• View in gallery
    Figure 1—

    Box-and-whiskers plots for concentrations of IFN-γ (A), GM-CSF (B), IL-2 (C), and IL-10 (D) in lymphocytes (obtained from 21 clinically normal cats) cultured with medium alone, medium that contained Con A alone, or medium that contained Con A in combination with cyclosporine (CyA), dexamethasone (Dex), a combination of cyclosporine plus dexamethasone (CyA-Dex), or human (hu)CTLA4-Ig. A feline-specific ELISA was used to measure cytokine concentrations in the supernatants. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line within each box represents the median, and the whiskers represent values between the 10th and 90th percentiles. Black circles represent outliers. *Median value differs significantly (P >0.05) from the median value for Con A–treated cells.

  • View in gallery
    Figure 2—

    Box-and-whiskers plots for concentrations of IFN-γ (A), GM-CSF (B), IL-2 (C), and IL-10 (D) in lymphocytes (obtained from 21 clinically normal cats and 5 cats that received a renal transplant) cultured with medium alone (left) or medium that contained Con A (right). Notice that the scale on the y-axis differs between culture conditions for each cytokine. *Within a culture condition, median value differs significantly (P >0.05) from the median value for the clinically normal cats. See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Graph of FACS results for CD4+ (A) and CD8+ (B) T cells obtained from a typical immunosuppressed cat (ie, a cat that underwent renal transplantation and was receiving immunosuppressive treatment that consisted of cyclosporine in combination with prednisolone). The CD4+ subset comprised a median of 18.7% (reference range, 17.4% to 49.5% [median, 32.3%]) and the CD8+ subset comprised a median of 20.8% (reference range, 6.5% to 39.3% [median, 18.7%]) of the total lymphocytes collected. Along the y-axis, SSC refers to side scatter. The intensity of side-scattered light is related to the granularity of the particle evaluated. Along the x-axis, PE is a fluorescent dye used for cell surface labeling. The x-axis displays the increasing intensity of the fluorescence of CD4+ and CD8+ T cells.

  • View in gallery
    Figure 4—

    Proliferation profiles of CD4+ (A) and CD8+ (B) T cells obtained from a clinically normal cat (top row) and a renal transplant recipient cat (bottom row) and that were cultured in medium alone (right) and medium with the addition of Con A (left). Notice that the scale on the y-axis differs among culture conditions within lymphocyte subsets. In the clinically normal cat, 85.8% of CD4+T cells and 72.3% of CD8+T cells proliferated in the medium with Con A. In contrast, only 7.2% of CD4+ T cells and 48.2% of CD8+T cells proliferated in the renal transplant recipient cat, which was immunosuppressed because of long-term treatment with cyclosporine in combination with prednisolone. On the x-axis, CFSE is a fluorescent dye used to assess lymphocyte proliferation. A shift of the proliferation profile to the left indicates a decrease in fluorescence following each cell division.

  • 1.

    Katayama M, McAnulty JF. Renal transplantation in cats: techniques, complications, and immunosupression. Compend Contin Educ Pract Vet 2002; 24:874882.

    • Search Google Scholar
    • Export Citation
  • 2.

    Katayama M, McAnulty. Renal transplantation in cats: patient selection and preoperative management. Compend Contin Educ Pract Vet 2002; 24:868872.

    • Search Google Scholar
    • Export Citation
  • 3.

    Bernsteen L, Gregory CR, Kyles AE, et al. Renal transplantation in cats. Clin Tech Small Anim Pract 2000; 15:4045.

  • 4.

    Gregory CR. Renal transplantation in cats. Compend Contin Educ Pract Vet 1993; 15:13251338.

  • 5.

    Gregory CR. Renal transplantation. In: Bojrab MJ, ed. Current techniques in small animal surgery. 4th ed. Baltimore: Williams & Wilkins, 1998;434.

    • Search Google Scholar
    • Export Citation
  • 6.

    Kadar E, Sykes JE, Kass PH, et al. Evaluation of the prevalence of infections in cats after renal transplantation: 169 cases (1987–2003). J Am Vet Med Assoc 2005; 227:948953.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Bernsteen L, Gregory CR, Aronson LR, et al. Acute toxoplasmosis following renal transplantation in three cats and a dog. J Am Vet Med Assoc 1999; 215:11231126.

    • Search Google Scholar
    • Export Citation
  • 8.

    Nordquist BC, Aronson LR. Pyogranulomatous cystitis associated with Toxoplasma gondii infection in a cat after renal transplantation. J Am Vet Med Assoc 2008; 232:10101012.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Griffin AG, Newton AL, Aronson LR, et al. Disseminated Mycobacterium avium complex infection following renal transplantation in a cat. J Am Vet Med Assoc 2003; 222:10971101.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Wooldridge J, Gregory CR, Mathews KG, et al. The prevalence of malignant neoplasia in feline renal transplant recipients. Vet Surg 2002; 31:9497.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Schmiedt CW, Grimes JA, Holzman G, et al. Incidence and risk factors for development of malignant neoplasia after feline renal transplantation and cyclosporine-based immunosuppression. Vet Comp Oncol 2009; 7:4553.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Mathews KG, Gregory CR. Renal transplants in cats: 66 cases (1987–1996). J Am Vet Med Assoc 1997; 211:14321436.

  • 13.

    Schmiedt CW, Holzman G, Schwarz T, et al. Survival, complications and analysis of risk factors after renal transplantation in cats. Vet Surg 2008; 37:683695.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Kyles AE, Gregory CR, Griffey SM, et al. Evaluation of the clinical and histological features of renal allograft rejection in cats. Vet Surg 2002; 31:4958.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Amirzargar A, Lessanpezeshki M, Fathi A, et al. Th1/Th2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc 2005; 37:29852987.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Saalmuller A. New understanding of immunological mechanisms. Vet Microbiol 2006; 117:3238.

  • 17.

    Rostaing L, Puyoo O, Tkaczuk J, et al. Differences in type 1 and type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuprression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients. Clin Transplant 1999; 13:400409.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Mosmann TR, Fowell DJ. The Th1/Th2 paradigm in infections. In: Kaufman SHE, Sher A, Ahmed R, eds. Immunology of infectious diseases. Washington, DC: ASM Press, 2002;163174.

    • Search Google Scholar
    • Export Citation
  • 19.

    Andre S, Tough DF, Lacroix-Desmazes S, et al. Surveillance of antigen presenting cells by CD4+CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications. Am J Pathol 2009; 174:15751587.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Hashimoto M, Sakaguchi S. Contribution of Th-1, Th-2, TH-17 or regulatory T cells to connective tissue diseases [in Japanese]. Nippon Rinsho 2009; 67:482486.

    • Search Google Scholar
    • Export Citation
  • 21.

    Miyara M, Wing K, Sakaguchi S. Therapeutic approaches to allergy and autoimmunity based on FoxP3+ regulatory T cell activation and expansion. J Allergy Clin Immunol 2009; 123:749755.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Turka LA. Normal immune responses. In: Norman DJ, Suki WN, eds. Primer on transplantation. Thorofare, NJ: American Society of Transplant Physicians, 1998;720.

    • Search Google Scholar
    • Export Citation
  • 23.

    Basak U, Mitra DK, Panigrahi A, et al. Clinical relevance of monitoring cytokine production following living donor renal transplantation. Transplant Proc 2003; 35:404406.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Poole KL, Gibbs PJ, Evans PR, et al. Influence of patient and donor cytokine genotypes on renal allograft rejection: evidence from a single centre study. Transplant Immunol 2001; 8:259265.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Li L, Yuhai Z. The relationship between cytokines in MLC supernatants and acute rejection after renal transplantation. Transplant Proc 2000; 32:25312534.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Divate SA. Acute renal allograft rejection: progress in understanding cellular and molecular mechanisms. J Postgrad Med 2000; 46:293296.

