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Gene transfer of RANTES elicits autoimmune renal injury in MRL-Faslpr mice

1998; Elsevier BV; Volume: 53; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1755.1998.00911.x

1523-1755

Kathryn J. Moore, Takashi Wada, Susannah D. Barbee, Vicki Rubin Kelley,

Cell Adhesion Molecules Research

Infiltrating macrophages and T cells are instrumental in autoimmune kidney destruction of MRL-Faslpr mice. We report that the β-chemokine RANTES, a chemoattractant for macrophages and T cells, is up-regulated in the MRL-Faslpr kidney prior to injury, but not normal kidneys (MRL-++, C3H-++) and increases with progressive injury. Furthermore, we establish an association between RANTES expression in the kidney and renal damage using a gene transfer approach. Tubular epithelial cells genetically modified to secrete RANTES infused under the renal capsule incites interstitial nephritis in MRL-Faslpr, but not MRL-++ or C3H-++ mice. RANTES recruits predominantly macrophages (Mø) and CD4+ and CD8+ T cells. In contrast, gene transfer of CSF-1, another molecule up-regulated simultaneously with RANTES in MRL-Faslpr kidneys, promotes the influx of Mø, CD4+ T cells and the unique double-negative (DN) T cells (CD4-,CD8-), which are prominent in diseased MRL-Faslpr kidneys. Thus, RANTES and CSF-1 recruit distinct T cell populations into the MRL-Faslpr kidney. In addition, delivery of RANTES and CSF-1 into the kidney of MRL-Faslpr mice causes an additive increase in pathology. We suggest that the complementary recruitment of T cell populations by RANTES (CD4, CD8) and CSF-1 (CD4, DN) promotes autoimmune nephritis in MRL-Faslpr mice. Infiltrating macrophages and T cells are instrumental in autoimmune kidney destruction of MRL-Faslpr mice. We report that the β-chemokine RANTES, a chemoattractant for macrophages and T cells, is up-regulated in the MRL-Faslpr kidney prior to injury, but not normal kidneys (MRL-++, C3H-++) and increases with progressive injury. Furthermore, we establish an association between RANTES expression in the kidney and renal damage using a gene transfer approach. Tubular epithelial cells genetically modified to secrete RANTES infused under the renal capsule incites interstitial nephritis in MRL-Faslpr, but not MRL-++ or C3H-++ mice. RANTES recruits predominantly macrophages (Mø) and CD4+ and CD8+ T cells. In contrast, gene transfer of CSF-1, another molecule up-regulated simultaneously with RANTES in MRL-Faslpr kidneys, promotes the influx of Mø, CD4+ T cells and the unique double-negative (DN) T cells (CD4-,CD8-), which are prominent in diseased MRL-Faslpr kidneys. Thus, RANTES and CSF-1 recruit distinct T cell populations into the MRL-Faslpr kidney. In addition, delivery of RANTES and CSF-1 into the kidney of MRL-Faslpr mice causes an additive increase in pathology. We suggest that the complementary recruitment of T cell populations by RANTES (CD4, CD8) and CSF-1 (CD4, DN) promotes autoimmune nephritis in MRL-Faslpr mice. colony stimulating factor-1 double negative interleukin implantation macrophage phosphate buffered saline Regulated upon Activation, Normal T cell Expressed and Secreted tubular epithelial cells transforming growth factor-β tumor necrosis factor-α The MRL-Faslpr mouse is a particularly appealing model of human systemic lupus erythematosus, since renal disease is rapid, predictable and regulated by a single gene mutation[1SINGER G.G. CARRERA A.C. MARSHAK-ROTHSTEN A. MARTINEZ-A C. ABBAS A.K. Apoptosis, Fas and systemic autoimmunity: The MRL-lpr/lpr model.Curr Opin Immunol. 1994; 6: 913-920Crossref PubMed Scopus (128) Google Scholar]. Nephritis in MRL-Faslpr mice is complex, with glomerular, perivascular, and interstitial components, and the rapid disease progression is fatal (50% mortality at 6 months of age)[2KELLEY V.E. ROTHS J.B. Interaction of mutant lpr gene with background strain influences renal disease.Clin Immunol Immunopathol. 