RGS3 controls T lymphocyte migration in a model of Th2-mediated airway inflammation

Abstract

T cell migration toward sites of antigen exposure is mediated by G protein signaling and is a key function in the development of immune responses. Regulators of G protein signaling (RGS) proteins modulate G protein signaling; however, their role in the regulation of adaptive immune responses has not been thoroughly explored. Herein we demonstrated abundant expression of the G_i/G_q-specific RGS3 in activated T cells, and that diminished RGS3 expression in a T cell thymoma increased cytokine-induced migration. To examine the role of endogenous RGS3 in vivo, mice deficient in the RGS domain (RGS3^ΔRGS) were generated and tested in an experimental model of asthma. Compared with littermate controls, the inflammation in the RGS3^ΔRGS mice was characterized by increased T cell numbers and the striking development of perivascular lymphoid structures. Surprisingly, while innate inflammatory cells were also increased in the lungs of RGS3^ΔRGS mice, eosinophil numbers and Th2 cytokine production were equivalent to control mice. In contrast, T cell numbers in the draining lymph nodes (dLN) were reduced in the RGS3^ΔRGS, demonstrating a redistribution of T cells from the dLN to the lungs via increased RGS3^ΔRGS T cell migration. Together these novel findings show a nonredundant role for endogenous RGS3 in controlling T cell migration in vitro and in an in vivo model of inflammation.

g protein-coupled receptors (GPCRs) represent a family of heptahelical transmembrane receptors that promote the GTP binding and activation of the α-subunit of heterotrimeric G proteins. There are four main classes of G proteins, namely G_s, G_i, G_q, and G_12/13 (38). G_sα stimulates cAMP-dependent signaling through activation of adenylyl cyclase, whereas G_iα antagonizes G_sα signaling through inhibition of adenylyl cyclase (20, 45). In addition, G_iα-coupled Gβγ subunits, along with G_qα, activate PLC-β isoforms (44) and stimulate mitogenic signaling, including mitogen-activated protein kinase (22, 28), Akt (27, 32), and the small GTPases Rac and Cdc42 (4). Finally, Gα_12/13 stimulates activity of the small GTPase Rho, modulating the actin cytoskeleton and cell motility (21).

Inactivation of G protein signaling occurs, in part, through the RGS family that enhances the intrinsic GTPase activity of the Gα subunit, thereby functioning as GTPase-activating proteins (GAP) (5, 10, 12, 23). The unifying feature of RGS proteins is the presence of the RGS domain that is responsible for their GAP function. RGS3 belongs to the R4/B subfamily and selectively interacts with G_iα and G_qα₁₁, but not G_sα or Gα_12/13 (14, 40). Ectopic expression of RGS3 in various cell types demonstrated its ability to regulate G_i- or G_q-mediated signaling pathways induced by agonists such as gonadotropin-releasing hormone (7, 33, 34), lysophospohatidic acid (8, 19), endothelin-1 (14), carbachol and angiontensin II (47), acetylcholine (24, 25, 46), and various chemokines (6, 39). Functionally, expression of RGS3 inhibited LPA- and chemokine-induced migration in renal tubular cells (19) and lymphoid cells (6, 39), respectively.

Although the regulation of G protein signaling by RGS3 has been studied in vitro using overexpression assays, the control of physiological cell functions by endogenous RGS3 is poorly understood, and its in vivo role has not been examined. Herein, we found that RGS3 is abundantly expressed in T cells, and knockdown of RGS3 leads to an enhanced migration of T cells toward stroma-derived factor (SDF-1/CXCL12) or secondary lymphoid tissue cytokine (SLC/CCL21). To understand the function of RGS3 in T cells in vivo, we examined how a targeted deletion of the RGS domain of RGS3 (RGS3^ΔRGS) affected in vivo functions of T cell responses in a mouse. Our findings highlight a RGS3-specific function in the regulation of T cell migration without affecting the differentiation of T cells following exposure to a model of house dust mite (HDM)-induced airway inflammation.

