1 Antigen Presentation Research Group, Faculty of Medicine, Imperial College London, Middlesex, U.K
2 Department of Endocrinology and Diabetes, Northwick Park Hospital, Middlesex, U.K
3 Department of Clinical Biochemistry, Northwick Park Hospital, Middlesex, U.K
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ABSTRACT |
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Advanced glycation end products (AGEs) result from irreversible changes to long-lived proteins in the body. Reducing sugars and their metabolites react with free amino groups (the Maillard reaction) and sequentially alter to produce complex heterogeneous adducts termed AGEs. Carbonyl compounds from glycolytic intermediates of Maillard reactions (1), including glyoxal and methylglyoxal (MG), are major contributors to AGE formation. MG is the most reactive AGE precursor, and its concentration is increased in diabetic subjects (2). MG reacts irreversibly with lysine to form glycosylamine protein cross-links and with arginine to give imidazolone derivatives (3).
AGEs are associated with complications of rheumatoid arthritis, Alzheimers disease, and diabetes. Hyperglycemia in diabetes increases rate of formation of AGEs (4). Build up of adducts, and associated cross-links, on long-lived proteins such as collagen, crystallins, and vascular basement membranes may contribute to the complications of diabetes, including retinopathy, vascular hypertension, and nephropathy (5). AGEs are also measurable in blood in association with circulating proteins (6). Levels are low in healthy subjects but higher in subjects with diabetes. Elimination of AGE-modified protein is by catabolism and subsequent renal excretion, with levels being elevated in renal failure (7). Therefore, there is opportunity for AGE-modified proteins to come in contact with components of the immune system.
Dendritic cells (DCs) are antigen-presenting cells of the immune system, unique in their potency at stimulating B- and T-cells in primary immune responses (8). DCs and their precursors are present in blood. Blood and tissue DCs are regarded as being "immature." Tissue DCs acquire and process antigens, mature, and migrate to lymph nodes, where they can stimulate lymphocytes (9). Markers expressed by DCs include the antigen presentation molecules major histocompatibility class I, class II, and CD1; adhesion molecules such as CD54; and the costimulatory molecules CD80 and CD86. Another marker traditionally thought of as a marker of DC maturation is CD83, a 45-kDa type 1 membrane glycoprotein. CD83 is not seen on resting blood leukocytes at detectable levels but may be rapidly upregulated. However, it is not limited to DCs. Recent reports show expression in a range of cells including polymorphonuclear neutrophils (10) and B-cells (11). Neither is CD83 just a marker of activation; it is a sialic acid-binding lectin adhesion receptor that binds monocytes and a subset of activated CD8+ T-cells (12). Preventing surface CD83 expression inhibits DC-mediated T-cell stimulation (13) and CD4+ T-cell development in the thymus (14).
The best characterized AGE receptor is the receptor for AGE (RAGE). RAGE-ligand interaction results in a signaling cascade to release proinflammatory cytokines, growth factors, and tissue factors (15).
Glycation of albumin in vivo is consistent with only one to three glucose residues per protein molecule, making it appropriate to study such minimally glycated models in vitro (2). Proteins can be minimally glycated with MG to produce methylglyoxal-derived hydroimidazolone (MG-H) (16,17). The aim of this study was to use this adduct as a model to test for immunomodulatory effects of AGEs. The complications of diabetes are largely vascular, and vascular disease is accompanied by leukocyte activation and inflammation (18). Because DCs initiate and modulate inflammatory responses, we chose to investigate the interaction between AGEs and these primary antigen-presenting cells. In patients with diabetes, there is both an increase in DC numbers and an impaired ability of DCs to stimulate primary immune responses (1921). We studied both of these aspects and report that AGEs modify the numbers, phenotype, and function of DCs.
