Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and −2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (β₂) integrins, and the β₁ integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of β₂ integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these β₂ integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal β₂ integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1-dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.     

Supplementation of CXCL12 (CXCL12) induces homing of CD11c+ dendritic cells to the spleen and enhances control of Plasmodium berghei malaria in BALB/c mice

In malaria, parasitaemia is controlled in the spleen, a multicomponent organ that undergoes changes in its cellular constituents to control the parasite. During this process, dendritic cells (DCs) orchestrate the positioning of effector cells in a timely manner for optimal parasite clearance. We have recently demonstrated that CXCL12 [stromal cell‐derived factor‐1 (CXCL12)] supplementation partially restores the ability to control parasitaemia in Plasmodium berghei‐infected mice. In the present study, we investigated the nature of the DCs involved by flow cytometry and immunohistochemistry of CD11c⁺ cells. Flow cytometry of bone marrow cells showed that infection with P. berghei did not alter the proportion of CD11c⁺ cells present in this haematopoietic compartment, while CXCL12 supplementation of naïve uninfected mice induced only minor increases in the population of CD11c⁺ cells. In the spleen, P. berghei infection alone resulted in an increase in CD11c⁺ cells as compared with naïve animals. Exogenously administered CXCL12 in the absence of infection resulted in a significant expansion of the splenic CD11c⁺ population, and this effect was even more pronounced in infected and supplemented mice. Immunohistochemistry revealed that CD11c⁺ cells infiltrated the perivascular areas and marginal zone of the spleen in infected animals treated with CXCL12, suggesting that this chemokine induces homing of CD11c⁺ dendritic cells to the splenic compartment. Our results show that small amounts of CXCL12 supplementation are effective in recruiting DCs to the spleens of both uninfected and infected mice, suggesting the participation of CXCL12 and CD11c⁺ cells in the establishment of an adequate environment in the spleen for malaria control.     

Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen

Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-μ stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blast-stromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.     

The neglected brothers come of age: B cells and cancer

The opposing roles of innate and adaptive immune cells in suppressing or supporting cancer initiation, progression, metastasis and response to therapy has been long debated. The mechanisms by which different monocyte and T cell subtypes affect and modulate cancer have been extensively studied. However, the role of B cells and their subtypes have remained elusive, perhaps partially due to their heterogeneity and range of actions. B cells can produce a variety of cytokines and present tumor-derived antigens to T cells in combination with co-stimulatory or inhibitory ligands based on their phenotype. Unlike most T cells, B cells can be activated by innate immune stimuli, such as endotoxin. Furthermore, the isotype and specificity of the antibodies produced by plasma cells regulate distinct immune responses, including opsonization, antibody-mediated cellular cytotoxicity (ADCC) and complement activation. B cells are shaped by the tumor environment (TME), with the capability to regulate the TME in return. In this review, we will describe the mechanisms of B cell action, including cytokine production, antigen presentation, ADCC, opsonization, complement activation and how they affect tumor development and response to immunotherapy. We will also discuss how B cell fate within the TME is affected by tumor stroma, microbiome and metabolism.     

Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production

During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24 h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.     

Distinct roles for IL-6 and IL-12p40 in mediating protection against Leishmania donovani and the expansion of IL-10+ CD4+ T cells

Adoptive dendritic cell (DC) immunotherapy provides a useful experimental tool to evaluate immunoregulation in vivo and has previously been successfully used to enhance host resistance in a variety of experimental models of leishmaniasis. Here, we used this approach to identify IL-6 and IL-12p40 as critical cytokines that cooperate to mediate host protection to Leishmania donovani but which act independently to regulate expansion of IL-10(+) CD4(+) T cells, shown here for the first time to be associated with this infection. Adoptive transfer of LPS-activated bone marrow-derived DC (BMDC) from wild-type mice was therapeutically beneficial and led to enhanced resistance as measured by spleen parasite burden. In contrast, IL-6- or IL-12p40-deficient BMDC had no protective benefit, indicating that production of both cytokines was essential for the therapeutic efficacy of DC. IL-10 production by CD25(-) FoxP3(-) IL-10(+) CD4(+) T cells is a strong correlate of disease progression, and BMDC from wild-type mice inhibited expansion of these cells. Strikingly, IL-12-deficient BMDC could also inhibit the expansion of this T cell population whereas IL-6-deficient BMDC could not, indicating that IL-6 played a key role in this aspect of DC function in vivo. Breadth of cytokine production is thus an important factor when considering strategies for DC-based interventions.     

