Lectin binding sites on CD34+ human haematopoietic stem cells and lymphocytes from peripheral blood: an ultrastructural post-embedding study

This study was performed to obtain a better insight into the glycosylation pattern of human CD34⁺ haematopoietic stem cells and lymphocytes from peripheral blood using an ultrastructural post-embedding technique. Lectins applied were derived from Canavalia ensiformis (Con A), Triticum vulgare (WGA), Lycopersicon esculentum (LEA), Limulus polyphemus (LPA), Ulex europaeus-I (UEA-I), Bauhinia purpurea (BPA), Glycine max (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA) and Erythrina cristagalli (ECA). Our results showed almost identical staining patterns with both CD34⁺ cells and mature lymphocytes from peripheral blood. Con A displayed a prominent reactivity with the nuclear envelope and a weak staining of the plasma membrane. As demonstrated by an elaborate lectin double-labelling technique, WGA revealed an opposite staining pattern. Following neuraminidase treatment of sections, BPA, PNA and SBA exhibited a prominent staining of the plasma membrane in CD34⁺ cells and lymphocytes as well. Membrane reactivity with HPA was restricted to the majority of lymphocytes, presumably T-lymphocytes. Infrequently occurring dense cytoplasmic (lysosomal) bodies were reactive with a variety of lectins, and a weak diffuse nuclear labelling was observable with LPA, UEA-I, WGA and Con A. It is tempting to speculate that carbohydrate moieties on plasma membranes may be involved in the complex mechanisms characterizing cell-to-cell interactions (adhesion) and particularly in the so-called phenomenon of homing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study

Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19-OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib.     

Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining

In order to isolate and enrich bone marrow mononuclear phagocytes, we performed magnetic-activated cell sorting using beads coupled to a monoclonal antibody directed against the monocyte/macrophage surface molecule CD14. Co-localization of antigens in single cells was achieved by combining an alkaline phosphatase--anti-alkaline phosphatase and an avidin--biotin complex immunoassay, avoiding the use of peroxidase. Bone marrow macrophages were first labelled by the monoclonal antibody PG-M1 (anti-CD68). Subsequently, cytoplasmic and/or surface double staining by the monoclonal antibodies against HLA-DR and Mac-2 antigen or the lectin GSA-I-B4 was carried out. Whereas HLA-DR was co-expressed by the great majority of PG-M1+ macrophages (84.9% +/- 6.9%), only a subpopulation exhibited Mac-2 (69.9% +/- 5.9%) antigen or galactoside structures detected by GSA-I-B4 (65.0% +/- 6.7%). The latter result differed only slightly from the percentage of GSA-I-B+4 macrophages determined in a previous comparative immunomorphometrical study. Therefore, using our method of isolation and enrichment by magnetic-activated cell sorting, only a negligible portion of macrophages is apparently stimulated, as shown by GSA-I-B4 staining. This methodology seems to be a valuable tool for further studies on the monocyte--macrophage system. © 1998 Chapman & Hall     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.     

Studies of lectin binding to the human cervix uteri: I. Normal cervix

Summary Lectins ofBauhinia purpurea (BPA),Canavalin ensiformis (Con A),Griffonia simplicifolia I (GS I),Griffonia simplicifolia II (GS II),Maclura pomifera (MPA),Arachis hypogaea (PNA),Glycine max (SBA),Ulex europaeus I (UEA I) andTriticum vulgaris (WGA) were used to evaluate cell surface carbohydrates in formalin-fixed paraffin-embedded tissue sections of normal human cervix uteri. Consistent patterns of staining of the squamous epithelium were obtained in all 30 cases with BPA, GS II, MPA, PNA, SBA and WGA. A variable distribution of lectin binding was seen in squamous epithelium with Con A, GS I and UEA I. The patterns of GS I and GS II binding reflected squamous epithelial maturation. Columnar epithelium did not stain with GS II, stained variably with Con A, and stained consistently with the remaining seven lectins in all cases. No association between lectin binding and blood group or phase of the menstrual cycle was found. These findings may be used as a baseline for evaluation of lectin binding in both preinvasive and invasive lesions of the cervix uteri.     

