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Corporate Research Business Area Dermatology (K.A.), Schering AG, Berlin, Germany; and Departments of Dermatology and Allergology (W.S.) and Institute of Medical Immunology (H.D.V.), University Hospital Charité, Humboldt University, Berlin, Germany
Abstract I. Introduction II. Interleukin-10 A. Interleukin-10 and Interleukin-10 Homologs B. Interleukin-10 Promoter and Interleukin-10 Polymorphisms C. Regulation of Interleukin-10 Secretion III. Interleukin-10 Receptors and Signaling A. Interleukin-10 Receptors and Other Cytokine Receptor Family Type 2 Members B. Interleukin-10 Receptor Polymorphisms C. Interleukin-10 Receptor Signaling IV. Immunobiology of Interleukin-10 A. Effects of Interleukin-10 on Immune Cells in Vitro 1. Effects on Myeloid Antigen-Presenting Cells. 2. Effects on T Cells. 3. Effects on Natural Killer Cells. 4. Effects on Other Immune Cells. 5. Effects on Epithelial Cells. B. Effects of Interleukin-10 in Animals/Animal Models 1. Interleukin-10 Knockout Mice. 2. Inflammation and Autoimmune Models. 3. Tumor Models. 4. Experimental Models of Infections. C. Interleukin-10 and Interleukin-10 Receptor Expression in Diseases 1. Expression in Malignant Diseases. a. Melanoma. b. Carcinoma. c. Lymphoma. d. Prognostic Value of Interleukin-10 Overexpression. 2. Autoimmune and Inflammatory Diseases. a. Systemic Lupus Erythematosus. b. Systemic Sclerosis. c. Bullous Pemphigoid. d. Psoriasis. e. Rheumatoid Arthritis. f. Allergic Contact Dermatitis and Other Non-Atopic Eczemas. g. Chronic Inflammatory Bowel Diseases. h. Multiple Sclerosis. i. Transplantation. 3. Expression in Atopic Disorders. a. Atopic Dermatitis. b. Allergic Asthma. 4. Expression in Infection. D. Interleukin-10 and Interleukin-10 Receptor Polymorphisms and Diseases V. Interleukin-10 As a Therapeutic Agent A. Phase I Trials in Healthy Volunteers B. Prevention of Cytokine Release in Transplant Patients and Jarisch-Herxheimer Reaction C. Therapy of Crohn's Disease D. Therapy of Rheumatoid Arthritis E. Therapy of Psoriasis F. Therapy of Viral Infections—Chronic Hepatitis C and Human Immunodeficiency Virus VI. Prospects of Interleukin-10/Interleukin-10 Receptor As a Therapeutic Target VII. Conclusions
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I. Introduction |
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,b).
Mosmann and coworkers (Fiorentino et al., 1989) first described a cytokine that is produced by T helper 2 (Th21) cell clones and inhibits interferon (IFN)-
synthesis in Th1 cell clones (Fiorentino et al., 1989). Today this "cytokine synthesis inhibiting factor (CSIF)" is known as interleukin (IL)-10, and although we also know that several immune cells produce IL-10, macrophages are the major source. Investigations during the last decade showed that this cytokine is of crucial importance for immunoregulation and led to its use in first clinical trials.
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II. Interleukin-10 |
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The human cytokine is a homodimer with a molecular mass of 37 kDa. Each monomer consists of 160 amino acids with a molecular mass of 18.5 kDa. Murine and human IL-10 exhibit a homology of about 80%. There are several viral IL-10 homologs: Epstein-Barr virus (BCRF1) (Hsu et al., 1990), herpes virus type 2 (Rode et al., 1994), cytomegalovirus (Kotenko et al., 2000; Spencer, 2002), and Orf virus (Fleming et al., 1997), with the EBV-derived BCRF1 being the most studied homolog. The structure of human IL-10 and BCRF1 (ebvIL-10) was studied by X-ray crystal-structure-analysis (Zdanov et al., 1995, 1997) (Fig. 1). Apart from marginal differences predominantly in the N-terminal part of the molecule, the structures of hIL-10 and ebvIL-10 are strikingly similar; the two identical intertwining polypeptide chains of 160 (hIL-10) or 145 (ebvIL-10) amino acids are rotated by 180o to each other, forming two domains oriented in a V-shaped structure. Each domain contains six helices, four (A–D) from one monomer and two (E + F) from the other (Spits et al., 1992; Moore et al., 2001). Such a topology has been described first for the interferon- homodimer (Ealick et al., 1991)—a cytokine with many biological properties antagonistic to those of IL-10.
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The capability for IL-10 production has been demonstrated for various cell populations; in addition to certain T cell subsets (Th2, Tc2, Tr1), also monocytes, macrophages, and several other cells may synthesize IL-10 (Table 1). Whether human keratinocytes really produce IL-10 like their murine counterparts is subject to contrary discussions (Enk and Katz, 1992; Kang et al., 1994; Enk et al., 1995; Grewe et al., 1995; Teunissen et al., 1997). The major source of IL-10 in vivo seems to be macrophages.
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Five new human molecules structurally related to IL-10 have been discovered (Jiang et al., 1995; Gallagher et al., 2000; Dumoutier et al., 2000a,b,c; Knappe et al., 2000; Blumberg et al., 2001). They are called IL-19, IL-20, IL-22, IL-24 (mda-7), and IL-26 (AK155). Similar to IL-10, they are 
-helical proteins with similar cysteine localizations, whose amino acid sequences are about 30% identical. Interestingly, in the human genome, the encoding genes are located in two clusters, one comprising the genes for IL-10, IL-19, IL-20, and IL-24 (mda-7) on chromosome 1q31-32, whereas the second cluster comprising the genes encoding IL-26 (AK155) and IL-22 is located on human chromosome 12q15 near the IFN- gene (12q14) (Dumoutier et al., 2000b; Blumberg et al., 2001) (Fig. 2). Taking into account the clear structural relation between the new IL-10 homologs and IL-10, all of these six molecules should be considered as (IL-10) family members.
