IL10

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IL-10 Rene de Waal Malefyt* Department of Molecular Biology, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104, USA * corresponding author tel: 650-496-1164, fax: 650-496-1200, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03005.

SUMMARY Interleukin 10 (IL-10) is a homodimeric cytokine, produced by activated monocytes and T cells, that is deeply involved in the regulation of inflammatory responses and immune reactions. Its main overall function is best described as dampener of immune responses, but IL-10 possesses stimulatory activities as well. Clearly, IL-10 is pleiotropic and research into its many functions and mechanisms of action is still very active. It affects not only the immune system, but through its modulation of growth factors, cytokines and other mediators, it can influence many physiological processes, including for example angiogenesis, tumorigenesis and infection. It is a key molecule that is able to ameliorate potential pathological autoimmune processes through the inhibition of many facets of the inflammatory response. In addition, the induction of regulatory T cells by IL-10 points towards a crucial role in the establishment of peripheral tolerance. All these qualities make IL-10 a desirable candidate to use in clinical intervention and studies examining the efficacy of IL-10 in several chronic inflammatory or autoimmune diseases are well underway.

BACKGROUND

Discovery IL-10 was discovered when Tim Mosmann and collegues at DNAX pursued a biological activity in the supernatants of activated murine TH2 cell clones that could modulate the effector function of TH1 cells (Fiorentino et al., 1989). The assay that was used to follow this biological activity measured a reduced

capacity of TH1 cells to produce cytokines, in particular IFN , hence the name cytokine synthesis inhibitory factor. An IL-10 cDNA clone was identified by both biochemical purification and expression cloning (Vieira et al., 1991). Subsequent expression of the protein and production of neutralizing monoclonal antibodies revealed that IL-10 was also active in assays that examined B cell, mast cells, and macrophage/monocyte functions. IL-10 is now well recognized as a dominant immunoregulatory molecule with important immunostimulatory and unique immunosuppressive and anti-inflammatory activities.

Alternative names Cytokine synthesis inhibitory factor (CSIF), mast cell growth factor III (MCSFIII), and B cell-derived T cell growth factor (B-TCGF) are alternative names that have been used.

Structure IL-10 belongs to the family of long-chain cytokines, which also encompasses IFN/ and IFN . Biologically active IL-10 is a homodimer.

Main activities and pathophysiological roles IL-10 affects many aspects of inflammatory and immune responses and acts on both hematopoietic and nonhematopoietic cells. It has dominant suppressive effects on the production of proinflammatory

166 Rene de Waal Malefyt cytokines by monocytes and neutrophils, and downregulates the expression of activating and costimulatory molecules on monocytes and dendritic cells, leading to its characterization as a macrophage deactivating factor. It also has important growth factor activities on B cells and mast cells, and inhibits or enhances the activities of T cells depending on their activation conditions and CD4 or CD8 subclass. By affecting these prominent players in immune and inflammatory reactions, it has an important regulatory function (Moore et al., 1993).

GENE AND GENE REGULATION

Accession numbers

dependent on NFB activation (Bondeson et al., 1999). Several polymorphisms have been observed in the human IL-10 gene 50 -flanking sequence. They include two areas of 6±11 (CA)n repeat microsatellite polymorphisms ÿ1109 kb and ÿ3942, as well as three linked point mutations at ÿ1082 (G/A), ÿ819 (C/ T ), and ÿ592 (C/A) (Eskdale et al., 1997a; Hurme et al., 1998). These polymorphisms have been linked to enhanced production of IL-10 by monocytes and T cells and are implicated in the levels of IL-10 production by patients suffering from or susceptible to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and meningococcal disease (Eskdale et al., 1997b; Westendorp et al., 1997; Mehrian et al., 1998; Crawley et al., 1999) and can be prognostic for graft rejection following renal transplantation (Sankaran et al., 1999).

IL-10 cDNA: M57627 IL-10 gene: U16720

Cells and tissues that express the gene

Chromosome location IL-10 is located on chromosome 1q. The human IL-10 gene spans 4.7 kb and consists of five exons separated by four introns.

Regulatory sites and corresponding transcription factors A TATA box is located 84 bp 50 of the first methionine codon. Other consensus transcription factorbinding sequences have been identified by computer searches using the transcription factor database (GenBank tfdb). These include CCAAT motifs at ÿ148, ÿ363, ÿ864, AP-1-binding sites at 29 and ÿ695, NF-IL6 consensus binding sites at ÿ338, ÿ532, ÿ777, ÿ1504, ÿ1551, ÿ2085, ÿ2793, ÿ2894, and ÿ2937. In addition a glucocorticoid response element (GRE) and a cyclic AMP response element (CRE) are located at ÿ250 and ÿ311. The CRE appears to be functional, as agents that raise intracellular cAMP levels enhance the expression of IL-10 in monocytes. Expression of the IL-10 gene in monocytes is not

IL-10 is expressed by naõÈ ve and memory T cells derived from both peripheral blood or cord blood, T cell clones belonging to TH1, TH2, and Tr1 subsets, NK cells, B cells derived from peripheral blood, tonsils or spleen, Epstein±Barr virus (EBV)-transformed B cell lines, Burkitt's lymphoma, AIDS lymphomas, monocytes, placental trophoblasts, bronchial epithelial cells, and certain tumor cells including melanomas and carcinomas of various origin (de Waal Malefyt and Moore, 1998).

PROTEIN

Accession numbers P22301 (g124292)

Sequence See Figure 1.

