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Late acute rejection in renal allograft recipients is mediated by inflammatory rather than by cytotoxic T-cell-dependent mechanisms
ELSEVIER
Late Acute Rejection in Renal Allograft Recipients Is Mediated by Inflammatory Rather Than by Cytotoxic T-Cell-Dependent Mechanisms S. Ode-Hakim,
W.-D. DGcke, S. Mutze, H.-D. Volk, and P. Reinke
A
CUTE Rx episodes can occur in a functional renal allograft even at a very late posttransplantation stage. Despite the fact that histopathology in early and late acute Rx (aRx) looks quite similar (similar composition of infiltrates, although the infiltrates are sometimes smaller in late aRx),‘,* the typical clinical indicators of early aRx, ie, inflammatory signs, are not observed in late aRx, and the increase in serum creatinine is more protracted.‘m4 The distinct clinical course suggests qualitatively different immune mechanisms in the pathogenesis of early and late aRx. Cytokines are thought to play a central role as inflammatory and allospecific components of aRx. Several studies have shown that measuring cytokine mRNA in experimental and human allograft tissues has the potential to detect activation of intracellular events at a very early stage and with a very high degree of sensitivity and specificity.‘-’ Early aRx of renal allografts is associated with upregulation of several cytokines, including IL-2, IFN-y, TNF-(u, IL-lo, etc, suggesting the presence of DTH-like mechanisms. The involvement of CTL in early aRx is supported by intragraft detection of CTL-specific serine protease and perforin gene expression which was shown to be highly restricted to biopsies in this category.7.8 In the present study, we demonstrate the upregulation of several cytokine transcripts in biopsies of long-term renal allograft recipients suffering from histologically proven late aRx. The lack of upregulation of granzyme A (GrnA), a CTL-specific serine protease, suggests involvement of DTH-like, but not CTL, mechanisms in the pathogenesis of late aRx. PATIENTS
AND
triple-drug therapy. Additionally, kidney biopsies were taken from seven nontransplant patients with glomerulonephritis. Methods
Total RNA from core biopsies was extracted by a matrix method as recommended by the manufacturer (Dianova, Hamburg, FRG) and reverse-transcribed as explained elsewhere.’ Relative quantification of specific cDNAs using a competitive RT-PCR was carried out as described in detail elsewhere.“‘,” Transcripts of the following cytokines were measured: IL-lp, IL-2, IL-4, IL-S, IL-lo, IFN-7, and TNF-(u. Additionally, the expression of the two IL-2Rs (~55 and ~75). and the serine protease, granzyme A (GrnA), were studied. RESULTS
AND
DISCUSSION
Fig 1 shows the quantification of intragraft cytokine gene expression. The following groups were studied: late aRx (group I, n = 16), chronic Rx (group II, n = 5), no Rx (group III, it = 3), and glomerulonephritis (not transplanted) (group IV, n = 7). Biopsies from patients with late aRx or chronic Rx (groups I and II) showed higher levels of proinflammatory cytokine transcripts, such as IL-l& IL-S, and TNF-(w, compared with graft biopsies without histologic signs of Rx and nontransplant controls, although the differences did not reach statistical significance. Additionally, gene expression of the probably macrophage-derived cytokine IL-10 was significantly elevated in the two rejection groups (I and II). In contrast to the macrophage-derived cytokine transcripts, the significant upregulation of the T-cell-derived cytokine transcripts, IL-2, IFN-y, and IL-4, was more restricted to the aRx group (group I). IL-2 (at a cutoff level of 5 AU) allowed for the best discrimination between late
METHODS
Patients
Our study included 16 renal allograft recipients suffering from histologically proven late aRx between years 2 and 13 posttransplant. The mean age of the grafts was 4 years. All patients were on triple-drug maintenance immunosuppression therapy except two who had received azathioprine/prednisone only during the last weeks before Rx episode. Renal long-term allograft recipients with histologically proven signs of chronic rejection (n = 5), or without significant complications (n = 3), were used as controls. All controls were on 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
From the Institute of Medical Immunology, Department of Radiology, and Department of Nephrologyflransplantation, Charit& Humboldt-University, Berlin, Germany. Supported in part by Deutsche Forschungsgemeinschaft (vo 489/l-2). This article was presented orally at the XVlth International Congress of the Transplantation Society, Barcelona, Spain, August 1996. Address reprint requests to Dr Petra Reinke, Klinik fi.ir lnnere Medizin, Abt Nephrologie/lntensivmedizin, Virchowklinikum der Humboldt-Universittit zu Berlin, D-l 3353 Berlin, Germany.