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In contrast to the extensively studied IL-10 (as described below and recently reviewed by Moore et al., 2001), the knowledge of the biology of the new IL-10 homologs is still fragmentary. The first functional data exists for IL-20, IL-22, and IL-24 (mda-7) (Fickenscher et al., 2002). Overexpression of IL-20 in transgenic mice induced neonatal lethality, psoriasis-like skin abnormalities, lack of adipose tissue, and elevated apoptosis of thymic lymphocytes (Blumberg et al., 2001). It has been suggested that IL-22 plays a role in inflammatory processes through the observation that it induces acute phase-reactant production in a hepatoma cell line and in vivo (Dumoutier et al., 2000b). Overexpression of mda-7 via adenoviral gene transfer induced growth inhibition in various tumor types (Jiang et al., 1996). Interestingly, the IL-24 (mda-7) mouse counterpart, called FISP, was postulated to be a Th2-specific protein (Schaefer et al., 2001). No function is known for IL-19 and IL-26 (AK155) to date. Table 2 summarizes the most important properties known so far (Volk et al., 2001).
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We recently investigated the expression of five new human IL-10-related molecules and their receptors in blood mononuclear cells (Wolk et al., 2002). IL-19 and IL-20 were found to be preferentially expressed in monocytes. IL-22 and IL-26 (AK155) expression was exclusively detected in NK and T cells, especially upon T1 polarization. IL-24 (mda-7) expression was restricted to monocytes and T cells. Secretion of these molecules by lymphocytes was predominantly linked to cellular activation. Regarding T cells, IL-26 was primarily produced by memory cells, and its expression was independent of costimulation. This data suggests that immune cells are a major source of the new IL-10 family members (Wolk et al., 2002).
B. Interleukin-10 Promoter and Interleukin-10 Polymorphisms
The human IL-10 gene is located on chromosome 1 and encodes for 5 exons (5.1 kb) (Spits and De Waal Malefyt, 1992). The IL-10 promoter is highly polymorphic with two informative microsatellites, IL10.G and IL10.R, 1.2 kb and 4 kb upstream of the transcription start site (Eskdale and Gallagher, 1995; Eskdale et al., 1996) and three frequent point mutations -1082(G/A), -819(C/T), and -592(C/A) (Eskdale et al., 1997a; Turner et al., 1997a; Hurme et al., 1998). Recently, several new single-nucleotide polymorphisms have been defined in the human IL-10 locus. A correlation of particular microsatellite polymorphisms with lipopolysaccharide (LPS)-induced IL-10 secretion by PBMC in vitro (presumably mostly from monocytes) was reported (Eskdale et al., 1998); the -1082(G) allele was associated with higher ConA-induced IL-10 production (likely both T cells and monocytes) (Turner et al., 1997a).
C. Regulation of Interleukin-10 Secretion
The IL-10 promoter contains several transcription factor-responsive elements (Platzer et al., 1994). Thus macrophages, the major source of IL-10, are stimulated to produce IL-10 by several endogenous and exogenous factors such as endotoxin (via Toll-like receptor 4, NF-
B dependent), tumor necrosis factor (TNF)- (via TNF receptor p55, NF-B-dependent), catecholamines, and cAMP-elevating drugs (both via protein kinase A, CREB-1/ATF-1 dependent) (Platzer et al., 1995, 1999, 2000; Meisel et al., 1996; Woichiechowsky et al., 1998; Riese et al., 2000).
In particular, the stress axis plays a significant role in regulating IL-10 expression in vivo. Inflammation of the central nervous system (particularly local IL-1 release following trauma, neurosurgery, or increase of intra-brain pressure) or indirect activation of the stress axis by endotoxemia/bacteremia triggers the release of catecholamines that up-regulate IL-10 production in macrophages, particularly in the liver (Barsig et al., 1995; Jilg et al., 1996; Woichiechowsky et al., 1998). Blocking the stress axis increases the susceptibility to endotoxemia-mediated shock. So the cross-talk between the central nervous system and (liver) macrophages controls systemic inflammation whereby the cAMP/protein kinase A/CREB-1/ATF-1 signaling pathway seems to play an essential role in inducing IL-10. On the other hand, systemic release of TNF- also induces IL-10 via a negative feedback by using a NF-B-dependent pathway (Barsig et al., 1995; Meisel et al., 1996). Recent data suggests that the p38 mitogen-activated kinase pathway also regulates the human IL-10 promoter via the activation of sp1 transcription factor (Ma et al., 2001).
