Uremia impairs monocyte and monocyte-derived dendritic cell function in hemodialysis patients
2007; Elsevier BV; Volume: 72; Issue: 9 Linguagem: Inglês
10.1038/sj.ki.5002425
ISSN1523-1755
AutoresWai H. Lim, Svjetlana Kireta, Emma Leedham, Graeme R. Russ, P Coates,
Tópico(s)HIV Research and Treatment
ResumoPatients with chronic renal failure maintained on intermittent hemodialysis have frequent infections and a suboptimal response to vaccinations. Dendritic cells are potent antigen-presenting cells essential for the initiation and maintenance of innate and adaptive immunity. In this study we used uremic sera from hemodialysis patients to measure its impact on monocyte and monocyte-derived dendritic cell function in vitro. Monocytes from healthy and uremic subjects were isolated using immunomagnetic beads and differentiated into dendritic cells in the presence of either complete sera or sera from hemodialysis patients. Dendritic cells from normal patients cultured in uremic sera had decreased endocytosis and impaired maturation. These cells, however, had enhanced IL-12p70 production and increased allogeneic T-cell proliferation compared to cells of normal subjects cultured in normal sera. Monocyte derived dendritic cells of hemodialysis patients cultured in either normal or uremic sera were functionally impaired for endocytosis and maturation but had enhanced IL-12p70 production and allogeneic T-cell proliferation only when cultured with uremic sera. High concentrations of urea in normal sera inhibited all aspects of normal dendritic cell function in vitro. Our study suggests that hemodialysis regimes tailored to remove uremic toxins more efficiently may improve immune functions of these patients. Patients with chronic renal failure maintained on intermittent hemodialysis have frequent infections and a suboptimal response to vaccinations. Dendritic cells are potent antigen-presenting cells essential for the initiation and maintenance of innate and adaptive immunity. In this study we used uremic sera from hemodialysis patients to measure its impact on monocyte and monocyte-derived dendritic cell function in vitro. Monocytes from healthy and uremic subjects were isolated using immunomagnetic beads and differentiated into dendritic cells in the presence of either complete sera or sera from hemodialysis patients. Dendritic cells from normal patients cultured in uremic sera had decreased endocytosis and impaired maturation. These cells, however, had enhanced IL-12p70 production and increased allogeneic T-cell proliferation compared to cells of normal subjects cultured in normal sera. Monocyte derived dendritic cells of hemodialysis patients cultured in either normal or uremic sera were functionally impaired for endocytosis and maturation but had enhanced IL-12p70 production and allogeneic T-cell proliferation only when cultured with uremic sera. High concentrations of urea in normal sera inhibited all aspects of normal dendritic cell function in vitro. Our study suggests that hemodialysis regimes tailored to remove uremic toxins more efficiently may improve immune functions of these patients. Immune protection and surveillance against pathogens in humans relies on a coordinated response of innate and adaptive immune systems.1.Fearon D. Locksley R. The instructive role of innate immunity in the acquired immune response.Science. 1996; 272: 50-53Crossref PubMed Scopus (1378) Google Scholar Chronic renal failure (CRF) patients have evidence of defects in both innate immunity (e.g. reduced polymorphonuclear leukocytes chemotaxis, phagocytosis, and bactericidal activity)2.Iida T. Umezawa K. Tanaka K. et al.Polymorphonuclear cells in chronic hemodialysis patients have intact phagocytic and impaired bactericidal activities.Nephron. 