Intracellular cytokines and their therapeutic modulation in immunological disorders

This study investigated abnormalities of cytokines principally in common variable immunodeficiency (CVID), and the effects of the immunomodulatory agent intravenous immunoglobulin (IVIG) on cytokine regulation. To this end, a rapid, small-volume assay of intracellular cytokine synthesis was established for lymphocytes. For 50 μl aliquots of heparinised blood diluted 1:2, the optimum culture conditions were: PMA (10 ng/ml), ionomycin (2 μmol/l), and monensin (3 μmol/l) cultured for 2 h for assessment of IFN-γ, TNF-α, IL-4 and CD69 expression, and 4 h culture for IL-2 expression. Validation was performed in a variety of settings where abnormalities of cytokine regulation are known to occur, and then extended to investigate additional situations where cytokine irregularities have not been formally established. The high IFN-γ expression by CD8+ T cells in CVID was confirmed, γβ T cell IFN-γ expression in sarcoidosis was similar to that of CD8+ cells. A Th2-bias was confirmed in hyper-IgE syndrome. The immunomodulatory effects of drugs on cytokine regulation in CVID and atopic eczema, were studied. IVIG, in vitro and at two different dose ranges in vivo, and ciprofloxacin in vitro, were studied. Both these diseases are treated with IVIG. The in vitro studies of IVIG showed a small but significant reduction in IFN-γ expression in CVID CD4+ cells with increasing concentrations of IVIG from 15±1.87% with no IgG down to 10.5±1.7% with 10 mg/ml IgG p=0.006) and 8.8±2.6% with 20 mg/ml IgG (p=0.04). The replacement dose in vivo study revealed IVIG could increase the potential of CD8+28- cells to make TNF-α, and CD4+ lymphocytes to make IL-2 in patients with CVID. NK cell IFN-γ and monocyte IL-12 expression was not affected by IVIG in vivo. High dose IVIG in eczema patients resulted in CD69 expression in both CD4+ and CD8+ cells declining during the six months of hdIVIG therapy to approximately 60% of baseline values. Ciprofloxacin in vitro had no discernible effect on cytokine expression. High T cell IFN-γ levels in CVID may be caused by abnormal monocytes. Overnight incubation of lymphocytes with monocytes from CVID patients increased IFN-γ expression, yet this effect was not totally abrogated by anti-IL-12, suggesting that additional cytokines may be involved in the high IFN-γ expression. Since different T lymphocyte populations responded differently to therapeutic immunomodulation, 4-colour flow cytometry was used to examine activation markers in T lymphocyte subsets in CVID and normal subjects. Both absolute counts of various subsets of T cells, and the expression of the activation markers CD25 and HLA-DR were examined in peripheral blood. Although serum CD25 levels are elevated in CVID, an increase in CD25+ cells was not detected. HLA-DR+ cells were increased however, particularly in CD4+28- cells. DR expression was lower in XLA than in normal controls. The proportions of CD28+ and CD45RA+ cells were decreased in CVID. These findings were used to construct a model for cytokine dysregulation in CVID, and to highlight potential new areas for therapeutic immunomodulation of CVID, including the use of high-dose IVIG, and co-administration of dopamine receptor modulating drugs