Single Cell Phospho-Protein Analysis Demonstrates That Immunosuppressive Drugs Inhibit MAPK and mTOR Signaling Molecules in Primary Monocytes After Transplantation
Nephrology and Transplantation, Dept. of Internal Medicine, Erasmus MC, Rotterdam, Netherlands.
Meeting: 2015 American Transplant Congress
Abstract number: C6
Keywords: FACS analysis, FK506, Kidney transplantation, Mononuclear leukocytes
Session Information
Session Name: Poster Session C: Antigen Presenting Cells in Alloimmune Responses/B Cells and Antibody in Alloimmune Responses
Session Type: Poster Session
Date: Monday, May 4, 2015
Session Time: 5:30pm-6:30pm
Presentation Time: 5:30pm-6:30pm
Location: Exhibit Hall E
Monocytes have been identified as key players driving rejection processes. It is therefore surprising that there are almost no data available about the impact of immunosuppressive drugs on monocyte activation. Here, we studied the cell signaling state to obtain valuable information about signaling events and as a measure of how monocytes interact with immunosuppression.
By phospho-specific flowcytometry we measured the phosphorylation levels of the signaling molecules NF-κB, MAPK (p38, ERK) and mTOR (AKT) in peripheral blood samples of kidney transplant patients (N=14) taken in the first 6 months after transplantation. Both the in vivo phosphorylation levels and the phosphorylation capacity after PMA/ionomycin stimulation were determined in CD14+ monocytes. Patients received tacrolimus, mycophenolate mofetil and prednisone in combination with basiliximab induction therapy during the first 4 days.
Before transplantation the in vivo phosphorylation levels of p38MAPK, ERK and AKT but not of NF-κB are highly expressed by monocytes (MFI: 2258, 486, 1176 and 255 respectively) compared to isotype controls (MFI: 683, 185, 602 and 248 respectively; p<0.001 for p38MAPk, ERK and AKT). In addition, these monocytes have significantly higher p38MAPK phosphorylation levels than cells of healthy controls (p<0.05). After transplantation lower levels of these phosphorylated signaling molecules were measured (p<0.05 for p38MAPK, ERK and AKT) and reached the baseline level of the negative controls. Whole-blood stimulation with PMA/Ionomycin resulted in high phosphorylation levels of p38MAPK, ERK and AKT. In these stimulated samples, the phosphorylation levels inversely correlated with tacrolimus predose concentrations (p38MAPK: p<0.05; ERK: p<0.05; AKT: p<0.01) and with prednisolone dosages (p38MAPK: p<0.05; ERK: p=0.10; AKT: p<0.01). No correlation was found between phosphorylated p38MAPK, ERK and AKT levels and kidney function, i.e. serum creatinine or eGFR levels.
These data show that before transplantation, monocytes of patients are more activated than monocytes of healthy controls. The decreased phosphorylation levels of the MAPK (p38, ERK) and mTOR (AKT) after transplantation demonstrate that current immunosuppression protocols adequately suppress early monocyte activation.
To cite this abstract in AMA style:
Kannegieter N, Hesselink D, Kraaijeveld R, Graav Gde, Betjes M, Weimar W, Baan C. Single Cell Phospho-Protein Analysis Demonstrates That Immunosuppressive Drugs Inhibit MAPK and mTOR Signaling Molecules in Primary Monocytes After Transplantation [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/single-cell-phospho-protein-analysis-demonstrates-that-immunosuppressive-drugs-inhibit-mapk-and-mtor-signaling-molecules-in-primary-monocytes-after-transplantation/. Accessed May 16, 2025.« Back to 2015 American Transplant Congress