Cell-enriched Preparations Research Articles

Human conjunctival mast cell responses in vitro to various secretagogues

Human conjunctiva was enzymatically digested into monodispersed cell preparations using collagenase and hyaluronidase. The preparations were enriched for mast cells using Percoll gradient centrifugation prior to challenge with various secretagogues. Mast cell percentage (9.7 ± 2.9%) and viability (96 ± 3%) were determined using toluidine blue staining and vital dye exclusion, respectively. The mast cell enriched preparations were challenged with calcium ionophore A(23187') compound 48/80, concanavalin A, anti-IgE, morphine and substance P. Mediator release was quantified by radioimmunoassay. The calcium ionophore A(23187), compound 48/80, concanavalin A and anti-IgE stimulated histamine release from the conjunctival mast cell preparations, with EC(30) values of 0.028, 62.5, 24.4 and 1.9 ϵg/ml, respectively. Morphine but not substance P challenge induced significant but low levels of histamine release. Tryptase, sulfidopeptide leukotriene, and prostaglandin D(2) levels in cell supernatants were also increased following stimulation. The profile of mediator release observed with these cells compared to that reported from human lung and human skin mast cells suggest that human conjunctival mast cells may have unique biological responses.

Presence of eosinophilic precursors in the human thymus: Evidence for intra‐thymic differentiation of cells in eosinophilic lineage

The distribution of myeloid cells in the human thymus was investigated by light and electron microscopy, immunohistochemistry, and/or flow cytometry. A series of 74 thymic samples, from newborn to 37 year old patients, were studied. By light microscopy, aggregates of mononuclear cells were frequently present in intralobular septa and outer medulla. Among those cells, eosinophilic precursors (promyelocyte, myelocytes and metamyelocytes) were readily identified. These immature granular cells were present in all pre-involutional thymi, and were particularly frequent in the thymi of patients who were younger than 5 years of age. The cells made up 30-50% of the total eosinophilic population and were frequently observed as a group of cells at various stages of differentiation, suggesting that they differentiate from pre-existing precursors in the thymus. These eosinophilic precursors were mostly located in the intralobular septa and fibroreticular network at the corticomedullary junction, while mature eosinophils were scattered throughout the thymus. Flow cytometric analyses, using stem cell-enriched preparations, showed that cells expressing CD33 or CD34 constituted on average 2.55% and 3.33% (0.09% and 0.12% of the total cells), respectively. CD33+/CD34+ coexpressors were also identified, and they constituted 0.36% of the analyzed cells (0.01% of the total cells). No statistical difference in the proportions of CD33+ and/or 34+ cells was noted between any age groups. It is concluded that eosinophilic precursors present in the thymus differentiate into cells in the eosinophilic lineage in particular areas such as the intralobular septa and fibroreticular network of the outer medulla in preinvolutional human thymi.

Secretion of Cytokines (Interleukins-la, -3, and -6 and Granulocyte-Macrophage Colony-Stimulating Factor) by Normal Human Bone Marrow Megakaryocytes

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.

Distribution of transforming growth factor alpha precursors in the mouse uterus during the periimplantation period and after steroid hormone treatments.

Temporal and cell-type specific distribution of transforming growth factor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mouse uterus during the periimplantation period, and after steroid hormone treatments of ovariectomized adult mice by immunohistochemistry using antibodies that recognize the precursor forms of the growth factor. These studies were complemented by immunoblot analysis of proTGF alpha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was targeted for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTGF alpha primarily in luminal and glandular epithelial cells on Day 1 of pregnancy or pseudopregnancy followed by little or no accumulation on Days 2 and 3. However, immunoreactive proTGF alpha started to reappear in the luminal epithelium on the morning of Day 4 and became more prominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immunostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonreceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the immunohistochemistry results, Western blot analysis detected two species of precursor proteins (14.5 and 17 kDa) in isolated luminal epithelial cell-enriched preparations on Day 4, but not on Day 5, of pseudopregnancy. The results suggest that proTGF alpha accumulates in the luminal epithelium of the receptive uterus prior to implantation. The effects of ovarian steroids on uterine accumulation of proTGF alpha were examined in ovariectomized adult mice by immunohistochemistry and immunoblotting. Whereas an injection of estradiol-17 beta (E2) or progesterone (P4) had little or a modest effect on epithelial accumulation of proTGF alpha, P4 priming for several days resulted in distinct accumulation of proTGF alpha in epithelial cells. The superimposition of an E2 treatment on P4 priming showed a biphasic response, with an initial gradual loss of immunostaining through 12 h followed by a return by 24 h of E2 treatment. The combined hormone treatment schedule employed here is similar to the situation of inducing implantation with E2 in P4-primed delayed implanting mice. The results suggest a paracrine/"juxtacrine" role for this growth factor in implantation.

