Inositol 1,4,5-trisphosphate synthesis in mononuclear white blood cells of malignant hyperthermia-susceptible and normal human beings, following in vitro exposure to halothane, caffeine and ryanodine.

Despite a plethora of findings associated with the pathophysiology of malignant hyperthermia (MH), the in vitro contracture test (IVCT) is the only reliable test for diagnosis of this heterogeneous syndrome in man. An increase of 1,4,5-IP3 (inositol 1,4,5-trisphosphate), a second messenger involved in cellular calcium homeostasis, has been observed in muscle tissue of MH susceptible (MHS) patients. The aim of this study was to evaluate if the known differences of 1,4,5-IP3 content in muscle tissue might be reproduced in mononucleated white blood cells (MWBCs). Subsequently, MWBCs of 23 healthy controls and 12 patients with a clinical suspicion for MH disposition were isolated and screened for 1,4,5-IP3 content. An IVCT according to the protocol of the European Malignant Hyperpyrexia Group (EMHG) was performed on muscle specimens of 12 patients. Eight MHN and four MHS individuals were diagnosed. Additionally, 1,4,5-IP3 synthesis in MWBCs was detected following in vitro exposure to IVCT test substances halothane (2%), caffeine (1-30mM), and ryanodine (1-5 microM). A broad inter-individual variability of 1,4,5-IP3 content was observed in MWBCs of all volunteers, but no differences were detected between MHS and MHN individuals. These findings are in strong contrast to those observed in muscle tissue. In vitro exposure of isolated MWBCs to halothane, caffeine and ryanodine yielded no statistically significant differences between groups. A time- and concentration-dependent increase in cellular 1,4,5-IP3 content could be induced in some but not all individuals of both groups. Since no correlation was obtained between induction of 1,4,5-IP3-synthesis following in vitro exposure of MWBCs to MH test substances and MH disposition, this study was terminated. We conclude from our data that the detection of 1,4,5-IP3 synthesis in MWBCs is not suitable for diagnosis of MH disposition. It remains questionable whether an altered 1,4,5-IP3 metabolism in MWBCs is involved in pathologic cascades of MH. Therefore, other cell tissues should be evaluated in further studies to clarify the role of the 1,4,5-IP3 metabolism in MH.

Cytokine-mediated effects of peripheral blood mononuclear cells from patients with atopic eczema on keratinocytes (HaCaT) in a new coculture system.

Interactions between keratinocytes and mononuclear cells via cytokines and adhesion molecules are thought to play a crucial part in inflammatory skin diseases. The cytokine-mediated effects of peripheral blood mononuclear cells (PBMC) from patients with atopic eczema (AE) and healthy individuals on keratinocytes (HaCaT) were investigated in vitro. A new coculture model (Transwell system) which consists of a lower and an upper compartment, which are separated by a polycarbonate-treated membrane, was established. 3[H]thymidine incorporation of keratinocytes and lymphocytes, as well as IL-6, IL-8 and IFN-gamma synthesis, were measured. Keratinocyte proliferation was significantly enhanced in the presence of PBMC from patients with AE. In contrast, PBMC from normal donors did not enhance HaCaT cell proliferation when they were cocultured. Lymphocytes from patients with AE showed a significantly enhanced proliferation after coculture with keratinocytes. However, PBMC from normal donors did not proliferate in the presence of HaCaT cells. Keratinocyte supernatants incubated with PBMC from either atopic or normal volunteers induced a suppression of lymphocyte 3[H]thymidine incorporation. In supernatants from cocultures of PBMC from patients with AE and keratinocytes, significantly enhanced amounts of IL-6 and IL-8, compared with normal donor's lymphocytes and HaCaT cells, were measured. No differences in IFN gamma production were observed. When PBMC were cultured without HaCaT cells, supernatants contained equal levels of IL-6, IL-8 and IFN-gamma in normal donors and in patients with AE. Interestingly, HaCaT cells spontaneously secrete measurable amounts of IL-6, IL-8 and IFN-gamma. Blocking experiments with neutralizing antibodies against these interleukins showed a complete inhibition of keratinocyte proliferation when PBMC from normal donors were used whereas the proliferative potency of PBMC supernatants from patients with AE on keratinocytes remained. Our data indicate that (i) PBMC from patients with AE stimulate keratinocyte proliferation via soluble factor(s) that are different from IL-6, IL-8 and IFN-gamma; (ii) probably, HaCaT cells spontaneously produce lymphocyte/monocyte inhibitory soluble factors and IL-6, IL-8 as well as IFN-gamma; and (iii) secretion and/or activity of keratinocyte-derived inhibitory mediators is regulated via cytokines of PBMC infiltrating inflammatory skin.

Staphylococcal enterotoxin B affects in vitro IgE synthesis, interferon-gamma, interleukin-4 and interleukin-5 production in atopic eczema.

Peripheral blood mononuclear cells from patients with atopic eczema (AE) stimulated with the 'superantigen' Staphylococcus enterotoxin B (SEB) secreted significantly more interleukin (IL)-4 and IL-5 as well as IgE, and markedly less interferon-gamma than those from healthy controls. Our results support the assumption that SEB produced by S. aureus colonizing the skin of patients with AE may induce expansion of IL-4- and IL-5-producing Th2 clones, leading to increased IgE synthesis and eosinophil activation.