| Abstract|| |
A large body of evidence suggests the existence of functionally polarized human T-helper responses based on their profile of cytokine secretion. Human T-helper cell clones can be divided into two mutually exclusive subsets, T-helper cell 1 (Th1) and Thelper cell 2 (Th2). Substantial work in several animal models has demonstrated that allograft rejection is associated with enhanced Th1 activity and tolerance with enhanced Th2. Some studies have not been consistent with this association. In this study, gamma interferon (INF-y) and interleukin 4 (IL-4) levels (as indicators of Th1 and Th2 activity, respectively) were assayed in supernatant of cultured peripheral lymphocytes. The levels of these cytokines were compared between a study group of 26 stable kidney transplant recipients immunosuppressed with cyclosporine A, corticosteroids and azathioprine or mycophenolate mofetil, and a control group of 10 healthy blood donors. The mean INF-γ and IL-4 levels in the control group were considered as the cutoff levels for comparison. Our results showed that 25/26 of the study patients (96%) had low levels of INF-γ compared to 4/10 of the control subjects (40%), (P<0.05). However, the IL-4 level was high in 10/26 of the study patients (38%) and 3/10 of the control subjects (30%), not a statistically significant difference, (P>0.05). In conclusion: These results suggest that wellestablished graft tolerance may be mediated via depressed Th1 activity rather than enhanced Th2 activity.
Keywords: Th1/Th2, INF-γ/IL-4, Kidney transplantation.
|How to cite this article:|
Hassan A A, Zaki M, Shoukeir M, Khalil R. T-Helper Subsets Cytokine Production in Kidney Transplant Recipients: Diverting Influences and Impact on Graft Outcome. Saudi J Kidney Dis Transpl 2000;11:161-6
|How to cite this URL:|
Hassan A A, Zaki M, Shoukeir M, Khalil R. T-Helper Subsets Cytokine Production in Kidney Transplant Recipients: Diverting Influences and Impact on Graft Outcome. Saudi J Kidney Dis Transpl [serial online] 2000 [cited 2019 Jul 17];11:161-6. Available from: http://www.sjkdt.org/text.asp?2000/11/2/161/36672
| Introduction|| |
Following the allotment of the regulatory CD4+ T-cells in 1986 by Mosmann et al  and Arthur and Mason (1986),  the T-helper cell 1 (Th1)/T-helper cell 2 (Th2) paradigm rapidly became established as a dominant theme in immunoregulation.  In organ transplantation, as in most other forms of chronic immunopathological disorders, the damage to tissues is regarded as the result of the activation of Th1 cells producing gamma interferon (INF-γ) (also interleukin 2 (IL-2), and a few other cytokines), which in health are balanced by the counter activity of Th2 producing interleukin 4 (IL4) (and other cytokines). 
Immunofluorescent double staining of intracellular INF- γ and IL-4 in combination with flowcytometry (FACS) revealed that most of the emerging Th cells produced either INF- γ or IL-4, and only a few double producers could be detected.  This view is now known to be over-simplified, principally because the number of cytokines has grown as far as IL-18, with great diversity in their patterns of expression among CD4+ cells, and because the key cytokine knockouts do not behave as expected. Substantial work in several animal models has demonstrated that allograft rejection is associated with enhanced Th1 activity and suppression of Th2.  Levy and Alexander  found that direct infusion of IL-4 (but not IL-10, that initiates the differentiation of naive CD4+ T cells to Th2), prolonged graft survival in experimental animals. Meanwhile, Sayegh et al  demonstrated that administration of recombinant IL-2 tended to precipitate allograft rejection. On the other hand, genetic manipulation did not have the expected effect, since the knockout of IL-2 gene does not delay,  or IL-4 gene accelerate rejection.  The existence of functionally polarized human Th1 and Th2 responses based on their profile of cytokine secretion in CD4+ T helper cells has been established. 
Several factors are involved in the Th cell differentiation into the polarized Th1 or Th2 pathway. Such factors include the cytokine profile of ' natural immunity' evoked by different offending agents, the nature of the peptide ligand, and the activity of some costimulatory molecules and microenvironmentally secreted hormones, in the context of different host genetic back grounds. 
