IL-11 James C. Keith Jr* The Genetics Institute, 1 Burtt Road, Andover, MA 01810, USA * corresponding author tel: 978-247-1372, fax: 978-247-1333, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.06004.
SUMMARY
BACKGROUND
IL-11, initially described in 1990 as a bone marrow stroma-derived hematopoietic cytokine, is a highly conserved, 178 amino acid cationic protein thought to exist as a four-helix bundle. The human and the nonhuman primate genes for IL-11 have been localized to the long arm of chromosome 19 at band 19q13.3q13.4. IL-11 is a member of the IL-6 ligand family since it utilizes gp130 as the signaling component of its receptor, and amino acid residues 59 (methionine), 41 (lysine), and 98 (lysine) are critical for the function of the IL-11 protein, being completely conserved in the mouse, nonhuman primate, and human. IL-11 has multiple activities that continue to be characterized. Initially, in vitro and in vivo studies demonstrated a hematopoietic activity, largely manifest as thrombopoiesis in humans. rhIL-11 has been developed and recently approved by the US Food and Drug Administration for use in the prevention of severe thrombocytopenia occurring after cancer chemotherapy. Studies of cells and tissues from other organ systems indicate that IL-11 is produced by a variety of other cell types and has activity in protection and restoration of the gastrointestinal mucosa, major effects as an immunomodulating agent, and activity in bone metabolism. Developmental investigations in mice indicate a widespread distribution of IL-11 expression in the embryo. Of great interest is the finding, from IL-11 receptor chain knockout mice, that IL-11 signaling is an absolute requirement for normal development of placentation and survival to birth.
Discovery Interleukin-11 (IL-11) was initially described in 1990 as a structurally unique, bone marrow stroma-derived hematopoietic cytokine. Subsequent research has demonstrated that it is a multifunctional cytokine, derived from many cell and tissue types.
Alternative names The chemical name for recombinant human interleukin-11 (rhIL-11) is oprelvekin. The rhIL-11 drug product, Neumega1, was approved and marketed in late 1997 for the prevention of severe thrombocytopenia caused by cancer chemotherapy.
Structure The mature IL-11 protein is a highly conserved, 178 amino acid cationic protein that is thought to exist as a four-helix bundle. The protein contains no cysteine residues, yet it is thermally stable. The mature human and murine proteins share 88% homology at the amino acid level, while the human and nonhuman primate proteins share 94% homology. Amino acid residues 59 (methionine), 41 (lysine), and 98 (lysine) are critical for function of the protein, and these residues are completely conserved in the mouse, nonhuman primate, and human proteins.
566 James C. Keith Jr
Main activities and pathophysiological roles IL-11 has multiple activities that continue to be characterized. Initially, in vitro and in vivo studies demonstrated a hematopoietic activity for IL-11, largely manifest as thrombopoiesis in humans, and rhIL-11 has been developed and recently approved by the US Food and Drug Administration for use in the prevention of severe thrombocytopenia occurring after cancer chemotherapy. Studies of cells and tissues from other organ systems indicate that IL-11 has: activity in protection and restoration of the gastrointestinal mucosa; major effects as an immunomodulating agent; and activity in bone metabolism. Developmental investigations in mice indicate a widespread distribution of IL-11 expression in the embryo, but of great interest is the finding that IL-11 signaling is an absolute requirement for normal development of placentation and survival to birth.
GENE AND GENE REGULATION
Accession numbers IL-11 was originally identified as an activity in conditioned medium from an IL-1 -stimulated primate bone marrow stromal cell line, PU-34, which supported long-term hematopoiesis in cell culture (Paul et al., 1990). The human cDNA, GenBank database accession number M37006, was cloned from a human fetal lung fibroblast cell line (MRC5) that was treated with phorbol 12-myristate 13-acetate (PMA) and IL-1 . The nonhuman primate IL-11 cDNA sequence is found at database accession number M37007, while the murine cDNA is found at UO3421 (Morris et al., 1996). The nucleotide sequence of the human IL-11 gene is seen in Figure 1 (from Schendel and Turner, 1998). It consists of five exons and four introns that contain 7 kbp of genomic DNA.
Figure 1 The nucleotide sequence of the human IL-11 gene. The 50 and 30 untranslated regions are underlined. From Schendel and Turner (1998).
IL-11 567
Chromosome location The human and the nonhuman primate genes for IL11 have been localized to the long arm of chromosome 19 at band 19q13.3-q13.4 (McKinley et al., 1992), while the murine IL-11 gene is found on chromosome 7 (Morris et al., 1996). A high degree of homology exists in this portion of murine chromosome 7 and human chromosome 19 (Stubbs et al., 1996). On human chromosome 19 the IL-11 gene is found close to the telomere, while on mouse chromosome 7 the topology is reversed as the gene is close to the centromere.
Relevant linkages In both the mouse and human genomes, the IL-11 gene is in close proximity to the gene that codes for the subunit of protein kinase C, as well as the genes for several zinc finger proteins (Stubbs et al., 1996).
Regulatory sites and corresponding transcription factors Binding sites for several transcription factors, including AP-1, SP-1, CTF/NF-1, and EF/C are found in the 50 region of the gene. Interferon-inducible and phorbol ester PMA-inducible elements are also
located in this region of the gene. Two polyadenylation sites exist at the 30 end of the gene, and several Alu repetitive sequences are also found within this region (Paul et al., 1990).
Cells and tissues that express the gene The IL-11 gene is expressed by a wide variety of cells and tissues throughout the body and its distribution has been reviewed previously (Du and Williams, 1997; Dorner et al., 1997; Schendel and Turner, 1998). The expression is relatively low as mRNA is only detected by RT-PCR in most tissue or cell types (Dorner et al., 1997). Table 1 lists the sites of IL-11 production (from Du and Williams, 1997).
PROTEIN
Accession numbers IL-11: 186273
Sequence The comparative 199 amino acid sequences of the murine, nonhuman primate, and human gene products are shown in Figure 2 (from Morris et al., 1996).
Figure 2 The amino acid sequences of the murine, nonhuman primate, and human IL-11 protein. The 21 amino acid leader sequence is underlined. Reprinted from Experimental Hematology 24, Morris et al. Molecular cloning and characterization of murine interleukin-11, 1369±1376,# 1996, with permission from Elsevier Science.
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Description of protein IL-11 is a 19 kDa, highly conserved, 199 amino acid precusor protein. The first 21 amino acids serve as a typical leader sequence for secreted proteins and are removed when the mature 178 amino acid protein is released from the cell. The protein contains no cysteine residues, but yet is thermally stable. The mature human and murine proteins share 88% homology at the amino acid level, while the human and nonhuman primate proteins share 94% homology. Amino acid residues 59 (methionine), 41 (lysine), and 98 (lysine) are critical for receptor binding of the protein, and these residues are completely conserved in the mouse, nonhuman primate, and human (Czupryn et al., 1995a). There are no sites for N-linked glycosylation and the naturally occurring protein has little, if any, carbohydrate modification. IL-11 is an unusually basic cytokine with a calculated isoelectric point of 11.7, due to the high content in proline (12%), leucine (23%), and positively charged amino acid residues (14%). The recombinant human protein is produced in Escherichia coli by genetic engineering and is 177 amino acids long, differing from the naturally occurring protein only by the absence of the N-terminal proline residue.
Figure 3 The hypothesized four-helix bundle structure of rhIL-11. (a) Helical regions are represented by rectangles drawn approximately to scale. Arrows indicate N- to C-terminal orientation of helices. (b) Helical wheel projections of 18 residues from each of the helices expected to form the protein core. Hydrophobic faces (shaded) consist mainly of leucine residues; hydrophilic faces contain all charged residues (in shaded circles). Reproduced with permission from Czupryn et al. (1995b).