1985; 37: 220-229Crossref PubMed Scopus (145) Google Scholar]. By contrast, renal disease in congenic MRL-++ mice lacking the Faslpr mutation is indolent, and is not evident until the second year of life. Thus, the Faslpr mutation accelerates renal disease in the predisposed autoimmune MRL strain[2KELLEY V.E. ROTHS J.B. Interaction of mutant lpr gene with background strain influences renal disease.Clin Immunol Immunopathol. 1985; 37: 220-229Crossref PubMed Scopus (145) Google Scholar]. Fas mediates apoptosis of autoreactive T cells in the periphery. Mutation of this gene (Faslpr) results in the accumulation of a unique subset of “double negative” (DN) T cells (CD4-, CD8-, B220+) in MRL-Faslpr mice[1SINGER G.G. CARRERA A.C. MARSHAK-ROTHSTEN A. MARTINEZ-A C. ABBAS A.K. Apoptosis, Fas and systemic autoimmunity: The MRL-lpr/lpr model.Curr Opin Immunol. 1994; 6: 913-920Crossref PubMed Scopus (128) Google Scholar]. Macrophage (Mø) and T cell infiltration is a prominent component of the renal pathology in MRL-Faslpr mice[3YUI M.A. BRISSETTE W.H. BRENNAN D.C. WUTHRICH R.P. KELLEY V.E. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.Am J Pathol. 1991; 139: 255-261PubMed Google Scholar,4BLOOM R.D. FLORQUIN S. SINGER G.G. BRENNAN D.C. KELLEY V.R. Colony stimulating factor-1 in the induction of lupus nephritis.Kidney Int. 1993; 43: 1000-1009Abstract Full Text PDF PubMed Scopus (84) Google Scholar]. Multiple T cell subsets are present in renal lesions; equal numbers of CD4+ and DN T cells accumulate in perivascular, glomerular, and interstitial lesions, while fewer CD8+ T cells accumulate, predominantly in the interstitium. However, the molecules responsible for recruiting and activating these inflammatory cells in the kidney have not been clearly defined. MRL-Faslpr mice have multiple cytokine abnormalities prior to the onset of autoimmune injury that are thought to promote disease progression. We and others have reported up-regulation of colony stimulating factor-1 (CSF-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, transforming growth factor-Β (TGF-β), and IL-6 in MRL-Faslpr mice[3YUI M.A. BRISSETTE W.H. BRENNAN D.C. WUTHRICH R.P. KELLEY V.E. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.Am J Pathol. 1991; 139: 255-261PubMed Google Scholar, 4BLOOM R.D. FLORQUIN S. SINGER G.G. BRENNAN D.C. KELLEY V.R. Colony stimulating factor-1 in the induction of lupus nephritis.Kidney Int. 1993; 43: 1000-1009Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 5YOKOYAMA H. KREFT B. KELLEY V.R. Biphasic increase in circulating and renal TNF-α in MRL-lpr mice with differing regulatory mechanisms.Kidney Int. 1995; 47: 122-130Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 6BOSWELL J.M. YUI M.A. BURT D.W. KELLEY V.E. Increased tumor necrosis factor and IL-1β expression in the kidneys of mice with lupus nephritis.J Immunol. 1988; 141: 3050-3054PubMed Google Scholar, 7TANG B. MATSUDA T. KISHIMOTO T. Age-associated increase in interleukin-6 in MRL/lpr mice.Int Immunol. 1991; 3: 273-278Crossref PubMed Scopus (140) Google Scholar, 8KREFT B. YOKOYAMA H. NAITO T. KELLEY V.R. Dysregulated transforming growth factor-beta in neonatal and adult autoimmune MRL-lpr mice.J Autoimmun. 1996; 9: 463-472Crossref Scopus (14) Google Scholar]. To define the importance of a single cytokine in the pathogenesis of kidney disease, ideally the cytokine should be expressed in a selected cell type in a specific location in the mature kidney. Gene transfer strategies that tailor cytokine expression in selected sites in the kidney have been limited by the lack of tissue specific promoters, and short-lived and variable expression of the transferred molecules[9BOSCH R. WOOLF A.S. FINE L.G. Gene transfer into mammalian kidney: Direct retrovirus-transduction of regenerating tubular epithelial cells.Exp Nephrol. 1993; 1: 49-54PubMed Google Scholar, 10ISAKA Y. FUJIWARA Y. IMAI E. Glomerulosclerosis induced by in vivo transfection of transforming growth factor-β or platelet-derived growth factor gene into the rat kidney.J Clin Invest. 