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Characterization of Dendritic Cell Subsets in Patients Undergoing Renal Transplantation
Characterization of Dendritic Cell Subsets in Patients Undergoing Renal Transplantation J. Fangmann, C. Wegmann, A. Hoppe, P. Martin, U. Sack, J. Harms, S. Faber, and F. Emmrich
ABSTRACT Dendritic cells (DCs) play a key role in transplantation tolerance and immune reactions to transplants. In order to ascertain whether DC levels are predictive for rejection, we examined the levels and expression patterns of DCs of renal transplant patients following immunosuppressive and/or surgical interventions. Myeloid (HLA-DR⫹/CD11c⫹) and plasmacytoid (HLA-DR⫹/CD123⫹) DCs were characterized by flow cytometry over 28 days. We demonstrated that myeloid DCs and plasmacytoid DCs in peripheral blood were discernable and dramatically decreased following renal transplantation and immunosuppression. Furthermore, the expression of CD62L was significantly up-regulated (P ⫽ .032), while CD86 was significantly down-regulated (P ⫽ .008) on myeloid but not plasmacytoid DCs. Although DC levels alone were not predictive for the occurrence of a rejection episode, in combination with other factors they may be indicative of rejection, thereby sparing the patient a biopsy.
D
ENDRITIC CELLS (DCs), which serve as professional antigen-presenting cells (APC)1 for CD4⫹ and CD8⫹ T cells, play a key role in the initiation of specific immune responses2 via the direct or the indirect pathway of antigen recognition. DCs exist in both immature and mature phenotypes. Mediated by CD62L, they home to lymph nodes. Immature DCs take up, present, and process antigens but only have a weak ability to stimulate an immune response. They express low levels of MHC, CD80, and CD86. Mature DCs, which become mature through the actions of nuclear factor (NF)B, have decreased endocytotic activity and an increased expression of MHC, CD80, and CD86.3 Recent studies have shown 2 separate cell lines of human DCs4 –7: myeloid DCs (mDCs), which are derived from myeloid precursors and express CD13 and CD33; and plasmacytoid DCs (pDCs), which are characterized by a lack of myeloid cell markers and express the ␣-chain of the IL-3 receptor (CD123). In this study, mDCs and pDCs in renal transplant recipients were measured and characterized by flow cytometry over 28 days. It has been postulated that tolerogenic DCs are responsible for tolerance induction towards allografts. Mazariegos et al8 suggested that an increased pDC/mDC ratio indicates a tolerogenic condition among liver transplant recipients who no longer take immunosuppressive medications.
Since the manipulation of the immunological function of DCs has been used to try to induce allograft tolerance,8 we examined whether DCs were detectable in peripheral blood, and whether there was a change in the number and/or the expression pattern during the observation period under standard immunosuppressive therapies. Furthermore, we explored the influence of various parameters on DC levels, including induction therapy, surgical trauma, living versus deceased donation. and dialysis therapy in end-stage renal disease patients compared with healthy controls. Finally, we investigated how DCs react during rejection episodes, whether they were predictable, if rejection induces alterations in DC surface expression in cases of infection as opposed to rejection episodes, and whether there may be a clinical utility of DC monitoring. To this end, DCs from peripheral blood isolated from kidney transplant patients maintained under standard immunosuppressive therapy were analyzed for CD62L, CD80, and CD86 expression by flow cytometry.
From the Department of Surgery II, Clinic for Transplant Surgery, Leipzig, Germany. Address reprint requests to Josef Fangmann, MD, Clinic for Transplant Surgery, Liebigstr. 20, Leipzig, Sachsen 04103, Germany.