    • Search Google Scholar
    • Export Citation
  • 27.

    McLean AG, Hughes D, Welsh KI, et al. Patterns of graft infiltration and cytokine gene expression during the first 10 days of kidney transplantation. Clin Transplant 1997; 63:374380.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Sadeghi MI, Daniel V, Weimer R, et al. Differential early post-transplant cytokine responses in living and cadaver donor renal allografts. Transplantation 2003; 75:13511355.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Gibbs PJ, Sadek SA, Cameron C, et al. Immunomonitoring of renal transplant recipients in the early posttransplant period by analysis of cytokine gene expression in peripheral blood mono-nuclear cells. Transplant Proc 2001; 33:32853286.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970; 169:10421049.

  • 31.

    Najafian N, Sayegh MH. CTLA4-Ig: a novel immunosuppressive agent. Exp Opin Invest Drugs 2000; 9:21472157.

  • 32.

    Schaub M, Stadlbauer THW, Chandraker A, et al. Comparative strategies to induce long term graft acceptance in fully allogeneic renal versus cardiac allograft models by CD28-B7 T cell costimulatory blockade: role of thymus and spleen. J Am Soc Nephrol 1998; 9:891898.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997; 94:87898794.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Kurlberg G, Haglind E, Schon K, et al. Blockade of the B7-CD28 pathway by ctla4-Ig counteracts rejection and prolongs survival in small bowel transplantation. Scand J Immunol 2000; 51:224230.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Turka LA, Linsley PS, Lin H, et al. T cell activation by CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc Natl Acad Sci USA 1992; 89:1110211105.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Sayegh MH, Zheng XG, Magee C, et al. Donor antigen is necessary for the prevention of chronic rejection in CTLA4-Ig treated murine cardiac allograft recipients. Transplantation 1997; 64:16461650.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Russell ME, Hancock WW, Akalin E, et al. Chronic cardiac rejection in the LEW to F344 rat model. Blockade of CD28-B7 costimulation by CTLA4-Ig modulates T cell and macrophage activation and attenuates arteriosclerosis. J Clin Invest 1996; 97:833838.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Azuman H, Chandraker A, Nadeau K, et al. Blockade of T cell costimulation prevents development of experimental chronic renal allograft rejection. Proc Natl Acad Sci U S A 1996; 93:1243912444.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Lin H, Bollong SF, Linsley PS, et al. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4-Ig plus donor specific transfusions. J Exp Med 1993; 175:18011806.

    • Search Google Scholar
    • Export Citation
  • 40.

    Chandraker A, Azuma H, Nadeau K, et al. Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection. J Clin Invest 1998; 101:23092318.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Akalin E, Chandraker A, Russell ME, et al. CD28-B7 T cell costimulatory blockade by CTLA4-Ig in the rat renal allograft model. Transplantation 1996; 62:19421945.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol 1993; 11:191212.

  • 43.

    Aronson LR, Drobatz KJ, Hunter CA, et al. Effects of CD28 blockade on subsets of naïve T cells in cats. Am J Vet Res 2005; 66:483492.

  • 44.

    Boyum A. A one stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1g gravity field. Scand J Clin Lab Invest Suppl 1968; S97:5176.

    • Search Google Scholar
    • Export Citation
  • 45.

    Gregory CR, Taylor NJ, Willits NH, et al. Response to isoantigens and mitogens in the cat: effects of cyclosporin A. Am J Vet Res 1987; 48:126130.

    • Search Google Scholar
    • Export Citation
  • 46.

    Haczku A, Alexander A, Brown P, et al. the effect of dexamethasone, cyclosporine and rapamycin on T-lymphocyte proliferation in vitro: comparison of cells from patients with glucocorticoid-sensitive and glucocorticoid-resistant chronic asthma. J Allergy Clin Immunol 1994; 93:510519.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Wells AD, Gudmundsdottir H, Turka LA. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J Clin Invest 1997; 100:31733183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Walker C, Malik R, Canfield PJ. Analysis of leucocytes and lymphocyte subsets in cats with naturally-occurring cryptococcosis but differing feline immunodeficiency virus status. Aust Vet J 2006; 72:9397.

    • Search Google Scholar
    • Export Citation
  • 49.

    Saggi BH, Fisher RA, Bu RA, et al. Intragraft cytokine expression and tolerance induction in rat renal allografts. Transplantation 1999; 67:206210.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Jiang H, Wynn C, Pan F, et al. Tacrolimus and cyclosporine differ in their capacity to overcome ongoing allograft rejection as a result of their differential abilities to inhibit interleukin-10 production. Transplantation 2002; 73:18081817.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Daniel V, Naujokat C, Sadeghi M, et al. Association of circulating interleukin (IL)-12 and IL-10 producing dendritic cells with time posttransplant, dose of immunosupression, and plasma cytokines in renal-transplant recipients. Transplantation 2005; 79:14981506.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Wang SC, Zeevi A, Jordan ML, et al. FK506, rapamycin and cyclosporine: effects on IL-4 and IL-10 mRNA levels in a T helper 2 cell line. Transplant Proc 1991; 23:29202922.

    • Search Google Scholar
    • Export Citation
  • 53.

    Salicru AN, Sams CF, Marshall GD. Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells. Brain Behav Immun 2007; 21:913920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54.

    Kuga K, Nishifuji K, Iwasaki T. Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells. J Vet Med Sci 2008; 70:10111016.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55.

    Kim W, Cho ML, Kim SI, et al. Divergent effects of cyclosporine on Th1/Th2 type cytokines in patients with severe, refractory rheumatoid arthritis. J Rheumatol 2000; 27:324331.

    • Search Google Scholar
    • Export Citation
  • 56.

    Halloran PF, Leung Lui S. Approved immunosupressants. In: Primer on transplantation. Thorofare, NJ: American Society of Transplant Physicians, 1998;93102.

    • Search Google Scholar
    • Export Citation
  • 57.

    Kahan BD, Yoshimura N, Pellis NR, et al. Pharmacodynamics of cyclosporine. Transplant Proc 1986; 18(suppl 5):238251.

  • 58.

    Bessler H, Kagazanov S, Punsky I, et al. Effect of dexamethasone on IL-10 and Il-12p40 production in newborns and adults. Biol Neonate 2001; 80:262266.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 59.

    Torres KCL, Antonelli LRV, Souza ALS, et al. Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC. J Neuroimmunol 2005; 166:144157.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 60.

    Visser J, van Boxel-Dezaire A, Methorst D, et al. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood 1998; 91:42554264.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 61.

    Martinez OM, Villanuera JC, Lawrence-Miyasaki L, et al. Viral and immunologic aspects of Epstein-Barr virus infection in pediatric liver transplant recipients. Transplantation 1995; 59:519524.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 62.

    Stylianou E, Aukrust P, Kvale D, et al. IL-1 in HIV infection: increasing serum IL-10 levels with disease progression-down regulatory effects of potent anti-retroviral therapy. Clin Exp Immunol 1999; 116:115120.

    • Search Google Scholar
    • Export Citation
  • 63.

    Reuter H, Burgess LJ, Carstens ME, et al. Characterization of the immunological features of tuberculosis pericardial effusions in HIV positive and HIV negative patients in contrast with non-tuburculosis effusions. Tuberculosis 2006; 86:125133.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 64.

