- Email: [email protected]
Is TGF-β1 the key to suppression of human asthma?
Research Update
Acknowledgements
We thank M. Duarte, E. Santiago and M.L. Subirá for suggestions and scientific discussion, and M. Mendez, J. Vidal, M.J. Huarte, CICYT and Antibióticos Farma SA for generous grants. References 1 Trinchieri, G. (1998) Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70, 83–243 2 Bramson, J.L. et al. (1996) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and longlasting immunity that is associated with highly localized expression of interleukin-12. Hum. Gene Ther. 7, 1995–2002 3 Sgadari, C. et al. (1996) Inhibition of angiogenesis by interleukin-12 is mediated by the interferoninducible protein 10. Blood 87, 3877–3882 4 Barajas, M. et al. (2001) Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for IL-12. Hepatology 33, 52–61 5 Addison, C.L. et al. (1998) Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors. Gene Ther. 5, 1400–1409 6 Di Carlo, E. et al. (2000) The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement. J. Immunol. 165, 3111–3118
TRENDS in Immunology Vol.22 No.3 March 2001
7 Osaki, T. et al. (1999) Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18). Gene Ther. 6, 808–815 8 Emtage, P.C. et al. (1999) Adenoviral vectors expressing lymphotactin and interleukin 2 or lymphotactin and interleukin 12 synergize to facilitate tumor regression in murine breast cancer models. Hum. Gene Ther. 10, 697–709 9 Narvaiza, I. et al. (2000) Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy. J. Immunol. 164, 3112–3122 10 Zitvogel, L. et al. (1996) Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors. Eur. J. Immunol. 26, 1335–1341 11 Sun, Y. et al. (2000) Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone. Hum. Gene Ther. 11, 127–138 12 Martinet, O. et al. (2000) Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long-term remission of liver metastases in a mouse model. J. Natl. Cancer Inst. 92, 931–936 13 Chen, S.H. et al. (2000) Rejection of disseminated metastases of colon carcinoma by synergism of
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IL-12 gene therapy and 4-1BB costimulation. Mol. Ther. 2, 39–46 14 Mazzolini, G. et al. (2000) Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level. Hum. Gene Ther. 11, 113–125 15 Melero, I. et al. (1999) Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas. Gene Ther. 6, 1779–1784 16 Nishioka, Y. et al. (1999) Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res. 59, 4035–4041
Ignacio Melero* Guillermo Mazzolini Iñigo Narvaiza Cheng Qian Jesús Prieto The Gene Therapy Division, Dept of Medicine, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain. *e-mail: [email protected] Lieping Chen Dept of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
β1 the key to suppression of human asthma? Is TGF-β Atsuhito Nakao β1) is Transforming growth factor β1 (TGF-β produced by many types of cells that are activated in the asthmatic response. Recent studies have highlighted this cytokine as an important negative regulator in an experimental model of asthma. Although β1 in human asthma the role of TGF-β remains obscure, data derived from animal models have encouraged the further investigation of such suppression mechanisms in order to develop novel therapies for asthma.
Asthma is a complex disorder characterized by airway hyperresponsiveness (AHR) and airway inflammation. Evidence has accumulated regarding factors that promote the asthma phenotype1, but the mechanisms by which the asthma phenotype is suppressed are largely unclear. Recently, a pleiotropic cytokine, transforming growth factor β1 (TGF-β1), has been reported to function as a negative regulator of AHR and airway
inflammation in an experimental model of asthma2–5. Here, we consider this evidence and discuss possible roles of TGF-β1 in suppression of human asthma. Current understanding of the pathophysiology of asthma
Asthma is a complex disorder consisting of various cellular and/or cytokine/chemokine networks1. The presentation of inhaled allergens to CD4+ T cells in the lungs of susceptible individuals results in the production of cytokines such as interleukin 4 (IL-4), IL-5, IL-9 and IL-13, which orchestrate the differentiation, recruitment and activation of eosinophils and mast cells in the airway mucosa1,6,7. Such effector cells release inflammatory mediators that cause acute bronchial constriction, disruption of the airway epithelial layer, alterations in neural control of airway tone, increased mucus production and increased smooth muscle mass. These consequences of the inflammatory process
induce AHR. Recent studies have shown that not only T cells but also a variety of other cells, including mast cells, bronchial epithelial cells and smooth muscle cells, which are activated by various mediators, contribute to the development of AHR in part through the secretion of cytokines or chemokines that generate tissue inflammation8,9. β elicit its biological effects? How does TGF-β
TGF-β1 is a member of the TGF-β superfamily, which comprises a large number of cytokines including TGF-βs, bone morphogenetic proteins (BMPs) and activins. These cytokines carry out a wide range of biological functions including cell proliferation, differentiation and apoptosis. TGF-β1 is inhibitory for inflammatory cells such as T cells, B cells, dendritic cells, mast cells and eosinophils, and also modifies the functions of structural cells such as bronchial epithelial cells, fibroblasts and bronchial smooth muscle
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TGF-β
P TGF-β receptor
P I
P II
II
P I
P
P
Smad7 –
Smad2
Smad3
DNA-binding partner
P P
P P
Smad4
Transcription
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Fig. 1. Transforming growth factor β (TGF-β) signaling by Smad proteins. TGF-β receptors consist of type I and type II receptors with intrinsic serine/threonine kinase activity. TGF-β binds to the type II receptor, which recruits the type I receptor, leading to the assembly of a heterotetrameric receptor complex in which the type II receptor phosphorylates (P) and activates the type I receptor. The pathway-restricted Smads (R-Smads) Smad2 and Smad3 are phosphorylated by the serine/threonine kinase of the type I receptor, leading to hetero-oligomeric complexes with a common-mediator Smad (Co-Smad), Smad4. The complexes then translocate to the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins and recruitment of transcriptional co-activators or co-repressors. An inhibitory Smad (I-Smad), Smad7, associates with the activated type I receptor and interferes with the activation of Smad2 and Smad3 by competing with their receptor interaction and thereby blocking their phosphorylation, resulting in TGF-β-mediated transcriptional responses. Expression of Smad7 is upregulated by various cytokines, including TGF-β. Smad7 might thus act in a negative-feedback loop to control TGF-β responses.