Differential expression of keratinocyte growth factor and its receptor in the human uterus

The expression of mRNAs for keratinocyte growth factor (KGF) (also called FGF-7) and its receptor was evaluated in normal human endometrium and myometrium as well as in myoma and in endometrial adenocarcinoma cell lines using reverse transcriptase polymerase chain reaction. Both KGF and its receptor mRNA are expressed in the human endometrium throughout the menstrual cycle, whereas fibroblast growth factor receptor 2 (FGFR-2) mRNA expression is low in this tissue. In endometrial stromal cell enriched preparations KGF mRNA dominates with little expression of KGF receptor (KGFR) and FGFR-2, whereas in the epithelial cell-enriched fraction the KGFR mRNA dominates. Human myometrium and myoma express mRNA for KGF, but not for KGFR. FGFR-2 is expressed in both myometrial and myoma tissues. None of the five endometrial adenocarcinoma cell lines studied expressed KGF mRNA, whereas all cell lines expressed mRNA for either KGFR or FGFR-2 or for both receptors. The results show a selective expression of KGFR and the closely related FGFR-2 in the human uterus with the former being expressed in the endometrium and the latter predominantly in the adjacent myometrium. In the endometrial tissue, selective expression of KGF in stromal cells and KGFR in epithelial cells supports the paracrine function of KGF in epithelial tissue.

Lymphohemopoietic reconstitution using wheat germ agglutinin-positive hemopoietic stem cell transplantation within but not across the major histocompatibility antigen barriers.

Nonadherent (NA), low density (LD), wheat germ agglutinin-positive (WGA+) murine hemopoietic stem cell-enriched preparations (HSCPs) were tested for the capability to reconstitute lymphohemopoietic elements in lethally irradiated mice. HSCPs from BALB/c mice reconstituted lethally irradiated, major histocompatibility complex (MHC)-matched DBA/2 mice to normal histology of the thymus and spleen and normal humoral and cellular immune functions. By contrast, lethally irradiated B6 mice could not be reconstituted after transplantation with NA, LD, WGA+ cells from MHC-mismatched BALB/c mice. We previously observed frequent survival, stable chimerism, and normally vigorous functioning immune systems in B6 mice transplanted with T-cell-depleted bone marrow from both BALB/c and B6 donors. To extend these findings to a stem cell transplantation system, lethally irradiated B6 mice were transplanted with NA, LD, WGA+ cells from both BALB/c and B6 mice. These mixed stem cell-enriched preparations did not reconstitute the lethally irradiated, MHC-mismatched mice. By contrast, such HSCPs from BALB/c plus DBA/2 into DBA/2 mice reconstituted the hematologic and lymphoid tissues and functional immune systems when the donor and the recipient pairs were matched at MHC and mismatched at multiminor histocompatibility barriers. These purified blood progenitors thus appear to lack certain cells/factors essential for engraftment and reconstituting recipients in a fully allogeneic environment.

