Cytokines involved in the downregulation of allergic airway inflammation

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Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin. J. Exp. Med., 183, 2379-2384. Wallaert, B., Desreumaux, P., Copin, M.C., Tillie, I., Benard, A., Colombel, J.F., Gosselin, B., Tonnel, A.B. & Janin, A. (199.5), Immunoreactivity for interleukin-3, -5 and granulocyte-macrophage colony stimulating factor of intestinal mucosa in bronchial asthma. J. Exp. Med., 182, 1897-1904. Walsh, G.M., Hartnell, A., Wardlaw, A.J., Kurihara, K., Sanderson, C.J. & Kay, A.B. (1990). IL-5 enhances the in vitro adhesion of human eosinophils, but not neutrophils, in a leucocyte integrin (CD1 l/l 8)-dependent manner. immunology, 7 1, 258-265.

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Wardlaw, A.J., Symon, F.S. & Walsh, G.M. (1994), Eosinophil adhesion in allergic inflammation. J. Allergy Clin. Immunol.,94, 1163-1171. Ying, S., Taborda-Barata, L., Meng, Q., Humbert, M. & Kay, A.B. (1995), The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 and RANTES in the skin of human atopic subjects : relationship to eosinophil, T cell, and macrophage recruitment. J. Exp. Med., 181, 21532159. Yousefi, S., Hemmann, S., Weber, M., Holzer, C., Hartung, K., Blaser, K. & Simon, H.U. (1995) IL-8 is expressed by human peripheral blood eosinophils. J. Immunol., 154, 548 l-5490.

Cytokines involved in the downregulation of allergic airway inflammation M. Pretolani and M. Goldman Unite’ de Pharmacologic Cellulaire, Unite’ Associe’eInstitut Pasteur/INSERM U28.5, Institut Pasteur, 75724 Paris Cedex 15 (France), and Dkpartement d’Immunologie, H&pita1 Erasme, Brussels (Belgium)

Introduction Blood and tissue eosinophilia characterize atopic allergy and bronchial asthma (Kroegel et al., 1994). The local generation of highly charged eosinophil-

derived cationic proteins is claimed to play a role in the development of a long-term tissue damage, as demonstrated in bronchial biopsies from asthmatics in which degranulated eosinophils are observed in the vicinity of airway epithelial shedding (Bousquet et al., 1990). The infiltration and maintenance of eosinophils in the inflamed tissues is regulated mostly by T-cell-derived cytokines, including interleukin (IL3, IL5 and granulocyte-macrophage colony-stimulating factor (GM-CSF)). These cytokines act on the proliferation and differentiation of eosinophils, promote chemotaxis of mature cells and prime them for their responses to exogenous stimuli (Lopez et al., 1992). While the biological activities of IL3 and GM-CSF extend to other cell types, IL5 primarily influences the eosinophil functions (San-

Received December

10, 1996.

derson, 1992), thus making this cytokine an important target for new therapeutic agents aimed at treating eosinophilic disorders. The preferential production of IL4 and IL5, but not interferon-y (IFNy), by broncholaveolar T cells from asthmatics supported the hypothesis of a Th2 pattern involved in the onset and maintenance of tissueinflammation characteristic of this disease(Robinson et al., 1992). Animal models are extensively used for investigating the inflammatory component of allergic diseases.Thus, studies performed with IL4 or IL5 gene-deleted mice (Coyle et al., 1995 ; Foster et al., 1996), or treatment of the animals with a specific monoclonal antibody directed against IL5 (Nakajima et al., 1992) further supported the concept that allergic eosinophilia dependson Th2 cytokines. Accordingly, the correction of this cytokine imbalance is now considered as one of the main objectives for the treatment of allergic disorders (International Consensus on Diagnosis and Treatment of Asthma, 1992).

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Here, we report recent findings concerning the antiinflammatory profiles of some T-celYmonocytemacrophage-derived cytokines in the context of atopic diseases, particularly bronchial asthma, and discuss their potential use as antiallergic molecules.

