Effect of Advanced Glycosylation End Products on the Induction of Nitric Oxide Synthase in Murine...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 358–362 (1996)ARTICLE NO. 1180

Effect of Advanced Glycosylation End Products on the Induction ofNitric Oxide Synthase in Murine Macrophages

Armando Rojas,1 Luis Caveda, Cheyla Romay,* Eduardo Lopez, Sandra Valdes,Julio Padron, Luis Glarıa, Ofelia Martınez, and Rene Delgado

Pharmacology and Toxicology Department, Center of Pharmaceutical Chemistry, POB 6990, Havana, Cuba; and*Biochemistry Lab., National Center for Scientific Research, POB 6880, Havana, Cuba

Received July 4, 1996

We studied the role of advanced glycosylation end products on the induction of nitric oxide synthase inperitoneal mouse macrophages previously exposed to modified BSA. A dose-dependent increment in thenitric oxide production induced by LPS and IFN-g was observed when cell cultures were pretreated withmodifed BSA for 48 hours. In addition, the up regulation of nitric oxide production was also time-dependent,being maximal at 24–48 hours. Experiments carried out in the presence of neutralizing antibodies to IL-1and TNF-a, suggested that up regulation was not due to the capacity of modified BSA to induce bothproinflammatory signals. The up regulation of nitric oxide production was paralelled with an increase iniNOS mRNA. q 1996 Academic Press, Inc.

Reducing sugar such as glucose can react nonenzymatically with the amino groups ofproteins to form complex structures which are called advanced glycosylation end-products(AGEs) (1).

This process goes by as a function of protein half-life and ambient glucose levels and it isthought to lead to irreversible tissue damage not only by protein cross-linking but also inducingproinflammatory cytokines such as TNF-a and IL-1 in monocytes (2) and IFN-g in T-lympho-cytes (3), through a membrane receptor system which mediates the binding and internalizationof AGE-modified macromolecules.

In normal aging and in some pathological conditions such as diabetes the excesive accumula-tion of AGEs could lead to tissue dysfunction, and particularly in the development of diabeticnephropathy in the latter (4). Both aging and diabetes are commonly acompanied by athereoscl-erosis.

Nitric oxide (NO) is synthetized from L-arginine by the enzyme nitric oxide synthase (NOS,EC 1.14.13.39). Thus far, three distinct NOS isoforms have been cloned and characterized. Twoof them are constitutive and calcium-dependent and mainly present in endothelial cells (ecNOS)and neurons (nNO), playing important roles in vascular hemostasis and neurotrassmission, respec-tively (5,6). These two isoforms constrated with a calcium-independent isoform which is notexpressed unless cells are previously exposed to some cytokines, microbes and bacterial productsand thus it is termed inducible NOS (iNOS) which exerts its main roles in cytostatic and cytotoxicprocessess in tissues as well as in certain pathogens and tumor cells (7).

Several experimental evidences support the fact that an enhanced NO production is evendetected in the early onset of diabetes, where infiltrating macrophages play a protagonic role(8). The production of NO by infiltrating macrophages has been also implicated in inflammatoryglomerulonephritis (9). Infiltration of the arterial subendothelial intima by macrophages is alsoan important event preceeding the development of the atherosclerotic lesion.

1 To whom correspondence should be addressed.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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AID BBRC 5153 / 6906$$$$81 07-29-96 07:03:27 bbrca AP: BBRC

Vol. 225, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Given the spontaneous and progressive acumulation of AGEs within aging and diabetictissues and particularly in glomeruli (10) and subendothelial intima (11), as well as the ubiquityof macrophages in any inflammatory process we investigated whether AGEs may have anyeffect on the production of nitric oxide by murine macrophages.

MATERIALS AND METHODS

Materials. All reagents were from Sigma Chemicals (St. Louis, MO) unless otherwise noted. IFN-g was a kindgift of Dr F.Y. Liew (Glasgow University, Scotland). Antiserum against IL-1 and TNF-a were kindly supplied byDrs P. Ghezzi and A. Mantovani (Istituto di Ricerche Farmacologiche ‘‘Mario Negri’’, Milano, Italy), respectively.