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.088
Transplantation Proceedings, 39, 3101–3104 (2007)
3101
3102
FANGMANN, WEGMANN, HOPPE ET AL
MATERIALS AND METHODS
Patient Statistics
Between November 2003 and November 2004, 2 types of DCs were examined in 4 patient groups: (1) 10 healthy volunteers; (2) 25 kidney transplant patients; (3) 5 dialysis patients with end-stage renal disease; and (4) 6 patients admitted for general surgery not requiring immunosuppression, namely, abdominal aorta aneurysm resection, inguinal or incisional hernia repair. Blood samples were analyzed using antibodies specific for mDCs (against HLA-DR⫹/ CD11c⫹) and pDCs (against HLA-DR⫹/CD123⫹). All samples were then further analyzed for the surface markers CD62L, CD80, and CD86. The healthy volunteers provided venous 2.7 mL EDTA monovette blood samples which were used to obtain normal percent values of DCs in blood as a basis of comparison with the other 3 groups. To gain further information regarding the possible influence of dialysis and surgical trauma on mDC levels, 2.7 mL EDTA monovette blood samples were taken from 5 patients with end-stage renal disease and 6 patients routinely operated on preoperative day 0 and on postoperative day 1. Blood samples from patients in all 4 groups were processed according to standard flow cytometric stainings (BD Bioscience, San Jose, Calif, United States). Control measurements to determine DCs levels were performed in a 4-color flow cytometer FACS Calibur (CellQuestPro Becton Dickinson) in 100,000 leukocytes. If the DCs reached values of at least 20/100,000 leukocytes, then subtyping for surface molecules took place on 250,000 leukocytes. Finally, the DCs were classified as lin-/HLA-DR⫹/CD11c⫹ (mDCs) and lin-/HLA-DR⫹/CD123⫹ (pDCs) cells. All results are given as average values ⫾ standard deviations.
The median patient age was 50.5 ⫾ 17.6 years. Six patients were females and 19, males. Among the 25 kidney allografts, 7 were from living donors and 18 from deceased donors. Patients displayed a variety of underlying diagnoses including glomerulonephritis, IgA nephropathy, diabetes mellitus, and hypertension. At the time of the study, all patients were administered immunosuppression with cyclosporine/tacrolimus, mycophenolate mofetil, and prednisolone. In 17 patients induction therapy with basiliximab or daclizumab was additionally administered. Complications included urinary tract infections, 3 HSV infections, 1 CMV infection, and 1 case of pneumonia. One patient exhibited histologically confirmed acute rejection and 3 others displayed signs of rejection which were not biopsyconfirmed. Graft loss occurred in 2 patients due to major vascular complications.
RESULTS Control Groups
Blood samples from healthy volunteers were used to determine the average percent and standard deviation of DCs in the total leukocyte population. Healthy volunteers showed upper limits between 171 and 349 mDC/100,000 leukocytes for mDCs and lower values between 103 and 226 pDC/ 100,000 leukocytes for the pDCs. Significant differences (P ⬍ .05) were derived with a Sigma plot t test. The average mDC level among patients with end-stage renal disease was 360 ⫾ 179 mDC/100,000 leukocytes, which was clearly above the upper limit of normal of 349 mDC/100,000 leukocytes. However, these values demonstrated no significant change compared with healthy volunteers. The level for pDCs among dialysis patients was on average 64 ⫾ 43 pDC/100,000 leukocytes, which was below the lower standard level range of 103 pDC/100,000 leukocytes (P ⫽ .006) and thus also lower than that of patients with healthy kidneys. Among operated patients, the average level of mDCs preoperatively was 322 ⫾ 165 mDC/100,000 leukocytes, which was within the normal value range. mDCs sank on postoperative day 1 (182 ⫾ 116 mDC/100,000 leukocytes); however, the decrease was not significant. For pDCs, the average value before the operation was 96 ⫾ 37 pDC/ 100,000 leukocytes. This level decreased significantly on postoperative day 1 to 25 ⫾ 29 pDC/100,000 leukocytes (P ⬍ .001).
