Nobuyuki Onai

Pivotal Role of CCR1-Positive Leukocytes in Bleomycin- Induced Lung Fibrosis in Mice

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-1α 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.

Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K–Rac–lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K–Rac–lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

Blockade of Secondary Lymphoid Tissue Chemokine Exacerbates Propionibacterium acnes-Induced Acute Lung Inflammation

Chemokine-chemokine receptor interaction plays an essential role in leukocyte/dendritic cell (DC) trafficking in inflammation and immune responses. We investigated the pathophysiological roles of secondary lymphoid tissue chemokine (SLC; CCL21) and macrophage inflammatory protein-2 (MIP-2) in the development of acute pulmonary inflammation induced by an intratracheal injection of Propionibacterium acnes in mice. Immunohistochemical studies revealed that SLC was constitutively expressed in the peribronchial areas and perivascular lymphatics in normal mice. MIP-2-positive cells were observed in alveolar spaces in mice challenged with P. acnes. Both neutralization Abs against MIP-2 and CXC chemokine receptor 2 alleviated the P. acnes-induced pulmonary inflammation when injected before P. acnes Ag challenge. On the other hand, polyclonal anti-SLC Abs (pAbs) exacerbated the pulmonary inflammation. The numbers of mature DCs (MHC class II +, CD11c+, and CD86+) as well as macrophages and neutrophils in the P. acnes Ag-challenged lungs were increased, whereas the number of CD4+ T cells, including memory T cells, was decreased. The numbers of mature and proliferating CD4+ T cells (bromodeoxyuridine+CD4+) in regional lymph nodes were decreased in mice injected with anti-SLC pAbs compared with those in mice treated with control Abs. An in vitro proliferation assay confirmed the impairment of the Ag-specific T cell response in regional lymph nodes of mice treated with anti-SLC pAbs. These results indicate for the first time a regulatory role for SLC-recruited mature DCs in bridging an acute inflammatory response (innate immunity) and acquired immunity in the lung.

Increased circulating CD11b+CD11c+ dendritic cells (DC) in aged BWF1 mice which can be matured by TNF-α into BLC/CXCL13-producing DC

Dendritic cells (DC) play a pivotal role in regulating immune responses. We previously reported aberrant high production of B lymphocyte chemoattractant (BLC/CXCL13) by DC in aged BWF1 mice, amurine model for systemic lupus erythematosus (SLE). We describe here that CD11b+CD11c+ cells were markedly increased in the peripheral blood (PBL‐DC) in aged BWF1, but not in similarly aged NZB or NZW mice. Part of PBL‐DC showed a typical dendritic morphology and expressed MHC class II molecules, and had a weak, but significant antigen‐presenting ability in mixed lymphocytereaction. PBL‐DC were chemoattracted to several chemokines in vitro including secondary lymphoid tissue chemokine (SLC), liver and activation‐regulated chemokine (LARC), RANTES, macrophageinflammatory protein‐1α, whereas splenic mature DC from aged BWF1 mice were preferentially chemoattracted towards SLC. BLC production was induced when PBL‐DC were cultured in the presence of TNF‐α for 3u2004days. BLC expression was also induced in bone marrow‐derived DC when they were differentiated into mature DC in the presence of TNF‐α and IL‐1β, while both IFN‐α and IFN‐γ failed to induce BLC expression in bone marrow‐derived DC. Since TNF‐α expression is increased in aged BWF1 mice, DC recruitment in the circulation and maturation into BLC‐producing DC by TNF‐α may play a pivotal role in the development of systemic autoimmune diseases.

Asthma severity is associated with an increase in both blood CXCR3+ and CCR4+ T cells.

Objectives:u2003 A predominance of type 2 helper T cells (Th2) in the bronchoalveolar space and peripheral blood is a well‐accepted feature of bronchial asthma. However, the relationship between peripheral blood Th2 cells and asthma severity has not been thoroughly investigated.

Immune parameters associated with early treatment effects of high-dose intravenous methylprednisolone in multiple sclerosis

To determine the immunological effects of high-dose intravenous methylprednisolone (IVMP) and elucidate immune measurements used for evaluation of its therapeutic effect, we analyzed lymphocyte subsets and humoral immune parameters in peripheral blood and cerebrospinal fluid (CSF) samples, before and within 2 weeks of treatment during 19 acute exacerbations in 16 relapsing-remitting multiple sclerosis (MS) patients. In addition to decreases in CSF albumin and IgG levels, treatment resulted in an increase of CD8(+)CXCR3(+) cells as well as a decrease in CD4(+) subsets expressing CD25, CD29, and CCR4 in the CSF. Further, the percentage of circulating CD4(+)CXCR3(+) Th1 cells also decreased. Clinical improvement was achieved following 15 of the 19 treatment occasions. Early (<2 weeks of treatment) clinical improvement was significantly associated with a decrease in CSF CD4(+)CD29(+) helper inducer T cells, whereas they were nearly unchanged in four patients who showed no improvement. Changes in other parameters following IVMP treatment were not different between the responder and non-responder groups.

