SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Current immunosuppressive strategies are aimed at abrogating the allospecific T-cell response against donor tissues or organs. However, little information is yet available on the potential influences of these drugs on innate immune responses. In order to address this, we have employed a whole blood model. Human whole blood was pretreated with sirolimus, cyclosporine A or tacrolimus in therapeutic as well as supra therapeutic doses, and subsequently stimulated with lipopolysaccharide (LPS), peptidoglycan (PepG) or lipoteichoic acid (LTA). Plasma cytokine analyses revealed a potent inhibitory effect of sirolimus on interleukin(IL)-10 production induced by all bacterial products tested. In contrast, cyclosporine A and tacrolimus inhibited the tumour necrosis factor (TNF)-α production in response to LPS, but not to PepG and LTA. Using a quantitative mRNA analyses, we also observed that sirolimus significantly decreased the IL-10 mRNA accumulation to sub-basal levels in peripheral blood mononuclear cells (PBMC). This suggests that the sirolimus inhibits IL-10 production by interfering with the IL-10 gene transcription. However, the molecular mechanism of this inhibition remains unclear. Based on the present study and observations by others, we postulate that the clinical use of the sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Sepsis frequently complicates the recovery of transplant recipients [1,2]. A successful outcome of organ transplantation depends on effective suppression of the T-cell response to the graft. Cyclosporine A (CyA), the cornerstone of modern immunosuppression, and tacrolimus inhibit the host T cell immune response by interfering with the T cell receptor(TCR)-signalling pathway. Sirolimus, a potent immunosuppressive and antiproliferative agent currently studied in clinical trials, inhibits the IL-2 signal transduction pathway [3,4]. A recent phase II multicentre study documents the efficacy and safety profile of sirolimus, and suggests this agent as an alternative to CyA for the prevention of acute rejection [5]. Furthermore, sirolimus inhibits the development of transplant vasculopathy [6] and the generation of graft versus host disease (GVHD) and graft versus leukemia causing T cells by interfering with the production of Th1-derived cytokines [7]. Little information is, however, yet available of potential effects of sirolimus on innate immune mechanisms.

The innate recognition of bacterial products constitutes an evolutionary important defence mechanism against infection. Cells of the monocytic lineage play a crucial role in the innate immunity, orchestrating the host response after recognition of pathogen-associated molecular patterns (PAMPs) [8]. The recognition of PAMPs is mediated by a set of diverse pattern recognition receptors (PRRs), e.g. the CD14 molecule. Sepsis denotes the overwhelming systemic response to infection, and is characterized by innate systemic cellular activation and cytokine release [9]. Gram negative (–) and Gram positive (+) bacteria are equally important in the pathogenesis of sepsis and its complications [10,11]. LPS, a Gram– PAMP, initiates sepsis through complex formation with the plasma protein LPS-binding protein (LBP) enabling the binding to CD14 molecules [12], a PRR preferentially expressed on cells of monocytic lineage [13,14]. Gram + cell walls, as well as the PepG and LTA purified from Gram + bacteria, trigger the release of cytokine in vitro and in vivo[15–19]. Like LPS, both PepG and LTA bind to CD14 [13,20,21], and signal through toll like receptors (TLR), which are a recently characterized family of PRR associated with CD14 [21,22].

The proinflammatory cytokines (TNF)-α and IL-6 serve to amplify the innate immune response. These effects are counteracted by the concomitant release of the anti-inflammatory cytokine IL-10 that inhibits cytokine synthesis [23,24]. IL-10 was recently identified as the functional repressor of monocyte activation in plasma from patients suffering fulminant meningococcal sepsis [25]. The role of IL-10 in sepsis caused by Gram + bacteria is still not well documented.

The present study was undertaken to determine whether CyA, tacrolimus or sirolimus interfere with innate immune mechanisms in blood, as measured by the production of TNF-α, IL-6 and IL-10 in response to bacterial products. We demonstrate that sirolimus, but not CyA or tacrolimus, strongly inhibits the IL-10 expression by interfering with the IL-10 gene activation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Materials and reagents Peripheral venous blood was obtained from healthy staff members. The blood was collected in vacutainers containing 0.129 m CitNa (Becton Dickinson Vacutainer Systems Eur, Meylan Cedex, France). Escherichia coli 026:B6 LPS (Difco Laboratories, Detroit, MI, USA) was suspended in pyrogen free sterile saline and 10 ng/ml LPS was added directly to the blood samples in microcentrifuge tubes. The same reference batch was used throughout the study. Lipoteichoic acid (LTA) and polymyxin-B-sulfate were purchased from Sigma Chemicals (St. Louis, MO, USA).

Purification of PepG Peptidoglycan (PepG) was isolated from Staphylococcus aureus as previously described [26]. Covalently attached proteins were removed by treatment with 2 mg/ml pronase for 1 h at 60 °C [27]. Anionic polymers were removed from the PepG by the treatment of purified cell walls (10 mg dry wt/ml) with hydrofluoric acid (48% v/v) for 24 h at 4 °C. The insoluble PepG was then washed by centrifugation (14 000 × g for 5 min) and resuspention, once in 100 ml Tris-HCl (pH 8.0) and five times in distilled water until the pH was neutral. The PepG was then recovered by centrifugation as above and resuspended in saline (0.9% wt/vol) prior to sterilization by autoclaving and storage at − 20 °C.

