Expression of cytotoxic mediators (perforin, granzyme B, FAS, and FAS-l) in renal allograft biopsies
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ORIGINAL ARTICLE
Expression of cytotoxic mediators (perforin, granzyme B, FAS,
and FAS-l) in renal allograft biopsies
Expressão de mediadores citotóxicos (perforina, granzima B, FAS e FAS-l) em biópsias de
aloenxerto renal
Therezinha Gauri Leitão 1, Luís Eduardo Becker2, Ivone Braga de Oliveira3, Flávia Ramos de Siqueira4, Maria Regina
Teixeira Araújo5, João Egídio Romão Junior 6, Hugo Abensur7, Irene de Lourdes Noronha8
ABSTRACT
Objectives: To analyze the in situ expression of perforin, granzyme
B, FAS -L and FAS in renal allograft biopsies by means of
immunohistochemistry and correlate these findings with the degree
of histologic rejection and allograft outcome. Methods: Ninety-six
allograft biopsies were divided into three groups: acute rejection (n
= 56), chronic rejection (n = 31), and cases with stable renal function
(no rejection; n = 9). The expression of perforin, granzyme B, FAS-L,
and FAS was evaluated by immunohistochemistry. Results: A
significantly higher expression of perforin and granzyme B was
observed in acute rejection biopsies (4.83 ± 0.65 and 30.05 ± 7.93
cells/mm2) compared to chronic rejection biopsies (0.71 ± 0.13 and
11.4 ± 3.84 cells/mm2; p < 0.001, and p <0.05, respectively), but this
was not the case for FAS-L (24.44 ± 5.56 in acute rejection versus 18.87
± 6.83 in chronic rejection). Perforin, granzyme B, and FAS-L expression
was significantly higher in the acute rejection group compared to the no
rejection and control groups. FAS expression was similar in all groups. A
modest correlation between perforin expression and the severity of AR
was observed (r = 0.28, p = 0.05). Perforin was the most reliable marker
for acute rejection diagnosis, with 80% sensitivity and 84.3% specificity.
Conclusion: The in situ expression of perforin, granzyme B, and FAS-L
in AR reflects the presence of an active cytotoxic process. Additional
allograft biopsies are necessary in order to evaluate the usefulness of
these markers for allograft rejection monitoring.
Keywords: Kidney transplantation/immunology; Kidney/pathology;
Immunohistochemistry; Antigens, CD95; Graft rejection
RESUMO
Objetivos: Analisar por imunoistoquímica a expressão in situ de
perforina, granzima B, FAS-L e FAS em biópsias de enxerto renal,
correlacionando esses achados com o grau histológico de rejeição e
prognóstico do enxerto. Métodos: Biópsias renais (n = 96) foram
divididas em três grupos: rejeição aguda (n = 56), rejeição crônica
(n = 31), função renal estável (sem rejeição; n = 9). A expressão de
perforina, granzima B, FAS-L e FAS foi avaliada por imunoistoquímica.
Resultados: A expressão de perforina e granzima B foi
significativamente maior na rejeição aguda (4,84 ± 0,65 e 30,05 ±
7,93) quando comparada à rejeição crônica (0,71 ± 0,13 céls./mm2
e 11,40 ± 3,84; p < 0,001 e p< 0,05 respectivamente), mas não de
FAS-L (24,44 ± 5,56 na rejeição aguda vs. 18,87 ± 6,83 na rejeição
crônica). As marcações de perforina, granzima B e FAS-L na rejeição
aguda foram significativamente maiores na rejeição aguda que nos
casos sem rejeição e controle. A expressão de FAS foi semelhante
entre os grupos. Houve modesta correlação entre a expressão de
perforina e a gravidade da rejeição aguda (r = 0,28, p = 0,05). A
perforina foi o marcador mais confiável para o diagnóstico de rejeição
aguda, com 80% de sensibilidade e 84,4% de especificidade.
Study carried out at Hospital Beneficência Portuguesa de São Paulo (SP), Brazil.
This study was financially supported by The State of São Paulo Research Foundation (FAPESP), process n. 98/16558-9.
1
Master’s Degree in Experimental Pathophysiology from the Universidade de São Paulo - USP, São Paulo (SP), Brazil.
