Aging phenotype(s) in kidneys of diabetic mice are p66ShcA dependent.

The p66ShcA protein controls cellular responses to oxidative stress, senescence, and apoptosis. Here, we test the hypothesis that aging phenotype(s) commonly associated with the broad category of chronic kidney disease are accelerated in diabetic kidneys and linked to the p66ShcA locus. At the organ level, tissue stem cells antagonize senescent phenotypes by replacing old dysfunctional cells. Using established methods, we isolated a highly purified population of stem cell antigen-1-positive mesenchymal stem cells (Sca-1+ MSCs) from kidneys of wild-type (WT) and p66 knockout (p66 KO) mice. Cells were plated in culture medium containing normal glucose (NG) or high glucose (HG). Reactive oxygen species (ROS) metabolism was substantially increased in WT MSCs in HG medium in association with increased cell death by apoptosis and acquisition of the senescent phenotype. DNA microarray analysis detected striking differences in the expression profiles of WT and p66 KO-MSCs in HG medium. Unexpectedly, the analysis for p66 KO-MSCs revealed upregulation of Wnt genes implicated in self-renewal and differentiation. To test the in vivo consequences of constitutive p66 expression in diabetic kidneys, we crossed the Akita diabetic mouse with the p66KO mouse. Homozygous mutation at the p66 locus delays or prevents aging phenotype(s) in the kidney that may be precursors to diabetic nephropathy.

DC-specific ICAM-3-grabbing nonintegrin mediates internalization of HIV-1 into human podocytes.

Human immunodeficiency virus (HIV)-1 has been demonstrated to contribute to the pathogenesis of HIV-associated nephropathy. In renal biopsy studies, podocytes have been reported to be infected by HIV-1. However, the mechanism involved in HIV-1 internalization into podocytes is not clear. In the present study, we evaluated the occurrence of HIV-1 internalization into conditionally immortalized human podocytes and the mechanism involved. Human podocytes rapidly internalized R5 and X4 HIV-1 primary strains via an endocytosis-dependent pathway, without establishing a productive infection. The HIV-1 internalization was dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) receptor mediated. The role of DC-SIGN was confirmed by using specific blocking antibodies and transfection with small interfering (si) RNA/DC-SIGN. Since podocyte HIV-1 trafficking was not altered by pH-modulating agents, it appeared that HIV-1 routing occurred through nonacid vesicular compartments. Interestingly, transfection of podocytes with neither siRNA/caveolin-1 nor siRNA/clathrin heavy chain inhibited podocyte viral accumulation. Thus it appears that clathrin-coated vesicles and caveosomes may not be contributing to HIV-1-associated membrane traffic.

Lymphopenia in dialysis patients: a preliminary study indicating a possible role of apoptosis.

Lymphopenia is a common finding in dialysis patients. Since infection rate and mortality associated with infection are high in dialysis patients, lymphopenia may be one of the contributing factors. In the present study, we evaluated the mechanism responsible for lymphopenia in these patients. Lymphocytes isolated from dialysis patients showed increased apoptosis (p < 0.001) when compared to lymphocytes isolated from healthy subjects (healthy subjects, 0.5 +/- 0.2% vs. dialysis patients, 8.8 +/- 0.7% apoptotic cells/field). Sera from dialysis patients promoted lymphocyte apoptosis in a time- and dose-dependent manner. These sera also enhanced lymphocyte DNA fragmentation into multiple integers of 180 base pairs in the form of a ladder pattern. Cellulose acetate membranes promoted T cell apoptosis when compared to polysulfone membranes and to control. Cellulose acetate dialysis membranes also appear to promote lymphocyte FasL expression. Similarly, dialysis sera enhanced T cell Fas as well as FasL expression. Neither the cellulose acetate nor polysulfone membranes could induce FasL expression on B cells. Similarly, dialysis sera failed to induce FasL expression on B cells. On the other hand, anti-FasL antibodies attenuated dialysis sera-induced apoptosis in T as well as B cells. Interestingly, dialysis serum showed a 5-fold increase in FasL content when compared with control serum. These results suggest that dialysis-associated factors can induce autocrine death in T cells but the help of activated T cells is required to induce death in B cells.