Cytokines which inhibit allergic airway inflammation: cellular mechanisms and in viva effects Interleukin-10 IL 10 was originally characterized as a factor generated by mouse Th2 cells which inhibits cytokine synthesis by Thl cells (Fiorentino et al., 1989). However, several other cell types have been further identified as a source of this cytokine, including CD4+ and CD8’ T lymphocytes, monocytes/macrophages, mast cells, keratinocytes, eosinophils and various tumour cells (Moore et al., 1993). The concept that IL10 acts as an antiinflammatory molecule emerged primarily from studies showing downregulation of the synthesis of a broad spectrum of proinflammatory cytokines by different cells, particularly of the monocytic lineage (Moore et al., 1993). Interestingly enough, the antiinflammatory properties of IL10 are not restricted to the inhibition of cytokine production, since its addition to purified monocytes or neutrophils promotes the release of the IL1 receptor antagonist (Cassatella et al., 1994), which displays antiinflammatory activities on its own. The observation that IL10 downregulates the production of IL5 by both human resting T cells (Schandene et al., 1994) and by human ThO and Th2 clones (Del Prete et al., 1993) suggested for the first time the potential antiallergic activity of this cytokine. While IL10 interferes directly with B7/CD28dependent activation signals in resting T cells, its inhibitory effect on ThO and Th2 clones was only observed in antigen-presenting cell (APC)-dependent activation systems, suggesting that the action of IL10 was indirect and related to functional inhibition of APC. As shown in human Th2 clones, IL10 suppressed IL5 synthesis by purified CD4+ T lymphocytes stimulated in an antigen-specific APC-dependent manner, but not in response to solid-phase anti-CD3 monoclonal antibody, indicating a primary activity on APC. A direct effect of IL10 on eosinophil functions has also been demonstrated. Thus, Takanaski et al. (1994) have shown that IL10 suppresses LPSinduced GM-CSF generation and the consequent increase in eosinophil survival, suggesting that IL10 may play an important role in the removal of excessive numbers of eosinophils from inflamed tissues. Recent findings indicate that human eosinophils express a functional CD40 at their surface and that its ligation with a specific antibody or with its natu-

IN IMMUNOLOGY ral ligand, CD4OL, prolongs their survival (Ohkawara et al., 1996). Low concentrations of IL10 were practically as active as the potent synthetic glucocorticosteroid budesonide in decreasing CD40 expression and in accelerating eosinophil death (Ohkawara et al., 1996), again supporting a role for IL10 in the resolution of eosinophilic inflammation. Like the eosinophil, the mast cell is also considered as an important effector cell implicated in the allergic response. This results mostly from its ability to generate a number of cytokines acting directly and indirectly on eosinophil recruitment and activation in target tissues, particularly IL3, IL4, IL5, GM-CSF and TNFa (Warner and Kroegel, 1994). Recently, IL10 has been shown to inhibit TNFa (Arock et al., 1996 ; Marshall et al., 1996) and IL6 (Marshall et al., 1996) generation from mouse bone-marrowderived mast cells and rat peritoneal mast cells, respectively, in response to IgE cross-linking with a specific antigen. Since IL10 also prevented the release of GM-CSF (Arock et al., 1996), a cytokine directly involved in the homing and activation of eosinophils and neutrophils in inflamed tissues, it was suggested that IL10 may influence antigendependent mast cell activation and thus interfere with the initiation of leukocytic inflammation. The physiopathological relevance of these observations was confirmed using two in vivo models of allergic inflammation in the mouse (Zuany-Amorim et al., 1995, 1996). First, we have demonstrated that the intranasal instillation of ILIO, along with ovalbumin, protected sensitized mice from allergic airway eosinophilia and neutrophilia by a mechanism probably involving the inhibition of local TNFol formation (Zuany-Amorim et al., 1995). TNFa generated in the airways may interfere directly with leukocyte chemoattraction, activation and survival, but also indirectly, via the expression of specific adhesion molecules (Tracey and Cerami, 1993). More recently, using a model of antigen-induced peritoneal eosinophilia in sensitized mice, we confirmed the effectiveness of IL10 in preventing allergic eosinophil accumulation and demonstrated that IL10 also suppressed IL5 generation in the peritoneal cavity which precedes eosinophil recruitment (Zuany-Amorim et al., 1996). In this case, IL10 acts most likely by deactivating APC. Together, these in vitro and in vivo findings indicate that IL10 may exert potent antiallergic activities, by inhibiting the release of those cytokines directly and indirectly involved in the onset and maintenance of tissue eosinophilia, such as IL5, GM-CSF and TNFa, and by acting on different cell types involved in allergic inflammation, particularly mast cells and the eosinophils themselves. Recently, the safety and immunomodulatory activities of systemic administrations of IL10 have been proven in healthy human volunteers (Chernoff et al., 1995 ;