1993; 92: 2597-2601Crossref PubMed Scopus (489) Google Scholar, 11KITAMURA M. TAYLER S. FINE L.G. Gene transfer into the rat renal glomerulus via a mesangial cell vector: Site-specific delivery, in situ amplification, and sustained expression of an exogenous gene in vivo.J Clin Invest. 1994; 94: 497-505Crossref PubMed Scopus (128) Google Scholar]. To overcome these obtacles, we devised an ex vivo gene transfer system using retrovirally modified tubular epithelial cells (TEC) as a carrier cell to deliver cytokines into the kidney[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar]. Syngeneic tubular epithelial cells, genetically engineered ex vivo to express a cytokine, are infused under the renal capsule, where they remain localized and provide stable, sustained delivery of cytokine into the kidney. Using this gene transfer approach, we established that CSF-1, but not TNF-α or IL-6, elicits renal injury in MRL-Faslpr mice[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar, 13MOORE K.J. NAITO T. MARTIN C. KELLEY V.R. Enhanced response of macrophages to CSF-1 in autoimmune mice. A gene transfer strategy.J Immunol. 1996; 157: 433-440PubMed Google Scholar, 14MOORE K.J. YEH K. NAITO T. KELLEY V.R. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.J Immunol. 1996; 157: 427-432PubMed Google Scholar, 15NAITO T. YOKOHAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. A gene transfer approach establishes IL-6 neither promotes nor suppresses renal injury.Am J Physiol. 1996; 271: F603-F609Google Scholar]. In spontaneous disease, CSF-1 is up-regulated in MRL-Faslpr, but not congenic MRL-++ mice, normal strains or other strains with the Faslpr mutation, well in advance of renal disease, and increases in proportion to the severity of kidney damage[3YUI M.A. BRISSETTE W.H. BRENNAN D.C. WUTHRICH R.P. KELLEY V.E. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.Am J Pathol. 1991; 139: 255-261PubMed Google Scholar,16BRENNAN D.C. JEVNIKAR A.J. BLOOM R.D. BRISSETTE W.H. SINGER G.G. RUBIN-KELLEY V.E. Cultured mesangial cells from autoimmune MRL-lpr mice have decreased secreted and surface M-CSF.Kidney Int. 1992; 42 (abstract): 279Abstract Full Text PDF PubMed Scopus (19) Google Scholar]. Our finding that introduction of CSF-1 initiates renal damage in MRL-Faslpr mice is supported by compelling evidence from experiments in which single nephritic MRL-Faslpr kidneys transplanted into MRL-++ mice resulted in a loss of CSF-1 expression, the disappearance of Mø and the reversal of renal lesions[17NAITO T. GRIFFITHS R.C. COFFMAN T.M. KELLEY V.R. Transplant approach establishes that kidneys are responsible for serum CSF-1 but require a stimulus in MRL-lpr mice.Kidney Int. 1996; 49: 67-74Abstract Full Text PDF PubMed Scopus (20) Google Scholar]. Taken together, we conclude that CSF-1 is responsible for attracting and fostering Mø and T cells in the MRL-Faslpr kidney. This prompted us to determine whether other molecules in addition to CSF-1 contributed to the accumulation of Mø and T cells in MRL-Faslpr kidneys. The migration of leukocytes into an inflammatory site requires the generation of a chemotactic gradient by cells of the extravascular compartment. The β-chemokine, RANTES (Regulated upon Activation, Normal T cell Expressed and Eecreted), is chemotactic for monocytes and T cells, and induces cellular activation of these leukocytes[18TAUB D.D. OPPENHEIM J.J. Chemokines, inflammation and the immune system.Ther Immunol. 1994; 1: 229-246PubMed Google Scholar, 19SCHALL T.J. BACON K. TOY K.J. GOEDDEL D.V. Selective attraction of monocytes and T lymphocytes of the memory phenotype by the chemokine RANTES.Nature. 1990; 347: 669-671Crossref PubMed Scopus (1234) Google Scholar, 20WANG J.M. MCVICAR D.W. OPPENHEIM J.J. KELVIN D.J. Identification of RANTES receptors on human monocytes: Competition for binding and desensitization by homologous chemotactic cytokines.J Exp Med. 1993; 177: 699-705Crossref PubMed Scopus (106) Google Scholar]. RANTES is expressed by renal mesangial and tubular epithelial cells (TEC)[21Robson RL, Westwick J, Brown Z: Differential regulation of RANTES in response to IL-1α and TNF-α in human mesangial cells. J Am Soc Nephrol (abstract)Google Scholar, 22HEEGER P. WOLF G. MEYERS C. Isolation and characterization of cDNA from renal tubular epithelium encoding murine RANTES.Kidney Int. 1992; 41: 220-225Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 23THAISS F. HELMCHEN U. ZAHNER G. HABERSTROH U. RADOUNIKLI N. SCHOEPPE W. STAHL R.A. muRANTES mRNA expression in glomeruli isolated from rats with autologous nephrotoxic serum nephritis.J Am Soc Nephrol. 