Robert C. Atkins

IL-1 Up-Regulates Osteopontin Expression in Experimental Crescentic Glomerulonephritis in the Rat

Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days -1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules (<5%) and absent from glomeruli. During the development of rat anti-GBM disease (days 7 to 21), there was substantial up-regulation of OPN mRNA and protein expression in glomeruli (>5 cells per glomerular cross-section) and tubular epithelial cells (50-75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (/75-85%, P < 0.001) and tubules (/45-60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury.

Differential control of mesangial cell proliferation by interferon-gamma.

Rat mesangial cells were shown to be sensitive to recombinant interferon‐gamma (IFN‐γ). IFN‐γ reduced thymidine uptake by these cells and inhibited cell proliferation. Incubation of the cells with 1000 U/ml IFN‐μ decreased thymidine uptake by up to 64% and cell numbers were decreased by 17%. The effects of IFN‐y were dose and time dependent and were partially reversible by the anti‐IFN‐γ monoclonal antibody DB‐I. This lymphokine did not reduce incorporation of RNA and protein precursors however. Measurements of 3H‐uridine and 3H‐leucine incorporation indicated significant increases in RNA and protein synthesis (37% and 45%, respectively) on a per cell basis. The mitogenic effects of IL‐1 and platelet‐derived growth factor (PDGF) were also susceptible to IFN‐γ‐mediated inhibition but the mitogenic response to epidermal growth factor (EGF) was much less sensitive. Weconclude that while IFN‐γ may act to modulate the mitogenic signals provided by some factors such as IL‐1 and PDGF, the response to EGF appears to be unaffected.

Up‐regulation of macrophage migration inhibitory factor in acute renal allograft rejection in the rat

Recent studies have identified a key role for macrophage migration inhibitory factor (MIF) in a number of immune cell‐mediated diseases. The current study investigated the potential role of MIF in acute allograft rejection. Lewis rats underwent bilateral nephrectomy and then received an orthotopic DA renal allograft or an orthotopic Lewis renal isograft. Groups of six animals were killed at day 1 or 5 after transplantation. No immunosuppression was used. Animals receiving a renal allograft exhibited severe rejection on day 5, as shown by high levels of serum creatinine, very low rates of creatinine clearance, and severe tubulitis with a dense macrophage and T cell infiltrate. In contrast, isografts had normal renal function on day 5 with no histological evidence of rejection. Northern blotting showed that renal MIF mRNA expression was unchanged at day 1, but was increased 3.5‐fold on day 5. In situ hybridization showed a marked increase in MIF mRNA expression by tubular cells and MIF mRNA expression by many infiltrating mononuclear cells in day 5 allografts. Immunostaining confirmed an increase in tubular MIF protein expression, particularly in areas of severe tubular damage with prominent leucocytic infiltration. Double staining showed that many infiltrating macrophages and T cells expressed the MIF protein in day 5 allografts. There was only a minor increase in MIF expression in day 5 isografts, demonstrating that neither surgical injury nor stress cause significant up‐regulation of MIF expression in allograft rejection. In conclusion, this study has demonstrated that local MIF production is specifically increased in acute renal allograft rejection. These results suggest that MIF may play an important role in the cellular immune response mediating acute allograft rejection.

Increased incidence of benign breast disease in female renal transplant patients receiving cyclosporin

Background: Unlike other cancers, breast cancer does not occur at increased frequency in renal transplant patients but fibroadenomata may be more common as a result of exposure to cyclosporin. In order to determine the incidence of benign breast disease in renal transplant patients at Monash Medical Centre, current female patients were studied.

Novel approaches to the treatment of progressive renal disease

Diabetes and hypertension are major contributors to the increasing incidence of progressive renal disease. In addition to more potent antihypertensive agents that block the renin-angiotensin system, drugs that modulate other pathogenetic pathways are also in development. Recent preclinical studies indicate that compounds that interfere with the formation and action of advanced glycation end products may have a role in the treatment and prevention of diabetic nephropathy, as may agents targeting the activity of protein kinase C.

