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Department of Pharmacokinetics and Drug Delivery, University of Groningen, Groningen, The Netherlands (H.R., C.R.-S., A.v.L.-W., K.P., L.B.); and Department of Nuclear Medicine, University Medical Center, Groningen, The Netherlands (M.N.L.-d.H.)
(Received October 25, 2006; accepted February 16, 2007)
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release) and antifibrotic (collagen deposition and degradation) activities. Biodistribution studies with radiolabeled M6P-IL10 and IL10 in rats with liver fibrosis showed that modification with M6P groups induced a shift in the distribution from the kidneys (IL10) to the liver (M6P-IL10). Hepatocellular binding of M6P-IL10 occurred via M6P/IGFII receptors and scavenger receptors, indicating that not only HSCs but also Kupffer and endothelial cells are target cells. IL10 did not bind to these receptors. We conclude that we prepared an active and liver-specific form of the cytokine IL10 that can be evaluated for its efficacy to treat liver diseases.
). The use of cytokines for the therapy of fibrosis may be beneficial because these compounds are very potent (picomolar to nanomolar range) and they play essential roles in the pathogenesis. With regard to liver cirrhosis, IL10 is relevant because potent anti-inflammatory and antifibrotic activities of this cytokine are described. The antifibrotic effects of IL-10 are thought to be mediated by modulation of extracellular matrix deposition via a down-regulation of collagen production or up-regulation of the expression of collagenase, in particular, matrix metalloproteinase-13 (MMP-13) (Reitamo et al., 1994; Thompson et al., 1998; Wang et al., 1998; Yamamoto et al., 2001; Mathurin et al., 2002). IL10 was tested in clinical trials in patients with hepatitis C virus-induced liver fibrosis (Nelson et al., 2000, 2003), but major drawbacks related to the systemic immunosuppressive effects became apparent. This was noted by a flare-up of viral levels in these patients, as well as the short half-life in serum with rapid elimination by the kidneys (Rachmawati et al., 2004a), meaning that the exposure time of this drug to liver cells is short, hampering its therapeutic use. Dose escalation to overcome this short plasma half-life or strategies to improve the circulation time (polyethylene glycol coupling) inevitably will lead to increased side effects, most probably, increased immunosuppressive effects on immune cells, i.e., B- and T-cells.
To improve the therapeutic effects of IL10, changes in the pharmacokinetic profile, in particular, the distribution in the body, are necessary (Brown, 2005). A specific delivery to the diseased target cells, thereby enhancing the concentration at the target site, will improve their efficacy in vivo because of an enhanced exposure to the diseased cells, a diminished excretion rate, and a reduced accumulation in the rest of the body. For IL10, the most relevant target cell with regard to fibrosis is the hepatic stellate cell. This is the major profibrotic cell within the liver that produces collagens, and this cell type expresses IL10 receptors (Mathurin et al., 2002; Rachmawati et al., 2004a). In vitro studies demonstrated that IL10 attenuated profibrotic activities of HSCs (Wang et al., 1998; Mathurin et al., 2002). Therefore, the pharmacokinetics of IL10 should be changed to obtain increased concentrations in this cell type to increase the antifibrotic activities of this cytokine.
