontaneous cytokine production was also found for the Th2-type cytokines IL-4 and IL-5, and the majority of determinations fell below detection limit of the assays.
IL-5
200
100
100
0
0
Figure 1. Spontaneous cytokine production by atopic dermatitis patients (AD), inhalation allergic patients (IA) and non-atopic donors (NA). Cells were cultured without added stimuli and harvested at 24 h (for determination of IL-1α, IL-4, IL-5, IL-6, TNF-α and TNF-β or at 72 h (for derermination of IL-2 and IFN-γ). Cytokine concentrations were determined by ELISA or immunoradiometric assay (IL-4) as described in Materials and Methods. Atopic dermatitis patients: (d), allergic patients: (.), non-allergic individuals: (r), Medians for each donor group: (–).
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 653
TABLE 1. Culture periods and assay characteristics for the cytokines tested in this study Main Culture period Assay cellular source (h) type*
IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
Monocytes T-lymphocytes T-lymphocytes T-lymphocytes Monocytes T-lymphocytes Monocytes T-lymphocytes
24 72 24 24 24 72 24 24
Sensitivity of assay (pg/ml)
E E I E E E E E
8 8 100 100 30 15 30 15
Cytokine profiles at optimal stimulations Figure 3 shows a comparison between the three donor groups, when cultures are stimulated optimally, as described above. IL-1α is not included in this figure, because no consistent treatment could be identified, which increased the level significantly above the spontaneous production. Compared to the two other groups, the AD group showed a lower production of IL-6 and TNF-α, but this was only statistically significant in the case of TNF-α. For IFN-γ, however, there was a significantly lowered production in the AD group. The IFNγ production was somewhat heterogenous, as two subjects expressed levels comparable to the IA and NA groups, the remainder being severely depressed. These two individuals did not otherwise express any aberrant cytokine production profile compared to the rest of the AD-group. The IA-group, on the other hand, showed
*E: ELISA, I: Immunoradiometric assay.
tures. The outcome of these experiments is depicted in Figure 2, where the median changes in cytokine production of the normal donors are illustrated as bars (error bars represent quartiles). The levels of IL-2, IL5, IL-6, IFN-γ, TNF-α and TNF-β could all be significantly increased by one or more of the stimuli. For IL-1α and IL-4, the stimuli did not induce significant changes compared to the spontaneous production, whereas PMA with or without PHA, actually decreased the levels of IL-6 in the supernatant. IL-1α
200 0
20 000 0
∗∗ ∗∗
∗∗
60 000 40 000
–20 000
20 000
–400
–40 000
0
IFN-γ
10 000
3000 2000
0
∗∗ ∗∗ ∗∗ 500
∗∗
∗
IL-4
1500
100
∗∗
∗
∗∗ ∗∗
IL-5
1250 ∆ pg/ml
∆ pg/ml
200
400
0
∗∗ ∗∗ ∗∗
400 300
600
200
1000 ∗∗
TNF-β
800 ∆ pg/ml
∆ pg/ml
20 000
∗∗ ∗∗ ∗∗ ∗∗ ∗∗
1000
4000
30 000
0
IFN-γ
5000
TNF-α
80 000
–200
40 000 ∆ pg/ml
IL-6
40 000 ∆ pg/ml
∆ pg/ml
400
∆ pg/ml
Cytokine
The stimulations in the two other groups (results not shown) generally coincided with the results shown in Figure 2, and produced a parallel pattern with the NA group. The conditions producing the highest and lowest levels for each of the cytokines are summarized in Table 2.
1000 750 500 250
0
Figure 2.
0 ∗∗ Alterations in cytokine levels induced by exogenous stimuli of normal donor cell culture.
Cultures and supernatants were otherwise treated as described in legend to Figure 1. The cytokine production is expressed relatively to the spontaneous cytokine production, i.e. the levels obtained after spontaneous production has been subtracted for each individual donor. Bars represent medians, error bars represent 1st and 3rd quartile. Stimuli (from left to right): PHA (u), Ionomycin (j), PMA , PHA 1 PMA , Ionomycin 1 PMA . *P , 5%; **P , 1%.
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CYTOKINE, Vol. 8, No. 8 (August 1996: 651–657)
TABLE 2. Summary of optimal stimulus for in vitro production of cytokines Stimulus producing lowest response
Cytokine IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
Same pattern in the three donor groups?