Enhancement of testosterone secretion by normal adult human Leydig cells by co‐culture with enriched preparations of normal adult human Sertoli cells

An in-vitro method was developed to study Sertoli-Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial Cytochrome C Oxidase II Messenger Ribonucleic Acid is Expressed in Pachytene Spermatocytes at High Levels and in a Stage-Dependent Manner during Spermatogenesis in the Rat

Screening of a rat seminiferous tubule library (stages VI-VIII) resulted in isolation of several clones to the same abundant mRNA. Sequencing revealed that these encoded all or part of the second subunit of cytochrome C oxidase (COX II), the terminal enzyme in the electron transport chain located in mitochondria. The pattern of expression of the COX II mRNA in the testis was examined by Northern blot and in situ hybridisation combined with specific depletion of pachytene spermatocytes or Leydig cells by treatment with methoxyacetic acid (MAA) or ethane dimethane sulphonate (EDS), respectively. COX II mRNA was found to be strongly expressed in pachytene spermatocytes in adult and prepubertal rats, with the highest levels of expression at stages VII-VIII of the spermatogenic cycle. Although this is the androgen-dependent stage, Northern analysis of total testicular RNA showed that expression of COX II mRNA was not altered detectably by EDS-induced androgen withdrawal nor was expression altered detectably by 24-h treatment with high doses of FSH. Treatment of rats with MAA, which selectively depletes seminiferous tubules at most stages of pachytene spermatocytes, resulted in a marked reduction in COX II mRNA. In the absence of pachytene spermatocytes, COX II mRNA was visualized in preleptotene spermatocytes and in Sertoli cell cytoplasm. The expression of COX II in Sertoli cells was confirmed by examination in situ of a testis devoid of most germ cells and by Northern analysis of RNA from an adult Sertoli cell-enriched preparation. However, using either approach the amount of mRNA in Sertoli cells was considerably lower than that in pachytene spermatocytes. Cytochrome C oxidase has thirteen subunits, three of which (I-III) are encoded on the mitochondrial genome. The physiological significance of the high levels of expression of COX II mRNA in pachytene spermatocytes and its stage-dependent changes is unknown but they presumably reflect requirements for alterations in energy demand as these cells enter the final stages of meiosis. The stage-dependent differences in expression of COX II mRNA in pachytene spermatocytes may also explain differences in the susceptibility of these cells to depletion by MAA.

Human Sertoli cells in vitro. Lactate, estradiol-17 beta and transferrin production.

Human Sertoli cell parameters, namely lactate, estradiol-17 beta, and transferrin production, were determined after a 24-hour incubation with either human follicle stimulating hormone (FSH) or dbcAMP in the presence or absence of testosterone plus a phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine; MIX). Testicular tissues were obtained from 10 young patients (mean age, 29 years); using a 3-step enzymatic treatment, Sertoli cell enriched preparations (> 92%) were studied after 4 days as primary cultures. No significant changes in lactate, estradiol-17 beta, and transferrin outputs have been observed according to age in patients ranging in age from 16 years to 47 years. Sertoli cell production of the compounds is controlled by testosterone plus MIX; FSH (or dbcAMP) treatment only slightly improves their synthesis. It is suggested that human Sertoli cell function, as far as the parameters measured in this study are concerned, is likely regulated by cAMP-dependent and independent pathways.

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.

Leukotriene C 4 formation by enriched human basophil preparations from normal and asthmatic subjects

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C 4 formation, histamine release, and prostaglandin D 2 formation. No prostaglandin D 2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C 4 formation or histamine release. A23187 and anti-IgE induced leukotriene C 4 formation were in the range of 1–20 and 0.6–4.8 pmol/10 6 basophils respectively.