Plasma membrane alterations of maturing goat (Capra indicus) spermatozoa: Lectin-binding and freeze-fracture study

Summary A qualitative and quantitative analysis of lectin-binding sites has been undertaken on spermatozoa recovered from different regions of the epididymis of the goat (Capra indicus) using fluorescein isothiocyanate-linked lectins (Bauhinia purpurea BPA, Concanavalin A Con A, Dolichos biflorus DBA, Maclura pomifera MPA, Arachis hypogaea or peanut agglutinin PNA, Glycine max or soyabean agglutinin SBA, Ulex europaeus UEA, and Triticum vulgaris or wheat-germ agglutinin WGA), in conjunction with scanning and transmission electron microscopy, and freeze-fracture techniques. Flow cytometric analysis has also been used to quantitize binding affinity. Spermatozoa from caput to cauda epididymidis show no significant variation in lectinbinding ability, but the samples removed from the corpus epididymidis contain a greater number of binding sites. The passage of spermatozoa through the epididymidis is accompanied by a redistribution of the plasma membrane lectin-receptors covering the sperm head and tail. Receptors for BPA, DBA, PNA and SBA are specifically restricted to the anterior region of the acrosome in caudal spermatozoa. Freeze-fracture replicas, examined to study changes in organisation of intramembranous particles of the plasma membrane during sperm maturation, reveal distinct changes in their distribution in the acrosome, post-acrosome and spermatozoon tail, especially in the corpus and cauda epididymidis.     

Identification of macrosialin (CD68) on the surface of host macrophages as the receptor for the intercellular adhesive molecule (ICAM-L) of Leishmania amazonensis

The intercellular adhesive molecule, ICAM-L, of Leishmania amazonensis is known to block the attachment as well as internalisation of Leishmania for infection in host macrophages. We employed monoclonal antibodies (mAb) to the surface molecules of a macrophage to block the attachment of ICAM-L to the macrophage surface and identified that CD68 macrosialin is likely the receptor molecule on the macrophage for ICAM-L. We then demonstrated physical interaction between ICAM-L and macrosialin by co-immunoprecipitation of macrosialin with ICAM-L or vice versa. Finally, macrosialin is expressed in macrosialin-negative murine fibroblast cell line NCTC clone 2555 and demonstrates that both ICAM-L and promastigotes of L. amazonensis can bind to the CD68 transfectant. We thus conclude that CD68 macrosialin is the receptor on host macrophages for ICAM-L. Also, involvement of ICAM-L-macrosialin interaction in other Leishmania species and other mammalian macrophages were demonstrated, indicating the biological relevance of this ligand–receptor interaction.     

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of .DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1⁺/SP-A⁺ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

Impaired tumor antigen processing by immunoproteasome-expressing CD40-activated B cells and dendritic cells

Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1_26?C34 (M26) and Cyp1B1_239?C247 (Cyp239)] and one derived from the influenza A viral antigen [FluM1_58?C66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.     

Localization of tartrate-resistant acid phosphatase in human placenta

Summary Tartrate-resistant acid phosphatase is an inducible marker of cell differentiation and activation expressed by specialized cells of macrophage lineage and some activated lymphocytes. Clinically, this phosphatase is a diagnostic marker for hairy cell leukaemia and osteoclast activity. The cDNA for this enzyme has been cloned from a placental expression library, yet the cell(s) expressing the enzyme protein has not been determined with certainty. Our laboratories have developed a monoclonal antibody, 9C5, suitable for immunohistochemical localization of tartrate-resistant acid phosphatase in paraffin sections. The purpose of this study was to use antibody 9C5 to identify cells expressing tartrate-resistant acid phosphatase in sections of paraffin-embedded, normal, full-term placenta and to determine if those cells expressed other macrophage markers including CD68(PG-M1 antibody), LN5, lysozyme ₁-antitrypsin and ₁-antichymotrypsin. Histochemical localization of activity in frozen sections was compared with immunohistochemical localization in paraffin sections of the same tissue specimens. The activity and antigenicity of this enzyme were detected in decidual cells, syncytiotrophoblast, and some macrophages distributed throughout maternal and embryonic tissues, but not in neutrophils. Unlike other tissues previously examined, placenta contains significant numbers of the phosphate-positive cells that are not of macrophage origin.     