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RESEARCH DESIGN AND METHODS |
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Peptide (2.4 mmol/l) was dissolved in sodium phosphate buffer (1 ml, 0.02 mol/l, pH 7.8) containing MG (500 mmol/l) under aseptic conditions. The reaction mixture (RM) was incubated at 37°C for 24 h in a 2-ml glass vial and stored in the reaction vessel at 4°C. An endotoxin test revealed insignificant contamination in these reagents (Charles River Endosafe LAL test kit; Wilmington, MA).
Mass spectrometric analysis of RMs.
The RMs (2 µl) were diluted 1:5 with distilled water, further diluted with 0.1% trifluoroacetic acid in water, mixed with freshly prepared -matrix (
-cyano-4-hydroxycinnamic acid, 100 mg/ml in 0.1% trifluoroacetic acid in water/acetonitrile/methanol, 1:1:1), and applied to the target plate. Samples were analyzed on the Micromass TofSpec 2E MALDI-MS.
Isolation of peripheral blood mononuclear cells.
Heparinized human peripheral blood was obtained after informed consent was granted, diluted with medium (Dutch modified RPMI 1640 containing 100 units/ml penicillin/streptomycin and 2 mmol/l L-glutamine [Sigma]), layered onto an equal volume of Ficoll Paque (Amersham Biosciences, Chalfont St. Giles, U.K.), and centrifuged (626gav, 30 min, 20°C). Mononuclear cells were aspirated from the interfaces, washed twice (medium containing 10% FCS [Sigma]), spun at 600g for 5 min), counted (trypan blue exclusion), and suspended at 3.5 million/ml.
For binding studies using MALDI-MS, and as stimulator cell populations for mixed leukocyte reactions, DCs were enriched from nonadherent mononuclear cell populations, after overnight incubation, by centrifuging at 600g over a 14.5% wt/vol metrizamide gradient (Sigma).
Exposure of cells.
Freshly isolated peripheral blood mononuclear cells (PBMCs) were exposed to sodium phosphate buffer (5 µl/ml), peptide solution (2.4 mmol/l solution in sodium phosphate buffer [5 µl/ml]), or minimally glycated peptide RM at 2 and 5 µl/ml. Samples were incubated (37°C, 5% CO2 in air, humidified incubator) for 2.5 h unless otherwise stated.
Antibodies.
Antibodies to CD14-FITC (MP9) and CD11c-PE (Leu-MS) (BD Biosciences, San Jose, CA); CD40-PE (MCA1590) and CD86-PE (MCA1118) (Serotec Oxford, U.K.); CD80-PE (L3074), CD83-PE (HB15e), and CD45-PE (HI30) (BD-Pharmingen, San Diego, CA); and CD3-ECD (UCHT 1), CD19-ECD (J4.119), and HLA-DR-PC5 (Immu-357) (Coulter Immunotech High Wycombe, U.K.) were used. Isotype-matched controls were obtained from the same manufacturers.
Monoclonal mouse anti-human RAGE was purchased from R&D systems (Abingdon, U.K.), and polyclonal anti-mouse Ig was purchased from BD-Pharmingen.
Flow cytometry.
Antibody labeling was performed on cell samples centrifuged (265gav, 5 min) and resuspended in PBS supplemented with 0.2% BSA and 0.02% sodium azide (fluorescence-activated cell sorting buffer). Aliquots of 50 µl cell suspension were labeled with monoclonal antibodies (3 µl each antibody, 15 min, room temperature), washed (2 ml fluorescence-activated cell sorting buffer, 265gav, 5 min), and resuspended and fixed in 1% paraformaldehyde in PBS (500 µl). Data were acquired uncompensated (40,000100,000 events) in the lymphocyte and monocyte light scatter region, using a Beckman Coulter EPICS XL-MCL flow cytometer, and analyzed using WinList 5.3D software (Verity). Single-color cell samples stained with either CD8 or CD3 were also acquired for objective compensation in WinList. Relative cell counts were obtained by simultaneous acquisition of Flow-count fluorospheres (Coulter Immunotech). Dead cells and debris were excluded on the basis of the light scatter (forward scatter/side scatter). Using four-color analysis, the HLA-DR+ DCs and monocyte/macrophage populations were defined by excluding CD3+ (T-cells) and CD19+ (B-cells) and differentiating on CD14 expression. CD14-FITC was detected in fl (fluorescence channel) 1, CD3- and CD19-ECD in fl 3, HLA-DR-PC5 in fl 4, and other phycoerythrin-conjugated markers in fl 2.
