转基因2C小鼠的局部口腔耐受性

Margenthaler Julie A, Flye M Wayne

Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA.

Surgery. 2005 Aug;138(2):141-9. doi: 10.1016/j.surg.2005.05.010.

BACKGROUND

Antigen injected subcutaneously (SQ) results in a strong systemic immune response, whereas antigen infused orally or by the portal vein tends to induce tolerance. Nontransgenic C57BL/6J (H-2(b)), or B6, mice and transgenic 2C mice (that express a cytotoxic T cell receptor against major histocompatibility complex Class I L(d)) were used to investigate the regional and systemic responses to oral antigen.

METHODS

B6 (H-2(b)) and 2C (H-2(b)) mice were given either saline or BALB/cByJ (H-2(d), L(d+)) (BALB/c) spleen cells (SCs) (25 x 10(6)) by oral gavage (PO) on day 0. Injection of 10 x 10(6) BALB/c SCs SQ was performed after 7 days, followed by a footpad injection of 10 x 10(6) BALB/c SC on day 14. Specific footpad swelling was measured 24 hours later by means of a micrometer. Mixed lymphocyte culture was performed on splenic and mesenteric lymph node (MLN) lymphocytes. The percentage of splenic and MLN CD4+ and CD8+ T cells was quantitated by fluorescence activated cell sorter. Cytokine messenger RNA (mRNA) and protein was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay.

RESULTS

Whereas B6 mice were shown to have specifically decreased responsiveness to Balb/c cells in vivo and in vitro after oral Balb/c, 2C mice did not demonstrate any downregulation in delayed-type hypersensitivity responsiveness or splenic lymphocyte proliferation. However, MLN lymphocytes from 2C mice did demonstrate decreased proliferation against Balb/c after oral Balb/c gavage (P < .01). Subset percentages of naïve B6 (n=8) spleen and MLN lymphocytes were 50% to 52% for CD4+ and 46% to 47% for CD8+ T cells. These percentages were unchanged in B6 mice after PO Balb/c. Subset percentages of naïve 2C (n=6) spleen and MLN lymphocytes were 2% to 5% for CD4+ and 61% to 64% for CD8+ T cells. After PO Balb/c, MLN CD4+ and CD8+ T cells were unchanged, whereas splenic CD8+ T cells decreased to 44% to 48% and CD4+ T cells increased to 32% to 33% (P < .05, P < .05 vs naïve 2C spleen). After PO Balb/c, inflammatory interleukin (IL)-2 and interferon gamma mRNA and protein were increased in 2C spleen cells, whereas they were decreased in 2C MLN. Anti-inflammatory cytokine IL-4 and IL-10 mRNA and protein were increased in 2C MLN after PO Balb/c, whereas they were not detectable in 2C spleen cells.

CONCLUSIONS

Systemic oral tolerance can be induced in B6 mice, whereas only regional mesenteric lymph node tolerance develops in transgenic 2C mice. The inability to induce systemic oral tolerance in 2C mice appears to be due to a significant increase in peripheral (splenic) CD4+ T cells.

背景

皮下注射（SQ）抗原会引发强烈的全身免疫反应，而口服或经门静脉注入抗原则倾向于诱导免疫耐受。使用非转基因C57BL/6J（H-2(b)），即B6小鼠和转基因2C小鼠（表达针对主要组织相容性复合体I类L(d)的细胞毒性T细胞受体）来研究口服抗原后的局部和全身反应。

方法

在第0天，通过口服灌胃（PO）给B6（H-2(b)）和2C（H-2(b)）小鼠给予生理盐水或BALB/cByJ（H-2(d)，L(d+)）（BALB/c）脾细胞（SCs）（25×10⁶）。7天后进行10×10⁶个BALB/c SCs的皮下注射，然后在第14天进行10×10⁶个BALB/c SC的足垫注射。24小时后用千分尺测量特异性足垫肿胀情况。对脾和肠系膜淋巴结（MLN）淋巴细胞进行混合淋巴细胞培养。通过荧光激活细胞分选仪对脾和MLN中CD4⁺和CD8⁺ T细胞的百分比进行定量。通过逆转录聚合酶链反应和酶联免疫吸附测定法测量细胞因子信使核糖核酸（mRNA）和蛋白质。

结果

口服BALB/c后，B6小鼠在体内和体外对Balb/c细胞的反应性均特异性降低，而2C小鼠在迟发型超敏反应性或脾淋巴细胞增殖方面未表现出任何下调。然而，2C小鼠的MLN淋巴细胞在口服BALB/c灌胃后对Balb/c的增殖确实降低（P <.01）。未接触过抗原的B6（n = 8）脾和MLN淋巴细胞的亚群百分比，CD4⁺为50%至52%，CD8⁺ T细胞为46%至47%。口服Balb/c后，B6小鼠的这些百分比没有变化。未接触过抗原的2C（n = 6）脾和MLN淋巴细胞的亚群百分比，CD4⁺为2%至5%，CD8⁺ T细胞为61%至64%。口服Balb/c后，MLN中的CD4⁺和CD8⁺ T细胞没有变化，而脾中的CD8⁺ T细胞降至44%至48%，CD4⁺ T细胞升至32%至33%（与未接触过抗原的2C脾相比，P <.05，P <.05）。口服Balb/c后，2C脾细胞中炎性白细胞介素（IL）-2和干扰素γ的mRNA和蛋白质增加，而在2C的MLN中则减少。口服Balb/c后，抗炎细胞因子IL-4和IL-10的mRNA和蛋白质在2C的MLN中增加，而在2C脾细胞中未检测到。