METHODS

RGS3 knockdown Lentivirus encoding short-hairpin RNA (shRNA) against RGS3 (5′-GGACCTCATTAACCAGAAGAA-3′) coexpressing a green fluorescent protein (GFP) selection marker was generated by America Pharma Source (Rockville, MD). A control lentivirus encoding scrambled, nontargeting shRNA was also generated. EL4 thymoma cells (ATCC) were transduced with desired lentiviruses for 3 days, and then GFP-positive cells were selected by fluorescence-activated cell sorting (FACS) and expanded in culture. Cells were maintained in DMEM supplemented with 10% FBS, 2 mM l-glutamine, and 100 U/ml penicillin/streptomycin in a humidified cell culture incubator kept at 37°C, with 5% carbon dioxide.

T cell isolation and skewing.

T cells from spleens of naïve C57bl/6 mice were enriched by nylon wool nonadherence purification. CD4 T cells were then isolated by magnetic purification using a CD4⁺ CD62L⁺ T cell selection kit from Miltenyi Biotec (130–093-227). Recovery was >99% pure CD4 T cells. For T cell skewing, cells were activated in vitro with anti-CD3 (2C11) and anti-CD28 (PV-1) and then incubated under skewing conditions. T cells were incubated with 1 μg/ml anti-IL-4 (11b11) and 2 ng/ml recombinant IL-12 (no. 14–8121-80; eBiosciences) to promote Th1 skewing, and T cells were incubated with 2 ng/ml recombinant IL-4 (no. 504306; Biolegend) and 2 ng/ml anti-IFNγ (R4-GA2) to promote Th2 skewing. On day 3 and day 6 cells were collected and split with fresh media with 20 U/ml rIL-2 (no. 575406; Biolegend). Th1 and Th2 cells were used for experimentation on day 8 after stimulation. Antibodies used for skewing experiments were purified by the University of Chicago Monoclonal Antibody facility.

Western blotting.

Cells were lysed in the ice-cold buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM NaF, 200 μM sodium orthovanadate, and protease inhibitor cocktail (Sigma Aldrich). The insoluble material was precipitated by centrifugation at 20,000 g for 5 min, and the cleared lysates were boiled in Laemmli buffer for 5 min. The samples were subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting with primary antibodies against RGS3 (14), GFP (Calbiochem), and β-actin (Sigma-Aldrich), followed by corresponding HRP-conjugated secondary antibodies, and developed by an enhanced chemiluminescence reaction (Pierce). The digital chemiluminescent images were taken by a Luminescent Image Analyzer LAS-4000 (Fujifilm).

In vitro migration assay.

EL4 cells were harvested by centrifugation, washed one time in PBS, and resuspended in DMEM containing 1% FBS. Cells (5 × 10⁵) in 100 μl media were seeded to the upper chamber of 5-μm-pore Transwell inserts designed for 24-well plates (BD Biosciences, San Jose, CA). Six hundred microliters of the same media with or without desired chemokines were added to the lower chambers (in triplicates). Cells were allowed to migrate for 6 h in a humidified cell culture incubator at 37°C, with 5% carbon dioxide. Following the incubation period, the media with migrated cells were collected from the bottom chambers, washed by centrifugation, and resuspended in PBS. The cell suspension used for the experiment was prepared in the same manner to serve as the input control. Approximately 5 × 10⁴ CountBright Absolute counting beads (Invitrogen, Grand Island, NY) were added to each sample, and the samples were analyzed by FACS. The transduced EL4 cells were detected by GFP fluorescence, and the counting beads were detected using APC-Cy7. For each sample, 2,000 beads were counted, and the number of cells collected in that duration was determined. The number of cells migrated in each condition was then normalized to the number of cells counted in the input cell suspension.

Targeted disruption of the RGS3 gene.

A targeting construct was produced that resulted in disruption of exons 30–32 that encode the RGS domain of RGS3. Linearized construct DNA was electroporated into embryonic stem cells derived from 129/SvJ mice. ES cells were screened for incorporation of the construct DNA by their ability to grow in the presence of G418 and then confirmed by PCR/Southern blotting. Positive stem cells were used for blastocyst injection in C57Bl/6J embryos and implanted in pseudopregnant females. Chimeric offspring were bred, and germline transmission was achieved. Mice were backcrossed (>6X) with wild-type (WT) C57Bl/6J mice obtained from the Jackson Laboratory before experimentation. Progenies were screened by tail-prep PCR. All mice were bred and housed in specific pathogen-free facilities maintained by the University of Chicago Animal Resource Center. The studies described conform to the principles set forth by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of laboratory animals in biomedical research.