    Maxwell L, Singh JL. Abatacept for rheumatoid arthritis. Cochrane Database Syst Rev 2009; 7:CD007277.

  • 65.

    Ponder KP, Wang B, Wang P, et al. Mucopolysaccharidosis I cats mount a cytotoxic T lymphocyte response after neonatal gene therapy that can be blocked with CTLA4-Ig. Mol Ther 2006; 14:513.

    • Crossref
    • Search Google Scholar
    • Export Citation

Advertisement

Effect of cyclosporine, dexamethasone, and human CTLA4-Ig on production of cytokines in lymphocytes of clinically normal cats and cats undergoing renal transplantation

Lillian R. AronsonDepartments of Clinical Studies-Philadelphia

Search for other papers by Lillian R. Aronson in
Current site
Google Scholar
PubMed
Close
 VMD
,
Jason S. StumhoferPathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Search for other papers by Jason S. Stumhofer in
Current site
Google Scholar
PubMed
Close
 PhD
,
Kenneth J. DrobatzDepartments of Clinical Studies-Philadelphia

Search for other papers by Kenneth J. Drobatz in
Current site
Google Scholar
PubMed
Close
 DVM
, and
Christopher A. HunterPathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Search for other papers by Christopher A. Hunter in
Current site
Google Scholar
PubMed
Close
 PhD

Abstract

Objective—To evaluate effects of cyclosporine, dexamethasone, and the immunosuppressive agent human CTLA4-Ig on cytokine production by feline lymphocytes in vitro and to assess patterns of cytokine production for 5 immunosuppressed renal transplant recipient cats.

Animals—21 clinically normal cats and 5 immunosupressed renal transplant recipient cats.

Procedures—Peripheral blood mononuclear cells were isolated from clinically normal cats and stimulated with concanavalin A (Con A; 10 μg/mL) alone or Con A with cyclosporine (0.05 μg/mL), dexamethasone (1 × 10−7M), a combination of cyclosporine-dexamethasone, or human CTLA4-Ig (10 g/mL). Cells from transplant recipients were stimulated with Con A alone. An ELISA was performed to measure production of interferon (IFN)-γ, granulocyte macrophage–colony stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, and IL-10. Proliferation of CD4+ and CD8+T cells from immunosuppressed cats were also evaluated. Pairwise comparisons were performed via a Wilcoxon signed rank test or Wilcoxon rank sum test.

Results—Cyclosporine, dexamethasone, cyclosporine-dexamethasone combination, and CTLA4-Ig caused a significant decrease in IL-2, IFN-γ, and GM-CSF production. Cyclosporine and cyclosporine-dexamethasone, but not human CTLA4-Ig, caused a significant decrease in IL-10 production. High basal concentrations of IL-2 and IL-10 were identified in transplant recipients, and IL-10 was significantly increased in stimulated cultures. In immunosuppressed cats, there was a decrease in frequency of responders and proliferative capacity of CD4+ and CD8+T cells.

Conclusions and Clinical Relevance—CTLA4-Ig successfully inhibited proinflammatory cytokines while sparing cytokines critical for allograft tolerance. These data may be useful for developing better strategies to prevent rejection while sparing other immune functions.

Abstract

Objective—To evaluate effects of cyclosporine, dexamethasone, and the immunosuppressive agent human CTLA4-Ig on cytokine production by feline lymphocytes in vitro and to assess patterns of cytokine production for 5 immunosuppressed renal transplant recipient cats.

Animals—21 clinically normal cats and 5 immunosupressed renal transplant recipient cats.

Procedures—Peripheral blood mononuclear cells were isolated from clinically normal cats and stimulated with concanavalin A (Con A; 10 μg/mL) alone or Con A with cyclosporine (0.05 μg/mL), dexamethasone (1 × 10−7M), a combination of cyclosporine-dexamethasone, or human CTLA4-Ig (10 g/mL). Cells from transplant recipients were stimulated with Con A alone. An ELISA was performed to measure production of interferon (IFN)-γ, granulocyte macrophage–colony stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, and IL-10. Proliferation of CD4+ and CD8+T cells from immunosuppressed cats were also evaluated. Pairwise comparisons were performed via a Wilcoxon signed rank test or Wilcoxon rank sum test.

Results—Cyclosporine, dexamethasone, cyclosporine-dexamethasone combination, and CTLA4-Ig caused a significant decrease in IL-2, IFN-γ, and GM-CSF production. Cyclosporine and cyclosporine-dexamethasone, but not human CTLA4-Ig, caused a significant decrease in IL-10 production. High basal concentrations of IL-2 and IL-10 were identified in transplant recipients, and IL-10 was significantly increased in stimulated cultures. In immunosuppressed cats, there was a decrease in frequency of responders and proliferative capacity of CD4+ and CD8+T cells.

Conclusions and Clinical Relevance—CTLA4-Ig successfully inhibited proinflammatory cytokines while sparing cytokines critical for allograft tolerance. These data may be useful for developing better strategies to prevent rejection while sparing other immune functions.

Renal transplantation is the most successful treatment option for cats with end-stage renal disease, but its success is dependent on the use of global immunosuppression to prevent allograft rejection.1,2 Immunosuppressive treatment currently used in cats consists of the calcineurin inhibitor cyclosporine in combination with the corticosteroid prednisolone.3–5 Similar to its use in human medicine, this treatment regimen is complicated because it can also impair host defense mechanisms and result in the emergence of various opportunistic infections as well as cancer.6–11 Investigators have evaluated the prevalence of infection and cancer in cats after renal transplantation. Forty-seven infections (bacterial, viral, fungal, protozoal, or a mixture of these) were detected in 43 of 169 cats,6 and the incidence of cats that developed cancer after surgery was 10%.10,11 Additionally, despite modern immunosuppressive treatment, episodes of acute rejection in cats of between 13% and 26% have been reported for clinical studies.12,13 Thus, there is a need to understand how to prevent rejection without compromising the immunosurveillance required to limit opportunistic infections and cancer.

The clinical and histologic features of renal allograft rejection have been characterized in cats.14 Immunohistochemical analysis performed on rejected kidneys revealed that similar to humans, the inflammatory infiltrate was dominated by lymphocytes with subpopulations consisting mostly of CD4+ (Th) cells and a few CD8+ (T-cytotoxic) cells. A sparse number of macrophages was also evident in areas of inflammation. Activated Th cells can be subdivided into multiple categories (Th1, Th2, Th17, and Treg) on the basis of function and the cytokines they produce.15–22 Several cytokines (eg, IFN-γ, IL-2, IL-4, IL-10, and GM-CSF) have been implicated in experimental animals and in humans as playing a role in acute and chronic allograft rejection.23–28 The Th1 cytokines (including IFN-γ and IL-2) contribute to rejection by activating the cellular immune response (including cytotoxic T cells, monocytes, and natural killer cells). It is believed that IL-2 is a driving force for acute cellular rejection; however, it is only 1 component of the rejection process. In contrast, IL-4 and IL-10 are pivotal in humoral responses and may play a role in the vasculopathy and fibrosis associated with chronic rejection or allograft tolerance.24,25,29

