Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF) and soluble TNF receptor (sTNF-R) before and after exposure to irradiation and interferon (IFN) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R_p55 and sTNF-R_p75 ELISA. The tumour cell lines released minimal amounts of TNF, prominent amounts of sTNF-R_p55 (7/12 cell lines) and no sTNF-R_p75. Exposure to irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF release without changing sTNF-R_p55 and sTNF-R_p75 levels. Priming of cultures with recombinant human IFN (rhIFN) markedly enhanced TNF secretion in the radiation-responsive cell lines and had no influence on sTNF-R_p55 and sTNF-R_p75 levels. rhIFN affected the magnitude rather than the sensitivity of the radiation response. The TNF secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A₂) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated -irradiation-stimulated TNF release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited -irradiation-mediated TNF production. Collectively our findings indicate that IFN priming potentiates the secretion of bioactive TNF by ES/pPNET cells in response to irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the -irradiation-mediated intracellular signalling pathway leading to TNF production.     

Antiproliferative effects of interferonγ in combination withα-difluoromethylornithine on human carcinoma cell cultures

The antiproliferative effects of human recombinant interferon (IFN) in combination with -difluoromethylornithine (DFMO) or as single agents were assessed on human cell cultures derived from carcinomas of the breast (MCF-7), the ovary (EFO-27) or the kidneys (EGI-4). Results were obtained in proliferation assays by direct cell counting. The cell lines differed considerably in their sensitivities to the antiproliferative effect of IFN as compared by the 50% inhibition doses of the growth (ID₅₀). In contrast to the findings with IFN, similar antiproliferative effects resulted from the application of comparable doses of DFMO. While IFN induced cytotoxic effects in EGI-4 cells, DFMO produced only cytostatic actions in the cell lines analyzed. Synergistic growth inhibition resulted from the combined application of IFN and DFMO in EFO-27 cell cultures. This finding was most pronounced after treatment with IFN or DFMO doses below the respective ID₅₀ values. However, antagonistic effects occurred in cells of the line EGI-4 after DFMO had been combined with IFN at concentrations below the cytotoxic dose range. Within the sensitivity of our proliferation assay, no synergistic interactions were found in MCF-7 cell cultures. In the cell lines tested, no relation between the sensitivity for the single agents and the effectivity of the drug combination was identified. Despite promising synergistic effects in the moderately IFN-sensitive ovarian carcinoma cell line EFO-27, the efficacy of the IFN/DFMO combination was restrained by possible antagonistic effects as demonstrated in the highly IFN-sensitive EGI-4 renal carcinoma cell cultures. We conclude that the differential interaction patterns in the cell cultures analyzed preclude general suggestions for clinical studies using IFN and DFMO.Abbreviations IFN interferon - DFMO difluoromethylornithine This work was part of the doctoral thesis of Mariam Klouche     

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon- (IFN-), tumornecrosis factor- (TNF-), andinterleukin-1 (IL-1) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN- and TNF-, but not afterexposure to LPS, SEB or IL-1. The adhesion inducedby either IFN- or TNF- was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN- or TNF-,and peaked after 12 hr of incubation with TNF-and after 24 hr with IFN-. These results indicatethat IFN- and TNF- induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.     

Serum concentrations of resistin-like molecules β and γ are elevated in high-fat-fed and obese db/db mice, with increased production in the intestinal tract and bone marrow

Aims/hypothesis Resistin and the resistin-like molecules (RELMs) comprise a novel class of cysteine-rich proteins. Among the RELMs, RELM and RELM are produced in non-adipocyte tissues, but the regulation of their expression and their physiological roles are largely unknown. We investigated in mice the tissue distribution and dimer formation of RELM and RELM and then examined whether their serum concentrations and tissue expression levels are related to insulin resistance.Methods Specific antibodies against RELM and RELM were generated. Dimer formation was examined using COS cells and the colon. RELM and RELM tissue localisation and expression levels were analysed by an RNase protection assay, immunoblotting and immunohistochemical study. Serum concentrations in high-fat-fed and db/db mice were also measured using the specific antibodies.Results The intestinal tract produces RELM and RELM, and colonic epithelial cells in particular express both RELM and RELM. In addition, RELM and RELM were shown to form a homodimer and a heterodimer with each other, in an overexpression system using cultured cells, and in mouse colon and serum. Serum RELM and RELM levels in high-fat-fed mice were markedly higher than those in mice fed normal chow. Serum RELM and RELM concentrations were also clearly higher in db/db mice than in lean littermates. Tissue expression levels revealed that elevated serum concentrations of RELM and RELM are attributable to increased production in the colon and bone marrow.Conclusions/interpretation RELM and RELM form homo/heterodimers, which are secreted into the circulation. Serum concentrations of RELM and RELM may be a novel intestinal-tract-mediating regulator of insulin sensitivity, possibly involved in insulin resistance induced by obesity and a high-fat diet.     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)     

