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The Modulation of Cytokine and IgE Production by Tumor Necrosis Factor-β in Atopic Dermatitis

2000; Elsevier BV; Volume: 114; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.2000.00847.x

1523-1747

Orathai Jirapongsananuruk, Heather L. Donahue, Anne E. Trumble, Donald Y.M. Leung,

Urticaria and Related Conditions

SummaryAtopic dermatitis (AD) is associated with increased IL-4, IL-5, and IL-13 but decreased IFN-γ production. This cytokine profile may account for the atopic features of this illness, including IgE upregulation. Recent studies have demonstrated that tumor necrosis factor (TNF)-β is produced by Th1-like cells, but the cytokine modulation by TNF-β and the clinical significance of this cytokine in AD is not known. Therefore, this study was carried out to determine the potential role of TNF-β in AD. In this study, we cultured peripheral blood mononuclear cells from patients with AD and normal subjects with anti-CD3 monoclonal antibodies and investigated the production of TNF-β by ELISA. The mean ± SEM of TNF-β production in AD was significantly lower than normal subjects (p = 0.03). The effect of TNF-β on cytokine production was investigated by culturing peripheral blood mononuclear cells with anti-CD3 monoclonal antibodies in the presence or absence of TNF-β. Compared with medium control, TNF-β significantly decreased IL-5 (p = 0.0004) and IL-13 (p = 0.008) but increased IFN-γ (p = 0.001) production. The effect of TNF-β on IgE production was determined by culturing peripheral blood mononuclear cells in the IL-4- and anti-CD40-induced IgE production system. Interestingly, TNF-β significantly decreased IgE (p = 0.02), but not IgG production compared with medium control. Our study demonstrates that TNF-β production is downregulated in AD. This cytokine increases IFN-γ production but decreases IL-5, IL-13, as well as IgE production. These findings suggest a potential role for TNF-β in the pathogenesis of AD. Atopic dermatitis (AD) is associated with increased IL-4, IL-5, and IL-13 but decreased IFN-γ production. This cytokine profile may account for the atopic features of this illness, including IgE upregulation. Recent studies have demonstrated that tumor necrosis factor (TNF)-β is produced by Th1-like cells, but the cytokine modulation by TNF-β and the clinical significance of this cytokine in AD is not known. Therefore, this study was carried out to determine the potential role of TNF-β in AD. In this study, we cultured peripheral blood mononuclear cells from patients with AD and normal subjects with anti-CD3 monoclonal antibodies and investigated the production of TNF-β by ELISA. The mean ± SEM of TNF-β production in AD was significantly lower than normal subjects (p = 0.03). The effect of TNF-β on cytokine production was investigated by culturing peripheral blood mononuclear cells with anti-CD3 monoclonal antibodies in the presence or absence of TNF-β. Compared with medium control, TNF-β significantly decreased IL-5 (p = 0.0004) and IL-13 (p = 0.008) but increased IFN-γ (p = 0.001) production. The effect of TNF-β on IgE production was determined by culturing peripheral blood mononuclear cells in the IL-4- and anti-CD40-induced IgE production system. Interestingly, TNF-β significantly decreased IgE (p = 0.02), but not IgG production compared with medium control. Our study demonstrates that TNF-β production is downregulated in AD. This cytokine increases IFN-γ production but decreases IL-5, IL-13, as well as IgE production. These findings suggest a potential role for TNF-β in the pathogenesis of AD. Atopic dermatitis (AD) is a common allergic skin disease associated with elevated IgE synthesis (Hanifin, 1982Hanifin J.M. Atopic dermatitis.J Am Acad Dermatol. 