Preparation of AGE-modified bovine serum albumin. AGE-BSA was prepared by incubating BSA in PBS with 50mM glucose at 37 7C for 6 weeks in the presence of PMSF (1.5nM) , EDTA (1mM) and antibiotics (100 mg/mlpenicillin and 40mg/ml gentamicin) as described (3). Control BSA was incubated under the same conditions withoutglucose. AGE-specific fluorescence was determined at 450 nm upon excitation at 390 nm using a fluorescent spectrome-ter. After 6 weeks AGE-BSA contained 85 arbitarry fluorescent units (AFU) / 100 mg of protein, whereas unmodifiedBSA maintained background levels (5 AFU/ 100m g of protein). To verify the effect of reducing early glycosylationproduct on nitric oxide production, AGE-BSA was incubated with 200-molar excess NaHB4 for 10 min at 47C asdescribed (3).

Cell culture. Peritoneal macrophages were obtained from Balb/c mice that were injected intraperitoneally with 1mL of 3 % Brewer’s thioglicolate medium. Peritoneal exudate cells were harvest after 3 days and then plated in 4mls of RPMI 1640 with 10 % FBS in 6-wells plates at 6 1 106 cells/well and allowed to adhere for 2 hours at 377C.After removing non adherent cells, BSA-AGE or unmodified BSA were added at different concentration and cultureswere incubated for 48 hours. After several washes with PBS, adherent monolayers were stimulated with LPS (1mg/ml) and IFN-g (50 U/ml). Cell-free culture supernatants were monitored for nitrite levels after further 24 hours.

NO2 determination. Cell-free culture supernatants were mixed with equal amounts of Griess reagent (sulfanilamide1%, naphtylethylenediamine 0.1% in phosphoric acid 2.5%). Samples were incubated at 25 7C for 10 min andabsorbance was measured at 550 nm using a microplate reader.

Northern blot analysis for iNOS mRNA. Total cellular RNA was extracted from 107 peritoneal macrophages by theguanidium thiocyanate phenol-chloroform method (12). Electrophoresis of RNA samples (10 mg/lane) was performedin 1.2 % agarose/2.2 M formaldehyde gels and the gels subsequently blotted onto nylon membranes by capillaryaction and then baked for 2 hours (80 C ) prior to prehybridization. Fluorescein labelling of a 645 bp HincII-BamHI fragment of mouse iNOS cDNA, hybridization and detection were performed according to the intructions ofGeneImage system (Amersham). Blots were subsequently rehybridize with b-actin cDNA probe as an internal control.

RESULTS

As depicted in Figure 1, the preincubation of peritoneal macrophages with different concen-trations of AGE-modified BSA was able to up regulate in a dose-dependent manner the nitriterelease induced by LPS and IFN-g. No effects on nitric oxide synthesis was achieved whencells were pretreated with unmodified BSA even at 250 mg/ml. In addition, the incubation ofcells with either AGE-BSA or unmodified BSA up to 250 mg/ml without further stimulationdid not produce any increase in the nitrite levels (data not shown). As expected, L-NMMA(300 mM), an specific inhibitor of nitric oxide synthase reduced significantly the LPS/IFN-g -induced nitric oxide synthesis.

Reduction with sodium borohydride did not affect the ability of AGE-BSA to up regulatethe LPS/IFN-g-induced nitric oxide production, suggesting that this proccess is not mediatedby early glycosylation products, such as a Schiff base or the Amadori product (data not shown).

In addition, the effect of AGE-BSA on the induction of nitric oxide synthase was also time-dependent (Figure 2), being maximal at 24 hours, in lesser extension at 12 hours and no effectswere observed when added at the same time of stimuli (time 0 hour).

Although cells were stimulated with LPS and IFN-g only after extensive cell culture washes,we also carried out some experiments in the present of neutralizing polyclonal antibodiesagainst TNF-a and IL-1, to ensure that this upregulation was not due to the capacity of AGEsto induce the synthesis and release of both cytokines as previously described, which couldthen act as synergistic signals to the stimuli used. Both polyclonal antibodies were added atconcentration previously tested to neutralize both TNF-a and IL-1 biological activities, as

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FIG. 1. Dose-dependent up regulation of nitrite level in cell culture supernatant of elicited peritonal macrophagespreincubated with different concentrations of AGE-BSA (0, 50, 100, 150, and 250 mg/mL ) for 48 hours and thenstimulated with LPS (1 mg/mL) and IFN-g (50 U/mL) and further incubated for 24 hours. Control cultures werepreincubated with unmodified BSA at 250 mg/mL and further treated with the stimuli. Data are means { SEM ofthree independent experiments. * põ0.05, ** põ0.01 by Student’s t test.

measured by the TNF-mediated L929 cell toxicity and the thymocyte costimulation bioassays,respectively. As can seen, there were no differences in the LPS- and IFN-g-induced nitricoxide production by macrophages pretrated with AGE-BSA in the presence or absence ofneutralizing antibodies.

