Cli. 8iochem, Vol. 24, pp. 75-80, 1991
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Printed in Canada. All rights reserved.
Immunological Monitoring in Cyclosporine-Treated Patients RACHEL M. McKENNA 1 and TIMOTHY J. SCHROEDER 2 1Transplant Program, Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada and ~'Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH, USA The immunosuppressive action of cyclosporine (CsA) in vivo is thought to primarily involve its inhibitory effect on lymphokine production by T lymphocytes. Most efforts to assess immunosuppression in CsA-treated patients have concentrated on measuring some aspect of activated T cell function. These have included monitoring of lymphocyte subsets and the appearance of activated T cell markers, assaying the production of lymphokines and the direct measurement of lymphokines in serum and urine, and most recently measurement of soluble Interleukin-2 receptor (SlL2R). Using a new microparticle enzyme immunoassay (MEIA) in a preliminary study of 12 CsA-treated renal transplant recipients, we found significant increases in serum SIL2R levels in patients with rejection and we conclude that MEIA may have some use in the monitoring of CsA-treated patients.
KEY WORDS: cyclosporine; immunological monitoring; Interleukin-2 receptors; T lymphocyte subsets; lymphokines; neopterin; transplantation.
Introduction C
yclosporine (CsA) is the major immunosuppressive drug in solid organ transplantation in North America and Europe at the present time. Drug dosing is empirical and drug levels can be measured by a variety of assays (1). There are currently no standard means of assessing the immunosuppressive action of CsA in vivo other than changes in graft function, and it can be difficult to distinguish the common causes of graft dysfunction: rejection, nephrotoxicity, and infection. This is especially so in renal transplantation where an increase in serum creatinine can signal rejection or CsA nephrotoxicity.
Immunosuppressive action of CsA Experimental studies have shown that a major
Correspondence: Dr. Rachel McKenna, Transplant Immunology Laboratory, GG549 Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba R3A 1R9, Canada. Manuscript received May 28, 1990; revised July 26, 1990; accepted August 13, 1990. CLINICALBIOCHEMISTRY,VOLUME 24, FEBRUARY1991
effect of CsA on the immune system in vitro is its ability to inhibit lymphokine production by T lymphocytes (2,3). CsA has been shown in vitro to inhibit production of Interleukin-2 (IL-2) (4,5), IL-3 (6), IL-4 (7) and Interferon-gamma (IFN-g) (8) at the transcriptional level. Lymphokines can be considered as the hormones of the immune system, and are crucial to many of the differentiated functions of immune cells, including B lymphocytes and monocytes as well as T cells. There is evidence that lymphokines such as IL-2, IFN-g, lymphotoxin and tumor necrosis factor are involved in graft rejection and autoimmunity probably by promoting the recruitment, expansion, and activation of appropriate effector cells. It is therefore reasonable to assume that the beneficial effect of CsA in the treatment of allograft rejection and autoimmunity is related to its inhibition of lymphokine production. In the methods reviewed below that have been used to assess immunological status in CsA-treated patients, all measure some aspect of T cell function, as this is the cell considered to be most directly affected by the drug. L y m p h o c y t e subset m o n i t o r i n g Monoclonal antibodies to cell surface glycoproteins on T lymphocytes can distinguish between different subpopulations. Using the technique of flow cytometry, correlations between changes in the percent, absolute number, and ratios of T cell subsets and clinical events have been sought. Some reports suggested that a decrease in the ratio of Cluster Designation (CD) 4 (helper T cells) to CD8 (suppressor/cytotoxic T cells) was an indication of good immunosuppression, while an increase in the CD4:CD8 ratio towards normal values was associated with rejection (9,10). Other studies failed to find correlations between the percent or ratio of CD4 and CD8 T cell subsets and clinical events (11-13). Another approach has been to look for changes in the percent of activated T cells. It is assumed that T cells that have encountered antigen (i.e., the allograft) will now express T cell activation markers 75
McKENNA AND SCHROEDER
TABLE 1 Soluble Interleukin-2 Receptor Levels in Renal Transplant Recipients .SIL2R2 Mean -- SD
Group
n
Status
Normals Hemodialysis patients Transplant patients 1 " " "
20
--
8
--
2691
-
12 17 16 12 18 15
Pre-transplant Stable transplant 7 Days pre-R 5 2-3 Days pre-R Diagnosis R Post-R
3192 1604 2434 2529 3315 2819
-- 929 s'4 -- 415 -+ 12463.4 -+ 12533,4 ± 18713"4 ± 19663'4
"
"
877 --- 311 4283'4
Range 572-1572 2201--3675 1741-4866 927-2558 907-5815 1227-5613 1327-7937 1054-6470
1All patients were receiving CsA and Prednisone for maintenance immunosuppression. 2SIL2R was measured using the MEIA technique developed by Abbot Diagnostics, expressed as arbitrary unita/mL of serum. ~p < 0.01 versus normals. 4p < 0.01 versus stable transplants. 5R = Rejection, diagnosed by a rise in serum creatinine concentration that responded to treatment. such as HLA Class II antigens and receptors for IL-2. Increases in the percent of HLA-DR positive and IL-2 receptor positive T cells have been reported in both the periphery and the grafts of CsA-treated renal, heart and liver allograft recipients during episodes of rejection (14-18).