Drugs Cyclosporine A (Sandimmune 50 mg/ml, Novartis, Basel, Switzerland) used in this study was diluted in 0.9% NaCl to the desired concentrations. Tacrolimus (Fujisawa GmbH, Munich, Germany) was dissolved in ethanol to 10 mg/ml and then Tween 80 (Sigma Chemical Co.) was added to 1/5 volume of ethanol solution. Further dilutions were obtained with 0.9% NaCl to the desired concentrations. Sirolimus powder (Wyeth-Ayerst Research, Princeton, NJ, USA) was dissolved in ethanol to a 2-mm stock solution which was stored at − 70 °C. Further dilutions were then obtained with 0.9% NaCl to the desired concentrations.

Whole blood experiments The whole blood model was used as previously described [28], with some modifications. Whole blood was aliquoted into 1.5 ml microcentrifuge tubes (Sorenson, BioScience Inc., Salt Lake City, UT, USA). Prior to stimulation with LPS, PepG or LTA, the blood was preincubated at 37 °C with the immunosuppressants at therapeutic (CyA: 250 ng/ml, tacrolimus: 10 ng/ml and sirolimus: 10 ng/ml) and supra therapeutic (CyA: 5 µg/ml, tacrolimus: 250 ng/ml and sirolimus: 500 ng/ml) concentrations with slow rotation for 4 h (G24 Environmental Incubator Shaker, New Brunswick Scientific Co., NJ, USA). Blood samples were then stimulated with 10 ng/ml of LPS, 10 µg/ml PepG or 100 µg/ml LTA for time periods as indicated. The blood was subsequently cooled on ice and centrifuged (14 000 ×g for 1 min), and plasma pipetted off for cytokine protein detection. As controls, equivalent volumes of 0.9% NaCl were added instead of the bacterial products. Basal cytokine levels were investigated immediately after blood drainage. Preliminary studies have shown that PepG and LTA give peak values of TNF-α 6 h after stimulation, compared to 4 h with LPS stimulation. Accordingly, 4 h of LPS stimulation and 6 h of PepG and LTA stimulation were chosen. Because IL-10 has been shown to have a more slow kinetics, experiments comparing the effect of sirolimus on the cytokine response at 4 and 12 h stimulation with LPS and PepG were also performed.

Elisa technique Plasma concentrations of TNF-α, IL-6 and IL-10 were determined with a commercially available solid-phase sandwich elisa kit (PeliKine Compact, CLB Labs, Amsterdam, the Netherlands) according to the manufacturer's instructions. The detection limit of the TNF-α, IL-6 and IL-10 elisa was 3, 0.4 and 3 pg/ml, respectively. The plates were read at 450 nm in an elisa reader (Thermo max microplate reader, Molecular Devices, Menlo Park, CA, USA).

TNF-a bioassay Biological activity of TNF-α was determined by assessing its cytotoxic effect on the fibrosarcoma cell line WEHI 164 subclone 13 [29], using the WST-1 substrate according to the protocol provided by the manufacturer (Boehringer Mannhein GmbH, Mannheim, Germany). Briefly, the cell line was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco, Paisley, UK), 2 mm glutamine and garamycin 0.05 mg/ml. Plasma samples were diluted 1 : 250 and 1 : 1250 in culture medium, and 50 µl of samples were applied to wells in triplicates. After trypsinating the WEHI 164 cells, they were resuspended in culture medium and adjusted to a cell concentration of 0.6 × 106/ml. Actinomycin D (Sigma Chemical Co.) was added (4 µl/ml) and 50 µl of the cell suspension was added to each well. After incubating the plates at 37 °C in a humidified 5% CO2 incubator (Cellstar, Queuesystems Inc., Asheville, NC, USA)for 20 h, 10 µl of WST-1 substrate was added to each well, before incubating the plates further for 3 h. The reaction was read at 450 nm in an elisa reader as described above, and the results were derived from a standard curve established with human recombinant TNF-α (Sigma Chemical Co.). The detection limit of the assay was 2 pg/ml.

Isolation of cells At the end of each incubation period, CD14 positive (+) cells were positively isolated from 500 µl blood using CD14-antibody (mouse immunoglobulin (Ig)G2a monoclonal antibodies (MoAb)) coated magnetic beads (Dynabeads M-450 CD14, Dynal AS, Oslo, Norway) according to the protocol supplied by the manufacturer. Subsequently the cells were lysed by adding lysis/binding buffer. PBMC were isolated from whole blood by density centrifugation over Ficoll Hypaque (Lymphoprep, Nycomed, Oslo, Norway).

Isolation of RNA Messenger RNA was isolated from lysed CD14 + cells by adding 50 µl Dynabeads Oligo (dT)25 (Dynal AS) according to the manufacturer's instructions. Total RNA from PBMC was isolated by a one-step method by Trizol according to protocol provided by the manufacturer (Gibco BRL, Life Technologies, Paisley, UK).