HDM allergy model.

Mice were sensitized on day 0 with 40 μg of total HDM protein (no. B84 Dermatophagoides pternyssinus; Greer Labs) and 4 mg aluminum hydroxide (Sigma) in PBS injected intraperitoneally. On days 7 and 14 after sensitization, mice were intratracheally instilled with 100 μg HDM in PBS. Mice were killed on day 18 (4 days after final challenge) and assayed for development of inflammation. At death, bronchoalveolar lavage (BAL) was performed by delivering 0.8 ml cold PBS in the airway via a trachea cannula and gently aspirating the fluid. The lavage was repeated four times. Lungs were dissociated by mechanical mincing followed by digestion with 150 U/ml collagenase I (Invitrogen) in 10 ml of medium for 30 min. Samples were washed, and red blood cells were lysed. Spleens and lymph nodes were collected, and the cells liberated by mechanical dissociation. Cells were counted using a hemacytometer, and cellularity was determined using flow cytometry.

Histological analysis.

Lung, lymph node, spleen, and thymus were collected and fixed in tissue cassettes in 10% formalin for 48 h. Samples were embedded in paraffin, cut into 5-μM sections, and stained for hematoxylin and eosin (H&E) or immunohistochemistry for B220 staining by the University of Chicago Histology Core Facility.

Flow cytometry.

In brief, 5 × 10⁵ total cells from lung, BAL, spleen, or lymph node were suspended in 50 μl of FACS buffer (PBS containing 0.1% sodium azide and 1% BSA) and labeled with specific antibodies. Antibodies used include anti-CD3 (145–2C11), CD4 (GK1.5), CD8 (53–6.7), Gr1 (RB6–8C5), CD62L (MEL-14), CCR7 (4B12), (eBiosciences), and CCR3 (83101) (R&D Systems). Flow cytometric data were collected on a LSRFortessa or LSRII (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star). Instruments are maintained by the University of Chicago flow cytometry core facility. T cells were identified by CD3 staining and then separated into CD4 and CD8 subsets. Macrophages were identified by FSC^high, SSC^high, and autofluorescence^high, and neutrophils were identified by autofluorescence^low, SSC^high, and Gr1⁺ expression, whereas eosinophils were identified by autofluorescence^low, SSC^high, and CCR3⁺ expression.

Proliferation assay.

5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma) was used to stain freshly isolated T cells for 5 min at 37°C. Cells were washed repeatedly and then plated on anti-CD3- and -CD28-coated plates. T cells were allowed to proliferate for 3 days when they were collected and assayed for CFSE (Invitrogen) dilution by flow cytometry following the manufacturer's instructions.

Cytokine secretion.

T cells from the lungs of mice were specifically restimulated in vitro by anti-CD3 (2C11)-coated plates for 48 h. Plates were frozen, and supernatant was measured for cytokines by multiplex bead arrays (Millipore).

Statistical analysis.

Statistics were calculated using GraphPad Prism software. All data are represented as means ± SE, where P values <0.05 were considered significant (P < 0.05, P < 0.01, and P < 0.001 or not significant). Student's t-tests were used to analyze data sets with only two groups.

RESULTS

RGS3 regulates T cell migration in vitro.

RGS3 was previously reported to be expressed in Jurkat cells, a T lymphocyte-derived cell line, but the role of RGS3 in T cell function has not been explored (40). Western blotting with RGS3-specific antibodies showed expression of RGS3 protein in splenic CD4 T cells that is further augmented upon activation and skewing toward Th1 or Th2 lineages (Fig. 1A). RGS3 was also expressed in the T cell thymoma cell line EL4 (Fig. 1B). Given that G_i signaling mediates migration of T cells toward chemokines, we sought to examine the role of endogenous RGS3 in T cell migration using shRNA-mediated knockdown of RGS3. EL4 cells were transduced with lentivirus encoding RGS3 shRNA or scramble control with GFP as selection marker. A significant reduction of RGS3 expression was observed in the GFP-positive cells transduced with shRGS3 compared with control or those with scramble shRNA (Fig. 1B). RGS3 knockdown resulted in an increased basal migration, and chemotaxis induced by SDF-1 was dramatically enhanced over control EL4 migration (Fig. 1C). Interestingly, control EL4 cells failed to migrate to the chemokine SLC/CCL21, but shRGS3-transduced EL4 cells demonstrated potent migration to this chemokine (Fig. 1C). Thus, endogenous RGS3 can regulate both the magnitude and the specificity of the T cell migratory response to chemokines.