Lymphokine production in CsA-treated patients As CsA has a major inhibitory effect on lymphokine production in vitro, several investigators have attempted to monitor immunosuppression in CsAtreated patients by looking at the ability of peripheral blood mononuclear cells from such patients to produce lymphokines. Mitogen-induced IL-2 production was found to be significantly reduced compared to controls in stable CsA-treated renal allograft recipients for up to one year post-transplant, at which time it returned to normal values (19,20). Yoshimura and K a h a n (21) found t h a t increased IL-2 production in renal transplant recipients correlated with rejection episodes. In contrast, in a serial study of eight renal transplant recipients where IL-2 production was measured every 2-3 days posttransplant, we failed to find any correlation between increases in IL-2 production and rejection episodes (22). In a further group of patients in whom IL-2 production was measured at the time of diagnosis of rejection and after t r e a t m e n t for rejection, IL-2 production decreased with a return to stable graft function only in some patients (22). There have been several reports of decreased IFN-g production by peripheral blood lymphocytes of CsA-treated patients with stable graft function,
76
and in these studies correlations with rejection were not sought (19,23). We found no association between IFN-g or lymphotoxin production and rejection in a study of renal allograft recipients (22), and we concluded from these studies and the IL-2 results cited above t h a t the usefulness of monitoring lymphokine production in CsA-treated patients for the diagnosis of allograft rejection was at best questionable.
Lymphokine levels in CsA-treated patients The direct assay of circulating cytokines might be a more useful measure of immune events in these patients, since assays of lymphokine production require removal of the cells from the source of immunosuppression, and manipulation in vitro, which may not accurately reflect in vivo events. With the availability of monoclonal antibodies to IL-2, there have been several reports on the use of monitoring plasma and urine IL-2 levels to assess graft function. Simpson et a/.(24) found t h a t plasma and urine IL-2 were almost undetectable in normal subjects and stable renal graft recipients, but increased with rejection and infection although not with CsA toxicity. Georgi et al. (25) also found a good correlation between clinical diagnosis of rejection and increases in plasma and u r i n a r y IL-2 levels in pancreas allograft recipients. Another candidate m a r k e r of rejection in the serum and urine of transplanted patients is neopterin, a pyrazine-pyrimidine compound derived from GTP. It is released from monocytes and macrophages t h a t have been stimulated by IFN-g. This, in turn, is released from T cells stimulated by a vari-
CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
I M M U N O L O G I C A L M O N I T O R I N G IN CsA-TREATED PATIENTS
& &
I
O
m &
X
3.0
&
@
•
t/] _J bJ
&
2.0
M
fir t'N
I
8
1.0
_J
-,
NORMALS
DIALYSIS
(n =
(n-- 8)
20)
PRE-TX
(n-- 12)
STABLE-I'X
(n =
17)
Figure 1--SIL2R values in normal subjects and renal transplant recipients. Results are expressed in arbitrary SIL2R units/mL and were assayed in serum using the MEIA technique. Pre-Tx: Pre-transplant. Stable-Tx: Stable transplant. *p < 0.01 vs. normals. ety of immune stimuli including alloantigens (26,27), and neopterin can therefore be considered an indirect measure of T cell function. Both serum and urine neopterin, which can be measured by RIA or HPLC, are increased in CsA-treated renal, heart and liver transplant recipients during episodes of rejection (28-33). However, neopterin levels are also increased during infection, and in some reports with kidney transplant recipients, high levels of neopterin have been found with renal graft dysfunction not associated with rejection (34-36). Thus, the specificity of this marker for diagnosing graft rejection m a y be poor.
