RESUMEN

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcgamma receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.

Asunto(s)

Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Oncogénicas/metabolismo , Fagocitosis/fisiología , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/fisiología , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Monocitos/citología , Monocitos/metabolismo , Fagosomas/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Proteína del Síndrome de Wiskott-Aldrich , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Six macrophage cell lines, each derived from a bone marrow macrophage colony grown in soft agar, were established by expansion of the macrophage clones in liquid culture until spontaneous transformation occurred. Four lines originated from the LPS(d) nonresponder mouse strain C3H/HeJ and two from the LPS(n) responder strain CBA/J. The cell lines adhered to plastic and glass surfaces and displayed typical macrophage functions such as phagocytosis and nonspecific esterase activity. Flow cytometry analyses showed that the lines expressed the macrophage surface markers CD11b, CD13, CD32/16, F4/80, and BM8 constitutively. A moderate expression of the adhesion receptor CD11a, but only a very low expression of its ligand CD54, was observed. A minor fraction of the cells in each line constitutively expressed MHC class II antigen, and its expression could be up-regulated in each cell line by treatment with interferon-gamma (IFN-gamma). Secretion of the inflammatory mediators nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) after induction by three bacterial derivatives, heat-killed Salmonella typhimurium (HKS), lipopolysaccharide (LPS), and the Mycoplasma fermentans-derived amphiphilic lipid MDHM, were examined in detail. Not only did the lines differ in the amounts of mediators secreted in response to any one stimulus, but the doses of MDHM or LPS required for 50% maximal induction of NO varied up to 10-fold among the four LPS(d) cell lines, suggesting considerable functional heterogeneity between the clones. Secretion of large amounts of TNF-alpha was induced in all the cell lines by HKS. Although it could be shown that exogenously added TNF-alpha acted synergistically with IFN-gamma to induce NO release from the cell lines, an autocrine role for TNF-alpha during HKS-IFN-gamma induction of NO synthesis could not be substantiated. Neutralization of TNF-alpha with a specific antibody completely blocked NO induction by exogenous TNF-alpha but did not abrogate NO release either by HKS-IFN-gamma-induced cells or by macrophages treated with supernatant from an HKS-IFN-gamma-activated cell line. These results indicate that the clones are arrested in distinct stages of differentiation and retain some properties of normal untransformed macrophages. They should be helpful tools for investigations into macrophage function.

Asunto(s)

Células de la Médula Ósea , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Animales , Anticuerpos/farmacología , Antígenos de Superficie/metabolismo , Línea Celular Transformada , Células Clonales , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Interferón gamma/farmacología , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Óxido Nítrico/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The monoclonal antibody U5, which is a potent inducer of proliferation in human T-cells, was found to bind to an alkali-sensitive derivative of ganglioside GD3. Using immunochemical and spectroscopic methods, the structure of the U5 antigen was determined as 7-O-acetyl-GD3. The antibody U5 did not react with 9-O-acetyl-GD3 and bound severalfold more stronger to 7-O-acetyl-GD3 than to GD3. U5 is the first antibody known to detect preferentially 7-O-acetyl-GD3. Flow cytometric analysis showed that each major class of human leukocytes contained a significant fraction of cells binding the U5 antibody.

Asunto(s)

Anticuerpos Monoclonales , Gangliósidos/análisis , Gangliósidos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Secuencia de Carbohidratos , Bovinos , Femenino , Gangliósidos/química , Humanos , Immunoblotting , Cinética , Leucocitos/inmunología , Activación de Linfocitos/efectos de los fármacos , Espectrometría de Masas , Leche/química , Datos de Secuencia Molecular , Linfocitos T/efectos de los fármacos

