[show abstract][hide abstract] ABSTRACT: To determine whether there is an association between intracellular cytokine profiles and the expression of surface antigens, we performed a simultaneous flow cytometric analysis of these laboratory parameters in 11 healthy volunteers. Peripheral blood lymphocytes were double-stained for CD4 or CD8, as well as CD11a, CD25, CD26, CD29 and CD45RA or the chemokine receptors CCR3, CCR4, CCR5 or CXCR3. Portions of the cell samples were cultured for 4 h in the presence of 1 microm monensin and 20 microg/ml brefeldin A with or without stimulation by phorbol myristate acetate plus ionomycin for the detection of intracellular interferon-gamma (IFN-gamma), interleukin-2 (IL-2), tumour necrosis factor (TNF)-alpha, and IL-4. As a result, CD4+CD29high helper inducer T cells were closely associated with IFN-gamma and TNF-alpha producing CD4+ cells, while CD4+CXCR3+ cells showed a negative correlation with IL-4-producing cells, suggesting that both of these CD4+ subsets consist mainly of Th1 cells. In contrast, CD4+CD45RA+ cells were correlated inversely with IFN-gamma and TNF-alpha-producing cells, and CD8+CD11ahigh killer effector and total CCR5+ cells showed an inverse correlation with IL-2 producing cells, suggesting an immunoregulatory role for these three subsets in non-pathological conditions. Therefore, monitoring of lymphocyte subsets that express functional surface antigens could provide additional information concerning immune deviation, as assessed by the production of Th1/Th2 type cytokines. Further, this type of combined study may provide clues for the pathogenesis of immune-mediated disorders.
[show abstract][hide abstract] ABSTRACT: Th2 cells are thought to be involved in eosinophilic inflammation of the lung. CC chemokine receptor 4 (CCR4) has been identified as a specific receptor for both thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), and is preferentially expressed on Th2 cells.
Our aim was to evaluate the role of Th2 cells in the lung of patients with eosinophilic pneumonia (EP).
The concentrations of TARC, MDC, and interleukin (IL)-5 were measured in bronchoalveolar lavage fluid (BALF) by ELISA. Proportion of CCR4-expressing CD4+ T cells (CCR4+ CD4+ T cells) was determined by flow cytometry.
TARC and MDC concentrations in BALF were higher in patients with EP than in normal subjects. The proportion of CCR4-expressing cells among CD4+ T cells was higher in BALF than in peripheral blood of patients with EP. There was a significant correlation between the number of CCR4+ CD4+ T cells and the levels of TARC, MDC, and IL-5 in BALF of patients with EP.
Our data suggest that Th2 cells, which express CCR4 and its ligands (TARC and MDC), contribute to the pathogenesis of EP in the lung.
[show abstract][hide abstract] ABSTRACT: Several chemokine receptors are expressed selectively on the surface of T cells depending on their polarization. The aim of this study was to characterize chemokine receptor expression in peripheral blood memory T cells in Crohn's disease (CD) and ulcerative colitis (UC), and to correlate the expression with disease activity. Peripheral blood mononuclear cells (PBMCs) were obtained from 24 patients with CD, 30 patients with UC, 24 normal controls and 10 disease controls. PBMCs were stained by anti-CCR3, CCR4, CCR5, CXCR3, CD4, CD8, CD45RO and beta 7 integrin, and the expression of the chemokine receptors were determined by flow cytometry. CCR4 expression on memory T cells was significantly lower in UC than in CD or normal controls, and that of memory CD4+ T and beta 7(high) memory CD4+ T cells was significantly higher in CD than in UC or normal controls. CCR4 expression on memory CD4+ T cells exhibited significant positive correlation with disease activity in CD, and this decreased significantly after treatment. Such a decrease was not found in the disease controls. CCR5 and CXCR3 expression on memory CD8+ T cells was significantly lower in CD than in normal controls. CXCR3 expression on beta 7(high) memory CD4+ T and CXCR3 expression on memory CD8+ T cells were lower in UC than in normal controls. These findings suggest that in peripheral blood memory T cells, chemokine receptor expression is different between CD and UC. Enhancement of CCR4 and suppression of CCR5 and CXCR3 seem to be the characteristic chemokine receptor profile in peripheral blood memory T cells of CD.
[show abstract][hide abstract] ABSTRACT: Th2 and Th1 cells have been suggested to express CCR3/CCR4 and CCR5/CXCR3, respectively.
We examined CCR3, CCR4, CCR5 and CXCR3 expression and cytokine production in peripheral blood CD4+ T cells from patients with atopic dermatitis (AD), which has been postulated to be a Th2-type cell-mediated disease, and then analysed the possible correlation between these values and the levels of several clinical parameters.
