Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice.

Infiltrating leukocytes may be responsible for autoimmune disease. We hypothesized that the chemokine monocyte chemoattractant protein (MCP)-1 recruits macrophages and T cells into tissues that, in turn, are required for autoimmune disease. Using the MRL-Fas(lpr) strain with spontaneous, fatal autoimmune disease, we constructed MCP-1-deficient MRL-Fas(lpr) mice. In MCP-1-intact MRL-Fas(lpr) mice, macrophages and T cells accumulate at sites (kidney tubules, glomeruli, pulmonary bronchioli, lymph nodes) in proportion to MCP-1 expression. Deleting MCP-1 dramatically reduces macrophage and T cell recruitment but not proliferation, protects from kidney, lung, skin, and lymph node pathology, reduces proteinuria, and prolongs survival. Notably, serum immunoglobulin (Ig) isotypes and kidney Ig/C3 deposits are not diminished in MCP-1-deficient MRL-Fas(lpr) mice, highlighting the requirement for MCP-1-dependent leukocyte recruitment to initiate autoimmune disease. However, MCP-1-deficient mice are not completely protected from leukocytic invasion. T cells surrounding vessels with meager MCP-1 expression remain. In addition, downstream effector cytokines/chemokines are decreased in MCP-1-deficient mice, perhaps reflecting a reduction of cytokine-expressing leukocytes. Thus, MCP-1 promotes MRL-Fas(lpr) autoimmune disease through macrophage and T cell recruitment, amplified by increasing local cytokines/chemokines. We suggest that MCP-1 is a principal therapeutic target with which to combat autoimmune diseases.

Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis.

Monocyte chemoattractant protein-1 (MCP-1) is upregulated in renal parenchymal cells during kidney disease. To investigate whether MCP-1 promotes tubular and/or glomerular injury, we induced nephrotoxic serum nephritis (NSN) in MCP-1 genetically deficient mice. Mice were analyzed when tubules and glomeruli were severely damaged in the MCP-1-intact strain (day 7). MCP-1 transcripts increased fivefold in MCP-1-intact mice. MCP-1 was predominantly localized within cortical tubules (90%), and most cortical tubules were damaged, whereas few glomerular cells expressed MCP-1 (10%). By comparison, there was a marked reduction (>40%) in tubular injury in MCP-1-deficient mice (histopathology, apoptosis). MCP-1-deficient mice were not protected from glomerular injury (histopathology, proteinuria, macrophage influx). Macrophage accumulation increased adjacent to tubules in MCP-1-intact mice compared with MCP-1-deficient mice (70%, P < 0.005), indicating that macrophages recruited by MCP-1 induce tubular epithelial cell (TEC) damage. Lipopolysaccharide-activated bone marrow macrophages released molecules that induced TEC death that was not dependent on MCP-1 expression by macrophages or TEC. In conclusion, MCP-1 is predominantly expressed by TEC and not glomeruli, promotes TEC and not glomerular damage, and increases activated macrophages adjacent to TEC that damage TEC during NSN. Therefore, we suggest that blockage of TEC MCP-1 expression is a therapeutic strategy for some forms of kidney disease.

Detection of intracellular interleukin-10 by flow cytometry.

Detection of cytokines is limited to measurements of the secreted molecule. To circumvent this problem we permeabilized the cell membrane with digitonin and localize cytokine expression using antibodies by flow cytometry. In this report we demonstrate that we can detect specific intracellular interleukin-10 (IL-10) in the HT-2 T cell line only after membrane permeabilization. With this technique intracellular cytokines are readily detectable.

Delayed omega-3 fatty acid supplements in renal transplantation. A double-blind, placebo-controlled study.