Analyses of cytokine mRNA All reagents for reverse transcription–polymerase chain reaction (RT-PCR) were purchased from Roche Molecular Systems Inc. (Branchburg, NJ, USA). RT-PCR was carried out in a Perkin-Elmer/Cetus thermal cycler 9600 (Perkin-Elmer Corp., Norwalk, CT, USA) as described by Solberg et al. [30]. Two µl mRNA product attached to the oligo (dT)25 was reverse transcribed in a 20-µl reaction volume containing 1 × RT buffer (10 mm KCl, 50 mm tris-HCl), 5 mm MgCl2, 1 mm dNTP, 1 U/µl RNase inhibitor and 2.5 U/µl MuLV reverse transcriptase. The RT was carried out for 1 h at 37 °C, followed by denaturation at 99 °C for 5 min. Subsequently, the PCR was performed on 10 µl of RT/cDNA mix and amplified using 25 pmol RT-PCR Amplifier Sets for IL-6 and IL-10 (Clontech Laboratories, Palo Alto, CA, USA). TNF-α and β-actin primers were manufactured by Pharmacia Biotech (Uppsala, Sweden). The PCR was carried out in a 50-µl solution containing 1.25 U AmpliTaq Gold polymerase (Perkin Elmer), 0.5 µm each of 3′ and 5′ primers specific for human TNF-α, IL-6, IL-10 and β-actin, 1 × PCR buffer (10 mm KCl, 50 mm tris-HCl) and 2 mm MgCl2. The conditions used for all reactions were polymerase activation at 94 °C for 12 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min 30 s, and polymerization at 72 °C for 1 min 30 s followed by 10 min elongation at 72 °C after the last cycle. Following RT-PCR, 18 µl of the reaction mixture was electrophorezed in 1% TAE-agarose gel containing 0.5 µg/ml ethidium bromide and visualized on an UV screen. The gel was then documented on polaroid film. Correct product lengths of the different PCR products (TNF-α, 443 base pairs (bp); IL-6, 628 bp; IL-10, 328 bp; and β-actin, 660 bp) were verified using a 100-bp DNA Ladder (Life Technologies, Gaithersburg, MD, USA). Total RNA was analyzed for the presence of IL-10 mRNA by Quantikine mRNA kit (R & D, Abdington, UK), according to the protocol provided by the manufacturer. In these experiments, 55 µg total RNA was applied to each well.

Statistical analysis Data are presented as means ± standard error of the mean (SEM). The data were analyzed with a 1-way analysis of variance (anova) followed by the Tukey test. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The leukocyte count and viability remained high throughout the experiments. Addition of the immunosuppressive drugs did not significantly influence these parameters (data not shown). Basal cytokine levels at the start of each experiment and in unstimulated blood incubated for 8–16 h were negligible (≤ detection limit).

Sirolimus strongly attenuates the IL-10 production induced by LPS, PepG and LTA

In whole blood stimulated with LPS (10 ng/ml) for 4 h the mean plasma concentration of IL-10 from 12 donors was 0.16 ng/ml. Pretreatment of whole blood for 4 h with both therapeutic (10 ng/ml) and supra-therapeutic (500 ng/ml) doses of sirolimus prior to stimulation with LPS (10 ng/ml) strongly inhibited the release of IL-10 by 70% (P = 0.013) and 80% (P = 0.004), respectively (Table 1). Neither CyA nor tacrolimus had any significant influence on the IL-10 production induced by LPS.

Table 1.  Influence of cyclosporine A, tacrolimus and sirolimus on the IL-10 production in response to LPS, PepG and LTA in human whole blood
  Cyclosporine ATacrolimusSirolimus
StimulantsUntreated250 ng/ml5 µg/ml10 ng/ml 250 ng/ml10 ng/ml500 ng/ml
  •  Whole blood from healthy donors was preincubated with cyclosporine A, tacrolimus or sirolimus at doses as indicated for 4 h. The blood was subsequently stimulated with LPS for 4 h (n = 12), PepG for 6 h (n = 7) or LTA for 6 h (n = 7). Plasma samples were analyzed for the presence of IL-10 using an elisa method. Data represent mean values (ng/ml) ± SE of results from 12 (LPS) or 7 (PepG/LTA) blood donors. In the absence of stimulants no IL-10 was detectable.

  • * 

    Significantly different from stimulant alone (P < 0.05).

LPS 10 ng/ml0.16 ± 0.040.16 ± 0.050.08 ± 0.020.16 ± 0.040.14 ± 0.040.05 ± 0.01*0.03 ± 0.01*
PepG 10 µg/ml0.68 ± 0.130.71 ± 0.280.46 ± 0.180.71 ± 0.210.90 ± 0.160.15 ± 0.08*0.07 ± 0.04*
LTA 100 µg/ml1.08 ± 0.181.17 ± 0.400.77 ± 0.341.11 ± 0.351.11 ± 0.440.28 ± 0.15*0.14 ± 0.05*

The mean plasma concentrations of IL-10 from seven donors stimulated with pep (10 µg/ml) or LTA (100 µg/ml) for 6 h were 0.6 ng/ml and 1.1 ng/ml, respectively. Four h pretreatment with sirolimus strongly inhibited the IL-10 response to these doses of both PepG and LTA (Table 1). At therapeutic doses the average inhibition was 78% to the PepG response (P = 0.005) and 74% to the LTA response (P = 0.005). A dose-dependent inhibition was observed also for the Gram + products, as sirolimus (500 ng/ml) inhibited the IL-10 release by more than 85% in both treatment groups. Neither CyA nor tacrolimus had any significant influence on the IL-10 production in response to Pep or LTA.