Fig. 1.

Regulator of G protein signaling (RGS) 3 is expressed in activated T cells and regulates migration of thymoma cells in vitro. A: lysates from resting splenic CD4⁺ T cells or Th1- or Th2-skewed T cells were assayed for RGS3 protein. B: RGS3 and green fluorescent protein (GFP) expression were assayed in EL4 thymoma cells following transduction with scrambled short-hairpin (sh) RNA-GFP or shRGS3-GFP. WT, wild type. C: in vitro migration of the control or RGS3 knockdown EL4 cells toward media, stroma-derived factor (SDF)-1, or secondary lymphoid tissue cytokine (SLC). Data represent means ± SE from one (of 3) independent experiment performed in triplicate (***P < 0.001; ns, not significant).

Naïve RGS3^ΔRGS mice show no immune defects compared with WT control.

To examine the in vivo function of endogenous RGS3 specifically in regulation of G protein signaling, we generated a mouse with targeted deletion of the RGS domain of RGS3 (RGS3^ΔRGS) (Fig. 2A). RGS3 contains a large NH₂-terminal region that regulates protein activity or has G protein-unrelated functions, but the COOH-terminal RGS domain in RGS3 is required for GAP function (13, 14, 35, 48). Validation of homologous recombination of the targeting construct was demonstrated by Southern blot and PCR analysis of ES cells (Fig. 2B). Western blotting of T cell lysate from WT and RGS3^ΔRGS T cells with an antibody against the NH₂-terminus of RGS3 (14) showed the presence of a single band corresponding to the electrophoretic mobility WT RGS3 and a smaller band corresponding the size of the COOH-terminal truncated of RGS3^ΔRGS (Fig. 2C). Normal architecture was found in lung, spleen, and thymus of RGS3^ΔRGS mice compared with WT littermates (Fig. 3A). RGS3^ΔRGS mice also showed no significant changes in T or B lymphocytes in spleen or lymph nodes (Fig. 3B and data not shown). Similarly, expression of various chemokine receptors and activation molecules remained comparable between T cells from RGS3^ΔRGS and WT littermates, including CCR7, CD62L, CXCR4, CD44, and CD25 (Fig. 3C and data not shown). Overall, naïve RGS3^ΔRGS mice showed no observed signs of immune or lung-specific defects compared with littermate control animals.

Fig. 2.

Development of an RGS3^ΔRGS mouse. A: schematic of RGS3^ΔRGS targeting vector with a PGK neocassette for the deletion of the RGS domain in exons 30–32 of the mouse RGS3 gene. B: mouse embryonic stem cells were electroporated and screened for incorporation of the targeting vector by Southern blot and PCR. C: Western blot of splenocytes from WT or RGS3^ΔRGS mice confirms loss of RGS domain in RGS3^ΔRGS through detecting a truncated RGS3 protein.

Fig. 3.

Characterization of RGS3^ΔRGS mouse immune compartment. A: histology sections of lung, spleen, and thymus from naïve WT or RGS3^ΔRGS mice. Images were taken at ×4 (lung) or ×10 (spleen and thymus). B: proportions of CD4 and CD8 T cells in the spleen and lymph nodes of unchallenged RGS3^ΔRGS mice compared with WT littermates was measured by flow cytometry. C: expression of CCR7 and CD62L on splenic CD4 T cells from unchallenged RGS3^ΔRGS mice compared with WT littermates.

Regulation of airway inflammation by RGS3.

