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* Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas,
Hospital Universitario La Princesa,
Hospital Universitario La Paz, and
¶ Hospital Universitario Gregorio Marañón, "Grupo de Estudios Peritoneales de Madrid" del Instituto de Investigaciones Nefrológicas Reina Sofía de la Fundación Renal Iñigo Alvarez de Toledo, Madrid, Spain;
# Baxter Healthcare Corporation, Deerfield, Illinois; and
Robarts Research Institute and The University of Western Ontario, London, Canada
Correspondence: Dr. Angel L. Corbí, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain. E-mail: acorbi{at}cib.csic.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin (IL)-6 and IL-12 p70, and nuclear factor (NF)-
B activation] revealed that currently used PD solutions differentially inhibit the lipopolysaccharide (LPS)-induced maturation of MDDC, an inhibition that correlated with their ability to impair the LPS-stimulated NF-B activation. Evaluation of PD components revealed that sodium lactate and glucose-degradation products impaired the acquisition of maturation parameters and NF-B activation in a dose-dependent manner. Moreover, PD solutions impaired monocyte-MDDC differentiation, inhibiting the acquisition of DC markers such as CD1a and DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (CD209). These findings have important implications for the initiation of immune responses under high lactate conditions, such as those occurring within tumor tissues or after macrophage activation.
Key Words: NF-B • IL-12 • IL-6 • TNF- • immune response
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), IL-1] or pathogen-associated molecules [e.g., lipopolysaccharides (LPS), CpG DNA] set off a maturation process, whereby DC lose their antigen-capturing ability; migrate into secondary lymphoid organs; up-regulate adhesion (CD54), major histocompatibility complex (MHC), and costimulatory molecules (CD80, CD83, CD86, CD40); and display the unique capacity to activate "naive" T lymphocytes [1
2
3
4
5
6
, 7
]. Depending on the maturation stimulus, mature DC can also synthesize and release IL-12, which critically contributes to T helper cell type 1 (Th1) lymphocyte polarization [3
]. The acquisition of all these maturation parameters is absolutely dependent on the activity of the nuclear factor (NF)-B family of transcription factors [8
].
DC have been identified within the peritoneal cavity and characterized morphologically and phenotypically [5
, 9
10
11
]. DC account for 1–2% of the total peritoneal cells [9
, 10
], and peritoneal monocytes/macrophages acquire DC phenotypic properties during transport of exogenous agents into adjacent lymph nodes [5
]. In the steady state, mesothelial cells could affect DC differentiation and maturation in the peritoneum through their capacity to secrete GM-CSF [12
], IL-1 [13
], and TNF- [14
].
Continuous ambulatory peritoneal dialysis (CAPD) has become an effective replacement therapy for treating patients with end-stage renal failure. However, CAPD duration is limited, as some patients exhibit a gradual loss of peritoneal membrane functionality and the periodic occurrence of peritonitis [15 , 16 ], which is the leading cause of discontinuation of PD [17 ]. Adequate, immunologic responses within the peritoneal cavity are vital to eradicate infection. The instillation of PD solutions interferes with the local immune and inflammatory response in the peritoneal cavity [18 , 19 ] and negatively affects the involvement of mesothelial cells and macrophages in the peritoneal immune response against invading pathogens [20 ]. In fact, PD solutions alter the viability, phenotype, and function of peritoneal cells (e.g., mesothelial cells and macrophages) in a phenomenon commonly referred to as bioincompatibility [21 ]. Furthermore, PD solutions are acidic and hypertonic and contain high concentrations of glucose and glucose degradation products (GDPs), and these four parameters are major determinants for the peritoneal membrane damage that results in altered peritoneal permeability and loss of ultrafiltration capacity [15 , 21 22 23 ].
PD solutions inhibit cytokine production by mesothelial cells and monocytes/macrophages [14
] and consequently, might impair normal DC development. In this regard, it has been shown that PD solutions inhibit TNF- mRNA expression and NF-B DNA-binding activity in LPS-treated macrophages [14
]. The present study was undertaken to analyze the influence of currently used PD solutions on monocyte-derived DC (MDDC) maturation and mature DC-effector functions. Analysis of PD solutions and their components revealed that sodium lactate and GDPs have a profound, negative impact on several phenotypic and functional maturation parameters. These findings have important implications for the initiation of primary immune responses within the peritoneal cavity and imply that DC maturation might be impaired in tissues exhibiting high concentrations of lactate (e.g., tumors) or GDPs (uremic or hyperglycemic conditions).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(at 100 U/ml) or Escherichia coli 055:B5 LPS (10 ng/ml) and were allowed to mature for 48 h.