S o l u b l e interleukin-2 r e c e p t o r levels in CsA-treated patients The interaction of T cells with an antigen for which they have a specific receptor results in de novo expression of genes regulating IL-2 and IL-2 receptors. The subsequent binding of IL-2 to its high-affinity receptor leads to cell division and the clonal expansion of the original antigen-specific T cells (37). The IL-2 receptor (IL-2R) is composed of two chains, alpha (55 kDa) and beta (75 kDa) which, when associated together on the cell surface, result in a high-affinity receptor for IL-2 which
8.0
O
I O
~-
6.0
X
_J L~
4.0
_J
I
_.1
2.O
STABLE
REJECTION
(N=18) Figure 2--SIL2R values in 18 episodes of rejection in 10 patients. SIL2R levels shown are prior to rejection when graft function was stable and at the time of diagnosis of rejection. CLINICAL BIOCHEMISTRY,VOLUME24, FEBRUARY1991
77
McKENNA AND SCHROEDER mediates IL-2-induced proliferation (38,39). It has been shown in vitro that stimulation of T cells by mitogen, antigen, or alloantigen leads to release of a soluble form of the receptor SIL2R into the culture supernatants (40,41). Increased levels of SIL2R have been detected in the serum of patients with active autoimmune disease (42), and high levels of SIL2R m a y therefore be considered as a marker of immune cell activation. Controversy exists as to whether CsA directly inhibits the expression of IL-2R on activated T lymphocytes or whether down-regulation of IL-2 results in reduced IL-2R expression (43-45), i.e., an indirect effect of the drug. Several studies have evaluated the use of SIL-2R levels in monitoring CsA-treated transplant recipients for rejection. Increases in SIL2R levels have been found in the serum, plasma and urine of renal transplant recipients with rejection and infection, as well as during treatment with OKT3, b u t not during CsA toxicity (46-48). One study suggested that urinary SIL2R levels may be more helpful in the diagnosis of rejection than plasma levels (46), and other studies have reported that monitoring changes in SIL2R levels are more useful than absolute values (48,49). A rise in the level of serum SIL2R has also been reported in heart transplant recipients with rejection; however, the specificity of this rise was poor as infections also caused an increase in SIL2R levels (50,51). In liver transplant recipients with rejection, increases in serum SIL2R levels have been detected as well as in infection, although increases found in the bile of these patients seem specific for rejection (52,53). A recent report that examined SIL2R levels in pancreatic duodenal allografts concluded that the value of SIL2R monitoring may be in indicating the best time for allograft biopsy (54). All of the studies above used a commercially available ELISA (T Cell Sciences, Cambridge, MA, USA). We recently undertook a study of SIL2R in renal transplant recipients using a new assay system to detect SIL2R. Serum SIL2R was measured using a semiautomated SIL2R assay under development by Abbott Laboratories (N. Chicago, IL, USA) which utilizes microparticle enzyme immunoassay technology (MEIA) and a rabbit antihuman IL-2R antibody, and which can be run on an automated analyser (IMx, Abbott Laboratories). The advantage of the assay is the fast turnaround time and its automation, which is in contrast to most of the methods discussed above which are very labour-intensive or require 24 h or more for a result. In a preliminary prospective study, we measured serum SIL2R levels in 12 renal transplant recipients treated with CsA and prednisone for maintenance immunosuppression. Samples were taken pre-transplant and every 2-3 days post-transplant for a month, and then once a week for 4 months. There were 18 episodes of rejection in 10 out of 12 patients, as diagnosed by a rise in serum creatine that responded to therapy.
78
Three of the 12 patients lost their grafts to rejection in the first month post-transplant. Serum SIL2R levels were significantly higher pre-transplant and in 8 hemodialysis patients compared to normal controls (p < 0.01, see Table 1 and Figure 1). Statistical analysis was performed using two-way ANOVA after log transformation of the data. SIL2R levels decreased post-transplant during periods of stable graft function b u t increased significantly at the time of diagnosis of rejection (Figure 2). To assess the predictive value of this assay, we analyzed SIL2R levels at 2-3 days and at 1 week prior to the diagnosis of rejection, and found a significant increase in the mean SIL2R at both time points compared to patients with stable graft function (Table 1). Using a change of 15% or greater as a cut-off, we found that SIL2R increased in 13 out of 17 episodes of rejection as defined by a rise in creatinine, and it decreased 13 out of 15 times when graft function returned to normal. Only one patient had cytomegalovirus infection, and SIL2R levels were very high in this patient during infection (>7000 units/mL). We conclude from this small study that monitoring of SIL2R using the MEIA m a y have some use in h u m a n renal transplantation, and further studies are underway to assess the specificity and sensitivity of this assay.
Acknowledgements We wish to acknowledge the help of Dr. John Jeffery for providing the clinical data on the patients in the SIL2R study, the excellent technical assistance of Ms. Denise Pochinco in gathering the samples and helping in the analysis of the data, and Dr. Karen Muth of Abbott Laboratories (N. Chicago, IL, USA) for providing the reagents for the SIL2R study. This work was supported in part by the PHT Thorlakson Foundation.
References 1. Vine, W. Bowers LD. Cyclosporine: structure, pharmacokinetics and therapeutic drug monitoring. Crit Rev Clin Lab Sci 1987; 25: 275-311. 2. Hess AND,Tutschka PJ, Pu A, Santos GW. Effect of cyclosporine A on human lymphocyte responses in vitro. IV. Production of T cell stimulatory growth factors and development of responsiveness to these growth factors in CsA treated primary MLR cultures. J Immunol 1982; 128: 360-7. 3. Granelli-Piperno A, Andrus L, Steinman RM. Lymphokine and nonlymphokine mRNA levels in stimulated human T cells: kinetics, mitogen requirements and effects of cyclospsorine A. J Exp Med 1986; 163: 922-37. 4. Elliot JF, Lin Y, Mizel SB, Bleackley RC, Harnish DG, Paetkau V. Induction of Interleukin-2 messenger RNA inhibited by cyclosporine A. Science 1984; 226: 1439-41.