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Huhn er al., 1996). It is of interest that peripheral blood mononuclear cells from ILlO-treated subjects show a sustained decrease in ILl, IL6 and TNFcl production in response to LPS (Huhn et al., 1996), suggesting that a single IL10 administration may be sufficient to exert a long-lasting antiinflammatory activity. Interferons IFNy is the major cytokine responsible for macrophage activation in the context of non-specific cell-mediated host defence and it may regulate the proliferation and function of T lymphocytes (Liu and Janeway, 1990). A number of recent observations suggested that IFNy may modulate allergic diseases. First, IFNy was shown to antagonize IL4mediated Ig isotype switching to IgE in murine and human B cells (Snapper and Paul, 1987 ; Pene et al., 1988), and to inhibit IL3-driven IgE synthesis by human B cells (De Vries and Zurawaski, 1995). Furthermore, IFNy selectively prevents Th2 cell proliferation and favours the differentiation of ThO cells into cells with a Thl phenotype (Bradley et al., 1995). In murine models of allergic airway inflammation, systemic (Iwamoto et al., 1993) or aerosol (Nakajima et al., 1993) administrations of IFNy prevent antigen-induced eosinophil infiltration into the tracheal tissue. It was suggested that this effect resulted from the inhibition of the influx of CD4+ T lymphocytes, a major source of eosinophilotactic cytokines, such as IL5, in the airways. Also, chronic exposure of sensitized mice to nebulized, but not intraperitoneal, IFNy before and during the immunization period reduced the formation of antigenspecific IgE, attenuated the development of airway hyperreactivity and decreased the frequency of IL4producing cells, both in the peribronchial lymphoid tissue and in the spleen (Lack et al., 1994). Using IFNy receptor-deficient mice, Coyle et al. (1996) recently demonstrated prolonged airway eosinophilia in response to antigen challenge, which was accompanied by a sustained capacity of lung T lymphocytes from those animals to secrete IL4 and IL5 This suggests that lack of IFNy signalling leads to a defective resolution of eosinophilic inflammation, probably linked to the maintenance of a local Th2 response. These results, however, are in conflict with the in vitro capacity of IFNy to prolong the survival of human peripheral blood eosinophils, to increase their cytotoxicity (Valerius et al., 1990) and to upregulate ICAMexpression on their surface (Czech et al., 1993). These effects may be partially accounted for by the capacity of IFNy to stimulate the synthesis of IL3 by the eosinophils themselves (Fujisawa et al., 1994). In an attempt to attenuate allergic inflammation, a number of clinical trials have been performed with

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IFNy. Subcutaneous administration of IFNy to patients with severe atopic dermatitis improved clinical symptoms and reduced the number of circulating eosinophils (Boguniewicz et al., 1990). However, a similar treatment in asthmatic corticosteroidsensitive individuals failed to modify pulmonary function (Boguniewicz et al., 1993). It was concluded from this study that local delivery of IFNy would be a more appropriate strategy for improving its bioavailability, avoiding side-effects and targeting its activities. This hypothesis was confirmed by Martin et al. (1993), who demonstrated that aerosolized IFNy to normal subjects over 12 days is clinically safe and represents an effective mode of delivery of this cytokine. Besides their well-known antiviral properties and their ability to stimulate the expression of MHC molecules and NK cells, type I interferons, which include IFNa and IFNp, also exert major biological actions on T cells (Belardelli and Gresser, 1996). In the context of allergic inflammation, it is almost exclusively IFNIX which has been considered as a potential therapeutic agent, although it is likely that IFNP might provide similar benefits, since both type I interferons bind to the same receptor (Langer and Pestka, 1988). In this context, the most relevant effects of IFNcl on T cells are (1) the inhibition of the differentiation of naive CD4+ T cells into Th2 cells (Parronchi et al., 1992), (2) the promotion of Thl-type responses, as shown in vivo in mice (Finkelman et al., 1991), (3) the inhibition of IL5 synthesis by differentiated Th2 cells (Schandene et al., 1996), and (4) the upregulation of IL10 synthesis by CD4+ T cells (Aman et al., 1996; Schandene et al., 1996). Interestingly, the inhibition of IL5 synthesis and the upregulation of IL10 production were observed not only on in vitro generated human Tcell clones, but also on Th2-like cells directly isolated from the peripheral blood of patients with the hypereosinophilic syndrome; indeed, the reduction in blood eosinophil count during IFNa therapy in those patients was associated with a decrease in serum IL5 levels (Schandene et al., 1996). The beneficial effects of IFNa administration in patients with the hypereosinophilic syndrome (Zielinski and Lawrence, 1990; Prin et al., 1991) and in atopic dermatitis (MacKie, 1990) might also involve direct effects of the cytokine on eosinophils and their precursors. As a matter of fact, IFNa was shown to inhibit the proliferation of human marrow granulocyte-macrophage progenitor cells (CFUGM) and their engagement in the eosinophil lineage, at least in part by inhibiting the action of IL3 (Broxmeyer et al., 1983; Sillaber et al., 1992). Moreover, there is evidence that IFNa inhibits the release of the eosinophil cationic protein (ECP) and the eosinophil-derived neurotoxin (EDN) by activated eosinophils as well as their cytotoxic potential in vitro