To assess the effect of PD fluids on DC maturation, immature MDDC were subjected to the standard maturation protocol [6
, 24
] and were cultured in a 1:1 dilution of each PD solution in complete medium. To that end, 3 ml of the corresponding PD solution was added onto 3 ml cultures of immature MDDC (5x105 cells/ml), and after a 1-h incubation, TNF- (100 U/ml) or LPS (10 ng/ml) was added to the culture medium. In all cases, the pH of the resulting media was within the 7.2–7.6 range. After 24–48 h, cells were harvested for phenotypic and functional analysis, and cell supernatants were collected for determination of cytokine levels. As a control, immature MDDC were diluted 1:1 with RPMI. Evaluation of the influence of sodium lactate and GDPs on MDDC maturation was performed by addition of the different reagents onto the wells, where immature MDDC had been differentiated. Similarly, the effect of PD fluids on DC differentiation was done by culturing monocytes with GM-CSF and IL-4 (both at 1000 U/ml) in a 1:1 dilution of each PD solution in complete medium, and phenotypic measurements were performed after 48 h.
Flow cytometry and antibodies
Twenty four or 48 h after TNF- or LPS stimulation, cells were collected, washed in ice-cold phosphate-buffered saline (PBS), and processed for indirect immunofluorescence. Cells were resuspended in 100 µl complete medium containing 50 µg/ml human immunoglobulin G (IgG) and were incubated for 15 min at 4°C to prevent binding through the Fc portion of the antibodies. Then, 100 µl of a solution containing 10 µg/ml of the indicated monoclonal antibodies (mAb) was added and incubated for 30 min on ice. After three washing steps in PBS, cells were resuspended in 100 µl complete medium containing fluorescein isothiocyanate (FITC)-labeled F(ab')2 rabbit anti-mouse IgG, kept on ice for 30 min, washed, and resuspended in 200 µl PBS for flow cytometry. mAb included anti-CD1a (BL6), anti-CD83 (HB1/5; Immunotech, Marseille, France), anti-CD86 (B-T7; Diaclone Research, Besancon, France), anti-CD49d (ALC1/6.3), anti-CD40 (MAB89), anti-MHC class II (TS1/2), anti-CD11a (TS1/11), anti-CD151 (kindly provided by Dr. Francisco Sánchez-Madrid, Hospital de la Princesa, Madrid), and anti-CD209 (MR-1). Cells were also incubated with isotype-matched control antibodies to determine the basal level of fluorescence. Flow cytometry analysis was performed with an EPICS-CS (Coulter Científica, Madrid, Spain) using log amplifiers.
Measurement of MDDC phagocytic activity
Mannose receptor-mediated endocytosis was measured after 24 or 48 h of maturation. Mature MDDC (5x105 cells/ml) were incubated in 200 µl solution buffered with 25 mM HEPES and containing 1 mg/ml FITC-dextran (Sigma). After a 1-h incubation at 37°C, cells were washed four times in ice-cold PBS and analyzed in a EPICS flow cytometer. As control, cells from each culture condition were maintained in the same solution for 1 h at 4°C.
Determination of cytokine levels
The level of IL-12 (p70) released in each culture condition was determined using the IL-12 Eli-pair system from Diaclone Research (capture mAb B-T21; detection biotynilated antibody B-P24) or the OptiEIA human IL-12 p70 set (PharMingen, San Diego, CA), following the manufacturer’s recommendations. Based on preliminary experiments, supernatants from MDDC were assayed, undiluted or diluted 1:3 in complete medium. Culture supernatants were assayed in triplicate for TNF- and IL-6 by enzyme-linked immunosorbent assay with Ready-SET-Go sets (eBioscience, San Diego, CA).
MDDC-induced allogeneic T lymphocyte proliferation
Allogeneic lymphocytes were obtained from the peripheral blood of healthy adults after density centrifugation on Lymphoprep (Nycomed Pharma) and monocyte removal by an adherence step. Lymphocytes (2x105) were stimulated in a flat-bottom 96-well plate with 200, 400, 103, 2 x 103, or 5 x 103 allogeneic, irradiated (150 rads/min for 10 min) MDDC, matured under the distinct culture conditions. Each stimulator/responder ratio was assayed in triplicate. After a 6-day incubation period, [3H]thymidine was added (1 µCi/well) during the last 16 h of coculture, and thymidine incorporation was determined to evaluate the level of T cell proliferation.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed essentially as described [25
], using 5 µg nuclear extracts prepared according to Schreiber et al. [26
]. For competition experiments, unlabeled oligonucleotide (100-fold molar excess) was preincubated with nuclear extracts at 4°C for 30 min before the addition of the probe. EMSA probe was NF-B-CONS (5'-AGTTGAGGGGACTTTCCCAGGC-3'), which contains a consensus-binding site for NF-B.
