Identification of an alternative Gα_q-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes

Chemokine receptors can be divided into CD38-dependent and -independent subclasses

We previously showed that the in vitro chemotaxis of mouse formyl peptide receptor (mFPR) 1–expressing mouse bone marrow neutrophils to the chemoattractant N-formyl methionyl leucyl phenylalanine (fMLF) is dependent on CD38 and at least one of its enzymatically generated products, cADPR (28, 31). Indeed, neither WT neutrophils pretreated with the membrane-permeant cADPR antagonist 8Br-cADPR nor Cd38^−/− neutrophils migrate in transwell chambers in response to an optimal concentration of fMLF (Fig. 1 A). In contrast, chemotaxis of Cd38^−/− neutrophils and 8Br-cADPR–treated WT neutrophils to the CXC chemokine receptor (CXCR) 1/CXCR2 ligand IL-8 (Fig. 1 B) is equivalent to chemotaxis of normal WT neutrophils, indicating that there must be at least two subclasses of chemokine receptors that can be distinguished from one another based on their requirement for CD38.

In subsequent analyses of mouse and human leukocytes, we found additional examples of chemokine receptors that signal in either a CD38/cADPR-dependent or -independent manner (28, 29, 31). However, when we compared the chemotactic response of peripheral (isolated from spleen and LNs) mouse DCs and T cells with the same CC chemokine receptor (CCR) 7 ligand, CCL19, we found that the peripheral Cd38^−/− DCs were unable to migrate in response to the CCL19 gradient (Fig. 1 C), whereas CD4 T cells purified from the same tissues of the same Cd38^−/− mice migrated normally in response to CCL19 (Fig. 1 D). Likewise, peripheral WT DCs pretreated with the cADPR antagonist 8Br-cADPR made a defective chemotactic response to CCL19 (Fig. 1 C), whereas the chemotactic response of WT T cells pretreated with 8Br-cADPR was equivalent to that observed for the untreated WT T cells (Fig. 1 D). Collectively, these data showed that chemokine receptors can be divided into different subclasses and that the subclass of the chemokine receptor is variable and dependent on the cell type expressing the chemokine receptor.

CD38-dependent chemokine receptors couple to Gα_q

Our data suggested that there was considerably more diversity or heterogeneity in the molecular signals that regulate chemotaxis than previously appreciated. To better understand the diversity between chemokine receptors, we examined the response of WT and Cd38^−/− neutrophils to platelet-activating factor (PAF), a ligand of the PAFR. We chose to analyze signaling through this receptor, as it is one of the few known chemoattractant receptors that can induce calcium release in a PTx-independent fashion, indicating that it must functionally couple to other G proteins in addition to those containing Gα_i (34–36). Therefore, we loaded bone marrow WT and Cd38^−/− neutrophils with calcium-sensing fluorescent dyes, stimulated the cells with PAF, and measured accumulation of intracellular free calcium by FACS. As previously reported for human neutrophils (34), PAF-stimulated WT bone marrow neutrophils made a bimodal calcium response with a rapid rise in intracellular free calcium levels that was followed by a second phase of sustained calcium mobilization (Fig. 2 A). The first phase of calcium release was largely caused by calcium release from intracellular stores, as it was not blocked in the presence of EGTA, whereas the second phase was caused by calcium entry, as it was inhibited when EGTA was added to the external medium (unpublished data). Interestingly, the first calcium release from intracellular stores was intact in the PAF-activated Cd38^−/− neutrophils; however, minimal calcium entry was observed during the second sustained phase of the response (Fig. 2 A). Similar results were observed with 8Br-cADPR–treated WT neutrophils and with PAF-stimulated bone marrow–derived Cd38^−/− DCs (unpublished data). Thus, calcium signaling through the PAFR appears to be regulated by CD38 and cADPR.