1997; 75: 41-47Crossref PubMed Scopus (24) Google Scholar,3.Vanholder R. Ringoir S. Polymorphonuclear cell function and infection in dialysis.Kidney Int. 1992; 38: S91-S95Google Scholar and adaptive immunity (reduced T-cell function) that contribute to generalized immunodeficiency.4.Girndt M. Kohler H. Schiedhelm-Weick E. et al.T-cell activation defect in hemodialysis patients: evidence for a role of the B7/CD28 pathway.Kidney Int. 1993; 44: 359-365Abstract Full Text PDF PubMed Scopus (72) Google Scholar,5.Gerez L. Madar L. Shkolnik T. et al.Regulation of interleukin-2 and interferon-gamma gene expression in renal failure.Kidney Int. 1991; 40: 266-272Abstract Full Text PDF PubMed Scopus (35) Google Scholar Infection is a leading cause of morbidity and mortality in CRF patients (including hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients) that may be attributed to these underlying immune defects in the innate system.6.No authors listed U.S. Renal Data System: USRDS 1998 annual data report.Am J Kidney Dis. 1998; 32: S150-S162Google Scholar,7.McDonald S. Deaths.in: Russ G. Australia and New Zealand Dialysis and Transplant Registry. Australia and New Zealand Transplant Registry, Adelaide, South Australia2003: 19Google Scholar In addition, CRF patients display suboptimal responses to therapeutic vaccinations (e.g. hepatitis B) characterized by low antibody titers, and short duration of protection typical of impairment of adaptive immunity.8.Kohler H. Arnold W. Renschin G. et al.Active hepatitis B vaccination of dialysis patients and medical staff.Kidney Int. 1984; 25: 124-128Abstract Full Text PDF PubMed Scopus (140) Google Scholar,9.Coates P.T.H. Wilson R. Patrick G. et al.Hepatitis B vaccines: assessment of the seroprotective efficacy of two recombinant DNA vaccines.Clin Therap. 2001; 23: 392-403Abstract Full Text PDF PubMed Scopus (126) Google Scholar These deficiencies observed in CRF patients may be attributed to defects in the number and function of antigen-presenting cells, in particular dendritic cells (DC),10.Hasselink D. Betjes M. Verkade M. et al.The effects of chronic kidney disease and renal replacement therapy on circulating dendritic cells.Nephrol Dial Transplant. 2005; 20: 1868-1873Crossref PubMed Scopus (38) Google Scholar but their role in the pathogenesis of innate and adaptive immune dysfunction in CRF patients has not been defined clearly. DC are a group of rare, heterogenous population of professional antigen-presenting cells that initiate primary immune responses, and regulate both innate and adaptive immunity.11.Banchereau J. Briere F. Caux C. et al.Immunobiology of dendritic cells.Ann Rev Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5419) Google Scholar, 12.Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-251Crossref PubMed Scopus (11832) Google Scholar, 13.Coates P.T.H. Thomson A.W. Dendritic cells, tolerance induction and transplant outcome.Am J Transplant. 2002; 2: 299-307Crossref PubMed Scopus (57) Google Scholar DC are critical in stimulating naïve T-cell responses to captured foreign antigens.14.Inaba K. Inaba M. Naito M. et al.Dendritic cell progenitors phagocytose particulates, including Bacillus Calmette–Guerin organisms, and sensitize mice to mycobacterial antigens in vivo.J Exp Med. 1993; 23: 1595-1601Google Scholar,15.Hsu F. Benike C. Fagoni F. et al.Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.Nature Med. 1996; 2: 52-58Crossref PubMed Scopus (1647) Google Scholar Following antigen capture and processing, DC undergo maturation and migrate to secondary lymphoid tissues where they present processed antigen/peptide coupled to major histocompatibility complexes, which allow for selection and expansion of antigen-specific CD4+ T-helper cells. DC may be differentiated in vitro from monocyte precursors and provide a model system whereby these otherwise rare antigen-presenting cells may be generated in sufficient numbers for studies.16.Leon B. Lopez-Bravo M. Ardavin C. Monocyte-derived dendritic cells.Semin Immunol. 2005; 17: 313-318Crossref PubMed Scopus (90) Google Scholar DC-based vaccination has been utilized successfully in certain clinical conditions to generate immunity to certain pathogens.17.Bozza S. Perruccio K. Montagnoli C. et al.A dendritic cell vaccine against invasive aspergillosis in allogenic hematopoietic transplantation.Blood. 2003; 102: 3807-3814Crossref PubMed Scopus (188) Google Scholar, 18.Richards J. Ampel N. Lake D. Reversal of coccidioidal anergy in vitro by dendritic cells from patients with disseminated coccidioidomycosis.J Immunol. 