Because RGS3 was upregulated in T cells following activation and regulated T cell migration in vitro (Fig. 1), we sought to determine the role of RGS3 in a model of HDM-induced airway inflammation. HDM models of allergic inflammation are widely studied and are known to elicit a strong Th2 cell-dependent eosinophilic response in the airways and lungs (16, 37). Mice received an intraperitoneal sensitization of HDM/Alum and two local pulmonary instillations of HDM alone on day 7 and day 14. Mice were killed at the peak of the inflammatory response and assayed for inflammation. RGS3^ΔRGS mice developed a significant increase in overall cellular infiltration as observed in H&E-stained histological lung sections compared with WT littermates (Fig. 4, A and B). The airways in both WT and RGS3^ΔRGS mice showed dramatic metaplasia of the bronchial epithelium and even smooth muscle thickening around the airways, characteristic of a strong Th2-mediated inflammatory responses (Fig. 4, C and D). Interestingly, RGS3^ΔRGS mice developed strikingly increased cellular infiltration directly surrounding vessels (Fig. 4, E and F). Increased infiltration around the bronchi and vessels in RGS3^ΔRGS mice was confirmed by blinded peribronchial and perivascular scoring of inflamed animals (Fig. 4G). This pathology resembled an organized induced bronchus associated lymphoid tissue (BALT) structure; therefore, we stained by immunohistochemistry for B220 to identify B cells and found a dramatic increase in B cells in the RGS3^ΔRGS mice after challenge (Fig. 4, H–K). T and B cells were localized into semiorganized structures directly surrounding the vessels, similar to pathology found in BALT.

Fig. 4.

RGS3^ΔRGS mice develop bronchus-associated lymphoid tissue (BALT) following house dust mite (HDM)-induced inflammation model. A–F: lungs from sensitized and challenged mice were sectioned and stained with hematoxylin and eosin (H&E). A and B: ×5 magnification images showing across a large portion of the lungs. C–F: ×20 magnification images showing representative airway (C and D) and blood vessel (E and F) areas. G: lung sections were blindly scored for peribronchial and perivascular infiltration. H–K: lung sections were stained for the presence of B cells by B220 immunohistochemistry staining. **P < 0.01. H and I: ×5 magnification shows a wide image of lung sections. J and K: ×20 magnification showing areas around vessels.

The dramatic histological observations were supported by flow cytometric analysis of the BAL fluid and lungs from sensitized and challenged mice that showed a significantly increased total number of CD4 and CD8 T cells in RGS3^ΔRGS mice compared with WT littermates (Fig. 5, A and B). Surprisingly, the increased number of T cells in the RGS3^ΔRGS mice did not lead to changes in the recruitment of eosinophils that are characteristic of Th2-mediated responses, whereas the neutrophil and macrophage responses were slightly augmented in the lungs or airways of RGS3^ΔRGS mice compared with WT mice. Furthermore, the percentage of T cells responding in the lungs following HDM challenge increased from 17% in WT to 26% of total lung cells in RGS3^ΔRGS mice following challenge, suggesting that the increased T cell recruitment in the lungs and airways likely drives the phenotypic changes in histopathology (Fig. 5C). These in vivo data clearly show an important role for RGS3 for regulating the infiltration of T cells in the lungs and airways following inflammatory stimulation.

Fig. 5.

RGS3^ΔRGS mice have increased T cell infiltration in the lungs and airways following HDM-mediated Th2 inflammation. Bronchoalveolar lavage (BAL, A) and lung (B) recovery of total cells, CD4 T cells, CD8 T cells, eosinophils, neutrophils, and macrophages after sensitization and challenge of WT (closed circles) and RGS3^ΔRGS (open circles) mice. C: percentages of cells recovered from the lungs of sensitized and challenged mice. Data represent means ± SE.

RGS3 does not control T cell effector function or proliferation.

To examine whether RGS3^ΔRGS influenced T cell differentiation in vivo, T cells from the lungs of HDM-challenged RGS3^ΔRGS mice and WT littermates were restimulated and assayed for cytokine production profiles. As shown in Fig. 6, total normalized cytokine production by T cells from the lungs of HDM-challenged RGS3^ΔRGS and WT mice showed no significant changes in the production of Th2-specific cytokines (IL-4, IL-5, and IL-13), and only slight trends in increased production of Th1- and Th17-specific cytokines (IFNγ and IL-17). These results might explain the in vivo inflammation data in terms of there being similar numbers of eosinophils in the lungs, which are recruited by Th2 cells, but slightly augmented neutrophils, which are recruited by Th17 cells. Overall, these findings show that RGS3^ΔRGS mice do not show signs of disruption of T cell differentiation.