Percentage positive events for cell surface markers on gated populations were determined by histogram subtraction of values for isotype-matched controls using Winlist software.
Mixed leukocyte reaction.
Stimulator DC populations were metrizamide enriched as described previously. Either sodium phosphate buffer or AI solution at 5 µl/ml, or one of two concentrations of AI-AGE RM (2 or 5 µl/ml), was added to the cells (at 100,000/ml). Suspensions were incubated for 2.5 h (37°C/5% CO2 in air).
Freshly isolated PBMCs from a different donor were suspended in medium containing 10% FCS at 5 million/ml. Triplicate cultures containing 2,50050,000 PBMCs per well were set up in Terasaki plates. Low-density cells (LDCs) were added at a concentration of either 1,000 or 500 cells per well to give a final culture volume of 20 µl. Control wells contained PBMCs alone, PBMCs + Concanavalin A (5 µg/ml), and exposed LDCs alone. Plates were cultured over saline (37°C/5% CO2 in air) for 3 days, wells were pulsed with [3H]thymidine (1 µg/ml; specific activity 37 kBq/µl) for 2 h, and cells were harvested onto damp filter paper. Papers were washed with saline, 5% trichloro acetic acid, and methanol. When fully air-dried, incorporation of [3H]thymidine was assayed using a Molecular Dynamics phosphor screen and Imager SI.
Culture for cell count.
PBMCs (3.5 million/ml) were treated with buffer, AI, or AI-AGE RM (2 µl/ml each); placed in a sterile six-well plate; and incubated at 37°C/5% CO2 in air. At times of 2, 4, 24, 48, and 69 h after addition of controls or AGE, 250-µl aliquots were analyzed using flow cytometry. The same protocol was followed for culture with ACTH (fragment 410) and ACTH-AGE. Addition of 50-µl count beads to each sample before acquisition allowed calculation of relative cell numbers.
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RESULTS |
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Expression of CD83 on AGE-exposed cells is dose dependent.
Among the DC population, expression of surface CD83 was inversely proportional to concentration of AGE RM added to culture, within standard errors (Fig. 5A). Monocyte expression of CD83 did not drop until a higher concentration of AGEs had been added, and the decrease was less gradual than for those in the DC region (data not shown).
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Specificity of effect of AGEs.
PBMCs were isolated and exposed to controls or AGEs and to controls or AGEs with added lipopolysaccharide (1 µg/ml) to upregulate surface markers. Cultures were left for 2, 4, or 24 h at 37°C/5% CO2 in air. Cells were stained with monoclonal antibodies to CD40, CD80, CD45, and HLA-DR alongside the usual cocktail for population discrimination.
Neither DCs nor monocytes showed a significant difference in percentage expression of any markers after AGE exposure, except for lower CD80 expression (50% less) after 24 h compared with controls. CD80 was not expressed significantly after 2 or 4 h.
AGE-exposed DCs have reduced capacity to stimulate a mixed leukocyte reaction.
Cells treated with AI or buffer equally stimulated allogeneic lymphocytes (Fig. 6). LDCs exposed to 2 µl/ml AGE RM produced a low level of allogeneic stimulation, but proliferation was significantly higher than that in responders alone (P = 0.05), whereas those incubated with 5 µl/ml AGE showed no stimulatory capacity (P = 0.78). Concanavalin A responses are shown as a high positive control. Survival of DCs, measured by flow cytometry, was similar in AI-AGE- and buffer-exposed cultures.
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All changes were calculated relative to added count beads and thus were indicative of real changes in cell number.
Effect of AGEs is not RAGE or CD83 mediated.