With the many drawbacks associated with the immunosuppressive treatments currently used, research in the field of transplantation immunology has focused on the development of strategies that would prevent graft rejection but that do not cause global immunosuppression associated with increased susceptibility to infection. It currently is proposed that naïve T cells require 2 distinct signals for activation. The first signal is through the interaction of the T-cell receptor with its cognate antigen presented in the context of the major histocompatibility complex on the surface of APCs.30–32 The second, or costimulatory, signal is not antigen specific and is between various receptors and their ligands.31 Many molecules have been identified that can serve as receptors for costimulatory signals, including the CD28 molecule on T cells and its counter receptors, which include the B7–1 (CD80) and B7–2 (CD86) molecules on APCs.31,32 The interaction between B7 on APCs with CD28 on T cells is one of the most important costimulatory interactions for T-cell activation. Activated T cells then direct immune responses through the production of cytokines, which in turn induce the activation and growth of other immune effector cells. The T cells that receive the first signal without receiving the second signal become anergic and are not able to respond to their cognate antigen. Therefore, 1 promising strategy to limit T-cell responses is the transient blockade of costimulatory signals that are essential for activation of naïve T cells required for graft rejection.30

The novel immunosuppressant human CTLA4-Ig is a potent antagonist of the B7-CD28 interaction. In animals experimentally used to evaluate transplantation, human CTLA4-Ig has been found to inhibit acute rejection and significantly prolong survival of transplanted organs.33–42 Additionally, in a study43 conducted by our research group, we found that compared with the effects of cyclosporine, human CTLA4-Ig had a sparing effect on antigen-specific proliferation of both CD4+ and CD8+ memory cells. These results suggest that human CTLA4-Ig may prevent immune responses to novel antigens such as grafts but that it may leave memory responses to previously encountered pathogens intact. In the aforementioned study43 conducted by our research group, we focused on T-cell proliferation in cats; however, to our knowledge, the effects of immunosuppressive drugs on cytokine production have not been evaluated. The purpose of the study reported here was to determine the effect of immunosuppressive drugs on cytokine production by lymphocytes of clinically normal cats and to assess whether these variables are altered in cats that have undergone renal transplantation.

Materials and Methods

Animals–-Twenty-one healthy domestic shorthair cats that ranged from 1 to 3 years of age and 5 client-owned cats that underwent renal transplantation at the University of Pennsylvania School of Veterinary Medicine because of chronic renal failure were included in the study. The 5 cats that underwent renal transplantation included 3 castrated male domestic shorthair cats, 1 castrated male Siamese, and 1 spayed female domestic shorthair cat; these cats ranged from 9 to 15 years of age, and all 5 of them were receiving immunosuppressive treatment consisting of administration of cyclosporine in combination with prednisolone. Approval by the Institutional Animal Care and Use Committee at the University of Pennsylvania for the study as well as owner consent for participation of the 5 renal transplant recipients was obtained.

Blood samples were obtained via jugular venipuncture. Samples (3 to 4 mL) were collected into tubes containing EDTA, and PBMCs were isolated via density gradient centrifugation by use of a technique described elsewhere,44 with slight modifications. Briefly, PBMCs were isolated by use of Ficoll gradient solutiona (density, 1.077) and centrifugation at 250 × g for 20 minutes at 22°C. The mononuclear cell layer was washed twice in DPBSS without calcium and magnesiumb and centrifuged at 184 × g for 10 minutes. The pellet was resuspended in 800 μL of DPBSS.

Lymphocyte culture and analysis–-Culture medium consisted of RPMI mediumc (250 mL) supplemented with 2.5% fetal calf serumd (50 mL) as well as 100 U of penicillin/mL and 0.1 mg of streptomycin/mL. Stock solutions of a costimulation-dependent mitogen (Con Ae [200 μg/mL]) and the immunoglobulin fusion protein human CTLA4-Igf (1 mg/mL) were used at a concentration of 10 μg/mL. Cyclosporineg (50 mg/mL) was used at a concentration of 0.05 μg/mL, and dexamethasone (4 mg/mL) was used at a concentration of 1 × 10−7M. Dosages were based on results of other studies.45,46

The PBMCs obtained from the clinically normal cats were plated in triplicate at a concentration of 2 × 106 cells/mL; 100 μL (2 × 105 cells) was placed in each well. Cells were cultured in medium alone, stimulated with Con A alone, or stimulated with Con A and human CTLA4-Ig, cyclosporine, dexamethasone, or a combination of cyclosporine plus dexamethasone. Cells from cats that underwent renal transplantation were labeled with CFSEh (0.8mM), as described in another study.47 These cells were plated in triplicate at concentrations of >1 × 106 cells/mL (4 cats) or 2 × 106 cells/mL (1 cat). Cells were plated with medium alone or were stimulated with Con A.

Flow cytometry–-Cells from cats that underwent renal transplantation were labeled with mouse anti-feline CD4-R-phycoerythrin conjugatei (0.1 mg/mL) or mouse anti-feline CD8-R-phycoerythrin conjugatei (0.1 mg/mL). Briefly, cells were washed with FACS buffer (1% BSA and 0.01% NaN3 in 1× PBS solution) and then incubated to block Fc receptors (5 μL of rat immunoglobulin, 890 μL of FACS buffer, 5 μL of mouse serum, and 100 μL of an FcR blocker [24G2]) for 30 minutes at 4°C. Cells were labeled with anti-CD4+ or anti-CD8+ monoclonal antibodies by incubation for 20 minutes at 4°C; cells then were washed once and analyzed with an FACS cytometer.j The percentage of cells proliferating in culture (responder frequency) and the mean number of daughter cells produced per proliferating cell (proliferative capacity) were determined for both CD4+ and CD8+ T cells, as described elsewhere.43

Cytokine measurement–-A feline-specific ELISAk was used to measure concentrations of IFN-γ, IL-2, IL-10, IL-4, and GM-CSF in tissue culture supernatants. An ELISA plate for each antibody was coated with a capture antibody diluted in 1× PBS solution specific for each cytokine. Wells containing the capture antibody were allowed to incubate overnight at 4°C; then, each plate was washed 5 times with wash buffer.k Each plate was blocked by the addition of a block bufferk (1% BSA in DPBSS) specific for each cytokine and incubation for 2 hours at 37°C. Plates were washed as previously described. Samples (100 μL) and standards (100 μL) in reagent diluentk (1% BSA in PBS solution for IL-2, IL-10, and GM-CSF; 0.1% BSA and 0.05% Tween 20 in tris-buffered saline solution for IL-4 and IFN-γ) were added to appropriate wells, and plates were incubated overnight at 4°C. Plates then were washed as previously described. Detection antibodyk (100 μL) diluted in reagent diluent was placed in each well, and plates were incubated for 2 hours at 22°C. Plates were washed as previously described. Streptavidin–horseradish peroxidasek (100 μL) was added to each well, and the plates were covered for 30 minutes at 22°C. Plates then were washed as previously described. Substrate solutionl (100 μL) was added to each well, and plates were incubated in the dark for 20 minutes at 22°C. Stop solutionl (100 μL) was added to each well. Plates were evaluated by use of an ELISA plate reader and commercial software.m Optimal time for cytokine evaluation was 24 hours after start of culture for IL-2 and 96 hours after start of culture for IFN-γ, IL-10, IL-4, and GM-CSF.

Statistical analysis–-Pairwise comparisons were performed by use of a Wilcoxon signed rank test or a Wilcoxon rank sum test. Significance was set at values of P >0.05. For the first part of the study, results for blood obtained from 21 cats were used. Power analysis revealed that for 21 cats, a difference of 1 SD could be detected between groups (99% power).

