Transgenic tubular cell expression of class II is insufficient to initiate immune renal injury.

Autoimmune disease in mouse models of lupus nephritis is associated with enhanced renal tubular epithelial cell (TEC) expression of major histocompatibility complex (MHC) class II (Ia) molecules. It is unknown whether de novo TEC expression of syngeneic la alone can initiate immune attack or whether expression is secondary to cytokines released by infiltrating lymphocytes. To establish if the expression of high levels of self-MHC molecules alone can initiate immune renal injury in the adult animal, kidneys from transgenic C57BL/6 (B6) mice (Ins-I-E) bearing constitutively high levels of I-Eb on proximal TEC were transplanted into nephrectomized male B6 x C3H F1 hybrid mice (I-Eb/k). Control mice received kidneys from I-Eb negative, nontransgenic B6 mice, and all transplant recipient mice were evaluated for renal disease. At the end of the study (> 8.3 months mean survival), the transgenic transplant recipients did not become proteinuric (< 1+ urinary protein) and had normal serum creatinine levels (control = 95 +/- 8 versus transgenic transplants = 116 +/- 23 mumol/L; N = four/group), and the kidneys remained histologically normal. These results establish that the expression of high levels of transgenic MHC class II molecules on TEC is insufficient to initiate autoimmune injury in this model. It is suggested that, in addition to MHC class II molecules, other signals or accessory molecules are required from TEC to initiate immune renal injury.

Interleukin 2 receptor targeted fusion toxin (DAB486-IL-2) treatment blocks diabetogenic autoimmunity in non-obese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) is strikingly similar in the non-obese diabetic (NOD) mouse and humans. In IDDM, the systematic autoimmune destruction of insulin-producing beta cells within the pancreas is dependent on autoreactive T cells. This autoimmune process can be accelerated by transferring spleen cells from diabetic donors into irradiated syngeneic NOD mice. In a previous study we established that interleukin 2 receptor (IL 2R)-bearing cells propagated from pre-diabetic NOD mice promote IDDM. Therefore, we reasoned that specific elimination of IL 2R+ T cells should abort the diabetogenic process. T cell expressing IL 2R can be selectively destroyed with a diphtheria toxin-related IL 2 fusion protein (DAB486-IL-2). We set DAB486-IL-2 the challenging task of preventing fulminant IDDM accelerated by the adoptive transfer of diabetic spleen cells. Eight weeks after the adoptive transfer only 10% and 20% of NOD mice treated with 10 and 5 micrograms/day of DAB486-IL-2, respectively, became diabetic while 100% control mice (vehicle buffer) became diabetic within 5 weeks. A dose of 1 microgram/day of DAB486-IL-2 had no protective effect. Although the protection conferred by DAB486-IL-2 is not permanent, it is maintained for at least 4 weeks following cessation of treatment. Furthermore, even though these NOD mice do eventually become diabetic, the tempo of expression and severity of diabetes, as assessed by the level of hyperglycemia, is dramatically reduced. Although histologic examination of pancreas revealed minimal degree of mononuclear infiltrate within the islets in both groups, the vehicle control mice had fewer islets per section indicating many islets had already been destroyed. In addition, spleen cells from diabetic NOD mice which were pre-treated with DAB486-IL-2 (10 micrograms/day) for 1 week lost their ability to transfer disease. Taken together, these studies strongly support the concept that IL 2R-bearing T cells are essential for the induction of IDDM and suggest that DAB486-IL-2 would be a promising therapeutic approach in the treatment of human IDDM.

Immunotoxins and cytokine toxin fusion proteins.

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or "magic bullets" through the fusion of cell-specific antibodies and toxins. In practice it has proven difficult to create safe and effective "magic bullets." In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell- or lineage-specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.

Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.

