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Dokumen 1 dari 1 Targeting genetically modified macrophages to the glomerulus Link dokumen ProQuest Abstrak: Macrophages are key players in the development of the majority of renal diseases and are thereforeideal cellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can begenetically modified using viral vectors ex vivo then re-introduced into the body where they can home to thediseased site. This review summarises current experience in efficiently targeting modified macrophages to theinflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfermethods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy forglomerular disease. Teks lengkap: Headnote Key Words Macrophages * Inflammation * Glomerulonephritis . Adenovirus . Gene Abstract Macrophages are key players in the development of the majority of renal diseases and are therefore idealcellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can begenetically modified using viral vectors ex vivo then re-introduced into the body where they can home to thediseased site. This review summarises current experience in efficiently targeting modified macrophages to theinflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfermethods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy forglomerular disease. Introduction Macrophage infiltration of renal tissue is a feature of many forms of nephrological disease ranging from classicalinflammatory diseases such as rapidly progressive glomerulonephritis to more indolent 'non-inflammatory'processes such as diabetic nephropathy. The classical view has been that these cells are primarily injurious,and that inhibiting their function or preventing entry into renal tissue is the main goal of therapy. Macrophagesare, however, heterogeneous and perform distinct functions; increasingly their role in the down-regulation andresolution of inflammation has been identified [1]. This is most clearly seen in skin wounds where inhibition ofmacrophage entry prolongs inflammation and delays healing [2]. This raises the potential to utilise macrophagesas therapeutic cells to down-regulate renal inflammation and restore normal function. Critical to achieving this isto establish what determines their function, what factors control macrophage localisation to inflamed renaltissue, and how their properties can be modified to ameliorate inflammation. Functions of Macrophages in Renal Inflammation Macrophages arc critical adaptors in the immune response, able to both drive the immune response by antigenpresentation to T cells and respond to cytokines produced by the specific immunity; they are both conductor andplayer in the inflammatory orchestra. Macrophage infiltration into renal tissue is an early feature ofglomerulonephritis and contributes to the development of necrosis, crescent formation and subsequent scarring.Macrophage function in the early stages of inflammation is geared towards promoting inflammation as within theinflamed environment macrophages are exposed to a wide range of stimuli including pro-inflammatory cytokinessuch as IL-1[beta], TNF-[alpha] and IFN-[gamma], lipid mediators, reactive oxygen and nitrogen products,chemokines and necrotic and apoptotic cells. The importance of a number of these factors in experimentalmodels of glomerular inflammation has been established with inhibition of cytokines such as IL-1[beta], TNF-[alpha] and macrophage migration inhibitory factor (MIF) reducing disease severity. This pro-inflammatoryactivation of macrophages renders them unresponsive to anti-inflammatory cytokines such as transforming

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growth factor-[beta] (TGF-[beta]) or IL-4 [3], and re-orientating this response is a critical approach to resolveinflammation. The crosstalk between infiltrating macrophages and their environment is important ininflammation. Suto et al. [4] have demonstrated that injection of activated macrophages into glomeruli resultedin the up-regulation of matrix metalloproteinase 9 (MMP-9) in normal glomeruli but expression was down-regulated when activated macrophages were injected into regenerating glomeruli from rats with Thy-1 nephritiswhere TGF-[beta] production was high. Thus, local expression of TGF-[beta] from resident