Significant elevation of serum levels of eotaxin-3/CCL26, but not of eotaxin-2/CCL24, in patients with atopic dermatitis: serum eotaxin-3/CCL26 levels reflect the disease activity of atopic dermatitis

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of T cells, eosinophils and macrophages in lesional skin. Recently, eotaxin-2/CCL24 and eotaxin-3/CCL26 were identified as CC chemokines that signal exclusively via the CCR3 receptor and have eosinophil-selective chemoattractant activity, as does eotaxin/CCL11. We previously reported that serum levels of thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 were correlated with the severity of AD. In this report, we investigated the participation of eotaxin-2/CCL24 and eotaxin-3/CCL26 in AD, first measuring the serum levels of eotaxin-2/CCL24 and eotaxin-3/CCL26 in 30 patients with AD, 20 patients with psoriasis vulgaris and 20 healthy controls. The serum levels of eotaxin-3/CCL26 (but not eotaxin-2/CCL24) were significantly higher in patients with AD than in either healthy controls or patients with psoriasis vulgaris; furthermore, the eotaxin-3/CCL26 levels in patients with moderate and severe AD were significantly higher than eotaxin-3/CCL26 levels in patients with mild AD. The serum eotaxin-3/CCL26 levels tended to decrease after treatment, but there was no significant difference between groups. Moreover, the serum eotaxin-3/CCL26 levels were significantly correlated with the serum TARC/CCL17 and MDC/CCL22 levels, eosinophil numbers in peripheral blood and the scoring AD (SCORAD) index. Our study strongly suggests that serum levels of eotaxin-3/CCL26, but not of eotaxin-2/CCL24, have a notable correlation with disease activity of AD and that eotaxin-3/CCL26, as well as TARC/CCL17 and MDC/CCL22, may be involved in the pathogenesis of AD.     

Human β defensin‐3 induces chemokines from monocytes and macrophages: diminished activity in cells from HIV‐infected persons

Human β defensin‐3 (hBD‐3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD‐3 and, for comparison, Pam3CSK4 and LL‐37 to induce co‐stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including monocyte chemoattractant protein 1 (MCP‐1), Gro‐α, macrophage‐derived chemokine (MDC) and macrophage inflammatory protein 1β (MIP1β), while only hBD‐3 and Pam3CSK4 significantly induced the angiogenesis factor, vascular endothelial growth factor (VEGF). Human BD‐3 induced similar chemokines in monocyte‐derived macrophages and additionally induced expression of Regulated upon activation normal T‐cell expressed and presumably secreted (RANTES) in these cells. Comparison of monocytes from HIV⁺ and HIV^– donors indicated that monocytes from HIV⁺ donors were more likely to spontaneously express certain chemokines (MIP‐1α, MIP‐1β and MCP‐1) and less able to increase expression of other molecules in response to hBD‐3 (MDC, Gro‐α and VEGF). Chemokine receptor expression (CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV⁺ donors compared with cells from HIV^– donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset (CD14⁺ CD16⁺⁺) of HIV⁺ donors. These observations implicate chemokine induction by hBD‐3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV‐infected persons could influence cell migration and modify the effects of hBD‐3 at sites of inflammation.     

Interleukin‐13 directly promotes oesophagus production of CCL11 and CCL24 and the migration of eosinophils

Background Eosinophilic oesophagitis (EE) is a clinico‐pathologically defined oesophageal disorder that is characterized by eosinophil migration into oesophageal tissues. There is growing support for EE being an allergic disease and for a contribution of T‐helper type 2 (Th2)‐associated cytokines in disease pathogenesis. The respiratory system has been shown to be critical in driving the development of EE in animal models. However, the mechanisms underlying the recruitment of eosinophils into the oesophagus remain unclear. Objective We sought to investigate the influence of Th2‐associated cytokines on the production of eosinophil‐specific chemokines from the oesophagus directly. Methods In order to eliminate the potential involvement of the lung, we utilized isolated oesophageal rings. These were treated in vitro with IL‐4 or IL‐13 and the expression and production of CCL11 and CCL24 were determined. Results Our data demonstrate that IL‐13 is a potent and direct inducer of both CCL11 and CCL24 production from the oesophagus, as is IL‐4 also. The expression of CCL11 precedes CCL24 by several hours but then diminishes over time, as well as at high concentrations of IL‐13. We demonstrate that there is an up‐regulation of the inhibitory IL‐13 receptor, IL‐13Rα2 but that IL‐13Rα1 remains unaltered. Oesophagus rings isolated from STAT6^?/? mice were unable to produce CCL11 or CCL24 upon IL‐13 treatment. Lastly, we demonstrate that oesophageal production of CCL11 and CCL24 upon IL‐13 stimulation is sufficient to promote eosinophil migration. Conclusions IL‐13 is capable of directly stimulating oesophageal tissue to produce eosinophil‐attracting chemokines and drive eosinophil migration. Cite this as: C. V. Neilsen and P. J. Bryce, Clinical & Experimental Allergy, 2010 (40) 427–434.     