RESUMEN

The Forssman glycolipid antigen (Fo) has been shown to be a differentiation marker for mouse macrophages both in vivo and in vitro. In order to determine whether or not there is a relationship between stage of differentiation and Fo expression, we have analyzed the kinetics of Fo expression during the growth of cultured mouse bone marrow-derived macrophages (BMDM). BMDM were grown in serum free medium to avoid the possible influence of undefined serum factors. In this medium they could be maintained over a period of up to 20 days with cell yields comparable to those obtained with serum-supplemented media. Fo antigen was assayed with a specific antibody using both a whole cell ELISA and immunocytochemical staining of cells grown on slides. With increasing age in culture, BMDM showed a gradual quantitative increase in Fo expression and parallel increase in the Fo+ BMDM fraction from about 10% Fo+ cells on the 10th day of culture to a maximum of 50%-60% Fo+ cells between the 17th and 19th days. The temporal control over the development of the Fo+ cell fraction was intrinsic to BMDM maturation but was specific for Fo. During the same time period expression of MHC class II (Ia) remained consistently low, whereas expression of both Mac-1 (C3bR) and the macrophage-specific marker ER-BMDM-1 was always high. The interleukins IL-4 and especially IL-6 induced a premature expression of Fo at earlier stages of BMDM culture, but neither could promote further Fo expression once the intrinsically occurring maximum had been reached. No evidence in support of an autocrine regulation of Fo expression by IL-6 could be obtained, nor could a connection between cell cycle status and Fo expression be established. These data provide further evidence that Fo is a temporally regulated differentiation marker for a mouse macrophage subpopulation and for modulation of its expression by lymphokines.

Asunto(s)

Médula Ósea/metabolismo , Antígeno de Forssman/biosíntesis , Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales , Células de la Médula Ósea , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Ensayo de Inmunoadsorción Enzimática , Femenino , Antígeno de Forssman/análisis , Inmunohistoquímica , Interleucina-4/farmacología , Interleucina-6/farmacología , Cinética , Células L , Antígeno de Macrófago-1/análisis , Antígeno de Macrófago-1/biosíntesis , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Factores de Tiempo

RESUMEN

The CD22 antigen, although present in the cytoplasm of early and immature B cells, first appears on the cell surface of mature B lymphocytes. The phenotypic patterns and some functional properties of the surface-expressed CD22 antigen are well described, but little is known about its molecular structure. We have therefore investigated the relationship of the two CD22 glycoproteins (140/130 kd), and the influence of their complex N-glycosylation on surface expression and antibody recognition. Comparative peptide mapping of the 100 and 80 kd protein cores, obtained by endoglycosidase F treatment, revealed a common structure shared by both protein cores. In pulse-chase experiments the mature glycoproteins originated from two separate precursor molecules, indicating that the two proteins may be generated by different RNA processing. In cell lysates a CD22 specific polyclonal anti-serum recognized both molecules the size of the protein cores and glycosylated CD22 molecules, whereas in membrane preparations only the glycosylated forms were detected. The CD22 mAb HD39 reacted exclusively with the glycoprotein froms in either cellular preparation. The influence of glycosylation on surface expression and epitope recognition was investigated in more detail by applying various inhibitors of the glycosylation pathway. 1-Deoxymannojirimycin and Swainsonine, which block glycosylation at the high-mannose and hybrid-type stages respectively, modulated the CD22 antigen but did not alter its surface expression. Tunicamycin blocked de novo glycosylation and led to reduced surface recognition of the CD22 antigen. Together, these results suggested that comparatively simple oligosaccharide structures of high-mannose type are sufficient for surface expression of the CD22 antigen and for epitope recognition by mAb HD39. It is most likely that glycosylation is required to stabilize epitopes in the protein moiety recognized by CD22 mAb. Finally, we demonstrated the presence of glycosylated, cytoplasmic CD22 antigen in CD22 surface negative B-lineage ALL cells. This finding led us to conclude that complex glycosylation does not provide the determining signal for the switch from cytoplasmic to surface expression of the CD22 antigen.