It has been previously demonstrated that PTx-treated PAF-activated cells produce IP₃ (34–36), indicating that the PAFR must couple to one or more members of the Gq family of G proteins that are capable of activating PLCβ and inducing IP₃ formation (32, 33). To assess whether the PAFR couples to Gα_q, we purified bone marrow neutrophils from chimeric mice that lacked Gα_q (Gnaq^−/−) expression in the bone marrow–derived hematopoietic cells. We then stimulated the cells with PAF and measured intracellular calcium accumulation. Similar to the previous experiments that used PTx-treated human neutrophils (34), the immediate calcium release from IP₃-gated intracellular stores was reduced, although not absent, in the PAF-stimulated Gnaq^−/− neutrophils relative to WT neutrophils (Fig. 2 A). Furthermore, in a manner identical to what we observed with Cd38^−/− neutrophils, the PAF-induced calcium response was not sustained in the Gnaq^−/− neutrophils (Fig. 2 A), suggesting that Gα_q regulates both intracellular calcium release and calcium influx in these cells.

These results suggested that CD38 and Gα_q might both be involved in activating calcium influx in chemokine-stimulated cells. If this conclusion is correct, then we postulated that Gα_q, like CD38, might also regulate calcium influx in fMLF-stimulated neutrophils. To test this hypothesis, we measured the calcium response of fMLF-activated bone marrow neutrophils isolated from WT, Cd38^−/−, and Gnaq^−/− mice. As shown earlier (28, 31), we observed a bimodal calcium response in the fMLF-activated WT neutrophils and a significantly reduced calcium response in the fMLF-stimulated Cd38^−/− neutrophils (Fig. 2 B). Interestingly, the calcium response of the fMLF-activated Gnaq^−/− neutrophils was very similar to that of the Cd38^−/− neutrophils and significantly reduced relative to the WT neutrophils (Fig. 2 B). We observed the most pronounced reduction in the calcium response of the fMLF-activated Gnaq^−/− neutrophils during the second calcium influx phase of the response (28), suggesting a prominent role for Gα_q in regulating calcium entry rather than intracellular calcium release in this response. To bolster this conclusion, we next examined the calcium response of IL-8–activated Gnaq^−/− neutrophils, as the calcium response to IL-8 in mouse bone marrow neutrophils is almost entirely caused by IP₃-induced intracellular calcium release (28). In agreement with our hypothesis, the calcium response of IL-8–stimulated neutrophils was largely unaffected by the loss of Gα_q (Fig. 2 C), again very similar to what we observed with the Cd38^−/− neutrophils (Fig. 2 C).

Collectively, these data suggested a strong positive correlation between CD38 and Gα_q, at least with respect to calcium influx in response to chemokine receptor ligation. Given the requirement for both CD38 and calcium influx in neutrophil chemotactic responses to chemoattractants such as fMLF (28), we postulated that Gα_q was also likely to be required for chemotaxis to chemokines that signal in a CD38-dependent fashion. To test this possibility, we examined the in vitro chemotaxis of Gnaq^−/− bone marrow neutrophils to chemokines that signal in a CD38- and calcium influx–independent (IL-8) or a CD38- and calcium influx–dependent (fMLF and CCL3) fashion (28). As shown in Fig. 2 D, in vitro chemotaxis of the Gnaq^−/− neutrophils to IL-8 was completely normal. However the Gnaq^−/− cells were unable to migrate in response to either fMLF or CCL3 (Fig. 2 D). This impaired chemotactic response was not caused by an altered responsiveness to the chemoattractants, as the Gnaq^−/− neutrophils made a defective chemotactic response at all doses of fMLF (see Fig. 3 E) or CCL3 (not depicted) tested. Instead, the data demonstrate the existence of a novel Gα_q- and CD38-dependent chemokine receptor signaling pathway that is engaged by some, but not all, chemokine receptors.

Signaling through a subset of chemokine receptors requires both Gα_i and Gα_q

The results showing that chemotaxis of neutrophils to mFPR1 and CCR1 ligands is Gα_q dependent were surprising, as the Gi inhibitor PTx is known to block chemotaxis of essentially all leukocytes (17). Because we could not find any reports testing the effect of PTx on mouse bone marrow–derived neutrophils, we thought it important to test this assumption experimentally. Therefore, we treated WT neutrophils with PTx and then measured the calcium response to IL-8 and fMLF. Similar to published data (37), the calcium responses were largely, although not entirely, ablated in the PTx-treated bone marrow neutrophils stimulated with either IL-8 (Fig. 3 A) or fMLF (Fig. 3 B). To confirm the PTx results, we also measured intracellular calcium levels and chemotaxis in WT, Gα_i2-deficient (Gnai2^−/−), and Gnaq^−/− neutrophils that were activated with either IL-8 or fMLF. Similar to the PTx-treated cells, Gnai2^−/− neutrophils made a very reduced calcium response after exposure to either IL-8 or fMLF (Fig. 3, C and D). However, the calcium response of the Gnaq^−/− neutrophils to IL-8 was normal (Fig. 3 C), and the calcium response of the Gnaq^−/− cells to fMLF was only partially impaired (Fig. 3 D). Not surprisingly, Gnai2^−/− neutrophils were unresponsive to both IL-8 and fMLF in the in vitro chemotaxis assay (Fig. 3 E). In contrast, the chemotactic response of Gnaq^−/− neutrophils to fMLF was ablated, whereas the response to IL-8 was completely intact (Fig. 3 E). Collectively, the data indicate that some chemokine receptors expressed by mouse bone marrow neutrophils, such as CXCR1/2, couple to G proteins containing Gα_i2 but not Gα_q. However other receptors, such as mFPR1 and CCR1, couple to two different classes of G proteins, Gα_i2 and Gα_q.

Gα_q regulates calcium influx in chemokine-stimulated neutrophils

The “signature” activity of the Gq class of G proteins is to activate one or more PLCβ isoforms, which catalyze production of IP₃ and promote intracellular calcium release (32, 33). This activity is performed by the α subunit of the trimeric Gα_q-containing proteins (32, 33). Therefore, it was surprising to find that the largest alteration in the calcium response of the fMLF-stimulated Gnaq^−/− neutrophils appeared during the later calcium entry phase of the response rather than during the initiation of the calcium response. To better understand how Gα_q regulates chemokine receptor signal transduction, we examined the calcium response of chemokine-stimulated Gnaq^−/− mouse bone marrow neutrophils in more detail. Initially, we stimulated Gnaq^−/− neutrophils with fMLF in the presence or absence of EGTA (to chelate the extracellular calcium) so that we could assess the relative contribution of Gα_q to intracellular calcium release and extracellular calcium influx. Identical to our earlier results, the calcium response of fMLF-stimulated Gnaq^−/− neutrophils initiated apparently normally but was not sustained and rapidly dropped to background levels (Fig. 4 A). Indeed, as shown in Fig. 4 B, the calcium response of the EGTA-treated fMLF-activated Gnaq^−/− neutrophils was very similar to that seen in equivalently treated WT neutrophils, indicating that Gα_q is not obligatorily required for intracellular calcium release, at least in this cell type. Because intracellular calcium release in chemokine receptor–stimulated leukocytes is IP₃ dependent, we asked whether we could block the remaining calcium response in Gnaq^−/− neutrophils by treating the fMLF-activated Gnaq^−/− cells with the IP₃ inhibitor 2-aminoethoxydiphenylborate (2-APB). As shown in Fig. 4 C, the calcium response was ablated in the 2-APB–treated Gnaq^−/− neutrophils, similar to that observed with fMLF-activated Gnai2^−/− neutrophils (Fig. 3).

