IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics
© Stanciu et al. 2005
Received: 07 February 2005
Accepted: 07 July 2005
Published: 07 July 2005
Virus infections are the major cause of asthma exacerbations. CD8+ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8+ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8+ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8+ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.
Peripheral blood CD8+ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.
Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8+ T cells from mild atopic asthmatics.
These data suggest that during a respiratory virus infection activated CD8+ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.
Asthmatic airways are characterised by high levels of IL-4 due to excess Th2 responses to common aeroallergens [1–3]. Respiratory viruses are associated with 80 to 85% of asthma exacerbations in children and with 60% in adults and rhinovirus (RV) is common in all age groups [4–6]. We have found that atopic asthmatic subjects suffer from more severe and longer lasting lower respiratory tract symptoms when infected with RV than do normal subjects , but the mechanisms are not known. We proposed that one mechanism could be that immune responses to virus infection are type 1 deficient/type 2 augmented if they take place in a milieu rich in the type 2 cytokine IL-4 as occurs in atopic asthma . Optimal CD8+ T cell responses to respiratory viruses are predominantly type 1 and over exuberant type 2 responses are detrimental . Animal studies suggest that immune responses to virus infections are characterized by an increase in the frequency of type 2 cytokine-producing T cells if they take place in an allergic environment [10, 11]. Data from human studies also show that an excessive type 2/deficient type 1 cytokine response to RSV-infection in the respiratory tract is associated with a more severe clinical course .
We have published data showing that human CD8+ T cells from both normal and asthmatic subjects have the capacity to produce type 2 cytokines [13, 14]. We have recently shown that stimulation of CD8+ T cells from normal, healthy subjects in an IL-4 rich milieu significantly increased the number of IL-5-positive CD8+ T cells . Due to excess Th2 immune response to allergens, CD8+ T cells from atopic asthmatic subjects are continuous exposed to IL-4 . Here we investigated whether exogenous IL-4 present during TCR/CD3 stimulation modulates cell growth, surface phenotype (activation markers and adhesion molecules) and type 1 (IL-2 and IFN-γ) and type 2 (IL-4, -5, -13) cytokine production in CD8+ T cells from atopic asthmatic subjects. We found that the presence of exogenous IL-4 during the activation of mild atopic asthmatic CD8+ T cells enhanced cell growth and increased IL-5 and IL-13 production.
Characteristics of subjects
Asthma subjects (n = 10)
PC20 methacholine (mg/mL) *
Geometric mean (range)
5.33 (1.5 to 7.9)
Serum IgE (IU/mL)
100 (81 to 442)
Antibodies and other reagents
"CD8+ T cell isolation" kits were from Miltenyi (Miltenyi Biotec GmbH, Germany). Mouse anti-human CD3 Abs was purified from the OKT-3 hybridoma (American Type Culture Collection, Rockville, USA). rIL-2 and rIL-4 were purchased from Genzyme (Genzyme, West Malling, Kent, UK). Leu 4 (CD3)-Peridinin Chlorophyll Protein conjugate, Leu 3a (CD4)-FITC and -phycoerythrin (PE), Leu 2a (CD8)-FITC and -PE, CD14-PE, CD16-PE, CD19-PE, CD25-FITC, CD11a (LFA-1)-FITC and CD49d (VLA-4)-PE were from Becton Dickinson (Becton Dickinson, Mountain View, CA). CD54 (ICAM-1)-FITC was from Serotec (Serotec Ltd, Oxford, UK). CD30-FITC and CD154 (CD40 ligand, CD40L)-PE were from PharMingen (PharMingen, San Diego, CA). ELISA kits were obtained from Biosource (Biosource Europe S.A., Fleurus, Belgium).
CD8+ T cell purification and culture
CD8+ T cells were enriched by negative immunomagnetic selection using the MACS system . After passage through MACS columns, between 0.25 and 2.3 × 106 CD8+ T cells were recovered from 1 mL of blood. The resulting cell populations were routinely >98% CD3+ and >97% CD8+ and contained no detectable CD4+, CD16+, CD19+, CD14+ cells as determined by flow cytometry.