Stimulus producing highest response
No significant differences between stimuli PMA higher in IA Spontaneous PHA 1 PMA and ION 1 PMA Yes Spontaneous ION 1 PMA Only in IA Spontaneous ION 1 PMA Yes PMA PHA Yes Spontaneous ION 1 PMA Yes Spontaneous PHA 1 PMA and ION 1 PMA Yes Spontaneous PHA 1 PMA Yes
no sign of impaired IFN-γ production, but instead IL-4 and IL-5 levels were increased. IL-4 was also slightly, but not significantly increased in the AD group. Interdependency of cytokines Table 3 shows the coefficients of correlation between the spontaneous and the maximal production of the cytokines. Only combinations producing signifi-
cant correlations are shown. For the spontaneous production the two monokines IL-6 and TNF-α correlated as did the Th1 cytokines IL-2 and TNF-β. When stimulated to maximal production, a strikingly different pattern emerged: IL-4, IL-5, TNF-α and TNF-β were closely interrelated, i.e. the production of each cytokine correlated to the three others; moreover, the production of IFN-γ was correlated to both IL-5 and IL-6.
IL-6 600 000 400 000 200 000 80 000
TNF-α
//
60 000
pg/ml
pg/ml
80 000
40 000
60 000 40 000
20 000
20 000 0
0
P = 0.03
40 000
4000
30 000
3000
20 000
IFN-γ
TNF-β
pg/ml
5000
pg/ml
pg/ml
50 000
IL-2
2000
10 000
1000
0
0
1400 1200 1000 800 600 400 200 0
P = 0.01 IL-4
IL-5 3000
800
2500 pg/ml
pg/ml
600 400 200
1500 1000 500
0
0 P = 0.009
Figure 3.
2000
P = 0.02
Optimal cytokine production by the groups of donors.
Cultures were cultured with the optimal stimulus (as described in Table 2) and harvested for cytokine determination as described in Fig. 1. Stimulation with PHA was used for IL-6, stimulation with PHA 1 PMA for TNF-β and ION 1 PMA for TNF-α, IL-2, IFN-γ, IL-4 and IL-5. Atopic dermatitis patients: (d), allergic patients: (.), non-allergic individuals: (r), Medians for each donor groups: (–).
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 655
TABLE 3. Spearman’s coefficients of correlation between spontaneous and maximal production of cytokines Spontaneous
Maximal
IL-1α IL-1α IL-2 IL-4 IL-5 IL-6 IFN-γ TNF-α TNF-β
IL-2
IL-4
IL-5
IL-6
IFN-γ
TNF-α
TNF-β
— —
0.37* — 0.49*
—
0.43* 0.44*
0.49* 0.63** 0.59**
— 0.45*
0.39* — — 0.53**
—
*P , 0.05; **P , 0.01. All donors combined.
Correlation of IL-4, IL-5 and IFN-γ with IgE levels and eosinophil levels A negative correlation (r 5 20.42, P 5 0.022) was found between the level of serum IgE and the maximal IFN-γ production, but this correlation mainly reflected the differences between serum IgE and IFN-γ production for the AD group compared to the two other groups. No significant correlations were found between serum IgE and the maximal production of IL-4 and IL-5. A clear correlation was found between the spontaneous production of IL-1α and eosinophil number (r 5 0.63, P 5 0.00013). Moreover, correlations were found between IL-1α production and the levels of the eosinophilic proteins, eosinophilic cationic protein (ECP), (r 5 0.75, P , 1025) and eosinophilic protein X (EPX) (r 5 0.64, P 5 0.00012), released to serum during a clotting time of 2 h. Eosinophil numbers were not correlated to the spontaneous production of other cytokines.