Intracellular cytokine production and chemokine receptor expression in peripheral blood CD4+ T cells from 40 AD patients and 20 sex- and age-matched healthy control subjects were studied by flow cytometry.
The frequencies of IL-4- and IL-13-producing CD4+ T cells from patients with AD were significantly higher than those from healthy control subjects (IL-4:3.9 +/- 2.1% vs. 1.6 +/- 0.7%, P = 0.0005, IL-13:4.0 +/- 2.1% vs. 1.8 +/- 0.8%, P = 0.0023), whereas the frequencies of IL-2- and IFN-gamma-producing CD4+ T cells were significantly decreased in AD patients (IL-2:38.1 +/- 10.3% vs. 51.3 +/- 6.3%, P = 0.0003, IFN-gamma: 9.9 +/- 3.5% vs. 26.4 +/- 4.6%, P < 0.0001). The percentage of CCR4+ cells in CD4+ CD45RO+ T cells in AD patients was significantly higher than that in healthy control subjects (24.4 +/- 8.0% vs. 10.9 +/- 2.3%, P < 0.0001) and was correlated positively with the total serum IgE, serum lactic dehydrogenase (LDH) level, eosinophil number, eruption score, and IL-4 and IL-13 secretion in CD4+ T cells, and inversely with IL-2 and IFN-gamma secretion in CD4+ T cells. In contrast, CCR3 was not detected on circulating CD4+ T cells even in AD patients. On the other hand, the percentage of CCR5+ or CXCR3+ cells in CD4+ CD45RO+ T cells in AD patients was significantly decreased (CCR5:23.2 +/- 7.0% vs. 28.4 +/- 5.4%, P = 0.023, CXCR3:29.9 +/- 11.4% vs. 38.5 +/- 6.7%, P = 0.028) and was positively correlated with eruption score (P < 0.05). Multiple regression analyses showed that the percentage of CCR4 expression highly correlated with serum IgE, LDH, eosinophil number and eruption in AD patients.
CCR4+ cells might be involved in the aetiopathogenesis of AD.
[show abstract][hide abstract] ABSTRACT: Corticosteroids are widely used in bronchial asthma, but their mechanism of action is not fully understood. The in vitro studies have proposed that human T helper cells, type 1 (Th1) favor expression of CXCR3, whereas Th2 cells favor CCR4. In this study we investigated whether oral prednisolone modulates the balance of peripheral blood CXCR3+ and CCR4+ T cells. We analyzed the T-cell subsets in 28 patients with stable atopic asthma and 13 normal control subjects before and after 2 wk of treatment with prednisolone, 20 mg/d, or placebo in a randomized, double-blind, parallel group study. The numbers of CXCR3+ and CCR4+ memory T cells were measured with a flow cytometer, and expressed as percentages in CD4+/CD45RO+ memory T cells. In the steroid-treated asthma group, there was a decrease in CCR4+ T cells (from 29.3% to 20.3%, p < 0.0001), and an increase in CXCR3+/ CCR4+ ratio (from 1.86 to 2.89, p = 0.0047), whereas there was no change in CXCR3+ T cells. However, the percentages of CCR4+ cells did not change after steroid therapy in normal control subjects. These results suggest that short-term oral corticosteroid modulates the balances of CXCR3+ and CCR4+ cells in patients with asthma.
American Journal of Respiratory and Critical Care Medicine 10/2001; 164(5):754-8. · 11.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies indicate that Th1 and Th2 cells differ in their chemokine receptor expression and their responsiveness to various chemokines. Therefore, selective Th2 cell recruitment in Th2-predominant inflammatory diseases such as atopic dermatitis may be under the influence of some chemokines. It is reported that CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells whereas CXC chemokine receptor (CXCR) 3 is selectively expressed on Th1 cells. In this study we examined CCR4 and CXCR3 expression on peripheral blood CD4+ and CD8+ T cells obtained from adult atopic dermatitis subjects, and compared the results with those from patients with psoriasis vulgaris and healthy controls. CCR4 was preferentially expressed on CD4+ T cells from atopic dermatitis subjects and CXCR3 was preferentially expressed on CD4+ T cells from psoriasis vulgaris subjects. This CCR4 expression was prominent especially in severe atopic dermatitis subjects. CCR4 expression on CD4+ T cells in severe atopic dermatitis subjects decreased on improvement of disease activity. CD25 was preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. Cutaneous lymphocyte-associated antigen was also preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. CD4+ T cells in atopic dermatitis skin lesions were predominantly CCR4+ cells. Taken together, this study strongly indicates that CCR4+CD4+ T cells reflect disease activity and suggests that CCR4 expression is important for T cell infiltration into atopic dermatitis lesions. Thus, CCR4 may be a possible target for therapy of atopic dermatitis in the future.