CLINICAL BIOCHEMISTRY,VOLUME 24, FEBRUARY1991
I M M U N O L O G I C A L M O N I T O R I N G IN CsA-TREATED PATIENTS
5. Kronke M, Leonard WJ, Depper JM et al. Cyclosporine inhibits T cell growth factor gene expression at the level of mRNA transcription. Proc Natl Acad Sci USA 1984; 81: 5214-8. 6. Palacios R. Cyclosporine inhibits antigen and lectininduced but not constitutive production of Interleukin 3. Eur J Immunol 1985; 15: 204--6. 7. Shevack EM. The effect of cyclosporine A on the immune system. A n n u Rev Immunol 1985; 3: 397423. 8. Reem GH, Cook LA, Vilcek J. Gamma Interferon synthesis by human thymocytes and T lymphocytes inhibited by cyclosporine A. Science 1983; 221: 63-4. 9. Kerman RH, Van Buren CT, Payne W, Flechner S, Kahan BD. Monitoring of T cell subsets and immune events in renal allograft recipients. Transplant Proc 1983; 15: 1170-2. 10. Ellis TM, Berry CR, Mendez-Picon G, et al. Immunological monitoring of renal allograft recipients using monoclonal antibodies to human T lymphocyte subpopulations. Transplant 1982; 33: 317-9. 11. Shen SY, Weir MR, Kosenko A, et al. Re-evaluation of T cell subset monitoring in cyclosporine-treated renal allograft recipients. Transplantation 1985; 40: 620--3. 12. Guttman RD, Poulsen RS. Fluorescence activated cell sorter analysis of lymphocyte subpopulations after renal transplantation. TransplantProc 1983; 15:11602. 13. Fiche M, Soulillou JP, Bignon JD, Bitlaudel S, Guenel J. T Lymphocyte monitoring in kidney transplant recipients undergoing cytomegalovirus infection or rejection episodes. Transplantation 1984; 37: 421-3. 14. Henny, FC, Weening JJ, Baldwin WM, Et al. Expression of HLA-Dr antigens on peripheral blood T lymphocytes and renal graft tubular epithelial cells in association with rejection. Transplantation 1986; 42: 479--83. 15. Thomas JM, Gross U, Cunningham P, Thomas FT. Two colour fluorescence activated flow cytometric analysis of T cells in fine needle aspirates for diagnosis of rejection. Transplant Proc 1989; 21: 3611-3. 16. Bishop GA, Waugh J, Hall BM et al. Diagnosis of renal allograft rejection by analysis of fine-needle aspiration biopsy specimens with immunostains and simple cytology. Lancet 1986; 2: 645-9. 17. Hammer C, Klanke D, Lersch C, et al. Cytoimmunologic Monitoring (CIM) for differentiation between cardiac rejection and viral, bacterial or hmgal infection: its specificity and sensitivity. Transplant Proc 1989; 21: 3631-3. 18. Winkler M, Schwinzer R, Wonigeit K. Patterns of MHC Class I antigen expression (HLA-DRf, DP and DQ) on peripheral blood T lymphocytes in patients after liver transplant. TransplantProc 1989; 21: 36278. 19. Dupont E, Huygen K, Schandene L, Vandercruys M, Palfliet K, Wybran J. Influence of in vivo immunosuppressive drugs on production of lymphokines. Transplantation 1985; 39: 143-6. 20. Guillou PJ, Giles GR, Ramsden CW. Natural killercell activity, Interferon-Alfa 2 production and Interleukin-2 production in cyclosporine-treated and conventionally immunosuppressed human allograft recipients. J Clin Immunol 1986; 6: 373-80. 21. Yoshimura N, Kahan BD. Pharmacodynamic assess-
CLINICAL BIOCHEMISTRY, V O L U M E 24, F E B R U A R Y 1991
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ment of the in vivo cyclosporine effect on Interleukin2 production by lymphocytes in kidney transplant recipients. Transplantation 1985; 40: 661--6. McKenna RM, Rush DN, Bakkestad-Legare P, Jeffery JR. Interleukin-2, interferon and lymphotoxin in renal transplant recipients. Transplantation 1988; 45: 76-81. Weimar W, Kramer P, Bijnen AB, Dekkers-Bijma AM, Westbroek DL, Jeekal J. Conversion from azathioprine to cyclosporine therapy enhances interferon production capacity. Transplant Proc 1985; 18: 118890. Simpson MA, Madras PN, Cornaby AJ, et al. Sequential determinations of urinary cytology and plasma and urinary lymphokines in the management of renal allograft recipients. Transplantation 