mDC Levels in Kidney Transplant Recipients Dropped Sharply in the Postoperative Period
Prior to transplantation, mDCs in kidney transplant recipients showed levels which were on average far above those for the volunteers with healthy kidneys (day 0, 560 ⫾ 383 mDC/100,000 leukocytes; Fig 1). The decrease in mDCs from preoperative day 0 to postoperative day 1 was highly significant (P ⬍ .001); the level of 37 ⫾ 39 mDC/100,000 leukocytes was far below normal levels. On postoperative day 28, the levels (125 ⫾ 76 mDC/100,000 leukocytes) still did not reach the normal range. Therefore, the levels of mDCs at the end of the observation period amounted to about 22% of the initial level at day 0. The rise in mDCs between postoperative days 1 and 28 was significant (P ⬍ .001), as was the decrease in mDC levels between the preoperative and the postoperative day 28 values (P ⫽ .004). Altogether it can be said that after the dramatic decrease in mDCs immediately following kidney transplantation, the DC levels again rose slightly, but did not reach levels in the normal range.
Fig 1. Quantity of mDCs in kidney transplant recipients over time showed a dramatic decrease immediately after transplantation.
DC SUBSETS IN RENAL TRANSPLANT PATIENTS
3103
Costimulatory Molecules on mDCs in Kidney Transplant Recipients Decreased Over the Postoperative Period
The percent of CD86-positive mDCs in kidney transplant recipients decreased between preoperative day 0 (98% ⫾ 1%) and postoperative day 1 (93% ⫾ 6%; P ⫽ .036). The percentages of mDCs expressing other surface molecules (CD62L, CD80, and CD83) did not exhibit changes. If one looks at the expression density of the surface molecules on mDCs, from preoperative day 0 (473 ⫾ 331) to postoperative day 28 (976 ⫾ 821), there was a demonstrable (P ⫽ .032) up-regulation of adhesion molecules and of the “homing receptor” CD62L (Fig 2). No changes were demonstrated in the expression of the costimulatory molecules CD80 and CD83. The costimulatory molecule CD86 was down-regulated on mDCs over the course of 4 weeks (Fig 3). The values obtained from preoperative day 0 (306 ⫾ 143) to postoperative day 1 (169 ⫾ 56; P ⫽ .011) sunk and then remained steady up to 28 days after the operation (163 ⫾ 48). Altogether, the down-regulation from preoperative day 0 to postoperatively day 28 remained significant (P ⫽ .008).
Fig 3. Expression of CD86 on mDCs in kidney transplant recipients decreased over time.
Costimulatory Molecules on pDCs
The percentages of cells expressing CD62L, CD80, and CD86 remained unchanged. DISCUSSION
Like the mDCs, the pDCs demonstrated a dramatic decrease from the preoperative level which was in the normal range (day 0, 127 ⫾ 182 pDC/100,000 leukocytes) to postoperative day 1 (2 ⫾ 2 pDC/100,000 leukocytes; P ⬍ .001; Fig 4). During the 4-week observation period, pDCs in the peripheral blood were barely detected; however, they rose significantly (15 ⫾ 10 pDC/100,000 leukocytes; P ⬍ .001) between postoperative days 1 and 28. The eventual level was, however, still far below the appropriate normal level of 103 pDC/100,000 leukocytes, was, corresponding to approximately 12% of the initial preoperative level. The fall from preoperative day 0 to postoperative day 28 was also highly significant (P ⬍ .001).
With this study we demonstrated that mDCs and pDCs in peripheral blood are both discernable and dramatically decreased following renal transplantation and immunosuppression. Furthermore, the expression of CD62L was significantly (P ⫽ .032) up-regulated, while CD86 was significantly down-regulated (P ⫽ .008) on mDCs but not pDCs. They did not reach normal levels until the end of the 28-day observation period. The remarkable decline in the DC cell number was primarily visible during the high-dose glucocorticoid application9 in the perioperative phase. Hesselink et al10 demonstrated that the DC number dropped not only in kidney transplant recipients, but also strongly among kidney donors following donation, whereas the decrease was transient with normalized levels by the third month after donation. Womer et al11,12 showed that 4 months after successful kidney transplantation, when immunosuppression was being reduced, levels of DCs rose again to
Fig 2. Expression of CD62L on mDCs in kidney transplant recipients increased over time.