Discussion of crystal structure Although the crystal structure of IL-11 has not been solved, the protein is thought to exist as a four-helix bundle as predicted by computer modeling based on the primary amino acid structure, and by the use of alanine-scanning mutagenesis of the mature protein (Figure 3) (from Czupryn et al., 1995a,b).
Important homologies Although IL-11 is considered a member of the IL-6 ligand family, since it utilizes gp130 as the signaling component of its receptor, the IL-11 gene shares no homology with IL-6 at the nucleotide level. Several regions of the 50 flanking sequence of the gene do contain small nucleotide sequences that share approximately 70% homology to other cytokines, such as IL-2, IL-3, IL-6, IL-9, and GM-CSF (McKinley et al., 1992).
Posttranslational modifications The cDNA for human IL-11 codes for a 199 amino acid polypeptide. Northern analysis reveals the
presence of two transcripts: a 2.5 kb mRNA and a minor 1.5 kb mRNA. Both transcripts encode the same IL-11 protein. The transcript size difference occurs because of alternative use of the two polyadenylation sites located within the 30 noncoding region of the gene, resulting in different lengths of the 30 noncoding region (Paul et al., 1990; McKinley et al., 1992). The initial 21 amino acids serve as the leader sequence, typical of secreted proteins. Although the mechanism is not well characterized, the leader
IL-11 569 sequence is removed as the IL-11 leaves the cell, so that the mature IL-11 protein is 178 amino acids. Since there are no N-linked glycosylation sites in the protein, IL-11 is not thought to have carbohydrate modification.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Many cell and tissue types throughout the body are capable of producing IL-11, dependent on the local environment of the cell or tissue (Table 1). Basal and inducible IL-11 mRNA expression can be detected in fibroblasts, epithelial cells, chondrocytes, synoviocytes, keratinocytes, endothelial cells, osteoblasts, and Table 1 Partial list of the sites of IL-11 production Tissue
Cell type/cell lines
CNS
Hippocampal neurons (H19-7) Spinal motor and sympathetic neurons Astrocytic glioblastomas (U373, U87)
Thymus
Myeloid cells? (T2)
Lung
Fibroblasts (MRC5, CCL202) Epithelial cells (9HTE, A549) Muscle cells
Bone
Fibroblasts (P-34, KM102) Osteosarcoma cell lines Osteoblasts
Connective tissues
Chondrocytes Synoviocytes Vein endothelial cells
Uterus
Fibroblasts Trophoblasts (TPA30-1) Endometriotic and endometrial tissues
Skin
Keratinocytes Melanoma cell lines Sarcoidosis (multinucleated giant cells)
Testis
Round spermatids
Adapted from Du and Williams (1997).
certain tumor cells and cell lines (Du and Williams, 1997; Dorner et al., 1997; Schendel and Turner, 1998).
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Agents known to induce IL-11 mRNA expression and protein include: IL-1 , TGF , parathyroid hormone, retinoic acid, histamine, PMA, or infection with a variety of respiratory viruses, including respiratory syncytial virus (RSV ) (reviewed in Du and Williams, 1997; Dorner et al., 1997). Transcriptional induction of multiple cytokines by human RSV requires activation of NFB and is inhibited by sodium salicylate and aspirin (Bitko et al., 1997). Infection of the lung epithelial cell line A549 by RSV resulted in the elevated synthesis of IL-11 mRNA, and nuclear run-on assays showed a direct effect of RSV on IL-11 gene transcription. Mutational analysis of the IL-11 promoter identified a region 720 nucleotides upstream of the mRNA start site in the transcriptional induction of IL-11 by RSV. In this region, 10-nucleotide-long sequences GGGGTCTCCC and GGGTCTCCCC were identified that resembled the NFB consensus motif. Mutation of either sequence decreased RSV-evoked induction of IL-11 promoter activity. NFB sites in IL-1 , IL-6, and IL-8 promoters are also required for RSV-mediated induction of transcription of these promoters. Sodium salicylate and aspirin, inhibitors of NFB activation, abolished transcriptional induction of all these cytokines by RSV. Recently, Mino et al. (1998) studied the combined effect of IL-1 and TNF , major cytokines in the rheumatoid synovium, on the production of IL-11 by cultured rheumatoid synovial fibroblasts (RSFs). IL1 and TNF synergistically induced phospholipase A2 (cPLA2) and COX-2, and hence prostaglandin E2 (PGE2), that stimulated RSF production of IL-11 mRNA and protein. The effect was completely inhibited by indomethacin, but inhibition was prevented by exogenous PGE2. Induction of IL-11 by IL-1 and TNF was inhibited by a potent inhibitor of all isoforms of protein kinase C (PKC), GF109203X, but PMA, which induced a downregulation of all PKC isoforms except atypical PKC, did not block the induction of IL-11. Fresh synovial cells from rheumatoid arthritis patients (Taki et al., 1998) produced high levels of IL-11. Indomethacin inhibited the production of IL-11 by about 55%, while dexamethasone inhibited the production of IL-11 by more than 80%.
570 James C. Keith Jr Exogenous PGE2 completely prevented the inhibition by indomethacin, but the inhibition caused by dexamethazone was not affected by PGE2. IFN inhibited the production of IL-11 from IL-1 stimulated cultured RSFs, but not the production of IL-11 by fresh synovial cells. Therefore, production of IL-11 by rheumatoid synovia was differentially regulated by PGE2 and IFN , and treatment with indomethacin or dexamethasone decreased the level of IL-11 in cells derived from inflamed joints in patients with rheumatoid arthritis. Immunoreactive IL-11 (0.08±24.65 ng/mL) was detected in the cultures of synovial membrane tissue obtained from rheumatoid arthritis patients who underwent synovectomy or joint replacement surgery (Hermann et al., 1998). Inhibition of IL-11 activity in synovial membrane mononuclear cell cultures with neutralizing antibody resulted in a 2-fold increase in TNF production, and inhibition of IL-10 with neutralizing antibody resulted in a 3-fold increase in TNF production. However, inhibition of both IL-10 and IL-11 activities resulted in a greater increase in TNF production (23-fold) than blocking either cytokine alone (Figure 4). INF downregulates IL-1-induced IL-11 in human bone marrow stromal cultures (Aman et al., 1996). Using northern blots, expression of IL-11 mRNA was reduced in the presence of INF , and the reduction was prevented by the addition of cycloheximide. A dose-dependent inhibition of IL-1-stimulated IL-11
Figure 4 The effects of endogenous cytokine inhibition, by neutralizing antibodies to IL-10 and IL-11, on TNF production can be seen in synovial membrane mononuclear cell cultures from rheumatoid arthritis patients. Neutralization of both IL-10 and IL-11 results in a marked elevation of TNF . Reproduced with permission from Hermann et al. (1998).
protein production by INF began at 10 U/mL as measured in stromal cell supernatants by ELISA. The role of second messenger molecules in parathyroid hormone (PTH)- and IL-1-induced IL-6 and IL-11 production has been studied in human bone marrow stromal cells (Kim et al., 1997). rhPTH(1-34) and rhIL-1 dose-dependently stimulated IL-6 and IL-11 production from human bone marrow stromal cells (hBMSCs). Agonists for protein kinase A (PKA) (forskolin), and protein kinase C (PKC) (PMA) also stimulated IL-6/IL-11 production. Stable isomers of cAMP that are inhibitors of PKA significantly inhibited PTH-stimulated IL-6/IL-11 production, but did not inhibit IL-1-stimulated IL-6/IL-11 production. Inhibitors of PKC suppressed IL-1-stimulated, but not PTH-stimulated, IL-6/IL-11 production. Estrogen antagonized IL-1-stimulated IL-6 production, but PTH-stimulated IL-6 production and IL-1- and PTHstimulated IL-11 production were not affected. Similar inhibition of PMA-stimulated IL-6 production, but not forskolin-stimulated IL-6/IL-11 production or PMA-stimulated IL-11 production, was produced by estrogen. Regulation of cytokine production by estrogen in hBMSCs was selective, in that only the IL-1-induced IL-6 production mediated by the PKC pathway was inhibited, but PTH-induced IL-6 production and PTH/IL-1-induced IL-11 production were not inhibited by estrogen. Human fibroblasts produce osteoclastogenesis inhibitory factor (OCIF) that specifically inhibits osteoclastogenesis (Tsuda et al., 1997). OCIF inhibited in a dose-dependent manner osteoclastogenesis elicited through three distinct signaling pathways stimulated by 1 ,25-dihydroxyvitamin D3, PTH, and IL-11, in a dose range of 1±40 ng/mL (IC50=4± 6 ng/mL). TGF induces both IL-11 and OCIF (Takai et al., 1998).