1993; 4: 638Google Scholar], and by T lymphocytes in vitro[24CONLON K. LLOYD A. CHATTOPADHYAY U. LUKACS N. KUNKEL S. SCHALL T. TAUB D. MORIMOTO C. OSBORNE J. OPPENHEIM J. YOUNG H. KELVIN D. ORTALDO J. CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein-1α, interleukin-8 and RANTES.Eur J Immunol. 1995; 25: 751Crossref PubMed Scopus (97) Google Scholar]. In addition, RANTES can promote the adhesion of invading leukocytes to matrix components of glomeruli[25GILAT D. HERSHKOVIZ R. MEKORI Y.A. VIODAVSKY L. LIDER O. Regulation of adhesion of CD4+ T lymphocytes to intact or heparinase-treated subendothelial matrix by diffusible or anchored RANTES or MIP-1α.J Immunol. 1994; 153: 4899-4906PubMed Google Scholar]. Thus, the release of RANTES by intrinsic renal cells may initiate the influx of leukocytes into the kidney and promote autoimmune inflammatory renal injury. Therefore, we tested the hypothesis that RANTES initiates Mø and T cell recruitment into the MRL-Faslpr kidney. We now report that RANTES is expressed in the kidney of MRL-Faslpr mice prior to the onset of autoimmune renal injury. Using a retroviral gene transfer strategy, the consequence of RANTES expression in the recruitment and propagation of inflammatory cells was evaluated in the MRL-Faslpr kidney. RANTES fostered the accumulation of a distinct subset of T cells (CD4+ >CD8+ >DN), and initiates renal damage. By contrast, CSF-1, which is simultaneously up-regulated, promoted the influx of DN and CD4+ T cells, but few CD8+ T cells. Thus, we propose that RANTES and CSF-1 are complementary in promoting autoimmune nephritis by their recruitment of Mø and different T cell subsets into the MRL-Faslpr kidney. MRL/MpJ-++ (MRL-++), MRL/MpJ-Faslpr/Faslpr (MRL-Faslpr) and C3H-HeJ (C3H-++) mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA) or bred in our pathogen-free animal facility. Tubular epithelial cells (TEC) derived from MRL-Faslpr, MRL-++ and C3H-++ mice one to two months of age were isolated as previously described[26WUTHRICH R.P. GLIMCHER L.H. KELLEY V.E. MHC class II, antigen presentation and tumor necrosis factor in renal tubular epithelial cells.Kidney Int. 1990; 37: 783-792Abstract Full Text PDF PubMed Scopus (233) Google Scholar]. Briefly, kidneys were removed and renal cortices were minced, dispersed in collagenase solution, and passed through a series of steel sieves (250, 150, 75 and 38 μm). Cells were collected, resuspended in modified K1 medium (50% DMEM, 50% Ham’s F12, 10% FCS, 100 U/ml each of penicillin and streptomycin, 10 mM HEPES), and incubated for one hour at 37°C. Non-adherent cells were transfered to Primaria tissue culture plates (Falcon Labware) and grown to confluence. Prior to retroviral infection, TEC were trypsinized and plated at 1 × 106 cells/10-cm plate for 18 hours. CRIP packaging cell lines producing helper-free recombinant retroviruses carrying cytokine genes were generated as previously described[27DANOS O. MULLIGAN R.C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.Proc Natl Acad Sci USA. 1988; 85: 6460-6464Crossref PubMed Scopus (793) Google Scholar]. Briefly, the DNA sequence encoding RANTES was subcloned into the MoMuLV-based MFG vector containing the long terminal repeat viral promoter and the ψ sequence necessary for viral RNA packaging. The MFG vector carrying the RANTES gene was transfected into a 3T3 packaging cell line (CRIP) containing the proviral sequences necessary for encapsidation of the recombinant viral RNA. The resulting virus-producing cell line elaborates recombinant retroviruses that stably