CXCL12 在母胎界面的表达及其促进BeWo滋养细胞系的体外迁移作用

目的研究CXCL12在早孕期母胎界面的表达情况及其对BeWo细胞的体外迁移作用.方法免疫组织化学法检测早孕期绒毛及蜕膜组织中CXCL12的表达情况;用Transwell细胞侵入系统检测CXCL12对BeWo细胞迁移的影响.结果早孕期绒毛及蜕膜组织均有CXCL12的表达,分布于绒毛柱、滋养细胞及间质血管内;CXCL12对BeWo细胞具有趋化作用,浓度达10ng/ml时趋化效应最强.结论 CXCL12可促进BeWo细胞的迁移,因此早孕期母胎界面产生的CXCL12可能参与滋养细胞的迁移,对于维系正常妊娠发挥了一定作用.     

人脐带间充质干细胞分泌白细胞介素6促进骨肉瘤细胞增殖和迁移

 目的: 探讨人脐带间充质干细胞(hUC-MSCs)对骨肉瘤Saos-2细胞增殖和迁移的作用及分子机制。方法: 组织块贴壁法分离培养hUC-MSCs,流式细胞术鉴定细胞表面标记物;CCK-8法和细胞计数法检测hUC-MSCs条件培养基(CM)、重组人白细胞介素6(rhIL-6)及IL-6中和抗体对Saos-2细胞增殖的作用;ELISA检测hUC-MSCs分泌IL-6的量;RT-PCR检测增殖相关基因增殖细胞核抗原(PCNA)、cyclin D1和survivin的转录水平;Transwell实验检测hUC-MSCs和Saos-2细胞迁移能力的变化。结果: hUC-MSCs可向Saos-2细胞迁移;hUC-MSCs-CM含有高浓度的IL-6,可达(1 835.5±134.1)ng/L;hUC-MSCs-CM和rhIL-6均能促进Saos-2细胞增殖和迁移,IL-6中和抗体能明显削弱hUC-MSCs-CM的促Saos-2细胞增殖和迁移作用;RT-PCR显示hUC-MSCs-CM和rhIL-6均能上调Saos-2细胞增殖相关基因PCNA、cyclin D1和survivin的表达,而IL-6中和抗体则削弱了这一作用。结论: 脐带间充质干细胞能向骨肉瘤Saos-2细胞迁移,并通过分泌IL-6促进其增殖和迁移。     

Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells,follicular dendritic cells and a third superficial stromal cell type

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271⁻ and CD271^+/− stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27⁺ and switched B lymphocytes, but also scattered IgD⁺⁺ B lymphocytes and variable numbers of CD4⁺ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far.     

精脒和腐胺对人胎儿脾LAK活性的影响

本研究表明,精脒、腐胺本身均不能使人胎脾单个核细胞(FSMC)转化为LAK细胞,但与5u/ml重组白细胞介素2(rIL-2)伍用,可以增强人胎脾LAK细胞活性;精脒,腐胺不能直接刺激细胞增殖,但能协同亚适剂量Con A诱导淋巴细胞增殖;精脒或腐胺本身可刺激FSMC的IL-2受体(IL-2R)的表达,并能协同rIL-2进一步提高人胎脾LAK细胞IL-2R的表达;精脒、腐胺均可使FSMC及胎脾LAK细胞内鸟氨酸脱羧酶(ODC)活性增强,而以腐胺的作用尤为显著。     

IL-23与IL-12诱导NK细胞功能的异同及机制初探

目的探讨细胞因子IL-23与IL-12对NK细胞功能的影响及可能的机制。方法密度梯度离心法分离人外周血单个核细胞(PBMCs)或磁珠纯化NK细胞,不刺激或用IL-23或IL-12刺激,用流式细胞术和ELISA法检测NK细胞产生IFN-γ的情况;以K562或Jurkat细胞作为靶细胞,用流式细胞术检测NK细胞的杀伤功能并分析NK细胞在不同的刺激条件下杀伤相关分子的表达情况及pSTAT的表达情况。结果与未刺激组相比,IL-23和IL-12均可以诱导NK细胞呈剂量和时间依赖方式产生IFN-γ;但IL-12而非IL-23可以增强NK细胞对靶细胞K562或Jurkat细胞的杀伤功能。进一步研究表明,IL-12而非IL-23可以诱导杀伤相关分子TRAIL及CD107a/b的表达。此外,IL-12诱导NK细胞表达更高水平的pSTAT4,而IL-23诱导NK细胞表达更高水平的pSTAT3。结论与IL-12相比,IL-23亦可以诱导NK细胞产生细胞因子但不能增强NK细胞的杀伤功能,IL-23不能诱导杀伤相关分子TRAIL及CD107a/b的表达,IL-23可以诱导低水平的pSTAT4但高水平的pSTAT3的表达。     

IL-12 reverses anergy to T cell receptor triggering in human lung tumor-associated memory T cells

Memory T cells in human non-small cell lung cancer are unresponsive to progressing tumors. T cells were evaluated at the single cell level by imaging the nuclear translocation of NF-kappaB and NFAT via immunofluorescence confocal microscopy as an early measure of responsiveness to T cell receptor triggering. Little translocation of NF-kappaB or NFAT occurred in tumor-associated T cells in response to CD3+CD28 cross-linking under conditions which led to maximal translocation in normal donor peripheral blood T cells. TNF-alpha induced maximal NF-kappaB translocation in these T cells, indicating that they remain receptive to alternative signaling pathways, and pulsing with IL-12 prior to TCR triggering reversed their apparent anergy. T cells from additional chronic inflammatory microenvironments demonstrated a similar refractoriness to TCR activation, suggesting either that a common regulatory mechanism present within the microenvironment controls these cells or that with continuous antigen exposure, they remain refractory to activation via the TCR.     

CD4+ NK cells can be productively infected with HIV, leading to downregulation of CD4 expression and changes in function

NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1α partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a viral reservoir of HIV in vivo.     

Regulation of B lymphocytes and plasma cells by innate immune mechanisms and stromal cells in rheumatoid arthritis

B cells mediate multiple functions that influence immune and inflammatory responses in rheumatoid arthritis. Production of a diverse array of autoantibodies can happen at different stages of the disease, and are important markers of disease outcome. In turn, the magnitude and quality of acquired humoral immune responses is strongly dependent on signals delivered by innate immune cells. Additionally, the milieu of cells and chemokines that constitute a niche for plasma cells rely strongly on signals provided by stromal cells at different anatomical locations and times. The chronic inflammatory state therefore importantly impacts the developing humoral immune response and its intensity and specificity. We focus this review on B cell biology and the role of the innate immune system in the development of autoimmunity in patients with rheumatoid arthritis.     

人IL-12的克隆、表达及生物活性的鉴定

目的：克隆中国人ＩＬ－２ｐ４０基因,构建ＩＬ－１２的真核基因表达载体。方法：采用ＲＴ－ＰＣＲ从北京地区人脐血树突状细胞（ＤＣ）中克隆ＩＬ－１２ｐ４０ｃＤＮＡ基因,并进行序列分析。利用ｐｃＤＮＡ３．１和ｐＬＸＰＸＳＮ构建ＩＬ－１２表达载体,转染人肝癌细胞后对其进行生物学和免疫学分析。结果：北京地区人ＤＣ中ＩＬ－１２ｐ４０ｃＤＮＡ基因２１０位密码子有独特的结构,为ＧＣＣ（Ａｌａ）。并且２３９和２９１位     

Invariant NKT cells and CD1d+ cells amass in human omentum and are depleted in patients with cancer and obesity

Invariant NKT (iNKT) cells recognize lipid antigens presented by CD1d and respond rapidly by killing tumor cells and release cytokines that activate and regulate adaptive immune responses. They are essential for tumor rejection in various mouse models, but clinical trials in humans involving iNKT cells have been less successful, partly due to their rarity in humans compared with mice. Here we describe an accumulation of functional iNKT cells in human omentum, a migratory organ with healing properties. Analysis of 39 omental samples revealed that T cells are the predominant lymphoid cell type and of these, 10% expressed the invariant Vα24Jα18 TCR chain, found on iNKT cells, higher than in any other human organ tested to date. About 15% of omental hematopoietic cells expressed CD1d, compared with 1% in blood (p<0.001). Enriched omental iNKT cells killed CD1d⁺ targets and released IFN‐γ and IL‐4 upon activation. Omental iNKT‐cell frequencies were lower in patients with severe obesity (p=0.005), and with colorectal carcinoma (p=0.004) compared with lean healthy subjects. These data suggest a novel role for the omentum in immune regulation and tumor immunity and identify it as a potential source of iNKT cells for therapeutic use.     

Dendritic cells modified to express fractalkine/CX3CL1 in the treatment of preexisting tumors

Fractalkine (CX3CL1) is a unique membrane-bound CX3C chemokine that serves as a potent chemoattractant for lymphocytes. The hypothesis of this study is that dendritic cells (DC) genetically modified ex vivo to overexpress fractalkine would enhance the T cell-mediated cellular immune response with a consequent induction of anti-tumor immunity to suppress tumor growth. To prove this hypothesis, established tumors of different mouse cancer cells (B16-F10 melanoma, H-2b, and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of bone marrow-derived DC that had been modified in vitro with an RGD fiber-mutant adenovirus vector expressing mouse fractalkine (Ad-FKN). In both tumor models tested, treatment of tumor-bearing mice with Ad-FKN-transduced DC gave rise to a significant suppression of tumor growth along with survival advantages in the treated mice. Immunohistochemical analysis of tumors treated with direct injection of Ad-FKN-transduced DC demonstrated that the treatment prompted CD8+ T cells and CD4+ T cells to accumulate in the tumor milieu, leading to activation of immune-relevant processes. Consistent with the finding, the intratumoral administration of Ad-FKN-transduced DC evoked tumor-specific cytotoxic T lymphocytes, which ensued from in vivo priming of Th1 immune responses in the treated host. In addition, the anti-tumor effect provided by intratumoral injection of Ad-FKN-transduced DC was completely abrogated in CD4+ T cell-deficient mice as well as in CD8+ T cell-deficient mice. These results support the concept that genetic modification of DC with a recombinant fractalkine adenovirus vector may be a useful strategy for cancer immunotherapy protocols.     

IL-6 promotes immune responses in human ulcerative colitis and induces a skin-homing phenotype in the dendritic cells and Tcells they stimulate

Dendritic cells (DCs) control the type and location of immune responses. Ulcerative colitis (UC) is considered a Th2 disease mediated by IL-13 where up to one third of patients can develop extraintestinal manifestations. Colonic biopsies from inflamed and noninflamed areas of UC patients were cultured in vitro and their supernatants were used to condition human blood enriched DCs from healthy controls. Levels of IL-13 in the culture supernatants were below the detection limit in most cases and the cytokine profile suggested a mixed profile rather than a Th2 cytokine profile. IL-6 was the predominant cytokine found in inflamed areas from UC patients and its concentration correlated with the Mayo endoscopic score for severity of disease. DCs conditioned with noninflamed culture supernatants acquired a regulatory phenotype with decreased stimulatory capacity. However, DCs conditioned with inflamed culture supernatants acquired a proinflammatory phenotype with increased expression of the skin-homing chemokine CCR8. These DCs did not have decreased T-cell stimulatory capacity and primed T cells with the skin-homing CLA molecule in an IL-6-dependent mechanism. Our results highlight the role of IL-6 in UC and question the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology.     

DEC-205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.