Abnormal macrophages in MRL-lpr mice are implicated in the pathogenesis of autoimmune disease. These mice die of lupus nephritis by 5 to 6 months of age. This study reports that MRL-lpr mice have an increased level of circulating macrophage colony-stimulating factor (M-CSF) detectable as early as 1 week of age. Macrophage colony-stimulating factor decreased between 2 and 4 months and then steadily increased beginning at 4 months of age. In contrast, M-CSF was not detected in sera from congenic MRL-++ mice, normal C3H/FeJ mice, two other mouse strains with the lpr gene (B6-lpr and C3H-lpr), or another lupus model, the NZB/W mouse. These observations indicate that the lpr gene alone is not responsible for inducing this growth factor, and elevated M-CSF is not required for all forms of murine lupus. The entire source of serum M-CSF is not clear. The unique T cells regulated by the lpr gene are not responsible for the increased serum M-CSF levels, as no M-CSFs could be detected in supernatants from cultured lymph nodes from MRL-lpr mice, and the steady-state levels of M-CSF mRNA in lymph nodes and spleens in MRL-lpr, C3H-lpr mice and in their respective congenic strains were similar. The steady-state M-CSF mRNA transcripts in liver, lung, and bone marrow in MRL-lpr, MRL-++, and C3H/FeJ mice were also similar. Macrophage colony-stimulating factor transcripts were clearly elevated in the kidneys of MRL-lpr mice, suggesting a renal source of circulating M-CSF. The increase of M-CSF might be responsible for the increased numbers and enhanced functions of macrophages, which in turn cause tissue destruction in MRL-lpr mice.

Stimulated kidney tubular epithelial cells express membrane associated and secreted TNF alpha.

Tumor necrosis factor-alpha (TNF alpha) is a pleiotropic, pro-inflammatory peptide cytokine which promotes immune renal injury, and participates in T cell activation. It is produced by macrophages, T cells, and some non-hematopoietic cells, and is cytotoxic in picogram quantities. As renal tubular epithelial cells (TEC) bearing MHC class II (Ia) antigens and adhesion molecules (ICAM-1) can act as immune accessory cells, the ability of TEC to produce costimulatory cytokines could augment TEC accessory capacity in vivo. We report that transformed TEC express low levels of TNF alpha in response to LPS or IL-1 alpha as a secreted product and as a cytotoxic membrane associated molecule displayed on the cell surface. Surface labelling and immunoprecipitation studies of TEC detect a number of bands including a prominent 26 kD protein, which is the predicted size of TNF alpha precursor. TNF alpha mRNA transcripts were also detected by in situ hybridization in cortical tubules of C3H/FeJ mice injected with LPS, demonstrating the capacity of normal tubular epithelial cells to express TNF alpha in vivo. This report demonstrates for the first time the ability of kidney tubular cells to express TNF alpha protein and that membrane associated TNF alpha is not limited to hematopoietic cells. The function of small amounts of TNF displayed on the surface of tubular cells may be amplified by the abundance of these cells within the renal cortex, and may allow TEC to modulate immune responses within the kidney during inflammation.

Antigen presentation by renal tubular epithelial cells.

The interaction between immune effector cells such as T lymphocytes and parenchymal cells in organ-specific immune injury is dynamic. It is now appreciated that the specificity, intensity, and eventual destructive effects of such interactions can be greatly influenced by responses of the target issue. Renal tubular cells are particularly well suited to participate in such immune collaborations. (1) They are exposed to innumerable potentially immunogenic peptides from blood and glomerular filtrate and have pathways to further process these peptides; (2) they express surface molecules which facilitate their engagement to T cells; and (3) they can produce proinflammatory cytokines. In the models of immune-mediated tubulointerstitial injury that are currently studied, there has been a great interest in defining the T lymphocytes that initiate, accelerate, or suppress disease. Surprisingly, there has been relatively little attention on defining the tubular cell responses that regulate these immune-mediated processes. This review will therefore focus on this intriguing aspect of immune tubular injury and relate what is known about antigen presentation by tubular cells in autoimmune renal disease.

Islet-infiltrating T cell clones from non-obese diabetic mice that promote or prevent accelerated onset diabetes.

In humans and non-obese diabetic mice (NOD), insulin-dependent diabetes mellitus (IDDM) results from a spontaneous T cell-dependent autoimmune destruction of the insulin-producing pancreatic beta cells. Previous data suggest that a delicate balance between autoaggressive T cells and suppressor-type immune phenomena determine whether expression of autoimmunity is limited to insulitis or progresses to IDDM. To resolve the cellular basis of this intricate network of pathogenic CD4+ and CD8+ T cells and the role of T cells in suppressive immune phenomena. T cell clones were propagated directly from islets of NOD mice at the onset of insulitis. Insofar as insulitis, but not IDDM, is universal in NOD mice, we have screened for the in vivo effects of the islet-infiltrating T cell clones upon expression of IDDM, not insulitis. A CD4+ T cell clone, IS-3S7D, proliferates in response to islet antigen(s) and its transfer into prediabetic NOD mice promotes the rapid onset of IDDM. An interleukin 2 (IL 2)-dependent noncytolytic, V beta 11+ CD8+. T cell clones IS-2.15, prevents an accelerated onset diabetes in two distinct models. The present study, which documents the presence of CD4+ diabetogenic T cell clones and CD8+ T cell clones that dampen autoimmunity, gives tangible evidence that opposing autoimmune processes may determine whether an autoimmune-prone host develops frank disease.