mesangial cells maysuppress activated macrophages and macrophage factors may influence the regulation of genes in residentglomerular cells [5]. Factors Controlling Macrophage Localisation to Inflamed Renal Tissue The first stage of monocyte/macrophage involvement in glomerular inflammation is adhesion to activatedendothelium and transmigration in response to a chemotactic gradient. There is increasing evidence thatdifferent vascular beds and different forms of inflammation utilise distinct molecules to enable macrophagelocalisation and these need to be understood for glomerular inflammation to allow efficient homing of geneticallymodified inflammatory cells. Initial work demonstrated that blockade of classical integrin molecules, intracellularadhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM) by antibodies decreasedinflammation in nephrotoxic nephritis (NTN) [6], however, there was no consistent evidence of a reduction inglomerular macrophage numbers and the reduction in injury may have reflected alteration of trafficking ofinflammatory cells in other organs such as spleen or regional lymph nodes. Recent studies have focused ondirect visualisation of leukocyte adhesion. De Vrcise et al. [7] showed that inhibition of selectins did not affectglomerular leukocyte adhesion, while inhibition of [beta]^sub 2^ integrin CD11b (counter-receptor to ICAM-1)prevented glomerular leukocyte accumulation. Recently, chemokines have also been show to have roles in adhesion. Fractalkine is a CX3C chemokine thatexists as a soluble chemotactic agent but it also has a transmembrane domain and is expressed on activatedendothelium. Inhibition of fractalkine receptor (CX3CR1) in NTN reduced glomerular macrophage infiltration andattenuated histological and biochemical measures of injury [8]. The chemokine GRO-[alpha] can also beimmobilised to cell surface proteoglycans to support adhesion of monocytes to mesangial cells in vitro. Inaddition, blockade of GRO-[alpha] or fractalkine receptors on macrophages injected directly into the renal arteryof rats with NTN suppressed their localisation to inflamed glomeruli [9]. Thus, adhesion to glomerularendothelium in experimental models appears to involve chemoadhesin interaction followed by integrin-mediatedadhesion. Once adherent to endothelium, macrophages transmigrate across the glomerular basement membrane inresponse to chemokines. A wide range of approaches have been used to study which chemokines are pre-eminent in the control of renal inflammation [reviewed in 10] and as with adhesion, it appears to depend on boththe nature of injury and at what point during the immune response they are assessed. Two chemokines forwhich the clearest role is defined are monocyte chemotactic protein-1 (MCP-1 - binds to CCR2) and regulatedon activation normal T cell expressed and secreted, RANTES (binds to CCR1, 3 and 5). Early studies showedthat inhibition of MCP-1 by anti-bodies reduced macrophage glomerular infiltration and proteinuria in NTN andexpression of MCP-1 and CCR2 are early features of immune complex glomerulonephritis, NTN and murinelupus nephritis. We have also demonstrated that direct inhibition of CCR2 receptor on macrophages reducedtheir glomerular localisation in rats with NTN [9]. Similarly, RANTES is found in many models of glomerularinflammation and inhibition by the antagonists met-RANTES or aminoxypentane-RANTES reduces macrophageinfiltration in NTN and anti-Thy-1 nephritis. Expression of chemokines has also been identified in human renal biopsies and a recent study by Segerer et al.[11] showed that in crescentic glomerulonephritis, CCR2 was mainly expressed on glomerular macrophageswhile CCR5 was found mainly on interstitial T cells. This suggests that specific chemokine expression directsmacrophages and T cells to separate compartments with distinct functional consequences. Thus, at present,