In figure 3 is observed the effect of AGE-BSA pretreatment of macrophages on the levelof iNOS mRNA induced by LPS and IFN-g showing that the up regulation of NO production,measured as nitrite, is paralleled by an enhanced iNOS mRNA level.

DISCUSSION

In this study we have shown that nitric oxide synthase induction is upregulated in murinemacrophages when cells were pretreated with AGEs. Previous studies have demonstrated thatAGEs can induce macrophage activation as demostrated by the release of proinflammatorycytokines such as TNF-a and IL-1 (2). The contribution of these cytokines to the upregulationof the nitric oxide synthase induction is unlikely given the results obtained in the presence ofneutralyzing antibodies against IL-1 and TNF-a.

Very recently, it has been reported an enhanced expression of iNOS in cultured murinemacrophages by elevated glucose levels (13). This up regulation is achieved even after incuba-tion times as shorter as 24 hours, therefore this fenomenon is unlikely to be related to AGEs.Furthermore, the results obtained after reduction with sodium borohydride confirm this assump-tion. At present these findings are the first to documentated an effect of AGEs on the nitricoxide synthase induction in murine macrophages.

AGEs are able to increase mRNA of collagen type IV and laminin in mouse mesengialcells (14) and therefore it is thought to play an important role in the accumulation of extracellu-

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FIG. 2. Time-dependent up regulation of LPS and IFN-g -induced nitric oxide synthesis by AGE-modified BSA.Panel A. Cell cultures were pretreated with 250 mg/mL of AGE-BSA for 12, 24, and 48 hours and further stimulatedto produce nitric oxide by the addition of LPS (1mg/mL) and IFN-g (50 U/mL) for 48 hours. Panel B. Cells weretreated as in Panel A, but in the presence (/) and absence (0) of neutralizing antibodies against IL-1 and TNF-aadded at the same time of AGE-BSA. Data are means { SEM of three independent experiments. * põ0.05, ** põ0.01by Student’s t test.

lar matrix components that practically obliterate the glomerular vascular loops. On the otherhand, increased renal production of NO in glomerular diseases may reduce the productionand accumulation of matrix components and thus contributing to attenuate the severity ofglomeruloesclerosis (15). In this context, the up-regulation of nitric oxide production exerted

FIG. 3. Northern analysis (10 mg of total RNA/lane) of cell cultures preincubated with AGE-BSA at 250 mg/mlfor 48 hours and then stimulated with LPS (1 mg/mL) and IFN-g (50 U/mL) for 6 hours. Control cultures were treatedwith LPS and IFN-g, and with AGE-BSA without any further stimulation. Similar results were obtained in threeseparate experiments.

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by AGEs may represent a regulatory element to the accumulation of extracellular matrixcomponents induced by them-self.

NO apperars to be a double-edged sword because it can protect cells and tissues fromdamage, but also it is an important proinflammatory molecule. In fact, NO has been reportedto be protective by preventing the hypoperfusion of several organs, including the kidneys,during acute endotoxemia (16). On the contrary, high amounts of NO produced in the glomeru-lus either by infiltrating macrophages or resident mesangial cells may account for tissue damagewhich is seen in several models of glomerulonephritis (17,18).

In connection with the biological relevance of these findings to the diabetes-related athero-sclerosis, AGEs have been previously found to induce transendothelial monocyte chemotaxis(11) and high levels of AGEs immunoreactivity has been observed within atheroscleroticplaques in blood vessels from diabetic patients (19). Furthermore, inducible nitric oxide activityhas been reported very recently in atherosclerotic vessels (20).

Although the molecular mechanism(s) by which AGEs exerted its action on nitric oxidesynthase induction remains to be established, these findings may represent new insights intothe deleterious effect of AGEs accumulation in the progression of diabetes-related processess,such as atherosclerosis and glomerular injury.

ACKNOWLEDGMENTSThe authors thank Dr. Q.w. Xie and C.F. Nathan for the generous gift of the iNOS probe.

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Am. J. Physiol. 266, H1558–H1564.17. Jansen, A., Cook, T., Taylor, M., Largen, P., Riveros-Moreno, V., Moncada, S., and Catell, V. (1994) Kidney

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