Western blot
Total cell lysates were obtained in 50 mM HEPES, pH 7.5, 250 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5 mM dithiothreitol, 10 mM NaF, 1 mM Na3VO4, 20 mM Pefabloc, and 2 µg/ml aprotinin, antipain, leupeptin, and pepstatin. Each sample (10 µg) was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and was transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking the unoccupied sites with 5% bovine serum albumin in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20, IB detection was accomplished with an anti-IB polyclonal antiserum from Santa Cruz Biotechnology (Santa Cruz, CA), a horsereadish peroxidase-labeled goat anti-rabbit antiserum (Dako A/S, Denmark), and the Supersignal West Pico chemiluminescent system (Pierce, Rockford, IL).
Statistical analysis
Data from individual representative experiments were presented as mean values ± SD, and data from multiple experiments were represented as mean group values ± SD. Statistical significance of the observed differences between groups was determined by ANOVA and paired Student’s t-test, and P < 0.05 values were considered significant. Statistical analysis of the data was performed with MPM III 1.34 (BioRad, Hercules, CA) and GraphPad Prism (GraphPad Software, San Diego, CA) software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(100 U/ml) or LPS (10 ng/ml). Addition of the D1 solution prevented the expression of maturation markers CD83 and CD49 (Fig. 1A
) and impaired the loss of mannose receptor-mediated endocytosis, an effect that could be observed 24 and 48 h after LPS addition (Fig. 1B
; and data not shown). More importantly, D1 significantly inhibited the DC-mediated, allogenic T cell proliferation at two distinct effector:target ratios (Fig. 1C)
and blocked the maturation-dependent production of IL-12 p70 (Fig. 1D)
. Therefore, maturation of MDDC in response to TNF- or LPS is inhibited by the D1 PD solution.
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or LPS and in the presence of PD solutions differing in their glucose, lactate, and GDP content (Table 1)
. Except for the D3 solution, MDDC viability was not affected by exposure to PD solutions, as propidium iodide and annexin V staining revealed that more than 90% of the cells remained viable for up to 48 h (data not shown). In the case of the D3 solution, 70% of the cells remained viable cells after 48 h, and flow cytometry analysis was performed after gating out dead cells. As shown in Table 2
, all PD solutions inhibited the LPS-triggered production of IL-12 p70 and differentially affected the expression of maturation markers and costimulatory molecules and the loss of FITC-dextran phagocytosis. More importantly, allogenic T cell proliferation induced by mature DC was also differentially inhibited by the tested PD solutions. Comparison of the relative composition of the distinct PD fluids and their inhibitory effects revealed that heat-sterilized, high lactate-containing solutions (D1, D3) produced higher inhibitory effects than heat-sterilized, low lactate solutions (P1, P3), and filtered, GDP-free solutions (D1f, D3f) always exerted a lower inhibitory effect than heat-sterilized, GDP-rich counterparts (D1, D3; Table 2
). Altogether, these experiments indicated that PD solutions differentially inhibit the acquisition of MDDC maturation parameters and suggested that lactate and GDPs are major contributors to the inhibitory effect of PD solutions on MDDC maturation.
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B activity and IL-6 [27
], and PD solutions were analyzed for their influence on the maturation-dependent release of both cytokines. MDDC released less TNF- when maturation took place in the presence of high lactate-containing solutions (D1, D3, D1f, D3f), whereas solutions with lower levels of lactate (15 mM; P1, P3) did not affect cytokine production (Fig. 2A
). For IL-6, a strong, inhibitory effect was also observed for the D1 and D3 solutions, and P1 and D1f solutions exerted a weak inhibition (Fig. 2A)
. Therefore, PD solutions differentially inhibit the production of TNF- and IL-6 by LPS-matured MDDC, and high lactate-containing solutions exhibited a higher inhibitory effect.