An earlier reported trial suggests that omega-3 fatty acids in fish oil supplements at 6 g/day with administration commencing at the time of engraftment may reduce acute CsA renal dysfunction. When started at the time of renal transplant, there are improvements in renal hemodynamics and blood pressure, and a decrease in rejection episodes. To examine the effect of later introduction of omega-3 fatty acids, 133 cadaver renal transplant recipients received CsA, prednisone, and AZA for 16 weeks (period 1). If patients were stable without rejection or infection activity, they were randomized to 9 g of eicosapentanoic acid (EPA), 18 g of EPA, 9 g of corn oil, or 18 g of corn oil in 1-g capsules as supplements. Glomerular filtration rate, renal blood flow, number of rejection episodes, blood pressure, and episodes of CsA nephrotoxicity were followed for 26 weeks in a double-blind manner (period 2). Ninety patients were evaluable and completed the protocol. There were 50 corn oil placebo patients, 22 low dose EPA patients, and 18 high dose EPA patients. In period 1, there were 27 rejection episodes in 21 patients without differences among subsequent treatment groups. In period 2, there were 13 rejection episodes in 4 patients. No patient with an EPA level in plasma statistically higher than placebo had a rejection episode. All allografts functioned for the entire 6 months with none lost to rejection. All 5 episodes of acute CsA nephrotoxicity occurred in placebo-treated patients without differences in whole blood CsA among toxic patients, other placebo patients, and EPA-treated recipients. At the end of the study, there were no differences in glomerular filtration rate, renal blood flow, or creatinine clearance among groups. Diastolic blood pressure fell by 9 mmHg during period 2 in high dose fish oil recipients and by 10 mmHg in low dose fish oil recipients (P < 0.05), while it rose by 2 mmHg in placebo patients. No serious adverse effects of EPA supplements were noted, although compliance based on plasma EPA was erratic. Based on our experience and that in the literature, administration of omega-3 fatty acids for purposes of kidney protection would seem to be most useful when started early after surgery. Late administration in our study was associated with minor clinical benefits.

Cytokines in the pathogenesis of systemic lupus erythematosus.

Cytokines, chemokines, and growth factors are overexpressed by renal parenchymal cells and by infiltrating mononuclear cells in human and experimental lupus nephritis. The importance of cytokines in the pathogenesis of lupus nephritis has been established using spontaneous mouse models of systemic lupus erythematosus (SLE). The actions of these cytokines are complex. There is a growing appreciation that the cytokine level and stage of kidney disease determines whether cytokine protects or promotes further tissue injury. This article identifies potential therapeutic targets and strategies that might halt progressive renal injury in patients with SLE.

Lupus nephritis: update on pathogenesis and disease mechanisms.

Immune-mediated nephritis is a common complication of systemic lupus erythematosus (SLE). It is now clear that multiple and independent mechanisms contribute to disease onset and pathogenesis, which may explain the remarkable phenotypic and histopathological heterogeneity observed in human SLE. Identification and characterization of disease-relevant autoantibodies, cellular effectors, and soluble immune elements have provided crucial insight into the immunologic interactions that promote renal immune injury. It is now clear that nephritogenic autoantibodies of diverse specificity localize to the kidney by a variety of mechanisms. They are accompanied by activated macrophages and T cells recruited in part through enhanced and abnormal production of macrophage growth factors and cytokines. These pathways provide novel targets for therapeutic intervention to prevent or ameliorate the aggressive autoimmune nephritis that characterizes SLE.

Enhanced tumor necrosis factor in anti-CD3 antibody stimulated diabetic NOD mice: modulation by PGE1 and dietary lipid.