To evaluate whether this inhibitory effect of sirolimus is valid also at later time points after stimulation, we next examined the influence of sirolimus on the IL-10 production subsequent to 4 and 12 h stimulation with LPS and Pep. As seen in Fig. 1, sirolimus (10 ng/ml) significantly (P < 0.05) inhibited the IL-10 production by 75% after 12 h stimulation, as potently as seen after 4 h. This inhibitory effect was equally strong for LPS (10 ng/ml) and PepG (10 µg/ml) stimulation.

image

Figure 1. Time dependent effect of sirolimus on lipopolysaccharide(LPS)-induced interleukin (IL)-10 and tumour necrosis factor (TNF)-α production. Whole blood from six donors were pretreated for 4 h with 10 ng/ml sirolimus or the equivalent volume of saline, and subsequently stimulated with 10 ng/ml LPS. Following 4 and 12 h of incubation, the plasma samples were analyzed for the presence of IL-10 and TNF-α by elisa as described in Materials and methods. The columns indicate mean relative cytokine value + SEM. * indicates significant difference from stimulated blood not treated with sirolimus (P < 0.05).

Download figure to PowerPoint

CyA and tacrolimus, but not sirolimus, inhibit the TNF-α production induced by LPS

The mean plasma concentration of immunodetectable TNF-α in whole blood from 12 donors stimulated with LPS (10 ng/ml) for 4 h was 6.6 ng/ml. Four h pretreatment of whole blood with CyA (250 ng/ml or 5 µg/ml) significantly inhibited the LPS induced TNF-α release by 36% (P = 0.014) and 33% (P = 0.04), respectively (Table 2). Tacrolimus also significantly inhibited the TNF-α production in response to the LPS by 30% (10 ng/ml tacrolimus, P = 0.05) and 32% (250 ng/ml tacrolimus, P = 0.04). Interestingly, in the sirolimus-treated groups a trend towards higher immunodetectable TNF-α plasma values in response to LPS was observed, however, the increase did not reach statistical significance.

Table 2.  Influence of cyclosporine A, tacrolimus and sirolimus on the TNF-α production in response to LPS, PepG and LTA in human whole blood
  Cyclosporine ATacrolimusSirolimus
StimulantsUntreated250 ng/ml5 µg/ml10 ng/ml 250 ng/ml10 ng/ml500 ng/ml
  •  Whole blood from healthy donors was preincubated with cyclosporine A, tacrolimus or sirolimus at doses as indicated for 4 h. The blood was subsequently stimulated with LPS for 4 h (n = 12 in ELISA, n = 6 in bioassay), PepG for 6 h (n = 7) or LTA for 6 h (n = 7). Plasma samples were analysed for the presence of TNF-α using elisa or bioassay. Data represent mean values (ng/ml) ± SE. In the absence of stimulants no TNF-α was detectable.

  • * 

    Significantly different from stimulant alone (P < 0.05).

LPS 10 ng/ml (elisa)6.6 ± 0.84.2 ± 0.4*4.4 ± 0.6*4.6 ± 0.6*4.5 ± 0.5*6.7 ± 0.67.0 ± 0.5
LPS 10 ng/ml (Bioassay)3.5 ± 0.82.6 ± 1.12.0 ± 0.72.1 ± 0.81.8 ± 0.53.7 ± 0.87.8 ± 1.4
PepG 10 µg/ml (elisa)1.3 ± 0.31.2 ± 0.21.3 ± 0.21.4 ± 0.20.9 ± 0.21.4 ± 0.21.2 ± 0.2
LTA 100 µg/ml (elisa)1.4 ± 0.31.2 ± 0.31.3 ± 0.41.1 ± 0.20.9 ± 0.21.3 ± 0.41.7 ± 0.6

The mean plasma concentration of bioactive TNF-α in whole blood from six donors stimulated with LPS (10 ng/ml) for 4 h was 3.4 ng/ml. None of the drugs had any significant influence on the release of bioactive TNF-α in response to LPS, however, a trend toward higher values in the sirolimus group was noted (Table 2).

None of the immunosuppressants influenced the TNF-α production induced by PepG or LTA.