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B transcription factors, whose activity increases during the process [3
, 8
, 27
28
29
]. As PD solutions impair MDDC functional maturation, their influence on NF-B activity during LPS-induced MDDC maturation was determined. As expected, immature MDDC exhibited a moderate level of NF-B DNA binding, and a great increase in NF-B-dependent DNA binding was found in mature MDDC (Fig. 2B)
[28
]. However, MDDC maturation in the presence of certain PD solutions resulted in reduced levels of NF-B activity (Fig. 2B)
. The maturation-associated increase in NF-B activity was strongly reduced by D1 and moderately inhibited by P1, and GDP-free solutions showed a minor inhibitory effect (D1 vs. D1f and D3 vs. D3f; Fig. 2B
), thus implying that high lactate and GDPs have a detrimental effect on the increase in NF-B DNA-binding activity during MDDC maturation. Previous studies have also shown increased expression of IB in mature MDDC [28
]. The level of IB was also greatly reduced in MDDC matured in the presence of D solutions, and GDP-containing solutions (P3 and D3) had a stronger inhibitory effect than the corresponding GDP-free solution (D3f; Fig. 2C
). These results indicate that the differential, inhibitory effect of PD solutions on MDDC maturation reflects their distinct ability to prevent the LPS-induced increase in NF-B activity.
Inhibitory effect of lactate and GDPs on the phenotypic and functional maturation of MDDC
As lactate and GDPs were potentially responsible for the observed, inhibitory effect of PD solutions, their individual effects on MDDC maturation were evaluated. In the case of lactate, experiments were performed using 15 or 40 mM sodium lactate, and the pH of the lactate-containing media remained within the 7.4–7.6 range. Lactate dose-dependently inhibited the LPS-induced up-regulation of CD83 and weakly affected the CD86 up-regulation but had no influence on the CD151 expression (Fig. 3A
). Lactate also inhibited IL-12 p70 production in a dose-dependent manner: IL-12 p70 production was reduced 5–10 times in the presence of 15 mM lactate, and 40 mM lactate reduced IL-12 p70 production to undetectable levels (Fig. 3B)
. Sodium lactate significantly inhibited the LPS-induced production of TNF-, had no effect on the maturation-dependent production of IL-6 (Fig. 3C)
, and reduced NF-B activation in immature and LPS-treated MDDC (Fig. 3D)
. As a whole, these results demonstrate that sodium lactate impairs the LPS-induced phenotypic and functional MDDC maturation by inhibiting NF-B activation in a dose-dependent manner.
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B activity during MDDC maturation. 3-DG impaired the increase in NF-B during MDDC maturation, with a higher inhibitory effect than lactate, and acetaldehyde had no effect (Fig. 4C)
. Finally, experiments were set up to evaluate the influence of lactate and 3-DG on the acquisition of T cell-stimulatory activity. Lactate moderately inhibited the T cell stimulatory activity, and 40 mM lactate caused a 25% reduction (Fig. 5
). The parallel analysis of 3-DG revealed that it reduced allogenic T cell proliferation to a higher extent than 40 mM lactate, as it brought allogenic proliferation down to 50% at a 1:40 MDDC:T ratio, and it caused an almost complete blockade at a 1:200 ratio (Fig. 5)
. Altogether, these results demonstrate that lactate and 3-DG negatively affect LPS-induced MDDC maturation and might contribute to the inhibitory action of PD solutions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activation induced by maturation agents. Although the experimental conditions used in the current studies may not mimic the changing kinetics of different solutes in clinical conditions, they provide a clear demonstration that different PD solutions and certain PD solution constituents (sodium lactate, GDPs) can inhibit differentiation programs at the genetic and functional levels. In addition, the protocols implemented in our studies may serve to illustrate the compounded effect of these solutions upon cyclical exposure. To the best of our knowledge, the present manuscript provides the first experimental evidence that currently used PD solutions exert a potent and differential inhibitory effect on DC differentiation and maturation.
Recent studies have investigated the presence and biological relevance of DC within the peritoneal cavity. DC account for 1–2% of the total peritoneal cell pool [5
, 10
, 11
] and participate in the initiation of immune responses against bacteria [34
]. However, the number of functionally effective peritoneal DC could be considerably higher in an infectious situation, as monocytes and macrophages can differentiate in vivo into fully mature DC [5
, 11
]. Previous studies have also illustrated the capacity of murine peritoneal cells to differentiate in vivo along the dendritic pathway in the presence of GM-CSF and/or TNF-, leading to the proposal that peritoneal cells constitute a reservoir of late DC precursors [35
], and the number of functionally mature DC is dramatically increased in the peritoneal cavity after Flt3L treatment [36
]. Our results indicate that PD solutions impair monocyte differentiation toward DC, affecting cellular morphology and the acquisition of the DC markers CD1a and DC-SIGN, which not only mediates transient DC–T interactions and DC rolling on endothelial cells by interacting with ICAM-3 and ICAM-2 [32
, 37
] but is also involved in HIV and Ebola virus capture [38
, 39
] and pathogen recognition [40
] and can be considered as a pathogen-recognition receptor. Conversely, the CD1 family of antigen-presenting molecules binds and presents a variety of mammalian and microbial glycolipids for specific recognition by T cells [41
]. Therefore, PD solutions, which inhibit the differentiation-associated induction of CD1a and DC-SIGN, might disrupt peritoneal immunocompetence by impairing the acquisition of the antigen-presenting and the pathogen-recognition capabilities normally exhibited by DC. In consequence, pharmacological treatment with differentiation- or maturation-promoting cytokines might improve peritoneal immunocompetence during exposure to PD fluids.
