Ultrastructural characterization of human large granular lymphocyte subsets defined by the expression of HNK-1 (Leu-7), Leu-11, or both HNK-1 and Leu-11 antigens

A peroxidase-colloidal gold double labeling system in immunoelectron microscopy was used to investigate the ultrastructural features of human large granular lymphocytes (LGL) subpopulations. Three subsets of LGL, Leu-7+-Leu-11-, Leu-7+-Leu-11+, Leu-7- -Leu-11+, were characterized using combinations of the monoclonal antibodies anti-Leu-7 and anti-Leu-11. They showed different ultrastructural patterns. In fact, Leu-7+-Leu-11- cells showed a high nuclear/cytoplasmic ratio (N/C), a round nucleus, a cytoplasm with few organelles, and a rather even surface. Moreover, most of them lacked electron-dense granules. On the other hand, Leu-11+ cells displayed a low N/C, an irregular-shaped nucleus, and a cytoplasm containing a well-developed Golgi apparatus, many mitochondria, vacuoles, vesicles, and numerous electron-dense granules. Moreover, they exhibited an irregular cell surface. Thus, Leu-7+-Leu-11- cells seemed to represent an immature form of LGL, while cells expressing the Leu-11 antigen showed a fine structure specific for functional NK cells. Our findings suggest that the expression of HNK-1 (Leu-7) and Leu-11 antigens respectively represents subsequent stages in NK cell differentiation.     

Characterization of HNK-1+ (Leu-7) human lymphocytes: III. Interferon effects on spontaneous cytotoxicity and phenotypic expression of lymphocyte subpopulations delineated by the monoclonal HNK-1 antibody

Interferon (IFN) augments the cytotoxic function of natural killer (NK) and killer (K) cells. We have previously shown that all NK- and K-cell function resides in the HNK-1⁺ population of granular lymphocytes. The present experiments demonstrated that IFN significantly augmented the efficiency of purified HNK-1⁺ cells to perform both NK- and K-cell function. In contrast, HNK-1^? cells could not lyse target cells even in the presence of IFN. IFN did not generate new HNK-1⁺ cells from the pool of HNK-1^? cells. We then examined the possibility that IFN might induce the maturation of immature NK cells previously defined as having an HNK⁺T3⁺ phenotype and a paucity of cytoplasmic granules. However, no changes were observed either in the proportion of HNK-1⁺ cells expressing the T3 antigen or in the number of granules within each HNK-1⁺ cell even after an 18-hr incubation with IFN. While fresh HNK-1^? cells lack NK-cell function, they can acquire NK-like activity without expressing the HNK-1 antigen after incubation with either alloantigens or mitogens. When incubated further with IFN, these alloantigen- or PHA-activated HNK-1^? cells with NK-like activity demonstrated relatively little augmentation of their cytotoxicity. It is concluded that interferon exerts its influence on restricted subpopulations of cells, primarily HNK-1⁺ cells. Its mechanism appears to concern the cytotoxic event rather than influencing cellular maturation.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

The fine structure of HNK-1 (Leu7) positive cells

Summary The ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

The monoclonal antibody HNK-1 reacts with a human peripheral nerve ganglioside

The monoclonal HNK-1 antibody, a marker for human natural killer cells, strongly reacted with human peripheral nerve gangliosides in the enzyme-linked immunosorbent assay. Autoradiography after the binding of HNK-1 to thin-layer chromatograms of peripheral nerve gangliosides followed by radioiodinated goat anti-mouse IgM revealed that HNK-1 was reacting with a minor ganglioside that chromatographed between GM1 and GD1a. The antigen was insensitive to digestion with neuraminidase and pronase.     

Leu-7(HNK-1)在人胃肠内分泌细胞中的表达

Leu-7(HNK-1)是一种分子量为110KD的糖蛋白,它是人类NK和K细胞特异性的表面抗原。Leu-7单克隆抗体可特异性识别NK和K细胞表面抗原。本文应用ABC免疫组织化学染色技术研究了Leu-7在人胃肠道的定位和分布。结果发现,Leu-7免疫反应(Leu-7-IR)细胞主要分布于胃腺和肠腺的底部。偶见于肠绒毛上皮中,呈典型内分泌细胞的形态特征。相邻切片法免疫双标记证明多数胆囊收缩素(CCK-8),生长抑素(SS),胃泌素(G34)或5-羟色胺(5-HT)免疫反应阳性细胞呈Leu-7阳性。我们的发现支持了在神经,内分泌和免疫系统间存在相同或相似抗原决定簇的假说。     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein

Mouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of Mr 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.     

Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides     

The fine structure of HNK-1 (Leu7) positive cells. A study using an immunoperoxidase technique

The ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.     

