Reduction In Chemotaxis Research Articles

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells

BackgroundDevelopment of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. MethodsWe compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FindingsThe PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. InterpretationConsistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FundingMRC (UK) Project Grant (P028160).

Growth and chemotaxis of nematodes reduced upon exposure to Third Fork Creek surface water

BackgroundThird Fork Creek is a historically impaired urban stream that flows through the city of Durham, North Carolina. Caenorhabditis elegans (C. elegans) are non-parasitic, soil and aquatic dwelling nematodes that have been used frequently as a biological and ecotoxicity model. We hypothesize that exposure to Third Fork Creek surface water will inhibit the growth and chemotaxis of C. elegans. Using our ring assay model, nematodes were enticed to cross the water samples to reach a bacterial food source which allowed observation of chemotaxis. The total number of nematodes found in the bacterial food source and the middle of the plate with the water source was recorded for 3 days.ResultsOur findings suggest a reduction in chemotaxis and growth on day three in nematodes exposed to Third Fork Creek water samples when compared to the control (p value < 0.05). These exploratory data provide meaningful insight to the quality of Third Fork Creek located near a Historically Black University.ConclusionsFurther studies are necessary to elucidate the concentrations of the water contaminants and implications for human health. The relevance of this study lies within the model C. elegans that has been used in a plethora of human diseases and exposure research but can be utilized as an environmental indicator of water quality impairment.

Rosa canina – Rose hip pharmacological ingredients and molecular mechanics counteracting osteoarthritis – A systematic review

BackgroundThe successful use of rose hip for the treatment of osteoarthritis is well documented. Several randomized placebo controlled double-blind studies, as mono or combination therapy, have demonstrated treatment efficacy as well as excellent tolerability. PurposeThis review focuses on the molecular mechanism underlying the clinical effects of rose hip in osteoarthritis (OA). MethodsThe database Medline was screened – using the search term “Rosa canina” or “rose hip” – for publications on pharmacological or mechanistic studies with relevance to OA; in addition for findings on pharmacologically active constituents as well as clinical studies. The screening results were complemented by following-up on cited literature. ResultsIn particular, 24 pharmacological studies on Rosa canina or preparations thereof were considered relevant. Potent antioxidant radical scavenging effects are well documented for numerous rose hip constituents besides Vitamin C. Furthermore, anti-inflammatory activities include the reduction of pro-inflammatory cytokines and chemokines, reduction of NF-kB signaling, inhibition of pro-inflammatory enzymes, including COX1/2, 5-LOX and iNOS, reduction of C-reactive protein levels, reduction of chemotaxis and chemoluminescence of PMNs, and an inhibition of pro-inflammatory metalloproteases. ConclusionThe antioxidant and anti-inflammatory effects of Rosa canina match its clinical action – especially considering new findings on the pharmacological disease pattern of OA. The entirety of several compounds including phenolics, terpenoids, galactolipids, carotenoids, fruit acids and fatty oils can be considered responsible for the observed pharmacological and clinical effects. Further research is needed to eludicate how and in which manner single rose hip compounds interact with their molecular pharmacological targets.

On the road to new treatments for multiple sclerosis: targeting dendritic cell migration into the central nervous system.

On the road to new treatments for multiple sclerosis: targeting dendritic cell migration into the central nervous system.

Two citrate chemoreceptors involved in chemotaxis to citrate and/or citrate-metal complexes in Ralstonia pseudosolanacearum