The calcium responses of the Gnaq^−/− neutrophils were very similar to our previous findings using Cd38^−/− neutrophils (28, 31), suggesting to us that Gα_q and CD38 likely coregulate the same calcium entry pathway in the fMLF-activated neutrophils. To test this possibility, we stimulated Gnaq^−/− neutrophils with fMLF in the presence or absence of the cADPR antagonist 8Br-cADPR and measured the calcium response. As predicted, the calcium signaling profiles were identical between the untreated and 8Br-cADPR–treated Gnaq^−/− cells (Fig. 4 D). Collectively, the data indicate that Gα_i2 controls the IP₃-gated calcium release, and that Gα_q and CD38 coordinately sustain the calcium response by activating calcium entry in fMLF-activated mouse bone marrow neutrophils. The data further suggest that the Gα_i2-dependent calcium release is necessary for the initiation of the sustained calcium response in the fMLF-activated bone marrow neutrophils. However, it is also possible that Gα_i2 additionally regulates the CD38- and Gα_q-dependent calcium influx response by other mechanisms unrelated to intracellular calcium release.

Gα_q differentially regulates chemokine receptor signaling in DCs and T lymphocytes

Because Gα_q and CD38 are both required for productive mFPR1 and CCR1 signaling in mouse neutrophils, we postulated that Gα_q would also be required for chemotaxis of DCs to CXCR4 and CCR7 ligands, as signaling through these receptors is CD38 and cADPR dependent (Fig. 1). Furthermore, we predicted that signaling through CXCR4 and CCR7 in T cells would not be Gα_q dependent, as CD38 is not required for the chemotaxis of these cells to the CXCR4 and CCR7 ligands CXCL12 and CCL19 (Fig. 1). Instead, we predicted that T cell chemotaxis to these ligands would require Gα_i2. To test these predictions, we measured the in vitro chemotaxis of DCs and T cells from WT, Gnaq^−/−, and Gnai2^−/− mice to CCL19 and CXCL12. As expected based on previously published experiments using the Gi inhibitor PTx (38–40), the chemotaxis of T cells and DCs to both chemokines requires Gα_i2 (Fig. 5, A and C). However, chemotaxis of Gnaq^−/− T cells to the same two chemokines was completely normal (Fig. 5 B), indicating that CCR7 and CXCR4 signaling is Gα_i2, but not Gα_q, dependent in T cells. In striking contrast, the chemotaxis of Gnaq^−/− DCs to CXCL12 and CCL19 was ablated (Fig. 5 D). The impaired chemotaxis of the Gnaq^−/− DCs was not caused by defective chemokine receptor expression, at least for CXCR4, as this receptor was expressed at equivalent levels in the WT, Gnai2^−/−, and Gnaq^−/− DCs (Fig. 5 E). Thus, Gα_q, like CD38, regulates CCR7 and CXCR4 signaling in DCs but not in T cells. Collectively, these data indicate that the same chemokine receptor can couple to the Gα_q/CD38-dependent signaling pathway in one cell type but not necessarily in all cell types.

Inflammation-induced migration of DCs and monocytes requires Gα_q

Previous results showed that Gα_q regulates the in vitro chemotaxis of DCs but not B cells (not depicted) and T cells (Fig. 5). Based on these data, we predicted that Gα_q would also be required for the in vivo migration of DCs to secondary lymphoid organs. To test this hypothesis, we first examined the composition and cellularity of the skin, LNs, and spleens of Gnaq^−/− chimeras. As shown in Table I, the total cellularity of the various LNs (axillary and mesenteric), spleen, and skin epidermis was similar between WT and Gnaq^−/− mice. Likewise, the numbers of T cells present in these tissues were indistinguishable between WT and Gnaq^−/− bone marrow chimeras (Table I). Finally, we did not observe any reductions in the numbers of DCs present in the LNs and spleens of the Gnaq^−/− mice (Table I). Indeed, if anything, some DC populations were elevated in the spleen and mesenteric LNs of the Gnaq^−/− mice. Therefore, these data indicate that Gα_q is not required for the migration of DCs or T cells to secondary lymphoid organs under homeostatic conditions.

