Libin Yang1, Shulei Li2, Yan Tan3, Shufen Xu3, Xiumei Duan3, Yanqiu Fang3, Lihua Liu3, Yuanyuan Che3, Lei Liu3, Liwei Zhou3

1Department of Pediatrics, First Hospital, Jilin University, Changchun  130021, Jilin Province, China
2Department of Histology and Embryology, Bethune Medical School, Jilin University, Changchun  130021, Jilin Province, China
3Central Laboratory, First Hospital, Jilin University, Changchun  130021, Jilin Province, China

the Program of the Key Laboratory of Health Department of Jilin Province, No. 2006079*; the Fortieth National Post-Doctoral Scientific Foundation, No. 20060400893*

Abstract
BACKGROUND: Human tumor necrosis factor-like molecule 1A (hTL1A) is a strong T helper cell type 1 (Th1) co-stimulator. Guillain-Barre syndrome (GBS) is an autoimmune disorder of the nervous system, which is mediated by Th1 cells.
OBJECTIVE: To determine hTL1A expression in peripheral blood T lymphocytes of acute GBS children and the effects of hTL1A on secretion of interferon-γ.
DESIGN, TIME AND SETTING: A randomized, controlled, neuroimmunological in vitro study was performed at the Central Laboratory of First Hospital of Jilin University, China from November 2005 to November 2007.
MATERIALS: Venous blood samples were obtained from 6 healthy donors, aged 6–12 years (all routine blood examination items were normal), and 6 additional children with acute GBS, aged 6–12 years. The GBS children fell ill within 1 week and were not treated with hormones or immunoglobulin. Purified recombinant human soluble tumor necrosis factor-like molecule 1A (rhsTL1A, 1 mg/mL, relative molecular mass 22 000, 6 × His tag, soluble form) was supplied by the Central Laboratory of First Hospital of Jilin University, China.
METHODS: Peripheral blood mononuclear cells were isolated from healthy donors using the stan-dard Ficoll gradient centrifugation and were incubated in 96-well culture plates. The cells were as-signed to the following groups: control (2 μg/mL phytohemagglutinin), 2 μg/mL phytohemagglutinin + 25, 100 and 400 ng/mL rhsTL1A. T cell proliferation was quantified using the tritiated thymidine (3H-TdR) method. Serum interferon-γ levels in acute GBS children were detected by enzyme-linked immunosorbent assay (ELISA). The ratio of hTL1A-positive T cells to CD3-positive T cells in pe-ripheral blood of acute GBS children was determined using flow cytometry. Following in vitro pre-activation of peripheral blood mononuclear cells by 2 μg/mL phytohemagglutinin, the peripheral blood mononuclear cells were treated with 400 ng/mL exogenous rhsTL1A. Finally, peripheral blood mononuclear cell-secreted interferon-γ levels were measured by ELISA.
MAIN OUTCOME MEASURES: The following parameters were measured: rhsTL1A stimulation index to stimulate proliferation of T cells; the serum interferon-γ levels in acute GBS children; the ratio of hTL1A-positive cells to CD3-positive cells; the levels of interferon-γ secreted by periph-eral blood mononuclear cells in acute GBS children, as well as rhsTL1A-stimulated interferon-γ levels.
RESULTS: T cell proliferation assay revealed that the stimulation index in each rhsTL1A group was greater than the control group. The stimulation index of the 400 ng/mL rhsTL1A group was the greatest. Serum interferon-γ levels in acute GBS children were significantly greater than the control group (P < 0.05). The ratio of hTL1A+ CD3+ T cells to CD3+ T cells in acute GBS children was sig-nificantly greater than the control group (P < 0.01). Phytohemagglutinin stimulated peripheral blood mononuclear cells to a greater extent than 400 ng/mL rhsTL1A in the acute GBS group, and the secreted interferon-γ levels were significantly increased (P < 0.05).
CONCLUSION: In T cells pre-activated with 2 μg/mL phytohemagglutinin, proliferation was effec-tively increased with 400 ng/mL rhsTL1A treatment. Expression of hTL1A was increased in activated T cells from peripheral blood of acute GBS children, followed by increased interferon-γ secretion. These mechanisms are considered to be part of the pathological process that induces the secretion of inflammatory cytokines in GBS syndrome.

Key Words: human tumor necrosis factor-like molecule 1 A; cell proliferation; Guillain-Barre syndrome; interferon-γ

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