Journal of Investigative Dermatology 09/2001; 117(2):188-96. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: We observed here that the expression of B lymphocyte chemokine (BLC/CXCL13) was markedly enhanced in the thymus and kidney in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, but not in similarly aged NZB and NZW mice. BLC-positive cells were present in the cellular infiltrates in the target organs with a reticular pattern of staining. CD11b+CD11c+ dendritic cells were increased in the thymus and spleen in aged BWF1 mice and identified as the major cell source for BLC. CD4+ T cells as well as B cells were dramatically increased in the thymus in aged BWF1 mice, whereas no increase was observed in aged NZB and NZW mice. B1/B2 ratio in the thymus was significantly higher than those in the spleen and peripheral blood in aged BWF1 mice. Interestingly, BLC showed preferential chemotactic activity for B1 cells derived from several mouse strains, including nonautoimmune mice. Cell surface CXCR5 expression on B1 cells was significantly higher than that on B2 cells. Thus, aberrant high expression of BLC by myeloid dendritic cells in the target organs in aged BWF1 mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B cells in the development of murine lupus.
Journal of Experimental Medicine 07/2001; 193(12):1393-402. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The immunosuppressive and anti-inflammatory cytokine IL-10 inhibits the phenotypic and functional maturation of dendritic cells (DC) and has been reported to confer tolerogenic properties on these important professional APC. Here, we exposed murine bone marrow-derived myeloid DC to either mouse (m) or viral (v) IL-10 early during their in vitro generation in response to GM-CSF and IL-4. Both mIL-10 and vIL-10 down-regulated the expression of CCR7 mRNA determined by RT-PCR, while mIL-10 up-regulated the expression of CCR5 transcripts. These changes in CCR7 and CCR5 expression were associated with inhibition and augmentation, respectively, of DC chemotaxis toward their respective agonists, macrophage inflammatory proteins 3beta and 1alpha, while in vivo homing of DC from peripheral s.c. sites to secondary lymphoid tissue of syngeneic or allogeneic recipients was significantly impaired. Anti-mIL-10R mAb reversed the effects of mIL-10 on CCR expression and restored DC homing ability. Retroviral transduction of mIL-10- and vIL-10-treated DC to overexpress transgenic CCR7 partially restored the cells' lymphoid tissue homing ability in allogeneic recipients. However, CCR7 gene transfer did not reinstate the capacity of IL-10-treated DC to prime host naive T cells for ex vivo proliferative responses or Th1 cytokine (IFN-gamma) production in response to rechallenge with (donor) alloantigen. These findings suggest that in addition to their capacity to subvert DC maturation/function and confer tolerogenic potential on these cells, mIL-10 and vIL-10 regulate DC migratory responses via modulation of CCR expression.
The Journal of Immunology 07/2001; 166(12):7136-43. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of TH2-type cells in lesional skin. Thymus and activation-regulated chemokine (TARC/CCL17) is a chemokine that attracts CC chemokine receptor 4-positive (CCR4+) or CCR8+ cells.
The purpose of this study was to investigate the participation of TARC in AD.
We measured serum TARC levels in 40 patients with AD, 20 healthy control subjects, and 20 patients with psoriasis. We also examined disease activity by using SCORAD score; serum soluble E-selectin, soluble IL-2 receptor, IgE, and GM-CSF levels; and eosinophil numbers in peripheral blood, as well as correlations between TARC levels and these factors. The positivity of CCR4 of CD4+CD45RO+ cells in PBMCs was examined by using FACS analysis. Immunohistochemical staining of TARC and GM-CSF was performed in the lesional skin of patients with AD.
The serum TARC levels of patients with AD were significantly higher than those of healthy control subjects and patients with psoriasis. The serum TARC levels significantly correlated with eosinophil number (r = 0.61), SCORAD score (r = 0.60), and serum soluble E-selectin levels (r = 0.58) and weakly correlated with serum soluble IL-2 receptor levels (r = 0.34) in patients with AD. The TARC levels of patients with AD decreased after the treatment in accordance with the improvement of clinical symptoms. The CCR4 positivity of CD4+CD45RO+ cells in PBMCs of patients with AD was also higher than that of healthy control subjects. Immunohistochemical staining revealed that TARC was positive in keratinocytes in the epidermis and in vascular endothelial cells, T cells, and dendritic cells in the dermis.