2002; 169: 2020-2025Crossref PubMed Scopus (31) Google Scholar, 19.Shao C. Qu J. He L. et al.Dendritic cells transduced with an adenovirus vector encoding interleukin-12 are a potent vaccine for invasive pulmonary aspergillosis.Genes Immun. 2005; 6: 103-114Crossref PubMed Scopus (35) Google Scholar Monocytes cultured in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 for 5 days differentiate into immature monocyte-derived DC (MoDC) characterized by low expression of major histocompatibility complex class II molecules, costimulatory molecules (CD40, CD80, and CD86), and the DC maturation marker, CD83. Bacterial lipopolysaccharide (LPS), tumor necrosis factor-α, and CD40 ligand can be used for the induction of DC maturation without cell proliferation.20.Sallusto F. Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4396) Google Scholar,21.Ardavin C. Martinez del Hoyo G. Martin P. et al.Origin and differentiation of dendritic cells.Trends Immunol. 2001; 22: 691-700Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar In a previous study, we established that circulating myeloid DC precursors from HD patients displayed functional impairment with reduced cell-surface costimulatory molecule expression following maturational signals.22.Lim W.H. Kireta S. Russ G. et al.Renal transplantation reverses functional deficiencies in circulating dendritic cell subsets in chronic renal failure patients.Transplantation. 2006; 81: 160-168Crossref PubMed Scopus (36) Google Scholar We hypothesized that the presence of uremic toxins in the serum of HD patients are responsible for the observed impairment in DC functions. In this study, we used MoDC system to assess the impact of uremic sera from HD patients on monocyte and MoDC functions. HD patients have high levels of IL-12p70. The serum concentration of bioactive IL-12p70 was significantly higher in the sera of HD patients (52.8±18.1 pg/ml), CAPD patients (12.6±3.8 pg/ml), and CRF patients (49.6±5.5 pg/ml) when compared to healthy controls and pooled human AB serum (both below the level of detection; Mann–Whitney U-test P<0.001). Uremic medium inhibits normal monocyte function. To determine the effect of uremic serum on monocyte function, we studied normal monocytes cultured in uremic medium (UM). LPS-stimulated normal monocytes cultured in UM demonstrated reduced expression of costimulatory molecules (CD40, CD80, and CD86) compared to normal monocytes in complete medium (CM) (Figure 1a and Table 1). Consistent with this observation, UM-cultured monocytes displayed reduced allo-stimulatory capacity (Mann–Whitney U-test, P<0.0001; Figure 1b) when compared to monocytes in CM. The amount of IL-12p70 present in CM and UM was below the level of detection and 13.5±3.2 pg/ml, respectively (Mann–Whitney U-test, P<0.001). Monocytes cultured in CM or UM produced no additional IL-12p70 in excess of background amount of IL-12p70 in mediums (sera of 5 HD patients used).Table 1Costimulatory molecule expression of normal and uremic monocytes in complete and uremic mediaMonocytesCD40 MFI (mean±s.d.)*CD80 MFI (mean±s.d.)*CD86 MFI (mean±s.d.)*Normal monocytes in CM#,†8.7±1.62.7±0.63.3±0.9Normal monocytes in UM#2.3±0.61.9±1.12.1±1.5Uremic monocytes in CM†3.3±1.11.9±0.52.2±0.7Uremic monocytes in autologous UM0.0±0.00.0±0.00.0±0.0CM, complete medium; MFI, mean fluorescent intensity; s.d., standard deviation; UM, uremic medium.*P<0.05 Kruskal–Wallis test.†P<0.05 Student's t-test (CD40 only).#P<0.05 Mann–Whitney U-test (CD40 only). Open table in a new tab CM, complete medium; MFI, mean fluorescent intensity; s.d., standard deviation; UM, uremic medium. *P<0.05 Kruskal–Wallis test. †P<0.05 Student's t-test (CD40 only). #P<0.05 Mann–Whitney U-test (CD40 only). Uremic monocytes from HD patients are functionally abnormal. Freshly isolated uremic monocytes from HD patients exhibited reduced fluorescein isothiocyanate (FITC)-dextran uptake compared to normal monocytes (representative of three HD patients, mean fluorescent intensity (MFI) 9.2±2.3 and 25.4±5.2, respectively; Student's t-test, P=0.02). Following