Nonspecific Esterase Staining Research Articles

Wright‐Giemsa and Nonspecific Esterase Staining of Cells

AbstractThis appendix provides two protocols for staining cells. The stains used in the Wright‐Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two‐stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteris termediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.

Immune cell populations in the equine corpus luteum throughout the oestrous cycle and early pregnancy: an immunohistochemical and flow cytometric study.

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.

Commitment to the Monocytic Lineage Occurs in the Absence of the Transcription Factor PU.1

AbstractMice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.

Commitment to the Monocytic Lineage Occurs in the Absence of the Transcription Factor PU.1

Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.

Bovine monocytoid cells transformed to proliferate cease to exhibit lineage-specific functions

Bovine cell lines of the monocyte-Mφ lineage were tested for surface marker expression and were characterized with respect to functions. Cell lines tested encompassed an SV40-transformed cell line (Bo-Mac), a spontaneously emerging monocytoid cell line (M617), and T. annulata-transformed lines derived from bovine Mφ. All lines failed to express surface markers expressed by 1° Mφ, with the exception of CD44, WC9 and the DH59 myleoid cell marker. T. annulata-derived lines expressed, in addition, CD45 and MHC-class-II molecules. Except for nonspecific esterase staining, none of the typical macrophage functions were expressed by any of the cell lines. These included phagocytosis of opsonized E. coli bacteria and of IgG-treated erythrocytes, eliciting of an oxidative burst, the ability to express type-I-interferon (IFN) and to respond to lipopolysaccharide, as determined by four different effector functions (nitric oxide synthesis, tumor necrosis factor (TNF) secretion, IFN production and procoagulant activity upregulation). When transformation induced by T. annulata was reversed by chemical elimination of the parasite, cells ceased to proliferate but started to acquire some of the phenotypic characteristics of Mφ. This suggests that regardless of their origin, exponentially growing bovine cells of the monocyte-Mφ lineage poorly represent a lineage-specific phenotype and should be used with caution in immunological studies.

A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells.

A "sequential culture step" system was devised to study osteoclast differentiation from newborn porcine bone marrow cells. Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process. The osteoclastic nature of the cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the first 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a specific antibody directed against M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast formation, accompanied by a significant increase in a mononuclear TRAP-positive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/ or fusion into multinucleated cells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble osteoclast-inducing factors affecting different stages of osteoclast development.

Production of a macrophage growth factor(s) by a goldfish macrophage cell line and macrophages derived from goldfish kidney leukocytes

Werecently established a spontaneously proliferating macrophage cell line from the goldfish (GMCL), and in this report demonstrate the production of a macrophage-specific growth factor(s) (MGFs) by these cells. The supernatants from GMCL cultures induced proliferation and differentiation of macrophage-like cells from kidney hematopoietic tissues of goldfish. Kidney leukocytes cultured at 6.25×10 4 cells\\ml in the presence of GMCL-derived MGFs proliferated during two weeks of cultivation, whereas those cultured without the MGFs did not. Leukocytes cultured at higher densities (2.5×10 5 cells\\ml) proliferated in the absence of exogenous growth factor, but not to the same extent as those stimulated with GMCL-derived MGFs, suggesting that kidney leukocytes may produce endogenous MGFs. At higher cell density (1×10 6 cells\\ml), kidney leukocytes multiplied extensively over a two-week cultivation period in the absence of exogenous GMCL-derived MGFs. The supernatants from these cultures restored the proliferative ability of leukocytes cultured at low densities, providing direct evidence of MGFs production by kidney leukocytes. The predominant cell-type in cultures grown in the presence of GMCL or kidney leukocyte-MGFs was the macrophage based on the following criteria: [1] non-specific esterase staining; [2] morphologic similarity to GMCL; [3] phagocytosis of the bacterium, A.salmonicida; [4] production of reactive oxygen and nitrogen intermediates in response to stimulation with macrophage activating factors and\\or bacterial lipopolysaccharide; and [5] flow cytometric analyses. Both in vitro-derived kidney macrophage (IVDKM) and GMCL cultures contained three distinct populations of cells, (determined by flow cytometry), suggesting that these macrophage cultures are comprised of cells arrested at distinct differentiation junctures in macrophage development. Production of MGFs by macrophages and kidney leukocytes may play an important role in regulating macrophage hematopoiesis in fish.

