The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 439 -448
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hwang, I.-Y.
Right arrow Articles by Kehrl, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hwang, I.-Y.
Right arrow Articles by Kehrl, J. H.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

Impaired Trafficking of Gnai2+/– and Gnai2–/– T Lymphocytes: Implications for T Cell Movement within Lymph Nodes

Il-Young Hwang, Chung Park and John H. Kehrl1

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signals generated by the engagement of chemoattractants with their cognate receptors orchestrate lymphocyte movements into and out of lymphoid organs and sites of inflammation. Yet, the role of chemokines in organizing lymphocyte movements in lymphoid organs is controversial. Recent evidence suggests that the extensive network of fibroblastic reticular cells within the T cell areas helps guide T cells. The expression of adhesion molecules and chemokines by fibroblastic reticular cells most likely facilitates their influence on T cell movements. Consistent with this hypothesis, CD4 T cells with defective chemokine receptor signaling move very differently within lymph nodes than do normal cells. For the imaging studies, we used CD4 T cells prepared from Gnai2–/– mice, which lack G{alpha}i2 expression. We first demonstrate that CD4 as well as CD8 T cells from these mice are markedly defective in chemokine receptor signaling. Gnai2–/– T cells have profound defects in chemokine-induced intracellular calcium mobilization, chemotaxis, and homing, whereas Gnai2+/– T cells exhibit modest defects. Intravital imaging revealed that within the inguinal lymph nodes Gnai2–/– CD4 T accumulate at the cortical ridge, poorly accessing the lymph node paracortex. They also lack the customary amoeboid-like cell movements and active membrane projections observed with normal CD4 T cells. These results demonstrate the importance of G{alpha}i2 for T lymphocyte chemokine receptor signaling and argue that local chemoattractants regulate the movement of CD4 T cells in lymph nodes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Proper functioning of the immune system depends upon changing intracellular contacts and cellular localization both within immune organs and within the body. Chemoattractants act as signposts to recruit and position lymphocytes and dendritic cells in lymphoid organs and inflammatory sites (1, 2). Most chemoattractants and chemokines signal through G protein-coupled receptors (GPCRs)2 that use the heterotrimeric G protein Gi to activate downstream effectors (3, 4). The binding of ligand activates receptors triggering G{alpha}i subunits to exchange GTP for GDP, resulting in the dissociation of the G{alpha} subunit from its associated Gbeta{gamma} heterodimers (5, 6). The release of Gi-associated Gbeta{gamma} subunits is necessary for triggering directional migration (3, 4, 7). Because G{alpha} subunits possess an intrinsic GTPase activity, GTP hydrolysis leads to the reassembly of heterotrimeric G protein, causing signaling to cease (5, 6). Lymphocytes strongly express two members of the Gi subfamily, G{alpha}i2 and G{alpha}i3 (8). Gnai3–/– mice are reportedly without a phenotype (9); however, Gnai2–/– mice exhibit defective B cell chemokine receptor signaling, as evidenced by depressed B cell chemotaxis, defective B cell homing to lymph nodes, poor B cell adherence to lymph node high endothelial venules (HEVs), and decreased B cell motility within lymph node follicles (8).

The impact of Gnai2 deficiency on T cell chemotaxis and T cell trafficking has not been reported. Yet defective T cell function has been documented. Gnai2–/– mice develop a Th1-mediated inflammatory colitis reminiscent of human ulcerative colitis, whose penetrance depends upon the genetic background of the mice (10). Gnai2–/– CD4 T cells exhibit augmented responses to TCR signaling with enhanced intracellular calcium release and cytokine production; in contrast, Gnai3–/– T cells respond normally to TCR signaling (9). A relative increase in mature thymocytes in the thymus has also been noted in the Gnai2–/– mice (10).

To characterize the impact of Gnai2 deficiency on T cell chemotaxis and T cell trafficking, we have examined CD4 and CD8 T cells from wild-type, Gnai2+/–, and Gnai2–/– mice. We find that Gnai2–/– T cells exhibit significant defects in chemokine receptor signaling and lymphocyte trafficking, suggesting that signals that modulate the level of G{alpha}i2 present in lymphocytes directly affect the capacity of lymphocytes to respond to chemokines. The Gnai2–/– mice have pronounced defects in chemokine receptor signaling, indicating that Gnai3 and Gnai1 poorly compensate for the loss of Gnai2 and that CXCR5 and CCR7 predominantly couple to Gi{alpha}2 in T lymphocytes. Finally, these results argue that GPCR signaling significantly impacts the movement of T cells within the lymph node.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The generation of Gnai2–/– mice has been previously described (10). The mutation was backcrossed onto a C57BL/6 background six times. Mice heterozygotic or homozygotic for the mutation were produced by crossing heterozygotic mice. Wild-type littermates were used as controls. C57BL/6 mice were purchased from The Jackson Laboratory. All mice used in this study were 8–14 wk of age. Mice were housed under specific pathogen-free conditions and used in accordance with the guidelines of the Institutional Animal Care Committee at the National Institutes of Health.

Reagents

Abs against mouse CD11a, CD11c, GR-1, CD49d, CXCR4, CXCR5, CCR5, CD4, CD8a, B220, and CD62L were purchased from BD Pharmingen; CCR7 from BioLegend; and G{alpha}i1, G{alpha}i2, and G{alpha}i3 from Santa Cruz Biotechnology. Streptavidin conjugated to PE was purchased from BD Pharmingen. Pertussis toxin was purchased from Calbiochem. The 5-(and 6-)(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine (CMTMR) and 5-chloromethylfluorescein diacetate (CMFDA) were purchased from Molecular Probes. Murine CCL19, CXCL12, and CXCL13 were purchased from R&D Systems.

Cells

Splenic T cells were isolated by negative depletion using biotinylated Abs to B220, GR-1, and CDllc and Dynabeads M-280 streptavidin (Dynal Biotech), as previously described (11). The addition of a biotinylated Ab to CD4 or CD8 allowed isolation of CD8 or CD4 T cells, respectively. The cell purity was greater than 95%. Cells were placed in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-ME.

