Human mast cell carboxypeptidase. Selective localization to MCTC cells

Two murine mAb were prepared against human mast cell carboxypeptidase (HMC-CP) purified from human skin, and were termed CP1 and CP2, respectively. Double immunohistochemical labeling of Carnoy's-fixed sections of human skin, lung, and gastrointestinal tissue with CP1 and CP2, respectively, and with a murine monoclonal antitryptase antibody demonstrated that HMC-CP was selectively present in a subset of human mast cells. Double labeling experiments with CP1 and CP2, respectively, and a murine anti-chymase mAb demonstrated the presence of HMC-CP in the tryptase-positive, chymase-positive mast cell type (MCTC) only. Immunohistochemical labeling of peripheral blood leukocytes resulted in staining of monocytes with CP2 but not with CP1. In addition to chymase and a cathepsin-G like proteinase, HMC-CP is another neutral protease that is selectively present in the MCTC tryptase-positive, chymase-positive mast cells type of mast cell, thus extending the biochemical definition of human mast cell heterogeneity.     

Evaluation of human peripheral blood leukocytes for mast cell tryptase

Murine monoclonal and goat polyclonal antibodies against tryptase, the dominant neutral protease and protein component in secretory granules of human mast cells, were used to assess the presence of tryptase in peripheral leukocytes. Carnoy's fluid-fixed cytocentrifuge preparations of enriched populations of lymphocytes, monocytes, eosinophils, and neutrophils showed no reactivity with anti-tryptase antibodies by a sensitive indirect immunoperoxidase procedure. Dispersed human lung mast cells showed strong granular cytoplasmic staining with both antibodies, whereas only approximately 50% of the peripheral blood basophils detectable with Wright's stain were detected with anti-tryptase antibodies, and these showed a staining pattern that was faint, granular, and cytoplasmic at high concentrations of antibody. At lower antibody concentrations mast cell staining was still intense, whereas basophils were not stained. Extracts of neutrophils and lymphocytes of up to 90% purity had undetectable amounts of tryptase by an ELISA sandwich immunoassay, as well as undetectable enzymatic activity with tosyl-L-gly-pro-lys-p-nitroanilide (a sensitive substrate for tryptase) in the presence of soybean trypsin inhibitor. Extracts of basophil-enriched (6 to 50% purity) preparations contained 0.046 +/- 0.013 pg of tryptase per basophil by the immunoassay along with 2 X 10(-9) +/- 0.8 X 10(-9) U of tryptase-like enzyme activity per basophil, compared with corresponding values of 12 pg, 480 X 10(-9) U of tryptase per human lung mast cell. Thus very small amounts of tryptase are present in human basophils (approximately 0.4% of that found in mast cells), but not in other peripheral leukocytes.     

An antibody raised against in vitro-derived human mast cells identifies mature mast cells and a population of cells that are Fc epsilon RI(+), tryptase(-), and chymase(-) in a variety of human tissues.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.     

In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

虎纹蛙消化道肥大细胞类胰蛋白酶免疫组化研究

研究采用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体AA1,应用ElivisionTM plus免疫组化染色法对虎纹蛙(Rana tigrina rugulosa)消化道组织中类胰蛋白酶阳性肥大细胞存在的可能性进行研究。研究发现单克隆抗体AAl可与中性缓冲福马林液固定的虎纹蛙组织的肥大细胞获得良好的交叉反应,类胰蛋白酶阳性细胞胞浆染成棕黄色,证实虎纹蛙肥大细胞胞浆颗粒中也存在类胰蛋白酶。虎纹蛙组织中AA1免疫染色阳性细胞的分布,与AB/SO和改良甲苯胺兰染色阳性细胞的分布存在较大的差异:虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,主要见于黏膜型肥大细胞(MMC)分布区域,如消化道黏膜上皮下方和固有层,少量分布于肠绒毛基底部及食管腺和胃腺周围。而在结缔组织型肥大细胞(CTMC)分布区域,如消化道黏膜下层结缔组织中却未见类胰蛋白酶阳性细胞。AB/SO和改良甲苯胺兰染色阳性细胞数量多,广泛分布于消化道黏膜固有层、黏膜下层、腺体之间、肌间及外膜结缔组织,说明并不是所有的虎纹蛙肥大细胞都含有类胰蛋白酶。很有可能是虎纹蛙MMC中含有类胰蛋白酶,而CTMC中不含类胰蛋白酶。虎纹蛙类胰蛋白酶阳性细胞数量很少,且阳性反应比人胃癌间质肥大细胞弱,说明虎纹蛙肥大细胞胞浆颗粒类胰蛋白酶含量较少,虎纹蛙属于低等脊椎动物,可能与生物进化水平较低有关,有待进一步研究。       

Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells

Two murine monoclonal antibodies were prepared against tryptase, the major neutral protease and protein component of human mast cells. The antibodies were termed G5 (IgG2B-kappa) and H4 (IgG1-kappa). They were specific for tryptase by an enzyme-linked immunosorbent assay and an immunotransblot technique. The latter procedure showed that H4 and G5 each bind to the 35,000 and 37,000 m.w. subunits of tryptase, indicating immunologic cross-reactivity between the subunits. The monoclonal antibodies reacted only with tryptase subunits in an extract of dispersed lung cells. By immunofluorescence microscopy, tryptase was further identified to be present only in cytoplasmic granules of Alcian Blue-stained mast cells in dispersed pulmonary cell preparations. No evidence for a mast cell subtype lacking tryptase was detected. In addition, a procedure for the purification of tryptase to homogeneity from dispersed pulmonary cells containing less than 10% mast cells was developed; this procedure involved high salt extraction, ammonium sulfate precipitation, and sequential chromatography with decyl-agarose, DEAE-agarose, and heparin-agarose. The procedure resulted in a higher yield even with less pure starting material than reported previously. Tryptase is a selective marker for mast cells in dispersed pulmonary cells, and can be detected with specific anti-tryptase antibodies.     

Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens

The expression of complement receptors by human follicular dendritic cells (FDC) was investigated by immunohistochemical techniques by using polyclonal and monoclonal antibodies to antigenic determinants of CR1, CR2, and CR3. Upon optical immunohistochemical examination of frozen sections from human reactive lymph nodes and tonsils by a three-step immunoperoxidase technique, a strong staining of cell bodies and cytoplasmic extensions of FDC was observed in germinal centers with anti-CR1 and anti-CR2 antibodies. Staining for these antigens was also found on cytoplasmic extensions of FDC in the mantle zone and on the plasma membrane of B cells in the entire follicles. Staining of FDC with anti-CR2 antibody was more intense than that of B lymphocytes. Monoclonal antibodies directed against epitopes of the alpha-chain of CR3 weakly stained FDC in follicles in a similar pattern to that which was observed on adjacent sections with mouse monoclonal antibody KIM4 that only recognizes FDC in human lymph nodes. Immunoelectron-microscopy was performed on frozen sections of a lymph node involved with a centroblastic centrocytic B malignant lymphoma and a reactive tonsil with the use of rabbit F(ab')2 anti-CR1 antibodies and mouse monoclonal anti-CR2 antibody. All the plasma membrane of the cell body and cytoplasmic extensions of FDC in germinal centers and in the mantle zones homogeneously stained for CR1 and CR2 antigens. Fibroblastic reticulum cells were negative. The plasma membrane of tumoral B lymphocytes strongly stained with anti-CR1 and weakly stained with anti-CR2 antibodies. The presence of CR1, CR2, and CR3 on FDC is a unique surface characteristic of these cells that should optimally allow the cells to bind antigen/antibody complexes bearing any type of C3 fragment.     

Localization of a novel integrin of the beta 1 subfamily in human tissues.

We have previously described a novel integrin composed of a beta 1-chain non-covalently linked to an alpha-chain which is biochemically different from those known so far (i.e., alpha 1-alpha 7 and alpha v). This molecule has been identified with a monoclonal antibody (MAb) termed 10.1.2 raised against long-term cultured human thymic epithelial cells (TEC). In this study we analyzed the immunohistochemical distribution of this new integrin in a variety of human tissues. MAb 10.1.2 stains several types of endothelial and epithelial cells. Among the endothelia, a strong reaction was detected in the HEV of lymphoid organs including thymus, lymph node, tonsil, and mucosa-associated lymphoid tissue. Epithelial localizations of note were those in the basal layer of the epidermis and of other stratified squamous epithelia, where the lateral and apical but not the deep surfaces of most cells were stained. A variety of water-electrolyte transporting cells in sweat glands, salivary glands, and kidney were also stained at their deep surface. The latter findings suggest that this molecule may subserve other functions in addition to those related to cell adhesion.     