Fig 4. Quantity of pDCs in kidney transplant recipients over time showed a dramatic decrease immediately after transplantation.
pDC Levels in Kidney Transplant Recipients Decreased Following Kidney Transplantation
3104
preoperative values. Additional induction therapy with IL-2 receptor antibodies did not result in a change in DC levels. However, an inhibitory effect on the maturation and function of DCs has been shown for IgG in vitro13,14 and for an ATG (rabbit Ig) induction therapy. Surgical trauma alone as well as dialysis treatment led to significant decreases in pDCs. mDCs showed a moderate increase among dialysis patients, which contradicted the results seen in the study of Womer et al.12 Reductions in mDCs caused by both operation and dialysis were not significant; therefore, changes in the mDC level must have been caused by immunosuppression and/or the kidney transplantation.15 Furthermore, no differences in DC levels between living donor and deceased donor kidney transplantations were detected. In one patient with a biopsy-proven acute rejection episode, we demonstrated a decreased CD62L expression and increased CD86 expression. These changes were observed during the rejection episode, demonstrating the inability to use DC levels, as obtained in this study, as a simple predictive indicator. Neither infection nor rejection episodes had any consistent influence on DC levels in this study. However, to definitively confirm a relationship between changes in DC level and rejection or infections, we must examine increased group sizes with more closely spaced blood draws around the times of events. As a future diagnostic marker, DC levels in combination with the lack of infectious markers (eg, leukocytes, C-reactive protein, proof of infection), and changes in DC expression pattern (decrease of CD62L and increase of CD86) as well as the clinical values of the patient (eg, increase of serum creatinine) might yield a picture to diagnose a rejection episode sparing the patient a renal transplant biopsy. UNCITED REFERENCES
This section comprises references that occur in the reference list but not in the body of the text. Please position each reference in the text or delete it. Any reference not dealt with will be retained in this section:3,15
FANGMANN, WEGMANN, HOPPE ET AL
REFERENCES 1. Morelli AE, Thomson AW: Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol Rev 196:125, 2003 2. Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271, 1991 3. Lutz MB, Schuler G: Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23:445, 2002 4. Banchereau J, Briere F, Caux C, et al: Immunobiology of dendritic cells. Annu Rev Immunol 18:767, 2000 5. Moser M, Murphy KM: Dendritic cell regulation of TH1-TH2 development. Nat Immunol 1:199, 2000 6. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245, 1998 7. O’Doherty U, Peng M, Gezelter S, et al: Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 82:487, 1994 8. Mazariegos GV, Zahorchak AF, Reyes J, et al: Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant recipients. Am J Transplant 3:689, 2003 9. Suda T, Chida K, Matsuda H, et al: High-dose intravenous glucocorticoid therapy abrogates circulating dendritic cells. J Allergy Clin Immunol 112:1237, 2003 10. Hesselink DA, Vaessen LM, Hop WC, et al: The effects of renal transplantation on circulating dendritic cells. Clin Exp Immunol 140:384, 2005 11. Womer KL, Peng R, Patton PR, et al: The effects of renal transplantation on circulating precursor dendritic cells. Transplant Proc 37:3, 2005 12. Womer KL, Peng R, Patton PR, et al: The effects of renal transplantation on peripheral blood dendritic cells. Clin Transpl 19:659, 2005 13. Bayry J, Lacroix-Desmazes S, Delignal S, et al: Intravenous immunoglobulin abrogates dendritic cell differentiation induced by interferon-alpha present in serum from patients with systemic lupus erythematosus. Arthritis Rheum 48:3497, 2003 14. Bayry J, Lacroix-Desmazes S, Carbonneil C, et al: Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin. Blood 101:758, 2003 15. Hackstein H, Thomson AW: Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nat Rev Immunol 4:24, 2004
ABSTRACT Dendritic cells (DCs) play a key role in transplantation tolerance and immune reactions to transplants. In order to ascertain whether DC levels are predictive for rejection, we examined the levels and expression patterns of DCs of renal transplant patients following immunosuppressive and/or surgical interventions. Myeloid (HLA-DR⫹/CD11c⫹) and plasmacytoid (HLA-DR⫹/CD123⫹) DCs were characterized by flow cytometry over 28 days. We demonstrated that myeloid DCs and plasmacytoid DCs in peripheral blood were discernable and dramatically decreased following renal transplantation and immunosuppression. Furthermore, the expression of CD62L was significantly up-regulated (P ⫽ .032), while CD86 was significantly down-regulated (P ⫽ .008) on myeloid but not plasmacytoid DCs. Although DC levels alone were not predictive for the occurrence of a rejection episode, in combination with other factors they may be indicative of rejection, thereby sparing the patient a biopsy.