Isolated LDCs were exposed to monoclonal anti-RAGE antibody before AI-AGE RM. Whole-cell samples were prepared for analysis by MALDI-MS with blank (control) cells and cells exposed to just anti-RAGE antibody or AI-AGE RM. The use of whole cells allowed visualization of cell surface components in the absence of a large mass of intracellular components. Added AI-AGE was seen to bind quickly to the cell surface from the supernatant and was evident for several hours. Within 24 h, AI-AGE was not detectable in either the supernatant or on the cell surface. Cells exposed to anti-RAGE antibody alone looked equivalent to control cells (Figs. 8A and C). Added AGEs could be seen equivalently on the cells with and without pretreatment with anti-RAGE antibody (Figs. 8B and D). Similar results were found when blocking was performed with the anti-CD83 antibody.
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DISCUSSION |
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Minimally glycated proteins are biologically significant, making it appropriate to investigate such models in vitro (2). We prepared two peptides, minimally glycated with MG, which had profound effects on PBMCs. We chose ACTH fragment 410 and AI as our starting peptides, as each contained only one arginine residue with a free amino group. This choice predicted that each peptide molecule would host only one AGE adduct, reducing complicating factors in studying its effect. The AGE adduct MG-H was detected by mass spectrometry as the major product, 54 mU above the starting peptide, within the AI-AGE reaction mixture. However, the ACTH (fragment 410) RM was a less simple mixture, the spectrum containing many different peaks above that of the start material, including the +54-mU peak expected; another site on this peptide may be reactive other than the considered arginine.
Both AGE peptides caused a reduction of CD83 expression in peripheral blood DCs and monocyte/macrophages, defined here as the DR+/(CD3 and CD19)/CD14 and DR+/(CD3 and CD19) /CD14+ populations, respectively. ACTH-AGE changed other surface markers, but we suggest that this was due to its toxicity, as shown by cell death and deterioration. Because AI-AGE was less toxic to cells and the mass spectrum showed only one adduct present, it was used predominantly for most tests.
Putative DCs in PBMCs cultured with AI-AGE showed a lower percentage expression of CD83 than corresponding buffer- or peptide-exposed cells after 2.5 h at 37°C/5% CO2 in air. The AGE-induced lack of CD83 was not a permanent effect, and recovery, like the initial effect, was dose dependent. The effect on monocytes differed, with reduction in CD83 expression occurring after a higher dose of AGEs. This difference suggests a varied response to AGEs, depending on cell type. We were initially unsure whether the effect of AGEs on CD83 was a downregulation, or a lack of upregulation, because freshly isolated PBMCs express no CD83, but studies indicated that it was probably the latter. Within monocytes, there was evidence of real downregulation of CD83, again suggesting a differing response according to cell type.
CD83 is quoted to have an average receptor turnover time of 90 min (12) and may show effects before other surface receptors with slower recycling systems. Samples incubated for longer times, both with and without lipopolysaccharide in the culture, showed little change in other receptors except for a lack of upregulation of CD80 after culture for 24 h. The AGE-related phenomena may not be CD83 specific.
Preventing surface expression of CD83 can inhibit DC-mediated T-cell stimulation (22). Loss in the capacity of DCs treated with AGEs to stimulate primary T-cell responses could be related to the loss of both CD83 and the costimulatory molecule CD80, particularly during the critical early events of lymphocyte activation by DCs. AGEs do not appear to bind directly to CD83 and seem to be consumed by the cells, suggesting that other indirect signaling pathways may contribute to functional changes.
Culturing peripheral blood cells with AGEs for >4 h caused an explosion in the DR+/(CD3 and CD19)/CD14 population. This effect on putative DCs was observed twice with AI-AGE and repeated once with ACTH-AGE to prove that the effect was not peptide specific. It is hard to prove a significant change in populations to account for our observations because of the small numbers of DCs compared with other cells in culture. However, known maturation of CD14+ monocytes into DCs in culture (23), together with loss of monocytes, make it likely that these are the source of the increased DC population in culture.