Asunto(s)

Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Linfocitos B/inmunología , Lectinas , Anticuerpos Monoclonales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/citología , Secuencia de Carbohidratos , Carbohidratos/inmunología , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/metabolismo , Epítopos , Glicosilación , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Mapeo Peptídico , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Células Tumorales Cultivadas/inmunología

RESUMEN

The neutral glycosphingolipid (GSL) globotriaosylceramide (Gb3) of the globo-series was recently defined as the CD77 antigen. This B cell-associated antigen is characterized by its specific expression on germinal center B cells. In order to study the potential relation of the CD77 antigen and other GSLs to B cell activation we have performed a comprehensive analysis of the synthesis and expression of neutral GSL in tonsillar B lymphocytes. Monoglycosylceramide (GL1) and lactosylceramide (LacCer) comprised the largest portion of GSL in tonsillar B lymphocytes as detected by HPLC analysis. GSLs of the globo-series Gb3 and globotetraosylceramide (Gb4), were found in smaller amounts. Since other GSLs, like gangliotriaosylceramide (Gg3) and gangliotetraosylceramide (Gg4), could only be detected using highly sensitive antibody reactions, we assume that these GSLs occur in B cells only in minor amounts. When tonsillar B cells were density fractionated on Percoll, the light density cells, which correspond to activated cells, contained and expressed more of both globo-GSLs than cells in the higher density fraction. When the dense fraction of tonsillar B cells was activated in vitro by anti-mu/BCGF, synthesis of GL1, LacCer, Gb3, and Gb4 was biphasic, with maxima at 12 and 84 h. Surface expression of the CD77 antigen on the denser cells was strongly induced by anti-mu/BCGF during the first 24 h of cultivation followed by a rapid decline thereafter, mimicking synthesis. PMA treatment of this cell fraction caused an even stronger expression of the CD77 antigen, which lasted over 48 h of cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Antígenos CD , Linfocitos B/metabolismo , Glicoesfingolípidos/metabolismo , Lactosilceramidos , Activación de Linfocitos , Anticuerpos Monoclonales , Linfocitos B/inmunología , Cerebrósidos/inmunología , Cerebrósidos/metabolismo , Globósidos/inmunología , Globósidos/metabolismo , Glicoesfingolípidos/inmunología , Humanos , Técnicas In Vitro , Tonsila Palatina/citología , Trihexosilceramidas/inmunología , Trihexosilceramidas/metabolismo

RESUMEN

Forssman (Fo) glycolipid antigen, as detected by a monoclonal antibody (mAb), is expressed by a subpopulation of murine macrophages in the spleen and peripheral lymph nodes. The histological distribution of Fo antigen in spleen and lymph nodes was studied by immunostaining of cryosections, and was compared with the staining pattern of four other mAbs known to recognize macrophage subpopulations: F4/80, Mac-1, MOMA-1, and ERTR-9. Fo+ macrophages were found exclusively in the red pulp of the spleen and the medulla of inguinal and axial lymph nodes. Macrophages in the other lymphoid organs were Fo-. Besides macrophages, reticular cells in T-dependent areas of spleen and lymph nodes were Fo+. Attempts to grow colonies of Fo+ macrophages from either bone marrow or spleen precursors were negative. While the usual number of F4/80+ colonies was obtained, only a few, small clusters of Fo+ macrophages were formed, which speaks against an early commitment of precursors to express Fo.

Asunto(s)

Globósidos/análisis , Glicoesfingolípidos/análisis , Histiocitos/inmunología , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Bazo/inmunología , Animales , Anticuerpos Monoclonales , Médula Ósea/inmunología , Células Cultivadas , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos CBA , Distribución Tisular

RESUMEN

Mouse bone marrow cells were grown in liquid culture in microtiter plates in the presence of different colony-stimulating factors (CSF). Growth was assayed using the tetrazolium salt MTT, which is reduced in the mitochondria of viable cells to a water-insoluble blue formazan dye. Two technical problems have limited the use of this assay: the solubilization of the dye crystals and the necessity to acidify the phenol red in the culture medium. Both could be solved here by the use of a developing solution of 5% formic acid in isopropanol. Using manual mixing combined with a short sonication by floating the plates in a sonic bath, the crystals were dissolved within minutes. There was no flocculation of protein, even using medium with 20% serum. The color remained stable for at least 4 h. This enabled the semi-automatic measurement of large numbers of cultures directly in the microtiter plates. Growth and differentiation of myelopoietic precursor cells in the liquid cultures was shown to be comparable to that in soft agar. Cell growth was CSF-dependent. The calculated cell yield per colony forming cell (CFC) seeded was within the range of the average cell number per colony found in soft agar, and the spectrum of mature cells obtained reflected the type of CSF used as stimulus. Using the combined culture and assay systems, it was possible to perform detailed kinetic studies of myelopoiesis. This technique should be useful for studying the mechanisms of action of pharmacological modulators of myelopoiesis.