Th1 is effective in defense against intracellular pathogens and Th2 against intestinal nematodes. They are also responsible for different types of immunopathological reactions. Th1- dominated responses may be involved in the pathogenesis of acute allograft rejection, organ specific autoimmune disorders, unexplained recurrent abortions, contact dermatitis and some chronic inflammatory disorders of unknown etiology. Th2-type responses are responsible for transplantation tolerance, graft versus host disease, some systemic autoimmune diseases, Omenn 's syndrome, reduced protection against intracellular pathogens, and some atopic disorders. 
In this study, production of INF- γ and IL-4 from T-helper cells of renal transplant recipients was evaluated as markers of Th1 or Th2 in an attempt to highlight their role in influencing the course of renal allografts.
| Subjects and Methods|| |
This study was conducted on 26 (7 females, 19 males) stable kidney transplant recipients attending the Nile International Kidney Center. The mean age of these patients was 37.4 ± 5.2 years. They were all immunosuppressed with cyclosporine A (CyA), corticosteroids, and either azathioprine (Aza) or mycophenolate mofetil (MMF). Ten apparently healthy, age and sex matched, blood donors were used as a control group.
Twenty-ml blood samples were collected on preservative-free heparin (200 µL) from all participants in the study. These blood samples were diluted with equal volumes of HBSS (Gibco, Europe), then carefully overlaid on a Ficoll-Hypaque (Flow Lab; USA) gradient solution without allowing the solutions to mix. This was applied in a ratio of two volumes of gradient solution to three volumes of diluted blood. The layered blood was centrifuged in a swing-out rotator at 2700 rpm at 20 o C for 30 minutes. Mononuclear cells layered on the upper meniscus of the gradient solution were collected using a fine Pasteur pipette and washed twice in HBSS at 2700 rpm for 10 minutes
For the activation step, 10 µL phytohemagglutinin (Sigma, USA) were added to each ml of count-adjusted cell suspension, and the mix was incubated at 37 o C and 5% CO 2 for four hours. After four hours incubation, the cell suspension was centrifuged at 1500 rpm for five minutes, and supernatants of the cell culture media were aliquoted into several aliquots (500 µL each), and kept in the deep freezer at minus 70 o C in dry clean Epindorph tubes.
Cell culture supernatants were brought to room temperature immediately before starting cytokine assay procedure using Quantikine kits (R & D Systems, Europe). Two hundred µL of freshly thawed cell culture medium supernatants were added to each well of a microtiter plate precoated with a murine monoclonal antibody specific for INF- γ and to wells of another microtiter plate precoated with a murine monoclonal antibody specific for IL-4. The plates were incubated for two hours at room temperature. Each well was aspirated and washed three times. Two hundred µL of polyclonal antibody against IL-4 conjugated to horseradish peroxidase was added to each well and incubated for two hours at room temperature. Each well was aspirated and washed three times. Two hundred μL of substrate solution was added to each well and incubated for 20 minutes at room temperature. Fifty µL of stop solution was added to each well. Determination of cytokines concentration using programmable microtiter plate reader was carried out within 30 minutes (a standard curve was obtained using a serial dilution of the standard solution provided with the kit).
Medical records of all the study patients were reviewed for certain relevant data such as date of last graft; immunosuppressive protocol; mean serum creatinine; and mean CyA dose and trough level. Furthermore, we reviewed various factors incriminated in influencing the diversion of Th cells into either Th1 or Th2 and various immunological challenges and risk factors. Such factors included type and duration of dialysis, number of previous blood transfusions, number of previous grafting, number of graft rejections, number of previous pregnancies (including abortions), ABO blood groups, and seropositivity for human acquired deficiency virus (HIV) and hepatitis B and C (HBV and HCV respectively).
| Statistical Analysis|| |
All the data were transferred to IBM card, using IBM-PC with statistical program SPSS-under windows V-6.21 to obtain descriptive statistics (mean, standard deviation (SD), and range), and analytical statistics (Mann Whitney U-test, Spearman rank order correlation, and Chi-square test. Data were graphically represented using HGW program.
| Results|| |
INF-γ and IL-4 levels (as indicators for Th1 and Th2 activity respectively) were assayed in supernatants of cultured peripheral mononuclear cells obtained from the study patients and control subjects. The mean ± SD, minimum, and maximum levels of INF-γ and IL-4 in the control group are summarized in [Table - 1]. The study group was subdivided into two subgroups: subgroup-1 were patients immunosuppressed with CyA + Aza + corticosteroids while subgroup-2 were the patients immunosuppressed with CyA+ MMF+ corticosteroids. Results of the subgroups are summarized in [Table - 2],[Table - 3].