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and interferon-
to endothelial cells of angiogenic blood vessels in tumors (Curnis et al., 2000, 2005). To that end, the cytokine TNF was fused with a targeting moiety recognizing the vß3 receptor to improve the therapeutic index of the cytokine. Also, interferon-ß has been targeted to inflammatory sites using a complicated strategy that involves the local degradation of a shell around the cytokine by matrix proteinases (Adams et al., 2003). So far, no reports are available on the chemical modification of IL10 or on the use of cell-specific targeted cytokines to the fibrotic liver. The targeting of IL10 has not been tried before, although it may be highly relevant in view of the important effects of IL10 on fibrogenic cells. Only viral delivery of the IL10 gene was tested and beneficial therapeutic results were obtained in various diseased models (Arai et al., 2000; Choi et al., 2003; Hung et al., 2005). In the present study, we aim to modify the IL10 protein with homing devices that recognize receptors present on the diseased (target) cells in the liver. In the past, we modified albumin with mannose 6-phosphate (M6P) groups and obtained a protein with affinity for the mannose 6-phosphate/insulinlike growth factor II (M6P/IGFII) receptors on the membrane of HSCs (Beljaars et al., 1999). This receptor is highly up-regulated on the designated target cells, myofibroblasts and activated HSCs, during liver fibrosis (De Bleser et al., 1995; Weiner et al., 1998; Greupink et al., 2006). In the present study, we explored the possibility of chemically modifying IL10 with M6P groups, as we aim to retain the biological activities of IL10 and to improve the pharmacokinetic features of this cytokine. |
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Modification of IL10 with M6P Groups. Recombinant human IL10 (rhIL10; obtained from TeBu-Bio, Heerhugowaard, The Netherlands) was modified with M6P groups in a manner similar to the synthesis of M6P-albumin (Beljaars et al., 1999). In brief, phosphorylated p-nitrophenyl--D-mannopyranoside (Sigma, St. Louis, MO) was synthesized and activated with thiophosgene. Activated M6P was subsequently coupled to IL10 (Fig. 1, A). Each time, 10-µg batches of rhIL10 were reacted with activated M6P in a molar ratio of M6P/IL10 of 400:1. The reaction was carried out in sodium carbonate buffer, pH 9.5 at room temperature for 1 h and proceeded at 4°C for 24 h. Free M6P and all buffer components were removed by extensive filtration with Nanosep centrifugal devices (10 kDa, Omega; Pall Corp., East Hills, NY), according to the manufacturer's instructions. The purified products were stored at –20°C until use.
Characterization of the protein content of the preparations was performed with NanoDrop (ND-1000 UV-visible spectrophotometer; NanoDrop Technology, Wilmington, DE) with a mini-Bradford method according to the manufacturer's instructions. This method allows quantification of very low amounts of protein. The coupling of M6P groups to IL10 was characterized by Western blot analysis with a rabbit polyclonal IL10 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) as a primary antibody and horseradish peroxidase-conjugated goat polyclonal anti-rabbit IgG (1:2000; DakoCytomation, Glostrup, Denmark) as a secondary antibody. The secondary antibody was visualized with 3,3'-diaminobenzidine (Sigma).
Anti-inflammatory Effects of IL10 and M6P-IL10 in Vitro. RAW 264.7 cells (TiB-71; American Tissue Culture Collection, Manassas, VA), a mouse embryonic macrophage cell line, were cultured in a humidified atmosphere at 37°C and 5% CO2 in Dulbecco's Modified Eagle's medium (DMEM; Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium) supplemented with 10% fetal calf serum (FCS; BioWhittaker), 60 µg/ml gentamicin (Invitrogen, Paisley, UK), 2 mM L-glutamine (Invitrogen), and 0.48 M L-arginine (Sigma). Cells up to passage number 20 were used for experiments.
RAW 264.7 cells (1.5 x 105 cells/well, detached by cell scraping) were cultured overnight in 96-well plates in complete medium. Then, the cells were preconditioned for 1 h at 37°C in 200 µl of serum-free DMEM. Subsequently, the cells were preincubated for 30 min with various concentrations of IL10 or M6P-IL10 (0–250 ng/ml) in 0.1 ml of serum-free medium containing 0.5% normal mouse serum. At time zero, 25 ng/ml lipopolysaccharide (LPS) of Escherichia coli (List Biological Laboratories, Campbell, CA) was added to the wells. Control cells were incubated without LPS. At 6 h, the culture media were collected for determination of TNF- levels.
TNF- was determined with a TNF- enzyme-linked immunosorbent assay sandwich method as described previously (Bentala et al., 2002). In brief, enzyme-linked immunosorbent assay plates were coated overnight with the monoclonal rat anti-mouse TNF- (BD PharMingen, San Diego, CA) in Na2HPO4 buffer (0.1 M, pH 6.0). Then, the samples and the standard mouse TNF- (BD PharMingen) were added to the plate. After addition of the capture antibody, biotinylated rabbit anti-mouse-TNF- (BD PharMingen), streptavidin-horse radish peroxidase, and the substrate o-phenylenediamine dichloride (Sigma) were subsequently added and the absorbance was measured at 490 nm.
