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Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors

Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal–related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12–16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

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Neutrophil Chemotactic Activity of Sputum From Patients With COPD

Neutrophilic inflammation is a major feature of COPD.  Several factors in bronchial secretions have been identified as chemoattractants for neutrophils. The present study was designed to assess the contribution of interleukin (IL)-8 and leukotriene B4 (LTB4) to neutrophil chemotaxis evoked by sputum obtained from patients with established COPD.
Design: Sputum supernatant of 20 patients with COPD was used as chemoattractant in a 96-well
chemotaxis chamber, with subsequent quantification of migrated cells by a luminescence assay.
The contribution of IL-8 and LTB4 to chemotaxis was determined by addition of a neutralizing
antibody and a selective receptor antagonist, respectively.
Measurements and results: COPD sputum caused neutrophil chemotaxis in a concentration dependent manner, with a maximum response evoked with a 10-fold dilution of the original
sample. Pretreatment of sputum or neutrophils with either an anti–IL-8 antibody or the LTB4
antagonist, SB 201146, led to a concentration-dependent inhibition of sputum-induced neutrophil chemotaxis, with a maximum suppression (mean  SEM) of 29.2  4.9% (p < 0.001) from baseline by 100 ng/mL of anti–IL-8 antibody, and 45.6  7% (p < 0.02) by 10 mol/L of SB
201146. The combination of the anti–IL-8 antibody and SB 201146 inhibited neutrophil
chemotaxis, but this was not significantly greater than the effect of SB 201146 or anti–IL-8 alone.
Conclusions: These data confirm the importance of IL-8 and LTB4 as chemoattractants for
neutrophils in bronchial secretions from patients with COPD, and suggest that specific inhibitors
may have therapeutic potential in COPD.

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A rapid, multiwell colorimetric assay for chemotaxis

This paper describes a colorimetric assay for the rapid quantification of chemotaxis in multiple samples. In this assay, cells that have migrated through polycarbonate membrane filters are collected onto the bottom wells of a chemotaxis chamber after centrifugation then the number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT). The degree of MTT reduction, which corresponds to the relative cell number, is measured automatically with an ELISA reader. The MTT method of quantitation is as sensitive as the standard manual method, is especially useful for large numbers of samples and requires no specialized laboratory equipment.

Shi Y, Kornovski BS, Savani R, Turley EA.

Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.

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