RECEPTOR UTILIZATION Although IL-11 utilizes the gp130 molecule as the signaling component of its receptor, the specific effects of this ligand are conferred through the use of a unique IL-11 chain receptor that must interact with the ligand and the gp130 complex.
IN VITRO ACTIVITIES
In vitro findings Hematopoiesis/ThromboPoiesis/Lymphocyte Proliferation rhIL-11, in synergy with either IL-3, IL-4, IL-6, or SCF supports murine primitive hematopoietic cell
IL-11 571 development (reviewed in Goldman, 1995), and Leary et al. (1992) have also found that rhIL-11 acts synergistically with either IL-3 or SCF in supporting human blast cell colony formation. Cultures of early hematopoietic cells from mice, monkeys, and humans have again demonstrated rhIL-11's synergistic activity with IL-11. Increased maturation of late human megakaryocyte progenitors and of megakaryocytes from gpIIb-IIIa (CD41) bone marrow cells was produced by rhIL-11, revealing that the isolated megakaryocytes were larger and had higher DNA ploidy (Bruno et al., 1991; Teramura et al., 1992). Therefore, in human cells rhIL-11 regulates megakaryocytopoiesis at several maturation levels, from the earliest recognizable megakaryocyte progenitor to the level of the nondividing megakaryocyte. Erythroid and monocyte lineages may also be affected. No mitogenic activity of primary murine thymocytes or of human peripheral T cells was caused by rhIL-11, nor has rhIL-11 supported T cell differentiation in culture. These results are in contrast to the effects of IL-6, which is known to be an important regulator of T cell activation and differentiation. Hepatocytes Protein synthesis by hepatocytes during the acute phase response is influenced by various cytokines, including IL-1, IL-6, and TNF . rhIL-11 can induce production of the same acute phase proteins as IL-6 (Baumann and Schendel, 1991). In rat hepatoma, H-35 cells, rhIL-11 increased production of fibrinogen, 1-antitrypsin, and 2-macroglobulin, and the effect was enhanced by dexamethasone. The increased production of the IL-1-regulated proteins, including complement C3, haptoglobin, and hemopexin, also occurred, but rhIL-11 had no effect on albumin synthesis. rhIL-11 appeared to be less potent than IL-6 in inducing acute phase protein synthesis. rhIL11 also increased the level of microsomal heme oxygenase, the rate-limiting enzyme in heme catabolism, in HepG2 cells (Fukuda and Sassa, 1993). The production of heme oxygenase may contribute to the immunomodulatory effects since it appears to protect tissues against oxidative stress (Willis et al., 1996). Adipocytes The effects of rhIL-11 on production of adipocytes were initially demonstrated by Kawashima et al. (1991) as inhibition of adipocyte differentiation mediated through inhibition of lipoprotein lipase. Other cytokines, including IL-6 and TNF , have similar activities.
Bone Metabolism rhIL-11 also acts on osteoclasts, another cell type commonly believed to be derived from the monocyte lineage. However, the in vitro effects appear to be different from those seen with therapeutic administration of rhIL-11 in in vivo. IL-1, IL-6, IL-11, and TNF can stimulate osteoclast development and affect bone resorption (reviewed in Manolagas, 1995). Increased IL-6 has been implicated in excessive osteoclastic bone resorption, typically associated with loss of either ovarian or testicular function. It is unclear whether IL-6 is the sole pathogenetic factor or whether IL-1, TNF , and IL-11 may also be involved, but the latter three cytokines do not appear to be directly regulated by sex steroids. P. A. Hill et al. (1998) used in vitro model systems of bone resorption to study the effects of IL-11 on osteoclasts and osteoblasts. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10ÿ10 M, approximately the same activity as 1,25-dihydroxyvitamin D3. IL-11 also caused a dose-dependent increase in osteoblast-mediated type I collagen degradation. The effect was less than that produced by D3 and was prevented by an inhibitor of matrix metalloproteinases (MMPs), but not those blocking arachidonic acid metabolism. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts, but it did dose-dependently increase osteoclast-like multinucleate cells' lacunar resorption, even though the effects were less than that of 1,25dihydroxyvitamin D3. Resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. Thus, the in vitro results suggest that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Matrix metalloproteinases and products of arachidonic acid metabolism are also involved. IL-11 is produced by human bone-derived endothelial cells (BDECs) in response to IL-1 and TNF and its role in the formation of osteolytic bone metastasis has been postulated based on in vitro studies (Zhang et al, 1998). Established cultures of BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting IL-11 when stimulated by several inflammatory cytokines. The production of IL-11 in BDECs was caused by conditioned medium from human melanoma A375M cell cultures. Since A375M cells formed osteolytic bone metastasis in vivo, the authors speculated that BDECs could contribute
572 James C. Keith Jr to pathological osteolysis by producing IL-11. Thus, endothelial cells in bone may be important in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions. IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast (OCL) formation was examined using a coculture of mouse osteoblasts and bone marrow cells (Romas et al., 1996). IL-1, TNF , PGE2, PTH, and 1 ,25-dihydroxyvitamin induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R ) and gp130. In cocultures, multinucleated osteoclastlike cell formation in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dosedependently inhibited by rat monoclonal anti-mouse gp130 antibody. Anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 ,25dehydroxyvitamin D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R , as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R mRNA, both bone-forming and boneresorbing cells are potential targets of IL-11. With age, bone marrow adipose tissue increases as bone turnover declines. The P6 strain of senescence-accelerated mice (SAM) have an early decrease in bone mass and a reduction in bone remodeling. Decreased osteoblastogenesis and osteoclastogenesis and enhanced adipogenesis are seen in the bone marrow. Since IL-11 can inhibit adipogenesis and stimulate osteoclast formation, the effect of IL-11 on bone marrow cells from SAM was studied (Kodama et al., 1998). Reduced production of osteoblasts and osteoclasts was restored and enhanced formation of adipocytes was decreased by 10 pM rhIL-11, while IL-6 and leukemia inhibitory factor (LIF) did not produce these effects. This group went on to analyze the entire coding region of IL-11 cDNA from SAMP6 bone marrow stromal cells, but they found no mutations. However, constitutively produced IL-11 protein, and its TGF -evoked mRNA expression were lower in bone marrow stromal cells cultures from SAMP6 than from control mice. Thus, the expression of IL-11 was reduced in bone marrow cells of SAMP6, suggesting that a reduction in IL-11 synthesis impairs both osteoblastogenesis and osteoclastogenesis. These data suggest
that decreased IL-11 levels or activity may be associated with the age-related impairment of bone metabolism. Macrophages rhIL-11 has direct effects in reducing proinflammatory mediator production by LPS-activated macrophages (Trepicchio et al., 1996). Pretreatment of murine peritoneal macrophages with 10 or 100 ng/mL rhIL-11 resulted in 50±90% inhibition of LPS-induced TNF , IL-6, IL-1 , IL-12 p40, and nitric oxide production in the conditioned medium, when assessed by protein or nitrate levels, respectively. Pretreatment of primary murine alveolar macrophages with 100 ng/mL rhIL11 also caused a 65% reduction in LPS-stimulated TNF production (Redlich et al., 1996). However, rhIL-11 did not render the macrophage insensitive to LPS stimulation since rhIL-11 did not inhibit LPSinduced cell surface expression of ICAM-1, MHC class II molecules, B7.2 antigen, or the LPS-binding protein receptor CD14 (Trepicchio et al., 1996). Treatment with rhIL-11 also did not alter the levels of IL-10 or TGF in the conditioned medium as assessed by ELISA or bioassay. These results indicate that rhIL-11 can act directly on activated macrophages to reduce the production of proinflammatory mediators including TNF . This activity was not mediated by inducing increased expression of either TGF or IL-10. IL-11 inhibits human macrophage IL-12 production (Leng and Elias, 1997). IL-11 did not alter the IL-12 (p70) production of unstimulated THP1 monocytic cells or human blood monocytes. It did, however, inhibit, in a dose-dependent fashion, the IL-12 production of IFN Staphylococcus aureus Cowan strain 1-stimulated THP1 cells and stimulated blood monocytes. Inhibition of IL-12 protein production was associated with a proportionate decrease in IL-12 p35 and p40 mRNA accumulation. Nuclear run-on assays revealed comparable decreases in IL-12 p35 and p40 gene transcription. IL-11 did not similarly regulate monocyte/macrophage production of IL-8 or MIP-1 , and IL-6 did not similarly inhibit IL-12 elaboration. These studies demonstrate that IL-11 is a potent inhibitor of monocyte/macrophage IL-12 production and that this inhibitory effect is, at least in part, transcriptionally mediated. Analysis of rhIL-11 effects on transcription factors that activate proinflammatory cytokines revealed that the level of LPS-induced NFB binding activity in the nucleus of rhIL-11-treated peritoneal macrophages was significantly reduced (Trepicchio et al., 1997). The block to NFB nuclear translocation was related to the ability of rhIL-11 to maintain or increase
IL-11 573 protein levels of the inhibitors of NFB, IB- , and IB- following LPS treatment. Treatment of LPSstimulated macrophages resulted in significant elevation of IB- and IB- mRNA levels. These results suggest that the anti-inflammatory activity of rhIL-11 is mediated in part by inhibition of NFB-dependent transcriptional activation and demonstrate for the first time the regulation of IB- by an anti-inflammatory cytokine.