Administration of OKT3 anti-CD3 monoclonal antibody (mAb) to patients for transplant rejection, is associated with a distinct and often severe clinical syndrome related to massive cytokine release. Previous reports have similarly demonstrated increased levels of serum tumor necrosis factor alpha (TNF alpha) in normal mice following administration of 1452-C11 anti-CD3 mAb. In this study, we compared serum TNF alpha levels at baseline and after anti-CD3 stimulation among three groups of mice: normal BALB/c controls, pre-diabetic non-obese diabetic (NOD) mice, and diabetic NOD mice. Baseline serum TNF alpha levels, as measured by L929 cell bioassay, were 2xhigher in diabetic NOD and 3xhigher in pre-diabetic NOD compared with BALB/c. Ninety minutes after anti-CD3 mAb stimulation, serum from BALB/c controls and pre-diabetic NOD contained 2- to 8-fold higher levels of TNF-alpha as compared to untreated control mice. In contrast, following anti-CD3 mAb, there was a dramatic 20-fold increase in serum TNF alpha in diabetic NOD mice (levels > 5000 pg/ml). Additionally, anti-CD3 mAb increased the steady-state TNF alpha mRNA transcripts. Spleens from diabetic mice given anti-CD3 mAb had higher steady-state TNF alpha mRNA than spleen from normal mice similarly treated. The enhanced release of circulating TNF alpha after anti-CD3 mAb in diabetic NOD mice was abrogated by pre-treatment of mice with prostaglandin E1 (PGE1) 30 min prior to anti-CD3 mAb stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal tubular epithelial and T cell interactions in autoimmune renal disease.

Interaction between epithelial cells and T cells may initiate autoimmune tissue destruction. Renal tubular epithelial cells may participate in such immune interactions since they: (1) can be induced to express surface molecules which facilitate engagement with T cells; (2) secrete and express membrane bound cytokines; (3) are exposed to peptides from blood and the glomerular filtrate and are capable of processing these potentially immunogenic peptides. We have recently established T cell clones captured from the interstitium of MRL-lpr mice with lupus nephritis. These T cell clones are unique and are regulated by the lpr gene. They express the alpha/beta T cell receptor, and beta cell markers, but do not display CD4 or CD8 on their surface. These T cell clones proliferate to renal tubular cells but not to cells from other tissues and secrete IFN-gamma which induces class II and ICAM-1 on renal tubular epithelial cells. Expression of class II and ICAM-1 induced by IFN-gamma renders these epithelial cells capable of triggering T cell hybridomas to proliferate and secrete IL-2. Therefore, renal tubular epithelial cells are capable of processing and presenting antigen. This review will focus on the dynamic interaction of renal epithelial cells and T cells and discuss its importance in the initiation of autoimmune renal injury.

Self-regulation of autoreactive kidney-infiltrating T cells in MRL-lpr nephritis.

MRL-lpr kidney-infiltrating (KI) T cell clones (CD3+, TCR alpha/beta+, B220+, CD4-, CD8-) are autoreactive, exclusively proliferate to renal tissues, and secrete interferon-gamma (IFN-gamma). We now report that IFN-gamma treatment of tubular epithelial cells (TEC) decreases their ability to induce KI T cell proliferation. The decreased ability of IFN-gamma-treated TEC to induce T cell proliferation is evident by 24 hours and can be restored by re-exposure to TEC not treated with IFN-gamma. IFN-gamma-treated TEC supernatant does not diminish KI T cell proliferation and IFN-gamma-treated TEC fixed with glutaraldehyde remain less capable of inducing KI T cell proliferation. Although we have not identified the TEC surface molecule(s) modified by IFN-gamma, neither class I, class II, ICAM-1 nor IFN-gamma bound to the surface of TEC are responsible. In conclusion, IFN-gamma induces a surface alteration(s) on TEC capable of limiting their ability to induce KI T cell proliferation. The ability of autoreactive KI T cells to release IFN-gamma represents a self-regulatory mechanism for limiting T cell expansion.

The antigen presentation function of renal tubular epithelial cells.