DISCUSSION Atopic patients have been reported to have an aberrant cytokine production profile.5–8,13,14 Looking at the spontaneous production of cytokines, similar and low levels were found of the cytokines that are primarily T-cell derived. Monokines, however, were produced in considerable amounts, and increased levels of IL-1α were noted in the AD group. Because cytokine production is highly inducible, different forms of non-specific in vitro stimulations of PBMC were examined. It is interesting that the optimal conditions for cytokine production varied even between cytokines that are believed to be produced by the same cells. This was especially prominent for IL-1α, IL-6 and TNF-α. Thus, IL-1α production was not increased by any stimuli; the IL-6 response was optimally potentiated by PHA, and TNF-α was optimally induced by PHA or ionomycin in combination with PMA. IL-6 was the only cytokine
exhibiting a reduced response when PMA was added. It should be emphasized that the concentrations of cytokines in culture supernatants are dependent not only on production but also on degradation and consumption. It is likely, for example, that the different stimuli induce different ratios between a cytokine and its receptor. The kinetics in the system may also be important. Thus, in accordance with these results, Tang et al. found optimal IL-4 and IFN-γ production at 24 h and 72 h, respectively,6 while Reinhold et al. found optimal IFNγ production at 24 h.5 Takahashi et al. found optimal concentrations of the cytokines IL-4, IL-2, IFN-γ and TNF-α at 24 h.13 Looking at the cytokine profile under optimal conditions, it is clear that two patient groups express different abnormalities. While the IA group has increased expression of the Th2-type cytokines IL-4 and IL-5, the AD group demonstrates an impaired IFN-γ production, i.e. a Th1 defect. It is worth noticing, however, that the latter impairment is not accompanied by a reduction in IL-2 or TNF-β production. Several investigators have focused on the ratio between IL-4 and IFN-γ,6 and from this study it is clear that both patient groups will have an increased IL-4: IFN-γ ratio. It is important to recognize, however, that the two groups differ in their IgE response. While the IA group has a relatively large fraction of their total IgE directed to distinct inhalation allergens,15,16 the AD patients are probably subject to a polyclonal activation. This could be interpreted in the way that IA patients have relatively few, but allergenspecific Th2-type cells, which will help production of large amounts of allergen-specific IgE. AD patients, however, have a reduced level of Th1-type cells, and a larger panel of antigen-specificities may escape the otherwise very tight control of the IgE synthesis exerted by a large number of inhibiting T-cells. Because T-cell development is believed to be controlled by professional antigen presenting cells, it was of interest to try to identify differences between the three groups for the monokine production profiles.
656 / Poulsen et al.
The production of TNF-α was significantly lowered in the AD group, whereas IL-6 was reduced although not significantly. Kapp et al. have likewise reported a reduced production of TNF-α in AD patients—a reduction that was not seen for psoriasis patients;14 the findings in AD patients were confirmed by Takashi et al.13 A close correlation between the production of TNF-α and the Th2-type cytokines, IL-4 and IL-5 was observed. This correlation only became significant, however, if all three groups were combined. The additional finding that the spontaneous production of IL-1α was increased in the AD group could suggest that an increased IL-1α:TNF-α ratio might be of importance for the relative deficiency in IFN-γ production. In considering a possible role for IL-1α in atopic diseases, it is interesting that allergic patients express a much lower frequency of autoantibodies to IL-1α compared to normals.10 There was also a close correlation between the spontaneous IL-1α production and the number of eosinophil counts or the levels of eosinophil counts and the serum levels of the eosino-phil proteins, ECP and EPX/Eosinophil-derived neurotoxin (EDN), high coefficients of correlation were achieved (r 5 0.63; 0.75; and 0.64; P , 0.0001, respectively). It is tempting to speculate that IL-1α plays an important role in the development of Th2-like cells, but a more direct association between the proinflammatory cytokine and eosinophils should also be kept in mind. In conclusion, a marked difference in cytokine production profiles between patients with atopic dermatitis and inhalation allergies has been demonstrated. Whereas a deficiency in the ability to produce TNF-α and IFN-γ upon stimulation is an important feature in the AD group, the group of inhalation allergic patients suffers from an excessive production of “atopic” cytokines like IL-4 and IL-5.