Serum TARC levels are associated with disease activity of AD, and TARC may play an important role in the pathogenesis of AD.
Journal of Allergy and Clinical Immunology 04/2001; 107(3):535-41. · 12.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both SDF-1 and CXCR4 disruption are lethal to mice at the embryonic stage and cause abnormalities in B lymphopoiesis, myelopoiesis, cardiogenesis, vasculogenesis, and cerebellar development. To investigate the role of SDF-1 and CXCR4 in hematopoiesis during the adult stage, mice reconstituted with bone marrow-derived hematopoietic progenitor cells transduced with either the SDF-1 or a genetically modified SDF-1-intrakine gene using a retroviral expression vector were analyzed. Flow cytometric (FCM) analysis showed a dramatic reduction of CXCR4 expression on the cells of intrakine-transduced mice, whereas CCR7 and CCR1 expression was unchanged or marginally decreased on splenocytes. Migration of splenocytes and bone marrow cells to SDF-1 was markedly suppressed in intrakine-transduced mice. FCM analysis of bone marrow cells of intrakine-transduced mice exhibited decreased numbers of pro-B (B220(+) CD43(+)), pre-B (B220(+) CD43(-)), and immature B (B220(+) IgM(+)) cells and a decreased number of granulocytes/myeloid (Gr1(+) CD11b(+)) cells. Impaired B lymphopoiesis and myelopoiesis in intrakine-transduced mice were confirmed by an in vitro colony-forming assay of bone marrow cells. In contrast, B lymphopoiesis and myelopoiesis were enhanced in SDF-1-transduced mice. Interestingly, T-cell maturation in the thymus was impaired both in intrakine- and SDF-1-transduced mice, suggesting that SDF-1 and CXCR4 play an important role in T lymphopoiesis as well as in B lymphopoiesis and myelopoiesis in adults. These results demonstrate an essential role of CXCR4 and its ligand SDF-1 in adult hematopoiesis, and they indicate the intrakine method as a powerful tool for functional analysis of chemokines/chemokine receptors in vivo and as a potential therapeutic approach for acquired immunodeficiency syndrome.
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DCs) are very potent antigen-presenting cells and play critical roles in regulating immune responses in cancer. The migrating of DCs from the tumor site to the lymphoid organs is believed to be one of the critical events. To examine this important DC function in tumor situations, bone marrow-derived DCs, cultured for 6 days with granulocyte macrophage colony-stimulating factor and interleukin 4, were inoculated at the tumor site. We have shown (Y. Nishioka et al., Cancer Res., 59: 40354041, 1999) that DCs can migrate from tumor site to the draining lymph nodes within 24 h (approximately 0.1% of administrated DCs). The DCs then form clusters with adjacent lymphoid cells, which produce IFN-gamma (1500-3200pg/10(6) cells/48 h) in response to tumor stimulation. The number of the DCs migrating into lymph nodes were greater when they were inoculated into the tumor rather than the skin. Coculture of DCs and apoptotic tumor cells resulted in decreased expression of CC chemokine receptor (CCR) 1 and increased CCR7 expression at mRNA level without alteration in other phenotypical markers on DCs. Chemotaxis assay showed that CCR7 ligands, macrophage inflammatory protein 3beta and secondary lymphoid-tissue chemokine significantly (P < 0.05) induced the migration of DCs when cocultured with apoptotic tumor cells. To directly examine the involvement of CCR7 expression in DC migration, we investigated the functions of DCs genetically modified to express high levels of CCR7. CCR7 transduction promotes DC migration in response to relevant ligands in vitro and in vivo. These results suggest that the CCR7 expression of DCs is enhanced with direct contact with apoptotic tumor cells and may have a critical role for DC migrating to regional lymph nodes. The means to promote DC delivery to tumor and to nodal sites represent novel targets for the biological therapy of cancer.
Cancer Research 05/2000; 60(8):2209-17. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have investigated the involvement of chemokine receptor CCR1-positive cells in bleomycin-induced lung injury, a model of pulmonary fibrosis. After bleomycin challenge in C57BL/6J mice, the expression of CCR1 mRNA increased and peaked at day 7, which paralleled to the expression of its ligands, macrophage-inflammatory protein-1 alpha and RANTES. Immunohistochemical study showed that CCR1-positive cells accumulated in the interstitial inflammatory site. Furthermore, the treatment of anti-CCR1 Ab significantly reduced the accumulation of inflammatory cells and collagen deposition, resulting in dramatic improvement of survival. These results suggest that CCR1-positive cells play significant roles in the pathogenesis of pulmonary fibrosis subsequent to bleomycin-induced lung injury, and that CCR1 could be a novel molecular target for intervention therapy against pulmonary fibrosis.
