Vancomycin down-regulates lipopolysaccharide-induced tumour necrosis factor alpha (TNFα) production and TNFa-mRNA accumulation in human blood monocytes

Immunophannacology

!,t,'w ELSEVIER

Immunopharmacology35 (1997) 265-271

Short communication

Vancomycin down-regulates lipopolysaccharide-induced tumour necrosis factor alpha (TNFa) production and TNFc -mRNA accumulation in human blood monocytes Maciej Siedlar a, *, Antoni Szczepanik b, Jerzy Wi~ckiewicz Anna Pituch-Noworolska

a

a,

Marek Zembala a

a Department of Clinical Immunology, Polish-American Children Hospital, Medical Faculty, Jagiellonian University, Wielicka 265 str., 30-663 Cracow, Poland b 1st Department of Surgery, Medical Faculty, Jagiellonian University, Kopernika 40 str., Cracow, Poland

Received 4 March 1996;revised 22 July 1996; accepted 31 July 1996

Abstract The cytokines play an important role in the cascade of the pathological events leading to septic shock. The TNFa produced by monocytes/macrophages upon stimulation with bacterial fragments may contribute to induction of this cytokine cascade. Moreover, the antibiotics used for antimicrobial therapy may cause the increase of TNFot production due to massive bacterial killing and exposure of monocytes/macrophages to bacterial cell constituents. To investigate the effect of Vancomycin on TNFt~ production, an in vitro model of LPS-stimulated monocytes was used. The level of TNFt~ protein or TNF biological activity were tested in the culture supernatants of monocytes with LPS. Vancomycin down-regulated, in dose-dependent manner, the TNFa production. Vancomycin also inhibited TNFt~-mRNA accumulation in LPS-stimulated monocytes, as assessed by fluorescence in situ hybridization (FISH) in cell suspension. The down-regulation of TNFa production in LPS-stimulated monocytes may indicate that inhibition of this cytokine release is one of the important therapeutic effects of Vancomycin in sepsis. Keywords: Monocytes;Vancomycin;Tumour Necrosis Factor alpha; Lipopolysaccharide

Abbreviations: BSA, bovine serum albumine; cDNA, complementaryDNA; DIG, digoxygenin; dNTP, deoxynucleotides;dsDNA, double stranded DNA; dTTP, 2-deoxythymidine5-tdphosphate; dUTP, 2-deoxyuridine5-triphosphate; EDTA, ethylenediaminetetraacetic acid; FCS, foetal calf serum; FISH, fluorescence in situ hybridization;FITC, fluoresceinisothiocyanate;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide;mAb, monoclonal antibodies; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCR, polimerase chain reaction; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline solution; PG, peptidoglycan; SSC, standard saline citrate; TA, teichoic acid; TNFa, turnout necrosis factor alpha * Corresponding author. Phone/fax: + (48-12) 58-24-86. 0162-3109/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. PH S0162-3 109(96)00156-7

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1. Introduction

The treatment of severe infections and associated septic syndrome is still unsatisfactory, despite the use of target-matched antibiotic therapy. Cytokines play an important role in the cascade of pathological events leading to septic shock (Suter et al., 1992; Zabel and Schade, 1994). The cytokine that induces the cascade--tumour necrosis factor alpha--is produced by monocytes/macrophages following stimulation with bacterial products (Beutler and Cerami, 1988; Mohler et al., 1994). In a case of Gram-negative bacterial sepsis, lipopolysaccharide (LPS) induces release of a large amount of TNFa, but also in mixed Gram-positive/negative sepsis, the fragments of Gram-positive bacteria cell wall--teichoic acid (TA) and peptidoglycan (PG)--are able to trigger the release of TNFc~ and interleukin-6 from monocytes (Mattsson et al., 1993; Heumann et al., 1994). The elevated serum level of TNFa in septic patients correlates with the fatal outcome (Debets et al., 1989). Current strategy of treatment of the bacterial sepsis is based on antimicrobial and antiinflammatory therapy (Tracey and Cerami, 1994). The antiinflammatory therapy is targeted to the inhibition of cytokine biological activity at the level of producing cells, circulating cytokines, and target organs (liver, lungs). Treatment with antibiotics in therapy may cause the release of TNFct that is due to massive bacterial killing and exposure of macrophages to the increased amount of circulating bacterial products. This was observed in many experimental models and in vitro studies (Hurley, 1992). The release of endotoxin depends on the mode of action of antibiotics. This effect is observed during ceftriaxone, imipenem-cilastatine, ampicillin, cefotaxime, ciprofloxacine and piperacilin therapy (Hurley, 1992; Crosby et al., 1994). Vancomycin is the bactericidal antibiotic used in severe Gram-positive, predominantly staphylococcal infections, i.e., endocarditis, septic complications of intravascular devices and endoprothesis or staphylococcal sepsis in immunocompromised patients (Cooper and Given, 1986). The postantibiotic peak of TNFa was observed in experimental bacterial endocarditis treated with other as Vancomycin antibiotics (Mohler et al., 1994). However, as far as we know,