LPS stimulation, uremic monocytes cultured in CM, but not when cultured in autologous UM, upregulated cell-surface expression of costimulatory molecules CD40, CD80, and CD86, but not to the same extent of LPS-stimulated normal monocytes cultured in CM (Supplementary Figures, Legend to Supplementary Figures and Table 1). Consistent with this observation, uremic monocytes in CM showed greater allo-stimulatory capacity compared to uremic monocytes cultured in autologous UM (Mann–Whitney U-test, P<0.0001; Supplementary Figures, Legend to Supplementary Figures). Uremic monocytes cultured in CM or autologous UM produced no detectable levels of IL-12p70 in excess of background levels in mediums (representative of three experiments). Download .ppt (.1 MB) Help with ppt files Supplementary Figures Download .doc (.02 MB) Help with doc files Legend to Supplementary Figures UM does not affect the differentiation of monocytes into MoDC but inhibits MoDC functions. To determine the effects of uremic serum on the differentiation of monocytes into MoDC and MoDC function, we studied normal monocytes/MoDC cultured in UM. Compared to normal immature MoDC in CM, normal immature MoDC cultured in UM exhibited reduced FITC-dextran uptake (sera of 10 separate HD patients used, MFI 45.2±6.8 and 15.5±7.2, respectively; Mann–Whitney U-test, P<0.05) and reduced cell-surface expression of CD206 (sera of 10 separate HD patients used, MFI 60.5±10.2 and 8.2±2.5; Mann–Whitney U-test, P<0.01). The differentiation of normal monocytes cultured in CM or UM into MoDC was complete and unaffected by the presence of uremic serum. LPS-stimulated MoDC derived from normal monocytes cultured in UM demonstrated reduced cell-surface expression of costimulatory (CD40, CD80, and CD86) and maturation (CD83) markers (Figure 2a and Table 2).Table 2Costimulatory molecule expression and viability of normal and uremic monocyte-derived dendritic cells in complete and uremic (sera of hemodialysis patients) mediumsMoDCCD40 MFI (mean±s.d.)*CD80 MFI (mean±s.d.)*CD83 MFI (mean±s.d.)*CD86 MFI (mean±s.d.)*Apoptotic/necrotic cells (% total cells)#Normal MoDC in CM†,‡189.1±18.912.1±4.56.6±2.320.1±6.541.1±10.2Normal MoDC in UM†33.9±10.27.2±3.24.1±1.27.9±2.382.4±12.9Uremic MoDC in CM‡7.9±3.20.4±0.30.2±0.10.5±0.3—Uremic MoDC in autologous UM1.1±0.50.1±0.00.3±0.10.3±0.3—CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells; s.d., standard deviation; UM, uremic medium.*P<0.05 Kruskal–Wallis test.#P<0.05 Student's t-test.†P<0.05 Mann–Whitney U-test (except CD83).‡P<0.05 Mann–Whitney U-test. Open table in a new tab CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells; s.d., standard deviation; UM, uremic medium. *P<0.05 Kruskal–Wallis test. #P<0.05 Student's t-test. †P<0.05 Mann–Whitney U-test (except CD83). ‡P<0.05 Mann–Whitney U-test. A greater proportion of normal LPS-stimulated MoDC cultured in UM compared to CM were apoptotic and/or necrotic (sera of three separate HD patients used; Student's t-test, P<0.05; Table 2). CD95 expression was similar in normal LPS-stimulated MoDC cultured in UM and CM (sera of 10 separate HD patients used, MFI 4.8±2.2 and 3.4±1.8, respectively; P=NS). Normal LPS-stimulated MoDC cultured in UM produced a greater amount of IL-12p70 (78.9±24.4 pg/ml) when compared to cells cultured in CM (32.2±10.2 pg/ml, sera of 10 separate HD patients used; Mann–Whitney U-test, P=0.01). The amount of IL-12p70 present in CM and UM was below the level of detection and 9.5±2.1 pg/ml, respectively (Mann–Whitney U-test, P<0.01). Normal LPS-stimulated MoDC cultured in UM demonstrated enhanced allo-stimulatory capacity compared to cells in CM (Mann–Whitney U-test, P=0.009; Figure 2b). In order to investigate the impact of small MW uremic toxins on MoDC functions, the effect of urea, a recognized small MW uremic toxin present at increased concentrations in the uremic sera of HD patients on normal MoDC functions was assessed. High concentration of urea inhibits normal MoDC function. Increasing concentrations of urea were added to CM containing normal monocytes in levels equivalent to CRF patients. However, only normal monocytes cultured