cells8–10. Interestingly, these cells develop the capacity to produce TGF-β1 following their activation10. Thus, it appears that TGF-β1 acts in a negative-feedback loop to suppress activation of these cells. Signaling by members of the TGF-β superfamily occurs through the Smad family of proteins (Fig. 1)11. The activated TGF-β receptors induce phosphorylation of Smad2 and Smad3, which are pathway-restricted Smads (R-Smads), and these form hetero-oligomeric complexes with Smad4, a commonmediator Smad (Co-Smad). The complexes then translocate to the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins and recruitment of transcriptional co-activators or corepressors. By contrast, Smad6 and Smad7, which are inhibitory Smads (I-Smads), block TGF-β-mediated http://immunology.trends.com
transcriptional responses. Recent studies suggest that Smad7 might be more potent as an inhibitor of TGF-β signaling than Smad6. Smad7 is also known to inhibit activin- and BMP-mediated signaling. Smad7 associates with the activated TGF-β receptors and interferes with the activation of Smad2 and Smad3 by competing with their receptor interaction, thereby blocking their phosphorylation. β1 can suppress AHR and airway TGF-β inflammation in a murine model of asthma
Given that TGF-β1 downregulates or modifies the functions of several cell types10, it would be reasonable to postulate that TGF-β1 might also suppress AHR and airway inflammation in asthma through downregulation of cells that are involved in asthma (Fig. 2). Recently, Hansen et al. provided evidence that TGF-β does suppress AHR
and airway inflammation in an experimental model of asthma2. They showed that transfer of ovalbumin (OVA)-specific T helper (Th) cells engineered in vitro to express latent TGF-β abolished AHR and airway inflammation induced by OVA-specific Th2 cells in severe combined immunodeficient (SCID) and BALB/c mice. The effect of TGF-β-producing T cells was antigen specific; it was also shown to be dependent on the secretion of TGF-β because the inhibitory effect was observed only when OVA was challenged, and a neutralizing monoclonal antibody to TGF-β abolished the inhibitory effect. Regulatory T cells that are generated after antigen feeding or inhalation play an important role in peripheral T-cell tolerance; this is known as ‘oral or respiratory tolerance’ through TGF-β1 secretion12. Previously, Haneda et al. showed that TGF-β1-producing T cells generated after oral or respiratory tolerance ameliorated antigen-induced eosinophilic airway inflammation3,4. They induced oral or respiratory tolerance by feeding or intratracheal administration of high-dose OVA in BALB/c mice. The adoptive transfer of CD4+ lymph node T cells from the OVA-tolerant mice inhibited OVA-induced eosinophilic inflammation in the airways of OVA-sensitized mice. The CD4+ T cells from the tolerant mice produced TGF-β1 upon OVA challenge, and neutralizing antibody against TGF-β1 abrogated the inhibitory effect by the transfer of CD4+ T cells from the tolerant mice. Thus, they showed that TGF-β1 produced by T cells in a particular situation of peripheral T-cell tolerance prevented airway inflammation in asthma. Taken together, it appears clear that TGF-β1 can suppress AHR and airway inflammation in a murine model of asthma. These findings suggest that TGFβ1 produced in the lung upon allergen challenge is sufficient to suppress the asthmatic phenotype in mice. But how does TGF-β1 produced in the airways suppress AHR and airway inflammation in an experimental model of asthma? β1 acts on T cells TGF-β
One possible mechanism by which TGF-β1 suppresses AHR and airway inflammation has been suggested by a recent study using transgenic mice in
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T cells Smooth muscle cells Dendritic cells
Mast cells
TGF-β
Eosinophils
Fibroblasts
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to inhibit activation of many types of cells that are involved in AHR and airway inflammation. Alternatively, as TGF-β has been implicated in tissue repairing14, the high levels of TGF-β1 in asthmatic airways might be involved in repairing processes of asthmatic airways that are damaged by inflammation. TGF-β1 in the airways of asthmatics could thus function as a healing molecule in the airways of asthmatics, promoting the process of tissue repair and diminishing AHR and airway inflammation. Persistent activity of TGF-β1 induced by chronic inflammation, which might be caused by repeated stimulation with allergens, might lead to the detrimental effect of TGF-β1 known as tissue fibrosis or airway remodeling, which results in chronic airflow obstruction. Conclusion
Bronchial epithelial cells
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Fig. 2. Transforming growth factor β1 (TGF-β1) in the airways of asthmatic patients might play a central role in the regulation of airway hyperresponsiveness (AHR) and airway inflammation. A variety of specific and nonspecific stimuli trigger T cells in the asthmatic airways, leading to production of cytokines/chemokines, and thereby the recruitment and activation of eosinophils, mast cells, bronchial epithelial cells, fibroblasts and smooth muscle cells. Major cellular sources of TGF-β1 in asthmatic airways include activated eosinophils, bronchial epithelial cells and fibroblasts, but mast cells, smooth muscle cells and T cells are also known to produce TGF-β1 upon activation. As TGF-β1 is inhibitory for these cells, TGF-β might act in a negative-feedback loop to regulate AHR and airway inflammation in asthma. Inhibitory effects of TGF-β1 on T cells appear to be critical determinants in regulating AHR and airway inflammation. Other cells described in the figure could also be targets of TGF-β1.