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III. Interleukin-10 Receptors and Signaling |
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IL-10 activity is mediated by its specific cell surface receptor complex, which is expressed on a variety of cells, in particular immune cells. Only a few copies of the IL-10R are expressed on the surface of the cells (Carson et al., 1995; Jurlander et al., 1997). The expression is variable, but so far only a few regulating factors are known. Endotoxin increases the expression of IL-10R on fibroblasts (Weber-Nordt et al., 1994). After T cell stimulation with anti-CD3-antibodies or phorbol ester, a decrease of IL-10R gene expression has been found (Liu et al., 1994). It has been demonstrated that dermatological therapeutic agents such as glucocorticoids, vitamin D3, and calcipotriol significantly increase IL-10R expression (Michel et al., 1997a,b). The IL-10 receptor is composed of two different chains, (Ho et al., 1993) and 
(CRFB4) (Kotenko et al., 1997), both members of the class II cytokine receptor family. The interaction of hIL-10R with hIL-10 has been characterized recently and seems to be highly complex (Ho et al., 1993; Tan et al., 1993; Reineke et al., 1998, 1999). The IL-10R chain is essential for IL-10-mediated effects and CRFB4-deficient mice display the same phenotype as IL-10 deficient mice (Spencer et al., 1998). Interestingly, for cells that only express IL-10R, no IL-10/IL-10R complexes are formed suggesting that only IL-10/IL-10R complexes interact with the -chain. Only in cells expressing both the IL-10R and chains is the characteristic STAT transcription factor activation pattern for IL-10 signaling observed (Kotenko et al., 1997; Spencer et al., 1998).
As for IL-10, all the receptors of the new molecules from the IL-10 family known so far belong to the cytokine receptor family type 2 (CRF2) (Kotenko and Pestka, 2000). They are generally transmembrane glycoproteins whose extracellular domains consist of about 210 amino acids comprising two tandem fibronectin type III domains and having several conserved amino acid positions important for the secondary structure. More recently, it has been discovered that some of the human IL-10 homologs share single receptor chains and even whole receptor complexes (Dumoutier et al., 2000c, 2001; Xie et al., 2000; Kotenko et al., 2001). One receptor from the family is soluble (Gruenberg et al., 2001). Overall, the interaction of IL-10 homologs with their receptors is quite complex (Fig. 3) and so far only partially understood. Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different CRF2. This leads, through the activation of STAT factors, to diverse biological effects.
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We recently investigated the expression of the receptors for the five new human IL-10-related molecules in blood mononuclear cells (Wolk et al., 2002). In contrast to the high expression of receptors for IL-10 homologs in different tissues and cell lines, immune cells (monocytes, NK, B, and T cells) showed only expression of IL-10R1, IL-10R2, and IL-20R2. In these cells, IL-20R2 might be part of a still unknown receptor complex. Immune cells, therefore, may represent a major source but a minor target of the new IL-10 family members (Fig. 3).
B. Interleukin-10 Receptor Polymorphisms
Polymorphisms within the human IL-10 receptor cDNA gene sequence have been described (Tanaka et al., 1997). However, their biological relevance is not clear so far.
C. Interleukin-10 Receptor Signaling
IL-10/IL-10R interaction in immune cells results in transcriptional activation of several hundred genes, some of them are more than 50-fold up-regulated. IL-10 down-regulates expression of far fewer genes (M. Jung, R. Sabat, J. Krätschmar, K. Wolk, C. Schönbein, S. Schütt, M. Freidrich, W. D. Asadullah, H. D. Volk, and G. Grütz, submitted). There is only limited knowledge, however, regarding the IL-10 intracellular signal transduction pathway to date. The IL-10/IL-10R interaction activates the tyrosine kinases Jak1 and Tyk2, which are associated with the IL-1R1 and IL-10R2, respectively (Moore et al., 2001). The receptor engagement and tyrosine phosphorylation activates the cytoplasmically localized inactive transcription factors STAT 1, 3, and 5, resulting in translocation and gene activation (Finbloom et al., 1995). The evidence for the key role of these signaling molecules for the inhibitory effects of IL-10 have been excellently reviewed recently (Moore et al., 2001).
How does IL-10 signaling result in the inhibition of immune functions? IL-10 controls inflammatory processes by suppressing the expression of proinflammatory cytokines, chemokines, adhesion molecules, as well as antigen-presenting and costimulatory molecules in monocytes/macrophages, neutrophils, and T cells (Moore et al., 2001). As all of these inflammatory proteins are transcriptionally controlled by NF-B it was suggested that IL-10 may exert a significant part of its anti-inflammatory properties by inhibiting this transcription factor. In fact, a number of studies were able to demonstrate that IL-10 blocks nuclear translocation of the classic NF-B p65/p50 heterodimer in monocytes/macrophages (Wang et al., 1995; Clarke et al., 1998). It has been recently shown that IL-10 inhibits NF-B activity through dual mechanisms: 1) it blocks NF-B nuclear translocation by inhibiting IKK activity; and 2) IL-10 blocks DNA-binding of NF-B already present in the nucleus (Fig. 4). Since the inhibition of nuclear NF-B could not be explained by an increase of nuclear levels of its inhibitor IB (Schottelius et al., 1999), the mechanisms underlying this observation needs to be further investigated. Our recent unpublished findings suggest that IL-10 exerts its anti-inflammatory activity, in part, by a selective induction of p50 nuclear translocation while blocking translocation of the classical p65-p50 heterodimer (F. Driessler, R. Sabat, K. Asadullah, and A. J. G. Schottelius, submitted).
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Recent reports (Ito et al., 1999; Yamaoka et al., 1999; Moore et al., 2001) demonstrated that IL-10 inhibits IFN-induced gene transcription (e.g., IP-10, ISG-54), which correlated with the IL-10-mediated inhibition of IFN-induced STAT1 phosphorylation. Moreover, IL-10 inhibition can be overcome by increasing IFN concentrations suggesting competitive interaction between the two cytokine pathways. This interaction at the STAT1 activation level results in inhibition of IFN-mediated antiviral effects by IL-10 (Ichikawa et al., 2002).