Renal tubular epithelial cells (TEC) have the capacity to function as antigen-presenting cells (APC). The processing of native antigen and the presentation of peptides bound to major histocompatibility complex (MHC) class II products on TEC results in engagement of the T cell antigen receptor (TCR). TEC also express a variety of adhesion molecules and cytokines which may enhance their interaction with T cells. The expression of intercellular adhesion molecules (ICAM-1) and vascular cell adhesion molecules (VCAM-1) on TEC enhances their adherence to T cells. Immune-activated TEC display and/or secrete numerous cytokines including tumor necrosis factor alpha (TNF alpha) which may provide accessory signals for T cells and upregulate TEC adhesion molecule receptors. We review the two signals required for T cell activation and suggest a model whereby T cell interaction with TEC can have two possible sequelae: (1) T cell activation or (2) T cell unresponsiveness. TEC and other parenchymal cells could potentially be important in maintaining peripheral tolerance to tissue-specific self-antigens if Ia (signal 1) and costimulatory signals (signal 2) are not induced simultaneously. Conversely, coordinate expression of signals 1 and 2 would inevitably lead to organ-specific immune injury. Studies to further elucidate the nature of T cell interactions with parenchymal cells are clearly essential for a more complete understanding of the pathogenesis of autoimmunity.

A paradigm 1994: the allograft response.

This review presents our concepts regarding the mechanisms of rejection and the pathway leading to graft tolerance. Allograft rejection is a T-cell-dependent process. Accordingly, we discuss the current understanding of T cell activation and cytokine production and then apply these concepts to the mechanisms that cause rejection and tolerance. We describe the ability of renal epithelial cells to either induce T cell activation or anergy. Finally, we review potential mechanisms responsible for the maintenance of tolerance.

Gene transfer in the kidney.

Gene transfer approaches offer the promise of revolutionizing medicine. In this review, we focus on the current and future prospects of somatic gene transfer into the kidney. The advantages and disadvantages of current vector systems are described, and the ex vivo and in vitro approaches applicable to the kidney are reviewed. We discuss uses of gene transfer approaches to dissect the pathogenesis of kidney disease and the future directions and applications of gene transfer to combat kidney destruction.

Dysregulated transforming growth factor-beta in neonatal and adult autoimmune MRL-lpr mice.

Transforming growth factor- beta (TGF- beta) is a cytokine that promotes inflammatory processes and prevents tissue injury. Autoimmune destruction of the kidney in MRL-lpr mice is spontaneous, rapid, fatal and consists of glomerular damage and an influx of lymphocytes surrounding vessels and in the interstitium. In MRL-lpr mice, cytokine dysregulation is apparent in neonates and continues throughout the life span. Circulating levels of tumour necrosis factor (TNF- alpha) and colony stimulating factor-1 (CSF-1) are detected in neonatal mice and progressively increase in proportion to the loss of renal function. We now report elevated intracellular expression of distinct isoforms of TGF- beta (TGF- beta 3, TGF- beta 2, and TGF- beta 1) detected immunohistochemically in MRL-lpr kidneys and other tissues including the liver and thymus. Enhanced TGF- beta 3 and TGF- beta 2 isoforms are detectable in neonatal mice within the renal tubular epithelial cells (TEC) and vascular smooth muscle cells (VSMC). In MRL-lpr mice 4-6 months of age, TGF- beta 2 and TGF- beta 1 are detected in TEC, VSMC, glomerular epithelial cells (GEC) and in perivascular infiltrating cells. By comparison, TGF- beta is minimally detectable in the normal kidneys of age and sex matched MRL(-)+2 or C3H/Fej mice. Paradoxically, in vitro cultured TEC and VSMC from MRL-lpr mice secrete less TGF- beta than TEC and VSMC isolated from MRL(-)+2 or C3H/FeJ mice. TNF- alpha, but not IL-6, CSF-1, or IFN- gamma stimulated the secretion of TGF- beta in TEC and VSMC. Our data demonstrate the dysregulation of TGF- beta isoforms in neonatal and adult MRL-lpr mice prior to and after the onset of autoimmune renal disease. We suggest that TNF- alpha and/or other molecules increase TGF- beta expression in MRL-lpr mice. We speculate that enhanced expression of TGF- beta promotes autoinmune renal injury in MRL-lpr mice.

