Mononuclear-cell subsets in human idiopathic crescentic glomerulonephritis (ICGN): Analysis in tissue sections with monoclonal antibodies

Mononuclear inflammatory cells (MIC) were analyzed in renal biopsies from 16 patients with ICGN (7 with glomerular immune complex deposits, 3 with anti-GBM disease, and 6 without immune deposits) by the avidin-biotin-immunoperoxidase technique utilizing monoclonal antibodies to cell surface antigens: T11 (total T), T4 (inducer/helper T), T8 (suppressor/cytotoxic T), B1 (B cells), M1 (monocytes/granulocytes), and Leu 7 [natural killer (NK) cells]. Total MIC were significantly increased in both glomeruli and interstitial tissues of the patients. Interstitial MIC consisted mainly of lymphocytes (80%) and monocytes (19%), with small numbers of B and NK cells present. In contrast, MIC in renal glomeruli of patients with ICGN were composed of monocytes (65%) rather than T lymphocytes (34%). A majority of T lymphocytes found in renal tissues of patients and controls had the helper/inducer phenotype. Tissue T4/T8 ratios were not significantly different in the glomeruli and interstitium. Monocytes and T lymphocytes accumulating in renal tissues of patients with ICGN may mediate glomerular injury in all forms of human ICGN.Presented in part at the 15th Annual Meeting of the American Society of Nephrology, Chicago, Illinois, 1982.     

巨噬细胞移动抑制因子在非IgA系膜增生性肾小球肾炎肾组织中的表达

目的 探讨巨噬细胞移动抑制因子（MIF）在非IgA系膜增生性肾小球肾炎患者肾组织中的表达及其意义。方法 应用免疫组织化学双标记技术检测正常对照组及不同病变程度非IgA系膜增生性肾小球肾炎患者肾组织内MIF和人巨噬细胞标记抗原CD68的表达。结果 在正常对照组和IgA系膜增生性肾小球肾炎患者轻度组仅有少量MIF和CD68表达。随着非IgA系膜增生性肾小球肾炎病变程度的加重,MIF、CD68的表达逐渐增强,重度病变时,MIF、CD68的表达最强,且MIF表达水平与CD68表达呈正相关（r=0.87,P〈0.01）。结论 肾组织内MIF表达上调所导致的巨噬细胞浸润增加可能是非IgA系膜增生性肾小球肾炎进展的重要机制之一。     

Monocytes secrete factors regulating glycosaminoglycan synthesis in mesangial cells in vitro.

The present study was conducted to determine the manner in which monocytes increase mesangial matrices, particularly glycosaminoglycans (GAGs) which interact with various other matrix components such as collagens, laminin, fibronectin and lipoproteins. A supernatant of human peripheral blood monocyte cultures activated by lipopolysaccharide (LPS) contains stimulating factors for glycosaminoglycan synthesis in rat mesangial cells (MCs). The culture supernatant in this study was concentrated and fractionated by gel chromatography and the GAG-stimulatory factor was found to have a molecular weight of 10-17 kD. This factor was shown to be present in fractions different from that of IL-1. Gel and ion-exchange chromatography studies of GAGs synthesized by MCs indicated the elution patterns of GAGs in the presence and absence of the monocyte culture supernatant to be essentially the same. Local infiltration of monocytes into the glomerulus, often seen in various types of glomerular injury, may be an important factor in the accumulation of the mesangial matrix.     

Intraglomerular expressions of IL-1 alpha and platelet-derived growth factor (PDGF-B) mRNA in experimental immune complex-mediated glomerulonephritis.

Acute lung injury frequently develops following haemorrhage, and is characterized by increased proinflammatory cytokine levels and massive neutrophil accumulation in the lung. Blood loss produces rapid increases in tumour necrosis factor-alpha (TNF-alpha) mRNA expression among pulmonary cell populations which precede the development of lung injury. In order to examine the role of TNF-alpha in producing acute inflammatory lung injury, we treated mice following haemorrhage and resuscitation with a TNF antagonist, composed of soluble dimeric human p80 TNF receptor linked to the Fc region of human IgG1 (sTNFR:Fc). Therapy with sTNFR:Fc prevented the post-haemorrhage increases in circulating and pulmonary TNF-alpha levels normally found following blood loss. Administration of sTNFR:Fc also diminished the increase in IL-1 beta, IL-6, TNF-alpha and interferon-gamma (IFN-gamma) mRNA normally found in the lungs following haemorrhage. However, therapy with sTNFR:Fc was not associated with improvement in the histologic parameters of post-haemorrhage lung injury, such as neutrophil infiltration and interstitial oedema. In contrast to the effects of sTNFR:Fc on cytokine mRNA levels among intraparenchymal pulmonary mononuclear cells, such therapy following haemorrhage was associated with increased amounts of mRNA for TNF-alpha among peripheral blood mononuclear cells, as well as increased IFN-gamma titres in serum and bronchoalveolar lavage (BAL) specimens. These results indicate that therapy with sTNFR:Fc in the post-haemorrhage period, although capable of decreasing proinflammatory cytokine expression in the lungs, does not prevent the development of acute lung injury in this setting.     