Figure 1 Amino acid sequence for IL-10. MHSSALLCCL VKTFFQMKDQ QDPDIKAHVN EKGIYKAMSE

VLLTGVRASP LDNLLLKESL SLGENLKTLR FDIFINYIEA

GQGTQSENSC THFPGNLPNM LRDLRDAFSR LEDFKGYLGC QALSEMIQFY LEEVMPQAEN LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ YMTMKIRN

IL-10 167

Description of protein The human IL-10 gene encodes a protein of 178 amino acids which includes 18 amino acids of hydrophobic signal sequence. The mature hIL-10 protein consists, therefore, of 160 amino acids and Ser1 was confirmed as the N-terminal residue by protein sequencing (Windsor et al., 1993). The predicted molecular weight of hIL-10 is 18,647 Da and it runs as a single species with an apparent molecular weight of 17 kDa in SDS-PAGE (Vieira et al., 1991). hIL-10 contains four cysteine residues which form two intramolecular disulfide bonds, with Cys12 pairing to Cys108 and Cys62 to Cys114 (Windsor et al., 1993). The isoelectric point (pI) of IL-10 is 7.92, which is close to its predicted value of 7.8. hIL-10 is acidic sensitive and exposure to a pH below 5.5 will result in biologically inactive material. IL-10 exists as a noncovalently linked homodimer and has a apparent molecular weight of 37 kDa in solution (Syto et al., 1998).

Discussion of crystal structure Structurally, IL-10 belongs to the family of longchain cytokines characterized by a four  helix bundle (Sprang and Bazan, 1993). hIL-10 has been crystallized and the resulting IL-10 crystals were tetragonal, of space group P43 21 2 (Trotta and Windsor, 1994). The crystal structure was resolved at 3.5, 2.0, 1.8, and 1.6 AÊ resolution following X-ray diffraction (Walter and Nagabhushan, 1995; Zdanov et al., 1995). IL-10 is a dimer composed of identical polypeptide chains related by a 2-fold axis forming a V-shaped structure. Each half of the dimer consists of six  helices, four originating from one subunit, and two from the other. The topology of the helices resemble that of IFN . The resolution map of 1.6 AÊ was used to generate a computer model of a complex of IL-10 with its two soluble receptors showing excellent complementary of polar and hydrophobic interactions (Zdanov et al., 1996). The crystal structure of viral IL-10 has also been resolved and shows a very comparable topology (Zdanov et al., 1997).

Important homologies Viral homologs of IL-10 have been found in Epstein± Barr Virus (EBV), equine herpes virus type 2 (EHV2), and poxvirus Orf genomes (Moore et al., 1990; Rode et al., 1993; Fleming et al., 1997). The EBV-encoded IL-10 (viral IL-10) is located on the BCRF sequence

and the protein has maintained some, but not all the biological activities of its cellular counterpart. hIL-10 has 90% homology at the amino acid level with viral IL-10 and 73% with murine IL-10. The greatest diversions between these IL-10 species are found in the signal sequence and the first 21 amino acids of the N-terminus (Vieira et al., 1991). The human IL-10 protein is active on murine cells but the murine cytokine is not active on human cells. IL-10 sequences for pig-tailed macaque, mangabey, rhesus, and owl monkeys, rat, guinea pig, Syrian hamster, rabbit, woodchuck, gerbil, opossum, cat, dog, cow, sheep, red deer, pig, horse, and killer whale are also deposited in GenBank.

Posttranslational modifications hIL-10 contains one possible N-linked glycosylation site at Asn115, but this site is unoccupied (Trotta and Windsor, 1994).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-10 is expressed by naõÈ ve and memory T cells derived from both peripheral blood or cord blood, T cell clones belonging to TH1, TH2, and Tr1 subsets, NK cells, B cells derived from peripheral blood, tonsils, or spleen, EBV-transformed B cell lines, Burkitt's lymphoma, AIDS lymphomas, monocytes, microglia (Chabot et al., 1999), placental trophoblasts, bronchial epithelial cells, keratinocytes (mouse) and certain tumor cells including melanomas and carcinomas of various origin (de Waal Malefyt and Moore, 1998). Activation is generally required for expression for nontransformed cells of hematopoietic origin. IL-10 is produced by dendritic cells derived from Peyer's patches, but not from dendritic cells derived from spleen (Iwasaki and Kelsall, 1999).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The inducing agents that have been used in various cell types are listed in Table 1. IL-10 production by T cells can be enhanced by IL-12, IL-4, IFN, and IL-10 itself can induce a population of T regulatory

168 Rene de Waal Malefyt

Table 1 Inducing agents used in various cell types Cell type

Inducing agent

T cells

Antigen presented by antigen-presenting cells (APCs) Anti-CD3 mAbs and PMA Combinations of anti-CD3 and anti-CD28 mAbs Mitogenic combination of anti-CD2 mAbs Concavalin A Mitogenic lectins Calcium ionophores and PMA (weak) Superantigens SEA, SEB Gangliosides GD1a, GM3 (Kanda, 1999) ICOS (Hutloff et al., 1999)

B cells

Staphylococcus aureus Cowan strain I (SAC) EBV Anti-IgM mAbs Anti-CD40 mAbs

NK cells

IL-2, IL-2 + IL-12 (Mehrotra et al., 1998)

Monocytes

Lipopolysaccharide (LPS) SAC Anti-CD40 mAbs Immune complexes (Sutterwala et al., 1998) CD23 crosslinking