Double Esterase Staining andOther Neutrophilic Granule Abnormalities in 237 Patients with the Myelodysplastic Syndrome Studied by the Cancer and Leukemia Group B

We investigated double (specific and nonspecific) esterase (DE) staining in marrow cells of 237 patients with the myelodysplastic syndromes (MDS). Additional abnormalities of neutrophilic granules were examined cytochemically and immunocytochemically for myeloperoxidase activity and antigen elastase, lactoferrin and CD15 granule-membrane glycoproteins. Abnormal DE staining (≥3% of all nucleated marrow cells) was present in 27% of patients with no difference among different MDS subtypes. However, the prevalence of high abnormal DE staining (≥10%) was significantly lower in refractory anemia with excess blasts in transformation (1%) compared to other MDS subtypes (12–15%; p = 0.004). The prevalence of other granule abnormalities was not statistically different in the DE normal and DE abnormal groups. Abnormal DE staining is relatively common among all MDS subtypes. High DE staining may identify a subgroup of patients with a lower grade MDS.

Induction of abundant osteoclast-like multinucleated giant cells in adjuvant arthritic rats with accompanying disordered high bone turnover.

The development of an in vivo system for investigating osteoclast differentiation is important because molecular events occurring in vivo can be observed during the differentiation of the authentic osteoclasts. In adjuvant arthritic rats, an experimental model of human rheumatoid arthritis, extensive bone resorption is observed in the distal diaphysis of the tibia. In the area of extensive bone resorption, it is always accompanied with clusters of numerous multinucleated giant cells (MGCs) as well as bone-resorbing osteoclasts. Here we characterized the morphological properties of these MGCs with the use of enzymehistochemical and immunohistochemical techniques. Extensive destruction but also a marked formation of the inner and outer bone surfaces were the predominant features in the tibiae of such arthritic rats 4 weeks after the adjuvant injection. Numerous MGCs were frequently clustered in the bone marrow spaces located apart from the bone matrices. Although the MGCs lacked ruffled borders, these cells were rich in mitochondria and vacuoles. These multinucleated cells revealed a positive reaction for tartrate-resistant acid phosphatase but a negative reaction for non-specific esterase staining. Most of these MGCs expressed the Kat 1-antigen, an immunological marker specifically expressed on the cell surface of rat osteoclasts. In a dentin resorption experiment using a cluster of MGCs excised from the bone marrow tissues of the tibial distal diaphyses of rats with adjuvant arthritis, many resorption lacunae were formed on dentin slices after a 3-day culture. These results suggest that the majority of the MGCs are osteoclasts but not macrophage polykaryons.

Origin, nature, and some functional considerations of intraventricular macrophages, with special reference to the epiplexus cells.

Intraventricular macrophages encompass the supraependymal, free-floating, and epiplexus (Kolmer) cells; the supraependymal cells lie in close apposition to the ventricular ependyma, the epiplexus cells are closely associated with the choroid plexus epithelium, and the free-floating cells are at a variable distance from the epithelial surface. Although the three cell types are regarded as one cellular entity, the epiplexus cells preponderate. On scanning electron microscopy, the epiplexus cells display diverse morphological forms, ranging from round to bipolar to stellate, and bear a variable number of cytoplasmic processes. Transmission electron microscopy shows the presence of large numbers of lysosomes. The phagocytic nature of epiplexus cells is shown by their intense staining for nonspecific esterase and active uptake of tracers, e.g., horseradish peroxidase and rhodamine isothiocynate, administered intravenously or intraperitoneally. The mode of entry of these tracers in the cerebral ventricles is by way of transepithelial transport. In rats, the population of intraventricular macrophages increases steadily after birth until 17 days of age; thereafter, their cell population remains relatively unchanged. The early upsurge is attributed to proliferation of residential cells and/or influx of circulating monocytes/stromal macrophages through the process of "emperipolesis." The immunophenotypic features of intraventricular macrophages are consistent with other mononuclear phagocytes being immunoreactive for OX-42, OX-18, OX-6, and OX-1 and ED1 for the detection of CR3 receptors, MHC class I and II antigens, leucocyte common antigen, and macrophage antigen, respectively. The expression of these antigens is noticeably enhanced following the injection of lipopolysaccharide (LPS) into postnatal rats. Remarkably, the intraventricular macrophages are induced to express MHC class II (Ia) antigen after LPS or interferon-gamma injections. Furthermore, the expression of transferrin receptors as detected with OX-26 is also upregulated after these treatments. Epiplexus cells are also elicited to display a de novo expression of nitric oxide synthase-like immunoreactivity following intracerebral injection of LPS. They also respond vigorously to a single nonpenetrative blast. Results of our series of studies suggest that, besides their primary function as scavenger cells, the intraventricular macrophages partake in possible immunological responses and iron regulation in the ventricular system or the brain as a whole.