The inhibitory effect of PD solutions on the maturation-dependent release of IL-12 p70 and IL-6 is another relevant finding. IL-12 p70 is critically involved in the process of polarization of naive T lymphocytes into the Th1 lymphocyte differentiation pathway and the cellular branch of the immune response [27
, 42
]. Consequently, if the inhibitory action of lactate and 3-DG takes place in vivo, peritoneal DC from CAPD patients would be deficient in their Th1-polarizing capability and would preferentially trigger pro-tolerogenic or Th2-mediated humoral-immune responses [1
, 3
, 42
], thus impairing the initiation of cellular immune responses within the peritoneal cavity. In this regard, it is worth mentioning that Th cells from CAPD patients have been found to show a dysregulated, differentiation profile characterized by a major increase in the percentage of Th2 cells [43
]. Conversely, IL-6 production is more strongly inhibited in the presence of PD solutions containing high levels of GDP (Fig. 2A)
, which suggests a role for GDPs in IL-6 inhibition. However, direct evaluation of the individual effects of 3-DG and acetaldehyde on the maturation-dependent IL-6 production revealed no effect (data not shown). Several GDPs have been shown previously to weakly inhibit IL-6 production by human peritoneal mesothelial cells but only when six distinct GDPs were present [44
]. Therefore, although GDPs might influence IL-6 production, their inhibitory action might require the combination of various GDPs or might even require the concomitant presence of other components of the PD solutions (including glucose and lactate). Along the same line, the inhibitory effect of lactate on NF-B activity and its differential effect on the production of TNF- and IL-6 (Fig. 3C)
suggest that their expression is differentially affected by NF-B inhibition. In fact, although TNF- expression is extremely sensitive to NF-B inhibition, the IL-6 promoter in DC is positively regulated by additional transcription factors such as activated protein-1, CCAAT/enhancer binding protein (C/EBP), or cyclic adenosine monophosphate response element-binding protein [45
]. These factors, whose sensitivity to lactate is currently under investigation, might compensate for the diminished NF-B activity and explain the lack of effect of lactate on the maturation-dependent IL-6 production. Alternatively, lactate might interfere with signaling pathways differentially affecting the expression of IL-6 and TNF- and whose involvement in the stimulus-specific MDDC maturation has been recently demonstrated [46
].
As a metabolite from activated macrophages, physiologically relevant concentrations of lactate (10–30 mM) are similar to those found to inhibit CD1a expression and MDDC maturation (15–45 mM) and have been previously found to modulate T cell receptor signaling [47
]. However, the negative effect of lactate on MDDC maturation has additional implications in other pathological situations. High lactate concentrations are normally found within solid tumors, and lactate concentration correlates with the incidence of metastasis [48
]. As tumor-associated DC usually have low allostimulatory capacity [49
] and are phenotypically immature [50
], it is tempting to speculate that lactate might contribute to the defective, functional maturation of DC in cancer patients. By contrast, GDP concentrations in PD fluids (500 µM for 3-DG in 3.86% glucose-containing solutions) are higher than those found in plasma under physiological conditions (1 µM) or even under diabetic conditions (7–10 µM) [51
]. Although we have demonstrated its inhibitory effect on NF-B activation, it remains to be established whether 3-DG also impairs MDDC maturation through its glycation capabilities, as 3-DG accelerates advanced glycation end-product formation [52
] and induces growth-factor gene expression at 500 µM [53
]. Regardless of the mechanism, 3-DG and sodium lactate appear to contribute to the inhibitory activity of PD fluids and consequently, might directly impair the onset of adequate immune responses within the peritoneal cavity and its surrounding tissues, a hypothesis that deserves further investigation.
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ACKNOWLEDGEMENTS |
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Received September 11, 2002; revised December 24, 2002; accepted January 13, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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