Hugh R. Brady

Cutting Edge: Lipoxins Rapidly Stimulate Nonphlogistic Phagocytosis of Apoptotic Neutrophils by Monocyte-Derived Macrophages

Lipoxins (LX) are lipoxygenase-derived eicosanoids generated during inflammation. LX inhibit polymorphonuclear neutrophil (PMN) chemotaxis and adhesion and are putative braking signals for PMN-mediated tissue injury. In this study, we report that LXA4 promotes another important step in the resolution phase of inflammation, namely, phagocytosis of apoptotic PMN by monocyte-derived macrophages (Mφ). LXA4 triggered rapid, concentration-dependent uptake of apoptotic PMN. This bioactivity was shared by stable synthetic LXA4 analogues (picomolar concentrations) but not by other eicosanoids tested. LXA4-triggered phagocytosis did not provoke IL-8 or monocyte chemoattractant protein-1 release. LXA4-induced phagocytosis was attenuated by anti-CD36, αvβ3, and CD18 mAbs. LXA4-triggered PMN uptake was inhibited by pertussis toxin and by 8-bromo-cAMP and was mimicked by Rp-cAMP, a protein kinase A inhibitor. LXA4 attenuated PGE2-stimulated protein kinase A activation in Mφ. These results suggest that LXA4 is an endogenous stimulus for PMN clearance during inflammation and provide a novel rationale for using stable synthetic analogues as anti-inflammatory compounds in vivo.

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mm versus 5 mm) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor β1 (TGF-β1), and addition of TGF-β1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-β1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFβ1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFβ1-driven matrix production within a diabetic milieu.

Lipoxins, Aspirin-Triggered Epi-Lipoxins, Lipoxin Stable Analogues, and the Resolution of Inflammation: Stimulation of Macrophage Phagocytosis of Apoptotic Neutrophils In Vivo

Lipoxins (LX) are eicosanoids with antiinflammatory activity in glomerulonephritis (GN) and inflammatory diseases, hypersensitivity, and ischemia reperfusion injury. It has been demonstrated that LXA(4) stimulates non-phlogistic phagocytosis of apoptotic polymorphonuclear neutrophils (PMN) by monocyte-derived macrophages (Mphi) in vitro, suggesting a role for LX as endogenous pro-resolution lipid mediators. It is here reported that LXA(4), LXB(4), the aspirin-triggered LX (ATL) epimer, 15-epi-LXB(4), and a stable synthetic analogue 15(R/S)-methyl-LXA(4) stimulate phagocytosis of exogenously administered excess apoptotic PMN by macrophages (M phi) in vivo in a classic model of acute inflammation, namely thioglycollate-induced peritonitis. Significant enhancement of phagocytosis in vivo was observed with 15-min exposure to LX and with intraperitoneal doses of LXA(4), LXB(4), 15(R/S)-methyl-LXA(4), and 15-epi-LXB(4) of 2.5 to 10 micro g/kg. Non-phlogistic LX-stimulated phagocytosis by M phi was sensitive to inhibition of PKC and PI 3-kinase and associated with increased production of transforming growth factor-beta(1) (TGF-beta(1)). LX-stimulated phagocytosis was not inhibited by phosphatidylserine receptor (PSR) antisera and was abolished by prior exposure of M phi to beta 1,3-glucan, suggesting a novel M phi-PMN recognition mechanism. Interestingly, the recently described peptide agonists of the LXA(4) receptor (MYFINITL and LESIFRSLLFRVM) stimulated phagocytosis through a process associated with increased TGF-beta(1) release. These data provide the first demonstration that LXA(4), LXB(4), ATL, and LX stable analogues rapidly promote M phi phagocytosis of PMN in vivo and support a role for LX as rapidly acting, pro-resolution signals in inflammation. Engagement of the LXR by LX generated during cell-cell interactions in inflammation and by endogenous LXR peptide agonists released from distressed cells may be an important stimulus for clearance of apoptotic cells and may be amenable to pharmacologic mimicry for therapeutic gain.

Neutrophil apoptosis is delayed in patients with inflammatory bowel disease.

Delayed neutrophil apoptosis is a feature of persistent acute inflammation. Neutrophil-mediated damage has been shown to be associated with the development of inflammatory bowel disease (IBD). Persistence of these cells both at the colonic site and circulation may further contribute to IBD. The aims of this study were to determine whether neutrophils isolated from IBD patients delay apoptosis and to investigate possible mechanisms involved in this delay. We studied 20 patients with IBD, 13 with Crohns disease, and 7 with ulcerative colitis, all of whom were undergoing intestinal resection for symptomatic disease. Seventeen patients undergoing elective resection of colon cancer acted as operative controls. Systemic, mesenteric arterial, and mesenteric venous blood was harvested. Neutrophils isolated from patients with IBD showed decreased spontaneous apoptosis compared to cancer patients. Mesenteric venous serum of IBD patients contributed to this delay, which contained higher concentrations of interleukin-8 (IL-8). Pro-caspase 3 expression was also reduced in IBD neutrophils, which may contribute to decreased spontaneous and Fas antibody-induced apoptosis. Neutrophil apoptosis may be altered in Crohns disease and ulcerative colitis through release of anti-apoptotic cytokines and altered caspase expression. The alterations in cell death mechanisms may lead to persistence of the inflammatory response associated with IBD.