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis

The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.     

Glomerular expression of cell-cycle-regulatory proteins in human crescentic glomerulonephritis

To elucidate the mechanism underlying crescentic formation, we assessed the phenotypic characterization and cell-cycle protein expression in human crescentic glomerulonephritis (CRGN). Kidney tissue specimens taken from CRGN patients (10 patients with pauci-immune type rapidly progressive glomerulonephritis (RPGN), 2 patients with Henoch-Schönlein purpura nephritis, and 1 patient with IgA nephropathy) were examined immunohistochemically. Most of the cellular components of the crescents expressed cytokeratin, whereas few cells expressed PHM-5. CD68-positive cells were minor components of cellular crescents, indicating that the major principal cellular component of the crescents is made up of cells with the parietal glomerular epithelial cell (PEC) phenotype. Additionally, serial section analysis revealed that Ki-67-positive cells in the crescents were frequently cyclin-A positive and Bcl-2 positive, but seldom cyclin-B₁ positive. Moreover, the expression of cyclin-dependent kinase inhibitor p27^Kip1 was low in the cellular crescents, despite being exclusively positive in podocytes within the same section. We concluded that the major component of the cellular crescents is made up of PECs and that apparent expression of cyclins and Bcl-2 and restrained expression of p27^Kip1 may be synergistically associated with the development of cellular crescents in human CRGN.     

Interferon-gamma (IFN-gamma) and IL-4 expressed during mercury-induced membranous nephropathy are toxic for cultured podocytes.

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.     

Mononuclear cells in glomeruli and cytokines in urine reflect the severity of experimental proliferative immune complex glomerulonephritis.

Immunohistochemical methods were used to investigate the role of macrophages in the progression of proliferative immune complex glomerulonephritis. The mononuclear cell component of glomerular inflammation was analysed in three different stages of chronic serum sickness, each of which was clearly distinguished by criteria of kidney function. Urinary excretion of the macrophage secretory products interleukin-1 and tumour necrosis factor was also evaluated in relation to the functional severity of kidney disease. T lymphocytes and macrophages began to accumulate in glomeruli at the onset of proteinuria, but not before. Urinary excretion of interleukin-1 also began with proteinuria. Proteinuria increased in direct correlation with increases in the number of glomerular macrophages. Development of the most severe stage of glomerulonephritis, characterized by cachexia, declining kidney function, and necrotizing glomerular pathology, was accompanied by the disappearance of T cells from glomeruli and the expression of highly abnormal phenotypes by most macrophages. In addition, there was a switch from urinary excretion of interleukin-1 to excretion of tumour necrosis factor. The progression of proliferative immune complex glomerulonephritis was associated with qualitative as well as quantitative changes in glomerular macrophage populations. Differentiation and/or activation of those glomerular macrophages may have resulted from local T cell-mediated immunoregulation. Measurements of urinary cytokine excretion provided a reliable means of monitoring disease progression. The local action of tumour necrosis factor probably contributed to declining kidney function in the most severe stage of disease.     

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage-dependent glomerulonephritis.

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(s)-15-methyl PGE1 (M-PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage-dependent glomerular injury. M-PGE1 ameliorated proteinuria (day 4; 61 +/- 13 mg/24 h, n = 9; vehicle treated, 164 +/- 17 mg/24 h, n = 11; P less than 0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44 +/- 9/glomerulus; vehicle treated, 119 +/- 15/glomerulus; P less than 0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8 +/- 5%; vehicle treated 25 +/- 4% P less than 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M-PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M-PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6 +/- 1.5/glomerulus; vehicle treated, 13.2 +/- 1.3/glomerulus) or alter Ia antigen expression (4.1 +/- 0.2 Ia + cells/glomerulus; vehicle treated, 3.9 +/- 0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage-mediated inflammation.