Keratinocytes

UV irradiation (mouse) (Enk et al., 1995) CD23 ligation (human) (Becherel et al., 1997)

cells that produces very high levels of IL-10 upon antigen-specific activation. LFA-1/ICAM-1 costimulatory signals are more potent in the induction of IL-10 by anti-CD3 activation than ICAM-2 or ICAM-3 costimulation (Bleijs et al., 1999). Cyclosporin inhibits the production of IL-10 by T cells, but paradoxically can enhance IL-10 production by monocytes. LPS-induced IL-10 production by monocytes is enhanced by TNF, and agents that increase intracellular cAMP levels such as melanocyte-stimulating hormone, prostaglandin E2, cyclic nucleotide phosphodiesterase type IV inhibitors, adenosine, catecholamines (epinephrine), and chlorpromazine (dopamine receptor type I agonist), histamine (Elenkov et al., 1998), vasointestinal peptide (VIP) (Delgado et al., 1999), and is inhibited by IL-4, IL-10 itself, IL-13, IFN , and TGF . The inhibition of IL-10 by IL-4 monocytes is dependent on expression of the common chain (Bonder et al., 1999). LPS-induced IL-10 production by monocytes is dependent on endogenous TNF

and IL-1 and mediated by p38 MAP kinases (Foey et al., 1998). The release of IL-10 by monocytes induced by catecholamines following brain injury may represent a common pathway for immunodepression following brain injury and stress (Woiciechowsky et al., 1998). IL-10 production by monocytes is relatively insensitive to glucocorticoids (Visser et al., 1998). Murine cytomegalovirus induced IL-10 expression in murine macrophages (Redpath et al., 1999). The production of IL-10 following UV irradition by keratinocytes may be dependent on the production of IL-6 by Langerhans cells (Nishimura et al., 1999).

RECEPTOR UTILIZATION The IL-10 dimer interacts with two heterodimeric receptor complexes consisting of IL-10R and IL-10R subunits. This interaction, as well as the resulting signaling pathways, are discussed under the IL-10 receptor.

IL-10 169

IN VITRO ACTIVITIES

In vitro findings Effects of IL-10 on Monocytes, Macrophages, and Dendritic Cells IL-10 significantly modulates expression of cytokines, soluble mediators, and cell surface molecules on cells of myeloid origin. These changes have important consequences for the functions of these cells in activating and maintaining immune and inflammatory responses. IL-10 strongly inhibits the production of IL-1, IL-1 , IL-6, IL-8, IL-10 itself, IL-12, GMCSF, G-CSF, M-CSF, TNF, MIP-1, MIP 1 , MIP-2, RANTES, and LIF by activated monocytes/ macrophages (de Waal Malefyt et al., 1991a, 1993a; Fiorentino et al., 1991a; D'Andrea et al., 1993; Gruber et al., 1994; Berkman et al., 1995; MarfaingKoka et al., 1996). Inhibition of production of certain cytokines, like KC, results from destabilization of mRNA mediated by AU-rich elements in the 30 untranslated region of sensitive genes (Kim et al., 1998; Kishore et al., 1999). Alternative mechanisms for cytokine production are also described as inhibition of IL-12 occurs at the transcriptional level and is dependent on new protein synthesis (Aste-Amezaga et al., 1998) and inhibition of TNF production occurs by transcriptional and posttranscriptional mechanims but is independent of the ability of IL-10 to inhibit NFB activation (Clarke et al., 1998). IL-10 induced and enhanced the expression of a novel chemokine that is chemotactic for monocytes called HCC4 (Hedrick et al., 1998) and IL-10 enhanced expression of CCR5 (Sozzani et al., 1998). In addition, IL-10 enhanced production of IL-1Ra (Cassatella et al., 1994; de Waal Malefyt et al., 1991a; Jenkins et al., 1994) and expression of soluble p55 and p75 TNFR (Hart et al., 1996; Joyce and Steer, 1996; Linderholm et al., 1996; Dickensheets et al., 1997), indicating that IL-10 induces a shift from production of proinflammatory to anti-inflammatory mediators. IL-10 also inhibited expression of MHC class II antigens, CD54 (ICAM-1), CD80 (B7), and CD86 (B7.2) on monocytes, even following induction of these molecules by IL-4 or IFN (de Waal Malefyt et al., 1991b; Ding et al., 1993; Kubin et al., 1994; Willems et al., 1994). Downregulation of these stimulatory or costimulatory molecules significantly affected the T cell-activating capacity of monocyte APCs (de Waal Malefyt et al., 1991b; Fiorentino et al., 1991b; Ding and Shevach, 1992). IL-10 inhibits MHC class II expression by monocytes through a posttranscriptional mechanism involving inhibition of transport of mature, peptide-loaded MHC class II

molecules to the plasma membrane (Koppelman et al., 1997). IL-10 selectively enhanced the expression of HLA-G on trophoblasts and monocytes, which may play a role in maternal/fetal tolerance (Moreau et al., 1999). In contrast, IL-10 upregulated expression of FcR on monocytes, including CD16 and CD64 (te Velde et al., 1992; de Waal Malefyt et al., 1993a; CalzadaWack et al., 1996) but downregulated the expression of IL-4-induced CD23 (Morinobu et al., 1996). Upregulation of CD64 correlated with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) (te Velde et al., 1992). In contrast, IL-10 inhibited tumor cytotoxicity (Nabioullin et al., 1994). Upregulation of FcR by IL-10 enhanced the capacity of monocytes/macrophages to phagocytose opsonized particles, bacteria, or fungi (Capsoni et al., 1995; Spittler et al., 1995), but IL-10 reduced the ability of the cells to kill the ingested organisms by decreasing the generation of superoxide anion (O2ÿ) and nitric oxide (NO) (Bogdan et al., 1991; Cunha et al., 1992; Gazzinelli et al., 1992; Niiro et al., 1992, 1995; Cenci et al., 1993; Wu et al., 1993; Kuga et al., 1996; Roilides et al., 1997). The inhibitory effects of IL-10 on production of NO by mouse macrophages can occur by an indirect mechanism involving inhibition of cytokine synthesis (Oswald et al., 1992a, 1992b; Flesch et al., 1994). IL-10 inhibited production of prostaglandin E2, another proinflammatory mediator (Niiro et al., 1994). IL-10 inhibited the ability of monocytes/macrophages to modulate turnover of extracellular matrix through its inhibitory effects on production of gelatinase and collagenase, and its ability to enhance production of tissue inhibitor of metalloproteinases (Lacraz et al., 1995; Mertz et al., 1994). IL-10 enhanced the expression of the fMLP receptor and the PAF receptor on monocytes, making the cells more responsive to these chemotactic factors (Thivierge et al., 1999). IL-10 also inhibited C-reactive protein (CRP)-induced and LPS-induced, but not CD40-induced tissue factor (TF) expression by human monocytes, which leads to a reduction in procoagulant activity (Jungi et al., 1994; Pradier et al., 1993, 1996; Ramani et al., 1993, 1994). The effects of IL-10 on cytokine production and function of human macrophages are generally similar to those on monocytes, although a little less pronounced (Berkman et al., 1995; Nicod et al., 1995; Wilkes et al., 1995; Armstrong et al., 1996; Park and Skerrett, 1996; Thomassen et al., 1996; Zissel et al., 1996). Furthermore, IL-10 inhibited production of IL-12 and expresssion of costimulatory molecules on various types of dendritic cells (Macatonia et al., 1993; Peguet-Navarro et al., 1994; Buelens et al., 1995; Ludewig et al., 1995; Mitra et al., 1995), which