Chemokine receptors associated with immunity within and outside the central nervous system in early relapsing–remitting multiple sclerosis

Thirty-four patients with early relapsing-remitting multiple sclerosis (RRMS) were studied to clarify the differences in chemokine receptor usage by blood and cerebrospinal fluid (CSF) lymphocytes relevant to the pathogenesis of MS. A total of 45 examinations (33 active and 12 inactive stages) revealed that circulating CD4+CXCR3+ T helper 1 (Th1) cells were increased in active MS patients and correlated with the number of gadolinium-enhanced lesions on magnetic resonance (MR) images. In contrast, CSF samples obtained during active stages were characterized by a decrease in the percentage of CD8+CXCR3+ T cells, which was inversely correlated with CSF cell count and intra-blood-brain barrier (BBB) IgG production.

Type1 and type2 memory T cells imbalance shown by expression of intrahepatic chemokine receptors relates to pathogenesis of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by the immunopathologic destruction of interlobular bile ducts. Generally, immunereaction induced by the antigenetic stimulation is divided to cellular and humoral reactions, which are regulated by type1 and type2 memory T cells, respectively. The relative strength of type1 and type2 responses is an important factor in the pathophysiology of immune-mediated diseases. Chemokine receptors are disproportionally expressed in type1 and type2 T cells, providing a basis for tissue-specific recruitment of helper and cytotoxic T cell subsets. In this study, the expression of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 4 (CCR4), which are preferentially expressed on type1 and type2, respectively, were examined to clarify their imbalance in the PBC liver tissue (23 cases). As a control, 16 livers of chronic viral hepatitis (CVH) were used. Reverse transcription-polymerase chain reaction and semiquantitative analysis revealed that the relative ratio of CXCR3/CCR4 mRNAs was higher in early PBC (0.76+/-0.56) rather than in advanced PBC (0.25+/-0.11), and was comparable to that in active CVH (0.67+/-0.43). By immunohistochemistry, the ratio of CXCR3-/CCR4-positive mononuclear cells in portal tracts of early PBC (4.77+/-1.70) is significantly higher than that of advanced PBC (3.11+/-1.26), and also than that of mild and of active CVH. In early PBC, mononuclear cells positive for CXCR3 were dense around the damaged bile ducts. These data suggest that the enhanced shift toward type1 occurs in portal tracts of early PBC and that the imbalance of type1/type2 changes during the course of PBC. This study provides further support for the state of enhanced type1 response in early PBC and potentially offers a possibility to suppress the bile duct lesions and to prevent the progression of PBC from early to advanced stages by reversing the type1/type2 imbalance.

Circulating lymphocyte subsets linked to intracellular cytokine profiles in normal humans

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 1u2003µm monensin and 20u2003µg/ml brefeldin A with or without stimulation by phorbol myristate acetate plus ionomycin for the detection of intracellular interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), tumour necrosis factor (TNF)‐α, and IL‐4. As a result, CD4+CD29high helper inducer T cells were closely associated with IFN‐γ and TNF‐α 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‐γ and TNF‐α‐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.

Chemokine-mediated thymopoiesis is regulated by a mammalian Polycomb group gene, mel-18.

Multiple chemokines are made in the thymus, and they are likely to function in the fine control of cellular migration and regulation of thymic T cell development. Mice lacking the gene mel-18, a member of the mammalian Polycomb group genes, displayed impaired thymic T cell development. Here we report that expression of chemokine receptors CXCR4 and CCR9 are regulated by mel-18 and that CXCL12/SDF-1- and CCL25/TECK-mediated chemotactic activities are also affected by the loss of mel-18. In mel-18-/- mice, high expression of CXCR4 on CD4-CD8- cells might lead to trapping in the SDF-1 rich subcapsular region, while low expression of CCR9 on CD4+CD8+ cells might reduce cell migration to the medulla. Therefore, this member of the Polycomb group genes plays a role in thymic T cell migration and differentiation via the chemokine system.