which TGF-β signaling is blocked specifically in mature T cells5. Transgenic mice have been established expressing Smad7 under the control of a distal Lck promoter, which specifically directs high Smad7 expression in peripheral T cells5. Peripheral T cells in the transgenic mice showed high expression of Smad7, resulting in the suppression of TGF-βinduced Smad2 phosphorylation and growth inhibition. The development of the immune system in the transgenic mice was normal and the mice survived into adulthood, suggesting that TGF-β signaling in T cells was not essential for normal T-cell homeostasis. Importantly, both OVA-induced AHR and airway inflammation was enhanced in the Smad7-transgenic mice when compared with the wild-type littermates. The enhanced AHR and airway inflammation was associated with high production of Th2-type cytokines in the bronchoalveolar lavage fluid (BALF) of the transgenic mice. TGF-β1 levels in the BALFs after inhaled OVA challenge were http://immunology.trends.com
comparable between the transgenic mice and the wild-type littermates. These findings indicated that blockade of TGF-β signaling in T cells enhanced AHR and airway inflammation in mice, suggesting that TGF-β1 acting on T cells suppressed the asthmatic phenotype in mice. β1 in the airways of Possible roles of TGF-β asthmatics
Although the inhibitory effects of TGF-β1 on the asthmatic phenotype in mice appear clear, the roles of TGF-β1 in human asthma remain obscure and require further investigation. The levels of TGF-β1 in the airways of asthmatics are reported to be higher than in normal subjects13. Inflammatory cells such as eosinophils and bronchial epithelial cells are major cellular sources of TGF-β1 in asthmatic airways. These findings suggest some roles of TGF-β1 in the pathophysiology of human asthma. On the basis of data from animal studies, the elevated levels of TGF-β1 in asthmatic airways might be a result of compensation
Although human asthma is clearly not the same as in experimental mouse models, recent data from such models encourage the further investigation of the roles of TGF-β1 in human asthma. A study of Smad3-knockout mice suggests that there are complexities in understanding the various roles of the TGF-β signaling pathway15, indicating that further study will be necessary before the precise role of this cytokine is known. Accumulating knowledge on TGF-β signal transduction will help reveal the detailed mechanisms by which TGF-β1 elicits its effects on target cells in the airways of the experimental models, eventually suppressing AHR and airway inflammation. It is hoped that when the roles of TGF-β1 in human asthma are clearer, the modification of TGF-β1 activity inside or outside the cells, possibly via molecular approaches such as using anti-sense Smad7 oligonucleotide, will offer some attractive avenues for potential therapeutic intervention for asthma. Acknowledgements
I thank P. ten Dijke, I. Iwamoto, Y. Saito, H. Nakajima, H. Tomioka, T. Tokuhisa, H. Ogawa, K. Okumura, C. Ra, H. Ushio, T. Imamura, M. Kawabata, K. Miyazono, A. Ishisaki, S. Itoh and C-H. Heldin for support; I. Nakao for illustration; and E. Kamijima for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Science
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and Culture, Japan, the Uehara Memorial Foundation for Biomedical Science, Japan, and the Foundation of Human Science, Japan. References 1 Wills-Karp, M. (1999) Immunological basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17, 255–281 2 Hansen, G. et al. (2000) CD4 T helper cells engineered to produce latent TGF-β1 reverse allergen-induced airway hyperreactivity and inflammation. J. Clin. Invest. 105, 51–70 3 Haneda, K. et al. (1997) TGF-β induced by oral tolerance ameliorates experimental tracheal eosinophilia. J. Immunol. 159, 4484–4490 4 Haneda, K. et al. (1999) Transforming growth factor-β secreted from CD4 T cells ameliorates antigen-induced eosinophilic inflammation. A novel high-dose tolerance in trachea. Am. J. Respir. Cell Mol. Biol. 21, 268–274
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5 Nakao, A. et al. (2000) Blockade of TGF-β/Smad signaling by overexpression of Smad7 in T cells enhances antigen-induced airway inflammation and airway reactivity. J. Exp. Med. 192, 151–158 6 Busse, W.W. (1998) Inflammation in asthma: the corner stone of the disease and target of therapy. J. Allergy Clin. Immunol. 102, S17–S22 7 Temann, U.A. et al. (1998) Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J. Exp. Med. 188, 1307–1320 8 Holgate, S.T. (2000) Epithelial damage and response. Clin. Exp. Allergy 30, 37–41 9 Hirst, S.J. (2000) Airwary smooth muscle as a target in asthma. Clin. Exp. Allergy 30, 54–59 10 Wahl, S.M. (1992) Transforming growth factor-β in inflammation: a cause and a cure. J. Clin. Immunol. 12, 61–74 11 Piek, E. et al. (2000) Specificity, diversity, and regulation of TGF-β superfamily signaling. FASEB J. 13, 2105–2124
12 Holt, P.G. and McMenamin, C. (1989) Defence against allergic sensitization in the healthy lung: the role of inhalation tolerance. Clin. Exp. Allergy 19, 255–262 13 Redington, A.E. et al. (1997) Transforming growth factor-β1 in asthma. Measurement in bronchoalveolar lavage fluid. Am. J. Respir. Crit. Care Med. 156, 642–647 14 Singer, A.J. and Clark, R.A.F. (1999) Cutaneous wound healing. New Engl. J. Med. 341, 738–746 15 Ashcroft, G.S. et al. (1999) Mice lacking Smad3 show accelerated would healing and an impaired local inflammatory response. Nat. Cell Biol. 1, 260–266