Antifibrotic Effects of IL10 and M6P-IL10. HSCs were freshly isolated from livers of normal rats (>500 g). In brief, the liver was digested with pronase (Merck, Darmstadt, Germany), collagenase P (Roche Diagnostics, Mannheim, Germany), and DNase (Boehringer) by in situ perfusion. After several centrifuge steps, the cell suspension was subjected to a Nycodenz (Nyegaard, Norway) gradient to collect the HSCs. The purity after isolation was confirmed by phase contrast microscopy and by staining of the cells with markers for all hepatic cell types. The isolated cells were cultured in DMEM containing 10% FCS, 100 U/ml penicillin (Sigma), and 100 µg/ml streptomycin (Sigma). After 6 days in culture, the cells displayed an activated phenotype as assessed by light microscopy and staining for -smooth muscle actin (mouse monoclonal antibody clone 1A4 from Sigma) (Rockey et al., 1992; Geerts, 2001). Staining of the cell cultures for IL10 receptor and M6P/IGFII receptor was performed with a rabbit polyclonal antibody against IL10 receptor (Santa Cruz Biotechnology) and a goat polyclonal M6P/IGFII receptor (Santa Cruz Biotechnology) according to standard indirect immunoperoxidase methods.
PCR analysis. To assess the effects of IL10 and M6P-IL10 on matrix deposition, HSCs at 6 days after isolation were seeded in six-wells plates (2.5 x 105 cells/well) and adhered overnight. The cells were preconditioned in FCS-free medium for 1 h before incubation with 0.5 ml of either IL10 or M6P-IL10 (12.5 and 25 ng/ml) for 1 h. Control cells were preincubated with FCS-free medium. Subsequently, the cells were stimulated with TGF-ß1(5 ng/ml; Roche Diagnostics, Indianapolis, IN). After 24 h, cell fractions were harvested and used for PCR analysis. The mRNA fraction was isolated with the QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. The mRNA concentration was determined with Nano-Drop. A reverse transcriptase reaction (Sensiscript RT kit; Qiagen GmbH, Hilden, Germany) was performed with 25 ng total RNA in 20 µl of reaction volume to obtain 20 µl of cDNA. cDNA was examined with the following primers: collagen 1a1 (5'-AGCCTGAGCCAGCAGATTGA-3' and 5'-CCAGGTTGCAGCCTTGGTTA-3'), MMP-13 (5'-AGGCCTTCAGAAAAGCCTTC-3' and 5'-GAGCTGCTTGTCCAGGTTTC-3'), tissue inhibitor of metalloproteinase (TIMP)-1 (5'-ACAGCTTTCTGCAACTCG-3' and 5'-CTATAGGTCTTTACGAAGGCC-3'), and GAPDH (5'-CCATCACCATCTTCCAGGAG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'). PCR for MMP-13 was performed in a total volume of 25 µl containing 1.5 µl of cDNA, 50 mM MgCl2, 2.5 µl of 10x TaqDNA polymerase buffer, 10 mM deoxynucleoside-5'-triphosphates, 0.5 U of TaqDNA polymerase (Eurogentec, Seraing, Belgium), and 50 pmol/µl of each primer. The MMP-13 reaction was performed with 30 cycles and an annealing temperature of 56°C for 30 s, whereas GAPDH PCR was performed with 26 cycles and annealing temperature of 58°C for 30 s. The band intensity of the PCR products was quantified with ImageJ (NIH Image Software, Bethesda, MD), and the gene expressions were normalized to the signal of the housekeeping gene GAPDH. For collagen 1a1 and TIMP-1, cDNA was amplified by quantitative real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the reaction was performed in an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Data were analyzed using the comparative threshold cycle (CT) method as described in User Bulletin 2 of the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Data are expressed as -fold induction of the gene of interest compared with the control condition as calculated by the formula 2–
CT.
Immunohistochemical analysis. The effects of M6P-IL10 on type I collagen production was studied in cultured HSCs, 6 days after isolation. After trypsinization, HSCs were cultured (5 x 103 cells/well) in eight-well cover slip chamber slides (Lab-Tek; Nalge Nunc International, Rochester, NY) for 24 h. Subsequently, cells were preconditioned in FCS-free medium for 2 h. After this, cells were incubated with 0.1 ml of either IL10 or M6P-IL10 (12.5 and 25 ng/ml) for 24 h. One hour after addition of IL10, cells were stimulated with TGFß-1. Control cells were incubated with FCS-free medium in parallel wells. Type I collagen deposition in the wells was detected immunohistochemically with a goat polyclonal antibody against type I collagen (Southern Biotechnology Associates Inc., Birmingham, AL), with horseradish peroxidase-conjugated rabbit polyclonal anti-goat immunoglobulin (DakoCytomation) as a secondary antibody, and visualized with 3-amino 9-ethyl carbazole (Sigma).
