Faculty Bibliography

Macrophage expression of matrix degrading metalloproteinases (MMPs) in human atheroma has been found to occur in rupture-prone areas of plaques. To investigate the effect of metalloproteinase activity on plaque stability, we attempted to generate mice that expressed a stromelysin-1 (MMP-3) transgene specifically in macrophages. Promoter sequences taken from a macrophage-tropic lentivirus (visna) were used to drive transgene expression. The transgene construct was expressed in macrophages in vitro and its autoactivation was established by casein zymography. Transgenic mice generated with this construct died at or before birth. No gross anatomical changes were observed in these mice. Embryos arising from a second round of oocyte injections with the transgene were examined at day 16 of gestation. Of the products of conception, approximately 40% resulted in vacant conceptuses. Only one animal of 38 examined carried the transgene and its expression of MMP-3 mRNA at E16 was faintly detected by RT-PCR. When a non-toxic reporter gene, luciferase, was substituted for the MMP-3 cDNA, healthy transgenic mice were produced that expressed the reporter gene in a wide variety of tissue macrophages, including those located in the brain, testis, lung, and thymus. These studies suggest that constitutive expression of MMP-3 in diverse populations of tissue macrophages leads to prenatal or neonatal death in the mouse. It appears likely that more sophisticated transcriptional control of MMP-3 expression will be required in order to generate stromelysin-1 transgenic mice that could be useful models for studying overexpression of this metalloproteinase's activity in the lesional macrophages of atherosclerotic plaques

The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPAR gamma, is known to promote fat cell differentiation in vitro. Whether PPAR gamma is required for this process in vivo has remained an open question because a viable loss-of-function model for PPAR gamma has been lacking. We demonstrate here that mice chimeric for wild-type and PPAR gamma null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPAR gamma for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPAR gamma gene dosage. These data provide direct evidence that PPAR gamma is essential for the formation of fat.

Monocytes/macrophages (Mo) appear to play a critical role in the initiation and progression of atherosclerotic lesions. In this study, we characterized in vitro-differentiated embryonic stem (ES) cell macrophages as a model system for studying atherosclerosis-associated Mo functions. Using immunofluorescence staining and Western analysis, we demonstrate that ES Mo express typical macrophage cell surface markers, as well as the known receptors for modified forms of low density lipoprotein (LDL), including the Mo scavenger receptors (SR-A type I and type II), CD36, and CD68. Differentiated ES Mo specifically bind and degrade 125I-labeled acetylated LDL with high affinity, and their incubation with acetylated LDL (15 microg/mL) for 48 hours produces characteristic 'foamy' Mo, as visualized by oil red O staining. ES Mo also express matrix-degrading metalloproteinases (MMP-3, MMP-9), which have been implicated in collagen breakdown in the fibrous cap of atherosclerotic plaques, and secrete cytokines (tumor necrosis factor-alpha, interleukin-6) in response to inflammatory stimuli. Transfection experiments, using a green fluorescent protein reporter gene, driven by the myeloid-specific promoter, CD11b, demonstrated that ES Mo can also be used to study macrophage-restricted gene expression in vitro. Taken together, these data demonstrate that ES Mo exhibit many properties typical of arterial lesion macrophages. Its ease of genetic manipulation makes it an attractive system for investigations of macrophage functions in vitro

We report that the beta-chemokine RANTES, a chemoattractant for macrophages and T cells, is up-regulated in the MRL-Fas(1pr) 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-Fas(1pr), but not MRL-++ or C3H-++ mice. RANTES recruits predominantly macrophages (M phi) and CD4+ and CD8+ T cells. In contrast, gene transfer of CSF-1, another molecule up-regulated simultaneously with RANTES in MRL-Fas(1pr) kidneys, promotes the influx of M phi, CD4+ T cells and the unique double-negative (DN) T cells (CD4-, CD8-), which are prominent in diseased MRL-Fas(1pr) kidneys. Thus, RANTES and CSF-1 recruit distinct T cell populations into the MRL-Fas(1pr) kidney. In addition, delivery of RANTES and CSF-1 into the kidney of MRL-Fas(1pr) 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-Fas(1pr) mice.

