IFN-gamma Concentrations In Supernatants Research Articles

Alternative screening assay to beryllium lymphocyte proliferation test for identification of beryllium sensitization – Pilot study

Sarcoidosis is a rare interstitial lung disorder of unknown aetiology. In contrast, chronic beryllium disease (CBD), which is clinically indistinguishable from sarcoidosis, is induced by occupational beryllium exposure. In both disorders the primarily affected organ is the lung. The only applicable hallmark to differentiate sarcoidosis from CBD is the presence of beryllium specific T cells defined as beryllium sensitization (BeS) which is a prerequisite for clinical manifestation of CBD. To date the only assay to detect BeS is the beryllium lymphocyte proliferation test (Be-LPT) which measures the capability of T cell lymphocytes in peripheral blood or bronchoalveolar lavage to proliferate ex-vivo in response to beryllium. Since this cumbersome Be-LPT harbours a huge number of technical parameters critical for the final result interpretation the Be-LPT is performed only in a limited number of expert laboratories world wide. We think a robust, cheap and simple assay for detection of BeS is needed to cover the increasing demand for appropriate occupational surveillance in referring occupational settings. To develop an alternative assay we measured beryllium induced IFN-gamma concentrations in supernatants of cultivated peripheral blood cells of beryllium exposed and unexposed individuals after defined time intervals and compared the results with those of simultaneously performed Be-LPTs. Up to date we included 37 assays which had been performed at two independent laboratories in Borstel and Freiburg following an identical work flow. Simultaneous performance of the alternative assay and the Be-LPT revealed consistent findings 76.9%. To date 23.1% of the findings differed between the tests compared. Of interest is the result that individuals with a positive Be-LPT show significantly increased IFN-gamma levels compared with those with a negative finding. This was true for all Beryllium concentrations included and time intervals tested. In conclusion the data presented in this pilot study are promising. However, the numbers of tests have to be increased significantly to improve the statistical power of this study.

Paired immunoglobin-like receptors A and B are new targets for inducing dendritic cells tolerance in mice

The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor beta1 (TGF-beta1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was applied for measurement of PIR-A and CD80, CD86, MHC-II mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using (3)H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR from distinct groups was analyzed by ELISA. The results showed that PIR positive rate was (28.65+/-8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21+/-6.34)%, (58.78+/-4.70)%, (48.24+/-6.75)% respectively for IL-10, TGF-beta1 and LPS induction (P<0.01), but there was no significantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-beta1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-II were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactivated T cell proliferation and down-regulated the IFN-gamma secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and enhanced the IFN-gamma secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-II expression, which might be the molecular mechanism for the T-DC.

Distribution of interferon-gamma mRNA-positive cells in oral lichen planus lesions.

The present study investigated the potential involvement of interferon-gamma (IFN-gamma)-producing cells in the pathogenesis of oral lichen planus (OLP). On biopsies from 10 OLP patients, an in situ hybridization technique was employed to determine the topographical distribution of cells expressing IFN-gamma mRNA. It was estimated that approximately 1% or fewer lesional cells were IFN-gamma mRNA-positive. These cells were mainly encountered lining the basal membrane in a majority of the patients, or were in a few cases circumscribing the infiltrate, but were more seldom localized to the center of the lesion. A slightly higher, but not statistically significant, number of phytohemagglutinin (PHA)-induced IFN-gamma-producing cells, in vitro, was found in blood from 11 other OLP patients compared with blood from matched controls. Equal concentrations of IFN-gamma in supernatants from PHA-stimulated blood cells were detected in the two groups. Similarly, the IFN-gamma response towards C. albicans was alike in OLP and in healthy control (HC) blood cells, indicating normal immunological memory function in the OLP patients. A small set of cells with spontaneous IFN-gamma production was found in OLP and in HC peripheral blood. The data suggest that T-lymphocyte activation and cytokine production act locally and are not reflected in peripheral blood. The localization of the IFN-gamma mRNA-positive cells indicates that the antigenic peptides are presented at the periphery of the mononuclear cell infiltrate. Furthermore, the low frequency of IFN-gamma mRNA-positive cells in the lesions suggests that the disease is maintained by a small number of antigen-specific T cells.