The most well-defined receptor for AGE is RAGE. We found that DC populations did not express RAGE, whereas monocytes expressed it highly. The effects of AGEs on DCs cannot therefore be RAGE mediated, as also suggested from the lack of blocking seen using anti-RAGE antibody in the spectrometric assay of AGE-exposed LDCs. Studies of alternative known AGE-binding sites such as macrophage scavenger receptors type I and II, galectin-3, and other scavenger receptors such as CD36, plus intracellular signaling pathways, will be required to determine the route by which the effects of AGEs are mediated.
Most long-term complications of diabetes can be related to vascular damage to either large vessels or microcirculation. Mortality associated with diabetes can be ascribed principally to large-vessel disease (24). Vessel disease in the diabetic and nondiabetic population is associated with recruitment and activation of monocytes and a subset of T-cells, with increased production of tumor necrosis factor-, leading to raised levels of C-reactive protein and interleukin-6, both well-defined markers of inflammation (18,25,26). Specific vascular DCs are increased in number at sites of vascular damage and may modulate T-cell activation (27). Damage of vascular endothelium, leading to this inflammation, may be caused by one or more of the following factors: modified LDL, free radicals, hypertension, and infectious microorganisms, such as cytomegalovirus, Chlamydia pneumoniae, Helicobacter pylori, and herpes simplex virus (18,28,29). AGEs must now be considered as another possible factor. Although AGEs are endogenously produced products, binding to RAGE leads to a signaling cascade for activation of nuclear factor-
B and increased expression of vascular cell adhesion molecule-1 and interleukin-6 (30), which in turn cause inflammation and tissue damage. From our studies, it is unlikely that RAGE is the sole mechanism by which AGEs mediate their effects on the immune system. These long-lasting modified proteins can also stimulate a more classic immune response, because antibodies to AGEs are present in diabetic sera and correlate with related complications (31). DCs are likely to be involved in the initiation of these responses.
AGE peptide increased numbers of DCs, but these DCs did not upregulate the maturation marker CD83 or the costimulatory molecule CD80. The reduced capacity of AGE-exposed DCs to stimulate primary T-cell proliferation is compatible with the concept that the DCs remained immature in the presence of AGEs. Increased production of immature DCs that fail to mature and migrate appropriately into tissues could perhaps predispose to accumulation of such cells in vascular epithelium and consequent local innate inflammatory effects. Although we presently have no data to show a direct function of DCs in initiating such inflammation, suggestion of reduced CD83 (32), increased DC numbers (19), and impaired stimulatory capacity of DCs (20,21) in patients with diabetes support the idea that effects reported here may have some counterpart in vivo. In normal subjects, the MG-H adduct is present on up to 2% of all arginine residues (33). Levels in diabetes are likely to be higher because of raised MG concentrations. Assuming 1020% conversion of AI to AI-AGE in our RM (judging by mass spectrum peak intensity), we can predict that addition of RM to cell culture, containing serum and other proteins, will result in a total arginine-MG-H concentration comparable to that in diabetic subjects.
In conclusion, changes to the maturation, phenotype, and function of DCs have been shown in response to AGEs. We have identified a novel pathway that may affect immunological activity in patients, particularly those with diabetes, who have high circulating levels of AGE proteins.
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ACKNOWLEDGMENTS |
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Address correspondence and reprint requests to Prof. S.C. Knight, Antigen Presentation Research Group, Faculty of Medicine Imperial College London, Northwick ParkSt Marks Campus, Level 7W, St. Marks Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K. E-mail: s.knight{at}imperial.ac.uk
Received for publication November 7, 2003 and accepted in revised form February 26, 2004
ACTH, adrenocorticotrophic hormone; AGE, advance glycation end product; AI, angiotensin I; DC, dendritic cell; LDC, low-density cell; MG, methylglyoxal; MG-H, methylglyoxal-derived hydroimidazolone; PBMC, peripheral blood mononuclear cell; RAGE, receptor for AGE; RM, reaction mixture
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REFERENCES |
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