The Actions of Calcitonin on the TM3 Leydig Cell Line and on Rat Leydig Cell‐Enriched Cultures

Studies demonstrating calcitonin receptors on Leydig cells have suggested that these cells may be one of the many sites affected by this peptide. To investigate this possibility, the effect of synthetic salmon calcitonin on the TM3 Leydig cell line (derived from immature mouse Leydig cells) and on primary Leydig cell-enriched preparations was examined. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in TM3 cells. In addition, the hormone stimulated the basal secretion of testosterone in both TM3 cell- and Leydig cell-enriched cultures and potentiated the action of hCG on Leydig cell-enriched cultures. Synthetic salmon calcitonin also increased the concentration of androgen and estrogen receptors in cultured TM3 Leydig cells by 2- and 4-fold, respectively, when added to the culture medium (1 micrograms/ml). The fact that 8-bromo-cyclic AMP decreased both androgen and estrogen receptor concentrations suggested that the effect of calcitonin on sex steroid receptors is not mediated by its effect on cyclic AMP in these cells. The possibility that the action of calcitonin on steroid receptors might be mediated by another messenger such as calcium (Ca2+) was therefore considered. Progressively lowering the concentration of Ca2+ in the culture medium of the cells from 1.5 mM to less than 0.01 mM decreased the concentration of both androgen and estrogen receptors. Returning the Ca2+ concentration to normal levels (1.5 mM) restored steroid receptor levels. Receptor levels were also decreased when the extracellular Ca2+ concentration was lowered to 0.5 mM, and treatment with the Ca2+ ionophore, A23187 (1 microM), restored receptor levels to normal. The calcium channel blocker, verapamil, decreased the androgen receptor concentration but unexpectedly increased the concentration of estrogen receptors. It was concluded that calcitonin stimulates cAMP formation and testosterone secretion, and increases the concentration of sex steroid receptors. These observations provide evidence that the previously demonstrated calcitonin receptors on Leydig cells may be coupled to several biologic responses in this cell type.

Characterization and Localization of in Vivo Phospholipid Methylation in the Hamster Testis

Although previous studies have demonstrated that phospholipid methylation occurs in the testis and may be involved in Leydig cell function, phospholipid methylation in spermatogenic cells has not been characterized. In this study we describe the occurrence, time course, and localization of phospholipid methylation in the hamster testis following intratesticular injection of radioactive methyl precursor. Adult and pubertal (seven day old) hamsters were injected intratesticularly with [3H-methyl]-methionine and sacrificed 10min. to 31 hours thereafter. The testes were then removed and homogenized or dispersed into cell suspensions. Spermatogenic cell and Leydig cell enriched preparations were isolated from the dispersed cell preparations using elutriation and Percoli gradient centrifugation and assayed for methylated phospholipids and proteins. These experiments demonstrated that 1) phospholipid methylation occurs in the hamster testis at a level seven-fold greater than protein methylation, 2) the incorporation of radioactivity associated with phospholipid methylation is progressive over time, and 3) in vivo, spermatogenic cell preparations enriched with pachytene spermatocytes have an almost four-fold higher level of measurable phospholipid methylation when compared to whole testis preparations. Taken together, these results suggest that phospholipid methylation may play an important stage-specific role in spermatogenesis.

Highly enriched preparations of cultured myocardial cells for biochemical and physiological analyses

Cultures of enzyme-dissociated myocardial cells contain myocytes as well as other cell types (e.g. fibroblasts); therefore, cell separation is necessary to interpret accurately biochemical measurements of substances or physiological measurements of processes common to more than one type of cell. Previous methods of cell separation have resulted in cultures enriched with, at most, about 80% myocytes. In the present study, a monoclonal antibody to cell-surface adhesion factors, CSAT, was used to obtain four highly enriched preparations: cultures of 7-day embryonic chick ventricular cells that contained either 99.1 +/- 0.2% (n = 10) myocytes or 96.5 +/- 0.8% (n = 6) fibroblasts; and cultures of 17-day chick cells that contained 99.1 +/- 0.2% (n = 11) myocytes or 92.5 +/- 0.8% (n = 8) fibroblasts. Following the removal of CSAT from myocyte-enriched cultures, 96.9 +/- 0.4% (n = 7) of the myocytes obtained from 7-day embryos and 93.8 +/- 1.4% (n = 6) of the myocytes obtained from 17-day embryos attached to culture dishes. Using these four preparations, we found the sialic acid content of myocytes to be significantly less than that of fibroblasts at both 7 and 17 days of development. In fact, the sialic acid content of myocytes did not change during this period, whereas that of fibroblasts increased significantly. These differences underscore the necessity of using highly enriched cell preparations. The antibody/differential adhesion method described provides an opportunity to evaluate and correlate the biochemical, physiological and pharmacological properties of myocytes without interference from other cell types.