Animal Model of Liver Fibrosis. Rats (weighing 250–300 g) were subjected to bile duct ligation (BDL) (Kountouras et al., 1984) under anesthesia with 40% O2/60% N2O combined with 0.5% isoflurane (Abbott Laboratories Ltd., Berkshire, UK). Three weeks after the ligation (BDL-3), the rats were used for further experiments. At that time, the fibrosis in the liver was in a late stage of disease. See also Fig. 6A, in which frozen sections are stained with a polyclonal antibody against collagen type III (Southern Biotechnology Associates Inc.) as described in the previous paragraph.
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; Mather and Ward, 1987). For gamma camera studies, these proteins were labeled with 123I (GE Healthcare, Chalfont St. Giles, UK) (Greenwood et al., 1963). Before each experiment, free iodine in the preparations was removed by gel filtration with a PD-10 column (GE Healthcare) to obtain dosing preparations with less than 5% free iodine.
Quantitative biodistribution studies. After anesthetizing BDL-3 rats (n = 4 per group) with O2/N2O/isoflurane, [125I]IL10 and [125I]M6P-IL10 (±500,000 cpm per rat, diluted in saline) was intravenously injected in the penile vein. After 10 min, blood and major organs were harvested to determine the amount of radioactivity. The total radioactivity in each tissue was measured with a gamma counter (Riastar Gamma Counting System; PerkinElmer Life and Analytical Sciences, Boston, MA) and then corrected for blood-derived radioactivity in that tissue. This correction factor was calculated from biodistribution studies with HSA, a protein that remains in the circulation during the time frame of this experiment, as described previously (Beljaars et al., 2000).
To assess the receptor-mediated uptake of the modified cytokine, 5 min before the intravenous injection of the radiolabeled [125I]IL10 or [125I]M6P-IL10, 5 mg/kg of either M6P-modified human serum albumin (M6PHSA; n = 4), succinylated human serum albumin (SucHSA; n = 6), a mixture of SucHSA and M6PHSA (n = 4), or HSA (n = 4) was administered intravenously. M6PHSA and SucHSA were prepared and characterized according to standard procedures (Swart et al., 1996; Beljaars et al., 1999).
Gamma camera studies. The body distribution of IL10 and M6P-IL10 was visualized in BDL-3 rats using gamma camera analysis (n = 3 per group). Anesthetized rats were placed on a low-energy all-purpose collimator of a gamma camera and received intravenously a tracer amount of 123I-labeled proteins. The radioactivity was dynamically recorded from 0 to 30 min with a frame rate of one total body scan per minute.
Statistical Analysis. Data are presented as mean ± S.D. All data were subjected to an unpaired, two-tailed distribution Student's t test. Differences were considered significant at p < 0.05.
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production, from 79 ± 49 pg/ml for control wells (without LPS) to 10,845 ± 746 pg/ml in the presence of LPS (p < 0.01). Coincubation of LPS with various concentrations of IL10 significantly attenuated the TNF production of the RAW cells starting at 15 ng/ml concentrations. The TNF levels were reduced to 4292 ± 452 pg/ml at a concentration of 250 ng/ml IL10 (p < 0.01). Also, the chemically modified form of IL10 (M6P-IL10) reduced the TNF- response of the macrophages in a dose-dependent manner, starting also at 15 ng/ml concentrations. At a concentration of 250 ng/ml, the TNF production in M6P-IL10 incubations was significantly reduced to levels similar to those with unmodified IL10 (4784 ± 160 pg/ml; p < 0.01).