1982; 6: 1-13Abstract Full Text PDF PubMed Scopus (132) Google Scholar;Leung, 1995Leung D.Y.M. Atopic dermatitis: the skin as a window into the pathogenesis of chronic allergic diseases.J Allergy Clin Immunol. 1995; 96: 302-318Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). The IgE molecule is thought to play an important role in allergen-driven responses and allergen presentation by CD23-positive B cells in AD (van der Heijden et al., 1993van der Heijden F.L. van Neirvan R.J.J. van Katwijk M. Bos J.D. Kapsenberg M.L. Serum IgE facilitated allergen presentation in atopic disease.J Immunol. 1993; 150: 3643-3650PubMed Google Scholar). Skin infiltrating cells in acute lesions and peripheral blood T cells from patients with AD express high levels of IL-4 and IL-5 but not IFN-γ consistent with the T helper 2 (Th2)-type cytokine pattern of synthesis (van der Heijden et al., 1991van der Heijden F.L. Wierenga E.A. Bos J.D. Kapsenberg M.L. High frequency of IL-4-producing CD4 + allergen-specific T lymphocytes in atopic dermatitis lesional skin.J Invest Dermatol. 1991; 97: 389-394Abstract Full Text PDF PubMed Google Scholar;Jujo et al., 1992Jujo K. Renz H. Abe J. Trumble A. Gelfand E.W. Leung D.Y.M. Decreased gamma interferon and increased interleukin-4 production promote IgE synthesis in atopic dermatitis.J Allergy Clin Immunol. 1992; 90: 323-330Abstract Full Text PDF PubMed Scopus (262) Google Scholar;Renz et al., 1992Renz H. Jujo K. Bradley K.L. Domenico J. Gelfand E.W. Leung D.Y.M. Enhance IL-4 production and IL-4 receptor expression in atopic dermatitis and their modulation by interferon-gamma.J Invest Dermatol. 1992; 99: 403-408Abstract Full Text PDF PubMed Google Scholar;van Reijsen et al., 1992van Reijsen F.C. Bruijnzeel-Kooman CAFM Kalthoff F.S. et al.Skin derived aeroallergen-specific T cell clones of TH-2 phenotype in patients with atopic dermatitis.J Allergy Clin Immunol. 1992; 90: 184-192Abstract Full Text PDF PubMed Scopus (336) Google Scholar;Hamid et al., 1994Hamid Q. Boguniewicz M. Leung D.Y.M. Differential cytokine gene expression in acute vs. chronic atopic dermatitis.J Clin Invest. 1994; 94: 870-876Crossref PubMed Scopus (706) Google Scholar, Hamid et al., 1996Hamid Q. Naseer T. Minshall E.M. Song T.L. Boguniewicz M. Leung D.Y.M. In vivo expression of interleukin-12 and interleukin-13 in atopic dermatitis.J Allergy Clin Immunol. 1996; 98: 225-231Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Tumor necrosis factor (TNF)-β (lymphotoxin, LT) is a T helper 1 (Th1)-type cytokine (Romagnani, 1995Romagnani S. Biology of human Th1 and Th2 cells.J Clin Immunol. 1995; 15: 121-129Crossref PubMed Scopus (359) Google Scholar) that is primarily produced by T and B lymphocytes (Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). Although TNF-β has been isolated together with TNF-α from human sources, there are many more reports on TNF-α than TNF-β (Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar) . These two cytokines have structural homology and share the same TNF receptor (TNFR) but demonstrate differences with respect to their cellular sources, receptor binding properties (Andrews et al., 1990Andrews J.S. Berger A.E. Ware C.F. Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes: TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4 + T cell hybridoma.J Immunol. 