170 Rene de Waal Malefyt correlates with its ability to inhibit primary alloantigen-specific T cell responses (Bejarano et al., 1992; Caux et al., 1994). Furthermore, IL-10 treatment of dendritic cells may induce a state of anergy in alloantigen- or peptide antigen-activated T cells (Steinbrink et al., 1997). IL-10 also inhibits the generation of monocyte-derived dendritic cells cultured with GMCSF and IL-4 (Allavena et al., 1998) and induced apoptosis of plasmacytoid dendritic cells (DC2) (Rissoan et al., 1999).

expression which results in increase prostaglandin D2 production (Ashraf et al., 1996). IL-10 is produced constitutively by mucosal type mast cells but needs to be induced by IL-3 in connective tissue type mast cells (Marietta et al., 1996). IL-10 inhibits the expression of c-kit on mouse bone marrow-derived mast cells, resulting in a reduced production of IL-13 and TNF in response to SCF (Mirmonsef et al., 1999). Effects of IL-10 on B Cells

Effects of IL-10 on Granulocytes and Eosinophils Granulocyte function is also affected by IL-10. LPS-induced cytokine and chemokine production, generation of superoxide anions, and survival by polymorphonuclear leukocytes or neutrophils were inhibited by IL-10, whereas production of IL-1Ra was enhanced (Cassatella et al., 1993, 1994, 1997; Kasama et al., 1994; Wang et al., 1994; Chaves et al., 1996; Cox, 1996; Marie et al., 1996). IL-10 also inhibited LPS-induced survival and cytokine production by purified eosinophils (Takanaski et al., 1994). IL-10 reduced neutrophil migration in IgG complexinduced lung injury and LPS- or antigen-induced pulmonary inflammation (Shanley et al., 1995; Zuany-Amorim et al., 1995; Cox, 1996), which may be related to its regulation of chemokine expression. On the other hand, neutralization of IL-10 led to enhanced survival in murine models of Klebsiella pneumoniae, Streptococcus pneumoniae, and Mycobacterium avium infections, since killing of phagocytosed bacteria was partly suppressed by endogenous IL-10 (Denis and Ghadirian, 1993; Greenberger et al., 1995; van der Poll et al., 1996b). Inhibition by IL-10 of production of chemokines, proinflammatory cytokines, and mediators of granulocyte survival no doubt collectively limit the duration of inflammatory responses, as these agents perpetuate the inflammatory response. Effects of IL-10 on Mast Cells IL-10 is a cofactor for the IL-3- or IL-4-induced proliferation of mucosal-type mouse mast cells (Thompson-Snipes et al., 1991; Rennick et al., 1995). In addition, it induces the expression of two mast cellspecific proteases, mMCP-1 and mMCP-2 (Ghildyal et al., 1992a, 1992b, 1993). These activities of IL-10 may contribute to immune responses against helminth infections (Behnke et al., 1993). IL-10 also has an inhibitory effect on mast cell TNF and GM-CSF production following triggering of the high-affinity IgE receptor (Arock et al., 1996). However, the same activation induces prolonged cyclooxygenase (Cox-2)

IL-10 induced enhanced expression of MHC class II antigens and survival of resting mouse B cells (Go et al., 1990). In contrast, IL-10 inhibited motility and IL-5-induced antibody production against thymusindependent type I and II antigens (produced by B-1 B cells), but not against thymus-dependent antigens. This inhibition was reversed by IL-4 (Clinchy et al., 1994; Pecanha et al., 1992, 1993). However, serum immunoglobulin levels and development of B-1 B cells were normal in IL-10ÿ/ÿ mice (Kuhn, 1993). Furthermore, B cells do not seem to play a role in the inflammatory bowel disease that develops in these animals (Davidson et al., 1996). Current data suggest that the in vivo role of IL-10 in murine B cell responses may be limited. More extensive activities of IL-10 have been described on human B cells. IL-10 enhanced the survival of normal human B cells (depending on their activation state), which correlated with increased expression of the anti-apoptotic protein Bcl-2 (Levy and Brouet, 1994; Itoh and Hirohata, 1995). IL-10 strongly enhanced proliferation of human B cell precursors and mature B cells activated by anti-IgM monoclonal antibodies, Staphylococcus aureus strain Cowan I (SAC), or crosslinking of CD40 (Rousset et al., 1992; Saeland et al., 1993). Both IL-2 and IL-4 further enhanced B cell proliferation activated by IL-10 with anti-CD40. The synergistic effects of IL2 correlated with IL-10-enhanced expression of highaffinity IL-2 receptor on B cells (Fluckiger et al., 1993). B cell-derived and exogenous IL-10 also affected B cell differentiation and isotype switching (Burdin et al., 1995). Isotype committed B cells, activated by SAC or anti-CD40 mAbs, produced large amounts of IgM, IgG1±3, and IgA in the presence of IL-10, and IgG4 and IgE in the presence of both IL-10 and IL-4 (Garrone et al., 1994; Nonoyama et al., 1993; Punnonen et al., 1993; Rousset et al., 1992; Uejima et al., 1996). Activation (anti-CD40 and IL-10) of sIgD‡ naõÈ ve B cells also resulted in production of IgM, IgG1±3, and IgA (Defrance et al., 1992; Briere et al., 1994b). Furthermore, anti-CD40 and IL10 stimulation of sIgD‡ B cells induced isotype

