Identification of an alternative G{alpha}q-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes

Abstract

CD38 controls the chemotaxis of leukocytes to some, but not all, chemokines, suggesting that chemokine receptor signaling in leukocytes is more diverse than previously appreciated. To determine the basis for this signaling heterogeneity, we examined the chemokine receptors that signal in a CD38-dependent manner and identified a novel "alternative" chemokine receptor signaling pathway. Similar to the "classical" signaling pathway, the alternative chemokine receptor pathway is activated by Galpha(i2)-containing Gi proteins. However, unlike the classical pathway, the alternative pathway is also dependent on the Gq class of G proteins. We show that Galpha(q)-deficient neutrophils and dendritic cells (DCs) make defective calcium and chemotactic responses upon stimulation with N-formyl methionyl leucyl phenylalanine and CC chemokine ligand (CCL) 3 (neutrophils), or upon stimulation with CCL2, CCL19, CCL21, and CXC chemokine ligand (CXCL) 12 (DCs). In contrast, Galpha(q)-deficient T cell responses to CXCL12 and CCL19 remain intact. Thus, the alternative chemokine receptor pathway controls the migration of only a subset of cells. Regardless, the novel alternative chemokine receptor signaling pathway appears to be critically important for the initiation of inflammatory responses, as Galpha(q) is required for the migration of DCs from the skin to draining lymph nodes after fluorescein isothiocyanate sensitization and the emigration of monocytes from the bone marrow into inflamed skin after contact sensitization.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.