A differential response elicited in macrophages on interaction with lectins

The interaction of several lectins, both native and chemically modified, with mouse peritoneal macrophages was studied. Surface distribution and interiorization of the lectins was assessed quantitatively using their radioactively-labeled derivatives, and qualitatively by employing fluorescein-labeled lectins. On the basis of their effect on the macrophages, the lectins tested fall into two classes: lectins that induce vacuole formation in the cells (concanavalin A (ConA), wax bean agglutinin (WBA) and wheat germ agglutinin (WGA)) and lectins that in their native form do not induce vacuolation (soybean agglutinin (SBA), peanut agglutinin (PNA) and the lectin from Lotus tetragonolobus (LT)). Neuraminidase treatment of the cells did not change their response to the lectins, though in the case of SBA and PNA binding was observed only with neuraminidase-treated macrophages. Incubation of the latter cells with SBA and subsequently with ConA resulted in significantly higher vacuolation than that observed with ConA alone. Glutaraldehyde-crosslinked polymers of SBA and of PNA, which are multivalent with respect to sugar binding, induced vacuolation in neuraminidase-treated macrophages. On the other hand, succinylation of ConA, which reduces the number of sugar binding sites per mole from four to two, abolished its ability to induce vacuole formation. These data suggest that multivalency of lectins and probably also their size are important factors in inducing vacuolation, by causing extensive crosslinkage of membrane receptors which is prerequisite for triggering of vacuole formation. Quantitative binding and internalization data indicate that vacuole formation is not directly related to the number of lectin receptors on the macrophages nor to the extent of their internalization.     

Development of a bispecific F(ab′)2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity

 A bispecific F(ab′)₂ antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and a variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab′ fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5′-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of “clump-like” cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively. Received: 2 May 1996 / Accepted: 21 May 1996     

Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Human Mycobacterium tuberculosis CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library

Identification of CD8⁺ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8⁺ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8⁺ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8⁺ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ_24–34, B3905-restricted PE9_53–67, and B3514-restricted PE_PGRS42_48–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8⁺ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.     

Decidual macrophages and their roles at the maternal-fetal interface

The semi-allogeneic fetus, whose genome consists of maternally and paternally inherited alleles, must coexist with an active maternal immune system during its 9 months in utero. Macrophages are the second most abundant immune cell at the maternal-fetal interface, although populations and functions for these populations remain ill defined. We have previously reported two distinct subsets of CD14(+) decidual macrophages found to be present in first trimester decidual tissue, 20 percent CD11c(HI) and 68 percent CD11c(LO). Interestingly, CD11c(HI) decidual macrophages express genes associated with lipid metabolism, inflammation, and antigen presentation function and specifically upregulate CD1 molecules. Conversely, CD11c(LO) decidual macrophages express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The large abundance of CD11c(HI) decidual macrophages and their ability to process antigens more efficiently than CD11c(LO) macrophages suggests that CD11c(HI) macrophages may be important antigen processing and presenting cells at the maternal-fetal interface, while CD11c(LO) macrophages may perform necessary homeostatic functions during placental construction. Thus, macrophage heterogeneity may be an important and necessary division of labor that leads to both an induction of maternal immune cell tolerance to fetal antigens as well as basic homeostatic functions in human pregnancy.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Roles of macrophages in programmed cell death and remodeling of tail and body muscle of Xenopus laevis during metamorphosis

 Examination was made of the involvement of macrophage phagocytosis in programmed cell death of tail and body muscle of the frog, Xenopus laevis, during metamorphosis by electron microscopy and immunohistochemical analysis. Electron microscopic observation revealed that macrophages were often found to be present in body and tail muscles at the most active stage of metamorphosis and to actively phagocytose apoptotic muscle fragments. Developmental changes in macrophages were examined using the macrophage-specific antibody, HAM56. Macrophages initially appeared in the early climax stage (stage 59), when the triiodothyronine (T₃) level was high, increased rapidly during the process of muscle cell death, and assumed their greatest number at the late climax stage (stage 63/64). They decreased after stage 65/66, with a decrease in T₃. Distribution and change in the number of macrophages were the same as those of muscle apoptotic bodies (sarcolytes) during metamorphosis, which suggests an interactive mechanism between macrophages and dying muscle cells. For clarification of this, study was made of the expression of HAM 56 antigens that were X. laevis homologs of mouse attachmin, non-specific adhesion proteins in macrophages. The expression of HAM56 antigens in macrophages was found to increase with macrophage phagocytosis at the late climax stage, thus, macrophage differentiation would appear to take place during metamorphosis and HAM56 antigens may be essential for macrophage–dying muscle cell interactions. Accepted: 29 May 1997