The inhibitory effect of sirolimus on the IL-10 production prompted us to investigate whether sirolimus would influence on the LPS- and PepG-induced TNF-α production at later time points. Interestingly, we observed an increase in TNF-α plasma values after a 12 h stimulation in blood treated with sirolimus (10 ng/ml) compared to untreated blood (Fig. 2). The LPS- and PepG-induced TNF-α plasma levels increased by 25% and 69%, respectively, in blood treated with sirolimus.

image

Figure 2. Time dependent effect of sirolimus on PepG-induced IL-10 and TNF-α production. Whole blood from six donors were pretreated for 4 h with 10 ng/ml sirolimus or the equivalent volume of saline, and subsequently stimulated with 10 µg/ml PepG. Following 4 and 12 h of incubation, the plasma samples were analyzed for the presence of IL-10 and TNF-α by elisa as described in Materials and methods. Columns indicate mean relative cytokine value + SEM. * indicates significant difference from stimulated blood not treated with sirolimus (P < 0.05).

Download figure to PowerPoint

The IL-6 production is not influenced by sirolimus, CyA or tacrolimus

The mean plasma concentration of IL-6 in whole blood stimulated with LPS was 16.9 ng/ml. We did not observe any influence on the LPS-induced IL-6 production of any of the drugs used (Table 3). The mean plasma concentrations of IL-6 when stimulated with PepG and LTA were 7.2 and 15.7 ng/ml, respectively, and this was not affected by any of the drugs.

Table 3.  Influence cyclosporine A, tacrolimus and sirolimus on the IL-6 production in response to LPS, PepG and LTA in human whole blood
  Cyclosporine ATacrolimusSirolimus
StimulantsUntreated250 ng/ml5 µg/ml10 ng/ml 250 ng/ml10 ng/ml500 ng/ml
  1.  Whole blood from healthy donors was preincubated with, tacrolimus or sirolimus at doses as indicated for 4 h. The blood was subsequently stimulated with LPS for 4 h (n = 12), PepG for 6 h (n = 7) or LTA for 6 h (n = 7). Plasma samples were analyzed for the presence of IL-6 using an elisa. Data represent mean values (ng/ml) ± SE of results from 12 (LPS) or 7 (PepG/LTA) blood donors. In the absence of stimulants no IL-6 was detectable.

LPS 10 ng/ml16.9 ± 3.016.5 ± 2.517.4 ± 2.614.5 ± 1.8 15.3 ± 2.515.4 ± 2.513.2 ± 2.4
PepG 10 µg/ml7.2 ± 0.67.8 ± 1.08.6 ± 0.97.4 ± 0.86.2 ± 0.46.9 ± 0.55.6 ± 0.4
LTA 100 µg/ml15.7 ± 1.115.2 ± 1.615.3 ± 1.514.9 ± 1.014.5 ± 1.116.3 ± 2.214.6 ± 0.7

Sirolimus inhibits the IL-10 gene activation

In order to investigate whether the ability of sirolimus to inhibit IL-10 formation would be associated with a decrease in IL-10 mRNA production, we first analyzed for the presence of IL-10 mRNA in CD14 + cells. Positive isolation of CD14 + with magnetic beads almost completely depleted the blood for monocytes as measured by flow cytometry (data not shown). Analyses of isolated mRNAs from these cells by RT-PCR revealed that IL-10 mRNA was indeed expressed in CD14 + cells from whole blood treated with sirolimus (n = 4), as was TNF-α and IL-6 mRNA (Fig. 3). Low basal expressions of TNF-α and IL-10 mRNA at time zero were observed, with slightly stronger expression after 8 h incubation with saline only. No basal expression of IL-6 mRNA was detected at time zero nor after 8 h with saline only. LPS strongly upregulated the expression of TNF-α, IL-6 and IL-10 mRNA in CD14 + cells after 4 h, however, we could not detect any influence on cytokine mRNA expression in these cells with any of the immunosuppressants. The β-actin controls indicated similar levels of expression of this transcript, independent of LPS stimulation and drug interventions.

image

Figure 3. Influence of CyA, tacrolimus and sirolimus on the LPS-induced expression of TNF-α, IL-6 and IL-10 mRNA in CD14 + cells. Whole blood from healthy donors was pretreated with CyA (lane 4, 250 ng/ml, and lane 5, 5 µg/ml), tacrolimus (lane 6, 10 ng/ml, and lane 7, 250 ng/ml) and sirolimus (lane 8, 10 ng/ml, and lane 9, 500 ng/ml) for 4 h before stimulation with LPS (10 ng/ml). Subsequent to 4 h of LPS stimulation, CD14 + cells were isolated with the Dynabead technique. Messenger RNA was isolated by oligo(dT)25 beads and subjected to reverse transcription–polymerase chain reaction (RT-PCR). (Α) 443 bp TNF-α, (B) 628 bp IL-6, (C) 328 bp IL-10, and (D) 660 bp β-actin fragments were identified using specific primer pairs. Lane 1–3 represent basal levels of cytokine mRNA immediately after blood drainage (lane 1), 8 h incubation with saline only (lane 2) and LPS (10 ng/ml) alone (lane 3). Lanes noted – and + represent negative and positive controls. The data shown are from a representative experiment that was repeated with similar results in six donors.

