Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

Deep splenic lymphatic vessels in the mouse: a route of splenic exit for recirculating lymphocytes

A study of pathways of lymphocyte migration through mouse spleen revealed lymphatic channels closely following arteries in trabeculae and white pulp. Because there is no detailed record of the layout of deep splenic lymphatics in the mouse, or other species, we present our observations in this paper, relating our findings to normal migratory pathways of lymphocytes through the spleen. Lymphatics draining the spleen are so inconspicuous that they often are not mentioned in anatomical discussions. The data presented clearly demonstrate 1) the existence and layout of deep lymphatic vessels in the mouse spleen, and 2) that migrating lymphocytes exit white pulp via these lymphatic vessels. CD4+ and CD8+ T cell subsets migrated proximally along the central artery from distal (dPALS) to proximal periarterial lymphatic sheaths (pPALS) and exited via deep lymphatic vessels that originate there. B cells migrated from dPALS to enter lymphatic nodules (NOD), thus segregated from T cells. B cells then migrated toward and exited via deep lymphatics. The appearance of labelled lymphocytes in lymph coincided with their disappearance from white pulp compartments. Labelled T cells were observed in splenic lymphatics as early as 1 hr after intravenous infusion but took, on average, about 6 hr. B cells took somewhat longer. Thus T and B cells entered and left white pulp through shared pathways, but took divergent intermediate routes through dedicated zones, pPALS for T cells, NOD for B cells.     

The ratio of splicing variants of MGC-24/CD164, a sialomucin, correlates with the metastatic potential of colorectal carcinomas

MGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation.     

Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed. presenilin 1-associated protein-mitochondrial carrier homolog 1; mitochondria; apoptosis; presenilin; Alzheimer's disease     

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.     

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.     

带γδ和αβ受体的T细胞（TCR）在人胎儿组织内分布的研究

本文用细胞免疫化学方法,在冰冻切片上,检测了胎儿不同组织和器官内带γδ和αβ受体的Ｔ细胞（ＴＣＲ）的分布,结果发现ＴＣＲ细胞的分布与,般Ｔ细胞不同,有相对固定的分布区,如胸腺内ＴＣＲ细胞主要分布在皮筋质交界处和髓质部;脾脏内的γδＴ主要位于边缘区,而αβＴ主要位于动脉周围淋巴鞘,在红髓和血窦两种细胞共存;淋巴结内只有少数ＴＣＲ细胞位于滤泡间或副皮质,滤泡内则未见。消化管内的ＴＣＲ细胞主要分布在小肠的固有膜,而胃、大肠和阑尾的固有膜内很少见;肝内ＴＣＲ细胞主要集中在血管和血窦周围;皮肤切片内的少数ＴＣＲ细胞见于真皮内,表皮基底层细胞内未见。这些细胞在胎儿期的免疫皮应及其生理功能还不清楚。     

Alternative splicing of mRNA of mouse interleukin-4 and interleukin-6

Interleukin-4 and interleukin-6 are multifunctional regulatory proteins, which participate both in haemopoiesis and in immunopoiesis. The alternative splicing of these interleukins in humans is known to proceed in a tissue-specific manner. Additionally, changes in splicing can also be dependent on tissue pathology. In this work, we report on the presence of alternatively spliced mRNA (IL-4delta2mRNA), lacking exon 2, in mouse bone marrow and spleen cells. We find that in unstimulated cells IL-4mRNA levels strongly dominate over IL-4delta2mRNA levels. Both increase in response to stimulation, with the concentration of the alternative variant rising earlier and faster than that of the full-length variant. In all other tissues studied dominance of IL-4delta2mRNA over the full-length variant was not observed. In addition, we find expression of three forms of IL-6 mRNA: the full-length IL-6 mRNA, IL-6Delta3 mRNA, and IL-6Delta5 mRNA in the second and third trimester placenta tissue and in the spleen of mice immunized with a high dose of sheep erythrocytes. It is anticipated that translation of these mRNA variants can generate proteins capable of binding to some subunits of the IL-6 receptor, thus possessing effector function. Alternative splicing is discussed as a source of cytokines with new regulatory properties.     