Asunto(s)

Células de la Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Hematopoyesis , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Factores Estimulantes de Colonias/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Sustancias de Crecimiento/farmacología , Hematopoyesis/efectos de los fármacos , Interleucina-3/farmacología , Factor Estimulante de Colonias de Macrófagos , Ratones , Oxidación-Reducción , Sales de Tetrazolio , Tiazoles

RESUMEN

Methods for the production of high titers of interleukin-2 (IL-2) from human buffy coat lymphocytes, and subsequent purification of the IL-2 are described. 50 buffy coats containing 1 X 10(11) leukocytes were first depleted of erythrocytes by batchwise leukapheresis using a Haemonetics model 15 blood wash centrifuge. Further lymphocyte enrichment was achieved using a one-step sedimentation in the presence of hydroxyethyl starch, which produced suspensions of more than 90% lymphocytes. This degree of lymphocyte purity was important since phagocytes were inhibitory to 12-O-tetradecanoylphorbol 13-acetate/calcium ionophore (TPA/A23187)-induced IL-2 production when their concentration exceeded 15% of the total cells. Cell culture was performed in stirred fermenters. Using TPA/A23187 induction, up to 500 micrograms of IL-2 per liter were produced. The IL-2 was purified by absorption from the supernatants onto controlled pore glass and elution with 50% ethylene glycol, followed by Fractogel chromatography, and then preparative high-performance liquid chromatography (HPLC) using an RP-6 column and elution with a gradient of n-propanol. A final HPLC rechromatography step using an analytical RP-6 column gave a homogeneous preparation with specific activity of 1.2 X 10(7) U/mg and a recovery from the starting supernatant of 22%.

Asunto(s)

Interleucina-2/aislamiento & purificación , Linfocitos/análisis , Calcimicina/farmacología , Separación Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Sinergismo Farmacológico , Humanos , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

To improve immune functions in an interleukin-2 (IL-2) deficient hemophiliac AIDS patient suffering from severe Pneumocystis carinii pneumonia, treatment with IL-2 was started in addition to standard antimicrobial therapy. Highly purified IL-2 was administered subcutaneously and then repeatedly intralymphatically in a manner similar to pedal lymphography. No toxicity was observed. The patient temporarily improved clinically as well as with regard to immunological functions. Particularly the in vitro response to phytohemagglutinin (PHA) could partly be restored, and skin tests revealed improved response to recall antigens. These findings indicate that IL-2 can be administered safely and effectively by the intralymphatic route and may--in addition to antibiotics--be of value in AIDS patients with severe opportunistic infections.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/terapia , Hemofilia B/inmunología , Interleucina-2/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adulto , Citotoxicidad Inmunológica , Humanos , Inyecciones Intralinfáticas , Interleucina-2/biosíntesis , Activación de Linfocitos , Masculino , Neumonía por Pneumocystis/terapia

RESUMEN

Patients with opportunistic infections during the course of acquired immunodeficiency syndrome (AIDS) were analyzed for cellular immune functions and found to be severely immunocompromised. In particular, interleukin 2 (IL 2) production appeared to be defect not only qualitatively but also quantitatively. In some of these patients, exogenous IL 2 improved immune response in vitro. Intralymphatically administered highly purified natural IL 2 was given repeatedly (over a time period of ten days) to three of these patients. In two cases, such a treatment course was repeated later. Clinical response - at least in some patients - appeared to be of temporary benefit. Shortly after termination of IL 2 application in two patients an increase of lectin responsiveness as well as improved reactivity in skin testing was noted, encouraging further exploration of IL 2 as an immunostimulatory drug in AIDS patients.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/terapia , Interleucina-2/administración & dosificación , Adulto , Antígenos de Diferenciación de Linfocitos T , Antígenos de Superficie/análisis , Humanos , Células Asesinas Naturales/inmunología , Ganglios Linfáticos , Activación de Linfocitos , Infecciones Oportunistas/terapia , Neumonía por Pneumocystis/terapia , Linfocitos T/clasificación , Linfocitos T/inmunología