Download figure to PowerPoint

We then employed a quantitative mRNA analyses. When total RNA from PBMC (n = 4) stimulated with PepG (10 µg/ml) in whole blood was subjected to quantitative IL-10 mRNA analyses, a mean of 1.6-fold increase compared to untreated blood was found (Fig. 4). Pre-treatment with sirolimus (10 ng/ml) significantly reduced the IL-10 mRNA to sub-basal levels.

image

Figure 4. Sirolimus significantly inhibits the PepG-induced expression of IL-10 mRNA in whole blood peripheral blood mononuclear cells (PBMC). Whole blood from healthy donors was pretreated with sirolimus (10 ng/ml) for 4 h before stimulation with PepG (10 µg/ml). After 6 h of PepG stimulation PBMC was isolated by density centrifugation, and total RNA from PBMC was isolated by a one-step method by Trizol and analyzed for the presence of IL-10 mRNA by the Quantikine mRNA kit. Data are means ± SEM of five independent experiments.* indicates P < 0.05.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this paper we demonstrate that sirolimus potently inhibits the formation of IL-10 in whole blood induced by LPS as well as with PepG and LTA. To the best of our knowledge, this is the first report demonstrating that sirolimus interferes with innate immune responses to bacterial products. Sirolimus has previously been shown to inhibit IL-10 production in human T cells stimulated via CD3 [31]. Sirolimus also inhibits the IL-10 production in human monocytes stimulated with the human immundeficiency virus (HIV)-1 associated protein gp41 [32]. In contrast, Blazar et al. [7] showed in a mice in vivo model a higher frequency of IL-10 mRNA expressing T cells and a higher IL-10 protein content of mixed lymphocyte reaction (MLR) supernatants in sirolimus-treated animals compared with controls. We can not make any firm conclusions as to the source of IL-10 in our experiments. However, the design of the experiments should allow for LPS stimulation only through its CD14 high affinity receptor, mainly expressed on cells of monocyte lineage [14]. Both T and B cells are, however, capable of producing IL-10, but these cells lack expression of CD14 molecules [33,34]. A soluble form of CD14 (sCD14) in the serum has been shown to bind LPS and induce signals in cells lacking CD14 [35,36]. The possibility that sCD14 might play a role in signal-transducing pathways for LPS, PepG and LTA in lymphocytes, however, remains to be determined. The Toll genes have recently been found to code for the LPS receptor in murine B cells [37], and both TLR2 and TLR4 genes are expressed in mouse T cells [38]. Thus, we cannot rule out the possibility that LPS, PepG and LTA directly affect B and T cells by binding to TLRs. A CD14 surface expression is also found on polymorphonuclear neutrophils [12], but these leukocytes do not produce IL-10 [39].

We were unable to observe any influence of sirolimus on IL-10 mRNA levels by RT-PCR. This may, however, be owing to the vast amplification of transcripts that occurs in the PCR reaction, which make this method unsuitable to detect subtle differences in mRNA levels. Thus, we employed a new quantitative mRNA assay based on a colorimetric detection of specific mRNAs bound to labelled probes. Using this technology, we demonstrated that sirolimus reversed the PepG-induced IL-10 mRNA accumulation to sub-basal levels. This suggests that sirolimus regulates IL-10 at the transcriptional level.

However, the molecular mechanisms by which sirolimus inhibits the IL-10 production in monocytes in response to bacterial products is not clear. In T cells, sirolimus has only limited effects on cytokine production, but blocks the proliferative response to a variety of cytokines [40]. Through interference with the target molecule mTOR, sirolimus inhibits the activation of a p70 S6 kinase involved in the phosphorylation of the S6 ribosomal protein believed to play a role in the translation of critical cell cycle regulating proteins [40]. It is not known whether mTOR is involved in signalling events in monocytes. However, activation of the p70 S6 kinase is one alternative pathway through which HIV gp41 triggers an IL-10 µpregulation in monocytes [32]. The IL-10 synthesis in cultured monocytes induced by LPS, however, has been reported to involve the activation of p38 mitogen-activated protein (MAP) kinase [41], a pathway not believed to be influenced by sirolimus.

The action of cytokines with anti-inflammatory properties has increasingly been recognized to be of importance in the co-ordinated response to inflammatory stimuli. In this respect, the inhibitory effect of sirolimus on IL-10 production shown in this paper is very interesting. The IL-10 inhibits the synthesis of several pro-inflammatory cytokines and downregulates the class II major histocompatibility complex (MHC) expression by human monocytes [23]. IL-10 is also recognized as the functional repressor of the monocyte activation in plasma from patients with meningococcal sepsis [25]. Moreover, studies with IL-10-deficient mice have demonstrated that the lethal dose of LPS is 20-fold lower than that for wild type mice [42]. Thus, the strong inhibitory effect of sirolimus on IL-10 production observed in our experiments may be associated with an increased risk of hyperinflammation and sepsis. Our observation that higher plasma TNF-α was found in blood treated with sirolimus is in agreement with this assertion. In further support of this notion the Sirolimus European Renal Transplant Study Group [5] recently reported a higher frequency of sepsis and bacterial infections in patients treated with sirolimus compared to the standard CyA therapy. The authors speculate that delayed wound healing subsequent to inhibitory effects on growth factors could explain this discrepancy. We believe, however, that the inhibition of the IL-10 expression may have contributed to the increased rate of the bacterial infection in the sirolimus-treated group.