The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells

Summary An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MC_T) or chymase together with tryptase (MC_TC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MC_T were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MC_T of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7–67%, average 30%), while this was not the case in the small intestinal mucosa (5–26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MC_T therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.     

用免疫荧光标记对人组织中肥大细胞亚型的分选与形态观察

方法:利用中性蛋白酶成分、特征性酶抗体的免疫荧光染色和流式细胞仪确定分选肥大细胞亚型,以激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒。结果:三种免疫表型被确定:肥大细胞的类胰蛋白酶阳性(MCT)、类糜蛋白酶阴性;类糜蛋白酶阳性(MCC)、类胰蛋白酶阴性和类胰蛋白酶阳性、类糜蛋白酶阳性(MCTC)。肥大细胞内分泌颗粒分散或聚集存在,分泌颗粒突起分泌或以分散的方式释放。分泌颗粒大范围释放后,肥大细胞的形态结构发生了改变。结论:利用肥大细胞的特征性酶抗体、免疫荧光标记和流式细胞仪可将人组织中的肥大细胞分选纯化为三种亚型;以共聚焦显微镜显示肥大细胞含有丰富的分泌颗粒,它说明肥大细胞具备了为人体I型变态反应提供快速反应的物质基础。     

Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.     

Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

Common antigens of oval cells and cholangiocytes in the mouse. Their detection by using monoclonal antibodies

Monoclonal antibodies (MAb) were produced against antigens (Ag) of oval cells isolated from the preneoplastic murine liver. To suppress the immune response to major antigens common with hepatocytes, the principle of anti-idiotype immunization was employed. Characteristics of three MAb reacting selectively with the foci of oval cell proliferation are described. MAb A6 and G7 detected two different antigens (Ag A6 and Ag G7, respectively) common for oval cells and cholangiocytes. Ag A6 was also found in normal parenchyma (in membranes of single hepatocytes adjacent to portal veins), in the preneoplastic liver (in hepatocytes formed de novo) and in some hepatoma cells. Ag G7 was not detected in hepatocytes. MAb E5 stained the matrix in the areas adjacent to oval cells and large bile ducts. All the three Ag were widely distributed in normal tissues of mice. The significance of the detected Ag as markers of murine liver epithelial cell lines and stages of their differentiation is discussed as well as the possible relationship between Ag A6 and Ag of human blood groups.     

Monoclonal antibody of IgG isotype against a cross-reactive lipopolysaccharide epitope of Chlamydia and Salmonella Re chemotype enhances infectivity in L-929 fibroblast cells

A murine monoclonal antibody (MAb) 202D7 of IgG3 isotype recognizes a lipopolysaccharide (LPS) epitope of Chlamydia spp. and cross-reacts with the Re chemotype LPS of Salmonella and Escherichia coli. The antibody exhibits strong complement activating properties and stimulates phagocytosis of Salmonella enterica serovar Minnesota Re mutant by murine macrophages. Salmonella Re mutants are non-invasive for cell monolayers but still can enter and replicate in L-929 murine fibroblast cells. The entry of bacteria within the cells increases five-fold in the presence of MAb 202D7. The antibody mediates attachment and enhances five-fold the infectivity of Chlamydia pneumoniae into L-929 cells, which suggests a possible IgG-mediated mechanism of entry and survival of the pathogen in fibroblast cells.     

Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis

The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.     