Antifibrotic Activities of IL10 and M6P-IL10 in Vitro. The antifibrotic activities of IL10 and M6P-IL10 were studied in fibroblast cultures. Primary rat HSCs were used because these myofibroblasts are our target cells in the liver, and after culturing them for 7 days in a plastic flask, they display an activated phenotype as reflected by -smooth muscle actin staining (data not shown). At this time point, we confirmed by immunostaining of the HSC cultures that these cells displayed the IL10 receptor as well as the target M6P/IGFII receptor (Fig. 3).
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; Ricke et al., 2002), we assessed that IL10 and M6PIL-10 both significantly enhanced this ratio (Fig. 4). Compared with control TGFß-treated cells, this ratio was increased 2.4 times and 1.8 times, respectively, at 25 ng/ml IL10 and M6P-IL10. This means that the collagenolytic activity of these cells was enhanced by both types of IL10. Collagen 1a1 mRNA expression of the HSCs was not significantly altered by M6P-IL10 or IL10 (data not shown).
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In addition, we tested whether IL10 and M6P-IL10 influenced total collagen deposition by staining HSC cultures for collagen type I, which is the major interstitial collagen produced by HSCs in fibrogenesis. Type I collagen was present in HSC cultures at 7 days after isolation. The collagen staining was diminished by incubation of the cells with IL10 (Fig. 5). M6P-IL10 reduced type I collagen staining in these cultures at similar concentrations.
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Immunohistochemical examination of the hepatocellular localization of modified IL10, similar to previous studies with modified albumin (Beljaars et al., 1999), was not possible, because this technique is not sensitive enough to detect the microgram dose of rhIL10 that was injected. Therefore, we performed radioactive studies aiming to block the receptor-mediated uptake to assess which receptors are responsible for the uptake. In addition to the IL-10 receptor and the M6P/IGFII receptor, we also tested whether scavenger receptors were able to bind M6P-IL10. It is known that the scavenger receptor type A is involved in the binding of polyanions (Terpstra et al., 2000), and our construct is negatively charged, as reflected by zeta potential measurements. Figure 7 shows that coadministration of an excess of M6PHSA, a ligand for the M6P/IGFII receptor, resulted in a 54% reduction in the hepatic accumulation of [125I]M6P-IL10. In addition, an excess of SucHSA, which is a ligand for the scavenger type A receptor, also significantly reduced the distribution to the liver by about 46%. Blocking the receptor-mediated uptake of [125I]M6P-IL10 by administration of a mixture of SucHSA and M6PHSA did not further reduce the hepatic radioactivity compared with M6PHSA alone (data not shown). The decrease in liver uptake of M6P-IL10 by coadministration of M6PHSA or SucHSA led to higher plasma concentrations of the cytokine at the same time point (data not shown). Administration of the control protein HSA did not display any effect on the hepatic accumulation of [125I]M6P-IL10. The distribution of [125I]IL10 was not influenced by M6PHSA, SucHSA, or HSA, which indicated that the M6P/IGFII receptor and the scavenger receptor were not involved in the hepatic uptake of unmodified IL10 (Fig. 7).
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Finally, gamma camera studies were used to visualize the body distribution. As early as 2 min after i.v. injection, high levels of [123I]M6P-IL10 were observed within the liver region, with very low levels in the kidneys. Hepatic levels remained high for at least 30 min. In contrast, native [123I]IL10 rapidly accumulated in the kidneys with low uptake in liver (Fig. 8), which is in agreement with previous studies (Andersen et al., 1999; Rachmawati et al., 2004a). Coupling of M6P groups to IL10 induced a shift in biodistribution of IL10 from the kidneys to the fibrotic liver.
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; Greupink et al., 2006). Previously, M6PHSA has been prepared and successfully applied as a drug carrier to HSCs in vitro and in vivo (Beljaars et al., 1999, 2001). In the present study, we used this approach for the stellate cell targeting of the antifibrotic cytokine IL10. A recombinant human IL10 was used in the present studies, because human IL10 is active on both rodent and human cells (Moore et al., 2001), and because clinical studies have been done (Nelson et al., 2000) and will be done in patients, the human form is the most suitable in preclinical studies.
