OK432-induced killer cell activity: potential method for monitoring immunological complications after renal transplantation

Masaharu Nishikido, Tatsuo Kiyohara, Shigehiko Koga, Kazuhiko Shindo, Fukuzo Matsuya, Yutaka Saito and Hiroshi Kanetake

Department of Urology, Nagasaki University School of Medicine, Nagasaki University Hospital, Nagasaki, Japan



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Various clinical and biochemical parameters are currently in use for monitoring allograft rejection. However, the mechanism of allograft rejection is complex and it is frequently difficult to obtain a prompt and accurate diagnosis. We examined the usefulness of OK432-induced killer cell activity as an immunological monitoring system for acute renal rejection after renal transplantation.

Methods. Twenty-four renal transplant recipients, seven patients on haemodialysis, and 10 normal volunteers were enrolled in our study. The killer cell activity of peripheral blood mononuclear cells was induced by culturing these cells with the immunopotentiator, OK432, a heat and penicillin-treated lyophilized powder of the Su-strain of Streptococcus pyogenes.

Results. The OK432-induced killer cell activity of renal transplant recipients without acute rejection (stable recipients) was significantly lower than in normal volunteers. In four renal transplant recipients with acute rejection, the killer cell activity was significantly higher than in stable recipients. In three recipients suffering from opportunistic infections, killer cell activity was significantly suppressed compared with stable recipients.

Conclusions. Our new test utilizing OK432-induced killer cell activity is potentially useful for monitoring the immunological state and complications after renal transplantation.

Keywords: cytotoxic activity; OK432; peripheral blood mononuclear cells; renal transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Allograft rejection is diagnosed through the evaluation of various clinical, biochemical, and pathological parameters. However, the underlying mechanisms of rejection are complex and not completely understood.

OK432, a heat and penicillin-treated lyophilized powder of the Su-strain of Streptococcus pyogenes, is one of the biological response modifiers (BRMs) developed by Okamoto et al. [1]. Several studies have demonstrated that anti-tumour effects of OK432 may be due to the activation of a variety of effector cells such as macrophages, natural killer (NK) cells, autotumour killer cells and cytotoxic T lymphocytes [25]. In the present study, we examined the usefulness of OK432-induced killer cell activity as a new system for monitoring immunological complications after renal transplantation.



   Subjects and methods
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Patients and protocol
We studied three groups of subjects: 24 renal transplant recipients (18 men and 6 women, aged 18–48 years, median 31 years; 19 living-related kidneys and 5 cadaveric kidneys), 7 patients on haemodialysis (5 men and 2 women, aged 35–56 years, median 45 years), and 10 normal volunteers (7 men and 3 women, aged 26–58 years, median 43 years). Blood samples were taken from patients in a stable state, 3–4 h after administration of immunosuppressive drugs. Blood samples from volunteers and haemodialysis patients were taken between 09.00 and 14.00 h. Blood samples from patients showing acute rejection were taken just before biopsy, and blood samples from patients with opportunistic infections were obtained following the appearance of clinical signs and symptoms of infection. All acute rejection episodes were confirmed histologically and sequential blood sampling was performed after biopsy. Renal transplant recipients were treated with a combination of cyclosporine A (CsA) and methylprednisolone (MP). Acute rejection was treated with steroid pulse therapy. The study protocol was approved by the Human Ethics Review Committee of Nagasaki University School of Medicine and signed consent was obtained from each subject.

Effector cells
Peripheral blood mononuclear cells (PBMNCs) from renal transplant recipients or normal volunteers were isolated by Ficoll–Hypaque gradient centrifugation. Adherent cells were removed on plastic dishes pre-coated with autologous plasma by 1 h incubation at 37°C in a humidified atmosphere of 5% CO2 in air. OK432 was kindly donated by the Chugai pharmacological company (Tokyo, Japan). Plastic non-adherent PBMNCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and OK432 (2.0 µg/ml). These OK432-induced killer cells were harvested at 24 h after incubation, except for the time kinetics experiments examining OK432-induced killer cell activity. These cells were washed three times and used as effector cells.

Target cells
Natural killer cell-resistant human renal cell carcinoma ACHN cells were used in this study. This cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 µmol/l L-glutamine, and 0.5% Na-bicarbonate. Viability was assessed by the trypan blue dye exclusion test and always exceeded 95%.