There was no significant correlation between INF-γ or IL-4 levels and the type of immunosuppressives used, so we recombined the two subgroups of the study patients.
The mean INF- γ and IL-4 levels in the control group (124.2 pg/ml and 15.13 pg/ml respectively) were considered the cutoff levels.
Our results showed that 25/26 of the study patients (96%) had low levels of INF- γ compared to 4/10 of the control subjects (40%), (P < 0.05). However, the IL-4 level was high in 10/26 of the study patients (38%) and 3/10 of the control subjects (30%), not a statistically significant difference, (P > 0.05).
There was no correlation between INF- γ and IL-4 levels. Also, there was no correlation between INF- γ or IL-4 levels and age, gender, blood group, type or duration of pre-transplant dialysis, pre-transplant blood transfusion, number of pregnancies, serum creatinine, blood urea, type of kidney donor, number of mismatches, or associated infections (HBV, HCV, HIV, CMV).
| Discussion|| |
Th1-dependent delayed-type-hypersensitivity and cytotoxic T-lymphocyte (CTL) activity play a central role in acute graft rejection.  Proteins and/or transcripts for intragraft INF-γ and IL-2 have consistently been detected in the rejecting allografts.  Accordingly, CD4+ cell clones generated from kidney allografts during the acute phase of rejection showed a clear cut Th1 profile.  The production of Th2 type cytokines was also demonstrated in the induction and maintenance of allograft tolerance.  Several in vivo studies that examined the pattern of cytokine expression during tolerance induction have consistently shown a dramatic decrease in the expression of INF-γ and IL-2, with increased levels of IL-4 and IL-10 transcripts. 
In our study, INF-γ and IL-4 levels (as indicators of Th1 and Th2 activity respectively) were assayed in supernatants of cultured peripheral lymphocytes obtained from a group of stable kidney transplant recipients and compared with those of the control group.
The level of INF-γ was lower than the cutoff level in a significantly higher percentage of the study patients than the control group. On the other hand, the level of IL-4 was higher than the cutoff level in a comparable percentage of patients in the study group compared with the controls.
Nickerson et al,  found a decrease of INF-γ and an increase of IL-4 levels, while in our study, we found no significant increase in the IL-4 levels compared to the controls. Weimer et al,  who investigated the association of acute and chronic kidney graft rejection with pre-transplant T-cell invitro cytokine production using enzymelinked immunosuppressive assay (ELISA) technique, found that low pre-transplant Th2-cell responses were significantly associated with a low incidence of acute rejection episodes (7% versus 47%; P < 0.01) and better 1- and 3-year graft function (P < 0.05). They also found that graft loss due to acute irreversible rejection, chronic rejection and recurrence of the original disease occurred only in patients with normal Th2-cell function. From these results they concluded that decreased pretransplant Th2 responses could predict low risk of kidney graft rejection and a better 1and 3-year graft survival. They also found that functions of B cells and monocytes were not of predictive value.
Integrating the results of these two studies, one may conclude that although enhanced pre-transplant Th2 responses are predictive of graft tolerance, yet the situation may change post-transplantation with administration of various immunosuppressive protocols. Cyclosporine A, the key immunosuppressive drug in most currently applied protocols, is known to inhibit the proliferation of both Th1 and Th2 and the production of cytokines including IL-2, IL-4, IL-5, and INF- γ, irrespective of the mode of stimulation.  It is also known that IL-4 production (Th2 response) is as sensitive to CyA as INF- γ (Th1 response).  Therefore, it is unlikely that the observed preferential suppression of Th1 response could be explained by CyA inhibition of cytokine production. It also seems that with established immunosuppressive protocols the preferential suppression of either Th1 or Th2 could be at least partly responsible for the post-transplant tolerance and rejection respectively.