Atsuhito Nakao Allergy Research Center, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. e-mail: [email protected]
Effector and regulatory T cells in allergic contact dermatitis Andrea Cavani, Cristina Albanesi, Claudia Traidl, Silvia Sebastiani and Giampiero Girolomoni Allergic contact dermatitis is a prototypic T-cell-mediated disease that has a socioeconomic impact in industrialized countries. Here, Andrea Cavani and colleagues highlight recent developments in the T-cell-based effector and regulatory mechanisms of this common skin disorder.
Allergic contact dermatitis (ACD) results from a T-cell response to harmless, lowmolecular-weight chemicals (haptens) applied to the skin. In the sensitization phase, haptens penetrating the skin are collected by resident dendritic cells that migrate to the regional lymph nodes to activate and clonally expand specific T-cell precursors1. Re-exposure to the relevant hapten initiates the efferent phase and clinical expression of ACD, characterized by the rapid recruitment and activation of specific T cells at the sites of hapten challenge. In spite of the longlasting persistence of the hapten in the skin, the reaction is self-limited, suggesting that regulatory mechanisms are actively involved in the termination of ACD. T-cell recruitment in ACD
The recruitment of T cells in the skin is regulated by the expression of proper
homing receptors, such as the cutaneous lymphocyte-associated antigen (CLA), which mediates rolling of T cells over activated endothelial cells expressing E-selectin1. More recently, chemokine receptors have been proposed as important regulators of the tissue targeting of T cells. In line with this concept, it has been shown that skinseeking CLA+ T cells co-express CC chemokine receptor 4 (CCR4), the ligand for thymus and activation-regulated chemokine [TARC; CC chemokine ligand 17 (CCL17)] and macrophage-derived chemokine (MDC; CCL22). CCR4 triggered by TARC exposed on the endothelial cell surface during inflammatory skin disorders is thought to augment integrin-dependent firm adhesion of T cells to endothelial intercellular adhesion molecule 1 (ICAM-1)2. T-cell migration into peripheral tissues mostly depends on their chemokine receptor profile. Owing to the high expression of CCR5 and CXCR3, T helper 1 (Th1) cells preferentially migrate to the respective ligands, macrophage inflammatory protein 1β (MIP-1β; CCL4) and interferon γ (IFN-γ)-inducible protein 10 [IP-10; CXC chemokine ligand 10
(CXCL10)]. By contrast, T helper 2 (Th2) cells are mostly attracted by eotaxin (CCL11), TARC and MDC, and I-309 (CCL1), because of the high levels of CCR3, CCR4 and CCR8, respectively3. Epidermal keratinocytes have been extensively investigated as a source of inflammatory mediators for the initiation and amplification of skin immune responses, and T-cell-derived lymphokines are among the most potent activators of keratinocytes. Treatment with IFN-γ or IFN-γ plus tumor necrosis factor α (TNF-α) induces keratinocytes to express ICAM-1 and mature MHC class II molecules, and to release a vast array of growth factors, chemokines and cytokines such as interleukin 1 (IL-1), TNF-α and granulocyte–macrophage colonystimulating factor (GM-CSF)4,5. IL-17, a cytokine produced by skin-infiltrating Th1 and Th2, but not CD8+ T cells, reinforces many of the effects induced by IFN-γ (Ref. 4). Interestingly, the Th2 cytokine IL-4 acts synergistically with IFN-γ to enhance keratinocyte ICAM-1 expression and release of the CXCR3 agonistic chemokines, IP-10, monokine induced by IFN-γ (Mig; CXCL9) and IFN-inducible T-cell α-chemoattractant (I-TAC;
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Acknowledgements
We thank M. Duarte, E. Santiago and M.L. Subirá for suggestions and scientific discussion, and M. Mendez, J. Vidal, M.J. Huarte, CICYT and Antibióticos Farma SA for generous grants. References 1 Trinchieri, G. (1998) Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70, 83–243 2 Bramson, J.L. et al. (1996) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and longlasting immunity that is associated with highly localized expression of interleukin-12. Hum. Gene Ther. 7, 1995–2002 3 Sgadari, C. et al. (1996) Inhibition of angiogenesis by interleukin-12 is mediated by the interferoninducible protein 10. Blood 87, 3877–3882 4 Barajas, M. et al. (2001) Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for IL-12. Hepatology 33, 52–61 5 Addison, C.L. et al. (1998) Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors. Gene Ther. 5, 1400–1409 6 Di Carlo, E. et al. (2000) The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement. J. Immunol. 165, 3111–3118
TRENDS in Immunology Vol.22 No.3 March 2001
7 Osaki, T. et al. (1999) Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18). Gene Ther. 6, 808–815 8 Emtage, P.C. et al. (1999) Adenoviral vectors expressing lymphotactin and interleukin 2 or lymphotactin and interleukin 12 synergize to facilitate tumor regression in murine breast cancer models. Hum. Gene Ther. 10, 697–709 9 Narvaiza, I. et al. (2000) Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy. J. Immunol. 164, 3112–3122 10 Zitvogel, L. et al. (1996) Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors. Eur. J. Immunol. 26, 1335–1341 11 Sun, Y. et al. (2000) Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone. Hum. Gene Ther. 11, 127–138 12 Martinet, O. et al. (2000) Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long-term remission of liver metastases in a mouse model. J. Natl. Cancer Inst. 92, 931–936 13 Chen, S.H. et al. (2000) Rejection of disseminated metastases of colon carcinoma by synergism of
