Category: Knowledgebase

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Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028771

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Small Molecule Disruption of G Protein βγ Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

G protein βγ subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) γ catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gβγ-dependent signaling, including Gβγ-dependent activation of PI3-kinase γ and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gβγ inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gβγ-regulation may be an effective anti-inflammation strategy.

http://molpharm.aspetjournals.org/content/73/2/410.full

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Tumorigenicity of Human Breast Cancer Is Associated with Loss of the Ca21- activated Chloride Channel CLCA2

The human Ca21-activated chloride channel-2 (CLCA2) is expressed in
normal breast epithelium but not in breast tumors of different stages of
progression. Northern analysis of nontransformed and transformed
breast epithelial cell lines revealed CLCA2 expression in the nontransformed
cell line MCF10A and the nontumorigenic cell line MDA-MB-453,
whereas all tumorigenic cell lines were negative (MDA-MB-231, MDAMB-
435, MDA-MB-468, and MCF7). When stably reintroduced into
CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression
reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic
tumors of MDA-MB-231 cells in nude mice. Our results suggest that
CLCA2 may act as a tumor suppressor in breast cancer.

cancerres.aacrjournals.org/content/59/21/5488.full.pdf

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A Rapid in Vitro Assay for Quantitating the Invasive Potential of Tumor Cells

We have reconstituted a matrix of basement membrane onto a filter in
a Boyden chamber and assessed the ability of various malignant and
nonmalignant cells to penetrate through the coated filter. Cells from all
the malignant cell lines tested were able to cross the matrix in 5-6 h,
whereas human fibroblasts as well as mouse 3T3 and lOT’/i cell lines,
which are not tumorigenic, were not invasive. In addition, normal primary
prostate epithelial cells and benign prostatic hyperplasia cells were not
invasive when tested in this assay, whereas malignant prostate carcinoma
cells were highly invasive. Parallel experiments with these prostatic cells
using the intrasplenic assay for metastasis detection in the nude mouse
confirmed the benign behavior of the former cells and the metastatic
phenotype of the latter ones. These results suggest that this in vitro test
allows the rapid and quantitative assessment of invasiveness and a means
to screen for drugs which alter the invasive phenotype of tumor cells.

.pdf downloadable at http://www.ncbi.nlm.nih.gov/pubmed/2438036

Note: do not autoclave acrylic chambers as this article recommends.

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Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis–cell migration chamber. 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), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner
histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAOaffected
horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P 5 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAO affected horses.

http://vdi.sagepub.com/content/18/3/257.abstract (link to pdf)

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Insulin-like Growth Factor (IGF)-binding Protein-4 Inhibits Colony Formation of Colorectal Cancer Cells by IGF-independent Mechanisms

Effects of insulin-like growth factor-binding protein-4 (IGFBP-4) on proliferation, colony formation, and cell migration were assessed in IGF-sensitive and -insensitive colorectal cancer cell lines. In IGF-insensitive Isreco-1 cells, overexpression of IGFBP-4 reduced colony formation but not cell proliferation and migration, whereas exogenous IGF-II had no effect. In IGF-dependent LS1034 cells, IGFBP-4 inhibited all parameters of growth tested, whereas IGF-II partially restored reduced proliferation and cell migration only. In Isreco-2 cells, which lack endogenous IGF expression but are IGF sensitive, colony formation was also reduced by IGFBP-4. Therefore, specific parameters of malignant progression of colon carcinoma cells are distinctly affected by IGF-dependent and IGF-independent effects of IGFBP-4.

http://cancerres.aacrjournals.org/content/64/5/1600.full

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CD28 Signaling in Neutrophil Induces T-Cell Chemotactic Factor(s) Modulating T-Cell Response

ABSTRACT: We previously reported that human peripheral
blood neutrophils express CD28 and interact
with macrophage B7 to generate CD28 signaling through
PI-3 kinase. Here, we demonstrate that crosslinking of
CD28 on neutrophils results in the release of IFN-,
which restricts amastigote growth and modulates CD4
T cells cytokine secretion. CD28 crosslinking also induces
a T-cell chemotactic factor (TCF) that induces chemotactic
migration of CD4 T cells. Based on our previous and
the current set of data, we propose an operational model
explaining how neutrophils are involved in Leishmania
infection and how the reported effect of neutrophils on the
control of infection is mediated by alteration of T-cell
function. Human Immunology 64, 38–43 (2003). ©
American Society for Histocompatibility and Immunogenetics,
2003. Published by Elsevier Science Inc.

www.chemotaxis.sote.hu/…/Venuprasad-K-HumImmunol-2003.pdf

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Tumorigenicity of Human Breast Cancer Is Associated with Loss of the Ca21- activated Chloride Channel CLCA2

The human Ca21-activated chloride channel-2 (CLCA2) is expressed in
normal breast epithelium but not in breast tumors of different stages of
progression. Northern analysis of nontransformed and transformed
breast epithelial cell lines revealed CLCA2 expression in the nontransformed
cell line MCF10A and the nontumorigenic cell line MDA-MB-453,
whereas all tumorigenic cell lines were negative (MDA-MB-231, MDAMB-
435, MDA-MB-468, and MCF7). When stably reintroduced into
CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression
reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic
tumors of MDA-MB-231 cells in nude mice. Our results suggest that
CLCA2 may act as a tumor suppressor in breast cancer.

cancerres.aacrjournals.org/content/59/21/5488.full.pdf

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Granulocyte Chemotactic Activity in Exhaled Breath Condensate of Healthy Subjects and Patients With COPD

Several chemoattractants have been measured in exhaled breath condensate (EBC) from
patients with COPD. The aim of this study was to compare the eosinophil and neutrophil chemotactic activity contained in EBC from healthy subjects and patients with COPD.
Methods: EBC collected using a commercially available condenser (EcoScreen; Erich Jaeger Viasys; Hoechberg, Germany) was compared in 45 COPD patients and 65 healthy subjects. EBC chemotactic activity for eosinophils and neutrophils was assessed using microchambers (Boyden; Neuro Probe; Gaithersburg, MD). Chemotactic index (CI) was used to evaluate cell migration.
Results: EBC from patients with COPD (CI, 2.210.16 [meanSEM]) and healthy subjects (CI, 1.670.11) displayed significant neutrophil chemotactic activity (p<0.0001 for both), which was however higher in patients with COPD (p<0.001). Healthy smokers had a significantly raised CI for neutrophils by comparison with healthy nonsmokers (p<0.01) and ex-smokers (p<0.05). Likewise, current COPD smokers tended to have greater neutrophil CI than COPD who stopped smoking (p0.08). COPD ex-smokers had raised chemotactic activity by comparison with healthy ex-smokers (p<0.05). Anti–interleukin-8 (106 g/mL) antibodies reduced neutrophil chemotactic activity by 35.2% (p<0.05). EBC also contained significant eosinophil chemotactic activity in healthy subjects (CI, 1.680.09; p<0.0001) and patients with COPD (CI, 1.230.07; p<0.01), with a significantly lower CI in patients with COPD as compared to healthy subjects (p<0.001). Smoking did not influence eosinophil
chemotactic activity in healthy subjects or patients with COPD.

http://chestjournal.chestpubs.org/content/131/6/1672.full.html

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Small Molecule Disruption of G Protein βγ Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

G protein βγ subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) γ catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gβγ-dependent signaling, including Gβγ-dependent activation of PI3-kinase γ and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gβγ inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gβγ-regulation may be an effective anti-inflammation strategy.

http://molpharm.aspetjournals.org/content/73/2/410.full