RESUMEN

The mouse lymphoma cell line Eb, its highly metastasizing variant ESb, and an unrelated metastasizing tumor MDAY-D2, were shown to produce colony-stimulating factor (CSF) constitutively both in vitro and in vivo in ascites. For each tumor, the amounts of CSF produced on a per-cell basis in vitro and in vivo were similar. The findings were substantiated using two different methods for CSF determination, a colony assay and an isotope incorporation test. Elevated levels of CSF in serum of mice with tumors were also found. Examination of blood from tumor-bearing mice revealed that whereas total leukocyte counts remained within the normal range, all three tumors caused a reversal of the normal neutrophil-lymphocyte ratio. The severity of the reversal correlated with the propensity of the tumor to elevate serum CSF rather than with in vitro CSF-producing capacity. Thus, whereas production of CSF in vivo was not related to the ability of the tumors to metastasize, it could be causative in creating an imbalance in normal hematopoiesis.

Asunto(s)

Factores Estimulantes de Colonias/biosíntesis , Granulocitos/citología , Leucemia L5178/metabolismo , Leucemia Experimental/metabolismo , Linfoma/metabolismo , Animales , Línea Celular , Células Cultivadas , Leucemia L5178/sangre , Recuento de Leucocitos , Linfoma/sangre , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación

RESUMEN

Absolute capacity for IL2 production by human peripheral blood mononuclear cells (PBMC) was studied using 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187, which act synergistically, to induce lymphokine synthesis. Culture parameters were optimized for the TPA/A23187 stimulation such that maximal IL2 titers were produced with a high degree of reproducibility. Thus, using a synthetic medium, TPA/A23187 at 20/50ng/ml respectively, and a cell concentration of 2.5 X 10(6)/ml, IL2 titers in the cultures increased linearly over a period of 96h, reaching values at least 15-fold higher than with lectin stimulation. This allowed for determinations of IL2 synthetic capacity in individual blood samples. Large fluctuations in normal IL2 production (range 1775-10654 BRMP U/ml at 48h) were observed among 23 normal persons. A statistically significant lower IL2 productive capacity was observed in the age group above 40 as compared to those under 40. The lower rates of IL2 synthesis in a group of patients with Hodgkin's disease was seen only among those who had undergone immunosuppressive therapy; newly diagnosed cases fell within the normal range.

Asunto(s)

Interleucina-2/biosíntesis , Leucocitos/inmunología , Linfocinas/biosíntesis , Calcimicina/farmacología , Enfermedad de Hodgkin/inmunología , Humanos , Técnicas In Vitro , Fitohemaglutininas/farmacología , Valores de Referencia , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Neutral glycosphingolipids (neutral GSLs) of the human myeloid leukemia cell lines ML-2, ML-3, HL-60 and THP-1-0 were metabolically labeled with [3H]galactose and [3H]glucosamine, and analyzed by high-performance liquid chromatography. They were compared with unlabeled neutral GSLs from purified human granulocytes and monocytes. Neutral GSLs were identified by retention times and the structures were further confirmed by degradation with specific exoglycosidases. Two neutral GSLs of the globoseries, globotetraosylceramide and globotriaosylceramide were found in monocytes and the monoblastic leukemia line THP-1-0. The leukemia-derived cell-lines, ML-3 and HL-60, representing successively earlier stages of myeloid differentiation, contained respectively less neutral GSLs of the globoseries and an increasing proportion of (neo)lacto neutral GSLs. Granulocytes and the cell line ML-2 contained almost exclusively neutral GSLs of the (neo)lacto series.

