Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Gene expression profiling of human mesenchymal stem cells chemotactically induced with CXCL12

In situ tissue engineering is a promising approach in regenerative medicine, with the possibility that adult stem or progenitor cells will be guided chemotactically to a tissue defect and subsequently differentiate into the surrounding tissue type. Mesenchymal stem cells (MSC) represent attractive candidate cells. Chemokines such as CXCL12 (SDF-1α) chemoattract MSC, but little is known about the molecular processes involved in the chemotaxis and migration of MSC. In this study, MSC recruitment by CXCL12 was investigated by genome-wide microarray analysis. The dose-dependent migration potential of bone-marrow-derived MSC toward CXCL12 was measured in an in vitro assay, with a maximum being recorded at a concentration of 1,000 nM CXCL12. Microarray analysis of MSC stimulated with CXCL12 and non-stimulated controls showed 30 differentially expressed genes (24 induced and six repressed). Pathway analysis revealed 11 differentially expressed genes involved in cellular movement and cytokine-cytokine receptor interaction, including those for migratory inducers such as the chemokines CXCL8 and CCL26, the leukocyte inhibitory factor, secretogranin II, and prostaglandin endoperoxide synthase 2. These results were confirmed by real-time polymerase chain reaction for selected genes. The obtained data provide further insights into the molecular mechanisms involved in chemotactic processes in cell migration and designate CXCL12 as a promising candidate for in situ recruitment in regenerative therapies. Stefan Stich and Marion Haag contributed equally to this work. This study was supported by the Investitionsbank Berlin and the European Regional Development Fund (grant: 10128098), Deutsche Forschungsgemeinschaft (grant: DFG SI 569/7–1), and the Bundesministerium für Bildung und Forschung (Bioinside: 13N9817).     

Human periosteum-derived progenitor cells express distinct chemokine receptors and migrate upon stimulation with CCL2, CCL25, CXCL8, CXCL12, and CXCL13

For bone repair, transplantation of periosteal progenitor cells (PCs), which had been amplified within supportive scaffolds, is applied clinically. More innovative bone tissue engineering approaches focus on the in situ recruitment of stem and progenitor cells to defective sites and their subsequent use for guided tissue repair. Chemokines are known to induce the directed migration of bone marrow CD34(-) mesenchymal stem cells (MSCs). The aim of our study was to determine the chemokine receptor expression profile of human CD34(-) PCs and to demonstrate that these cells migrate upon stimulation with selected chemokines. PCs were isolated from periosteum of the mastoid bone and displayed a homogenous cell population presenting an MSC-related cell-surface antigen profile (ALCAM(+), SH2(+), SH3(+), CD14(-), CD34(-), CD44(+), CD45(-), CD90(+)). The expression profile of chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that PCs express receptors of all four chemokine subfamilies CC, CXC, CX(3)C, and C. Migration of PCs and a dose-dependent migratory effect of the chemokines CCL2 (MCP1), CCL25 (TECK), CXCL8 (IL8), CXCL12 (SDF1alpha), and CXCL13 (BCA1), but not CCL22 (MDC) were demonstrated using a 96-multiwell chemotaxis assay. In conclusion, for the first time, here we report that human PCs express chemokine receptors, present their profile, and demonstrate a dose-dependent migratory effect of distinct chemokines on these cells. These results are promising towards in situ bone repair therapies based on guiding PCs to bone defects, and encourage further in vivo studies.     

Differential binding of chemokines to macrophages and neutrophils in the human inflamed synovium

In chronic inflammatory foci, such as the rheumatoid joint, there is enhanced recruitment of phagocytes from the blood into the tissues. Chemokines are strongly implicated in directing the migration of these cells, although little is known regarding the chemokine receptors that could mediate their chemotaxis into the joint tissue. Therefore the objective of the study was to identify chemokine binding sites on macrophages and neutrophils within the rheumatoid synovium using radiolabeled ligand binding and in situ autoradiography. Specific binding sites for CCL3 (macrophage inflammatory protein-1alpha), CCL5 (RANTES), CCL2 (monocyte chemoattractant protein-1) and CXCL8 (IL-8) were demonstrated on CD68+ macrophages in the subintimal and intimal layers. The number and percentage of intimal cells that bound chemokines were greater in inflamed regions compared to noninflamed regions. The intensity of intimal binding varied between chemokines with the rank order, CCL3 > CCL5 > CCL2 > CXCL8. Neutrophils throughout the synovium bound CXCL8 but did not show any signal for binding CCL2, CCL3 or CCL5. Immunohistochemistry showed that both CXCR1 and CXCR2 are expressed by macrophages and neutrophils in the rheumatoid and nonrheumatoid synovia, suggesting that both of these receptors are responsible for the CXCL8 binding. The chemokine binding sites described on phagocytes may be involved in the migration of these cells into the inflamed joint.     