1990; 144: 2582-2591PubMed Google Scholar), p75 TNFR dissociation rate (Medvedev et al., 1996Medvedev A.E. Espevik T. Ranges G. Sundan A. Distinct roles of the two tumor necrosis factor (TNF) receptors in modulating TNF and lymphotoxin α effects.J Biol Chem. 1996; 271: 9778-9784Crossref PubMed Scopus (86) Google Scholar), and signal transduction (Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). TNF-β is expressed primarily by lymphocyte, whereas TNF-α is expressed by macrophages, natural killer cells, lymphocytes, polymorphonuclear cells, mast cells, and a variety of tumor cells (Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). TNF-α is known to induce Th1-type cytokines such as IL-1, TNF-α, and IFN-γ (Goh, 1990Goh C.R. Tumor necrosis factors in clinical practice.Ann Acad Medicine. 1990; 19: 235-239PubMed Google Scholar;Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). Whether TNF-β can induce Th1-type cytokine remains to be determined. In AD, it has been demonstrated that TNF-α is downregulated along with IFN-γ in in vitro studies of human peripheral blood mononuclear cells (PBMC) (Kapp et al., 1990Kapp A. Textor A. Krutmann J. Moller A. Immunomodulating cytokines in atopic dermatitis and psoriasis: production of tumor necrosis factor and lymphotoxin by mononuclear cells in vitro.Br J Dermatol. 1990; 122: 587-592Crossref PubMed Scopus (20) Google Scholar;Poulsen et al., 1996Poulsen L.K. Bindslev-Jensen C. Diamant M. et al.Biomolecular regulation of the IgE immune response III. Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors.Cytokine. 1996; 8: 651-657Crossref PubMed Scopus (14) Google Scholar). In this study, therefore, we studied the production of this cytokine from PBMC of AD patients compared with normal control subjects and investigated the effect of TNF-β on Th1/Th2-type cytokines and IgE production. We obtained blood from 16 patients (six males and 10 females, aged 27–45 y) with moderate to severe AD (skin involvement more than 20%) diagnosed according to well-defined criteria (Hanifin and Rajka, 1980Hanifin J.M. Rajka G. Diagnostic features of atopic dermatitis.Acta Dermatol Venerol (Stockh). 1980; 92: 44-47Google Scholar). None of the patients had previously used systemic corticosteroids. Topical corticosteroids were withheld for greater than 48 h before blood collection. Sixteen normal healthy adults who were skin prick test negative for a panel of common allergens were used as the healthy control group (eight males and eight females, aged 27–36 y). Informed consent was obtained from all subjects before entry into the study. Recombinant (r) human IL-4 was a kind gift from Schering Research Institute (Bloomfield, NJ). A monoclonal antibody (MoAb) against human CD40 was purchased from Pharmingen (San Diego, CA). A MoAb against human CD3 was purchased from Ortho Diagnostic Systems (Raritan, NJ). Recombinant (r) human TNF-β was purchased from R&D Systems (Minneapolis, MN) PBMC were isolated from heparinized venous blood from study subjects by density gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden), and washed three times in Hanks' balanced salt solution (Gibco, Grand Island, NY). For cytokine studies, PBMC (at 1 × 106 cells per ml) were cultured in RPMI 1640 (Bio-Whitaker, Walkersville, MD) supplemented with 10% heat inactivated fetal calf serum (Gibco), 20 mM HEPES buffer (Gibco), antibiotics, and 2 mM L-glutamine (Gibco) in flat-bottomed 24 well plates (Costar, Cambridge, MA) at 37°C in a humidified 5% CO2 atmosphere with antihuman CD3 MoAb (20 ng per ml) for 72 h. Culture supernatants were collected and stored at - 20°C until assayed for TNF-β by ELISA. In selected experiments, rTNF-β (500 ng per ml) was added in the culture system to examine the effects of TNF-β on cytokine production. For immunoglobulin (Ig) studies, PBMC (at 1 × 106 cells per ml) were cultured in Iscove's modified Dulbecco's medium (Gibco) supplemented with 10% heat inactivated fetal calf serum (Gibco), 0.5% bovine serum albumin, 50 μg human transferrin per ml, 5 μg bovine insulin per ml, 1 μg of each oleic, linolenic, and palmitic acid (Sigma, St Louis, MO) per ml as previously described (Yssel et al., 1984Yssel H. deVries J.E. Koken M. Blitterawijk W.V. Spits H. Serum free medium for generation and propagation of functional human cytotoxic and helper T cell clones.J Immunol Methods. 