Testing the “Cytokine-ome” Theory: A Longitudinal Pilot Study in Renal Transplant Recipients

Abstract

Cytokines function within a complex network. Their function relates to specific immunological processes that also promote autoimmunity, alloimmunity, chronic inflammation, and tissue destruction (1–6). Here, we tested the hypothesis that stable cytokine milieus (“cytokine-omes”) might define “signatures” of the cytokine network associated with pathology. To do so, we first measured the frequency of peripheral blood mononuclear cells (PBMCs) secreting the regulatory cytokine interleukin (IL)-10 in 10 healthy blood donors (7, 8). Two of 10 healthy individuals had a frequency of IL-10-secreting PBMCs of >1300/106 cells, and 8 of 10 healthy individuals had a frequency <600/106 cells (Fig. 1A). Next, the frequency of IL-10-secreting PBMCs was related to levels of homeostatic secretion of the inflammatory cytokines IL-1β, IL-8, IL-12, tumor necrosis factor (TNF)-α, and IL-2 (8). Secretion of these cytokines, with the exception of IL-8, was very low, and no co-segregation of IL-8 with the frequency of IL-10 secreting PBMCs was observed (data not shown).FIGURE 1.: A, Frequency of constitutive IL-10-secreting PBMCs. The frequency of IL-10-secreting PBMCs was polarized both in healthy donors (upper panel) and in kidney transplant recipients (lower panel), separating 2 nonoverlapping groups (Wilcoxon Sum-of-Ranks tests). After transplantation, the frequency of IL-10-secreting PBMCs decreased slightly in high-frequency individuals (as analyzed with the Student’s t-test); however, the groups remained well separated (Wilcoxon Sum-of-Ranks tests). B, Inflammatory cytokines in high vs. low IL-10-frequency kidney transplant recipients. Constitutive secretion of the cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, GM-CSF, and IL-2 was compared before and after transplantation in individuals with a high vs. a low frequency of IL-10-secreting PBMCs (Wilcoxon Sum-of-Ranks tests). Note the selective inverse correlation of IL-8 secretion and frequency of IL-10-secreting PBMCs.We then assessed the frequency of spontaneously IL-10-secreting PBMCs in a cohort of kidney transplant recipients. Patient characteristics relevant to this study are summarized in Supplementary Table 1, available for viewing online only. In these experiments, the following observations were made: (1) analogous to healthy donors, 4 of 12 patients had a pretransplant frequency of IL-10-secreting PBMCs of >1300/106 cells and 8 of 12 patients a pretransplant frequency <600/106 cells, (2) in each of the 4 transplant recipients with a high frequency of IL-10-secreting PBMCs, the frequency decreased after transplantation but remained high (posttransplant frequency: always >1300/106 cells), and (3) transplant recipients with a low frequency of IL-10-secreting PBMCs before transplantation had a stable and low frequency after transplantation (Fig. 1A). No association between the frequency of IL-10-secreting cells and total monocyte- or lymphocyte-counts was observed (Supplementary Table 1, available for viewing online only). Also, no correlation of acute-phase reaction (as reflected by levels of C-reactive protein) or frequency of IL-10 secreting cells was detected (Supplementary Table 1, available for viewing online only). At these same time points, the frequency of IL-10-secreting PBMCs was related to levels of homeostatic secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, granulocyte monocyte colony stimulating factor (GM-CSF), and IL-2 (8). Contrasting healthy donors, 2 patterns of cytokine secretion were observed: the group of patients with a high frequency of IL-10-producing PBMCs produced high amounts of IL-1β, IL-6, TNFα, and GM-CSF. Production of these cytokines was not influenced by immunosuppression. By contrast, patients with a low frequency of IL-10-producing PBMCs secreted large amounts of IL-8, whereas production of IL-1β, IL-6, TNF-α, and GM-CSF was low. Again, this cytokine-pattern was similar before and after transplantation. With respect to the constitutive production of IL-2 and IL-12, transplant patients did not differ (Fig. 1B). Together, these data suggest the existence of immunological milieus, exclusively observed in patients that developed end-stage renal disease, wherein inflammatory cytokines (usually thought of as a group) separate. Defining such immunological milieus (“cytokine-omes”) may prove to be an effective—and largely unexplored—approach when aiming at dissecting the “anatomy” of the complex cytokine network. Furthermore, knowledge of such differing cytokine milieus may prove relevant when interpreting cytokine levels both in physiology and pathology. ACKNOWLEDGMENTS We thank Matthias Mehling for technical assistance. O.G. is supported by the Swiss Foundation for Medical and Biological Grants and S.S. and C.H. are supported by grants from the Swiss National Science Foundation (3200B0-109302 and PP00B-114850, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Olivier Gasser Partners AIDS Research Center Massachusetts General Hospital Boston, MA Christoph Berger Gabriela Zenhaeusern Ineke Oehri Christoph Hess Immunobiology Laboratory University of Basel Basel, Switzerland Hanno Elsässer Ineke Oehri Stefan Schaub Christoph Hess Clinic for Transplantation-Immunology and Nephrology University Hospital Basel Basel, Switzerland