The role of HIF-1α in transcriptional regulation of the proximal tubular epithelial cell response to hypoxia

Epithelial cells of the kidney represent a primary target for hypoxic injury in ischemic acute renal failure (ARF); however, the underlying transcriptional mechanism(s) remain undefined. In this study, human proximal tubular epithelial cells (HK-2) exposed to hypoxia in vitro demonstrated a non-lethal but dysfunctional phenotype, closely reflective of the epithelial pathobiology of ARF. HK-2 cells exposed to hypoxia demonstrated increased paracellular permeability, decreased proliferation, loss of tight junctional integrity, and significant actin disassembly in the absence of cell death. Microarray analysis of transcriptomic changes underlying this response identified a distinct cohort of 48 genes with a closely shared hypoxia-dependent expression profile. Within this hypoxia-sensitive cluster were genes identified previously as hypoxia-inducible factor-1 (HIF-1)-dependent (e.g. vascular endothelial growth factor and adrenomedullin) as well as genes not previously known to be hypoxia-responsive (e.g. stanniocalcin 2). In hypoxia, HIF-1 bound to evolutionarily conserved hypoxia-response elements (HRE) in the promoters of these genes as well as to the HRE consensus motif. A further subset of these genes, not associated with transcriptional regulation by HIF-1, was also present, suggesting alternative HIF-1-independent pathways. Overexpression of HIF-1α in normoxia induced the expression of a significant number of the hypoxia-dependent genes; however, it did not induce the pathophysiologic epithelial response. In summary, hypoxia-elicited alterations in renal proximal tubular epithelial cells in vitro closely resemble the epithelial pathophysiology of ARF. Our data indicate that although this event may rely heavily on HIF-1-dependent gene transcription, it is likely that separate hypoxia-dependent transcriptional regulators also play a role.

15-Epi-16-(Para-Fluorophenoxy)-Lipoxin A4-Methyl Ester, a Synthetic Analogue of 15-epi-Lipoxin A4, Is Protective in Experimental Ischemic Acute Renal Failure

Lipoxins are endogenous lipoxygenase-derived eicosanoids, generated during inflammatory, hypersensitivity, and vascular events, that display vasodilatory, antiinflammatory, and pro-resolution activity. Here, we evaluated the efficacy of 15-epi-16-(para-fluorophenoxy)-lipoxin A(4)-methyl ester (15-epi-16-(FPhO)-LXA(4)-Me), a stable synthetic analogue of aspirin-triggered 15-epi-lipoxin A(4) in ischemic acute renal failure (ARF) in NIH Swiss mice. ARF was induced by 30-min crossclamping of renal pedicles and was associated with elevated serum creatinine, morphologic injury, polymorphonuclear leukocyte (PMN) recruitment, and increased mRNA levels for adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]), chemokines (growth regulated oncogene-1 [GRO1]), and cytokines (interleukin-1beta [IL-1beta] and IL-6) after 24-h reperfusion. A single bolus of 15-epi-16-(FPhO)-LXA(4)-Me afforded striking functional (mean +/- SEM creatinine in mg/dl: sham-operated, 0.77 +/- 0.04; ARF + vehicle, 2.49 +/- 0.19; ARF + 15-epi-16-(FPhO)-LXA(4)-Me, 0.75 +/- 0.12; P < 0.001) and morphologic protection and reduced PMN infiltration. Treatment with 15-epi-16-(FPhO)-LXA(4)-Me was also associated with lower IL-1beta, IL-6, and GRO1 mRNA levels, whereas ICAM-1 and VCAM-1 mRNA levels were unchanged. Compatible with these results, LXA(4) blunted chemoattractant-stimulated PMN migration across HK-2 renal epithelial cell monolayers in vitro, but it did not inhibit cytokine-induced HK-2 ICAM-1 expression or adhesiveness for PMN. Interestingly 15-epi-16-(FPhO)-LXA(4)-Me-treated animals also displayed increased renal mRNA levels for suppressors of cytokine signaling-1 (SOCS-1) and SOCS-2, but not CIS-1, endogenous inhibitors of cytokine-elicited Jak/Stat-signaling pathways. These results indicate that 15-epi-16-(FPhO)-LXA(4)-Me is protective in renal ischemia reperfusion injury in vivo, at least partially by modulating cytokine and chemokine expression and PMN recruitment, and provides a rationale for further exploration of the efficacy of LXA(4) structural analogues in ischemic ARF and other renal diseases.

Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation

The lipoxygenase‐derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4‐induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF‐ and LTD4‐stimulated proliferation through modulation of platelet‐derived growth factor receptor β (PDGFRβ) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRβ, a process associated with c‐src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4‐induced c‐src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4‐stimulated activation of the PDGFRβ and that LXA4 modulates PDGF‐BB‐stimulated tyrosine phosphorylation of the PDGFRβ and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum‐stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross‐talk between G protein‐coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti‐inflammatory and proresolution bioactions of LX.

Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co‐stimulation by lipopolysaccharide (LPS) or granulocyte macrophage‐colony stimulating factor (GM‐CSF) on these events. Fas engagement by an agonistic anti‐Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM‐CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co‐stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.

Clinical features, predictors of disease progression and results of renal transplantation in fibrillary/immunotactoid glomerulopathy

BACKGROUND The clinical manifestations of fibrillary-immunotactoid glomerulopathy are still being appreciated. It is unclear whether fibrillary-immunotactoid glomerulopathy actually represents two distinct clinicopathological entities, fibrillary glomerulopathy (FG) and immunotactoid glomerulopathy (ITG), or a single disease with different ultrastructural variants. METHODS To address these issues, we analysed the clinical features of 186 patients with fibrillary-immunotactoid glomerulopathy referred to our institutions (25 patients) or reported in the literature (161 patients). In separate analyses, patients were subclassified as having either fibrillary glomerulopathy (FG) or immunotactoid glomerulopathy (ITG) according to fibril diameter (FG<=30nm, ITG>30 nm) or arrangement (FG, random; ITG, focally organized). RESULTS Proteinuria (FG approximately 100%, ITG approximately 100%), nephrotic syndrome (FG approximately 71%, ITG approximately 82%), haematuria (FG approximately 71%, ITG approximately 64%), hypertension (FG approximately 67%, ITG approximately 45%), and renal insufficiency (FG approximately 54%, ITG approximately 42%) were frequent clinical correlates of both FG and ITG, irrespective of the ultrastructural criteria for diagnosis. Twenty-five patients presenting to our institutions (24 FG, 1 ITG) were divided into three groups based on rate of decline of GFR (mean slope of 1/serum creatinine versus time: group 1 -0. 103+/-0.238; group 2 0.121+/-0.040; group 3 0.466+/-0.318) in an attempt to identify clinical predictors of progression at presentation. Rapid progressors (Group 3) had an increased incidence of nephrotic syndrome and tended to have higher blood pressure than patients with milder disease, but did not differ from other groups in age, prevalence of haematuria or degree of renal insufficiency. The number of patients requiring dialysis was 0/10 in group 1, 2/6 in group 2, and 2/4 in group 3 over a follw-up period 47+/-46, 55+/-32, and 19+/-19 months respectively; two predialysis deaths being recorded in group 3. Four patients received five renal allografts (one patient being transplanted twice) and were followed for 4-11 years. Whereas recurrence of FG was documented in three allografts undergoing post-transplant biopsy, the rate of deterioration of GFR was invariably slower in allografts than native kidneys (mean slope of 1/Cr versus time: 0.036+/-0.01 versus 0. 0301+/-0.18 respectively). The strength of association between FG-ITG and lymphoproliferative malignancy varied depending on whether patients with monoclonal-gammopathy-associated fibrillary deposits were included or excluded from the analysis. CONCLUSIONS We contend that patients presenting with Congo-red-negative fibrillary deposits on renal biopsy should be evaluated carefully for monoclonal-gammopathy and cryoglobulins, but there is insufficient published data, as yet, to justify subclassification of FG and ITG as distinct clinical entities. Indeed, we argue that it remains to be determined if FG-ITG represents a unique condition or a forme fruste of cryoglobulin- or gammopathy-associated renal disease. Although the optimal treatment for FG-ITG has not been determined, renal transplantation appears an attractive option in patients with end-stage renal failure.

Atrial natriuretic peptide(31-67) inhibits Na+ transport in rabbit inner medullary collecting duct cells. Role of prostaglandin E2.

Atrial natriuretic peptide (ANP)(31-67), a portion of the atrial peptide prohormone, circulates in humans, and its plasma level varies with atrial pressure. Like the more widely studied carboxy-terminal fragment ANP(99-126), ANP(31-67) stimulates natriuresis and diuresis. We examined the mechanism of this natriuresis by measuring the effects of ANP(31-67) on Na+ transport in cells of the rabbit inner medullary collecting duct (IMCD). ANP(31-67) (10(-8) M) caused a 26 +/- 4% inhibition of oxygen consumption (QO2); half-maximal inhibition occurred at 10(-11) M, suggesting a physiologic effect. This effect was not additive with either ouabain or amiloride, suggesting that it reflected inhibition of Na+ transport-dependent QO2. ANP(31-67) reduced the amphotericin-induced stimulation of QO2 consistent with inhibition by this peptide of the Na(+)-K(+)-ATPase. In addition, ANP(31-67) reduced ouabain-sensitive 86Rb+ uptake under Vmax conditions. Several lines of evidence indicated that PGE2, a known endogenous IMCD Na(+)-K(+)-ATPase inhibitor, mediates pump inhibition by ANP(31-67). Thus, ANP(31-67) inhibits Na+ transport by inhibiting the Na(+)-K(+)-ATPase of IMCD cells, an effect mediated by the generation of PGE2.