there is no evidence for enhanced TNFo~ production in septic patients treated with a bactericidal glycopeptide antibiotic--Vancomycin. In this study the effect of Vancomycin on TNFa production by LPSstimulated monocytes was investigated. This preliminary report indicates that Vancomycin can down-regulate, at the level of transcription, TNFa production by human monocytes treated with LPS.

2. Materials and methods

2.1. Isolation of monocytes

Human peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood obtained from healthy donors by standard Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) gradient. Monocytes were separated by counter-current centrifugal elutriation using Beckman JE-5.0 elutriation system (Beckman, Palo Alto, CA) equipped with 5 ml Sanderson separation chamber, as previously described (Zembala et al., 1994). Isolated monocytes were > 90% pure as judged by FACS analysis (FACScanV, Becton-Dickinson, Mountain View, CA) using anti-CD14 (Leu-M3) monoclonal antibody (mAb) (Becton-Dickinson). Monocytes were suspended in RPMI-1640 medium (Biochrom, Berlin, Germany) supplemented with antibiotics (100 /zg/ml streptomycin, 100 U / m l penicillin, 25/~g/ml gentamycin), 2 mM glutamine (Gibco, Paisley, UK), 10% foetal calf serum (FCS, Biochrom) at 1 X 106/ml and used for experiments. 2.2. Monocytes stimulation and culture

Monocytes were cultured with LPS from E. coli, serotype 0127:B8 (0.01-1 /xg/ml; Sigma, St. Louis, MO) in flat-bottom microplates (Nunc, Roskilde, Denmark) in 200 /zl of RPMI 1640 medium, with different concentrations (1-1000 /xg/ml) of Vancomycin (kindly provided by Lilly, Warsaw, Poland). In some experiments, monocytes were incubated with anti-CD14 mAb (10 /xg/ml; Becton-Dickinson) together with LPS (1 /zg/ml). The cells were cultured for 18 h at 37°C in CO 2 humidified atmosphere, then the supernatants were harvested and tested for TNFa content. For the hybridization, monocytes were incubated in 1 ml of RPMI 1640 medium for 4 h with

M. Siedlar et al. / Immunopharmacology 35 (1997) 265-271

Vancomycin a n d / o r 1 /zg/ml of LPS, then washed twice in PBS and fixed for 30 min. at room temperature in 1% formaldehyde (Merck, Darmstadt, Germany) in PBS containing 5 mM MgC12. The cells were centrifuged, washed in 1 × SSC with 0.5 m g / m l of BSA (SSC-BSA), spun down and resuspended in 70% ethanol at the concentration of 4 × 106/ml and stored at - 7 0 ° C until use. To exclude the possible toxic effect of Vancomycin or LPS, the viability of monocytes was checked by trypan blue exclusion test. The viability of the cells after incubation was in range of 85-90%.

2.3. Determinantion of TNFa in the culture supernatants TNFa protein was determined by ELISA (TNFa-alpha EASIA, Medgenix, Fleurus, Belgium) or TNF activity was established in a bioassay using actinomycin D-pretreated mouse L929 cell line, as previously described (Zembala et al., 1993).

