CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

CCL28 induces the migration of IgA Ab-secreting cells (ASCs) via CCR10 and also displays a potent antimicrobial activity in vitro. To explore the role of CCL28 in vivo, we generated CCL28-deficient mice. The mice exhibited a significant reduction and abnormal distribution of IgA ASCs in the lamina propria of the colon. The concentrations of total and Ag-specific IgA in the fecal extracts of CCL28-deficient mice were also drastically reduced. The average amount of IgA secreted by a single IgA ASC derived from the colon was also substantially reduced in CCL28-deficient mice. Furthermore, CCL28 was found to significantly increase the average amount of IgA secreted by a single IgA ASC derived from the colon in vitro. In contrast, the generation of IgA ASCs in Peyer's and cecal patches was not significantly impaired in CCL28-deficient mice. We also found a relative increase in the Class Bacilli in the fecal extracts of CCL28-deficient mice and demonstrated a potent antimicrobial activity of CCL28 against Bacillus cereus and Enterococcus faecalis, both of which belong to Class Bacilli. Thus, CCL28 may also suppress the outgrowth of some bacterial species by its direct antimicrobial activity. Finally, CCL28-deficient mice exhibited a highly aggravated dextran sodium sulfate-induced colitis that was ameliorated by pretreatment with antibiotics. Collectively, CCL28 plays a pivotal role in the homing, distribution, and function of IgA ASCs in the colon and may also affect the intestinal microbiota through its direct antimicrobial activity.

c-Maf suppresses human T-cell leukemia virus type 1 Tax by competing for CREB-binding protein.

Latent infection of human T-cell leukemia virus type 1 (HTLV-1) is considered to be preferentially associated with CCR4(+) CD4(+) T cells. Here we report that c-Maf, one of the critical transcription factors for Th2 differentiation, suppresses the transcriptional activity of HTLV-1 Tax by competing for CREB-binding protein. Notably, c-maf expression is selectively induced in a fraction of CCR4(+) CD4(+) T cells upon activation. Furthermore, c-Maf significantly decreases Tax-induced HTLV-1 envelope gp46 gene expression from an infectious HTLV-1 molecular clone and tax expression in a cell-free HTLV-1 infection system. Collectively, c-Maf may play a role in latent infection of HTLV-1 in CCR4(+) CD4(+) T cells by negatively regulating Tax activity.

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1.

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.

CXCR7 is inducible by HTLV-1 Tax and promotes growth and survival of HTLV-1-infected T cells.

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia (ATL), encodes the potent transcriptional activator Tax, which is required for HTLV-1-induced immortalization of T cells. CXCR7 is an atypical chemokine receptor frequently expressed by tumor cells and known to promote cell growth and survival. We found that HTLV-1-immortalized T cells expressing Tax consistently expressed CXCR7. Induction of Tax in JPX-9 upregulated CXCR7. Wild-type Tax efficiently activated the CXCR7 promoter via a proximal NF-kappaB site, while a mutant Tax selectively defective in NF-kappaB activation did not. CCX754, a synthetic CXCR7 antagonist, inhibited cell growth and increased apoptosis of HTLV-1-immortalized T cells. Knockdown of CXCR7 by small interfering RNA also reduced cell growth. Stable expression of CXCR7 in a CXCR7-negative ATL cell line promoted cell growth and survival. Taken together, CXCR7 is inducible by Tax and may play an important role in HTLV-1-induced immortalization of T cells by promoting growth and survival of HTLV-1-infected T cells.

An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation.

Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin-mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin-mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1-deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1-deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1-deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1-independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.

CD43 plays both antiadhesive and proadhesive roles in neutrophil rolling in a context-dependent manner.

As the first step in the recruitment of neutrophils into tissues, the cells become tethered to and roll on the vessel wall. These processes are mediated by interactions between the P- and E-selectins, expressed on the endothelial cells of the vessel wall, and their ligands, expressed on the neutrophils. Recently, we reported that CD43 on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with P-selectin glycoprotein ligand-1 (PSGL-1), a major P- and E-selectin ligand. Here, we examined whether CD43 on neutrophils also functions as an E-selectin ligand. CD43 was precipitated with an E-selectin-IgG chimera from mouse bone marrow neutrophils. A CD43 deficiency diminished the E-selectin-binding activity of neutrophils when PSGL-1 was also deficient. Intravital microscopy showed that the CD43 deficiency significantly increased leukocyte rolling velocities in TNF-alpha-stimulated venules blocked with an anti-P-selectin mAb, where the rolling was mostly E-selectin dependent, when PSGL-1 was also absent. In contrast, in venules with trauma-induced inflammation, where the rolling was largely P-selectin dependent, the CD43 deficiency reduced leukocyte rolling velocities. Collectively, these observations suggest that CD43 generally serves as an antiadhesive molecule to attenuate neutrophil-endothelial interactions, but when E-selectin is expressed on endothelial cells, it also plays a proadhesive role as an E-selectin ligand.

P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes.