Isolation and culture of ram rete testis epithelial cells: structural and biochemical characteristics.

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.

Biosynthesis and secretion of clusterin by ram rete testis cell-enriched preparations in culture.

Rabbit polyclonal antibodies, directed specifically against clusterin purified from ram rete testis fluid, were employed in an investigation of the biosynthesis of clusterin by cultures of rete testis epithelial cells and by Sertoli cells prepared from testes of adult rams. Cells in serum-free medium were incubated in the presence of either [35S]methionine, [3H]leucine, or [3H]glucosamine, and radiolabeled proteins secreted were immunoprecipitated. The pellet was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the gels were then fluorographed. In other experiments, protein bands were transferred to nitrocellulose and visualized immunochemically. Under non-reducing conditions, a single band was detected, having a molecular weight of 80,000. Under reducing conditions, doublet bands were detected, having approximate molecular weights of 40,000 (major band) and 37,000 (minor band). These properties were indistinguishable from those obtained with authentic samples of pure clusterin subjected to gel electrophoresis and Western immunoblot procedures. Amounts of clusterin synthesized by rete testis cells in culture, quantitatively determined with a sandwich enzyme-linked immunosorbent assay procedure, were approximately 4 micrograms/micrograms cell DNA/48 h. Immunocytochemical localization investigations, using monoclonal antibodies against clusterin, revealed the presence of clusterin in the perinuclear of juxtanuclear regions, in both rete testis epithelial cells and Sertoli cells in culture. The possible functions of clusterin produced by rete testis epithelial cells and by Sertoli cells are discussed.

Dual fluorochrome analysis of human B lymphocytes: phenotypic examination of resting, anti-immunoglobulin stimulated, and in vivo activated B cells.

B cell-enriched preparations were prepared from human peripheral blood and lymphoid tissues by the depletion of T cells and monocytes. Only B cells by virtue of their staining with anti-B1 conjugated to fluorescein were additionally examined. Dual fluorescence staining and flow cytometric analysis demonstrated that the majority of "resting" human peripheral blood and splenic B cells co-express the B cell-restricted B1 and B2 antigens and lack B5, a B cell-restricted activation antigen, and interleukin 2 receptor (IL 2R). In contrast, nearly 2/3 and 1/3 of B1+ cells isolated from lymph node expressed IL 2R and B5 antigens, respectively. When B1+ B cells from peripheral blood and spleen were "activated" by anti-Ig, they lost the B2 antigen and acquired the B5 and/or IL 2R antigens. 2/3 of B1+ cells strongly expressed IL 2R, and up to 1/2 of B1+ cells co-expressed B5. Delineation of increased numbers of B1+ cells that co-express B5 and/or IL 2R within lymphoid tissues obtained from patients with diseases characterized by "activated" B cells provides in vivo confirmation that these phenotypic changes correlate with B cell activation. We believe that the identification and isolation of these and similar subsets of cells defined by differing cell surface phenotypes should further our understanding both of normal B cell activation and the pathophysiology of B cell disease states.

IgE receptors on human lymphocytes. III. Expression of IgE receptors on mitogen-stimulated human mononuclear cells.