Monocyte apoptosis in dialysis patients is Fas ligand-mediated.

BACKGROUND: The mononuclear phagocyte system plays an important role in host defense. Since dialysis patients have been reported to show enhanced leukocytes apoptosis, we evaluated the mechanism of increased apoptosis of monocytes in dialysis patients. METHODS: Apoptotic studies were carried out on monocytes isolated from dialysis patients as well as healthy subjects. The effect of dialysis sera and membranes was evaluated on monocyte apoptosis as well as monocyte expression of proapoptotic proteins such as Fas and FasL. To confirm the role of FasL, we evaluated the effect of activated secretory products on T cell apoptosis. In addition, we studied FasL content of dialysis sera and supernatants of activated monocytes. RESULTS: Monocytes isolated from dialysis patients (MDP) showed a greater magnitude of apoptosis when compared to monocytes isolated from healthy subjects (MHS) (MHS, 3.6 +/- 1.1% vs. MDP, 24.3 +/-1.4%). Sera of hemodialysis patients (SHD) promoted (p < 0.001) apoptosis of MHS when compared to pooled control sera (HPS) (HPS, 0.8 +/- 0.5% vs. SHD, 11.5 +/- 0.5% apoptotic cells/field). Dialysis membranes, cellulose acetate membranes in particular, promoted monocyte apoptosis. Interestingly, anti-FasL antibodies partly inhibited dialysis sera-induced monocyte apoptosis. Dialysis membranes also modulated monocyte expression of both Fas and FasL. Secretory products of activated monocytes also promoted T cell apoptosis. Dialysis sera and activated monocyte secretory products showed increased FasL content. CONCLUSIONS: These results suggest that dialysis patients have an increased rate of monocyte apoptosis, which is mediated through a uremic milieu (serum factors). One of these serum factors seems to be FasL. In addition, dialysis membranes seem to promote apoptosis independent of the uremic milieu. The present study provides a mechanistical insight into the enhanced apoptosis of monocytes in dialysis patients.

Morphine-induced degradation of the host defense barrier: role of macrophage injury.

The effect of morphine on the degradation of the host defense barrier in rats and mice was studied. Mice received either 3 or 11 doses of morphine. Mice receiving 11 doses of morphine showed gram-negative bacteremia and bacterial growth in samples of peritoneal fluid (PF), liver, spleen, kidneys, heart, and lungs; PF and tissue samples from only 1 control mouse showed bacterial growth, and no control mice had bacteremia. Mice receiving 11 doses also had suppressed bone marrow macrophage colony formation. Monocytes and peritoneal macrophages harvested from morphine-treated mice showed greater injury than did those from control mice. Pretreatment of mice with naloxone inhibited morphine-induced macrophage injury and degradation of the host defense barrier. In in vitro studies, morphine attenuated the killing of bacteria phagocytosed by macrophages and also facilitated their escape. This study indicates that morphine-induced monocyte and macrophage injury may be linked to degradation of the host defense barrier.

Fas-mediated apoptosis of neutrophils in sera of patients with infection.

