Functional expression of the C-X-C chemokine receptor CXCR4 by human bronchial epithelial cells: regulation by proinflammatory mediators

CXCR4 and its ligand stromal cell-derived factor 1alpha (SDF-1alpha) have recently been implicated in the development of airway inflammation in a mouse model of allergic airway disease. Here we report, for the first time, the expression of a functional CXCR4 in primary human normal bronchial epithelial cells and the regulation of CXCR4 gene expression by proinflammatory mediators. Both bradykinin (BK) and IL-1beta induced an accumulation of CXCR4 mRNA in normal bronchial epithelial cells in a time-dependent manner, with peak levels of CXCR4 mRNA reached between 4 and 24 h after stimulation. Ligand activation of CXCR4 in airway epithelial cells resulted in the activation of the extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun amino-terminal kinase signaling pathways and calcium mobilization. Pretreatment of airway epithelial cells with BK or IL-1beta enhanced SDF-1alpha induced phospho-extracellular signal-regulated kinase and calcium mobilization, in addition to increasing the level of CXCR4 protein. Finally, we describe the expression of CXCR4 mRNA and its regulation by BK in vivo in human nasal tissue. CXCR4 mRNA levels are significantly higher in the nasal tissue of symptomatic allergic rhinitis subjects compared with normal subjects. Moreover, BK challenge significantly increased CXCR4 mRNA levels in nasal tissue of mild allergic rhinitis subjects in vivo, but not normal controls. In conclusion, this study demonstrates that human airway epithelial cells respond to proinflammatory mediators by up-regulating the chemokine receptor CXCR4, thus enabling the cells to respond more effectively to constitutively expressed SDF-1alpha. This may lead to enhanced activation of intracellular signaling pathways resulting in the release of mediators involved in inflammatory allergic airway disease.     

An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

Inflammatory angiogenesis and vascular remodeling play key roles in the chronic inflammatory skin disease psoriasis, but little is known about the molecular mediators of vascular activation. Based on the reported elevated mRNA levels of the angiogenic chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in psoriasis, we investigated the relevance of the SDF-1/CXCR4 axis in two experimental models of chronic psoriasis-like skin inflammation. The cutaneous expression of both SDF-1 and CXCR4 was upregulated in the inflamed skin of K14-VEGF-A transgenic mice and in imiquimod-induced skin inflammation, with expression of CXCR4 by blood vessels and macrophages. Treatment with the CXCR4 antagonist AMD3100 potently inhibited skin inflammation in both models, associated with reduced inflammatory angiogenesis and inflammatory cell accumulation, including dermal CD4⁺ cells and intraepidermal CD8⁺ T cells. Similar anti-inflammatory effects were seen after treatment with a neutralizing anti-SDF-1 antibody. In vitro, inhibition of CXCR4 blocked SDF-1-induced chemotaxis of CD11b⁺ splenocytes, in agreement with the reduced number of macrophages after in vivo CXCR4 blockade. Our results reveal an important role of the SDF-1/CXCR4 axis in skin inflammation and inflammatory angiogenesis, and they indicate that inhibition of the SDF-1/CXCR4 axis might serve as a novel therapeutic strategy for chronic inflammatory skin diseases.     

CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1beta or TNF-alpha. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1alpha (SDF-1alpha) indicated that the CXCR4 receptors were functional. Incubation with SDF-1alpha resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene alpha. RPE cells also migrated in response to SDF-1alpha. As SDF-1alpha expression by RPE cells was detected constitutively, we postulate that SDF-1-CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.     

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.     

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.     

Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4

CXCR4 is a chemokine receptor used by some strains of HIV-1 as an entry coreceptor in association with cell surface CD4 on human cells. In human immunodeficiency virus type 1 (HIV-1)-infected individuals, the appearance of viral isolates with a tropism for CXCR4 (T tropic) has been correlated with late disease progression. The presumed natural ligands for CXCR4 are SDF-1alpha and SDF-1beta, which are proposed to play a role in blocking T-tropic HIV-1 cell entry. Here, we demonstrate that addition of an N-terminal methionine residue to SDF-1beta (Met-SDF-1beta) results in a dramatically enhanced functional activity compared to that of native SDF-1beta. Equivalent concentrations of Met-SDF-1beta are markedly more inhibitory for T-tropic HIV-1 replication than SDF-1beta. A comparison of the biological activities of these two forms of SDF-1beta reveals that Met-SDF-1beta induces a more pronounced intracellular calcium flux yet binds with slightly lower affinity to CXCR4 than SDF-1beta. Down-modulation of CXCR4 is similar after exposure of cells to either chemokine form for 2 h. However, after a 48-h incubation, the surface expression of CXCR4 is much lower for cells treated with Met-SDF-1beta. The enhanced blocking of T-tropic HIV-1 by Met-SDF-1beta appears to be related to prolonged CXCR4 down-modulation.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.     

Selection of a 2-azabicyclo[2.2.2]octane-based alpha4beta1 integrin antagonist as an inhaled anti-asthmatic agent

The alpha4beta1 integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infiltration and activation. From a number of potent alpha4beta1 antagonists that we evaluated for safety and efficacy, 1 was selected as a lead candidate for anti-asthma therapy by the inhalation route. We devised an optimized stereoselective synthesis to facilitate the preparation of a sufficiently large quantity of 1 for assessment in vivo. Administration of 1 to allergen-sensitive sheep by inhalation blocked the late-phase response of asthma and abolished airway hyper-responsiveness at 24h following the antigen challenge. Additionally, the recruitment of inflammatory cells into the lungs was inhibited. Administration of 1 to ovalbumin-sensitized guinea pigs intraperitoneally blocked airway resistance and inhibited the recruitment of inflammatory cells.     

The chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.     

Stromal cell-derived factor-1 increase alphavbeta3 integrin expression and invasion in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. The stromal cell-derived factor 1 (SDF-1), constitutively secreted by human lung epithelium cells, has been shown to function in a key role for recruitment of neutrophils. Here, we found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of CXCR4 (SDF-1 receptor), which was higher than normal cartilage and human chondrocyte. SDF-1alpha and lung epithelium cells conditioned medium (LECM) induced the invasiveness of chondrosarcoma cells. SDF-1 siRNA inhibited LECM-induced invasion of chondrosarcoma cells and SDF-1alpha also directly induced the cell surface expression of alphavbeta3 but not alpha2beta1 and alpha5beta1 integrin. Activations of ERK and NF-kappaB pathways after SDF-1 treatment was demonstrated, and SDF-1alpha-induced expression of alphavbeta3 integrin and invasion activity was inhibited by the specific inhibitor and mutant of ERK and NF-kappaB cascades. Taken together, our results indicate that lung derived-SDF-1alpha enhances the invasiveness of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.     

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells

The interaction between the stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1α axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1α levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1α significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1α interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1α gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1α axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.     

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.     

Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK

Chemokines are critical in controlling lymphocyte traffic and migration. The CXC chemokine CXCL12/SDF-1alpha interacts with its receptor CXCR4 to induce the migration of a number of different cell types. Although an understanding of the physiological functions of this chemokine is emerging, the mechanism by which it regulates T cell migration is still unclear. We show here that the Tec family kinase ITK is activated rapidly following CXCL12/SDF-1alpha stimulation, and this requires Src and phosphatidylinositol 3-kinase activities. ITK regulates the ability of CXCL12/SDF-1alpha to induce T cell migration as overexpression of wild-type ITK-enhanced migration, and T cells lacking ITK exhibit reduced migration as well as adhesion in response to CXCL12/SDF-1alpha. Further analysis suggests that ITK may regulate CXCR4-mediated migration and adhesion by altering the actin cytoskeleton, as ITK null T cells were significantly defective in CXCL12/SDF-1a-mediated actin polymerization. Our data suggest that ITK may regulate the ability of CXCR4 to induce T cell migration.     

CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway

Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response. The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1beta/SDF-1alpha or by the HIV envelope protein, gp120. We have studied the role of the proteasome pathway in the down-regulation of these receptors. Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the zeta subunit of proteasome. Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1beta stimulation. The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1beta induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy. The proteasome inhibitors also inhibited the SDF-1alpha and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells. However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor. These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1alpha-induced p44/42 MAP kinase or MIP-1beta-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors. These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity.     

Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation

Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.