Adult porcine islets produce MCP-1 and recruit human monocytes in vitro

Abstract:  Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4⁺ T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1β and TNF-α. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of α- and β-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1β nor TNF-α were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1β and TNF-α were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.     

Cellular immunity in IgA nephropathy

Summary: A comprehensive study on the role of various cytokines in the regulation of IgA synthesis and progression of glomerular damage in IgA nephropathy was attempted. Semi-quantitative PCR for IL-I, IL-2, IL-4, IL-6, IL-10, IL-12, interferon (IFN)-γ, transforming growth factor (TGF)-β, tumour necrosis factor (TNF)-α, platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) was performed. In parallel studies, protein production of some cytokines was also determined. It was demonstrated that IL-4, IFN-γ and presumably IL-12 expressed predominantly in peripheral blood mononuclear cells in patients with IgA nephropathy. Some positive correlations between the mRNA expression of these cytokines and the degree of tissue damage were observed. It was concluded that these cytokines may play some role in the alteration of cellular immunity in this disease.     

肾间质泡沫细胞MCP-1/CCR2的表达与肾小管-间质损害的关系

目的：检测Alport综合征、膜增生性肾小球肾炎、局灶节段硬化性肾小球肾炎、特发性膜性肾病及IgA肾病患者肾间质区域泡沫细胞浸润与MCP-1/CCR2蛋白的表达及小管-间质损害的相关性。方法：选取诊断明确的Alport综合征患者5例、膜增生性肾小球肾炎患者28例、局灶节段硬化性肾小球肾炎患者35例、特发性膜性肾病患者36例、IgA肾病患者34例作为研究对象。采用免疫组织化学法检测各例患者肾间质区域（除肾小管）MCP-1与CCR2的表达并分析其与肾小管-间质损害积分的相关性。结果：（1）肾小球疾病患者肾间质区域浸润的泡沫细胞上均可见MCP-1和CCR2蛋白的同时表达。（2）在各类原发性肾小球疾病患者中,泡沫细胞浸润组肾间质区域MCP-1和CCR2蛋白的表达明显高于无泡沫细胞组（P〈0.05）。（3）MCP-1和CCR2蛋白的阳性表达值与肾小管-间质损害积分呈正相关（P〈0.05）。结论：各种病理类型的肾小球疾病间质区域浸润的泡沫细胞可同时分泌产生MCP-1和CCR2蛋白,参与了肾小管-间质损害的发生。抑制泡沫细胞的浸润和阻断MCP-1/CCR2通路可延缓肾脏疾病的进展。     

Renal cytokine profile in an endotoxemic porcine model

Introduction: In animals exposed to acute endotoxemia with lipopolysaccharide (LPS), high levels of cytokines are found in the kidney. The objective of this study is to determine whether the high renal content of TNF-α, IL-1β, IL-10 and IL-1 receptor antagonist (IL-1ra) is due to glomerular filtration and reabsorption, or whether the cytokines are produced locally in the kidney.
Methods: Eighteen anesthetized and mechanically ventilated pigs (35–43 kg) were randomized into two groups: Group 1 ( n =12) LPS infusion for 360 min and Group 2 ( n =6) control pigs, no treatment. At 360 min, the pigs were euthanized and tissue samples from the kidneys were obtained. Localization of the cytokines was determined by immunohistochemistry and double immunofluorescence (dIF).
Results: Pigs exposed to endotoxemia showed increased accumulation of leukocytes and increased protein expression of TNF-α and IL-1β when compared with controls. dIF showed that TNF-α-positive cells co-localized with both endothelial and mesangial cells in the glomeruli. Furthermore, the endothelial cells of the cortical arterioles were positive for IL-1β. TNF-α and IL-1β staining were absent in renal tubular cells. A positive signal for IL-10 was detected at the tubular brush border while IL-1ra was detected in the glomerulus and in the tubular cells.
Conclusion: LPS-induced endotoxemia increased TNF-α and IL-1β protein expression and leukocyte accumulation in the kidneys. The results indicate that the increased levels of the pro-inflammatory cytokines TNF-α and IL-1β are caused by a local production in the kidneys while the anti-inflammatory cytokines IL-10 and IL-1ra are filtrated and reabsorbed in the tubuli.     