In the presence of infection, neutropenia is considered to be a marker of poor prognosis; conversely, neutrophilia may not be a determinant of a better prognosis. Since apoptotic neutrophils are compromised functionally, we evaluated the effect of infection on neutrophil apoptosis. The rate of apoptosis was greater for neutrophils isolated from patients with infection than for healthy controls. Escherichia coli did not directly modulate the rate of neutrophil apoptosis. However, sera from infected patients promoted (P < 0.001) neutrophil apoptosis. Interestingly, the sera of patients with different types of infection (gram negative, gram positive, or culture negative) exerted a more or less identical response on neutrophil apoptosis. Sera of infected patients showed a fivefold greater content of FasL compared to controls. Moreover, anti-FasL antibody partly attenuated the infected-serum-induced neutrophil apoptosis. In in vitro studies, E. coli enhanced monocyte FasL expression. Moreover, conditioned media prepared from activated macrophages from control mice showed enhanced apoptosis of human as well as mouse neutrophils. On the contrary, conditioned media prepared from activated macrophages isolated from FasL-deficient mice induced only a mild degree of neutrophil apoptosis. These results suggest that neutrophils in patients with infection undergo apoptosis at an accelerated rate. Infection not only promoted monocyte expression of FasL but also increased FasL content of the serum. Because the functional status of apoptotic cells is compromised, a significant number of neutrophils may not be participating in the body's defense. Since neutrophils play the most important role in innate immunity, their compromised status in the presence of infection may transfer the host defense burden from an innate response to acquired immunity. The present study provides some insight into the lack of correlation between neutrophilia and the outcome of infection.

Puromycin aminonucleoside induces glomerular epithelial cell apoptosis.

Glomerular epithelial cell (GEC) injury has been considered to play an important role in puromycin aminonucleoside (PAN)-induced nephrosis. We studied the effect of PAN on rat as well as human GEC apoptosis. Morphogic evaluation of GEC apoptosis and necrosis was carried out by staining with H-33342 and propidium iodide. GEC apoptosis was further confirmed by DNA fragmentation assay (by both agarose gel electrophoresis and end-labeling). To determine the dose- and time-response effect of PAN, GECs were treated with variable concentrations of PAN (10 to 500 microg/ml) for variable time periods (6 to 48 h). To determine the role of gene synthesis, we studied the effect of actinomycin D (a transcriptional inhibitor) on PAN-induced GEC apoptosis. To determine the role of free radicals, we evaluated the effect of superoxide dismutase (SOD), dimethylthiourea (DMTU), and catalase on PAN-induced GEC apoptosis. PAN induced GEC apoptosis in a dose- and time-dependent manner. PAN at a high concentration (PAN, 100 microg/ml) also induced a moderate degree of GEC necrosis. In DNA fragmentation assays PAN-treated GECs showed the classic ladder pattern. PAN-induced GEC apoptosis was partly attenuated with free radical scavengers, such as SOD, DMTU, and catalase. In addition, actinomycin D attenuated PAN-induced GEC apoptosis. PAN induces GEC apoptosis, which may be mediated through the generation of reactive oxygen species.

Role of 14-3-3epsilon, c-Myc/Max, and Akt phosphorylation in HIV-1 gp 120-induced mesangial cell proliferation.

Focal glomerulosclerosis (FGS) is the predominant glomerular lesion in patients with human immunodeficiency virus (HIV)-associated nephropathy. Initial mesangial cell hyperplasia and subsequent hypoplasia are common features of FGS. In the present study we evaluated the effect of HIV-1 glycoprotein (gp) 120 on human mesangial cell (HMC) growth. HIV-1 gp 120 stimulated HMC proliferation at lower concentrations, whereas it suppressed cell proliferation at higher concentrations. In parallel to the modulation of cell growth, gp 120 at low concentrations resulted in an increase in the expression of c-Myc, Max, and 14-3-3epsilon proteins and phosphorylation of ATP-dependent tyrosine kinases (Akt) at Ser(473). However, the expression of these proteins decreased with increasing concentrations of gp 120. Furthermore, gp 120 also exhibited a dose-dependent inhibition of Akt phosphorylation at Ser-473 without any significant alteration of Akt expression. Little or no effects of gp 120 were observed on the expression of extracellular signal-regulated kinase (ERK), phospho-ERK, Bcl-2, and Bax proteins. At a higher concentration, gp 120 not only promoted HMC apoptosis but also enhanced expression of Fas and FasL. These results suggest that HIV-1 gp 120 induces alterations in conflicting survival signaling pathways that contribute to the potential dual effects of gp 120 in promoting or inhibiting HMC proliferation.