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IL-12 gene therapy and 4-1BB costimulation. Mol. Ther. 2, 39–46 14 Mazzolini, G. et al. (2000) Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level. Hum. Gene Ther. 11, 113–125 15 Melero, I. et al. (1999) Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas. Gene Ther. 6, 1779–1784 16 Nishioka, Y. et al. (1999) Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res. 59, 4035–4041
Ignacio Melero* Guillermo Mazzolini Iñigo Narvaiza Cheng Qian Jesús Prieto The Gene Therapy Division, Dept of Medicine, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain. *e-mail: [email protected] Lieping Chen Dept of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
β1 the key to suppression of human asthma? Is TGF-β Atsuhito Nakao β1) is Transforming growth factor β1 (TGF-β produced by many types of cells that are activated in the asthmatic response. Recent studies have highlighted this cytokine as an important negative regulator in an experimental model of asthma. Although β1 in human asthma the role of TGF-β remains obscure, data derived from animal models have encouraged the further investigation of such suppression mechanisms in order to develop novel therapies for asthma.
Asthma is a complex disorder characterized by airway hyperresponsiveness (AHR) and airway inflammation. Evidence has accumulated regarding factors that promote the asthma phenotype1, but the mechanisms by which the asthma phenotype is suppressed are largely unclear. Recently, a pleiotropic cytokine, transforming growth factor β1 (TGF-β1), has been reported to function as a negative regulator of AHR and airway
inflammation in an experimental model of asthma2–5. Here, we consider this evidence and discuss possible roles of TGF-β1 in suppression of human asthma. Current understanding of the pathophysiology of asthma
Asthma is a complex disorder consisting of various cellular and/or cytokine/chemokine networks1. The presentation of inhaled allergens to CD4+ T cells in the lungs of susceptible individuals results in the production of cytokines such as interleukin 4 (IL-4), IL-5, IL-9 and IL-13, which orchestrate the differentiation, recruitment and activation of eosinophils and mast cells in the airway mucosa1,6,7. Such effector cells release inflammatory mediators that cause acute bronchial constriction, disruption of the airway epithelial layer, alterations in neural control of airway tone, increased mucus production and increased smooth muscle mass. These consequences of the inflammatory process
induce AHR. Recent studies have shown that not only T cells but also a variety of other cells, including mast cells, bronchial epithelial cells and smooth muscle cells, which are activated by various mediators, contribute to the development of AHR in part through the secretion of cytokines or chemokines that generate tissue inflammation8,9. β elicit its biological effects? How does TGF-β
TGF-β1 is a member of the TGF-β superfamily, which comprises a large number of cytokines including TGF-βs, bone morphogenetic proteins (BMPs) and activins. These cytokines carry out a wide range of biological functions including cell proliferation, differentiation and apoptosis. TGF-β1 is inhibitory for inflammatory cells such as T cells, B cells, dendritic cells, mast cells and eosinophils, and also modifies the functions of structural cells such as bronchial epithelial cells, fibroblasts and bronchial smooth muscle
http://immunology.trends.com 1471–4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(00)01827-5
Research Update
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TGF-β
P TGF-β receptor
P I
P II
II
P I
P
P
Smad7 –
Smad2
Smad3
DNA-binding partner
P P
P P
Smad4
Transcription
TRENDS in Immunology
Fig. 1. Transforming growth factor β (TGF-β) signaling by Smad proteins. TGF-β receptors consist of type I and type II receptors with intrinsic serine/threonine kinase activity. TGF-β binds to the type II receptor, which recruits the type I receptor, leading to the assembly of a heterotetrameric receptor complex in which the type II receptor phosphorylates (P) and activates the type I receptor. The pathway-restricted Smads (R-Smads) Smad2 and Smad3 are phosphorylated by the serine/threonine kinase of the type I receptor, leading to hetero-oligomeric complexes with a common-mediator Smad (Co-Smad), Smad4. The complexes then translocate to the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins and recruitment of transcriptional co-activators or co-repressors. An inhibitory Smad (I-Smad), Smad7, associates with the activated type I receptor and interferes with the activation of Smad2 and Smad3 by competing with their receptor interaction and thereby blocking their phosphorylation, resulting in TGF-β-mediated transcriptional responses. Expression of Smad7 is upregulated by various cytokines, including TGF-β. Smad7 might thus act in a negative-feedback loop to control TGF-β responses.