Cytotoxicity assay
Cytotoxicity was measured by 18-h 51Cr release assay. Target cells (1x106) were radiolabelled with 100 µCi Na2CrO4 (Amersham International, Aylesbury, UK) at 37°C for 1 h. They were then washed three times and finally resuspended at 5x104 cells/ml in complete medium. Chromium-labelled target cells (5x103/0.1 ml) were deposited into the wells of 96-well V shape bottomed microtitre plates (Costar, Cambridge, MA, USA), and graded numbers of effector cells in 100 µl complete medium were then added to yield effector cell to target cell ratios (E : T) of 50 : 1, 25 : 1, 12.5 : 1 and 6.25 : 1. Each assay was performed in triplicate. The plates were centrifuged for 5 min at 1000 rpm, and then incubated for 18 h at 37°C in a humidified atmosphere of 5% CO2 in air. After incubation and recentrifugation, aliquots (100 µl) of the supernatant were removed from each well, and their radioactivity was determined in a {gamma}-counter (ARC-500, Aloka, Tokyo). Data were expressed as the percentage of 51Cr release and were calculated according to the formula:


(001)
Spontaneous release was the radioactivity released in supernatants from target cells incubated in complete medium only, and maximal release was determined by counting an aliquot of resuspended target cells. Lytic unit (LU) was calculated from cytotoxic titration curves. One LU was defined as the number of effector cells required to cause 20% lysis of 5x103 target cells and the data were presented as LU/107 effector cells [6].

Statistical analysis
Data were expressed as means±SD. Differences between groups were examined using the Mann–Whitney test. P values less than 0.05 were considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
OK432-induced killer cell activity
Normal human PBMNCs were cultured with OK432 and tested by 18-h 51Cr-release assay against ACHN cells. At 2 µg/ml, OK432 showed optimal induction of killer cell activity (data not shown). Figure 1Go shows that killer cell activity was induced on day 1 of culture and that activity remained stable thereafter.



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Fig. 1. OK432-induced killer cell activity in normal human PBMNCs. PBMNCs were cultured with OK432 (2 µg/ml) and tested in an 18-h 51Cr-release assay against NK-resistant ACHN cells.

 

Inhibition of OK432-induced killer cell activity by immunosuppressants
The blood concentration of CsA ranged from 0.1 to 1.0 µg/ml and the serum concentration of MP varied between 0.01 and 0.1 µg/ml in patients treated for immunosuppression. At the beginning of culture, the addition of immunosuppressants (CsA or MP) to the medium containing OK432 (2 µg/ml), at doses equivalent to those used clinically, resulted in inhibition of OK432-induced killer cell activity against ACHN cells (Figures 2AGo and 2BGo).



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Fig. 2. Inhibition of OK432-induced killer cell activity by immunosuppressants. (A) Cyclosporine A and (B) MP were added to PBMNCs culture medium containing OK432 and the killer cell activity in PBMNCs against ACHN cells was assayed.

 

OK432-induced killer cell activity in renal transplant recipients with complications
We compared OK432-induced killer cell activity after one-day of culture with OK432 from renal transplant patients with and without acute rejection (Figure 3Go). The 24 transplant patients presented did not include individuals that were counted twice. The killer cell activities of normal volunteers and haemodialysis patients without renal transplantation were not significantly different (normal volunteers: n=10, 384±165 LU vs haemodialysis patients: n=7, 430±1600 LU, P=0.58). In contrast, the killer cell activity of recipients without acute rejection (stable recipients) was significantly lower than in normal volunteers (normal volunteers: n=10, 384±165 LU vs stable recipients: n=17, 52.4±28.8 LU, P<0.001) (Figure 3Go).



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Fig. 3. OK432-induced killer cell activity in normal volunteers, haemodialysis patients, stable recipients and recipients with acute rejection. PBMNCs were cultured with OK432 for one day and assayed against ACHN. Data are expressed as means±SD. *P<0.001.

 
In four renal transplant recipients with acute rejection, the killer cell activity (180±44 LU, P<0.001) was significantly higher than that of stable recipients (P<0.001). All patients with acute rejection improved after steroid pulse therapy, which was associated with a decrease in killer cell activity. Figure 4Go shows the time course of cell killer activity in the 4 patients with acute rejection.



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Fig. 4. Sequential changes in OK432-induced killer cell activity in four patients with acute rejection. Pre-Tx, before transplantation; Pre-AR, before acute renal rejection; AR, acute renal rejection; 2 and 4–8 weeks Post-AR, 2 and 4–8 weeks after acute renal rejection. Data are expressed as means±SD. *P<0.001.

 
Opportunistic infections by Pneumocystis carinii were found in one patient and by cytomegalovirus in two patients. The mean killer cell activity in these recipients was significantly lower than in stable recipients (stable recipients: n=17, 52.4±28.8 LU vs infection group: n=3, 13.3±11.5, P=0.036) (Figure 5Go).