We also detected slight differences between the two subgroups of patients, yet these differences did not mount to statistical significance probably due to small numbers within each subgroup. Patients on MMF showed lower mean INF- γ level compared to the mean level in patients on Aza. Patients on MMF also showed higher mean IL-4 level compared to the mean level in controls, whereas patients on Aza showed slightly lower mean IL-4 level compared to the mean level in controls. According to our assumption, these differences if proved valid may point out to the additional benefit gained by adding MMF to the immunosuppressive regimen by not only suppression of diversion of Th cells into Th1 but also enhancing diversion towards Th2 responses.
We conclude that, although its validity is not yet proved, the Th1/Th2 paradigm may represent the basis for understanding the mechanisms of rejection and tolerance in transplantation. It also seems that graft tolerance is probably mediated via depressed Th1 activity rather than enhanced Th2 activity.
| References|| |
|1.||Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T-cell clone I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348-57. |
|2.||Arthur RP, Mason D. T-cells that help B cell responses to soluble antigen are distinguishable from those producing interleukin 2 on mitogenic or allogeneic stimulation. J Exp Med 1986;163:774-86. [PUBMED] |
|3.||Abbas AK, Murphy KM, Sher A. Functional diversity of helper T-lymphocytes. Nature 1996;383:787-93. [PUBMED] [FULLTEXT]|
|4.||Gimsa U, Sieper J, Braun J, Mitchison NA. Type II collagen serology: a guide to clinical responsiveness to oral tolerance? Rheumatol Int 1997;16:237-40. [PUBMED] [FULLTEXT]|
|5.||Palm N, Germann T, Goedert S, et al. Codevelopment of naive CD4+ cells towards T helper type 1 or T helper type 2 cells induced by a combination of IL-12 and IL4. Immunobiology 1996-97;196:475-84. |
|6.||Seder RA, Prussin C. Are differentiated human T helper cells reversible? Int Arch Allergy Immunol 1997;113:163-6. [PUBMED] |
|7.||Levy AE, Alexander JW. Administration of intragraft interleukin-4 prolongs cardiac allograft survival in rats treated with donor-specific transfusion/cyclosporin. Transplantation 1995;60:405-6. [PUBMED] |
|8.||Sayegh MH, Akalin E, Hancock WW, et al. CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2. J Exp Med 1995;181: 1869-74. [PUBMED] |
|9.||Steiger J, Nickerson PW, Steurer W, Moscovitch Lopatin M, Strom B. IL-2 knockout recipient mice reject islet cell allografts. J Immunol 1995;155:489-98. |
|10.||Nickerson P, Zheng X, Steiger J, et al. Prolonged islet allograft acceptance in the absence of interleukin 4 expression. Transpl Immunol 1996;4:81-5. |
|11.||Harada Y, Watanabe S, Yssel H, Arai K. Factors affecting the cytokine production of human T cells stimulated by different modes of activation. J Allergy Clin Immunol 1996;98(6 Pt. 2):S161-73. |
|12.||Romagnani S, Parronchi P, D 'Elios MM, et al. An update on human Th1 and Th2 cells. Int Arch Allergy Immunol 1997; 113:153-6. |
|13.||Romagnani S. Th1 and Th2 in human diseases. Clin Immunol Immunopathol 1996;80(3):225-35. |
|14.||Nickerson P, Steurer W, Steiger J, Zheng X, Steele AW, Strom TB. Cytokines and the Th1/Th2 paradigm in transplantation. Curr Opin Immunol 1994;6:757-64. [PUBMED] |
|15.||Del Prete G, De Carli M, Almerigogna F, et al. Preferential expression of CD30 by Human CD4+ T cells producing Th2-type cytokines. FASEB J 1995;9:81-6. [PUBMED] [FULLTEXT]|
|16.||Weimer R, Zipperle S, Daniel V, Carl S, Staehler G, Opelz G. Superior 3-year kidney graft function in patients with impaired pre-transplant Th2 responses. Transpl Int 1998;11(Suppl 1):S350-6. [PUBMED] [FULLTEXT]|
A A Hassan
Nile International Kidney Center, 18 Khalifa El-Maamoun Street, Heliopolis, Cairo
[Table - 1], [Table - 2], [Table - 3]