2.4. Determination of mRNA for TNFa by fluorescence in situ hybridization (FISH) in cell suspension 2.4.1. Labeling of probes The pGEM1 plasmid vector containing 1138 bp PstI-PstI fragment of human TNFa cDNA (gift from W. Fiers, Ghent Univ., Belgium) was used for preparation of the dsDNA probe (318 bp) by the PCR DIG-11-dUTP amplification technique. The primers used were: 5'-TAG ATG GGC TCA TAC CAG GG-3' (antisense) and 5'-AGC CCA TGT TGT AGC AAA CC-3' (sense). As control, dsDNA probe coding for GAPDH enzyme was synthetized directly on human genomic DNA sample using the following primers: 5'AGC GTC AAA GGT GGA GGA GT-3' (antisense) and 5'-ACC CAG AAG ACT GTG GAT GG-3' (sense) overlapping the fragment (342 bp) of exon 8 of the human gene for GAPDH enzyme. For the first step we amplified cDNAs to obtain the templates stocks from which the DIG-11-dUTP labeled probes were synthetized. The PCR mixture were prepared in the final volume of 100/zl containing: PCR buffer (Gibco-BRL); 1.5 mM MgC12; 20 /~M of each of dNTPs; 100 ng of linearized plasmid DNA; 0.1 /zM of each primer; 2.5 units of TaqI DNA polymerase (Gibco-BRL). The DNA amplifica-

267

tion was carried out in GeneAmp PCR 9600 Thermal Cycler (Perkin-Elmer, Norwalk, CT) at the following conditions: initial denaturation--5 min at 94°C; 55 cycles of 20 s at 95°C, 20 s at 64°C and 30 s at 72°C; final extension--15 min at 72°C. For the second step of procedure the PCR amplification and simultaneous labeling required a partial substitution of the dTTP in the reaction mixture by a ratio of 35% DIG-11-dUTP and 65% dTTP, and a purified product of the previous standard PCR was used as a template.

2.4.2. In situ hybridization For mRNA determination the cells were spun down, resuspended in SSC-BSA, centrifuged and fluid aspirated by micropipette. The prehybridization solution was prepared from: 2 /~1 of 20 × SSC; 2 /xl of 100 ×Denhardt's solution; 1 /~1 of 100 mM sodium phosphate buffer at pH 7.0; 10 /zl of deionized formamide (Sigma); 1 /zl of Baker's yeast tRNA (10 mg/ml, Sigma); and 2 /zl of redistilled diethyl pyrocarbonate-treated water. Eighteen /zl of freshly prepared prehybridization mixture were added to each cell sample, resuspended and incubated at 42°C for 1 h in the water bath shaker. Then 2 /zl (app. 30 ng) of DIG-labeled probe were added to the cell suspension and incubated 3 h at 42°C with shaking. After hybridization 80 /xl of 50% formamide with 0.1 × SSC-BSA (0.5 m g / m l ) was added and cells were incubated for 1 h at 42°C and for further 30 min after addition of 400 /~1 of 0.1 × SSC-BSA (0.5 /zg/ml). The cells were spun down and incubated with 200 /zl of 0.1 × SSC-BSA (0.5 m g / m l ) for 30 min at 42°C. Subsequently, the cells were washed twice in 200/zl of PBS-BSA (0.5 m g / m l ) and 0.1% sodium azide, centrifuged and resuspended in 100 /xl of the same solution, and incubated at 4°C overnight. After centrifugation cells were resuspended in 50/xl of the same solution with anti-DIG/FITC-labeled mAb (Sigma) diluted 1:1000 and the samples were incubated for 30 min at 4°C. The cells were spun, washed three times in PBS-BSA (0.5 m g / m l ) and resuspended in 300/zl of the same buffer. The control sample of cells without the probe was run in parallel. The cells were analyzed in FACScan V (Becton-Dickinson) flow cytometer and the mean channel of fluorescence intensity (MCF) was determined.