IL-10 induces the suppressor of cytokine synthesis (SOCS)-3 probably via a STAT3-dependent pathway (Cassatella et al., 1999; Ito et al., 1999; Moore et al., 2001). There is indirect (Donnelly et al., 1999) and more direct (Berlato et al., 2002) evidence that SOCS-3 plays a key role as mediator of the inhibitory effects of IL-10 on macrophage activation. Very recently, it was shown (Shen et al., 2000) that the IL-10-mediated attenuation of IFN-activated STAT1 is also dependent on SOCS-2 and SOCS-3.
Recent data suggest that IL-10 induces heme oxygenase-1 (HO-1), a heat-shock protein, in murine macrophages via a p38 mitogen-activated protein kinase-dependent pathway (Lee and Chau, 2002). This stress protein degrades heme to carbon monoxide, free iron (that induces ferritin), and biliverdin/bilirubin (Buelow et al., 2002) and plays an essential role in controlling tissue homeostasis in inflammation by inhibiting proinflammatory cytokine synthesis and inducing antiapoptotic processes. Blocking HO-1 by zinc protoporphyrin attenuated the IL-10-mediated protection against endotoxin-induced septic shock in mice, suggesting HO-1 might be an important downstream effector of IL-10 (Lee and Chau, 2002). Induction of HO-1 by cobalt protoporphyrin is associated with up-regulation of SOCS-3 and STAT3 supporting this connection (K. Kotsch, R. Buelow, U. Janssen, and H. D. Volk, submitted).
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IV. Immunobiology of Interleukin-10 |
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Antigen-presenting cells and lymphocytes are the primary targets of IL-10. Direct effects on these populations explains the major immunological impact of this cytokine, including the regulation of the Th1/Th2 balance (Fig. 5). Th1 cells are known to be essential for effective cell-mediated immunity [cytotoxic T cell lysis (CTL), cell-mediated inflammation, complement/Fc-R binding antibodies] in particular against intracellular organisms, whereas a Th2 (or type 2) cytokine pattern is especially responsible for effective production of IgE, IgA, and noncomplement/Fc-R binding IgG in particular for neutralizing microorganisms and their toxins and for mucosal immunity (Romagnani, 1995). IL-10 promotes the development of a type 2 cytokine pattern by inhibiting the IFN- production of T lymphocytes particularly via the suppression of IL-12 synthesis in accessory cells. According to this, IL-10 costimulates the proliferation and differentiation of B cells, which is important in the adequate defense against intestinal parasites, neutralization of bacterial toxins, and in local mucosa defense (Romagnani, 1995). Moreover, IL-10 suppresses proinflammatory cytokine production and the antigen-presenting capacity of monocytes/macrophages and dendritic cells (De Waal Malefyt et al., 1991a,b; Fiorentino et al., 1991a,b; Romagnani, 1995). Therefore, IL-10 represents a substantial suppressor of the cellular immunity (Spits and De Waal Malefyt, 1992). Important effects of IL-10 on immune cells are summarized in Table 3 and have recently been reviewed by Moore et al., 2001.
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1. Effects on Myeloid Antigen-Presenting Cells.
Peripheral blood monocytes are very sensitive to IL-10 presence. These cells are not a finely differentiated population. After their 24- to 48-h residence in the circulation, they migrate into the stromal tissues where, depending on the micromilieu, they develop into more specialized cell populations, into either macrophages (M
) or type 1 dendritic cells (DC1) (Randolph et al., 1998). IL-10 is able to prevent monocyte differentiation into DC1, which are the most important antigen-presenting cells (APC) especially for primary immune responses (Buelens et al., 1997; Allavena et al., 1998; Banchereau and Steinman, 1998). During DC1 development, the influence of IL-10 on these cells decreases. This is associated with a decrease of cellular IL-10R expression (R. Sabat, unpublished). In contrast, IL-10 supports monocyte maturation to M, and the sensitivity of M to IL-10 is comparable to that of monocytes (Allavena et al., 1998; R. Sabat, unpublished). The functions of monocytes and M that are regulated by IL-10 can be divided into three groups: 1) production of soluble immunomediators regulating inflammation and tissue repair; 2) antigen presentation; and 3) phagocytosis. In general, IL-10 inhibits all those activities that favor the inflammatory or specific cellular immune response and enhances those activities that are associated with the induction of tolerance in adaptive immunity as well as with scavenger function. More concretely, IL-10 inhibits the production of proinflammatory mediators by monocytes and M, such as endotoxin- and IFN--induced release of IL-1, IL-6, IL-8, G-CSF, GM-CSF, and TNF- (de Waal Malefyt et al., 1991a; Fiorentino et al., 1991a). In addition, it enhances the production of anti-inflammatory mediators such as IL-1RA and soluble TNF- receptors (Jenkins et al., 1994; Joyce et al., 1994; Hart et al., 1996). IL-10 inhibits the capacity of monocytes and M to present antigen to T cells. This is realized by down-regulation of constitutive and IFN--induced cell surface levels of MHC class II, of costimulatory molecules such as CD86 and of some adhesion molecules such as CD58 (de Waal Malefyt et al., 1991b; Willems et al., 1994; Creery et al., 1996). Moreover, IL-10 inhibits the monocytic production of IL-12, an essential mediator for the development of specific cellular immune defense (D'Andrea et al., 1993). Beside these suppressive activities, IL-10 favors the phagocytic activity of monocytes and M (Buchwald et al., 1999). This is mediated via up-regulation of specific receptors that are essential for the uptake of opsonized and nonopsonized microorganisms. Indeed, IL-10-treated monocytes and M display an enhanced expression of IgG-Fc receptors (CD16, CD32, and CD64) as well as scavenger receptors (CD163 and CD14) (te Velde et al., 1992; Spittler et al., 1995; Calzada-Wack et al., 1996; Ritter et al., 1999). Interestingly, IL-10 simultaneously diminishes the killing of ingested microorganisms (Roilides et al., 1998). The up-regulated expression of scavenger receptors seems to be responsible for the observation that IL-10-treated monocytes and M more strongly ingest apoptotic cells (W. D. Döcke and R. Sabat, unpublished), whereas the chemotaxis of monocytes is only marginally impaired by IL-10 (Vicioso et al., 1998).