In the past, some cytokines have been chemically modified to obtain products with an improved pharmacokinetic profile (Rachmawati et al., 2004b; Schrama et al., 2006) or an improved therapeutic profile (Curnis et al., 2000, 2005; Adams et al., 2003). The most well known therapeutic cytokine with regard to liver diseases is Pegasys, which is interferon 2a modified with polyethylene glycol (PEG) groups. Substitution of cytokines with PEG moieties prevents rapid renal elimination, which results in compounds with prolonged plasma levels (Harris and Chess, 2003; Veronese and Pasut, 2005). This strategy to improve pharmacokinetics has also been applied to some other cytokines, such as IL2, IL6, granulocyte-macrophage colony-stimulating factor, and TNF, but not for IL10. However, this strategy only prevents rapid clearance. An even more powerful way is to improve the therapeutic profile of cytokines by enhancing the accumulation at the target site based on the delivery to specific target receptors. With this strategy, also applied in the current study, side effects in other parts of the body will be reduced, because elevated amounts of cytokine in the blood will be present only for a short time after injection. In contrast, after PEGylation of proteins, elevated plasma concentrations can be measured for several hours. That delivery of IL10 may be promising can be derived from a few studies exploring the therapeutic potential of IL10 after viral delivery (Arai et al., 2000; Choi et al., 2003; Hung et al., 2005). In these gene-targeting studies, viral-produced IL10 significantly attenuated the fibrotic process.
The present study shows successful coupling of M6P to IL10, which was confirmed by chemical characterization with Western blot analysis. The sugar moiety was attached to the 
NH2 groups present in the lysine residues in the protein backbone of IL10. Human IL10 contains 13 lysine residues in total. Not all these residues are readily accessible for chemical modification. With our characterizations, we estimated that approximately four molecules of M6P were attached to IL10. Of note, the lysines at position 34 and 57 in the IL10 protein are predicted to interact with the IL10 receptor and are therefore involved in IL10 signaling (Reineke et al., 1998). Coupling of M6P groups to these lysine amino acids might therefore block the biological activities of this cytokine. With our in vitro systems to examine anti-inflammatory and antifibrotic activities of IL10, we assessed that M6P-IL10 was still biologically active. First, we examined the effect of M6P-IL10 on RAW cells that constitutively express the IL10 receptor but have no M6P/IGFII receptors. These receptor expressions were confirmed immunohistochemically (data not shown). RAW cells are highly responsive to LPS, leading to their activation followed by the release of proinflammatory mediators such as TNF-. Inhibition of TNF- by M6P-IL10 was observed at concentrations similar to those used for IL10. This indicates that M6P-IL10 is still able to bind to IL10 receptors.
Second, we tested the antifibrotic effect of this conjugate on the target cell; that is, the activated HSC. This cell contains both IL10 and M6P/IGFII receptors as tested immunohistochemically. With respect to this testing, relevant parameters are TIMP-1, MMP-13, and type I collagen, because these key parameters of the fibrotic process are regulated by IL10 (Mathurin et al., 2002; Schaefer et al., 2003). In advanced liver fibrosis, a complex process resulting from an imbalance between matrix deposition and degradation leads to enhanced collagen deposition. The fibrillar collagen type I is one of the most abundant fibrous matrix constituents. During fibrogenesis, the activity of MMP-13, the principal protease capable of cleaving fibrillar collagens, decreases. At the same time, TIMP-1, the inhibitor that blocks MMP-13 activity, is overexpressed. Under conditions in which TIMP-1 levels are increased and MMP13 levels are decreased, there is an increased deposition of fibrillar collagens in the liver. The imbalance between MMP-13 and TIMP-1 can be expressed as the MMP-13/TIMP-1 ratio (Vaillant et al., 2001; Ricke et al., 2002). In this study, we observed antifibrotic effects of both IL10 and M6P-IL10 via the regulation of MMP-13 and TIMP-1 mRNA expressions. In addition, we found an effect of IL10 and M6P-IL10 on the type I collagen deposition by HSCs in culture. These results indicate that M6P-IL10 is pharmacologically active and exerts antifibrogenic effects on activated HSCs.