Asunto(s)

Glicoesfingolípidos/sangre , Leucemia Mieloide/sangre , Monocitos/metabolismo , Diferenciación Celular , Línea Celular , Cromatografía Líquida de Alta Presión , Galactosa/metabolismo , Glucosamina/sangre , Glicoesfingolípidos/clasificación , Glicoesfingolípidos/aislamiento & purificación , Granulocitos/metabolismo , Humanos

RESUMEN

This report describes a clinical trial with Interleukin 2 (IL-2) on a 17-month old male child with combined immunodeficiency (Nezelof's syndrome). IL-2 was prepared from conditioned media of phytohemagglutinin-stimulated leukocytes from buffy coats. The purification of IL-2 involved chromatography on Matrex Blue A sepharose and gel filtration chromatography. The preparation was free of macrophage cytotoxicity factor, macrophage migration inhibition factor and colony-stimulating factor. It contained negligible activity of interferon-gamma. IL-2 activity was adjusted to 1600 U/ml, which corresponds to about 0.8 micrograms homogeneous IL-2/ml. The patient was treated over a 50-day period with a total dose of 20,000 U IL-2, which was injected subcutaneously. IL-2 was well tolerated. Within 3 weeks, the treatment led to a normalization of a lymphocytosis which had prevailed for the previous 3 months. A pronounced eosinophilia also improved but did not reach normal levels. The most striking effect was a normalization of the OKT4+/OKT8+ ratio with a concomitant relative increase in OKT3+ cells in the peripheral blood. No effects were seen on E rosette formation, B cell counts or serum Ig levels. Also NK or ADCC activity remained high, as before the treatment. Infectious episodes and requirement for antibiotic treatment were less frequent during IL-2 therapy. Some effects of IL-2 were transient, e.g., the counts of OKT4+ and OKT3+ cells which returned to pathological values a few weeks after the treatment was discontinued.

Asunto(s)

Síndromes de Inmunodeficiencia/terapia , Interleucina-2/administración & dosificación , Linfocitos/inmunología , Humanos , Inmunidad Celular , Síndromes de Inmunodeficiencia/inmunología , Inmunoterapia , Lactante , Células Asesinas Naturales/inmunología , Linfocitos/clasificación , Masculino , Linfocitos T/inmunología

RESUMEN

Biochemical analyses of murine lymphocytes have shown that the glycosphingolipid globoside (Glo) is present exclusively on alloantigen-stimulated murine T lymphocytes (Gruner, K. R., Van Eijk, R. V. W. and Mühlradt, P. F., Biochemistry 1981. 20: 4518). An anti-Glo antibody has now been raised in rabbits immunized with purified antigen. Most activity was recovered in the IgM fraction. The specificity of the antibody was ascertained in an enzyme-linked immunosorbent assay with purified glycosphingolipids bound to the solid phase. In antibody-dependent complement lysis experiments the anti-Glo eliminated about 20% of nylon wool-nonadherent splenic T cells of CBA/J mice. To determine the functional identity of these Glo+ cells, the effects of Glo+ cell elimination on mitogen stimulation with concanavalin A and lipopolysaccharide, as well as the effects on the mixed lymphocyte culture (MLC) reaction and cell-mediated lympholysis with mitomycin-treated DBA/2 splenocytes as stimulator cells were studied. Whereas lipopolysaccharide stimulation was not affected by elimination of Glo+ cells, there was a slight inhibitory effect on the concanavalin A stimulation, and a severe inhibition of the MLC reaction and the generation of H-2d-specific cytolytic T lymphocytes. Addition of interleukin 2 increased the MLC reaction, but interleukin 2-saturated cultures were also severely inhibited by anti-Glo and complement treatment. Combined treatment with anti-Glo and anti-Lyt-1 or anti-Lyt-2 antibodies, and determination of cytolytic T lymphocyte precursor frequencies in limiting dilution cultures after Glo+ cell elimination showed that a large proportion of T cells proliferating in a primary MLC are Lyt-1+,2+,3+Glo+, whereas in secondary MLC they are Lyt-1+,2-,3-,Glo+. Fifty % of the cytolytic T lymphocyte precursors in primary as well as secondary MLC are Glo+. The Glo marker is lost upon differentiation to cytolytic T lymphocyte effector cells. It is discussed herein that Glo is a marker for alloantigen-stimulated precursor T lymphocytes of both helper and cytolytic T cells.