2. Effects on T Cells.
Besides the dominating indirect impact via the APC (Fig. 5), IL-10 also exerts some direct effects on T cells. In particular, inhibitory effects have been described on CD4+ T cells. IL-10 inhibits the proliferation as well as the cytokine synthesis of these cells. Concerning the latter, it affects their IL-2 and IFN- as well as their IL-4 and IL-5 production, which has been induced by various stimuli (Del Prete et al., 1993; Groux et al., 1996). At least in the human system, IL-10, therefore, seems to inhibit both the Th1-type and the Th2-type responses, although the effect on Th1 cells appears to be stronger (Asadullah et al., 1998). Whereas naive CD4+ T cells are targeted by IL-10, activated and memory T cells seem to be rather insensitive toward this cytokine. This might be related to the down-regulation of IL-10R upon T cell activation (Liu et al., 1994). However, we observed a similar SOCS-3 induction in activated and resting T cells following IL-10 incubation, making a functional receptor down-regulation less likely (M. Schroeder, unpublished). The presence of IL-10 during the activation of CD4+ T cells results in the development of a regulatory phenotype of these cells (Groux et al., 1997; Zeller et al., 1999; Levings et al., 2001a,b). It is characterized by weak proliferation, absence of IL-2 production, and a specific cytokine profile (IL-10+, IFN-g+, IL-4-, IL-5-) after repeated stimulation. Typically these cells also have the capacity to transfer this phenotype to other T cells with the same antigen specificity. This transfer may not be dependent on soluble mediators but on cell surface molecules (Jonuleit et al., 2000). Whether the influence of IL-10 on CD4+ T cells or on APC is more important in vivo or the generation of such regulatory cells remains to be clarified. In vitro, both pathways have been demonstrated. IL-10 does not exert potent direct inhibitory effects on CD8+ T cells. It can even activate CD8+ T cells under certain conditions (Groux et al., 1998; Santin et al., 2000).
3. Effects on Natural Killer Cells.
The effect of IL-10 on NK cells is mainly stimulatory. IL-10 favors the cytotoxic activity of these cells. It increases the IL-2-induced production of cytokines such as IFN-, GM-CSF, and TNF-. Furthermore, it amplifies the IL-2-induced proliferation of the CD56-bright NK cell subpopulation (Carson et al., 1995). Moreover, IL-10 augments the ability of IL-18 to stimulate NK cells (Cai et al., 1999).
4. Effects on Other Immune Cells.
IL-10 has various but weak stimulatory effects on B cells. It prevents apoptosis and enhances the proliferation and differentiation toward plasma cells as well as the IgM synthesis (Levy and Brouet, 1994; Rousset et al., 1995). It also plays a role in the Ig switch. In combination with IL-4, it induces IgG4 but inhibits IgE production; in combination with TGF-, IL-10 induces IgA1 and IgA2 secretion (Defrance et al., 1992; Jeannin et al., 1998).
Very similar to monocytes and M, in granulocytes IL-10 inhibits the production of proinflammatory (TNF-, IL-1) and induces the production of anti-inflammatory (IL-1RA) mediators. Moreover, it inhibits the release of various chemokines by neutrophils (Cassatella et al., 1993; Kasama et al., 1994). The synthesis of cyclooxygenase-2 as well as the production of prostaglandin E2 is also inhibited by IL-10 (Niiro et al., 1997). Another effect of IL-10 is the inhibition of LPS-induced synthesis of proinflammatory mediators in eosinophils and mast cells (Takanaski et al., 1994; Arock et al., 1996). In combination with IL-3 and IL-4, however, IL-10 favors the growth of mast cells (Lin and Befus, 1997).
5. Effects on Epithelial Cells.
It has been shown that several epithelial cells express the IL-10R. IL-10 exerts direct effects on these cells (Bourreille et al., 1999; Denning et al., 2000; Parry et al., 2001). The capability of IL-10 to target keratinocytes (KC) is still a matter of debate. When we analyzed the biological effects of IL-10 on KC in vitro, we did not find any evidence for IL-10 effects on KC proliferation, cytokine formation, and expression of surface molecules with impact on immunoregulation. No effects on unstimulated or stimulated KC were observed in primary human KC or on cultured HaCaT cells (Seifert et al., 2000). These results are in line with other observations (Chatelain et al.,1998) showing that IL-10 inhibits intercellular adhesion molecule-1 (ICAM-1) expression on Langerhans cells but not on KC, but do not support some earlier observations regarding a certain in vitro effect of IL-10 in KC. So it was reported that IL-10 inhibits the cytokine synthesis of TNF- and IL-6 (Bécherel et al., 1995) as well as proliferation (Michel et al., 1997) of KC. This discrepancy might result from impure primary KC cultures (for example contamination with fibroblasts), other culture conditions, different IL-10 proteins used (LPS contamination?), or differences in the experimental proceedings, but overall the reasons remain unclear (Seifert et al., 2000). Recently, we investigated further the direct effects of IL-10 on keratinocytes and addressed the reason for potential IL-10 unresponsiveness using the keratinocyte-like cell line HaCaT as well as primary foreskin keratinocytes. Using real time reverse transcription-polymerase chain reaction, we demonstrated that IL-10 is neither able to induce its typical early gene product SOCS-3 nor to modulate the IFN--induced expression of SOCS-1 and -3. Although flow cytometric analyses showed binding of biotin-labeled IL-10 to HaCaT cells, blocking experiments indicated that this resulted from unspecific binding, which may explain discrepancies to some earlier observations (Michel et al., 1997a,b). Moreover, scattered plot analyses excluded specific binding to primary KC and HaCaT cells. Finally, real-time mRNA analyses demonstrated that the absence of any specific binding results from the lack of IL-10R1 (-chain) expression, whereas the IL-10R2 (-chain) is constitutively expressed. This indicates that IL-10 unresponsiveness of keratinocytes could be explained by a lack of IL-10R1 expression and suggest that any IL-10 effects on these cells observed in vivo are indirectly mediated (Seifert et al., 2003).