Identification, synthesis and properties of a consensus peptide recognized by a monoclonal antibody directed to various type I cytokine receptors

Previous works demonstrated that the monoclonal antibody (MAb) called R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones as well as interleukins 2 and 6 (IL-2 and IL-6). The MAb inhibited the biological effects of those hormones and cytokines by impairing their binding to receptors. It is known that the receptors for growth hormones (GH), prolactins (PRL), IL-2, and IL-6 pertain to the type I cytokine receptor family, sharing the common motif WSXWS or the homologous F(Y)GEFS. Thus, a set of 34 decapeptides corresponding to diverse receptors containing those sequences were synthesized by the PEPSCAN method and their reactions with MAb R7B4 were measured by ELISA. The MAb significantly recognized 21 peptides, allowing us to establish the consensus sequence HGYWSEWSPE as a portion of the R7B4 epitope. The consensus peptide was synthesized and purified by conventional methods, and its capacity to bind to MAb R7B4 paratope confirmed. Moreover, polyclonal Ab to the peptide elicited in mice were able to inhibit the hGH binding to lactogenic, somatogenic and human specific liver receptors. This fact suggests that the consensus peptide could be used as an immunogen to produce anti-hGH receptor Ab behaving as hormone or cytokine antagonists in certain pathological conditions.     

Deficiency of the tryptase-positive, chymase-negative mast cell type in gastrointestinal mucosa of patients with defective T lymphocyte function

The distribution and concentration of human T (tryptase-positive, chymase-negative) and TC (tryptase-positive, chymase-positive) mast cells were examined in Carnoy's-fixed specimens of the gastrointestinal tract of normal individuals, patients with inflammatory bowel diseases, and patients with immunodeficiency disorders. In normal specimens, T mast cells predominated in the mucosa (89%), with a mean concentration of 17,850 +/- 4,998 per mm3 (+/- SD, n = 16), whereas TC mast cells predominated in the submucosa (90%) with a mean concentration of 7,516 +/- 1,227 per mm3 (+/- SD, n = 16). The concentrations of T and TC mast cells in specimens of ileum from five patients with active Crohn's disease and of colon from three patients with active ulcerative colitis were not significantly different (p greater than 0.4) from normal values. Three patients with combined immunodeficiency disorders demonstrated a marked decrease in the concentration of the T mast cells in the intestinal mucosa, to 540 +/- 630, and a corresponding decrease in the percentage of T mast cells to 9%. Concentrations of TC mast cells were unchanged, both in the mucosa and in the submucosa. In three patients with acquired immunodeficiency syndrome, a similar deficiency of the T mast cell type was observed in the ileal mucosa, with a mean concentration of 788 +/- 534 T mast cells per mm3, but not in the appendiceal and colonic mucosa of one of the three patients. These findings indicate a role for functional T lymphocytes in the development of the T mast cell type in humans, and suggest divergent pathways for development of T and TC mast cells.     

Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines

EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.     

Human dendritic reticulum cells of lymphoid follicles: their antigenic profile and their identification as multinucleated giant cells

The B-dependent areas of human lymphoid tissue contain non-lymphoid, non-phagocytic cells known as dendritic reticulum cells (DRC). These cells can be detected only very occasionally in routinely stained histologic sections. Recently we were able to overcome this limitation by preparing a monoclonal antibody, termed R 4/23, that reacts selectively with DRC. Thus by using an optimized immunoperoxidase method applied to frozen sections, it is possible to detect DRC in situ. To determine the antigenic profile of DRC, serial frozen sections of human tonsils were immunostained with R 4/23 and a large panel of other monoclonal antibodies or conventional antisera. In addition, touch imprints of tonsils and cytocentrifuge slides of cell suspensions with increased concentrations of DRC were immunostained with these reagents. DRC proved to be positive for mu, gamma, alpha, kappa and lambda chains, complement component C3b, C3b receptors, C3d receptors, HLA-A,B,C antigens, human Ia-like antigens, common ALL antigen (cALLa), and antigens that are characteristic of the monocyte/macrophage lineages. DRC did not express delta chains, T cell antigens, or antigens that are expressed on interdigitating reticulum cells (IDC) and Langerhans cells. DRC in touch imprints and suspensions prepared from hyperplastic tonsils were found to be giant cells often with 10 or more nuclei. In certain cases of follicular hyperplasia and of centroblastic-centrocytic lymphoma, DRC with several nuclei were also detectable in situ. These results show that (1) the phenotype of DRC differs from that of all other cell types in lymphoid tissue, (2) this phenotype most nearly resembles that of cells of the monocyte/macrophage series, thus suggesting that DRC are related to these cell lineages, and (3) DRC are multinucleated giant cells.