IL-10 171 switching to IgG1 and IgG3 (Malisan et al., 1996) and, in combination with TGF , to IgA (Defrance et al., 1992). IL-10 induced production of IgA by antiCD40-activated B cells of patients suffering from IgA deficiency, although no defects in the production of IL-10 were observed in these patients (Briere et al., 1994a). Long-term culture of B cells stimulated by either anti-CD40 mAbs or by activated T cells expressing CD40 ligand in the presence of IL-10 resulted in differentiation of B cells into plasma cells (Merville et al., 1995; Rousset et al., 1995). Such effects of IL-10 may play a role in pathogenesis of systemic lupus erythematosus (SLE) where positive correlations between serum IL-10 levels and severity of disease, and between the production of IL-10 and autoantibodies by SLE patient's B cells were demonstrated (al-Janadi et al., 1996; Hagiwara et al., 1996; Houssiau et al., 1995; Llorente et al., 1994, 1995). A role for IL-10 in development of SLE was suggested by the delayed onset of autoimmunity in lupus-prone NZB mice treated with anti-IL-10 mAbs (Ishida et al., 1994). These results suggested that IL-10 antagonists could be useful therapeutics in SLE. Direct Effects of IL-10 on T Cells IL-10 strongly inhibited cytokine production and proliferation of T cells and T cell clones activated in the presence of APCs. This effect of IL-10 is due mostly to its downregulatory effects on APC functions (de Waal Malefyt et al., 1991b; Fiorentino et al., 1991b; de Waal Malefyt and de Vries, 1996). However, T cells do express IL-10R (Liu et al., 1994, 1997) and IL-10 modulates T cell function. IL-10 inhibits production of IL-2 and TNF, but not IL-4 and IFN when T cells were stimulated without APCs (de Waal Malefyt et al., 1993b; Taga and Tosato, 1992). In addition, inhibition of IL-2 production may also indirectly inhibit CD28- and PMA-induced production of IL-5, which is dependent on IL-2 (Schandene et al., 1994). Interestingly, activation of T cells in the presence of IL-10 can induce a longlasting state of nonresponsiveness/anergy, which cannot be reversed by IL-2 or stimulation by antiCD3 and anti-CD28 (Groux et al., 1996). A role for IL-10 in induction and maintenance of nonresponsiveness or anergy was suggested by studies of antitumor cell responses, UV-induced tolerance, hapten-specific tolerance, parasitic and HIV infections, and superantigen-induced hyporesponsiveness (Flores et al., 1993; Enk et al., 1993, 1994; Becker et al., 1994; Flores Villanueva et al., 1994; Suzuki et al., 1995; King et al., 1996; Schols and De Clercq, 1996; Sundstedt et al., 1997) and can be induced by specific immunotherapy (Akdis et al., 1998) or by

continuous antigenic challenge in vivo (Buer et al., 1998). IL-10 inhibited apoptosis of normal or infectious mononucleosis T cells following growth factor deprivation which correlated with upregulation of Bcl-2 (Taga et al., 1993, 1994; Cohen et al., 1997), and has chemotactic activity on CD8‡ T cells (Jinquan et al., 1993). In addition, IL-10 induced proliferation of CD8‡ T cells (Groux et al., 1998; Rowbottom et al., 1999). Stimulatory activities of IL-10 on T cells were also described in the mouse where it acts as a thymocyte growth factor and augments the outgrowth cytotoxic T cell precursors (MacNeil et al., 1990; Chen and Zlotnik, 1991). Effects of IL-10 on NK Cells IL-10 directly or indirectly affects the function of NK cells in several ways. IL-10 inhibited monocytedependent, IL-2-enhanced production of IFN and TNF by human NK cells. This effect was indirect and mediated via inhibition of production of monokines such as IL-1 and IL-12 (D'Andrea et al., 1993; Hsu et al., 1992). Inhibition by IL-10 of IFN production following infection of mice by intracellular parasites occurred through similar mechanisms (Tripp et al., 1993; Hunter et al., 1994; Cardillo et al., 1996). IL-10 also inhibited production of IL-5 by human NK cell clones stimulated by tumor cells and IL-2 (Warren et al., 1995). In contrast, IL-10 enhanced IL-2-induced proliferation of CD56 bright NK cells and cytokine production and cytotoxicity by CD56 bright and CD56 dim subsets (Carson et al., 1995). Studies with IL-10 also revealed a role for NK cells in antitumor responses. At low concentrations, IL10's inhibition of tumor metastasis in experimental or spontaneous models correlated with localization of high numbers of NK cells at the tumor site. These findings were repeated in T and B cell-deficient SCID mice, but not in NK cell-deficient mice (Zheng et al., 1996). Furthermore, administration of IL-10 or transduction of tumor cells with IL-10 expression plasmids resulted in NK cell-dependent inhibition of tumor growth/tumorigenity and in the establishment of protective memory responses (Giovarelli et al., 1995; Gerard et al., 1996; Kundu et al., 1996). Interestingly, IL-10 transfection of a B cell lymphoma resulted in inhibition of MHC class I expression and a change in phenotype from CTL-sensitive, NK-resistant to CTL-resistant, NK-sensitive (Salazar-Onfray et al., 1995). It is possible that the absence of MHC class I on these cells abrogated the inhibition of target cell lysis mediated by KIR (killer inhibitory receptors) on NK cells (Lanier, 1997). Downregulation of MHC class I antigens on human melanoma cells and

172 Rene de Waal Malefyt EBV-LCL following treatment of these targets with IL-10 also increased sensitivity to NK cell cytotoxicity and inhibited CTL activity (Matsuda et al., 1994).