0041-l 345f97/$17.00 PII SO041 -1345(96)00019-X
93
Transplantation
Proceedings,
29, 93-95
(1997)
ODE-HAKIM,
94
DOCKE, MUTZE ET AL
IFN-y
IL-2
:“--j-j
S’;E ll=16 I
n=3 II
II=15 IV
III
I
n=S
n=3
11
111
$-y----J
fz !iiI!I,L(
“=I4 I
n=3 II
111
Granzyme
Fig 1. Quantitative FIT-PCR from kidney biopsies. Group I: transplant recipients, late acute rejection (n = 16). Group II: transplant recipients, chronic rejection (n = 5). Group Ill: transplant recipients, no rejection (n = 3). Group IV: nontransplant recipients, glomerulonephritis (n = 7). The cytokine mRNA levels are expressed in absolute units (see “Patients and Methods”) as mean (-t-SEM).
IV
IL-10
IL-4
5
n=l
IL-2Rp55
aRx and the other groups, with a sensitivity of 83% (10 of 12) and a specificity of 93% (14 of 15). Another sign of T-cell activation in biopsies of late aRx was the significant upregulation (P < .05) of IL-2R ~55. The levels of intragraft IL-2R ~75 transcripts were elevated in all three transplant groups (Fig 1). Our data are in agreement with what was found in the early aRx of renal allografts, ie, upregulation of IFN-7, TNF-a, and IL-2 at the mRNA and protein level.‘-a This
A
II=5 IV
n=7
ll=5 I
11
Ill
N
TNF-ci
IL-2Rp75
suggests that cell-mediated inflammation (DTH reaction) may play an important role in aRx. However, we did not find significantly elevated GrnA levels in the biopsies taken during late aRx compared with the controls, although the mean values were about 1.5 to 2 times higher than in the control groups. Despite the expression of GrnA, mRNA was not absent in late aRx, and its involvement in the pathogenesis of this type of aRx is doubtful as similar levels of GrnA expression were found in
LATE ACUTE REJECTION
well-functioning grafts of long-term transplant recipients and in kidneys from patients with glomerulonephritis. In contrast to our observations in late aRx, Lipman et al emphasize the pathogenetic role of CTL, as they reported a strong upregulation of CTL-specific serine proteases and perforin transcripts in early aRx (>lOO-fold) that correlated much closer with aRx than those of several cytokine genes.7,x A plausible hypothesis explaining the different clinical courses of early and late aRx would be: DTH (CD4 T-cell-dependent) mechanisms are involved in the pathogenesis of both early and late aRx. The additional involvement of CTL activity in early aRx, but not late, aRx results in quicker graft injury in the former, reflected by the more rapid rise of serum creatinine. Explanations for the differences in pathogenesis may be the lower immunogenicity of adapted allografts and/or different pathways of alloantigen presentation. Direct allopresentation by donor-derived antigen-presenting cells, which is a very strong stimulator of T cells (in vitro model: mixed lymphocyte reaction), dominates during the early course posttransplantation. If most of the “passenger” leukocytes are dead, the indirect presentation of allopeptides by the recipient’s own antigen-present-
95
ing cells should be more important immunogenic than the former.