Intravital two-photon microscopy

Splenic CD4 or CD8 T cells prepared from wild-type, Gnai2+/–, or Gnai2–/– were labeled for 15 min at 37°C with 2.5~5 µM CMTMR or CMFDA (8). Seven to 10 million labeled cells of each population in 200 µl of PBS were adoptively transferred by tail vein injection into 6~10-wk-old recipient mice. Recipient mice were anesthetized with isoflurane (Baxter). The isoflurane was used at 2.5% for induction and 1–1.5% for maintenance vaporized in an 80:20 mixture of oxygen and air. The inguinal lymph node was prepared microsurgically for intravital microscopy using a slightly modified published surgical technique (8). The mouse was placed on a heating pad, with the skin flap raised on a glass plate over a heating pad. The inguinal lymph node was surgically exposed on a skin flap, and the surrounding tissue was attached to the base of Microwell Dish with veterinary-grade glue (Nexaband), and 3 ml of prewarmed PBS at 37°C was added. The temperature of PBS, tissue near the lymph node, and the surrounding closed air were monitored and maintained at 36.5 ± 0.5°C. Inguinal lymph node was intravitally imaged from the capsule (100~200 µm deep). Two-photon imaging was performed with an inverted Leica TCS-SP2 MP confocal microscope (Leica Microsystems) equipped with x20 water-immersion objective, 0.5 W U-V-I (immersion medium used Dulbecco’s PBS). Two-photon excitation was provided by a Mai Tai Ti:Sapphire laser (Spectra Physics) with a 10 W pump, tuned to 800 nm. For four-dimensional analysis of cell migration, stacks of 12 section (z step = 3 µm) were acquired every 30 s to provide an imaging volume of up to 33 µm in depth. Emitted fluorescence was collected using a two-channel nondescanned detector. Wavelength separation was through a dichroic mirror at 560 nm, followed by 525/50 nm, and 610/75 nm emission filters. Sequences of image stacks were transformed into volume-rendered four-dimensional movies using Imaris software v.5.0.1 (Bitplane), and the spot analysis was used for semiautomated tracking of cell motility in three dimensions by using the parameters of autoregressive motion as an algorithm, 7 µm spot diameter, 2 µm background object diameter, and 15 µm maximum distance (every 30 s). The data set was corrected for tissue drift by the Imaris software. Calculations of the cell velocity, track straightness, and cell displacement were performed using the Imaris software. The cell polarity was calculated by measuring long and short axes of individual cells using the point-to-point measurement feature in Imaris software. Blood vessels within the inguinal lymph node were revealed by i.v. injection of 0.05 mg of tetramethylrhodamine dextran (3 kDa; Molecular Probes) and 0.1 mg of FITC-dextran (150 kDa; Sigma-Aldrich) into the anesthetized mouse immediately before T cell imaging. For three-dimensional analysis, stacks of 267 section (z step = 1 µm) were acquired to provide an imaging volume of up to 266 µm in depth. Individual slices and three-dimensional (3-D) reconstruction was done using the Surpass feature of the Imaris software.

Homing assays

Splenic CD4 or CD8 T cells from wild-type, Gnai2+/–, or Gnai2–/– mice were labeled with either 1 µM CMFDA or 2.5 µM CMTMR for 15 min at 37°C, and 7–20 million cells of each population were injected i.v. to recipient mice. After 2 h, spleen, inguinal lymph nodes, and popliteal lymph nodes were removed, and gently dissociated into single-cell suspensions. Peripheral blood was collected by retro-orbital eye bleeding. After removing RBC with Tris-NH4Cl, the cells were resuspended in PBS containing 1% BSA at 4°C. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star). Forward and side scatter parameters were used to gate on live cells. Alternatively, the labeled cells were injected into the footpad of recipient mice, and 12 h later the ipsilateral popliteal and inguinal lymph nodes and the contralateral popliteal lymph node were removed and processed, as above. In some instances, the T cells were pre incubated with 100 ng/ml pertussis toxin for 2 h at 37°C before transfer.

Lymph node transit assay

The assay was performed as previously described (8). Splenic CD4 T cells from wild-type or Gnai2–/– mice were labeled with either 2 µM CMFDA or CMTMR for 15 min at 37°C, and 7–20 million cells of each population were injected i.v. to recipient mice. Two hours later, the mice were injected i.v. with either PBS or anti-L-selectin Ab (100 µg/mouse). After 12 h, inguinal and popliteal lymph nodes were removed and gently dissociated into single-cell suspensions. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using the FlowJo software (Tree Star). Forward and side scatter parameters were used to gate on live cells.

Chemotaxis

Chemotaxis assays were performed using a Transwell chamber, as previously described (8, 11). In some experiments, T cells were incubated with 100 ng/ml pertussis toxin for 2 h at 37°C. The cells were washed twice, resuspended in complete RPMI 1640 medium, and added in a volume of 100 µl to the upper wells of a 24-well Transwell plate with a 5-µm insert. Lower wells contained various doses of chemokines in 600 µl of complete RPMI 1640 medium. The number of cells that migrated to the lower well following a 2-h incubation was counted using a flow cytometer.

Determination of changes in intracellular Ca2+ concentration ([Ca2+]i)

Cells were seeded at 105 cells per 100 µl loading medium (RPMI 1640, 10% FBS) into poly(D-lysine)-coated 96-well black-wall, clear-bottom microtiter plates (Nalge Nunc International). An equal volume of assay loading buffer (FLIPR Calcium 3 assay kit; Molecular Devices) in HBSS supplemented with 20 mM HEPES and 2 mM probenecid was added. Cells were incubated for 1 h at 37°C before adding chemokine, and then the calcium flux peak was measured using a FlexStation (Molecular Devices). The data were analyzed with SOFT max Pro (Molecular Devices). Data are shown as fluorescent counts, and the y-axis is labeled as Lm1.

Immunoblotting

Cell lysates were prepared as previously described (8). The detergent-insoluble materials were removed, and equal amounts of protein were fractionated by 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in Tween 20 plus PBS for 1 h and then incubated with an appropriate dilution of the primary Ab in 5% BSA in Tween 20 plus PBS for 2 h. The blots were incubated with biotinylated Ab for 1 h, and further incubated with streptavidin conjugated to HRP for 1 h. The signal was detected by ECL (Amersham Biosciences).

Statistics

In vivo results represent samples from three to six mice per experiment. In vitro results represent mean values of sextuplet samples. All experiments were performed more than three times. SD and p values were calculated with Student’s t test using Microsoft Excel software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Gnai2+/– and Gnai2–/– T lymphocytes express reduced levels of Gi{alpha}2 compared with wild-type cells and the effects of T cell activation on Gi{alpha}2 levels

T cell lysates from splenic T cells prepared from wild-type, Gnai2+/–, and Gnai2–/– mice were immunoblotted for the presence of Gi{alpha}1, Gi{alpha}2, and Gi{alpha}3. The T cells prepared from Gnai2+/– mice displayed ~50% less Gi{alpha}2 than did wild-type T cells, whereas no detectable Gi{alpha}2 was found in the T cells prepared from the Gnai2–/– mice. As we had previously observed with Gnai2–/– B lymphocytes (8), Gnai2–/– T cells express very low levels of Gi{alpha}1 and an increased level of Gi{alpha}3 compared with wild-type T cells (Fig. 1A). To explore the impact of T cell activation on Gnai1, Gnai2, and Gnai3 expression, we performed RT-PCR using RNA samples prepared from wild-type CD4 and CD8 T cells stimulated for various durations with a mAb directed at CD3 and IL-2. We found a 2-fold enhancement in Gnai2 and Gnai3 expression and no change in the low level of Gnai1 after 24 h of stimulation; however, when we immunoblotted for Gi{alpha}2 and Gi{alpha}3 we did not detect a significant difference between the stimulated and unstimulated cells (data not shown).


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 1. Expression of Gi{alpha} isoforms and chemokine receptors in T cells from Gnai2+/– and Gnai2–/– mice. A, Immunoblot of cell lysates prepared from wild-type, Gnai2+/–, and Gnai2–/– T cells for expression of Gi{alpha}1, Gi{alpha}2, Gi{alpha}3, and actin. B, Flow cytometry of splenic CD4 or CD8 T cells prepared from either wild-type, Gnai2+/–, or Gnai2–/– mice. Wild-type profiles are nonshaded, Gnai2+/– profiles are shaded in light gray, and Gnai2–/– are shaded in dark gray. Isotype controls are shown with a light gray line. Cell type and marker are indicated in the upper right-hand corner of each profile. Representative results are from four separate experiments.