Receptor mediated endocytosis by mesangial cells modulates transmigration of macrophages.

BACKGROUND: Accumulation of immune complexes in the mesangium is a common finding. Since migration of macrophages (Mphi) in the mesangium has been demonstrated to be an important event in the development of glomerular lesions, we studied the role of immune complexes and mesangial cell (MC) interaction in the transmigration (Tm) of Mphi. METHODS: To determine the effect of MC and immune complexes (aggregated IgG, IgGAg) on transmigration of Mphi. MC were incubated with or without IgGAg in the lower compartment of a modified Boyden Chamber. To determine the effects of the secretory products (as a result of endocytosis of IgGAg by mesangial cells), MC-IgAg conditioned media was prepared and placed in the lower compartment of the Boyden chamber. We evaluated the effects of MC alone, MC + IgGAg, or MC-IgGAg conditioned media on the transmigration of macrophages across a filter. To determine the effect of free radicals on MC-IgAg endocytosis-induced Mphi migration we evaluated the effect of free radical scavengers such as dimethyl thiourea (DMTU) and tetramethylthiourea (TMTU) in MC-IgAg endocytosis-induced Mphi migration. To determine the role of chemokines in MC-IgAs endocytosis-induced Mphi migration we evaluated the effect of ani-MCP-1 antibodies on MC-IgAg endocytosis-induced Mphi migration, and also studied the effects of IgAg on MC mRNA expression of MCP-1 and RANTES. In addition, we evaluated the role of Fc receptors and actin cytoskeleton of MC in transmigration of Mphi. RESULTS: Mesangial cell endocytosis of IgG aggregates (IgGAg) is associated with enhanced (P < 0.001) transmigration of Mphi (control, 11.2 +/- 0.2 vs. MC + IgGAg, 22.1 +/- 0.9 migrated Mphi/field). IgGAg also induced MC mRNA expression for RANTES and MCP-1 on MC. DMTU and TMTU attenuated (P < 0.001) the MC + IgGAg-induced migration of Mphi as well as IgGAg-induced mRNA expression for RANTES and MCP-1. MC and IgGAg interaction products (MC-IgGAg conditioned media) also increased (P < 0.01) transmigration of Mphi (control, 18.3 +/- 1.7 vs. MC-IgGAg conditioned media, 30.7 +/- 0.6 Mphi/field). This effect of MC-IgGAg conditioned media on the migration of macrophages was dose dependent. Anti-MCP-1 antibody partially inhibited MC-IgGAg-induced migration of macrophages. MC and monomeric IgG (MIgG) interaction (MC-MIgG conditioned media) showed a lower (P < 0.05) migration of Mphi, when compared to the MC-IgGAg conditioned media. MC-IgGAg conditioned media prepared from cytochalasin B pretreated MCs also showed a lower (P < 0.001) migration of Mphi when compared with MC-IgGAg conditioned media-induced migration. CONCLUSIONS: These results indicate that MC-IgGAg conditioned media-induced transmigration of macrophages may be mediated through the generation of RANTES and MCP-1 by MC.

Morphine stimulates mesangial cell TNF-alpha and nitrite production.