The bacterial wilt pathogen Ralstonia pseudosolanacearum Ps29 exhibited chemotactic responses to citrate. This pathogen expresses 22 putative chemoreceptors. In screening a complete collection of mcp single-gene deletion mutants of Ps29, none showed a significant decrease in response to citrate compared with the wild-type strain. Analysis of a collection of stepwise- and multiple-deletion mutants of Ps29 revealed that the RS_RS07350 homolog (designated McpC) and McpP (chemoreceptor mediating both positive chemotaxis to phosphate and negative chemotaxis to maleate) are chemoreceptors for citrate. Double deletion of mcpC and mcpP markedly reduced the response to citrate, indicating that McpC and McpP are major chemoreceptors for citrate. Wild-type Ps29 was attracted to both free citrate and citrate complexed with divalent metal cations such as magnesium and calcium. The mcpC mcpP double-deletion mutant also showed significant reduction in chemotaxis to Mg2+- and Ca2+-citrate complexes. Introduction of a plasmid harboring the mcpC gene (but not the mcpP gene) restored the ability to respond to these citrate-metal complexes, demonstrating that McpC can sense complexes of citrate and metal ions such as Mg2+ and Ca2+ as well as free citrate. Thus, R.pseudosolanacearum Ps29 expresses two chemoreceptors for citrate. In plant infection assays using tomato seedlings, the mcpC and mcpP single- and double-deletion mutants of the highly virulent R.pseudosolanacearum MAFF106611 strain were as infectious as the wild-type strain, suggesting that citrate chemotaxis does not play an important role in infection of tomato plants in this assay system.

O30 Natural compounds affecting neutrophil migration

Hydrogen peroxide (H 2 O 2 ) has recently been identified as the earliest messenger released by damaged tissues and is crucial for the chemoattraction of neutrophils to sites of injury and infection. However, how neutrophils sense and respond to H 2 O 2 remains unresolved. Recently, we have identified the transient potential receptor melastatin-2 ion channel (TRPM2) as having a key role in directing the chemotaxis of neutrophils towards hydrogen peroxide (H 2 O 2 ), in both in vitro and in vivo models. We isolated neutrophils from wild-type and TRPM2 −/− mice and we observed that the genetic deletion of TRPM2 results in a reduction in both the speed and directionality of the neutrophil migration up a gradient of H 2 O 2 . We tested a diverse group of natural products, including a range of terpenes, alkaloids and flavonoids, for their effects on this H 2 O 2 -induced neutrophil chemotaxis using the Ibidi-chemotaxis system, which allows live-cell imaging in vitro of migration over time. From our screen, we have identified: beta-carotene, artemisinin, ferulic acid and N-acetylcysteine as compounds which reduce neutrophil chemotaxis in a comparable way to TRPM2 deletion, leading to the suggestion that they may be acting as TRPM2 inhibitors. Moreover, our compounds induce a more pronounced reduction in chemotaxis than the currently available TRPM2 inhibitors, which lack potency and selectivity. The natural compounds we have tested may have clinical applications; for example, in the treatment of inflammatory conditions, such as sepsis, which are characterised by excessive and damaging neutrophil migration.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in β-thalassemia mice and patients

