D. De Groote

Tumour necrosis factor (TNF) gene polymorphism influences TNF-α production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans

TNF‐α is involved in infectious and immuno‐inflammatory diseases. Different individuals may have different capacities for TNF‐α production. This might determine a predisposition to develop some complications or phenotypes of these diseases. The aims of our study were to assess the inter‐individual variability of TNF‐α production and to correlate this variability to a single base pair polymorphism located at position −308 in TNF gene. We studied 62 healthy individuals. TNF‐α production after LPS stimulation was evaluated using a whole blood cell culture model. The TNF gene polymorphism was studied by an allele‐specific polymerase chain reaction. Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured. Among the 62 individuals, 57 were successfully genotyped. There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes. TNF‐α production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNF1 homozygotes (929 pg/ml (480–1473 pg/ml) versus 521 pg/ml (178–1307 pg/ml); P < 0.05). This difference was even more significant after correction of TNF‐α production for CD14 expression on blood cells. In conclusion, the single base pair polymorphism at position −308 in the TNF gene may influence TNF‐α production in healthy individuals.

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.

Increased production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by inflamed mucosa in inflammatory bowel disease

Inflammatory bowel diseases (IBD) are characterized by a sustained inflammatory cascade that gives rise to the release of mediators capable of degrading and modifying bowel wall structure. Our aims were (i) to measure the production of matrix metalloproteinase‐3 (MMP‐3), and its tissue inhibitor, tissue inhibitor of metalloproteinase‐1 (TIMP‐1), by inflamed and uninflamed colonic mucosa in IBD, and (ii) to correlate their production with that of proinflammatory cytokines and the anti‐inflammatory cytokine, IL‐10. Thirty‐eight patients with IBD, including 25 with Crohn’s disease and 13 with ulcerative colitis, were included. Ten controls were also studied. Biopsies were taken from inflamed and uninflamed regions and inflammation was graded both macroscopically and histologically. Organ cultures were performed for 18 h. Tumour necrosis factor‐alpha (TNF‐α), IL‐6, IL‐1β, IL‐10, MMP‐3 and TIMP‐1 concentrations were measured using specific immunoassays. The production of both MMP‐3 and the TIMP‐1 were either undetectable or below the sensitivity of our immunoassay in the vast majority of uninflamed samples either from controls or from those with Crohn’s disease or ulcerative colitis. In inflamed mucosa, the production of these mediators increased significantly both in Crohn’s disease (P < 0·01 and 0·001, respectively) and ulcerative colitis (P < 0·001 and 0·001, respectively). Mediator production in both cases was significantly correlated with the production of proinflammatory cytokines and IL‐10, as well as with the degree of macroscopic and microscopic inflammation. Inflamed mucosa of both Crohn’s disease and ulcerative colitis show increased production of both MMP‐3 and its tissue inhibitor, which correlates very well with production of IL‐1β, IL‐6, TNF‐α and IL‐10.

Direct stimulation of cytokines (IL-1β, TNF-α, IL-6, IL-2, IFN-γ and GM-CSF) in whole blood: II. Application to rheumatoid arthritis and osteoarthritis

Abstract Rheumatoid arthritis (RA) is an immune disease in which the pathological immune reaction is thought to be initiated by the presentation of an (auto) antigen or superantigen by MHC class II positive cells to CD4 T cells. These successive immunological events can be studied by the cytokines produced at the different stages. Cytokine secretion by stimulated cells in autologous diluted whole blood has allowed the study of the immune profile characteristic of rheumatoid arthritis. The pattern of RA patient whole blood cells cultured in autologous blood is characterized by hyperactivity of the mononuclear cells with high secretion of IL-1β, TNF-α and IL-6 and low production of IFN-γ, in comparison with the normal (N) and osteoarthrosis (OA) populations. The IL-2 secretion pattern is unique, arising from production followed by consumption. This production-consumption turnover is the most elevated in the RA group. The T cells are indeed activated in rheumatoid arthritis but regulatory events suppress some of their functions. A correlation was found between the inflammatory proteins and mediators of cellular immunity and macrophagic function: IL-1β and the sedimentation rate; IL-6 and fibrinogen; TNF-α and the number of blood monocytes. The secretion of OA-stimulated whole blood cells was similar to RA for two monokines (overproduction of TNF-α and IL-6) and different for IL-1β, not different from normal in OA. Stimulated whole blood cell cytokine secretion profile from RA and OA groups, was the same as previously observed in synovial fluid. This parallel in cytokine production between synovial fluid and whole blood stimulated cells, in RA, can be explained by immune induction occurring in the extraarticular lymphatic system, the immune reaction becoming systemic and articular expression being one of the targets of the stimulated immune cells.