Human peripheral blood mononuclear cells (PBMC) were tested for the expression of Fc epsilon-receptor (Fc epsilon R) after stimulation with various mitogens in the absence of IgE. Fc epsilon R were found on virtually all the cells from 19 Epstein-Barr virus-transformed B cell lines including those derived from cord blood, from one agamma-globulinemic patient and VDS-0 pre-B cells. Hence, the data clearly indicate that Fc epsilon R may be expressed on very immature B cells. PBMC cultures stimulated with either pokeweed mitogen (PWM), phytohemagglutinin (PHA) or concanavalin A displayed an early increase of their content in Fc epsilon R-bearing cells followed by a decrease to levels below those of control cultures. After fractionation of the PWM-stimulated cultures into T and B cell-enriched preparations, most of the Fc epsilon R+ cells were in the in the B cell fractions and the same low levels of Fc epsilon R+ cells were found in the T cell fractions isolated from the PWM-stimulated and from the control cultures. Double-labeling experiments, employing biotinylated F(ab')2 monoclonal antibody to FcR and either fluorescein isothiocyanate-conjugated B1 or Mo2 monoclonal antibodies, indicated that PWM mainly exerted its effect on B cells and on monocytes. This effect was T cell dependent and it was mediated by soluble factors of T cell origin. At the peak of the PHA or concanavalin A response, most of the Fc epsilon R-bearing cells were found in the B cell fraction but the T cells isolated from mitogen-stimulated cultures contained significant more Fc epsilon R+ cells than those from the control cultures, suggesting that T cell mitogens had increased the expression of Fc epsilon R on some T cells. This view was supported by the finding of a higher proportion of Fc epsilon R+ cells in PHA-stimulated than in control cultures of highly purified T cells with a maximum response at the end of the culture period. Double-labeling experiments at the peak (day 2) of the peripheral blood mononuclear cell response indicated that the expression of Fc epsilon R was increased on B cells (B1+) and on monocytes (Mo2+). By using the same approach at the peak of the T cell response (day 7), it was found that T cells isolated from PHA-stimulated cultures expressed more Fc epsilon R than those isolated from control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Two-dimensional membrane protein patterns of myeloid cells, at various stages of differentiation

In order to detect differentiation linked and leukemia associated surface proteins, twodimensional polyacrylamide gel electrophoresis patterns after lactoperoxidase labelling were made from normal promyelocyte enriched cell preparations, immature and mature myeloid cells, peripheral blood granulocytes and cells from patients with acute myeloid leukemia. Three proteins were found to be more expressed in the patterns of promyelocyte enriched cell preparations. These proteins were also seen with leukemic myeloblasts and are therefore associated with immature myeloid cells. Five surface proteins were expressed preferentially on mature myeloid cells, but not or only weakly on promyelocyte enriched preparations and absent in leukemic myeloblasts. Seven surface proteins were present at all stages of maturation and also on leukemic myeloblasts. Three surface proteins were found only in leukemic myeloblasts; these could be associated with an early stage of maturation or specifically linked with leukemia.

Inhibition of macrophage C3b-mediated ingestion by syphilitic hamster T cell-enriched fractions.

Macrophages are important for host defense against syphilitic infection. Our results show that C3b-mediated ingestion (C3bMI), a characteristic of activated macrophages, was inhibited in vitro by nonadherent cells from hamsters infected with Treponema pallidum subspecies endemicum. When macrophage target cells from normal, syphilitic, or lipopolysaccharide-treated animals were co-cultured with nonadherent cells derived from normal or syphilitic hamsters, noticeable differences were detected. Nonadherent syphilitic cells significantly suppressed macrophage C3bMI, whereas normal nonadherent cells displayed little or no suppressive activity. In general, macrophage C3bMI was reduced by nonadherent cells obtained throughout the course of syphilitic infection, although it eventually began to recover. The syphilitic nonadherent cells with maximum suppressive effect were those obtained from hamsters infected for 3 to 6 wk. The addition of treponemal antigens additionally inhibited C3bMI. The inhibitory effect of syphilitic nonadherent cells on either lipopolysaccharide-treated or syphilitic macrophages was sustained even when the nonadherent cells were removed from the cultures, and the effect continued for at least 72 hr thereafter. Fractionation of syphilitic nonadherent cell populations by two independent methods produced T cell-enriched preparations with significantly more suppressive activity than non-T cell-enriched preparations. These observations may account for the chronicity of syphilitic infection.