Interleukin‐4, interferon‐gamma and interleukin‐5 in peripheral blood of children with moderate atopic asthma

In asthmatic inflammation, TH2 cells play an important role. TH2 cells specifically secrete cytokines like IL-4 and IL-5. IL-4 stimulates IgE production and IL-5 is involved in hemopoiesis, chemotaxis, priming and activation of eosinophils. IFNgamma, produced by TH1 cells, has an inhibitory action on IgE production. To investigate the TH1/TH2-cell pattern in the cytokine production of peripheral blood of asthmatic children. We determined IL-4, IFNgamma and IL-5 in serum and in supernatants of unstimulated and stimulated (24 h with Concanavaline A) cultures of peripheral blood mononuclear cells (PBMCs) in 22 children with moderate asthma (mean age 9.3 years) and in 17 healthy controls (mean age 10.3 years). All children visited the out-patient department (OPD) where history taking, physical examination and blood sampling took place. Children younger than 8 years of age performed symptom and peak flow registration during 1 week after the visit to the OPD. The number of eosinophils were significantly higher in children with asthma, compared with healthy controls. The concentration of IFNgamma in supernatants of cultures of stimulated PBMCs was significantly lower and the ratio of IL-4/IFNgamma was significantly higher in children with asthma compared with healthy controls. The FEV1 was directly and IgE was inversely related to the concentration of IFNgamma in supernatants of cultures of stimulated PBMCs. IFNgamma may play an important role in the pathophysiology of childhood atopic asthma.

Analysis of Cytokine Producing Activity of Intestinal Intraepithelial T Cells from TCR β‐Chain and 5‐Chain Mutant Mice

Intestinal intraepithelial T cells (IELs) expressing either gammadelta TCR or alphabeta TCR have been proposed to play an important role in the regulation of intestinal epithelia by producing cytokines that directly influence the adjoining intestinal epithelial cell (IEC) functions. To illuminate this issue, we utilized TCR mutant mice to obtain gammadelta IELs, alphabeta IELs and mixed gammadelta and alphabeta IELs from corresponding alphabeta T-cell-deficient (beta-/-), gammadelta T-cell-deficient (delta-/-) and wild-type (WT) littermate mice. The production of IFN-gamma by these IELs as well as the mRNA for IFN-gamma, TGF-alpha, TGF-beta1, TNF-alpha and TNF-beta in these IELs, in conjunction with the effect of produced cytokines on the expression of class II MHC molecules by the in vitro cell line IEC-6, were investigated. IFN-gamma and TNF-alpha [corrected] specific mRNA were detectable in all freshly isolated gammadelta, alphabeta and WT IELs. In addition to the IFN-gamma and TNF-alpha [corrected] mRNA, alphabeta and WT IELs that had been activated in culture plates coated with anti-CD3 mAb contained mRNA for TGF-beta1 and TNF-beta proteins. In the cultured gammadelta IELs, however, the signals for IFN-gamma and TNF-alpha [corrected] transcripts were weak, and mRNA for the latter two cytokines was almost undetectable. Supernatants from in vitro culturing of alphabeta and WT IELs but not gammadelta IELs induced class II MHC gene expression in IEC-6, whereas, in the presence of anti-IFN-gamma mAb, the same culture supernatants failed to do so. In fact, the concentration of IFN-gamma in supernatants from alphabeta and WT IEL cultures was ten- to twentyfold higher than that in the supernatant from the gammadelta IEL culture. Finally, TGF-alpha specific mRNA was not detectable in the gammadelta and alphabeta IELs even after in vitro activation. These results indicate that alphabeta IELs are superior to gammadelta IELs in the ability to produce IFN-gamma, TGF-beta1, TNF-alpha [corrected] and TNF-beta through TCR crosslinking primary in vitro stimulation.

Altered synthesis of interferon-gamma and expression of interferon-gamma receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis.

Previously we reported disease-specific interaction between interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon-gamma (IFN-gamma) and expression of IFN-gamma receptor (IFN-gamma R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN-gamma concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-gamma R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-gamma R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4+ T cells, CD8+ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN-gamma or IFN-gamma R mRNA expression by the semiquantitative RT-PCR method. In vitro IFN-gamma synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-gamma R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN-gamma system might be involved in the development of CGN.