1989; 47: 21823. Georgi BA, Dempsey RA, Corry RJ. Interleukin-2 assay in serum and urine as a means of monitoring pancreatic allograft rejection. Transplant Proc 21: 2784-5. Huber C, Fuchs D, Hausen A, Margretter R, Reibnegger G, Spielberger M, Wachter H. Pteridines as a new marker to detect human T cells activated by allogeneic or modified self major histocompatibility complex (MHC) determinants. J Immunol 1983; 130: 1047-50. Huber C, Batchelor JF, Fuchs D, et al. Immune response-associated production of neopterin. Release from macrophages primarily under control in Interferon-gamma. J Exp Med 1984; 160: 310-6. Woloszczuk W, Schwarz M, Havel M, Laczkovics A, Muller MM. Neopterin and Interferon gamma serum levels in patients with heart and kidney transplants. J Clin Chem Clin Biochem 1986; 24: 729-34. Woloszczuk W, Troppmair J, Leiter E, et al. Relationship of Interferon-gamma and neopterin levels during stimulation with alloantigens in vivo and in vitro. Transplantation 1986; 41: 716-9. Kovarik J, Woloszczuk W, Schwarz M, Pohanka E, Mayer G, Stummroll HK. Serum gamma interferon and neopterin in the immunologic monitoring of kidney transplant recipients. Transplant Proc 1986; 18: 1315-27. Aulitzky WE, Tilg H, Niederwieser D, et al. Comparison of serum neopterin levels and urinary neopterin excretion in renal allograft recipients. Clin Nephrol 1988; 29: 248--52. Tilg H, Vogel W, Aulitzky WE et al. Neopterin excretion after liver transplantation and its value in differential diagnosis ofcomplications. Transplantation 1989; 48: 594-9. Schafer AJ, Daniel V, Dreikorn K, Opelz G. Assessment of plasma neopterin in clinical kidney transplantation. Transplantation 1986; 41: 454-9. Giraud P, Tafani JAM, Calot M, Durand D, Bouissou F. Serum neopterin radioimmunoassay for kidney transplant monitoring: relationship with renal function and acute allograft rejection. Transplant Proc 1985; 17: 2518-20. Magalini SC, Sambo A, Agnes S, et al. Monitoring of kidney allograft rejection with urinary neopterin. Transplant Proc 1986; 118: 1063-6. Backman L, Ringden O, Bjorkhem I. Monitoring of serum neopterin levels in renal transplant recipients: increased values during impaired renal function and
79
McKENNA
AND SCHROEDER
cytomegalovirus infection. Nephron 1987; 46: 319-22. 37. Greene WC. The human interleukin-2 receptor. A n n u Rev Immunol 1986; 4: 69-95. 38. Weissman AM, Harfard JB, Svetlik PB, et al. Only high affinity receptors for Interleukin-2 mediate internalization ofligand. Proc Natl Acad Sci USA 1986; 83: 1463-6. 39. Warf HM, Smith KA. The Intorleukin-2 receptor. Functional consequences of its bimolecnlar structure. J Exp Med 1987; 166: 1055-69. 40. Rubin LA, Kurkman CC, Fritz ME, et al. Soluble Interleukin-2 receptors are released from activated human lymphoid cells in vitro. J Immunol 1985; 135: 3172-7. 41. Nelson DL, Rubin LA, Kruman CC, Fritz ME, Boutin B. An analysis of the cellular requirements for the production of soluble Interleukin-2 receptors in vitro. J Clin Immunol 1986; 6: 114-20. 42. Manoussakis MN, Papdopoulos GK, Drosos AA, Moutsopoulos HM. Soluble Interleukin-2 receptor molecules in the serum of patients with autoimmune diseases. Clin Immunol Immunopathol 1989; 50: 32132. 43. Reed JC, Abidi AH, Alpers JD, Hoover RG, Robb RJ, Nowel PC. Effect of cyclosporine A and dexamethasone on Interleukin-2 receptor gene expression. J Immunol 1986; 137: 150-4. 44. Solbach W, Heeg KD, Von Steldern DU, Rollinghoff M, Wagner H. On the partial suppression of IL-2 receptor expression and the prevention of lectin-induced lymphoblast formation by cyclosporine A. Clin Exp Immunol 1985; 60: 501-8. 45. Caillat-Zucman S, Chatenoud L, Bach JF. In vitro and in vivo action of cyclosporine A on the induction of human Interleukin-2 receptor and chains. Clin Exp Immunol 1989; 77: 184-90. 46. Louvin RB, Ruller TC, MacKeen L, Kung PC, Ip SH,
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47.