Variability in chemokine-induced adhesion of human mesenchymal stromal cells

BACKGROUND AIMS. Intravenously applied mesenchymal stromal cells (MSC) are under investigation for numerous clinical indications. However, their capacity to activate shear stress-dependent adhesion to endothelial ligands is incompletely characterized. METHODS. Parallel-plate flow chambers were used to induce firm adhesion of MSC to integrin ligand vascular cell adhesion molecule (VCAM)-1. Human MSC were stimulated by chemokine (C-C motif) ligand (CCL15)/macrophage inflammatory protein (MIP-5), CCL19/MIP-3β chemokine (C-X-C motif) ligand (CXCL8)/interleukin (IL)-8, CXCL12/ stromal derived factor (SDF-1) or CXCL13/B lymphocyte chemoattractant (BLC). RESULTS. Two MSC isolates responded to three chemokines (either to CCL15, CCL19 and CXCL13, or to CCL19, CXCL12 and CXCL13), two isolates responded to two chemokines (to CCL15 and CCL19, or to CCL19 and CXCL13), and one isolate responded to CCL19 only. In contrast, all tested MSC isolates responded to selectins (P-selectin and E-selectin) or integrin ligand VCAM-1, as visualized by a velocity reduction under flow. CONCLUSIONS. Inter-individual variability of chemokine-induced integrin activation should be considered when evaluating human MSC as cellular therapies.     

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL‐6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP‐1 secretion was reduced by CCL15/CCR3. In conclusion, this in‐vitro study identifies chemokine receptor‐ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL‐6 and HGF and reducing the secretion of TIMP‐1.     

Differential binding of chemokines to macrophages and neutrophils in the human inflamed synovium

In chronic inflammatory foci, such as the rheumatoid joint, there is enhanced recruitment of phagocytes from the blood into the tissues. Chemokines are strongly implicated in directing the migration of these cells, although little is known regarding the chemokine receptors that could mediate their chemotaxis into the joint tissue. Therefore the objective of the study was to identify chemokine binding sites on macrophages and neutrophils within the rheumatoid synovium using radiolabeled ligand binding and in situ autoradiography. Specific binding sites for CCL3 (macrophage inflammatory protein-1α), CCL5 (RANTES), CCL2 (monocyte chemoattractant protein-1) and CXCL8 (IL-8) were demonstrated on CD68⁺ macrophages in the subintimal and intimal layers. The number and percentage of intimal cells that bound chemokines were greater in inflamed regions compared to noninflamed regions. The intensity of intimal binding varied between chemokines with the rank order, CCL3 > CCL5 > CCL2 > CXCL8. Neutrophils throughout the synovium bound CXCL8 but did not show any signal for binding CCL2, CCL3 or CCL5.     

Proinflammatory chemokine gene expression influences survival of patients with non-Hodgkin's lymphoma

The migration, survival and proliferation of cells is the basis for all physiologic and pathologic processes in the human body. All these reactions are regulated by a complex chemokine network that guides lymphocytes homing, chemotaxis, adhesion and interplay between immunologic system response cells. Chemokines are also responsible for metastatic dissemination of cancers, including Hodgkin's and non-Hodgkin's lymphomas. The purpose of this study was to determine chemokine gene expression (CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5) in lymphoma lymph nodes compared to their expression in reactive lymph nodes. We also analyzed the influence of chemokine gene expression on the survival of lymphoma patients. Chemokine gene expression was evaluated in 37 lymphoma lymph nodes and in 25 samples of reactive lymph nodes. Gene expression of chemokines CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5 was measured using the PCR method. Statistical analysis was performed using CSS Statistica for Windows (version 7.0) software. Probability values 〈 〈 0.05 were considered statistically significant and those between 0.05 and 0.1 as indicative of a trend. We found lower CXCL8 and CXCL10 gene expression in lymphoma lymph nodes compared to reactive lymph nodes. In the cases of CCL2 and CCL3, expression in lymphomas was higher than in reactive lymph nodes. Patients with high expression of CCL2 and CXCL10 had shorter survival.     