A High Serum Concentration of Interleukin-6 Is Predictive of Relapse in Quiescent Crohn's Disease

Background/aims: Relapses of Crohns disease are difficult to predict. We assessed the value of serum level of interleukin‐6, tumour necrosis factor alpha (TNF‐&agr;) and soluble TNF receptors as predictors of relapse in quiescent Crohns disease. Patients/methods: Thirty‐six patients with inactive Crohns disease, treated or not, were included. Various clinical and biological parameters, including interleukin‐6, TNF‐&agr; and soluble TNF receptors serum levels were measured at inclusion in the study and the patients were followed clinically for 1 year. The relapse was defined as a Crohns Disease Activity Index (CDAI) greater than 150 with an increase greater than 100 compared to the inclusion value. We analysed the ability of these parameters to predict relapse in parallel to clinical characteristics and other laboratory parameters. Results: Among the 32 variables tested, interleukin‐6 serum level had the greatest ability to predict the time‐to‐relapse, with 17‐fold chance of relapse over a 1‐year period for patients with an interleukin‐6 serum level greater than 20 pg/ml than for patients with a lower level (P<0.001). A high serum level of the soluble TNF receptors p55 and p75 also had significant predictive value, in contrast to TNF‐&agr; serum levels. An interleukin‐6 serum level greater than 20 pg/ml and either an acid &agr;‐1glycoprotein level greater than 1.1 g/l or a soluble interleukin‐2 receptor serum level greater than 95 pM/l were risk factors selected by a stepwise multivariate analysis. In both models a good prognosis group was defined by the absence of the two risk factors, a bad prognostic group by the presence of the two risk factors and an intermediate in between. With both models, the good prognosis group included 17 patients who experienced no relapse over the 1‐year follow‐up, whereas all patients (seven with the first model and six with the second) in the bad prognosis group had a relapse during the follow‐up. Looking specifically at two homogeneous subgroups including either naturally/5‐aminosalicylic acid (5‐ASA) quiescent or corticoid quiescent patients, a very good predictive value for interleukin‐6 serum concentration was also found. Conclusion: Interleukin‐6 serum level alone or in association with other biological parameters such as acid &agr;‐1‐glycoprotein or the soluble interleukin‐2 receptor serum level may be useful for predicting the course of the disease in patients with quiescent Crohns disease.

Effects of three avocado/soybean unsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes.

The in-vitro effects of avocado and soybean unsaponifiable residues on neutral metalloproteinase activity, cytokines and prostaglandin E2 (PGE2) production by human articular chondrocytes were investigated. Avocado and soybean unsaponifiable residues were mixed in three ratios: 1∶2 (A1S2), 2∶1 (A2S1) or 1∶1 (A2S2). Freshly isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1β (IL-1β) (17 ng/ml), with or without unsaponifiable residue mixtures at a concentration of 10 μg/ml. A/S unsaponifiable residues were also tested separately at concentrations of 3.3, 6.6 and 10 μg/ml. All A/S unsaponifiable mixtures reduced the spontaneous production of stromelysin, interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) by chrondrocytes. At concentrations of 3.3 and 6.6 μg/ml, A/S residues, tested separately, were potent inhibitors of the production of IL-8 and PGE2. Nevertheless, only avocado residue inhibited IL-6 production at these concentrations. A/S unsaponifiable mixtures had a more pronounced inhibitory effect on cytokine production than avocado or soybean residues added alone. As anticipated, IL-1β induced a marked release of collagenase, stromelysin, IL-6, IL-8 and PGE2. A/S unsaponifiable mixtures partially reversed the IL-1 effects on chrondrocytes. These findings suggest a potential role for A/S unsaponifiable extracts in mitigating the deleterious effects of IL-1β on cartilage.