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Cosimi AB. Plasma Interleukin-2 receptor levels in renal allograft recipients. Clin Immunol Immunopathol 1987; 43: 273-6. Simpson MA, Madras PN, Cornaby AJ, et al. Sequential determinations of urinary cytology and plasma and urinary lymphokines in the managements of renal allograft recipients. Transplantation 1989; 47: 218-23. Smith AY, Citterio F, Wesh M, Kerman RH, Kahan BD. Interleukin-2 receptor as an immunodiagnostic tool to differentiate rejection from nephrotoxicity. Transplant Proc 1989; 21: 1462-4. Colvin RB, Preffer F, Fuller TC, et al. A critical analysis of serum and urine Interleukin-2 receptor assays in renal allograft recipients. Transplantation 1989; 48: 800-4. Southern JT, Fallon JT. A comparison of serum Interleukin-receptor levels and endomyocardial biopsy grades in the monitoring of cardiac allograft rejection. J Heart Transplant 1986; 5:370 (Abstr. 29). DeMaria R, Zuccelli GC, Masini S, et al. Non specific increase of Interleukin-2 receptor serum levels during immune events in heart transplantation. Transplant Proc 1989; 21: 440-1. Adams DH, Wang L, Hubscher SG, Elias E, Neuberger JM. Soluble Interleukin-2 receptors in the serum and bile of liver transplant recipients. Lancet 1989; 1: 469-71. Perkins JD, Nelson DL, Rakela J, Grambsch PM, Krom RAF. Soluble Interleukin-2 receptor level as an indicator of liver allograft rejection. Transplantation 1989; 47: 77-81. Perkins JD, Munn SR, Barr D, Ferguson DC, Carpenter H. Evidence that the soluble Interleukin-2 receptor level may determine the optimal time for cystoscopically-directed biopsy in pancreaticoduodenal allograft recipients. Transplantation 1990; 49: 363-6.
CLINICAL BIOCHEMISTRY,VOLUME 24, FEBRUARY 1991
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Printed in Canada. All rights reserved.
Immunological Monitoring in Cyclosporine-Treated Patients RACHEL M. McKENNA 1 and TIMOTHY J. SCHROEDER 2 1Transplant Program, Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada and ~'Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH, USA The immunosuppressive action of cyclosporine (CsA) in vivo is thought to primarily involve its inhibitory effect on lymphokine production by T lymphocytes. Most efforts to assess immunosuppression in CsA-treated patients have concentrated on measuring some aspect of activated T cell function. These have included monitoring of lymphocyte subsets and the appearance of activated T cell markers, assaying the production of lymphokines and the direct measurement of lymphokines in serum and urine, and most recently measurement of soluble Interleukin-2 receptor (SlL2R). Using a new microparticle enzyme immunoassay (MEIA) in a preliminary study of 12 CsA-treated renal transplant recipients, we found significant increases in serum SIL2R levels in patients with rejection and we conclude that MEIA may have some use in the monitoring of CsA-treated patients.
KEY WORDS: cyclosporine; immunological monitoring; Interleukin-2 receptors; T lymphocyte subsets; lymphokines; neopterin; transplantation.
Introduction C
yclosporine (CsA) is the major immunosuppressive drug in solid organ transplantation in North America and Europe at the present time. Drug dosing is empirical and drug levels can be measured by a variety of assays (1). There are currently no standard means of assessing the immunosuppressive action of CsA in vivo other than changes in graft function, and it can be difficult to distinguish the common causes of graft dysfunction: rejection, nephrotoxicity, and infection. This is especially so in renal transplantation where an increase in serum creatinine can signal rejection or CsA nephrotoxicity.
Immunosuppressive action of CsA Experimental studies have shown that a major
Correspondence: Dr. Rachel McKenna, Transplant Immunology Laboratory, GG549 Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba R3A 1R9, Canada. Manuscript received May 28, 1990; revised July 26, 1990; accepted August 13, 1990. CLINICALBIOCHEMISTRY,VOLUME 24, FEBRUARY1991
effect of CsA on the immune system in vitro is its ability to inhibit lymphokine production by T lymphocytes (2,3). CsA has been shown in vitro to inhibit production of Interleukin-2 (IL-2) (4,5), IL-3 (6), IL-4 (7) and Interferon-gamma (IFN-g) (8) at the transcriptional level. Lymphokines can be considered as the hormones of the immune system, and are crucial to many of the differentiated functions of immune cells, including B lymphocytes and monocytes as well as T cells. There is evidence that lymphokines such as IL-2, IFN-g, lymphotoxin and tumor necrosis factor are involved in graft rejection and autoimmunity probably by promoting the recruitment, expansion, and activation of appropriate effector cells. It is therefore reasonable to assume that the beneficial effect of CsA in the treatment of allograft rejection and autoimmunity is related to its inhibition of lymphokine production. In the methods reviewed below that have been used to assess immunological status in CsA-treated patients, all measure some aspect of T cell function, as this is the cell considered to be most directly affected by the drug. L y m p h o c y t e subset m o n i t o r i n g Monoclonal antibodies to cell surface glycoproteins on T lymphocytes can distinguish between different subpopulations. Using the technique of flow cytometry, correlations between changes in the percent, absolute number, and ratios of T cell subsets and clinical events have been sought. Some reports suggested that a decrease in the ratio of Cluster Designation (CD) 4 (helper T cells) to CD8 (suppressor/cytotoxic T cells) was an indication of good immunosuppression, while an increase in the CD4:CD8 ratio towards normal values was associated with rejection (9,10). Other studies failed to find correlations between the percent or ratio of CD4 and CD8 T cell subsets and clinical events (11-13). Another approach has been to look for changes in the percent of activated T cells. It is assumed that T cells that have encountered antigen (i.e., the allograft) will now express T cell activation markers 75