Origin, nature, and some functional considerations of intraventricular macrophages, with special reference to the epiplexus cells

Intraventricular macrophages encompass the supraependymal, free-floating, and epiplexus (Kolmer) cells; the supraependymal cells lie in close apposition to the ventricular ependyma, the epiplexus cells are closely associated with the choroid plexus epithelium, and the free-floating cells are at a variable distance from the epithelial surface. Although the three cell types are regarded as one cellular entity, the epiplexus cells preponderate. On scanning electron microscopy, the epiplexus cells display diverse morphological forms, ranging from round to bipolar to stellate, and bear a variable number of cytoplasmic processes. Transmission electron microscopy shows the presence of large numbers of lysosomes. The phagocytic nature of epiplexus cells is shown by their intense staining for nonspecific esterase and active uptake of tracers, e.g., horseradish peroxidase and rhodamine isothiocynate, administered intravenously or intraperitoneally. The mode of entry of these tracers in the cerebral ventricles is by way of transepithelial transport. In rats, the population of intraventricular macrophages increases steadily after birth until 17 days of age; thereafter, their cell population remains relatively unchanged. The early upsurge is attributed to proliferation of residential cells and/or influx of circulating monocytes/stromal macrophages through the process of “emperipolesis.” The immunophenotypic features of intraventricular macrophages are consistent with other mononuclear phagocytes being immunoreactive for OX-42, OX-18, OX-6, and OX-1 and ED1 for the detection of CR3 receptors, MHC class I and II antigens, leucocyte common antigen, and macrophage antigen, respectively. The expression of these antigens is noticeably enhanced following the injection of lipopolysaccharide (LPS) into postnatal rats. Remarkably, the intraventricular macrophages are induced to express MHC class II (Ia) antigen after LPS or interferon-γ injections. Furthermore, the expression of transferrin receptors as detected with OX-26 is also upregulated after these treatments. Epiplexus cells are also elicited to display a de novo expression of nitric oxide synthase-like immunoreactivity following intracerebral injection of LPS. They also respond vigorously to a single nonpenetrative blast. Results of our series of studies suggest that, besides their primary function as scavenger cells, the intraventricular macrophages partake in possible immunological responses and iron regulation in the ventricular system or the brain as a whole. Microsc. Res. Tech. 41:43–56, 1998. © 1998 Wiley-Liss, Inc.

Reduced CD4 and HLA-DR Expression in Neonatal Monocytes

Three-color flow cytometry was used to assess the immunophenotypic characteristics of normal cord blood monocytes after labeling with a variety of antibodies against myeloid/monocyte-specific markers. Monocytes both in cord blood and in peripheral blood from normal adults were defined in a backgating procedure as cells with the light-scattering characteristics of monocytes that also expressed CD14. The percentage of monocytes, defined in this fashion, that also displayed CD4 receptors was significantly lower in cord blood (mean ± SD = 29.3 ± 13.9%) than in peripheral blood from normal adult controls (mean ± SD = 68.9 ± 13%) (P< 0.005). Similarly, HLA-DR expression was found on only 86 ± 6.6% of monocytes in cord blood but on 99 ± 1% of monocytes in adults (P< 0.005). The percentage of monocytes displaying CD16 receptors in cord blood did not show any significant difference in comparison with adult monocytes. When coexpression of CD14, CD16, and CD4 was assessed, cord blood showed a predominant population of monocytes bearing the phenotype CD14+/CD16−/CD4−. Similarly, approximately 10% of CD14+ monocytes in cord blood expressed neither CD4 nor HLA-DR. Cytochemically, monocytes from cord blood revealed intense granular staining for PAS and marginal or absent staining for nonspecific esterase (NSE). These results raise the possibility that reduced expression of CD4 and HLA-DR receptors on cord blood monocytes may contribute to their impaired immune response. Additionally, the high percentage of CD14+/CD16−/CD4− cells in cord blood suggests that these cells may represent a phenotypically immature population of monocytes. Likewise, the unusual cytochemical staining patterns suggest that these cells are biochemically immature as well.

Infiltration of peroxidase-producing eosinophils into the lamina propria of patients with ulcerative colitis

Little information is available to explain the pathogenesis of ulcerative colitis (UC). In this study, we focused on eosinophils in the lamina propria of the mucosa of patients with UC in the active phase. Biopsy specimens were taken from 17 patients with UC in the active phase, 17 in the inactive phase, and 20 control patients, and submitted for histochemical staining for peroxidase and chloroacetate esterase for microscopic examination. Both peroxidase-producing and chloroacetate esterase-producing cells in the lamina propria increased markedly in the active phase (8.3 +/- 3.1/0.01 mm2 and 6.6 +/- 2.7/0.01 mm2, respectively), compared with values in the inactive phase (0.8 +/- 0.6/0.01 mm2 and 1.3 +/- 0.6/0.01 mm2) or in the controls (1.3 +/- 0.8/0.01 mm2 and 1.3 +/- 0.4/0.01 mm2). Triple staining for peroxidase, chloroacetate esterase, and nonspecific esterase in the specimens revealed that the peroxidase-producing cells constituted a different population from that of neutrophils, macrophages/monocytes, or basophils. A monoclonal antibody specific for eosinophil peroxidase stained almost all infiltrated peroxidase-producing cells. These results indicated that eosinophils with strong peroxidase activity had infiltrated the lamina propria in UC, suggesting an allergic background and the involvement of released peroxidase in the mucosal damage characteristic of UC.