IL-11 and IL-6 and not to other human cytokines using the same gp130 transducer chain or to other human interleukins. IL-11-induced proliferation of B9-11 cells was unaffected by anti-murine IL-6 receptor monoclonal antibody but was inhibited by anti-gp130 monoclonal antibody. Half-maximal proliferation of B9-11 cells occurred with 30 pg/mL of rIL-11. Commercially produced ELISA kits are also available for measurement of IL-11 in human and murine samples.
Intestinal Epithelial Cells Treatment of intestinal epithelial IEC-6 cells in culture with rhIL-11 (1±100 ng/mL) has been shown to reduce the cellular growth rate (Booth and Potten, 1995; Peterson et al., 1996). This direct antiproliferative effect of rhIL-11 on the IEC-6 cell line was not mediated through induction of endogenous TGF 1 production, and was associated with transient cell cycle arrest, mediated through inhibition of pRb phosphorylation. Tumor Cells rhIL-11 did not alter the growth of the human colonic epithelial transformed cell lines SW620 and HT-29 (Booth and Potten, 1995). Nor did rhIL-11 have any effect on the proliferation of three human melanoma cell lines derived from primary lesions or four lines derived from metastatic tumors (Paglia et al., 1995). The response of rhIL-11-treated tumor cells exposed to cytotoxic therapies has been evaluated (Teicher et al., 1996). rhIL-11 treatment did not alter the response of HT-29 cells to radiation in culture or to 5-fluorouracil treatment in vivo or ex vivo. rhIL-11 treatment did not alter the ex vivo survival of EMT-6 mammary carcinoma cells following treatment with melphalan or cyclophosphamide. In in vivo tumor growth delay experiments, rhIL-11 treatment did not adversely affect treatment with melphalan, thiotepa, cyclophosphamide, or carboplatin. The data from these studies indicate that for tumor cells of nonhematopoietic origin, rhIL-11 does not stimulate proliferation or alter the tumor response to cytotoxic cancer therapies when assessed in vitro, in vivo, or ex vivo.
Bioassays used Levels of IL-11 activity and protein can be quantified by the use of the B9, B9-11, and T10 bioassays (Quesniaux et al., 1993; Lu et al., 1994; Yang, 1993). A subclone (B9-11) from the B9 hybridoma was used to develop a sensitive bioassay for human IL-11 (Lu et al., 1994). B9-11 cells responded only to human
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Limited knowledge concerning the exact physiologic roles of IL-11 is available since the ligand-null mouse has yet to be produced. However as reviewed in the preceding section, production of IL-11 appears to be evoked by several proinflammatory mediators. When this fact is combined with the inhibitory activities produced by exogenous rhIL-11 or the synergistic antiinflammatory activity of endogenous IL-11 with IL-10 (Hermann et al., 1998), a strong rationale for immunomodulating effects of IL-11 can be constructed.
Species differences Daily in vivo administration of rhIL-11 elevated the number of circulating neutrophils and platelets and increased the number of megakaryocytes in normal mice (Quesniaux et al., 1993). However, in nonrodent species only the thrombopoietic activities of rhIL-11 have been reported (Goldman, 1995).
Knockout mouse phenotypes The targeted disruption of the IL-11 gene has not been performed to date. See the IL-11 receptor chapter for a discussion of the IL-11 chain receptor knockout mouse.
Transgenic overexpression The human IL-11 gene has been expressed in the lung, and targeted continuous expression of IL-11 in the murine airway causes an inflammatory response and
574 James C. Keith Jr airway obstruction (Tang et al., 1996). The Clara cell 10 kDa protein promoter was used to express human IL-11 in the airways. In contrast to transgene (ÿ) littermates, the airways of IL-11 transgene () animals lesions observed included nodular peribronchiolar mononuclear cell infiltrates and bronchiolar remodeling with subepithelial fibrosis. The infiltrates contained large numbers of B220 cells and MHC class II cells and lesser numbers of CD3 cells, CD4 cells, and CD8 cells. The areas of fibrosis contained types III and I collagen, increased numbers of smooth muscle actin- and desmin-containing cells, and the stromal cells included fibroblasts, myofibroblasts, and smooth muscle cells. Studies of airway function demonstrated that 2-month-old transgene () mice had increased airway resistance and nonspecific airway hyperresponsiveness to methacholine when compared with their transgene (ÿ) littermates. The fibrosis seen may have been related to the continued production of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) since IL-11 is known to induce TIMP-1 (Maier et al., 1993; Hermann et al., 1998). Regulated overexpression of IL-11 in the lung was then employed to differentiate development-dependent and -independent phenotypes (Ray et al., 1997). The Clara cell 10 kDa protein (CC10) promoter and the reverse tetracycline transactivator (rtTA) were used to create a lung-specific, regulatable, overexpression transgenic system. This system was used to express human IL-11 in murine respiratory structures at various times. Gene activation could be evoked in utero, in neonates, and in adult animals, and then expression could be turned off by removal of the inducing stimulus. When gene activation was initiated in utero and continued into adulthood, subepithelial airway fibrosis, peribronchiolar mononuclear nodules, and alveolar enlargement (emphysema) were typical histologic findings. Evoked transgene expression in the mature lung caused airway remodeling and peribronchiolar nodules, but no alveolar enlargement. However, induction in utero and during the first 2 weeks after birth caused alveolar enlargement without airway remodeling or peribronchiolar nodules. The IL-11 overexpression-induced abnormalities appeared to be dependent (large alveoli) and independent (airway remodeling, peribronchiolar nodules) of lung growth and development. Targeted lung expression of human IL-11 enhanced murine tolerance of 100% oxygen and decreased hyperoxia-induced DNA fragmentation (Waxman et al., 1998). IL-11 markedly increased survival time in 100% oxygen with 100% of transgene (ÿ) animals dying within 72±96 hours and > 90% of transgene () animals living for more than 10 days. Protection was
associated with significant decreases in alveolarcapillary protein leak, reduced endothelial and epithelial membrane injury, lipid peroxidation, and less pulmonary neutrophil recruitment. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene () as compared with (ÿ) mice after 100% oxygen exposure, and IL-11 reduced hyperoxiainduced expression of IL-1 and TNF . Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (ÿ) animals, and IL-11 reduced this cell death response.
