B. M. E. Von Blomberg

Secretion of Tumour Necrosis Factor α and Lymphotoxin α in Relation to Polymorphisms in the TNF Genes and HLA-DR Alleles. Relevance for Inflammatory Bowel Disease

The genes for tumour necrosis factor alpha (TNFα) and lymphotoxin alpha (LTα; TNFβ) are tandemly arranged in the central region of the MHC. They may, therefore, be of importance for the aetiology of MHC‐associated diseases. The authors have prospectively studied the secretion of TNFα and LTα in relation to polymorphisms at positions ‐308 and ‐238 in the TNFα gene (TNFA), and two polymorphisms in the first intron of the LTα gene (LTA), as well as HLA‐DR in 30 patients with chronic inflammatory bowel diseases (IBD) and 12 healthy controls. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF‐haplotypes: TNF‐C, ‐E, ‐H, ‐I, and ‐P. Significant associations between TNF haplotypes and TNFα and LTα secretion were found when PBMC were cultured with T‐cell activators, irrespective of disease. Mean TNFα secretion of individuals carrying the HLA‐DR3 associated TNF‐E haplotype was significantly higher, as compared to individuals without this haplotype (26 441 pg/ml versus 19 629 pg/ml; P = 0.014). Individuals carrying the TNF‐C haplotype produced the lowest amount of TNFα (17 408 pg/ml; P = 0.022). The TNF‐C and TNF‐E haplotypes differ only at position ‐308 in the promoter of TNFA. Individuals carrying the HLA‐DR1 associated TNF‐I haplotype produced significantly less LTα when compared to those who lack this haplotype (1979 pg/ml versus 3462 pg/ml; P = 0.006). As the TNF‐I haplotype is also associated with low TNFα secretion, this haplotype thus defines a ‘low secretor phenotype’. In conclusion, this is the first study to show associations between TNF haplotypes and TNFα and LTα secretion when T‐cell stimulators are used. These findings will contribute to define disease heterogeneity in IBD and may be of relevance for understanding the pathogenesis of autoimmune diseases.

Survival in Refractory Coeliac Disease and Enteropathy associated T cell Lymphoma: Retrospective evaluation of single centre experience

Background: Coeliac disease may be regarded as refractory disease (RCD) when symptoms persist or recur despite strict adherence to a gluten-free diet. RCD may be subdivided into types I and II with a phenotypically normal and aberrant intraepithelial T-cell population, respectively. RCD I seems to respond well to azathioprine/prednisone therapy. RCD II is usually resistant to any known therapy and transition into enteropathy-associated T-cell lymphoma (EATL) is common. Aim: To provide further insight into RCD and the development of EATL, by reporting on long-term survival and risk of transition of RCD into EATL in a large cohort of patients with complicated coeliac disease. Design and Methods: Retrospective comparison of responses to therapy in four groups of patients with complicated coeliac disease: 43, RCD I; 50, RCD II (total), of whom 26 with RCD II developed EATL after a period of refractoriness to a gluten-free diet (secondary EATL) and 13 were EATL patients without preceding history of complicated coeliac disease (de novo EATL). Results: No coeliac-disease-related mortality was recognised in the RCD I group. The overall 5-year survival in the RCD I group it was 96%; in the RCD II (total) group was 58%; and in the RCD II group after developing EATL it was only 8%. The 2-year survival in the de novo EATL group was 20% versus 15% in secondary EATL group (p = 0.63). Twenty-eight (56%) of the 50 patients with RCD II died, 23 (46%) due to EATL, 4 due to a progressive refractory state with emaciation and 1 from neurocoeliac disease. Conclusion: Remarkably, no patient with RCD I developed RCD II or EATL within the mean follow-up period of 5 years (range 2–15 years). A total of 52% of the RCD II patients developed EATL within 4–6 years after the diagnosis of RCD II. More aggressive and targeted therapies seem necessary in RCD II and EATL.

