Functional evaluation of the role of C-type lectin domain family 16A at the chromosome 16p13 locus.

The type 1 diabetes-associated 16p13 locus contains the CLEC16A gene. Its preferential immune cell expression suggests involvement in autoimmunity. Given its elevated expression in dendritic and B cells - known professional antigen-presenting cells (APCs) - we hypothesize that C-type lectin domain family 16 member A (CLEC16A) may be involved in T cell co-stimulation and consequent activation and proliferation. We also sought to identify CLEC16A's subcellular localization. The effect of the CLEC16A knock-down (KD) on B cell co-stimulation and activation of T cells was tested in human lymphoblastoid cell lines (LCLs) by co-culture with CD4(+) T cells. T cell activation and proliferation were determined by flow-cytometric analysis of CD69 and CD25 expression and carboxyfluorescein succinimidyl ester (CFSE) dilution, respectively. CLEC16A subcellular localization in K562 cells was examined by immunofluorescence. We show that the CLEC16A KD did not affect the tested indices of lymphoblastoid cell line (LCL) APC capacity. Additionally, the percentage of activated T cells following LCL co-culture was not affected significantly by the CLEC16A KD. T cells co-cultured with KD or control LCLs also exhibited similar cell division profiles. CLEC16A co-localized with an endoplasmic reticulum (ER) marker, suggesting that it may be an ER protein. In conclusion, CLEC16A may not be involved in T cell co-stimulation. Additional studies on CLEC16A, accounting for its ER localization, are needed to uncover its biological role.

Class III alleles of the variable number of tandem repeat insulin polymorphism associated with silencing of thymic insulin predispose to type 1 diabetes.

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic beta cells. The insulin gene (INS) is also expressed in human thymus, an ectopic expression site likely involved in immune tolerance. The IDDM2 diabetes susceptibility locus maps to a minisatellite composed of a variable number of tandem repeats situated 0.5 kb upstream of INS. Chromosomes carrying the protective long INS variable number of tandem repeats alleles (class III) produce higher levels of thymic INS mRNA than those with the predisposing, short class I alleles. However, complete silencing of thymic INS transcripts from the class III chromosome was found in a small proportion of heterozygous human thymus samples. We hypothesized that the specific class III alleles found on these chromosomes silence rather than enhance thymic insulin expression. To test the prediction that these alleles are predisposing, we developed a DNA fingerprinting method for detecting two putative "silencing" alleles found in two thymus samples (S1, S2). In a set of 287 diabetic children and their parents we found 13 alleles matching the fingerprint of the S1 or S2 alleles. Of 18 possible transmissions, 12 of the S1-S2 alleles were transmitted to the diabetic offspring, a frequency of 0.67, significantly higher than the 0.38 seen in the remaining 142 class III alleles; P = 0.025. This confirms our prediction and represents an additional level of correlation between thymic insulin and diabetes susceptibility, which supports a thymic enhancer effect of the INS variable number of tandem repeats as the mechanism of IDDM2 and refines the contribution of IDDM2 genotyping to diabetes risk assessment.

In vitro effects of 2 antirheumatic drugs on the synthesis and expression of proinflammatory cytokines in synovial membranes from patients with rheumatoid arthritis.

OBJECTIVE: To compare the effects of tenidap, a new antirheumatic drug, with a nonsteroidal anti-inflammatory drug, naproxen, on the synthesis and expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interleukin-6 (IL-6) in rheumatoid synovium. METHODS: Human synovial membrane explants from patients with rheumatoid arthritis (RA) were incubated in the absence or presence of 20 micrograms/ml lipopolysaccharides (LPS) and tenidap at 50, 20 (therapeutic concentration), and 5 micrograms/ml or naproxen at 90 (therapeutic concentration) and 30 micrograms/ml. The levels of IL-1 beta, TNF-alpha, and IL-6 in the culture medium were measured by specific enzyme linked immunosorbent assays. The cytokine mRNA levels were quantitated by Northern blotting. RESULTS: In the absence of LPS, tenidap at 20 micrograms/ml produced a significant (p < 0.04) decrease in the IL-1 synthesis level. Under LPS stimulation, IL-1 beta synthesis was inhibited by tenidap at all concentrations tested (p < 0.01) and by naproxen at only 90 micrograms/ml (p < 0.01). Very small amounts of TNF-alpha could be detected only when the synovial membranes were stimulated with LPS. Tenidap significantly reduced LPS stimulated TNF-alpha synthesis; the maximum inhibition was noted at 20 micrograms/ml (69%, p < 0.002). Naproxen, at 90 micrograms/ml, reduced TNF-alpha synthesis by about 40% (p < 0.03) and values were similar to those with subtherapeutic concentrations (5 micrograms/ml) of tenidap. The spontaneous and LPS induced synthesis of IL-6 was significantly inhibited by tenidap at all concentrations tested, whereas neither concentration of naproxen demonstrated a significant effect. Tenidap induced a somewhat similar reduction pattern of IL-1 beta and IL-6 mRNA to that observed for cytokine synthesis. Naproxen only slightly reduced the LPS induced expression of IL-6, while enhancing the IL-1 beta expression. CONCLUSION: Tenidap and naproxen showed differences in their effects on cytokine synthesis and mRNA expression. Tenidap, at the therapeutic concentration, was most clearly differentiated from naproxen by its inhibition of IL-6, but was also a more potent modulator of IL-1 beta and TNF-alpha in RA synovial explants. The significance of these findings lies in the possible therapeutic benefit of proinflammatory cytokine suppression in joint disease.