in CM supplemented with 40 mmol/l of urea inhibited monocyte differentiation into immature MoDC and exhibited reduced expression of costimulatory molecules and CD83 on LPS-stimulated MoDC. Monocytes cultured in CM alone or CM supplemented with lower concentrations of urea (5 and 20 mmol/l) displayed appropriate monocyte differentiation into immature MoDC and subsequent maturation following LPS stimulation (Figure 3a and Table 3). Normal immature MoDC cultured in CM containing 40 mmol/l urea demonstrated reduced FITC-dextran uptake (MFI 39.6±15.2) and expression of CD206 (MFI 8.8±3.2) when compared to immature MoDC cultured in CM alone (MFI 68.5±10.2 and 40.5±6.2, respectively, representative of six separate experiments; Mann–Whitney U-test, P<0.05). Immature MoDC cultured in CM containing lower concentrations of urea exhibited similar endocytic capacity (FITC-dextran uptake) and CD206 expression as cells cultured in CM alone (representative of six experiments, data not shown).Table 3Costimulatory molecule expression and viability of normal monocyte-derived dendritic cells in complete and uremic (sera of peritoneal dialysis and chronic renal failure patients) mediumsMoDCCD40 MFI (mean±s.d.)CD80 MFI (mean±s.d.)CD83 MFI (mean±s.d.)CD86 MFI (mean±s.d.)*Apoptotic/necrotic cells (% total cells)Normal MoDC in CM104.2±54.37.5±3.63.7±2.823.1±4.336.4±12.2Normal MoDC in UM (PD)111.5±24.63.2±2.60.8±0.511.1±4.754.5±15.2Normal MoDC in UM (CRF)110.2±64.34.3±2.71.4±0.915.2±7.250.1±23.2CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells; s.d., standard deviation; UM (CRF), uremic medium containing sera of chronic renal failure patients; UM (PD), uremic medium containing sera of peritoneal dialysis patients.*P<0.05 Kruskal–Wallis test. Open table in a new tab CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells; s.d., standard deviation; UM (CRF), uremic medium containing sera of chronic renal failure patients; UM (PD), uremic medium containing sera of peritoneal dialysis patients. *P<0.05 Kruskal–Wallis test. A greater proportion of LPS-stimulated normal MoDC cultured in CM and 40 mmol/l urea were apoptotic and/or necrotic when compared to cells cultured in CM alone. LPS-stimulated MoDC cultured in CM containing lower concentrations of urea had similar percentage of apoptotic and/or necrotic cells as MoDC cultured in CM alone (representative of three experiments, P=NS; Table 3). CD95 expression on LPS-stimulated normal MoDC was not affected by the presence of any concentrations of urea in CM (representative of six experiments, data not shown). Increasing concentrations of urea imparted a dose-dependent inhibition in the ability of LPS-stimulated normal MoDC to produce IL-12p70 detected by enzyme-linked immunosorbent assay (ELISA). LPS-stimulated MoDC cultured in CM alone produced 35.7±4.6 pg/ml of IL-12p70, whereas MoDC cultured in CM containing 5 mmol/l (28.4±6.9 pg/ml), 20 mmol/l (26.1±4.9 pg/ml), and 40 mmol/l (below the level of detection) of urea produced less IL-12p70 (representative of eight separate experiments; Kruskal–Wallis test, P<0.05). There was no detectable IL-12p70 in CM±urea. However, only LPS-stimulated normal MoDC cultured in CM and 40 mmol/l urea significantly inhibited allogeneic T-cell proliferation compared to MoDC cultured in the absence/lower concentrations of urea in CM (Figure 3b; Kruskal–Wallis test, P=0.1). Uremic sera of CAPD and CRF patients also affect MoDC function and survival. In order to investigate the impact of uremia and dialysis modality on MoDC functions, the effect of the sera of CAPD and CRF patients on normal MoDC functions was assessed. Compared to normal immature MoDC in CM, normal immature MoDC cultured in UM containing either sera of CAPD and CRF patients demonstrated similar capacity for FITC-dextran uptake (sera of three separate CAPD and CRF patients used, MFI 113.2±15.8, 145.6±89.2, and 123.6±10.5, respectively; P=NS). LPS-stimulated MoDC derived from normal monocytes cultured in UM (containing either sera of CAPD and CRF patients) demonstrated slightly reduced cell-surface expression of CD80 and CD86, but not CD40 and CD83. The