Distribution of four polymorphisms in the tumour necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD)

In 153 patients with IBD, 64 with Crohns disease (CD), and 89 with ulcerative colitis (UC), as well as in 54 healthy controls (HC), the frequencies of four known di‐allelic polymorphisms in the genes for TNF‐α and lymphotoxin alpha (LTα) were investigated. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF haplotypes: TNF‐C, ‐E, ‐H, ‐I, ‐P. Furthermore, the relation with the presence of perinuclear anti‐neutrophil cytoplasmic autoantibodies (P‐ANCA) was studied. A small, but statistically significant, association between the polymorphism at position ‐308 in the promoter region of the TNF‐α gene and UC was found. The frequency of the uncommon TNF‐α ‐308 allele 2 was found to be decreased in patients with UC compared with HC (allele frequency of allele 2 in UC patients 0±15 versus 0±25 in HC, P= 0±044). No significant differences in distribution of the TNF haplotypes were found between IBD patients and HC, although there was a tendency towards a higher frequency of the TNF‐C haplotype in UC patients compared with controls (haplotype frequency 22%versus 13%; P= 0±19). No statistically significant differences in distribution of the TNF haplotypes were observed between P‐ANCA‐positive and P‐ANCA‐negative UC patients. The strength of the associations indicates that TNF genes are not markers for the predisposition to suffer from IBD. They may, however, be markers of subsets of patients with UC and CD.

Serum antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease are mainly associated with ulcerative colitis. A correlation study between perinuclear antineutrophil cytoplasmic autoantibodies and clinical parameters, medical, and surgical treatment.

Perinuclear antineutrophil cytoplasmic antibodies have recently been demonstrated in the sera of patients with inflammatory bowel disease. Three hundred and sixty six sera obtained from 120 patients with ulcerative colitis, 105 patients suffering from Crohns disease and 49 non-inflammatory bowel disease controls were tested in two laboratories, using an indirect immunofluorescence assay. In addition, a fixed-neutrophil enzyme linked immunoadsorbent assay (ELISA) was evaluated in one of the two laboratories. The results in the immunofluorescence test showed a high degree of correlation between the two laboratories (Kappa coefficient = 0.8). Ninety five of the 120 (79%) ulcerative colitis patients had a positive test whereas only 14 of the 105 (13%) patients with Crohns disease were positive. Sera from four patients suffering from primary sclerosing cholangitis were positive as well as four of the 45 control sera (9%). The sensitivity of the perinuclear antineutrophil cytoplasmic antibody immunofluorescence test for the diagnosis of ulcerative colitis was 0.75 with a specificity of 0.88 and a positive predictive value of 0.88 (all sera). In the ELISA technique 37 of 94 ulcerative colitis sera and one of the 68 Crohns disease sera were positive. In the control group only one of the patients suffering from primary sclerosing cholangitis reacted positively (32 non-inflammatory bowel disease sera tested). The ELISA technique had a high specificity (0.97), but a low sensitivity (0.39). There was no relation of perinuclear antineutrophil cytoplasmic antibodies in ulcerative colitis patients or in Crohns disease patients with disease activity, duration of illness, localisation, extent of disease, previous bowel operations or medical treatment. The clinical significance of perinuclear antineutrophil cytoplasmic antibody positive and negative subsets in both groups of patients thus remains unexplained. Our study confirms that determination of serum antineutrophil cytoplasmatic antibodies in patients with inflammatory bowel disease may differentiate ulcerative colitis from Crohns disease. Further immunological studies are needed to explain the absence of these antibodies in a subset of ulcerative colitis patients and their role in the pathogenesis of the disease.

Induction of cytokine (interleukin-1alpha and tumor necrosis factor-alpha) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure.

Abstract:  The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)].

Analysis of effector and regulatory immune reactivity to nickel

Background Diagnostic patch testing in allergic contact dermatitis faces the risk of boosting existing hypersensitivities or active sensitization. Risk‐free and reliable in vitro assays using peripheral blood are, therefore, wanted.

Human T lymphocyte priming in vitro by haptenated autologous dendritic cells

Dendritic cells (DC), generated from adherent peripheral blood mononuclear cells (PBMC) by culturing with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐4, were used to study in vitro sensitization of naive, hapten‐specific T cells and to analyse cross‐reactivities to related compounds. DC were hapten‐derivatized with nickel sulphate (Ni) or 2‐hydroxyethyl‐methacrylate (HEMA), followed by tumour necrosis factor‐alpha (TNF‐α)‐induced maturation, before autologous T cells and a cytokine cocktail of IL‐1β, IL‐2 and IL‐7 were added. After T cell priming for 7 days, wells were split and challenged for another 7 days with Ni or HEMA, and potentially cross‐reactive haptens. Hapten‐specificity of in vitro priming was demonstrated by proliferative responses to the haptens used for priming but not to the unrelated haptens. Highest priming efficiencies were obtained when both IL‐4 and IL‐12 were added to the cytokine supplement. Marked interferon‐gamma (IFN‐γ) release (up to 4 ng/ml) was found when IL‐12 was included in the cultures, whereas IL‐5 release (up to 500 pg/ml) was observed after addition of IL‐4 alone, or in combination with IL‐12. Nickel‐primed T cells showed frequent cross‐reactivities with other metals closely positioned in the periodic table, i.e. palladium and copper, whereas HEMA‐primed T cells showed distinct cross‐reactivities with selected methacrylate congeners. Similar cross‐reactivities are known to occur in allergic patients. Thus, in vitro T cell priming provides a promising tool for studying factors regulating cytokine synthesis, and cross‐reactivity patterns of hapten‐specific T cells.

Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10

Background  Whereas T lymphocytes are widely accepted as effector cells determining the pathogenesis of allergic contact dermatitis, contradictory results have been found regarding the roles of different T‐cell subsets. The use of various experimental models, involving long‐term cultured T‐cell lines or clones, may explain these contradictory results.

Neutrophil cytoplasmic antibodies (p-ANCA) in ulcerative colitis.

AIMS--To study ulcerative colitis associated neutrophil cytoplasmic antibodies (p-ANCA) in respect of class and subclass distribution, antigen specificity, and (sub)cellular localisation of the antigen(s) to which these antibodies are directed. METHODS--p-ANCA positivity was determined using the standard indirect immunofluorescence test (IIFT). The immunoglobulin (Ig) subclass distribution of p-ANCA was investigated using monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4. Intracellular antigen localisation studies were performed on (fractionated) neutrophils using antigen-specific antibodies. RESULTS--In contrast to vasculitis associated ANCA, ulcerative colitis p-ANCA are mainly of IgG1 and IgG3 subclass and lack IgG4. Ulcerative colitis p-ANCA are myeloid specific. IIFT data indicate that the related antigen(s) seem(s) to be located not in the cytosol, but in the granules (most likely the azurophil granules) of the neutrophil. CONCLUSIONS--p-ANCA in ulcerative colitis have a different immunoglobulin subclass distribution than the ANCA of systemic necrotising vasculitis and necrotising and crescentic glomerulonephritis. This may point to differences in immune regulation between these diseases. Both cathepsin G and lactoferrin are recognised by a subpopulation of ulcerative colitis p-ANCA. In our series, eight out of 36 (22%) of ulcerative colitis associated p-ANCA react with lactoferrin and seven (19.5%) other sera with cathepsin G. None of them recognised both antigens. The main target antigen(s) of ulcerative colitis p-ANCA still remain(s) to be identified.

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses.

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand α-galactosylceramide (αGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using αGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-γ production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8+ CTL response, which was dependent on iNKT cell-derived IFN-γ. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as αGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-γ-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with αGC.