A monoclonal antibody that recognizes both leb and Y (Ley) antigens

A hemagglutinating monoclonal antibody has been obtained from a mouse/mouse hybridoma after immunisation with the le^b-active oligosaccharide, lacto-N-difucohexaose I, coupled to edestin. The antibody agglutinated human red cells regardless of Lewis phenotype. Blood group O cells were strongly, agglutinated, and progressively weaker agglutination was observed with A₂, B and A₂B cells. Blood group A₁ and A₁B cells were not agglutinated.By examining the binding of the antibody to glycolipids and oligosaccharides it was shown that the Le^b and Y (Le^y)-haptens bind to a similar extent. Full binding activity was dependent on the presence of, both fucosyl residues.Abbreviations LND l lacto-N-difucohexaose l - IV²Fuc,lll⁴FucLcOse₄ LND l-OL, lacto-N-difucohexaitol l     

Demonstration of glucuronic acid on brain glycoproteins which react with HNK-1 antibody

Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.     

Lymphoma-selective antibody Lym-1 recognizes a discontinuous epitope on the light chain of HLA-DR10

 The selectivity of Lym-1 for malignant B lymphocytes makes this monoclonal antibody a promising candidate for the delivery of toxic agents to malignant B cells. The original immunogen used for the development of Lym-1 was Raji Burkitt’s lymphoma cell nuclei [Epstein A. L., Marder R. J., Winter J. N., Stathopoulos E., Chen F. M., Parker J. W., Taylor C. R. (1987) Cancer Res 47: 830]. The Lym-1 antigen was characterized at that time as a polymorphic HLA-DR variant. We prepared an affinity column using immobilized Lym-1 to isolate the Lym-1 antigen from Raji cell lysate. Immunological characterization of the immunoaffinity-purified Lym-1 antigen on Western blots led to the conclusion that the antigen is the β chain of HLA-DR10. This was confirmed by Edman sequencing of the isolated polypeptide chain. Western blots further show that the Lym-1 epitope is only recognized if the β chain disulfide bonds are intact. These results imply that Lym-1 binds a discontinuous epitope on the β chain of HLA-DR10. Received: 22 February 1996 / Accepted: 28 June 1996     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Enhanced expression of fibrillin-1, a constituent of the myocardial extracellular matrix in fibrosis

Fibrillin-1 localization in the myocardium and the modulation of its expression in cardiac fibrosis were examined. In normal rat hearts, fibrillin-1 was abundant throughout the myocardium as thin fibers that crossed over the perimysium and around arteries. After cardiac fibrosis was induced in rats by either 14-day ANG II infusion or 21-day DOCA-salt treatment [a high endothelin-1 (ET-1) model], fibrillin-1 immunostaining was stronger in the interstitium (2.8-fold and 4.4-fold increases, respectively, in each model), extended between myocytes, and accumulated in microscopic scars and in the perivascular area of both ventricles. mRNA analysis confirmed its enhanced ventricular expression in both groups of rats (2.5-fold and 6.6-fold increments, respectively, in each model). In 1B normotensive and 2C hypertensive transgenic mice, two lines expressing an ANG II fusion protein in cardiac myocytes, strong fibrillin-1 immunoreactivity was observed in the interstitium and around arteries (3.7-fold and 7-fold increases, respectively). ANG II and transforming growth factor-beta1 enhanced fibrillin-1 synthesis by cardiac fibroblasts. Some fibrillin-1 fragments interacted with RGD-dependent integrins, including alpha(8)beta(1)-integrin, of cardiac fibroblasts but not necessarily through the RGD motif. Our findings illustrate that fibrillin-1 is an important constituent of the myocardium. In vitro and in vivo evidence suggests that ANG II can directly induce fibrillin-1 expression in cardiac fibroblasts. This protein can thus contribute to reactive and reparative processes.     

Jellyfish mucin (qniumucin) extracted with a modified protocol indicated its existence as a constituent of the extracellular matrix

Jellyfish (JF) mucin (precisely, a mucin-type glycoprotein named qniumucin: Q-mucin) first discovered in JF is mainly composed of highly O-glycosylated domains, and its unique structure suggests its wide applications as a smart material. In this study, the standard protocol used to date was thoroughly reinvestigated because the processing of raw JF was rather difficult and continuous production from frozen sources was also indispensable. Finally, we concluded that Q-mucin is involved not in mucus but in the mesoglea, i.e., the extracellular matrix (ECM), as a part of a very large polymer complex. We added a treatment procedure with a chelate reagent (e.g. EDTA) to inactivate endogenous proteases that induce the spontaneous decomposition of the collagens in ECM. The amino acid composition (AAC) of each precipitate formed upon EtOH addition indicated that Q-mucin dissociates from the biopolymer complex as a constituent highly soluble in deionized water. Since the remaining portion of ECM still seemed to contain a large amount of the precursor of Q-mucin even after the extraction with water is completed, the yield of Q-mucin is expected to increase markedly if an innovative method to decompose EtOH precipitates is developed. The existence of Q-mucin in ECM seems to be described in parallel with that of proteoglycans (PG) in mammalian cartilage because they resemble each other.