but it is actually less
REFERENCES
1. Reinke P, Fietze E, Dacke WD, et al: Transplantation 58:35, 1994 2. Mahoney JF, Sheil AGR: In Morris PJ, Tilney NL (eds): Transplantation Reviews. New York: Grune & Stratton, 1987, p 47 3. Rao KV, Kasiske BL, Bloom PM: Transplantation 47:290, 1989 4. Prieto C, Pulido F, Rodriguez-Paternina E, et al: Transplant Proc 24~35, 1992 5. Dallman MJ, Larsen CP, Morris PJ: J Exp Med 174:493, 1991 6. Morel D, Nordmand E, Lemoine C. et al: Transplantation 55773, 1993 7. Lipman ML, Stevens AC, Bleckley RC, et al: Transplantation 53~73, 1992 8. Lipman ML, Stevens AC, Strom TB: J Immunol 1.52:5120, 1994 9. Platzer C, Richter G, oberla K, et al: Eur J Immunol22:1179, 1992 10. Platzer C, Ode-Hakim S, Reinke P, et al: Transplantation 58:264, 1994 11. Ode-Hakim S, Diicke WD, Kern F, et al: Transplantation 61:1233, 1996
Late Acute Rejection in Renal Allograft Recipients Is Mediated by Inflammatory Rather Than by Cytotoxic T-Cell-Dependent Mechanisms S. Ode-Hakim,
W.-D. DGcke, S. Mutze, H.-D. Volk, and P. Reinke
A
CUTE Rx episodes can occur in a functional renal allograft even at a very late posttransplantation stage. Despite the fact that histopathology in early and late acute Rx (aRx) looks quite similar (similar composition of infiltrates, although the infiltrates are sometimes smaller in late aRx),‘,* the typical clinical indicators of early aRx, ie, inflammatory signs, are not observed in late aRx, and the increase in serum creatinine is more protracted.‘m4 The distinct clinical course suggests qualitatively different immune mechanisms in the pathogenesis of early and late aRx. Cytokines are thought to play a central role as inflammatory and allospecific components of aRx. Several studies have shown that measuring cytokine mRNA in experimental and human allograft tissues has the potential to detect activation of intracellular events at a very early stage and with a very high degree of sensitivity and specificity.‘-’ Early aRx of renal allografts is associated with upregulation of several cytokines, including IL-2, IFN-y, TNF-(u, IL-lo, etc, suggesting the presence of DTH-like mechanisms. The involvement of CTL in early aRx is supported by intragraft detection of CTL-specific serine protease and perforin gene expression which was shown to be highly restricted to biopsies in this category.7.8 In the present study, we demonstrate the upregulation of several cytokine transcripts in biopsies of long-term renal allograft recipients suffering from histologically proven late aRx. The lack of upregulation of granzyme A (GrnA), a CTL-specific serine protease, suggests involvement of DTH-like, but not CTL, mechanisms in the pathogenesis of late aRx. PATIENTS
AND
triple-drug therapy. Additionally, kidney biopsies were taken from seven nontransplant patients with glomerulonephritis. Methods
Total RNA from core biopsies was extracted by a matrix method as recommended by the manufacturer (Dianova, Hamburg, FRG) and reverse-transcribed as explained elsewhere.’ Relative quantification of specific cDNAs using a competitive RT-PCR was carried out as described in detail elsewhere.“‘,” Transcripts of the following cytokines were measured: IL-lp, IL-2, IL-4, IL-S, IL-lo, IFN-7, and TNF-(u. Additionally, the expression of the two IL-2Rs (~55 and ~75). and the serine protease, granzyme A (GrnA), were studied. RESULTS
AND
DISCUSSION
Fig 1 shows the quantification of intragraft cytokine gene expression. The following groups were studied: late aRx (group I, n = 16), chronic Rx (group II, n = 5), no Rx (group III, it = 3), and glomerulonephritis (not transplanted) (group IV, n = 7). Biopsies from patients with late aRx or chronic Rx (groups I and II) showed higher levels of proinflammatory cytokine transcripts, such as IL-l& IL-S, and TNF-(w, compared with graft biopsies without histologic signs of Rx and nontransplant controls, although the differences did not reach statistical significance. Additionally, gene expression of the probably macrophage-derived cytokine IL-10 was significantly elevated in the two rejection groups (I and II). In contrast to the macrophage-derived cytokine transcripts, the significant upregulation of the T-cell-derived cytokine transcripts, IL-2, IFN-y, and IL-4, was more restricted to the aRx group (group I). IL-2 (at a cutoff level of 5 AU) allowed for the best discrimination between late
METHODS
Patients
Our study included 16 renal allograft recipients suffering from histologically proven late aRx between years 2 and 13 posttransplant. The mean age of the grafts was 4 years. All patients were on triple-drug maintenance immunosuppression therapy except two who had received azathioprine/prednisone only during the last weeks before Rx episode. Renal long-term allograft recipients with histologically proven signs of chronic rejection (n = 5), or without significant complications (n = 3), were used as controls. All controls were on 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
From the Institute of Medical Immunology, Department of Radiology, and Department of Nephrologyflransplantation, Charit& Humboldt-University, Berlin, Germany. Supported in part by Deutsche Forschungsgemeinschaft (vo 489/l-2). This article was presented orally at the XVlth International Congress of the Transplantation Society, Barcelona, Spain, August 1996. Address reprint requests to Dr Petra Reinke, Klinik fi.ir lnnere Medizin, Abt Nephrologie/lntensivmedizin, Virchowklinikum der Humboldt-Universittit zu Berlin, D-l 3353 Berlin, Germany.