 
Gnai2+/– and Gnai2–/– T cells have altered chemokine receptor expression

To explore whether haploinsufficiency or the lack of Gnai2 expression impacted chemokine receptor expression and other receptors involved in lymphocyte trafficking, we examined freshly isolated splenic CD4 and CD8 T cells from wild-type and the mutant mice for their levels of CCR5, CCR7, CXCR4, L-selectin, CD11a, and CD49d expression by flow cytometry. We found either a very modest or no change in receptor expression on both CD4 and CD8 T cells from the Gnai2+/– mice, and mild to modest decreases of each of the tested receptors on CD4 and CD8 T cells prepared from the Gnai2–/– mice (Fig. 1B). In contrast, the expression levels of CD4 and CD8 on T cells from Gnai2+/– and Gnai2–/– mice were similar to wild-type mice. The mechanism accounting for the reductions in chemokine receptor expression in the Gnia2–/– T cells is not known. Whether the reduction in G{alpha} subunit expression impacts the expression of GPCRs that couple to that G{alpha} subunit is not an issue that has been addressed to our knowledge. Alterations in peripheral T cells trafficking receptors may also arise from altered thymocyte development and thymocyte egress that most likely occurs as a consequence of the absence of Gi{alpha}2. The reduction in L-selectin levels noted in the Gnai2–/– CD4 and CD8 T cells most likely impacts the homing of these cells to lymphoid organs because T cells from L-selectin+/– mice exhibit a greater than 50% reduction in homing to lymph nodes (12).

Gnai2+/– T cells have diminished in vitro responses to chemokines and mildly impaired in vivo trafficking

To determine whether a reduction in Gnai2 expression altered the responsiveness of T cells to chemokine stimulation, we first assessed responses in standard chemotaxis assays using increasing concentrations of CCL19 and CXCL12. Both CD4 and CD8 T cells from Gnai2+/– mice migrated at reduced frequency compared with wild-type cells at each tested concentration of CXCL12 and CCL19 (Fig. 2A). The reduction averaged 25–30% for both chemokines. We also tested B cells from Gnai2+/– mice, and they also showed a reduction in responsiveness to chemokines with decreases of 40 and 50%, when stimulated with optimal concentrations of CXCL12 and CCL19, respectively (data not shown). Pretreatment with pertussis toxin blocked the chemotaxis of both wild-type and Gnai2+/– T and B cells (Fig. 2A).


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 2. Gnai2+/– T cells have reduced responses to chemokines and exhibit mild defects in homing. A, Chemotaxis assays. CD4 (first and second panels) and CD8 (third and fourth panels) T cells purified from wild-type (WT, Figure 2) and Gnai2+/– ({blacksquare}) mice were subjected to a 2-h chemotaxis in response to CXCL12 or CCL19, as indicated. The percentages of cells responding to 10, 30, and 100 ng/ml CXCL12 or CCL19 are shown. Results are mean and SE of sextuplet samples from four experiments (*, p < 0.01 vs wild type). In some instances, the cells were pretreated with pertussis toxin (100 ng/ml) for 2 h and washed, before being subjected to a chemotaxis assay. B, Homing of i.v. transferred CD4 T cells. Ten million labeled Gnai2+/– T cells and the same number of labeled wild-type T cells were i.v. transferred to recipient mice (six experiments each with three pairs of mice; *, p < 0.01 vs wild type). Two hours after transfer, cells from blood, spleen, inguinal lymph nodes, and popliteal lymph nodes of recipient mice were analyzed using flow cytometer. Shown are the actual number of transferred CD4 T cells found in blood (per 10 µl), spleen (x104 cells), inguinal lymph nodes (x102 cells), and popliteal lymph nodes (x10 cells). C, Homing of footpad-injected CD4 T cells. Ten million labeled Gnai2+/– T cells and the same number of labeled wild-type T cells were injected into the footpad of recipient mice (four experiments each with three pairs of mice). Twelve hours after transfer, cells from ipsilateral and contralateral popliteal and ipsilateral inguinal lymph nodes of recipient mice were analyzed using flow cytometer. Shown are the percentages of recovered cells. D, Velocity profiles. Ten million labeled Gnai2+/– T cells and the same number of labeled wild-type T cells were transferred i.v. to wild-type recipients. Twenty-four hours after cell transfer, inguinal lymph nodes were imaged intravitally using multiphoton microscope. Velocity profile of wild-type ({square}) and Gnai2+/– ({blacksquare}) cells. The average speeds are indicated with arrows. E, Mean velocity of wild-type and Gnai2+/– T cells. Speed was calculated from three independent movies by spot analysis using Imaris software.

 
To assess the consequence of reducing Gnai2 expression on T cell trafficking in vivo, we examined the homing of transferred CD4 T cells and their motility in the inguinal lymph node of recipient mice by intravital imaging. We performed six experiments that examined CD4 T cell homing to the spleen, inguinal, and popliteal lymph nodes. In each experiment, fewer Gnai2+/– CD4 T cells homed to the lymph nodes as compared with wild-type cells simultaneously transferred, a result consistent with the previous chemotaxis assays. On average 30 and 24% fewer cells entered the popliteal and inguinal lymph nodes, respectively (Fig. 2B). In addition, we noted a modest increase in the number of Gnai2+/– CD4 T in the spleen and peripheral blood of the recipient mice as compared with wild-type cells. We did not check the efficiency at which the Gnai2+/– CD4 T cells entered the splenic white pulp, although we would expect a modest reduction similar to that observed in lymph node entry. We also determined whether Gnai2+/– CD4 T cells trafficked normally to draining lymph nodes. We transferred labeled wild-type and Gnai2+/– CD4 T into the footpads of recipient mice and determined the percentage of transferred cells that entered into the ipsilateral popliteal and inguinal lymph nodes and the contralateral popliteal lymph node. We noted a slight reduction in the percentage of the recovered Gnai2+/– CD4 T in the ipsilateral popliteal lymph node compared with wild-type cells (Fig. 2C). We also assessed Gnai2+/– CD4 T cell motility by intravital imaging. We again performed six experiments, each of which revealed a slight decrease in T cell motility compared with wild-type cells. The velocity profile of the Gnai2+/– T cells was slightly shifted to the right compared with T cells from wild-type mice; however, the difference was not statistically significant (Fig. 2D). The average velocity of the Gnai2+/– CD4 T cells was ~10% less than that observed with simultaneous transferred wild-type CD4 T cells (Fig. 2E).

