Gα_q and cADPR coregulate calcium influx in fMLF-stimulated neutrophils. (A–D) Bone marrow neutrophils from WT (blue) or Gnaq^−/− (red, green, and black) mice were preincubated in media (Gnaq^−/−, red; WT, blue), 100 μM 8Br-cADPR (green), or 100 μM 2-APB (black) for 20 min. The cells were loaded with Fluo-3 and Fura-red and stimulated with 1 μM fMLF. Relative intracellular calcium levels were measured by FACS and are reported as the ratio of Fluo-3/Fura-red. In B, the extracellular calcium was chelated with 2 mM EGTA immediately before stimulation. Arrows indicate when the stimulus was added to the cells. The data shown are representative of at least three independent experiments.

Signaling through CD38-dependent chemokine receptors also requires Gα_q. (A–C) Bone marrow neutrophils from WT (blue), Cd38^−/− (green), and Gnaq^−/− (red) mice were loaded with the calcium-detecting dyes Fluo-3 and Fura-red and stimulated with 100 nM PAF (A), 1 μM fMLF (B), or 100 nM IL-8 (C). Relative intracellular calcium levels were measured by FACS and are reported as the ratio of Fluo-3/Fura-red. Arrows indicate when the stimulus was added to the cells. (D and E) Bone marrow neutrophils from WT and Gnaq^−/− mice were placed in transwells containing media (nil), fMLF (D, 1 μM; E, 0.1–5 μM), 100 nM IL-8, or 50 ng/ml CCL3. The cells that migrated to the bottom chamber in response to the chemokine gradient were collected after 1 h and enumerated by FACS. The results are expressed as the mean ± SD of the CI (see Materials and methods for description) of triplicate cultures. The data shown are representative of four or more independent experiments. *, P ≤ 0.0001; or **, P < 0.03 between WT and Gnaq^−/− neutrophils.

Signaling through a subset of chemokine receptors is dependent on Gα_i2 and Gα_q. (A–D) Bone marrow neutrophils from WT (blue or black), Gnaq^−/− (red), or Gnai2^−/− (green) mice were preincubated in media or 500 ng/ml PTx (black) for 4 h. The cells were loaded with Fluo-3 and Fura-red and stimulated with 100 nM IL-8 (A and C) or 1 μM fMLF (B and D). Relative intracellular calcium levels were measured by FACS and are reported as the ratio of Fluo-3/Fura-red. Arrows indicate when the stimulus was added to the cells. (E) Bone marrow neutrophils from WT, Gnaq^−/−, or Gnai2^−/− mice were placed in transwells containing media (nil), 1 μM fMLF, or 100 nM IL-8. The cells that migrated to the bottom chamber in response to the chemokine gradient were collected after 1 h and enumerated by FACS. The results are expressed as the mean ± SD of the CI of triplicate cultures. The data shown are representative of four or more independent experiments. *, P ≤ 0.0001 between WT neutrophils and the indicated groups. ns, not significant.

DC migration to CCR7 and CXCR4 ligands is regulated by both Gα_q and Gα_i2, but only Gα_i2 is necessary for the migration of T cells to the same ligands. (A and B) Spleen cells were isolated from WT (blue), Gnai2^−/− (green), and Gnaq^−/− (red) mice and placed in transwell chambers containing media (nil), 300 ng/ml CCL19, or 300 ng/ml CXCL12. The number of CD4⁺ T cells placed in the top chamber and the number of CD4⁺ T cells that migrated to the bottom chamber after 2 h were determined by FACS. The percentage of CD4⁺ T cells that migrated in response to the chemokine gradient was calculated, and the CI was determined. The data are shown as the mean ± SD of the CI of triplicate cultures. (C–E) CD11c⁺ cells were purified from collagenase-digested spleens and LNs of WT, Gnai2^−/−, and Gnaq^−/− mice and placed in transwell chambers containing media (nil), 50 ng/ml CCL19, or 100 ng/ml CXCL12. After 2 h, the cells that migrated to the bottom chamber in response to the chemokine gradient were collected and enumerated by FACS. The results are expressed as the mean ± SD of the CI of triplicate cultures. (E) CXCR4 expression levels on purified CD11c⁺ cells were determined by FACS. *, P ≤ 0.004 between WT cells and the indicated groups. The data shown are representative of at least three independent experiments. ns, not significant.

Differential control of leukocyte chemotaxis by the CD38/cADPR signaling pathway. (A and B) Bone marrow neutrophils from C57BL/6J (WT and WT + 8Br-cADPR) and Cd38^−/− mice were preincubated for 20 min in media (white and black bars) or 100 μM 8Br-cADPR (gray bars) and placed in transwell chambers containing media (nil), or 1 μM fMLF (A) or 100 nM IL-8 (B) in the bottom chamber. The cells that migrated to the bottom chamber in response to the chemokine gradient were collected after 1 h and enumerated by FACS. (C and D) Splenic and LN CD11c⁺ DCs and splenic CD4⁺ T cells were purified from WT and Cd38^−/− mice. The DCs (C) and T cells (D) were preincubated for 20 min in media or 8Br-cADPR (as described for A and B) and placed in transwell chambers containing media or CCL19 (50 ng/ml for DCs and 300 ng/ml for T cells). The number of cells that migrated to the bottom chamber after 2 h was determined by FACS. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of four or more independent experiments. *, P ≤ 0.0007 between WT cells and the indicated groups. ns, not significant.

The in vivo migration of DCs and monocytes is dependent on Gα_q. (A and B) The abdominal skin of WT (A) or Gnaq^−/− (B) mice was shaved and painted with FITC. The draining inguinal LNs were removed 18 h after sensitization, and frozen sections were prepared for immunohistology. The sections were stained with anti-CD11c and anti-CD90.2 to identify DCs and T cells, respectively. FITC⁺ cells and cells that were FITC⁺ and expressed CD11c (orange cells, indicated with yellow arrowheads) were found primarily within the T cell zone of the LN. Bar, 100 μm. (C) The inguinal LNs were isolated 20 h after sensitization with FITC, collagenase digested, counted, stained with antibodies to CD11c and MHCII I-Ab, and analyzed by FACS. The number of FITC^hiCD11c⁺ClassII⁺ DCs is indicated. n = 3 mice per group. *, P ≤ 0.008 between WT and Gnaq^−/− mice. The data are representative of three independent experiments with at least three mice per group. (D) Lethally irradiated CD45.1⁺ C57BL/6J hosts were reconstituted with either CD45.2⁺ WT bone marrow (WT chimeras) or CD45.2⁺Gnaq^−/− bone marrow (Gnaq^−/− chimeras). Resident LCs are radiation resistant and remain of host origin (reference 41). All other hematopoietic cells, including DCs and monocytes, are replaced by the donor bone marrow (reference 41). (E) 8 wk after reconstitution, the epidermal surface of the ears of the chimeric mice were either sensitized with DNFB (day 4 after DNFB) or left untouched (day 0). The number and origin (either host derived [CD45.1] or donor derived [CD45.2]) of the CD11c⁺ClassII⁺ cells in the epidermal sheet isolated from the ears of the chimeric mice were determined by cell counting and FACs analysis. The mean ± SD is shown (n = 3 mice per group per time point). **, P < 0.0001 between the number of donor-derived WT CD11c⁺ClassII⁺ cells and donor-derived Gnaq^−/− CD11c⁺ClassII⁺ cells in the inflamed skin. There was no statistical difference in the number of host-derived WT resident LCs in the epidermis of either group at either time point. The data are representative of two independent experiments.