Results

To assess the effects of the currently used immunosuppressive treatment as well as human CTLA4-Ig on cytokine production, lymphocytes from 21 clinically normal cats were incubated in medium alone (unstimulated cultures) or in medium containing Con A with or without cyclosporine, dexamethasone, a combination of cyclosporine plus dexamethasone, or human CTLA4-Ig. In unstimulated cultures, basal concentrations of all cytokines ranged from 0 to 0.088 ng/mL. Stimulation with Con A alone did not result in significant production of IL-4 (data not shown). However, stimulation with Con A alone induced the production of IFN-γ (mean, 0.609 ng/mL), IL-2 (mean, 0.615 ng/mL), IL-10 (mean, 0.256 ng/mL), and GM-CSF (mean, 0.685 ng/mL). The addition of cyclosporine, the combination of cyclosporine plus dexamethasone, or human CTLA4-Ig to these cultures led to significant suppression of IFN-γ, IL-2, and GM-CSF concentrations. Addition of dexamethasone alone caused significant suppression of only GM-CSF concentrations.

In contrast, although the addition of human CTLA4-Ig led to significant suppression of IFN-γ, IL-2, and GM-CSF concentrations, human CTLA4-Ig did not significantly suppress IL-10 concentrations. Moreover, whereas cyclosporine and the combination of cyclosporine plus dexamethasone caused a significant decrease in IL-10 production, addition of dexamethasone alone significantly enhanced production of IL-10 from feline lymphocytes (Figure 1). Cyclosporine alone and the combination of cyclosporine plus dexamethasone had a significantly greater effect, compared with the effect for human CTLA4-Ig, on decreasing the production of IL-10, GM-CSF, IFN-γ, and IL-2 in the cell cultures in response to Con A. These results are consistent with the hypothesis that the immunosuppressive treatment currently in use in cats has global inhibitory effects on cytokine production.

Figure 1—
Figure 1—

Box-and-whiskers plots for concentrations of IFN-γ (A), GM-CSF (B), IL-2 (C), and IL-10 (D) in lymphocytes (obtained from 21 clinically normal cats) cultured with medium alone, medium that contained Con A alone, or medium that contained Con A in combination with cyclosporine (CyA), dexamethasone (Dex), a combination of cyclosporine plus dexamethasone (CyA-Dex), or human (hu)CTLA4-Ig. A feline-specific ELISA was used to measure cytokine concentrations in the supernatants. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line within each box represents the median, and the whiskers represent values between the 10th and 90th percentiles. Black circles represent outliers. *Median value differs significantly (P >0.05) from the median value for Con A–treated cells.

Citation: American Journal of Veterinary Research 72, 4; 10.2460/ajvr.72.4.541

Analysis of the aforementioned results revealed the effect of short-term exposure of these immunosuppressants on in vitro cytokine production. To determine whether these inhibitory effects were reflected in vivo, the PBMC response and cytokine production of the 5 cats that underwent renal transplantation because of chronic renal failure and were receiving cyclosporine in combination with prednisolone were compared with results for the 21 clinically normal cats. All transplant recipients were receiving cyclosporine (dose range, 1.2 to 8.9 mg/kg, q 24 h) and prednisolone (0.18 to 0.75 mg/kg, q 24 h) to provide immunosuppression and aid in prevention of transplant rejection, and 4 of the 5 cats had cyclosporine blood concentrations of 158 to 324 ng/mL (therapeutic range, 250 to 500 ng/mL). One cat had a blood concentration of <3,000 ng/mL. Duration of immunosuppressive treatment for these cats ranged from 3 to 25 months.

A CFSE dilution was used to assess lymphocyte proliferation. Following staining with CFSE, cells were plated with medium alone or were stimulated with Con A, and cytokine concentrations were evaluated in the supernatant at 24 (IL-2) or 96 (IFN-γ, IL-10, IL-4, and GM-CSF) hours after the start of culture. Because of differences in the number of lymphocytes available for plating, data of all cats were normalized and expressed as pg of cytokine/1,000 cells. High basal concentrations of IL-2 were identified in cats that underwent transplantation; however, for stimulation with Con A, there was no significant difference between clinically normal and immunosuppressed cats (Figure 2). Additionally, compared with results for clinically normal cats, IL-10 production was significantly increased in both unstimulated and stimulated cultures for cats that underwent renal transplantation. Finally, there was no significant differences between clinically normal and immunosuppressed cats with regard to the production of IFN-γ and GM-CSF in both unstimulated and stimulated cultures.

Figure 2—
Figure 2—

Box-and-whiskers plots for concentrations of IFN-γ (A), GM-CSF (B), IL-2 (C), and IL-10 (D) in lymphocytes (obtained from 21 clinically normal cats and 5 cats that received a renal transplant) cultured with medium alone (left) or medium that contained Con A (right). Notice that the scale on the y-axis differs between culture conditions for each cytokine. *Within a culture condition, median value differs significantly (P >0.05) from the median value for the clinically normal cats. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 4; 10.2460/ajvr.72.4.541

In addition to the cytokine data, CFSE labeling and flow cytometry were used to assess CD4+ and CD8+ T-cell responses for cats receiving long-term immunosuppressive treatment. For comparison purposes, information from the 5 transplant recipients was compared with results for clinically normal cats reported in another study.48 In the 5 immunosuppressed cats, the CD4+ subset comprised 18.2% to 31.9% (median, 22.9%; reference range, 17.4% to 49.5% [median, 32.3%]) and the CD8+ subset comprised 12.1% to 31.9% (median, 21.5%; reference range, 6.5% to 39.3% [median, 18.7%]) of the total lymphocytes collected (Figure 3). Although percentages for both CD4+ and CD8+ subsets were within the respective reference ranges, a mean of only 15.6% (range, 8.4% to 20%) of the CD4+ T cells and 19.1% (range, 9.2% to 44.8%) of the CD8+ T cells proliferated in response to stimulation with Con A (Figure 4). Additionally, for stimulation with Con A, there was a decrease in the responder frequency and proliferative capacity of both CD4+ (responder frequency, 8.5%; proliferative capacity, 2 daughter cells) and CD8+ (responder frequency, 13.3%; proliferative capacity, 2 daughter cells) T cells in immunosuppressed cats, compared with values for CD4+ (responder frequency, 34%; proliferative capacity, 3 daughter cells) and CD8+ (responder frequency, 33%; proliferative capacity, 6 daughter cells) T cells in clinically normal cats. The effect on both CD4+ and CD8+ subsets suggested that immunosuppressive treatment currently used for feline renal transplant recipients resulted in global immunosuppression.

Figure 3—
Figure 3—

Graph of FACS results for CD4+ (A) and CD8+ (B) T cells obtained from a typical immunosuppressed cat (ie, a cat that underwent renal transplantation and was receiving immunosuppressive treatment that consisted of cyclosporine in combination with prednisolone). The CD4+ subset comprised a median of 18.7% (reference range, 17.4% to 49.5% [median, 32.3%]) and the CD8+ subset comprised a median of 20.8% (reference range, 6.5% to 39.3% [median, 18.7%]) of the total lymphocytes collected. Along the y-axis, SSC refers to side scatter. The intensity of side-scattered light is related to the granularity of the particle evaluated. Along the x-axis, PE is a fluorescent dye used for cell surface labeling. The x-axis displays the increasing intensity of the fluorescence of CD4+ and CD8+ T cells.

Citation: American Journal of Veterinary Research 72, 4; 10.2460/ajvr.72.4.541

Figure 4—
Figure 4—

Proliferation profiles of CD4+ (A) and CD8+ (B) T cells obtained from a clinically normal cat (top row) and a renal transplant recipient cat (bottom row) and that were cultured in medium alone (right) and medium with the addition of Con A (left). Notice that the scale on the y-axis differs among culture conditions within lymphocyte subsets. In the clinically normal cat, 85.8% of CD4+T cells and 72.3% of CD8+T cells proliferated in the medium with Con A. In contrast, only 7.2% of CD4+ T cells and 48.2% of CD8+T cells proliferated in the renal transplant recipient cat, which was immunosuppressed because of long-term treatment with cyclosporine in combination with prednisolone. On the x-axis, CFSE is a fluorescent dye used to assess lymphocyte proliferation. A shift of the proliferation profile to the left indicates a decrease in fluorescence following each cell division.