Biphasic increase in circulating and renal TNF-alpha in MRL-lpr mice with differing regulatory mechanisms.

Tumor necrosis factor (TNF)-alpha contributes to expansion of lymphocytes in neonatal mice and can accelerate renal injury. T cells induced by the lpr gene promote renal injury. However, the lpr gene alone is insufficient to cause renal damage, since MRL-lpr, but not C3H-lpr mice develop lupus nephritis. In this study, we examined the temporal expression of TNF-alpha in the kidney and circulation of mice (MRL and C3H) with the lpr gene and their congenic counterparts (++). We measured a bioactive TNF-alpha using L929 cells and tissue expression with an avidin-biotin immunoperoxidase method. A biphasic increase in circulating TNF-alpha in MRL-lpr mice was detected. There was an initial peak in neonatal mice (703 +- 208 pg/ml) which normalized by two months of age (87 +- 13 pg/ml) and reascended proportional to the severity of renal injury (non-proteinuric 570 +/- 87, proteinuric; 1255 +/- 135 pg/ml). In addition, there was only a single peak in neonatal C3H-lpr mice (1270 +/- 318 pg/ml) with a nadir by six weeks of age (434 +/- 52 pg/ml). In contrast, serum TNF-alpha was low in MRL-(++) and C3H-(++) mice (80 +/- 3 and 95 +/- 30 pg/ml), respectively. TNF-alpha expression in kidneys paralleled the serum pattern in MRL-lpr mice. Enhanced TNF-alpha expression was restricted to tubular epithelial cells (TEC) in neonatal MRL-lpr and C3H-lpr mice, and not detected in congenics. In adult mice, intrarenal TNF-alpha expression was more ubiquitous and was detected in glomeruli, vascular smooth muscle and perivascular infiltrating cells as well as TEC.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of genetically engineered tubular epithelial cells in kidney disease.

We constructed an ex vivo gene transfer system to deliver cytokines into the kidney and circulation using genetically modified renal tubular epithelial cells (TEC). TEC were infected with recombinant retroviruses expressing macrophage (Mphi) growth factors using a retroviral Moloney murine leukemia virus-based MFG vector. These infected TEC have the capacity to secrete stable and sustained amounts of cytokines for prolonged periods (>4 months) in vitro and in vivo (>28 days). Implanting genetically modified TEC secreting Mphi growth factors under the kidney capsule initiates severe local renal injury in mice with a deficiency in Fas (Faslpr gene). This system offers a novel and powerful approach to probe for the impact of sustained cytokine expression in the progression of kidney destruction.

Colony stimulating factor-1 in the induction of lupus nephritis.

In this study we examine the role of colony stimulating factor-1 (CSF-1) in the induction of lupus nephritis. The purpose of the study was to establish the relationship of CSF-1 to the prominent influx of macrophages (M phi) in the glomeruli of MRL-lpr mice with autoimmune lupus nephritis. The kidneys of MRL-lpr mice were examined before (< 12 weeks of age) and after (> 12 weeks of age) renal injury for CSF-1 transcripts by in situ hybridization. CSF-1 mRNA was detected at four weeks of age within glomeruli and increased with disease severity. To examine whether glomerular M phi (glom M phi) required CSF-1 we isolated M phi from the kidneys of MRL-lpr mice. Two types of glom M phi (with morphological and growth characteristics which correlated with the presence or absence of proteinuria) were isolated. Under CSF-1-free culture conditions, where the viability of glom M phi from proteinuric mice was maintained, glom M phi from pre-proteinuric mice were unable to survive. Neutralization of CSF-1 in the media reduced viability of pre-proteinuric glom M phi (5 to 6 x), while viability of proteinuric glom M phi was diminished < 1.5 x. Additionally, CSF-1 supplementation induced a 10 x proliferation of pre-proteinuric glom M phi when compared to CSF-1-free medium. In contrast, proteinuric glom M phi did not proliferate in response to CSF-1. These studies suggest that CSF-1 induces macrophage proliferation and differentiation within glomeruli and, in turn, renal injury.