Efficacy of Continuous Subcutaneous Infusion Therapy Using Interferon α and the Possible Prognostic Indicator of TNF-α in Renal Cell Carcinoma

Background :
In an attempt to improve efficacy by escalating the dose and maintaining higher serum concentrations over a long period of time, this study examines continuous interferon α (IFNα) subcutaneous infusion therapy in patients with renal cell carcinoma (RCC).
Methods :
Seven of 11 patients with RCC had evaluable metastatic lesions. A highly purified natural human IFNα was injected subcutaneously via an infuser pump for 5 consecutive days, followed by a 2-day rest period (25 million IU/week). The treatment was continued for a period of 15 weeks. Serum concentrations of IFNα, IL-1α, IL-1β, TNF-α, and IFNγ were measured at intervals throughout the study period.
Results :
Two of the 7 patients with evaluable lesions achieved a partial response (overall response rate, 29%), while 1 achieved a partial response only to lung metastasis. These 3 cases were defined as responders. No difference was found in the concentration of serum IFNα between responders and nonresponders, however, a significantly higher concentration of serum TNF-a was observed in responders (P<0.05, Mann-Whitney U test). Five cases (45%) had moderate to severe adverse effects, including depression (n = 1), eyeground hemorrhage (n = 2), and general fatigue (n = 2).
Conclusion :
Appropriate patient selection may be necessary for subcutaneous continuous infusion therapy for the treatment of metastatic RCC. Also, the serum concentration of TNF-α measured during the course of treatment reflected well on the outcome of IFNα therapy.     

Effect of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on human sperm motility, viability and motion parameters

Male genital tract infections and non-specific inflammatory conditions may be associated with unexplained infertility. Previous studies have shown the presence of cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the semen of infertile men. However, the mechanism of effect of these cytokines on human sperm function is still controversial. The present study was undertaken to investigate the in-vitro effects of TNF-α and IFN-γ on human sperm motion, viability and the hypoosmotic swelling test (HOST).
Washed spermatozoa from normal volunteers ( n =9) were incubated in the presence/absence of TNF-α (1 μg/mL) plus IFN-γ (0.1 μg/mL). Sperm motility, viability, HOST, and video sequences were recorded at different time intervals (0, 30, 60 and 180 min). Sperm motion parameters were analysed using computer-assisted semen analysis. There was a time-dependent negative effect of TNF-α plus IFN-γ on sperm motility, viability, HOST, and lateral-head displacement (ALH). The maximum decrease was observed between 60 and 180 min for sperm motility (50.8 ± 5.6%), viability (52.8 ± 4.0%), HOST (38 ± 2%) and ALH (4.7 ± 0.1 μm) compared to control samples (62.2 ± 2.8, 62.4 ± 2.9, 58 ± 4, and 5.3 ± 0.4, respectively; All p  < 0.05). There was no significant effect on sperm straight-line velocity and mean linearity when compared to control.
These data suggest that the common inflammatory cytokines TNF-α plus IFN-γ have only partial detrimental effects on sperm motility, viability, membrane integrity and lateral head displacement, which may contribute to the poor fertilizing potential of human spermatozoa during inflammatory conditions.     

Prostacyclin and prostaglandin E2 inhibit proinflammatory cytokine-induced macrophage colony-stimulating factor production in cultured human glomerular mesangial cells

Summary: Monocyte/macrophages within the mesangium plays some important roles in the progression of renal glomerular injury in which prostanoids exert a broad range of actions. We have examined the production of macrophage colony-stimulating factor (M-CSF), a monocyte-specific cytokine, by human glomerular mesangial cells (MC) and its regulation by prostacyclin and prostaglandin E₂ (PGE₂). the MCSF production by MC under non-stimulatory conditions was below detectable levels by ELISA, and was also at a trace level in the steady-state M-CSF mRNA expression. Proinflammatory cytokines, interleukin-1β (IL-1β) or tumour necrosis factor-α (TNF-α) induced the M-CSF production in the protein and mRNA levels. Both beraprost, a stable analogue of prostacyclin, and PGE₂ attenuated the IL-1β- or TNF-α-driven M-CSF production. Indomethacin, a non-selective cyclooxygenase inhibitor, enhanced the IL-1β- or TNF at-induced M-CSF production. Beraprost and PGE₂ showed similar inhibitory effects in the presence of indomethacin. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP decreased the M-CSF production. These results indicate that: (i) human MC have capacity to produce M-CSF; (ii) exogenous prostacyclin and PGE₂ downregulate the IL-1β- or TNF-α-driven M-CSF production possibly by an increase of intracellular cAMP; and (iii) endogenous prostanoids can exert on the M-CSF production.     