Interleukin‐10 and interferon‐γ modulate surface expression of fractalkine‐receptor (CX3CR1) via PI3K in monocytes

The membrane‐anchored form of the chemokine fractalkine (CX₃CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX₃CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX₃CL1 can be cleaved from the cell membrane to induce chemotaxis of CX₃CR1‐expressing leucocytes. Recently, marked variations in CX₃CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX₃CR1 in monocytes during basal or inflammatory/anti‐inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX₃CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX₃CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin‐10 and interferon‐γ treatment abrogated CX₃CR1 down‐modulation, through a phosphatidylinositol 3 kinase‐dependent pathway. Most importantly, CX₃CR1 membrane expression correlated with monocyte CX₃CL1‐dependent function. Taken together, our data demonstrate that CX₃CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX₃CL1‐dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.     

Differentiation of a human monocytic cell line associated with increased production of Rift Valley fever virus by infected cells

Rift Valley fever (RVF) virus is a cause of significant human and animal disease in many parts of Africa. In some cases, it causes a hemorrhagic fever, which is frequently fatal. Prior studies have shown that RVF virus productively infects peritoneal macrophages from susceptible rat strains. The U937 human monocytic cell line was used to determine the effect of monocytic cell differentiation on the degree of viral production by cell cultures infected with RVF virus. Differentiation of U937 cells to more mature monocytic cells by phorbol ester resulted in production of 10 times more infectious virions in comparison with undifferentiated cells. These studies imply that monocytic cell differentiation increases permissiveness for RVF virus production.     

Reversal of P‐glycoprotein‐mediated multi‐drug resistance by the E3 ubiquitin ligase Cbl‐b in human gastric adenocarcinoma cells

P‐glycoprotein (P‐gp)‐mediated multi‐drug resistance (MDR) is a major barrier to the effective chemotherapy of many cancers. Recent studies have shown that inhibition of the PI3K/Akt signalling pathway can reverse P‐gp‐mediated MDR. We investigated the expression of activated Akt (p‐Akt) in 124 human gastric carcinoma tissue samples. Ubiquitous p‐Akt expression was recorded in the majority (88/124). There was a significant correlation between p‐Akt expression and the expression of P‐gp. In the adriamycin‐resistant MDR gastric carcinoma cell line SGC7901/ADR, p‐Akt expression was increased in comparison with the parental cell line SGC7901. Treatment of SGC7901/ADR cells with the PI3K inhibitor LY294002 reduced the expression of both p‐Akt and P‐gp. To explore the role of ubiquitin ligase Cbl‐b in this regulatory pathway, SGC7901/ADR cells were transfected with a plasmid overexpressing wild‐type Cbl‐b. This down‐regulated the expression of both p‐Akt and P‐gp. Furthermore, resistance to chemotherapeutic drugs was partially reversed. These results demonstrate an important role for Cbl‐b in reversing P‐gp‐mediated gastric cancer MDR through suppression of the PI3K/Akt signalling pathway and the down‐regulation of P‐gp expression. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Effect of oestradiol on PAMP-mediated CCL20/MIP-3 alpha production by mouse uterine epithelial cells in culture

The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3 alpha (CCL20/MIP-3 alpha) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3 alpha with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3 alpha increased rapidly (4 hr) in both apical and basolateral secretions. Time-course studies indicated that responses to PAMPs added to the apical surface persisted for 12-72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3 alpha. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3 alpha. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3 alpha secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3 alpha is produced in response to PAMPs. Since CCL20/MIP-3 alpha is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3 alpha production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.     

BDCA3 expression is associated with high IFN‐λ production by CD34+‐derived dendritic cells generated in the presence of GM‐CSF,IL‐4, and/or TGF‐β

High BDCA3 expression is associated with a specific human IFN‐λ‐producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34⁺ hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a⁺ preDCs, and CD14⁺ preDCs, respectively. Here, we identified BDCA3 expression in this well‐known GM‐CSF/TNF‐α‐driven culture system and described the effect of IL‐4 and/or TGF‐β on induction of BDCA3 expression. In control or TGF‐β cultures, BDCA3 was only detected on CD14⁺ preDC‐derived intDCs. IL‐4 induced BDCA3 expression in both CD14⁺‐derived and CD1a⁺‐derived cultures. TGF‐β and IL‐4 together further increased CD14⁺‐derived and CD1a⁺‐derived BDCA3⁺ DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3^highCLEC9A⁺ DC subset in vivo. Importantly, BDCA3⁺ cells, but not BDCA3^? cells, in this system produced high IFN‐λ levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN‐λ‐producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN‐λ production by DCs.     

PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.     

Enrichment of Oct3/4‐positive cells from a human bronchial epithelial cell line

Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti‐tumor drug 5‐fluorouracil (5‐FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5‐FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5‐FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and β‐catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5‐FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5‐FU‐treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5‐FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5‐FU‐treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5‐FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4‐positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5‐FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of β‐catenin. Furthermore, 5‐FU‐treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4‐positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5‐FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5‐FU and serum‐free medium as a new method for isolation of stem‐like cells from the HBE cell line.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.