cells8–10. Interestingly, these cells develop the capacity to produce TGF-β1 following their activation10. Thus, it appears that TGF-β1 acts in a negative-feedback loop to suppress activation of these cells. Signaling by members of the TGF-β superfamily occurs through the Smad family of proteins (Fig. 1)11. The activated TGF-β receptors induce phosphorylation of Smad2 and Smad3, which are pathway-restricted Smads (R-Smads), and these form hetero-oligomeric complexes with Smad4, a commonmediator Smad (Co-Smad). The complexes then translocate to the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins and recruitment of transcriptional co-activators or corepressors. By contrast, Smad6 and Smad7, which are inhibitory Smads (I-Smads), block TGF-β-mediated http://immunology.trends.com
transcriptional responses. Recent studies suggest that Smad7 might be more potent as an inhibitor of TGF-β signaling than Smad6. Smad7 is also known to inhibit activin- and BMP-mediated signaling. Smad7 associates with the activated TGF-β receptors and interferes with the activation of Smad2 and Smad3 by competing with their receptor interaction, thereby blocking their phosphorylation. β1 can suppress AHR and airway TGF-β inflammation in a murine model of asthma
Given that TGF-β1 downregulates or modifies the functions of several cell types10, it would be reasonable to postulate that TGF-β1 might also suppress AHR and airway inflammation in asthma through downregulation of cells that are involved in asthma (Fig. 2). Recently, Hansen et al. provided evidence that TGF-β does suppress AHR
and airway inflammation in an experimental model of asthma2. They showed that transfer of ovalbumin (OVA)-specific T helper (Th) cells engineered in vitro to express latent TGF-β abolished AHR and airway inflammation induced by OVA-specific Th2 cells in severe combined immunodeficient (SCID) and BALB/c mice. The effect of TGF-β-producing T cells was antigen specific; it was also shown to be dependent on the secretion of TGF-β because the inhibitory effect was observed only when OVA was challenged, and a neutralizing monoclonal antibody to TGF-β abolished the inhibitory effect. Regulatory T cells that are generated after antigen feeding or inhalation play an important role in peripheral T-cell tolerance; this is known as ‘oral or respiratory tolerance’ through TGF-β1 secretion12. Previously, Haneda et al. showed that TGF-β1-producing T cells generated after oral or respiratory tolerance ameliorated antigen-induced eosinophilic airway inflammation3,4. They induced oral or respiratory tolerance by feeding or intratracheal administration of high-dose OVA in BALB/c mice. The adoptive transfer of CD4+ lymph node T cells from the OVA-tolerant mice inhibited OVA-induced eosinophilic inflammation in the airways of OVA-sensitized mice. The CD4+ T cells from the tolerant mice produced TGF-β1 upon OVA challenge, and neutralizing antibody against TGF-β1 abrogated the inhibitory effect by the transfer of CD4+ T cells from the tolerant mice. Thus, they showed that TGF-β1 produced by T cells in a particular situation of peripheral T-cell tolerance prevented airway inflammation in asthma. Taken together, it appears clear that TGF-β1 can suppress AHR and airway inflammation in a murine model of asthma. These findings suggest that TGFβ1 produced in the lung upon allergen challenge is sufficient to suppress the asthmatic phenotype in mice. But how does TGF-β1 produced in the airways suppress AHR and airway inflammation in an experimental model of asthma? β1 acts on T cells TGF-β
One possible mechanism by which TGF-β1 suppresses AHR and airway inflammation has been suggested by a recent study using transgenic mice in
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to inhibit activation of many types of cells that are involved in AHR and airway inflammation. Alternatively, as TGF-β has been implicated in tissue repairing14, the high levels of TGF-β1 in asthmatic airways might be involved in repairing processes of asthmatic airways that are damaged by inflammation. TGF-β1 in the airways of asthmatics could thus function as a healing molecule in the airways of asthmatics, promoting the process of tissue repair and diminishing AHR and airway inflammation. Persistent activity of TGF-β1 induced by chronic inflammation, which might be caused by repeated stimulation with allergens, might lead to the detrimental effect of TGF-β1 known as tissue fibrosis or airway remodeling, which results in chronic airflow obstruction. Conclusion
Bronchial epithelial cells
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Fig. 2. Transforming growth factor β1 (TGF-β1) in the airways of asthmatic patients might play a central role in the regulation of airway hyperresponsiveness (AHR) and airway inflammation. A variety of specific and nonspecific stimuli trigger T cells in the asthmatic airways, leading to production of cytokines/chemokines, and thereby the recruitment and activation of eosinophils, mast cells, bronchial epithelial cells, fibroblasts and smooth muscle cells. Major cellular sources of TGF-β1 in asthmatic airways include activated eosinophils, bronchial epithelial cells and fibroblasts, but mast cells, smooth muscle cells and T cells are also known to produce TGF-β1 upon activation. As TGF-β1 is inhibitory for these cells, TGF-β might act in a negative-feedback loop to regulate AHR and airway inflammation in asthma. Inhibitory effects of TGF-β1 on T cells appear to be critical determinants in regulating AHR and airway inflammation. Other cells described in the figure could also be targets of TGF-β1.