1984; 72: 219-227Crossref PubMed Scopus (333) Google Scholar;Hofer et al., 1995Hofer M.F. Lester M.R. Schlievert P.M. Leung D.Y.M. Effect of bacterial toxins on IgE synthesis.Clin Exp Allergy. 1995; 25: 1218-1227Crossref PubMed Scopus (58) Google Scholar) in round-bottomed 96 well plates (Costar) at 37°C in a humidified 5% CO2 atmosphere. Recombinant human IL-4 (400 μ per ml) and anti-CD40 MoAb (1 μg per ml) were added to promote Ig synthesis. Varying concentrations of TNF-β were added to investigate the effect of this cytokine on Ig synthesis. Culture supernatants were collected after 14 d and stored at - 20°C until assayed. The IgE assay was carried out as previously described (Jujo et al., 1993Jujo K. Renz H. Abe J. Trumble A. Gelfand E.W. Leung D.Y.M. Pokeweed mitogen induced IgE synthesis in the presence of a blocking antibody to the interferon-_ receptor.J Allergy Clin Immunol. 1993; 91: 1201-1216Abstract Full Text PDF Scopus (12) Google Scholar). Ninety-six-well microtiter plates (Dynatech, Chantilly, VI) were coated with 0.1 ml of a 1:1 mixture of purified monoclonal antihuman IgE (4.15 and 7.12, a kind gift from Dr. A. Saxon, University of California, Los Angeles, CA) diluted in 0.1 M NaHCO3 at pH 9.6, at a final concentration of 20 μg per ml after overnight incubation at 4°C. The wells were blocked with 0.1% gelatin in 0.1 M NaHCO3 at room temperature for 1 h. Serial dilutions of culture supernatants were incubated in duplicate for 2 h at room temperature and overnight at 4°C, with parallel human IgE standard controls (Pharmacia). The plates were then washed, a 1:100 dilution of affinity-purified biotinylated goat antihuman IgE (Vector, Burlingame, CA) was added, and plates were incubated for 90 min at 37°C. After a subsequent wash, wells were incubated with a 1:1500 dilution of streptavidin-alkaline phosphatase (Tago, Burlingame, CA) for 90 min at 37°C. The wells were then developed with 2 mM p-nitrophenyl phosphate substrate (Sigma) and the optical density was read at 405 nm on an Emax microplate reader (Molecular Devices, Menlo Park, CA). The concentrations of IgE in the supernatants were read from an IgE standard curve. The lower limit of sensitivity of this assay was 0.50 ng per ml. The protocol for IgG assay (Jujo et al., 1993Jujo K. Renz H. Abe J. Trumble A. Gelfand E.W. Leung D.Y.M. Pokeweed mitogen induced IgE synthesis in the presence of a blocking antibody to the interferon-_ receptor.J Allergy Clin Immunol. 1993; 91: 1201-1216Abstract Full Text PDF Scopus (12) Google Scholar) was identical to that for IgE except that the initial capture antibody was an affinity-purified polyclonal goat antihuman IgG antibody (Tago) diluted in 0.1 M NaHCO3 at a final concentration of 10 μg per ml. The second antibody was biotinylated goat-antihuman IgG (Vector). The IgG standards were obtained from Sigma. The lower limit of sensitivity of this assay was 1 ng per ml. Cytokines assays were measured by a commercially available enzyme immunoassay kit. IFN-γ and IL-5 (Endogen, Woburn, MA) had a lower sensitivity limit of 2 pg per ml. IL-13 (Endogen) had a lower sensitivity limit of 7 pg per ml. TNF-β (R&D Systems) had a lower sensitivity limit of 7 pg per ml. Data were expressed as individual values and the mean for each subject group. Statistical comparison were made using an unpaired Student t test to compare different groups of study subjects, and a paired Student t test to compare the results from the same subjects. Geometric mean was used for IgE and