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages.

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.

Cutaneous toxicity of the benzidine dye direct red 28 applied as mechanistically-defined chemical mixtures (MDCM) in perfused porcine skin

Complex chemical mixtures at hazardous waste sites can potentially consist of a marker chemical and several other chemicals, each of which can have different modulating actions on the dermatotoxicity of the marker chemical and/or other components in the mixture. A total of 16 mixtures, consisting of a marker chemical direct red 28 (DR28), a solvent (80% acetone or DMSO in water), a surfactant (0 or 10% sodium lauryl sulfate, SLS), a vasodilator (0 or 180 μg methyl nicotinate, MN) and a reducing agent (0 or 2% stannous chloride, SnCl 2) were selected. Isolated perfused porcine skin flaps (IPPSFs), which have been proven to be an in vitro model for assessing absorption and toxicity, were utilized. These mixtures did not cause severe dermatotoxicity. However, light microscopic observations depicted minor alterations (intracellular and intercellular epidermal edema) with DMSO mixtures than with acetone mixtures. The presence of SLS caused an alteration in the stratum corneum. Enzyme histochemical staining for alkaline phosphatase (ALP) and nonspecific esterase (NSE) revealed no significant treatment effects, but increased staining for acid phosphatase (ACP) in the stratum basale was significant when associated with SLS or SLS+MN in DMSO mixtures. At 8 h post-dose, only DMSO mixtures containing SL+MN, SL+SnCl 2, or SLS+MN+SnCl 2 significantly increased transepidermal water loss. In conclusion, this study demonstrated that various mixtures, especially those containing SLS alter the epidermal barrier differently with complex interactions occurring simultaneously.

Long-term stromal cultures produce dendritic-like cells.

A stromal cell-dependent long-term culture (LTC) system has been developed from spleen which continuously generates non-adherent cells with dendritic cell (DC) characteristics. Bioassays using factor-dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)-3 and IL-6-like growth factors. Conditioned medium from LTC also contains factors which appear to be unrelated to IL-3 and induces growth of stromal cells from bone marrow. Non-adherent cells generated in LTC were not T or B lymphoid cells or granulocytes, nor were mast cells detectable. Morphological and electron microscopic examination has also excluded the presence of macrophages (Mo). Cells with DC morphology have been detected by both light and electron microscopy. The majority of cells in the non-adherent population were found to have multiple membrane pseudopodia, with a small acentric nucleus. These appear to be the precursors of DC. They expressed cell surface markers detectable with DC-specific antibodies and antibody specific for major histocompatibility complex Class II molecules. A proportion of cells also expressed myeloid markers, but since this expression was not supported by histochemical staining for myeloperoxidase or non-specific esterase, it was concluded that the cells produced are not typical of the myeloid lineage. Cells generated in LTC were shown to be potent stimulators for allogeneic and syngeneic MLR and for antigen-specific T-cell proliferation.

Human interdigitating dendritic cells directly stimulate CD40-activated naive B cells.

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.

Wright‐Giemsa and Nonspecific Esterase Staining of Cells

This appendix provides two protocols for staining cells. The stains used in the Wright-Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two-stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteristic ability to split esters under particular conditions. In the staining method given here, the substrate, a-naphthyl butyrate, is incubated with cells under conditions in which esterases present in monocytes/macrophages split the substrate to yield an intermediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.

Isolation and characterization of murine Kupffer cells and splenic macrophages

A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 × 10 6/g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages bases on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-α (TNF-α) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.

In vitro characterization of a novel avian haemopoietic growth factor derived from stromal cells

To better define the role of the chicken haemopoietic microenvironment in supporting haemopoiesis, a continuous cell line was generated by RSV transformation of avian spleen stromal cells (SSL-1). Super-natants from this line were found to contain haemopoietic growth factor activity as measured by the ability to induce proliferation and differentiation of precursor cells present in embryonic and post-hatched haemo-poietic tissues. Comparison of cultures grown in the presence of cMGF and SSL-1 conditioned media (CM) revealed that both cytokine sources induced similar types of cell populations. Both sources supported the proliferation of predominantly macrophage-like cells based on colony morphology, differential staining, non-specific esterase staining, and phagocytosis activity. Interestingly, SSL-1 does not express any message for cMGF, nor does it secrete any IL-2 or Interferon activities suggesting that the growth factor activities seen in SSL-1 are novel.