transfer the recombinant viral genome containing the RANTES gene upon infection of a suitable host cell. Producer cells were grown in DMEM complete medium (10% FCS, 100 U/ml each of penicillin and streptomycin, 10 mM HEPES) to subconfluence (5 × 106 cells/10 cm dish), replenished with 8 ml of fresh medium and incubated for 18 hours. The virus-containing cell culture supernatant was harvested, filtered through a 0.45 μm membrane, and applied to TEC cultures as previously described[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar]. Following retroviral infection, TEC were replenished with K1 medium, and grown to confluence. RANTES gene transfer into TEC was verified and quantitated by Northern analysis as described above. The retroviral transfer of the CSF-1 and TNF-α gene into TEC has been previously established in our laboratory and characterized in detail[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar, 13MOORE K.J. NAITO T. MARTIN C. KELLEY V.R. Enhanced response of macrophages to CSF-1 in autoimmune mice. A gene transfer strategy.J Immunol. 1996; 157: 433-440PubMed Google Scholar, 14MOORE K.J. YEH K. NAITO T. KELLEY V.R. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.J Immunol. 1996; 157: 427-432PubMed Google Scholar]. Genetically modified cytokine producing TEC retain the ability to produce cytokine in vitro for at least 10 months, and upon implantation, persist under the renal capsule for up to 28 days[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar]. Prior to infusion under the renal capsule, production of CSF-1 by transduced TEC was verified by bone marrow proliferation assay, and production of TNF-α was verified by ELISA assay (Endogen, Woburn, MA, USA) as previously described[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar,14MOORE K.J. YEH K. NAITO T. KELLEY V.R. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.J Immunol. 1996; 157: 427-432PubMed Google Scholar]. In this manuscript, we abbreviate TEC transduced with the RANTES, CSF-1, and TNF-α genes as RANTES-TEC, CSF-1-TEC, or TNF-TEC, respectively. RNA was extracted and Northern blot analyses were performed as previously described[3YUI M.A. BRISSETTE W.H. BRENNAN D.C. WUTHRICH R.P. KELLEY V.E. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.Am J Pathol. 1991; 139: 255-261PubMed Google Scholar]. Total RNA was isolated from cultured TEC or dissected renal cortices using RNAzol B (Tel-Test Inc., Friendswood, TX, USA), which is a modification of the guanidium thiocyanate-phenol-chloroform method[28CHOMCZYNSKI P. SACCHI N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar]. Total RNA (20 μg) was electrophoresed through a 1% agarose-formaldehyde gel, blotted to nylon membrane and hybridized in 50% formamide with 32P-labeled nick-translated probes at 42°C. Hybridized membranes were washed in 2 × SSC, 0.1% SDS at room temperature and 0.2 × SSC, 0.1% SDS at 60°C. Autoradiography was performed using a PhosphoImager (Molecular Dynamics Corp., Sunnyvale, CA, USA). The RANTES probe was kindly provided by Dr. E. Neilson (University of Pennsylvania Medical Center, PA, USA) and consisted of a 450 kb fragment of the PBS KS plasmid containing the cDNA. Blots were reprobed with β-actin (Pst-1 fragment of pBA-1) as an internal control for quantity and integrity of RNA. Densitometry analysis was performed to quantitate mRNA expression. TEC were cultured in chamber slides (2 × 104 cells/well) overnight and treated with 5 μM monensin (Boehringer Mannheim) for three hours prior to staining. TEC were fixed in acetone and incubated for 30 minutes with 2.5% goat serum, followed by goat anti-human RANTES Ab (20 μg/ml; 1 hr; R & D Systems), which is weakly cross-reactive for mouse RANTES. Cells were rinsed three times with phosphate buffered saline (PBS) and immunoreactivity was visualized by incubating with rabbit anti-goat IgG-FITC conjugated Ab for one hour. Cell culture supernatants (48 hr) were collected from 5 × 106 MRL-Faslpr and C3H-++ RANTES-TEC or uninfected TEC and concentrated fourfold using