Adrenergic control of the adaptive immune response by diurnal lymphocyte recirculation through lymph nodes

Abstract

Various aspects of the immune system display circadian rhythms. Although lymphocyte trafficking has been suggested to show diurnal variations, the mechanisms and influences on immune responses are unclear. Here, we show in mice that inputs from adrenergic nerves contribute to the diurnal variation of lymphocyte recirculation through lymph nodes (LNs), which is reflected in the magnitude of the adaptive immune response. Neural inputs to β₂-adrenergic receptors (β₂ARs) expressed on lymphocytes reduced the frequency of lymphocyte egress from LNs at night, which was accompanied by an increase of lymphocyte numbers in LNs. Immunization during the period of lymphocyte accumulation in LNs enhanced antibody responses. The diurnal variation of the humoral immune response was dependent on β₂AR-mediated neural signals and was diminished when lymphocyte recirculation through LNs was stopped. This study reveals the physiological role of adrenergic control of lymphocyte trafficking in adaptive immunity and establishes a novel mechanism that generates diurnal rhythmicity in the immune system.

Figure 1.

Diurnal oscillation of lymphocyte numbers in circulation and LNs. (A–D) Numbers of B cells and CD4⁺ T cells in blood (A), lymph (B), and peripheral LNs (the sum of inguinal, axillary, and brachial LNs; C), and the noradrenaline content in peripheral LNs (D) of WT mice at the indicated ZTs. Data in A–D are representative of two experiments and shown as the mean ± SD of four mice. (E–H) Noradrenaline levels in peripheral LNs (E) and numbers of B cells and CD4⁺ T cells in blood (F), lymph (G), and peripheral LNs (H) of vehicle- and 6-OHDA–treated mice at the indicated ZTs. (I–K) Numbers of B cells and CD4⁺ T cells in blood (I), lymph (J), and peripheral LNs (K) of WT (Adrb2^+/+ or Adrb2^+/− littermates) and Adrb2^−/− mice at the indicated ZTs. Data are pooled from two (E and K) or three (F–J) experiments. Each symbol represents an individual mouse (at least six mice per group) and bars indicate means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. P-values were obtained by one-way ANOVA with Dunnett’s post-hoc test in comparison with the trough (A–D) or two-way ANOVA with Tukey’s post-hoc test (E–K).

Figure 2.

Diurnal variation of lymphocyte egress from LNs. (A) Experimental design. Mice were treated with neutralizing antibodies against α4 and αL integrins at ZT1 or ZT13. Fractions of lymphocytes remaining in peripheral LNs at 12 h after the integrin blockade were determined as ratios relative to those at 0 h. (B–D) Fractions of B cells and CD4⁺ T cells remaining in peripheral LNs of WT mice (B), vehicle- and 6-OHDA–treated mice (C), and BM chimeras generated with WT (Adrb2^+/+ or Adrb2^+/−) or Adrb2^−/− mice as donors and/or recipients (D). (E) B cells or CD4⁺ T cells from Adrb2^+/+ or Adrb2^−/− mice were labeled with CFSE and transferred into WT mice. Fractions of transferred cells remaining in peripheral LNs were determined at 12 h after the integrin blockade. Data are pooled from two (B and C) or eight (D) experiments, or two experiments for each cell type (E). Each symbol represents an individual mouse (at least six mice per group) and bars indicate means. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. P-values were obtained by unpaired Student’s t test (B) or two-way ANOVA with Tukey’s post-hoc test (C, D, and E).

Figure 3.

Diurnal variation of the humoral immune response. (A and B) WT mice were immunized in the ear with NP-CGG at ZT5 or ZT17. Serum titers of NP-specific IgM and IgG1 at the indicated times after immunization were measured by ELISA (A). The numbers of IgM⁺ and IgG1⁺ NP-specific GC B cells, and total Tfh cells generated in the ear-draining cervical LN were measured by flow cytometry at 7 and 14 d after immunization (B). (C–F) Serum antibody titers (C and E) and the generation of GC B cells and Tfh cells (D and F) were measured at the indicted time after immunization in mice treated with the vehicle or 6-OHDA (C and D), and in WT (Adrb2^+/+ or Adrb2^+/− littermates) and Adrb2^−/− mice (E and F). Data are representative of three (A) or two (C and E) experiments, or pooled from two experiments (B, D, and F). Antibody titers are shown as the mean ± SD of at least four mice (A, C, and E). Cell numbers are shown as the mean of at least four mice with symbols representing individual mice (B, D, and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. P-values were obtained by unpaired Student’s t test (A–C and E) or two-way ANOVA with Tukey’s post-hoc test (D and F).

Figure 4.

Contribution of lymphocyte recirculation through LNs to the diurnal variation of humoral immunity. (A) Experimental design. WT mice were treated with FTY720 (FTY) and a neutralizing antibody against CD62L (αCD62L) at ZT1, and then immunized with NP-CGG in the ear at ZT5 or ZT17. Or mice were treated with FTY and αCD62L at ZT13, and then immunized at ZT17 or ZT5 on the next day (ZT5′). (B and C) Numbers of B cells and CD4⁺ T cells in peripheral LNs of unimmunized mice were measured at the indicated ZTs after treatment with saline or FTY plus αCD62L at ZT1 (B) or ZT13 (C). (D–G) Serum titers of NP-specific antibodies (D and F) and the generation of NP-specific GC B cells and total Tfh cells in the ear-draining cervical LN (E and G) were analyzed in mice immunized at the indicated ZTs after treatment with FTY and αCD62L at ZT1 (D and E) or ZT13 (F and G). Data are representative of two experiments (D and F) or pooled from two experiments (B, C, E, and G). Antibody titers are shown as the mean ± SD of four mice (D and F). Cell numbers are shown as the mean of at least four mice with symbols representing individual mice (B, C, E, and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. P-values were obtained by two-way ANOVA with Tukey’s post-hoc test (B, C, E, and G) or unpaired Student’s t test (D and F).