Citation: American Journal of Veterinary Research 72, 4; 10.2460/ajvr.72.4.541

Discussion

It has been established in human medicine and through the use of experimental models that cytokines play an important role in immune defense, immunopathologic processes, and allograft rejection. Although information exists for animals used in transplantation experiments37,38,49,50 as well as for in vivo15,17,23,28,29,51 and in vitro25,52,53 studies in humans regarding the effects of immunosuppressive treatment on cytokines critical to the rejection process, limited information currently exists for these effects in cats. In 1 report,54 the effects of cyclosporine on the expression of inflammatory cytokines for PBMCs in 5 cats was evaluated by the use of Con A as a stimulus. Real-time PCR assay was used to determine that expression of mRNA for IL-2, IL-4, IFN-γ, and tumor necrosis factor-α was inhibited by cyclosporine in a dose-dependent manner.54 Those results are in agreement with results of the study reported here in which we found that the addition of cyclosporine to Con A-stimulated cultures led to significant suppression of IFN-γ and IL-2 concentrations. In addition, other cytokines critical to the rejection process as well as cytokines from cats that underwent renal transplantation and were receiving long-term immunosuppressive treatment were evaluated in the present study.

Cyclosporine in combination with corticosteroids has been an essential component in many immmunosuppressive protocols for organ transplantation in humans and cats. Cyclosporine acts by inhibiting calcineurin, thus preventing the activation of transcription factors that regulate the expression of cytokines (including IL-2, IL-4, IFN-γ, tumor necrosis factor-α, and GM-CSF) and genes with a role in allograft rejection.55–57 Although these cytokines are also inhibited by corticosteroids, the exact mechanism of action of corticosteroids is not fully understood. The cytokines IFN-γ, IL-2, and GM-CSF are potential therapeutic targets for renal transplantation. Interferon-γ and IL-2 are Th1 cytokines that are thought to play a critical role in acute allograft rejection in humans. In addition, GM-CSF is a cytokine that functions as a WBC growth factor that stimulates the proliferation and differentiation of hematopoeitic stem cells into granulocytes and macrophages responsible for responding to foreign tissue (eg, an allograft). Inhibition of these cytokines by immunosuppressive treatment is thought to be critical to graft survival. In the study reported here, which involved the use of lymphocytes from clinically normal cats, cyclosporine caused a significant decrease in the production of IFN-γ, IL-2, and GM-CSF, compared with results for Con A alone. Similar to results for humans, our data support a similar mechanism of action for cyclosporine in cats. Dexamethasone alone suppressed the production of only GM-CSF but not the production of IFN-γ or IL-2. These latter results are consistent with results in another study43 conducted by our research group in which it was found that dexamethasone alone did not significantly suppress lymphocyte proliferation in cats. Results from that study43 and the study reported here suggest that a different mechanism of action for dexamethasone may exist in cats. Immunosuppressive treatment used in the cats of the present report consisted of prednisolone in combination with cyclosporine. When dexamethasone was combined with cyclosporine in vitro, a significant decrease in the production of IFN-γ, IL-2, and GM-CSF was detected.

Compared with results for other cytokines, the results for IL-10 did not match with an expected profile. Cyclosporine caused a significant decrease in IL-10 production in the cats, although not to the degree of the aforementioned cytokines. This is in contrast to the finding in many reports49–52 in humans that indicated cyclosporine does not suppress IL-10 production. Interleukin-10 is a cytokine with immunosuppressive and anti-inflammatory properties that, in some transplantation studies,49,51 has been thought to play a role in allograft tolerance. The ability to suppress proinflammatory factors while sparing IL-10 production would be beneficial for renal transplant recipients. However, an increase in IL-10 expression in allografts undergoing rejection has been identified in some studies.24,29 Conflicting information also exists in the literature with regard to corticosteroids; some studies58,59 have revealed an inhibition of IL-10 production, whereas other studies53,60 have found the opposite effect. In the study reported here, dexamethasone alone did not suppress IL-10 production; however, when dexamethasone was combined with cyclosporine, there was a significant decrease in the production of IL-10.

Similar to findings reported in the human literature, the addition of human CTLA4-Ig to feline lymphocytes resulted in a significant decrease in the production of IL-2, IFN-γ, and GM-CSF but not IL-10. These results support findings in another study43 conducted by our research group in which we found a greater effect of cyclosporine alone and cyclosporine in combination with dexamethasone on lymphocyte proliferation, compared with the effects for human CTLA4-Ig. Because of the potential role in allograft tolerance, sparing of IL-10 in feline renal transplant recipients while suppressing proinflammatory cytokine production may be beneficial to allograft survival and provides justification for evaluation of human CTLA4-Ig as an immunosuppressant for cats. It is unclear as to how sparing of IL-10 would affect the immune response to opportunistic infections in feline renal transplant recipients. Studies in human transplant patients61 as well as patients with HIV62,63 have detected elevated serum IL-10 concentrations in patients with disease progression or those with stable disease.

Currently, human CTLA4-Ig (abatacept) is being used in humans for the treatment of rheumatoid arthritis.64 In a study65 conducted to evaluate the efficacy of neonatal gene therapy for the treatment of mucopolysaccharidosis I in cats, short-term (4 doses over a 2-week period) administration of human CTLA4-Ig to kittens at the time of gene therapy had no adverse effects and resulted in long-term tolerance to the canine enzyme α-L-iduronidase. Additionally, cats with mucopolysaccharidosis I that were treated with human CTLA4-Ig prior to vaccination with a killed parvovirus vaccine still developed a 16-fold increase in anti-parvovirus titers. These results suggest an appropriate memory response was generated with vaccination in the face of CTLA4-Ig.65 In contrast to the fact that the currently used immunosuppressive treatment caused global immuno-suppression, human CTLA4-Ig appears to have a sparing effect on antigen-specific proliferation of memory CD4+ and CD8+ T cells. Although unproven, the suppression of both naïve T-cell function as well as memory T-cell function by current immunosuppressive treatment may be associated with an increased incidence of opportunistic infections and neoplasia in cats that have undergone renal transplantation.

Cyclosporine and corticosteroids are immunosuppressive drugs used commonly in veterinary medicine to treat a variety of disorders; however, minimal information currently exists regarding their mechanisms of action in patients. Renal transplantation has been performed for <20 years in cats, and cyclosporine and corticosteroids are the mainstay of immunosuppressive treatment to prevent allograft rejection. It might be expected that cytokine production for cats receiving long-term immunosuppressive treatment would mirror results from the first part of the present study in which the combination of cyclosporine and dexamethasone was added to naïve cells from clinically normal cats; however, this was not the case. Concentrations of Th1 cytokines, including IL-2 and IFN-γ, that are thought to play a key role in acute allograft rejection were not significantly decreased in cats receiving long-term immunosuppressive treatments. Interestingly, IL-10 concentrations were significantly increased in both unstimulated and stimulated cultures. The increase in expression of IL-10 identified in our stable allograft patients suggests that IL-10 may be associated with allograft tolerance in cats. Our results do not support findings of elevated IL-10 concentrations in patients having a rejection episode.24

Finally, compared with results for clinically normal cats, evaluation of CD4+ and CD8+ T-cell responses for cats receiving long-term immunosuppressive treatment revealed a decrease in both the CD4+ and CD8+ T-cell subsets. Additionally, there was a decrease in the responder frequency of CD4+ but not CD8+ T cells. These results support findings in humans that the immunosuppressive agents cyclosporine and prednisolone have an additive effect in suppression of T lymphocytes and their subsets and that cyclosporine more selectively inhibits CD4+ T cells than it does CD8+ T cells.56,57

Results of the study reported here further elucidate the mechanism of action of immunosuppressive treatment currently used in feline renal transplant recipients. The immunosuppressive treatment currently used has many shortcomings and can result in an increase in morbidity and mortality rates as well as potentially decreasing life expectancy of the graft and recipient. Strategies targeted at blocking T-cell costimulation are novel approaches to immunoregulation. In the present study, human CTLA4-Ig was successful in inhibiting cytokines critical to the rejection process and sparing cytokines potentially critical for allograft tolerance. Data from this study support further investigation of human CTLA4-Ig in clinical patients.