BACKGROUND: Intravenous opiate abusers are susceptible to develop heroin and HIV-associated nephropathies; however, the role of opiates in the development of these kidney lesions is not clear. Patients with opiate addiction are prone to recurrent infections. METHODS: The effect of morphine was studied on the generation of TNF-alpha with or without LPS (lipopolysaccharide) by cultured mouse mesangial cells. In addition, the effect of morphine was evaluated on mesangial cell nitrite production. To evaluate the role of opiate receptors, we studied the effect of naloxone and naltrexone on mesangial cell TNF-alpha and nitrite production. To determine the role of TNF-alpha on mesangial cell nitrite production, we examined the effect of anti-TNF-alpha antibody on morphine-induced nitrite production. Assay of TNF-alpha and nitrite production was carried by ELISA and Griess method respectively. RESULTS: Morphine alone did not enhance the generation of TNF-alpha by mesangial cells, however, an enhanced (P < 0.001) TNF-alpha production was observed when mesangial cells were first treated with morphine for 18 h and then activated further with LPS. Maximum release of TNF-alpha was seen at a concentration of 10(-12) M of morphine. Opiate receptor antagonists (naloxone and naltrexone) inhibited the effect of morphine. Morphine also amplified (P < 0.0002) the effect of LPS on mesangial cell nitrite production. Anti-TNF-alpha antibody attenuated morphine induced nitrite generation. CONCLUSION: We conclude that morphine stimulates the generation of TNF-infinity by LPS-activated mesangial cells. This effect of morphine seems to be opiate receptor mediated and has a downstream effect in the form of mesangial cell nitrite generation. The present in vitro study provides the basis for a hypothesis that morphine may be playing a role in the development of heroin and HIV-associated nephropathies.

Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta.

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta antibody inhibited (P < 0.001) the morphine-induced apoptosis in J774 cells (control 0.7 +/- 0.4%; 10-6 M morphine 23.5 +/- 0.7%; anti-TGF-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta by morphine-treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine-induced bax expression was inhibited by anti-TGF-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.

Vasoactive agents modulate migration of monocytes across glomerular endothelial cells.

BACKGROUND: Macrophages seem to play an important role in the development of glomerulosclerosis. In both human and experimental animal models of focal glomerulosclerosis (FSGS), infiltration of macrophages in the mesangium has been considered key in the development of FSGS. METHODS: In the present study, we evaluated the effect of vasoactive agents on the migration of monocytes across a filter in a modified Boyden chamber as well as across a cultured glomerular endothelial cell layer (in vitro model of glomerular mesangium). Both light as well as scanning electron microscopic studies were performed. We evaluated the effect of vasoactive agents including histamine, prostaglandin (PG) E2, angiotensin II, endothelin-1, platelet-activating factor, and interleukin-1 (IL) on the migration of monocytes/macrophages across an endothelial cell layer as well as a gelatin-coated filter. In addition, we evaluated the effect of cyclic adenosine 3',5' cyclic monophosphate (cAMP) and PGE2 on vasoactive-induced migration of monocytes. RESULTS: Histamine increased (P < 0.003) the migration of monocytes across the filter. This effect of histamine was dose-dependent. Histamine at concentrations of 10(-8) to 10(-5) mol/L induced optimal migration across the filter (control, 16.6 +/- 1.1 vs histamine, 10(-8) mol/L, 40.9 +/- 0.9 monocytes/high power field). Cimetidine, an H2 receptor blocker, attenuated (P < 0.001) the effect of histamine on the migration of monocytes. PGE2 inhibited the migration of monocytes in a dose-dependent manner. Histamine increased (P < 0.001) the passage of monocytes across the glomerular endothelial cell layer (control, 1012 +/- 37 vs 1711 +/- 163 cpm/well). Histamine also increased the migration of murine macrophages across the glomerular endothelial cell layer. PGE2 inhibited the migration of monocytes across the endothelial cell layer under basal as well as histamine-stimulated states. Dibutyryl cyclic (DBc) AMP also attenuated the migration of monocytes under basal as well as histamine-stimulated states. Both PGE2 and DBcAMP also attenuated the IL-1 beta-stimulated migration of monocytes. Angiotensin II, endothelin-1, and platelet-activating factor did not modulate the migration of monocytes. CONCLUSIONS: Vasoactive agents directly modulate the transmigration of monocytes. The present in vitro study provides a basis for a hypothesis that vasoactive agents may also be modulating the migration of monocytes across the glomerular endothelial cell layer (into the mesangium).

HIV-1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells.