Asunto(s)

Globósidos/análisis , Glicoesfingolípidos/análisis , Isoantígenos/inmunología , Linfocitos T Citotóxicos/citología , Animales , Especificidad de Anticuerpos , Proteínas del Sistema Complemento/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Lípidos de la Membrana/análisis , Ratones , Ratones Endogámicos , Conejos , Bazo/citología

RESUMEN

Interleukin 2 was purified 100 000-fold to apparent homogeneity from the supernatants of mitogen-stimulated human blood leukocytes. A sequence of three purification steps was used: affinity chromatography on the bound dye cibacron blue, gel filtration on Ultrogel AcA44, and reversed-phase high-performance liquid chromatography on hexyl phase. The resulting interleukin 2 had a specific activity of 2 X 10(6) U/mg protein, and was free of pyrogenicity in the rabbit test. The final purification product showed two bands in sodium dodecyl sulfate/polyacrylamide gels with apparent molecular masses of 15 kDa and 17 kDa respectively. Both bands were biologically active.

Asunto(s)

Interleucina-2/aislamiento & purificación , Leucocitos/análisis , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Medios de Cultivo/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Peso Molecular

RESUMEN

A new method is presented for assaying mouse colony stimulating factor (CSF) based on the presumption that one of the earliest events after the induction of differentiation or maturation is an increase in synthesis and/or turnover of glycoconjugates in the target cell plasma membranes which may be detected as an increase in incorporation of [3H]galactose into acid precipitates of these cells. Using mouse bone marrow cells cultured in microtiter plates, we show that addition of CSF indeed results in dose-dependent stimulation of incorporation of galactose into their membrane glycoconjugates. Maximum stimulation of galactose incorporation occurs between 16 and 24 h of culture in the presence of CSF. A comparison of the standard colony test with the galactose incorporation assay showed similar dose-response patterns with 2 different CSFs. In a 3-step separation of bone marrow cells, there was a parallel enrichment of target cells for both assays. Furthermore, [3H]galactose-labeled marrow cells from our assay formed a discrete subpopulation which banded at a density of 1.065 g/cm3 on a linear gradient, and which contained all the identifiable CFU-c when further cultured in soft agar. The galactose incorporation assay does not require special medium or serum, and is simpler and quicker than the colony test. In comparative experiments it is demonstrated that this new 24 h assay is also reliable for screening CSF during purification procedures.

Asunto(s)

Células de la Médula Ósea , Factores Estimulantes de Colonias/fisiología , Glicoproteínas/biosíntesis , Hematopoyesis , Proteínas de la Membrana/biosíntesis , Animales , Médula Ósea/metabolismo , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Galactosa/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA

RESUMEN

Three cell lines, isolated from different patients with Hodgkin's disease, were analyzed for their neutral glycosphingolipids by high performance liquid chromatography. On two of these lines gangliotriaosylceramide (asialo GM2) was identified by the comparison of retention time to authentic standards and by degradation with specific glycosidases. This glycosphingolipid was also serologically detected with a specific monoclonal antibody of the IgM class. The third line, which according to other criteria such as the Epstein Barr virus nuclear antigen differs phenotypically from the first two, did not exhibit gangliotriaosylceramide. This glycosphingolipid is an unusual marker for human cells and could not be detected on the peripheral blood leukocytes of healthy donors.

Asunto(s)

Gangliósido G(M2)/inmunología , Gangliósidos/inmunología , Enfermedad de Hodgkin/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/análisis , Línea Celular , Cromatografía Líquida de Alta Presión , Antígenos Nucleares del Virus de Epstein-Barr , Humanos , Ratones

RESUMEN

The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event.

Asunto(s)

Médula Ósea/fisiología , Hematopoyesis/efectos de los fármacos , Lipopolisacáridos/farmacología , Lipoproteínas/farmacología , Peptidoglicano/farmacología , Médula Ósea/metabolismo , Adhesión Celular , Galactosa/metabolismo , Proteus mirabilis/fisiología , Timidina/metabolismo