The inhibitory effect of CyA and tacrolimus on the TNF-α release subsequent to LPS stimulation is in agreement with the observations made by Dawson et al. [43] and Nguyen et al. [44] suggesting an inhibitory effect of CyA on TNF-α directly on the macrophages, but contradicting those by Andersson et al. [45] who concluded that neither CyA nor tacrolimus had any influence on TNF-α production by monocytes stimulated with LPS. In T cells, both CyA and tacrolimus have been shown to inhibit several transcriptional regulatory proteins involved in the activation of cytokine genes [46]. Less information is available on the effects on the various transcription factors in human monocytes and macrophages. LPS activates nuclear factor kappa B (NF-κB), an ubiquitous transcription factor involved in the activation of several pro-inflammatory cytokines [47] and both CyA and tacrolimus have been shown to inhibit the induction of this factor [48]. Other factors, as the recently described LPS-induced TNF-alpha factor (LITAF) [49] may potentially be less influenced by CyA and tacrolimus.

In our experimental setting, none of the drugs influenced on the IL-6 release, and no effect on IL-6 mRNA expression in CD14 + cells was seen. This finding is in accordance with observations made by others [43,45].

In summary, we have shown that sirolimus, but not CyA or tacrolimus, interfere with the innate response to bacterial products by attenuation of IL-10 production. We demonstrate that sirolimus decrease the PepG induced IL-10 mRNA accumulation to sub-basal levels, however, the molecular mechanisms for this inhibition remains unclear. Based on these data and observations by others, we therefore postulate that the clinical use of sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We are indebted to Dr K. Murato, Fujisawa GmbH, Munich, Germany and Dr S. Sehgal, Wyeth-Ayerst Research, Princeton, NJ, USA for kindly providing the tacrolimus and sirolimus, respectively. The fibrosarcoma cell line WEHI 164 subclone 13 was a kind gift from T. Espevik, University of Trondheim, Trondheim, Norway.