Fig. 6.

Loss of RGS3 function does not lead to defects in cytokine production. T cells from WT or RGS3^ΔRGS mice were isolated from the lungs of sensitized and challenged WT (closed circles) and RGS3^ΔRGS (open circles) mice and restimulated for cytokine production. Cytokine secretion at 48 h was measured by Millipore multiplex assay.

To examine if RGS3 knockout affected proliferation of T cells, we examined cell division of CFSE-labeled splenic CD4 T cells in vitro when activated with anti-CD3 antibody. As shown in Fig. 7, T cells from RGS3^ΔRGS mice have similar proliferation kinetics to WT T cells, suggesting RGS3 does not influence T cell proliferation. Together, these two findings suggest that RGS3 regulates the number of T cells that infiltrate into the airway and lungs in a model of HDM-induced lung inflammation through mechanisms independent from T cell differentiation or proliferation.

Fig. 7.

RGS3^ΔRGS T cells do not have defects in proliferation. T cells from the spleen of WT or RGS3^ΔRGS mice were stained for 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) and stimulated with anti-CD3 antibody for 5 days. Cells were collected and assayed for dilution of CFSE. No. of dilutions was calculated and represents the no. of rounds of proliferation by each cell. Data represent means ± SE from 3 independent experiments.

RGS3^ΔRGS mice have reduced T cells in the lung draining lymph node following HDM challenge.

During allergic airway responses, T cells are normally activated by dendritic cells that have taken up allergens in the lungs and traffic to the local draining lymph nodes. For pulmonary responses, the mediastinal lymph node is the primary site of antigen presentation and T cell proliferation (31). Once T cells are primed, they undergo active GPCR-mediated exit and migrate toward sites of inflammation. Because we found that RGS3 regulated migration but not proliferation in vitro, we hypothesized that the increased T cell number found in the lungs of inflamed RGS3^ΔRGS mice after HDM challenge might be at the expense of T cells present in the lung draining lymph nodes. Following HDM-induced inflammation, the mediastinal lymph nodes from RGS3^ΔRGS mice were found to have dramatically decreased total cellularity compared with WT mediastinal lymph nodes (Fig. 8B). This observed cellular decrease appeared to be a direct result of a severe reduction in T cell populations in the lymph nodes of the RGS3^ΔRGS mice. In contrast, after HDM challenge, the spleens from RGS3^ΔRGS mice were found to have similar cellularity compared with WT mice, showing that RGS3 did not affect systemic immune tissues (Fig. 8B). These findings support previous in vitro and in vivo experiments and suggest that RGS3 plays a critical role in regulating the localization of activated T cells during inflammatory responses.

Fig. 8.

RGS3^ΔRGS mice have increased T cell migration from lung draining lymph nodes. A: total cell, CD3 T cell, CD4 T cell, and CD8 T cell recovered from the draining mediastinal lymph node of sensitized and challenged WT and RGS3^ΔRGS mice. B: total spleen cell count recovered from sensitized and challenged WT or and RGS3^ΔRGS mice.

DISCUSSION

In this study, we demonstrate for the first time that RGS3 is expressed in primary T cells and T cell lines. In the absence of the RGS domain of RGS3, HDM-challenged mice recruited augmented T cell numbers in the perivascular regions of the lung, leading to the development of organized lymphoid-like structures. Interestingly, loss of the RGS domain of RGS3 did not affect the development of T cell compartments of unchallenged mice. Furthermore, we found no evidence of RGS3 regulating T cell differentiation or in vitro proliferation but that increased T cell infiltration in the lung was associated with decreased T cells present in the priming lymph node. These data and our findings that RGS3 controls chemotaxis of a T cell line in vitro emphasize a nonredundant role for RGS3 and suggest that RGS3 in T cells controls trafficking and cellular localization following activation independent of other RGS proteins.