Specific delivery to target receptors will yield compounds with an enhanced accumulation at the target site because of receptor-mediated removal of the compound from the circulation. Coupling of M6P groups to IL10 resulted in a modified protein that was rapidly eliminated from the blood, as illustrated by the fact that less than 10% was present in plasma at 10 min after intravenous injection, and most of the compound (54 ± 6% of the radioactive dose) was traced back in the fibrotic liver and, more importantly, in the target cells; that is, in HSCs. M6P-modified albumin displayed a similar distribution pattern and plasma elimination rate (Beljaars et al., 1999). As previously reported (Rachmawati et al., 2004a), the cytokine IL10 itself was rapidly cleared by the kidneys through glomerular filtration, with only minor uptake in the liver. As shown in Fig. 7, we found that the hepatic accumulation of M6P-IL10 is mediated by several hepatic receptors. In addition to the M6P/IGFII receptor and the IL10 receptor present on HSCs, the scavenger receptor appears to be involved in the removal of this cytokine from the blood, because succinylated albumin, a well known ligand for this scavenger receptor, attenuated the liver uptake of M6P-IL10. This is not surprising since, after coupling of M6P to NH2 groups of lysine, a protonated group (NH +3) is replaced by a negatively charged group (PO 3–4). Scavenger receptors are found on Kupffer and sinusoidal endothelial cells, and also on activated HSCs (Schneiderhan et al., 2001; Adrian et al., 2006). The combination of SucHSA and M6PHSA did not induce a complete blockage of liver uptake, even after administration of very high doses. This residual hepatic uptake likely reflects IL10 receptor-mediated uptake. Because high amounts of IL10 cannot be administered, complete IL10 receptor blockage cannot be achieved. However, studies with RAW cells show that our construct does bind to the IL10 receptor, and this receptor is expressed in fibrotic livers (Rachmawati et al., 2004a). Thus, we conclude that M6P-recognizing receptors, scavenger receptors, and IL10 receptors are involved in the hepatic uptake of M6P-IL10. From this, we can deduce a distribution of M6P-IL10 to Kupffer cells, endothelial cells, and hepatic stellate cells. This distribution is, from a therapeutic point of view, beneficial with regard to the anti-inflammatory and antifibrotic activities of M6P-IL10 in vivo.
Delivery of IL10 to different receptors creates a complex situation, because only the IL10 receptor is relevant for the biological effects of M6P-IL10, and binding to the other receptors will not induce any IL10-mediated effect. We propose that delivery of high amounts of IL10 in the vicinity of the IL10 receptor may be beneficial. IL10 receptor expression is low, even after its up-regulation during fibrosis. Scavenger receptor and M6P/IGFII receptor expression is relatively very high, in particular, during fibrosis. M6P-IL10 bound in high concentrations to the target cell or to neighboring cells may trigger the IL10 receptor via contact between adjacent cells or via receptor cross talk during membrane perturbations, similar to the situation of a cytokine coupled to an antibody as the targeting device (Penichet and Morrison, 2001; Schrama et al., 2006). A study of pharmacological effects of M6P-IL10 in rats with liver fibrosis would clarify whether M6P-IL10 is able to induce effects in vivo after binding to the intrahepatic receptors. The assessment of in vivo efficacy and improvement by targeting of IL10 will be the ultimate goal of our studies. To demonstrate this, long-term studies are required, and at the moment, we are up-scaling the synthesis to examine the effects in animal models of liver fibrosis.
In summary, we successfully prepared a novel liver-selective form of IL10, M6P-IL10, which displays the biological activities of IL-10. M6P-IL10 is efficiently distributed to relevant cell types in the liver; that is, to HSCs, endothelial cells, and Kupffer cells. The chemical modification of the cytokine IL10 may be a novel approach in the use of cytokines for diseases.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: IL10, interleukin-10; rhIL10, recombinant human IL10; HSC, hepatic stellate cell; TNF, tumor necrosis factor; M6P, mannose 6-phosphate; M6P/IGFII receptor, mannose 6-phosphate/insulin-like growth factor II receptor; M6P-IL10, interleukin-10 modified with mannose 6-phosphate groups; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PCR, polymerase chain reaction; MMP-13, matrix metalloproteinase-13; TIMP-1, tissue inhibitor of metalloproteinase-1; TGFß, transforming growth factor ß; HSA, human serum albumin; SucHSA, HSA modified with succinic acid groups; M6PHSA, HSA modified with mannose 6-phosphate groups; BDL, bile duct ligation; PEG, polyethylene glycol.
Address correspondence to: L. Beljaars, Dept. of Pharmacokinetics and Drug Delivery, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: E.Beljaars{at}rug.nl
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