Gene expression profile of adult human bone marrow-derived mesenchymal stem cells stimulated by the chemokine CXCL7

A variety of chemokines has been shown to recruit human bone marrow-derived mesenchymal stem cells (MSC) and may be potential candidates for chemokine-based tissue regeneration approaches. The aim of our study was to determine whether the chemokine CXCL7 stimulates migration of human bone marrow-derived MSC and to analyze the effect of CXCL7 on the recruitment of MSC on the broad molecular level. Chemotaxis assays documented that high doses of CXCL7 significantly recruited MSC. Gene expression profiling using oligonucleotide microarrays showed that MSC treated with CXCL7 differentially expressed genes related to cell migration, cell adhesion and extracellular matrix remodeling. Pathway analysis showed that CXCL7 induced the expression of all chemokines binding the interleukin (IL) receptors A and B, CXCR1 and CXCR2, as well as the IL6 signal transducer (gp130) and its ligands IL6 and leukemia inhibitory factor (LIF). Induction of differentially expressed chemokines CXCL1-3, CXCL5, and CXCL6 as well as LIF and gp130 in MSC by CXCL7 was verified by real-time polymerase chain reaction. Immunoassay of cell culture supernatants confirmed elevated levels of the interleukins 6 and 8 in MSC upon treatment with CXCL7. Chemotaxis assays showed that interleukin 6 did not recruit MSC. In conclusion, CXCL7 significantly stimulates the migration of human MSC in vitro. Pathway analysis suggests that recruitment of human MSC by CXCL7 is supported by the induction of ligands of the interleukin 8 receptors, synergistically activating the respective signaling pathways.     

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3α, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFκB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.     

Profiling heparin-chemokine interactions using synthetic tools.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.     

Non-enzymatic dissociation of human mesenchymal stromal cells improves chemokine-dependent migration and maintains immunosuppressive function

Background aimsHuman bone marrow–derived mesenchymal stromal cells (MSC) can suppress inflammation; therefore their therapeutic potential is being explored in clinical trials. Poor engraftment of infused MSC limits their therapeutic utility; this may be caused by MSC processing before infusion, in particular the method of their detachment from culture.MethodsEnzymatic methods of detaching MSC (Accutase and TrypLE) were compared with non-enzymatic methods (Cell Dissociation Buffer [CDB], ethylenediamine tetra-acetic acid and scraping) for their effect on MSC viability, chemokine receptor expression, multi-potency, immunomodulation and chemokine-dependent migration.ResultsTrypLE detachment preserved MSC viability and tri-lineage potential compared with non-enzymatic methods; however, this resulted in near complete loss of surface chemokine receptor expression. Of the non-enzymatic methods, CDB detachment preserved the highest viability while retaining significant tri-lineage differentiation potential. Once re-plated, CDB-detached MSC regained their original morphology and reached confluence, unlike with the use of other non-enzymatic methods. Viability was significantly reduced with the use of ethylenediamine tetra-acetic acid and further reduced with the use of cell scraping. Addition of 1% serum during CDB detachment led to higher MSC numbers entering autophagy and increased MSC recovery after re-plating. TrypLE and CDB-detached MSC suppressed CD3⁺CD4⁺CD25⁻ T-cell proliferation, although TrypLE-detached MSC exhibited superior suppression at 1:20 ratio. CDB detachment retained surface chemokine receptor expression and consequently increased migration to CCL22, CXCL12 and CCL4, in contrast with TrypLE-detached MSC.ConclusionsThis study demonstrates that non-enzymatic detachment of MSC with the use of CDB minimizes the negative impact on cell viability, multipotency and immunomodulation while retaining chemokine-dependent migration, which may be of importance in MSC delivery and engraftment in sites of injury.     

Human intestinal mast cells are a potent source of multiple chemokines

Mast cells are key effector cells of immediate type allergic reactions. Upon activation they release a broad array of pre-stored and de novo synthesized mediators including immunoregulatory cytokines and chemokines. Here, we analyzed the chemokine profile expressed by mature human mast cells. Human mast cells were isolated from intestinal tissue and cultured with stem cell factor (SCF) in the presence or absence of IL-4 for 10d. Cells were stimulated by cross-linking of the high affinity IgE receptor (FcεRI) and/or by SCF. Chemokine and chemokine receptor mRNA expression was determined by real-time RT-PCR and chemokine release was measured by multiplex bead immunoassay. Out of 43 chemokines and 19 chemokine receptors human intestinal mast cells express 27 chemokines and nine chemokine receptors. Twelve chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL18, CCL20, CXCL2, CXCL3, CXCL8, and XCL1) were more than four-fold up-regulated in response to FcεRI cross-linking. Combination of pre-culture with IL-4 and/or stimulation with SCF in addition to FcεRI cross-linking further increased the antigen-dependent expression of mRNA for most chemokines. In contrast, the expression of CCL20, CXCL2, and CXCL3 was strongly inhibited by IL-4 treatment. In conclusion, human intestinal mast cells express a broad spectrum of different chemokines underlining their important role as immunoregulatory cells. Furthermore, combined treatment with IL-4 and SCF increases the antigen-mediated expression and release of multiple chemokines, but IL-4 priming inhibits the expression of CCL20, CXCL2, and CXCL3.     

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.     