Markedly reduced chemotaxis and [Ca2+]i mobilization following exposure of Gnai2–/– lymphocytes to CXCL12 or CCL19

Examination of the increases in [Ca2+]i following chemokine exposure of Gnai2+/– and Gnai2–/– CD4 T cells revealed a significant and more marked reduction in their responses, respectively, as compared with wild-type CD4 T cells (Fig. 3A). This indicates that the major mechanism by which CXCL12 and CCL19 increase [Ca2+]i levels is directed by Gbeta{gamma} subunits associated with Gi{alpha}2. We have previously reported markedly reduced chemotactic responses of Gnai2–/– B cells to CXCL12 and CCL19. The Gnai2–/– CD4 T and CD8 T were slightly less impaired than the Gnai2–/– B cells, but all three cell types migrated poorly to CXCL12 and CCL19 at each concentration tested. The specific migratory responses (percentage migrated in the presence of chemokine minus percentage migrated in the absence of chemokine) of the Gnai2–/– CD4 and CD8 T cells at the highest concentration of CXCL12 and CCL19 tested were reduced by 4- to 7-fold compared with wild-type T cells (Fig. 3B). Similar to what we had observed with the Gnai2–/– B cells, the majority of the residual responses of the Gnai2–/– CD4 and CD8 T cells to CCL19 and CXCL12 were resistant to pretreatment with pertussis toxin (Fig. 3B). In contrast, pertussis toxin pretreatment completely inhibited the chemotaxis of wild-type T cells.


Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 3. Markedly impaired chemokine-triggered responses in Gnai2–/– T cells. A, Measurement of changes in [Ca2+]i. Splenic T cells from wild-type, Gnai2+/–, or Gnai2–/– mice were incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12 or CCL19 (100 ng/ml). Changes in [Ca2+]i were monitored over 3 min. The data were analyzed with SOFT max Pro and are shown as fluorescent counts, and the y-axis is labeled as Lm1. Experiment was performed twice with similar results. B, Chemotaxis assays. CD4 (upper panels) and CD8 (lower panels) T cells purified from wild-type (WT, Figure 3) and Gnai2–/– ({blacksquare}) mice were subjected to a 2-h chemotaxis in response to CXCL12 or CCL19, as indicated. Results are the mean of sextuplet samples from four experiments performed (*, p < 0.01 vs wild type). In some instances, the cells were pretreated with pertussis toxin (100 ng/ml) for 2 h and washed, before being subjected to a chemotaxis assay.

 
Impaired lymph node homing of T cells from Gnai2–/– mice

Next, we compared the homing of transferred CD4 T cells and CD8 T cells from wild-type and Gnai2–/– mice to peripheral lymph nodes, spleen, and blood. The various lymphocyte populations showed similar defects in homing with markedly reduced homing to popliteal and inguinal lymph nodes compared with wild-type cells. We recovered ~6.5-fold more wild-type CD4 T cells and 4-fold more wild-type CD8 T cells from the two sets of lymph nodes than we did the corresponding Gnai2–/– populations (Fig. 4A). By way of comparison, the recovery of wild-type B cells from the inguinal and popliteal lymph nodes exceeded by 5.5-fold the recovery of the Gnai2–/– B cells. These results are similar to what we observed with the Gnai2+/– cells, although the magnitude of the impairment was greater with the Gnai2–/– cells.


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 4. Gnai2–/– T cells home poorly to lymph nodes, but have no impairment in lymph node transit. A, Homing of i.v. transferred CD4 and CD8 T cells. Ten million labeled CD4 T cells, CD8 T cells, or B cells prepared from Gnai2–/– mice along with the same number of differentially labeled cells from wild-type mice were i.v. transferred to recipient mice (results are from six experiments each with three pairs of mice; *, p < 0.01 vs wild type). Two hours after transfer, cells from blood, spleen, inguinal lymph nodes, and popliteal lymph nodes of recipient mice were analyzed using flow cytometer. Shown are the actual number of transferred cells found in blood (per 10 µl), spleen (x104 cells), inguinal lymph nodes (x102 cells), and popliteal lymph nodes (x10 cells). B, Homing of footpad-injected CD4 T cells. Ten million labeled Gnai2–/– CD4 T cells and the same number of labeled wild-type CD4 T cells were injected into the footpad of recipient mice (results from four experiments each with three pairs of mice; *, p < 0.01 vs wild type). Twelve hours after transfer, cells from ipsilateral and contralateral popliteal and ipsilateral inguinal lymph nodes of recipient mice were analyzed using flow cytometer. Shown are flow cytometry results from the ipsilateral and contralateral popliteal lymph nodes and a chart of the percentages of recovered cells. C, Pertussis toxin effects on the homing of CD4 T cells. Similar experiment as B, but the wild-type, Gnai2+/–, or Gnai2–/– CD4 T cells were pretreated with pertussis toxin or not. Data are shown as percentages of wild-type, nonpertussis toxin treated. D, T cell transit through lymph nodes. Ten million labeled Gnai2–/– T cells and the same number of differentially labeled wild-type cells were i.v. transferred to recipient mice (results from four experiments each with three pairs of mice; *, p < 0.01 vs wild-type). Anti-L-selectin Ab (100 µg/mouse, {blacksquare}) was injected i.v. 2 h after cell transfer. Control mice were injected with PBS (Figure 4). Cells were isolated from inguinal and popliteal lymph nodes and analyzed using flow cytometer 24 h after Ab injection. Actual numbers of transferred cells recovered from the inguinal (left panel) and popliteal (right panel) nodes are shown. E, The ratios between Ab- and PBS-treated control groups are shown.

 
We also examined the ability of wild-type and Gnai2–/– T cells to migrate from peripheral tissues into draining lymph nodes. We injected each number of differentially labeled wild-type and Gnai2–/– CD4 T cells into the footpad of a wild-type mouse and determined the relative percentage of cells in the ipsilateral and contralateral popliteal and inguinal lymph nodes. We did not recover transferred cells from the inguinal lymph nodes or from the contralateral popliteal lymph node, but could recover cells from the ipsilateral popliteal lymph node. In the ipsilateral popliteal lymph node, we recovered ~2.5-fold more transferred wild-type cells than we did Gnai2–/– cells (Fig. 4B). To assess the effect of pertussis toxin on T cell migration from peripheral tissues into draining lymph nodes, we treated either wild-type, Gnai2–/+, or Gnai2–/– CD4 T cells with pertussis toxin before transfer. The pretreatment completely inhibited the appearance of wild-type and Gnai2–/+ CD4 T cells. In contrast, the reduced homing of the Gnai2–/– CD4 T cells was only modestly affected by prior pertussis toxin treatment (Fig. 4C).

For several reasons, the duration that Gnai2–/– T cells remain within the lymph node might be different from that of wild-type T cells. First, the reduced motility of Gnai2–/– T cells may impact their retention rate. Second, the presence of chemokines within the lymph node cortex may normally retain T lymphocytes there, or conversely, chemokines located at exit sites may promote T cell migration to those sites. In the absence of Gi{alpha}2, those signals would be greatly weakened. Third, sphingosine 1-phosphate (S1P) receptors signal lymph node egress via the activation of Gi, and the absence of Gi{alpha}2 could reduce the strength of an egress signal, thereby slowing Gnai2–/– T lymphocyte egress (13, 14, 15). A previous study has shown that pertussis toxin treatment inhibits lymphocyte egress from lymph nodes and that S1P1+/– cells have reduced lymph node exit efficiency (15). To compare wild-type and Gnai2–/– T cell retention in peripheral lymph nodes, we transferred differentially labeled cells to wild-type recipients, and 2 h later blocked further lymphocyte ingress by the i.v. injection of an L-selectin-specific Ab (Fig. 4D). We compared the number of wild-type and Gnai2–/– T cells in lymph nodes 24 h after Ab injection. We also plotted the data as the ratio between the recovered cells in the presence or absence of Ab. The treatment with anti-L-selectin Ab significantly reduced lymph node sizes and the recovery of lymphocytes from them. As expected, fewer Gnai2–/– T cells entered peripheral lymph nodes; however, based on the ratio indicated above, the Gnai2–/– T cells exited lymph nodes as well or better than did wild-type cells (Fig. 4E).