MATERIALS AND METHODS Allergic and non-allergic donors Three groups of donors were admitted to the study: (1) Patients with atopic dermatitis with hyper serum-IgE (AD): 11 individuals with serum IgE . 5000 IU/ml. All patients in this group demonstrated atopic dermatitis according to the criteria of Hanifin and Rajka;18 (2) Allergic patients (IA): 10 individuals with total IgE in the range of 200–2000 IU/ml, and inhalant allergy to pollen, house dust mites or animal dander. The diagnosis was established by case history, positive skin prick test and presence of allergen-specific IgE; (3) Non-allergic donors (NA): 10 individuals with total IgE , 100 IU/ml and no history of allergy. None of the donors had received systemic glucocorticoid treatment or cytostatic treatment within 8 weeks of the study, and no patients were undergoing allergen specific immunotherapy. The age profiles were similar in the three groups (mean 6 SD: 36 6 11, 39 6 11 and 35 6 6 years) as were the distri-
CYTOKINE, Vol. 8, No. 8 (August 1996: 651–657)
bution between the two sexes (F/M): 7/4, 3/7 and 6/4. Total serum IgE in the three groups were: 12 000 6 9200, 485 6 416 and 32 6 30 IU/ml.9 Apart from the eosinophilic counts (0.51 3 106/ml, 0.18 3 106/ml and 0.13 3 106/ml) no abnormal or varying haematological parameters were observed.17
Blood sampling, isolation of cells and culture conditions Blood was drawn in Vacutainers (Labco Inc., Bucks, UK) with heparin for isolation of mononuclear cells. PBMC were isolated from heparinized blood using LymphoPrep™ (Specific density: 1.077) (Nycomed, Oslo, Norway). Cells were grown in 96-well plates (200 µl at 2 3 106/ml) in the medium described by Yssel19 at 37°C, in a humidified atmosphere containing 5% CO2. Cytokine levels were determined in supernatants of cultures with or without addition of various combinations of phytohaemagglutinin (Difco, Detroit, MI) (PHA, 1%), calcium ionophore (Ionomycin, Calbiochem, La Jolla, CA) (ION 1 µM), and phorbol myristate acetate (Sigma, St Louis, MO) (PMA, 50 ng/ml). Preliminary experiments had revealed maximum levels of IL-4 and IL-5 in 24 h cultures vs higher levels of IL-2 and IFN-γ in 72 h cultures, and these two culture periods were subsequently used. Based on kinetic studies, IL-1α, IL-6, TNF-α and TNF-β were determined in the 24 h cultures. Determination of cytokines IL-1α, IL-2, IL-4, IL-5, IL-6, TNF-α, TNF-β and IFN-γ Analytical sensitivities and references to detailed descriptions of the cytokine assays are given in Table 1.
Immunoradiometric assay for IL-4 An antiserum towards IL-4 was produced by immunizing rabbits with recombinant human IL-4 (a kind gift from Dr Jan de Vries, DNAX, Palo Alto, CA).9 IL-4 was determined in culture supernatants by an immunoradiometric assay using the IgG fraction of the rabbit anti-IL-4 antiserum which were coated onto “Maxisorp” tubes. Samples or standard (Genzyme, Cambridge, MA) were diluted in RPMI 1640 1 10% fetal calf serum, and the detecting antibody was radioiodinated rabbit anti IL-4 IgG. There was no cross-reactivity between the seven other natural cytokines or recombinant IL2, IL-5 and IFN-γ. The inter-assay coefficient of variation was 15%.
ELISA for IL-5 IL-5 was measured by a sandwich enzyme-linked immunoabsorbent assay (ELISA) using monoclonal rat antibodies. Briefly, Maxisorp plates (NUNC, Roskilde, Denmark) were coated with mAb rat anti-mouse/human IL-5 (clone TRFK5) (Pharmingen, San Diego, CA) and non-attached sites were blocked with 1% human serum albumin (Behringwerke, Marburg, Germany) in phosphate-buffered (10 mM, pH 7.4) saline (150 mM) (PBS). Samples and standard (recombinant human IL-5, Pharmingen) were diluted in RPMI 1640 1 10% fetal calf serum. Biotinylated monoclonal rat anti-human IL-5 (clone JES-A1 Pharmingen, cat no 18522
Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors / 657
D) and the streptavidin system were used to detect IL-5. Interassay variation was 10%. The ELISA did not show any crossreactivity with recombinant IL-2, IL-4 or IFN-γ.
ELISAs for IL-1α, IL-2, IL-6, TNF-α, TNF-β and IFN-γ IL-1α, IL-2, IL-6, TNF-α, TNF-β and IFN-γ was measured by a double sandwich ELISA using monospecific, polyclonal rabbit antibody.20 Immuno-Maxisorp plates (NUNC) were coated with protein A affinity-purified IgG. Nonattached sites were blocked with 5% human serum albumin in PBS. Samples were diluted in phosphate buffered saline (PBS) supplemented with 2% normal rabbit serum (DAKO, Glostrup, Denmark), EDTA 10 mM, aprotinin 2000 KIE/ml. The assays were calibrated with international standards of the respective cytokines (National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK). Biotinylated rabbit antibodies were used as reported along with streptavidin peroxidase (Kirkegaard and Perry La., Gaithersburg, MD). Development was caried out with 1,2phenylendiamine dihydrochloride and measured at 492 nm. The inter- and intra-assay coefficients of variation were less than 15%. The detection limits of these ELISAs are shown in Table 1.