Purified CD8+ T cells were cultured in an antigen presenting cell-free system as previously described . In brief, 0.5 × 106 cells/mL were stimulated for 3 days with plate-bound anti-CD3 Abs (10 μg/mL) in 96-well microtiter plates in RPMI 1640 culture medium supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, antibiotics (all from Gibco BRL, Life Technologies, Uxbridge, UK), IL-2 (50 U/mL) with or without added IL-4 (10 ng/mL) (stimulation). After 3 days, the cells were harvested, washed, adjusted to 0.2 × 106/mL and cultured for 4 days in culture medium containing IL-2 (50 U/mL) (expansion). After every cycle of stimulation or expansion the cells were harvested and the number of live cells was determined by trypan blue exclusion and counting. After the second cycle of stimulation and expansion the cells were harvested, washed, adjusted to 1 × 106/mL and analysed.
Cell surface markers
Following the second cycle of stimulation/expansion, the cells were harvested and washed. PerCP-, FITC-, and PE-conjugated antibodies were added to 50 μl of suspensions (0.05 × 106 cells) and incubated for 30 min at 4°C. After washing in PBS, stained cells were resuspended in 500 μl PBS. Flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, CA). The FACScan program was used to acquire data of 10,000 events and the data were analysed by using Lysys II software. Live lymphocytes were gated by the forward scatter and side scatter pattern. Percentage in dot plots and mean fluorescence intensity (MFI) in histograms were analysed on CD3+CD8+ cells for the molecules of interest.
After two cycles of stimulation/expansion, CD8+ T cells (1 × 106/mL) were restimulated for 24 hours with phorbol myristate acetate (PMA) (20 ng/mL) and ionomycin (2 μg/mL) or with immobilised anti-CD3 Ab. Supernatants were microfuged for 5 minutes at 400 g to remove cell debris and IL-2, IL-4, IL-5, IL-13, and IFN-γ levels were measured by ELISA using commercial assay kits (Biosource, Europe SA). The limits of sensitivity of these assays were 3 pg/mL for IL-4, 4 pg/mL for IL-5 and IFN-γ, 5 pg/mL for IL-2, and 12 pg/mL for IL-13.
Paired Student t-test was used to analyse normally distributed data such as growth of CD8+ T cells cultured with or without IL-4. The Wilcoxon signed rank test for paired data was used for comparison of surface markers and for cytokine levels in culture supernatants. P values less than 0.05 were chosen for rejection of the null hypothesis.
IL-4 amplifies the growth of CD8+ T cells from atopic asthmatic subjects
IL-4 decreases surface expression of adhesion molecules in CD8+ T cells from atopic asthmatic subjects
Surface molecules on repeatedly stimulated CD8+ T cells*
97 ± 1
98 ± 0.5
97 ± 1
97 ± 1
0.2 ± 0.2
0.3 ± 0.2
2 ± 1
2 ± 1
7 ± 6
4 ± 1
53 ± 9
68 ± 9
23 ± 7
40 ± 6
14.5 ± 5
14.5 ± 6
444 ± 97
283 ± 72
280 ± 51
194 ± 25
37 ± 10
27 ± 7
IL-4 enhances type 2 cytokine production by CD8+ T cells from atopic asthmatic subjects
Type 2 cytokine production by repeatedly stimulated CD8+ T cells from atopic asthmatic subjects was increased by the presence of IL-4 during stimulation (Fig. 2). There was a trend towards an increase in IL-4 (median and [range]) production by CD8+ T cells stimulated in the presence of IL-4 (without IL-4, 146 pg/mL [20 to 793 pg/mL]; with IL-4, 389 pg/mL [45 to 4309 pg/mL], P < 0.1). IL-5 levels were significantly increased by the presence of IL-4 (without IL-4, 494 pg/mL [56 to 1612 pg/mL]; with IL-4, 1251 pg/mL [391 to 10936 pg/mL], P < 0.02). IL-13 levels were also significantly increased by the presence of IL-4 during CD8+ T cell stimulation (without IL-4, 1045 pg/mL [378 to 1392 pg/mL]; with IL-4, 1455 pg/mL [580 to 5820 pg/mL], P < 0.01).
Excess IL-4 during repeated stimulation of CD8+ T cells did not significantly alter the levels of IL-2 (without IL-4, 2000 pg/mL [1000 to 4000 pg/mL]; with IL-4, 2135 pg/mL [1000 to 2900 pg/mL]) and IFN-γ (without IL-4, 22 ng/mL [2 to 44 ng/mL]; with IL-4, 23 ng/mL [1 to 44 ng/mL] released in culture by CD8+ T cells.
In this study, we show that excess exogenous IL-4 during in vitro CD8+ T cell stimulation amplified the proliferation of CD8+ T cells from atopic asthmatic subjects. Furthermore, excess IL-4 resulted in a decrease in the expression of the adhesion molecules VLA-4 and LFA-1 on CD8+ T cells, along with increased production of the type 2 cytokines IL-5 and IL-13. These data indicate that the activation of CD8+ T cells from asthmatic subjects in the presence of IL-4 increases their capacity to produce type 2 cytokines but not IFN-γ or IL-2. These effects may be important in promoting adverse CD8+ T cell responses to virus infections in asthmatic subjects.
The presence of exogenous IL-4 during TCR/CD3 stimulation of peripheral blood CD8+ T cells induced expansion of cell numbers. It has previously been reported that IL-4 may act as a growth factor for human CD4+ and CD8+ T cell clones and that there is a synergy between IL-2 and IL-4 during CD8+ T cell proliferation . This suggests that stimulation of CD8+ T cells in an environment rich in IL-4 may result in the expansion of CD8+ T cells when exogenous IL-2 is present.
CD8+ T cells exposed to repeated stimulation in the presence of exogenous IL-4 decreased their expression of the α4β1 integrin (VLA-4, CD49d/CD29) and αLβ2 integrin (LFA-1, CD11a/CD18). VLA-4 is a surface marker associated with the capacity of T cells to migrate towards sites of antigen stimulation  and it has been previously reported that CD49d membrane expression on Th1 and Th2 clones is markedly reduced by activation . The reduction in the levels of adhesion molecules as a result of activation of CD8+ T cells may play a role in the retention of activated cells at sites of antigenic stimulation. Recently, in an animal model it was reported that type 2 or type 0 CD8+ T cells had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro and consequently reduced antiviral activity in vivo . The presence of exogenous IL-4 during stimulation of CD8+ T cells also tended to increase the frequencies of CD30-positive cells. The expression of CD30, a member of the TNF receptor family, by T cells has been previously reported to be primarily regulated by IL-4  and preferentially expressed by type 2 and type 0 cells . These data collectively suggest that the presence of IL-4 in the microenvironment induces a phenotype of cells similar to tissue-resident cells and induces patterns of expression of adhesion molecules associated with adoption of type 2 function. These data suggest our in vitro observations on induction of type 2 functions more likely to truly reflect events occurring in the lungs in vivo.
We have found that peripheral blood CD8+ T cells from atopic asthmatic individuals, when stimulated in the presence of exogenous IL-4, released higher levels of IL-5 and IL-13 as compared with CD8+ T cells stimulated in the absence of IL-4. It has previously been reported that exogenous IL-4 increased production of IL-4 and IL-5 in stimulated neonatal CD8+ T cells . However, it is now known that neonatal T cells are skewed towards a type 2 phenotype and may, therefore, not be representative of adult human CD8+ T cells . We have recently reported induction of type 2 activities in CD8+ T cells from healthy adult subjects during TCR stimulation by the presence of exogenous IL-4 . However, we observed only a tendency to increased IL-5-production and no increased IL-13-production in CD8+ T cells from normal subjects [ and unpublished data]. In the present study we extend these findings to confirm that induction by IL-4 of type 2 activities also occurs in CD8+ T cells from asthmatic subjects and demonstrate the deviation in phenotype towards type 2 is more extensive with increases in IL-5 and IL-13 production.
Respiratory virus infections are frequently associated with asthma exacerbations and CD8+ T cells are important in anti-viral immune responses. Given the above data in our in vitro model, we suggest that stimulation of CD8+ T cells during viral infections in an environment enriched in IL-4 as occurs in asthma is likely to favour the induction of type 2 cytokine synthesis by CD8+ T cells. Production of type 2 cytokines by CD8+ T cells during immune responses to viral infections in asthma has the potential to augment allergic inflammation by a number of different mechanisms including each of the type 2 cytokines measured in this study. This hypothesis is lent credence by our recent observations that virus infection and allergen exposure are synergistic risk factors for acute exacerbations of asthma .