Intravital imaging of wild-type and Gnai2–/– CD4 T cells

Finally, we wanted to compare the distribution and motility of Gnai2–/– CD4 T cells with that of normal cells. To determine whether wild-type and Gnai2–/– CD4 T cells localized similarly within lymph nodes, we transferred differentially labeled wild-type and Gnai2–/– CD4 T cells into a recipient mouse and directly imaged the cells in the inguinal lymph node by intravital multiphoton microscopy acquiring a single stack of images at 1-µm intervals to a depth of 266 µm. Just before imaging, we injected a mixture of green and red fluorescent dextran to delineate the microcirculation of the lymph node. Five images are shown, the most superficial image acquired (0), and images from 50, 100, 200, and 265 µm deep (Fig. 5A; see supplemental movie 1).3 The most superficial image shows vessels near the surface of the lymph node, fat pads on the right side of the image, and a lymph node follicle. At a depth of 50 µm, the lymph node follicle is well visualized and along the edge of the follicle wild-type (green) and Gnai2–/– CD4 T cells (red) are seen. Despite reduced lymph node homing, more Gnai2–/– CD4 T cells localized along the edge of the lymph node follicle than did wild-type cells. This was even more evident at a depth of 100 µm, in which the ratio of Gnai2–/– CD4 T cells to wild-type T cells exceeded 2:1. In contrast, at a depth of 200 and 265 µm, the number of wild-type cells far outnumbers Gnai2–/– CD4 T cells, although several small groups of Gnai2–/– CD4 T cells were present. Also shown is a x,z projection of 20 µm compressed along the y-axis. Individual images of wild-type (green), Gnai2–/– CD4 T cells (red), and the composite image are shown (Fig. 5B). This again demonstrates the differing localization of the wild-type and Gnai2–/– CD4 T cells. Next, we acquired a series of 3-D image stacks sequentially every 30 s at depths between 100 and 150 µm (see supplemental movies 2 and 3).3 We noted a marked difference in the morphology of the wild-type and the Gnai2–/– CD4 T cells. The majority (~70%) of the wild-type T cells had a polarized morphology, whereas <10% of the Gnai2–/– T cells did so in the imaging files. Using the ratio of the cell length to cell width as a measure of cell polarity, the wild-type CD4 T cells had an average polarity index of 1.8, whereas the Gnai2–/– CD4 T cells had an index of 1.1 (Fig. 5C). Time-lapse videos compressed along the z-axis revealed that the wild-type and Gnai2–/– CD4 T cells moved within the lymph node; however, the customary amoeboid-like cell movements and active membrane projections normally observed were less evident with the Gnai2–/– CD4 T cells (see supplemental movies 2–4).3 Tracking individual cells over a 20-min interval revealed that the Gnai2–/– CD4 T cells exhibited oscillatory movements and rather short tracks as compared with the longer and less erratic movement of the wild-type CD4 T cells (Fig. 5D; supplemental movies 4 and 5).3 Randomly chosen tracks of CD4 T cells prepared from the wild-type or Gnai2–/– mice and plotted from the same origin show a striking difference in the length of the individual tracks (Fig. 5E). From the tracking data, we extracted the displacement of individual cells over 20 min of imaging. These data are shown by arrows beginning when individual cells are first visualized and ending when they leave the imaging space or the imaging terminated. Displacement of wild-type (green) and Gnai2–/– (red) CD4 T cell mice is shown separately (Fig. 5F, left and right panels). The displacements of 132 wild-type and Gnai2–/– CD4 T cell tracks acquired over a 10-min interval were sorted by length and plotted (Fig. 5G). One of the typical approaches to study motion paths of individual cells relies on fitting mean square displacements to a persistent random walk function. Focusing on 15 cells that remained in the tracking volume for 4 min, we plotted the mean squared displacement vs time. Both the wild-type and the Gnai2–/– CD4 T cells exhibited a linear relationship, although the slope, which is a measure of cell motility, differed substantially (Fig. 5G). The slope of the wild-type cells was 275, whereas that of the Gnai2–/– CD4 T cells was 17. A measure of directional persistence is track straightness (displacement/track length). Twelve randomly chosen tracks were sorted on the basis of their track straightness and plotted (Fig. 5H). Despite their slow velocity, there was little difference between the wild-type and Gnai2–/– CD4 T cells with the exception of three wild-type cells that exhibited relatively straight tracks. When we analyzed the tracks of randomly chosen wild-type and Gnai2–/– CD4 T cells from one movie, we found that the wild-type cells moved with an average speed of 8.8 µm/min, whereas the Gnai2–/– CD4 T cells moved with an average speed of 4.2 µm/min (Fig. 5I, left panel). More Gnai2–/– CD4 T cells moved with an average speed below 5 µm/min as compared with wild-type cells, whereas relatively few of them moved with average speed above 10 µm/min. An examination of three movies from separate imaging experiments revealed a similar difference between wild-type and the Gnai2–/– CD4 T cells (Fig. 5I, right panel). We also acquired time-lapse videos compressed along the z-axis from deeper in the lymph node (~250 µm) of the wild-type and Gnai2–/– CD4 T cells (supplemental movie 6).3 Relatively few Gnai2–/– CD4 T cells are present compared with wild-type CD4 T cells (Fig. 6A). Tracking individual cells showed that the motility of the Gnai2–/– CD4 T cells was less impaired than observed more superficially in the lymph node (Fig. 6B). The wild-type T cells moved with an average speed of 10 µm/min, whereas the Gnai2–/– CD4 T cells moved at an average speed of 7.8 µm/min (data not shown). The lesser impairment may be secondary to the selection of those Gnai2–/– CD4 T best able to respond to chemoattractants. Nevertheless, a comparison of individual tracks from those cells that remained within the imaging space for similar duration (of 6–8 min) of the 30-min imaging period revealed a significant decrease in the motility of the Gnai2–/– CD4 T cells compared with wild-type cells (Fig. 6B).