ABBREVIATIONS

APC

Antigen presenting cell

BSA

Bovine serum albumin

CFSE

Carboxyfluoroscein succinimidyl-ester

Con

A Concavalin A

DPBSS

Dulbecco PBS solution

FACS

Fluoresence-activated cell sorting

GM-CSF

Granulocyte macrophage-colony stimulating factor

IFN

Interferon

IL

Interleukin

PBMC

Peripheral blood mononuclear cell

Th

T helper

a.

Histopaque, Sigma-Aldrich, St Louis, Mo.

b.

Mediatech Inc, Herndon, Va.

c.

Life Technologies, Gaithersburg, Md.

d.

HyClone Laboratories, Logan, Utah.

e.

Sigma-Aldrich Co, St Louis, Mo.

f.

Bristol Myers Squibb Research Institute, Princeton, NJ.

g.

Neoral, Novartis, East Hanover, NJ.

h.

Molecular Probes, Eugene, Ore.

i.

Southern Biotechnology Associates Inc, Birmingham, Ala.

j.

FACSCaliber flow cytometer, BD Biosciences, Mansfield, Mass.

k.

R&D systems, Minneapolis, Minn.

l.

KPL, Gaithersburg, Md.

m.

Soft Max Pro software, Molecular Devices Inc, Sunnyvale, Calif.

References

  • 1.

    Katayama M, McAnulty JF. Renal transplantation in cats: techniques, complications, and immunosupression. Compend Contin Educ Pract Vet 2002; 24:874882.

    • Search Google Scholar
    • Export Citation
  • 2.

    Katayama M, McAnulty. Renal transplantation in cats: patient selection and preoperative management. Compend Contin Educ Pract Vet 2002; 24:868872.

    • Search Google Scholar
    • Export Citation
  • 3.

    Bernsteen L, Gregory CR, Kyles AE, et al. Renal transplantation in cats. Clin Tech Small Anim Pract 2000; 15:4045.

  • 4.

    Gregory CR. Renal transplantation in cats. Compend Contin Educ Pract Vet 1993; 15:13251338.

  • 5.

    Gregory CR. Renal transplantation. In: Bojrab MJ, ed. Current techniques in small animal surgery. 4th ed. Baltimore: Williams & Wilkins, 1998;434.

    • Search Google Scholar
    • Export Citation
  • 6.

    Kadar E, Sykes JE, Kass PH, et al. Evaluation of the prevalence of infections in cats after renal transplantation: 169 cases (1987–2003). J Am Vet Med Assoc 2005; 227:948953.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Bernsteen L, Gregory CR, Aronson LR, et al. Acute toxoplasmosis following renal transplantation in three cats and a dog. J Am Vet Med Assoc 1999; 215:11231126.

    • Search Google Scholar
    • Export Citation
  • 8.

    Nordquist BC, Aronson LR. Pyogranulomatous cystitis associated with Toxoplasma gondii infection in a cat after renal transplantation. J Am Vet Med Assoc 2008; 232:10101012.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Griffin AG, Newton AL, Aronson LR, et al. Disseminated Mycobacterium avium complex infection following renal transplantation in a cat. J Am Vet Med Assoc 2003; 222:10971101.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Wooldridge J, Gregory CR, Mathews KG, et al. The prevalence of malignant neoplasia in feline renal transplant recipients. Vet Surg 2002; 31:9497.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Schmiedt CW, Grimes JA, Holzman G, et al. Incidence and risk factors for development of malignant neoplasia after feline renal transplantation and cyclosporine-based immunosuppression. Vet Comp Oncol 2009; 7:4553.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Mathews KG, Gregory CR. Renal transplants in cats: 66 cases (1987–1996). J Am Vet Med Assoc 1997; 211:14321436.

  • 13.

    Schmiedt CW, Holzman G, Schwarz T, et al. Survival, complications and analysis of risk factors after renal transplantation in cats. Vet Surg 2008; 37:683695.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Kyles AE, Gregory CR, Griffey SM, et al. Evaluation of the clinical and histological features of renal allograft rejection in cats. Vet Surg 2002; 31:4958.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Amirzargar A, Lessanpezeshki M, Fathi A, et al. Th1/Th2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc 2005; 37:29852987.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Saalmuller A. New understanding of immunological mechanisms. Vet Microbiol 2006; 117:3238.

  • 17.

    Rostaing L, Puyoo O, Tkaczuk J, et al. Differences in type 1 and type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuprression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients. Clin Transplant 1999; 13:400409.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Mosmann TR, Fowell DJ. The Th1/Th2 paradigm in infections. In: Kaufman SHE, Sher A, Ahmed R, eds. Immunology of infectious diseases. Washington, DC: ASM Press, 2002;163174.

    • Search Google Scholar
    • Export Citation
  • 19.

    Andre S, Tough DF, Lacroix-Desmazes S, et al. Surveillance of antigen presenting cells by CD4+CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications. Am J Pathol 2009; 174:15751587.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Hashimoto M, Sakaguchi S. Contribution of Th-1, Th-2, TH-17 or regulatory T cells to connective tissue diseases [in Japanese]. Nippon Rinsho 2009; 67:482486.

    • Search Google Scholar
    • Export Citation
  • 21.

    Miyara M, Wing K, Sakaguchi S. Therapeutic approaches to allergy and autoimmunity based on FoxP3+ regulatory T cell activation and expansion. J Allergy Clin Immunol 2009; 123:749755.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Turka LA. Normal immune responses. In: Norman DJ, Suki WN, eds. Primer on transplantation. Thorofare, NJ: American Society of Transplant Physicians, 1998;720.

    • Search Google Scholar
    • Export Citation
  • 23.

    Basak U, Mitra DK, Panigrahi A, et al. Clinical relevance of monitoring cytokine production following living donor renal transplantation. Transplant Proc 2003; 35:404406.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Poole KL, Gibbs PJ, Evans PR, et al. Influence of patient and donor cytokine genotypes on renal allograft rejection: evidence from a single centre study. Transplant Immunol 2001; 8:259265.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Li L, Yuhai Z. The relationship between cytokines in MLC supernatants and acute rejection after renal transplantation. Transplant Proc 2000; 32:25312534.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Divate SA. Acute renal allograft rejection: progress in understanding cellular and molecular mechanisms. J Postgrad Med 2000; 46:293296.

    • Search Google Scholar
    • Export Citation
  • 27.