D
ENDRITIC CELLS (DCs), which serve as professional antigen-presenting cells (APC)1 for CD4⫹ and CD8⫹ T cells, play a key role in the initiation of specific immune responses2 via the direct or the indirect pathway of antigen recognition. DCs exist in both immature and mature phenotypes. Mediated by CD62L, they home to lymph nodes. Immature DCs take up, present, and process antigens but only have a weak ability to stimulate an immune response. They express low levels of MHC, CD80, and CD86. Mature DCs, which become mature through the actions of nuclear factor (NF)B, have decreased endocytotic activity and an increased expression of MHC, CD80, and CD86.3 Recent studies have shown 2 separate cell lines of human DCs4 –7: myeloid DCs (mDCs), which are derived from myeloid precursors and express CD13 and CD33; and plasmacytoid DCs (pDCs), which are characterized by a lack of myeloid cell markers and express the ␣-chain of the IL-3 receptor (CD123). In this study, mDCs and pDCs in renal transplant recipients were measured and characterized by flow cytometry over 28 days. It has been postulated that tolerogenic DCs are responsible for tolerance induction towards allografts. Mazariegos et al8 suggested that an increased pDC/mDC ratio indicates a tolerogenic condition among liver transplant recipients who no longer take immunosuppressive medications.
Since the manipulation of the immunological function of DCs has been used to try to induce allograft tolerance,8 we examined whether DCs were detectable in peripheral blood, and whether there was a change in the number and/or the expression pattern during the observation period under standard immunosuppressive therapies. Furthermore, we explored the influence of various parameters on DC levels, including induction therapy, surgical trauma, living versus deceased donation. and dialysis therapy in end-stage renal disease patients compared with healthy controls. Finally, we investigated how DCs react during rejection episodes, whether they were predictable, if rejection induces alterations in DC surface expression in cases of infection as opposed to rejection episodes, and whether there may be a clinical utility of DC monitoring. To this end, DCs from peripheral blood isolated from kidney transplant patients maintained under standard immunosuppressive therapy were analyzed for CD62L, CD80, and CD86 expression by flow cytometry.
From the Department of Surgery II, Clinic for Transplant Surgery, Leipzig, Germany. Address reprint requests to Josef Fangmann, MD, Clinic for Transplant Surgery, Liebigstr. 20, Leipzig, Sachsen 04103, Germany.