Figure 5
View larger version (68K):
[in this window]
[in a new window]

  		FIGURE 5. Intravital imaging of CD4 T cells from Gnai2–/– mice reveals major defects in T cell motility. A, Imaging CD4 T cells within the inguinal lymph node. Twenty-four hours after cell transfer, inguinal lymph nodes were imaged intravitally using multiphoton microscope immediately following injection of mixture of red and green fluorescent dextrans (Gnai2–/– CD4 T cells, red; wild-type T cells, green). A 750 µm x 750-µm image was collected every second at 1-µm intervals to a depth of 266 µm. The first image acquired is shown in the left panel, and additional images acquired at depths of 50, 100, 200, and 265 µm are also shown, as indicated. The red arrow identifies a small group of Gnai2–/– CD4 T present among the much more numerous wild-type T cells. B, An XZ plane projection of the imaging data. Following a 3-D reconstruction of the imaging data, an XZ projection is shown, which was compressed along 20 µm y-axis at the level indicated by the white line in the first panel in A. Green (top, wild-type cells), red (middle, Gnai2–/– CD4 T cells), and composite images (bottom) are shown. Because of the lower resolution in the z-axis, the cells are elongated along the z-axis. C, Decreased polarity of Gnai2–/– CD4 T. Twenty-four hours after cell transfer, inguinal lymph nodes were imaged intravitally using a multiphoton microscope (Gnai2–/– CD4 T cells, red; wild-type T cells, green). The long and short axes of 20 randomly chosen wild-type and Gnai2–/– CD4 T cells were measured, and a polarity index was calculated by dividing the long axis by the short axis. The mean and SD of the data are shown. D, Cell tracking. Individual cells were tracked for 20 min using the Surpass feature of Imaris software within a volume of 300 µM x 300 µM x 30 µM. Individual wild-type (green) and Gnai2–/– CD4 T cell tracks (red) are shown (left panel). E, Direct comparison of cell tracks. Ten randomly chosen wild-type (middle panel) and Gnai2–/– (right panel) CD4 T cell tracks from cells that persisted within the imaging space are shown. The initial location of each cell was set to the origin of the graphs. F, CD4 T cell displacements within the lymph node. The displacement of wild-type (green) and Gnai2–/– (red) CD4 T cells tracked from when they first appeared in the imaging space to when they exited or the imaging was terminated. The arrows originate where the cell was first imaged. Wild-type and Gnai2–/– CD4 T cells are shown separately (electronically zoomed x5). G, A direct comparison of wild-type and Gnai2–/– CD4 T cell displacements. The displacements of 132 randomly chosen wild-type and Gnai2–/– CD4 T cells during a 20-min imaging session were sorted by length and plotted (left panel). Fifteen wild-type and Gnai2–/– CD4 T cells that remained in the tracking volume for 4 min of the 20-min imaging session were tracked. The mean squared displacement was plotted vs time (right panel). H, Comparison of straightness indices. Twelve randomly chosen tracks were sorted on the basis of their track straightness (track displacement/track length) and plotted. I, Velocity profiles of transferred CD4 T cells. Data show the frequency histograms of 3-D velocities of wild-type ({square}) and Gnai2–/– ({blacksquare}) CD4 T cells. Results from a representative experiment are shown. Individual cells were tracked by spot analysis using Imaris software. The average speeds of the tracked cells are indicated with arrows. The mean velocity of wild-type and Gnai2–/– CD4 T cells calculated from three independent movies from distinct experiments is shown in the right panel.

 

Figure 6
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 6. Comparison of wild-type and Gnai2–/– CD4 T cells imaged in the T cell cortex. A, Representative image from intravital microscopy acquired at a depth between 252 and 267 µm. Twenty-four hours after cell transfer, the inguinal lymph node was imaged intravitally using multiphoton microscope (Gnai2–/– CD4 T cells, red; wild-type T cells, green) every 30 s for a total of 30 min (zoomed x3). B, Tracking wild-type and Gnai2–/– CD4 T at a depth between 252 and 267 µm. Individual wild-type and Gnai2–/– CD4 T cell tracks from 30 min of imaging. Tracks are from those 19 cells that remained within the imaging space a minimum of 6 min (the average duration of imaging of the wild-type cells was 441 s, and the Gnai2–/– CD4 T cells 465 s). C, Direct comparison of cell tracks. Ten randomly chosen wild-type (bottom panel) and Gnai2–/– (top panel) CD4 T cell tracks from cells that persisted within the imaging space for a minimum of 7 min are shown. The initial location of each cell was set to the origin of the graphs. The average duration of imaging of the wild-type cells was 429 s, and the Gnai2–/– CD4 T cells was 441 s.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Naive T cells preferentially recirculate between the blood and secondary lymphoid organs, whereas memory and effector T cells can enter into nonlymphoid tissues and inflammatory sites, returning to lymph nodes via the afferent lymph. Pertussis toxin has proven to be a valuable reagent in discerning the role of Gi proteins in the entrance of lymphocyte into lymph nodes via either route (16, 17, 18). Pretreatment of T cells with pertussis toxin before i.v. transfer to a recipient mouse completely blocks the entrance of cells into lymph nodes. Intravital imaging of transferred lymphocytes within HEVs has shown that pretreatment with pertussis toxin blocks firm adhesion to HEVs, thereby preventing the exit of lymphocytes from the blood and their entrance into lymph nodes (19). Pretreatment of mouse splenocytes with pertussis toxin also completely inhibits the migration of T cells from peripheral tissues to draining lymph node (20).

Yet the analysis of pertussis toxin-treated cells masks any differential role of the various Gi{alpha} isoforms, all of which are sensitive to pertussis toxin. T lymphocytes predominantly express Gnai2 and Gnai3 with little expression of Gnai1. The marked reduction in chemokine signaling and T cell homing to peripheral lymph nodes noted with Gnai2–/– T cells occurs despite their enhanced expression of Gnai3. In addition, the residual responsiveness of Gnai2–/– T cells to chemokines in vitro and in vivo is relatively insensitive to pertussis toxin treatment, arguing that those Gnai2–/– T cell chemokine receptors that have coupled to Gi{alpha}3 can no longer link to the signaling pathways needed for chemotaxis. This could result from an inability of the chemokine receptors to trigger Gi{alpha}3 GDP/GTP exchange or because those Gbeta{gamma} subunits paired with Gi{alpha}3 do not activate the appropriate effectors. A precedent for divergent roles for Gi{alpha}2 and Gi{alpha}3 in linking GPCR activation to downstream signaling pathways has been described in cardiac myocytes (21, 22).

The pertussis toxin resistance of the residual chemotaxis and lymph node homing of the Gnai2–/– T cells indicates that CXCR4 and CCR7 in the Gnai2–/– mice have most likely coupled to a nonpertussis toxin-sensitive heterotrimeric G protein such as Gq or G12, which has some capacity to trigger chemotaxis. This raises the possibility that under certain circumstances, T cell chemokine receptors may couple to nonpertussis toxin-sensitive G proteins. In fact, during T cell stimulation by APCs, T cell chemokine receptors coupled to Gq were shown to be recruited to the immunological synapses (23). Our results contrast to studies examining the requirement for Gi isoforms in C5a receptor-triggered chemotaxis of a mouse macrophage cell line (24). Although pertussis toxin blocks C5a receptor-triggered chemotaxis, the reduction of either Gnai2 expression (90% knockdown) or Gnai3 expression (80% knockdown) by lentiviral delivery of small interfering RNA did not. This suggests that the C5a receptor couples to both Gi isoforms, and each is capable of triggering chemotaxis.

Despite the reduced migration of Gnai2–/– CD4 T cells into the deeper lymph node cortex and their reduced motility, we found that the Gnai2–/– CD4 T cells transited through the lymph node as well, if not better than did wild-type cells. Normally, entering T cells scan cortical ridge-dendritic cells searching for specific Ag (25). If they fail to find Ag, they pass through the cortical ridge, enter the deeper cortical zones, and migrate toward the medulla, where they exit into the efferent lymphatics. The presence of high levels of S1P within the efferent lymph facilitates lymphocyte egress (13, 14, 15). Our data indicate that the Gnai2–/– T lymphocyte can readily access the medullary region to exit into lymphatics. It is worth noting that the cortical ridge directly connects to the medulla, perhaps providing an avenue for Gnai2–/– T cell egress. Our data also argue that S1P receptors can couple to Gi{alpha}3 to trigger egress.

Two competing theories explain how GPCRs (i.e., chemokine receptors) transfer information after activation (26). The first postulates that freely diffusible GPCRs collide with membrane-associated heterotrimeric G proteins, which, following GDP/GTP exchange, localize with their primary effectors. The other and more recently favored theory postulates that individual GPCRs are precoupled to heterotrimeric G proteins, which are in turn preassembled with their effectors. A key feature of this physical scaffolding is fast activation and deactivation of the signaling pathway as well as specificity. Because individual heterotrimeric G protein most likely competes for precoupling, the mass ratio between the cell’s repertoire of GPCRs and heterotrimeric G proteins and the relative affinities of different GPCRs for heterotrimeric G proteins, various combinations of G{alpha}, Gbeta, and G{gamma}, determine the outcome of ligand exposure (27). In this context, the impaired responsiveness of the Gnai2+/– T cells to chemokine stimulation can be explained as a consequence of an altered balance between Gi{alpha}2 and other heterotrimeric G proteins expressed within lymphocytes. A decrease in Gi{alpha}2 allows the precoupling of other heterotrimeric G proteins, thereby altering the outcome of receptor activation. These observations suggest that changes in the expression levels of G{alpha} subunits, which may occur as consequence of lymphocyte development, differentiation, or activation, may significantly alter the outcome of chemokine receptor signaling.

Initial imaging studies examining lymphocyte trafficking concluded that the movement of T and B cells within lymph nodes is best modeled by a "random walk" behavior, that is lymphocytes lack any organized directional movement (28, 29, 30, 31). Recently, an alternative explanation for the T cell imaging data has been put forth postulating that T cell movements within the lymph node are controlled by the presence of many small local attractors, such as dendritic cells secreting chemokines (32). As suggested, this issue is of some importance because productive dendritic-T cell interactions are more likely to occur if there is mechanism to bias naive T cells toward interacting with Ag-bearing DCs. Supporting this model in immunogen-draining lymph nodes, naive CD8+ T cells up-regulated their expression of CCR5, which led to their attraction by the chemokines CCL3 and CCL4 produced at sites of Ag-specific dendritic cell-CD4+ T cell interaction (33). A more recent study has expanded this model by showing that T lymphocytes migrate along fibroreticular cells (FRC) within the T cell zone (34). Because FRC not only secrete CCR7 ligands, but are also decorated with them, solid-phased chemokines may provide haptotactic guidance clues and promote chemokinesis (35). The evaluation of the imaging data of the Gnai2–/– CD4 cells has some bearing on these issues. First, we found that the Gnai2–/– CD4 T cells clustered around the HEVs and migrated poorly into the deeper cortical region. The impaired responsiveness of Gnai2–/– CD4 T cells to chemokines may explain their improper localization and failure to normally follow the FRC network into the T cell zone. Second, we found that the Gnai2–/– CD4 T cells failed to adopt the normal polarized morphology observed with wild-type CD4 T cells. This indicates an inability to respond to environmental clues, such as chemokines, that trigger a polarized phenotype. Third, the velocity profile of the Gnai2–/– CD4 T cells was significantly shifted to the left with the average velocity of the Gnai2–/– T cells reduced nearly 50% compared with wild-type cells. The velocities of the Gnai2–/– T cells are most likely artifactually elevated due to cell shape change and oscillatory movements that change the center of the cell mass affecting the spot analysis. The reduced velocities of the Gnai2–/– CD4 T cells can in part be explained by their preferential localization in regions surrounding HEVs, where T cells are known to have slower motility. However, those Gnai2–/– CD4 T cells that managed to enter the deeper cortical region also showed a reduced velocity profile compared with wild-type cells. Because the Gnai2–/– T cells retain some responsiveness to chemokines and those cells entering the T cell zone are most likely to be those cells most responsive, their movement would most likely be further impaired were that not the case. These results indicate that within the confines of the lymph node, T cells are subject to Gi{alpha}2-dependent signals that enhance their motility and control their localization.

Many questions need answering before a comprehensive understanding of chemokine receptor signaling and its role in lymphocyte trafficking and motility is fully achieved. The analysis of chemokine receptor signaling and lymphocyte trafficking of T cells isolated from Gnai2+/– and Gnai2–/– mice has illustrated that the effect of chemokines on lymphocyte trafficking cannot be understood solely on the basis of the pattern of cognate receptor expression. Our results provide a rationale for a comprehensive analysis of the effects of other signaling pathways, such as those triggered by Ag, Toll, or cytokine receptors on heterotrimeric G protein expression and function as well as upon the proximal regulators of these proteins. Finally, the analysis of the movement of those Gnai2–/– CD4 T cells that enter into the inguinal lymph node supports a role for local directional clues in organizing the movement of CD4 T cells within lymph nodes.


   Acknowledgments
 
We thank Mary Rust for excellent editorial assistance; Dr. Lutz Birnbaumer of the National Institute of Environmental Health Sciences for the Gnai2–/– mice; Dr. Owen Schwartz, Dr. Juraj Kabat, and Dr. Mewggan Czapiga of the Research Technology Branch of the National Institute of Allergy and Infectious Diseases for their support in the acquisition and analysis of the multiphoton microscopy data; and Dr. Anthony Fauci and the National Institute of Allergy and Infectious Diseases for their continued support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. John H. Kehrl, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11B08, 10 Center Drive MSC 1876, Bethesda, MD 20892. E-mail address: jkehrl{at}niaid.nih.gov Back

2 Abbreviations used in this paper: GPCR, G protein-coupled receptor; 3-D, three-dimensional; [Ca2+]i, intracellular Ca2+ concentration; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and 6-)(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine; FRC, fibroreticular cell; HEV, high endothelial venule; S1P, sphingosine 1-phosphate. Back