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Fig. 5. OK432-induced killer cell activity in three recipients who developed opportunistic infections caused by Pneumocystis carinii and cytomegalovirus. PBMNCs from stable recipients were cultured with OK432 for one day and assayed against ACHN. Data are expressed as means±SD. *P=0.036.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
A prompt confirmation of acute rejection is important because of diminishing graft function. However, it is often difficult to obtain a rapid and accurate diagnosis. Several groups have investigated various immunological parameters that are clinically useful for monitoring immunosuppression and for predicting graft rejection. Previously studied parameters include the peripheral CD4/CD8 ratio [7,8], proportion of interleukin-2 (IL-2)-receptor-positive cells [9], mitogen-stimulated IL-2 production [10], donor-specific cytotoxic T lymphocyte tests [11], and NK and killer cell response to interferon [12]. Despite this work, a reliable and useful parameter for indicating graft rejection has yet to be determined.

In the present study, we demonstrated that OK432-induced killer cell activity may potentially be used to monitor the immune system. The cytotoxic activity against NK-resistant ACHN cells was induced in PBMNCs of renal transplant recipients after one-day culture with OK432. Several investigators have suggested that the antitumour effects of OK432 may be related to immunological responses. For example, Uchida et al. [3] demonstrated an augmentation by OK432 of autologous tumour killing activity by tumour-associated NK cells. However, Saito et al. [4,5] suggested that NK cells did not greatly contribute to the OK432 effect observed during treatment with anti-asialo GM1 antibodies in mice. Instead, activated macrophages were responsible for the tumour inhibitory effect. Subsequently, Yamashita et al. [13] reported that macrophages activated by OK432 caused increased T cell responses to alloantigen without major histocompatibility (MHC) restriction and stimulated induction of T cells responding to minor histocompatibility antigens. In the present study, non-adherent cells were used and no other enrichment methods were performed. Although the observed cytotoxicity was mainly due to NK-like cells, we cannot exclude the possible involvement of other cell types in the preparation, such as CD8 T cells.

Previous studies have shown that OK432 also induces the production of several cytokines such as interferon-{alpha} (IFN-{alpha}), IFN-{gamma}, IL-1, IL-2, IL-6, IL-12, tumour necrosis factor-{alpha} (TNF-{alpha}) and TNF-ß. These cytokines may have interactions with immunological responses [14]. For example, IL-2 and IFN-{gamma} have been shown to strongly modulate immune system function. IL-2, which is produced by CD4 T cells, stimulated the growth of NK cells and enhanced their cytotoxic function by inducing lymphokine-activated killer (LAK) cells. IFN-{gamma} is produced by CD4 T cells and by nearly all CD8 T cells when T cells are activated by stimulation with specific antigens. IFN-{gamma} is also produced by NK cells. This cytokine acts as a powerful stimulator of macrophage activation and cytotoxic activity in NK cells. Furthermore, IFN-{gamma} amplifies the cognitive phase of the immune response by promoting the activation of class II related CD4 T cells [15]. These findings suggest two mechanisms of OK432 induced killer cell activity: a cell-mediated pathway and a cytokine-mediated pathway. Methylprednisolone and CsA may either diminish the killer cell activity or the capacity to produce IL-2 and IFN-{gamma}.

Our results demonstrated that OK432-induced killer cell activity was significantly higher in recipients showing acute rejection than in stable recipients, and that activity was suppressed after steroid pulse therapy. Furthermore, changes in killer cell activity correlated with changes in the clinical state. However, our method has several limitations. First, our population sample was too small for drawing major conclusions regarding the clinical applicability of this new method. Second, the PBMNCs had to be incubated with OK432 for 24 h followed by 18 h of cytotoxicity assay, rendering our test rather slow compared with histological examination. At present, histological examination is faster and more accurate than our test for acute rejection. However, the determination of killer cell activity may be useful for predicting the success of antirejection therapy and for detecting marked immunological suppressive states. Finally, before our OK432-induced killer cell activity test could be used clinically, further studies are needed to examine its sensitivity and specificity. In future studies, the cut-off level of LU may be calculated if larger numbers of patients are tested.

OK432-induced killer cell activity was also markedly suppressed during opportunistic infection. Cytomegalovirus infection leads to increased expression of cell surface antigens, MHC class II antigens on CD8 cells [16] and MHC class I and class II antigens on kidney cells [17]. Cytomegalovirus infection causes both an immunosuppressive and immunostimulatory state. When certain parameters reflecting T cell activation are used, the differentiation between rejection and cytomegalovirus infection may be difficult. Our patients with cytomegalovirus infection showed low OK432-induced killer cell activity. Thus, further studies will be required to determine whether OK432-induced killer cell activity is suppressed in renal transplant patients with cytomegalovirus infection.

In conclusion, although the mechanisms of OK432-induced killer cell activity are complex, this activity may provide a useful method for immunological monitoring of renal allograft rejection and opportunistic infections.



   Notes
 
Correspondence and offprint requests to: Masaharu Nishikido, MD, Department of Urology, Nagasaki University School of Medicine, Nagasaki City, Nagasaki 8528501, Japan. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 8.00
Revision received 14. 5.01.



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