Interferon‐gamma stimulates the secretion of IL‐1, but not of IL‐6, by glomerular mesangial cells

IL‐1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon‐gamma (IFN‐γ) was measured by a thymocytc proliferation assay. While IFN‐γ alone had no effect on the secretion or the intracellular pool of IL‐1, the enhancement by IFN‐γ of IL‐1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti‐IL‐l antibody. At least 4–h incubation with IFN‐γ and LPS was required to detect enhancing effect of I FN‐γ. The addition of as little as 1 U/ml IFN‐γ significantly increased IL‐1 secretion in the presence of 10 μg/ml LPS. The IL‐6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL‐6‐dependent cell line (MH60.BSF2). Mesangial cells secreted IL‐6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL‐1 production, no effect of IFN‐γ on IL‐6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL‐1 release is associated with la expression on mesangial cells. IFN‐γ alone and the combination with LPS induced marked expression of la antigen, whereas LPS alone did not. We conclude that IFN‐γ stimulates the production of IL‐1, but not IL‐6, by mesangial cells and suggest an important role of IFN‐γ in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL‐1 and the accessory cell function of mesangial cells.

Subclassification of Leukemia Using Monoclonal Antibodies

The rapid proliferation of monoclonal antibodies since 1975 (1) has resulted in vast numbers of antibodies to leukocyte antigens being made available for typing of leukemia. We have collected a panel of monoclonal antibodies selected from those which we have found most specific (2) in the First International Workshop on Leucocyte Differentiation Antigens and complemented these antibodies with others available locally or internationally, but not tested in the First Workshop. The panel of 44 antibodies is listed in Table 29.1. This panel of antibodies has been tested against leukemic cells from patients with childhood acute lymphocytic leukemia (ALL) and myeloid leukemia as well as T-lymphoma cell suspensions from patients with nodal disease. The results obtained have been compared to the normal ranges of reactivity in the tissues being tested and to results from patients in remission.

Monoclonal anti-T-cell antibodies react with circulating myeloid leukemia cells and normal tissue macrophages.

&NA; Rabbit and monoclonal antibodies to human myeloid leukemia cells, monocytic leukemia cells and human thymocytes have shown the existence of common T‐cell/myeloid/monocyte antigens. For this reason, the specificity of a series of monoclonal antibodies to human T‐cells (OKT 1, 3, 4, 5, 6, 8, 9, 10; and NA1/34) was tested by immunofluorescence (cytofluorograph) and complement‐mediated cytotoxicity against human myeloid leukemia and normal blood cells and leukemic cell lines. In addition, an immunohistological analysis of the specificity of OKT4, 9.3, Leu 3a, OKT3 and NA1/34 antibodies was performed using normal lymphoid tissues and a sensitive immunoperoxidase technique. Normal human peripheral blood mononuclear cells reacted with OKT3 (“pan T‐cell”, mean 54%), OKT4 (“helper T‐cell”, mean 35%) and OKT 5/8 (“suppressor T‐cell”, mean 18%) as previously reported. However, 0KT3 reacted with the cell lines K562 (myeloid), RC2a and THP‐1 (monocytoid) and U937 (macrophage) as well as with cells from 9/65 myeloid leukemia patients. 0KT4 reacted with the cell lines HL60 (promyelocyte), RC2a and U937 and also with cells from 6/60 myeloid leukemia patients. OKT5 reacted with the cell lines K562 and THP‐1. OKT1 (“pan T‐cell”) reacted with THP‐1 and with myeloid and monocytic leukemia samples (5/32) as did 0KT6 (“cortical thymocyte”) (3/32). OKT10 (“common thymocyte”) reacted with a range of leukemia cell lines (B‐cell, pre‐ B‐cell and macrophage) as well as 7/21 myeloid leukemia samples. In tissue sections Leu 3a, (9.3 and OKT4 to a lesser extent), stained paracortical lymphocytes, plus subcapsular and medullary macrophages, and dendritic cells present within the paracortex. In addition, Leu 3a also stained Kupffer cells in the liver. These results demonstrate the anti‐T‐cell monoclonal antibodies reaction with antigens expressed on tissue macrophages, and with human myeloid and monocytic leukemia cells.