Several members of the R4 family have been shown to be expressed in different populations of T cells isolated from various tissues, including RGS1 (1, 15, 18), RGS2 (3, 15, 36), RGS9 (1), RGS10 (1, 17), RGS13 (15), RGS14 (9), and RGS16 (1, 3, 15, 29, 42). The inverse relation between the migration and the expression levels of RGS1, RGS9, RGS13, and RGS16 in various T cell populations has been observed in several studies (1, 15). Overexpression of RGS1 in Jurkat cells results in a decreased migration toward CXCL12 and CCL19, whereas targeted deletion of rgs1 gene increases migration of intestinal T cells toward these chemokines (18). Likewise, overexpression of RGS16 in T cells leads to inhibition of CXCL12-induced signaling and migration (29), whereas T cells from RGS16 knockout mice show enhanced migration toward CXCL9 and CCL17 (42). These data are consistent with the regulation of G protein signaling by RGS proteins in T cells. Interestingly, RGS16 also regulates Th2 cytokine production (IL-5, IL-10, and IL-13) (42), whereas RGS2 promotes T cell proliferation and IL-2 production (36). The latter functions of RGS16 and RGS2 may be unrelated to the regulation of G protein signaling as was shown for a number of RGS proteins (41).

Our experiments find RGS3 expressed basally in naïve murine CD4 T cells at a low level and strongly upregulated following activation. Several splice isoforms of RGS3 with variable tissue distribution have been described, including PDZ-RGS3, which contains PDZ domain NH₂-terminal to RGS3, as well as a short RGS3S that lacks a large portion of the RGS3's NH₂-terminal region, but all splice variants contain an identical RGS domain (26). Interestingly, PDZ-RGS3 is known to use the PDZ region of RGS3 to regulate migration of cerebellar granule cells and modulate Wnt signaling through interaction with and inhibition of glycogen synthase kinase 3β (30, 43). However, in our study, using antibodies against NH₂-terminus of RGS3 (14), we failed to detect a high-molecular-weight PDZ-RGS3 band in T cells by Western blotting. On the other hand, RGS3 in T cells does contain the Smad-interacting domain NH₂-terminal to the RGS domain that can modulate transforming growth factor-β signaling independent of its role in regulation of G protein signaling (48); however, in our asthma model the RGS3^ΔRGS mice had no alteration in Treg numbers (data not shown). Together, RGS3 is dynamically regulated in T cells, and the RGS protein we detect by Western blotting contains both the canonical RGS domain and the noncanonical Smad-binding regions.

G_iα signaling downstream of GPCRs is a critical regulatory point in the migration of T cells following their activation in response to pathogens. We tested whether RGS3 controlled in vitro EL4 thymoma cell migration and found that RGS3 was a potent regulator of T cell chemokinesis (ligand-independent) and chemokine-mediated chemotaxis. The functional significance of other RGS proteins in regulation of T cell migration toward chemokines was suggested by either the inverse relation between migration and expression of RGS proteins (RGS13/16) (15) or was confirmed by knockdown/knockout of RGS1 (18) or RGS16 (42). Given that all of these identified RGS proteins control G_iα- and/or G_qα-mediated signaling, it is remarkable that the knockdown of the RGS3 alone was able to promote EL4 T cell migration toward SDF-1 and SLC (Fig. 1C), suggesting the nonredundant role of RGS3 in this process.

Because T cell motility and localization are primary components of effective immunity, it is not surprising that a variety of RGS proteins play critical roles in immune responses; RGS4, RGS13, RGS16, and now RGS3 are known to independently regulate allergic inflammation (11). RGS2 expression in T cells is required for effective T cell proliferation, IL-2 production, and antiviral immunity in vivo (36). RGS13 has RGS domain-independent functions necessary for control of IgE:FcεRI-mediated anaphylaxis in mast cells and RGS domain functions in regulating mast cell localization (2). In vivo, RGS16 modulates T cell localization, differentiation, and the development of overall airway inflammation (29). Furthermore, in nonallergic models of inflammation, recent in vivo studies suggest a role for RGS1 in regulation of the intestinal inflammation (18) and of RGS16 in inhibition of the Schistosoma mansoni-induced airway inflammation (42) through their effects on T cell trafficking. In our study, we find that RGS3 controls T cell localization during allergic inflammatory responses through its RGS domain. Importantly, these mice express normal levels of truncated RGS3 protein containing intact Smad-binding domain. The importance of this domain on T cell function will need to be examined in future assays. Even though the HDM model of inflammation is CD4 driven, we found dramatic increases in both CD4 and CD8 T cells. Importantly, in these mice, we did not observe a significant difference in RGS16 expression between RGS3^ΔRGS and WT T cells, whereas RGS1 and RGS2 were actually slightly increased in RGS3^ΔRGS mice (data not shown).