By antagonising the production of IFN-γ, type 2 cytokines may also have effects on the capacity of CD8+ T cells to clear virus. In our culture conditions, even though exogenous IL-4 did not significantly decrease the levels of IFN-γ, the levels remained stable and the ratio of IL-4/IFN-γ was increased (data not shown). It is quite possible that during respiratory viral infections in asthmatic relative to normal subjects in vivo, where the stimulation conditions are different, IFN-γ production is depressed as a result of these mechanisms. This hypothesis is supported by the observation in mice that elevation of IL-4 levels concurrent with viral infections suppresses or delays activation of virus-specific CD8+ T cells and leads to delayed viral clearance [26–28]. We have previously reported a significant increase in submucosal CD4+ and CD8+ lymphocytes and in epithelial eosinophils in both normal and asthmatic subjects with experimental rhinovirus infections, and in asthmatic subjects eosinophil numbers remained elevated during convalescence . These data suggest that modulation by IL-4 of CD8+ virus-specific immune responses towards a type 2 phenotype may occur in humans in vivo, and that this may be an important mechanism in virus-induced exacerbations of asthma acting both by augmenting allergic inflammation and by diminishing viral clearance. A recent rhinovirus experimental infection study has supported this hypothesis by observing impaired virus clearance and increased symptoms in subjects with increased IL-5/IFN-γ mRNA ratios in induced sputum .
In conclusion, our data demonstrate induction of type 2 cytokine production and a tissue-resident phenotype by exogenous IL-4 in CD8+ T cells from atopic asthmatic subjects. Given the excess IL-4 observed in asthma we believe that these mechanisms may play an important role in impairing virus clearance and augmenting allergic inflammation in virus-induced asthma. We are currently testing this possibility in vivo in human experimental virus infection models.
S.L. Johnston and L.A. Stanciu thank the National Asthma Campaign and British Lung Foundation for their generous support.
- Robinson DS, Hamid Q, Yin S, Tsicopoulos A, Barkans J, Bentley AM, Corrigan C, Durham SR, Kay AB: Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992, 326:298–304.View ArticlePubMedGoogle Scholar
- Walker C, Bauer W, Braun RK, Menz G, Braun P, Schwarz F, Hansel TT, Villiger B: Activated T cells and cytokines in bronchoalveolar lavages from patients with various lung diseases associated with eosinophilia. Am J Respir Crit Care Med 1994, 150:1038–1048.View ArticlePubMedGoogle Scholar
- Shahid SK, Kharitonov SA, Wilson NM, Bush A, Barnes PJ: Increased interleukin-4 and decreased interferon-gamma in exhaled breath condensate of children with asthma. Am J Respir Crit Care Med 2002, 165:1290–1293.View ArticlePubMedGoogle Scholar
- Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, Symington P, O'Toole S, Myint SH, Tyrrell DAJ, Holgate ST: Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. Br Med J 1995, 310:1225–1229.View ArticleGoogle Scholar
- Nicholson KG, Kent J, Ireland DC: Respiratory viruses and exacerbations of asthma in adults. Br Med J 1993, 307:982–986.View ArticleGoogle Scholar
- Wark PA, Johnston SL, Moric I, Simpson JL, Hensley MJ, Gibson PG: Neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma. Eur Respir J 2002, 19:68–75.View ArticlePubMedGoogle Scholar
- Corne JM, Marshall C, Smith S, Schreiber J, Sanderson G, Holgate ST, Johnston SL: Frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study. Lancet 2002, 359:831–834.View ArticlePubMedGoogle Scholar
- Papadopoulos NG, Stanciu LA, Papi A, Holgate ST, Johnston SL: A defective type 1 response to rhinovirus in atopic asthma. Thorax 2002, 57:328–332.View ArticlePubMedPubMed CentralGoogle Scholar
- Woodland DL: Cell-mediated immunity to respiratory virus infections. Curr Opin Immunol 2003, 15:430–435.View ArticlePubMedGoogle Scholar
- Coyle AJ, Erard F, Bertrand C, Walti S, Pircher H, Le Gros G: Virus-specific CD8 + cells can switch to interleukin 5 production and induce airway eosinophilia. J Exp Med 1995, 181:1229–1233.View ArticlePubMedGoogle Scholar