Up-regulation of the tumour-associated marker CD44V6 in experimental kidney disease

CD44 is an adhesion molecule involved in a wide range of cell–cell and cell–matrix interactions. The standard form of CD44 (CD44S) is a 85–90‐kD glycoprotein, but alternative splicing of RNA encoding 10 variable exons (V1‐V10) can give rise to many different CD44 variant protein isoforms of higher molecular weight. CD44 isoforms containing the V6 exon play a crucial role in tumour metastasis and lymphocyte activation. However, the role of CD44V6 in the kidney is unknown. The aim of this study was to examined renal CD44V6 expression in health, disease and in vitro. Immunohistochemistry staining with the V6‐specific 1.1ASML antibody identified constitutive CD44V6 expression by occasional cortical tubular epithelial cells and medullary tubules in normal rat kidney. In immune‐induced kidney disease (rat anti‐glomerular basement membrane glomerulonephritis), there was a marked increase in CD44V6 expression by cortical tubules, particularly in areas of tubulointerstitial damage, which was associated with focal macrophage infiltration. There was also a marked increase in CD44V6 expression by damaged tubules in a model of non‐immune kidney disease (unilateral ureteric obstruction). Reverse transcription‐polymerase chain reaction revealed a complex pattern of CD44V6‐containing mRNA isoforms in normal rat kidney. This pattern of CD44V6 splicing was essentially unaltered in disease. The NRK52E normal rat kidney tubular epithelial cell line expresses both CD44S and CD44V6. Stimulation of NRK52E cells with IL‐1 or transforming growth factor‐beta 1 induced a two‐to‐five‐fold increase in the expression of both CD44S and CD44V6. Furthermore, triggering of NRK52E cells by antibodies to CD44S or CD44V6, but not isotype control antibodies, induced secretion of monocyte chemoattractant protein‐1. In conclusion, this study has identified expression of the tumour‐associated marker CD44V6 in tubular epithelial cells in normal and diseased rat kidney, and suggests that signalling through the CD44V6 molecule may participate in the pathogenesis of experimental kidney disease.

Interleukin 1 induces renal CD44 expression in vivo and in vitro: role of the transcription factor Egr‐1

SUMMARY: Tubular expression of the cell‐surface adhesion molecule CD44 is upregulated in both human and experimental glomerulonephritis, and this is associated with interstitial leucocytic infiltration. However, we know little of the mechanisms by which renal CD44 expression is upregulated in disease. This study focuses on the potential role of the proinflammatory cytokine interleukin 1 (IL‐1) and the IL‐1‐inducible transcription factor early growth response factor‐1 (Egr‐1) on tubular CD44 expression in vivo and in vitro. Immunostaining identified constitutive CD44 expression by occasional glomerular cells and a small number of cortical tubules in normal rat kidney. Glomerular and tubular CD44 expression was markedly upregulated in rat crescentic anti‐glomerular basement membrane glomerulonephritis. Blocking IL‐1 activity by administration of the IL‐1 receptor antagonist significantly reduced glomerular and tubular CD44 expression in this disease model. In vitro studies found that the NRK52E rat tubular epithelial cell line constitutively expresses CD44 mRNA and protein. Stimulation with 10U/mL IL‐1 caused upregulation in both CD44 mRNA and protein expression. A role for the transcription factor Egr‐1 in this process was suggested by a time course study in which IL‐1 stimulation of NRK52E cells caused a rapid and transient increase in Egr‐1 mRNA, peaking at 30–60 min, which preceded the increase in CD44 mRNA that was evident at 3–6h. A direct link between Egr‐1 and CD44 expression was provided by the ability of an Egr‐1 antisense oligonu‐cleotide, but not sense or scrambled control oligonucleotides, to inhibit IL‐1‐induced upregulation of CD44 protein in NRK52E cells. These data demonstrate that IL‐1 is an important stimulus for upregulation of tubular CD44 expression in vivo and in vitro. Furthermore, in vitro studies have shown that IL‐1‐induced upregulation of CD44 expression operates, at least in part, via the transcription factor Egr‐1.