The Journal of Immunology 04/2000; 164(5):2745-51. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe here that lineage phenotype- negative (Lin)(-)c-kit(+) hematopoietic progenitor cells (HPCs) from day 13 postcoitus (dpc) murine fetal liver (FL) can generate dendritic cell (DC) precursors when cultured in vitro in the presence of PA6 stromal cells plus granulocyte/macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + Flt3 ligand (Flt3L) for 12 to 14 days, and develop into mature DCs when stimulated with GM-CSF plus mouse tumor necrosis factor alpha (mTNFalpha) for an additional 3 to 5 days. A transwell culture system showed that the generation of DC precursors depended on the support of PA6 cell-secreted soluble factor(s). The mature DCs derived from 13 dpc FL Lin(-)c-kit(+) HPCs showed characteristic morphology and function of DCs and expressed high levels of Ia, CD86, and CD40 molecules, low levels of DEC205, E-cadherin, and F4/80 molecules, but barely detectable CD11c antigen. Once FL-derived HPCs were cultured without GM-CSF, NK1.1(+) cells developed in the presence of PA6 cells + SCF + Flt3L. These NK1.1(+) cells could develop into DC precursors at an earlier stage of differentiation by reculturing with PA6 cells + SCF + Flt3L + GM-CSF, but they would be irreversibly committed to NK cell precursors without GM-CSF after 3 days, suggesting that GM-CSF plays a critical role in controlling the transition of DC and NK cell precursors from 13 dpc FL-derived Lin(-)c-kit(+) HPCs. This study represents the first success in generating mature DCs in vitro from murine FL HPCs. (Blood. 2000;95:138-146)
[show abstract][hide abstract] ABSTRACT: Bisbenzylisoquinoline alkaloids are known to affect immune responses as well as inflammatory responses, and have been used for the treatment of inflammatory symptoms in China. This study is aimed at elucidating the inhibitory effects of two alkaloids, fangchinoline and isotetrandrine, on the induction of the proinflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha), by Staphylococcus aureus Cowan 1 (SAC)-stimulated human peripheral blood mononuclear cells. These two alkaloids inhibited cytokine production in a dose-dependent manner, and they inhibited it by more than 90% at 10 micrograms/ml at every time point examined. Of note was that these two alkaloids appeared to inhibit IL-1 beta production more effectively than IL-1 alpha production. When the levels of cytokine mRNA were measured by semiquantitative RT-PCR, these alkaloids reduced the levels of the mRNAs of IL-1 beta and TNF-alpha, but not that of beta 2-microglobulin, suggesting that these alkaloids may suppress cytokine transcription selectively.
[show abstract][hide abstract] ABSTRACT: To understand the role of endogenous AP-1 activity in cellular transformation induced by oncogenes, we have made use of a fos mutant (supfos-1) and a jun mutant (supjun-1), either of which can function as a transdominant inhibitor of AP-1-mediated transcriptional regulation. Chicken embryo fibroblasts (CEF) infected with a series of transforming retroviruses were doubly infected with retrovirus carrying supfos-1 or supjun-1, and suppression of cellular transformation was monitored in terms of reversion to normal cellular morphology or acquisition of anchorage-dependent growth. Cellular transformation induced by several exogenously expressed transforming genes of the fos or jun family was efficiently suppressed, as expected. CEF transformed by v-src, v-yes, v-fps, c-Ha-ras, and N-terminally truncated c-raf were also induced to revert to the normal phenotype by these transdominant mutants, suggesting that functional transcription factor AP-1 activity is essential for the cellular transformation induced by these oncogenes. The suppression is not attributable to nonspecific inhibition of cellular proliferation, because CEF transformed by v-ros or v-myc were not induced to revert to the normal phenotype. We next analyzed changes in all known components of chicken AP-1 induced by v-src, c-Ha-ras, or activated c-raf transformation. The levels of both Fra-2 and c-Jun expression were elevated two- to fourfold, and hyperphosphorylation of Fra-2 was also observed. We further showed that Fra-2-c-Jun heterodimer is mainly responsible for the elevated AP-1 DNA-binding activity in these transformed cells, and we propose that this heterodimer play a crucial role in the transformation induced by these oncogenes.
Journal of Virology 07/1994; 68(6):3527-35. · 5.08 Impact Factor