0041-l 345f97/$17.00 PII SO041 -1345(96)00019-X
93
Transplantation
Proceedings,
29, 93-95
(1997)
ODE-HAKIM,
94
DOCKE, MUTZE ET AL
IFN-y
IL-2
:“--j-j
S’;E ll=16 I
n=3 II
II=15 IV
III
I
n=S
n=3
11
111
$-y----J
fz !iiI!I,L(
“=I4 I
n=3 II
111
Granzyme
Fig 1. Quantitative FIT-PCR from kidney biopsies. Group I: transplant recipients, late acute rejection (n = 16). Group II: transplant recipients, chronic rejection (n = 5). Group Ill: transplant recipients, no rejection (n = 3). Group IV: nontransplant recipients, glomerulonephritis (n = 7). The cytokine mRNA levels are expressed in absolute units (see “Patients and Methods”) as mean (-t-SEM).
IV
IL-10
IL-4
5
n=l
IL-2Rp55
aRx and the other groups, with a sensitivity of 83% (10 of 12) and a specificity of 93% (14 of 15). Another sign of T-cell activation in biopsies of late aRx was the significant upregulation (P < .05) of IL-2R ~55. The levels of intragraft IL-2R ~75 transcripts were elevated in all three transplant groups (Fig 1). Our data are in agreement with what was found in the early aRx of renal allografts, ie, upregulation of IFN-7, TNF-a, and IL-2 at the mRNA and protein level.‘-a This
A
II=5 IV
n=7
ll=5 I
11
Ill
N
TNF-ci
IL-2Rp75
suggests that cell-mediated inflammation (DTH reaction) may play an important role in aRx. However, we did not find significantly elevated GrnA levels in the biopsies taken during late aRx compared with the controls, although the mean values were about 1.5 to 2 times higher than in the control groups. Despite the expression of GrnA, mRNA was not absent in late aRx, and its involvement in the pathogenesis of this type of aRx is doubtful as similar levels of GrnA expression were found in
LATE ACUTE REJECTION
well-functioning grafts of long-term transplant recipients and in kidneys from patients with glomerulonephritis. In contrast to our observations in late aRx, Lipman et al emphasize the pathogenetic role of CTL, as they reported a strong upregulation of CTL-specific serine proteases and perforin transcripts in early aRx (>lOO-fold) that correlated much closer with aRx than those of several cytokine genes.7,x A plausible hypothesis explaining the different clinical courses of early and late aRx would be: DTH (CD4 T-cell-dependent) mechanisms are involved in the pathogenesis of both early and late aRx. The additional involvement of CTL activity in early aRx, but not late, aRx results in quicker graft injury in the former, reflected by the more rapid rise of serum creatinine. Explanations for the differences in pathogenesis may be the lower immunogenicity of adapted allografts and/or different pathways of alloantigen presentation. Direct allopresentation by donor-derived antigen-presenting cells, which is a very strong stimulator of T cells (in vitro model: mixed lymphocyte reaction), dominates during the early course posttransplantation. If most of the “passenger” leukocytes are dead, the indirect presentation of allopeptides by the recipient’s own antigen-present-
95
ing cells should be more important immunogenic than the former.
but it is actually less
REFERENCES
1. Reinke P, Fietze E, Dacke WD, et al: Transplantation 58:35, 1994 2. Mahoney JF, Sheil AGR: In Morris PJ, Tilney NL (eds): Transplantation Reviews. New York: Grune & Stratton, 1987, p 47 3. Rao KV, Kasiske BL, Bloom PM: Transplantation 47:290, 1989 4. Prieto C, Pulido F, Rodriguez-Paternina E, et al: Transplant Proc 24~35, 1992 5. Dallman MJ, Larsen CP, Morris PJ: J Exp Med 174:493, 1991 6. Morel D, Nordmand E, Lemoine C. et al: Transplantation 55773, 1993 7. Lipman ML, Stevens AC, Bleckley RC, et al: Transplantation 53~73, 1992 8. Lipman ML, Stevens AC, Strom TB: J Immunol 1.52:5120, 1994 9. Platzer C, Richter G, oberla K, et al: Eur J Immunol22:1179, 1992 10. Platzer C, Ode-Hakim S, Reinke P, et al: Transplantation 58:264, 1994 11. Ode-Hakim S, Diicke WD, Kern F, et al: Transplantation 61:1233, 1996