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)-associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV-1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein-treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0. 001) the growth of HGEC. To evaluate the mechanism of gp120 protein-induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti-gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120-induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c-fos and at higher concentrations promoted mRNA expression of c-jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes.

Morphine promotes apoptosis in Jurkat cells.

Patients with intravenous heroin addiction are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose-dependent manner. DAGO, a specific mu receptor agonist, also promoted Jurkat cell apoptosis. DNA isolated from morphine-treated Jurkat cells and T lymphocytes also showed integer multiples of 200 base pairs. Superoxide dismutase (SOD) enhanced lymphocyte apoptosis; whereas catalase attenuated the morphine-induced apoptosis of Jurkat cells as well as of T lymphocytes. Morphine-treated Jurkat cells also showed a decreased expression of bcl-2 and an enhanced expression of bax. In addition, morphine-treated Jurkat cells showed activation of caspase-3. These results indicate that morphine-induced T lymphocyte apoptosis may be mediated through the generation of reactive oxygen species. The change in ratio of bax and bcl-2 seems to tilt the balance toward apoptosis, leading to the activation of caspase-3. This study provides further support for the hypothesis that morphine may be directly compromising immune function by enhancing apoptosis of T lymphocytes in patients with heroin addiction.

Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3.

Aging is associated with altered immune function. We previously reported that splenocytes and thymocytes undergo apoptosis with aging in rats. In the present study, we examined the expression of genes associated with apoptosis in splenocytes and thymus in aging rats. We evaluated the expression of bax, interleukin 1-beta-converting enzyme (ICE)/ced-3 protease family, caspase-3 and tumor suppressor gene p53. Rats in age groups of 6, 24, 48, and 96 weeks were sacrificed; thymocytes and splenocytes were isolated followed by lysis in a modified RIPA buffer containing protease inhibitors. Western blot analysis of proteins was performed by probing immunoblots with antibodies against p53, bax and PARP (poly ADP-ribose polymerase). Increased aging was associated with enhanced expression of bax, p53 and cleavage of PARP by Caspase-3. The expression of p53 and cleavage of PARP indicates the presence of damaged DNA; nevertheless, the cleavage of PARP or activation of caspase-3 may be playing an important role in the initiation of early events in apoptosis. These results suggest that aging of splenocytes and thymocytes is associated with the expression of cell death genes. The present study provides an insight into age-associated altered immune function.

Ethanol-induced macrophage apoptosis: the role of TGF-beta.

Both clinical and laboratory reports indicate that ethanol addicts are prone to recurrent infections. We hypothesize that ethanol promotes macrophage apoptosis, thus compromising the efficiency of the mononuclear phagocyte system in dealing with infection. We studied the effect of ethanol on macrophage apoptosis. Human monocytes isolated from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 1.2 +/- 0.3% vs after, 28.4 +/- 3.7% apoptotic cells/field). Peritoneal macrophages harvested from ethanol-treated rats also showed increased (p < 0.0001) apoptosis. DNA isolated from peritoneal macrophages of ethanol-treated rats displayed integer multiples of 200 base pairs (ladder pattern). Furthermore, macrophages harvested from ethanol-treated rats had an enhanced expression as well as accumulation of TGF-beta. In in vitro studies, ethanol promoted apoptosis of human monocytes as well as rat peritoneal macrophages. In addition, ethanol enhanced apoptosis of murine macrophages (J774) in a time-dependent manner. The ethanol-induced apoptosis was amplified by LPS and partly attenuated (p < 0.001) by anti-TGF-beta Ab. TGF-beta also promoted macrophage apoptosis in a dose-dependent manner. Moreover, ethanol enhanced TGF-beta protein production by macrophages. These results indicate that ethanol promotes macrophage apoptosis. This effect of ethanol seems to be partly mediated through the generation of TGF-beta by macrophages.

Matrix-mesangial cell interaction modulates migration of macrophages.