    McLean AG, Hughes D, Welsh KI, et al. Patterns of graft infiltration and cytokine gene expression during the first 10 days of kidney transplantation. Clin Transplant 1997; 63:374380.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Sadeghi MI, Daniel V, Weimer R, et al. Differential early post-transplant cytokine responses in living and cadaver donor renal allografts. Transplantation 2003; 75:13511355.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Gibbs PJ, Sadek SA, Cameron C, et al. Immunomonitoring of renal transplant recipients in the early posttransplant period by analysis of cytokine gene expression in peripheral blood mono-nuclear cells. Transplant Proc 2001; 33:32853286.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970; 169:10421049.

  • 31.

    Najafian N, Sayegh MH. CTLA4-Ig: a novel immunosuppressive agent. Exp Opin Invest Drugs 2000; 9:21472157.

  • 32.

    Schaub M, Stadlbauer THW, Chandraker A, et al. Comparative strategies to induce long term graft acceptance in fully allogeneic renal versus cardiac allograft models by CD28-B7 T cell costimulatory blockade: role of thymus and spleen. J Am Soc Nephrol 1998; 9:891898.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997; 94:87898794.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Kurlberg G, Haglind E, Schon K, et al. Blockade of the B7-CD28 pathway by ctla4-Ig counteracts rejection and prolongs survival in small bowel transplantation. Scand J Immunol 2000; 51:224230.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Turka LA, Linsley PS, Lin H, et al. T cell activation by CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc Natl Acad Sci USA 1992; 89:1110211105.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36.

    Sayegh MH, Zheng XG, Magee C, et al. Donor antigen is necessary for the prevention of chronic rejection in CTLA4-Ig treated murine cardiac allograft recipients. Transplantation 1997; 64:16461650.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Russell ME, Hancock WW, Akalin E, et al. Chronic cardiac rejection in the LEW to F344 rat model. Blockade of CD28-B7 costimulation by CTLA4-Ig modulates T cell and macrophage activation and attenuates arteriosclerosis. J Clin Invest 1996; 97:833838.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Azuman H, Chandraker A, Nadeau K, et al. Blockade of T cell costimulation prevents development of experimental chronic renal allograft rejection. Proc Natl Acad Sci U S A 1996; 93:1243912444.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Lin H, Bollong SF, Linsley PS, et al. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4-Ig plus donor specific transfusions. J Exp Med 1993; 175:18011806.

    • Search Google Scholar
    • Export Citation
  • 40.

    Chandraker A, Azuma H, Nadeau K, et al. Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection. J Clin Invest 1998; 101:23092318.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Akalin E, Chandraker A, Russell ME, et al. CD28-B7 T cell costimulatory blockade by CTLA4-Ig in the rat renal allograft model. Transplantation 1996; 62:19421945.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Linsley PS, Ledbetter JA. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol 1993; 11:191212.

  • 43.

    Aronson LR, Drobatz KJ, Hunter CA, et al. Effects of CD28 blockade on subsets of naïve T cells in cats. Am J Vet Res 2005; 66:483492.

  • 44.

    Boyum A. A one stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1g gravity field. Scand J Clin Lab Invest Suppl 1968; S97:5176.

    • Search Google Scholar
    • Export Citation
  • 45.

    Gregory CR, Taylor NJ, Willits NH, et al. Response to isoantigens and mitogens in the cat: effects of cyclosporin A. Am J Vet Res 1987; 48:126130.

    • Search Google Scholar
    • Export Citation
  • 46.

    Haczku A, Alexander A, Brown P, et al. the effect of dexamethasone, cyclosporine and rapamycin on T-lymphocyte proliferation in vitro: comparison of cells from patients with glucocorticoid-sensitive and glucocorticoid-resistant chronic asthma. J Allergy Clin Immunol 1994; 93:510519.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Wells AD, Gudmundsdottir H, Turka LA. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J Clin Invest 1997; 100:31733183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Walker C, Malik R, Canfield PJ. Analysis of leucocytes and lymphocyte subsets in cats with naturally-occurring cryptococcosis but differing feline immunodeficiency virus status. Aust Vet J 2006; 72:9397.

    • Search Google Scholar
    • Export Citation
  • 49.

    Saggi BH, Fisher RA, Bu RA, et al. Intragraft cytokine expression and tolerance induction in rat renal allografts. Transplantation 1999; 67:206210.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Jiang H, Wynn C, Pan F, et al. Tacrolimus and cyclosporine differ in their capacity to overcome ongoing allograft rejection as a result of their differential abilities to inhibit interleukin-10 production. Transplantation 2002; 73:18081817.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Daniel V, Naujokat C, Sadeghi M, et al. Association of circulating interleukin (IL)-12 and IL-10 producing dendritic cells with time posttransplant, dose of immunosupression, and plasma cytokines in renal-transplant recipients. Transplantation 2005; 79:14981506.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Wang SC, Zeevi A, Jordan ML, et al. FK506, rapamycin and cyclosporine: effects on IL-4 and IL-10 mRNA levels in a T helper 2 cell line. Transplant Proc 1991; 23:29202922.

    • Search Google Scholar
    • Export Citation
  • 53.

    Salicru AN, Sams CF, Marshall GD. Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells. Brain Behav Immun 2007; 21:913920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54.

    Kuga K, Nishifuji K, Iwasaki T. Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells. J Vet Med Sci 2008; 70:10111016.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55.

    Kim W, Cho ML, Kim SI, et al. Divergent effects of cyclosporine on Th1/Th2 type cytokines in patients with severe, refractory rheumatoid arthritis. J Rheumatol 2000; 27:324331.

    • Search Google Scholar
    • Export Citation
  • 56.

    Halloran PF, Leung Lui S. Approved immunosupressants. In: Primer on transplantation. Thorofare, NJ: American Society of Transplant Physicians, 1998;93102.

    • Search Google Scholar
    • Export Citation
  • 57.

    Kahan BD, Yoshimura N, Pellis NR, et al. Pharmacodynamics of cyclosporine. Transplant Proc 1986; 18(suppl 5):238251.

  • 58.

    Bessler H, Kagazanov S, Punsky I, et al. Effect of dexamethasone on IL-10 and Il-12p40 production in newborns and adults. Biol Neonate 2001; 80:262266.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 59.

    Torres KCL, Antonelli LRV, Souza ALS, et al. Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC. J Neuroimmunol 2005; 166:144157.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 60.

    Visser J, van Boxel-Dezaire A, Methorst D, et al. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood 1998; 91:42554264.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 61.

    Martinez OM, Villanuera JC, Lawrence-Miyasaki L, et al. Viral and immunologic aspects of Epstein-Barr virus infection in pediatric liver transplant recipients. Transplantation 1995; 59:519524.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 62.

    Stylianou E, Aukrust P, Kvale D, et al. IL-1 in HIV infection: increasing serum IL-10 levels with disease progression-down regulatory effects of potent anti-retroviral therapy. Clin Exp Immunol 1999; 116:115120.

    • Search Google Scholar
    • Export Citation
  • 63.

    Reuter H, Burgess LJ, Carstens ME, et al. Characterization of the immunological features of tuberculosis pericardial effusions in HIV positive and HIV negative patients in contrast with non-tuburculosis effusions. Tuberculosis 2006; 86:125133.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 64.

    Maxwell L, Singh JL. Abatacept for rheumatoid arthritis. Cochrane Database Syst Rev 2009; 7:CD007277.

  • 65.

    Ponder KP, Wang B, Wang P, et al. Mucopolysaccharidosis I cats mount a cytotoxic T lymphocyte response after neonatal gene therapy that can be blocked with CTLA4-Ig. Mol Ther 2006; 14:513.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Address correspondence to Dr. Aronson (laronson@vet.upenn.edu).