Mechanical stretch induces monocyte chemoattractant activity via an NF-kappaB-dependent monocyte chemoattractant protein-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone

Hemodynamic abnormalities are important in the pathogenesis of the glomerular damage in diabetes. Glomerular macrophage infiltration driven by the chemokine monocyte chemoattractant protein-1 (MCP-1) is an early event in diabetic nephropathy. The thiazolidinedione rosiglitazone ameliorates albumin excretion rate in diabetic patients with microalbuminuria and has anti-inflammatory properties, raising the possibility of a relationship between its renoprotective and anti-inflammatory activity. Investigated was whether mesangial cell stretching, mimicking in vitro glomerular capillary hypertension, enhances MCP-1 expression and monocyte chemoattractant activity. The effect of the combination of stretch with high glucose on MCP-1 production was studied and, finally, the effect of rosiglitazone on these processes was assessed. Stretching of human mesangial cells significantly enhanced their monocyte chemoattractant activity. This effect was mediated by MCP-1 as it was paralleled by a significant rise in both MCP-1 mRNA and protein levels and was completely abolished by MCP-1 blockade. Combined exposure to both stretch and high glucose further increased MCP-1 production. Stretch activated the IkappaB-NF-kappaB pathway, and NF-kappaB inhibition, with the use of the specific inhibitor SN50, completely abolished stretch-induced MCP-1, indicating that stretch-induced MCP-1 was NF-kappaB dependent. The addition of rosiglitazone significantly diminished stretch-induced NF-kappaB activation, MCP-1 production, and monocyte chemotaxis. In conclusion, stretching of mesangial cells stimulates their monocyte chemoattractant activity via an NF-kappaB-mediated, MCP-1-dependent pathway, and this effect is prevented by rosiglitazone.     

The vascular ectonucleotidase ENTPD1 is a novel renoprotective factor in diabetic nephropathy

Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39) is the dominant vascular ectonucleotidase. By hydrolyzing ATP and ADP to AMP, ENTPD1 regulates ligand availability to a large family of P2 (purinergic) receptors. Modulation of extracellular nucleotide metabolism is an important factor in several acute and subacute models of vascular injury. We hypothesized that aberrant nucleotide signaling would promote chronic glomerular injury in diabetic nephropathy. Inducing diabetes in ENTPD1-null mice with streptozotocin resulted in increased proteinuria and more severe glomerular sclerosis compared with matched diabetic wild-type mice. Diabetic ENTPD1-null mice also had more glomerular fibrin deposition and glomerular plasminogen activator inhibitor-1 (PAI-1) staining than wild-type controls. In addition, ENTPD1-null mice showed increased glomerular inflammation, in association with higher levels of monocyte chemoattractant protein-1 (MCP-1) expression. Mesangial cell PAI-1 and MCP-1 mRNA expression were upregulated by ATP and UTP but not ADP or adenosine in vitro. The stable nucleotide analog ATPgammaS stimulated sustained expression of PAI-1 and MCP-1 in vitro, whereas the stable adenosine analog NECA [5'-(N-ethylcarboxamido)adenosine] downregulated expression of both genes. Extracellular nucleotide-stimulated upregulation of MCP-1 is, at least in part, protein kinase C dependent. We conclude that ENTPD1 is a vascular protective factor in diabetic nephropathy that modulates glomerular inflammation and thromboregulation.     

The TNF-α system after successful living-related kidney transplantation

Abstract The TNF-α system is thought to play a central role in the reduced immunity of haemodialysis patients. The imbalance between the high levels of soluble TNF receptors R1 and R2 and the low levels of immunoactive TNF-α results in an increased TNF-α buffering capacity leading to reduced immune responses. Apart from impaired renal clearance of the receptors, inefficient TNF-α production as a result of the uraemia may also contribute to the imbalance between this cytokine and its receptors. In patients receiving a living-related kidney transplant, renal function is nearly normalized in a very short period. This restoration of renal function may result in a state of better immunocompetence, either as a result of improved clearance of the receptors or as a result of reversal of the uraemic state. To differentiate between these two possibilities, we measured TNF-a protein, mRNA and the soluble TNF receptors R1     

Molecular analysis of glomerular diseases in renal biopsies

Summary: The purpose of this study was to determine the pattern of gene expression of type IV collagen alpha chains in several chronic human glomerular diseases using micro dissected glomeruli and assessment of mRNA by competitive polymerase chain reaction (PCR). After showing that the level of a2 type IV collagen mRNA was elevated in sclerotic glomeruli isolated from nephrectomies, we undertook a preliminary cross-sectional study of type IV collagen alpha chain mRNA in renal biopsies in two of the leading causes of glomerulosclerosis: (i) diabetic nephropathy; and (ii) membranous glomerulopathy. We found that glomerular type IV collagen mRNA levels alterations were disease-specific. the relative levels of the individual α-chains of type IV collagen depended on the anatomic site of the glomerular lesions. the α-2IV/α-3IV collagen mRNA ratio was high in diabetes mellitus, but not in membranous glomerulopathy. These data, coupled with that obtained in experimental animals, suggest that a defective basement collagen synthesis is associated with progressive glomerular scarring. If these conclusions are verified in studies of repeat biopsies, the risk of progressive glomerulosclerosis in individual patients could be estimated, leading to the means to assess therapeutic responses.     