BACKGROUND: Macrophages (Møs) have been demonstrated to play an important role in immune-mediated renal injury. Accumulation of macrophages in the mesangium has been reported to be a key event in the development of focal glomerulosclerosis. We hypothesized that mesangial cells (MCs) and matrix interaction may be a determinant for the migration of Møs into the mesangium. Therefore, we studied the effect of the interaction between matrix and MCs on the migration of Møs. METHODS: Mouse MCs were plated on Petri dishes coated either with buffer, collagen type I, III, IV, or Matrigel in media containing 1% fetal calf serum for 48 hours. Subsequently, supernatants were collected and stored. The effect of these supernatants (conditioned media) was evaluated on the migration of Møs across a filter in a modified Boyden chamber. RESULTS: Conditioned media from MCs grown on Matrigel (MC-Matrigel interaction products, MC-MGP) enhanced the migration of macrophages across a filter in a modified Boyden chamber when compared with conditioned media from MCs grown on plastic, collagen type I, type III, or type IV (MC-PP, MC-CI, MC-CIII, and MC-CIV). MC-MGP enhanced the migration of Møs in a dose dependent manner. Anti-MCP-1 antibodies attenuated (P < 0.05) the MC-MGP-induced Mø migration (MC-MGP, 16.8 +/- 2.5 vs MC-MGP + anti-MCP-1 antibody, 6.5 +/- 1.2 migrated macrophages/field, n = 12). Anti-TGF-beta antibodies did not attenuate MC-MGP-induced Mø migration. MCs grown on Matrigel showed a 5-fold increase of MCP-1 mRNA when compared with cells grown on plastic or collagen type IV. CONCLUSIONS: The present study suggests that matrix components may modulate the migration of Møs. This effect of MC-matrix interaction on macrophage migration may be mediated through the generation of MCP-1.

Metal-catalyzed oxidation of extracellular matrix increases macrophage nitric oxide generation.

BACKGROUND: Oxygen radicals are believed to play a significant role in glomerular disease. In part this may be due to oxidation of lipids, but protein oxidation may play a contributory role as well. We have demonstrated that the mesangial extracellular matrix is susceptible to metal-catalyzed oxidation and that this increases scavenger receptor-mediated adhesion of macrophages, cells which appear to be important participants in glomerular injury via their secretory products. As other scavenger receptor ligands can increase macrophage nitric oxide generation, we examined whether oxidation of matrix could increase the activity of macrophage inducible nitric oxide synthase (iNOS). METHODS: Extracellular matrix was oxidized using a metal-catalyzed oxidation system. Matrix oxidation was measured using carbonyl analysis, and iNOS activity in macrophages seeded onto the matrix was measured by nitrite determination and Western and Northern analyses for iNOS. RESULTS: Macrophages exposed to oxidized matrix demonstrated a significant enhancement of iNOS activity. This enhancement could be antagonized by cotreatment of matrix with the radical spin trap N-tert-butyl-a-phenylnitrone, resulting in a corresponding decrease in protein carbonyl content, a measure of protein oxidation. Seeding macrophages onto oxidized matrix and adding the scavenger receptor ligand polyinosinic acid further augmented iNOS activity, suggesting that additional scavenger receptors were available to bind ligand and that further augmentation of iNOS activity did not require an additional change in cell shape. Western blot analysis revealed an increase in iNOS protein expression as a consequence of interaction with the oxidized matrix, but there was no difference in iNOS mRNA expression by Northern analysis suggesting a post-transcriptional mechanism for enhanced iNOS activity. CONCLUSION: These data demonstrate that oxidation of extracellular matrix enhances macrophage nitric oxide generation, and suggest a previously undescribed role for extracellular matrix modification in the regulation of cellular function and possibly the mediation of glomerular injury.

Metal-catalyzed oxidation of immunoglobulin G impairs Fc receptor-mediated binding to macrophages.