MCP-1 and its counter-receptor CCR2 are prominent in the early stages of glomerular inflammation and providea target to direct macrophages to inflamed glomeruli. Further work is required to determine the orchestration ofchemokines during glomerulonephritis, particularly those expressed during the resolution of inflammation. Macrophage Manipulation Macrophages are ideally suited to alter inflammatory disease due to their preferential localisation to inflamedtissue, the properties they can develop and their interaction with other cells of the immune response. Thus far,work has focused on altering macrophage function ex vivo before re-administration into animals with renalinflammation. A number approaches exist to transduce macrophages to express specific transgenes includingviral vectors such as adenoviruses, retroviruses and lentiviruses as well as non-viral methods including cationicDNA carrier molecules, electroporation and receptor-mediated DNA uptake [reviewed in 12]. Non-viralapproaches give a low efficiency of transfection compared to viral methods, thus the latter have been favouredfor in vivo manipulation. Adenoviral vectors are at present the most popular method of genetically modifyingmacrophages; large genes (up to ~ 10 kb) can be transferred in standard vectors and a high level of expressionof the transgene achieved. Adenoviruses are relatively inefficient at transducing monocytes due to the lowexpression of adenoviral receptor integrins [alpha]^sub v^[beta]^sub 3^ and [alpha]^sub v^[beta]^sub 5^, butinfection rates can be increased to greater than 90% if primary cultures of monocytes are incubated withmacrophage colony-stimulating factor which up-regulates integrin expression. Adenoviral transduced primarycultures of bone-derived macrophages (BMDM) or macrophage cell lines such as RAW 267.4 and NR8383 cellsexpress high levels of transgene within 24 h of transduction and this remains stable for 7-10 days [13] allowingefficient short-term expression in vivo. Retroviruses transduce rapidly dividing cells by stably incorporating DNA into the host cell genome, however,the transduction efficiency is low due to the low rate of proliferation. To overcome the problem, CD34^sup +^bone marrow stem cells, precursors of macrophages, have been used that are capable of cell division andretroviral infection. This allows reconstitution with genetically modified bone marrow cells that permits long-lasting expression of the relevant transgene. Recombinant lentiviruses have a natural tropism for macrophagesand can stably transduce these cells. Despite their high level of gene transfer and expression, concernsregarding safety issues such as generation of replicon-competent virus during production of vectors have so farlimited their use to in vitro work. In vivo Localisation of Genetically Modified Macrophages Site specifically targeting genetically modified macrophages to the glomerulus and limiting expression in non-target tissues is a fundamental requirement for their use as vehicles for gene therapy. Macrophages have beeninjected intravenously or directly into the renal artery where they have a concentrated exposure to theinflammatory site. The main concern with intravenous injection is that only a small proportion of the modifiedmacrophages localise to inflamed glomeruli due to their hold up in the lung and trafficking to the spleen. Directinjection into the renal artery by contrast, allows highly efficient first-pass localisation and we have shown thatadenoviral transduced macrophages localise preferentially in the presence of glomerular inflammation in ratsand when macrophages are activated with lipopolysaccharide [13]. Yokoo et al. [14] have shown that CD11b^sup +^CD18^sup +^ (ligand for ICAM-1) bone marrow-derived cellstransduced with recombinant adenovirus then re-injected systemically into mice localise to glomeruli when theanimals were treated with lipopolysaccharide, which up-regulates ICAM-1 expression, and on induction ofglomerulonephritis [15]. Thus the state of activation of macrophages and the endothelium, as well as themethod of delivery, affects the efficiency of glomerular macrophage localisation. The transgene expressed bymacrophages can also affect the properties of cells as we found that transduction with adenoviruses expressingIL-4 markedly enhanced the ability of macrophages to localise to inflamed glomeruli compared to expression ofinert transgene [16]. The most likely mechanism for this is up-regulation of CXCR1 and 2 expression bymacrophages, which adhere to GRO-[alpha], a recognised chemoadhesin on glomerular endothelium. Thus,

glomerular-specific genetically modified macrophage localisation can be achieved effectively.