IFN-gamma is capable of enhancing and limiting inflammation. Therefore, an increase in IFN-gamma in autoimmune MRL-Fas(lpr) mice could exacerbate or thwart renal injury. We have established a retroviral gene transfer approach to incite interstitial nephritis in MRL-Fas(lpr) mice that is rapid, enduring, and circumscribed. Renal tubular epithelial cells (TEC) were genetically modified to secrete macrophage (Mphi) growth factors (CSF-1-TEC, GM-CSF-1-TEC) and infused under the renal capsule. To determine the impact of IFN-gamma in Mphi growth factor-incited renal injury, we constructed a MRL-Fas(lpr) IFN-gamma-receptor (IFN-gammaR)-deficient strain. Gene transfer of CSF-1 or GM-CSF incited more severe interstitial nephritis in IFN-gammaR-deficient than in IFN-gammaR-intact MRL-Fas(lpr) mice, consisting of an increase of Mphi. To determine the mechanism responsible for the increase in Mphi in IFN-gammaR-deficient MRL-Fas(lpr) mice, we evaluated Mphi proliferation, apoptosis, and recruitment. Proliferation of bone marrow Mphi from IFN-gammaR-intact MRL-Fas(lpr) costimulated with CSF-1 or GM-CSF and IFN-gamma was reduced twofold, while the IFN-gammaR-deficient MRL-Fas(lpr) bone marrow Mphi remained stable. Furthermore, we detected more proliferating and fewer apoptotic Mphi within the interstitium in IFN-gammaR-deficient MRL-Fas(lpr) mice. Using unilateral ureteral ligation we established that IFN-gammaR signaling does not alter Mphi recruitment into the kidney. Thus, the increase in Mphi elicited by Mphi growth factors in IFN-gammaR-deficient MRL-Fas(lpr) mice is a result of enhanced proliferation and decreased apoptosis, and is independent of recruitment. Taken together, we suggest that IFN-gamma provides a negative regulatory pathway capable of limiting Mphi-mediated renal inflammation.

Conflicting reports claim that circulating interleukin (IL)-6 promotes or suppresses renal disease. Although autoimmune MRL-lpr mice have an increase in serum IL-6, and kidneys can produce IL-6, the relevance of systemic and local exposure remains undefined. To investigate the impact of IL-6 on kidney disease, we constructed a gene transfer approach to deliver sustained, stable IL-6 into the kidney and circulation. We infused syngeneic genetically modified tubular epithelial cells (IL-6-TEC) under the renal capsule of autoimmune and nonautoimmune mice. IL-6-TEC did not incite renal injury in any strain. Furthermore, serum IL-6 levels, which were increased three- to fivefold by IL-6-TEC, did not alter the contralateral kidney. Therefore, neither local nor systemic exposure to IL-6 promoted renal injury. As opposed to IL-6, we previously established that granulocyte macrophage (GM)-colony-stimulating factor (CSF) initiates renal injury in autoimmune mice. To determine whether IL-6 could suppress GM-CSF-incited damage, we infused GM-CSF-TEC TEC along with IL-6-TEC. Local production of IL-6 into the kidney did not alter the tempo or severity of GM-CSF-induced injury. Thus neither local nor systemic delivery of IL-6 promotes or suppresses kidney disease.

Mice with the MRL background have a genetic propensity for autoimmune lupus nephritis. The lpr mutation on the MRL, but not the C3H background, induces rapid and fatal renal injury in which macrophages (M phi) are prominent. We previously established that CSF-1 accompanies M phi accumulation in the kidney of MRL-lpr mice. Furthermore, CSF-1 introduced into the kidney incites renal injury in mice with the lpr mutation, but not congenic strains. Notably, CSF-1 induces more severe tissue injury in MRL-lpr than in C3H-lpr mice. We hypothesized that M phi from the MRL background respond more readily to CSF-1 than normal strains. We establish herein the following: 1) glomerular M phi and bone marrow M phi (BMM phi) from MRL-lpr mice proliferate similarly to CSF-1; 2) MRL BMM phi proliferate more vigorously to CSF-1 than normal strains (C3H, BALB/c) or another strain with lpr (C3H-lpr); and 3) modulation of CSF-1 receptor expression by CSF-1 is more rapid in MRL than C3H BMM phi. We used a gene transfer strategy to deliver CSF-1 into the kidney to evaluate M phi response to CSF-1. We genetically modified tubular epithelial cells to produce CSF-1 (CSF-1-TECs) and placed these cells with BMM phi under the renal capsule. CSF-1-TEC + BMM phi caused a greater accumulation of M phi in the implant site and interstitium of MRL +/+ than C3H +/+ mice. Furthermore, CSF-1-TEC + BMM phi caused a lesion consisting of M phi in MRL +/+ mice, extending from the implant into the adjacent cortex. We suggest that the response of MRL M phi to CSF-1 is responsible for the notable accumulation of M phi in the MRL-lpr kidney.