Concomitant administration of hapten and IL-2-toxin (DAB486-IL-2) results in specific deletion of antigen-activated T cell clones.

During the proliferative burst after Ag recognition, T cells express cell-surface, high-affinity IL-2R. The importance of IL-2R+ T cells in supporting/mediating tissue injury has been documented by the ability of mAb anti-IL2R therapies to prevent allograft rejection and autoimmunity. The delayed-type hypersensitivity (DTH) response, an experimental model of T-dependent immunity, offers the possibility of studying responses mounted against defined Ag. We previously reported that the chimeric IL-2 toxin (DAB486-IL-2) prevents DTH responses and selectively eliminates activated IL-2R bearing CD4 and CD8 T cells from lymph nodes draining the site of inflammation. We have examined the duration of immunosuppression and relative specificity of action of DAB486-IL-2 and anti-CD3 mAb for Ag-activated clones in a murine model of DTH using two different noncross-reacting haptens. Treatment with DAB486-IL-2 generates a state of selective unresponsiveness to subsequent challenge with the hapten introduced during the therapeutic period. Immediately after cessation of DAB486-IL-2 therapy, immunization with an unrelated hapten induces a normal vigorous immune response. By comparison, anti-CD3 mAb treatment causes a broad immunosuppression because unrelated haptens introduced after anti-CD3 therapy do not evoke a vigorous immune response. After cessation of DAB486-IL-2 toxin treatment response to the hapten is eventually restored probably by cells trafficking from the thymus, because thymectomized hosts remain unresponsive to the hapten. Taken together these data reinforce the role of the IL-2R as an important target for immunosuppression in T cell-mediated immune reactions. DAB-486-IL-2 treatment confers highly selective immunosuppression.

Mesangial cell accessory functions: mediation by intercellular adhesion molecule-1.

Mesangial cell (MC) proliferation is an early pathologic alteration characteristic of many forms of immune mediated glomerulonephritis. The intracapillary position, contractile capacity, and production of cytokines and other inflammatory molecules place MC in a pivotal position to initiate, mediate, and direct glomerular damage. We as well as others have noted increased levels of cytokines including IFN gamma, TNF, and IL-1 and the cell surface MHC class II and ICAM-1 molecules in the kidneys of mice with lupus nephritis. MHC class II and ICAM-1 molecules are central to the interaction of T cells with antigen presenting cells (APC). Since cytokines can increase both MHC class II and ICAM-1 molecules, we investigated whether mesangial cells could function as APC or accessory cells after cytokine stimulation. For these studies we established a permanent MC line through transformation with origin-deficient SV40 DNA. Surface expression of ICAM-1 was similar in untransformed MC as well as SV40 transformed MC from normal mice and in untransformed cells from mice with lupus nephritis. Basal expression of ICAM-1 was upregulated rapidly by IFN gamma, TNF, and IL-1. MHC class II expression could not be induced with TNF or IL-1 alone but required prolonged stimulation with IFN gamma. MC adhered and presented antigen to an antigen specific IaK restricted T cell hybridoma. Anti-ICAM-1 mAb decreased adhesion and antigen presentation of cytokine stimulated MC. By comparison, MHC class II mAb abrogated antigen presentation by MC bearing MHC class II but did not block adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunotoxins and cytokine toxin fusion proteins.

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or 'magic bullets' through the fusion of cell specific antibodies and toxins. In practice it has proven difficult to create safe and effective 'magic bullets'. In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell or lineage specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.

Differing regulation and function of ICAM-1 and class II antigens on renal tubular cells.