Enhanced oxidative stress is a feature of inflammatory and infectious conditions. Proteins may be important targets of oxidation and this may alter their function. We evaluated whether metal-catalyzed oxidation of IgG could alter its ability to bind to Fc receptors on macrophages. Human IgG incubated with an FeCl3/EDTA/ascorbate metal-catalyzed oxidation system resulted in a significant increase in carbonyl content, a measure of protein oxidation, compared to IgG treated with EDTA alone (control). Western blot analysis using an antibody to oxidized protein revealed an increase in antibody binding to both the heavy (Fc portion-containing) and light chains of IgG treated with the oxidizing system. Western blot analysis of papain-digested IgG confirmed oxidative modification of the Fc portion. Binding studies carried out with J774.16 macrophages demonstrated significantly diminished ability of the oxidized IgG to bind to macrophage Fc receptors compared to control IgG. These data demonstrate that IgG is susceptible to metal-catalyzed oxidation and that this impairs its ability to bind to macrophage Fc receptors. Oxidation of IgG might play a role in modulating immune function in infection and disorders associated with immune complex formation by diminishing IgG binding to phagocytic cells.

HIV-1 gp120 envelope protein and morphine-tubular cell interaction products modulate kidney fibroblast proliferation.

BACKGROUND: Renal interstitial scarring is an important feature of HIV-associated nephropathy. Intravenous drug abuse has been demonstrated to be a risk factor for the development of HIV-associated nephropathy in patients with HIV infection. We studied the effect of tubular cell-morphine and/or HIV-1 gp120 envelope protein interaction products on kidney fibroblast (KF) proliferation and apoptosis. METHODS: Tubular cell-morphine and/or gp120 interaction products were prepared by incubating confluent human proximal tubular cells with buffer (TCP), morphine (10(-3) mol/L) (TCM-IP), gp120 (0.01 microgram/mL)(TC-120IP), or morphine (10(-3) mol/L) + gpl20 (0.01 microgram/mL) (TCM-120IP). To evaluate the effect of tubular cell interaction products (TCIP) on KF proliferation, growth arrested kidney fibroblasts were treated with variable concentrations (5%, 10%, 20%, 30%, and 50%) of TCP, TCM-IP, TC-120IP, or TCM-120IP for 48 hours. To evaluate the role of cytokines in TCIP-induced KF proliferation, cells were incubated with TCIP with or without cytokine neutralizing antibodies to TGF-beta, TNF-alpha, FGF, or IL-6 for 48 hours. Subsequently, cells were counted in a hemocytometer (n = 3). To evaluate the effect of TCIP on KF apoptosis, cells were treated with 50% TCP, 50% TCM-IP, 50% TC-120IP, or 50% TCM-120IP for 24 hours and stained with H-33342 and propidium iodide. In parallel experiments KFs were harvested under identical conditions, DNA was isolated and run on gel electrophoresis. To evaluate the role of early growth genes in TCM-120-induced KF proliferation, TCM-120IP-treated cells were probed with cDNA for c-fos and c-jun. RESULTS: TC-120IP at a lower concentration (20%) enhanced (P < 0.001) proliferation of KF when compared with TCP. TCM-IP did not stimulate KF proliferation. On the contrary, TCM-120IP at a lower concentration (20%) promoted (P < 0.001) KF proliferation when compared with TCP, TCM-IP and TC-120IP. TCM-120IP at a lower concentration (20%) also enhanced KF mRNA expression of c-fos and c-jun. TCM-120IP enhanced KF proliferation in a dose-dependent manner. All tubular cell interaction products at a higher concentration (50%) promoted apoptosis of KF. CONCLUSIONS: Tubular cell-gp120 interaction products stimulated KF proliferation. Morphine amplified the effect of tubular cell-gp120 interaction on the proliferation of KF. TCM-120IP-induced KF proliferation may be mediated through the expression of early growth genes; whereas TCM-120IP-induced KF growth suppression may be mediated through the induction of apoptosis.