which TGF-β signaling is blocked specifically in mature T cells5. Transgenic mice have been established expressing Smad7 under the control of a distal Lck promoter, which specifically directs high Smad7 expression in peripheral T cells5. Peripheral T cells in the transgenic mice showed high expression of Smad7, resulting in the suppression of TGF-βinduced Smad2 phosphorylation and growth inhibition. The development of the immune system in the transgenic mice was normal and the mice survived into adulthood, suggesting that TGF-β signaling in T cells was not essential for normal T-cell homeostasis. Importantly, both OVA-induced AHR and airway inflammation was enhanced in the Smad7-transgenic mice when compared with the wild-type littermates. The enhanced AHR and airway inflammation was associated with high production of Th2-type cytokines in the bronchoalveolar lavage fluid (BALF) of the transgenic mice. TGF-β1 levels in the BALFs after inhaled OVA challenge were http://immunology.trends.com
comparable between the transgenic mice and the wild-type littermates. These findings indicated that blockade of TGF-β signaling in T cells enhanced AHR and airway inflammation in mice, suggesting that TGF-β1 acting on T cells suppressed the asthmatic phenotype in mice. β1 in the airways of Possible roles of TGF-β asthmatics
Although the inhibitory effects of TGF-β1 on the asthmatic phenotype in mice appear clear, the roles of TGF-β1 in human asthma remain obscure and require further investigation. The levels of TGF-β1 in the airways of asthmatics are reported to be higher than in normal subjects13. Inflammatory cells such as eosinophils and bronchial epithelial cells are major cellular sources of TGF-β1 in asthmatic airways. These findings suggest some roles of TGF-β1 in the pathophysiology of human asthma. On the basis of data from animal studies, the elevated levels of TGF-β1 in asthmatic airways might be a result of compensation
Although human asthma is clearly not the same as in experimental mouse models, recent data from such models encourage the further investigation of the roles of TGF-β1 in human asthma. A study of Smad3-knockout mice suggests that there are complexities in understanding the various roles of the TGF-β signaling pathway15, indicating that further study will be necessary before the precise role of this cytokine is known. Accumulating knowledge on TGF-β signal transduction will help reveal the detailed mechanisms by which TGF-β1 elicits its effects on target cells in the airways of the experimental models, eventually suppressing AHR and airway inflammation. It is hoped that when the roles of TGF-β1 in human asthma are clearer, the modification of TGF-β1 activity inside or outside the cells, possibly via molecular approaches such as using anti-sense Smad7 oligonucleotide, will offer some attractive avenues for potential therapeutic intervention for asthma. Acknowledgements
I thank P. ten Dijke, I. Iwamoto, Y. Saito, H. Nakajima, H. Tomioka, T. Tokuhisa, H. Ogawa, K. Okumura, C. Ra, H. Ushio, T. Imamura, M. Kawabata, K. Miyazono, A. Ishisaki, S. Itoh and C-H. Heldin for support; I. Nakao for illustration; and E. Kamijima for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Science
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and Culture, Japan, the Uehara Memorial Foundation for Biomedical Science, Japan, and the Foundation of Human Science, Japan. References 1 Wills-Karp, M. (1999) Immunological basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17, 255–281 2 Hansen, G. et al. (2000) CD4 T helper cells engineered to produce latent TGF-β1 reverse allergen-induced airway hyperreactivity and inflammation. J. Clin. Invest. 105, 51–70 3 Haneda, K. et al. (1997) TGF-β induced by oral tolerance ameliorates experimental tracheal eosinophilia. J. Immunol. 159, 4484–4490 4 Haneda, K. et al. (1999) Transforming growth factor-β secreted from CD4 T cells ameliorates antigen-induced eosinophilic inflammation. A novel high-dose tolerance in trachea. Am. J. Respir. Cell Mol. Biol. 21, 268–274
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5 Nakao, A. et al. (2000) Blockade of TGF-β/Smad signaling by overexpression of Smad7 in T cells enhances antigen-induced airway inflammation and airway reactivity. J. Exp. Med. 192, 151–158 6 Busse, W.W. (1998) Inflammation in asthma: the corner stone of the disease and target of therapy. J. Allergy Clin. Immunol. 102, S17–S22 7 Temann, U.A. et al. (1998) Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J. Exp. Med. 188, 1307–1320 8 Holgate, S.T. (2000) Epithelial damage and response. Clin. Exp. Allergy 30, 37–41 9 Hirst, S.J. (2000) Airwary smooth muscle as a target in asthma. Clin. Exp. Allergy 30, 54–59 10 Wahl, S.M. (1992) Transforming growth factor-β in inflammation: a cause and a cure. J. Clin. Immunol. 12, 61–74 11 Piek, E. et al. (2000) Specificity, diversity, and regulation of TGF-β superfamily signaling. FASEB J. 13, 2105–2124