CCR9A and CCR9B: two receptors for the chemokine CCL25/TECK/Ck beta-15 that differ in their sensitivities to ligand

We isolated cDNAs for a chemokine receptor-related protein having the database designation GPR-9-6. Two classes of cDNAs were identified from mRNAs that arose by alternative splicing and that encode receptors that we refer to as CCR9A and CCR9B. CCR9A is predicted to contain 12 additional amino acids at its N terminus as compared with CCR9B. Cells transfected with cDNAs for CCR9A and CCR9B responded to the chemokine CC chemokine ligand 25 (CCL25)/thymus-expressed chemokine (TECK)/chemokine beta-15 (CK beta-15) in assays for both calcium flux and chemotaxis. No other chemokines tested produced responses specific for the cDNA-transfected cells. mRNA for CCR9A/B is expressed predominantly in the thymus, coincident with the expression of CCL25, and highest expression for CCR9A/B among thymocyte subsets was found in CD4+CD8+ cells. mRNAs encoding the A and B forms of the receptor were expressed at a ratio of approximately 10:1 in immortalized T cell lines, in PBMC, and in diverse populations of thymocytes. The EC50 of CCL25 for CCR9A was lower than that for CCR9B, and CCR9A was desensitized by doses of CCL25 that failed to silence CCR9B. CCR9 is the first example of a chemokine receptor in which alternative mRNA splicing leads to proteins of differing activities, providing a mechanism for extending the range of concentrations over which a cell can respond to increments in the concentration of ligand. The study of CCR9A and CCR9B should enhance our understanding of the role of the chemokine system in T cell biology, particularly during the stages of thymocyte development.     

Microcirculation in mouse spleen (nonsinusal) studied by means of corrosion casts

Corrosion casts of mouse spleen, examined by scanning electron microscopy, enabled vascular pathways of the arterial, intermediate, and venous circulations to be traced over considerable distances. The arterial tree is surrounded by white pulp immediately upon entering at the hilus, and relatively few arterioles extend into red pulp. A profusion of capillaries is present in both periarterial lymphatic sheaths and lymphatic nodules, arranged as bifurcating systems (rather than anastomosing networks) terminating in the marginal sinus (MS) and marginal zone (MZ). The MS, which is situated between white pulp and MZ, consists of a discontinuous layer of flattened anastomosing spaces which are up to six times as large as those in rat spleen. Extensive filling of the entire MZ took place before appreciable filling of surrounding red pulp occurred. Capillary terminations in red pulp are always continuous with reticular meshwork, i.e., no evidence for a “closed” circulation was found. Casts of the venous origins support the classification “pulp venules” rather than “venous sinuses” and show major morphological differences from the richly anastomosing system of sinuses in rat. In the subcapsular region of mouse spleen large anastomosing veins ramify over the surface, with reticular meshwork occupying extensive areas between adjacent veins. For in vivo microscopy this arrangement offers advantages over that found in rat spleen (accompanying paper), where almost the entire surface is densely covered with venous sinuses.     

From insulin synthesis to secretion: Alternative splicing of type 2 ryanodine receptor gene is essential for insulin secretion in pancreatic β cells

Increases in the intracellular Ca²⁺ concentration in pancreatic islets, resulting from the Ca²⁺ mobilization from the intracellular source through the ryanodine receptor, are essential for insulin secretion by glucose. Cyclic ADP-ribose, a potent Ca²⁺ mobilizing second messenger synthesized from NAD⁺ by CD38, regulates the opening of ryanodine receptor. A novel ryanodine receptor mRNA (the islet-type ryanodine receptor) was found to be generated from the type 2 ryanodine receptor gene by the alternative splicing of exons 4 and 75. The islet-type ryanodine receptor mRNA is expressed in a variety of tissues such as pancreatic islets, cerebrum, cerebellum, and other neuro-endocrine cells, whereas the authentic type 2 ryanodine receptor mRNA (the heart-type ryanodine receptor) was found to be generated using GG/AG splicing of intron 75 and is expressed in the heart and the blood vessel. The islet-type ryanodine receptor caused a greater increase in the Ca²⁺ release by caffeine when expressed in HEK293 cells pre-treated with cyclic ADP-ribose, suggesting that the novel ryanodine receptor is an intracellular target for the CD38-cyclic ADP-ribose signal system in mammalian cells and that the tissue-specific alternative splicing of type 2 ryanodine receptor mRNA plays an important role in the functioning of the cyclic ADP-ribose-sensitive Ca²⁺ release.     