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.088
Transplantation Proceedings, 39, 3101–3104 (2007)
3101
3102
FANGMANN, WEGMANN, HOPPE ET AL
MATERIALS AND METHODS
Patient Statistics
Between November 2003 and November 2004, 2 types of DCs were examined in 4 patient groups: (1) 10 healthy volunteers; (2) 25 kidney transplant patients; (3) 5 dialysis patients with end-stage renal disease; and (4) 6 patients admitted for general surgery not requiring immunosuppression, namely, abdominal aorta aneurysm resection, inguinal or incisional hernia repair. Blood samples were analyzed using antibodies specific for mDCs (against HLA-DR⫹/ CD11c⫹) and pDCs (against HLA-DR⫹/CD123⫹). All samples were then further analyzed for the surface markers CD62L, CD80, and CD86. The healthy volunteers provided venous 2.7 mL EDTA monovette blood samples which were used to obtain normal percent values of DCs in blood as a basis of comparison with the other 3 groups. To gain further information regarding the possible influence of dialysis and surgical trauma on mDC levels, 2.7 mL EDTA monovette blood samples were taken from 5 patients with end-stage renal disease and 6 patients routinely operated on preoperative day 0 and on postoperative day 1. Blood samples from patients in all 4 groups were processed according to standard flow cytometric stainings (BD Bioscience, San Jose, Calif, United States). Control measurements to determine DCs levels were performed in a 4-color flow cytometer FACS Calibur (CellQuestPro Becton Dickinson) in 100,000 leukocytes. If the DCs reached values of at least 20/100,000 leukocytes, then subtyping for surface molecules took place on 250,000 leukocytes. Finally, the DCs were classified as lin-/HLA-DR⫹/CD11c⫹ (mDCs) and lin-/HLA-DR⫹/CD123⫹ (pDCs) cells. All results are given as average values ⫾ standard deviations.
The median patient age was 50.5 ⫾ 17.6 years. Six patients were females and 19, males. Among the 25 kidney allografts, 7 were from living donors and 18 from deceased donors. Patients displayed a variety of underlying diagnoses including glomerulonephritis, IgA nephropathy, diabetes mellitus, and hypertension. At the time of the study, all patients were administered immunosuppression with cyclosporine/tacrolimus, mycophenolate mofetil, and prednisolone. In 17 patients induction therapy with basiliximab or daclizumab was additionally administered. Complications included urinary tract infections, 3 HSV infections, 1 CMV infection, and 1 case of pneumonia. One patient exhibited histologically confirmed acute rejection and 3 others displayed signs of rejection which were not biopsyconfirmed. Graft loss occurred in 2 patients due to major vascular complications.
RESULTS Control Groups
Blood samples from healthy volunteers were used to determine the average percent and standard deviation of DCs in the total leukocyte population. Healthy volunteers showed upper limits between 171 and 349 mDC/100,000 leukocytes for mDCs and lower values between 103 and 226 pDC/ 100,000 leukocytes for the pDCs. Significant differences (P ⬍ .05) were derived with a Sigma plot t test. The average mDC level among patients with end-stage renal disease was 360 ⫾ 179 mDC/100,000 leukocytes, which was clearly above the upper limit of normal of 349 mDC/100,000 leukocytes. However, these values demonstrated no significant change compared with healthy volunteers. The level for pDCs among dialysis patients was on average 64 ⫾ 43 pDC/100,000 leukocytes, which was below the lower standard level range of 103 pDC/100,000 leukocytes (P ⫽ .006) and thus also lower than that of patients with healthy kidneys. Among operated patients, the average level of mDCs preoperatively was 322 ⫾ 165 mDC/100,000 leukocytes, which was within the normal value range. mDCs sank on postoperative day 1 (182 ⫾ 116 mDC/100,000 leukocytes); however, the decrease was not significant. For pDCs, the average value before the operation was 96 ⫾ 37 pDC/ 100,000 leukocytes. This level decreased significantly on postoperative day 1 to 25 ⫾ 29 pDC/100,000 leukocytes (P ⬍ .001).
mDC Levels in Kidney Transplant Recipients Dropped Sharply in the Postoperative Period
Prior to transplantation, mDCs in kidney transplant recipients showed levels which were on average far above those for the volunteers with healthy kidneys (day 0, 560 ⫾ 383 mDC/100,000 leukocytes; Fig 1). The decrease in mDCs from preoperative day 0 to postoperative day 1 was highly significant (P ⬍ .001); the level of 37 ⫾ 39 mDC/100,000 leukocytes was far below normal levels. On postoperative day 28, the levels (125 ⫾ 76 mDC/100,000 leukocytes) still did not reach the normal range. Therefore, the levels of mDCs at the end of the observation period amounted to about 22% of the initial level at day 0. The rise in mDCs between postoperative days 1 and 28 was significant (P ⬍ .001), as was the decrease in mDC levels between the preoperative and the postoperative day 28 values (P ⫽ .004). Altogether it can be said that after the dramatic decrease in mDCs immediately following kidney transplantation, the DC levels again rose slightly, but did not reach levels in the normal range.
Fig 1. Quantity of mDCs in kidney transplant recipients over time showed a dramatic decrease immediately after transplantation.
DC SUBSETS IN RENAL TRANSPLANT PATIENTS
3103
Costimulatory Molecules on mDCs in Kidney Transplant Recipients Decreased Over the Postoperative Period
The percent of CD86-positive mDCs in kidney transplant recipients decreased between preoperative day 0 (98% ⫾ 1%) and postoperative day 1 (93% ⫾ 6%; P ⫽ .036). The percentages of mDCs expressing other surface molecules (CD62L, CD80, and CD83) did not exhibit changes. If one looks at the expression density of the surface molecules on mDCs, from preoperative day 0 (473 ⫾ 331) to postoperative day 28 (976 ⫾ 821), there was a demonstrable (P ⫽ .032) up-regulation of adhesion molecules and of the “homing receptor” CD62L (Fig 2). No changes were demonstrated in the expression of the costimulatory molecules CD80 and CD83. The costimulatory molecule CD86 was down-regulated on mDCs over the course of 4 weeks (Fig 3). The values obtained from preoperative day 0 (306 ⫾ 143) to postoperative day 1 (169 ⫾ 56; P ⫽ .011) sunk and then remained steady up to 28 days after the operation (163 ⫾ 48). Altogether, the down-regulation from preoperative day 0 to postoperatively day 28 remained significant (P ⫽ .008).
Fig 3. Expression of CD86 on mDCs in kidney transplant recipients decreased over time.
Costimulatory Molecules on pDCs
The percentages of cells expressing CD62L, CD80, and CD86 remained unchanged. DISCUSSION
Like the mDCs, the pDCs demonstrated a dramatic decrease from the preoperative level which was in the normal range (day 0, 127 ⫾ 182 pDC/100,000 leukocytes) to postoperative day 1 (2 ⫾ 2 pDC/100,000 leukocytes; P ⬍ .001; Fig 4). During the 4-week observation period, pDCs in the peripheral blood were barely detected; however, they rose significantly (15 ⫾ 10 pDC/100,000 leukocytes; P ⬍ .001) between postoperative days 1 and 28. The eventual level was, however, still far below the appropriate normal level of 103 pDC/100,000 leukocytes, was, corresponding to approximately 12% of the initial preoperative level. The fall from preoperative day 0 to postoperative day 28 was also highly significant (P ⬍ .001).
With this study we demonstrated that mDCs and pDCs in peripheral blood are both discernable and dramatically decreased following renal transplantation and immunosuppression. Furthermore, the expression of CD62L was significantly (P ⫽ .032) up-regulated, while CD86 was significantly down-regulated (P ⫽ .008) on mDCs but not pDCs. They did not reach normal levels until the end of the 28-day observation period. The remarkable decline in the DC cell number was primarily visible during the high-dose glucocorticoid application9 in the perioperative phase. Hesselink et al10 demonstrated that the DC number dropped not only in kidney transplant recipients, but also strongly among kidney donors following donation, whereas the decrease was transient with normalized levels by the third month after donation. Womer et al11,12 showed that 4 months after successful kidney transplantation, when immunosuppression was being reduced, levels of DCs rose again to
Fig 2. Expression of CD62L on mDCs in kidney transplant recipients increased over time.