McKENNA AND SCHROEDER
TABLE 1 Soluble Interleukin-2 Receptor Levels in Renal Transplant Recipients .SIL2R2 Mean -- SD
Group
n
Status
Normals Hemodialysis patients Transplant patients 1 " " "
20
--
8
--
2691
-
12 17 16 12 18 15
Pre-transplant Stable transplant 7 Days pre-R 5 2-3 Days pre-R Diagnosis R Post-R
3192 1604 2434 2529 3315 2819
-- 929 s'4 -- 415 -+ 12463.4 -+ 12533,4 ± 18713"4 ± 19663'4
"
"
877 --- 311 4283'4
Range 572-1572 2201--3675 1741-4866 927-2558 907-5815 1227-5613 1327-7937 1054-6470
1All patients were receiving CsA and Prednisone for maintenance immunosuppression. 2SIL2R was measured using the MEIA technique developed by Abbot Diagnostics, expressed as arbitrary unita/mL of serum. ~p < 0.01 versus normals. 4p < 0.01 versus stable transplants. 5R = Rejection, diagnosed by a rise in serum creatinine concentration that responded to treatment. such as HLA Class II antigens and receptors for IL-2. Increases in the percent of HLA-DR positive and IL-2 receptor positive T cells have been reported in both the periphery and the grafts of CsA-treated renal, heart and liver allograft recipients during episodes of rejection (14-18).
Lymphokine production in CsA-treated patients As CsA has a major inhibitory effect on lymphokine production in vitro, several investigators have attempted to monitor immunosuppression in CsAtreated patients by looking at the ability of peripheral blood mononuclear cells from such patients to produce lymphokines. Mitogen-induced IL-2 production was found to be significantly reduced compared to controls in stable CsA-treated renal allograft recipients for up to one year post-transplant, at which time it returned to normal values (19,20). Yoshimura and K a h a n (21) found t h a t increased IL-2 production in renal transplant recipients correlated with rejection episodes. In contrast, in a serial study of eight renal transplant recipients where IL-2 production was measured every 2-3 days posttransplant, we failed to find any correlation between increases in IL-2 production and rejection episodes (22). In a further group of patients in whom IL-2 production was measured at the time of diagnosis of rejection and after t r e a t m e n t for rejection, IL-2 production decreased with a return to stable graft function only in some patients (22). There have been several reports of decreased IFN-g production by peripheral blood lymphocytes of CsA-treated patients with stable graft function,
76
and in these studies correlations with rejection were not sought (19,23). We found no association between IFN-g or lymphotoxin production and rejection in a study of renal allograft recipients (22), and we concluded from these studies and the IL-2 results cited above t h a t the usefulness of monitoring lymphokine production in CsA-treated patients for the diagnosis of allograft rejection was at best questionable.
Lymphokine levels in CsA-treated patients The direct assay of circulating cytokines might be a more useful measure of immune events in these patients, since assays of lymphokine production require removal of the cells from the source of immunosuppression, and manipulation in vitro, which may not accurately reflect in vivo events. With the availability of monoclonal antibodies to IL-2, there have been several reports on the use of monitoring plasma and urine IL-2 levels to assess graft function. Simpson et a/.(24) found t h a t plasma and urine IL-2 were almost undetectable in normal subjects and stable renal graft recipients, but increased with rejection and infection although not with CsA toxicity. Georgi et al. (25) also found a good correlation between clinical diagnosis of rejection and increases in plasma and u r i n a r y IL-2 levels in pancreas allograft recipients. Another candidate m a r k e r of rejection in the serum and urine of transplanted patients is neopterin, a pyrazine-pyrimidine compound derived from GTP. It is released from monocytes and macrophages t h a t have been stimulated by IFN-g. This, in turn, is released from T cells stimulated by a vari-
CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
I M M U N O L O G I C A L M O N I T O R I N G IN CsA-TREATED PATIENTS
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Figure 1--SIL2R values in normal subjects and renal transplant recipients. Results are expressed in arbitrary SIL2R units/mL and were assayed in serum using the MEIA technique. Pre-Tx: Pre-transplant. Stable-Tx: Stable transplant. *p < 0.01 vs. normals. ety of immune stimuli including alloantigens (26,27), and neopterin can therefore be considered an indirect measure of T cell function. Both serum and urine neopterin, which can be measured by RIA or HPLC, are increased in CsA-treated renal, heart and liver transplant recipients during episodes of rejection (28-33). However, neopterin levels are also increased during infection, and in some reports with kidney transplant recipients, high levels of neopterin have been found with renal graft dysfunction not associated with rejection (34-36). Thus, the specificity of this marker for diagnosing graft rejection m a y be poor.