Multifactor-dimensionality reduction shows a two-locus interaction associated with Type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetes mellitus is a complex genetic disease, which results from interactions between multiple genes and environmental factors without any single factor having strong independent effects. This study was done to identify gene to gene interactions which could be associated with the risk of Type 2 diabetes.Methods We genotyped 23 different loci in the 15 candidate genes of Type 2 diabetes in 504 unrelated Type 2 diabetic patients and 133 non-diabetic control subjects. We analysed gene to gene interactions among 23 polymorphic loci using the multifactor-dimensionality reduction (MDR) method, which has been shown to be effective for detecting and characterising gene to gene interactions in case-control studies with relatively small samples.Results The MDR analysis showed a significant gene to gene interaction between the Ala55Val polymorphism in the uncoupling protein 2 gene (UCP2) and the 161C>T polymorphism in the exon 6 of peroxisome proliferator-activated receptor (PPAR) gene. This interaction showed the maximum consistency and minimum prediction error among all gene to gene interaction models evaluated. Moreover, the combination of the UCP2 55 Ala/Val heterozygote and the PPAR 161 C/C homozygote was associated with a reduced risk of Type 2 diabetes (odds ratio: 0.51, 95% CI: 0.34 to 0.77, p=0.0016).Conclusions/interpretation Using the MDR method, we showed a two-locus interaction between the UCP2 and PPAR genes among 23 loci in the candidate genes of Type 2 diabetes. The determination of such genotype combinations contributing to Type 2 diabetes mellitus could provide a new tool for identifying high-risk individuals.Abbreviations MDR Multifactor-dimensionality reduction - SNP single nucleotide polymorphism - CV cross-validation - UCP uncoupling protein - PPAR peroxisome proliferator-activated receptor - FABP fatty acid binding protein - ADRB3 3-adrenergic receptor - IRS1 insulin receptor substrate 1 - OR odds ratio     

Rheumatoid arthritis associated with transient gamma 2-heavy chain disease

Summary This paper describes the clinical history of a patient (F.-O.) with longstanding rheumatoid arthritis (RA), who subsequently developed a transient gamma 2-heavy chain disease (₂-HCD). Immunochemical studies comprised serial determinations of serum levels of intact IgG, the -HCD protein, IgA, and IgM. New applications of the rocket immunoselection and the radial immunodiffusion were used for the quantitation of the -HCD protein and intact IgG, respectively, in the presence of one another. Immunofluorescent microscopy on bone marrow cells showed cells containing -heavy chains but devoid of light chains. Protein studies of the isolated -HCD protein revealed a molecular weight of 72 000 in the dimeric form, a carbohydrate content of 9.7%, and a PCA-Val-Gln NH₂-terminal amino acid sequence. The literature on the rare coexistence of RA and -HCD in a single patient is reviewed.     

Cytokine Production by Intestinal Intraepithelial Lymphocyte Subsets in Celiac Disease

One of the earliest signs of mucosal immune activation in celiac disease (CD) is an increase in the intraepithelial lymphocyte (IEL) count in the small intestinal epithelium. Though most of those IELs express T cell receptor (TcR)- chains, CD is characterized by an increase in TcR- ⁺ IELs and by the loss of CD3^– IELs. There is currently little evidence that these changes in IEL subset distribution are of relevance in the pathogenesis of CD. We aimed to determine the pattern of cytokine production by IEL subsets isolated from duodenal biopsy specimens from control subjects and CD patients at different stages of the disease. We quantified the capacity of IEL subsets to produce IFN-, TNF-, IL-2, IL-4, and IL-10 by intracellular staining by flow cytometry. All IEL subsets studied displayed a type I cytokine profile in both CD and control subjects, with TcR-⁺ IELs being the main IFN- producers. Untreated CD exhibited a trend toward a superior accumulation of IFN- per cell but a reduced proportion of INF-⁺ cells in vitro in association with a significantly increased apoptotic rate of IELs. IL-4 was almost undetectable in all cases and IL-10 showed a tendency to increase in treated and silent celiac patients. IEL subsets have a similar Th1 profile in controls and CD patients, and the superior in vitro apoptosis of IELs from CD patients may reflect their superior in vivo activation. The induction of IL-10-dependent regulatory Tr1 responses may be of potential clinical significance in this disease and merits further investigation.     

Intratumoral injection of interferon-gamma gene-modified dendritic cells elicits potent antitumor effects: effective induction of tumor-specific CD8<Superscript>+</Superscript> CTL response

Purpose To examine the antitumor efficacy of intratumoral injection of interferon-gamma gene-modified dendritic cells (DC-IFN-) in a B16 melanoma model and to investigate its related immunological mechanisms.Methods C57BL/6 mice-derived DC were transfected with adenovirus encoding IFN- or -galactosidase (DC-LacZ). Secretion of IFN- and TNF- by DC was detected by ELISA. Nitric oxide (NO) production was measured by Griess reaction. Cytotoxicity of DC against tumor cell lines and activity of cytotoxic T lymphocytes (CTLs) were determined by ⁵¹Cr-release assay. TRP-2aa180–188-specific CD8⁺ CTLs in tumor-bearing mice with different treatment were determined by ELISPOT.Results DC-IFN- could secrete high levels of IFN-, NO and TNF-. DC-IFN- were cytolytic to B16 melanoma cells in vitro, but DC-LacZ and DC were not. Significant inhibition of tumor growth and prolonged survival were achieved in tumor-bearing mice intratumorally injected with DC-IFN- when compared with those in tumor-bearing mice intratumorally injected with DC, DC-LacZ, fibroblasts, IFN- gene-modified fibroblasts or PBS. After treatment with DC-IFN-, enhanced Th1 and decreased Th2 responses were observed, and B16 melanoma antigen TRP-2aa180–188-specific CD8⁺ CTLs were induced significantly in the tumor-bearing mice.Conclusions Intratumorally injected DC-IFN- can uptake tumor antigens in situ and cross-present tumor antigens to specific CD8⁺ T cells, hereby eliciting effective antitumor effects in murine model with preestablished B16 melanoma.     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.