Fig 4. Quantity of pDCs in kidney transplant recipients over time showed a dramatic decrease immediately after transplantation.
pDC Levels in Kidney Transplant Recipients Decreased Following Kidney Transplantation
3104
preoperative values. Additional induction therapy with IL-2 receptor antibodies did not result in a change in DC levels. However, an inhibitory effect on the maturation and function of DCs has been shown for IgG in vitro13,14 and for an ATG (rabbit Ig) induction therapy. Surgical trauma alone as well as dialysis treatment led to significant decreases in pDCs. mDCs showed a moderate increase among dialysis patients, which contradicted the results seen in the study of Womer et al.12 Reductions in mDCs caused by both operation and dialysis were not significant; therefore, changes in the mDC level must have been caused by immunosuppression and/or the kidney transplantation.15 Furthermore, no differences in DC levels between living donor and deceased donor kidney transplantations were detected. In one patient with a biopsy-proven acute rejection episode, we demonstrated a decreased CD62L expression and increased CD86 expression. These changes were observed during the rejection episode, demonstrating the inability to use DC levels, as obtained in this study, as a simple predictive indicator. Neither infection nor rejection episodes had any consistent influence on DC levels in this study. However, to definitively confirm a relationship between changes in DC level and rejection or infections, we must examine increased group sizes with more closely spaced blood draws around the times of events. As a future diagnostic marker, DC levels in combination with the lack of infectious markers (eg, leukocytes, C-reactive protein, proof of infection), and changes in DC expression pattern (decrease of CD62L and increase of CD86) as well as the clinical values of the patient (eg, increase of serum creatinine) might yield a picture to diagnose a rejection episode sparing the patient a renal transplant biopsy. UNCITED REFERENCES
This section comprises references that occur in the reference list but not in the body of the text. Please position each reference in the text or delete it. Any reference not dealt with will be retained in this section:3,15
FANGMANN, WEGMANN, HOPPE ET AL
REFERENCES 1. Morelli AE, Thomson AW: Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol Rev 196:125, 2003 2. Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271, 1991 3. Lutz MB, Schuler G: Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23:445, 2002 4. Banchereau J, Briere F, Caux C, et al: Immunobiology of dendritic cells. Annu Rev Immunol 18:767, 2000 5. Moser M, Murphy KM: Dendritic cell regulation of TH1-TH2 development. Nat Immunol 1:199, 2000 6. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245, 1998 7. O’Doherty U, Peng M, Gezelter S, et al: Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 82:487, 1994 8. Mazariegos GV, Zahorchak AF, Reyes J, et al: Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant recipients. Am J Transplant 3:689, 2003 9. Suda T, Chida K, Matsuda H, et al: High-dose intravenous glucocorticoid therapy abrogates circulating dendritic cells. J Allergy Clin Immunol 112:1237, 2003 10. Hesselink DA, Vaessen LM, Hop WC, et al: The effects of renal transplantation on circulating dendritic cells. Clin Exp Immunol 140:384, 2005 11. Womer KL, Peng R, Patton PR, et al: The effects of renal transplantation on circulating precursor dendritic cells. Transplant Proc 37:3, 2005 12. Womer KL, Peng R, Patton PR, et al: The effects of renal transplantation on peripheral blood dendritic cells. Clin Transpl 19:659, 2005 13. Bayry J, Lacroix-Desmazes S, Delignal S, et al: Intravenous immunoglobulin abrogates dendritic cell differentiation induced by interferon-alpha present in serum from patients with systemic lupus erythematosus. Arthritis Rheum 48:3497, 2003 14. Bayry J, Lacroix-Desmazes S, Carbonneil C, et al: Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin. Blood 101:758, 2003 15. Hackstein H, Thomson AW: Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nat Rev Immunol 4:24, 2004