3 The online version of this article contains supplemental material. Back

Received for publication September 25, 2006. Accepted for publication April 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314. [Medline]
  2. Kunkel, E. J., E. C. Butcher. 2002. Chemokines and the tissue-specific migration of lymphocytes. Immunity 16: 1-4. [Medline]
  3. Arai, H., C. L. Tsou, I. F. Charo. 1997. Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by beta{gamma} dimers released by activation of G{alpha}i-coupled receptors. Proc. Natl. Acad. Sci. USA 94: 14495-14499. [Abstract/Free Full Text]
  4. Neptune, E. R., H. R. Bourne. 1997. Receptors induce chemotaxis by releasing the beta{gamma} subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA 94: 14489-14494. [Abstract/Free Full Text]
  5. Hepler, J. R., A. G. Gilman. 1992. G proteins. Trends Biochem. Sci. 17: 383-387. [Medline]
  6. Neer, E. J.. 1995. Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80: 249-257. [Medline]
  7. Neptune, E. R., T. Iiri, H. R. Bourne. 1999. G{alpha}i is not required for chemotaxis mediated by Gi-coupled receptors. J. Biol. Chem. 274: 2824-2828. [Abstract/Free Full Text]
  8. Han, S. B., C. Moratz, N. N. Huang, B. Kelsall, H. Cho, C. S. Shi, O. Schwartz, J. H. Kehrl. 2005. Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity 22: 343-354. [Medline]
  9. Huang, T. T., Y. Zong, H. Dalwadi, C. Chung, M. C. Miceli, K. Spicher, L. Birnbaumer, J. Braun, R. Aranda. 2003. TCR-mediated hyper-responsiveness of autoimmune G{alpha}i2–/– mice is an intrinsic naive CD4+ T cell disorder selective for the G{alpha}i2 subunit. Int. Immunol. 15: 1359-1367. [Abstract/Free Full Text]
  10. Rudolph, U., M. J. Finegold, S. S. Rich, G. R. Harriman, Y. Srinivasan, P. Brabet, G. Boulay, A. Bradley, L. Birnbaumer. 1995. Ulcerative colitis and adenocarcinoma of the colon in G{alpha}i2-deficient mice. Nat. Genet. 10: 143-150. [Medline]
  11. Moratz, C., J. R. Hayman, H. Gu, J. H. Kehrl. 2004. Abnormal B-cell responses to chemokines, disturbed plasma cell localization, and distorted immune tissue architecture in Rgs1–/– mice. Mol. Cell. Biol. 24: 5767-5775. [Abstract/Free Full Text]
  12. Tang, M. L., D. A. Steeber, X. Q. Zhang, T. F. Tedder. 1998. Intrinsic differences in L-selectin expression levels affect T and B lymphocyte subset-specific recirculation pathways. J. Immunol. 160: 5113-5121. [Abstract/Free Full Text]
  13. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427: 355-360. [Medline]
  14. Allende, M. L., J. L. Dreier, S. Mandala, R. L. Proia. 2004. Expression of the sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration. J. Biol. Chem. 279: 15396-15401. [Abstract/Free Full Text]
  15. Lo, C. G., Y. Xu, R. L. Proia, J. G. Cyster. 2005. Cyclical modulation of sphingosine-1-phosphate receptor 1 surface expression during lymphocyte recirculation and relationship to lymphoid organ transit. J. Exp. Med. 201: 291-301. [Abstract/Free Full Text]
  16. Spangrude, G. J., B. A. Braaten, R. A. Daynes. 1984. Molecular mechanisms of lymphocyte extravasation. I. Studies of two selective inhibitors of lymphocyte recirculation. J. Immunol. 132: 354-362. [Abstract]
  17. Bargatze, R. F., E. C. Butcher. 1993. Rapid G protein-regulated activation event involved in lymphocyte binding to high endothelial venules. J. Exp. Med. 178: 367-372. [Abstract/Free Full Text]
  18. Cyster, J. G., C. C. Goodnow. 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182: 581-586. [Abstract/Free Full Text]
  19. Warnock, R. A., S. Askari, E. C. Butcher, U. H. von Andrian. 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187: 205-216. [Abstract/Free Full Text]
  20. Debes, G. F., C. N. Arnold, A. J. Young, S. Krautwald, M. Lipp, J. B. Hay, E. C. Butcher. 2005. Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nat. Immunol. 6: 889-894. [Medline]
  21. Foerster, K., F. Groner, J. Matthes, W. J. Koch, L. Birnbaumer, S. Herzig. 2003. Cardioprotection specific for the G protein Gi2 in chronic adrenergic signaling through beta2-adrenoceptors. Proc. Natl. Acad. Sci. USA 100: 14475-14480. [Abstract/Free Full Text]
  22. Nagata, K., C. Ye, M. Jain, D. S. Milstone, R. Liao, R. M. Mortensen. 2000. G{alpha}i2 but not G{alpha}i3 is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes. Circ. Res. 87: 903-909. [Abstract/Free Full Text]
  23. Molon, B., G. Gri, M. Bettella, C. Gomez-Mouton, A. Lanzavecchia, A. C. Martinez, S. Manes, A. Viola. 2005. T cell costimulation by chemokine receptors. Nat. Immunol. 6: 465-471. [Medline]
  24. Hwang, J. I., I. D. Fraser, S. Choi, X. F. Qin, M. I. Simon. 2004. Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA. Proc. Natl. Acad. Sci. USA 101: 488-493. [Abstract/Free Full Text]
  25. Katakai, T., T. Hara, J. H. Lee, H. Gonda, M. Sugai, A. Shimizu. 2004. A novel reticular stromal structure in lymph node cortex: an immuno-platform for interactions among dendritic cells, T cells and B cells. Int. Immunol. 16: 1133-1142. [Abstract/Free Full Text]
  26. Nobles, M., A. Benians, A. Tinker. 2005. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc. Natl. Acad. Sci. USA 102: 18706-18711. [Abstract/Free Full Text]
  27. Huang, J. S., L. Dong, G. C. Le Breton. 2006. Mass-dependent signaling between G protein coupled receptors. Cell. Signal. 18: 564-576. [Medline]
  28. Miller, M. J., S. H. Wei, I. Parker, M. D. Cahalan. 2002. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296: 1869-1873. [Abstract/Free Full Text]
  29. Miller, M. J., S. H. Wei, M. D. Cahalan, I. Parker. 2003. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc. Natl. Acad. Sci. USA 100: 2604-2609. [Abstract/Free Full Text]
  30. Wei, S. H., I. Parker, M. J. Miller, M. D. Cahalan. 2003. A stochastic view of lymphocyte motility and trafficking within the lymph node. Immunol. Rev. 195: 136-159. [Medline]
  31. Halin, C., J. Rodrigo Mora, C. Sumen, U. H. von Andrian. 2005. In vivo imaging of lymphocyte trafficking. Annu. Rev. Cell. Dev. Biol. 21: 581-603. [Medline]
  32. Germain, R. N., F. Castellino, M. Chieppa, J. G. Egen, A. Y. Huang, L. Y. Koo, H. Qi. 2005. An extended vision for dynamic high-resolution intravital immune imaging. Semin. Immunol. 17: 431-441. [Medline]
  33. Castellino, F., A. Y. Huang, G. Altan-Bonnet, S. Stoll, C. Scheinecker, R. N. Germain. 2006. Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction. Nature 440: 890-895. [Medline]
  34. Bajenoff, M., J. G. Egen, L. Y. Koo, J. P. Laugier, F. Brau, N. Glaichenhaus, R. N. Germain. 2006. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25: 989-1001. [Medline]
  35. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, J. G. Cyster. 2000. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97: 12694-12699. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
A. Zarbock, T. L. Deem, T. L. Burcin, and K. Ley
G{alpha}i2 is required for chemokine-induced neutrophil arrest
Blood, November 15, 2007; 110(10): 3773 - 3779.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hwang, I.-Y.
Right arrow Articles by Kehrl, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hwang, I.-Y.
Right arrow Articles by Kehrl, J. H.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS