Antibody blockade of c-fms suppresses the progression of inflammation and injury in early diabetic nephropathy in obese db/db mice.

AIMS/HYPOTHESIS: Macrophage-mediated renal injury plays an important role in the development of diabetic nephropathy. Colony-stimulating factor (CSF)-1 is a cytokine that is produced in diabetic kidneys and promotes macrophage accumulation, activation and survival. CSF-1 acts exclusively through the c-fms receptor, which is only expressed on cells of the monocyte-macrophage lineage. Therefore, we used c-fms blockade as a strategy to selectively target macrophage-mediated injury during the progression of diabetic nephropathy. METHODS: Obese, type 2 diabetic db/db BL/KS mice with established albuminuria were treated with a neutralising anti-c-fms monoclonal antibody (AFS98) or isotype matched control IgG from 12 to 18 weeks of age and examined for renal injury. RESULTS: Treatment with AFS98 did not affect obesity, hyperglycaemia, circulating monocyte levels or established albuminuria in db/db mice. However, AFS98 did prevent glomerular hyperfiltration and suppressed variables of inflammation in the diabetic kidney, including kidney macrophages (accumulation, activation and proliferation), chemokine CC motif ligand 2 levels (mRNA and urine protein), kidney activation of proinflammatory pathways (c-Jun amino-terminal kinase and activating transcription factor 2) and Tnf-alpha (also known as Tnf) mRNA levels. In addition, AFS98 decreased the tissue damage caused by macrophages including tubular injury (apoptosis and hypertrophy), interstitial damage (cell proliferation and myofibroblast accrual) and renal fibrosis (Tgf-beta1 [also known as Tgfb1] and Col4a1 mRNA). CONCLUSIONS/INTERPRETATION: Blockade of c-fms can suppress the progression of established diabetic nephropathy in db/db mice by targeting macrophage-mediated injury.

IL-10 induces mesangial cell proliferation via a PDGF-dependent mechanism.

Interleukin-10 (IL-10) is a mesangial cell growth factor in vivo and in vitro. However, the mechanism by which IL-10 exerts its mitogenic activity is not known. The aim of this study was to determine whether IL-10 induces mesangial cell proliferation in a PDGF-dependent or independent fashion. A well--characterized rat mesangial cell line (1097) was used in a series of cell proliferation experiments in which cells were serum-starved and then incubated with recombinant IL-10 in the presence or absence of STI 571 (a specific inhibitor of signalling via the PDGF-alpha and beta receptors) or a neutralizing anti-PDGF-AB antibody. IL-10 induced significant mesangial cell proliferation at 24 and 48 h after cytokine addition. This response was inhibited totally by the addition of STI-571, demonstrating that IL-10 mitogenic activity has an absolute requirement for signalling through the PDGF receptor. In further studies, it was found that STI-571 could be added 24 h after IL-10 stimulation and still exert a profound inhibition of IL-10 mitogenic activity. The ability of a neutralizing anti-PDGF-AB antibody to inhibit completely IL-10-induced mesangial cell proliferation confirmed that IL-10 acts via induction of an autocrine PDGF response rather than the possibility that IL-10 may transactivate the PDGF receptor in a PDGF-independent fashion. In conclusion, this study has demonstrated that IL-10 induces mesangial cell proliferation via an autocrine PDGF-mediated mechanism. Thus, therapies which antagonize PDGF signalling will also inhibit any contribution of IL-10 to mesangial proliferation.

Tubules are the major site of M-CSF production in experimental kidney disease: correlation with local macrophage proliferation.

BACKGROUND: Local proliferation of macrophages occurs within both the glomerulus and the interstitium in severe forms of human and experimental glomerulonephritis and plays an important role in amplifying renal injury. Macrophage colony-stimulating factor (M-CSF) is thought to be the growth factor driving this local macrophage proliferation. Previous studies have found that glomeruli are the predominant source of M-CSF production. However, this is difficult to reconcile with the prominent macrophage accumulation and proliferation seen in the interstitial compartment in glomerulonephritis. To address this issue, we localized M-CSF expression in rat models of glomerular versus tubulointerstitial injury and examined its relationship to local macrophage proliferation. METHODS: M-CSF expression (Northern blotting, in situ hybridization, immunostaining, Western blotting) and local macrophage proliferation (double immunostaining) was examined in normal rat kidney on days 1 and 14 of rat anti-glomerular basement membrane (anti-GBM) glomerulonephritis and on day 5 following unilateral ureteric obstruction. RESULTS: M-CSF mRNA and protein expression were identified in small numbers of glomerular podocytes, approximately 25% of cortical tubules, and most medullary tubules in normal rat kidney. Northern blotting showed a significant increase in whole kidney M-CSF mRNA in rat anti-GBM glomerulonephritis. Up-regulation of glomerular and, most prominently, tubular M-CSF production was confirmed by three independent methods: in situ hybridization, immunostaining, and Western blotting. The increase in M-CSF expression colocalized with local macrophage proliferation (ED1+PCNA+ cells) in both the glomerulus and tubulointerstitium. On day 5 after ureter ligation, there was a significant increase in tubular M-CSF mRNA and protein expression in the obstructed kidney, with no change in glomerular M-CSF. In parallel with M-CSF expression, macrophage accumulation and proliferation was prominent in the interstitium, but was absent from glomeruli. CONCLUSIONS: The tubular epithelial cell is the major site of M-CSF production within the injured kidney. Indeed, substantial macrophage accumulation and local proliferation can occur in the tubulointerstitium in the absence of glomerular inflammation. These results suggest that M-CSF production within the kidney, particularly by tubular epithelial cells, plays an important role in regulating local macrophage proliferation in experimental kidney disease.

CD44-mediated neutrophil apoptosis in the rat.

BACKGROUND: Apoptosis is an important mechanism by which neutrophils are removed from sites of inflammation, including the kidney. This study investigated whether ligation of the cell-surface adhesion molecule, CD44, can trigger neutrophil apoptosis. METHODS: The anti-rat CD44 antibody OX-50 was used to induce apoptosis of cultured blood neutrophils, as determined by flow cytometry using annexin V staining and by transmission electron microscopy. The functional consequences of OX-50-mediated neutrophil depletion were examined in a rat model of accelerated antiglomerular basement membrane glomerulonephritis. RESULTS: Flow cytometric analysis using the OX-50 antibody, which recognizes the common amino terminal domain of CD44, showed that rat blood neutrophils express very high levels of CD44. The addition of OX-50, but not control antibodies, rapidly induced neutrophil apoptosis in cultured rat blood leukocytes, as demonstrated by annexin V staining and by electron microscopy. Cross-linking of CD44 was essential since F(ab) fragments of the OX-50 antibody failed to induce neutrophil apoptosis. The CD44 ligand hyaluronan and an antibody to the CD44v6 isoform failed to induce neutrophil apoptosis, indicating that OX-50 antibody-mediated neutrophil apoptosis is epitope specific. This effect was specific to neutrophils since the OX-50 antibody did not induce apoptosis in other CD44-expressing cell types (lymphocytes, mesangial cells, or tubular epithelial cells). An injection of OX-50 antibody into normal rats caused a rapid and profound neutropenia, and apoptotic neutrophils could be seen in the blood by electron microscopy. Furthermore, the administration of OX-50 antibody abrogated neutrophil-dependent glomerular injury (proteinuria) on day 1 of rat antiglomerular basement membrane glomerulonephritis, whereas injury on day 10 of the disease (neutrophil independent) was largely unaffected. CONCLUSIONS: The cross-linking of specific epitopes of the CD44 molecule can rapidly induce neutrophil apoptosis in vitro and inhibit neutrophil-dependent renal injury in vivo. This finding suggests that physiological ligands of the CD44 molecule may play an important role in eliminating neutrophils from sites of inflammation, including inflammatory kidney disease.

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.

Universal and cultural features in the delusions of a black urban group.

Conflicting views as to the form and content of delusions among African psychotics, notably the influence of cultural factors in the preliterate, led us to devote special attention to this area in a recent study of Bantu psychiatric patients. Our sample was drawn chiefly from Baragwanath Hospital serving the South Western Townships of Johannesburg, and smaller samples were drawn from mental hospitals, mental health society clinics, an old age home, and a middle class township in the Witwatersrand area. The degree of systematization and fixity of delusions in the sample as a whole, and in schizophrenia and manic-depressive psychosis in particular, were assessed. On the side of delusion content, cultural features such as witchcraft, animals as instruments of witchcraft, familiars, and ancestor worship are described. In conclusion, the relationship of cultural content of delusion to class, education and income are evaluated, with special attention to the influence of an early tribal background in our urban professional group, as illuminating the general issue reflected in our title.