Intercellular adhesion molecule-1 (ICAM-1) and MHC class II (Ia) antigens are increased on proximal tubular epithelial cells (TEC) in autoimmune nephritis and transplant rejection. ICAM-1 is a member of the immunoglobulin gene superfamily that increases the avidity of T cell interactions with antigen-presenting cells bearing Ia antigens. Using an existing mAb and cDNA probe for Ia antigen, and a newly described mAb (YN1/1.7.4) and cDNA probe for murine ICAM-1, we compared mRNA transcript levels, surface expression and function of these molecules in transformed TEC derived from normal (C3H/FeJ) and autoimmune (MRL-lpr) mice. No differences were found on TEC between these strains of mice. Stimulation of TEC with interferon-gamma (IFN-gamma) resulted in the expression of Ia antigens, and a marked increase of ICAM-1 from basal levels. Increases in ICAM-1 levels occurred with concentrations of IFN-gamma 10 to 100 times lower (0.5 to 1.0 U/ml) than those required for Ia expression, and preceded Ia antigen expression by more than 48 hours. Anti-ICAM-1 mAb lowered the binding and antigen-presenting ability of TEC to the A2A2 T cell hybridoma, suggesting a role for ICAM-1 in immune interactions between TEC and T cells. Dexamethasone treatment of MRL-lpr mice abrogated the increase of Ia antigens found in the kidneys of nephritic mice yet did not reduce the expression of ICAM-1 in either kidneys or cultured stimulated TEC. We conclude that elevated ICAM-1 expression on TEC increases the immune accessory cell capability of TEC bearing Ia antigens, and is resistant to down-regulation by some immunosuppressive agents.

Dose-related mechanisms of immunosuppression mediated by murine anti-CD3 monoclonal antibody in pancreatic islet cell transplantation and delayed-type hypersensitivity.

Although anti-CD3 mAb therapy is used extensively in clinical transplantation, the dose-related effects and mechanisms of action are not clearly defined. We have examined the dose-related effects of an antimurine CD3 mAb, 145-2C11, in pancreatic islet cell allograft and the delayed type hypersensitivity reaction models of T-cell-dependent immunity. Low-dose anti-CD3 therapy (0.5 micrograms/day) administered over several days mediated superficially equal, effective clinical immunosuppression as a single high-dose intravenous injection (400 micrograms). T cells harvested from animals treated with high-dose anti-CD3 were unresponsive to in vitro restimulation. In contrast, T cells isolated from low-dose treated animals retained in vitro proliferative capacity when restimulated with polyvalent anti-CD3 mAb. The terminal complement components were not required to support in vivo immunosuppression mediated by anti-CD3 mAb as C5 deficient mice were immunosuppressed by the administration of this mAb. In some pancreatic islet cell allograft recipients, permanent engraftment, but not tolerance, was achieved. Replacement of donor leukocytes produced acute rejection in hosts bearing long-term, well-accepted grafts. Prolonged anti-CD3 mAb treatment may provide sufficient time for replacement or inactivation of donor leukocytes.

Prostaglandin E2 inhibits oxygen consumption in rabbit medullary thick ascending limb.

The effect of prostaglandin (PG) E2 on transport-dependent oxygen consumption (QO2) of rabbit medullary thick ascending limb (MTAL) cells was studied. Exogenous PGE2, at a concentration of 30 microM, inhibited ouabain-sensitive QO2 by 70%. Addition of either ouabain or bumetanide, after PGE2, further depressed QO2, whereas PGE2 had no effect when added after these transport inhibitors. There was no significant inhibition of QO2 by PGE2 in the absence of either Na or Cl. The QO2 of amphotericin-treated cells was inhibited by the addition of PGE2. Therefore the inhibitory effect of PGE2 was on the transport-dependent moiety of QO2 and was independent of Na entry. Other prostanoids had no significant effect on MTAL QO2. Suspensions of isolated MTAL cells accumulated PGE2 at about one-fifth the rate of outer medullary collecting duct cells. Finally, PGE2 caused an increase in intracellular adenosine 3',5'-cyclic monophosphate levels by approximately 100%. Although the precise mechanism of action is unclear, PGE2, which is synthesized by several cell types in the renal medulla, exerts an inhibitory effect on transport in rabbit MTAL.

The cascading, interrelated roles of interleukin-1, interleukin-2, and interleukin-6 in murine anti-CD3-driven T cell proliferation.