Bioassays used A most commonly used bioassay is the cytokine synthesis inhibitory assay, which measures the ability of IL-10 to inhibit the production of IFN by PHAor anti-CD3-activated peripheral blood mononuclear cells (Vieira et al., 1991). Alternatively, murine Baf/3 cells that are transfected with the human IL-10R chain can be used to measure short-term proliferation in response to IL-10 (Liu et al., 1997).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Knockout mouse phenotypes IL-10ÿ/ÿ mice have been produced and have a normal appearance at birth as well as normal numbers and distribution of T cell, B cells, and macrophages. However, IL-10ÿ/ÿ mice become anemic and runted after 4±6 weeks due to a spontaneously developed enterocolitis (Kuhn et al., 1993), with the appearance of adenocarcinomas at advanced stages (Berg et al., 1996). Enterocolitis in IL-10ÿ/ÿ mice could be prevented by administration of IL-10 from birth and by treatment with anti-IFN or anti-IL-12 mAbs. IL-10 ameliorated but could not completely cure established disease. CD4‡ T cells isolated from diseased colon produced high levels of IFN and TNF and could transfer disease, indicating that the dysregulated interaction between enteric flora and inflammatory cells in the absence of IL-10 leads to uncontrolled TH1 responses (Davidson et al., 1996).

Transgenic overexpression Four types of IL-10 transgenic mice have been produced, where IL-10 expression is driven by the IL-2 promoter, the MHC class II promoter, the human salivary amylase promoter, or the rat insulin promoter. Expression of IL-10 under islet-specific promoters accelerated the development insulin-dependent diabetes mellitus (IDDM) in transgenic NOD mice (Wogensen et al., 1993, 1994; Lee et al., 1994, 1996; Moritani et al., 1994, 1996; Balasa and Sarvetnick, 1996; Mueller et al., 1996). Localized

high level IL-10 expression in the pancreas, as compared to systemic administration or production at a different site, may activate endothelium and promote chemoattraction of lymphocytes and/or myeloid cells, thus predisposing the mice to development of disease. In contrast, transgenic mice expressing IL-10 under control of the IL-2 promoter (Hagenbaugh et al., 1997) do not become diabetic. Expression of IL-10 by the MHC class II promoter resulted in inhibitory effects on the generation of T cell responses to protein antigens but enhanced CTL responses to tumor antigens, in agreement with the stimulatory effects of IL-10 on CD8‡ cells. MBPspecific T cells could be generated in these mice following immunization, but were nonpathogenic, resulting in mice that are highly resistant to experimental allergic encephalomyelitis (EAE) induced by active immunization (Cua et al., 1999). On the other hand, these transgenic mice were highly susceptible to infection with intracellular pathogens, confirming the inhibitory effects of IL-10 on NO-dependent macrophage killing (Groux et al., 1999) and showed defects in the development of T cells (Rouleau et al., 1999). Transgenic expression of IL-10 in the salivary gland induced apoptosis of glandular tissue, destruction and lymphocyte infiltration consisting primarily of Fas ligand (FasL)‡ CD4‡ T cells, as well as in vitro upregulation of FasL expression on T cells, indicating that Fas/FasL bystander destruction plays a role in the development of the disease (Saito et al., 1999).

IN THERAPY

Preclinical ± How does it affect disease models in animals? Systemic Inflammation The effects of IL-10 on individual cell types suggested that it could have potent anti-inflammatory/immunosuppresive activities in vivo, which has been tested in a variety of experimental models. IL-10 rescued Balb/c and BDF1 mice from LPS- or SEB-induced toxic shock, which correlated with reduced serum levels of TNF (Bean et al., 1993; Gerard et al., 1993; Howard et al., 1993). Intratracheal IL-10 gene transfer protected mice from a lethal intraperitoneal endotoxin challenge and furthermore reduced pulmonary TNF levels and neutrophil infiltration following LPS challenge (Rogy et al., 1995). When administered at the time of challenge, IL-10 did not prevent lethality in Corynebacterium parvum-primed BDF1 mice following endotoxin challenge, but it