CD72几种新的剪切形式的发现及它们在红斑狼疮模型鼠中的差异表达

CD72是一个重要的B细胞特异性受体,它以多种选择性剪切形式存在.在小鼠脾细胞中发现并鉴定了8种新的CD72选择性剪切形式,这些剪切形式中包含有2种独特的插入片段,一种选择性剪切保留了一个内含子(intron1),而这个内含子被翻译成氨基酸序列后并没有改变前后外显子的读码框,另一种使用了一个位于内含子之内的3′剪切位点,从而产生移码,提前终止了蛋白质的开放读码框,称为3′AS(3′alternative splicingsite).比较了CD72所有剪切形式在BALB/C小鼠和NZB/W小鼠中的差异表达,发现:a.含有3′AS的剪切形式的表达都很少;b.WT, In1, In1-Ex3和-Ex3的表达在BLAB/C小鼠中比在NZB/W小鼠中高;c.没有ITIM2的-Ex2-Ex3剪切形式在NZB/W小鼠中有特异性高表达.这些结果提示,CD72的多种选择性剪切形式在调控B细胞受体信号转导过程中可能发挥着不同的作用,并与系统性红斑狼疮的发病密切相关.     

Tissue localization and CD8 accessory molecule expression of T gamma delta cells in humans

In this study, we used TCR isotype-specific antibodies to examine the frequency, phenotype, and histologic localization pattern of T gamma delta cells in humans. The TCR delta 1+ cells comprised an average of 15% of the splenic CD3+ cells and 7% of circulating T cells. The T gamma delta cells in these human tissues, like their avian counterparts, were often not "double-negative" for the CD4 and CD8 accessory molecules. Approximately 50% of the splenic delta+ cells expressed CD8, and 30% of the delta+ cells in blood were CD8+. T cells of both gamma delta and alpha beta TCR isotypes were exceedingly rare in the skin. The T gamma delta cells exhibited preferential homing to the sinusoidal areas (red pulp) of the spleen and into the epithelial layer of the intestine in humans, as had been previously noted in chickens. Although 80% of the T gamma delta cells in the human intestinal mucosa were localized in the epithelial layer, these cells represented only 5 to 10% of all the CD3+ T cells in this microenvironment. We conclude that T gamma delta cells represent a sizeable subpopulation of the T cells in human peripheral tissues. The phylogenetic conservation of the CD8 expression by peripheral T gamma delta cells and of their preferential homing pattern suggests a special role in bodily defense for this T cell subpopulation.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

The spleen and skin wound healing in Xenopus adults

In most vertebrates, the regenerative capacity to restore lost/damage tissues to original structure and functionality decreases at some time during ontogenesis. To evaluate the role of the acquired immunity in the decline of regenerative potential, we examined the cellular responses elicited in the spleen during skin repair in Xenopus adults. Modifications in the architecture were found to be induced and were remarkable 14 days postinjury when the spleen increased significantly in size. In white pulp, the periarteriolar lymphoid sheaths were associated with follicles having central light zones, morphologically similar to germinal centers. With the progress of healing, pigment‐containing cells were seen to accumulate in both white and red pulp regions. Moreover, compared to controls, the cells immunoreactive to anti‐cytokines (TNF‐α, TGF‐β1) and ‐iNOS increased from the first days after wounding. The 14th day, the positive cells formed a dense network of reticular cells in central regions of lymphoid follicles and more frequent reactive leukocytes were detected within the red pulp. A higher number of lymphoid cells immunostained with anti‐CD3ε were also observed in the perifollicular zone. The results suggest that the spleen of adult frogs is involved in skin wound healing with the expansion of lymphoid compartments. J. Morphol. 277:888–895, 2016. © 2016 Wiley Periodicals, Inc.