Pharmacological effects Subcutaneous treatment with rhIL-11 increases peripheral platelet counts when administered in vivo as a single agent to rodents, dogs, and nonhuman primates. The major effect of in vivo rhIL-11 administration to normal animals was stimulation of multiple phases of megakaryocytopoiesis and stimulation of thrombopoiesis. IL-11-treated megakaryocytes, while exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation (Philipp et al., 1998). Platelets from rhIL-11-treated nonhuman primates were functionally and ultrastructurally normal, supporting the contention that the physiologic process of platelet production was stimulated by rhIL-11 treatment (Dorner et al., 1997; Schlerman et al., 1996; Kaviani et al., 1996).
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects Information regarding the regulation of IL-11 expression in vivo in humans is limited. Under normal conditions, circulating serum levels of IL-11 are very low. This may be related, in part, to the unique and marked cationic nature of the protein that can predispose its binding with negatively charged surfaces throughout the body. IL-11 has been detected in the ovarian follicular fluid of women treated with gonadotropins in the setting of in vitro fertilization (Branisteau et al., 1997). However, the protein was not detected in the patients' sera.
IL-11 575
Role in experiments of nature and disease states IL-11 plasma levels are significantly elevated in patients with severe thrombocytopenia secondary to myeloablative therapy or in patients with immune thrombocytopenia (Chang et al., 1996). Platelet counts and endogenous IL-11 levels following myeloablative therapy were inversely related. In the same study, circulating IL-6 levels were not elevated in thrombocytopenic patients, decreasing the possibility that elevated IL-11 levels were due to a global induction of the family of gp130-interacting cytokines. Therefore, it is suggested that induction of IL-11 expression is a physiological response to thrombocytopenia in humans and that IL-11 plays a role in the regulation of megakaryocytopoiesis and thrombopoiesis during acute thrombocytopenia. Low, but detectable, levels of IL-11 (20 pg/mL) have been reported in a significant portion of patients with disseminated intravascular coagulation with or without sepsis (Endo et al.,1996). Recently, measurable serum levels of IL-11 have also been seen in rheumatoid arthritis patients (De Benedetti et al., 1997; Hermann et al., 1998). IL-11 has been found in seminal plasma and is elevated in seminal plasma of infertile patients with urogenital infection (Matalliotakis et al., 1998). The group normal spermiogram had a mean value for IL-11 of 51.83 pg/mL, while the abnormal spermiogram was 66.30 pg/mL. Immunoreactive IL-11 has also been reported in aseptic loosening of total hip replacement implants (Xu et al., 1998). Chronic inflammatory response to abrasion particles from total hip replacement (THR) implants is believed to cause osteolysis and to contribute to prosthetic loosening. The expression of IL-11 and its major cellular sources in the interface and pseudocapsular tissues obtained from total hip revisions performed for aseptic loosening were investigated. IL-11 was found in the interface and pseudocapsular tissues. IL-11-containing cells were more numerous in the interface (760 171 cells) and pseudocapsular tissues (684 171 cells) than in the control synovial tissue (235 68 cells). This is not an unexpected finding since inflammatory mediatorinduction of IL-11 has been previously discussed. IL-11 production is increased in organ cultures of lesional skin of patients with active plaque-type psoriasis as compared with nonlesional and normal skin (Ameglio et al., 1997). Levels of IL-11 and three other proinflammatory cytokines (IL-1 , IL-6, and IL-8) were determined in supernatants of skin from lesional and nonlesional psoriatic skin and in supernatants of biopsies from normal volunteers, using
commercially available ELISA kits. The amounts of IL-11 and the other three modulators were increased in lesional areas, and the levels of IL-11 were also correlated with the disease severity index. The synovial expression and serum levels of IL-6, IL-11, LIF, and oncostatin M (OSM) were determined in rheumatoid arthritis patients by RT-PCR and ELISA, respectively (Okamoto et al., 1997). Cells from the synovium of rheumatoid arthritis patients expressed mRNA for IL-6, IL-11, LIF, and OSM at higher levels than did synovial cells from osteoarthritis patients, and spontaneously released greater quantities of these proteins in culture. Fibroblast cell lines derived from rheumatoid arthritis synovium were able to produce IL-6, IL-11, and LIF, but not OSM, when stimulated with IL-1 and TNF . However, in this study only IL-6 was significantly elevated in the serum of rheumatoid arthritis patients and correlated with the serum CRP level, while other IL-6-type cytokines were not detected. Human renal cell carcinomas (RCC) produce IL-6, IL-10, IL-11, and TGF 1 in primary cultures and modulate T lymphocyte blast transformation (Knoefel et al., 1997). Immunomodulation is a common feature of RCC and in vitro these tumors produce IL-10 up to 20 ng/mL, IL-6 up to 35 mg/mL (> 250 kU/mL in the B9 system), IL-11 up to 15 mg/mL, and TGF 1 up to 22 ng/mL.
IN THERAPY
Preclinical ± How does it affect disease models in animals? Thrombopoietic Pharmacology rhIL-11 has been evaluated in murine models of chemotherapy- and/or irradiation-induced myelosuppression and in murine models of bone marrow transplantation (Goldman, 1995; Dorner et al., 1997). Although administration of rhIL-11 as a single agent to normal animals primarily stimulated megakaryocytopoiesis and thrombopoiesis, in myelosuppressed animals, rhIL-11 stimulated multilineage recovery of hematopoietic progenitor cells in many murine models. Multilineage stimulation of both bone marrow and splenic hematopoietic progenitors was seen in the peripheral circulation as significant improvements in the platelet nadir and in the red blood cell nadir, accompanied by a shorter period of thrombocytopenia and anemia. rhIL-11 treatment or retroviral-mediated expression of hIL-11 or mIL-11 consistently accelerated the recovery of platelets following transplantation in all models of bone marrow transplantation.
576 James C. Keith Jr In some models, IL-11 enhanced neutrophil and red blood cell recovery as well. Therapy with rhIL-11 was studied in carboplatin-induced myelosuppression in cynomolgus monkeys (Schlerman et al., 1996). Once daily, subcutaneous therapy with rhIL-11 beginning the day following chemotherapy, significantly improved platelet nadirs and recovery in this model of myelosuppression.
Figure 5 The survival curve for hamsters with 5fluorouracil-induced oral mucositis. All doses of rhIL11 increased animal survival. Reprinted from Oral Oncol., Eur. J. Cancer, 31B, Sonis et al. Alteration in the frequency, severity and duration of chemotherapyinduced mucositis in hamsters by interleukin-11, 261± 266, # 1995, with permission from Elsevier Science.
