PURPOSE. The purpose of this study was to examine expression of platelet-derived growth factor (PDGF) and PDGF receptors in the human cornea and to study the effects of the PDGF isotypes on proliferation and chemotaxis of human corneal fibroblasts. The effects of interleukin (IL)-lai, bone morphogenic protein (BMP)2, and BMP4 on chemotaxis of human corneal fibroblasts were also studied.
Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.
ABSTRACT: Pulmonary alveolar proteinosis (PAP) is a rare disease of unknown
aetiology characterized by accumulations of lipoproteinaceous material within the
alveoli. The alveolar macrophages become increasingly foamy, and are thought to
have a role in the pathogenesis of PAP. However, the mechanisms of macrophage
recruitment are unclear.
In the bronchoalveolar lavage fluid (BALF) of four patients with PAP and 20
normal control subjects, the following were examined: the monocyte chemotactic
activity due to the chemokine monocyte chemoattractant protein (MCP)-1 with the
use of a chemotactic chamber assay, the levels of MCP-1 by enzyme-linked immunosorbent
assay, and the MCP-1 expression on lavage cells by immunocytochemistry
and in situ hybridization.
The monocyte chemotactic activity in the BALF of the PAP patients was markedly
elevated, and the activity was completely absorbed by treatment with anti-MCP-1.
The MCP-1 levels in the BALF were surprisingly high in the PAP group (25,100472
pg.mL-1), whereas low levels of MCP-1 were detected in the normal control subjects
(mean: never smokers 4.8; smokers 10.4 pg.mL-1). MCP-1 protein and messenger
ribonucleic acid were expressed by macrophages from the PAP patients, and the
expression was reduced according to foaming of the cells; there were monocyte-like
macrophages with strong expression, small foamy cells with moderate expression,
large foamy cells with a faint expression of MCP-1, and ghost cells with no expression.
However, the increase of macrophage number in the PAP BALF was relatively small.
These data suggest that monocyte chemoattractant protein-1 expression by alveolar
macrophages represents an amplification mechanism for the recruitment of additional
macrophages to the alveoli in pulmonary alveolar proteinosis. It is possible that an
ingestion of an excess of alveolar materials in pulmonary alveolar proteinosis may
impair the macrophage function and the survival, resulting in the lack of a prominent
increase in the macrophage number in bronchoalveolar lavage fluid.
Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration.
Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 ± 88% (mean ± SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca2+] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5–10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.
Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of
interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80%
The purpose of a chemotaxis assay is to determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is the ability of a protein to direct the migration of a specific cell. This assay is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. If the agent is not chemotactic for the cell, then the majority of the cells will remain on the membrane. If the agent is chemotactic, then the cells will migrate through the membrane and settle on the bottom of the well of the chemotaxis plate.
We hypothesized that the priming and activation of maternal leukocytes in peripheral blood is a key component of parturition, and that inappropriate preterm priming of leukocytes might initiate preterm labour and delivery. The purpose of this study was to characterize peripheral blood leukocyte activation during human term and preterm labour. We obtained blood samples from pregnant women at term and preterm, both in labour and not in labour. Leukocytes were characterized according to cell subtype and cell surface marker expression. Additionally, we quantified leukocyte cytokine mRNA production, migratory ability and reactive oxygen species production of neutrophils and macrophages. We found that both term and preterm labour were associated with an increase in monocyte and neutrophil proportion or number—neutrophil migratory ability and cell surface marker expression indicating activation. Messenger RNA expression of IL-1β and IL-8, MCP-1 and TLR-2 was also increased. We conclude that leukocytes in peripheral blood are primed in preparation for activation during term and preterm labour, and that this may contribute to the pathophysiological events of parturition. These data may lead to novel therapies and diagnostic tools for the prevention and/or diagnosis of preterm birth.
Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) β-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) α as the critical signaling component that regulates the sorting of the PDGF β-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCα, using myristoylated inhibitory peptides or by knockdown of PKCα with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF β-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF β-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF β-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF β-receptors on early endosomes is regulated by sequential activation of PKCα and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.
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.
Full text available by subscription at: http://www.sciencedirect.com/science/article/pii/002217599390307S