Determination of Eosinophilic Cationic Protein (ECP) and Eosinophilic Protein X (EPX) ECP and EPX were determined in plasma and serum (clotting time: 2.0 h) as previously described.17
Statistics The Mann–Whitney test for unpaired and the Wilcoxon test for paired observations were applied for testing differences between groups. Coefficient of correlation was calcuated as Spearman’s ρ.
Acknowledgements This study was supported by the Danish Biotechnology Program. CM Reimert is a senior research fellow of the Danish Medical Research Council. The excellent technical assistance of Lena Baron, Ulla Minuva and Marianna Thomsen are gratefully acknowledged.
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2. Pene J, Rousset F, Briere F, Chretien I, Bonnefoy JY, Spits H, Yokota T, Arai N, Arai K, Banchereau J, de Vries JE (1988) IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferon γ and α and prostaglandin E2. Proc Natl Acad Sci USA 85:6880–6884. 3. Punnonen J, de Vries JE (1994) IL-13 induces proliferation, Ig isotype switching, and Ig synthesis by immature human fetal B cells. J Immunol 152:1094–1102. 4. Owen WF (1991) Cytokine regulation of eosinophil inflammatory disease. ACI News 3:85–89. 5. Reinhold U, Wehrmann W, Kukel S, Kreysel HW (1990) Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities. Clin Exp Immunol 79:374–379. 6. Tang M, Kemp A, Varigos G (1993) IL-4 and interferongamma production in children with atopic disease. Clin Exp Immunol 92:120–124. 7. Del Prete G, Tiri A, Maggi E, De Carli M, Macchia D, Parronchi P, Rossi ME, Pietrogrande MC, Ricci M, Romagnani S (1989) Defective in vitro production of gamma-interferon and tumor necrosis factor-alpha by circulating T cells from patients with hyperimmunoglobulin E syndrome. J Clin Invest 84:1830–1835. 8. Pene J, Rivier A, Lagier B, Becker WM, Michel FB, Bousquet J (1994) Differences in IL-4 release by PBMC are related with heterogeneity of atopy. Immunology 81:58–64. 9. Poulsen LK, Reimert CM, Bindslev-Jensen C, Hansen MB, Bendtzen K (1995) Biomolecular regulation of the IgE immune response. II. In vitro IgE synthesis and spontaneous production of cytokines. Int Arch Allergy Immunol 106:55–61. 10. Bendtzen K, Hansen MB, Ross C, Poulsen LK, Svenson M (1995) Cytokines and autoantibodies to cytokines. Stem Cells 13: 206–222. 11. Marshall JD, Wen Y, Abrams JS, Umetsu DT (1993) In vitro synthesis of IL-4 by human CD4 1 T cells requires repeated antigenic stimulation. Cell Immunol 152:18–34. 12. Kelso A (1995) Th1 and Th2 subsets: paradigms lost? Immunol Today 16:374–379. 13. Takahashi T, Sasaki Y, Hama K, Furue M, Ishibashi Y (1992) Production of IL-4, IL-2, IFN-gamma and TNF-alpha by peripheral blood mononuclear cells of patients with atopic dermatitis. J Dermatol Sci 3:172–180. 14. Kapp A, Textor A, Krutmann J, Moller A (1990) Immunomodulating cytokines in atopic dermatitis and psoriasis: production of tumour necrosis factor and lymphotoxin by mononuclear cells in vitro. Br J Dermatol 122:587–592. 15. Gleich GJ, Jacob GL (1975) Immunoglobulin E antibodies to pollen allergens account for high percentages of total immunoglobulin E protein. Science 190:1106–1108. 16. Poulsen LK, Pedersen MF, Malling H-J, Søndergaard I, Weeke B (1989) Maxisorp RAST. A sensitive method for detection of absolute quantities of antigen-specific IgE. Allergy 44:178–189. 17. Poulsen LK, Reimert CM, Bindslev-Jensen C (1994) In vitro release of eosinophil proteins in allergic and atopic dermatitis patients. Med Inflam 3:223–227. 18. Hanifin JM, Rajka G (1980) Diagnostic features of atopic dermatitis. Acta Derm Venereol (Stockh) 92:44–47. 19. Yssel H, de Vries JE, Koken M, van Blitterswijk W, Spits H (1984) Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J Immunol Methods 72:219–227. 20. Hansen MB, Svenson M, Bendtzen K (1991) Human antiinterleukin-1α antibodies. Immunol Lett 30:133–140.