IL-10 173 prevented lethality to endotoxin challenge when administered at priming (Smith et al., 1994, 1996). Although IL-10 reduced TNF and IFN levels in the former situation, synergistic effects of TNF and IFN still resulted in mortality in this model. IL-10 also reduced serum levels of TNF, IL-6, and IL-8 as well as neutrophil accumulation, elastase production, and cortisone levels in human volunteers challenged with a low dose of endotoxin (Pajkrt et al., 1997). Administration of endotoxin induced IL-10 production in mice, chimpanzees, baboons, and humans (Durez et al., 1993; van der Poll et al., 1994; Jansen et al., 1996; Pajkrt et al., 1997). Interestingly, LPSinduced IL-10 expression by monocytes in vitro occurs late after activation (de Waal Malefyt, 1991a), but IL-10 serum levels in vivo were already elevated 3±6 hours after endotoxin challenge (Durez et al., 1993; van der Poll et al., 1994; Jansen et al., 1996; Pajkrt et al., 1997), indicating that other factors or cells may contribute to IL-10 production in these circumstances. Endogenous IL-10 confers significant protection to endotoxin challenge and reduces TNF, IFN , and MIP-2 production (Marchant et al., 1994a; Standiford et al., 1995). This is also clearly observed in mice treated from birth with anti-IL-10 mAbs and IL-10ÿ/ÿ mice which are killed by 20-fold lower doses of LPS (Ishida et al., 1993; Berg et al., 1995a). Furthermore, IL-10ÿ/ÿ mice were extremely vulnerable to a generalized Swartzman reaction where prior exposure to a small amount of LPS primes the host for a lethal response to a subsequent, otherwise sublethal dose (Berg et al., 1995a). In a model of septic peritonitis in which mice undergo cecal ligation and puncture, endogenous IL-10 also had a protective effect (Kato et al., 1995; van der Poll et al., 1995). IL-10 also protected neonatal mice from lethal streptococcal B infections (Cusumano et al., 1996). Furthermore, endogenous IL-10 prevented lethality from SEB-induced shock, which is dependent on IL-2 and IFN production by T cells (Florquin et al., 1994). In humans, IL-10 is produced during septicemia and septic shock and serum levels correlate with intensity of the inflammatory response (Marchant et al., 1994b, 1995; Gomez-Jimenez et al., 1995; Sherry et al., 1996; van der Poll et al., 1997), which was especially evident in patients suffering from septic shock associated with meningococcal infections (Frei et al., 1993; Derkx et al., 1995; Lehmann et al., 1995; van Furth et al., 1995). The inhibitory effects of IL-10 on the endotoxininduced production of proinflammatory cytokines, including indirect effects on endothelial cell activation and adhesion (Pugin et al., 1993; Eissner et al., 1996), monocyte tissue factor expression (which contributes to procoagulant activity and ultimately disseminated

intravascular coagulation) (Pradier et al., 1993, 1996; Ramani et al., 1993, 1994; Jungi et al., 1994), as well as the observation that many strategies to intervene in sepsis affected IL-10 production (Mengozzi et al., 1994; Bourrie et al., 1995; Suberville et al., 1996; van der Poll et al., 1996a), indicate an important role for this cytokine in controlling systemic inflammatory responses. IL-10 in Localized Inflammatory Diseases Similarly, IL-10 exhibited protective effects in a variety of experimental models of local inflammation, such as pancreatitis (Van Laethem et al., 1995), uveitis (Rosenbaum and Angell, 1995; Li et al., 1996), keratitis (Tumpey et al., 1994), hepatitis (Arai et al., 1995), peritonitis (Kato et al., 1995; van der Poll et al., 1995; Cusumano et al., 1996), and lung injury (Mulligan et al., 1993; Shanley et al., 1995). In addition, IL-10 inhibited allergic airway inflammation and infiltration of neutrophils and eosinophils in sensitized mice (Zuany-Amorim et al., 1995). In IL10ÿ/ÿ mice, a role for IL-10 as a natural suppressor of cytokine production and inflammation in allergic bronchopulmonary aspergillosis was revealed, indicating that IL-10 suppresses not only inflammatory TH1, but also TH2-related responses (GruÈnig et al., 1997). IL-10 also inhibited both the sensitization and effector phases of delayed-type hypersensitivity (DTH) responses (Li et al., 1994; Schwarz et al., 1994) as well as the elicitation of contact hypersensitivity (CHS) reactions in sensitized mice (Ferguson et al., 1994; Kondo et al., 1994). IL-10 synergized with IL-4 to inhibit tuberculin-type DTH reactions in Balb/c mice that had recovered from a Leishmania major infection (Powrie et al., 1993b). The role of endogenous IL-10 in these responses is critical since reactions to irritants, DTH and CHS responses in IL-10ÿ/ÿ mice are exaggerated and prolonged (Berg et al., 1995b). Keratinocyte-derived IL-10 plays a major role in UVinduced suppression of DTH, CHS, and antitumor responses (Enk and Katz, 1992; Rivas and Ullrich, 1992, 1994; Enk et al., 1994; Beissert et al., 1995; Yagi et al., 1996), since IL-10ÿ/ÿ mice were completely resistant to UV-induced immunosuppression (Beissert et al., 1996). Inflammatory Bowel Disease IL-10 is also involved in the development of inflammatory bowel disease (IBD) as revealed by two in vivo models. Transfer of naõÈ ve CD45RBhi T cells (which develop T cells that produce high levels of IFN and TNF in response to enteric antigens)

174 Rene de Waal Malefyt from Balb/c into CB-17 SCID mice resulted in the spontaneous development of IBD. The disease could be prevented by coadministration of CD45RBlo cells (which contain regulatory T cells producing IL-10 and IL-4), treatment with anti-IFN or anti-TNF mAbs, or administration of IL-10 (Powrie et al., 1993a, 1994; Powrie, 1995). Interestingly, both CD45RBhi and CD45RBlo cells from IL-10ÿ/ÿ mice could induce IBD in Rag2ÿ/ÿ mice, suggesting that IL-10 is involved in the protective effects of CD45RBlo cells in normal mice (Rennick et al., 1997). In addition, IL-10ÿ/ÿ mice spontaneously develop enterocolitis (Kuhn et al., 1993). Rheumatoid Arthritis A possible beneficial role for IL-10 in pathogenesis of rheumatoid arthritis (RA) has been described. Production of IL-10 was demonstrated in sera, synovial fluid, and synovial explant cultures from RA patients (Katsikis et al., 1994; Llorente et al., 1994; Cush et al., 1995; al-Janadi et al., 1996; Bucht et al., 1996). IL-10 inhibited TNF, IL-1 , and IL-8 production by synovial macrophages and synoviocytes (Deleuran et al., 1994; Chomarat et al., 1995; Hart et al., 1995), and synergistic effects were observed in combination with IL-4 (Sugiyama et al., 1995). Endogenous IL-10 partly suppressed cytokine production from synovial cells, indicating that IL-10 in vivo may have a protective role (Katsikis et al., 1994). IL-10-producing cells in the joint include both synovial macrophages and T cells (Katsikis et al., 1994; Cohen et al., 1995). On the other hand, IL-10 production in RA has been linked to increased autoantibody production, serum factor, and B cell activation (Cush et al., 1995; Perez et al., 1995; al-Janadi et al., 1996). IL-10 is protective in animal models of RA since it reduced joint swelling, infiltration, cytokine production and cartilage degradation in collagen-induced arthritis and streptococcal cell wall-induced arthritis (Kasama et al., 1995; Persson et al., 1996; Tanaka et al., 1996; van Roon et al., 1996; Walmsley et al., 1996; Apparailly et al., 1998). IL-4 and IL-10 synergistically reduced joint inflammation in acute and chronic athritis models (Joosten et al., 1997). Role of IL-10 Models of Autoimmunity IL-10 has been tested in mouse and rat models of experimental autoimmune encephalomyelitis (EAE) which are in part representative of the pathology observed in multiple sclerosis in humans. Systemic administration of IL-10 during the initiation phase of