There is some evidence that IL-10 regulates collagen and DNA synthesis in activated hepatic stellate cells (Mathurin et al., 2002). Taken together, IL-10 is a pluripotent cytokine with potent effects on numerous cell populations, in particular circulating and resident immune cells, as well as epithelial and some other parenchymal cells. Whereas initial data after its discovery suggested that IL-10 mainly mediates suppressive functions, more recent data showed stimulatory properties on certain cell populations, too. Recent data suggests that the effects of IL-10 are quite complex and still considering IL-10 just as immunosuppressive and anti-inflammatory (as it was done in the past) might be an oversimplification. Considering IL-10 as immunoregulatory instead of immunosuppressive is supported by recent in vivo data.
B. Effects of Interleukin-10 in Animals/Animal Models
The data from investigations of IL-10 effects on immune cells suggests that the major physiological importance of IL-10 seems to be the limitation of inflammation, the prevention of uncontrolled nonadequate immunologic reactions, as well as the support of the humoral (Th2) immune responses (De Waal Malefyt et al., 1991a,b; Romagnani, 1995). This hypothesis was confirmed by experimental research in animals, including analyses of IL-10 knockout mice as well as by the effects of IL-10 observed in several inflammatory, autoimmune, and tumor models.
1. Interleukin-10 Knockout Mice.
IL-10-deficient mice develop lethal inflammation of the intestine, which can be stopped by application of IL-10 (Kuhn et al., 1993). Interestingly, IL-10-/- mice kept under germ-free conditions do not develop enterocolitis, which suggests that in the absence of the immunomodulatory effects of IL-10, an unrestricted intestinal inflammatory response develops toward normal enteric antigens (Rennick et al., 2000).
2. Inflammation and Autoimmune Models.
The observations in the IL-10-/- mice were the rationale for administering IL-10 in several animal models for colitis. The results of these studies clearly showed prevention of intestinal inflammation by IL-10, mainly by down-regulation of an intestinal proinflammatory Th1-like response. However, systemic IL-10 administration was successful only when administered before the initiation of colitis but was ineffective at reversing any established inflammation (Powrie et al., 1993; Herfarth et al., 1996, 1998; Barbara et al., 2000).
Effects of IL-10 application have been investigated in various other inflammatory animal models, too. It turned out that treatment with IL-10 is beneficial in models of experimental autoimmune encephalomyelitis (Rott et al., 1994), pancreatitis (Van Laethem et al., 1995), diabetes mellitus (Pennline et al., 1994), and experimental endotoxemia (Gerard et al., 1993). IL-10 was also effective in various animal models of arthritis, in reducing inflammation, in cellular infiltrates, and in joint destruction (Persson et al., 1996; Tanaka et al., 1996).
However, some of the data are conflicting. For examples, studies have shown both inhibition and exacerbation of experimental allergic encephalomyelitis (EAE) after systemic IL-10 administration. Different therapeutic outcomes are also dependent on the mode of delivery of IL-10 by gene therapeutic vectors (Broberg et al., 2001; Croxford et al., 2001; Cua et al., 2001). Thus the action of IL-10 may differ depending on the local micro-environment, the disease stage, and the IL-10 concentration.
The majority of experimental data suggests the IL-10 application might be beneficial in several inflammatory and organ-restricted autoimmune diseases. With regard to systemic autoimmune diseases a different picture is emerging. For example, anti-IL-10 mAb treatment of SCID mice injected with PBMC from systemic lupus erythematosus (SLE) patients strongly inhibits autoantibody production in vivo (Llorente et al., 1995); also, treatment of New Zealand black/white mice (mice that spontaneously develop a severe autoimmune disease that closely resembles SLE) with anti-IL-10 mAb substantially delayed onset of autoimmunity (Ishida et al., 1994). This may indicate that neutralizing IL-10 might be a new therapeutic option here. In contrast to the organ-specific autoimmunopathies, SLE is thought to be more a "B cell disease". The different pathogenesis might explain the opposite effects of IL-10.
3. Tumor Models.
Several animal experiments have been performed to analyze the role of IL-10 on tumor development. The data are complex, showing diverse effects regarding the influence of IL-10 on cancer. Dependent on the experimental model, IL-10 seems to favor or inhibit the existence and progression of tumors (Sabat and Asadullah, 2002).