- Makela MJ, Tripp R, Dakhama A, Park JW, Ikemura T, Joetham A, Waris M, Anderson LJ, Gelfand EW: Prior airway exposure to allergen increases virus-induced airway hyperresponsiveness. J Allergy Clin Immunol 2003, 112:861–869.View ArticlePubMedGoogle Scholar
- Legg JP, Hussain IR, Warner JA, Low JL, Johnston SL, Warner JO: Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med 2003, 168:633–639.View ArticlePubMedGoogle Scholar
- Stanciu LA, Shute J, Holgate ST, Djukanovic R: Production of IL-8 and IL-4 by positively and negatively selected CD4 + and CD8 + human T cells following a four-step cell separation method including magnetic cell sorting (MACS). J Immunol Methods 1996, 189:107–115.View ArticlePubMedGoogle Scholar
- Stanciu LA, Shute J, Promwong C, Holgate ST, Djukanovic R: Increased levels of IL-4 in CD8 + T cells in atopic asthma. J Allergy Clin Immunol 1997, 100:373–378.View ArticlePubMedGoogle Scholar
- Stanciu LA, Roberts K, Lau LCK, Coyle AJ, Johnston SL: Induction of type 2 activity in adult human CD8 Tcells by repeated stimulation an IL-4. Int Immunol 2001, 13:341–348.View ArticlePubMedGoogle Scholar
- American Thoracic Society: Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease and asthma. AmRev Respir Dis 1987, 136:225–244.View ArticleGoogle Scholar
- Stanciu LA, Djukanovic R: Isolation of T-cell subsets by magnetic cell sorting (MACS). In Methods in molecular biology. T cell protocols: Development and activation. Volume 134. Edited by: Kearse KP. New York: Humana Press; 1999:133–141.Google Scholar
- Spits H, Yssel H, Takebe Y, Arai N, Yokota T, Lee F, Arai K, Banchereau J, de Vries JE: Recombinant interleukin 4 promotes the growth of human T cells. J Immunol 1987, 139:1142–7.PubMedGoogle Scholar
- Nakajima H, Sano H, Nishimura T, Yoshida S, Iwamoto I: Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J Exp Med 1994, 179:1145–1154.View ArticlePubMedGoogle Scholar
- Almerigogna F, D'Elios MM, De Carli M, Del Prete G: Markers of Th1 and Th2 cells. Chem Immunol 1996, 63:30–50.View ArticlePubMedGoogle Scholar
- Wirth S, van der Broek M, Frossard CP, Hugin AW, Leblond I, Pircher H, Hauser C: CD8 + T cells secreting type 2 lymphokines are defective in protection against viral infection. Cell Immunol 2000, 202:13–22.View ArticlePubMedGoogle Scholar
- Nakamura T, Lee RK, Nam SY, Al-Ramadi BK, Koni PA, Bottomly K, Podack ER, Flavell RA: Reciprocal regulation of CD30 expression on CD4. J Immunol 1997, 158:2090–2098.PubMedGoogle Scholar
- Byun D-G, Demeure CE, Yang LP, Shu U, Ishihara H, Vezzio N, Gately MK, Delespesse G: In vitro maturation of neonatal human CD8 T lymphocytes into IL-4 and IL-5-producing cells. J Immunol 1994, 153:4862–4871.PubMedGoogle Scholar
- Prescott SL, Macaubas C, Smallacombe T, Holt BJ, Sly PD, Holt PG: Development of allergen-specific T-cell memory in atopic and normal children. Lancet 1999, 353:196–200.View ArticlePubMedGoogle Scholar
- Green RM, Custovic A, Sanderson G, Hunter J, Johnston SL, Woodcock A: Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study. BMJ 2002, 324:763.View ArticlePubMedPubMed CentralGoogle Scholar
- Moran TM, Isobe H, Fernandez-Sesma A, Schulman JL: Interleukin-4 causes delayed virus clearance in influenza virus-infected mice. J Virol 1996, 70:5230–5235.PubMedPubMed CentralGoogle Scholar
- Fischer JE, Johnson JE, Kuli-Zade RK, Johnson TR, Aung S, Parker RA, Graham BS: Overexpression of interleukin-4 delays virus clearance in mice infected with respiratory syncytial virus. J Virol 1997, 71:8672–8677.PubMedPubMed CentralGoogle Scholar
- Bot A, Holz A, Christen U, Wolfe T, Temann A, Flavell R, von Herrath M: Local IL-4 expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic T cell responses. Virology 2000, 269:66–77.View ArticlePubMedGoogle Scholar
- Fraenkel DJ, Bardin PG, Sanderson G, Lampe F, Johnston SL, Holgate ST: Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects. Am J Respir Crit Care Med 1995, 151:879–886.PubMedGoogle Scholar
- Gern JE, Vrtis R, Grindle KA, Swenson C, Busse WW: Relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection. Am J Respir Crit Care Med 2000, 162:2226–2231.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.