IgG, which were nonparametric data. These data were log-transformed prior to analysis to produce an approximately normal distribution. A series of repeated measure analysis of variance (Anova) models were performed. All pairwise comparisons between pairs of mean were made using the Tukey-Kramer multiple comparison procedure. All findings were considered significant at a p value of ≤ 0.05. A recent study demonstrated that lymphotoxin-α gene polymorphisms can be associated with the diagnosis of childhood asthma (Albuquerque et al., 1998Albuquerque R.V. Hayden C.M. Palmer L.J. et al.Association of polymorphisms within the tumor necrosis factor (TNF) genes and childhood asthma.Clin Exp Allergy. 1998; 28: 578-584Crossref PubMed Scopus (133) Google Scholar). Thus this cytokine may play a role in the pathogenesis of atopic diseases; however, there has been no study of genetic polymorphisms for this cytokine in AD. Although it has been demonstrated that TNF-α is downregulated in AD, there has been no study demonstrating the significance of TNF-β in the pathogenesis of AD. As T lymphocytes are a major source for TNF-β, we used anti-CD3 MoAb as an inducer for cytokine production from T cells in this study. PBMC from 16 AD patients and 16 normal donors were cultured for 72 h with antihuman CD3 MoAb. Supernatants were measured for TNF-β by ELISA. As shown in Figure 1, we demonstrated that TNF-β is downregulated in AD compared with control subjects. The mean ± SEM of TNF-β in AD was 488.94 ± 59.84 pg per ml versus 682.71 ± 60.03 pg per ml for normal subjects (p = 0.03). These findings support the concept that Th1-type cytokines are downregulated in AD (van der Heijden et al., 1991van der Heijden F.L. Wierenga E.A. Bos J.D. Kapsenberg M.L. High frequency of IL-4-producing CD4 + allergen-specific T lymphocytes in atopic dermatitis lesional skin.J Invest Dermatol. 1991; 97: 389-394Abstract Full Text PDF PubMed Google Scholar;Jujo et al., 1992Jujo K. Renz H. Abe J. Trumble A. Gelfand E.W. Leung D.Y.M. Decreased gamma interferon and increased interleukin-4 production promote IgE synthesis in atopic dermatitis.J Allergy Clin Immunol. 1992; 90: 323-330Abstract Full Text PDF PubMed Scopus (262) Google Scholar;Renz et al., 1992Renz H. Jujo K. Bradley K.L. Domenico J. Gelfand E.W. Leung D.Y.M. Enhance IL-4 production and IL-4 receptor expression in atopic dermatitis and their modulation by interferon-gamma.J Invest Dermatol. 1992; 99: 403-408Abstract Full Text PDF PubMed Google Scholar;van Reijsen et al., 1992van Reijsen F.C. Bruijnzeel-Kooman CAFM Kalthoff F.S. et al.Skin derived aeroallergen-specific T cell clones of TH-2 phenotype in patients with atopic dermatitis.J Allergy Clin Immunol. 1992; 90: 184-192Abstract Full Text PDF PubMed Scopus (336) Google Scholar;Hamid et al., 1994Hamid Q. Boguniewicz M. Leung D.Y.M. Differential cytokine gene expression in acute vs. chronic atopic dermatitis.J Clin Invest. 1994; 94: 870-876Crossref PubMed Scopus (706) Google Scholar, Hamid et al., 1996Hamid Q. Naseer T. Minshall E.M. Song T.L. Boguniewicz M. Leung D.Y.M. In vivo expression of interleukin-12 and interleukin-13 in atopic dermatitis.J Allergy Clin Immunol. 1996; 98: 225-231Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). TNF-α is known to induce IL-1, TNF-α, and IFN-γ, which are Th1-type cytokines (Aggarwal and Natarajan, 1996Aggarwal B.B. Natarajan K. Tumor necrosis factors: Developments during the last decade.Eur Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar). In contrast, there is very limited evidence that TNF-β induces Th1-type cytokines (Goh, 1990Goh C.R. Tumor necrosis factors in clinical practice.Ann Acad Medicine. 