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M. Siedlar et a l . / lmmunopharmacology 35 (1997) 265-271 1500-

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M. Siedlar et al. / lmmunopharmacology 35 (1997) 265-271

270

3. Results 3.1. Vancomycin inhibits TNFot release by monocytes stimulated with LPS

Vancomycin added to monocytes cultured in the presence of LPS inhibited the release of both bioactive TNF, as assessed in a bioassay (Fig. 1), and TNFa protein determined by ELISA (Fig. 2). This effect was dose dependent and reduction of TNF and TNFce release was clearly observed at Vancomycin doses of 10-1000 /.tg/ml. No toxic effects of LPS and Vancomycin were observed. The TNF or TNFa production by unstimulated monocytes was negligible. It was concluded that Vancomycin inhibited in vitro the release of bioactive TNF and TNFt~ protein by monocytes. The LPS-stimulated TNFc~ production was CD14 dependent, as TNFce release was blocked by anti-CD14 mAb (Fig. 3). 3.2. The effect of Vancomycin on TNF~-mRNA accumulation in LPS-stimulated monocytes

In order to determine whether Vancomycin exerted its effect at the level of transcription, hybridization in cell suspension using DIG-labeled TNFacDNA probe was used. At 4 h substantially stronger hybridization signal was detected in LPS-stimulated monocytes in comparison to unstimulated monocytes (Fig. 4A). This signal was significantly reduced when LPS-stimulated monocytes were cultured in the presence of 10 or 100 /zg/ml of Vancomycin (Fig. 4B). Vancomycin did not reduce or even slightly enTable 1 The values of mean channel of fluorescence of TNFot-mRNA and GAPDH-mRNA signals in monocytes cultured for 4 h in the presence of I / x g / m l LPS and with different doses of Vancomycin Exp.

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hanced hybridization signals with GAPDH-cDNA probe (Fig. 4C) that was used as control. This suggested that Vancomycin down-regulated, in dose dependent manner, TNFa-mRNA but not GAPDHmRNA accumulation in LPS-stimulated monocytes, that was confirmed in three independent experiments (Table 1).

4. Discussion

Induction of cytokine cascade is the major pathomechanism in Gram-negative sepsis and septic shock. The cell wall fragment lipopolysaccharide is one of the factors responsible for induction of excessive cytokine secretion, mainly TNFa (Beutler and Cerami, 1988). Similar cytokines may be induced by Gram-positive bacteria (Heymer et al., 1985; Dziarski, 1986). In the present studies, the in vitro stimulation of monocytes with LPS was used to assess potential effect of Vancomycin on TNFa production. Data showed that at 10-1000 /.tg/ml Vancomycin down-regulated TNFa production. The effect of Vancomycin was exerted probably at the level of transcription, as it reduced TNFa-mRNA accumulation in monocytes, that occurred following stimulation with LPS. Although in these studies LPS was used to stimulate monocytes for TNFa production, it should be noted that TA and PG from Grampositive bacterial cell walls also stimulate TNFa production (Mattsson et al., 1993; Heumann et al., 1994). It was recently shown that PG, similarly to LPS, induce expression of TNFa-mRNA and secretion of bioactive TNF (Gupta et al., 1995). Moreover, the both these agents may activate macrophages through the same cellular receptor, namely CD14 (Weidemann et al., 1994; Pugin et al., 1994). It should be also noted that responsiveness of monocytes to LPS depends on the source of the monocytes, and the level of TNFa production differs significantly between particular experiments, which was observed also by the others (Agarwal et al., 1995). This study shows that Vancomycin down-modulates LPS-induced TNFa production and TNFc~mRNA accumulation in human blood monocytes. Similar observations has been described after periph-

M. Siedlar et al. / lmmunopharmacology 35 (1997) 265-271

eral blood mononuclear cells stimulation with LPS in the presence of the other antibiotics (Foca et al., 1993). It would be interesting whether Vancomycin can inhibit TNF~ production in patients treated for a Gram-positive sepsis (for example burn wound staphylococcal sepsis) where Vancomycin is one of the drugs of choice, or in mixed Grampositive/negative sepsis treated by the multiantibiotic regimens. The down-regulation of TNFa production by monocytes may be one of the important therapeutical effects of Vancomycin.

Acknowledgements We thank Ms. M. Hyszko for skillful technical assistance and M. Wotoszyn for performing the bioassay for TNF. M.S. is supported by the Polish Science Foundation personal grant.

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