centricon concentrators-3 (Amicon, Beverly, MA, USA). Samples were heat denatured in the presence of Laemmli sample buffer containing 5% 2-mercaptoethanol and electrophoresed on a 15% linear gradient SDS-PAGE gel. Proteins were transfered to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) in the presence of Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) using a wet electrophoretic transfer apparatus. Membranes were blocked in TBS-T (Tris buffered saline/0.1% Tween-20) containing 5% (wt/vol) non-fat dry milk for one hour, and then incubated in fresh buffer containing rabbit anti-murine RANTES Ab (1:2000) for one hour. Membranes were washed in TBS-T and incubated with peroxidase conjugated anti-rabbit Ab (1:20000) in TBST-T/5% dry milk. Immunoreactive proteins were visualized using the enhanced chemiluminescence detection system (Dupont NEN, Boston, MA, USA) and exposed to X-ray film. Using our gene transfer strategy, we have previously established that delivery of CSF-1 under the renal capsule incites an accumulation of Mø and T cells in the kidney of MRL-Faslpr mice six weeks of age or older, but not in younger mice[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar]. Therefore, we delivered RANTES into the kidney of mice that were 6 to 8 weeks of age at the time of TEC implantation to study the impact of RANTES overexpression on the kidney. We placed RANTES-TEC, CSF-1-TEC, or uninfected TEC under the renal capsule of syngeneic MRL-Faslpr, MRL-++ or C3H-++ mice as previously described[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar]. We anesthetized mice with sodium pentobarbital and ether, and exposed the left kidney through a flank incision. A cell suspension of 1 × 106 TEC in 50 μl of HBSS was infused under the renal capsule, and the peritoneum and skin were sutured. Mice were sacrificed 7, 14 or 28 days post-implantation (post-Ix) and the implanted kidney and control contralateral kidney were removed for histological evaluation. To establish the combined effect of CSF-1 and RANTES on the MRL-Faslpr kidney, we placed equal numbers of (1 × 106) RANTES-TEC and CSF-1-TEC, or RANTES-TEC and uninfected TEC, or CSF-1-TEC and uninfected TEC under the renal capsule of MRL-Faslpr mice as described above. Mice were sacrificed at 28 days post-Ix and the implanted kidney and control contralateral kidney were removed for histological evaluation. Kidneys were halved and fixed in 10% phosphate-buffered formalin for paraffin-embedded sections or snap-frozen in OCT compound (Miles, Naperville, IL, USA). Paraffin-embedded tissue sections were stained by the hematoxylin and eosin (H & E) method and evaluated by light microscopy. Sequential frozen sections (6 μ) were taken every 250 μ and stained with toluidine blue to identify the area of largest cell accumulation in the implant site, as previously described[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar, 13MOORE K.J. NAITO T. MARTIN C. KELLEY V.R. Enhanced response of macrophages to CSF-1 in autoimmune mice. A gene transfer strategy.J Immunol. 1996; 157: 433-440PubMed Google Scholar, 14MOORE K.J. YEH K. NAITO T. KELLEY V.R. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.J Immunol. 1996; 157: 427-432PubMed Google Scholar]. The mean subcapsular cell accumulation in TEC implanted kidneys was evaluated by counting the cell layers in the area of accumulation under the capsule. Intrarenal lesions emanating from the subcapsular implant were scored according to the depth of infiltration into the kidney: 0 = none, 1 = cortex, 2 = inner cortex, 3 = medulla. To evaluate T cell and Mø accumulation in the kidney, we stained sequentially sectioned tissue specimens by the immunoperoxidase technique using purified rat anti-mouse Abs to F4/80 Ag (Mø), CD4 (T cell), CD8 (T cell), and CD21/35 (B cell) and the B220 determinant (B cell, DN T cell) (Pharmingen, San Diego, CA, USA) as previously described[12NAITO T. YOKOYAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. Macrophage growth factors introduced into the kidney initiate renal injury.Molec Med. 1995; 2: 297-312Google Scholar, 13MOORE K.J. NAITO T. MARTIN C. KELLEY V.R. Enhanced response of macrophages to CSF-1 in autoimmune mice. A gene transfer strategy.J Immunol. 