Abstractβ-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

CXCR7 Participates in Chemotaxis and Homing Mediated By CXCL12

Hematopoiesis is tightly orchestrated by a precise quiescence/cycling equilibrium that is required for the continuous controlled production of differentiated blood cells. Chemokines and their receptors play an essential role in maintaining the hematopoietic cells pool within cell niches. The CXCL12/CXCR4 axis has been identified as the central axis for migration, adhesion and homing of hematopoietic cells and for leukemic cells. Another CXCL12-binding receptor has recently, been identified, CXCR7, however the contribution of this receptor to CXCL12 - mediated effects in hematopoietic cells, is still controversial, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. We recently demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic (ALL) cells and that CXCR7 contributed to T-ALL cell chemotaxis induced by CXCL12 potentializing CXCR4 response. The mouse model with reduced expression of Arhgap21+/- exhibits a 71% downregulation of CXCR7 protein in membrane of T cells, probably due to lower recruitment by β-arrestin since GPCRs recycling to the membrane needs association with this protein. Moreover, this model showed reduction in chemotaxis, adhesion and homing of hematopoietic stem and progenitor cells suggesting that Arhgap21 may be a strong candidate for CXCL12/CXCR4 axis regulation. Thus, in the present study we aimed to investigate the involvement of CXCR7 in chemotaxis induced by CXCL12 and homing using U937 myeloid leukemic cell line in NOD/SCID mice and T-lymphocytes in Arhgap21+/- mice.The subcellular location of CXCR4 and CXCR7 by confocal microscopy evidenced both receptors in the membrane and cytoplasm of U937 cells. After CXCL12 induction, the blocked of CXCR4 by AMD3100 and/or the inhibition of CXCR7 by transduction with lentivirus mediated shRNA resulted in significant changes in U937 cells chemotactic response analyzed by transwell assay (U937 treated with AMD3100, p=0.0019; U937 shCXCR7, p<0.001;U937 shCXCR7 treated with AMD3100, p=0.0056; Mann-Whitney test) and homing assay to spleen of NOD/SCID mouse after 16 hours of transplant (U937 treated with AMD3100, p=0.0070; U937 shCXCR7, p=0.044 6; U937 shCXCR7 treated with AMD3100, p=0.0243, Student's t test). Bone marrow analysis of Arhgap21+/- mice showed a reduction of T lymphocytes (CD4+ andCD8+; p=0.02, Student's t test), erythroblasts (Ter119+, p=0.01) and myeloid cells (Gr1+ Mac+, p=0.02), but there was no difference however in B lymphocytes (B220+). Indeed, peripheral blood showed a significant reduction in CD8+ cells (p=0.04) but similar amounts of CD4+ cells. Thymus did not show difference in CD4+ and CD8+ populations. Herein, we show by transwell assay a lower response of T lymphocytes to CXCL12 (p=0.0050, Mann-Whitney test), corroborating to the reduced CXCR7 expression observed. No difference in CXCR4 expression was observed in any of the cell types.Taken together, our results suggest that CXCR7 is involved in chemotaxis and homing of T-lymphocytes and myeloid leukemic cells, potentalizing the CXCR4 response. The reduction of T-lymphocytes in bone marrow of Arhgap21+/- mice, may be related to a reduced homing due to the reduction of CXCR7 in the membrane of these cells, which leads to a lower attraction induced by CXCL12 ligant to bone marrow. Moreover, an inhibition of both receptors in myeloid leukemic cells could have a better effect in reducing their homing than the blocking of a single receptor. DisclosuresNo relevant conflicts of interest to declare.

McpQ is a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes.

Chemoreceptors are at the beginning of chemosensory pathways that mediate chemotaxis. Pseudomonas putida KT2440 is predicted to have 27 chemoreceptors, most of which uncharacterized. We have previously identified McpS as chemoreceptor for Krebs cycle intermediates. Citrate is primarily present in the environment as metal complex, which, however, is not recognized by McpS. We show here that the McpS paralogue McpQ recognizes specifically citrate and citrate/metal2+ complexes. The McpQ ligand binding domain (McpQ-LBD) binds citrate/metal2+ complexes with higher affinity than citrate. McpQ-LBD is present in a monomer-dimer equilibrium and citrate and particularly citrate/Mg2+ binding stabilize the dimer. The bacterium showed much stronger responses to citrate/Mg2+ than to citrate and mcpQ inactivation caused a dramatic reduction in chemotaxis. Responses to Krebs cycle intermediates are thus mediated by the broad range McpS and McpQ that responds specifically to an intermediate not recognized by McpS. Interesting parallels exist to the paralogous amino acid chemoreceptors of Pseudomonas aeruginosa and Bacillus subtilis. Whereas one paralogue recognizes most amino acids, the remaining paralogue binds specifically one of the few acids not recognized by the broad range receptors. Therefore, chemotaxis to compound families by the concerted action of broad and narrow range receptors may represent a general mechanism.