differentiation of MoDC from monocytes was not affected by the presence of uremic serum in cell culture (Supplementary Figures, Legend to Supplementary Figures and Table 4).Table 4Costimulatory molecule expression and viability of normal monocyte-derived dendritic cells in complete medium±urea solutionMoDCCD40 MFI (mean±s.d.)CD80 MFI (mean±s.d.)CD83 MFI (mean±s.d.)CD86 MFI (mean±s.d.)Apoptotic/necrotic cells (% total cells)Normal MoDC in CM*147.2±33.815.6±5.27.8±2.126.9±8.738.5±10.5Normal MoDC in CM+urea 5 mmol/l160.2±44.514.5±4.38.6±3.224.3±7.742.9±12.2Normal MoDC in CM+urea 20 mmol/l149.3±41.215.3±4.99.0±2.720.1±11.140.8±15.5Normal MoDC in CM+urea 40 mmol/l*99.0±15.67.3±3.33.9±2.111.1±4.456.6±20.2CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells.*P<0.05 Mann–Whitney U-test (except CD83). Open table in a new tab CM, complete medium; MFI, mean fluorescent intensity; MoDC, monocyte-derived dendritic cells. *P<0.05 Mann–Whitney U-test (except CD83). A greater proportion of normal LPS-stimulated MoDC cultured in UM compared to CM were apoptotic and/or necrotic (sera of three separate CAPD and CRF patients used, P=NS; Table 4). Normal LPS-stimulated MoDC cultured in UM produced a greater amount of IL-12p70 (12.5±4.7 pg/ml in UM containing sera of CAPD patients, and 155.5±25.9 pg/ml in UM containing sera of CRF patients) when compared to cells cultured in CM (undetectable; Kruskal–Wallis test, P<0.01). The amount of IL-12p70 present in CM and UM was both below the level of detection. Uremic MoDC of HD patients are functionally abnormal. Uremic monocytes from HD patients cultured in either CM or autologous UM were capable of differentiating into MoDC though there was a marginal increase in the number of undifferentiated monocytes (Figure 4a). Compared to normal immature MoDC cultured in CM, uremic immature MoDC cultured in CM and autologous UM exhibited reduced FITC-dextran uptake (50.8±6.7, 0.1±0.1, and 0.2±0.1, respectively, representative of three HD patients; Kruskal–Wallis test, P<0.01) and reduced expression of CD206 (108.5±15.8, 0.3±0.2, and 0.3±0.1, respectively, representative of three HD patients; Kruskal–Wallis test, P<0.0001). Uremic LPS-stimulated MoDC cultured in CM or autologous UM demonstrated reduced expression of costimulatory molecules and CD83 compared to normal MoDC cultured in CM (Figure 4a and Table 2). LPS-stimulated uremic MoDC cultured in autologous UM produced greater amount of IL-12p70 (34.2±10.2 pg/ml) than uremic MoDC cultured in CM (below the level of detection, representative of three HD patients; Student's t-test, P<0.01). The amount of IL-12p70 present in CM and UM was below the level of detection and 11.3±5.1 pg/ml, respectively (Student's t-test, P<0.01). As demonstrated with normal MoDC cultured in UM, uremic MoDC cultured in autologous UM exhibited greater allo-stimulatory capacity than uremic MoDC cultured in CM (Kruskal–Wallis test, P=0.001; Figure 4b). In this study, we demonstrated that uremic sera from HD patients inhibited normal monocytes and MoDC function. We established that the presence of uremic serum in cell culture prevented the upregulation of costimulatory molecule expression on monocytes (including CD40, CD80, and CD86) following LPS stimulation resulting in reduced allo-stimulatory capacity of monocytes in vitro. However, the presence of uremic serum did not considerably affect the differentiation of monocytes into MoDC in vitro but did affect the function of immature and mature normal MoDC post-differentiation. Immature MoDC generated in the presence of uremic serum displayed suboptimal capacity for endocytosis via macrophage mannose receptor pathway that may directly reflect a reduction of CD206 expression on the cell surface of these immature MoDC. Furthermore, the presence of uremic serum inhibited the maturation of MoDC demonstrated by lower expression of CD83 and costimulatory molecule expression despite LPS stimulation. However, despite the lack of phenotypic maturation displayed by LPS-stimulated MoDC cultured in UM, their allo-stimulatory capacity and ability to secrete IL-12p70 were paradoxically enhanced. A likely explanation for this unexpected finding is the presence of IL-12p70 in the uremic serum of HD patients. IL-12 is a proinflammatory cytokine that is an important regulator of Th1 response and is produced predominantly by monocytes and DC.23.Langrish C. McKenzie B. Wilson N. et al.IL-12 and IL-23: master regulators of innate and adaptive immunity.Immunol Rev. 2004; 202: 96-105Crossref PubMed Scopus (576) Google Scholar IL-12 is particularly efficient in the induction of interferon-γ by T cells, and both IL-12 and interferon-γ may provide a positive auto-regulatory feedback to T cells and DC further promoting interferon-γ and IL-12 production, respectively.24.Kang B. Kim E. Kim T. Regulatory mechanisms and their therapeutic implications of interleukin-12 production in immune cells.Cell Signal. 2005; 17: 665-673Crossref PubMed Scopus (40) Google Scholar Higher serum levels of IL-12 have been shown by others in the sera of chronic HD patients, and the overproduction of IL-12 has been associated with polarization of T-cell phenotype toward Th1 responses in these patients.25.Ishizuka T. Nitta K. Yokoyama T. et al.Increased serum levels of interleukin-12 may be associated with Th1 differentiation in hemodialysis patients.Nephron. 2002; 90: 503-504Crossref PubMed Scopus (10) Google Scholar,26.Sester U. Sester M. Hauk M. et al.T-cell activation follows Th1 rather than Th2 pattern in haemodialysis patients.Nephrol Dial Transplant. 2000; 15: 1217-1223Crossref PubMed Scopus (141) Google Scholar IL-12p70 present in uremic serum may stimulate MoDC via IL-12 receptor to enhance production of IL-12p70, which in turn augments T-cell activation and proliferation manifesting as enhanced by allo-stimulatory capacity of MoDC in the MoDC/T cell-mixed lymphocyte reaction (MLR) in our study. In addition, monocytes27.Heidenreich S. Schmidt M. Bachmann J. et al.Apoptosis of monocyte cultured from long-term hemodialysis patients.Kidney Int. 1996; 49: 792-799Abstract Full Text PDF PubMed Scopus (68) Google Scholar and T cells28.Meier P. Dayer E. Blanc E. et al.Early T-cell activation correlates with expression of apoptosis markers in patients with end-stage renal disease.J Am Soc Nephrol. 2002; 13: 204-212PubMed Google Scholar from uremic HD patients have also been demonstrated to undergo accelerated apoptosis. Consistent with these observations, our study established that uremic serum induces greater MoDC apoptosis and necrosis (and hence increased release of inflammatory mediators) that may directly result in enhanced T-cell proliferation induced by UM-cultured MoDC, further contributing to the immune dysfunction of HD patients. Unlike MoDC, LPS-stimulated monocytes cultured in either CM or UM did not produce IL-12 and may explain why MoDC but not monocytes cultured in UM exhibited enhanced allo-stimulatory capacity. The latter may perhaps reflect the lack of IL-12 receptor on monocytes29.Nagayama H. Sato K. Kawasaki H. et al.IL-12 responsiveness and expression of IL-12 receptor in human peripheral blood monocyte-derived dendritic cells.J Immunol. 2000; 165: 59-66Crossref PubMed Scopus (80) Google Scholar essential for positive auto-regulatory feedback and subsequent experiments are ongoing in our laboratory to determine the extent of IL-12 receptor expression on monocytes and MoDC (including in response to uremic serum) and whether the inhibition of IL-12 receptor on UM-cultured MoDC could inhibit their enhanced allo-stimulatory capacity and the ability to produce IL-12. Furthermore, neutralizing IL-12 activity in culture medium using a blocking monoclonal antibody may also help to distinguish which of the two factors (i.e. IL-12 or cell apoptosis) is predominantly responsible for the amplified allo-stimulatory capacity of MoDC cultured in UM. Both low and high MW uremic toxins contained within the uremic serum or plasma of dialysis-dependent patients have been implicated in the observed dysfunction of the innate immune system including polymorphonuclear leukocytes30.Masuda M. Komiyama Y. Murakami T. et al.Decrease of polymorphonuclear leukocyte membrane fluidity in
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