Costimulation by B7-1 and B7-2 is required for autoimmune disease in MRL-Faslpr mice.

Autoimmune lupus nephritis is dependent on infiltrating autoreactive leukocytes and Igs. B7 costimulatory molecules (B7-1 and B7-2) provide signals essential for T cell activation and Ig class switching. In MRL-Faslpr mice, a model of human lupus, although multiple tissues are targeted for autoimmune injury, nephritis is fatal. We identified intrarenal B7-1 and B7-2 expression, restricted to kidney-infiltrating leukocytes, before and increasing with progressive nephritis in MRL-Faslpr mice. Thus, we hypothesized that the B7 pathway is required for autoimmune disease in MRL-Faslpr mice. To investigate the role of B7 costimulatory molecules in this autoimmune disease, we generated a MRL-Faslpr strain deficient in B7-1 and B7-2. Strikingly, MRL-Faslpr mice lacking both B7 costimulators do not develop kidney (glomerular, tubular, interstitial, vascular) pathology, or proteinuria, and survive far longer. Intrarenal downstream effector transcripts (IFN-gamma, IL-12, monocyte chemoattractant protein-1, CSF-1) linked to nephritis remained at normal levels compared with wild-type mice. Skin lesions and lymphoid enlargement characteristic of MRL-Faslpr mice were diminished in B7-1/B7-2-deficient MRL-Faslpr mice. B7-1/B7-2-deficient MRL-Faslpr mice did not develop leukocytic infiltrates, elevated serum IgG and isotypes (G1,G2b,G3), autoantibodies, and intrarenal IgG deposits. Our findings demonstrate that B7-1 and B7-2 costimulatory pathways are critical to the pathogenesis of autoimmune lupus.

An anergic, islet-infiltrating T-cell clone that suppresses murine diabetes secretes a factor that blocks interleukin 2/interleukin 4-dependent proliferation.

The mechanism of peripheral immunological tolerance has not been fully established. While anergic T cells have been noted in tolerant hosts, the mechanism by which they contribute to the induction and maintenance of tolerance has not been defined. As we previously reported, an accelerated form of diabetogenic autoimmunity in nonobese diabetic mice can be blocked by passive transfer of a CD3+, CD8+, beta-chain variable region 11-positive islet-infiltrating T-cell clone (IS-2.15). In this report we examine the properties of this T-cell clone. We have established that this clone is unresponsive to mitogenic concentrations of anti-T-cell receptor or anti-CD3 monoclonal antibodies and is only weakly responsive to syngeneic islet and spleen cells. Moreover, these T cells secrete an inhibitory factor(s) that irreversibly inhibits interleukin (IL) 2/IL-4-driven proliferation of IL-2/IL-4 indicator T-cell lines. This noncytotoxic factor, which possesses an apparent size of 10-30 kDa, does not interfere with low-affinity IL-2 receptor expression. These data indicate that at least some anergic T cells can play an active role in peripheral tolerance by secreting suppressor factor(s) that regulate IL-2/IL-4-dependent proliferation.

IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice.

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Fas(lpr) mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-gamma, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-gamma-secreting T cells within the kidney, which, in turn, incites injury in MRL-Fas(lpr) mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Fas(lpr) tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Fas(lpr) mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-gamma-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 +/- 14%) as compared with 7 days (20 +/- 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60-70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-gamma was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Fas(lpr) kidneys genetically deficient in IFN-gamma receptors. Furthermore, IFN-gamma "carrier cells" elicited kidney injury in wild-type MRL-Fas(lpr) mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-gamma-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Fas(lpr) mice.