IL-10 is able to favor tumor growth both directly by affecting the tumor cells and indirectly by inhibition of immune cells. IL-10 can convert tumor cells to a CTL-resistant phenotype. Kiessling and coauthors reported an approximately 50% reduction of MHC class I expression in human melanoma cells after IL-10 treatment. This pretreatment resulted in a dose-dependent, and up to 100% inhibition of autologous CTL-mediated, tumor-specific lysis (Matsuda et al., 1994). This effect is mediated by reduced expression of the so-called transporter associated with antigen processing (TAP)-1 and -2, which results in reduced translocation of peptides to the endoplasmic reticulum and, therefore, in diminished MHC class I peptide loading and cell surface levels (Salazar-Onfray et al., 1997). However, the down-regulation of MHC class I expression results in higher sensitivity of these cells toward NK cell activity (Salazar-Onfray et al., 1995; and see below). The consequence of IL-10 presence and thereby the resulting inhibition of the antitumor immune reaction might be the uncontrolled development of cancers. This has been demonstrated in transgenic mice expressing IL-10 under control of the IL-2 promoter. These animals are unable to limit the growth of immunogenic tumors. However, administration of anti-IL-10 antibodies restored the anti-cancer response (Hagenbaugh et al., 1997).
The direct negative effect of IL-10 on tumor survival has been described by the Fulton group. They observed that the IL-10 gene transfer in murine mammary tumor cells was associated with increased expression of the inducible isoform of nitric-oxide synthase (iNOS). The activity of this enzyme was elevated as well. This can result in elevated levels of nitric oxide in transfected tumor cells (Kundu et al., 1998). Nitric oxide is known to show potent antitumor activity.
IL-10 can inhibit the generation of new vessels within the tumor both directly by acting on the tumor cells and indirectly by influencing infiltrating immune cells. IL-10 induced the tissue inhibitor of metalloproteinase 2 (TIMP-2) in primary human prostate cancer cells. Simultaneously, it reduced the secretion of matrix metalloproteinase (MMP)-2 and MMP-9 from these cells. The consequence was the inhibition of microvessel formation (Stearns et al., 1999). Interestingly, TGF- induced the expression of MMP-2, and this induction was prevented by IL-10. When primary human prostate cancer cells either expressing TGF- or IL-10 were implanted in SCID mice, TGF--promoted tumor growth, angiogenesis, and metastasis. In contrast, IL-10 reduced growth rates, angiogenesis, and metastasis. More importantly, none of the mice bearing TGF--expressing tumor cells survived compared with 80% of those expressing IL-10 (Stearns et al., 1999). IL-10 can also inhibit the angiogenesis by inhibiting tumor-resident macrophages. Bar-Eli and coauthors (Huang et al., 1996, 1999) reported that the transplantation of human melanoma cells that had been transfected with the murine IL-10 cDNA into nude mice resulted in fewer lung metastases and significant inhibition of tumor growth. The authors suggested that this was due to inhibition of angiogenesis by IL-10. They referred to the fact that IL-10 down-regulated the production of vascular endothelial growth factor in the tumor-associated macrophages. Other factors involved in neovascularization such as IL-1, TNF-, and IL-6 were also inhibited (Huang et al., 1996, 1999). Velu and coauthors (Gerard et al., 1996) demonstrated a loss of tumorigenicity of melanoma cells injected into syngeneic mice after the previous retroviral transfection of these cells with IL-10 cDNA. Host T cells and NK cells might be involved in the observed tumor eradication because IL-10-producing tumor cells grew in nude mice and in CD8+ T or NK cell-depleted mice (Gerard et al., 1996). Similar observations have been reported by the Fulton group. They described that injection of a murine mammalian tumor cell line in syngeneic mice resulted in progressive tumor growth and death from pulmonary metastases. In contrast, transfection of IL-10 cDNA in these cell lines resulted in complete inhibition of growth and metastatic disease. Interestingly, the antimeta-static activity of IL-10 is observed also in T cell-deficient mice but is lost when NK cell activity is suppressed (Kundu and Fulton, 1997).
4. Experimental Models of Infections.
It is well recognized that IL-10 can inhibit protective immune response to infections (Moore et al., 2001). It has been shown that the trauma-, burn-, and major surgery-induced immunodepression, which predispose to infectious complications, is related to IL-10 overexpression (Ayala et al., 1994; Woiciechowsky, 1998; Kobayashi et al., 2001). Prolonged IL-10 expression increases the risk for infectious complications, whereas neutralizing IL-10 >12 h post trauma reduces the immunodepression and infection-related mortality (Song et al., 1999).
Overexpression of IL-10 or other type 2 cytokines also modifies the immune response to intracellular bacteria and parasites as well as the susceptibility of mice to these infections (Yamakami et al., 2002).
On the other hand, IL-10 has also protective effects in infection because it prevents an uncontrolled inflammatory response to infectious triggers. IL-10-deficient mice show a prolonged inflammatory response to acute Pseudomonas challenge resulting in neutrophil accumulation in the lung. This observation suggests that IL-10 deficiency might contribute to prolonged inflammatory responses early in cystic fibrosis, a lung disease that is characterized by a neutrophilic infiltrate that is excessive relative to the burden of infection (Chmiel et al., 2002). Overexpression of IL-10 prevents mice from endotoxin or bacteria-induced septic shock whereas lack of IL-10 increases the susceptibility to toxin-related shock (Moore et al., 2001; Oberholzer et al., 2002). IL-10 also protects against experimental group B streptococcal arthritis (Puliti et al., 2002). Similar protective properties of IL-10 were observed for gastrointestinal helminth infections (Schopf et al., 2002).
C. Interleukin-10 and Interleukin-10 Receptor Expression in Diseases
Its considerable anti-inflammatory effects and ability to act as a main suppressor of cellular immunity (Spits and De Waal Malefyt, 1992) raises the question of the IL-10 expression under pathophysiological conditions. Both overexpression (e.g., in lymphoma) as well as IL-10 deficency were found (e.g., in inflammatory bowel disease, psoriasis) and seems to have a pathophysiological significance (Schreiber et al., 1995). Numerous studies have investigated the expression and suggested the importance of IL-10 dysregulation in different entities.