Gastrointestinal Mucositis Increased survival of rhIL-11-treated mice that had been subjected to severe cytoablative therapy has been reported (Du et al., 1994). In this model, mice received 5-fluorouracil (5-FU) followed 3 days later by sublethal whole-body irradiation. Extensive damage to the small intestinal mucosa resulted and was fatal for all vehicle-treated animals. Death was due to sepsis associated with gastrointestinal mucosal damage. Treatment with rhIL-11 (250 mg/kg/day) s.c., beginning on the day of irradiation, increased survival to 85% and was associated with a rapid recovery of small intestine. There was a reduction of bacterial foci in multiple organs, indicating that rhIL-11 treatment improved or maintained the integrity of the gastrointestinal epithelium, preventing bacterial translocation and subsequent sepsis. Further study of rhIL-11 treatment in this model demonstrated that rhIL-11 suppressed apoptosis in the small intestine following cytoablative therapy and increased villus length (Orazi et al., 1996). Therapy with rhIL-11 was also associated with increased mitosis in crypt cells. Thus, rhIL-11 has effects on crypt cell proliferation and apoptosis which facilitates mucosal recovery following insult. rhIL-11 reduced oral mucositis in a 5-FU hamster model (Sonis et al., 1995, 1997). Animals typically experience severe mucositis 7±10 days after 5-FU administration. Peak mucositis occurred by day 10 in vehicle-treated animals. rhIL-11 decreased the frequency, severity, and duration of mucositis in a dosedependent manner (Figure 5) and increased animal survival at all doses tested. Oral mucosal tissue damage was reduced when rhIL-11 was administered subcutaneously at the time of chemotherapy as well as when rhIL-11 administration was delayed until chemotherapy was complete. The efficacy of rhIL11 in this model appeared to be due to prevention of mucosal damage as well as to acceleration of healing. Combined with the data from Booth and Potten (1995) and Peterson et al.(1996), these results suggest that in models of mucositis using cell cycle-specific cytoablative agents, the activity of rhIL-11 may be due in part to inhibition of cell cycle progression in intestinal epithelial cells.
Radiation-induced damage to the murine small intestinal crypt clonogenic stem cells has been reduced by rhIL-11 (Potten, 1995). Mice receiving subcutaneous rhIL-11 before, or before and after, wholebody radiation exposure had increased numbers of surviving crypts. Crypt survival was increased 4-fold at the highest radiation dose. Since the regenerative capacity of the intestinal crypt stem cells following injury determines the survival of crypts, these results suggest another mechanism by which rhIL-11 can maintain mucosal integrity. Radiation Pneumonitis Administration of IL-11 was also shown to enhance survival in a murine model of radiation-induced pulmonary injury (radiation pneumonitis) in which TNF is involved in lung damage (Redlich et al., 1996). The reduced injury in rhIL-11-treated animals correlated with a decrease in TNF mRNA in lung tissue. The radioprotection was apparently specific for normal thoracic structures since rhIL-11 did not affect the development or radiosensitivity of EMT6 tumor cell lung metastases. Graft-versus-Host Disease An in vivo study was performed to investigate the pharmacologic properties of rhIL-11 in the setting of allogeneic bone marrow transplantation (BMT) (G.R. Hill et al., 1998). Female C57Bl/6 mice, 11±15
IL-11 577 weeks old, served as donors for allogeneic transplant to 11±15-week-old B6D2F1 mice across MHC and minor H antigen barriers. Some groups of mice received allogeneic BMT (5,000,000 bone marrow cells and 500,000 T cells) after 1500 cGy of wholebody irradiation. Mice received rhIL-11 (500 mg/kg s.c. divided by twice daily doses) or its diluent from day ÿ2 to 7 or 14. Graft-versus-host disease (GVHD) was severe, with mortality beginning on day 4. Treatment with rhIL-11 significantly decreased lethal GVHD (day 50 survival: 90 versus 20%, p < 0.001) when compared with treatment with diluent. Survival in syngeneic BMT recipients was 88% at day 45. In 9- to 11-week-old mice receiving syngeneic BMT, rhIL-11 therapy in the same regimen resulted in 100% long-term survival at 90 days versus 60% survival in diluent-treated animals. In separate experiments where mice received allogeneic BMT with a lower whole-body irradiation dose (1300 cGy) and more T cells (2,000,000), the same rhIL-11 therapy described previously increased survival from 35 to 70% at day 70. With rhIL-11 therapy, donor T cell cytokine responses to host antigen following BMT were polarized toward TH2 with a 10-fold increase in IL-4 occurring as 50% decreases in IFN and IL-2 were observed. Serum IFN levels were reduced, and decreased IL-12 production was present in mixed lymphocyte cultures. Histologic evaluation of the small bowel, large bowel, and liver at day 45 revealed that the total GVHD score was reduced from 27 3.2 in allogeneic control animals to 16.1 1.5 in rhIL-11-treated mice ( p < 0.02). The GVHD score in syngeneic recipients was 13.3 2, different from allogeneic animals ( p < 0.05) but not different from rhIL-11-treated mice. The therapy with rhIL-11 prevented small intestinal damage ( p < 0.01 versus allogeneic controls), and serum endotoxin levels were reduced by 80%. The serum TNF levels were lowered, and rhIL-11 decreased LPS-stimulated release of TNF from macrophages in vitro. These data indicate that therapy with rhIL-11 reduced mortality and morbidity following allogeneic BMT by polarizing T cells, protecting the small intestine and suppressing proinflammatory cytokine release. Models of Inflammatory Bowel Disease HLA-B27 transgenic Fischer 344 rats, expressing HLA-B27 and 2-microglobulin genes, exhibit lesions of the gastrointestinal system which are similar to those of Crohn's disease. Treatment with rhIL-11 decreased the clinical signs and histologic lesion scores of inflammatory bowel disease in these rats, either eliminating diarrhea or resulting in completely normal
Figure 6 The results of RT-PCR analysis of colonic tissue from normal Fischer 344 rats or HLA-B27 transgenic rats treated with vehicle or rhIL-11 twice weekly. Several proinflammatory mRNAs were decreased in the HLA-B27 rats with rhIL-11 treatment. From Peterson et al. (1998).
stool character (Keith et al., 1994; Peterson et al., 1998). RT-PCR analysis of colonic RNA revealed that treatment with rhIL-11 downregulated expression of proinflammatory cytokines (Figure 6) including TNF , IL-1 , and IFN . rhIL-11 also reduced the level of myeloperoxidase activity in the cecum, indicating reduced inflammation. After stimulation in vitro with anti-CD3 antibody, spleen cell cultures derived from rhIL-11-treated rats produced less IFN , TNF , and IL-2 than cultures derived from vehicle-treated rats. These findings suggest that rhIL11 acts to reduce inflammation through modulation of multiple proinflammatory mediators including products of activated T cells. The effects of rhIL-11 on acute colonic injury were assessed in acetic acid-induced colitis in rats (Keith et al., 1994). Animals treated with acetic acid usually develop diffuse colonic lesions, characterized by ulceration, hemorrhage, edema, depletion of goblet cells, and infiltration of leukocytes. The gross and histologic lesions of rhIL-11-treated animals were significantly reduced compared with vehicle control animals. Administration of rhIL-11 (10±1000 mg/kg s.c.) for only the first 5 days following acetic acid injury was as effective as administration for 14 days after injury (Figure 7). rhIL-11 was also protective or therapeutic in intracolonic administration of trinitrobenzene sulfonic acid (TNBS) colitis in rats (Qiu et al., 1996). Dosedependent reductions in colonic ulcer indexes of rhIL-11-treated rats were achieved. Myeloperoxidase
578 James C. Keith Jr Figure 7 The reduction (%) in colonic histologic lesions relative to vehicle-treated rats caused by different doses and schedules of rhIL-11 14 days after acetic acid injury of the colon. Each treatment group and associated vehicle group contained 10 animals. Data are shown as means SD. Note the lack of a dose response for the 5-day treatment schedule.