Effects of dexamethasone on the profile of cytokine secretion in human whole blood cell cultures

EXPERIMENTAL OBJECTIVES The interaction between the endocrine and immune systems is a very intriguing area. Endogenous glucocorticoids, as end-effectors of the hypothalamo-pituitary-adrenal axis, inhibit the immune and inflammatory responses and are used as immunosuppressive drugs in many inflammatory, autoimmune and allergic diseases. The aims of this study were to investigate the effects of dexamethasone on the profile of cytokine secretion in whole blood cell cultures from healthy subjects and to analyse the gender-related sensitivity to dexamethasone on each cytokine secretion. RESULTS There was a significant inhibition by dexamethasone (from 1 to 100 nM) on the secretion of monokines (IL-1beta, IL-6, IL-8 and TNF alpha) and lymphokines (IL-2, IL-4, IL-10 and IFN gamma), either after LPS or PHA stimulation (P < 0.01). Interleukin 4 and IL-10 were less inhibited than IFN gamma (P < 0.05 at 1 nM, P < 0.01 at 10 nM and P < 0.001 from 100 nM to 10 microM). No gender difference was observed in the rate of inhibition of the secretion of each cytokine. CONCLUSION This study shows that the inhibition of cytokine secretion by dexamethasone is more marked on Th1-type cytokines than on Th2-type cytokines. These data support the idea that glucocorticoids may induce a shift from the Th1 to Th2 profile of cytokine secretion.

Novel method for the measurement of cytokine production by a one-stage procedure

A new one-step culture-immunoassay procedure is described for testing cytokine production by immunocompetent cells in whole blood (WB) without the need for an isolation step. Briefly, WB samples or distilled water were added to RPMI medium containing specific anti-cytokine peroxidase-labelled monoclonal antibodies and incubated in micro-well plates coated with specific capture monoclonal antibodies, directed against distinct epitopes of the cytokine, and containing dried polyclonal activators (5.625 micrograms LPS + 1.125 micrograms PHA) or dried standards respectively. The optimalisation of the assay is described for an extended measurement range. The best compromise between sensitivity and linearity was obtained with the addition of 50 ng/well for TNF-alpha and IL-6 or 100 ng/well for IFN-gamma of unconjugate antibodies to the corresponding conjugate. The kinetics of individual production of each cytokine in WB of normal healthy donors showed values entering the standard range following incubation times of between 2 and 8 h for TNF-alpha, 2 and 4 h for IL-6, and 4 and 24 h for IFN-gamma. The sensitivity, the precision (intra-assay CVs) and the reproducibility (interassay CVs) of the assays were as follows: 70 pg/ml, < or = 14% and < or = 11% for TNF-alpha; 25 pg/ml, < or = 11% and < or = 16% for IL-6; 25 pg/ml, < or = 19% and < or = 20% for IFN-gamma. The accuracy (% of recovery) of the assays was in the order of 100% and between 40 and 60% in the absence or presence of polyclonal activators, reflecting the occurrence of an active production/consumption mechanism during the activation.

Cytokine production from peripheral whole blood in atopic and nonatopic asthmatics: relationship with blood and sputum eosinophilia and serum IgE levels

Background: The cytokine network is thought to be essential in orchestrating airway inflammation in asthma. Although evidence has accumulated to suggest that atopic asthma is a Th2 disease, much less is known about nonatopic asthma.

Cytokine Production by Human Thymic Epithelial Cells: Control by the Immune Recognition of the Neurohypophysial Self-Antigen

Oxytocin (OT) has been shown to be the dominant peptide of the neurohypophysial family expressed by thymic epithelial and nurse cells (TEC/TNC) in various species. Thymic OT is not secreted but, after translocation of a hybrid neurophysin/MHC class I protein, is integrated within the plasma membrane of TEC, thus allowing its presentation to pre-T cells. In order to further demonstrate that thymic OT behaves like a membrane antigen, we assessed the effect of mAbs to OT on cytokine productions by cultures enriched in human TEC. 75-85% pure TEC cultures were prepared from human thymic fragments. Using immunofluorescence and confocal microscopy, ir-OT, ir-interleukin-1 beta (IL-1 beta), ir-interleukin-6 (IL-6) and ir-leukemia inhibitory factor (LIF) could be detected in these TEC cultures. ir-OT was restricted to TEC, while some ir-IL-6 and ir-LIF were also seen in occasional fibroblasts. In basal conditions, ir-IL-6 and ir-LIF (but not ir-OT and ir-IL-1 beta) were detected in the supernatants of human TEC cultures. MAbs to OT induced a marked increase of ir-IL-6 and ir-LIF secretion in TEC cultures. No significant effect was observed using mAbs against vasopressin, mouse immunoglobulins, or control ascitic fluid controls. These data show that OT is fully processed and recognized by specific mAbs at the outer surface of TEC plasma membrane. They further support that thymic OT behaves as the self-antigen of the neurohypophysial family.