This work was supported by Harry W. Holms Foundation and Alexander Malthes Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Singh N. Infectious diseases in the liver transplant recipient. Semin Gastrointest Dis 1998;9:13646.
  • 2
    Paya CV & Hermans PE. Bacterial infections after liver transplantation. Eur J Clin Microbiol Infect Dis 1989;8:499504.
  • 3
    Abraham RT & Wiederrecht GJ. Immunopharmacology of rapamycin. Annu Rev Immunol 1996;14:483510.
  • 4
    Sehgal SN, Baker H & Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic. II Fermentation, isolation and characterization. J Antibiot (Tokyo) 1975;28:72732.
  • 5
    Groth CG, Backman L & Morales JM et al. Sirolimus (rapamycin) -based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal Transplant Study Group [see comments]. Transplantation 1999;67:103642.
  • 6
    Schmid C, Heemann U & Tilney NL. Factors contributing to the development of chronic rejection in heterotopic rat heart transplantation. Transplantation 1997;64:2228.
  • 7
    Blazar BR, Taylor PA, Panoskaltsis-Mortari A & Vallera DA. Rapamycin inhibits the generation of graft-versus-host disease- and graft-versus-leukemia-causing T cells by interfering with the production of Th1 or Th1 cytotoxic cytokines. J Immunol 1998;160:535565.
  • 8
    Medzhitov R & Janeway Ca Jr. Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997;9:2958.
  • 9
    Chaudry IH. Sepsis: lessons learned in the last century and future directions. Arch Surg 1999;134:9229.
  • 10
    Dahmash NS, Chowdhury NH & Fayed DF. Septic shock in critically ill patients: aetiology, management and outcome. J Infect 1993;26:15970.
  • 11
    Sriskandan S & Cohen J. Gram-positive sepsis. Mechanisms and differences from gram-negative sepsis. Infect Dis Clin North Am 1999;13:397412.
  • 12
    Ulevitch RJ & Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 1995;13:43757.
  • 13
    Dziarski R, Tapping RI & Tobias PS. Binding of bacterial peptidoglycan to CD14. J Biol Chem 1998;273:868090.
  • 14
    Ziegler-Heitbrock HW & Ulevitch RJ. CD14: cell surface receptor and differentiation marker [see comments]. Immunol Today 1993;14:1215.
  • 15
    Timmerman CP, Mattsson E & Martinez-Martinez L et al. Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans. Infect Immun 1993;61:416772.
  • 16
    Mattsson E, Verhage L, Rollof J, Fleer A, Verhoef J & Van Dijk H. Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. FEMS Immunol Med Microbiol 1993;7:2817.
  • 17
    Keller R, Fischer W, Keist R & Bassetti S. Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids. Infect Immun 1992;60:366472.
  • 18
    Heumann D, Barras C, Severin A, Glauser MP & Tomasz A. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes. Infect Immun 1994;62:271521.
  • 19
    Bhakdi S, Klonisch T, Nuber P & Fischer W. Stimulation of monokine production by lipoteichoic acids. Infect Immun 1991;59:461420.
  • 20
    Sugawara S, Arakaki R, Rikiishi H & Takada H. Lipoteichoic acid acts as an antagonist and an agonist of lipopolysaccharide on human gingival fibroblasts and monocytes in a CD14-dependent manner. Infect Immun 1999;67:162332.
  • 21
    Schwandner R, Dziarski R, Wesche H, Rothe M & Kirschning CJ. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 1999;274:174069.
  • 22
    Kopp EB & Medzhitov R. The Toll-receptor family and control of innate immunity. Curr Opin Immunol 1999;11:138.DOI: 10.1016/s0952-7915(99)80003-x
  • 23
    De Waal Malefyt R, Abrams J, Bennett B, Figdor CG & De Vries JE. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 1991;174:120920.
  • 24
    Moore KW, O'garra A, De Waal Malefyt R, Vieira P & Mosmann TR. Interleukin-10. Annu Rev Immunol 1993;11:16590.
  • 25
    Brandtzaeg P, Osnes L, Ovstebo R, Joo GB, Westvik AB & Kierulf P. Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes[published erratum appears J Exp Med 1996, November 1;184(5):2075]. J Exp Med 1996;184: 5160.
  • 26
    Foster SJ. Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis. J Bacteriol 1992;174:46470.
  • 27
    Atrih A, Zollner P, Allmaier G & Foster SJ. Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol 1996;178:617383.
  • 28
    Wang JE, Solberg R & Okkenhaug C et al. Cytokine modulation in experimental endotoxemia: Characterisation of an ex vivo whole blood model. Eur Surg Res 2000;32:6573.
  • 29
    Espevik T & Nissen-Meyer J. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods 1986;95:99105.
  • 30
    Solberg R, Scholz T, Videm V, Okkenhaug C & Aasen AO. Heparin coating reduces cell activation and mediator release in an in vitro venovenous bypass model for liver transplantation. Transpl Int 1998;11:2528.DOI: 10.1007/s001470050137
  • 31
    Cohen SB, Parry SL, Feldmann M & Foxwell B. Autocrine and paracrine regulation of human T cell IL-10 production. J Immunol 1997;158:5596602.
  • 32
    Barcova M, Speth C, Kacani L, Uberall F, Stoiber H & Dierich MP. Involvement of adenylate cyclase and p70 (S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1. Pflugers Arch 1999;437:53846.
  • 33
    Brophy VH & Sibley CH. Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3. Immunogenetics 1998;47:196205.DOI: 10.1007/s002510050348
  • 34
    Uchimura E, Watanabe N & Kobayashi Y. Modulation by lipopolysaccharide of inflammatory cytokine production by two T cell lines. Cytokine 1997;9:72733.DOI: 10.1006/cyto.1997.0230
  • 35
    Pugin J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ & Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci U S A 1993;90:27448.
  • 36
    Frey EA, Miller DS & Jahr TG et al. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 1992;176:166571.
  • 37
    Hoshino K, Takeuchi O & Kawai T et al. Cutting edge: Toll-like receptor 4 (TLR4) -deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:374952.
  • 38
    Matsuguchi T, Takagi K, Musikacharoen T & Yoshikai Y. Gene expressions of lipopolysaccharide receptors, toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes. Blood 2000;95:137885.
  • 39
    Reglier H, Arce-Vicioso M, Fay M, Gougerot-Pocidalo MA & Chollet-Martin S. Lack of IL-10 and IL-13 production by human polymorphonuclear neutrophils. Cytokine 1998;10:1928.DOI: 10.1006/cyto.1997.0272
  • 40
    Sehgal SN, Camardo JS, Scarola JA & Maida BT. Rapamycin (sirolimus, rapamune). Curr Opin Nephrol Hypertens 1995;4:4827.
  • 41
    Foey AD, Parry SL, Williams LM, Feldmann M, Foxwell BM & Brennan FM. Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF- alpha: role of the p38 and p42/44 mitogen-activated protein kinases. J Immunol 1998;160:9208.
  • 42
    Berg DJ, Kuhn R & Rajewsky K et al. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 1995;96:233947.
  • 43
    Dawson J, Hurtenbach U & MacKenzie A. Cyclosporin A inhibits the in vivo production of interleukin-1beta and tumour necrosis factor alpha, but not interleukin-6, by a T-cell- independent mechanism. Cytokine 1996;8:8828.DOI: 10.1006/cyto.1996.0118
  • 44
    Nguyen DT, Eskandai MK & DeForge LE et al. Cyclosporin a modulation of tumor necrosis factor gene expression and effects in vitro and in vivo. J Immunol 1990;144:38228.
  • 45
    Andersson J, Nagy S, Groth CG & Andersson U. Effects of FK506 and cyclosporin A on cytokine production studied in vitro at a single-cell level. Immunology 1992;75:13642.
  • 46
    Rao A, Luo C & Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 1997;15:70747.
  • 47
    Baeuerle PA & Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12:14179.
  • 48
    Frantz B, Nordby EC & Bren G et al. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. Embo J 1994;13:86170.
  • 49
    Myokai F, Takashiba S, Lebo R & Amar S. A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment. Proc Natl Acad Sci U S A 1999;96:451823.DOI: 10.1073/pnas.96.8.4518