A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family

Monoclonal antibodies in the Hermes family recognize a lymphocyte structure that participates in lymphocyte adhesion to endothelium and has been suggested to be the human homolog of the murine Mel-14 lymph node homing receptor. Recently, antibodies against the Hermes antigen, the polymorphic glycoprotein Pgp-1 antigen, and the broadly expressed CDw44 antigen have been shown to recognize the same structure. In this work, cDNA clones encoding the CDw44 antigen were isolated and expressed in COS cells. Two forms were identified: a lymphoid form expressed in hematopoietic cells, and an epithelial form weakly expressed in normal epithelium but highly expressed in carcinomas. The extracellular domain of CDw44 bears homology to cartilage link proteins and a related segment of proteoglycan core protein. However, comparison with the recently identified sequence of the Mel-14 antigen shows that CDw44 and Mel-14 are unrelated.     

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

E-cadherin和CD44V6在食管上皮癌变过程及癌组织中的表达

研究不同类型的食管上皮增生和癌组织的 E- cadherin (E- cad)和 CD44 V6的表达 ,并探讨其与食管癌发生和发展的关系。应用免疫组织化学 SABC法 ,观察 10例正常、 3例消化性溃疡、 2 5例单纯性增生、 15例不典型增生的食管粘膜上皮 ,5例食管原位癌与 5 4例浸润癌组织中的 E- cad和 CD44 V6蛋白的表达情况。结果显示正常食管鳞状上皮和高分化肿瘤细胞膜和细胞浆 E- cad和 CD44 V6染色 ,非典型增生、低分化肿瘤细胞两种蛋白抗体表达减弱或呈阴性。E- cad和 CD44 V6的表达与癌组织的组织学分级、类型和淋巴结转移有关 (P<0 .0 1,P<0 .0 5 ) ,与癌组织的浸润深度无关 (P>0 .0 5 )。提示E- cad和 CD44 V6表达减弱是癌组织低分化和高度恶性的生物学标志 ,但其与淋巴结转移的关系有待进一步研究     

乳腺癌细胞hMSH2 蛋白、CD4、CD8的表达及其与淋巴结转移的关系

目的:探讨乳腺癌细胞hMSH2蛋白、CD4和CD8表达与淋巴结转移的关系及临床意义.方法:采用免疫组织化学方法检测49例乳腺癌(淋巴结转移25例,无淋巴结转移24例)患者癌细胞hMSH2蛋白、CD4和CD8的表达,并分析其表达与淋巴结转移的关系.结果:hMSH2蛋白在乳腺癌细胞细胞核内表达,CD4和CD8在乳腺癌细胞细胞浆和细胞膜表达.淋巴结转移组与无淋巴结转移组hMSH2蛋白、CD4和CD8的表达率分别为29%和68%(P＜0.05)、58%和44%(P＞0.05)、29%和68%(P＜0.05).结论:在有淋巴结转移的乳腺癌细胞hMSH2蛋白、CD4、CD8表达的改变可能与乳腺癌淋巴结转移有关.     

Epithelial cell-specific laminin 5 is required for survival of early thymocytes

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.     

Cellular specificity for the activation of fibroblast growth factor-2 by heparan sulfate proteoglycan

Heparan sulfate proteoglycans (HSPGs) promote cellular proliferation through interaction with FGF-2. To examine the role of cellular specificity of HSPG in FGF-2 function, a recombinant soluble isoform of CD44 (rsCD44v3,8-10) was expressed in various cell types; 293 T fibroblasts, the epithelial carcinoma cell lines A431 and HOTZ, the myelomonocytic cell line THP-1, and the Ig-secreting B lymphoblast IM9. The capacity of the recombinant HSPGs expressed in these cell lines to bind and present FGF-2 to the high-affinity receptor FGFR1 was addressed. This novel approach showed a minor difference in the binding and in the FGF-2 stimulating activity of rsCD44v3,8-10 HSPGs from fibroblasts and epithelial cells. However, FGF-2 binding of rsCD44v3,8-10 from IM9 and THP-1 cells was significantly lower, and stimulation of FGF-2 by rsCD44v3,8-10 from these two cell types could not be detected. We tested the possibility that the differences among cell types were related to the functional profile of endogenous HSPGs. The initial survey of a wider panel of cell types revealed high levels of HSPGs synthesis on the surface of 293 T, epithelial and IM9 cells, but low levels on the surface of other cells of hematopoietic origin. Surprisingly, native HSPGs from fibroblasts and epithelial cell lines promoted FGF-2 biological activity to vastly different extents, and cell surface HSPGs from IM9 cells induced an FGF-2 response. Altogether, the results suggested a role for cell-specific HS modification in addition to synthesis as regulatory mechanisms for the cellular specificity of proteoglycan function.     

Expression and modulation of CD44 variant isoforms in humans

CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.     

CD44 is the principal cell surface receptor for hyaluronate.

CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracelular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluonate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.     

Inflammatory cytokines can enhance CD44-mediated airway epithelial cell adhesion independently of CD44 expression

In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (+/- 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited >95% (P < 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-alpha, IFN-gamma, IL-1 beta, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-gamma treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-gamma-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression.     

Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes.

Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. In this report, we demonstrate that the recently described alpha 6 beta 4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha 6 beta 4 is controlled by regulation of beta 4 expression, because alpha 6 was expressed by virtually all cells examined, paired with the beta 1 integrin chain to form VLA-6. During fetal ontogeny, beta 4 was highly expressed by 35% of day-13 thymocytes, 75% of day-14 to -15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta 4 expression was highest on CD4- CD8- CD3- and TCR(+)-gamma delta subsets. Correlation of IL-2R, CD44 and beta 4 on CD4- CD8- thymocytes revealed maximal levels on the intermediate CD44- IL-2R+ subset. Most CD4- CD8+ TCR- thymocytes and a significant fraction of CD4+ CD8+ thymocytes were beta 4lo, whereas the most mature J11d- single positive thymocytes were beta-4. Overall, down-regulation of beta 4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. alpha 6 beta 4 was undetectable on fetal liver or bone marrow cells, lymphocytes from lymph node, spleen, or blood, and mitogen-activated splenic T cells cultured up to 10 wk with IL-2. The data suggest that alpha 6 beta 4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells. The developmentally regulated pattern of expression and the prominence of alpha 6 beta 4 on day-13 to -16 fetal and adult CD4- CD8- CD3- thymocytes further suggest this unusual integrin may play a role in early T cell development, including stages before acquisition of the TCR.     

Histological study of stem-like cells in human colon adenocarcinoma at different stages of the disease

Abstract

The presence of stem-like cells in tumors reflects the invasive character of the disease; however, their identification is controversial. We investigated the distribution of CD133, CD44 and CD24 using histological sections and tissue microarrays (TMAs) of human colon adenocarcinoma obtained from patients with and without lymph node metastases and/or liver metastases. Immunohistochemical staining was combined with nuclear staining and evaluated quantitatively using image analysis software. Sections of normal colon mucosa, the primary tumor, lymph node, and liver also were analyzed qualitatively and compared to the quantitative method, which was more accurate. In most tissues, the expression of CD44 and CD24 was relatively low compared to CD133, with some variations. CD133 also was expressed in the normal colon mucosa and to a lesser degree in normal hepatic parenchyma. Liver metastases exhibited significantly greater CD133 staining compared to normal colon mucosa, primary tumor and lymph node metastases. Moreover, lymph node metastases obtained from patients with liver metastases expressed significantly greater CD133 staining than those obtained from patients without liver metastasis. Our data suggest that CD133 expression in lymph node metastases may be of value for prognosis of the disease.     

Phenotypic characteristics associated with the acquisition of HSV-specific CD8 T-lymphocyte-mediated cytolytic function in vitro.

Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.     

Schistosoma mansoni eggs induce antigen-responsive CD44-hi T helper 2 cells and IL-4-secreting CD44-lo cells. Potential for T helper 2 subset differentiation is evident at the precursor level.

Schistosoma mansoni eggs are potent inducers of biased Th2-like immune responses. Using a model system where mice are immunized with isolated schistosome eggs, we demonstrate that CD44 expression, up-regulation of which has been linked to Th cell development, is increased on Th2 cells. We also investigate the functional properties of CD44-lo Th cells recovered from the overtly Th2 environment constituted by lymph nodes draining sites of egg deposition. Production of high levels of IL-4, IL-5, and IL-10 by Th cells responding to egg Ag is shown to be the property of a subpopulation expressing CD44-hi. This population of Th cells cosegregates with a blasting subpopulation expressing more IL-4R (but similar amounts of IL-2R) than Th cells from normal mice. These results indicate that mature Th2 cells responding to schistosome eggs are CD44-hi and suggest that they use IL-4 as a growth factor. In contrast, CD44-lo cells sorted from lymph node populations responding to eggs are able to produce small amounts of IL-4 and IL-2, but no IL-5 or IL-10. This is surprising, because low expression of CD44 is considered a characteristic of Th cell naivite and concomitant ability to produce only IL-2. Thus, in lymph nodes responding to schistosome eggs, potential for Th2 subset differentiation is evident within the CD44-lo precursor Th subpopulation.     

Immunohistochemical assessment of the tumour-associated epitopes CD44v6 and E48 in tumour-free lymph nodes from patients with squamous cell carcinoma in the head-neck region.

We examined immunohistochemically 370 tumour-free lymph nodes from 41 patients with a head and neck squamous cell carcinoma (HNSCC) to clarify whether the tumour-associated epitopes CD44v6 and E48 are suitable for adjuvant postoperative immunotherapy. All the positively immunostained cells found were single cells. CD44v6+ cells were found in 55% of the lymph nodes, with their numbers increasing in pN>0-patients (62%). Only pN>0-patients had abundant to massive CD44v6+ cells. A comparison with mononuclear cells in lymphatic tissue from control patients suggested a similarity with activated T-cells. In the 41 cancer patients there were significantly fewer lymph nodes with E48+ cells (11%), but the number of E48+ cells increased in pN> 1-patients (29%) with predominantly abundant E48+ cells. We conclude from the comparison with the epithelial marker EMA that the E48+ single cells are epithelial in origin. Only a specific E48 peptide sequence appears suitable for adjuvant immunotherapy in patients with head-neck tumours.     

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions.     

The existence of epithelial-to-mesenchymal cells with the ability to support hematopoiesis in human fetal liver

The fetal liver is the major hematopoietic organ during mid-gestation, and it is also a source of stem cells that exist in a complex environment. In this study, we isolated a population of actively replicating cells with the characteristic of the epithelial-to-mesenchymal transition (EMT) from fetal liver. These cells were identified with the epithelial markers, including alpha-fetoprotein (AFP), albumin (ALB), cytokeratins (CK) 7, and CK18, as well as the mesenchymal markers, such as alpha-smooth muscle actin (ASMA), CD29, CD44, CD49, CD54, collagen I and osteopontin (OPN). Furthermore, they also expressed some hematopoiesis-related genes. In addition, the cell population had the ability to retain hematopoietic stem cells (HSCs) in an undifferentiated state in vitro during cytokine-stimulated proliferation. These results provide an insight about early human liver development and may also help to understand hematopoiesis in the fetal liver.