Next, to test whether Gα_q regulates DC migration to LNs in response to inflammatory stimuli, we sensitized the skin of WT and Gnaq^−/− mice with FITC in acetone/dibutyl phthalate and prepared frozen tissue sections of the draining LNs 18 h after sensitization. The sections were stained with CD11c and CD90.2 to identify DCs within the T cell zone of the LNs. As shown in Fig. 6 A, the FITC-bearing DCs were easily detected in the T zone of WT LNs but were largely absent from the T cell area of Gnaq^−/− LNs (Fig. 6 B). To determine whether this was caused by the inappropriate localization of the Gnaq^−/− DCs to other regions within the LN, we used FACS analysis to enumerate the total number of FITC^hiCD11c⁺ClassII⁺ DCs present in the draining LNs of the FITC-sensitized WT and Gnaq^−/− mice. The number of FITC-labeled DCs present in the Gnaq^−/− LN was reduced by >90% (Fig. 6 C), indicating that the DCs present in the skin of Gnaq^−/− mice did not accumulate in secondary lymphoid tissues in response to inflammatory challenge. This was not caused by a deficit of DCs in the epidermis of Gnaq^−/− mice, as the cells were present in equal numbers in the epidermis before stimulation (Table I). Nor did it appear that the Gnaq^−/− DCs were unable to mature in response to inflammatory stimuli, as bone marrow–derived Gnaq^−/− DCs matured equivalently to WT DCs in vitro in response to TNF-α (unpublished data). Thus, the data strongly suggest that Gnaq^−/− DCs are unable to migrate to LNs in response to inflammatory challenge.

We previously demonstrated that CD38 and cADPR regulate the trafficking of CCR2-expressing monocytes both in vitro and in vivo (29). Given that CD38 is expressed on monocytes (not depicted) and that CD38 and Gα_q each regulate the migration of mature DCs to draining LNs in response to inflammatory stimuli (Fig. 6 A) (29), we hypothesized that Gα_q would also be required for the migration of CCR2⁺ monocytes to the inflamed skin. To test this hypothesis, we took advantage of a previously described model to monitor the migration of monocytes to inflamed skin (41). In brief, we lethally irradiated WT CD45.1⁺ B6 mice and reconstituted the mice with congenic CD45.2⁺ WT (WT chimeras) or CD45.2⁺ Gnaq^−/− bone marrow (Gnaq^−/− chimeras; Fig. 6 D). 8 wk after reconstitution, we isolated cells from skin epidermal sheets (isolated from the ear) of the WT and Gnaq^−/− chimeras and stained the cells with antibodies to CD45.1, CD45.2, CD11c, and MHCII I-A^b. As previously reported for this model (41), the resident Langerhans cells (LCs; CD11c⁺ClassII⁺) in the skin were radiation resistant. Thus, the vast majority of LCs in the skin of both groups of chimeras were of host origin and expressed the CD45.1 allele (Fig. 6 E, day 0). In contrast, the hematopoietic cells isolated from all other sites (i.e., bone marrow, blood, and secondary lymphoid tissues) expressed the CD45.2 allele (unpublished data), indicating that these populations were radiation sensitive and had been replaced by cells derived from the donor (either WT or Gnaq^−/−) bone marrow.

Next, we treated the epidermis of the chimeric animals with the inflammatory agent dinitrofluorobenzene (DNFB), as we previously showed that this contact stimulant activates LCs to migrate to LNs and induces the subsequent influx of bone marrow– or blood-derived monocytes to the inflamed epidermis, where the cells up-regulate MHCII and CD11c (29). 4 d after DNFB exposure, we isolated cells from the inflamed epidermis and again stained the cells with antibodies to CD45.1, CD45.2, CD11c, and MHCII I-A^b. As expected (29), DNFB exposure promoted the egress of the host-derived resident WT CD45.1⁺ LCs from the skin, resulting in a large decrease in this population in the epidermis of both WT and Gnaq^−/− chimeras (Fig. 6 E, day 4). In the WT chimeras, the epidermis was largely repopulated with CD11c⁺ClassII⁺ cells that expressed the CD45.2 allele (Fig. 6 E, day 4), indicating that WT donor–derived monocytes were recruited to the inflamed skin. In striking contrast, the epidermis of the Gnaq^−/− chimeras was very deficient in CD11c⁺ClassII⁺ cells, and only a small number of CD45.2-expressing (Gnaq^−/−) CD11c⁺ClassII⁺ cells were detected (Fig. 6 E, day 4). These data therefore indicate that Gnaq^−/− monocytes were not efficiently recruited to the inflamed epidermis, despite the fact that the inflamed skin and initial resident LCs were of WT origin. Collectively, these data show that the alternative Gα_q-dependent chemokine receptor signaling pathway is critical for the in vivo trafficking of DCs and monocytes in response to inflammatory signals and indicates that this signaling pathway regulates innate immune responses and the cellular processes associated with inflammation.