T cell stimulation occurs following interaction of T cell receptor (TcR) with processed antigen presented by autologous accessory cells (AC). The effects of antigen stimulation on T cells are mimicked by monoclonal antibodies (Mabs) defining proteins of the TcR-CD3 complex. In this study, we examine the roles of T cell density, AC density, divalent and polyvalent forms of anti-CD3 Mab, and the cytokines interleukin (IL)-1, IL-2, and IL-6 in T cell activation and proliferation. Stringently AC-depleted T cells do not proliferate in response to Con A or divalent anti-CD3; however, polyvalent anti-CD3 provides a powerful signal for isolated resting T cell proliferation. Recombinant (r)IL-2 strongly amplifies T cell proliferation in response to anti-CD3, whereas rIL-1 exerts no direct effects on anti-CD3-stimulated T cells. In the presence of AC, however, rIL-1 augments T cell proliferation to anti-CD3. Recombinant IL-6 promotes T cell proliferation among T cells stimulated with polyvalent but not divalent anti-CD3. As deduced by Northern blot analysis, rIL-1 increases cytoplasmic levels of IL-6 mRNA in AC. Recombinant IL-6, in turn, amplifies the accumulation of stable IL-2 transcripts in purified T cells stimulated with polyvalent anti-CD3. Hence, IL-1, IL-6, and IL-2 support T cell proliferation through cascading effects at the level of gene transcription. The cytokines evaluated in this study, however, do not fully reconstitute AC functions in promoting anti-CD3 Mab T cell proliferation.

Intercellular adhesion molecule-1 (ICAM-1) expression is upregulated in autoimmune murine lupus nephritis.

Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface protein regulating interactions among immune cells. To determine whether altered expression of ICAM-1 occurs in autoimmune lupus nephritis, we studied ICAM-1 expression in kidneys of normal and autoimmune MRL-lpr and (NZBX NZW)F1 (NZB/W) mice. By immunoperoxidase staining, ICAM-1 is constitutively expressed at low levels in proximal tubules (PT), endothelium and interstitial cells in normal C3H/FeJ mice. In nephritic MRL-lpr and NZB/W kidneys, staining for ICAM-1 is increased in the PT, particularly in the brush border, and is prominent in the glomerular mesangium and the endothelium of large vessels. By Western blot analysis, ICAM-1 is not detected in the urine of normal BALB/c and C3H/FeJ or autoimmune MRL-lpr. By Northern blot analysis, nephritic MRL-lpr and NZB/W have a two- to fivefold increase in steady state levels of ICAM-1 transcripts in the kidney as compared with normal or prenephritic mice. This is paralleled by an increase in MHC class II transcripts. In cultured PT cells, ICAM-1 is expressed at basal levels in PT and is increased by the cytokines interferon-gamma, IL-1 alpha, and TNF-alpha. Thus cytokine-mediated upregulation of ICAM-1 in lupus nephritis may promote interaction of immune cells with renal tissue. The predominant apical expression of ICAM-1 opposite to the basolateral Ia expression suggests a novel role for this adhesion molecule in PT.

MHC class II, antigen presentation and tumor necrosis factor in renal tubular epithelial cells.

Proximal tubular (PT) epithelial cells express MHC class II (Ia) antigens in immunologically-mediated renal injury. To study the role of PT as accessory cells, we generated several murine PT-like epithelial cell lines by transformation with origin-defective SV40 DNA. These transformed cell lines display typical alkaline phosphatase and gamma-glutamyl-transpeptidase enzyme activity, proliferation to epidermal growth factor (EGF) and sodium-dependent glucose uptake. Clonal lines of transformed tubular cells from both normal C3H/FeJ and autoimmune MRL-lpr mice do not constitutively express Ia antigens or mRNA for class II. However, stimulation with recombinant interferon-gamma(rIFN-gamma) induces Ia mRNA and surface product in the cell lines. These Ia-positive cells can process and present hen egg-white lysozyme (HEL) to antigen-specific Iak-restricted T cell hybrids. Unstimulated tubular cells do not express detectable IL-1 alpha, IL-1 beta, TNA-alpha, or IL-6 mRNA. However, stimulation with IL-1 alpha or LPS induces TNF-alpha transcripts. We conclude that these cell lines have characteristics most consistent with a proximal tubular origin. They also bear characteristics of accessory cells such as processing and presentation of antigen and TNF-alpha gene expression. We speculate that PT have the capacity to participate in the pathogenesis of immune renal injury.