the disease suppressed disease induction, and reduced T cell proliferation to myelin basic protein and CNS infiltration (Rott et al., 1994). In addition, IL-10 prevented TNF-induced relapses in SJL mice that had partially or completely recovered from acute EAE. Furthermore, neutralization of endogenous IL-10 increased both the incidence and severity of SEB- or TNF-induced relapses, suggesting a protective role for IL-10 in the development of disease (Crisi et al., 1995). Elevations of IL-10 mRNA levels were observed in the recovery phase of acute EAE in mice and rats (Kennedy et al., 1992; Issazadeh et al., 1995), and a lack of IL-10 production was observed in chronic relapsing EAE (Issazadeh et al., 1996). IL-10 expression was detected in CSF mononuclear cells of multiple sclerosis patients (Navikas et al., 1995). IFN , which was successfully used to treat MS patients, induced expression of IL-10 in PBMCs of MS patients, indicating that IL-10 could potentially be useful for treatment of MS. IL-10 in Infectious Diseases A number of infectious diseases are characterized by a lack of cell-mediated immunity and hyporesponsiveness to the inducing pathogen. IL-10 contributes to immunosuppression observed in lepromatous leprosy, filariasis, visceral leishmaniasis, toxoplasmosis, listerisosis, schistosomiasis, Legionella pneumophila infection, trypanosoma infection, malaria, and HIV infection. Involvement in human infections is revealed in many studies by enhanced production of IL-10 by patients' PBMCs and the restoration of antigenspecific proliferative responses of patients' PBMCs in vitro by neutralizing anti-IL-10 mAbs (Sieling et al., 1993; Peyron et al., 1994; Carvalho, 1995; Mahanty and Nutman, 1995; Sieling and Modlin, 1995; King et al., 1996; Mahanty et al., 1996). The role of IL-10 in animal models of infection has also been clearly established. Neutralization of IL-10 leads to enhanced antigen-specific IFN production from CD4‡ T cells of mice infected with Leishmania major or Schistosoma mansoni, and in vivo use of IL10 or anti-IL-10 mAbs can convert susceptible strains into resistant or vice versa, respectively (Sher et al., 1991; Flores Powrie and Coffman, 1993; Villanueva et al., 1994, 1996; Reed et al., 1994). IL-10 acts in several ways to modulate immune responses to parasites, including inhibition of production of IFN , which is necessary to activate macrophages for intracellular killing, and of production of IL-12, which induces protective cellular immune responses. On the other hand, the complete absence of IL-10 in acute infections also results in pathology. Infection of IL-10ÿ/ÿ mice with Trypanosoma cruzi resulted in

IL-10 175 reduced parasitemia and enhanced IFN production (Abrahamsohn and Coffman, 1996). Furthermore, IL-10ÿ/ÿ mice infected with Toxoplasma gondii succumbed to the infection and had high levels of TNF, IFN , IL-1 , and IL-12 in serum and lung, implying an important role for endogenous IL-10 synthesis in downregulating monokine and IFN responses to acute intracellular infection, and preventing host immunopathology (Gazzinelli et al., 1996). These data imply a required balance in the production of IL-10, IL-12, and IFN which determines the outcome of infection.

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. No antibodies against IL-10 have been detected in single or multiple dose administration clinical trails.

Pharmacokinetics A randomized two-way crossover study was undertaken in 17 healthy volunteers in which recombinant human IL-10 was administered as 25 mg/kg s.c. and i.v. doses to study the pharmacokinetics and ex vivo leukocyte responses of recombinant human IL-10. rhIL-10 showed favorable PKPD characteristics, especially by the s.c. route of administration which produced prolonged suppression of cytokine production (ex vivo) (Radwanski et al., 1998).

Clinical results IL-10 has been tested in two phase I trials for its safety, tolerance, pharmacokinetics, and hematologic effects in healthy humans. IL-10 could be administered i.v. at doses up to 100 mg/kg with only mild to moderate flu-like symptoms at the highest doses while inhibiting production of IL-6, IL-1, and TNF in ex vivo stimulation of whole blood cells with LPS (Chernoff et al., 1995; Huhn et al., 1996). A randomized, double-blind, placebo-controlled, rising singledose study has been conducted to investigate the effects of recombinant human IL-10 in renal transplant patients who received OKT3 as induction therapy. Pretreatment with doses of up to 1 mg/kg rhIL-10 reduced the release of TNF induced by OKT3. However, higher doses might promote early sensitization to OKT3 and have reversible adverse effects on acceptance of the transplant (Wissing et al., 1997). An open label phase 2 trial of 10 psoriasis

patients indicated that the treatment for 7 weeks with 8 mg/kg daily or 20 mg/kg three times a week administered subcutaneously was well tolerated and possibly effective as a significant decrease of the psoriatic area and severity index were observed in 9/10 patients (Asadullah et al., 1998, 1999).

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