1. Expression in Malignant Diseases.
a. Melanoma.
Krüger-Kraskagakes et al. (1994) could demonstrate significant IL-10 mRNA expression in melanoma and melanoma metastases but not in healthy skin. Moreover, they found IL-10 mRNA and the biologically active protein in 3 of 13 melanoma cell lines. This suggests that melanoma cells themselves are contributing at least in part to the IL-10 overexpression in melanoma lesions. Similar results were reported by Dummer et al. (1996b) who also demonstrated IL-10 production by a high percentage of melanoma metastases and corresponding cell lines. This may be of particular pathogenetic importance, since IL-10 functions as autocrine growth factor for malignant melanoma and reduces the expression of HLA class I and II on melanoma cells (Yue et al., 1997).
b. Carcinoma.
There are reports on the overexpression of IL-10 in basal cell and squamous cell carcinoma (Kim et al., 1995). Cytotoxic T cell lines recognizing these tumors proliferated in the presence of the tumor cells only when IL-10 was neutralized by monoclonal antibodies. On the other hand, the intralesional injection of IFN- resulted in a tumor regression that was associated with the down-regulation of IL-10 mRNA expression. IL-10, therefore, seems to be an important mediator in evading the T cell-mediated immune response in these cutaneous malignancies (Kim et al., 1995). The Strieter group described increased levels of IL-10 protein in tissue homogenates of human bronchogenic carcinomas compared with normal lung tissues. Staining of these tumors illustrated primary localization of IL-10 protein to cancer cells. Furthermore, IL-10 protein was present in supernatants of several unstimulated human bronchogenic cell lines (Smith et al., 1994).
Interestingly, a correlation between IL-10 and vascular endothelial growth factor expression in esophageal cancer was demonstrated suggesting a relation between IL-10 and tumor-promoting angiogenic factor gene expression (Nagata et al., 2002).
c. Lymphoma.
Tumor cells from B, T, and NK cell lymphoma are able to produce biologically active IL-10 (Kitabayashi et al., 1995; Masood et al., 1995; Sjoberg et al., 1996; Beatty et al., 1997; Boulland et al., 1998; Jones et al., 1999). As early as 1993, Favrot and coauthors (Blay et al., 1993) investigated IL-10 serum levels using an ELISA, which detects both viral and human IL-10 in patients with active non-Hodgkin's lymphoma (NHL) and healthy volunteers. They described the detection of IL-10 in serum from about 50% of these patients but none of the control blood donors. IL-10 was detectable with a similar frequency in all subtypes of NHL and in all clinical stages, as well as in both EBV-seropositive and EBV-seronegative patients (Blay et al., 1993). One year later the Papa group (Stasi et al., 1994a) demonstrated similar results obtained in patients with aggressive non-Hodgkin's lymphoma. In the following years these observations were extended to Hodgkin's disease and other lymphoma species, and due to improved sensitivity of ELISA systems, it was possible to demonstrate that lymphoma patients had significantly higher serum levels of IL-10 than healthy volunteers (Cortes et al., 1995; Cortes and Kurzrock, 1997; Sarris et al., 1999; Bohlen et al., 2000; Vassilakopoulos et al., 2001; Fayad et al., 2001). An elevated local expression of IL-10 was detected in various cutaneous T cell lymphoma entities (CTCL). Dummer et al. (1996a) showed IL-10 production by malignant T cells in Sézary syndrome, a leukemic type of cutaneous T cell lymphoma. We demonstrated cutaneous IL-10 mRNA overexpression in mycosis fungoides (MF) lesions (Asadullah et al., 1996). Increasing IL-10 gene expression correlated with the tumor progression. An increased cutaneous IL-10 mRNA expression was also found in CD30+ pleomorphic T cell lymphomas (Asadullah et al., 1996; Yagi et al., 1996) and cutaneous B cell lymphomas (CBCL) (Asadullah et al., 2000b). The IL-10 overexpression in CTCL might contribute to a number of immunological abnormalities well known in these patients. These include eosinophilia and elevated IgE and IgA levels (Edelson, 1980). We recently observed a stage-dependent decrease in T cell activation of antigen expression suggesting impairment of tumor surveillance in advanced MF stages (Asadullah et al., 1997a). Such findings might result from the IL-10 overexpression that also might be responsible for the development of a systemic type 2 cytokine pattern in CTCL (Dummer et al., 1993).
d. Prognostic Value of Interleukin-10 Overexpression.
In different lymphomas, increased IL-10 production has been reported and a negative prognostic meaning of increased IL-10 plasma levels is being discussed (Blay et al., 1993; Stasi et al., 1994a,b; Cortes et al., 1995). Elevated IL-10 serum levels have been also described as a negative prognostic factor for responsiveness toward treatment, as well as the disease-free and overall survival by patients with melanoma and solid tumors, particularly with lung, gastrointestinal, and renal cell cancer. Several groups including ours reported on increased circulating IL-10 serum levels in gastric, colon, and renal-cell cancer patients (Ordemann et al., 2002). IL-10 serum levels commonly returned to normal in radically resected patients. Persistently elevated IL-10 serum levels after surgery predicted tumor recurrence (Galizia et al., 2002a,b; Uwatoko et al., 2002). Moreover, a further significant increase in IL-10 serum levels has been observed in nonresponders after chemotherapy (Wojciechowska-Lacka et al., 1996; De Vita et al., 1999, 2000a,b