Effect of Genetically Modified Macrophages on Renal Inflammation The transduction of macrophages to alter the development of inflammatory disease has thus far focused onexpression of cytokines with principally anti-inflammatory and/or regulatory effects. Systemic injection ofmacrophages transduced with recombinant adenovirus to express IL-1 receptor antagonist (IL-1ra) reduced theseverity of glomerular inflammation in mice with NTN and reduced interstitial macrophage infiltration in a modelof unilateral urcteric obstruction [15, 17]. We have studied the effects of macrophages transduced to expressanti-inflammatory cytokincs IL-4, IL-10 and TGF-[beta]. Initial work transduced the rat alveolar macrophage cellline NR8383 with adenovirus to express IL-4. The cells localised to glomeruli of rats with NTN, produced thecytokine in vivo and reduced the level of albuminuria, histological markers of glomerular inflammation andmacrophage infiltration [16] demonstrating that macrophage cell lines can be modified efficiently and havetherapeutic effects in vivo. By contrast, injection of NR8383 cells transduced to express active TGF-[beta] hadlimited impact on the development of renal injury in rats with NTN [unpubl. data]. The properties of macrophagecell lines arc different from native macrophages and may express transgenes in a different manner andsubsequent work focused on utilising primary cultures of BMDM from inbred strains of rats. Macrophagestransduced to express IL-10 localised preferentially to glomeruli of rats with NTN after renal artery injection andproduced a profound reduction in the severity of glomerular inflammation reducing both macrophage infiltrationand their state of activation as assessed by both MHC class II and ED3 expression [18]. Interestingly, in all these experiments, delivery of macrophages transduced with an inert gene ([beta]-galactosidase) to inflamed glomeruli resulted in a reduction in injury as assessed by albuminuria but only up to48 h after disease induction. The most likely mechanisms for this are either transduction of macrophagescauses them to release antiinflammatory molecules (e.g. IL-10) or that infusion of the transduced macrophagesprevents infiltration of pathogenic macrophages by competition for adhesion sites. This further highlights howgenetically modified macrophages have a complex interaction with glomerular inflammation. The injection of IL-4 and IL-10 expressing macrophages resulted in highly effective localisation to glomeruli of asingle kidney with transduced cells seen infrequently in the contralateral kidney. Despite this, we have foundthat injection into a single kidney markedly attenuated the development of inflammation in the contralateralkidney. This implies that the expression of cytokine expressing macrophages at one inflammatory site candecrease injury both at that site and at distant sites. These changes could not be mimicked by systemic

administration of IL-10 or IL-4 expressing macrophages and there was no evidence of increased systemic levelsof the relevant cytokines. In light of the specific requirement of glomerular localisation in producing thisresponse, we hypothesise the effect is due to high local concentrations of cytokines altering the properties ofmacrophages, dendritic cells and T cells trafficking through the glomerulus that consequently modify thedevelopment of the immune response or that the transduced macrophages themselves migrate to regionallymph tissue and regulate the systemic development of the immune response (fig. 1). As many autoimmunediseases arc multifocal, these results raise the potential that disease modified by macrophages at one site maybe able to ameliorate inflammation at distant sites, a potentially powerful tool in therapy. Regulation of Macrophage Gene Expression The work so far has demonstrated the feasibility of macrophages to down-regulate inflammation and has largelyused adenoviral transduction to achieve this. However, a critical issue in macrophage-mediated gene therapy ishow to control gene expression, in particular, how macrophage activation can lead to regulated transgeneexpression. Yokoo et al. [19] used double transduced macrophages that only express target gene at the site ofinflammation. To achieve this, macrophages were transduced with an adenovirus containing the IL-1[beta]promoter which controls expression of Cre recombinase that, in turn, excised DNA from a second targetadenovirus carrying a reporter gene under the control of strong viral promoter, thus leading to reporter geneexpression. Following injection, the vehicle cells were detected in various organs including the kidney but thereporter gene was expressed only in the kidney following the induction of nephritis. The double transductionrequirement for this method, however, reduce its general applicability and an alternative approach usingtetracycline sensitive promoters in a single vector could control transgene expression in vivo. An additional problem with use of adenovirus is that expression of the transgene is short-lived. Thus to continueto suppress ongoing inflammation would potentially