Intercellular adhesion molecule-1 and tumour necrosis factor-α expression in human glomerulonephritis

Summary: The relationship between renal expression of intercellular adhesion molecule-1 (ICAM-1), glomerular hypercellularity, renal function and renal tumour necrosis factor-α (TNF-α) expression was examined by immunohistochemistry staining in 64 cases of human glomerulonephritis. Glomerular anti-ICAM-1 antibody staining was increased in most cases of IgA nephropathy and lupus nephritis, but was unchanged compared to normal in membranous nephropathy and minimal change disease, and reduced in glomerular sclerosis. However, when taken together, patients with mild or no glomerular hypercellularity (group A) showed normal ICAM-1 expression, those with moderate to severe hypercellularity (group B) had increased glomerular ICAM-1 expression (P<0.001), while those with glomerular sclerosis (group C) had reduced glomerular ICAM-1 expression. Patients in groups B and C also showed a significant increase in tubular ICAM-1 expression (P<0.01) and interstitial infiltration of ICAM-1 ⁺ cells (P<0.001). Indeed, tubular ICAM-1 expression correlated with decreased creatinine clearance (r= -0.352; P<0.05). In situ hybridization demonstrated that increase in tubular ICAM-1 staining was due to de novo gene expression, rather than absorption of soluble ICAM-1 from the lumen. Focal expression of tumour necrosis factor-α was seen in areas of leucocyte infiltration and strong ICAM-1 expression. Indeed, TNF-α staining correlated with increased renal ICAM-1 expression in both glomerular and tubulointerstitial compartments (r=0.81; P<0.01). to confirm that TNF-α can directly stimulate renal ICAM-1 expression, TNF-α was shown to transiently increase ICAM-1 mRNA synthesis for 4-8 h and cause a progressive increase in ICAM-1 protein on the surface of cultured human mesangial cells. In summary; (i) increased glomerular ICAM-1 expression was restricted to cases of moderate to severe hypercellularity; (ii) tubular ICAM-1 expression correlated with both creatinine clearance and interstitial infiltration of ICAM-1⁺ cells; and (iii) TNF-α expression was shown to correlate with the degree of renal ICAM-1 expression, suggesting that local TNF-α plays an important role in the up-regulation of ICAM-1 in human glomerulonephritis.     

The expression of mRNA for tumour necrosis factor-α increases in the obstructed kidney of rats soon after unilateral ureteral ligation

Summary: Cytokines, including transforming growth factor (TGF)-β1, contribute to the tubulointerstitial fibrosis of ureteral obstruction. Tumour necrosis factor (TNF)-α, a proinflammatory cytokine produced by multiple cells including macrophages and resident renal cells, has a role in inflammatory cell recruitment in glomerular injury. We measured TNF-α mRNA in the renal cortex of rats at different times after the onset of unilateral ureteral obstruction (UUO) and determined whether angiotensin II (AngII) inhibition or total body irradiation affects the mRNA levels of TNF-α. Rats were killed at 1, 2, 4, 24, 72 and 120h after UUO. Levels of TNF-α mRNA increased significantly in the obstructed kidney at 1h (X 2), 2h (X 2.7), 4h (X 3.6), 24h (X 2.7), 72h (X 1.8) and 120h (X 2.8) after ureteral ligation when compared to the contralateral kidney of the same animals or to control (normal) kidneys. Tumour necrosis factor-α mRNA increased in renal cortical tubules but not in glomeruli. Treatment with enalapril, an angiotensin-converting enzyme (ACE) inhibitor, before and after UUO decreased TNF-α mRNA levels in the obstructed kidney by about 40% at 4h after the onset of UUO, but at 120h there was no difference in TNF-α levels in the obstructed kidney of treated and untreated animals. Total body irradiation, which depletes macrophages in the obstructed kidney, did not prevent the upregulation of TNF-α mRNA expression at 4 h after UUO. Thus, TNF-α may have a role in initiating tubulointerstitial injury in the obstructed kidney. Leucocytes infiltrating the renal interstitium of the obstructed kidney do not appear to contribute to the increased mRNA expression of TNF-α. Angiotensin II may contribute, at least in part, to the early increased expression of TNF-α mRNA in the obstructed kidney.     