Mice and bone marrow reconstitutions.

C57BL/6J (B6), CD45.1⁺ congenic B6, C57BL/6J.129 Cd38^−/− (N12 backcross to C57BL/6J) (28), Gnaq^−/− (N>5 backcross to C57BL/6J) (70), and Gnai2^−/− (N>5 backcross to C57BL/6J) (70) mice were bred and maintained in the Trudeau Institute breeding facility. Gnaq^−/− mice are born runted and often do not live to adulthood (71). Therefore, all in vitro chemotaxis and calcium signaling assays were routinely performed using cells isolated from lethally irradiated B6 recipients that were reconstituted with B6, Gnaq^−/−, or Gnai2^−/− bone marrow 8 wk previously. This approach allowed us to examine the impact of Gα_q protein deficiency specifically within hematopoietic lineage cells and permitted us to obtain cells from larger numbers of adult animals for the analyses. Bone marrow chimeric mice were generated by reconstituting lethally irradiated recipients (950 cGy from a 137Cs source) with 10⁷ total bone marrow cells isolated from appropriate donor mice. Mice were allowed to reconstitute for 8 wk before experiments. The Trudeau Institute Institutional Animal Care and Use Committee approved all procedures involving animals.

Reagents.

Reagents were obtained from R&D Systems (CXCL12, CCL3, and CCL19), Sigma-Aldrich (PAF, IL-8, fMLF, PTx, and DNFB), Calbiochem (2-APB), and BD Biosystems (all antibodies). 8Br-cADPR (provided by T. Walseth, University of Minnesota, Minneapolis, MN) was prepared as previously described (72). All reagents were used at the concentrations indicated in the text and figure legends.

Cell subset quantification, cell purification, and chemotaxis assays.

Cells were isolated from the spleen, LNs (axillary and mesenteric), and skin epidermis of either WT or Gnaq^−/− chimeric mice. The total number of viable cells was determined, the cells were stained with fluorochrome-labeled antibodies, and the total number of B cells (CD19⁺), T cells (CD3⁺ and CD4⁺ or CD8⁺), myeloid DCs (CD11c⁺CD11b⁺CD8α⁻), lymphoid DCs (CD11c⁺CD11b⁻CD8α⁺), plasmacytoid DCs (CD11c^intGR1⁺B220^int), and LCs (CD11c⁺I-Ab⁺Langerin⁺) was determined.