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(Aldebert et al., 1996). The decreasein ECP serum levels observed during IFNa treatment in patients with the hypereosinophilic syndrome (Desreumaux et al., 1993) suggeststhat the direct effects of IFNa on eosinophils are also operative in vivu. Finally, IFNa not only inhibits the differentiation and effector functions of T cells and eosinophils involved in allergic inflammation, but might also interfere with their homing properties. This is suggested by the observation that IFNCX prevents the antigen-induced recruitment of activated CD4+ Tcells and eosinophils in the airways of sensitized mice (Nakajima et al., 1994). Interleukin-12 IL12, a heterodimeric cytokine primarily produced by monocytes, macrophages, B cells and other accessory cells in response to infections, promotes cell-mediated immune functions, upregulates IFNy production and inhibits Th2-mediated responses(Trinchieri, 1994). The ability of IL12 to prevent IL4-mediated IgE production by human peripheral blood mononuclear cells (Kiniwa et al., 1992), its effectiveness in inhibiting the in vivo expression of Th2-derived cytokines as well as the associatedeosinophilia and IgE responseduring parasite infections (Finkelman et al., 1994; Wynn et al., 1994) have suggestedthe potential application of this cytokine in the treatment of allergic disorders. Recently, Gavett et al. (1995) and Kips ef al. (1996) have shown that systemically administered IL12 at the time of immunization or during antigen challenge inhibits bronchial hyperreactivity and eosinophi1 infiltration into the airways of sensitized mice. This was accompanied by a reduction in the levels of IL4 and IL5 mRNA in the lung and in the corresponding proteins in the bronchoalveolar lavage fluid (Gavett et al., 1995). Since the administration of an antibody directed against IFNy partially reversed the inhibition by IL12 of allergic airway eosinophilia, it was suggestedthat part of the protective effects of this cytokine are mediated through the endogenous production of IFNy (Gavett et al., 1995). Although these findings support the potential use of IL12 as antiinflammatory agent for the treatment of allergic diseases, additional studies are required to establish the safety, tolerance and bioavailability of this cytokine in humans to reach a firm conclusion on its possible clinical application. The potential place of antiinflammatory kines in the therapy of allergic diseases

cyto-

New treatments of allergic diseasesaim at alleviating the inflammatory lesions of target tissues.In the case of allergic asthma, the pharmacologic treatment

requires both bronchodilators to reverse airflow obstruction and antiinflammatory agents and to prevent chronic bronchial inflammation. Steroids are the most effective antiinflammatory drugs for controlling asthma and related diseasesand their effects have been demonstrated at different levels, particularly on the production of proinflammatory mediators and cytokines by a number of cell types, including T lymphocytes (Barnes, 1996). Although the benefits of corticosteroids in the treatment of the inflammatory component of allergic diseasesare well established, there is still some concern about local and systemic side-effects, resulting mostly from their long-term administration. Furthermore, a small subset of asthmatics are corticoresistant, since they do not respond to orally administered corticosteroids with clinical improvement (Carmichael et al., 1981). There is therefore a need for the development of new strategies aimed at treating Th2-mediated allergic inflammation and their consequent alterations of the bronchopulmonary function. One of these strategiesmight be the use of antiinflammatory cytokines. Due to their safety and already establishedbioavailability in normal human volunteers, IL10 and IFNy seem to have the most promising future as additiontialtemative therapies for the treatment of allergic disorders. Furthermore, the demonstrationsthat alveolar macrophages and CD4+ T-lymphocytes from asthmatic patients showed decreased IL10 and IFNy generation, respectively (Borish et al., 1996; Jung et al., 1995), suggestedthat defective production of these cytokines during allergic inflammatory processes might be compensatedfor by their exogenous administration. It is noteworthy, however, that the inflammatory reaction underlying allergic diseases,particular bronchial asthma, is rather an ongoing than an acute and reversible phenomenon, which makes the timing and the route of cytokines administration critical elementsto be considered for precisely determining their effectiveness.

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