Identification of a Chemoreceptor for C 2 and C 3 Carboxylic Acids

Chemoreceptors are at the beginnings of chemosensory signaling cascades that mediate chemotaxis. Most bacterial chemoreceptors are functionally unannotated and are characterized by a diversity in the structure of their ligand binding domains (LBDs). The data available indicate that there are two major chemoreceptor families at the functional level, namely, those that respond to amino acids or to Krebs cycle intermediates. Since pseudomonads show chemotaxis to many different compounds and possess different types of chemoreceptors, they are model organisms to establish relationships between chemoreceptor structure and function. Here, we identify PP2861 (termed McpP) of Pseudomonas putida KT2440 as a chemoreceptor with a novel ligand profile. We show that the recombinant McpP LBD recognizes acetate, pyruvate, propionate, and l-lactate, with KD (equilibrium dissociation constant) values ranging from 34 to 107 μM. Deletion of the mcpP gene resulted in a dramatic reduction in chemotaxis toward these ligands, and complementation restored a native-like phenotype, indicating that McpP is the major chemoreceptor for these compounds. McpP has a CACHE-type LBD, and we present data indicating that CACHE-containing chemoreceptors of other species also mediate taxis to C2 and C3 carboxylic acids. In addition, the LBD of NbaY of Pseudomonas fluorescens, an McpP homologue mediating chemotaxis to 2-nitrobenzoate, bound neither nitrobenzoates nor the McpP ligands. This work provides further insight into receptor structure-function relationships and will be helpful to annotate chemoreceptors of other bacteria.

Abstract 286: The Role of Gut Microbiota in Neointimal Hyperplasia After Vascular Injury

Introduction: There is increasing evidence that the gut microbiome regulates susceptibility to certain diseases through systemic effects of microbe-derived metabolites. Sodium butyrate is a short chain fatty acid that is produced by microbial fermentation of dietary fiber and has known anti-proliferative and anti-migratory effects on vascular smooth muscle cells (VSMC). We hypothesized that perturbation of the gut microbiome with antibiotics would alter systemic serum butyrate concentration and impact neointimal hyperplasia after vascular injury. Methods: 10-wk-old male Lewis Inbred rats were treated with vancomycin (“vanco”) in the drinking water (0.5mg/mL) ± sodium butyrate (“buty”, 0.5mg/mL) for 4 wks prior to undergoing left carotid angioplasty. Serum butyrate concentration was assessed by gas chromatography. Gut microbial composition was assessed by 16S rRNA gene surveys of fecal samples. VSMC were treated with butyrate (0-5mM) and assessed for cell proliferation using cell counting, cell migration using a transwell assay, and cell cycle progression using FACS. Results: Post-angioplasty carotid arteries from vanco-treated rats developed 38% more neointima than controls (0.032±0.004mm2 vs. 0.044±0.003 mm2, P=0.02), but vanco+buty treatment prevented this increase in intimal area (0.035±0.004 mm2, P=.62 vs. control). Analysis of gut microbial communities revealed unique shifts in bacterial clustering by treatment group, which correlated with changes in serum butyrate levels, with the lowest butyrate level detected in vanco-treated rats (0.54±0.1 μmol/mL control, 0.017±0.1 μmol/mL vanco, 0.45±0.1 μmol/mL vanco+buty, P=.008). In vitro, butyrate treatment inhibited VSMC proliferation at 24-48 hrs in a dose-dependent manner, which correlated with induction of G0/G1 cell cycle arrest (P=.001) and a reduction in chemotaxis (P=.03). Conclusions: Oral vancomycin treatment induced a shift in the gut microbial community that was associated with decreased serum butyrate levels and increased neointimal hyperplasia, both of which were reversed by oral butyrate supplementation. These data demonstrate proof-of-concept that there is a correlation between gut microbial dysbiosis and susceptibility to neointimal hyperplasia.

An oral preparation containing hylauronic acid (Oralvisc®) can normalize the turnover of hyaluronic acid in synovial fluid of osteoarthritic knee patients

An oral preparation containing hylauronic acid (Oralvisc®) can normalize the turnover of hyaluronic acid in synovial fluid of osteoarthritic knee patients

Cellular and Molecular Mechanisms Underlie the Anti-Tumor Activities Exerted by Walterinnesia aegyptia Venom Combined with Silica Nanoparticles against Multiple Myeloma Cancer Cell Types