To purify bone marrow neutrophils, bone marrow cells were first stained with biotinylated anti-GR1 antibody and MACS streptavidin microbeads (Miltenyi Biotec) and then positively selected on a MACS midi column. CD4 T cells were purified using CD4 microbeads (Miltenyi Biotec). To isolate DCs, single-cell suspensions were prepared from pooled spleens and mesenteric, inguinal, and axillary LNs that were perfused with collagenase D and incubated for 60 min at 37°C. The cells were purified by two rounds of positive selection using biotinylated anti-CD11c antibody and streptavidin microbeads. The purity of all cell populations was routinely >90%. In vitro chemotaxis assays were performed using 24-well transwell plates (Costar) with either a 3-μm (for neutrophils) or 5-μm (DCs and T cells) pore size polycarbonate filter. In some experiments, the isolated cells were first pretreated for 20 min with 100 μM 8Br-cADPR. After any pretreatments, the cells were added to the top chamber of the transwell (10⁵ DCs and 10⁶ T cells or neutrophils per well, respectively). Transwell plates were incubated for 45 min (neutrophils) or 2 h (DCs and T cells). The transmigrated cells were collected from the lower chamber, fixed, and counted on a flow cytometer. The absolute number of cells in each sample was determined by spiking each sample with a known number of 20-μm fluorescent microbeads that were simultaneously counted on the flow cytometer. In some cases, the results are expressed as the mean ± SD of the chemotactic index (CI). The CI represents the fold increase in the number of migrated cells in response to chemoattractants over the spontaneous cell migration (to control medium).

Calcium mobilization assays.

10⁷ bone marrow neutrophils and 10⁶ DCs per milliliter were loaded with a mixture of Fluo-3 AM and Fura-red AM, as previously described (28, 29). In some experiments, the cells were preincubated for 20 min in 8Br-cADPR or 2-APB. In other experiments, cells were pretreated with 500 ng/ml PTx for 4 h, washed, and stimulated with chemokines. The accumulation of intracellular free calcium was assessed by flow cytometry by measuring the fluorescence emission of Fluo-3 in the FL-1 channel and Fura-red in the FL-3 channel over time. Data were analyzed using FlowJo software (version 3.0; TreeStar, Inc.). Relative intracellular free calcium levels are expressed as the ratio between Fluo-3 and Fura-red mean fluorescence intensity.

In vivo DC activation and migration assays.

To measure the migration of monocytes to the skin, we followed the protocol described by Merad et al. (41). In brief, CD45.1⁺ B6 recipient mice were lethally irradiated and reconstituted with either CD45.2⁺ B6 bone marrow or with CD45.2⁺ Gnaq^−/− bone marrow. 8 wk after reconstitution, cells from skin epidermal sheets were isolated (73) and stained with antibodies to CD45.1, CD45.2, CD11c, and ClassII to determine whether the LCs (CD11c⁺ClassII⁺) were of donor (CD45.2⁺) or host (CD45.1⁺) origin. The remaining mice were then exposed to DNFB (0.5% applied to the ear). On day 4 after DNFB application, cells were isolated from skin epidermal sheets and stained with the same panel of antibodies. The number of donor (CD45.2⁺) and host-derived (CD45.1⁺) LCs (CD11c⁺ClassII⁺) present in the skin samples was then determined by cell counting and FACS.

To track DC migration from inflamed skin to LNs, 25 μl FITC (8 mg/ml in 1:1 acetone/dibutylphthalate) was applied to two shaved abdominal areas of B6 or Gnaq^−/− (nonchimeric) animals. Inguinal LNs were removed after 18–24 h, counted, stained with antibodies to class II and CD11c, and analyzed by flow cytometry. The total number of migrated FITC^hiCD11c⁺classII⁺ DCs was determined.

Immunohistology.

5-μm frozen sections were prepared from inguinal LNs isolated from FITC-sensitized mice at 18 h after exposure. Sections were stained with antibodies to CD11c (conjugated to Alexa Fluor 594; Invitrogen) and CD90.2 (conjugated to Alexa Fluor 350; Invitrogen). Slides were viewed at 200× magnification using a microscope (Axiophot 2; Carl Zeiss MicroImaging, Inc.), and images were captured with a digital camera (AxioCam; Carl Zeiss MicroImaging, Inc.) using Axiovision software (version 3.0; Carl Zeiss MicroImaging, Inc.). Images were cropped and scaled using Canvas software (version 9.0; ACD Systems).

Statistical analysis.

Datasets were analyzed using Prism software (version 4.0 for Macintosh; GraphPad Software). Student's t tests were applied to the datasets, and P < 0.05 was considered statistically significant. All data are shown as the mean ± SD.