PMC:4136538 / 14631-18771 JSONTXT

{"project":"2_test","denotations":[{"id":"25143871-18196515-63946024","span":{"begin":238,"end":239},"obj":"18196515"},{"id":"25143871-18196515-63946024","span":{"begin":238,"end":239},"obj":"18196515"},{"id":"25143871-18196515-63946025","span":{"begin":1397,"end":1398},"obj":"18196515"},{"id":"25143871-18196515-63946025","span":{"begin":1397,"end":1398},"obj":"18196515"},{"id":"25143871-18196515-63946026","span":{"begin":1778,"end":1779},"obj":"18196515"},{"id":"25143871-18196515-63946026","span":{"begin":1778,"end":1779},"obj":"18196515"},{"id":"T29409","span":{"begin":238,"end":239},"obj":"18196515"},{"id":"T51350","span":{"begin":238,"end":239},"obj":"18196515"},{"id":"T93801","span":{"begin":1397,"end":1398},"obj":"18196515"},{"id":"T23576","span":{"begin":1397,"end":1398},"obj":"18196515"},{"id":"T54619","span":{"begin":1778,"end":1779},"obj":"18196515"},{"id":"T14918","span":{"begin":1778,"end":1779},"obj":"18196515"}],"text":"Flow cytometric analysis of CD4+ T lymphocytes in peripheral lymphoid tissues and the lumbar spinal cord\nPreviously, we have shown that spinal cord CD4+ T lymphocytes contribute to L5Tx-induced maintenance of mechanical hypersensitivity [4]. To identify the phenotype of CD4+ T lymphocytes involved in the development of L5Tx-induced mechanical hypersensitivity, we examined CD4+ T lymphocytes in both peripheral lymphoid tissues and the lumbar spinal cord via flow cytometry with antibodies that identify the Th1 and Th2 subtypes. WT BALB/c mice were randomly assigned to naive (no surgery), L5Tx, and sham groups. Since 7 days post-surgery is the peak time of detecting spinal cord-infiltrating CD4+ T lymphocytes, samples were collected at day 7 post-surgery.\nTo examine CD4+ T lymphocytes in the periphery, spleens and lumbar LNs were collected and processed for intracellular flow cytometric analysis. CD4+ T lymphocytes were first identified (the CD45+CD3+CD4+ population), and then the expression of T-bet (transcription factor found in Th1, and not Th2, cells), GATA-3 (transcription factor found in Th2, and not Th1, cells), IFN-γ (one of the signature cytokines produced by Th1 cells), and IL-4 (one of the signature cytokines produced by Th2 cells) were examined within the CD4+ T lymphocytes (Figure 1A). Consistent with our previous findings regarding peripheral responses post-L5Tx [4], no significant differences in the percentages of T-bet+, GATA-3+, IFN-γ+, or IL-4+ CD4+ T lymphocytes were detected in either spleen or lumbar LNs when the naive, L5Tx and sham groups were compared (Figure 1B and 1C, one-way ANOVA, p\u003e0.05 for all data sets within each graph). Since signs of splenic CD4+ T lymphocyte activation were previously observed at day 3 post-surgery [4], similar experiments were performed to examine splenic CD4+ T lymphocytes at day 3 post-surgery. However, no significant differences were detected in any of the above populations assessed (data not shown). Thus, the data indicate a lack of a significant Th1- or Th2-dominant response in the periphery after either L5Tx or sham surgery.\nWe then examined the phenotype of the lumbar spinal cord-infiltrating CD4+ T lymphocytes in the naive, L5Tx, and sham groups. At day 7 post-surgery, lumbar spinal cord mononuclear cells were harvested and analyzed as described above via intracellular flow cytometric analysis (Figure 2). First, we examined the expression of the transcription factors T-bet and GATA-3 within the lumbar spinal cord-infiltrating CD4+ T lymphocytes. Within each set of experiments, the number of T-bet+CD4+ T lymphocytes in the L5Tx group was consistently higher than that of the naive and sham groups. As a result, there was a significant increase in the number of T-bet+CD4+ T lymphocytes in the L5Tx group compared to both the naive and sham groups. A slight, but not significant, increase in T-bet+CD4+ T lymphocytes was also observed in sham animals compared to naive mice (Figure 2B, one-way ANOVA, p\u003c0.05). On the other hand, GATA-3+ CD4+ T lymphocytes were below the level of detection. Thus, these data indicated that the L5Tx-induced lumbar spinal cord infiltrating CD4+ T lymphocytes were predominantly Th1 cells. We further examined the cytokine expression of the infiltrating CD4+ T lymphocytes by measuring the number of IFN-γ+, IL-4+, TNF-α+, and GM-CSF+ CD4+ T lymphocytes. TNF-α and GM-CSF are two other cytokines predominantly associated with Th1 rather than Th2 cells. There were L5Tx-induced significant increases in the number of IFN-γ+ and GM-CSF+ lumbar spinal cord infiltrating CD4+ T lymphocytes (Figure 2C and E, one-way ANOVA, p\u003c0.05). L5Tx induced similar, but not significant, changes in the number of TNF-α+ CD4+ T lymphocytes (Figure 2D, one-way ANOVA, p\u003e0.05; p=0.051, L5Tx vs. sham), while no significant changes in the number of IL-4+ CD4+ T lymphocytes were detected (data not shown). These data further support lumbar spinal cord-infiltrating Th1 CD4+ T lymphocytes mediating L5Txinduced maintenance of mechanical hypersensitivity, and also suggest that this mediation might involve multiple Th1 cytokines."}