1990; 19: 235-239PubMed Google Scholar), even though TNF-β can upregulate IFN-γ receptors and increase IFN-γ binding to its receptor (Raitano et al., 1991Raitano A.B. Scuderi P. Korc M. Upregulation of interferon-γ binding by tumor necrosis factor and lymphotoxin: Disparate potencies of the cytokines and modulation of their effects by phorbal ester.J Interferon Res. 1991; 11: 61-67Crossref PubMed Scopus (15) Google Scholar). In the following experiments, we found that TNF-β upregulated IFN-γ but downregulated IL-5 and IL-13. PBMC from 16 AD patients and 16 normal donors were cultured for 72 h with antihuman CD3 MoAb in the absence or presence of TNF-β. Supernatants were measured for IFN-γ, IL-5, and IL-13 by ELISA. Figure 2 demonstrates that in AD subjects, TNF-β significantly upregulated IFN-γ production. The mean ± SEM of IFN-γ productions in AD PBMC cultured with TNF-β was 4498.70 ± 682.62 pg per ml versus 2480.03 ± 391.80 pg per ml for medium control (p = 0.001). In contrast to IFN-γ, TNF-β significantly downregulated IL-5 and IL-13 (Figure 3, Figure 4). The mean ± SEM of IL-5 production in AD PBMC cultured with TNF-β was 32.95 ± 6.27 pg per ml versus 92.47 ± 16.00 pg per ml for medium (p = 0.0004). The mean ± SEM of IL-13 production in AD PBMC cultured with TNF-β was 81.12 ± 15.37 pg per ml versus 140.27 ± 23.67 pg per ml for medium (p = 0.008). The effect of TNF-β on cytokine production in normal subjects was the same as in AD patients (data not shown). These data suggest that TNF-β induces Th1-type cytokines and this effect can be seen in both atopic and normal subjects.Figure 3IL-5 synthesis is significantly decreased by TNF-β. PBMC from 16 AD patients were cultured for 72 h in the presence of anti-CD3 MoAb with or without TNF-β. Supernatants were measured for IL-5 by ELISA. Results from each individual and mean values are shown.View Large Image Figure ViewerDownload (PPT)Figure 4IL-13 synthesis is significantly decreased by TNF-β. PBMC from 16 AD patients were cultured for 72 h in the presence of anti-CD3 MoAb with or without TNF-β. Supernatants were measured for IL-13 by ELISA. Results from each individual and mean values are shown.View Large Image Figure ViewerDownload (PPT) To further demonstrate that TNF-β may play a role in the pathogenesis of AD, which is frequently associated with increased IgE synthesis, we investigated whether TNF-β had any impact in IgE synthesis. PBMC from five normal subjects were cultured with IL-4 and anti-CD40 MoAb to promote Ig production for 14 d. Varying concentrations of TNF-β were added in the culture system in order to investigate the effects of this cytokine on Ig production. Supernatants were measured for Ig by ELISA. As shown in Figure 5, IgE production was downregulated by TNF-β in a concentration-dependent fashion. In contrast, TNF-β did not have any effect on IgG production. In conclusion, this study demonstrates that TNF-β synthesis is downregulated in AD patients. This cytokine upregulated Th1-type cytokine but downregulated Th2-type cytokine. Furthermore, it suppressed IgE but not IgG production. The mechanism for the effects of TNF-β appears to be due to a direct action on target cells as anti-IFN-γ does not reverse the effects of TNF-β (data not shown). These findings suggest a potential role of TNF-β in the pathogenesis of AD and the possibility of using this cytokine as a therapeutic target in the future. Supported by HL 36577, AR 41256, HL 37260, 5MO1 RR00051, and an Endowed Fellowship Award from National Jewish Medical and Research Center. Dr. Jirapongsananuruk's work was supported in part by the Florence Myers Goldhamer Fellowship in Pediatric Allergy and Immunology funded through National Jewish Medical and Research Center. The authors wish to thank Maureen Plourd-Sandoval for preparation of this manuscript.