activity was increased during the TNBS-induced colitis and was reduced by rhIL-11 administration. Mucus production was enhanced with the rhIL-11 therapy. Pretreatment with s.c. rhIL-11 inhibits Clostridium difficile toxin A enterotoxicity in rat ileal loops, decreasing fluid secretion, epithelial permeability to mannitol, and histopathological damage (Castagliuolo et al., 1997). The release of rat mast cell protease II (RMCP II) from intestinal mast cells and of TNF and MIP-2 from lamina propria macrophages was also inhibited. rhIL-11 had no effect on cholera toxinmediated secretion and permeability. Models of Short Bowel and Intestinal Ischemia/Reperfusion IL-11 produces intestinal mucosal trophic effects in rats with experimental short bowel syndrome (Liu et al., 1996). After 90% small bowel resection, groups of animals were treated with rhIL-11 (125 mg/kg twice daily, s.c.) or 0.1% bovine serum albumin (BSA). The animals were weighed daily and were killed on day 2, 4, 6, or 8; the remaining small intestine was evaluated for villus height and crypt cell mitosis. The body weight of the animals that received IL-11 was significantly greater at the beginning of postoperative day 4 in comparison to that of the BSA groups. The rats treated with IL-11 also had significantly greater villus height and crypt cell mitotic rates. The effects of IL-11 and epidermal growth factor (EGF) on residual small intestine were compared after massive small bowel resection in rats (Fiore et al.,
1998). Body weight was similar at day 4 and day 8 after resection. Animals treated with rhIL-11 and IL-11/EGF had increased mucosal mass at days 4 and 8 when compared with controls and EGF. Muscle thickness was significantly increased in the EGF group. IL-11 had a trophic effect on small intestinal enterocytes, causing cell proliferation and increased mucosal thickness. EGF has a more generalized effect on intestine causing proliferation of both enterocytes and myocytes. IL-11, with or without EGF, may have clinical utility in instances of short bowel syndrome. Protective effects of IL-11 have been shown in a murine model of ischemic bowel necrosis (Du et al., 1997). Pretreatment with rhIL-11 in mice with bowel ischemia (induced by occluding the superior mesenteric artery for 90 minutes) significantly decreased morbidity and mortality. rhIL-11-treated mice demonstrated rapid recovery of intestinal mucosa, a concurrent increase in mitotic activity, a suppression of apoptosis in intestinal crypt cells, and an increased peripheral platelet and leukocyte count. Vehicle-treated mice developed thrombocytopenia after ischemia, but no rhIL-11-treated mice developed thrombocytopenia.
Models of Systemic Inflammatory Conditions rhIL-11 improves survival in the neutropenic rat model of Pseudomonas sepsis (Opal et al., 1998). rhIL11 (150 mg/kg) was given i.v. once daily for 3 days, beginning at the onset of fever in cyclophosphamidetreated rats that were colonized in the gastrointestinal tract with Pseudomonas aeruginosa 12.4.4 at the beginning of the experiment. rhIL-11-treated animals had a significant reduction in the quantitative level of lung infection; reduction in pulmonary edema, and marked reduction in injury to the gastrointestinal epithelium of the small and large intestine. Forty per cent of rhIL-11-, 60% of ciprofloxacin-, and 100% of rhIL-11/ciprofloxacin-treated animals survived, but none of the vehicle group animals survived. These results indicate that rhIL-11 supports mucous membrane integrity of the alimentary tract and decreases the systemic inflammatory response to experimental gram-negative infection in immunocompromised animals. In Balb/c mice given doses of D-galactosamine and Staphylococcus aureus enterotoxin B as a model of T cell-mediated toxic shock triggered by superantigen (Barton et al., 1996), rhIL-11 (5±500 mg/kg i.p.) given as a pretreatment 1 hour before superantigen decreased mortality in a dose-dependent fashion at 48 hours. At 500 mg/kg, mortality was 55% compared with 100% in BSA-treated animals.
IL-11 579 rhIL-11 has been evaluated in a rabbit model of endotoxemia (Misra et al., 1996). LPS was administered i.v. and 30 minutes later vehicle or rhIL-11 (100 mg/kg i.v.) was administered. Mean arterial pressure of the vehicle-treated group was 55% of baseline 5 hours after LPS administration, while that of rhIL-11-treated animals was 94% of baseline. Histologic evaluation of the ileum, cecum, and colon showed decreased hemorrhage, edema, and mucosal damage in rhIL-11-treated animals compared with vehicle-treated animals. rhIL-11-treated animals displayed significantly lower plasma nitrate/nitrite levels compared with vehicle-treated animals. These results showed that rhIL-11 can prevent hypotension that occurs as a consequence of endotoxemia and that this effect was associated with decreased plasma nitric oxide levels. In a murine model of endotoxemia the effect of rhIL-11 on serum levels of proinflammatory cytokines was evaluated (Trepicchio et al., 1996). Female C57Bl/6J mice were injected with PBS or rhIL-11 (500 mg/kg i.p.) and 4 hours later injected with PBS or LPS. Animals treated with rhIL-11 prior to LPS administration had significantly lower peak TNF , IFN , and IL-1 serum levels, ranging from 80 to 95% reductions, compared with animals which received only LPS. rhIL-11 pretreatment did not affect LPS-induced IL-6 or IL-10 production and rhIL-11 treatment alone did not result in detectable serum levels of IL-6 or IL-10. rhIL-11 also inhibited LPS-induced TNF production in the IL-6-deficient knockout mouse. Therefore, rhIL-11 can reduce proinflammatory cytokine production during a systemic inflammatory response in vivo and this activity is not dependent on induction of IL-10 or IL-6. IL-11 improved survival and reduces bacterial translocation and bone marrow suppression in a murine burns model (Schindel et al., 1997). The effect of IL-11 on survival, intestinal cytoarchitecture, bacterial translocation, and bone marrow suppression in a highly lethal murine burn model was assessed. C3H/HeJ, 8- to 10-week-old mice, underwent a standardized 32% total body surface area (TBSA) scald burn using a burn template. Mice were divided equally between groups receiving IL-11 (125 mg/kg, twice daily, s.c.) and control (BSA). Survival was calculated to 7 days postburn. At 24 hours postburn, IL-11-treated mice had less enteric bacteria in mesenteric lymph, increased intestinal crypt cell and intestinal villus height, increased peripheral platelet and lymphocyte counts, and an improved survival compared with controls. These data demonstrate that IL-11 improved survival, intestinal cytoarchitecture, reduced bacterial translocation, and reduced
bone marrow suppression after a 32% TBSA burn in mice. Osteoporosis The effect of subcutaneously administered rhIL-11 in doses between 10 and 200 mg/kg/day for 6 weeks into 6-month-old rats after ovariectomy has been studied (Verhaeghe et al., 1998). There was no difference between vehicle-treated and rhIL-11-treated rats in the ovariectomy-induced increase in the urinary excretion of pyridinoline and deoxypyridinoline. Neither was there a significant effect of rhIL-11 on the plasma concentrations of osteocalcin and on bone mineral density (BMD) measured at a metaphyseal area of the distal femur after 6 weeks. At all dosages tested, rhIL-11 increased the femoral diaphyseal area. In conclusion, IL-11 has no deleterious in vivo effect on biochemical parameters of bone remodeling and BMD in estrogen-deficient rats.
Pharmacokinetics Slight differences in relative potency between species appear to be related to size/metabolic rate determined differences in clearance. Allometric scaling pharmacokinetic estimates based on clearance indicate an approximate 10-fold difference in the relative potency between mice and humans. The pharmacokinetics of rhIL-11 in healthy male volunteers have been evaluated following s.c. and i.v. administration (Aoyama et al., 1997). rhIL-11 was infused intravenously at 10±50 mg/kg for 1 or 3 hours, or administered subcutaneously at 3±50 mg/kg. rhIL11 was also administered at 3 mg/kg s.c. once daily for 7 days. Plasma and urinary concentrations were measured by ELISA. Linear pharmacokinetics were seen after both i.v. infusion and s.c. administration. Comparison of half-life and mean residence values after i.v. administration with those after s.c. administration indicated that rhIL-11 s.c. pharmacokinetics were absorption rate-limited. Bioavailability in this study was about 65% after s.c. injection. Since rhIL11 was not detected in urine after a single 50 mg/kg s.c. dose, rhIL-11 was considered to be eliminated by renal metabolism. Further studies of the absorption and elimination of subcutaneously administered rhIL-11 in normal volunteers and in patients with renal failure have confirmed that the kidney is the site of elimination and metabolism since anephritic animal models and patients with renal failure have delayed elimination of rhIL-11 (Hutabarat et al., 1997).