12 Holt, P.G. and McMenamin, C. (1989) Defence against allergic sensitization in the healthy lung: the role of inhalation tolerance. Clin. Exp. Allergy 19, 255–262 13 Redington, A.E. et al. (1997) Transforming growth factor-β1 in asthma. Measurement in bronchoalveolar lavage fluid. Am. J. Respir. Crit. Care Med. 156, 642–647 14 Singer, A.J. and Clark, R.A.F. (1999) Cutaneous wound healing. New Engl. J. Med. 341, 738–746 15 Ashcroft, G.S. et al. (1999) Mice lacking Smad3 show accelerated would healing and an impaired local inflammatory response. Nat. Cell Biol. 1, 260–266
Atsuhito Nakao Allergy Research Center, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. e-mail: [email protected]
Effector and regulatory T cells in allergic contact dermatitis Andrea Cavani, Cristina Albanesi, Claudia Traidl, Silvia Sebastiani and Giampiero Girolomoni Allergic contact dermatitis is a prototypic T-cell-mediated disease that has a socioeconomic impact in industrialized countries. Here, Andrea Cavani and colleagues highlight recent developments in the T-cell-based effector and regulatory mechanisms of this common skin disorder.
Allergic contact dermatitis (ACD) results from a T-cell response to harmless, lowmolecular-weight chemicals (haptens) applied to the skin. In the sensitization phase, haptens penetrating the skin are collected by resident dendritic cells that migrate to the regional lymph nodes to activate and clonally expand specific T-cell precursors1. Re-exposure to the relevant hapten initiates the efferent phase and clinical expression of ACD, characterized by the rapid recruitment and activation of specific T cells at the sites of hapten challenge. In spite of the longlasting persistence of the hapten in the skin, the reaction is self-limited, suggesting that regulatory mechanisms are actively involved in the termination of ACD. T-cell recruitment in ACD
The recruitment of T cells in the skin is regulated by the expression of proper
homing receptors, such as the cutaneous lymphocyte-associated antigen (CLA), which mediates rolling of T cells over activated endothelial cells expressing E-selectin1. More recently, chemokine receptors have been proposed as important regulators of the tissue targeting of T cells. In line with this concept, it has been shown that skinseeking CLA+ T cells co-express CC chemokine receptor 4 (CCR4), the ligand for thymus and activation-regulated chemokine [TARC; CC chemokine ligand 17 (CCL17)] and macrophage-derived chemokine (MDC; CCL22). CCR4 triggered by TARC exposed on the endothelial cell surface during inflammatory skin disorders is thought to augment integrin-dependent firm adhesion of T cells to endothelial intercellular adhesion molecule 1 (ICAM-1)2. T-cell migration into peripheral tissues mostly depends on their chemokine receptor profile. Owing to the high expression of CCR5 and CXCR3, T helper 1 (Th1) cells preferentially migrate to the respective ligands, macrophage inflammatory protein 1β (MIP-1β; CCL4) and interferon γ (IFN-γ)-inducible protein 10 [IP-10; CXC chemokine ligand 10
(CXCL10)]. By contrast, T helper 2 (Th2) cells are mostly attracted by eotaxin (CCL11), TARC and MDC, and I-309 (CCL1), because of the high levels of CCR3, CCR4 and CCR8, respectively3. Epidermal keratinocytes have been extensively investigated as a source of inflammatory mediators for the initiation and amplification of skin immune responses, and T-cell-derived lymphokines are among the most potent activators of keratinocytes. Treatment with IFN-γ or IFN-γ plus tumor necrosis factor α (TNF-α) induces keratinocytes to express ICAM-1 and mature MHC class II molecules, and to release a vast array of growth factors, chemokines and cytokines such as interleukin 1 (IL-1), TNF-α and granulocyte–macrophage colonystimulating factor (GM-CSF)4,5. IL-17, a cytokine produced by skin-infiltrating Th1 and Th2, but not CD8+ T cells, reinforces many of the effects induced by IFN-γ (Ref. 4). Interestingly, the Th2 cytokine IL-4 acts synergistically with IFN-γ to enhance keratinocyte ICAM-1 expression and release of the CXCR3 agonistic chemokines, IP-10, monokine induced by IFN-γ (Mig; CXCL9) and IFN-inducible T-cell α-chemoattractant (I-TAC;
http://immunology.trends.com 1471–4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(00)01815-9