require repeat injections of modified macrophages.However, retroviral transformation of bone marrow stem cells leads to stable incorporation of the gene ofinterest into the chromosomal DNA and subsequent daughter cells. This approach has been used to generateCD34^sup +^ stem cells from mice expressing IL-1ra and following re-infusion in irradiated mice around 10-20%of bone marrow and spleen cells were derived from donor cells [20]. When NTN was induced in these micethere was a reduced severity of disease, although it is unclear to what extent this effect was a consequence ofIL-1ra expression by glomerular macrophages as opposed to effects elsewhere in the immune system.However, combining long-term expression with regulation of transgene activation would provide the optimumsystem to control genetically modified macrophages in inflammation. Future The experimental work to date has shown the potential for macrophages to ameliorate immune-mediatedglomerular disease (fig. 1). They provide a powerful tool to study the effects of both macrophages and cytokineson the evolution of inflammatory disease. Therapies using genetically modified macrophages are in thepreliminary stages and many problems need to be addressed before moving to the clinical setting. In spite ofthis, cell-based therapy to treat immune-mediated diseases is a clear objective. With the increasing use of stemcells and other bone marrow-derived cells in the treatment of autoimmune diseases, it could be feasible toaugment the anti-inflammatory and reparative properties of macrophages using genetic manipulation. Theadministration of such genetically enhanced macrophages could provide a powerful complimentary therapy forglomerulonephritis and other forms of inflammation. Sidebar Copyright (C) 2003 S. Karger AG, Basel Sidebar KARGER Fax + 41 61 306 12 34 E-Mail karger@karger.ch

www.karger.com (C) 2003 S. Karger AG, Basel 1660-2129/03/0944-0113$19.50/0 Accessible online at: www.karger.com/nee References References 1 Mosser DM: The many faces of macrophage activation. J Leuk Biol 2003;73:209-212. 2 Nagaoka T, Kaburagi Y, Hamaguchi Y, Hasegawa M, Takehara K, Steeber DA, Tedder TF, Sato S: Delayedwound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression. Am J Pathol2000;157:237-247. 3 Erwig LP, Stewart K, Rees AJ: Macrophages from inflamed but not normal glomeruli are unresponsive to anti-inflammatory cytokines. Am J Pathol 2000;156:295-301. 4 Suto TS, Fine LG, Shimizu F, Kitamura M: In vivo transfer of engineered macrophages into the glomerulus:Endogenous TGF-[beta]-mediated defence against macrophage-induced glomerular cell activation. J Immunol1997;159:2476-2483. 5 Kitamura M: Identification of an inhibitor targeting macrophage production of monocyte chemoattractantprotein-1 asTGF-[beta]^sub 1^. J Immunol 1997;159:1404-1411. 6 Allen AR, McHale J, Smith J, Cook HT, Karkar A, Haskard DO, Lobb RR, Pusey CD: Endothelial expressionof VCAM-1 in experimental crescentic nephritis and effect of antibodies to very late antigen-4 or VCAM-1 onglomerular injury. J Immunol 1999;162:5519-5527. 7 De Vriese AS, Endlich K, Elger M, Lameire NH, Atkins RC, Lan HY, Rupin A, Kriz W, Steinhausen MW: Therole of selectins in glomerular leukocyte recruitment in rat anti-glomerular basement membraneglomerulonephritis. J Am Soc Ncphrol 1999; 10:2510-2517. 8 Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P, Wilson CB, Harrison JK, Bacon KB: Prevention ofcrescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1. Kidney Int1999;56:612-620. 9 Zernecke A, Weber KS, Erwig LP, Kluth DC, Schropel B, Rees AJ, Weber C: Combinatorial model ofchemokine involvement in glomerular monocyte infiltration. J Immunol 2001; 166:5755-5762. 10 Segerer S, Nelson PJ, Schlondorff D: Chemokines, chemokine receptors and renal disease: From basicscience to pathophysiologic and therapeutic studies. J Am Soc Nephrol 2000; 11:152-176. 11 Segerer S, Cui Y, Hudkins KL, Goodpaster T, Eitner F, MacK M, Schlondorff D, Alpers CE: Expression of thechemokine monocyte chemoattractant protein-1 and its receptor chemokine receptor-2 in human crescenticglomerulonephritis. J Am Soc Nephrol 2000;11:2231-2242. 12 Burke BS: Macrophages in gene therapy: Cellular delivery vehicles and in vivo targets. J Leuk Biol2002;72:417-428. 13 Kluth DC, Erwig LP, Rees AJ: Gene transfer into inflamed glomeruli using macrophages transfected withadenovirus. Gene Ther 2000; 7:263-270. 14 Yokoo T, Utsunomiya Y, Ohashi T, Imasawa T, Kogure T, Futagawa Y, Kawamura T, Eto Y, Hosoya T:Inflamed site-specific gene delivery using bone marrow-derived CD11b^sup +^ CD18^sup +^ vehicle cells inmice. Hum Gene Ther 1998;9: 1731-1738. 15 Yokoo T, Ohashi T, Utsunomiya Y, Kojima H, Imasawa T, Kogure T, Hisada Y, Okabe M, Eto Y, KawamuraT, Hosoya T: Prophylaxis of antibody-induced acute glomerulonephritis with genetically modified bone marrow-derived vehicle colls. Hum Gene Ther 1999;10: 2673-2678. 16 Kluth DC, Ainslie CV, Pearce WP, Clarke D, Anegon I, Rees AJ: Macrophages transfected with adenovirusto express IL-4 reduce inflammation in experimental glomerulonephritis. J Immunol 2001;166:4728-4736.