RGS proteins have been reported to also influence the T cell differentiation and proliferation. RGS16 regulated Th2-cytokine production (IL-5, IL-10, and IL-13) (42), whereas RGS2 promotes proliferation of T cells (36). In contrast, our studies did not find RGS3 to significantly regulate T cell differentiation or in vitro proliferation. Although our model did not show statistically significant increases in cytokine production, there was an interesting correlation between augmented neutrophil/macrophage responses in the lungs and trends in augmented IL-17 and IFNγ responses following HDM challenge. Whereas these findings could be a direct result of aberrant T cell migration in the lungs, our current study does not exhaustively examine T cell differentiation toward these lineages. Future studies will examine whether RGS3 may regulate cellular differentiation using RGS3^ΔRGS mice in models of Th1- and Th17-mediated immune responses.

We report that RGS3 regulates the development of T cell and B cell aggregates in the perivascular space in HDM-challenged mice. Deletion of RGS16 promoted the development of eosinophilia and granuloma formation in a model of helminth infection, and, importantly, RGS16-deficient animals were reported to develop abnormal accumulation of T cells surrounding both perivascular and peribronchial spaces (42). These findings suggest that, in addition to direct migration and infiltration of T cells in the airways of inflamed animals, RGS proteins play a role in the discrete regulation of T cell distribution in inflamed tissues into perivascular and peribronchial spaces. Given the results that RGS3 and RGS16 each regulate T cell migration and distribution following HDM challenge, RGS3 and RGS16 may work in accord, and their combined deletion may result in a more severe inflammation and lymphoid structure formation.

Together, our findings along with others show that RGS proteins are dynamically expressed in T cells following activation, and each is capable of playing important regulatory functions in differentiation, proliferation, and localization following immune stimulation. With the identification of so many RGS proteins being expressed in T cells, it has yet to be exhaustively studied, and future studies will need to determine whether additional RGS proteins are expressed and function in T cells. Additional research into the expression patterns of RGS proteins in activated T cells and their redundant and nonredundant functional roles will provide a better understanding of the mechanisms by which GPCR signaling impacts immunity and inflammation.

GRANTS

This study was supported by National Institutes of Health Awards R01 GM-85058 (N. O. Dulin), R21-AI-094408 (A. I. Sperling), R01-AI-067697 (A. I. Sperling), T32-HL-007237 (J. W. Williams, D. Yau, and J. Kach), and T32-HD-009007 (N. Sethakorn) and American Heart Association Fellowships 0825868G (D. Yau), 10PRE2630163 (N. Sethakorn), and 10PRE4190120 (J. Kach).

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: J.W.W., D.Y., A.J.M., A.I.S., and N.O.D. conception and design of research; J.W.W., D.Y., N.S., J.K., E.B.R., T.V.M., J.C., X.J., H.X., A.J.M., and N.O.D. performed experiments; J.W.W., D.Y., N.S., J.K., E.B.R., T.V.M., J.C., X.J., H.X., A.J.M., A.I.S., and N.O.D. analyzed data; J.W.W., D.Y., N.S., J.K., E.B.R., T.V.M., J.C., X.J., H.X., A.J.M., A.I.S., and N.O.D. interpreted results of experiments; J.W.W., D.Y., N.S., J.K., T.V.M., J.C., X.J., H.X., A.J.M., and N.O.D. prepared figures; J.W.W. and N.O.D. drafted manuscript; J.W.W., A.I.S., and N.O.D. edited and revised manuscript; J.W.W., D.Y., N.S., J.K., E.B.R., T.V.M., J.C., X.J., H.X., A.J.M., A.I.S., and N.O.D. approved final version of manuscript.