Figure 7. β 2 AR-mediated signals... | Rockefeller University Press

Figure 7.

$Figure 7. β2AR-mediated signals inhibit LN egress of antigen-primed T cells. (A–C) WT OT-II T cells (CD45.2+) were transferred into WT mice (CD45.1+), and the recipients were immunized with OVA. 7 d later, the mice were subjected to continuous administration of saline or clenbuterol (40 mg/kg/day). (A) OT-II T cells recruited to the ear were quantified by flow cytometry 12 h after OVA challenge. The graph shows numbers of OT-II T cells per 105 cells in saline-injected or OVA-challenged ears. (B) Numbers of OT-II T cells in blood and lymph of immunized mice were enumerated after 12-h clenbuterol treatment. (C) The immunized mice were treated with integrin-neutralizing antibodies for 20 h during treatment with saline or the indicated doses of clenbuterol. Fractions of central memory (CD44hiCD62Lhi) and effector memory (CD44hiCD62Llo) OT-II T cells remaining in the draining inguinal LNs were determined as ratios relative to those of immunized mice treated with saline but not integrin-neutralizing antibodies. Data are shown as mean ± SEM for at least three mice. (D) Surface expression of CCR7 and CXCR4 on CD44hiCD62Lhi and CD44hiCD62Llo OT-II T cells in the inguinal LNs. (E) Adrb2 mRNA transcript abundance was measured by quantitative RT-PCR in naive (CD44loCD62Lhi), CD44hiCD62Lhi, and CD44hiCD62Llo OT-II T cells, and quantified relative to the abundance of Gapdh. Data are shown as mean + SD of triplicates. (F-J) CD45.1+ WT mice received a mixture of CD45.1+CD45.2+ WT and CD45.2+ Adrb2+/− OT-II T cells (Adrb2+/− mix) or mixture of CD45.1+CD45.2+ WT and CD45.2+ Adrb2−/− OT-II T cells (Adrb2−/− mix), and were immunized with OVA. 7 d later, the mice were subjected to analysis. Normalized ratios of CD45.2+ cells relative to CD45.1+ CD45.2+ cells were obtained by dividing observed ratios by the ratio of input populations (F, H, and I). (F) CD44hiCD62Lhi and CD44hiCD62Llo OT-II T cells were detected in the draining inguinal LNs. (G) Frequencies of IFN-γ–producing OT-II T cells were measured in the inguinal LNs. (H) Recruitment of OT-II T cells to the ear 12 h after OVA challenge was measured and shown. (I) OT-II T cells in blood and lymph of immunized mice were measured and shown. (J) The immunized mice were treated with integrin-neutralizing antibodies for 14 h, and fractions of CD44hiCD62Lhi and CD44hiCD62Llo OT-II T cells remaining in the inguinal LNs were determined as in C. Each symbol represents an individual mouse and bars indicate means (A, B, and F–J). Data are representative of two (D and E) experiments, or pooled from two (A–C, F, and H–J) or three (G) experiments. Sal, saline; Clen, clenbuterol. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. P-values were obtained by one-way ANOVA with Bonferroni’s post-test (A) or unpaired Student’s t test (B and F–J).$

β₂AR-mediated signals inhibit LN egress of antigen-primed T cells. (A–C) WT OT-II T cells (CD45.2⁺) were transferred into WT mice (CD45.1⁺), and the recipients were immunized with OVA. 7 d later, the mice were subjected to continuous administration of saline or clenbuterol (40 mg/kg/day). (A) OT-II T cells recruited to the ear were quantified by flow cytometry 12 h after OVA challenge. The graph shows numbers of OT-II T cells per 10⁵ cells in saline-injected or OVA-challenged ears. (B) Numbers of OT-II T cells in blood and lymph of immunized mice were enumerated after 12-h clenbuterol treatment. (C) The immunized mice were treated with integrin-neutralizing antibodies for 20 h during treatment with saline or the indicated doses of clenbuterol. Fractions of central memory (CD44^hiCD62L^hi) and effector memory (CD44^hiCD62L^lo) OT-II T cells remaining in the draining inguinal LNs were determined as ratios relative to those of immunized mice treated with saline but not integrin-neutralizing antibodies. Data are shown as mean ± SEM for at least three mice. (D) Surface expression of CCR7 and CXCR4 on CD44^hiCD62L^hi and CD44^hiCD62L^lo OT-II T cells in the inguinal LNs. (E) Adrb2 mRNA transcript abundance was measured by quantitative RT-PCR in naive (CD44^loCD62L^hi), CD44^hiCD62L^hi, and CD44^hiCD62L^lo OT-II T cells, and quantified relative to the abundance of Gapdh. Data are shown as mean + SD of triplicates. (F-J) CD45.1⁺ WT mice received a mixture of CD45.1⁺CD45.2⁺ WT and CD45.2⁺Adrb2^+/− OT-II T cells (Adrb2^+/− mix) or mixture of CD45.1⁺CD45.2⁺ WT and CD45.2⁺Adrb2^−/− OT-II T cells (Adrb2^−/− mix), and were immunized with OVA. 7 d later, the mice were subjected to analysis. Normalized ratios of CD45.2⁺ cells relative to CD45.1⁺ CD45.2⁺ cells were obtained by dividing observed ratios by the ratio of input populations (F, H, and I). (F) CD44^hiCD62L^hi and CD44^hiCD62L^lo OT-II T cells were detected in the draining inguinal LNs. (G) Frequencies of IFN-γ–producing OT-II T cells were measured in the inguinal LNs. (H) Recruitment of OT-II T cells to the ear 12 h after OVA challenge was measured and shown. (I) OT-II T cells in blood and lymph of immunized mice were measured and shown. (J) The immunized mice were treated with integrin-neutralizing antibodies for 14 h, and fractions of CD44^hiCD62L^hi and CD44^hiCD62L^lo OT-II T cells remaining in the inguinal LNs were determined as in C. Each symbol represents an individual mouse and bars indicate means (A, B, and F–J). Data are representative of two (D and E) experiments, or pooled from two (A–C, F, and H–J) or three (G) experiments. Sal, saline; Clen, clenbuterol. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. P-values were obtained by one-way ANOVA with Bonferroni’s post-test (A) or unpaired Student’s t test (B and F–J).

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