Lymphocyte homing to peripheral lymph nodes (LNs) requires L-selectin. Previous studies, however, suggest that there are L-selectin-independent mechanisms of lymphocyte homing. P-selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed in a selectin-binding form on myeloid cells and subsets of lymphoid cells. To discover whether PSGL-1 plays a role in lymphocyte homing, we examined leukocyte rolling and adhesion in the high endothelial venules (HEVs) of the subiliac LNs of wild-type and PSGL-1-deficient mice by intravital microscopy. There were no significant differences in blood velocity or wall shear stress between wild-type and PSGL-1-deficient mice. Although the leukocyte rolling fraction was not altered in PSGL-1-deficient mice, infusion of an anti-L-selectin mAb into these mice completely abolished leukocyte rolling, while the same treatment in wild-type mice inhibited 90% of the leukocyte rolling. This residual rolling in wild-type mice appears to depend on the PSGL-1-P-selectin interaction, since infusion of an anti-L-selectin mAb together with an anti-PSGL-1 mAb or anti-P-selectin mAb almost completely abolished the rolling. PSGL-1 deficiency also led to a higher rolling velocity, suggesting that PSGL-1 mediates leukocyte rolling at low velocities. P-selectin was found to be expressed on the HEVs of subiliac LNs under the conditions of intravital microscopy. Taken together, these results indicate that the interaction of PSGL-1 with P-selectin constitutes a second mechanism of leukocyte rolling in the HEVs of peripheral LNs.

CD43 collaborates with P-selectin glycoprotein ligand-1 to mediate E-selectin-dependent T cell migration into inflamed skin.

Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-selectin expressed on endothelial cells and their ligands on T cells. P-selectin glycoprotein ligand-1 (PSGL-1) has been the only E-selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.

CD43 functions as a ligand for E-Selectin on activated T cells.

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing alpha-1,3-fucosyltransferase VII and a core 2 beta-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.

A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage.

A new mouse monoclonal antibody (mAb), HUKT was raised against chicken peripheral blood thrombocytes. The mAb HUKT appeared to detect a specific marker on the surface of chicken thrombocytes. Flow cytometry (FCM) analysis revealed that it did not react with cells from the normal thymus, bursa of Fabricius, six kinds of chicken cell lines, chicken erythrocytes or human platelets. In addition, HUKT(+) cells in peripheral blood leukocytes (PBL) were CD45(low), Bu-1a(-) and CD3(-) cells. Immunoblotting analysis showed that the molecule recognized by HUKT is a monomer with an apparent molecular weight of 150 kDa under non-reducing and reducing conditions. Tissue distribution studies revealed that only cells of thrombocyte lineage in bone marrow and embryonic blood cells were stained by HUKT. The HUKT mAb presented here may be useful for both ontogenetic studies of thrombocyte lineage and immunological studies in the chicken.

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems.

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.

Genomic organization of the chicken T-cell receptor beta chain D-J-C region.

We examined overlapping genomic clones containing the chicken T cell receptor (TCR) Dbeta-Jbeta-Cbeta complex, which contains a single diversity segment, four joining segments and four exons that encode the constant region. This sequence comprised 18.3 kb. All four Jbeta sequences possessed typical recombination signal sequences (RSS) with intervening 12-bp spacers at their 5'-ends and splice sites at their 3'-ends. No Jbeta-pseudogenes were identified. TGTG sequences in the RSS heptamer sequences were well conserved, as is the case in mammals. A chicken repeat 1-like sequence was found in the intron region between Jbeta-1336 and Cbeta, and several small repeat sequences were identified in intron regions throughout this cloned genome. As germline sequences revealed complete Jbeta sequences, the CDR3 (complementarity-determining region) sequences of TCRbeta from non-immunized splenocytes were analyzed. Non-coding (N) and palindromic (P) nucleotides were frequently observed at the Dbeta-Jbeta recombination sites. There were differences in length of deletion at the 5'-end of each Jbeta. Deletion of the 5'-end of Jbeta-1280 was particularly short when compared with that of Jbeta-1336, but there were no changes in the length of the CDR3 using any of the four Jbeta sequences.

Flow cytometric analysis of chicken NK activity and its use on the effect of restraint stress.

Immune system is organized by the influence of both neural and endocrine systems. NK activity plays an important role in the innate immunity. In this study, we observed the effects of restraint stress on chicken peripheral blood NK activity. Viability of FITC-labeled RP9 was measured with PI after treatment with the effector cells. Chicken peripheral blood CD8alpha+ cells expressed strong cytotoxic activity, in contrast to thrombocytes, while peripheral blood CD3+ CD8alpha+ cells and CD4+ cells had little cytotoxic activity. Con A supernatant enhanced the cytotoxic activity of CD8alpha+ cells. Therefore, it is considered that these cytotoxic activities measured by flow cytometry (FCM) analysis are NK activity. When chickens were exposed to restraint stress, the levels of serum corticosterone increased transiently over a short period of time while the NK activity decreased. The decreased NK activity, however, did not recover to the intact levels for a long time, even once the serum corticosterone levels had recovered. These data indicate that chicken NK activity is able to be measured by flow cytometric analysis and that restraint stress causes severe damage to the chicken NK activity.

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo.

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.

Epidemiological correlation between chromium content in gallstones and cholesterol in blood.

The authors measured the chromium in gallstones and bile from patients in three areas (Kawasaki (a city adjacent to Tokyo) in Japan, Chiang Mai and Bangkok in Thailand) by means of neutron activation analysis. The chromium in three types of gallstones (cholesterol, pigment, and rare stones) and bile from patients living in Bangkok were evidently larger than those from patients living in Kawasaki and Chiang Mai. The high chromium intake by Bangkok patients continued from the start of gallstone formation until the time the stones were removed. The total-cholesterol, triglyceride, and hemoglobin A(1C) levels in the blood from Bangkok residents with high chromium intake over a long period were clearly lower than those of Japanese and Chiang Mai residents. The authors showed that the high dietary intake of chromium over a long period may play a role in the lowering of total-cholesterol, triglyceride, and hemoglobin A(1C) in blood.