S o l u b l e interleukin-2 r e c e p t o r levels in CsA-treated patients The interaction of T cells with an antigen for which they have a specific receptor results in de novo expression of genes regulating IL-2 and IL-2 receptors. The subsequent binding of IL-2 to its high-affinity receptor leads to cell division and the clonal expansion of the original antigen-specific T cells (37). The IL-2 receptor (IL-2R) is composed of two chains, alpha (55 kDa) and beta (75 kDa) which, when associated together on the cell surface, result in a high-affinity receptor for IL-2 which
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(N=18) Figure 2--SIL2R values in 18 episodes of rejection in 10 patients. SIL2R levels shown are prior to rejection when graft function was stable and at the time of diagnosis of rejection. CLINICAL BIOCHEMISTRY,VOLUME24, FEBRUARY1991
77
McKENNA AND SCHROEDER mediates IL-2-induced proliferation (38,39). It has been shown in vitro that stimulation of T cells by mitogen, antigen, or alloantigen leads to release of a soluble form of the receptor SIL2R into the culture supernatants (40,41). Increased levels of SIL2R have been detected in the serum of patients with active autoimmune disease (42), and high levels of SIL2R m a y therefore be considered as a marker of immune cell activation. Controversy exists as to whether CsA directly inhibits the expression of IL-2R on activated T lymphocytes or whether down-regulation of IL-2 results in reduced IL-2R expression (43-45), i.e., an indirect effect of the drug. Several studies have evaluated the use of SIL-2R levels in monitoring CsA-treated transplant recipients for rejection. Increases in SIL2R levels have been found in the serum, plasma and urine of renal transplant recipients with rejection and infection, as well as during treatment with OKT3, b u t not during CsA toxicity (46-48). One study suggested that urinary SIL2R levels may be more helpful in the diagnosis of rejection than plasma levels (46), and other studies have reported that monitoring changes in SIL2R levels are more useful than absolute values (48,49). A rise in the level of serum SIL2R has also been reported in heart transplant recipients with rejection; however, the specificity of this rise was poor as infections also caused an increase in SIL2R levels (50,51). In liver transplant recipients with rejection, increases in serum SIL2R levels have been detected as well as in infection, although increases found in the bile of these patients seem specific for rejection (52,53). A recent report that examined SIL2R levels in pancreatic duodenal allografts concluded that the value of SIL2R monitoring may be in indicating the best time for allograft biopsy (54). All of the studies above used a commercially available ELISA (T Cell Sciences, Cambridge, MA, USA). We recently undertook a study of SIL2R in renal transplant recipients using a new assay system to detect SIL2R. Serum SIL2R was measured using a semiautomated SIL2R assay under development by Abbott Laboratories (N. Chicago, IL, USA) which utilizes microparticle enzyme immunoassay technology (MEIA) and a rabbit antihuman IL-2R antibody, and which can be run on an automated analyser (IMx, Abbott Laboratories). The advantage of the assay is the fast turnaround time and its automation, which is in contrast to most of the methods discussed above which are very labour-intensive or require 24 h or more for a result. In a preliminary prospective study, we measured serum SIL2R levels in 12 renal transplant recipients treated with CsA and prednisone for maintenance immunosuppression. Samples were taken pre-transplant and every 2-3 days post-transplant for a month, and then once a week for 4 months. There were 18 episodes of rejection in 10 out of 12 patients, as diagnosed by a rise in serum creatine that responded to therapy.
78
Three of the 12 patients lost their grafts to rejection in the first month post-transplant. Serum SIL2R levels were significantly higher pre-transplant and in 8 hemodialysis patients compared to normal controls (p < 0.01, see Table 1 and Figure 1). Statistical analysis was performed using two-way ANOVA after log transformation of the data. SIL2R levels decreased post-transplant during periods of stable graft function b u t increased significantly at the time of diagnosis of rejection (Figure 2). To assess the predictive value of this assay, we analyzed SIL2R levels at 2-3 days and at 1 week prior to the diagnosis of rejection, and found a significant increase in the mean SIL2R at both time points compared to patients with stable graft function (Table 1). Using a change of 15% or greater as a cut-off, we found that SIL2R increased in 13 out of 17 episodes of rejection as defined by a rise in creatinine, and it decreased 13 out of 15 times when graft function returned to normal. Only one patient had cytomegalovirus infection, and SIL2R levels were very high in this patient during infection (>7000 units/mL). We conclude from this small study that monitoring of SIL2R using the MEIA m a y have some use in h u m a n renal transplantation, and further studies are underway to assess the specificity and sensitivity of this assay.
Acknowledgements We wish to acknowledge the help of Dr. John Jeffery for providing the clinical data on the patients in the SIL2R study, the excellent technical assistance of Ms. Denise Pochinco in gathering the samples and helping in the analysis of the data, and Dr. Karen Muth of Abbott Laboratories (N. Chicago, IL, USA) for providing the reagents for the SIL2R study. This work was supported in part by the PHT Thorlakson Foundation.
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