Effects of sivelestat, a new elastase inhibitor, on IL-8 and MCP-1 production from stimulated human alveolar epithelial type II cells

Purpose The alveolar epithelial cell type II (AEC-II) is itself able to amplify lung inflammation by producing inflammatory cytokines and chemokines, leading to the activation and recruitment of phagocytes. Sivelestat, a new neutrophil elastase inhibitor, has been shown to attenuate acute lung injury in animal experiments. In the current study, we assessed the effects of sivelestat on the production of chemokines from cultured A549 cells, a human AEC-II-like cell line. Methods A549 cells were stimulated with endotoxin or tumor necrosis factor-α in the presence of sivelestat (1–100 μg·ml⁻¹). Culture supernatant levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were determined by enzyme-linked immunosorbent assay. The expression of IL-8 and MCP-1 mRNAs in stimulated A549 cells in the presence of sivelestat (100 μg·ml⁻¹) was quantified by real-time polymerase chain reaction. Results Sivelestat, at 100 μg·ml⁻¹ reduced the accumulation of IL-8 and MCP-1 in the culture medium. The high dose of sivelestat significantly inhibited the expression of IL-8 mRNA in A549 cells. The drug also decreased MCP-1 mRNA expression, although not significantly. Conclusion These data suggest that a high dose of sivelestat regulates the production of IL-8 and MCP-1 in AEC-II.     

Adrenomedullin inhibits pressure-induced mesangial MCP-1 expression through activation of protein kinase A

BACKGROUND: In glomerular hypertension, monocyte chemoattractant protein-1 (MCP-1) has been implicated in glomerulosclerosis progression. High-pressure load and stretch on mesangial cells (MC) are two major effects of increased glomerular pressure. We previously reported that pressure per se could induce MCP-1 expression in cultured MC, suggesting the involvement of glomerular hypertension in renal disease progression through MCP-1 expression in MC. We also showed that adrenomedullin (AM) inhibited pressure-induced MC proliferation; however, it is not clear whether AM alters pressure-induced mesangial MCP-1 expression. In this study, we examined the effect of AM on pressure-induced MCP-1 expression in cultured rat MC and the mechanism of such action. Using compressed helium, pressure was applied to MC placed in a sealed chamber. AM inhibited pressure-induced MCP-1 mRNA expression, measured by reverse transcribed-polymerase chain reaction (RT-PCR), in a dose-dependent manner. This inhibition was in parallel to an increase in cellular cyclic AMP (cAMP) levels evoked by AM. The effects of forskolin and dibutyryl cAMP mimicked those of AM. Protein kinase A (PKA) inhibitor H-89 significantly weakened the effects of AM. AM significantly reduced the pressure-induced increase in MCP-1 protein in supernatants of cultured MC, measured by enzyme-linked immunosorbent assay (ELISA). Our results suggested that AM inhibits pressure-induced mesangial MCP-1 expression through PKA activation.     

Up-regulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy

BACKGROUND: We previously described that monocyte chemoattractant protein-1 (MCP-1) plays an important role in progressive glomerular and interstitial damage in inflammatory renal diseases. However, the expression of MCP-1 in diabetic nephropathy remains to be investigated. METHODS: We examined whether locally expressed MCP-1 participates in human diabetic nephropathy via recruiting and activating monocytes/macrophages (Mphi). Urinary and serum MCP-1 levels were measured by enzyme-linked immunosorbent assay in 45 patients with diabetic nephropathy. The presence of MCP-1 in diseased kidneys was determined by immunohistochemical and in situ hybridization analyses. RESULTS: Urinary MCP-1 levels were significantly elevated in patients with diabetic nephrotic syndrome and advanced tubulointerstitial lesions. Moreover, urinary levels of MCP-1 were well correlated with the number of CD68-positive infiltrating cells in the interstitium. In contrast, serum MCP-1 levels remained similar to those of healthy volunteers. Furthermore, we detected the MCP-1-positive cells in the interstitium of diabetic nephropathy via both immunohistochemical and in situ hybridization analyses. CONCLUSION: These observations suggest that locally produced MCP-1 may be involved in the development of advanced diabetic nephropathy, especially in the formation of tubulointerstitial lesions possibly through Mphi recruitment and activation. Moreover, up-regulation of MCP-1 may be a common pathway involved in the progressive tubulointerstitial damage in diabetic nephropathy as well as inflammatory renal diseases.