580 James C. Keith Jr
Toxicity Having recently been approved by the US Food and Drug Administration and marketed for the treatment of chemotherapy-induced thrombocytopenia, rhIL-11 has been studied in several preclinical and clinical trials. In patients or normal volunteers receiving thrombopoietic doses of rhIL-11 (50 mg/kg/day) a low incidence of adverse effects appear to be related to increases in plasma volume mediated by renal sodium retention and by induction of an acute phase response (Dykstra et al., 1997). Lower, less frequent doses of rhIL-11 (7.5 mg/kg twice weekly) in patients with Crohn's disease do not produce these changes (Sands et al., 1999). Administration of rhIL-11 has not been studied in pregnant women, but reproductive toxicology studies of rhIL-11 in rats have demonstrated that rhIL-11 can reduce ovulation numbers and cause early embryonic loss when administered at 2±20 times the human thrombopoietic dose. rhIL-11 does not demonstrate evidence of mutagenicity or teratogenicity in rabbits, but does cause maternal toxicity (decreased appetite) and abortion, increased embryonic and fetal deaths at 0.02±2 times the human thrombopoietic dose in rabbits (Neumega1, Oprelvekin, IL-11, Package Insert 12/97). However, IL-11 activity appears to be required for normal development of the placenta and implantation site since the secondary decidual response and subsequent successful placental formation fails to occur in the IL-11 R knockout mouse.
Clinical results Seven days of rhIL-11 subcutaneous administration at a dose of 25 mg/kg increased plasma levels of von Willebrand factor (vWF) and fibrinogen 94% and 140%, respectively, in normal volunteers (Kaye et al., 1994). vWF and fibrinogen are important proteins in the hemostatic process, as vWF mediates the attachment of platelets to basement membranes after injury, contributes to normal platelet aggregation, and complexes with coagulation factor VIII to protect it from proteolysis. This activity could be especially beneficial in the thrombocytopenic patient. The results of three rhIL-11 clinical trials in cancer patients have been recently reported (Gordon et al., 1996; Tepler et al., 1996; Vredenburgh et al., 1998). In a phase I study in patients with breast cancer (Gordon et al., 1997), rhIL-11, at doses ranging from 10 to 100 mg/kg, was administered once daily by s.c. injection to women for 14 days prior to chemotherapy (cycle 0). Patients then received up to four 28-day cycles of cyclophosphamide and doxorubicin,
followed by rhIL-11 therapy at their assigned dose on days 3±14. Treatment with rhIL-11 produced a dose-dependent increase in peripheral platelet counts in cycle 0. Patients receiving rhIL-11 at doses 25 mg/kg experienced less thrombocytopenia in the first two cycles of chemotherapy. Weight gain (3±5%) associated with edema was seen in all dose groups, but a capillary leak syndrome was not observed. A grade 3 neurological event was seen at a dose of 100 mg/kg and grade 2 constitutional symptoms (myalgias, arthralgias, and fatigue) were reported at a dose of 75 mg/kg. rhIL-11 had thrombopoietic activity at all doses tested and was well tolerated at doses of 10, 25, and 50 mg/kg. The effects of rhIL-11 were subsequently evaluated in a randomized placebo-controlled trial in cancer patients who had previously received platelet transfusions for severe chemotherapy-induced thrombocytopenia (Tepler et al., 1996). Ninety-three patients who had received platelet transfusions for nadir platelet counts 20,000/mL during the chemotherapy cycle immediately prior to entry into the study were randomized to receive placebo or rhIL-11 at doses of 25 or 50 mg/kg s.c. once daily for 14±21 days beginning one day after chemotherapy. Chemotherapy was continued during the study without dose reduction. Therapy with rhIL-11 reduced the need for platelet transfusions. Eight of 27 (30%) patients receiving rhIL-11 at a dose of 50 mg/kg did not require transfusions compared with 1 of 27 (4%) in the placebo group, and 5 of 28 (18%) patients receiving rhIL-11 at a dose of 25 mg/kg. Some patients experienced fatigue and cardiovascular symptoms, including a low incidence of atrial arrhythmias and syncope. This study demonstrated that rhIL-11 treatment at a dose of 50 mg/kg reduced the chance of platelet transfusion in subsequent chemotherapy cycles for patients who had required platelet transfusions for thrombocytopenia in a previous cycle. The safety and efficacy of rhIL-11 in decreasing platelet transfusion requirements in patients with breast cancer who were undergoing autologous bone marrow transplantation (ABMT) with peripheral blood progenitor cell (PBPC) support has been reported (Vredenburgh et al., 1998). After high-dose therapy with cyclophosphamide, cisplatin, and carmustine, 80 patients were randomized to one of three treatment groups: placebo (26), 25 mg/kg of rhIL-11 (28), and 50 mg/kg of rhIL-11 (26). In the placebo group, each patient received an average 12.4 (10.2) platelet transfusions versus 9.2 (5.0) in the 25 mg/kg rhIL-11 group ( p 0.17) and 9.9 (3.5) in the 50 mg/kg rhIL-11 group ( p 0.34). The imbalance in the number of patients already alloimmunized at study entry in the rhIL-11 groups (12) and in the placebo
IL-11 581 group (1) may have confounded the primary efficacy assessment. Most adverse events were related to the high-dose chemotherapy. Generally mild edema and minor conjunctival bleeding (grades 1 or 2) were statistically associated with rhIL-11 administration ( p < 0.04). Although no statistically significant association between rhIL-11 and the occurrence of atrial arrhythmias was achieved, there was a suggestion of an association with rhIL-11, 5 of 50 cases versus 1 of 25 in the placebo group. Two cardiovascular events, tachycardia and hypotension (grade 1 or 2), occurred in the 50 mg/kg rhIL-11 group. The number of patients who discontinued study drug dosing because of an adverse event was distributed across all treatment groups. rhIL-11 was safe and well tolerated in this study, but the results did not demonstrate that rhIL11 treatment significantly decreased platelet transfusion requirements after high-dose chemotherapy with ABMT and PBPC support. Based on the activity of rhIL-11 to protect or accelerate healing of the gastrointestinal epithelium following cytoablative injury, rhIL-11 is currently in phase 1 clinical trials for the prevention or treatment of oral mucositis in patients undergoing high-dose chemotherapy and peripheral stem cell support for the treatment of advanced breast cancer and in phase1 clinical trials for the prevention and amelioration of acute graft-versus-host disease following allogeneic BMT for myelodysplasia. Because of its effects on acute and chronic inflammation models clinical trials are also underway in Crohn's disease, rheumatoid arthritis, and psoriasis. The safety and tolerability of rhIL-11 in patients with Crohn's disease was investigated, and the effects of dose and schedule on platelet count and Crohn's disease activity were determined in a multicenter, double-masked, placebo-controlled, dose-escalation, phase 1 study of 76 patients with active Crohn's disease (Sands et al., 1999). Patients received subcutaneous placebo or rhIL-11 at doses of 5, 16, or 40 mg/kg given 2 or 5 times weekly for 3 weeks. rhIL11 generally was well tolerated. Lower, less frequently administered doses had little effect on platelet count. Patients receiving 16 mg/kg had the highest clinical response rates, with a response seen in 42% of patients receiving 5 weekly doses and 33% of patients receiving 2 weekly doses, compared with 7% of placebo patients.
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LICENSED PRODUCTS Neumega1 is available from the Genetics Institute, Cambridge, MA 02140-2387, USA.
