Intracellular cytokines in peripheral blood leucocytes in children with chronic renal failure.

We have previously shown that children with mild renal impairment show significant changes in leucocyte subsets and circulating cytokines, indicating that these patients show an increased inflammatory state. We hypothesised that measurement of intracellular cytokine production by lymphocytes and monocytes would more precisely define the immunological mechanism associated with the inflammatory state in children with pre-dialytic chronic renal failure. Blood was collected from children with chronic renal failure (CRF) who were not yet on dialysis and an age-matched control group. Leucocyte subsets and intracellular cytokine production were determined using flow cytometry. Children with CRF showed increased production of interleukin (IL)-12 by monocytes accompanied by decreased production of interferon (IFN)-gamma and increased production of IL-4 by T cells. There were no significant changes in the production of IL-8, IL-10, IL-6, IL-1alpha or tumour necrosis factor (TNF)-alpha by monocytes or in IL-2 or TNF-alpha production by T cells. There were no significant differences in total white cell count or lymphocyte count. There was a significant decrease in both B and NK cells. This study examines intracellular cytokine production in children with CRF in detail. It is the first to show that children with relatively mild renal failure display significant immunological changes of lymphocyte subsets and leucocyte cytokine production. These data provide a more accurate understanding of the immunological changes that may contribute to the clinical manifestations and progression of the disease.

Poststorage leuko-depleted plasma inhibits T-cell proliferation and Th1 response in vitro: characterization of TGFbeta-1 as an important immunomodulatory component in stored blood.

BACKGROUND: Poststorage, leuko-depleted blood transfusions have been associated with increased postoperative infections and improved allograft survival compared with prestorage leukocyte-depleted blood transfusion. Although the mechanism of this phenomenon remains to be fully elucidated, it is clear that the immunomodulatory effect is mediated by leukocytes/platelets or their products. METHOD: The aim of this study was to investigate the in vitro effects of pre- and poststorage leuko-depleted plasma (LDP) and buffy coat LDP on T-cell proliferation and cytokine synthesis using multiparameter flow cytometry. RESULTS: In cell cultures exposed to prestorage LDP and buffy coat LDP there were no significant changes compared with fresh blood. In cell cultures exposed to poststorage LDP, T-cells showed reduced expression of CD69, CD25 (IL-2Ralpha), CD122 (IL-2Rbeta) and CD132 (IL-2Rtau) and production of TNF-alpha and IL-2 but there was no significant alteration for IFN-tau or IL-4. Changes in cytokine/cytokine receptor synthesis and T-cell proliferation were shown to be directly proportional to poststorage LDP concentration. Some of these changes were characteristic of TGFbeta-1. Addition of TGFbeta-1 neutralising antibody to poststorage LDP, negated the immunosuppressive effect on PHA-stimulated PBMC cultures. CONCLUSIONS: The decrease in T-cell proliferation and Th1 cytokines TNF-alpha and IL-2, may be one basis of altered immunoregulation resulting in increased rates of certain types of infections and increased graft tolerance reported in patients receiving poststorage LD blood transfusions. TGFbeta-1 is a major immunomodulatory component of poststorage LD blood transfusions.

Changes in leukocyte subsets: clinical implications for children with chronic renal failure.

Increased systemic inflammation and an impaired immune response are features of adult chronic renal failure (CRF). These patients have increased rates of infection, cardiovascular disease, anemia, and malnutrition. We measured inflammatory and immunological markers in a group of children with pre-dialytic CRF. No prior studies have explored these markers even though children with non-dialysed CRF exhibit similar complications to those seen in adults with CRF. Blood was collected from children with mild, moderate, or severe CRF and an age-matched control group. Functional leukocyte subsets were determined using flow cytometry. Circulating levels of interleukin (IL)-1beta, IL-6, IL-8, IL-12, IL-10, and tumor necrosis factor-alpha were measured using a flow cytometric bead assay. Children with severe CRF showed significantly reduced total white cell count and absolute neutrophil and lymphocyte counts. Absolute numbers of CD3+/CD45RO+ memory T cells and CD3+/CD45RO+/CD62L+ memory Th2 cells were significantly reduced in all CRF groups versus controls. Children with severe CRF showed increased CD11b expression on neutrophils and monocytes. Some patients showed increases in pro-inflammatory cytokines that were not related to their level of residual renal function. As CD11b expression mediates leukocyte adhesion to vascular endothelium, upregulation may contribute to the increased endothelial dysfunction observed in children with CRF. L-selectin mediates extravasation of leukocytes into tissue and homing of peripheral blood lymphocytes to lymph nodes. The reduction in L-selectin may inhibit these actions and predispose patients to increased infection later in life. This is the first study to comprehensively investigate leukocyte functional molecules and inflammatory cytokine profiles in children with pre-dialytic CRF and provides new immunological evidence for the clinical manifestations associated with the disease.