Complement C3a enhances CXCL12 (SDF-1)-mediated chemotaxis of bone marrow hematopoietic cells independently of C3a receptor

Complement C3a promotes CXCL12-induced migration and engraftment of human and murine hemopoietic progenitor cells, suggesting a cross-influence between anaphylatoxin and chemokine axes. Here we have explored the underlying mechanism(s) of complement anaphylatoxin and chemokine cooperation. In addition to C3a, C3a-desArg and C4a but not C5a, are potent enhancers of CXCL12-induced chemotaxis of human and murine bone marrow (BM) stem/progenitor cells and B lineage cells. C3a enhancement of chemotaxis is chemokine specific because it is also observed for chemotaxis to CCL19 but not to CXCL13. The potentiating effect of C3a on CXCL12 is independent of the classical C3a receptor (C3aR). First, human BM CD34(+) and B lineage cells do not express C3aR by flow cytometry. Second, the competitive C3aR inhibitor SB290157 does not affect C3a-mediated enhancement of CXCL12-induced chemotaxis. Third, enhancement of chemotaxis of hemopoietic cells is also mediated by C3a-desArg, which does not bind to C3aR. Finally, C3a enhances CXCL12-induced chemotaxis of BM cells from C3aR knockout mice similar to BM cells from wild-type mice. Subsequent studies revealed that C3a increased the binding affinity of CXCL12 to human CXCR4(+)/C3aR(-), REH pro-B cells, which is compatible with a direct interaction between C3a and CXCL12. BM stromal cells were able to generate C3a, C3a-desArg, C4a, as well as CXCL12, suggesting that this pathway could function in vivo. Taken together, we demonstrate a C3a-CXCL12 interaction independent of the C3aR, which may provide a mechanism to modulate the function of CXCL12 in the BM microenvironment.     

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRalphabeta NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Valpha14i-positive and approximately 50% of Valpha14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Valpha14i-negative than Valpha14i-positive NKT cells. In spleen only, subsets of Valpha14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-gamma/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4(-) Valpha14i-negative NKT cells that were L-selectin(high) and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Valpha14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1(+) Valpha14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Valpha14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4(+) or CD4(-)CD8(-) and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Valpha14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.     

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.     

MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells

Stromal Derived Factor 1 (SDF1 or CXCL12), is a chemokine known to be critical for the migration of cells in several tissue systems including the homing of the hematopoietic stem cell (HSC) to its niche in the bone marrow. A comparative analysis of miRNA expression profiles of two stromal cell lines, distinguishable by function and by CXCL12 expression (CXCL12+ and CXCL12-), revealed that the CXCL12- cells expressed>40 fold more miR-886-3p than the CXCL12+ cells. Screening studies showed that when miR-886-3p was transfected into the CXCL12+ stromal cells, the expression of CXCL12 was down-regulated by as much as 85% when compared to appropriate controls, and results in the loss of CXCL12-directed chemotaxis. Similar reductions in CXCL12 were obtained with the transfection of miR-886-3p into primary stromal cell cultures. Additional studies showed that miR-886-3p specifically targeted the 3' untranslated region (UTR) of CXCL12 mRNA. These data suggest a role for miRNA in modulating the expression of CXCL12, a gene product with a critical role in hematopoietic regulation.     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

Peroxisome proliferator-activated receptor α agonists modulate Th1 and Th2 chemokine secretion in normal thyrocytes and Graves' disease

Until now, no data are present about the effect of peroxisome proliferator-activated receptor (PPAR)α activation on the prototype Th1 [chemokine (C–X–C motif) ligand (CXCL)10] (CXCL10) and Th2 [chemokine (C–C motif) ligand 2] (CCL2) chemokines secretion in thyroid cells.The role of PPARα and PPARγ activation on CXCL10 and CCL2 secretion was tested in Graves' disease (GD) and control primary thyrocytes stimulated with interferon (IFN)γ and tumor necrosis factor (TNF)α.IFNγ stimulated both CXCL10 and CCL2 secretion in primary GD and control thyrocytes. TNFα alone stimulated CCL2 secretion, while had no effect on CXCL10. The combination of IFNγ and TNFα had a synergistic effect both on CXCL10 and CCL2 chemokines in GD thyrocytes at levels comparable to those of controls. PPARα activators inhibited the secretion of both chemokines (stimulated with IFNγ and TNFα) at a level higher (for CXCL10, about 60–72%) than PPARγ agonists (about 25–35%), which were confirmed to inhibit CXCL10, but not CCL2.Our data show that CCL2 is modulated by IFNγ and TNFα in GD and normal thyrocytes. Furthermore we first show that PPARα activators inhibit the secretion of CXCL10 and CCL2 in thyrocytes, suggesting that PPARα may be involved in the modulation of the immune response in the thyroid.