2
Graduate Student of Nephrology, at the Faculdade de Medicina da Universidade de São Paulo - USP; Assistant Physician of the Nephrology Clinic and Member of the Renal and Renal-pancreatic
Transplant Clinic of the Hospital Beneficência Portuguesa de São Paulo (SP), Brazil.
3
Master’s Degree in Pharmacology from the Instituto de Ciências Biomédicas da Universidade de São Paulo - USP; Research collaborator at the Renal Pathophysiology Laboratory of the Faculdade de
Medicina da Universidade de São Paulo – USP, São Paulo (SP), Brazil.
4
Student of Scientific Initiation at the Renal Pathophysiology Laboratory of the Faculdade de Medicina da Universidade de São Paulo – USP, São Paulo (SP), Brazil.
5
PhD in Nephrology from the Faculdade de Medicina da Universidade de São Paulo - USP; Member of the Nephrology Clinic and Member of the Renal and Renal-pancreatic Transplant Clinic of the
Hospital Beneficência Portuguesa de São Paulo (SP), Brazil.
6
Post-Doctorate Degree in Nephrology from the Faculdade de Medicina da Universidade de São Paulo - USP; Member of the Nephrology Clinic and Member of the Renal and Renal-pancreatic Transplant
Clinic of the Hospital Beneficência Portuguesa de São Paulo (SP), Brazil.
7
Post-Doctorate Degree in Nephrology from the Faculdade de Medicina da Universidade de São Paulo - USP; Member of the Nephrology Clinic and Member of the Renal and Renal-pancreatic Transplant
Clinic of the Hospital Beneficência Portuguesa de São Paulo (SP), Brazil.
8
Post-Doctorate Degree in Nephrology from the Faculdade de Medicina da Universidade de São Paulo - USP; Head of the Immunohistochemistry Laboratory, Medical Investigation Laboratory-29, of the
Faculdade de Medicina da Universidade de São Paulo - USP; Member of the Nephrology Clinic and Coordinator of the Renal and Renal-Pancreatic Transplant Program of the Hospital Beneficência
Portuguesa de São Paulo (SP), Brazil.
Corresponding author: Irene de Lourdes Noronha - Laboratório de Nefrologia Molecular, Celular e Genética - Faculdade de Medicina, Universidade de São Paulo – Av. Dr. Arnaldo, 455 - 3º andar, sala
3342 - Cerqueira César - CEP 01246-903 - São Paulo (SP), Brazil - Tel.: 11 3068-9428 – e-mail: [email protected]
Received on Feb 15, 2006 - Accepted on Oct 16, 2006
einstein. 2006; 4(4):277-283
Expression of cytotoxic mediators (perforin, granzyme B, FAS, and FAS-l) in renal allograft biopsies
rejection with the subsequent appearance of chronic
rejection, suggesting that cytotoxic mediators might
have a role in the development of chronic rejection.
In addition, the presence of cytotoxic mechanisms in
chronic rejection situations suggests the need for a
reevaluation of the immunosuppressive regimen in these
cases(1,21,23). Although increasing relevance has been given
to the non-immune mechanisms involved in chronic
rejection, the most important risk factors leading to allograft
failure in this situation are the immunological risk factors.
The analysis of sensitivity and specificity of three of the
mediators studied (perforin, granzyme, and FAS-L)
regarding acute rejection showed that the
immunohistochemical technique is sensitive enough to reveal
the expression of these molecules when an active cytotoxicity
process is present. The immunohistochemical technique for
perforin, granzyme B, and FAS-L proved capable of
characterizing the destruction pathway of the allograft and
furthermore, identified the exact localization of this process
on the biopsy tissue. Thus, this study demonstrated that the
biopsy is still the most effective method for post-transplant
monitoring, since it clearly recognizes the cells involved in
the allograft rejection process.
CONCLUSION
This study demonstrated the expression of perforin,
granzyme B, and FAS-L in allograft biopsies, particularly
in cases with a clinical-histological diagnosis of acute
rejection. The presence of these markers in chronic
rejection cases, especially during early phases, reveals
the presence of an active cytotoxic process, suggesting,
in these cases, the need for a reevaluation of the
immunosuppression regimen. The data point to the
possibility of using these molecules as rejection markers
that could become valuable instruments in posttransplant monitoring.
283
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