The lpr mutation on the MRL background accelerates autoimmune nephritis in which macrophage (M phi) accumulation is prominent. Renal disease is absent in other strains with lpr. TNF-alpha and CSF-1 are increased in the kidney of MRL-lpr mice with loss of renal function. We have established that CSF-1 can incite renal injury in mice with the lpr mutation, and M phi from the MRL strain hyper-respond to this growth factor. We hypothesized that TNF-alpha enhanced the M phi response to CSF-1 in MRL-lpr mice. We now report that TNF-alpha enhanced CSF-1-induced bone marrow M phi proliferation in MRL-lpr mice, and not in congenic MRL +/+, normal C3H +/+, and BALB/c, or another strain with lpr (C3H-lpr). Using a gene transfer approach to deliver CSF-1 together with TNF-alpha into the kidney, we evaluated the impact on renal injury. Tubular epithelial cells genetically modified to produce CSF-1 (CSF-1-TEC) and TNF-alpha (TNF-TEC) placed under the renal capsule caused a greater accumulation of M phi in the implant site than CSF-1-TECs alone in MRL-lpr, but not MRL +/+ mice. We noted in tissues adjacent but not distal to the implanted TECs, an increase in M phi in the interstitium and surrounding glomeruli of MRL-lpr but not MRL +/+ mice. This indicated that CSF-1 and TNF-alpha released by TECs were responsible for promoting renal pathology. Taken together, these data suggest that the simultaneous expression of TNF-alpha and CSF-1 in the MRL-lpr kidney fosters M phi accumulation. We speculate that the increase in M phi in the kidney in response to CSF-1 and TNF-alpha is responsible for the rapid tempo of autoimmune renal injury in MRL-lpr mice.

BACKGROUND:CSF-1 expression precedes renal injury in autoimmune MRL-lpr mice and is responsible for macrophage (M phi) proliferation and survival in the kidney. By comparison, C3H-lpr mice do not express CSF-1 in the kidney, and despite the lpr mutation, kidneys remain normal. The purpose of this study was to test the capacity of local and systemic expression of M phi growth factor, CSF-1 to initiate renal injury in normal (C3H-(++), MRL-(++) and autoimmune (C3H-lpr, MRL-lpr) mice. MATERIALS AND METHODS/METHODS:We designed a gene transfer system to deliver cytokines into the kidney by transducing renal tubular epithelial cells (TEC) using retroviral vectors expressing CSF-1 or another M phi growth factor, GM-CSF. We placed transduced syngeneic cytokine-TEC under the renal capsule of normal and autoimmune prone mice prior to renal injury and evaluated renal pathology at 3, 7, 14, 28, and 90 days postimplant. RESULTS:CSF-1-TEC and GM-CSF-TEC, but not uninfected TEC, caused extensive local renal injury in strains with the lpr mutation. At 3-7 days the infiltrating cells were mainly M phi, and by 28 days they were predominantly lymphocytes. By comparison, the kidneys of MRL-(++) and C3H-(++) mice remained normal. Implanted genetically modified TEC caused a sustained increase of CSF-1 or GM-CSF in the circulation which did not modify the contralateral kidney. CONCLUSIONS:Gene transfer of M phi growth factors into the kidney initiates severe local renal injury in autoimmune prone mice with the lpr mutation, but does not compromise the kidney in nonautoimmune hosts. Of note, introduction of M phi growth factors into the kidney of C3H-lpr mice which do not spontaneously develop renal injury incites renal damage. These studies offer a gene transfer approach to explore the impact of local and systemic cytokine production on renal injury.