17 Yamagishi H, Yokoo T, Imasawa T, Mitarai T, Kawamura T, Utsunomiya Y: Genetically modified bonemarrow-derived vehicle cells site specifically deliver an anti-inflammatory cytokine to inflamed interstitium ofobstructive nephropathy. J Immunol 2001;166:609-616. 18 Wilson HM, Stewart K, Brown PAJ, Anegon I, Chettibi S, Rees AJ, Kluth DC: Bone marrow-derivedmacrophages genetically modified to produce IL-10 reduce injury in experimental glomerulonephritis. Mol Ther2002;6:710-717. 19 Yokoo T, Ohashi T, Utsunomiya Y, Shiba H, Shen JS, Hisada Y, Eto Y, Kawamura T, Hosoya T: Inflamedglomeruli-specific gene activation that uses recombinant adenovirus with the Cre/loxP system. J Am SocNephrol 2001;12: 2330-2337. 20 Yokoo T, Ohashi T, Utsunomiya Y, Shen JS, Hisada Y, Eto Y, Kawamura T, Hosoya T: Genetically modifiedbone marrow continuously supplies anti-inflammatory cells and suppresses renal injury in mouse Goodpasturesyndrome. Blood 2001;98:57-64. AuthorAffiliation H.M. Wilson D.C. Kluth Department of Medicine and Therapeutics, Institute of Medical Sciences, Foresterhill, Aberdeen, Scotland AuthorAffiliation H.M. Wilson Department of Medicine and Therapeutics Institute of Medical Sciences Foresterhill, Aberdeen AB25 2ZD (UK) Tel. +44 1224 55835, Fax +44 1224 555766, E-Mail d.c.kluth@abdn.ac.uk MeSH: Adenoviridae -- genetics, Animals, Cell Movement -- physiology, Gene Transfer Techniques, GeneticEngineering -- methods, Glomerulonephritis -- pathology, Glomerulonephritis -- therapy, Humans,Immunotherapy, Adoptive -- methods, Macrophages -- virology, Cell Movement -- genetics (utama), KidneyGlomerulus -- pathology (utama), Macrophage Activation -- genetics (utama), Macrophages -- physiology(utama), Macrophages -- transplantation (utama) Judul: Targeting genetically modified macrophages to the glomerulus Pengarang: Wilson, H M; Kluth, D C Judul publikasi: Nephron Volume: 94 Edisi: 4 Halaman: e113-8 Tahun publikasi: 2003 Tanggal publikasi: Aug 2003 Tahun: 2003 Bagian: Minireview Penerbit: S. Karger AG Tempat publikasi: Basel Negara publikasi: Switzerland Subjek publikasi: Medical Sciences

ISSN: 00282766 CODEN: NPRNAY Jenis sumber: Scholarly Journals Bahasa publikasi: English Jenis dokumen: Journal Article Nomor aksesi: 12972709 ID dokumen ProQuest: 274309492 URL Dokumen: http://search.proquest.com/docview/274309492?accountid=166961 Hak cipta: Copyright S. Karger AG Aug 2003 Terakhir diperbarui: 2014-04-18 Basis data: ProQuest Medical Library

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Wilson, H. M., & Kluth, D. C. (2003). Targeting genetically modified macrophages to the glomerulus. Nephron,94(4), e113-8. Retrieved from http://search.proquest.com/docview/274309492?accountid=166961

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