Multiple myeloma (MM) is a clonal disease of plasma cells that remains incurable despite the advent of several novel therapeutics. In this study, we aimed to delineate the impact of snake venom extracted from Walterinnesia aegyptia (WEV) alone or in combination with silica nanoparticles (WEV+NP) on primary MM cells isolated from patients diagnosed with MM as well as on two MM cell lines, U266 and RPMI 8226. The IC50 values of WEV and WEV+NP that significantly decreased MM cell viability without affecting the viability of normal peripheral mononuclear cells (PBMCs) were determined to be 25 ng/ml and 10 ng/ml, respectively. Although both WEV (25 ng/ml) and WEV+NP (10 ng/ml) decreased the CD54 surface expression without affecting the expression of CXCR4 (CXCL12 receptor) on MM cells, they significantly reduced the ability of CXC chemokine ligand 12 (CXCL12) to induce actin cytoskeleton rearrangement and the subsequent reduction in chemotaxis. It has been established that the binding of CXCL12 to its receptor CXCR4 activates multiple intracellular signal transduction pathways that regulate MM cell chemotaxis, adhesion, and proliferation. We found that WEV and WEV+NP clearly decreased the CXCL12/CXCR4-mediated activation of AKT, ERK, NFκB and Rho-A using western blot analysis; abrogated the CXCL12-mediated proliferation of MM cells using the CFSE assay; and induced apoptosis in MM cell as determined by PI/annexin V double staining followed by flow cytometry analysis. Monitoring the expression of B-cell CCL/Lymphoma 2 (Bcl-2) family members and their role in apoptosis induction after treatment with WEV or WEV+NP revealed that the combination of WEV with NP robustly decreased the expression of the anti-apoptotic effectors Bcl-2, BclXL and Mcl-1; conversely increased the expression of the pro-apoptotic effectors Bak, Bax and Bim; and altered the mitochondrial membrane potential in MM cells. Taken together, our data reveal the biological effects of WEV and WEV+NP and the underlying mechanisms against myeloma cancer cells.

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Abstract 780 Background:The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia.The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results:Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner.To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05).We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions:The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures:Chevalier:Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors invitro and their recovery in transplanted recipients invivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Interplay between signaling via the formyl peptide receptor (FPR) and chemokine receptor 3 (CCR3) in human eosinophils

Eosinophils express the chemoattractant receptors CCR3 and FPR. CCR3 binds several agonists such as eotaxin-1, -2, and -3 and RANTES, whereas the FPR binds the formylated tripeptide fMLP and a host of other ligands. The aim of this study was to investigate if there is interplay between these two receptors regarding the elicitation of migration and respiratory burst in human blood-derived eosinophils. Inhibition of the FPR with the antagonists CyH and boc-MLP abrogated the migration of eosinophils toward all of the CCR3 agonists. Similar results were seen when the FPR was desensitized with its cognate ligand, fMLP. In contrast, the respiratory burst triggered by eotaxin-1 was not inhibited by CyH. Thus, signals evoked via the FPR caused unidirectional down-regulation of CCR3-mediated chemotaxis but not respiratory burst in human eosinophils. The underlying mechanism was neither reduced ability of the CCR3 ligand eotaxin-1 to bind to CCR3 nor down-regulation of CCR3 from the cell surface. Finally, confocal microscopy and adFRET analysis ruled out homo- or heterodimer formation between FPR and/or CCR3 as an explanation for the reduction in chemotaxis via CCR3. Pharmacologic inhibition of signal transduction molecules showed that the release of free oxygen radicals in response to eotaxin-1 compared with fMLP is relatively more dependent on the p38 MAPK pathway.

Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells

The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

Targeted Deletion of Integrin-Linked Kinase Reveals a Role in T-Cell Chemotaxis and Survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.

Site-Directed Perturbation of Protein Kinase C- Integrin Interaction Blocks Carcinoma Cell Chemotaxis

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.

IL-2 and IL-12 Alter NK Cell Responsiveness to IFN-γ-Inducible Protein 10 by Down-Regulating CXCR3 Expression

Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-gamma-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression.