Activated killer cell activity in lymph nodes

The activated killer cell activity of cells from the perigastric lymph nodes (LNC) in patients with gastric carcinoma or benign lesions was assayed in comparison with that of peripheral blood mononuclear cells (PBM). The cytotoxic activity induced by phytohemagglutinin (PHA) activation in LNC from patients with either carcinoma or benign lesions was significantly decreased as compared to that in PBM, although the ability to produce interleukin 2 (IL 2) in LNC was significantly increased. Therefore, the ability to generate cytotoxic cells after activation with IL 2 was examined, and decreased capacity in LNC was observed. In LNC, the proportion of OKT3+ cells was similar to that in PBM, with a prevalence of OKT4+ cells over OKT8+ cells. Also, the percentage of Leu-11+ cells was remarkably reduced. The results indicated that decreased levels of the activated killer cell activity, which might be partially attributable to the composition of lymphocyte subsets, existed in lymph nodes, and this might facilitate tumor metastasis to the regional lymph nodes.     

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.     

Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1beta-converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1beta and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1(-/)- mice) had an oral S. typhimurium 50% lethal dose (LD(50)) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1(-/)- mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1(-/)- mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1(-/)- mice after an oral dose of S. typhimurium that was 100-fold higher than the LD(50). The increased resistance in casp-1(-/)- animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.     

Distribution of T cell subsets in human lymph nodes

A series of T cell-specific monoclonal antibodies was used to determine the location of T lymphocyte subpopulations in frozen sections of human lymph nodes by means of an immunoperoxidase technique. The majority of cells in the paracortical regions were reactive with anti-T1 and anti-T3 antibodies, which define all mature peripheral T cells. In contrast, the majority of cells within primary follicles were unreactive with anti-T1 and anti-T3 antibodies, but were reactive with anti-Ia and anti-IgM antibodies. In addition, a substantial number of T1+, T3+ cells were found in the germinal centers of secondary follicles on the capsular side. The vast majority of T1+, T3+ cells in the paracortex and the follicles were reactive with anti-T4 antibody, which defines inducer/helper T cells. Only a minority of cells in these areas were reactive with anti-T5 and anti-T8 antibodies, which define cytotoxic/suppressor cells. No lymphocytes were stained with anti-T6 antibody, which reacts with a majority of thymocytes but not with peripheral T cells. Scattered cells in the paracortex showed staining for Ia antigen in an irregular dendritic pattern. The findings demonstrate that the major T cell population found within human lymph node bears the mature T1+, T3+, T4+ phenotype characteristic of inducer T cells. Moreover, the location of this population indicates that they play a role in the induction of B cell differentiation in vivo.     

Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes

Natural interferon-producing cells (IPCs) are found in peripheral lymph nodes (PLNs), where they support NK cell, T cell, and B cell responses to pathogens. However, their route of entry and the adhesive mechanisms used to gain access to PLNs remain poorly defined. We report that IPCs can enter PLNs via a hematogenous route, which involves a multistep adhesive process, and that transmigration is enhanced by inflammation. Results indicate that L-selectin on IPCs is required for efficient attachment and rolling on high endothelial venules in vivo in both nonstimulated and inflamed PLNs. IPCs, however, also possess functional ligands for E-selectin that contribute to this process only in the latter case. In conjunction with selectin-mediated adhesion, both beta(1)- and beta(2)-integrins participate in IPC attachment to the inflamed vessel wall, whereas chemotaxis relies in part on the chemokine receptor CCR5. Identification of the adhesive machinery required for IPC trafficking into PLNs may provide opportunities to regulate immune responses reliant on the activity of these cells.     

FTY720 stimulates multidrug transporter- and cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes

FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and the leukotriene C(4) transporter Abcc1 (Mrp1). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S1P also require 5-lipoxygenase-mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL19, CCL21, VLA-4alpha, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb1, and Abcc1, and comparison of the effects of FTY720 with those of S1P, suggest a model of sequential engagement of Abcb1, SP1 receptors, 5-lipoxygenase, and Abcc1 to enhance T cell migration and homing.     

Preoperative workflow for lymph nodes staging

Purpose  Accurate staging of lymph nodes relies mainly on surgical exploration and manual palpation. We present a new non-invasive diagnostic approach: simulated palpation through virtual laparoscopic instruments. Methods  We set up a diagnostic process to extract lymph nodes shape and position from CTs and to analyze the trend of pixels intensities to determine tissue properties in order to feedback the force information. Results  We have integrated the model, obtained from both the morphological information and stiffness values, in our laparoscopy simulator and surgeons can virtually palpate, with a haptic device, the lymph nodes. We evaluated the workflow extracting lymph nodes from a case study: the feedback provided through the simulator greatly helps the surgeon in the correct staging. Conclusions  Results show the feasibility of the approach and in the future we will clinically evaluate this new diagnostic methodology. We are studying the possibility to integrate CTs with other imaging systems to increase the accuracy.     

Adenovirus vector-mediated gene transfer to regional lymph nodes

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.     

Identification of cell adhesion molecules in the human follicle-associated epithelium that improve nanoparticle uptake into the Peyer's patches

The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyer's patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker beta(1)-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence. Integrin targeting was studied by investigating the transport of Arg-Gly-Asp (RGD)-coated (integrin-binding), Arg-Gly-Glu (RGE)-coated (nonintegrin-binding), and uncoated nanoparticles across ileal specimens mounted in Ussing chambers. Both beta(1)-integrin and the cell adhesion molecule CD9 were more abundantly expressed in the model and human FAE compared with the Caco-2 control cells or villus epithelium (VE). Uncoated nanoparticles were not taken up across either FAE or VE. General integrin targeting with RGD improved the nanoparticle transport dramatically across the FAE and to a lower extent across the VE. Compared with RGE, RGD improved transport 4-fold across the FAE. There was no difference in the transport of RGD- and RGE-coated nanoparticles across the VE. In conclusion, beta(1)-integrin and CD9 were identified as targets in human FAE. The difference in RGD- and RGE-mediated transport across the FAE, but not the VE, suggests that a specific integrin interaction was the dominating mechanism for improved nanoparticle uptake across the FAE., whereas charge interaction contributed substantially to the improved VE uptake.     

累及全身淋巴结的滤泡树突状细胞肉瘤临床病理观察

目的 探讨滤泡树突状细胞肉瘤(FDCS)的临床病理学特征、生物学行为、治疗和预后.方法 对1例累及全身多处淋巴结的FDCS进行临床病理学分析、免疫组化染色及EBV编码的小RNA原位杂交检测,并结合文献进行讨论.结果 患者病变累及全身多处淋巴结,伴随全身荨麻疹和关节肿痛.瘤组织排列成束状、席纹状.瘤细胞胞膜欠清,胞质淡伊红染,胞核卵圆形,有异型,核膜清晰,呈空泡状,核仁明显,可见病理性核分裂象2～4个/10HPF.瘤组织中散在少量淋巴细胞、浆细胞和嗜酸性粒细胞.免疫组化瘤细胞clusterin、SNA和vimentin弥漫(+),CD21+35和S-100局部(+),CD1a、desmin、CK8/18和CD68(-).EBER原位杂交(-).结论 滤泡树突状细胞肉瘤是罕见的起源于淋巴结和结外淋巴组织间质细胞的恶性肿瘤,诊断依赖于组织病理学和免疫组化.     

Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.     

Natural killer cell behavior in lymph nodes revealed by static and real-time imaging

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice.

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Aspirin reduces lung cancer metastasis to regional lymph nodes

Background

Lung cancer is the main cause of cancer-related death worldwide. The high mortality is probably attributable to early metastasis; however, the mechanism underlying metastasis to regional lymph nodes is still unknown. Cyclooxygenase (COX)-derived prostaglandin E₂ (PGE₂) induces tumor growth and metastasis and is associated with a poor prognosis. The present study investigated the effect of an authentic COX inhibitor, aspirin, on regional lymph node metastasis during the development of lung cancer in mice.

Methods

An orthotopic intrapulmonary implantation model based on male C57BL/6 (6–8-weeks-old) mice was used. The lungs were injected with a solution containing Lewis lung carcinoma (LLC) cells overexpressing green fluorescent protein (GFP) and BD Matrigel^®. The effect of aspirin on mediastinal lymph node metastasis of LCC cells from the primary injection sites was then examined.

Results

The implantation process took approximately 30 s per mouse and operative mortality was 10%. Single pulmonary nodules developed at the implanted site in 95% of animals, and regional mediastinal lymph node metastasis was observed at 14 days post-LLC-GFP cell injection in all mice that formed a primary lung tumor. The mean survival time of mice injected with LLC-GFP cells was 15 ± 3 days (range, 12–22 days). Histopathological analysis revealed that no metastatic tumors developed in the regional mediastinal lymph nodes by Day 10–12 post-LLC-GFP cell injection and no metastasis to distant organs or distant lymph nodes was observed by Day 21 post-injection. Oral administration of aspirin (100 mg/kg, twice a day) after LLC-GFP cell injection inhibited metastasis to the regional lymph nodes, with no significant suppression of primary tumor growth in the lungs. Aspirin treatment led to a significant reduction in mortality (P < 0.0001).

Conclusions

The present lymph node metastasis model is useful for evaluating the efficacy of agents that inhibit tumor metastasis to the regional lymph nodes. Aspirin reduced the metastasis of LLC-GFP cells injection to the regional lymph nodes, with a significant reduction in mortality. These findings suggested that COX inhibitors have potential for preventing lymph node metastasis.