1996; 157: 433-440PubMed Google Scholar, 14MOORE K.J. YEH K. NAITO T. KELLEY V.R. TNF-α enhances colony-stimulating factor-1-induced macrophage accumulation in autoimmune renal disease.J Immunol. 1996; 157: 427-432PubMed Google Scholar, 15NAITO T. YOKOHAMA H. MOORE K.J. DRANOFF G. MULLIGAN R.C. KELLEY V.R. A gene transfer approach establishes IL-6 neither promotes nor suppresses renal injury.Am J Physiol. 1996; 271: F603-F609Google Scholar]. We identified the unique DN T cells (CD4-,CD8-, CD21/35-, B220+) by examining sequential sections. In brief, endogenous peroxidase (0.6% H2O2, 0.2% sodium azide; 10 min), and avidin and biotin (Avidin/Biotin blocking kit; Vector Laboratories, Burlingame, CA, USA) were blocked prior to incubating the tissue sections with primary Abs (5 μg/ml, 2 hr). Sections were washed in TBS/BSA 0.1% and incubated with biotinylated goat anti-rat IgG (mouse adsorbed) for one hour. We exposed the tissue sections to avidin-peroxidase complex (Vector Elite Kit; Vector Laboratories) for one hour, followed by 3-3′-diamino-benzidine (0.5 mg/ml in TBS containing 0.02% H2O2) for a chromogenic reaction and counterstained with methyl green/alcian blue. Specificity controls included replacement of primary Ab with normal rat IgG. Immunohistochemical staining was analyzed by counting the number of F4/80+, CD4+, CD8+, or CD21/35-B220+ in more than six random fields (×40) in the induced lesion. These analyses were performed independently by two investigators in a blinded fashion. Statistical analyses were performed using the Mann-Whitney μ test. By Northern analysis, we determined that RANTES is expressed in the MRL-Faslpr kidney prior to overt renal injury, and increases with advancing renal pathology Figure 1. RANTES mRNA is expressed in MRL-Faslpr kidney cortex as early as two months of age, and is highly up-regulated in MRL-Faslpr kidney cortex at six months of age during fulminant renal disease. In contrast, RANTES mRNA is absent in mice with normal kidneys, including congenic MRL-++ (2 and 10 months of age) and C3H-++ (5 months of age) strains. This pattern of RANTES up-regulation in the MRL-Faslpr kidney parallels that previously described for CSF-1 and TNF-α[3YUI M.A. BRISSETTE W.H. BRENNAN D.C. WUTHRICH R.P. KELLEY V.E. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.Am J Pathol. 1991; 139: 255-261PubMed Google Scholar, 4BLOOM R.D. FLORQUIN S. SINGER G.G. BRENNAN D.C. KELLEY V.R. Colony stimulating factor-1 in the induction of lupus nephritis.Kidney Int. 1993; 43: 1000-1009Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 5YOKOYAMA H. KREFT B. KELLEY V.R. Biphasic increase in circulating and renal TNF-α in MRL-lpr mice with differing regulatory mechanisms.Kidney Int. 1995; 47: 122-130Abstract Full Text PDF PubMed Scopus (90) Google Scholar]. Primary renal TEC were retrovirally transduced with the RANTES gene sequence, to create a cellular vehicle for the constitutive delivery of RANTES into the kidney. We verified that the transduced TEC expressed RANTES in culture Figure 2. Both MRL-Faslpr and C3H-++ TEC constitutively express high levels of RANTES mRNA after retroviral gene transfer Figure 2A. By contrast, uninfected MRL-Faslpr and C3H-++ TEC do not express endogenous RANTES mRNA. RANTES protein expression by transduced TEC was confirmed by staining for the presence of RANTES by immunohistochemistry Figure 2B. RANTES was detected in the cytoplasm of > 90% of MRL-Faslpr and C3H-++ RANTES-TEC, but not in uninfected TEC of either strain. Furthermore, MRL-Faslpr and C3H-++ RANTES-TEC secreted similar levels of RANTES into the culture media as determined by Western analysis Figure 2C. These data confirm that the RANTES gene was successfully integrated into the TEC genome resulting in constitutive expression, and that uninfected TEC do not express RANTES. To determine whether RANTES expre